THE 100 YEARS OF THE FUNGUS COLLECTION

MUCL
1894-1994

Fungal Taxonomy and Tropical Mycology:

Quo vadis ?

Taxonomy and Nomenclature of the Fungi

Grgoire L. Hennebert

Catholic University of Louvain, Belgium

 Notice of the editor
This document is now published as an archive
It is available on www.Mycotaxon.com
It is also produced on CD and in few paperback copies
G. L. Hennebert ed.

Published by Mycotaxon, Ltd.

Ithaca, New York, USA
December 2010
ISBN 978-0-930845-18-6 (www pdf version)
ISBN 978-0-930845-17-9 (paperback version)
DOI 10.5248/2010MUCL.pdf
 1894-1994 MUCL Centenary
CONTENTS

Lists of participants 8
Forword
John Webser 13

PLENARY SESSION
The 100 Year Fungus Culture Collection MUCL, June 29th, 1994
G.L. Hennebert, UCL
Mycothque de l'Universit Catholique de Louvain (MUCL) 17
D. Hawksworth, IMI, U.K.
Fungal genetic resource collections and biodiversity. 27
D. van der Mei, CBS, MINE, Netherlands
The fungus culture collections in Europe. 34
J. De Brabandere, BCCM, Belgium
The Belgian Coordinated Collections of Microorganisms. 40

Fungal Taxonomy and tropical Mycology

G.L. Hennebert, UCL
Introduction. Fungal taxonomy and tropical mycology: Quo vadis ? 41
C.P. Kurtzman, NRRL, USA
Molecular taxonomy in the yeast fungi: present and future. 42
M. Blackwell, Louisiana State University, USA
Phylogeny of filamentous fungi deduced from analysis of molecular characters:
present and future. 52
J. Rammeloo, National Botanical Garden, Belgium
Importance of morphological and anatomical characters in fungal taxonomy. 57
M.F. Roquebert, Natural History Museum, France
Possible progress of modern morphological analysis in fungal taxonomy. 63
A.J. Masuka, Forest Research Center, Zimbabwe
Mycofloristic studies in South Central Africa: status, constraints,
opportunities and strategies. 68
A. Peerally, University of Mauritius, Mauritius
Strategies for the development of tropical mycology. 83
E.J. Da Silva, MIRCEN, UNESCO, France
The role of the Microbiological Resources Centers (MIRCEN) in the
development of mycology in the tropics. 92

 Discussion and conclusion 96

POSTERS
Fungus Collections and Services

The Mycothque de l'Universit Catholique de Louvain (MUCL) 100 years

1894-1994 Grgoire L. Hennebert 115

MUCL, a Public Service Collection. C. Decock, Ch. Moulliard, L. Nlissen, P.

Massart, P. Evrard, N. Jamin, P. Charue and G.L. Hennebert 116

MUCL, a Fungus Culture Collection, service to African fundamental and applied

mycology. - Grgoire L. Hennebert 118

The activities and mycological collections of the Belgian National Botanical

Garden (BR) - Andr Fraiture 120

Classical Taxonomy of the Fungi

Anamorph-teleomorph connectionsin the Xylariaceae. - M.A. Whalley, G.P.
Sharples and A.J.S. Whalley 123

Anamorphs and the classification of xylariaceous fungi.- K. Van Der Gucht 133

First occurrence in nature of the teleomorph of Cochliobolus . - R. Raemaekers

and J. Coosemans 137

Pyxidiophora, a possible teleomorph of Pleurocatera acicularis. - Gnter R.W.

Arnold 142

Illustrations of the cultural features of Gyrodontium boveanum (Montagne) Maas

Geesteranus - Cony Decock and Anxious J. Masuka 145

The Lactarius gymnocarpus complex in tropical Africa - Annemieke Verbeken

(abstract) 146

Scorias spongiosa (Schw.)Fries from Kenya - R. Mibey (abstract) 147

An interesting species of Puccinia on Gentiana - Evangelia Kapsanaki-Gotsi

and I. Vovou (abstract) 147

Cultural characteristics and their taxonomic potential in Lentinellus species - Zapi

Gonou-Zagou and Maria Pantidou (abstract) 147

Responses to the thermal stress in the inter-sterility groups of Ganoderma australe

in Taiwan - Zuei-Ching Chen (abstract) 148

Molecular Taxonomy
18S rRNA and the fungal tree: evolutionary relationships among ascomycetes,
basidiomycetes, zygomycetes, and chytridiomycetes. - Yves Van de Peer,
Grgoire L. Hennebert and Rupert De Wachter 149

A rapid molecular technique to distinguish Cylindrocladium species. - P.W.

Crous, A. Korf and W.H. Van Zyl 153

Identification of basidiomycetous yeasts using whole-cell protein electrophoresis

Marc Vancanneyt, Grgoire L. Hennebert and Karl Kersters 158

Phytopathogenic filamentous (Ashbya, Eremothecium) and dimorphic fungi

(Nematospora) with falcate ascospores as new members within the Saccharo-
mycetaceae. H. Prillinger, R. Messner, M. Breitenbach, P. Briza, E.
Staudacher, O. Molnr, F. Weigang, K. Lopandic, M. Ibl & G. Himmler. 162

Genetic Organization of the Yeast Yarrowia lipolytica. - Serge Casaregola, Huu

Vang Nguyen, Chantal Feynerol, Monique Diez and Claude Gaillardin 168

Taxonomic revision of Saccharomyces sensu stricto strains of NCAIM. - J.

Tornai-Lehoczki., D. Dlauchy.and G. Peter 169

Phylogenic relationships within the Saccharomyces cerevisiae complex species. -

Dilnora E. Gouliamova and Grgoire L. Hennebert 174

What about the "species group" concept in Aspergillus flavus ? - Joelle Dupont,
Marie-France Roquebert and Evelyne Guho (abstract) 177

A comparative study of electrophoretic karyotypes in the yeast genera

Torulaspora Lindner and Zygosaccharomyces Barker - Guido Capriotti and Ann
Vaughan-Martini (abstract) 178

Fungal biodiversity
Biodiversity of Cylindrocladium on water-lilies at SSR Pamplemousses Botanic
Garden in Mauritius. - Abed Peerally 179

The occurrence of wood decaying fungi in Belgium houses - Cony Decock and
Grgoire L. Hennebert 185

Symbiotic fungi in the galleries of the striped bark beetle, Trypodendron lineatum)
(Coleoptera, Scolytidae). - G. Babuder and F. Pohleven 194

Cercosporoid Fungi associated with the Eyespot Disease of graminicolous Hosts. -

G.F. Campbell, B. Robbertse, P.W. Crous and B.J.H. Janse 198

 Diversity of Colletotrichum gloeosporioides pathogenic to Stylosanthes sp. in

tropical areas. - F. Munaut, N. Hamaide and H. Maraite 205

Diversity in non-specific foliar pathogens of wheat from non-traditional warm

areas. - Di Zinno T., Duveiller E., Longre H. and H. Maraite 208

Pathogenic diversity in Magnaporthe grisea, the rice blast pathogen. - J.B.

Bahama, J.L.Notteghem and H.Marate 211

Characterization of Polymyxa graminis involved in the transmission of Peanut

Clump Virus in tropical areas. - A. Legreve, B. Vanpee, P. Delfosse, D. V. R.
Reddy and H. Maraite 214

Practical Mycology
Practical Advice for collecting wood-rotting Fungi in the Tropics. Erast
Parmasto 219

The influence of preparation on ascospore measurements. - Bellis Kullman, Aivo

Jakobson and Mart Rahi 221

Microplate technique of physiological tests for Yeast identification - Pierre

Evrard and Grgoire L. Hennebert (abstract) 226

Cryopreservation of Aspergillus repens - David Smith (abstract) 227

Cultivation of the fungi

Activated charcoal-mediated sporophore initiation in Agaricus and Tricholoma -
Abed Peerally. 228

In vitro Culture of endomycorrhizal Fungi: why hat interest? - S. Declerck, T.

Diop, C. Plenchette and D.G. Strullu. 232

Enhancement of carpophore production and protein content of Pleurotus spp. by

cultivation on grass hay. - Kandanda Tshinyangu (abstract) 236

Is the rust of the Creeping Thistle (Cirsium arvense) the microsymbiont of its
VAM? - Hartmut Heilmann (abstract) 236

Morphological studies of mycelia from ectomycorhizian fungi cultivated in vitro -

J. Jeanfils and J.-J. Cuvelier (abstract) 237

Fungal Biotechnology
Yeast fungi and alcohol from bamboos. - Francis S.S. Magingo 238

Growth of Fusidium coccineum on solid Media. - Helen E. Smith and

Glyn Hobbs 241

 Strain polymorphism in PDR1, a transcription regulator of pleiotropic drug

resistance genes in Saccharomyces cerevisiae. - E. Carvajal, E. Balzil and A.
Goffeau (abstract) 246

Structural identification and biochemical mode of action of a mycotoxine isolated

from Cochliobolus sativus MUCL 18528. - D. Vilret, P. Goblet, J.
Hutschemackers, N. Cosyns, M. Nol and M. Briquet (abstract) 247

Characterization of the carbohydrate moiety on proteins secreted from different

fungi. - M. Maras, W. Laroy, W. Fiers, G.L Hennebert and R. Contreras
(abstract) 247

Specific and non-specific interactions in yeast cell flocculation. - P.B. Dengis,

L.R. Nlissen, and P.G. Rouxhet (abstract) 248

Modification of support surface properties to improve microorganism adhesion -

P.A. Gerin, M.-N. Bellon-Fontaine, M. Asther & P.G. Rouxhet (abstract) 249

Bacteriology
Identification of Lactic Acid Bacteria using RAPD-PCR. - A. Nol and J.
Decallonne 250

Gas Chromatographic Analysis of Cellular Fatty Acids in the Identification of

Microorganisms. - P. Wauthoz, M. El Lioui and J. Decallonne 252

LAST LECTURE, 5 December 1995

Taxonomy and Nomdenclature of the fungi:Reasons for a Mycotheque.

Grgoire L. Hennebert 257

 LIST OF PARTICIPANTS

Ainsworth A. Martyn, Applied Mycology Department, Xnova Ltd, Ipswich Road 545, Slough, SL1
4EQ, Bucks, United Kingdom.
Antoine Raymond, "Le Sous-Bois", Chemin des Vignerons 32, B-5100, Wpion, Belgium.
Aptroot Andr, Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740, AG Baarn, Netherlands.
Arnold Gnter, Friedrich-Schiller-Universitt Jena, Biologische Fakultt, Pilzkulturensammlung,
Freiherr-vom-Stein-Allee 2, 99425, Weimar, Germany.
Babuder Gorazd, Biotechnical Faculty, Department of Wood Science and Technology, University of
Ljubljana, Rozna dolina Cesta VIII/34, Vecna pot 2, 61000, Ljubljana, Slovenia.
Bahama Jean-Baptiste, Clinique des Plantes, Unit de Phytopathologie - FYMY, Dpartement BAPA,
Facult des Sciences Agronomiques, UCL, Place Croix du Sud 2 bte 3, B-1348, Louvain-la-
Neuve, Belgium.
Beblowska Marta, Zaklad Systematyki i Gaeografii Roslin, Warsaw University, Aleje Ujazdowskie 4,
00-478, Warsaw, Poland. Email: [email protected]
Belloch Carmela, Coleccion Espanola de Cultivos Tipo - CECT, Facultad de Ciencias Biologicas,
Departamento de Microbiologia, Universidad de Valencia, C/Dr. Moliner 50, 46100, Burjassot,
Spain. Email: [email protected]
Blackwell Meredith, Department of Botany, Louisiana State University, Life Sciences Building 502,
Baton Rouge, 70803-1705, Louisiana, United States. Email: [email protected]
Bonneff Eric, Laboratoire de Microbiologie Industrielle, Universit de Reims / Champagne-Ardenne,
U.R.C.A., UFR Sciences Exactes et Naturelles, Moulin de la Housse B.P. 347, 51062, Reims
Cdex, France.
Briquet Michel, Unit de Biochimie Physiologique FYSA, Dpartement CABI, Facult des Sciences
Agronomiques, UCL, Place Croix du Sud 2 Bte 20, B-1348, Louvain-la-Neuve, Belgium.
Buffin de Chosal Nathalie, Guido Bezellestraat 7, B-8970, Poperinge, Belgium.
Caeymaex Louis, Bureau Marcel van Dijk, Avenue Louise 250 Bote 14 , B-1050, Bruxelles, Belgium.
Cahagnier Bernard, Laboratoire de Microbiologie et Technologie Cralires, Centre de Recherches de
Nantes, Institut National de la Recherche Agronomique, B.P. 527, 44026, Nantes Cdex, France.
Casaregola Serge, Laboratoire de Gntique Molculaire et Cellulaire, Collection de Levures d'Intrt
Biotechnologique, Institut National de la Recherche Agronomique, 78850, Thiverval-Grignon,
France.
Castillo Cabello Gabriel, Dp. de Botanique (B.22), Universit de Lige, B-4000, Lige, Belgium.
Cerisier Yvanne, Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Rue du Docteur
Roux 25, 75015, Paris Cdex 15, France.
Chasseur Camille, Institut d'Hygine et d'Epidmiologie, IHE, Rue Juliette Wijtsman 14, B-1050,
Bruxelles, Belgium.
Chen Zuei-ching, The Mycological Society of the Republic of China, Mycology Laboratory, Department
of Botany, National Taiwan University, Roosevelt Road, Section 4 N1, Taipei, 10764, Taiwan,
China.
Cimerman Aleksa, Culture Collection of Fungi, Boris Kidric Institute of Chemistry, Hajdrihova 19, P.O.
Box 30, 61115, Ljubljana, Slovenia. Email: [email protected]
Contreras Roland, Laboratorium voor Moleculaire Biologie, Rijksuniversiteit Gent - RUG, K.L.
Ledeganckstraat 35, B-9000, Gent, Belgium. Email: [email protected]
Coosemans Jozef, Laboratorium Fytopathologie en Plantenbescherming, Faculteit der
Landbouwwetenschappen der Leuven, Katholieke Universiteit Leuven, Willem De Croylaan 42,
B-3001, Heverlee, Belgium.
Crous Pedro W., Department of Plant Pathology, University of Stellenbosch, Private bag X5018, 7600,
Stellenbosch, South Africa. Email: [email protected]
Cuvelier Jean-Jacques, Facult de Mdecine, Universit Mons-Hainaut, Avenue du Champ de Mars 8,
B-7000, Mons, Belgium
Danckaert Jan, Kabinet Wetenschapbeleid, Wetstraat 155/16, B-1040, Brussels, Belgium.,
De Brabandere Jan, Services Fdraux des Affaires Scientifiques, Techniques et Culturelles, Rue de la
Science 8, B-1040, Bruxelles, Belgium.
De Bruyne Erik, SES Europe s.a., Industriepark 15, B-3300, Tienen, Belgium.
De Wachter Rupert, Departement Biochemie, Fakulteit Wetenschappen, Universitaire Instelling
Antwerpen - UIA, Universiteitsplein 1, Btiment T, B-2610, Antwerpen, Belgium. Email:
[email protected]
Decallonne Jacques, Laboratoire de Bactriologie, Unit de Microbiologie MBLA, Dpartement CABI,
Facult des Sciences Agronomiques, UCL, Place Croix du Sud 2 Bte 12, B-1348, Louvain-la-
Neuve, Belgium.

Declerck Stphane, Laboratoire d'Agronomie, Institut National de la Recherche Agronomique - INRA,

B.P. 1540, Rue Sully 17, 21034, Dijon, France.
Decock Cony, MUCL, 2 Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgique, E-mail :
[email protected]
Degreef Jrme, Laboratoire d'Ecologie, Facult des Sciences Agronomiques de Gembloux, Passage des
Dports, B-5030, Gembloux, Belgium.
Delvaux Bruno, Unit des Sciences du Sol PEDO, Dpartement MILA, Facult des Sciences
Agronomiques, UCL, Place Croix du Sud 2 Bte 10, B-1348, Louvain-la-Neuve, Belgium.Phone:
010-473686, Email: [email protected]
Di Zinno Tonio, Clinique des Plantes, Unit de Phytopathologie - FYMY, Dpartement BAPA, Facult
des Sciences Agronomiques, Universit Catholique de Louvain - UCL, Place Croix du Sud 2 bte
3, B-1348, Louvain-la-Neuve, Belgium., Email: [email protected]
Diederich Paul, Rue de la Solidarit 22, 8020, Strassen, Luxembourg.
Doyle Alan, European Collection of Animal Cell Cultures, Pathology Division, PHLS Centre for Applied
Microbiology and Research, Porton Down, Salisbury, SP4 0JG, Wiltshire, United Kingdom.
Dupont Jolle, Laboratoire de Cryptogamie, Musum National d'Histoire Naturelle, Rue de Buffon 12, F-
75005, Paris, France
Fattori Maria, SES Europe s.a., Industriepark 15, B-3300, Tienen, Belgium
Fisch Colette, Laboratoire Intercommunal de Chimie et Bactriologie, Rue du Maelbeek 3, B-1040,
Bruxelles, Belgium.
Fraiture Andr, Jardin Botanique National de Belgique, Domaine de Bouchout, Brusselsesteenweg 8-26,
B-1860, Meise, Belgium
Gabor Peter, National Collection of Agricultural & Industrial Microorganisms, Department of
Microbiology and Biotechnology, University of Horticulture and Food Industry, Somloi ut 14-16,
1118, Budapest, Hungary.
Gams Walter, Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740, AG Baarn, Netherlands.
Email: [email protected]
Gigi Jacques, Cliniques Universitaires St-Luc, Laboratoire de Microbiologie, Universit Catholique de
Louvain - UCL, Avenue Hippocrate 10, B-1200, Bruxelles, Belgium.
Girlanda Mariangela, Centro di Studio sulla Micologia del Terreno, Dipartimento di Biologia Vegetale
dell'Universita, Consiglio Nazionale delle Ricerche - CNR, Viale Mattioli 25, 10125, Torino,
Italy.
Godden Bernard, Laboratoire de Microbiologie, Unit de Physiologie et d'Ecologie Microbienne, Facult
des Sciences, Universit Libre de Bruxelles - ULB, Institut Pasteur du Brabant, Rue Engeland
642, B-1180, Bruxelles, Belgium.
Gonou Zagou Zapi, Section of Ecology & Systematics, Department of Biology, University of Athens,
Panepistimiopolis, 15784, Athens, Greece.
Gouliamova Dilnora, MUCL, 2 Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgique,
Grler Blent, Centre for Research and Application of Culture Collections of Microorganisms,
Microbiology Department, Istanbul Faculty of Medicine, Temel Bilimler Binasi, 34390, Capa-
Istanbul, Turkey.
Halama Patrice, Institut Suprieur d'Agriculture, Rue du Port 41, 59046, Lille Cdex, France.
Hawksworth David L., International Mycological Institute - IMI, Bakeham Lane, Englefield Green,
Egham, TW20 9TY, Surrey, United Kingdom.
Hennebert Alexis-Jean, Clos des Salanganes 15, B-1150, Bruxelles, Belgium.
Hennebert Gregoire Laurent, MUCL, 2 Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgique,
Phone 010-473742, E-mail : [email protected]. Priv : 32 Rue de lElevage, 1340
0ttignies-LLN, Belgique
Hennebert Paul, Facult de Mdecine, UCL, Ophemstraat 81, B-3050, Oud-Heverlee, Belgium.
Hughes Stanley J., 360 Hamilton Avenue, Ottawa , K1Y 1C6, Ontario, Canada.
Ilnicka-Olejniczak Olga, Technical Microbiology and Biochemistry Department, Institute of Agro-Food
Biotechnology, Rakowiecka Street 36 , 02-532 , Warszawa, Poland.
Janssens Danielle, Laboratorium voor Microbiologie en Microbile - LMG, Rijksuniversiteit Gent - RUG
, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium., Email: [email protected]
Jeanfils Joseph, Facult de Mdecine, Universit Mons-Hainaut, Avenue du Champ de Mars 8, B-7000,
Mons, Belgium.
Jones Gareth E.B., School of Biological Sciences, University of Portsmouth, King Henry Building, King
Henry 1 Street , PO1 2DY, Portsmouth, Hants, United Kingdom.
Kapsanaki-Gotsi Evangelia, Department of Biology, Section of Ecology and Systematics, University of
Athens, Panepistimiopolis, 15784, Athens, Greece.

Kersters Karel, Laboratorium voor Microbiologie en Microbiele Genetica - LMG, Faculteit

Wetenschappen, Rijksuniversiteit Gent - RUG, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium.
Knuyt Walter, Services Fdraux des Affaires Scientifiques, Techniques et Culturelles, Rue de la
Science 8, B-1040, Bruxelles, Belgium.
Korf Richard P., College of Agriculture and Life Sciences, Plant Pathology Herbarium, Cornell
University, Plant Science Building 401, Ithaca, 14853-4203, New York, United States. Email:
[email protected]
Kost Gerhard, Fachbereich Biologie, Botanik/Mykologie, Philipps Universitt Marburg, 35032,
Marburg, Germany.
Kracht Manfred, Deutsche Sammlung von Mikroorganismen - DSM, Mascheroder Weg 1B, 3300,
Braunschweig, Germany.
Kullman Bellis, Institute of Zoology and Botany, Estonian Academy of Sciences, Vanemuise St. 21,
EE2400, Tartu, Estonia. Email: [email protected]
Kurtzman Cletus P., United States Department of Agriculture, Northern Regional Research Center U.S.,
Midwest Area, 1815 North University Street, Peoria, 61604, Illinois, United States
Laurent Serge, Fast Net sa, Avenue du Bempt 41, B-1190, Bruxelles, Belgium.
Legrve Anne, Clinique des Plantes, Unit de Phytopathologie - FYMY, Dpartement BAPA, Facult des
Sciences Agronomiques, UCL, Place Croix du Sud 2, B-1348, Louvain-la-Neuve, Belgium.
Email: [email protected]
Lehoczki Tornai Judit, National Collection of Agricultural & Industrial Microorganisms, Department of
Microbiology and Biotechnology, University of Horticulture and Food Industry, Somloi ut 14-16,
1118, Budapest, Hungary
Lierneux Pierre, Muse Royal de l'Arme et d'Histoire Militaire, Parc du Cinquantenaire 3, B-1040,
Bruxelles, Belgium.
Locci Romano, Dipartimento di Biologia Applicata alla Difesa delle Piante , Universita degli Studi di
Udine, Via delle Scienze 208, Area Rizzi, 33100, Udine, Italy.
Macau J., Research and Development Department, Citrique Belge, Pastorijstraat 249, B-3300, Tienen,
Belgium.
Magingo Francis S.S., Applied Microbiology Unit, Department of Botany, University of Dar Es Salaam,
P.O. Box 35060, Dar Es Salaam, Tanzania.
Maraite Henri, Clinique des Plantes, Unit de Phytopathologie - FYMY, Dpartement BAPA, Facult
des Sciences Agronomiques, UCL, Place Croix du Sud 2, B-1348, Louvain-la-Neuve, Belgium.,
Email: [email protected]
Maras Marleen, Laboratorium voor Moleculaire Biologie, Rijksuniversiteit Gent - RUG, K.L.
Ledeganckstraat 35, B-9000, Gent, Belgium. n
Marin Bernard, Laboratoire de Biotechnologie, ORSTOM, Avenue Agropolis 911, B.P. 5045, F-34032,
Montpellier Cedex 1, France.
Marson Guy , Muse National d'Histoire Naturelle, March aux Poissons, 2345, Luxembourg,
Luxembourg.
Martini-Vaughan Ann, Collezione del Lieviti Industriali, Dipartimento di Biologia Vegetale, Universita
degli Studi di Perugia, Borgno XX Giugno 74, 06121, Perugia, Italy.
Marvel Danielle, Rue Saint-Henri 65, 1200 Bruxelles.
Masuka Anxious J., Forest Research Centre, Biotechnology Kutsaga Research Station, Tobacco
Research Board Zimbabwe, P.O. Box HG 595, Highlands, Harare, Zimbabwe.
Melcion Dominique, Laboratoire de Microbiologie et Technologie Cralires, Centre de Recherches de
Nantes, Institut National de la Recherche Agronomique, B.P. 527, 44026, Nantes Cdex, France.
Mencinicopschi Gheorghe, Department of Biotechnology, Institute for Food Chemistry, Grlei Street 1,
71576, Bucharest, Romania;
Messner Robert, Universitt fr Bodenkultur, Nussdorfer Lnde 11, 1190, Wien, Austria. Email:
[email protected]
Mibey Richard K., Department of Botany, University of Nairobi, Chiromo, P.O. Box 30197, Nairobi,
Kenya.
Michiels Chris, Katholieke Universiteit Leuven - KUL, Kardinaal Mercierlaan 92, B-3001, Heverlee,
Belgium.
Miller Liliane, Somycel, Z.I. Sud, Route de Tours, B.P. 25, 37130, Langeais, France.
Montemartini Corte Aurora, Istituto botanico "Hanbury" ed Orto botanico dell'Universita, Universita di
Genova, Corso Dogali 1c, 16136, Genova, Italy.
Mouchacca Jean, Laboratoire de Cryptogamie, Musum National d'Histoire Naturelle, Rue Buffon 12,
75005, Paris, France.
MUCL, Laboratoire de Mycologie systmatique et applique, Facult des Sciences Agronomiques UCL,
2 Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgium. Pr.Grgoire L. Hennebert, Dr Cony

 Decock, Dr Dilnora Gouliamova, Dr .Kandanda Tshinyangu, Laurence Nlissen, Pascale

Massart; Ch. Mouilliard, Ph. Charue, P. Evrard, N. Jamin.
Munaut Franoise, Clinique des Plantes, Unit de Phytopathologie - FYMY, Dpartement BAPA,
Facult des Sciences Agronomiques, UCL, Place Croix du Sud 2 bte 3, B-1348, Louvain-la-
Neuve, Belgium.
Naveau Henry, Unit de Gnie Biologique - GEBI, Dpartement CABI, Facult des Sciences
Agronomiques, Universit Catholique de Louvain - UCL, Place Croix du Sud 2 Bte 19, B-1348,
Louvain-la-Neuve, Belgium. Phone: 010-473646, Email: [email protected].
Nguyen Huu-Vang, Laboratoire de Gntique Molculaire et Cellulaire, Collection de Levures d'Intrt
Biotechnologique, Institut National de la Recherche Agronomique, 78850, Thiverval-Grignon,
France. Email: [email protected]
Nol Anne, Laboratoire de Bactriologie, Unit de Microbiologie MBLA, Dpartement CABI, Facult
des Sciences Agronomiques, UCL, Place Croix du Sud 2 Bte 12, B-1348, Louvain-la-Neuve.
Nolard Nicole, Ministre de la Sant Publique, Institut d'Hygine et d'Epidmiologie - IHEM, Rue
Juliette Wytsman 14, B-1050, Bruxelles, Belgium.
Noordeloos Machiel E., Mycological Department, Rijksherbarium/Hortus Botanicus, P.O. Box 9514,
Schelpenkade 6, 2300, RA Leiden, Netherlands.
Oxenboell Karen M. , Novo Nordisk A/S, Novo All, 2880, Bagsvaerd, Denmark.
Parmasto Erast, Laboratory of Mycology, Institute of Zoology and Botany, 21 Vanemuise Street, 2400,
Tartu, Estonia. Email: [email protected]
Peerally Abed, School of Agriculture, Department of Natural Sciences, University of Mauritius, Reduit,
Mauritius.
Peloille Michle, Laboratoire d'Ecologie Parasitaire, Centre de Recherches Tours-Nouzilly, Institut
National de la Recherche Agronomique, 37380, Monnaie, France.
Penninckx Michel, Laboratoire de Microbiologie, Unit de Physiologie et d'Ecologie Microbienne,
Facult des Sciences, Universit Libre de Bruxelles - ULB, Institut Pasteur du Brabant, Rue
Engeland 642, B-1070, Bruxelles, Belgium.
Poppe Jozef, Centrum voor de studie van de Sierplantenteelt, I.W.O.N.L. Sect, Labo Fytopathologie,
Fakulteit van de Landbouwwetenschappen, Rijksuniversiteit Gent - RUG, Coupure Links 653, B-
9000, Gent, Belgium.
Poswick Pierre, Fast Net sa, Avenue du Bempt 41, B-1190, Bruxelles, Belgium.
Prieels Anne-Marie, Tech Know, Avenue de l'Observatoire 2, B-1180, Bruxelles, Belgium .
Prillinger Hansjrg, Institut fr Angewandte Mikrobiologie - IAM, Universitt fr Bodenkultur,
Nussdorfer Lnde 11, 1190, Wien, Austria. Email: [email protected]
Raemaekers Romain, Belgian Administration for Developement Cooperation - BADC, Marsveldplein
5/57, B-1050, Bruxelles, Belgium.
Rammeloo Jan, Jardin Botanique National de Belgique, Domaine de Bouchout, Brusselsesteenweg 8-26,
B-1860, Meise, Belgium.
Risede Jean-Michel, CIRAD, B.P. 153, 97202, Fort de France, Martinique
Roquebert Marie-France, Laboratoire de Cryptogamie, Musum National d'Histoire Naturelle, Rue de
Buffon 12, 75005, Paris, France.
Rosentuler Leon, Foreign Relations Department, Institute for Food Chemistry, Grlei Street 1, 71576,
Bucharest, Romania.
Rouxhet Paul, Unit de Chimie des Interfaces - CIFA, Dpartement CABI, Facult des Sciences
Agronomiques, UCL, Place Croix du Sud 1, B-1348, Louvain-la-Neuve, Belgium. Email:
[email protected]
Sedeyn Patrick, Mycoblank Bvba, Oude Gaversesteenweg 70, B-9820, Merelbeke, Belgium.
Simonart Paul, Guido Gezellelaan 10, B3090, Overijse, Belgium
Smith David, International Mycological Institute, Bakeham Lane, Englefield Green, Egham, TW20 9TY,
Surrey, United Kingdom.
Spencer-Martins Isabel, Portuguese Yeast Culture Collection, Instituto Gulbenkian de Ciencia, Rua da
Quinta Grande 6, Apartado 14, 2781, Oeiras Codex, Portugal Email: [email protected]
Stalpers Joost, Centraalbureau voor Schimmelcultures - CBS, Oosterstraat 1, P.O. Box 273, 3740, AG
Baarn, Netherlands., Email: [email protected]
Stegehuis Gerrit, Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740, AG Baarn, Netherlands.
Subramanian C.V. , Plot 885, Ramaswami Salai 62, K.K. Nagar, 600078, Madras, India.
Sugiyama Junta , Institute of Molecular and Cellular Biosciences, IAM Culture Collection, University of
Tokyo, Yayoi 1-1-1, Bunkyo-ku 113, Tokyo, Japan. Email: [email protected]
Suihko Maija-Liisa, VTT Collection of Industrial Microorganisms, VTT Biotechnical Laboratory, P.O.
Box 1504, 02044, Espoo, Finland. Email: [email protected]

Sulten Evelyne, Laboratoire de Bactriologie, Institut Royal des Sciences Naturelles de Belgique -
IRSNB, Rue Vautier 29, B-1040, Bruxelles, Belgium.
Swinne Danielle, Dpartement de Mycologie, Institut de Mdecine Tropicale, Nationalestraat 155, B-
2000, Antwerpen, Belgium.
Symoens Franoise, Institut d'Hygine et d'Epidmiologie - IHEM, Rue Juliette Wytsman 14, B-1050,
Bruxelles, Belgium.
Thiran Jean-Philippe, Laboratoire de Tlcommunications et Tldtection, Dpartement ELEC, Facult
des Sciences Appliques, Universit Catholique de Louvain - UCL, Place du Levant 2, B-1348,
Louvain-la-Neuve, Belgium.
Tshinyangu Kandanda, MUCL, 2 Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgique,
Uruburu Federico, Coleccion Espanola de Cultivos Tipo - CECT, Facultad de Ciencias Biologicas,
Departamento de Microbiologia, Universidad de Valencia, C/Dr. Moliner 50, 46100, Burjassot,
Spain. Email: [email protected]
Vancanneyt Marc, Laboratorium voor Microbiologie en Microbile Genetica - LMG, Faculteit
Wetenschappen, Rijksuniversiteit Gent - RUG, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium.
Van Cutsem Jan M.P., Department of Bacteriology and Mycology, Janssen Research Foundation,
Zenithlaan 17, B-2340, Beerse, Belgium.
Vandenput Olivier, Services Fdraux des Affaires Scientifiques, Techniques et Culturelles, Rue de la
Science 8, B-1040, Bruxelles, Belgium.
Van de Peer Yves, Universitaire Instelling Antwerpen - UIA , Universiteitsplein 1, B-2610, Wilrijk,
Belgium. Email: [email protected]
Van der Gucht Katleen, Laboratorium voor Morfologie Systematiek en Ecologie van de Planten,
Rijksuniversiteit Gent, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium.
Van der Mei Dirk, Centraalbureau voor Schimmelcultures, Oosterstraat 1, P.O. Box 273, 3740, AG
Baarn, Netherlands.
Vanderveken J. Professor, Laboratorium voor Morfologie Systematiek en Ecologie van de Planten,
Rijksuniversiteit Gent, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium.
Vandewyer Paul H., Research and Development Department, Citrique Belge, Pastorijstraat 249 , B-3300,
Tienen, Belgium.
Van Gestel Jef, Janssen Pharmaceutica n.v., Plant Protection Division, Turnhoutseweg 30, B-2340,
Beerse, Belgium.
Van Heetvelde Ingeborg, Research & Development Center, Vandemoortele n.v., Prins Albertlaan 79,
Postbus 40, B-8770, Izegem, Belgium.
Vanhonacker Katrien, Laboratorium voor Microbiologie en Microbile - LMG, Rijksuniversiteit Gent -
RUG , K.L. Ledeganckstraat 35, B-9000, Gent, Belgium. Email: [email protected]
Vanhoucke Martine, Laboratory of Molecular Biology - Plasmid Collection, Rijksuniversiteit Gent -
RUG, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium. Email: [email protected]
Van Langenhove Luk, Kabinet Wetenschapbeleid, Wetstraat 155/16, B-1040, Brussels, Belgium.
Verbeken Annemieke, Faculteit Wetenschappen, Rijksuniversiteit Gent - RUG, K.L. Ledeganckstraat 35,
B-9000, Gent, Belgium.
Verhoyen Michel, Laboratoire de Phytovirologie, Facult des Sciences Agronomiques, Universit
Catholique de Louvain - UCL, Place Croix du Sud 3, B-1348, Louvain-la-Neuve, Belgium.
Email: [email protected]
Viteri Ricardo, Xnova Ltd, Ipswich Road 545, Slough, SL1 4EQ, Bucks, United Kingdom.
Vivegnis Jacques, Laboratoire de Bactriologie, Unit de Microbiologie MBLA, Dpartement CABI,
Facult des Sciences Agronomiques, UCL, Place Croix du Sud 2 Bte 12, B-1348, Louvain-la-
Neuve, Belgium.
Vrijmoed Lilian L.P., Department of Biology and Chemistry, City Politechnic of Hong Kong, Tat Chee
Avenue 83, Kowloon, Hong Kong, Hong Kong. Email: [email protected]
Wauthoz Pascale, Laboratoire de Bactriologie, Unit de Microbiologie MBLA, Dpartement CABI,
Facult des Sciences Agronomiques, UCL, Place Croix du Sud 2 Bte 12, B-1348, Louvain-la-
Neuve, Belgium.
Webster John, Hatherly Laboratories, Department of Biological Sciences, University of Exeter, Prince of
Wales Road, EX4 4PS, Exeter, United Kingdom.
Whalley Anthony J.S., Department of Biomolecular Sciences, Laboratory of Microbiology, Liverpool
John Moores University, Byrom Street, L3 3AF, Liverpool, England, U.K.

10

 Forword

Centenary Celebration of the Mycotheque de l'Universit

Catholique de Louvain, Belgium, 1894-1994
JOHN WEBSTER

University of Exeter, United Kingdom

MUCL is a fungal culture collection founded in Louvain in 1894 by Philibert

Biourge. The centenary of the inauguration of this important collection is
celebrated at a special symposium attended by some 150 guests from many
countries at the Catholic University at Louvain-la-Neuve on Wenesday 29th June
1994. There are two sessions of presented papers, followed by a discussion, a
poster presentation and an exhibition tracing the history of the collecton.

The morning sesssion is openend by addresses of welcome from Dr H.

Naveau, Head of Applied Chemistry and Bioindustry Department of the Faculty of
Agricultural Sciences and Dr P. Rouxhet, Prorector of UCL. Dr Rouxhet explains
the philosophy behind the setting up of the University at Louvain-la-Neuve. It is
an integrated community of academic staff and their families, students, residents,
shops, private bisinesses and a science and industry park. Priority is given to
scientific research and development, advanced technology, etc., with spin-offs
likely to be benefit to the University. The Faculty of Agricultural Sciences, which
receives state support, includes graduate school forming chemists and engineers
able to apply their skills to the living world. The Prorector paid a large tribute to
the dedication and hard work of the present director of the culture collection,
Professor Grgoire Laurent Hennebert, and assured him that the collection would
continue to receive University support. Professor Hennebert then traced the history
of the culture collection over the past century since its foundation by Biourge, who
had studied with Pasteur. A large number of distinguished scientists had been on
the staff including Dierckx and Biourge who laid the foundation of the
classification of Penicillium, Vandendries who worked on sexuality of
Basidiomycetes, Wieldiers who discovered the "bios", a growth factor for yeast,
later found composed of the B vitamins, Cappuyns whose work with the strains of
Penicillium species and Aspergillus niger from the Biourge's collection established
the industrial process of citric acid p-fermentation, Simonart, former director of
the collection and present at the celebration, who worked with Raistrick of Oxford
on the chemistry of Penicillium griseofulvum and discovered the first antifungal
antibiotic the griseofulvin. The rest of the morning session is also devoted to
papers on the significance of fungus culture collections. Prof. D.L. Hawksworth
(Director of the International Mycolgical Institute, IMI) speaks about "Fungal
resource collections and biodiversity", Dr. D. van der Mei (Director of the
Centraalbureau voor Schimmelcultures, CBS) "The fungus culture collections in
Europe", and Dr J. De Brabandere (Federal Service for Scientific Affairs,
Belgium) "The Belgian Co-ordinated Collections of Microorganisms".
The afternoon session is a workshop entitled "Fungal taxonomy and tropical
mycology: Quo vadis?" After an introduction by Pr. Hennebert, Dr C.P. Kurtzman

11

(NRRL, USA) and Dr. M. Blackwell (President MSA, USA) express the benefits
of phylogenetic analysis for fungal taxonomy, Dr. J. Rammeloo, (National
Botanical Garden, Belgium) and Dr. M.-F. Roquebert (National Natural History
Museum, France) show the both the historical foundation up to the development of
new means of the morphological approach in fungal taxonomy, Dr. A.J. Masuka
(Forest Research Centre, Zimbabwe), Dr. A. Peerally (University of Mauritius)
and Dr. J. Mouchaca, in name of E.J. daSilva (MIRCEN, UNESCO, France) develop
the strategies in the development of mycological research and of fungus
collections in the Tropics. In the subsequent discussion much concern is expressed
about the lack of funding for taxonomic research on fungi and in the training of
future generations of fungal taxonomists not only in developing tropical countries
but also in developed temperate areas.
The exhibition of the history of MUCL was very interesting, including the
notesbooks of Biourge, some of the original apparatus and microscopes used in his
first laboratory, portraits, photographs, manuscripts, paintings and drawings, and
publications, and a most attractive display of colourful and stricking fungal
cultures.
The celebration ended with a reception in the grounds of a ruined Cistersian
monastery, followed by a banquet in the adjacent Hotel des Ruines. The guests
were entertained during the banquet by singers and dancers who were refugees
from Rwanda. The British Mycological Society President, Professor A. Walley,
presented Professor Hennebert with a ceramic sculpture of Polyporus squamosus
on behalf of the BMS Council, thanked him for his hospitality and for organizing
such a splendid celebration and gave the MUCL good wishes and continuing
success for the next century of their activities.

12

 PLENARY SESSION

13

 14

 PART 1

MYCOTHEQUE DE L'UNIVERSITE CATHOLIQUE DE LOUVAIN

(MUCL):
100 YEARS OF EXISTENCE AND ACTIVITY OF THE FUNGUS CULTURE
COLLECTION

Grgoire L. HENNEBERT

Mycothque of the Catholic University of Louvain, Place Croix du Sud, 1348 Louvain-la-Neuve, Belgium

The Mycotheque of the Catholic University of Louvain is a fungal culture

collection of filamentous fungi and yeasts.
It was started by Professor Philibert Biourge, microbiologist at the Brewery High
School of the University in 1892. From contaminated beers, malts and barleys he
purified many strains of beer yeasts and isolated wild yeasts, filamentous fungi and
bacteria that he maintained in the collection. The collection was publicly inaugurated
on July 8th, 1894.
After a well-known preliminary study of Penicillium Link by his assistant F.
Dierckx (1901), Biourge prepared a full monograph of the genus Penicillium (1920,
1923). Later Professor P. Simonart made biochemical investigations on the
Penicillium species in the collection and discovered the first antimycotic antibiotic
griseofulvin from Penicillium griseofulvum Dierckx (1937). After Biourge's death in
1942, Pr. Simonart named the collection "Mycothque Philibert Biourge".
In 1968, Pr. Simonart brought the collection together with the fungal collection
begun in 1956 by G.L. Hennebert, and which already included 11 000 strains and
herbarium specimens. In 1968 Pr. J. Meyer's fungus culture collection of
Hyphomycetes from Central Africa, and in 1984, the collection of beer yeasts
initiated by Biourge and maintained by the UCL Laboratory of Brewery Sciences
were also incorporated.
In 1968 the Collection was officially recognized as the "Mycothque de
l'Universit Catholique de Louvain" (MUCL) by the University, in 1969 by the
International Association of Plant taxonomy (IAPT), in 1972 by the World
Federation of Culture Collections (WFCC), and registered at the World Data Center
(WDC). Since 1983, the MUCL Collection has been financially supported by the
Science Policy Office of Belgium. It is a member of the Belgian Coordinated
Collections of Microorganisms (BCCM), together with the collection of medical
fungi at the Institute of Hygiene and Epidemiology (IHEM) in Brussels, the one of
bacteria from the Laboratorium for Microbiology, University of Ghent (LMG) and
the one of plasmids at the Laboratory for Molecular Biology, University of Ghent
(LMB). From 1983 MUCL also has been a member of the European Culture
Collections' Organization (ECCO) and from 1985, a partner in the Microbial
Information Network Europe (MINE).

The MUCL culture collection

The MUCL Collection currently holds 24000 strains from all groups of
filamentous and yeast fungi, including Oomycetes, Zygomycetes, Ascomycetes,
Basidiomycetes, Blastomycetes Hyphomycetes and Coelomycetes. Most of the living

15

strains originated from fungal specimens collected throughout the world and kept in
the MUCL mycological herbarium which includes 38000 specimens of which 24000
are as living strains: 22000 filamentous fungi and 2000 yeast fungi, representing
3500 species of fungi and yeasts. The second edition of the catalogue was issued in
1992 with about 8000 strains 1.
The MUCL strains are maintained in at least two different ways, depending on
the taxonomic group and on their effective sporulation. All strains are maintained on
agar slants under mineral (parafin) oil. Strains producing spores in culture are
lyophilized. Delicate and important strains are also maintained frozen at -80 C.
MUCL is a research-based culture collection. Its staff is currently carrying on
research in both systematic and applied mycology.
Systematic mycology:
- Collection and isolation of fungi in unexplored sites and substrates throughout the
world, particularly in extreme environments and in Africa.
- Morphotaxonomy, chemotaxonomy and nomenclature of yeasts and filamentous
fungi, particularly Blastomycetes, Hyphomycetes and Aphyllophorales.
- Morphogenesis and morphology of sexual and asexual reproduction.
- Floristics and ecology of fungi in their natural habitats (thermal waters, burned
sites, sea sand, rocks, dung, resins, leathers, dried fish, tar, all kinds of industrial
products and residues).
- Physiological, biochemical and biomolecular characterization of fungi and yeasts
for taxonomy and biotechnology, implemented into a new expert system of
identification.
- Biochemical and biomolecular taxonomy of yeasts and filamentous fungi: protein
and enzymic profiles, coenzyme Q, RFLP, RAPD mapping, rRNA and rDNA
sequencing, phylogenetic analysis.
- Development of a fungal data base on Macintosh (software 4D) following the
MINE formats.
Applied Mycology:
- Food contamination. Post-harvest deterioration in storage of cereals and legumes.
Flour and dairy contamination. Food fermentation and starter production (cheese,
kefirs, sourdoughs, tempeh, fermented cassava, beers and wines).
- Cultivation of edible mushrooms. Production of spawn. Improvement of production
and nutritional value. Domestication of edible non-cultivated tropical species.
- Industrial, agricultural and domestic waste biodegradation, agricultural composting
and industrial recycling.
- Characterization of wood-decaying fungi. Deterioration of material by fungi
(wood, paints, plastics, textiles, etc). Fungal resistance testing of materials.
Bioassays of fungicides. Diagnoses in industries and buildings. - Association of
termites and fungi.
- Monitoring of product contamination in agricultural and industrial environments.
Improvement of production and storage conditions.
- Characterization and production of mycorrhizae for reforestation in the tropics.
Production of starters for inoculation.
- Screening of fungi in search of particular properties for biosynthesis.

1
The Mycotheque preserves presently over 51.000 herbarium specimens and 35.000 living strains of
fungi

16

- Improvement of preservation techniques of living strains of fungi (freeze-drying

and cryopreservation).

References

Biourge Ph. 1920. Les moisissures du groupe Penicillium Link, tude monographique (Conference,
Louvain, 3 April 1916) Bull. Ass. Anc. El. Ec. Sup. Brass. Univ. Louv., 20(3): 99-127.
Biourge Ph. 1923. Les moisissures du groupe Penicillium Link. Etude monographique. La Cellule, 33: 5-
331.
Dierckx F. 1901. Essai de revision du genre Penicillium Link. Note prliminaire. Annales Soc. Scient.
Brux., 25: 83-89.
Hennebert G.L. 1979. Philibert Biourge, microbiologiste et mycologue, 1864-1942. In Les Sciences
Exactes et Naturelles l'Universit de Louvain de 1835 1940. IIIe Colloque d'Histoire des
Sciences, Louvain-la-Neuve, 17 Mars 1977. Recueil de Travaux d'Histoire et de Philologie, 6e Srie,
15: 61-98, 3pl. Louvain.
Hennebert G.L. 1985. Dierckx' Contribution to the Genus Penicillium. In Advances in Penicillium and
Aspergillus Systematics, Samson, R.A. and J.I. Pitt, eds., 9-21., 4 fig. Plenum Press N.Y. and
London.

17

 MUCL 1894-1994 step by step

1891-1892 Philibert Biourge, born on April 8, 1864, is Dr in Botany UCL, and is awarded a scientifi
research stay at the Pasteur Institute in Paris, under the direction of Louis Pasteur and of Drs Roux and
Duclaux on the pure culture of microorganisms.

1892 On return from Paris Ph. Biourge is

nominated assistent of Prof. Jean-Baptiste Carnoy
, Professor of Botany and Microbiology, UCL.
(photo) . Carnoy was author of "Recherches
morphologiqueset physiologiques sur les
champignons" (Bull. Soc. Roy. Bot. Belg.9-2:157-
311, 1870).

1892 Ph. Biourge makes a second study stay

in Denmark by E. Christian Hansen wo was
applying the monospore technique of Pasteur for
the purification of Saccharomyces cereviviae from the wild yeasts.

1892-1894 Ph. Biourge initiates a Culture Collection of Microorganisms, yeasts, filamentous fungi and
bacteria from beers and cereals, at the Laboratory of Zymotechny of the Brewery High School, Catholic
University of Louvain, in Louvain.

1894 Public inauguration of the Biourge's Culture Collection with exhibition of living cultures of
yeasts, filamentous fungi and bacteria, at the Brewers Congress, Antwerp, 8 July 1894 (Journal du Petit
Brasseur, 2 (19) suppl., p.7-11, July 8, 1894, photos).

18

1895 Biourge studies the microbial alterations of beers and of the products of alcoholic fermentation
at the Laboratoire de zymologie de l'Ecole Suprieure de Brasserie de l'UCL (photos) (Ph. Biourge, "Les
maladies microbiennes de la bire" Bull. Assoc. Anc. El. Ec. Sup. Brass. Univ. Louv., 1(1): 1-4.
"Recherches sur la fermentation alcoolique" La Cellule, 11: 93-109). Applying single cell culture method
from Pasteur and Hansen, Ph. Biourge is purifying the Belgian beer yeasts for breweries.

1896 Ph. Biourge is nominated Professor UCL.

1897 Ph. Biourge develops the culture collection of moulds, Penicillium and others, from malts.
Oscar Semal study fungal production of ammonium (La Cellule, 13(2): 284-312, 1897). Ren
Vandendries presents a PhD thesis on the "Matires colorantes azotes chez les
Champignons".Vandendries will be one pioneer in the sexual mechanism in the Basdiomycetes from 1923
to 1937,

1898-1901 Franois Dierckx (1863-1937) makes a taxonomic study of

Penicillium species of the Biourge's collection.

1899 Death of Prof. Jean-Baptiste Carnoy (1836-1899).

Prof. Ph. Biourge becomes head of the Laboratory of Microbiology, Faculty
of Sciences, UCL.

1900 F. Dierckx (photo) presents a PhD thesis on 26 new species of

Penicillium with drawings and publishes "Essai de revision du genre
Penicillium Link. Note prliminaire", Anns. Soc. scient. Brux., 25(1): 83-89,
1901. He showed his type cultures to P.A. Saccardo, on his way to Indonsia.
but lost all his belongings including the cultures in that country. Only one
duplicate type culture left in Louvain, Penicillium griseo-roseum Dierckx, could be recovered by Biourge.

1901 Ernest Wieldiers, Md Dr, another Biourge's collaborator, discovers the "Bios", an extract of
yeast that contains the required vitamins B, K and others for the growth of yeast. Wieldiers E. "Nouvelle
substance indispensable au dveloppement de la levure" La Cellule, 18(2): 311-333, 1901.

1902 The "Question of Bios" is a matter of dispute among scientists. A. Amand, collaborator of
Biourge, answers the dispute (La Cellule, 20(2): 223-251, 1902).

1903-1914 Ph. Biourge develops his collection of Penicillium strains. He collected again the species
described by Dierckx on basis of Dierckx drawings left in Louvain. He also gathers other Penicillium
types from Zaleski, Whemer, Thom, Bainier and Franz Krl Collections (Krl started in 1884 a private
collection of several hundreds of bacteria, yeasts and filamentous fungi at the Institute of Hygiene of the
Faculty of Medecine at the Prague University. It was one of the earliest collection in the world. After
Krl's death in 1911, the collection went to Vienna from where Ersnst Pribam got it in 1915 and published
a first catalogue of it in1919).

1910-1914 Alphonse Cappuyns (1887-1936) starts biochemical researches on bacteria and fungi.

19

1914-1918 World war: Ph. Biourge retires in his landhome where he works on a Penicillium monograph.
A. Cappuyns retires in a studio at Bruxelles where he selects a citric acid producing Penicillium strain
from Biourge's collection.

1919 First industrial production of citric acid from a Penicillium strain by A. Cappuyns in "s.a. Les
Produits Organiques de Tirlemont", Belgium, after the failure of such a production by the Fabrique de
Produits Chilmiques de Thann et
Mulhouse, France. (Maz P. & Perrier
A. 1904 Anns. Inst. Pasteur, 18:553-
575)

1920 Ph. Biourge publishes a

summary of his forthcoming
monograph "Les moisissures du
groupe Penicillium Link, tude
monographique", Bull. Assoc. Anc. El.
Ec. Sup. Brass. Univ. Louv., 20(3): 99-
127, 1920.

1923 Ph. Biourge publishes his

Penicillium monograph with 126
species "Les moisissures du groupe
Penicillium Link, tude
monographique", La Cellule, 33: 3-
331, 1923.
Ph. Biourge receives the Grand Prix de l'Exposition International du Centenaire de Pasteur Strasbourg
(photo)
A collaborator, Victor Estienne presents a PhD thesis on Aspergillus atro-ruber (Harzia
acremonioides).

1925 Ph. Biourge is also devoting much time to the study of the Dutch Elm disease: "La maladie des
ormes. J. Soc. cent. Agric. Belg., 73: 22-44, 1925.

1926 A. Cappuyns started a new selection of an acid-resistant citric acid

producing strain to substitute it to the too instable Penicillium strain in use:
"Sur la formation d'acide sulfurique libre dans les cultures de certains
microorganismes.", Anns. Soc. scient. Bruxelle, 45: 177-183, 1926).

1927 Biourge is working on the manuscript of a monogrraph of the

Aspergillus as "Brevis Conspectus generis Aspergillus Link" and a
preliminary paper entitled "Monographie culturale macroscopique des
Aspergillus" with 13 colour plates describing the colours of 208 strains on
two media, of which three original plates are preserved at MUCL.

1929 New and successful start of "La Citrique Belge" in Tirlemont by

A. Cappyuns (photo), from a selected strain of Aspergillus group niger from the Biourge's Collection.
Paul Simonart (1907-1998), collaborator of Biourge, starts biochemical studies of the lactic
fermentation.

1931-1934 P. Simonart is awarded with Prof. H. Raistrick in London. They publish the discovery of the
gentisic acid in the biochemistry of Penicillium griseofulvum Dierckx: Raistrick H & Simonart P. Studies
in the biochemistry of microorganisms 29. 2:5-Dihydroxybenzoic acid (gentisic acid) a new product of the
metabolism of glucose by Penicillium griseofulvum Dierckx. Biochem. J. 27(3): 628-633, 1933);

1932 Raoul Mosseray makes physiological and morphological studies on the Aspergillus group niger
for PhD: "Influence du zinc sur les Aspergillus de la srie niger et sur quelques autres." La Cellule 41: 11-
128

1933 Biourge provides in his paper "Sur les champignons dits moisissures. A quoi bon leur tude et
comment la faire.", Rev. Quest. scient., 52(103): 53-78, a justification of a fundamental and taxonomic
study of the fungi, in view of their large physiological and biochemical potentialities.

20

1934 P. Simonart defends his PhD thesis on "The biochemistry of Penicillium griseofulvum
Dierckx" at the University of London.
From the Aspergillus strains collected by Ph. Bourge and collected by himself in Africa, Raloul
Mosseray publishes the monographic study of "Les Aspergillus de la Section Niger Thom et Churh." La
Cellule, 43: 201-286. also Ann. Soc. scient. Brux. B, 54: 72-85. He also studies the "Races naturelles et
variations de culture chez divers Aspergillus." Ann. Soc. scient. Brux. B, 54: 161-189.
Ren Vandendries develops his study of the sexuality in Basidiomycetes
Paul Henrard develops a PhD study on the sexuality of Aspergillus nidulans and Eurotium
species, "Polarit, hrdit et variation chez diverses souches dAspergillus." La Cellule, 43(3): 350-324.

1935 P. Henrard studies further the sexuality of ascomycetes in "Observation sur le comportement
de souches monascospores de Xylaria polymorpha Pers. (Grev.)." Broteria 14(31): 79-83.
P. Simonart, in collaboration with A.E. Oxford and H. Raistrick
publishes the discovery of the "Fulvic acid, a new crystalline yellow pigment,
a metabolic product of Penicillium griseofulvum Dierckx, P. flexuosum Dale
and P. brefeldianum Dodge.and other species." Biochem. J. 29(5) 1102-1115.

1936 Ph. Biourge publishes his observations on Ophiostoma ulmi "Le

cycle du champignon de la maladie de l'orme." Ann. Soc. scient. Brux. 56:
130-193.
P. Simonart (photo) is nominated Professor UCL, and awarded
Advanced Fellow of the Belgian American Educational Foundation. With Dr.
Ch. Thom, in Washington, he is confronting the Biourge's Penicillium
Collection to that of Thom.

1938 Ph. Biourge is Emeritus Professor. Prof. P. Simonart is responsible of the Laboratory of
Microbiology and takes the Fungus Culture Collection in charge, but the Biourge's yeast collection is kept
under care at the Brewery School.

1939 Ph. Biourge, G. Van Cutsem & E. Bredo put forwards Ophistoma ulmi as the cause of a
humain infection, "Une mycose nouvelle: la graphiomycose." Rev. Belg. Scienc. Mdic., 11(5): 217-236.
P. Simonart, in collaboration with A.E. Oxford and H. Raistrick publishes the discovery of the
"Griseofulvin, C17H1706Cl, a metabolic product of Penicillium griseofulvum Dieckx." Biochem. J. 33(2):
240-248, the first antimycotic antibiotic.
The Fungus Culture Collection moves, together with the Laboratory of Microbiology, from
Carnoy Institute of Botany, Faculty of Sciences, to the new Institute of Agronomy. Prof. Simonart
denominates the Collection "Mycothque Philibert Biourge".

1940-1944 Second world war.

1942 Prof. Ph. Biourge passed away on April 19, 1942.

1945 The Mycothque Ph. Biourge within the Laboratory of

Microbiology, moves from the old Institute in Louvain city to the new
Institut Agronomique in the Parc van Arenberg in Heverlee near Louvain.

1949 Raymond Lambert (1923-1992), UCL assistant, then workleader

in Prof. Simonart's Microbiology laboratory, takes care of the Mycothque
Philibert Biourge up to 1960.

1950-1960 Joseph Meyer (1924-), Dr in Botany, studies soil microfungi, Hyphomycetes and others, of
Central Congo, in Yangambi, Congo, establishing a herbarium and a culture collection. (photo)

1956 Grgoire L. Hennebert (1929-), FNRS research fellow at UCL, initiates a personal Fungus
Culture Collection in the Plant Pathology Laboratory, UCL Faculty of Agronomy, and starts a research on
Botrytis and the Sclerotiniaceae with Prof. V. Estienne as promotor.

1960 Joseph Meyer, collaborator in Prof. Simonart's Laboratory, takes care of the Mycothque
Philibert Biourge, until 1969, as well as of his collection of African Hyphomycetes.
G.L. Hennebert presents the thesis "Recherches morphologiques sur le genre Botrytis Persoon",
for obtention of the PhD graduation at UCL.

21

 1960-1962 G.L. Hennebert is awarded by the Canadian National Research

Council a two-year postdoctoral fellowship at the Mycology Unit of the Plant
Research Institute, Ottawa, with Dr Stanley J. Hughes (photo).

1962 G.L. Hennebert Visiting Research Mycologist, Plant Pathology &

Mycology Dept., Cornel Univerity, Ithaca, NY.

1962-1965 G.L. Hennebert is awarded FNRS qualified researcher and

appointed UCL teaching assistant in Plant pathology, developing the Fungus
Culture Collection, at the Faculty of Agriculture, Heverlee-Leuven. (photo)

1963-1975 MUCL develops a close collaboration with the Central Bureau

voor Schimmelcultures CBS in Baarn.

1964 Visite of Dr.C.J. Alexopoulos from to the Collection

(24.08.1964).

1965 Visit of Pr. Dr. C.V. Subramanian from Madras to the

Collection (23.06.1965).
G.L. Hennebert and J. Meyer are nominated UCL
Professors.

1966 Visit of Pr. DR. R.P. Korf from Cornell University,

Ithaca, NY. to the Collection (13.06.1966)

1967 G.L. Hennebert, visiting lecturer, University Lovanium, Kinshasa, Congo.

1968 The G.L.H. Collection is composed of 12,000 herbarium

specimens and 7,000 living strains (Anonymous 1968. Centre d'Etudes: le
Laboratoire de Mycologie Systmatique et Applique. Nouvelles Brves,
REUL, Rel. ext. Univ. Louvain, 18 Juin: 3-4)
Prof. Simonart transfers the Mycothque Philibert Biourge to the
G.L. Hennebert Fungus culture Collection. The Mycoth.que Biourbe consists
of 683 Penicillium strains and 122 Aspergillus strains, a number of them
having been returned to Pr. Simonart by Thom (USA) as earlier received from
Biourge.
The UCL authorities agrees upon the new denomination of the
integrated Collection of Fungi as the "Mycothque de l'Universit Catholique
de Louvain (MUCL)", which becomes internationally recognized by the
International Association for Plant Taxonomy (IAPT Regnum vegetabile
1969).

1969 Pr. J. Meyer contributed his Collection of African soil fungi to the Mycothque.
Visit of DR. J.A. von Arx, CBS, Baarn, The Netherlands (19.04.1969)

1969-1986 G.L. Hennebert is effective member of the Special Committee for the Nomenclature of
Fungi and Lichens of the IAPT-N.

1970 G.L. Hennebert is visiting Professor, Laval Univerity, Qubec, and invited Professor
Tchechoslovakian Academy of Sciences, Praha.

1970-1994 MUCL is member of the International Biodeterioration Research Group (IBRG) and of the
Internationa Association for Wood Preservation (IRGWP)

1971 G.L. Hennebert is nominated UCL Professor.

Second Visit of Pr. Dr. C.V. Subramanian, India (28.09.1971).

1972 MUCL becomes affiliate of the World Federation of Culture Collections (WFCC Directory ed.
1972). A first catalogue of the included species represented by living strains is then provided.

22

 1972-1973 Prof. Richard P. Korf is awarded Fulbright-Hays

Senior Research Scholar and stay six months at MUCL and the UCL
Faculty of Agriculture (photo). He delivered a special conference on "A
new classification of the Operculate Discomycetes"

1973 Visit of Dr. Stanley J.

Hughes at MUCL (photo) for
discussion on the classification of
Fungi Imperfecti and collaborative
work on Sporoschismopsis (3-
17.05.1973).

1974 Creation and first issues

of MYCOTAXON, an international
journal for the taxonomy of fungi and lichens, by Dr R.P. Korf and
G.L. Hennebert.

1975 Move of the MUCL Fungus Culture Collection to new laboratories of the Faculty of
Agricultural Sciences on the new UCL campus in Louvain-la-Neuve, 30km south-east of Brussels.

1977 Publication of "Anamorph, teleomorph and holomorph, terms for forms of fungi, their names
and types", defined by Hennebert and Weresub, Mycotaxon 6: 207-211.

1981-1990 Creation of the Laboratory of Microbiology at the

University of Burundi, Faculty of Agronomical Sciences,
Bujumbura, Burundi and establishment of the LMUB Culture
Collection.

1981 G.L. Hennebert, Invited Professor at the

Potchefstroom Univerity, South Africa

1982-1990 G.L. Hennebet, Visiting Professor in

Microbiology, University of Burundi, Bujumbura.

1983 MUCL becomes a member of the European Culture Collection

Organization (ECCO) and take part to the second annual ECCO meeting in
Paris.
MUCL is a contractual partner, together with LMG and IHEM, of
the Belgian Coordinated Collections of Microorganisms (BCCM) newly
created by the Belgian Science Policy Office (SPPS) and supported by the
Belgian State.
MUCL within BCCM, IMI and CBS with support of the SPPS, at
Bangkok, are deciding the initiation of the project of the Microbial
Information Network Europe (MINE) aiming the harmonization and
computerization on line of the strain data available in European collections.

1985 The project MINE is approuved by the CEC with MUCL, LMG and ICP in BCCM, CBS,
CMI, DSM, IP, LCP as first partners. Among them MUCL, LMG and CBS
elaborate the standard format for the database and published catalogue (photo)
of the fungi and bacteria.

1986 G.L. Hennebert, Visiting Professor, Institut Agronomique et

Vtrinaire d'Agadir, Maroc.

1987 Visit of Dr. M. van Uden (IGC, Portugal), Dr. S. Udagawa (NHL,
Japan), DR. R.P. Korf (CUP, USA), Dr. L. Blaine (ATCC, USA); Dr. R.
Haruenkit (TISTR, Thailand) to MUCL in Louvain-la-Neuve.

1989 First edition of the "MUCL List of Cultures Fungi-Yeasts 1989" of

almost 5000 strains (photo).

23

1990 MUCL is elected responsible of the MINE Committee for the harmonization of the
computerized strain data on Fungi and Yeasts, under the BRIDGE EEC Programme.

Publication of "La Mrule, Science Technique et Droit" par G.L.

Hennebert, Ph. Boulenger et Fr. Balon, Ed. Ciaco, 198 p. over the dry rot fungus
problem.

1992 Second edition of the "MUCL Catalogue Fungi-Yeasts 1992"

containing almost 8000 strains.(photo)
MUCL, and the other BCCM Collections, are elected as International
Depository Authority (IDA) for the deposit of fungus and yeast strains for patent
purposes under the Budapest Treaty by Belgian state decision..

1994 July 8. The Mycothque de l'Universit Catholique de Louvain is

since 100 Years a public collection.

24

 FUNGAL GENETIC RESOURCE COLLECTIONS AND BIODIVERSITY

David L. HAWKSWORTH
International Mycological Institute, Bakeham Lane, Egham, Surrey TW20 9TY, UK

Abstract: Institutions or activities must reconsider and reinterpret their objectives in the language of the
day; failure may endanger their survival. The single act of relabelling "Culture Culture Collections" as
"Microbial Genetic Resource Collections" immediately makes a link with agendas of the 156 government
signatories to the Convention on Biological Diversity. In addition, collections must be poised to answer
fundamental questions: Who needs the collections and why? How effective is the present system? How
many strains of a species are needed ? In what ways should collections collaborate to maximize
effectiveness? And how can national and regional collections contribute to the sustainable use of the
Earth's resources? In order to realize the potential of the collections, a fuller integration in which national,
regional, and international facilities have complementary roles must be sought.

Introduction

In order to prosper, an institution or activity must repeatedly reconsider and

reinterpret its objectives in the language of the day. And also in accordance with the
agendas of its users and funding sources. However much a service is seen as
invaluable and essential to those intimately involved in it, failure to be seen to relate
to topical issues risks increasing marginalization, and a falling away of the very
constituency essential to its survival. Microbial collections have no special
exemption, and heed should be taken of the experience of some of our major
museums and botanical herbaria.
Founded in 1894, the Mycothque de l'Universit Catholique de Louvain, whose
centenary we are celebrating, is the oldest collection of fungal cultures still
operating. If the Mycothque had not been able to adapt to changing policy
requirements, we would not be here today.
In the mid-1990s, demands for justification and accountability have become
universal. In this climate, it is imperative that those involved in culture collection
development and management are poised to present their case in a manner which is
in harmony with the issues of today. Here, I pose questions that need to be addressed
at levels from the individual collection to the national, regional, and international.

What should culture collections be called?

The label "culture collection" is a barrier to communication. It has hindered the

forging of links with the rise of interest and funding devoted to the ex-situ
conservation of plants and animals during the last two decades. Interdisciplinary
meetings and reports on "genetic resources" have only exceptionally considered
microorganisms, and it is proving a slow process to change the perception that fungi
and microorganisms are something apart from "genetic resources".
Article 2 of the Convention on Biological Diversity defines genetic resources as
"genetic material of actual or potential value"; "genetic material" being "any
material of plant, animal and microbial or other origin containing functional units of
heredity" (UNEP 1992). Article 9 (b) directs the contracting parties to the
Convention to "Establish and maintain facilities for ex-situ conservation of and
research on plants, animals and micro-organisms, preferably in the country of origin
of genetic resources". A dictionary definition of a resource is "a source or possibility

25

of help"; that immediately conveys a positive message in contrast to "culture" which

makes no value statement.
The phrase "culture collection" does not appear in the Convention. The single act
of relabelling "culture collections" as "microbial genetic resource collections" makes
an instant link with the agendas of the 156 governments that are signatories to the
Convention; and also to other programmes starting to be developed as the ripples
from Rio extend ever wider (e.g. Kapoor-Vijay 1992).

Who needs the collections and why?

Fungal genetic resource collections have a diverse customer base. However,

while they are crucial at different points in time in many areas, a single customer
will often use their services only rarely. The key needs are:
- to provide named material for industry and both pure and applied research in a
timely and cost effective manner.
- to safeguard genetic resources as an insurance policy through holding stocks in
long-term storage for future needs.
- for comparative purposes to make identifications and maintain type cultures *
that fix the application of names.
- to maintain patent and industrial strains of microorganisms.
- to use in education and training.
Common myths must also be dispelled. There is a frequent misconception that
fungi and other microorganisms are everywhere and just wait to be isolated. Were
that true, desired fungi could be isolated from nature on demand. While that thesis
may hold for opportunistic moulds associated with humans and their products, and
perhaps some elements of the soil mycobiota (Gams 1992), such statements have to
be kept in perspective. Quotations such as "everything is everywhere and the
environment selects" (Baas-Becking 1934) can be profoundly damaging if not placed
in context. In reality, around 70 % of the fungi are obligately associated with
particular host plants or animals (including insects) as pathogens, commensals,
mutualists, or specialized saprobes. Their distributions will thus tend to be closely
correlated with those of their hosts.
In the case of fungi which are not visible to the unaided eye or with a hand lens,
and which are isolated from nature by dilution-plate or similar methods (Gams
1992), the probability of refinding a species the spores of which occur at low
frequencies is so remote as to be impractical.
Further, many fungi which can be seen by eye, forming macroscopic fruit-bodies
or otherwise visible colonies, have only once or exceptionally been successfully
brought into culture. In some cases this may not be technically difficult provided that
isolations are conducted while the material is in a fresh condition - for example,
isolated close to the point of collection before the specimen dries out. An example of
what can be achieved by developing particular isolation strategies is a recent project
involving lichen-forming fungi. Although around one fifth of all fungi have this
biology, lichen-forming fungi are scarcely present in microbial collections. Yet 427

*
* Although living cultures of fungi are not acceptable as nomenclatural types under the International
Code of Botanical Nomenclature (ICBN), the latest edition of the Code (Greuter et al. 1994) makes clear
by an example that cultures "permanently preserved in a metabolically inactive state", such as by
lyophilization or storage in liquid nitrogen are acceptable; the Code recommends that cultures resurrected
from such permanently preserved cultures are referred to as ex-holotype, ex-isotype, etc.

26

(42 %) of 1021 lichen-forming and lichen-inhabiting species attempted were

successfully isolated, including representatives of many genera, families, and even
orders never previously obtained in culture (Crittenden et al. in prep.).
Fungal genetic resource collections can maintain such rarely or not otherwise
cultured strains so that they are available for both research and evaluation for
potential applications. Pragmatically, the collections are the only source of pure
living isolates of many fungi.

How effective is the present system?

One measure of the effectiveness of the present system is the number of species
of fungi available in the world's service collections. In 1990 the collective holdings
were estimated at 11 500 fungi, about 17 % of the 69 000 then described
(Hawksworth 1991) *. If the conservative working estimate of 1.5 million fungi on
Earth is accepted, something for which there is growing evidence (Hawksworth
1993), that means only about 0.8 % of those actually existing are currently in a
service collection.
Fungi, as catalogued in the Index of Fungi, are currently described at the rate of
about 1 700 species per year. Precise data are not available, but only about 30 % of
these are known in culture. As the number of known species increases, on current
trends the proportion represented in the service collections can be forecast to
decrease rather than to increase. This situation could be at least partly rectified by
mycologists endeavouring to prepare cultures immediately after collection, as
strongly advocated by Huhtinen (1994), although major targeted isolation
programmes (see above) may be necessary to make a significant impact on the
current situation.

How many strains of a species are needed?

At 351 263, the number of individual strains of fungi available in the world's
service collections at first seems impressive; on average that is 17 strains under each
name indexed (Sugawara et al. 1993). However, this statistic does not automatically
imply an enormous duplication in the genetic resource held. The seed banks of major
cereals count their accessions in 100 000s, for example, with 333 413 accessions for
Oryza sativa in 1991 (Groombridge 1992) in order to preserve the range of the
genetic resources within a single species. Fungi also exhibit substantial variation
within a species. The extent of infraspecific variation in a particular fungus is often
unclear, due to the lack of investigation, but well-studied examples show that this
can be expected to be considerable.
The mycelia from morphologically defined species are often unable to fuse
together, forming "vegetative incompatability groups" in both sexually and asexually
reproducing species (Brasier 1987). For example, Ploetz (1990) found 11
incompatability groups in 96 strains of Fusarium oxysporum sp. f. cubense. Stable
aneuploids and polyploids may be a key factor in some cases, but chromosomes

* *
* The most recent edition of the World Directory of Collections of Cultures of Microorganisms
(Sugawara et al. 1994) lists 19 392 names of fungi implying that a much higher proportion of species is
held. Further, of the names of fungi listed, 9 539 are indicated as held in only one. However, these figures
are inflated as they do not allow for synonyms, separately named anamorphs, alternative taxonomies, nor
different spelling variants used in the collections submitting data.

27

receive scant attention from mycologists; indeed, aneuploidy was recognized in

Penicillium only recently (Bridge et al. 1986).
The extent of variation at the molecular level can also be exemplified by F.
oxysporum, where seven mtDNA RFLP patterns may occur within a single special
form (Jacobsen & Gordon 1991). In the particularly well-studied Neurospora crassa,
genetic stock collections maintain some 3000 different genetic variants of this single
species (Fungal Genetics Stock Center 1988).
Against the background of this genetic variation, it is sobering to note that a
study of the holdings of the various living fungal collections demonstrates that a
considerable number of fungi are represented only by single isolates. An indication
of this is provided by data compiled in the World Directory of Collections of
Microorganisms (Sugawara et al. 1993) where 49% of the taxa are listed as
represented in only a single collection. In the case of the International Mycological
Institute (IMI) collection, 2541 (56%) of the 4541 species maintained are
represented by only one isolate.
There is at present no general answer to the question "How many strains of a
species are needed?" in fungi, but we can be confident that the figure is considerable.
Were a modest 10 strains to be maintained for all 72 000 known fungi, that 720 000
would require a doubling of the collective holdings of all the world's service
collections.

In what ways should collections collaborate to maximize effectiveness?

The World Directory of Collections of Cultures of Microorganisms lists 246

collections of fungi (including yeasts) world-wide (Sugawara et al. 1993). While this
work does not include industrial and other research collections, this number
approximates to those actively involved in the preservation and supply of fungus
cultures today. These collections vary enormously in size, from a few hundred to
more than 45 000 strains. Some are general in scope, but many specialize in fungi
relevant to particular applied areas, for example plant pathology, medical mycology,
genetic stocks, edible fungi, or brewing.
The collections are not evenly distributed geographically. Only 39 of the 142
countries wholly or partly located in the tropics have collections registered with the
World Directory; while 107 collections are involved, 52 (49 %) are in one of two
countries, Australia and India (Hawksworth & Ritchie 1993).
It has to be accepted that: (a) no single collection is ever likely to achieve the
level of funding necessary to serve as a genetic resource collection for all groups of
fungi; and (b) many countries are likely to remain without national collections for
the foreseeable future unless appropriate funds can be released. If an adequate
service to the world is to be provided, one which is able to fulfil all the criteria
identified above, and further satisfy national needs, collaborative mechanisms must
be sought.
The need for collaboration was foreseen in the Convention, the signatory
governments agreeing under Article 9 (e), to "Cooperate in providing financial and
other support for ex-situ conservation". This call was not a new one; the UN
Conference on the Human Environment, meeting in Stockholm in 1972, had already
recommended action "in respect of micro-organism germ plasms, [to] co-operatively
establish and properly fund a few large regional collections" (United Nations 1972).
Co-operative action on the level required has not taken place, and nor will it now

28

unless the collections themselves develop a workable strategy in discussion with the
donor community and national governments.
An indication of what could be achieved is provided by the International Plant
Genetic Resources Institute (IPGRI; formerly the International Board for Plant
Genetic Resources, IBPGR). IPGRI, founded in 1974, established a network of
centres specializing in storing the seeds of particular crops in order to further the
collection, documentation and use of germplasm for crop species (Williams 1988).
In the case of microbial collections, organizations and networks exist which can
be used to ascertain what strains are held, including the European Culture Collection
Organization (ECCO), the World Federation for Culture Collections (WFCC),
Microbial Strain Data Network (MSDN), Microbial Information Network Europe
(MINE), and various national organizations. The Biodiversity Information Network
21 (BIN 21; Canhos et al. 1992) also promises to ease the exchange of data on
holdings. However, these bodies do not have the resources or mandate to control or
coordinate their member collections. The UNESCO/UNEP-sponsored
Microbiological Resource Centres (MiRCENs) scheme, establishing centres,
especially in less developed countries (Da Silva 1991), is very much on the lines
required but has been undersourced and will need to adapt to the changed situation
with regard to property rights.

How can national and regional collections contribute to the sustainable use of
the earths resources?

Article 10 of the Convention on Biological Diversity places emphasis on "the

sustainable use of components of biological diversity" and not only on its
conservation. In addition to the general roles of genetic resource collections of fungi,
they can contribute to sustainability at the national and regional level by:
- safeguarding the resource, especially from disappearing habitats.
- making the resource available for screening and use on the terms of the country
of origin.
- utilizing the resource for biocontrol, waste degradation, and food production.
- maintaining mycorrhizal mutualists important for forest re-establishment.
- providing a source of reference strains to speed the diagnosis of plant
pathogens.
Making the fungal resource available in a controlled way for screening purposes
which will bring maximum income into a country is of especial significance. The
issues are complex (Reid et al. 1993), and national legislations have yet to evolve.
However, payments on a per strain basis linked to royalty agreements are possible.
When the question of how to use a particular hitherto uncut forest in a sustainable
way for the benefit of the local population is posed, isolating and selling on fungi is
an option not to be discounted. In cases where an individual country cannot provide
a national facility, agreements on a regional basis should be sought.

Conclusion

Microbial resource collections are the way of making fungal biodiversity

available for exploitation in support of sustainable development. They are the
catalyst required to turn potential into reality.

29

 However, fulfilment of that goal will require both collaboration and the
development of mechanisms to enhance the complementarity between collections.
Collaborative networks at a variety of levels are emerging as the logical approach to
the provision of biosystematic services in speciose groups (Janzen 1993, Jones
1994). Genetic resource collections have demonstrated, through MINE and other
initiatives, that they can successfully combine and exchange data. We should not be
complacent, and recognize the need to move to a second level of co-operation
beyond that of networking data; to a fuller integration in which national, regional,
and international facilities have complementary roles. As such a proposal would be
in accord with current concerns as to the conservation and sustainable use of
biodiversity, we should not be afraid of making major funding proposals. "Big
science" funds with no immediate benefits to humankind are secured because of a
unanimous clamour from the enthusiastic scientists involved; we need to follow suit
and convey our vision of the potential to the donor community.

Acknowledgement

I am indebted to Dr David Smith for his contribution to the statistics and also discussion and
comments during the preparation of this article, and to Dr H. Sugawara for a detailed analysis of some of
the data in the World Directory.

References

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[Not seen.]
Brasier C.M. 1987. The dynamics of fungal speciation. In Evolutionary Biology of the Fungi, Rayner
A.D.M., Brasier C.M. and Moore D. eds., Cambridge: Cambridge University Press. pp. 231-260
Bridge P.D., Hudson L., Hawksworth D.L. and Bridge D.A. 1986. Variation in nuclear DNA content in an
ex-type isolate of Penicillium measured by continuous flow microfluorimetry. FEMS Microbiology
Letters 37: 241-244.
Canho V., Lange D., Kirsop B.E., Nandi S. and Ross E. 1992. Needs and Specifications of a Biodiversity
Information Network. Nairobi: United Nations Environment Programme.
Crittenden P.D., David J.C., Hawksworth D.L. and Campbell F.S.1995. Isolation and culturing success in
a broad spectrum of lichen-forming and lichenicolous fungi. Mycol. Res. 106: 788-795.
Fungal Genetics Stock Center. 1988 Catalogue of Strains. Second edition. Kansas City, Mo.: University
of Kansas Medical Center.
Da Silva E.J. 1991. Biotechnologies, microbes and the environment. Nature and Resources 27(3): 23-29.
Gams W. 1992. The analysis of communities of saprophytic microfungi with special reference to soil
fungi. In Fungi in Vegetation Science Winterhoff W. ed., Handbook of Vegetation Science, 19(1):
183-223.. Dordrecht: Kluwer Academic Publishers.
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Nomenclature (Tokyo Code). Regnum Vegetabile No. 131. Knigstein: Koeltz Scientific Books.
Groombridge B. ed. 1992. Global Biodiversity. Status of the Earth's Living Resources. London: Chapman
and Hall.
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Mycological Research 95: 641-655.
Hawksworth D.L. 1993. The tropical fungal biota: census, pertinence, prophylaxis, and prognosis. In
Aspects of Tropical Mycology. Isaac S., Frankland J.C., Watling R. and Whalley A.J.S. eds.
Cambridge: Cambridge University Press. pp. 265-293.
Hawksworth D.L. and Ritchie, J.M. 1993. Biodiversity and Biosystematic Priorities: Microorganisms and
Invertebrates. 120 pp. Wallingford: CAB International.
Huhtinen S. 1994. Traditional discomycete taxonomy: should we also shift to a second gear. In
Ascomycete Systematics: Problems and Prospects in the Nineties. Hawksworth D.L. ed.: New York:
Plenum Press. pp. 295-302.

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Jacobsen D.J. and Gordon, T.R. 1991. Fusarium oxysporum f.sp. melonis: a case study of diversity within
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Janzen D.H. 1993. Taxonomy: universal and essential infrastructure for development and management of
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31

 THE FUNGAL CULTURE COLLECTIONS IN EUROPE:

CHANCES, CHANGES AND CHALLENGES
D. VAN DER MEI

Centraalbureau voor Schimmelcultures, Baarn, the Netherlands

The celebration of the centennial of the "Mycothque" of the Catholic University

of Louvain-la-Neuve is in itself reason enough to congratulate the University and the
"Mycothcaire", Gregoire Hennebert. I could leave it at this.
This centennial jubilee, however, concerns an Institution within a University, and
a collection of fungi. And these two aspects ask for a somewhat deeper reflection.
To-day's universities are characterized by rapid changes, triggered by financial
problems and short-term policies more often than by long-term considerations in
which sciences should be embedded. This holds for most disciplines, but particularly
so for systematic biology with as its main component taxonomy and as its major
instrument the collections. Most universities, as a consequence, have in the recent
history been not very accommodating media for these two.
In the last two decades the number of taxonomic staff in universities all over
Europe and world-wide has dwindled, collections in particular being considered as
questionable assets.
One of the reasons is that collections are rather expensive to keep, while the
revenues, mostly measured as numbers of publications in highly esteemed
international journals, are only to be seen in the long term and are seldom directly
retraceable to the collection and the university that pays for it.
More important is that the scientific scene for many years has been dominated in
biology by the so-called "reductionistic approaches", with as main components
molecular and cell-biology. The great break-through, originating from these
disciplines, has grasped the imagination of the public domain, masterminding the
direction of research in virtually all biological subjects. The holistic approach, in
which the organisms themselves in relation to their surrounding environment, living
and dead, are the issue, has lagged behind.
The fact is that these two mainstreams, reductionism and holism, are still not
well-balanced, making biology a divided house. This is in contrast to physics and
chemistry. The holistic approach, of which collections - whether they contain living
or dead material - are to be seen as a major component, has to gain a lot of lost
ground in order to compete successfully for national and international shares of the
budget.
Well over 100 years ago the Swiss author Rudolph Tpfer created his
"Monsieur Cryptogame", internationalized as "Herrn Steckelbein" in Germany and
as "Mijnheer Prikkebeen" in the Netherlands. He personified the "Collectioner" as a
man deeply satisfied by the pleasure of sampling in Nature, and deeply unhappy with
womanhood, expressed by his pathological fear for his sister Ursula. A caricature he
was.
But when discussing collections and taxonomy with those who decide upon my
budget for next year, I still encounter the deep-felt suspicion that the only reason for
our existence is in playing Monsieur Cryptogame, the fear for sister Ursula maybe
replaced by the fear for bureaucrats, still mainly motivated by gathering as many

32

organisms as possible for the pleasure we get from them and not bothering about the
sense of it all.
The taxonomist, at the time of birth of Monsieur Cryptogame, stood at the cradle
of the unifying concept in biology, the theory of evolution. And he still stands in the
centre of evolutionary biology, looking for the roots and the history of biodiversity
as it presents itself to-day. And he has grasped the opportunities that are offered by
new techniques, especially in molecular biology, to tackle the problems more
adequately. In taxonomy, and directly in relation to it, in collections, a major effort is
undertaken to bridge the gap between reductionism and holism, for the benefit of
biology as a whole.
I hope that in the near future the taxonomy, within the context of systematic and
evolutionary biology, of life on the planet earth will reach on the scale of issues
worthwhile investigating the same level as the taxonomy of our stellar system, for
which billions of dollars are attributed easily in the construction and salvation of
Hubble-telescopes and the like.
The fact that Biodiversity has appeared on the agenda of the United Nations is a
milestone, as is the fact that taxonomy nowadays is firmly linked to the mainstream
in biology.
Let's not be over-optimistic and self-confident. There are some major hurdles
ahead, to mention a few:
1. Taxonomy and collections lack a strong organizational structure, that can act as a
sparring partner speaking with a voice that is clearly heard.
In the Netherlands, within the framework of the graduate school of biodiversity,
botanists and zoologists are always to be reminded that there are also
microorganisms and collections, besides botanical gardens and zoos, where
living organisms are kept in perfect shape. There seem to be three taxonomies,
that of animals, that of plants and that of microorganisms.
2. The expectations of those who put biodiversity on the political agenda may differ
greatly from insights, expectations, possibilities and capacities of taxonomists
and collections.
3. Biodiversity has the danger of becoming a fashion. Fashions in science may lead
to a proliferation of activities, based upon status and "getting a slice of the pie",
rather than upon sound scientific strategies and ideas. The fact that taxonomy is
at the basis of this fashionable term is often overlooked.
Nevertheless, there is a revival of taxonomy, and together with that, a keen
interest in what collections are and what they can do. It is up to us to show that we
are up to the challenges, national, European and world-wide.
A recent report of the Royal Netherlands Academy of Arts and Sciences, in
which its scientific priorities are reconsidered, still does not mention taxonomy as
one of the main issues.
For survival of the bacterial collections in the Netherlands money has to be found
exclusively on the free market and in contract-research.
These are also facts. Structural support has to come from reshuffling of budgets
invested in medicine, chemistry and physics, disciplines which have well-structured
organizations and lobbies. Without a fight we will not succeed.
My first words in this part of my lecture were to praise the Universities of
Leuven and Louvain-la-Neuve for fostering the "Mycothque" through the first 100
years. They have shown wisdom in doing so, and it is my sincere wish that they
continue to support the Mycothque for the next 100 years, as the only

33

"mycothque" among many just plain European fungal "collections". And we even
do not ask them to ensure that the "mycothcaire" should be allowed to go on till he
can celebrate his centennial. We know him well enough that - apart from any official
position - he will continue as the taxonomist in heart and soul that he has always
been.
The main theme given to me to talk about is.

Fungal collections in Europe.

My expertise on this subject should not be exaggerated. On first sight it seems to

be quite appropriate for someone who has been the coordinator of the Microbial
Information Network Europe (MINE) project, aiming at bringing together some
40 collections in 12 European countries in the endeavour to set up a common format
for the data on bacteria and fungi and to bring these data together at a Central Data
Node. I have to admit that my view on this matter is still a bit blurred, MINE not
always being illuminating in this respect. Notwithstanding the rather limited
financial support from EC, and an underestimation of the complexity of the project,
it is clear that:
1. There is great willingness to cooperate between collections.
2. The framework for optimal cooperation still has to be established.

In order to improve on this second factor, the good framework for cooperation, a
number of facts and conditions should be kept in mind.
1. Collection policy is mainly determined at the national level, not in Brussels. Any
cooperative approach should be based on a "Europe of Nations" not on a "Federal
Europe". In retrospect, without having participated during the whole preparatory
phase of MINE, I am convinced that this aspect has been neglected in the onset
of the MINE project. After all, we must realize that, however important Brussels
has become on the playground of subsidizing institutions in Europe, a role
recently illustrated by the acceptance of the 4th Framework in which some 12.3
billion ECU's are to be spent on research for the next four years, it still does not
cover more than 5% of the total input from public means in the countries of the
European Union. The major impact of Europe is in cooperation and in
stimulation, the necessary infrastructure still being a matter of the national
governments.
2. Without cooperation and presentation of initiatives that are considered worthwhile
and challenging in the pursuit of biotechnology, a sustainable use of natural
resources, and in the competition with Asian and American counterparts, major
inputs from the European Union in taxonomy and collections are, at least,
questionable. This support is needed to cope with expected technical and
methodological changes in collections.
3. Without cooperation and coordination, the diversity in European collections,
which can be considered a great asset, both in the variety of organisms kept and
in the expertise they represent, will become a hampering factor, not to be used to
the maximum in the world's biodiversity programs.
4. Any future cooperation must include Eastern Europe. The borders of the European
Union are not to be considered limitative.
5. Collections are fiercely competing organizations, not only in assets and their
scientific value, but also - and increasingly so - on the free market.

34

 This summary of statements proves that any framework for cooperation should
accommodate the deeply felt necessity for working together bearing in mind that
there are a number of hampering factors in this respect. There is a certain reluctance
to be encountered when discussing input from taxonomy and collections in
biodiversity research with EU officials. This was the case with MINE, and it can be
clearly seen in the cautious approach in the concerted action for microbial
biodiversity. And the underlying factor is mistrust in the potential for collections and
taxonomists to come forth with coherent and well-defined proposals. Maybe not
rightfully so, but the fact has to be acknowledged.
An overview of the major actors, professional groups as well as organizations, in
the fields of taxonomy, collections and biodiversity research is represented here in
the form of a human figure, the caption 'Quo Vadis?' indicating that the direction it
will take is not certain yet.
The trunk consists of the scientific community and it financing bodies. The arms
represent the major international organizations, the United Nations and the Unions of
the International Council of Scientific Unions.
The head, becoming rather large, represents the politicians, civil servants,
managers and lawyers.
The legs, symbolizing the European Culture Collections Organization (ECCO)
and MINE are in contrast rather thin.
The driving factors are, of course, to be found at the heart: the taxonomist and the
curator. They are supplied, symbolized by blood vessels and lungs, by enthralling
ideas from many other scientific disciplines such as molecular biology and
biotechnology, which in turn acknowledge the mighty impulses they get from the
heart, recently revived by the bypasses biodiversity and evolutionary biology.
The discussions between these groups are not always easy, as illustrated by some
imaginary conversations on biodiversity.
The molecular biologist has just received a DNA sample for identification from a
zoo in some faraway country. He compares it with some DNA obtained from his
colleague, the taxonomist. After one day he exclaims that in the country concerned
they have the atrocity to keep human beings in zoos, as the DNA sample matches
rather perfectly the DNA from the taxonomist. The taxonomist, who by nature is a
curious man, travels to the zoo, and returns quite disappointed: the sample was that
of a chimpanzee. Every one of us in this case is an expert: quite clearly the
chimpanzee and the human being are different species, one bothering about his
origins, the other, as far as we know, not.
However, when we are talking about microorganisms, the claim of the molecular
biologist that he is the one who can unequivocally discriminate between species by
applying his DNA trick is often acknowledged without dispute.
The biotechnologist and the taxonomist discuss biodiversity in a quantitative
way, the taxonomist stating that there are over six million species on earth. The
biotechnologist is hardly impressed. His biodiversity concept is governed by the
enzymes and he knows that in evolution, through all kingdoms, from archaebacteria
to the animals, has resulted in about 5000 different enzymes, subdivided in 60
groups and 6 classes. His biodiversity concept is much simpler, interested as he is in
regulatory systems that lead to the production of enzymes in higher quantities and
that do the tricks he wants them to perform, better. And then again he is primarily
interested in strains, not in species. The direct surrounding of the taxonomist thus is

35

one in which different paradigms are amalgamated in an enriching way. The least
that should come of it is a reconsideration of species concepts.
The discussions with the organizations constituting the rest of the body are quite
different, but not less important in character.
They have to do with selling ideas in such a way that the product, of which we
only are at the onset aware of its value, becomes attractive to those who should pay
for it.
This process takes place at an increasing number of levels and is to a certain
degree masterminded by politicians, managers and lawyers. It starts at the level of
universities and research councils and ends at conferences in Mexico and Nairobi. It
is sometimes tempting to look at these levels with some disdain, but fact is that all of
us are devoting more and more time to travel around the world and participate in the
circuit of buying and selling science. Without clear research-strategies the danger
that the outcome is mainly determined by the buyer, is evident. When looking at
biodiversity, the four main topics are: the scale, where? what? and how?

The scale
It is the world, but also the own back garden.
The most far-fetched proposal, the All Taxon Biodiversity Investigation may
raise severe doubts about feasibility and scientific value; it has the scale that can
grasp the mind of policy-makers, which surely can boost the impact of taxonomy,
together with collections as the main actors in the biodiversity field, a role that
seemingly cannot be attained by smaller projects.

Where?
The focus on the tropics is evident. There are, however, many other habitats that
are of great interest and about which much is still unknown.
They should not be overgrown by over-emphasizing, strongly supported by
public concern about its destination, the Rain Forest. The soil, the tundra, the sea are
habitats in the temperate zones that ask for special attention of taxonomists and of
collections in Europe too.

What?
Besides taxonomic issues about inventory, isolation and identification, evaluation
of species concepts, and nomenclature, the collections in particular are confronted
with questions on how to preserve, in situ and ex situ, what to preserve, and where.
This is accompanied by international questions about ownership of
microorganisms, resulting form the Biodiversity Convention.

How?
There is a limited number of taxonomists, and a rather large number of
collections, different in character and quality. Without good cooperation between
collections, resulting in well-defined forms of quality control and division of labour
and fields of activity, I am afraid that collections will find it even more difficult to
present themselves in the field of biodiversity research than the taxonomists in
general.
It is important to indicate strong - and consequently also weak - points in the
European collections, and this should be done by collections themselves.

36

 I have already stated that the legs are in bad shape: we have to worry about their
capacity to carry the whole body steadily. The message is clear: the legs have to be
strengthened.
Rather then finding new formulas, I am of the opinion that in principle the
existing organizational structures, ECCO for more conceptual thinking, MINE for
more technical matters should be adequate, on condition that they are well-equipped
for their supporting roles. And this asks for a rather fundamental discussion and
reshaping of both organizations.
ECCO should evolve from a yearly meeting of curators into an organization that
can speak up for collections and is not afraid to raise issues that may be of some
concern to the collections themselves.
MINE should be transferred into a consortium.
Most important is to find, for both organizations, a constitution that is recognized
by all collections as fair, with boards that are equipped with a clear mandate to work
with.

There is nothing new, only the form has changed and things are now more
complicated. CBS originated in 1903 as a result from a concerted action by the
international community of scientists to start something that is to the benefit of that
community.
I am sure that we also will celebrate our centennial as proof that international
cooperation pays off in the long run.
And if things become boring or too complicated we have always our inspiration
from the organism that governs our daily lives, the Fungus. In itself it is beautiful
and far more complicated than the structures I have been talking of.

37

THE BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS

BCCM
J. DE BRABANDERE

SSTC, Rue de la Science 8, 1040 Bruxelles

The 'Belgian coordinated Collections of Microorganisms, BCCM' form a

consortium of four complementary research-based culture collections at the service
of the international scientific and industrial community.
The Belgian Science Policy Office launched the BCCM-action in 1983 with the
following mission.
1. The characterization, inventory and preservation of the pool of available isolates,
reference strains and plasmids (over 41.000 items), to be expanded along the
lines of the specializations of each collection:
- the LMG bacteria-collection (Director: Prof. Dr. K. Kersters, Curator: Dr. D.
Janssens) holding over 13000 strains;
- the MUCL collection for fungi/yeasts of biodiversity and (agro)industrial interest
(Director/Curator: Prof. Dr. Ir. G.L. Hennebert) holding over 22000 strains;
- the IHEM collection for fungi/yeasts of biomedical interest (Director/Curator: Dr.
N. Nolard) holding over 5000 strains;
- the LMBP plasmid-collection (Director: Prof. Dr. Ir. W. Fiers, Curarator: Prof. Dr.
E, Remaut) holding over 1000 plasmids for use in bacteria, filamentous fungi,
yeasts and animal cells.

2 The distribution of the authenticated biological material and the corresponding

information to interested scientific and industrial circles.

3 The acceptance of patent deposits valid within the framework of the international
Budapest Treaty (N.B. The BCCM received in 1992 - and this as a consortium -
status of International Depositary Authority, IDA);

4 The valorization of the available skills in the field of both fundamental and applied
(micro)biology by providing professional and confidential ad hoc services and
contract-research to third parties (e.g. molecular characterization and typing of
industrial and medical strains, safe deposits of propriety strains, training,
screening for primary and secondary metabolites etc.).

All BCCM collections are active members of the World Federation for Culture
Collections, WFCC' and the European Culture Collections Organization, ECCO'.
Moreover, the BCCM are founding-members of the Microbial Information Network
Europe, MINE'. As such, they host the Data Integrating Node for bacteria at
Ghent and chair the Responsible Committees for bacteria and fungi/yeasts
The organization of the BCCM-consortium constitutes a rather unique model that
distinguishes itself from foreign examples by the stable funding from one common
financier that is also in charge of the central coordination and support of the main
policy issues (client approach, informatics, quality management etc.).

38

 PART 2

FUNGAL TAXONOMY AND TROPICAL MYCOLOGY: QUO VADIS ?

An Introduction

Grgoire L. HENNEBERT

Mycotheca of the Catholic University of Louvain, 2 Place Croix du Sud, 1348 Louvain-la-Neuve,
Belgium

Modern taxonomy, including mycotaxonomy, relies in an increasing degree on

new molecular approaches.
As such, one can observe that the traditional morphotaxonomic approach is more
and more complemented - or sometimes even substituted - by chemotaxonomic
methods or genome-based analyses. Questioning the value and mutual
complementarities of all available tools - both classical and modern - is essential to
be able to handle the systematic classification problem in an integrated and effective
way.

Molecular Classical
Taxonomy Taxonomy
Fungal
Biodiversity
&
Collections

Taxonomy in the Tropics

On the other hand, the greater part of the fungal biodiversity has still to be
discovered and described especially in the tropical regions. As these are mainly
situated in developing countries, one has to discuss - in the spirit of the Rio de
Janeiro earth summit - key topics such as training, technology transfer and the set-up
of local 'ex situ' collections.
During such an exercise, special attention has to be given to taxonomic and
preservation methods that are not only effective but also widely applicable, user-
friendly and robust.

39

 MOLECULAR TAXONOMY OF THE YEASTS: PRESENT AND FUTURE

CLETUS P. KURTZMAN

Microbial Properties Research, National Center for Agricultural Utilization Research, Agricultural
Research Service, U. S. Department of Agriculture, Peoria, Illinois 61604 U.S.A.

Key words. -- Yeast systematics, ribosomal RNA/DNA, molecular evolution, phylogeny

Introduction

The systematics of yeasts, as with other groups of fungi, is predominantly based

on a hierarchy constructed from morphological differences among the taxa. The
present system of classification relies heavily on types of budding or the occurrence
of fission, presence or absence of hyphae, morphology of ascospores, and
morphology of basidia and the manner in which basidiospores are produced. As a
consequence, these characters often define orders, families, genera, and even species.
Whether the characters reflect the evolutionary history of the yeasts is unknown, but
if systematics is to be based on phylogeny, i.e., a natural classification, the genetic
basis of currently used characters must be understood.
Application of molecular comparisons to questions in yeast classification offers
an unprecedented opportunity to reevaluate current taxonomic schemes from the
perspective of quantitative genetic differences. This review examines the impact of
molecular comparisons, notably ribosomal RNA/ribosomal DNA (rRNA/rDNA)
sequence divergence, on the current phenotypically defined classification of the
yeasts.

Placement of yeasts among the fungi

The yeasts represent a unique group of fungi characterized by vegetative growth

that is predominantly unicellular, and by the formation of sexual states which are not
enclosed in fruiting bodies. Saccharomyces cerevisiae has been recognized as an
ascomycete for well over a century, but it was not until much later that some yeasts
were thought to be basidiomycetes (Kluyver and van Niel 1927). This supposition
was confirmed by Banno (1967) with the description of the heterobasidiomycetous
species Rhodosporidium toruloides. With this finding came the realization that the
yeasts are phylogenetically quite divergent. In the following discussion,
relationships among ascomycetous and basidiomycetous taxa are examined from the
perspective of phylogeny determined from small and large (18S, 26S) subunit
rRNA/rDNA sequence divergence. Methodologies have been discussed by Bruns et
al. (1991), Kaltenboeck et al. (1992), Kurtzman (1992), O'Donnell (1992), and
White et al. (1990).

Ascomycetous yeasts
The phylogeny of the ascosporogenous yeasts has been vigorously debated since
the time of Guilliermond (1912) and before. Some have viewed the yeasts as
primitive fungi while others perceived them to be reduced forms of more evolved
taxa. Cain (1972) has been a proponent of this latter idea, arguing that hat (galeate)-
spored genera such as Pichia and Cephaloascus are likely to be reduced forms of the

40

perithecial euascomycete genus Ceratocystis. Redhead and Malloch (1977) and von
Arx and van der Walt (1987) accepted this argument and commingled yeasts and
mycelial taxa in their treatments of the Endomycetales and Ophiostomatales.
Examination of rRNA/rDNA sequence divergence from a limited number of taxa
indicated the ascosporogenous yeasts, with the exception of Schizosaccharomyces, to
form a monophyletic group (clade) distinct from the filamentous species (Barns et al.
1991, Bruns et al. 1991, Hausner et al. 1992, Hendriks et al. 1992, Kurtzman 1993a,
Nishida and Sugiyama 1993, Walker 1985, Wilmotte et al. 1993). Kurtzman and
Robnett (1994a) analyzed rRNA sequence divergence from type species of all
cultivatable ascomycetous yeasts and yeastlike taxa. This work demonstrated the
yeasts, as well as yeastlike genera such as Ascoidea and Cephaloascus, to comprise a
clade sister to the "filamentous" ascomycetes (euascomycetes). Eremascus, which
forms asci unenclosed in a fruiting body, aligned with the euascomycete clade and
may represent a genus close to the phylogenetic demarcation of the
hemiascomycetes (excluding Schizosaccharomyces) and the euascomycetes. These
results substantiate the long-held observation that yeasts cannot be defined solely on
the basis of presence or absence of budding. Such members of the yeast clade as
Ascoidea, Ashbya and Eremothecium show no typical budding, whereas
Aureobasidium, Phialophora and certain other genera of euascomycetes are usually
dimorphic. Budding is also a common mode of vegetative reproduction among many
basidiomycetous genera. Similarly, vegetative reproduction by fission is shared by
Dipodascus and Galactomyces, members of the yeast clade, as well as by the
distantly related genus Schizosaccharomyces. Sexual states of all members of the
yeast clade are characterized by asci unenclosed in a fruiting body. This feature is
shared by only a few taxa outside the yeast clade such as Eremascus and
Schizosaccharomyces. Myrioconium and Trichomonascus form unenclosed asci but
may be euascomycetes as well.
Phylogenetic relationships among the ascomycetous yeasts, calculated from
partial sequences of small and large subunit rRNAs, are depicted in Fig. 1. Although
there are insufficient phylogenetically informative sites to resolve many of the
genera, the comparison gives an overview of relationships. Tree topology is similar
to that presented by Wilmotte et al. (1993), who examined fewer species, but used
nearly complete 18S sequences. In the comparison of partial sequences, most taxa
having coenzyme Q with the same number of isoprene units tend to group, but this is
not true of the closely related genera Ashbya, Eremothecium, Holleya and
Nematospora which show a variation in coenzyme Q ranging from 5-9 isoprene
units. Some congruence is found between location of taxa on the rRNA gene tree and
the type of hyphal septal pore produced. The two known genera (Ambrosiozyma,
Hormoascus) that form dolipore-like septa are closely associated. However,
Arthroascus produces septa with a simple central pore whereas Guilliermondella has
multiperforate septa, yet the two genera closely cluster. These incongruities may be
resolved by sequencing the complete 18S molecule and by including all known
species for each of the genera under study.
rRNA/rDNA sequence comparisons have been quite helpful for understanding
species relationships within genera. For example, Schwanniomyces occidentalis
(Kurtzman and Robnett 1991), Wingea robertsii (Kurtzman and Robnett 1994b), and
the Pichia species, P. carsonii and P. etchellsii (Yamada et al. 1992), were found to
be members of the genus Debaryomyces on the basis of rRNA relatedness. The
initial assignment of these species to other genera resulted from misinterpretation of

41

the phylogenetic significance of ascospore morphology. Species originally placed in

Debaryomyces are characterized by spheroidal ascospores that are roughened by
wartlike or ridgelike outgrowths of wall material. An exception is D. marama which
has ellipsoidal ascospores with wartlike outgrowths and spiral ridges. In contrast,
Schwanniomyces forms spheroidal ascospores with surface projections and a
prominent equatorial ring, Wingea has smooth, lenticular ascospores, and the two
former Pichia species produce smooth, spheroidal ascospores. Furthermore,
Saturnospora and Williopsis both form saturnoid ascospores, yet the two genera are
only distantly related (Liu and Kurtzman 1991).
The impact of rRNA/rDNA comparisons on the taxonomy of ascomycetous
yeasts is just being felt and will require additional work to fully realize its potential.
Major findings to date include: 1) yeasts and yeastlike species are phylogenetically
separate from the euascomycetes, 2) the fission yeast genus Schizosaccharomyces is
phylogenetically distant from the "budding" yeast clade and from the euascomycetes,
resulting in the reassignment of the fission yeasts to a separate order, the
Schizosaccharomycetales (Eriksson et al. 1993, Kurtzman 1993a) and, 3) the
demonstration that many phenotypic characters such as ascospore morphology are
poor indicators of phylogeny.

Basidiomycetous yeasts
Anamorphic (asexual) basidiomycetous yeasts may be morphologically
indistinguishable from anamorphic ascomycetous yeasts. The discovery that the
inner cell walls of basidiomycetous yeasts are lamellar when viewed in thin section
under the transmission electron microscope, in contrast to the uniform inner layer of
ascomycetes, has provided a reliable means for separation of the two taxonomic
classes when sexual states are not found (Kreger-van Rij and Veenhuis 1971). A
second method of separation, more easily applied, is the Diazonium Blue B (DBB)
staining technique (van der Walt and Hopsu-Havu 1976). Colonies of
basidiomycetes stain a magenta color in the presence of DBB whereas colonies of
ascomycetes remain unstained. From these findings, it has become apparent that
basidiomycetes make up a large part of the yeast domain.
Basidiomycetous yeasts (Fell and Kreger-van Rij 1984, Boekhout et al. 1993). In
the first, teliospores are formed and germinate to produce a basidium that bears
basidiospores. This type of sexual cycle shows considerable similarity to the rust and
smut fungi. The second type of sexual state lacks teliospores. Basidia develop on
hyphae or yeast cells and give rise to basidiospores in a manner similar to the
Tremellales (jelly fungi).
Several other characteristics are added to the dichotomy of sexual states. Some
taxa produce carotenoids, and the presence of these pigments has been used as a
criterion for genus assignment. Ballistoconidia, forcibly ejected vegetative cells, are
common to some taxa and their presence is a defining character of genera.
Additionally, the hyphal septal pore of basidiomycetous yeasts may be either simple
or the ultrastructurally more complex dolipore. Finally, some taxa exhibit the
presence of cellular xylose, evidently arising from extracellular polysaccharides
(Golubev 1991), whereas other species do not.
Guho et al. (1989) presented an overview of the phylogeny of basidiomycetous
yeasts from measurements of divergence among partial sequences of large and small
subunit rRNAs. Three major groups were resolved; 1) teliospore formers with
hyphae having simple septal pores, 2) teliospore formers with hyphae having

42

dolipore septa and, 3) non-teliospore formers with hyphae having dolipore septa. On
the basis of 18S sequence comparisons, Suh and Sugiyama (1993) placed the smut
fungus Ustilago maydis, a teliospore former, near the clade comprising
representative genera of all basidiomycetous yeasts. In turn, the basidiomycetous
yeasts appear to be a sister group to the Agaricales (Berbee and Taylor 1993).
At present, the most extensive phylogenetic comparison of basidiomycetous
yeasts is that of Fell et al. (1992) who examined 117 species assigned to 23 genera.
A 247-nucleotide segment in the D2 region was sequenced; this region resolves
closely related species, but may have too few phylogenetically informative sites to
accurately assess more distant relationships. The phylogram from that work (Fig. 2)
shows the species to be divided between two major clades. The analysis generally
supports the concept that taxa assigned to the Tremellales are characterized by
dolipore septa and cellular xylose, whereas taxa placed in the Ustilaginales form
teliospores, have simple septal pores, and lack cellular xylose. Some exceptions are
apparent. The teleomorphic genus Erythrobasidium does not form teliospores as do
other members of the clade. Cystofilobasidium and Mrakia, both members of the
Tremellales, form teliospores. Another inconsistency concerns the genera located in
the lower portion of the Tremellales clade (Fig. 2, B). From what is known of their
septal pore structure and lack of cellular xylose, the genera would be expected to
group with the Ustilaginales. If their placement is correct, this would suggest that
the Tremellales arose from within an already highly diversified group that now
represents the Ustilaginales.
Ballistoconidia and carotenoids are found among many genera of the
basidiomycetous yeasts suggesting these traits to be ancestral, but not always
expressed, thus rendering them of little value for defining taxa. These conclusions
were also drawn by Nakase et al. (1993). Heterogeneity of coenzyme Q composition
occurs in many currently defined genera and will require additional study before its
taxonomic significance is fully understood. rRNA sequence analysis demonstrates
Rhodotorula, Sporobolomyces, Cryptococcus, and Bensingtonia to be polyphyletic,
further confirming that commonly used phenotypic characters are insufficient for
defining anamorphic genera.
A strong start has been made to understand the phylogeny of basidiomycetous
yeasts from rRNA/rDNA sequence comparisons, but additional sequencing must be
done to better resolve taxa. This will include sequences of greater length as well as
inclusion of all known species of a group. For example, the anamorphic genera
Tseuchiyaea and Ballistosporomyces, which were recently defined from differences
in partial sequences, may overlap with some earlier described genera (Fig. 2).

Reliability of rRNA/rDNA gene trees to infer phylogeny

With such great emphasis being placed on rRNA/rDNA gene trees to reconstruct
phylogenies, it must be asked if there is other evidence to corroborate the
conclusions drawn. There are presently only a few comparisons of other molecular
sequences that allow this question to be addressed. Sequence analysis of orotidine 5'-
monophosphate decarboxylase by Radford (1993) demonstrated the budding yeasts
and the euascomycetes to be sister groups as shown from rRNA/rDNA sequences.
One unusual aspect of this work was placement of the Mucorales as a sister group to
the basidiomycetes. Tsai et al. (1994) showed phylogenetic relationships among
species of Epichlo (Clavicipitaceae) were the same when analyzed from either

43

rDNA sequences or those from the -tubulin gene. Relationships among species of
Dekkera and its anamorph Brettanomyces were essentially identical when analyzed
from either nuclear rDNA sequences or from sequences of the mitochondrially
encoded cytochrome oxidase subunit II gene (Boekhout et al. 1994).
Another aspect of gene tree reliability is the method used for its construction.
Most investigators now analyze data using phylogeny inference programs based on
cladistic principles. These programs often include a statistics package to test the
robustness of competing phylogenetic trees. Several recent reviews address these
important issues (Avise 1989, Felsenstein 1988, Hillis et al. 1994, Saitou and
Imanishi 1989).

Rapid molecular methods for yeast

The specificity of nucleic acid sequences has prompted development of several

methods for rapid species identification that are directly applicable to the strains
maintained by culture collections. Because these techniques can detect single
nucleotide changes, they should first be tested on a large variety of genetically
defined strains to understand species variation because aberrant strains may
represent different species.

Restriction fragment length polymorphisms of rDNA

rDNAs occur in multiple copies and lend themselves to analysis based on
restriction fragment length polymorphisms (RFLP). Magee et al. (1987) treated
rDNAs from several medically important Candida species with a variety of
restriction endonucleases and concluded that Candida guilliermondii, C. tropicalis,
and C. albicans produced sufficiently different digestion patterns to allow
recognition of each species. Similar results were obtained by Vilgalys and Hester
(1990) for several species of the genus Cryptococcus. Lachance (1990) used RFLP
patterns to map the genetic profiles of 125 isolates of the cactus yeast Clavispora
opuntiae that had been collected worldwide. Nearly all of the restriction sites that
allowed discrimination of individual strains were located in the hypervariable
intergenic spacer region.
Data from the preceding studies show that RFLP patterns allow recognition of
individual species, as well as individual strains of a species. Consequently, the
method has considerable diagnostic value. Estimates of evolutionary relationships
from RFLP patterns have been reported for species assigned to Candida (Magee et
al. 1987) and Cryptococcus (Vilgalys and Hester 1990). Such estimates would be
expected to be less accurate than estimates derived from sequence comparisons
because as evolutionary distances increase, the extent of pattern similarities becomes
less certain. Bruns et al. (1991) listed some of the factors that require attention when
using RFLPs.

Random amplified polymorphic DNA (RAPD)

RAPDs represent another methodology that promises widespread application.
The technique is based on amplification of genomic DNA in the presence of one or
more short (ca. 10-15-mers) oligonucleotide primers of random sequence.

44

Fig. l. A phylogenetic tree derived from maximum parsimony analysis depicting the ascomycetous yeasts,
yeastlike fungi, and various reference species. The phylogram was calculated from combined small and
large subunit rRNA partial sequences as described by Kurtzman and Robnett (1994a). Branch lengths are
proportional to nucleotide differences, and the numbers given on branches are the percentage of
frequencies with which a given branch appeared in 100 bootstrap replications. Branches without numbers
had frequencies of less than 50%. Coenzyme Q data are from the compilation of Barnett et al. (1990) and
refer to the number of isoprene units in the sidechain on the parent molecule. Information on septal pores
is from Kreger-van Rij and Kurtzman (1984) and Barnett et al. (1990).
Cent = central pore, Multi = multiperforate septum, Doli = dolipore-like.

45

Fig. 2. A phylogenetic tree derived from maximum parsimony analysis depicting the basidiomycetous
yeasts.The phylogram, which represents the most parsimonious tree, was calculated from rRNA sequences
of the D2 region of the large subunit as described by Fell et al. (1992). Branch lengths are proportional to
the number of nucleotide differences. Bootstrap values were not determined. Clade A is comprised of taxa
placed in the Ustilaginales. Taxa in Clade B are predominantly assigned to the Tremellales, but see text
for discussion. The strain of the outlying species Rhodotorula buffonii that was examined was shown to be
a misidentified ascomycete. Genera preceded by a broken line were not in the original analysis and have
been placed in the present phylogram on the basis of other studies (Guho et al. 1989, Nakase et al. 1993,

46

Sugiyama and Suh 1993). Teleomorphic genus names are followed by the letter T and anamorphic genera
are designated with the letter A. Morphological and biochemical characters are from Barnett et al. (1990),
Boekhout et al. (1993) and Fell and Kreger-van Rij (1984). Simple = simple septal pore, Doli = dolipore
septum.

The amplified products are visualized on an agarose gel and strains identified from
matching band patterns. Hadrys et al. (1992) discussed details of this procedure and
also noted points of technical difficulty.

Species-specific PCR reactions

Peterson and Kurtzman (1991) and Kurtzman (1993b) have shown that the 5' end
of the large subunit rRNA/rDNA is sufficiently variable to detect even closely
related yeast species. Fell (1993) used this region to identify individual yeast species
by applying a three-primer, PCR-based technique. In this method, the reaction mix
includes genomic DNA, two external primers for the D1/D2 region of large subunit
rDNA, and a species-specific internal primer. The external primers allow
amplification of the ca. 600-nucleotide D1/D2 region, but in the presence of a
species-specific primer (third primer) the amplification product is shorter and easily
detected on an agarose gel. There is currently considerable activity in the field of
molecular probe development and rapid methods appear nearly ready for widespread
use.

Future prospects

rRNA/rDNA sequence comparisons have provided us with a genetic perspective

to the relationships among yeasts and other fungi. On the basis of limited
comparisons, rRNA/rDNA gene trees are generally congruent with phylogenies from
other molecular sequences, but additional studies should be done to verify this
preliminary assessment. In many cases, molecular comparisons are supportive of
relationships among the fungi determined from traditional morphological studies. In
other cases, the molecules have led us to new perspectives. One of the greatest
challenges facing the field of mycology is to use molecular data to develop a
phylogenetic system of classification.
The unique nature of molecular sequences promises to allow development of
species-specific molecular probes for rapid strain identification. Once this
technology becomes widespread, a realistic assessment of the earth's biodiversity
may finally be possible.

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Suh S.-O. and Sugiyama J. 1993. Phylogeny among the basidiomycetous yeasts inferred from small
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49

PHYLOGENY OF FILAMENTOUS ASCOMYCETES DEDUCED FROM

ANALYSIS OF MOLECULAR CHARACTERS: PRESENT AND FUTURE

MEREDITH BLACKWELL

Department of Plant Biology, Louisiana State University, Baton Rouge, LA 70803 USA

Abstract. Phylogenetic studies of ascomycetes dispersed by arthropods have benefited from the use of
rDNA sequence analysis. At higher taxonomic levels it has been necessary to compare a variety of taxa
that either lack corresponding morphological characters (Raffaelea, Ambrosiella, Laboulbeniales, yeasts)
or have characters that may be the result of convergent evolution (Ceratocystis, Ophiostoma,
Pyxidiophora). For example Ambrosiella has been found to be polyphyletic with one group of species
related to Ceratocystis and the other group allied with Ophiostoma. Species of Ambrosiella are asexual
and could not have been compared to sexual forms soley on the basis of morphology. Several genera with
evanescent asci and arthropod associations previously have been placed in Ophiostomatales. We now
know that Ophiostoma, Ceratocystis, and Pyxidiophora are not closely related, and, in fact, at least six
lineages of such fungi have been identified. One other taxonomically problematical group,
Laboulbeniales, lacks a number of morphological characters that can be compared with filamentous forms
(including even the hyphal filaments!). The hypothesis of a relationship with Pyxidiophora based on few
morphological characters now has support from rDNA sequence analysis. It is important to note that our
results could not have been obtained so easily were it not for the previous studies of the morphology, life
histories, and associations of the fungi. For the future it will be important for mycologists in distant
localities using different techniques to cooperate in discovering and classifing the Earth's fungal diversity.
In addition to the continuing development of phylogenetic techniques, another inovation is important.
Worldwide collaboration already is enhanced at the close of the twentieth century by our ability to
communicate rapidly and at will using computerized communication networks, and this technology must
be available to all mycologists throughout the world.

Previous attempts at phylogenetic classification of ascomycetes have been

confounded by the presence of convergent morphological features, the absence of
characters that extend across all taxa, and an inability to determine character
polarity. However, a non-morphological approach, specifically ribosomal DNA
(rDNA) sequence analysis has provided new characters that circumvent the problems
assciated with the use of morphological characters.
In our work with arthropod-associated fungi we have been particularly interested
in the ascomycetes with long necked perithecia and evanescent asci and the
possibility that these characters are the result of convergent evolution to facilitate
ascopore dispersal by arthropods. Some of this work has been published and will be
reviewed only briefly to provide an example of the usefulness of the technique. In
this case nuclear-encoded rDNA sequence was applied to the study of ascomycetes
previously placed in Ophiostomatales, or in some cases, never placed in an ordinal
level taxon. The taxa are all characterized by evanescent asci and, in most cases, are
arthropod dispersed.
The relationships hypothesized on the basis of rDNA analysis indicate that a
divergence of the fungal lineage gave rise to the basidiomycetes and ascomycetes.
Both groups presumably arose from a filamentous ancestor. An early ascomycete
divergence, or perhaps several, produced such forms as Taphrina,
Schizosaccharomyces, Protomyces, and Pneumocystis (Sugiyama and Nishida 1993,
Nishida and Sugiyama 1994, Kurtzman 1993). These fungi are marked by yeast-like
stages as well as mycelial growth. It is interesting that they often have been at the
center of taxonomic controversy and all have been considered basidiomycetes at

50

some time in their history.

The next major divergence of ascomycetes gave rise to two lineages, the yeasts
of Saccharomycetales and the filamentous ascomycetes. It is these two groups that
have numerous arthropod associations. In addition some of the species in both
groups have morphological and biochemical characters in common. For example the
galeate or hat-shaped ascopore morphology, yeast thallus, and various conidial
stages found among species of Ophiostoma and Ceratocystis as well as some yeasts
led to the appealing hypothesis that these forms might constitute a monophyletic
group (Redhead and Malloch 1977, von Arx and van der Walt 1987). With most of
the major groups of yeasts examined, it does not seem likely that any of them are
derived from the filamentous ascomycetes, and the characters typical of arthropod-
associated ascomycetes are convergent features.
One major accomplishment involving the filamentous ascomycetes has been the
resolution of the relationship of Ceratocystis and Ophiostoma. At times species of
both genera have been considered congeneric. We now have evidence that not only
are the two genera distinct, but also they occur within different subclades of
perithecial ascomycetes (Spatafora and Blackwell 1993). Other filamentous forms
with evanescent asci including Pyxidiophora, Kathistes, and Subbaromyces lie
outside of the perithecial ascomycetes, and each may be part of an independent
lineage. The yeast-like Symbiotaphrina kochii also is in this part of the tree.
Several years ago Dr. David Malloch and I (Blackwell and Malloch 1989) used
morphology and life cycle studies to propose a relationship between Pyxidiophora
and Laboulbeniales. This hypothesis was tested using rDNA analysis and the
evidence supports the previous conclusion of their close relationship (Blackwell
1994). However, the nearest relatives of the Pyxidiophora-Laboulbeniales,
Kathistes, Subbaromyces, and Symbiotaphrina lineages have not been identified
because this part of the tree is unstable. Clarification awaits addition of more taxa
from the discomycetes and loculoascomycetes, and, perhaps sequencing of other
DNA regions.
Our more recent work on arthropod-associated ascomycetes involves the analysis
of more rapidly evolving gene regions to develop species concepts for Pyxidiophora
and the asexual taxa associated with Ceratocystis and Ophiostoma. Of particular
interest is the finding that Ambrosiella is not a monophyletic group, but consists of
two groups of species, one related to Ceratocystis and the other allied with
Ophiostoma, both in association with scolytid beetles. Convergence of conidial
forms has followed the pattern of convergence in their sexual relatives (Cassar
1993). Another asexual lineage does appear to be monophyletic. Species of
Raffaelea, however, have diverged from an ophiostomatalean lineage as associates
of platypodid beetles.
I have discussed examples of the impact of recent technological advances in
helping to define monophyletic groups and clarify the phylogenetic relationships of
the ascomycetes. But there are other reasons why mycologists study fungi, and the
same methods are useful in these cases as well. Molecular technology has become
important to us in identifying particular fungi at a variety of taxonomic levels. From
the practical point of view, a knowledge of species that attack plants and animals is
necessary. Not only is an identification of species necessary, but it also has become
important to identify populations among species of some fungi.

51

 Balansia sclerotica
Claviceps paspali
Epichloe typhina
Hypocrea schweinitzii
Sphaerostilbella aureonitens
Hypomyces polyporinus
Nectria haematococca
Neocosmospora vasinfecta

Filamentous ascomycetes
Nectria cinnabarina
Melanospora zamiae
Melanospora fallax
Ceratocystis fimbriata
Ceratocystis virescens
Ambrosiella hartigii
Ambrosiella xylebori
Microascus trigonosporus
Petriella setifera
Halosphaeriopsis mediosetigera
Colletotricum gloeosporioides
Glomerella cingulata
Raffaelea tritirachium
Raffaelea santoroi
Raffaelea albimanens
Raffaelea arxii
Raffaelea canadensis
Ambrosiella sulcati
Ambrosiella sulfurea
Ambrosiella ips
Ambrosiella macrospora
Ophiostoma piliferum
Ophiostoma ulmi
Diaporthe phaseolarum
Leucostoma persoonii
Cercophora septentrionalis
Chaetomium globosum
Neurospora crassa
Daldinia concentrica
Hypoxylon atroroseum
Xylaria curta
Xylaria hypoxylon
Aspergillus fumigatus
Talaromyces flavus
Penicillium chrysogenum
Blastomyces dermititidis
Coccidioides immitis
Symbiotaphrina kochii
Y17065
Aureobasidium pullulans
Stictis radiata
Cladosporium cladosporioides
Pyxidiophora sp1
Pyxidiophora spo3
Rickia sp1
Subbaromyces splendens
Curvularia brachyspora
Leptosphaeria maculans
Ambrosiozyma platypodis
Saccharomyces cereviseae
Candida albicans
Candida tropicalis
Cephaloascus fragrans
Yeasts
Taphrina deformans Taphrinales
Athelia bombacina
Spongipellis unicolor
Cryptococcus neoformans
Basidiomycetes

Fig. 1. Phylogenetic hypothesis of the relationships of certain arthropod-associated ascomycetes based on

analysis of small subunit rDNA partial sequences using parsimony criteria. See Spatafora and Blackwell
(1993) and Blackwell (1994) for details.

52

 The identification of ectomycorrhizal fungi usually has depended on collection of

basidiocarps during seasonal fruiting periods to answer questions about the presence
and prevelance of individual mycorrhizal species. However, it now is possible to use
molecular methods to identify the fungi to family and, sometimes to species, from
samples of field collected roots at any time of the year (Bruns and Gardes 1993). The
technique already has shown that the most important mycorrhizal species in terms of
frequency are not those that are discovered to be common on the basis of fruiting.
The tools that are being developed soon will allow mycologists to determine the
precise ecological relationships of the symbioses.
Molecular techniques applied to population studies of Phyphthophtora infestans
(Drenth et al. 1993, Therrien et al. 1993), Ophiostoma ulmi (Brasier 1994), and
Cryphonectria parasitica (Anagnostakis 1993) are helping to determine the origin
and monitor the epidemiology of introduced pathogens, to determine the importance
of different modes of reproduction in the development of aggressive populations
and, perhaps, to discover the basis of speciation. All of this information depends
upon the use of molecular techniques to provide more characters than mycologists
have ever had available to them. However, in addition the characters are available at
all taxonomic levels, extend across all taxa, and can be polarized.
The polymerase chain reaction (PCR) has lead to rapid progress in molecular
methods. As the technology is simplified through continued improvements in
automated DNA sequencers and more biologists can use the techniques, the
associated costs will come down. In fact there are now rumors of automated
sequences that will become available within a few years that will cost no more than a
sophisticated light microscope.
It is important to recognize that our new-found ability as mycologists to address
problems that previously eluded us, should be viewed as contributing to a worldwide
revitalization of fungal studies that is even greater than the electron microscopy
revolution of the 1960's and 1970's. It also is possible to use morphological
characters with new confidence because their polarity and convergent nature can be
inferred with the help of molecular characters. If one looks at previous phylogenetic
hypothesis the inability to determine polarity and recognize the possibility of
convergence has most often caused mistaken interpretations.
It is obvious to me that mycology is poised to make progress in all areas.
Previous morphological studies have contributed to the basic body of knowledge that
provides the context for molecular applications, and they will continue to be
important in the discovery of the great fungal diversity (Hawksworth 1991). The
importance of isolation and culture is increased by the knowledge of the genetic
potential of diversity. In these endeavors all mycologists do not have to use all
techniques. Some may not have the inclination or the technology to apply molecular
methods. Those versed in molecular techniques may not have the means of acquiring
the training in morphology that may require years of concentrated study. However,
teams of mycologists with different backgrounds must cooperate, perhaps in
formally organized groups in distant geographical regions. These collaborations
already are practical because of another technological advance, the rapid
communication available through computer communications networks. It is
imperative that the mycologists of the world be united by "e-mail" before the end of
the twentieth century so that all may participate fully in the task at hand.

53

 Acknowledgements

I thank Drs. Kevin Jones and Joseph Spatafora and Mr. Steven Cassar for the use of unpublished data
and for many helpful discussions on fungi. Mss. Brandye Sawyer, Hester Johnson, and Aime Kinney
gave expert technical assistance. The National Science Foundation provided financial support (DEB
9208027) for the work reported.

Literature Cited

Anagnostakis S.L. 1992. Diversity within populations of fungal pathogens on perennial parts of perennial
plants. In: The Fungal Community. Carroll G.C.and Wicklow D. T. eds, Dekker, NY, USA., pp. 183-
192.
Blackwell M. 1994. Minute mycological mysteries: the influence of insects on the lives of fungi.
Mycologia 86:1-17.
Blackwell M. and Malloch D.W.. 1989. Pyxidiophora: a link between the Laboulbeniales and the hyphal
ascomycetes. Mem. N. Y. Bot. Gard. 49::23-32.
Brasier C.M. 1993. The genetic system as a fungal taxonomic tool: gene flow, molecular variation and
sibling species in the 'Ophiostoma piceae-Ophiostoma ulmi' complex and its taxonomic and
ecological significance. In: Ceratocystis and Ophiostoma. Taxonomy, Ecology, and Pathogenicity.
Wingfield M. J., Seifert K.A., and Webber J.F. eds., APS Press, St. Paul, MN, USA, pp. 77-92.
Bruns T.D., and Gardes M. 1993. Molecular tools for the identification of ectomycorrhizal fungi: taxon-
specific oligonucleotide probes for the suilloid fungi. Mol. Ecol. 2: 233-242.
Drenth A., Goodwin S. B., Fry W.E. and Davidse L.C. 1993. Genotypic diversity of Phytophthora
infestans in The Netherlands revealed by DNA polymorphisms. Phytopathology 83: 1087-1092.
Kurtzman C.P. 1993. Systematics of the ascomycetous yeasts assessed from ribosomal RNA sequence
divergence. Anton. v. Leeuw. 63: 165-174.
Nishida H. and Sugiyama J. 1993. Phylogenetic relationships among Taphrina, Saitoella, and other higher
fungi. Mol. Biol. Evol. 10:431-436.
Redhead R.A. and Malloch D.W. 1977. The Endomycetaceae: new concepts, new taxa. Canad. J. Bot. 55:
1701-1711.
Spatafora J.W. and Blackwell M. 1993. The polyphyletic origins of the ophiostomatoid fungi. Mycol. Res.
98: 1-9.
Sugiyama J. and Nishida H. 1994. Phylogenetic divergence of taphrinalean fungi: evidence from
molecules and morphology. International Mycological Congress (Abstract). Vancouver, Canada.
Therrien C.D., Tooley P.W., Spielman L.J., Fry W.E., Ritch D.L. and Shelly S.E. 1993. Nuclear DNA
content, allozyme phenotypes and metalaxyl sensitivity of Phytophthora infestan s from Japan.
Mycol. Res. 97: 945-950.
von Arx J.A. and van der Walt J.P. 1987. Ophiostomatales and Endomycetales. Stud. Mycol. 30: 166-183.

54

 IMPORTANCE OF MORPHOLOGICAL AND ANATOMICAL

CHARACTERS IN FUNGAL TAXONOMY

J. RAMMELOO

National Botanical Garden, Belgium

Introduction

Morphological and anatomical characters and their importance to the taxonomy

of fungi must be seen in a historical context. The reason is obvious: when taxonomic
classification started only macroscopical characters were taken into account (as it
was the case for the study of other organisms). Later anatomical and microscopical
characters were added as the science continued to develop. Today, physiology,
molecular biology, genetics, enzymology, and ecology all contribute to an
understanding of living organisms and their classification.
With this background in mind, I will discuss the following questions:

1. How has our knowledge at the most "basic level" of "species", the level used
in taxonomy, evolved; and how important are morphology and anatomy in this
conception?
2. How has "taxonomy" been interpreted over time?
3. What is the currrent situation, with an emphasis on morphology and anatomy?
4. Is the importance of morphology and anatomy similar for all groups of fungi?
5. With respect to this symposium's emphasis on tropical mycology: is the
situation in the tropics different from the one in temperate regions?

How has our knowledge at the most "basic level" of "species", the level used in
taxonomy, evolved; and how important are morphology and anatomy in this
conception?

The often very sudden appearance on fruit-trees of larger fungi as well as the
immediate impact of moulds on the everyday lives of human beings prompted the
initial study of fungi. To commence such a study, however, it was necessary to begin
the process of classifications.
In the case of fungi, the first observations were visual. This is apparent given that
the eating of macrofungi was important. As such, the first classifications were very
pragmatic. Classifying was done on the basis of edibility and morphology in the very
broad sense of the word.
It is to be expected then that the oldest classification, Clusius (1601), which made
the distinction between Fungi esculenti and Fungi noxii et perniciosi would be
adopted by Bauhin (1623) and Van Sterbeeck (1675) who also made use of these
criteria. Ray (1686-1704) added the morphological criterion of whether or not the
fungal fruitbodies were lamellate, as well as other ecological criteria. Magnol (1689)
continued in this line, though Dillenius (1718) added a phenological criterion, which
was subsequently refined by Micheli (1729).
Linaeus developed his system of classification on the grounds that (Micheli's)
system was not satisfactory. Gleditsch (1753) introduced, in turn, more
morphological characters, while Person (1794 and following) made a classification
55

system based on gross morphology that included very accurate descriptions of

serveral common microfungi. The most important contribution was made by Fries
who described 1860 species of agarics in twenty genera, using mainly
macroscopical characters.
I could go on, but that is not the aim. The important thing to remember is that
descriptions and classification schemes started as very empirical. From this simple
beginning, gross morphology was introduced and shortly thereafter microscopy was
added. In the beginning, microscopy was very basic, but it progessed to finer
microscopy. Later, developmental characters were introduced. Later still, the results
from experimental biology, ecology, physiology, and biochemistry were included as
characters for classifying fungi.
Let us look at the actual situation. I have selected a few characters from recent
publications, selecting them from different systematic groups. As a general
conclusion, it must be said that it was rather difficult to find descriptions of new
species which used characters other than morphology and anatomy. This situation is
easy to observe through a short analysis of vols. 46, 47, and 48 of Mycotaxon. Only
systematic treatments have been taken into account, and lichens were not included.

Volume 46 47 48

Basidiomycetes

morphology, anatomy, ecology: 8 10 10

morphology, anatomy, culture: - - 1
morphology, anantomy, experimental: - - 1
numeric: - - 1

Ascomycetes and deuteromycetes

morphology, anatomy: 13 12 2
morphology, anatomy, culture: 14 5 12
morphology, anatomy, culture, developmental: 3 4 3
morphology, anatomy, culture, physiology: - - 1
morphology, anatomy, culture, genetics: - 1 1
morphology, anatomy, culture, computer: - 2 -

It is obvious that even in today's practices morpholgy and anatomy remain the
basic characters in systematic research and taxonomy.
I did the same for Mycological Research, but as the bulk of the work is
experimental and not taxonomic, this exercise was not very valuable. Nevertheless, it
is clear that while Mycological Research explores many new fields, its studies do not
influence basic taxonomic research to a great extent. These fields of study are
excellent, however, as they force taxonimists to think about biological phenomenon
which they will have to account for eventually.
Descriptions and the delimitation of taxa remain very traditional and are based
primarily on morphology and anatomy. Description, however, demonstrate an
increasing degree of precision. The increased precision in describing morphological
and anatomical characters can be assessed easily by examining the introduction of
Singer's Agaricales in modern taxonomy (1975) or Kuhner's Les Hymenomyctes
agaricodes (1980), for example.

56

 Within this context it should be stressed that often small differences in

morphological or anatomical characters can be extrememly important for
understanding the biology of a given species. Good examples can be found in
experimental phytopathological work where it has been demonstrated that spore
form and spore dimension play a major role in the place where a spore will land on a
phanerogam in order to infect its host.
Still, morphology and anatomy will continue to play a major role in systematics
because our knowledge, gathered from previous generations, has created a
framework into which more recent observations are to be fitted. Such a framework
put together by so-called modern techniques is lacking for a large number of species.
Given the enormous investment in human and other resources needed to produce this
framework, it cannot be expected that this situatiuon will change very fast. Still, the
results from new techniques are very welcome. Quite often these recent inquiries
derive from studies of species or species groups that are easy to cultivate or to
collect, or from studies of fungi of high economic value.

How has "taxonomy" been interpreted over time?

Classification and taxonomy aim to order certain kinds of information about

living organisms. Whereas, on the whole, species and infraspecific categories can be
defined by the existence of the interbreeding groups of individuals, this is no longer
the case for the supraspecific categories used in taxonomy.
Genera and more universally supraspecific categories can be approached in
different ways:
- genera (and higher taxa) can be recognized in virtue of being objectively
recognisable in nature from patterns of morphological similarity (Kornet, 1994: 5);
- genera exist in virtue of there being a historical entity, possessing cohesion over
time as assured by relationships among its members (Kornet, 1994: 5).

In early classification systems, morphological and anatomical characters were

absolutely predominant in ordering information about living organisms: phenetic
similarity was the basis of these classification schemes. At that time, however, it was
often impossible to eliminate all background considerations in the classification
which were due to convergent or parallel evolution.
With the introduction of cladistic methods, this situation changed to a certain
extent. However, one of the major obstacles in using cladistic methods is the lack of
knowledge of most of the characters of organisms. In most instances the characters
described in the past were those which were considered as taxonomically important,
and it is these characters which have noted by ("phenetic") taxonomists.

What is the currrent situation,

with an emphasis on morphology and anatomy?

From a theroretical point of view, a phylogenetic methodology can give far

superior classifications than the phenetic approach. On the other hand, when looking
at the results of cladistic analyses in mycology, it is obvious that the interpretations
of the cladograms have been leading to interpretations which are cleary ambiguous.
This lack of clarity is partly due to the characters which were readily employed in
the studies.

57

 In order to address this ambiguity, then, if the group of species for which the
analysis will be made has not been treated monographically, data should be included
from a number of sources: different periods, different authors and different
taxonomic traditions. And yet, as can be seen in even recent monographs, it can be
difficult or even impossible to construct a solid data matrix as, for instance, phenetic
taxonomists have preselected certain characters in their descriptions on the basis of
their experience with the group. What this practical consideration highlights is that
taxonomists can easily prejudice their analyses, if they do not assess their taxonomic
characters adequately enough.
Taxonomist must use good descriptive terminology consistently, describing
character states very precisely. Given the examples I have seen, I must stress that the
collecting of basic data needs to be done more rigourously.
Going through the recent cladistic mycological literature, I have noticed that too
often character sets have been accummulated from the literature without any real
expertise in the group, which has led to the misinterpretation of characters and their
significance. Unfortunately, the results from such analyses cannot be expected to
improve our knowledge of systematics.
To avoid these sorts of problems from a practical point of view means that
ecologists, phytopathologists, medical mycologists as well as amateur mycologists
must have a common understanding of characters at the species level. Thus far,
experience shows that easily observable morphological and anatomical characters
have so far been the most readily available.

Is the importance of morphology and anatomy similar

in all groups of fungi?

Considering fungi as a whole, it is obvious that classification and systematics

have employed different starting philosophies. As a result, it can be expected that
systematics developed along different lines in the case of yeasts and yeast-like fungi
as compared to other groups of fungi.
The origin of systematics of yeasts and yeast-like fungi was in the first place an
applied study, having its roots in the fermenation industry. From its practical point of
view, selection of physiological properties was the most important character. As
systematics progressed along these lines, a new study developed which we now
know as bacteriology. In this new field, the use of morphological characters was
hardly possible because of the lack of a well-differentiated thallus that would have
been useful for distinguishing between groups of bacteria.
The systematics of the majority of fungi developed along the line of practice of
systematics for higher plants. They began as primarily morphological and progressed
to include anatomical characters. For a long time, systematic of both "types" of fungi
developed on these lines, and it was thought that yeasts were a heterogeneous groups
of organisms. But with the introduction of new techniques, links were made between
both groups when it became obvious that yeasts are unicellular, ontogenetic stages of
ascomycetes and basidiomycetes.
From this perspective, it is obvious that for the classification of yeasts and yeast-
like fungi an important contribution will be made by mycologists who are acquainted
with systematics of ascomycetes, basidiomycetes, endomycetes and ustomycetes.
One result may be that the virtual non-exception to the general rule that classification
is primarily based on morphology and anatomy will disappear. However, from a

58

practical point of view, the artificial classification of yeasts and yeast-like fungi will
continue to be valuable as an approach for specific purposes, though not as a general
approach for basic systematics.

With respect to this symposium's emphasis on tropical mycology:

is the situation in the tropics different from the one in temperate regions?

Basic knowledge of fungi from the tropics is extremely poor given the large
number of species which require describing. On the other hand, this situation can be
advantageous as mycologists can, by adapting their experience from temperate
regions, collect and describe fungi from the tropics according to the specific
requirements of new techniques, such as cladistics. Furthermore, since tropical
mycology is not as encumbered by nomenclatorial problems as mycology conducted
in temperate regions, this circumstance can be considered as an advantage.
Systematic mycologists can profit from this situation, though they need to be as
careful and consistent as possible when collecting and describing specimens. The
need for care is certainly the case where, because new techniques are more limited in
the tropics, systematics will continue to rely on morphology and anatomy.

Conclusion

Morphology and anatomy have been the primary characters used in the
systematics of fungi. Other types of characters which have been considered
outmoded have not always been adapted by taxonomists. At present, taxonomists
have high performance computers at their disposal, which have greatly enhanced the
possibility for networking and the exchange of data and results between scientists.
Even though taxonomy has evolved considerably from its early beginnings, it is still
very important that it continues to develop, to adapt new techniques and technologies
as they arise. The critical issue is that taxonomy remains a central discipline in the
study of fungi. This possibility is especially significant in view of the fact that
systematists work to synthesize everything that is known about organisms. For it is
through these syntheses that more complete schemes of relationships can be
developed. Though there is a distinct possibility that the importance of morphology
and anatomy will diminish with the development of these syntheses, from a practical
point of view, and this is especially true for tropical mycology, they will remain the
most important characters for separating adjacent taxa.

References
Bauhin J. 1620. Prodromus theatri botanici. Frankfurt, 4, 16 0 p.
Clusius C. 1601. Rariorum aliquot stirpium per Pannoniam, Austriam et vicinas. Fungi p.261-295.
Antwerp, 760 p.
Dillenius J.J. 1718. Catalogus plantarum circa Gissam sponte nascentium.
Gledistsch J.G. 1753. Methodus fungorum. Berlin 162 p.
Kornet D.J., Reconstructing species: demarcations in genealogical networks. PhD thesis. Institute
for Theoretical Biology and Rijksherbarium - Hortus Botanicus Leiden, Leiden.
Kuhner R. 1980. Les Hymenomyctes agaricodes. Bull. mens. Soc. Linn. Lyon 49: 1-1027.
Magnol P. 1684 Botanicum Monspeliense. Hamburg.
Micheli P.A. 1729. Nova plantarum genera. Florence, 4, 234 p.
Persoon C.H. 1794 . Neuer versuch einer systematische eintleitung der Schamme. Rmer's Neues Mag. f.
d. Botanik, pp.63-128.
Persoon C.H. 1795. Observationes mycologiciae. Usteri's Annal. d. Botan. 15: 1-39; 16: 1-33.
Persoon C.H. 1796. Observationes mycologicae. Leipsig, 1: 115 p.

59

Persoon C.H. 1799. Observationes mycologicae. Leipsig, 2: 106 p.

Persoon C.H. 1797. Tentamen dispositionis methodical fungorum. Leipsig, 76 p.
Persoon C.H. 1801. Synopsis methodical fungorum. Gtingen, 706 p.
Ray J. 1686. Historia plantarum. London, M. Clark, 1686: 1-983; 1688: 985-1944; 1704: 255p.
Singer R. 1975. The Agaricales in modern Taxonomy. 3d ed., J. Cramer, Vaduz, 912 p.
Van Sterbeek Fr. 1675. Theatrum fungorum. Antwerp.

60

 POSSIBLE PROGRESS OF MORPHOLOGICAL ANALYSIS IN FULGAL

TAXONOMY

M.F. ROQUEBERT

Laboratoire de Cryptogamie, MNHN, 12 rue Buffon 7500,Paris

Introduction

From its beginning, fungal taxonomy was based on morphology, first by means
of direct examination of the fungus and later by microscopical observations. Since
the launching of the new techniques by the pioneer microscopists of the 17th
Century such as Hook and Leeuwenhoek, optical microscopy progressed
considerably to the end of the 19th Century, when the cultural study of fungi was
introduced. This new perspective opened the possibility of a complementary
developmental approach to fungi.
Much later, around 1950, the use of the Transmission Electron Microscopy
enabled researchers to make comprehensive progress in the study of fungal structure
and ontogeny, while the use of Scanning Electron Microscoopy permitted more
detailed surface and three-dimensional observation of fungal specimens. Generally
speaking, data obtained by electron mircoscopy (Pegler and Young 1971, Cole and
Samson 1979) did not impair those obtained by good optical observations and
descriptions, rather it improved them. Accordingly, both macroscopical and
microscopical approaches have become increasingly useful and complementary.
Still later in the 1980's, the advent of molecular techniques was applied to the
taxonomy of fungi. This development gave raise to the belief that the descriptive
phase of taxonomy was drawing to a close (Hillis 1987). Nevertheless, it is
becoming more evident that these new techniques do not modify substantially the
results of traditional morphological taxonomy. Rather these techniques help to
clarify questions suggested by the limited resolution of morphological studies, such
as for example, the exact delimitation of taxa or the definition of holomorphic
relationships (Kohn 1992). In point, it is important to stress that these various
approaches should be considered as complementary and not exclusive of one
another. As such, microscopists should be encouraged to assist molecular biologists
in their efforts to order any new data in the context of taxonomy, and vice-versa
(Reynolds and Taylor 1993).
In this respect, while morphological analysis may be understood to lie at one
"end"of a phyllum, it remains still in a process of evolution. Given that such
techniques will continue to evolve, two axes need to be considered:

1. the improvement and simplification of observational techniques

2. the interpretation of multiple data sets

The improvement and simplification of observational techniques

At present, morphological studies are generally performed simultaneously

through direct observation in situ through optical and electron microscopical
examination. Of course, each technique has its limits, its advantages and
61

disadvantages. The performance and efficiency of optical microscopy does not need
to be demonstrated. Still, it is a stumbling block for fungal identification. Although it
is relatively inexpensive and thus readily available in all countries as well as easy to
use, magnification (x 2000) and resolution (0.2 m) can be limiting factors despite
the efficiency of this device.
Electron microscopy, on the other hand, permits high magnification (x 100000)
and resolution (3-10 m), but both transmission and scanning electron microscopes
necessitate observation under vacuum conditions which requires special preparation
of specimens (fixation, dehydration, sectioning, and staining). Recently,
considerable progress has been made in these areas particularly with the
development of low temperature fixations (Hock 1986). A low temperature scanning
microscope (LTSEM) is now widely available and allows for morphological
observation in hydrated states (Jeffree and Read, 1991).
Even if these various forms of electron microscopy are really useful for fungal
taxonomists, however, these techniques require heavy and expensive equipment
which is not always available to everyone in all parts of the world. An ideal situation
for the future would be the development of less expensive equipment that allows an
easy observation of the specimen at high magnification and resolution but does not
require that the specimen undergo preparation, like the use of vacuum.
New techniques are progressing in this direction. The most promising for
morphological analysis in fungal taxonomy is Confocal Microscopy. It bridges the
gap betweeen conventional optical and electron microscopes. (Laurent et al. 1992,
Shotton 1989). In this case, the image is produced by scanning a diffraction limited
laser over the specimen, exposing an extremely small volume of it to the incident
light, and thereby restricting sources of reflected light. The most novel imaging
mode accounts for vertical sectioning. Instead of scanning the specimen along the x-
y planes, this mode generates images by scanning optical sections that are parallel to
the axis of the microscope. This approach has the advantage of limiting out of focus
information from the observed image.
Confocal microscopy does not need any preparation of the specimen except for
fluorescent staining. With resolution down to 0.25 m, the technique is used
frequently in cytology, cytometry, and molecular biology (Shotton 1989).
Preliminary results of morphological observation obtained with Penicilium
chryogenum (Fig. 1) shows clearly the absence of out of focus blur in the confocal
images. These results also show the fine resolution in successive vertical planes
which allows for the exploration of thick structures (70-80 m).
Cell surfaces can also be investigated by other new kinds of microscopes: the
Scanning Probe and Scanning Tunnelling Microscopes (STM) as well as the Atomic
Force Microscope (AFM) (Guckenberger and Baumeister 1992). Research with
STM is still focused on exploring type of experiments that might be more feasible in
the future. At present, it is being tested principally on non-biological materials. In
contrast, AFM has already been used quite successfully for obtaining images of
biological specimens in aqueous solutions (Butt et al. 1990, Kasas 1992). The
principle technique applied in AFM involves scanning the surface of the specimen
with the tip of a very thin probe (diamond or silicium). The three dimensional
scanned displacements are recorded and analysed by a computer. AFM combines a
high resolution (80 m on blood cells) without the need for sample preparation as it
works in aqueous environments.

62

Figure 1. Penicillium chrysogenum Thom, Coloration: Fluorescein-dextran (negative coloration).

Confocal Microscope Biorad MRC 1000. A-B: Optical sections at different focal planes separated by 1
m. A: Non-confocal, B: Confocal optics. C-D: Detail of penicillus. C: Non-confocal, D: Confocal
optics.

63

 It is currently used for studying molecules (proteins, phospholipids, and DNA)

constituting organic matter. The advantage of the technique is that it permits
observations in environmental conditions with good magnification and resolution.
The lightness of this equipment also constitutes an advantage.
Even if these new microscopy tehniques are still in the research phase, we can
safely assume that these techniques, in particular confocal and atomic force
microscopy, will be used in the near future for fungal morphological observation and
taxonomy.

The interpretation of multiple data sets

To be effective in taxonomy, observations must be intrepreted and represented as

objectively as possible. Until now, line drawings and photographs have been very
effective for providing clear visual representations of macro- and microscopical
observations, but because they are man-made and interpretative, they are often very
expressive and artistic, and thus quite subjective.
On the other hand, quantitative morphological analysis provides a means to
translate visually apparent structures into numerical data that can be subjected to the
powerful methodologies of mathematical analysis. (Wagener 1990, Steer 1991).
Because of its capacity for decomposing and anaylsing a great number of images,
image analysis may be a very effective tool for limiting subjective interpretations.
The important point is to obtain the maximum information from visual images so
that the data can be interpreted with the greatest possible confidence. This possibility
may also be very helpful for taking into account the variability of specimens. One of
the advantages of the technique is that image analysers can be integrated directly into
the apparatus of microscope as they are already in confocal and scanning probe
microscopes.
In addition to image analysis, another way to exploit a large number of
morphological observations may be the use of morphometrics. The field of
morphometrics is traditionally concerned with methods for the description and
statistical analysis of shape variation within and among samples of organisms
adjusted to a common size, measuring certain landmarks of an organism (length,
width, etc.). An example of the impact that morphometric analysis can have on
fungal taxonomy is evident in the recent publication by Vanev (1993) on the conidia
of 14 type specimens of Discosia section Strobilina. Analyses of the shape as well as
the length and width measurements of the conidia enabled the author to reach
decisions about the taxonomic status of the specimens, that is, to gather data useful
for separating and grouping taxa.
Many methods agree on the nature of the differences among a set of shapes, but
fewer agree on the relative significance of the differences between these shapes. A
new trend in morphometrics is to incorporate data on the geometry of the structure
so as to distinguish changes due to mere differences of size from those which
describe purely homogeneous features (Zelditch et al. 1992). The ideal aim would be
to eliminate from the analysis, dimensional features that are due to the natural
variability of biological material, so that phenetic or cladistic relationships can be
estimated more accurately.

64

 Conclusion

Interesting progress is being made in the morphological observation and analysis

of biological material. The methods and equipments used in these techniques are
being simplified considerably without loss of performance. Even though the analysis
and the objective interpretation of data remain at the research phase, in part because
of the variety of biological material, great possibilities exist for these techniques as
they are effectively adapted to fungal taxonomy. These technical and analytic
approaches seem promising for the progress of fungal taxonomy, as they also are an
interesting subject for research in themselves.

References

Butt H.J., Wolff E.K., Gould S.A.C., Dixon-Northern B., Peterson C.M. and Hansma P.K. 1990. Imaging
cells with the atomic field miscroscope. Journal of Structural Biology 105: 54-61.
Cole G.T. and Samson R.A. 1979. Patterns of Development in Conidial Fungi. Pitman., London.
Guckenberger, R., Hartma n T., Wiegrabe W.and Baumeister. W. 1992. The Scanning tunnelling
microscope in Biology. In: Scanning Tunnellling Microscopy II . Wisendanger R. and Guntherodt
H.J. eds., Springer Series in Surface Science, 8: 51-98.
Hillis D.M. 1987. Molecular versus morpholical approaches to systemeatics. Ann. Review of Ecology and
Systematics 18: 23-42.
Hoch H.C. 1986. Freeze substitution of fungi. In: Ultrqstructures techniques for microorganisms. Aldrich
H.C. and Todd W.J.J. eds., Plenum. N.Y.
Jeffree, C.E. and Read N.D.. 1991. Ambient- and low-temperature scanning electron microscopy. In:
Electron microscopy of plant cells. Hall J.L. and Hawes C. eds. Academic Press, London, pp. 313-
413..
Kasas S. 1992. La microscopie force atomique dans les recherches en biologie. Mdecine/Science 8:
140-148.
Kohn, L.M. 1992. Developing new characters for fungal systematics: an experimental approach for
determining the rank of resolution. Mycologica 84: 139-153.
Laurent M., Johanin G., Le Guyader H. and Fleury A. 1992. Confocal scanning optical microscopy and
three dimensional imaging. Biol. Cell 76: 113-124.
Pegler D.N. and Young T.W.K.. 1971. Basidiospore morphology in the Agaricales. Beih. Nova Hedwigia
35.
Reynolds, D.R. and Taylor J.W. 1993. The fungal holomorph: mitotic, meiotic and pleomorphic speciation
in fungal systematics. Wallingsford, CAB International, 375 pp.
Shotton D.M. 1989. Confocal scanning optical microscopy and its application for biological specimens. J.
Cell Science 94: 175-206.
Steer M.W. 1991. Quantitative morphological analysis. In: Electron microscopy of plant cells. Hall
J.L.and Hawes C. eds., Academic Press, London, pp. 85-104..
Vanev S.G. 1993. Morphological variability of the conidia of the fungi from the genus Discosia section
Strobilina. Mycotaxon, 49: 199-207.
Wagener, M. 1990. Digitization in scanning electron microscopy. In: Electron microscopy of plant cells.
Hall J.L. and Hawes C. eds., Academic Press, London., pp. 297-305.
Zelditch M.L. F.L. Bokkstein and Lundrigan B.L.. 1992. Ontogeny of integrated skull growth in the cotton
rat Sigmodon fulviventer. Evolution 46: 1164-1180.

65

 MYCOFLORISTIC STUDIES IN SOUTH CENTRAL AFRICA: STATUS,

CONSTRAINTS, OPPORTUNITIES AND STATEGIES

A.J. MASUKA

Forest Research Centre, P.O. Box HG 595,

Highlands, Harare, Zimbabwe

Abstract. Mycofloristic studies of the South Central African region, comprising Malawi, Zambia and
Zimbabwe, have largely focussed on the identification of macrofungi, apart from fungi of agricultural
importance. The only relatively well studied group is the Aphyllophorales, with over 200 known species.
In addition to their studies on the Aphyllophorales, visiting scientists have contributed immensely to our
present knowledge of the Agaricales, Boletales, Cantharellales, Russullales and other non-basidiomycete
fungi. Termitomyces, Cantharellus species, edible boletes and species of Russula and Lactarius have a
unique contribution to the diets of many people during the rainy season. This has prompted publication of
country and regional guides to mushroom identification and mushroom poisoning. Most studies of the
larger fungi have been of a taxonomic nature, though mushroom productivity studies have just begun. The
major taxonomic approach has stressed morphological and anatomical characters; supplemented by
chemical, ecological, cultural and other characteristics. We find the approach to be pragmatic under our
circumstances, and applicable to a large range of fungi, though its shortcomings have been realised in
recent collaborative studies on African Armillaria. The genetic basis of species differentiation has largely
been of an academic interest. Unfortunately, taxonomic studies are still regarded as being peripheral to
agricultural and forest pathology/mycology and most identifications of fungi are conducted by institutions
outside Africa. There is a lack of qualified scientists, as well as adequate and continuous funding at some
established research institutions. There is need to undertake more fungal inventories and ecological
studies because of their importance to aspects of the conservation of fungal and ecosystem biodiversity.
Furthermore, with an estimated annual deforestation rate of over 250 000 hectares in the region, some
species will eventually be lost before they are known. Assistance in training local scientists must be
sought, and there should be a provision for suitable equipment and literature. Local, regional and
international support for such activities should be mobilised. The importance of herbaria for educational,
reference, conservation and other purposes should be publicised. These are our present challenges and
obligations.

Introduction

The South Central African Region

The South Central African region comprises Malawi, Zambia and Zimbabwe.
The region is bound by the latitudes 8 25 and 22 30 South and longitudes 23 and
36 East. The region is wholly land-locked, the nearest points to the oceans being
over 100 km to the Mozambique Channel and 900 km to the Atlantic Ocean. The
total land area is about 1.25 million km2. Climate is broadly divided into two distinct
seasons - a summer rainfall season starting November and ending in April, and a
prolonged dry winter season. This has a marked influence on the fruiting times of
fungi and the timing of collection forays - which in our region are best productive in
February and March. The mean annual rainfall is 635-1500 mm per annum. Mean
temperatures are 18-21 C in areas of a latitude range of 900 to 1200 m.a.s.l. (about
70% of the land area). Wild and Barbosa's (1967) Vegetation Map of the Flora
Zambesiaca Area presents a floristic and physiognomic classification of the region,
in addition to Mozambique and Botswana, the area draining the Zambezi River.
The characteristic vegetation type is that of the Leguminoseae genera
Brachystegia, Julbernardia and Isoberlina. Such dry deciduous forests are termed
miombo - they are widespread and predominant, covering up to half of the area of
the region, at medium to high altitude and rainfall areas, on soils derived from

66

granite, schist, quartzite, gabbro or shale. The miombo ecozone - or miombo region -
also extends into southern Zaire, northern Mozambique and parts of Tanzania and
Kenya. Other forest types found in the region are the Zambezi Teak Forests, in
which Baikiaea plurijuga is the dominant species (on the Kalahari Sands in western
Zimbabwe and south-western Zambia, where rainfall is generally low). These forests
also occur in parts of Angola and Botswana. There are small relic swamp and
evergreen forests in high rainfall areas, containing the only coniferous species of the
region (Juniperus, Podocarpus and Widdringtonia). Typical savanna woodlands are
associated with some impediment in drainage and species of Acacia, Combretum and
Terminalia predominate, though there are several secondary associates. In the lower
elevation areas Colophospermum mopane predominates, especially on calcareous
soils. Forested areas are interspersed with shallow depressions or drainage canals
that are seasonally waterlogged (dambo or vlei), forming grasslands. Considerable
development of plantations with exotic species (mainly Acacia, Cupressus,
Eucalyptus and Pinus) currently totalling 2 500 km2 has taken place within the last
4-5 decades
The assemblage of fungi in South Central Africa is large and heterogenous. It is
impractical and probably unadvisable, considering the variety of species and the
constellations of characters they posses, for one to sufficiently and efficiently
address aspects of their taxonomy, ecology, distribution etc. - floristics - in this
presentation. I will therefore, focus on the macrofungi, a group I have also studied
for some years now.

Importance of fungi
The importance of and uses of fungi are diverse, some being detrimental to
mankind, crops and animals. The presence and importance of fungi in agricultural
and forestry systems as pathogens, saprotrophs and symbionts is well recognised.
Their use in wine-making and beer-brewing industries, as food, medicines and
mycopesticides, and in other novel biotechnological applications have been tapped to
a variable extent in the region (Subramanian, 1992, Hawksworth, 1993, for the
general importance of mycology to development). The applied mycology aspects
attract the bulk of funding from public and private institutions. Generally, studies on
fungal taxonomy and ecology, are regarded as being of fundamental importance
only, and have received limited attention.

STATUS OF MYCOLOGY RESEARCH AND TAXONOMIC STUDIES

Meaningful mycology research is conducted by Universities, Departments of

Agriculture and Forestry, some parastatals and a few private organisations.

Parasites
Comprehensive lists of fungi of pathological importance in agriculture (Rothwell
1982, Angus 1962-66, Doidge 1950) and forestry (Masuka and Ryvarden 1992a)
have been compiled. Macrofungi on living forest trees (Table 1) have only been
collected for serious study recently. The fungi play an important role in tree
succession and hence stand and ecosystem dynamics. Fomitopsis widdringtoniae
Masuka & Ryv. is commonly found on Widdringtonia nodiflora in Malawi (Masuka
and Ryvarden, 1993a), and is associated with the widespread decline of the host
species. Armillaria, Amauroderma and Ganoderma species are the most common

67

fungal causes of mortality in indigenous tree species. Heart-rot in indigenous

merchantable timber species (e.g. B. plurijuga and P. angolensis) may reach an
incidence of up to 19% in logs (Masuka and Ryvarden 1993b). This has prompted
some recent studies (including those on the taxonomy and ecology of causal fungi)
on the subject in Zimbabwe.

Table 1. Macrofungi associated with living trees in South Central Africa.

--
Species Host Parasitised part2 (rot)

Abortiporus biennis (Bull.: Fr.)Sing Bu, root, butt

Amauroderma argenteofulvum (Van der Byl)Doidge Bs, Jg root
A. fuscoporia Wakef. Bs, Jg root
A. preusii (Henn.) Steyaert Bs, Jg root
Armillaria (Fr.) Kummer spp. Bs, Jg, Pc root
Aurificaria indica (Mass.) Reid Csp heart
Fomitopsis widdringtoniae Masuka & Ryv. W butt
Ganoderma australe (Fr.) Pat. Kn, Bg heart, butt
G. colossum (Fr.) Baker Cm root, butt
G. eminii (Henn.) Ryv. Bs, Jg root, butt
G. lucidum (Fr.) Karst. Bs, Jg, Pc, F root, butt
G. sculptrutum Lloyd Bs, Jg (Eg)* root, butt
Inonotus afromontanus Ryv. and Gilbn. Psp butt
Perenniporia martius (Berk.) Ryv. Kn heart
Laetiporus baudonii (Pat.) Ryv. Bs, Jg root
L. percisinus (Berk. & Curt.) Gilbn. Bu heart
Phellinus allardii (Bres.) Ryv. Bsp, Cm heart
P. fastuosus (Lev.) Ryv. Cm heart
P. lamaensis (Murr.) Ryv. Bu, Kn heart
P. merillii (Murr.) Ryv. Kn heart
P. rimosus (Berk.) Pil. Psp, Pa, Ba, Bp, Bs heart
P. robustus (Karst.) Bourd. & Galz. Bu, Bs, Cp heart
P. senex (Nees. & Mont.) Imaz. Bs, Jg heart
P. wahlbergii (Fr.) Reid Bu, Bs heart
--
1 Bp, Baikiaea plurijuga; Bg, Brachystegia glauscesens; Bs, Brachystegia spiciformis; Ba, Burkea
africana; Cm, Colophospermum mopane; Csp, Combretum sp.; F, Ficus spp.; Jg, Julbernardia globiflora;
Kn, Khaya nyasica; Pa, Pterocarpus angolensis; Pc, Parinari curatelifolia; Psp, Phillipia sp., W,
Widdringtonia nodiflora
.2 But-rot defined as up to 1 m above ground; higher than that the term heart-rot applies.
* The fungus has recently adapted onto Eucalyptus grandis in Zimbabwe and causes up 4% annual
mortality in high risk areas whose previous indigenous vegetation is B. spiciformis or J. globiflora
(Masuka and Nyoka, 1994).
--

Mushrooms and mushroom poisoning

Mushrooms are highly rated on the local gastronomic scale, have a unique
contribution to the diets of many people, and are extensively collected during the
rainy season (December-March). Country and regional guides to mushroom
identification and mushroom poisoning have been produced. Morris's (1987)
Mushrooms of Malawi and Storrs and Piearce's (1982) Do Not Eat These, Piearce
(1981a) and more recently Ryvarden, Piearce, Masuka's (1994) An introduction to
the Larger Fungi of South Central Africa are useful starting points for anyone
studying mushrooms in this part of the world. Throughout the region fungi are
normally equated with mushrooms (Khonga 1994, Masuka 1992a, Morris 1984,
68

1987) and traditional classification systems - passed orally from one generation to
the next - have existed to this day, though with urbanisation local, elderly people
remain the only repository of such knowledge. The classification system is based
principally on colour (there are 4-5 in the region), aroma, texture and taste. In
Nigeria the shape, texture, taste and maturation period are the major taxonomic
criteria employed by local people (Alabi 1994). A start has been made in Zimbabwe
to prepare a checklist of vernacular names of fungi (in different languages and
dialects) and to provide their English equivalents (where applicable) and scientific
names. There is substantial folklore associated with edible and poisonous
mushrooms though there are a few records of the use of macrofungi in traditional
medicine, or for ritual purposes, and fungi are rarely used in artwork (Masuka 1992a,
Piearce 1981a, Morris 1984, 1987). Perenniporia mundula (Wakef.) Ryv. is the only
medicinal fungus used throughout the region, for treatment of pleurisy and
impotence.
Termitomyces, Cantharellus, some species in the Russulales and Boletales are
regarded as delicacies. A representative number of these, and other macrofungi, have
been included in the book by Ryvarden et al (1994). Amanita zambiana Pegler &
Piearce, confined to South Central Africa and southern Zaire, is abundant and widely
collected for consumption. The hard-textured species such as Lentinus cladopus Lev.
and Polyporus tenuiculus (Beauv.) Fr. are edible but seldom collected; for
consumption during the off-season.
There are about seven common Termitomyces species among which four are
highly regarded. They have been studied in greater detail in Zambia (Piearce 1987).
There are approximately 1 800 termite species in Africa and South East Asia among
which some 100 species in six genera grow, among them, about 20 species of
Termitomyces (Bels and Pataragetvit 1982). The associated mounds can be invisible
to about 10 m high. The relationship between Termitomyces and termites is an
example of obligate symbiosis. The only other similar association of insects and
fungi is that of the Attini, confined to Central and South America, which have been
reported to grow a Leucocoprinus sp. (Holldobler and Wilson 1990). There is no
evidence that the complementary habits of Termitomyces and Leucocoprinus sp. are
mutually preemptive involving competitive exclusion, or accidental, reflecting the
rarity of evolution along that path.
Amanita, Cantharellus, Lactarius and Russula, and also Termitomyces species
cannot, as yet, be cultured. Their continued presence will depend on the protection of
their natural environments. Mushroom collecting is regarded as a women's task.
Mushroom productivity studies (counts by species and basidiocarps, basidiocarp
diameters, determination of fresh and dry weights and nutritive values) in miombo
forests in Zimbabwe have only been recently initiated. Aspects of mushroom
collection, processing and consumption (and utilisation) patterns; importance in rural
economy; and valuation, marketing and trade will also be considered. Such studies
should result in a further understanding of their taxonomy and ecology.
Several cases of mushroom poisoning are reported each year, and children are
most vulnerable. Most cases of poisoning reported generally fall within the class of
gastrointestinal irritants (Ryvarden et al. 1994). Species (authentically) associated
with poisoning are Chlorophyllum molybdites (Meyer: Fr.) Masse, and some
Amanita species which are commonly mistaken for Macrolepiota and Agaricus
species. Mushroom poisoning has attracted considerable attention from the print and
electronic media in the region. Publications on the subject are by Flegg (1981),

69

Gelfand and Harris, (1982), Robertson (1983) and Sharp (1983) for Zimbabwe;
Storrs and Piearce (1982) for Zambia, Morris (1987) for Malawi, and (Ryvarden et.
al. 1994) for the region.
Following introduction of exotic plantation species (Pinus spp.) poisonous
species of Amanita [A. muscaria (L.:Fr.) Hooker], but also edible species such as
Boletus edulis Bull.:Fr. and Suillus granulatus (L.:Fr.) O. Kuntze have become
naturalised in those plantations. Most locals regard macrofungi under exotic
plantations with very deep suspicion.

Fungi on timber in service

Recent studies in South Central Africa have also focussed on the identification of
decay fungi on timber in service, ascertaining their temperature and moisture
characteristics in culture, determining frequency of occurrence and extent of damage
they cause, and on aspects of their control (Masuka and Ryvarden 1992). There is
even greater scope for such studies as the construction of timber houses is now
permitted in Zimbabwe (Standards Association of Zimbabwe, 1990). Furthermore,
fungi responsible for deterioration of paints and other products may also be studied.
Some survey work on the fungi causing decay of mine timber has also been
conducted. The studies have revealed the presence of Gyrodontium boveanum
(Montagne) Maas G. as an important rotter of mine timber, Coniophora puteana
(Fr.) Karst., Antrodia vaillantii (Fr.) Ryv. and Gloeophyllum trabeum (Fr.) Murr. in
pine timber, and Coriolopsis polyzona (Pers.) Ryv. and Trametes vesrsicolor (Fr.)
Pil. in hardwood timber (Masuka 1992a).

Saprotrophic and mycorrhizal macrofungi

Most fungi in the region are saprotrophs on dead wood and on leaf litter, being
inconspicuous to the unaided eye to large and sometimes brightly coloured species.
The only well studied group is the Aphyllophorales (Masuka 1992b, Masuka and
Ryvarden 1992b, 1993c, Mswaka 1992). This is so because the fungi are easy to
collect and store. Collections in the region have yielded much information, and
currently the region is better explored mycologically than most parts of Africa.
Studies on the agarics and boletes have recently been resuscitated following
collaborative collection trips with institutions and individuals in the United States of
America.
The taxonomy of tropical fungi is in a continuing state of flux including that of
relatively well studied groups such as the polypores (Ryvarden, 1994). New species
continue to be discovered following thorough collecting (Masuka and Ryvarden
1993a) and many more polypores will be described soon from our region (Mswaka,
Masuka and Ryvarden, unpublished). Such new discoveries have added to the
instability of generic and species delimitations, whose descriptions are often based,
for historical reasons, on temperate species. Current work on novel Dichomitus
species from the region have repercussions on the generic delimitations of
Grammothele, Grammothelopsis and Megasporoporia.
Collections of Aphyllophorales have yielded very valuable information (Table 2)
and recent comparisons indicate that there are only 2-5% brown-rot fungi in the
Polyporaceae in the tropics compared to 28-30% in temperate countries (Ryvarden,
1994).

70

Table 2. Ecological roles of Aphyllophorales in Zimbabwe.

--
Family Number of Species
--
Mycorrhizal Parasitic Saprotrophic
--
Corticiaceae 0 0 7
Hymenochaetaceae 0 11 28
Lachnocladiaceae 0 0 6
Coniophoraceae 0 0 8
Ganodermataceae 0 8 11
Hericiaceae 0 0 11
Polyporaceae 1 11 106
Punctulariaceae 0 0 1
Schizophylaceae 0 0 2
Steccherinaceae 0 0 2
Stereaceae 0 2 4
Thelepohoraceae 1 0 0
--
source: Masuka and Ryvarden (1993c)

The majority of the recorded species found in a study by Masuka and Ryvarden
(1993c) had di- or tri-mitic systems of hyphae (Table 3), and there were significantly
(x2 = 23.24, P < 0.01) more monomitic species in higher rainfall areas (Afromontane
and moist ever-green forests) than in the drier areas (Baikiaea and Mopane forests).
Within the various forest types the species occurring there tend to be predictable.
Thus while Phellinus rimosus is a common species in mopane woodlands
Amauroderma and Ganoderma species do not occur there but predominate in
miombo woodlands.

Table 3. Hyphal system and vegetation type.

--
Forest Type Number of Species
--
Monomitic Di-/Tri-mitic
--
Afromontane 3 (38) 5
Baikiaea 3 (9) 29
Deciduous tree savanna 3 (10) 28
Eucalypts 7 (22) 25
Miombo 12 (15) 67
Moist evergreen 24 (26) 72
Mopane 3 (10) 26
Pine 22 (41) 32
--

The figures in parenthesis indicate the percentage of species with a monomitic

hyphal system.
An analysis of the species of the Coniophoraceae (Table 4) shows that only a few
have been found in Africa. The data was compiled from various sources including
Ginns (1976, 1978, 1982), Gilbertson and Ryvarden (1987) and Masuka and
Ryvarden (1991). These observations are based on the morphological species
concept. It is not known whether some of the species found in temperate

71

environments are present in the tropics in a vegetative state but fail to produce
basidiocarps because of adverse climatic and other conditions. One inherent flaw in
such comparative mycogeographical studies is the dilemma of distinguishing
between non collection and non occurrence of a species in an area.

Table 4. Representation of genera of the Coniophoraceae in Africa.

--
Genus Number of species in
--
Genus Africa
--
Coniophora 15 8
Corneromyces 1 1
Gyrodontium 3 2
Jaapia 2 0
Leucogyrophana 9 1
Meruliporia 1 0
Pseudomerulius 1 0
Serpula 3 1
--

The Myxomycetes are extremely common but have not been collected for serious
study in the region. Species of Lycogala are quite common in sheltered moist places.
Among the common Ascomycetes known here are Cookeinia (Sarcosomataceae)
though the whole group has been poorly studied. Daldinia, Hypoxylon and Xylaria
species (Xylariaceae) are also quite common but the species have not been identified
with certainty. Among the Heterobasidiomyectes only the common species of the
Auriculariales, in Auricularia, and the Dacrymycetales, such as the conspicuous
Dacryopinax, are known and often collected. The other order, the Tremellales, has
not been studied in this part of the world.
Studies on the Agaricales of the region have been conducted by Pegler (1982),
Pegler and Piearce (1980), Piearce (1981a, 1987), Morris (1987) and Ryvarden et al
(1994). Pegler's (1977, 1986), A Preliminary Agaric Flora of East Africa and Agaric
Flora of Sri Lanka have many applicable keys to agarics of South Central Africa and
have formed a basis on which to build local knowledge. Studies on agarics have been
most intensive in Zambia. However, a thorough and systematic inventory of the
whole group has not been undertaken. Among the notable species are the poisonous
Chlorophyllum molybdites, which can be mistaken for some Agaricus or
Macrolepiota species. Collybia aurea (Beeli) Pegler (Tricholonmataceae) is a
tropical African species whose dense and clustered habit makes it easily
recognisable. The Russullales have also not been studied in any great detail. A few
notable species in the genera Lactarius and Russula are however, confined to this
region. Lactarius kabansus Pegler & Piearce is only known as yet from Zimbabwe
and Zambia, in miombo woodlands. Perhaps the least studied group of macrofungi is
the boletes which occur scattered throughout miombo woodlands. Among the poorly
studied Aphyllophorales are resupinate species - the 'corticiums' including species in
Hymenochaete (Hymenochaetaceae). The gasteromycetes too have been poorly
documented, though some preliminary work was conducted in Zambia (Piearce
1981b). A notable species in the Lycoperdales (Broomeiaceae) is Broomeia
congregata Berk. only known in Africa south of the Sahara, often close to Acacia
tree species. Another distinctive fungus is Podaxis pistillaris (Linn.:Pers.) Fr.

72

(Podaxales) a tropical and sub-tropical species, in Southern Africa found mostly on

termite mounds.

Taxonomic approach
The biological species concept is the basis of taxonomy in fungi, though there
has been much debate on this subject (Clemencon 1977, Parmasto 1985, Boidin
1986, Hallenberg 1988). Morphological and anatomical characters of the
basidiocarp, type of rot, reaction with selected chemicals, host range and
specialisation and distribution (mycogeography) are routinely used in taxonomy.
The approach is simple, inexpensive and pragmatic under our conditions were a
greater number of species are unknown. As the taxonomic species concept
emphasises the importance of developmental criteria, this has necessitated the
analysis of the basic characteristics of the individual. Cultural, biochemical,
incompatibility and genomic studies, and phenetic and cladistic analyses have not
been used for fungi of the region to any great extent. Such studies could solve some
delimitation problems, and are especially useful in economically important species
and genera (e.g. Armillaria). A broader consideration of taxonomic characters is
often done in mycologically better known regions, where molecular studies have
gained prominence; and phenetic and cladistic analyses yield most valuable
information there. However, there is hardly need for competing taxonomic
approaches - the classical and culture-based methods - rather they should be
regarded as being complementary. Indeed, in several cases the modern methods have
corroborated delimitations arrived at using the classical or "conventional" taxonomic
methods.
Morphological characters are polymorphic and it is impossible to reveal
convergence phenomena or parallel evolution from their analysis. Funalia leonina
Kl. is primarily separated from Trametes by the presence of a thick mat of hairs on
the upper surface of the basidiocarp. The presence of this feature in the species might
just be an adaptation to predation by insects. Similarly, Coriolopsis species are
separated from Trametes species by the coloured vegetative hyphae, and Pycnoporus
is recognised as a distinct genus because of the presence of cinnabarin. Flavodon
flavus (Kl.) Ryv. is separated from Irpex by its yellow colour, but is
micromorphologically similar - also having simple-septate hyphae and encrusted
cystidia. An in depth analysis of the species' cultural and biochemical characters
might reveal their true affinities. Furthermore, the yellow colour often fades with
age.
Only a few species are known in culture (11 500 strains out of an estimated 254
000 strains in herbaria around the world or 17% of known - and only 0.8% of
estimated number of - species present in the world, see Hawksworth 1994a,b). The
rapid disappearance of species due to deforestation and other causes has given
priority to less tedious, but workable approaches to taxonomy, and to collection for
ex-situ conservation purposes in general.
In recent collaborative studies on the genus Armillaria the limitations of the
morphological species approach have been poignantly manifold. Various features
(and methods) are now being used to differentiate the Armillaria species in culture,
to complement classical methods (Table 5).

73

Table 5. Methods used to group Armillaria species from tropical Africa.

--
1.Fruiting in culture
2.Observation of morphology of basidiocarps, conditions conducive to fruiting, frequency of fruiting,
morphology of single-spore isolates
3. Pairing tests
4. Comparison of growth in culture at different temperatures
5. Comparison of subterranean rhizomorphs obtained in a mist case
6. Biochemical and molecular analysis of proteins: esterase, whole cell DNA by hybridisation of
restriction digests with mtDNA and random amplified polymorphic DNA analysis
Source: Mohammed and Guillaumin (1994).
-

Basidiocarps of the fungus are rarely produced under our conditions (Masuka,
1994). Studies by the "Zimbabwe Armillaria Group", under the EEC Project on
African
Armillaria coordinated by the Oxford Forestry Institute, focussed on growth
features and biochemical characteristics of the fungus in culture (Mwenje and Ride
1994) and on species screening for resistance and nursery pathogenicity experiments.

CONSTRAINTS

Priorities and funding

There is a lack of adequate, assured and continuous funding, as taxonomic
studies have been relegated from the priority list; and applied pathology/mycology,
including biotechnology, have gained prominence. It is however, important to note
that knowledge of biodiversity, the .raw material for biotechnologies for agricultural
development' (Boussingeut 1991), (which is heavily dependent on taxonomic
studies) is (still) poor and fragmentary. Among the reasons for the lack of taxonomic
studies is perhaps the opinion that taxonomic studies have no commercial value or a
place in applied research. The importance of these studies has been underplayed
leading to a marginalisation of the subject, hence taxonomy has become peripheral to
the fields of agricultural or forest pathology. There is competition with other fields
for funding, in addition to status and credibility. Only three out of 18 papers
presented at the First African Regional Mycology Conference in Mauritius in 1990
dealt with fungal taxonomy (Hennebert 1994).
Countries in the region are pursuing structural adjustment programmes. These
have generally, led to a reduction in public expenditure. In any budget cuts research
operations, by their nature, usually suffer first and most. The salaries budget for
some research institutions already account for 70% of the operational budget, further
reducing financial resources allocated to research work.

Personnel
There is a lack of qualified staff. Those that have training in taxonomy often
conduct taxonomic studies on a part-time basis, as there is no full-time taxonomist in
the region. There is also a lack of status, recognition and generally, inadequate career
paths for researchers in developing countries, and taxonomists are no exception. At
this symposium whose theme is "Tropical Mycology: quo vadis?" only two out of
the seven speakers are from the tropics. The respective number of permanent
scientists listed in the directory of African mycologists for Malawi, Zambia and
74

Zimbabwe are only 1, 3 and 7 (Buyck and Hennebert 1994). Only Zimbabwe has the
personnel capable of conducting systematic inventories of macrofungi.
Literature
There are no monographs for the region. One has to depend on literature from
elsewhere. The shortage of relevant information on tropical species has made any
pioneering study a frustrating experience. With regards to the polypores Ryvarden
and Johannson's (1980) Preliminary Polypore Flora of East Africa provides a
valuable starting point, so do Pegler's (1978, 1986) monographs of agarics.

Facilities
The facilities available do not allow for advanced methods to be employed, so
studies employing molecular approaches to taxonomy (enzyme, DNA analysis) can
only be conducted at Universities (there is a shortage of equipment there as well),
and will probably remain being of an academic interest only for the foreseeable
future.

In all three countries the macrofungi herbaria fall under different institutions: the
Zambian collection is at the Forestry Research Headquarters (ca. 2000 specimens),
in Malawi the collections (ca. 500 specimens) are at the National Herbarium and
Botanic Garden, while those in Zimbabwe are at the University of Zimbabwe (on
temporary transfer to the Forestry Commission, Forest Research Centre since 1989,
ca. 3000 specimens).

Methodology
Our priority in the region is to collect and document species before they
disappear. This calls for the morphological and anatomical species approach to
taxonomy. In well studied regions, studies have now concentrated on elucidating
relationships between taxa and solving delimitation problems. Sometimes our
approach (morphological) to species delimitation appears parallel to the biological
species concept, and problems have been encountered in conclusively identifying
species described on the basis of the latter concept. The shortage of equipment to
conduct the required further analyses compounds the problem.

Communication and networking

Because of the constraints imposed by a shortage of funding scientists in the
region do not travel often enough to meet and share ideas with peers. This has often
led to unnecessary duplication, thus retarding the pace of progress in some studies.
Joint project proposals are rarely made with institutions in the region. Opportunities
for networking have often come from exogenous factors, being spear-headed from
institutions outside the region.

WINDOWS OF OPPORTUNITY

The rate of deforestation on a global scale is estimated at 15 million hectares a

year (UNEP, 1987) with greater losses taking place in developing countries and at
present rates of deforestation about 40% of remaining forest cover might be lost by
the year 2000 (Organisation for Economic Cooperation and Development, 1985).
The estimated rate of deforestation is over 250 000 hectares annually in the region
(Table 6). The causes are shifting cultivation, expansion of agriculture, cattle

75

ranching, the growth of industrial mining, and timber and wood exploitation and
rapid urbanisation. These are exacerbated by high population growth rates of up to
3% per annum.
During the past ten years the need to preserve (plant) diversity has been
popularised culminating in the conniving of the Earth Summit (and Agenda 21) in
Brazil in 1992. Funding for conservation efforts has been mobilised and much of this
has targeted developing countries. However, the need for a holistic ecosystem
management approach has also illuminated the need to incorporate other
components.

Table 6. Deforestation rates in the region.

--
Country Total Area Rate of Deforestation
(x1000 km2) (% per annum)
--
Malawi 9408 0.93
Zambia 74072 0.53
Zimbabwe 38667 0.45
--
Source: Singh (1991)

Fungi are important components of forest ecosystems, recycling immobilised

nutrients and, through symbiotic associations, directly contributing to the well-being
of forests. However, at present rates of deforestation some species might even be lost
before they are known. A window of opportunity has been opened for conserving
and inventorying fungi, and to further study their ecology and biology. Indeed, more
than ever before, there is now scope for assessing absolute, generic, ecological and
mycogeographical diversity of fungi.
There is still a receptive audience (in both public and private institutions) willing
to cooperate, collaborate and fund joint studies, and to assist in training developing-
country scientists. The opportunity must not be missed.
There are vast opportunities for studying the mycorrhizal species in miombo
woodlands, for their contribution to plant growth, and for opportunities to create
manufacturing and processing industries to satisfy local and international demand for
mushrooms and mushroom products. These studies will gain prominence due to the
current wave of interest in non-timber forest products arising from a new realisation
that woodland management is an integral component of social forestry programmes
in the region.
A variety of national and regional literature has been published on mushroom
poisoning because the only sure way to know whether a mushroom is safe to eat is to
be able to identify it in advance accurately and with certainty (from own and others
experience and books). Mushroom poisoning will continue to be an important public
health concern. This should result in more taxonomic and ecological studies on
mushrooms and related fungi in the region.
The biotechnological applications of fungi will soon feature prominently in the
region, in concert with developments in other parts of the world. There is a search
for emtomopathogenic fungi for use against scale insects (Aspidoproctus sp.) which
have decimated extensive areas of Brachystegia-dominated forests in Zimbabwe.
Such studies will, undoubtedly, have taxonomic and ecological aspects - which
should contribute to a better inventory of microfungi of the region. Some work might

76

focus on elucidating the biochemistry of the curative properties of fungi such as

Perenniporia mundula, for the academic and practical challenges it presents. The
increasing use of genetically uniform crops and trees in monocultures could give rise
to more disease problems. That should keep mycofloristic studies of this important
group of fungi alive. In addition, biological control options in agriculture and
forestry will gain prominence in line with the need to employ environmentally
benign methods of pests and disease control. Finally, the search for secondary
metabolites for use in pharmaceutical and other industries should spur mycofloristic
studies in the region, as hitherto unknown or little explored habitats are thoroughly
inventoried. Indeed, the diversity of tropical fungi has been recognised together with
their potential use in biotechnology (Subramanian 1992), so an increase in their
inventorying and methodic study is envisaged.
Herbaria must be regarded as assets, and studies in these could provide a basis
for checklists, and indicate priority areas for further field collecting and
documentation studies.

STRATEGIES

Training
There is need to build capacity for fungal inventorying, taxonomic and other
studies. This can be accomplished by organising fungal identification training
courses. The International Mycological Association (IMA), International
Mycological Institute (IMI) and the Committee for the Development of Mycology in
Africa (CODMA) are planning to organise a Regional Fungal Identification Training
Course for Africans actively engaged in research in the fields of pathology and
mycology during the early part of 1995. Short courses and exchange visits foster the
sharing of information sharing, so should be encouraged.

Fungal inventorying
Intensified collection and documentation studies should be accorded the highest
priority, otherwise many species will disappear before they are collected. Little
explored habitats must also be inventoried. A series of ecological studies are either
in progress or shall be initiated soon.

Literature
Access to literature should be improved through increased subscription to
journals. Severe foreign exchange problems and the high subscription rates militates
against these efforts -ultimately only a handful of journals might be subscribed to
and, even fewer may be regularly available. Electronic literature databases such as
CD-ROM, Internet etc. should be provided to allow for rapid and accurate literature
searches. However, to enable this funding will need to be sought.

Mobilise, organise and educate

There is need to sensitise and organise schools into amateur clubs; organise
poster competitions in schools; create awareness and fungal conservation societies;
involve the media (regarding mushrooms); and publicise fungi through stamp and
other anniversary commemorations. The importance of fungi in the maintenance of
ecosystem diversity through symbiosis and saprotrophic activities, in biotechnology,
as food and medicines; and for aesthetic purposes, should be continually highlighted.

77

Environmental impact assessment studies should have a fungal component.

Mycologists should mobilise and organise themselves and others to lobby
governments, public and private organisations for this component to be included
wherever such studies are being conducted. The importance of fungi as
bio-indicators of change (habitat disturbance, pollution etc.), if clearly articulated,
should result in an increase in fungus-related environmental studies.

Herbaria
Herbaria should be publicised for their scientific, educational, conservation and
other purposes. This might lead to more collections being undertaken by individuals
and local societies. Presently herbaria are grossly underutilised. Herbaria are a
repository of myco-diversity - analogous to seed centres and gene banks - and must
be regarded and treated as assets.

Documentation
Mycologists in the region will need to devote a considerable proportion of their
time to documentation studies. A checklist of macrofungi recorded in Zimbabwe is
now being prepared. There are no pathology or mycology newsletters, nor journals
in the region. The launching of the African Mycology and Biotechnology Journal in
Cairo, Egypt, in 1992 should provide an outlet for relevant publications (and African
perspectives). Exploratory work on starting a regional newsletter has just
commenced.

Cooperation, collaboration and networkin

Cooperation, collaboration and networking (with individuals and institutions
within and outside the region) are prerequisites for any meaningful progress to be
achieved on mycofloristic studies of the region. Attendance at conferences,
symposia, workshops and seminars should further provide important fora for the
exchange of ideas and information. Meagre financial, human and physical resources
can then be appropriately deployed resulting in a sharpening of research focus and
rapid progress. Faster methods of communication are needed. International
collaborative/cooperative fungal inventorying efforts should be initiated, but need to
be clearly defined and well planned (in the wake of property rights) and, of
necessity, should encompass and build up and equip national centres in the
respective countries.

CONCLUSION

The success of strategies outlined above depends on the availability of funds;

local, regional and international support; and perhaps most importantly, the whims
and aspirations of scientists in the region. We must initiate intensive studies soon,
and even if we do not achieve our goals, in the words of Robert Louis Stevenson,
'...to travel hopefully is a better thing than to arrive, and the true success is to labour.'

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 STRATEGIES FOR THE DEVELOPMENT OF TROPICAL MYCOLOGY

A PEERALLY

Faculty of Science, University of Mauritius, Reduit, Mauritius

Introduction

The need to develop biosystematics generally and mycology specifically has

been the subject of some pertinent interventions during the last five to six years
(Hawksworth and Bisby 1988, Hawksworth 1991, Hawksworth 1993, Peerally 1994,
Haskell 1994, Rossman 1994, Zedan 1994, Bennett 1994, Jones 1994). Earlier
Subramanian (1982, 1985) had highlighted various manners by which mycology
could promote development in the tropics. Subramanian (1986) observed that there
was no organisation in South or South-East Asia which could help anyone in the
identification of fungi important in agriculture, forestry, medicine, public health and
industry. We could very much make a similar observation today as we approach
2000A.D. It must be said that the development of mycology is of relevance not only
to the tropical countries but to all industrialized countries as well. Any strategies for
mycological development inescapably have to take into account the synergistic
interaction between developing and developed countries.
This paper will attempt to review briefly the relevance of mycology to
development, the various efforts in the tropics and in the North aimed at promoting
biosystematics and/or mycology. These interventions and efforts already incorporate
recommendations for the development of mycology world-wide and what is now
needed really is to seek funds to support mycology in a more integrated and
organised manner.

Relevance of Mycology to Development

Hawksworth (1993) detailed out, prioritized and categorized the relevance of

mycology to economic development with particular reference to the tropics and a
summary of his ideas is as follows:

(a) Food Security

Diseases in the fields
Post-harvest losses
Disease diagnosis
Biocontrol
Quarantine
Chemical control
Plant breeding for disease resistance
Mycorrhizas
Edible fungi
Mycoprotein
Mycotoxicology
Fermented foods and beverages

81

(b) Waste Utilization

Upgrading of wastes into animal feeds
Composting
Biodegradation for paper making
Recycling of organic wastes
Bioremediation
Limitation of organic pollutants

(c) Public Health

Food safety
Mycotoxins
Mycoses
Fungi in AIDS
Fungi in allergy

(d) Biotechnology
Fungi as an unevaluated resource
Pharmaceuticals
Industrial enzymes
Pesticidal properties
Fermentation technologies
Flavours, fragrances
Delignification

(e) Maintenance of Biodiversity

Fungi play an important role in the maintenance of biodiversity through the
maintenance of ecosystem function. Thus fungi are useful to higher plants in
mycorrhizas, fungi and fungal enzymes play a key role in the digestive tracts of
many feeding insects and even in the stomach of ruminant mammals. Fungi are
important in nutrient cycling and natural biodegradation. Other fungi and various
lichens have roles as bioindicators of ecological continuity.

Status of Mycology in Tropical Countries

While a few tropical countries like Australia and South-Africa have attained at
least the critical mass in terms of applying mycology to development, most other
tropical countries significantly lag behind. Hawksworth in his keynote address at the
Second African Regional Mycology Conference (Hawksworth 1993) said:
"Mycology has an immense amount to contribute to key aspects of development, but
the subject is rarely accorded the attention it merits in national programmes aimed at
increased sustainability. In the Tropics, and less developed regions generally, this is
particularly true. Yet it is in those very areas that mycologists are generally few in
number, and have scant resources?"
All aspects of mycology need strengthening in tropical countries, especially in
view of the various continua such as mycology-biotechnology, mycology-
agriculture, mycology-human health, mycology-industry. Areas which need urgent
attention are crop diseases, post-harvest losses, new biological control possibilities,
mycorrhizas, food storage and mycotoxins, edible fungi, the microbiology of
fermented foods and beverages, waste utilization, environmental bioremediation,

82

fungi causing human diseases and the isolation of novel and desired strains.
(Hawkswort 1993).
In terms of constraints the major problems are the scant human resources,
information and reference collection resources and funding for research, training and
development work in mycology, as well as the inadequacy of appropriate career
structures in Universities and research institutions, government bodies and in the
private sector.
The International Mycological Directory (Hall and Hawksworth, 1990) lists 96
mycological organisations and institutions in the North, compared to 23 in the South
of which 11 are in Australia alone. Only Argentina, Australia, India, Mexico, and the
Republic of China have mycological societies. No African country has its own
mycological society. Most lack even basic check lists, only nine of 53 African
countries have any mycological collections included in the latest edition of Index
Herbariorum (Holmgren et al. 1990). Only five African countries have culture
collections listed in the current World Directory of Culture Collections (Takishima et
al. 1989). Checklists are virtually non-existent.
Hawksworth (1993) went on to say that mycologists in Africa are isolated, and
spatially remote from other mycologists, the major work in African mycology
continues to be authored by Europeans.
Although a few mycologists in Asia, Latin America and Africa have lived up to
international standards in their contribution, the magnitude of mycological problems
in the tropics remains totally out of proportion with the available resources. The
most poorly equipped are Africa, Central America and the Caribbean.
The IMA Regional Committees for Africa, Asia and Latin America are
attempting to popularize mycology in their respective regions, but the meagre
financial resources at their disposal barely allow meaningful activities to be
organised, except for the occasional regional conferences. Plans of action which
have been worked out as part of the deliberations of regional meetings have not
taken off in most cases due to problems of networking resulting from financial
constraints.

International and National Organisations/Institutions Promoting Mycology

Associations and Societies

In spite of the strong linkage in the continuum mycology-biotechnology,
mycology is a discipline which cannot muster the media coverage not the public or
private funding as biotechnology. There are good signs however that mycology,
which has a brighter future now than was felt say a decade ago, is becoming more
organised and coordinated. The International Mycological Association and D. L.
Hawksworth have contributed significantly to the popularisation of mycology. The
International Mycological Congress, formerly held every 6 years or so, is now
organised every four years. IMA Regional Committees in Europe, Asia, Latin
America and Africa are actively having their own regional conferences and some
other activities. Other subcommittees of the IMA have contributed significantly in
rationalizing nomenclatural problems.
The International Society for Human and Animal Mycology (ISHAM) formed in
1954 encourages the practice and study of all aspects of medical and veterinary
mycology, and had 842 members from 68 countries in 1990. It publishes the Journal
of Medical and Veterinary Mycology. It holds regular international meetings and

83

occasionally ad hoc meetings and workshops.

The International Society of Mushroom Science (ISMS) focuses on fungal
biotechnology, edible fungi, macromycetes and holds regular international
congresses, and symposia on specific topics.
Other international mycology organisations are the Mycology Division,
International Union of Microbiological Societies with interests in biotechnology,
medical and veterinary fungi, micromycetes, mycotoxins and yeasts; the
International Association of Lichenology and the International Pythium Group. The
recently established World Society of Mushroom Biology and Mushroom Products
(WSMBMP) is interested in all aspects of edible and non-edible, poisonous
macrocycetes including those having medicinal attributes.

Institutes

International Mycological Institute (IMI)

A major world centre of excellence for the systematics of filamentous fungi, it
provides specialist support and associated services in all aspects of applied
mycology, including information and training.

Centraalbureau voor Schimmelcultures (CBS)

An institute of the Netherlands Academy of Sciences, it deals with all aspects of
mycology, provides information and identification services and organises training
courses regularly.

Mycotheque Universit Catholique de Louvain (MUCL)

MUCL is part of the Belgian Coordinated Collection of Microorganisms
(BCCM) financed and coordinated by the Science Policy Office of Belgium. Besides
being a reputable fungal reference centre, MUCL encompasses systematic and
applied mycology. It also offers mycological services and customized training
facilities.

American Type Culture Collection (ATCC)

Its primary aim is to acquire ex-type cultures of all named taxa and strains of
scientific and commercial interests. It provides information and identification
services. It organises training courses and workshops regularly.

US National Fungus Collection (USDA)

It is dedicated to the systematic study of plant pathogenic fungi, those useful in
biological control and of plant quarantine significance. It has developed an important
computerized information system for the identification of pathogenic fungi.

Microbiological Resources Centres (MIRCEN)

The Microbiological Resources Centres consist of a network of centres of
excellence in both tropical and developed countries dedicated to the application of
microbiology to development, and emphasize bacteria as well as fungi. It is one of
the organisations dealing with mycology that are mainly funded through the UN
system. MIRCEN has also contributed significantly to training of personnel in
applied microbiology.

84

 The Biodiversity Debate

The realization that biological sciences, molecular biology and biotechnology in

particular, might hold the key to economic explosion and sustainable development in
the decades to come is, as we know, significantly behind the global debate about
biodiversity. The risk of environmental degradation with consequential loss of this
biodiversity has also been an important element.
According to Hawksworth (1993) as little as 5% of the world's fungi have yet
been described out of a mycobiota of some 1.5 million, of which the major part is
found in the tropics. This mycobiota is a largely untapped resource in terms of
biotechnology. Their exploitation for commercial applications has naturally raised
the important question of intellectual property and patent rights. The 1992
Biodiversity Convention of Rio, signed by 153 countries, clearly establishes:
(a) the sovereignty of Governments over their biodiversity,
(b) the principle of poor countries having a claim on the international community
to help meet some of the costs of conserving biodiversity for global benefit through
such mechanism as the Global Environment Facility (GEF), and
(c) the need for mutually sharing the benefits accruing from intellectual property
and genetic resources.
Hawksworth (1993) pointed out that the benefits from fungi and other biotic
materials exploited in the Northern Hemisphere largely accrue to the companies
found in the North. There is therefore a need for most tropical countries to
themselves become involved in the isolation of novel and desired strains, and to
regulate their supply to overseas companies under mutually agreed terms of payment
and royalty (Hawksworth 1993)

Some Recent Initiatives for the Promotion of Systematics and/or

Mycology

(1) The setting up of Regional Committees for the Development of Mycology by the
International Mycological Association provides new opportunities to create greater
awareness at the level of policy makers, industrialists, the scientific community and
even in the public at large.
(2) The meeting organized in 1987 at the Royal Society by the Systematics
Association on the occasion of its Golden Jubilee spearheaded a new vision about
systematics. The opening paper (Hawksworth and Bisby 1988) entitled "Systematics:
The Keystone of Biology" provided substantial proof of the indispensability of
systematics to biology.
(3) The Convention of Biological Diversity and the emergence of the concept of
sustainable development have had an important impact through the creation of a
world realization on the need to study and conserve biodiversity.
(4) A study conducted by CABI at the request of ODA on the priorities of
Biosystematics Research in Support of Biodiversity in Developing Countries with
particular emphasis on microorganisms and invertebrates was completed in 1993
(Hawksworth and Ritchie 1993). The report made some very useful
recommendations for the development of tropical systematics.
(5) The WEFSA (Workshop on the Ecological Foundation of Sustainable
Agriculture) has been another critical thrust. WEFSA I (Hawksworth 1991) drew
attention to the inadequate current capabilities in matters of systematics of

85

microorganisms and WEFSA III (Hawksworth 1994) examined this issue in depth
and suggested ways to alleviate them.
(6) The Darwin Initiative for the Survival of Species, announced by John Major in
Rio, focuses attention on strategies for the monitoring of biodiversity and on
conservation and sustainable development. The Darwin Fellowship Scheme which
includes awards mycology, is already operational.
(7) BIONET - International established by CABI at its 1993 Biennial Review
Conference is a network with a global dimension and currently deals with
arthropods, nematodes and microorganisms. BIONET is composed of subregional
networks or LOOPS (Jones, 1994). The LOOPS will pool together resources to
facilitate the pursuit of biosystematics endeavour using local resources. The LOOPS
will coordinate and obtain support from the Bionet Technical Secretariat and from
the world's major consortium of biosystematics institutions (BIOCON).
UNDP has already agreed to provide catalytic funding to BIONET International. The
first BIONET LOOP was established in December 1993 for the Caribbean subregion
(CARINET) involving 12 countries of the region. Similar LOOPS are in the pipeline
for India, Eastern and Southern Africa. BIONET has an International Consultative
Group with Dr. M. S. Swaminathan as its Chairman. CABI was asked by its 33
member countries at the Review Conference (14-17 June 1993) to do its utmost to
assist the implementation of BIONET International.
(8) The U.S. National fungus collection has compiled electronic data from about
500,000 fungal specimens including rusts, smuts, polypores, asexual fungi and now
ascomycetes as well. This database is important in providing identification of
agriculturally important fungi and is not only useful for primary identification
services, but also for substantiating new records (Rossman 1994).
(9) In terms of biochemical and molecular biosystematics, molecular studies are
beginning to reveal hidden taxonomic criteria, especially where confusion exists
with complexes of sibling species where molecular analysis may be usefully applied
as with the former Armillaria mellea complex. (Rossman 1994).

Reckoning with Some Constraints

In the current attempt to organize, rationalize and consolidate ourselves as a

world mycological community with common practices and objectives, we have to
engage in some kind of corporate planning and in so doing we cannot escape
analysing both strengths and weaknesses.
The following quotations speak for themselves:
1. "The practices of taxonomy and identification services are perceived as wrapped
in white coats, obscured by Latin words, clouded in mists of camphor and
formaldehyde, or even pickled in alcohol. They are seen as the preserve of an elite,
to some extent parasitic, epiphytic, or at best, saprophytic upon the resources of
other activities. Can this image be changed?" (Bennett 1994)
2. "Taxonomy must become an extrovert activity and escape the isolation of a Petri
dish of introversion. The full value of taxonomic studies can only be demonstrated as
a vital link in a process that creates income or achieves wider objectives". (Bennett,
1994)
3. "As a world community we have not come close to satisfy the most basic needs for
identification services and resources to agriculture". (Rossman 1994)

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4. "The identification and characterisation of important organisms depends directly

on the state of systematic knowledge of those organisms. At present, for many
groups of pest organisms, knowledge on which to base accurate identification and
characterisation and an understanding of the relationships of target organisms to
related species is lacking". (Rossman 1994)
5. "For both fungi and insects any attempt to identify agriculturally important
organisms can be unsatisfying in that an accurate identification is difficult. Between
25% and 50% of the specimens submitted to the USDA systematic laboratories
cannot be accurately identified to the species level". (Rossman 1994)
6. "Many of the organisms belong to large genera which have never been
monographed so that identification to the species level is impossible". (Rossman
1994)

The Strategies

Hawksworth (1993) has clearly emphasized that prospects for developing

mycology in the tropics are good provided the prerequisite organisation and planning
can be effected by 1995. These prospects arise mainly from the new opportunities
available through current awareness and interest in biodiversity and sustainable
development, and through the Biodiversity Action Plans as proposed under the aegis
of the Global Environment Facility.
It is evident from the points raised so far that the mycological scene globally is
fairly buoyant with prospects of a better future in the years to come. It has been
repeatedly emphasized how mycology hooks up with sustainable development,
agriculture, industry, biotechnology, human health, food security, maintenance of
biodiversity. Mycology is not and should not be sold as an applied science only.
Fundamental mycology is an important as applied mycology. Mycology has both its
Petri dish introversion and its biotechnological extroversion.
Another point should be emphasized. In the international, global outlook of
mycology, in contrast to areas like the materials and the electronic technologies and
others which are mainly the affairs of industrialized nations, an industrially useful
fungal strain, for example, can be found anywhere but we need to know its identity
and unravel its practical use.
The consolidation of mycology is naturally a priority for the tropical regions, but
strategies for the development of mycology in the tropics should not ignore the fact
that expanding facilities, research, infrastructure and services in the North will be of
enormous benefits to the tropics.
The main actors in strategies for mycological development are:
(1) The international societies and associations like the IMA, ISMS, IUBS,
WSMBMP and their regional committees, if any.
(2) The centres of excellence like IMI, ATCC, CBS, MUCL, USDA.
(3) Networks like BIONET, MIRCEN.
(4) Mycological laboratories in the North and South.
(5) National, regional and international funding agencies.
Strategies for promoting tropical mycology will have as main ingredients:
(a) Improving expertise of existing human resources through appropriate training
opportunities.
(b) Devising a Tropical Post Graduate Training Programme in Pure and Applied
Mycology.

87

 (c) Creating critical masses through national and regional pooling together of
resources in priority areas.
(d) Creating national and regional mycological societies.
(e) Funding existing international mycological associations to enable them and
their regional committees to operate efficiently.
(f) Encouraging the development of tropical centres of fungal identification
through North-South joint ventures. Hawksworth (1992) emphasized that both
national and regional capabilities in identification infrastructure are essential to meet
the needs of agricultural and environmental services.
(g) Establishing an international task force (call it Project Neo-Saccardo) for the
urgent preparation of monographs on important groups of fungi and available in
electronic form. This would be of very significant help to taxonomists and non-
taxonomists world-wide. In fact Rossman (1994) made the extremely pertinent
observation that, in order to ease the identification requirements of both small and
large scale countries, we need: (a) a world-wide network of primary contacts well
trained and with relevant information tools at hand, (b) a cadre of back-up experts
who provide identification of unusual species, and conduct research in systematic
and (c) a mechanism to upgrade, revise and modernize the identification tools
provided to primary contacts.
(h) Assisting developing countries to set up culture collections at least on a
regional basis.
(i) Encouraging the development of centres of excellence in mycology in the
tropics.
(j) Harnessing national and international support for established networks like
BIONET and MIRCEN.
(k) For the purpose of attracting better funding possibilities it might be desirable
to organize workshops/training seminars on topics which extrovert mycology: e.g.
Mycology and Human Health (WHO might fund), Mycology and Pollutant
Bioremediation (UNEP would be interested), Mycology and Industrial Development
(UNIDO might certainly be interested), Mycology and Biotechnology, Mycology
and the Maintenance of Biodiversity, Mycology and AIDS, Mycology and Allergy,
Mycology and Protein Production, Mycology and Animal Feeds, Mycology and
Fermented Foods, and many others.
The collective wisdom of the world's leading mycologists has been prominently
seen at work during the past ten years or so, and it has started to yield positive results
in terms of reasonable success in mobilising the scientific community, policy
makers, and international agencies. The MUCL Centenary Celebration which
focuses so much attention on tropical mycology is ample proof of the determination
of the world's mycology community to function for the common good of humanity.

References
Ainsworth A.M. and Hawksworth D.L. eds 1992. Biodiversity in the Caribbean. CAB International,
Wallingford, U.K
Bennett A. 1994. A review of existing identification services and future needs. In The identification and
Characterization of Pest Organisms. Hawksworth D. L. ed., CAB International, Wallingford, U.K.
pp. 57-68
Hall G.S. and Hawksworth D.L. eds l990. International Mycological Directory. 2nd edition. CAB
International, Wallingford, U.K.

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Haskell P.T. 1994. Are biosystematic services necessary, desirable and possible? In: The Identification
and Characterization of Pest Organisms. Hawksworth D.L. ed.,CAB International, Wallingford, U.K.
pp. 3-16.
Hawksworth D.L. ed. (l99l). The biodiversity of microorganisms and invertebrates: Its role in sustainable
agriculture. In The Identification and Characterization of Pest Organisms. CAB International,
Wallingford, U.K.
Hawksworth D.L. 1993. The relevance of Mycology to development. The African Journal of Mycology
and Biotechnology 1(1): 1-11.
Hawksworth D.L. ed. 1994. The Identification and Characterization of Pest Organisms. CAB
International, Wallingford, U.K
Hawksworth D.L. and Bisby F. A., (1988). Systematics: The keystone of Biology. In Prospects in
Systematics. Hawksworth D. L. ed.,Clarendon Press, Oxford.
Hawksworth D. L. and Ritchie J. M., eds. 1993. Biodiversity and biosystematic priorities.
Microorganisms and invertebrates. CAB International, Wallington, U.K.
Holmgren P.K., Holmgren N.H. and Barnett L.C. 1990. Index Herbariorum. Part 1: The Herbaria of the
World, 8th Edition. Regnum Vegatabile 120. New York: New York Botanical Garden.
Jones T. (1994). BIONET - International: A global network for biosystematics of arthropods, nematodes
and microorganisms. In The Identification and Characterization of Pest Organisms. Hawskworth
D.L. ed. CAB International, Wallingford, U.K. pp. 81-92.
Peerally A. 1994. Biodiversity: adding weight to the debate: Proceedings Seminar (1993) on Plant Genetic
Resources: Towards a National Strategy for Mauritius. IBPGR, University of Mauritius, Ministry of
Agriculture.
Rossman A.Y. 1994. The need for identification services in agriculture. In The Identification and
Characterization of Pest Organisms. Hawskworth D.L. ed. CAB International, Wallingford, U.K. pp.
35-46.
Subramanian C.V. 1982. Tropical mycology: future needs and development. Current Science 51: 321-325.
Subramanian C.V. 1986. Foreward UNESCO Workshop on Progress on Applied Mycological Research in
the Tropics, University of Singapore, May 1985. Proceedings of the Indian Academy of Sciences,
Plant Science 96: 333-34.
Takishima Y., Shimura J., Udagawa Y. and Sugawara H. 1989. Guide to World Data Center on
Microorganisms with a list of Culture Collections in the World. Saitama: World Data Center on
Microorganisms.
Zedan H. 1994. Pest organisms: number, ecosystem impact and developing country needs. In The
Identification and Characterization of Pest Organisms. Hawskworth D.L. ed. CAB International,
Wallingford, U.K. pp. 17-34.

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 THE ROLE OF THE MICROBIOLOGICAL RESOURCES CENTRES

(MIRCENs) IN THE DEVELOPMENT OF MYCOLOGY IN THE TROPICS 2

E.J. DA SILVA

Introduction

It is a pleasure to have the opportunity, albeit even in abstentia, to be associated

with the Centenary Celebration of the Mycothque de l'Universit Catholique de
Louvain. In this regards, UNESCO notes with satisfaction the herculean work
carried out over several decades under the inspiring leadership of Professor G;
Hennebert in building up this culture collection of fungi. While in the 1950's the
collection maintained a few hundred cultures of fungi, reference to the classical
World Directory of Collections of Microorganisms, produced in 1972 by John Wiley
& Sons, Inc., with the financial support of UNESCO, shows that MUCL then held
1183 species of fungi. Those were represented at the time by 14,500 cultures of
fungi and 500 cultures of yeasts. Moreover, the interests of the collection were
distinctly confined to agricultural and industrial mycology. Charging no fee for
distributing and exchange of cultures, providing research qnd teaching services,
MUCL had no published catelogue.
Twenty-two years later, the picture is different. From a one-time staff component
of 1, the total strength has grown to 9. Moreover, a fee is levied for the distribution
and exchange of 24,000 cultures of fungi, including 2000 cultures of yeasts. Storage,
identification, training and consultation services are provided in the management of
culture collection, propagation, culture and preservation methods, and in quaratine
and shipment regulations. Still again, the interests of the culture collection have
grown. These range from agriculture, applied and industrial microbiology,
fermentation, freeze drying, food science, forest microbiology, and biodeterioiation,
to systematics and taxonomy.Finally, in addition to sponsorship by the University,
there has been support from the Belgian Science Policy Office, and the then
Commission of the European Communities (CEC).
This admirable yet painstaking evolution of MUCL, in just two decades, from its
early humble existence into its present day acknowledged national and international
stature and status, is but a powerful reminder of the efforts and time required for the
development of such culture collections in the developing word. Furthermore, the
obstacles and challenges, encountered by mycologists in different regions of the
developing world, are virtually the same as those encountered by virologists,
protozoologists, phycologists and even bacteriologists.
Although well established fungal culture collections do exist, e.g. the
Centraalbureau voor Schimmelcultures in The Netherlands, the CAB International
Mycological Institute in the U.K., and the Mycological Culture Collection in the
USA, proposals for funding and setting up such collections have often received

2.Presented by Jean Mouchacca, Laboratoire de Cryptogamie, Musum National d'Histoire Naturalle,

Rue Buffon 12, 75005, Paris, France.3 I agree with Barr (1983) that we are in need of a suitable term
referring to fungi, in parallel to the terms "flora" and "fauna". However, from the two terms proposed by
her, I consider "funga" as more suitable than "mycota" (preferred by Barr 1983: 8), because the latter term
is also used as a suffix for naming taxa at the Division level.

90

token support. The stark reality of the scarcity of such resources in times of
economic repression and emerging competitive priorities such as environmental
management or greening of the deserts is to be found every time a current culture
collection changes status to that of being an endangered culture collection -- be it
mycological, viral, protozoal or bacterial in content.
This is evident from the excellent initiative of the World Federation of Culture
Collections (WFCC) in releasing "Living Resources for Biotechnology" which is a
series of practical books that provide primary data and guides to sources for
information on matters relating to the location and use of animal cells, bacteria,
filamentous fungi and yeasts. It is worth noting that each volune contains specialized
information together with material on general matters (information centres, patents,
consumer services, and international coordination of culture collection activities)
that is common to each group treated, i.e. animal cells, filamentous fungi, bacteria
and yeasts. Moreover, each volume also provides valuable information on resource
centres located world-wide.
Of late much concern has been voiced about the lack of attention given to these
research centres, and by consequence to specialized areas such as mycology,
protozoology, entomology, and the like. This concern has been eloquently
articulated in the run-up to and the follow-up of the 1992 U.N. Conference on
Environment and Development in the Rio de Janerio, Brazil (Hawskwoth and
Ritchie, 1993).
In order to address the topic of the assignment, a brief review of the MIRCEN
network appears to be useful.
The international support of culture collections -- the treasure houses of the
planet's microbial (and, of course, mycological) genetic heritage, can be traced back
to the early catalytic support provided by UNESCO, in 1946. Several of today's
well-known culture collections have been beneficiaries of such support (Da Silva et
al. 1977). Moreover, support was extended also to relevant publication such as the
Internatinal Bulletin of Bacteriological Nomenclature and Taxonomy.
In 1962, UNESCO's Twelfth General Conference adopted a resolution by the
Government of Japan to initiate and intensify research and training activities in
microbial biotechnology in view of the growing domestication and use, at that time,
of microbial resources in the sector of food, energy, industry, medicine, and
agriculture. As a consequence, UNESCO, in 1963, initiated a series of GIAM
conferences and in subsequent years, undertook joint collaborative activities with
the Panel on Microbiology of the International Cell research Organizations (ICRO),
the International Association for Microbiological Societies (now known as IUMS),
the International Organisation for Biotechnoloy and Bioengeneering (IOBB), and
the World Federation of Culture Collections (WFCC).
The 16th. Session of UNESCO's deliberations in 1970 were enriched by a
resolution from the Governments of Denmark, Finland, Norway and Iceland calling
for the establishment of specialized microbial research centres in developing
countries. This foresight by these Governments, in its historic perspective, was the
essence of what today is known as the MIRCEN network.
The origins of the MIRCEN network can be traced back to the early days of the
UNESCO/IRCO Panel on Mircobiology. Established in 1965, the Panel was
principally concerned with:
(i) the establishment of an international network for the preservation and
exchange of cultures;

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(ii) the promotion of the use of micro-organisms as a natural resource; and,

(iii) the world-wide training of microbiologists (Porter 1974, Bull and Da Silva
1983).
Following the United Nations Conference on the Human Environment at
Stocklohm, 1972, experts from UNEP, UNESCO, and the international scientific
community, represented in ICRO, met in 1974 and 1975 at the UNEP Secretariat at
Nairobi, to formulate jointly a world-wide programme aimed at the presevation of
microbial gene pools and to make them accessible to developing countries through
the establishment of a network of microbiological resources centres (MIRCENs).
Subsequently, a UNEP/UNESCO/ICRO project titled "Development of an
Integrated Programme in the Use and Preservation of Microbial Strains for
Deployment in Environmental Management" (July 1975 - October 1984) was
formulated and submitted by UNESCO/ICRO, and approved by UNEP for
implementation. The objectives of the MIRCEN network are:
(i) to provide a global intrastructure which would incorporate national,
regional, and inter-regional co-operating laboratories geared to the management,
distribution, and utilization of the microbial gene pools;
(ii) to reinforce the conversation of micro-organisms, with emphasis on
Rhizobium gene pools in developing countries with an agrarian base;
(iii) to foster the development of novel technologies native to specific regions;
(iv) to promote the economic and environmental applications of microbiology;
and,
(v) to serve as focal centres in the network for the training of manpoer and
diffusion of microbiological knowledge.
The first development in the World Network of Microbiological Resource
Centres was the development of the World Data Centre for Microorganisms
(WDCM), in 1975, at the University of Queensland, Brisbane, Australia. The
WDCM houses a master copy of the World Directory of Collections of Culture of
Microorganisms and serves as a pivotal point for fostering development of culture
collections in developing countries in an international cooperation (Da Silva 1986).
To help provide permanency and continuity to the work of the WDCM, the
World Data Centre, has been relocated, from July, 1986 to the Life Sciences
Research Information Section, RIKEN, Tokyo, Japan.
Today, the World Network of Microbial Resources Centres (MIRCENs) is
comprised of the BNF and Biotechnology MIRCENs. Culture collections continue
to occupy an integral role in the network and the emphasis is more on strengthening
current assets rather than funding new ventures.
To return to the theme of mycology, it is evident that there is a distinct need for
basic training especially at research sites and institutions in the developing world.
Emphasis to date has been on specialized training at established institutions in the
developed world. Important and valuable as training is, there needs to be a concerted
effort that focuses on the awareness and beneficial inputs of the mycological
sciences in improving the quality of life and by consequent the environment.
Emphasis should be on responding to the needs for food, health, and energy coupled
to the adaptation rather than the adoption of new skills and techniques for use by
researchers in the developing word.
Fungal collection such as that of the Mycothque de l'Universit Catholique de
Louvain and the IMI MIRCEN have important roles to play in taking the message
into developing countries by organizing training courses and participating as

92

"Professors" in these countries. Such pioneering roles help to create awareness in

university administrators, help update curricula and syllabi, and help attract
industrial support and participation. An example is the excellent contribution of
Mycothque de l'Universit Catholique de Louvain in the development of a basic
unit in the University of Burundi, Bujumbura, Burundi. The grass-roots approach
has never failed. One of the best examples is the case of the study of mushrooms
that has emerged on the international scientific scene as an important theme for
international co-operation. In fact, these "mushroomologists" have their own society,
their own computer network, their own newsletter, and their own programme of
scientific activities.
In wishing the Organizers of this meeting every success, UNESCO, through its
decentralized programme activities, and the Participation Programme for Member
States, in which national culture collections in Bangladesh, Iran, and Paskistan have
been strengthened, is ready to assist in the delivery of this message and would like,
publically to thank all those scientists who have strengthen the bonds of
international scientific co-operation.

References
Bull A. and Da Silva E.J. 1983. World networks for microbial technology. Society for General.
Microbiology Quarterly. 10, 6-7.
Da Silva E.J. 1986. Culture Collections, a field for international cooperation. Symbiosis 2(1): 249-263.
Da Silva E.J., Burgers A.C.J. and Olembo R.J. 1977. UNEP-UNESCO and the international community if
culture collections. Proc. Third Int. Conf. Culture Collections, Bombay, 1977, pp. 107-120.
Hawksworth D.L. and Ritchie J.M. 1993. Biodiversity and Biosystematic priorities: Microorganisms and
invertebrates. CAB International, Wallingford. 70 pp.
Porter J.R. 1974) UNESCO/ICRO Panel on Microbiology. A.J.M. News 40:259-265.

93

 DISCUSSION

FUNGAL TAXONOMY AND TROPICAL MYCOLOGY: QUO VADIS?

Hennebert. It is now the time for questions and discussion. We heard this afternoon
different options in approaching fungal taxonomy. In regard of the need developing
tropical mycology, we may question what kind of approach is now suitable.
Dr Kurtzman and Dr Blackwell showed that molecular analysis can provide
reasonable answers to problems left by the so-called classic - meaning morphology-
based taxonomy. But they made clear that such answers, if sometimes leading to
definite nomenclatural changes in yeast taxonomy, are most often indicating new
relationships between taxa that need further investigation before definite
nomenclatural changes are made like in the Ascomycetes.
In another hand, Dr Rammeloo and Dr Roquebert pointed out the fact that the two
century-old taxonomic framework of classification of the fungi is morphology-
based, and that is more practical to build on it or revise it that to construct another
one de novo. Dr Rammeloo also indicated the particular need for more precision,
consistency and coherence in morphological criteria and their expression. Dr
Roquebert showed promising perspectives in that direction with new types of
microscopy that may diminish subjective interpretations.
For mycologists wanting to cover as soon as possible the largest part of the still
unexplored and disappearing fungal diversity in mainly tropical areas, the question
of methodology is surely justified. Mycologists in those areas have not so many
facilities, as we heard from Dr Masuka and Dr Peerally. They are two taxonomic
approaches to fungal diversity. Which of these two taxonomic approaches should
they adopt? What option should we propose? Your opinions are welcome.

Webster. Let me ask Dr Kurtzman and Dr Blackwell if they feel that we are here
approaching a finality in ascomycete classification whether we are moving to a
stable state or we are moving to a phase that would become to develop much further.

Kurtzman. That is a very good question actually. I would like to think, on the basis
of ribosomal RNA and rDNA sequence comparisons, that we are placing our
systematics in a framework that would not change too much. And we have seen
from comparing these molecules with other molecular sequences that they are giving
pretty much the same outline. That is what we see now, and what we will see for
some time.

Blackwell. I agree with Dr Kurtzman. Some people have expressed that they believe
that some of these advances have been so fast that they have not been considered
using ribosome genes, and it does not seem that other genes could do it. This may be
pessimistic, but we have not studied enough taxa to know this. I think we are coming
very soon to something quite stable for most of the Ascomycetes. Also I tend to be
taxonomically very conservative, and I don't like to make taxonomic changes at this
point to confuse the issue for a time.

Hennebert. At what level do you see this?

Blackwell. At the order level, for now, but later, at other levels.

94

Hennebert. It seems that also morphologists are waiting to define orders within the
Ascomycetes.

Blackwell. The things that we have been doing and in what we believe, is that in the
Ophiostomales there has always been the same ravel. The genes we have done so far
gave the same story. And if you sequence a gene that is evolving very quickly, it
might be you will not get the same story. But, you will get a lot of noise that you
may not be able to distinguish it. We are making gene trees. By doing that, we are
making a lot of headeway from what has been made.

Hennebert. But doing such an approach is the classical taxonomy keeping some
reference?

Blackwell. In my work certainly. I am a classical taxonomist, you know for thirty

years. I am using morphology but it sometimes come to a certain point that I cannot
decide what is right or wrong. Then I look for gene analysis possibly to provide an
answer.

Prillinger. I think it is absolutely right that there is a necessary combination of

classical taxonomy and systematics with molecular analysis, especially taking into
account that there is in molecular biology a dependence on applied computer
programs. Indeed it is possible that depending on the program we use we get
different trees, so we have trees. Another aspect is that we need other characters,
other methods to prove our results. It is therefore absolutely necessary for all people
working on systematics not to use one method only either molecular analysis, or
morphology, or anatomy, but to combine theses methods.

Hennebert. There are indeed many different programs giving different trees and
neither the molecular biologists nor the fungal taxonomists know really how those
programs are constructed, on the basis of what algorithms, and what is the
significance and real value of these algorithms for our taxonomic purposes.
Perhaps, and that is not a joke, mycologists should be good computer scientists,
and not only good biologists, and, I should add, good molecular biologists.
Therefore, if things are such, the required training to become a mycologist should
change and be much broader.

Whalley. I have a question for Meredith Blackwell. Meredith, do you remember the
paper by Gowan and Vilgalys (1991, Am. J. Bot. 78:1603-1607). They looked at
Xylaria magnoliae and they looked at the ribosomal length polymorphisms from
different stromata taken from the same fruit and from the different fruits in the same
region and then from different localities. And they got quite a lot of variations in
their data. In fact they were getting more variations, I think, on the DNA material
that they would on the classical morphological characters. The question I ask is how
much molecular data do you need to be confident in the results?

Blackwell. It all depends. I do know the opposite problem too, with little rDNA
variation within large specimen variability. If I consider the biology of the organism,
questions are how is its dispersal, how it is getting here or there. If you count its

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population, is it really a population? And what is an effective population size? All

these things ought to be known before being able to interpret the molecular data.
Does it help?

Whalley. Thank you, Meredith. It does indicate that interpretation of molecular data
must be done with great care.

Prillinger. I feel there is also different information from ribosomal DNA. If we look
to a recent paper of John Taylor and Berbee, and to your paper of today, Mrs
Blackwell, and to the paper of Dr Kurtzman, we have different interpretations of the
Endomycetes. For John it is a side group of the Ascomycetes near the filamentous
ones. In the Kurtzman paper it is a primitive group of fungi. What do we have to
believe?

Blackwell. What I think of the Endomycetes? I think they are sister taxa to the
filamentous Ascomycetes, do'nt you? It is somewhat deceptive when you have
different arrangements from the tree made from different numbers of taxa. But I
think now, looking with clean eyes, that, with the increasing number of species
included in the trees, we see that the Endomycetales and the filamentous
Ascomycetes are sister taxa.

Kurtzman. These are indeed not far away.

Blackwell. Dr Sugiyama what do you think?

Sugiyama. I agree with Dr Blackwell. From phylogenetic analysis of the nuclear

small subunit rRNA (18S rRNA) sequence data, we found three major lineages
within the Ascomycetes. their distinction supported by very strong bootstrap
confidence. These are the Archiascomycetes (early Ascomycetes), the
Hemiascomycetes (yeast Ascomycetes) and the Euascomycetes (filamentous
Ascomycetes). The Archiascomycetes comprise Taphrina, Protomyces, Saitoella,
Schizosaccharomyces and Pneumocystis. The Archiascomycete lineage diverged
first among the ascomycetes, whereas the Hemiascomycetes and Euascomycetes as
sister taxa evolved more recently.

Korf. I like to shift a little bit from phylogeny and relationships of organisms, which
is indeed an example of the most exciting things that come out molecular work, to
look, in fact, at "fungal taxonomy and tropical mycology: quo vadis?", as our topic
today, specifically at biodiversity and particularly at biodiversity in the tropics.
The question I want to rise is how to go about training fungal taxonomists. That is
mostly only done in temperate areas by people that have been trained in temperate
areas also. My own experience with both temperate and tropical floras had led me to
realize that we have that tremendous history of a development of morpho-anatomical
systems primarily based upon temperate floras.
When I went to the tropics, I discovered something that made me change all my
viewpoints upon how one has to train a mycologist and I always insist on, and force
all my students to collect in the tropics. It is not only that the tropics are loaded with
a lot of species, and we know from the biodiversity information that the number of
species in the tropics may be usually in excess of that in the temperate areas, and

96

that they have been vastly understudied, as has already been said several times
today. What really strickes me as most amazing was that my generic concepts were
immediately challenged the moment I moved to the tropics. I kept finding things that
were neither this nor that, and when I hear of my colleagues here saying, we want to
identify at least to genus, I do not know how you can, given the way current
taxonomy is arranged. There are temperate genera. If you are going to work in the
tropics you will have to find that you are going to have all new systems, you are
going to have to develop them. And you are going to find lots of species that you
cannot put names on.
Now, I spent all my life travelling all over the world convincing the dean of my
College that I could not possibly study the fungi in Ithaca, New York, without going
to the Canary Islands, to Africa, to Asia, Not true? I could spend all my life studying
the fungi outside my door and I would still not have had all the answers because I
don't know two-thirds of the fungi outside my door. But the tropics really change
your perspective. And I am sorry for the fact that there are not many places in the
tropics where taxonomy is being per persued today, and if it is going to be persued it
has to be persued with the idea that the tropics have lots of unanswered questions,
lots of things to be described, and that we indeed must go there and collect these
things. Rammeloo, Masuka and Peerally have clearly shown that the traditional
morpho-anatomic methods are what is going to have to be done if we want to
document what is there, and to go and collect them in order to explore that tropical
flora.
Where we are going to find the mycologists being trained to monograph large
genera, or to find financial support for people producing large monographs? That is
a big and unsolved problem. And I want to sound an alarm. An alarm that does not
seem to have been voiced today. We must stress, no matter how exciting molecular
techniques are, and they are exciting - and if I was a young student again, I am sure I
would be a molecular taxonomist - they will not, and cannot, address the problem of
biodiversity or cataloguing of our tropical floras.
In addition, classical mycologists are losing out in these areas. When I look about
me, I see mycological taxonomic positions in numerous universities all over the
world, in museums, being replaced by either nobody or by molecular taxonomists
whose interests are not in producing monographs, or in cataloguing the tropical
floras that we know so poorly.
I just wanted to point out my worries for the future of our understanding of
biodiversity in the tropics.
(Applause.)

Webster. I truly share what Dr Korf has said.

Hughes. Grgoire, it was a surprise and a pleasure to be invited here. Yes, in order
to survive the Ottawa winters, I have been a long time in the tropics. When I think of
the tropics, I think of the tremendous leaf surface of the living leaves, and probably
in these land areas where the leaves and the nutrient within are separated from the
fungi outside by a thin cuticle only.
I stated as a novice at IMI in 1945. In 1946, Hansford's wonderful work on
'Foliicolous Ascomycetes and their parasites' appeared. The availability of this work
and the riches in herbarium IMI, followed by a 3 month foray in Ghana in 1949

97

further impressed upon me the enormous microfungus diversity even in the small
area that I covered.
Flat leaf surfaces in the tropics support a vas array of Ascomycetes. For the most
part theses have been assigned to well over 30 families in various orders. Some of
the fungi are entirely superficial and comprise several families of the pleomorphic
sooty moulds. Others are leaf parasites and, as Hansford has stressed, various
devices have evolved to tap the nutrient resources of the leaf. These fungi can cause
severe damage or may be benign parasites which cause minimal damge to the leaves,
such as the Meliolaceae, Asterinaceae and several other families. A third group of
common Ascomycetes comprises the mycoparasites of other foliicolous fungi.
But the numerous families of foliicolous Ascomycetes are by no means well
defined. There is a great need of studies on the hyphal behaviour on leaf surface,
perithecium ontogeny, development morphology of the type species as well as the
identification of thei anamorphs. Such studies can be carried out adequately with
ordinary microscope using current simple and straightforward techniques. We have
much to learn about, and from these beautiful Ascomycetes.

Subramanian. I am entirely agreeing with much of what has been said here. I feel
really happy that we are going to make great progress in the field of molecular
taxonomy. But I think, for the developing countries, which don't have a complete
inventory of their fungal resources, they have to rely on the ordinary light
microscope and the simple tools which are at their command.
And, to begin up, the collections and the herbaria from those regions, which
remain largely unexplored, have to be done by the people in these regions. Such a
task needs, of course, cooperation and help which they can get from mycologists in
the advanced countries.
Just to make an example. how pertinent observations can be made by ordinary
light microscope, my good friend Stan Hughes here, his work in his 1953 paper on
conidiogenesis and the classification of the Hyphomycetes was based entirely on
observations under the ordinary light microscope. And so was it earlier with
Vuillemin, Mason and other people. And we do know now how much of it has been
confirmed by studies with better tools, like the transmission electron microscope, the
scanning electron microscope and, maybe, studies on the biosynthesis of certain
metabolites may also confirm some of these things.
The important thing to do, is to use the ordinary light microcope, the phase
contrast microscope, in the best way, to study the fungi. They are so beautiful. They
are everywhere. Professor Korf said there are so many new things in the tropics.
Almost everything is new! But of course it is also interesting to see the old fellows
when you collect. Some beautiful things also occur in the tropics that are in the
temperate regions.
I think we ourselves must define our priorities in developing countries. And the
first thing to do is the inventory of the fungi using simple tools which may be easy to
get, the ordinary light microscope, and the literature. Getting past literature is more
difficult. But there again, things are better now. Things were not the same when
Saccardo compiled his Sylloge fungorum, we have better things today to come up
with lists of fungi. So I thing the first priority is the inventory.
The second priority is to have monographs. This has been expressed already.

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I believe it is up to us, people of the developing countries, to do this job. And I am

sure if we seek for cooperation of the mycologists in other countries, we will get it,
as I got it when I started my work in mycology.
(Applause)

Parmasto. I wish to comment about the importance of collecting and use of tropical
specimens.....
First, you make a data matrix, trying to make a system of the species you have
classified using phenetic or cladistic methods. Then you are adding data and many
other species and your cladogram is almost totally changing. That means that we not
only have need to know more species of the tropics, but also more various
collections of each species.
Secondly, if nowadays there are not many possibilities to use molecular methods in
tropical countries, it is possible to collect specimens in such a way that they will be
usable in future for such studies. That means that specimens must be collected and
preserved in such a way that their DNA are not damaged, f. i. by chemicals, high
temperature, or simply contaminations..As far as I know no one has been using well
preserved spore print on cellophane for molecular studies. One spore is enough for
sequencing. I hope that somebody shall do it and tell how it does work.
Third, it concerns molecular data as well as morphological ones. The molecular
data are mainly based on morphological classification of the species. They are meant
to check each other, inducing revision of the morphological classification, but also
revisiting the exact value of the numerical treatment of sequences. Only after that
process, they are to unite. It seems that it is not so important how to make the
sequencing. Half of the work comes after sequencing, how to use the data. An seen
on the posters, they are nice cladograms, made using neigbour-joining method.
When using cladistic methods, instead of one cladogram, one shall get 3466
cladograms of equal length. That implies that training young taxonomists must
include such courses as philosophy of sciences and methodology of sciences. We are
a little bit too old, including me, to understand what to do with all the data we have,
but let them have a little more "soil under their feet", a better background to
understand what they do.
The last not the least, seventy to eighty percent of new fungus species are based on
one collection. The percentage of one-specimen species has been very stable for the
last forty years. As a result, description of such new species does not give any
information about the variability of the species. Half of these new species may never
been found later again. Because they are specimens but not real species, they are
producing noise in data treatment. We should avoid that situation.
(Applause)

Masuka. Yes, it was good to get some corroboration on the most important things to
do, that is to go out, to collect, to document and to make inventoies. That is priority
for us. We accept, of course, what temperate scientists are saying on molecular
taxonomy.
But wa are convinced that the priority is finally to compile check lists of the fungi.
We know that check lists form the base for our studies. Then, of course we must do
monographs.
An important thing that was hightlighted is that, as Professor Perally said, in our
work agenda in developing countries we must seek for collaboration and cooperation

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with laboratories in the developed world. That has been so in the past and that must
continue.

Arnold. I would like to indicate that only one existing system of the fungi is a
complete one, the one of Saccardo, contructed on the base of all fungi known at his
time. All the following systems are incomplete, as constructed on only a part of the
fungi: Ascomycetes or Basidiomycetes or Fungi Imperfecti. If one wants to make a
system on the basis of molecular data, one must study about 75000 existing species.
You can imagine how many years such a work needs..
We have to combine all data on morphology, anatomy, biochemistry of these species
with the data of molecular biology. This would make a system, may be almost
natural.

Hawksworth. I feel the time has come to startt thinking in terms of different
practices. This is an issue that has been brought up many times over the years, and
initially by the mycologist J.H. Corner in 1946 who, having to cope with a tropical
flora of not only the fungi but the trees that are unknown, decided to monograph the
trees first, viz. the two volume 'Trees of Malaya'. Then he was able to continue work
on the fungi, something he is still doing. Corner's way of operating, drawing
carefully all that he saw, led him see things that others had not seen before, often
because they were looking with temperate trained eyes and with preconceived ideas.
A fresh look is very valuable, and a great deal of the work can be done locally.
We also have to consider making data available. Dr Huhtinen, at the First
Intensive Workshop on Ascomycetes Systematics, in Paris last year, pointed out that
also we have to think more about data capture, as well as trying to get fungi into
culture close to the site of collection while the specimens are still fresh. With the
resources we get, we can carry out the other work, make comprehensive drawings,
look at all possible characters and determine ways to exchange these data
electronically. We have been thinking about this at the IMI. This is also true in the
insects. We can not think just about conventional publications, as the volume of data
will be so great. In the case of insects, it has been estimated that something like 1-6
km of shelves would be needed to accommodate the literature on the Earth's insects
at the rate of two pages per species. The task is absolutely enormous, so we have to
develop new ways of working.
In the tropics, this means really developing a taxonomy as we go along. Dr
Subramanian, Dr Korf, and others have mentioned today that a vast number of fungi
are unknown. So it is not a matter of working with a key that exists, because we will
find very quickly a huge number of genera, even of families, not documented in the
keys. The same phenomenon arises in specialized habitats even in Europe, as some
of you will know.
What I have been very pleased to see florishing in the IMA, is the regional
committees. This is has been a noticeable development, especially in the African
Committee, that we have been hearing about today: two major conferences have
been held, a third one is being planned next year, together with a training course, a
new journal has been launched, and an African Center of Biotechnology and
Ecology in Cairo has recently managed to secure almost two millions dollars to
actually set it up.
This move should be linked with other programmes that have been mentioned
several times today. There are entomologists meeting at this very moment 20 km

100

from here in Belgium looking at the development of BioNET INTERNATIONAL in

Europe. BioNET is a concept that CAB INTERNATIONAl, as a peculiar
intergovernmental organization mainly owned by developing countries, has
developed in agreement with major institutions around the world, particularly in
North America and in Europe, to establish a number of regional biosystematics
loops which bring together the expertise in different regions. It is amazing how
many specialists there are in now developing countries, but who don't necessarily
know each other at all, being in different departments of a university or in different
institutions.
This networking can be linked with a building up of collection resources locally,
and there is money for this type of activity to be sought. I mentioned the year 1995
as a critical date for planning in mycology, in a paper I gave in 1992. That date still
stands, as it will be the watershed as to how the funding of the Global Environment
Facility will be used. The fund has a large amount of money available for
biodiversity and related actions, with the 2.1 billion dollars so far pledged to this
fund. The fund is being re-structured in November 1994. Mycologists need to think
how we actually tackle that funding source. And because the fund scale is so huge,
you cannot tackle it just as mycologists. We have to present ourselves as
systematists, linking with other groups.
The major problem is to increase recognition that, in order to support the
development and strengthen country capabilities, the training of people in the
developing countries is visible. Yet it is up to them to work on their priorities and
make their submissions for support. IMI has been fortunate in securing biodiversity
related money under the Danish Initiative scheme, not just for mycology but
including entomology and parasitology. Through this initiative, 22 fellowships for
systematists with some training are already established in positions in developing
countries and founded. Money is around if the right sort of proposalsis is made and
if we think much more as biologists and systematists, rather than only as
mycologists.

Demoulin. Just a small point, I wish would not be forgotten concerning tropical
mycology. Is it true that we need to put inventory as a priority? There is also a thing,
perhaps more experimental, but within reach of many laboratories, including those
in developing countries, which is quite interesting. It is ecophysiology and
autoecology of the species and of the strains and the study of the differences
between the tropics and the temperate zone. Just to give an example, with a case that
is studied in Papua New Guinea by several Belgian laboratories. We found the
North-temperate Polyporus squamosus in Papua New Guinea. The species has a well
defined North-temperate ecology concerning the optimum temperature. Although it
is definitely not well adapted in that tropical area, it is still fruiting there. There are
many similar cases that can be studied in an ordinary laboratory, with not much
equipment.

Hennebert. I shall try to conclude in few words. It comes out the discussion
particularly, that there is a priority on a classical approach of the large unexplored
diversity of fungi in tropical countries. A priority and urgent duty is to describe and
make the inventory of this mycoflora, using microscope and culture in situ, for
which well trained young mycologists are needed. And on that basis, elaboration of
monographs should provide the necessary tools to further investigations.

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 This does not at all exclude research in molecular taxonomy, to solve taxonomic
problems, afterwards. There are lots of problems arising every day in fungal
taxonomy in classifying tropical fungi. As Dr Korf and Dr Hawksworth said, when
collecting in tropical countries, we need almost to forget our temperate system of
fungi and be open to construct new concepts. Taxonomy is an evolving field where
revision is current.
Moleclar taxonomy does not supercede morpho-anatomical taxonomy. On the
contrary, they are complementary to each other. Good phylogeny based on
molecular data may demonstrate relationships that might lead one to amend the
system of the fungi at the highest ranks. But the molecular approach has not opposed
the species concept as defined by classical taxonomists as wrong.
Another issue from this discussion is the important need of a new training profile
with collaboration from both tropical and temperate regions. That training should
cover different disciplines, including computer science, cladistics, molecular
biology, ecophysiology, without neglecting at all the traditional but unavoidable and
basic methodologies of fungal taxonomy: collecting, microscopying, drawing,
isolating fungi into pure culture, studying type materail, interpreting past literature,
writing descriptions, constructing keys, writing monographs, building expert
systems of identification and managing herbaria and culture collections. These are
the first acts for a mycologist in tropical and unexplored areas of the world.
Thanks to all of you for your contribution.

102

 The founders of the Mycothque MUCL:

Pr. Jean-Baptiste Carnoy (1894), Pr. Philibert Biourge (1894-1934), Pr. Paul
Simonart (1934-1964).

The first public catalogues of the three Belgian Collections of Microoganisms

coordinated in the BCCM (1987)

103

 104

 Pr. Drs D.L. Hawksworth (IMI, UK), D. van der Mei (CBS, NL), M. Blackwell (MSA, USA), C.P.
Kurtzman (NRRL, USA), M.-F. Roquebert (CP, FR), J. Rammeloo (BR, BE), A.J. Masuka (FRC,
Zimbabwe ), A. Peerally (MU, Mauritius), J. De Brabandere (SPPS, BE), P. Rouxhet (Prorecteur UCL),
H. Naveau (Dept. Head, UCL) & G.L. Hennebert (UCL)

105

 Drs (1) Hughes, (2) Cerisier, Kersters, Korf, Gams, De Brabandere, (3) Webster,
Blackwell, van der Mei, (4) Masuka, Roquebert, Subramanian, Sugyama, Spencer
(5) Mrs Korf, Mrs Hennebert, Hawksworth, (6) Martini-Vaughan, Cimerman,
Uruburu, Parmasto, Fraiture, (7) Marate, Kurtzman, Marson, Martini-Vaughan,
Uruburu, (8) Verbeken, Walley.

106



Fig. 1. View on the exhibition of cultures from three
classes of fungi, original drawings of Hyphomycetes,
catalogues of culture collections and publications.
Fig. 2. Ancient equipments and manuscripts from Pr.
Dr Ph. Biourge.
Fig. 3. Evidence of the MUCL activity in applied
mycology.

107

 Openair lunch with breads, cheeses and beers from Wallony, at the Faculty in
Louvain-la-Neuve: Figs. 1, Dr Sughyama, Dr.Hughes; 2, Dr Hughes, Dr Korf;
3, Dr Peeraly, Dr Hawksworth; 4, Dr. Vanderveken; 5, Dr Nguyen, Hennebert,
Dr Parmasto; 6, Dr Walley, Dr. Kersters, Dr.Spencer.
7-8. African concert near the ruins of the Abbey of Villers-la-Ville

108

 The lunch, at the Faculty in Louvain-la-Neuve

The diner, in the Abbey htel of Villers-la-Ville

109

 The Faculties of Agronomy and of Biological Sciences, Louvain-la-Neuve

The Laboratory of Systematic and Applied Mycology (MUCL)

110

 POSTERS

111

 112

 FUNGUS COLLECTIONS AND SERVICES

__________
THE MYCOTHEQUE OF THE UNIVERSITE CATHOLIQUE DE LOUVAIN
(MUCL)
100 years 1894-1994

GREGOIRE L. HENNEBERT

MUCL, 3, Place Croix du Sud, 1348 Louvain-la-Neuve, Belgium

The genealogy of the MUCL culture collection is presented. Under the pulse of
Carnoy, Philibert Biourge inaugurated his young collection of yeasts, molds and
bacteria in 1894. He left the hand to his young colleague Paul Simonart in 1938
Simonart named the collection "Mycothque Philibert Biourge" in 1939. He
maintained the collection untill 1969, when he passed it to the author.

Jean-Baptiste Carnoy
(1836-1898)

Fr. Dierckx Philibert Biourge A. Cappuyns
R. Mosseray (1864-1942) E. Wieldersz
1894-1934
Penicillium-Aspergillus
yeasts (kept in School of Brewery)


Paul Simonart
(1907-1997)
1934-1969
Mycothque Biourge 1949-1960 Raymond Lambert
Joseph Meyer 1960-1969 300 strains
(1924-20--) Grgoire Hennebert
1950-1960 (1929-20--)
Fungi of Congo 1956-1969
450 strains \ Fungi of Europe
\ and North America
\ / 3200 strains
\ /
. \ /
Grgoire Hennebert
1969-1994
Mycothque de l'UCL
MUCL
1972: UCL-IATP-WFCC
1983: BCCM ECCO
1985: MINE
35000 strains

113

 Hennebert integrated the Biourge's Collection with his own one into the new
"Mycothque de l'Universit Catholique the Louvain" internationally recognized by
the IAPT in 1969 ant the WFCC in 1972. MUCL, in 1983, became one of the two
first collections of the Belgian Coordinated Collections of Microorganisms BCCM
and a member of the European Culture Collection Organization ECCO. In 1985, it is
a member of the Microbial information network, and in 1992, an International
Depository Authority IDA under the Budapest Treaty.

The Collection made important contributions to fungal taxonomy (Franois

Dierckx in Penicillium; Philibert Biourge in Penicillium; Ren Vandendries in
Basidiomycetes; Raoul Mosseray in tropical Aspergillus, Paul Henrard in tropical
Xylaria, Pr. Joseph Meyer in African soil Hyphomycetes, ....Others developed
applied mycology and biotechnology: Ernest Wieldiers, the Bios production by
yeast; Alphonse Cappuyns, the citric acid production by Aspergillus niger; Pr. Paul.
Simonart: the discovery of the griseofulvin among metabolites of Penicillium
griseofulvum; Pr. P. Simonart and Pr. R. Lambert, the quality control of dairy
products and the invention of the milk aseptisation by ultracentrifugation. From its
start, the collection has been oriented both to fungal taxonomy and to applied
mycology.

_____

MUCL, a Public Service Collection

C. DECOCK, CH. MOULLIARD, L. NELISSEN, P. MASSART, P. EVRARD, N. JAMIN, P. CHARUE AND G.L.
HENNEBERT

Mycothque de l'Universit Catholique de Louvain, Unit de Microbiologie, Facult des Sciences

Agronomiques, Place Croix du Sud 3, B-1348 Louvain-la-Neuve, Belgium

The MUCL culture collection is a partner of the Belgian Coordinated Collections

of Microorganisms (BCCM), the four complementary collections of fungi, yeast,
bacteria and plasmids in Belgium. Besides the mycological research, MUCL has
developed many services for the scientific and industrial community. The
Mycothque MUCL is indeed recognized as a public service collection. Its main
characteristics are the field collection, identification, isolation and preservation of a
constantly increasing patrimony of fungal strains (yeast and fungi), of taxonomic,
environmental, (agro)-industrial and biotechnological interest.

114

 Ascomycetes
24%

Deuteromycetes
Oomycetes
45%
1%

Basidiomycetes
Zygomycetes 28%
2%

As public collectiopn, MUCL is aimed to offer many confidential services

(Anomymous 1973, 1994) such as:
1) Identification of yeast and fungi by means of morphological, physiological,
biochemical and gene-sequencing analysis. Isolation and identification of fungi
from submitted products.
2) Material deterioration and material testing for resistance to fungicides and
bioassays of fungicides according international standards.
Survey and expertise of fungal decay and mycoflora in buildings and industries, with
recommendations of contrl.
3) Fungal analysis of agricultural and industrial food
products.
4) Screening and characterization of strains with particular
properties, and creation of strains with specific enhanced
property.
4) Distribution of strains. Biomass production;
5) Patent deposit under the Budapest Treaty. Confidential
safe deposit of industrial strains. Deposit of strains for the
MUCL public collection.
6) Training in general, taxonomic and applied mycology
with special attention to undergraduate and gradute
researchers from abroad.

The MUCL collection currently holds thirty thousands of strains of filamentous

and yeast fungi of all groups especially Zygomycetes, Ascomycetes, Hyphomycetes
and Basidiomycetes, and includes taxonomically and biotechnologically important
reference strains. Since 1992, the 2d edition of the MUCL catalogue containing 7300
strains of filamentous and yeast fungi is available on request at MUCL. A particular
attention has been put on the incorporation of reference strains for industry, for
identification and for bioassays and material testing.

Anonymous 1973. Laboratoire de Mycologie systmatique et applique. Revue des Fermentations et

Industries alimentaires 28(2): 80-81.
Anonymous 1994. La Mycothque de l'UCL a cent ans. Champignons: un sicle de passion. UCL
Quinzaine universitaire 36: 1.

P.S. In 1998, a third edition of the MUCL List of Cultures was issued with 8300 selected strains of
fungi.
_____

115

 MUCL, a Fungus Culture Collection,

a service to African fundamental and applied mycology.
GREGOIRE L. HENNEBERT

MUCL, 3, Place Croix du Sud, 1348 Louvain-la-Neuve, Belgium

It is the aim of the MUCL fungus culture collection to make its expertise in
fundamental taxonomy of fungi and in aspects of applied mycology available to
serve African countries. Such a contribution could be offered by means of
eveloped ence, exchange of materials, training and research assistance in situ in
Africa, training and research periods at MUCL. Training and research grants are
available from the Lom agreements, by the GD VIII and GD XII of the ECC, by the
National Agencies for Cooperation and Development of Switzerland and Canada,
and by international arrangements for higher education and development such as
Association des Universits Partiellement ou Entirement de Langue Franaise
AUPELF, Association des Universits Africaines, International Foundation of
Science, International Development Research Center, United Nations University,
World Bank, FAO, UNESCO and MIRCEM, and from Belgium by the General
Agency for Cooperation and Development and by the Commission for International
Cooperation of the Catholic University of Louvain.
MUCL has been collaborating with the University of Burundi, Faculty of
Agricultural Sciences, Bujumbura, in establishing a Laboratory of Microbiology
mainly oriented toward food microbiology.
During the last decade, the Laboratory of Microbiology LMUB (see photos) has
carried out, owing national and international financial support, several projects for
food sanitation and quality.

- Fungal deterioration of cereals and legumes in storage in Burundi (CEC)

- Improvement of storage conditions for cereals and legumes in Burundi (AGCD).
- Microbiological quality control of dried fish ndagala from Lake Tanganika (FED)
- Selection of an appropriate drying technology for optimal microbiological and
sanitary quality of dried fish (UB)
- Quality control of indigenous and imported food products as a current service of
the Laboratory LMUB since 1985.
MUCL is also eveloped research project in applied mycology for the Tropics,
particularly on food production with different African countries. With Brazzaville
and Matsoumba, Congo, MUCL investigated the solid fermentation of cassava by

116

Rhizopus oryzae for its protein enrichment. With Guinea, Zare and Cameroon,
diverse levels of collaboration, guidance, local experiments and PhD research,
concerned the domestication of native species of edible mushrooms, the evaluation
of local wastes as cultivation substrate and the improvement of yield and protein
content by appropriate substrate composition (Tshinyangu 1994).
The production of mycorrhizae is another field of interest for the Tropics.
Original mycorrhizogenic fungi, like Suillus cothurnatus from Perou, Pisolithus
arhizus from different continents, are cultured and used in the mycorrhization of
Pinus spp, both in laboratory experiments and in the field for reforestation.
Also MUCL provides skill and training in many aspects of fundamental and
applied mycology. In fungal taxonomy, it offers training in isolating, identifying,
describing and publishing local fungi. MUCL has contributed to the development of
an African Culture Collection of fungi and yeasts at the Laboratory of Microbiology,
University of Burundi, at Bujumbura. The Collection LMUB contains more than 800
strains. Duplicates are deposited in MUCL.
Robert et al. (1994) have set up an expert programme for the identification of
yeasts, which has been elaborated and tested on a large collection of yeasts isolated
by the first author from Burundi.
MUCL, for the I.M.A. International Committee for the Development of
Mycology in Africa, and with the financial support of the Centre Technique de
Coopration Agricole et Rurale ACP-CEE Convention de Lom, has published the
first Directory of African Mycology, a tool that should enhance mutual contacts and
collaboration between African mycologists themselves and with non-African
mycologists.

Ndenzako C. 1986. Les altrations fongiques du Sorgho en cours de stockage. Cas de la provinde de
Kirundo, Burundi. Universit du Burundi, Bujumbura, 143p.
Ngabonziza P. 1987. La croissance des moisissures de stockage et l'activit de l'eau. Le genre Aspergillus.
Universit du Burundi, Bujumbura, 59p.
Simbizi J. 1987. La contamination fongique du poisson sch au Burundi. Universit du Burundi,
Bujumbura, 68p.
Nyandwi A. 1987. Contamination fongique du Haricot avant et aprs la rcolte: recherche sur la flore du
stockage. Universit du Burundi, Bujumbura, 110p.
Baert W. 1988. Valorisation de la Bagasse de Canne sucre par la production de Pleurotes en Cte
d'Ivoire. Fac. Sc. Agronomiques, UCL, 52p.
Tshinyangu K. 1994. Production et valeur alimentaire du Pleurotes en fonction du substrat. PhD thesis
UCL, 255p.
Robert V.,De Bien J., Buyck B. and Hennebert G.L. 1994. ALLEV, a new program for computer-assisted
identification of yeasts. Taxon, 43: 433-439.
Buyck B. and Hennebert G.L. 1991. Directory of African Mycology.MUCL-IMA-CTA publ. Louvain-la-
Neuve,189p.

________

117

 THE ACTIVITIES AND MYCOLOGICAL COLLECTIONS

OF THE BELGIAN NATIONAL BOTANICAL GARDEN (BR)

ANDR FRAITURE

Belgian National Botanical Garden (BR), Brussels

The study of African Fungi

The first works of African mycology published by the Botanical Garden were
edited by . De Wildeman and T. Durand, who were both directors of the National
Botanical Garden in Brusseles (Bresadola and Saccardo, 1899; De Wildeman and
Durand, 1901; De Wildeman 1905-1907; De Wildeman 1903-1912). The study is
about collections which were carried out by various collectors (principally A.
Dewvre and H. Vanderyst) and identified by J. Bresadola, P. Hennings, P.-A.
Saccardo, and H. and P. Sydow. This materiel consists of more than 450 taxons of
which close to 400 are new to mycology.
The first mycologist from the Botanical Garden to have worked systematically
on the collections of African fungi was Maurice Beeli (1879-1957), who worked
there as a scientific collaborator for more than 30 years (Heinemann 1959).
Although never having stayed in Africa, he worked extensively to develop the study
of the mycoflora of this continent. Following on his advice, Mme Goossens-Fontana
started collecteing from 1919 numerous specimens for the Botanical Garden, while
also adding notes and watercolours (more than 900). This collection constitutes a
remarkable source of knowledge about African fungi. Between 1920 and 1940, M.
Beeli published close to 30 studies on the African fungi preserved in the Botanical
Garden, 11 of which are recognized in the "Fungi Goossensiani".
The efforts of M. Beeli to highlight these collections succeeded in the publication of
the "Iconographical Flora of Fungi from the Congo", the 17 volumes of which were
published between 1935 and 1970. This flora was enlarged by the "Illustrated Flora
of Central African Fungi", the 16th. volume of which is in the course of publication.
It presents descriptions and identifcation keys, accompaigned by line drawings and
colours prints, for differents groups of Basidiomycetes, Ascomycetes, and
Myxomycetes. Several Belgian (P. Heinemann, M. Beeli, B. Buyck, J. Rammeloo,
D. Thoen) and foreign mycologists (J. Boidin, E.J.H. Corner, R.W.G. Dennis, H.
Dissing, R. Heim, E. Horak, M. Lange, M. Le Gal, R.A. Maas Geesteranus, D.N.
Pegler, H. Romagnesi, R. Singer, R. Watling) collaborated in this work.
The presence of R. L. Steyaert (1905-1978) (Bienfait, 1979) at the Botanical
Garden also needs mentioning. He conducted the greatest part of his career in
Africa, then at the Commission for the Study of the Flora of the Belgian Congo and
Ruanda-Urundi. He was interested in African phytopathology and studied primarily
the genera Pestalotia, Monochaetia and Ganoderma. He left an abundance of
material at the Botanical Garden.

The Study of European Fungi

From the second half of the last century, collaborators with and personnel of the
Botanical Gardien became interested in the mycoflora of Belgium. . Bommer
(1832-1910) (Rousseau, 1910) and E. Rousseau ( 1845-1926) gathered a great deal
of materiel for the herbarium and published several floristic catalogues. Paul Nypels

118

(1865-1909) was interested in species of harmful plants. mile De Wildeman (1866-

1947) (Robyns, 1948) carried out the cryptogamic part of the "Prodrome of the flora
from Belgium" (De Wildeman 1898, 1899).
During the first half of this century, Beeli published several works and numerous
notes concerning the mycoflora of Belgium. In the 1960's, Steyaert published
several articles on the Ganoderma of the European flora.
The series "Icones Mycologicae" debuted in 1982. It presents high quality
illustrations and detailed descriptions of various species of fungi. In this same year,
130 species, mostly European, were described in this way.
The distribution of various species of fungi was studied as well. The
"Distributiones fungorum Belgii et Luxemburgi", the second volume which should
appear in 1994, presents annotated distribution maps. Today, a cartography
programme developed by A. Empain lets one produce distribution charts by
computer.
Finally, the Laboratory conducts public service activities as well. This service
consists mainly of identifying fungi and providing expertise about fungi which grow
in homes. The Laboratory is also consulted by the Centre of Anti-Poisons when
there are possible cases of mushroom poisoning.

The Fungus Herbarium

The Mycology Herbarium of the National Botanical Garden Botanique National
consists of about 126,000 specimens. It is divided into three parts, according to the
geographical source of the collection:
- the European Herbarium gathers together specimens collected in Europe and in the
countries of the mediterranean bassin; it contains some 88,000 specimens;
- the African Herbarium is constituted of specimens collected in Afrique, south of
the Sahara; and accounts for some 25,000 specimens;
- the General Herbarium unites the exsiccata coming from the other regions of the
world, and contains about 13,000 specimens.
Among these specimens figure close to 2,500 types (1,100 for the African
Herbarium, 900 for the European Herbarium and 500 for the General Herbarium).
The Herbarium consists of type species created notably by the following mycologist:
M. Beeli, J. Boidin, E. Bommer and E. Rousseau, H.F. Bonorden, G. Bresadola, B.
Buyck, E.J.H. Corner, G. De Notaris, R.W.G. Dennis, H. Dissing, L. Fuckel, C.G.
Hansford, R. Heim, P. Heinemann, P. Hennings, E. Horak, K. Kalchbrenner, P.
Karsten, J. Komarov, M. Lange, M. Le Gal, M.A. Libert, R.A. Maas Geesteranus,
m. Marchal, J. Moureau, V. Mouton, G. Passerini, C.H. Peck, D.N. Pegler, F.
Petrak, J. Rammeloo, H. Romagnesi, H. Rehm, H. Riess, C. Roumegure, L.
Ryvarden, P.A. Saccardo, R. Singer, R.L. Steyaert, P. Sydow, C. Torrend, F. Von
Thmen, R. Watling, G. Winter.
As well, the mycological herbarium contains several collections of watercolours
representing fungi. These watercolours were produced by several artist-mycologists;
the most important collections are those from Marthe Goosens-Fontana (African
mycoflora) and Omer Van de Kerckhove (European mycoflora), but also those from
M. Beeli, J. Bruylants, P. Heinemann, L. Imler, E. Klopfenstein, G. Malenon.
Also, the Laboratory possesses a collection of 6.000 slides, in addition to
numerous dossiers containing notes, descriptions, and optic- and electron-
microscope photographs, etc...

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 The Mycological Library

The Library of the Belgian National Botanical Garden contains close to 2000
books on mycology, among those which happened to be originals from Ch. D.
Badham, J.B. Barla, M.J. Berkeley, A.N. Berlese, . Boudier, J. Bresadola, J.B.F.
Bulliard, C. Clusius, M.C. Cooke, A.C.J. Corda, L. Forquignon, E.M. Fries, L.
Fuckel, C.C. Gillet, R. Hesse, H. Hoffman, D.G.F. Hoffmann, K. Kalchbrenner,
P.A. Karsten, J. Kickx, P. Kummer, E. Lambotte, C. Linnaeus, N. Patouillard, M.
Paulet, C.H. Persoon, L. Qulet, P.A. Saccardo, J.C. Schaeffer, J. Schroeter, L.
Secretan, C. Spegazzini, L.-R. & C. Tulasne, G. Van Sterbeeck, D. Viviani, G.D.
Westendorp, O. Wunsche. The library also contains a collection of 100 periodicals
that are strictly mycological, al though not all sets are complete, as well as numerous
other public periodicals that sporadically contain articles on mycology. Finally,
there is also a collection off-prints.

References
Bienfait A. 1979. Ren Lopold Steyaert (1905-1978). Bulletin of theNational Botanical Garden of
Belgium 49(1/2): 3-9.
Bresadola J. and Saccardo P.-A. 1899. Fungi congoenses. Bulletin of the Royal Botanical Society of
Belgium 38(2):152-168 + 5 pl. [Collection of Alfr. Dewvre, 87 taxons, 17 which are new; De
Wildeman and Durand 1901 made numerous references to this publication calling it, "Durand and De
Wildeman, Materials for the Flora from the Congo 5: 33-48 (1899)"].
De Wildeman . 1898. Prodrome of Belgian Flora. Pt.I, vol. 1-3, and Pt.II, vol. 4. Castaigne, Brussels.
543 + 160 pp.
De Wildman, . 1899. Prodrome of Belgian Flora. Pt.II, vol. 5 and 6. Castaigne, Brussels. pp. 161-480.
De Wildeman . and Durand T.. 1901. Reliquiae Dewevreanae. Annals of the Museum of the Congo.
Botanique, Series 3, 2: 269-287. [It consists of a reprise, assorted commentaries, and identifications
published by Bresadola and Saccardo (1899) about the collections of Alfr. Dewvre]
De Wildeman . 1905-1907. Mission mile Laurent 1903-1904. 3d ed., 2 vols. Brussels. CCXXV, 617 p.
+ 1 map and CLXXXV prints.
De Wildeman . 1903-1912. Studies of systematic and botanical geography on the flora of the Low- and
Middle-Congo. Annales of the Museum of the Congo, Botanique, Series 5, volumes 1-3.
Heinemann P. 1959 Maurice Beeli (1879-1957). Bulletin of the National Botanical Garden in Brussels
39(1):1-6 + 1 portrait.
Robyns W. 1948. mile De Wildeman (1866-1947). Bulletin of the National Botanical Garden in
Brussels 19: 1-35 + 1 portrait.
Rousseau E. 1910. Madame J.E. Bommer, ne Elisa Destre. Bulletin of the Royal Botanical Society of
Belgium 47(2):256-261 + 1 portrait.
______

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 CLASSICAL TAXONOMY OF THE FUNGI

____________

ANAMORPH-TELEOMORPH CONNECTIONS IN THE XYLARIACEAE

M.A. WHALLEY, G.P. SHARPLES AND A.J.S. WHALLEY

School of Biomolecular Sciences, Liverpool John Moores

University, Byrom Street, Liverpool L3 3AF, UK.

The Xylariaceae is a large family belonging to the ascomycotina and

encompassing perhaps as many as 40 genera (Eriksson and Hawksworth 1993). The
family is worldwide in distribution but exhibits its greatest diversity in the tropics
and subtropics (Rogers 1993, Whalley 1993). The majority of species occur on
angiospermous wood but there is also good representation on leaves, fruits, animal
dung, and even on abandoned insect nests (Whalley 1985). To date most species
investigated produce both teleomorphic and anamorphic states.
Traditionally, but also for convenience and sometimes through necessity, the
teleomorph has assumed the central role in taxonomic decision making and has been
the source of data for most identification keys. There are, however, situations where
a teleomorphic form is absent and under these conditions the anamorph inherits the
leading role for identification purposes.
The ability to relate anamorphs with known teleomorphs is dependent on the
taxon involved and the ecological situation in which it occurs. Thus in many of the
Xylariaceae anamorphs occur on the immature teleomorph and recognition of the
connection is readily made. In other taxa they can occur separated both in time or
space and connecting the two states now becomes more difficult and uncertain. A
further challenge is provided by the apparent widespread occurrence of these fungi
as endophytes of a wide range of plants where in most cases identification of the
fungus is limited to features of the anamorphic state (Petrini and Petrini 1985). To
what extent confident assignment to a known xylariaceous taxon can be based on the
anamorph alone is open to discussion although several authors have reported species
distinctive characteristics which enable identification in the absence of a teleomorph
(e.g. Greenhalgh and Chesters 1968, Jong and Rogers 1972, Petrini and Muller
1985, Petrini 1992).
In the case of these endophytic isolates many undoubtedly have xylariaceous
affinities but Rogers (1993) urged caution in making assignations without a clear
connection to a teleomorph stating "in my laboratory anamorph and teleomorph
connections are considered as definite only after ascospore cultures have been
established or after cultures from natural substrata have been equated with known
culture types that originated from ascospores". To what extent molecular methods
can be used to make anamorph-teleomorph connections in the Xylariaceae in the
absence of a teleomorph is unknown. Recent examination of ribosomal DNA
restriction patterns in single conidial cultures of X. magnoliae J.D. Rogers derived
from stromata from single host fruits, from different host fruits and from different
host tree localities indicate considerable variability in ribosomal DNA phenotypes
within localities and from single fruits (Gowan and Vilgalys 1991). Therefore the
amount of molecular data required before unequivocal identification of a particular
xylariaceous taxon can be achieved under a range of different situations is an
unknown quantity but is likely to be more extensive than originally expected.

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 122

Figs 1-8. Xylocladium anamorph of Camillea leprieurii (Mont.) Mont. and C. tinctor (Berk.) Laessoe,
Rogers & Whalley. Scale bar = 5 um. Figs. 1-3. C. leprieurii (applanate form). Figs. 1-2. Ampullae,
conidiogenous cells and conidia (arrowed). Fig. 3. Ampullae with conidiogenous cells. Figs. 4-6. C.
leprieurii (erect form). Fig. 4. Formation of secondary conidiogenous cells (arrowed). Fig. 5. Conidia.
Fig. 6. Ampullae bearing conidiogenous cells with scars from conidial production. Figs 7-8. C. tinctor.
Fig. 7. Ampullae with numerous conidia. Fig. 8. Ampullae with conidiogenous cells and conidia
(arrowed). Figs 1, 2, 7. Cryo SEM micrographs. Figs 3-6, 8. Differential interference contrast
microscopy.

Regardless of the form genera to which Xylariaceous anamorphs are assigned

conidiogenesis has been described as holoblastic coupled with a more or less
sympodial proliferation of the conidiogenous cell (eg. Greenhalgh and Chesters
1968, Jong and Rogers 1972, Petrini and Muller 1986). The conidia which are
produced singly characteristically possess a flattened base relating to their former
point of attachment to the conidiogenous cell. Corresponding succession scars on the
conidiogenous cell mark the production of single conidia. Since the taxonomic value
of the anamorphic state in the Xylariaceae was first recognised by Chesters and
Greenhalgh (1964) many xylariaceous taxa have been cultured from ascospores and
their anamorphs described and assigned to particular form genera (e.g. Martin 1967,
Jong and Rogers 1972, Petrini and Muller 1985). Initial attempts to relate anamorph
type with taxonomic groupings were apparently not considered particularly
successful at that time (Greenhalgh and Chesters 1968) but the subsequent
accumulation of additional data and re-organization of some of these taxonomic
groupings has resulted in the recognition of stable anamorph-teleomorph patterns in
many genera (Rogers 1985, 1993, Petrini and Muller 1986).
The allocation of particular anamorphs to form genera can in many cases be open
to personal interpretation but there is overall agreement that certain genera have
clear anamorph connections. Thus Camillea Fr. possesses a Xylocladium Sydow
(=Basidiobotrys Hohnel) anamorph (Figs 1-8) (Laessoe, Rogers and Whalley 1991)
whilst the dung inhabiting genera Poronia Willd. and Podosordaria Ellis. & Holw.
produce a Lindquistia Subram. & Chandrashekara state (Fig. 18) (Stiers, Rogers and
Russell 1973, Subramanianand Chandrashekara 1977, Rogers and Laessoe 1992). In
these three xylariaceous genera the assigned form genera are currently known to
exhibit an exclusive association.
However other anamorphic forms, although distinctively xylariaceous, are more
widely occurring and can be associated with a range of, all be it, in the main closely
related teleomorphic genera. Nodulisporium Preuss (Figs 9-14) is the typical
anamorphic form in Hypoxylon Bull. s. str., Daldinia Ces. & De Not., Biscogniauxia
O. Kuntze, Rhopalostroma Hawksw. and several other genera (Martin 1967,
Greenhalgh and Chesters 1968, Jong and Rogers 1972, Hawksworth and Whalley
1985). Geniculosporium Chesters & Greenhalgh (Figs 15-16) is associated with
Leprieuria Laessoe, Rogers & Whalley (Samuels and Muller 1980) Nemania S.F.
Gray, many species of Rosellinia Ces. & De Not. (Greenhalgh & Chesters 1968,
Martin 1967, Jong and Rogers 1972, Petrini and Muller 1986, Petrini 1992)
Phylacia Lev. (Rodrigues and Samuels 1989), Anthostomella Sacc. (Francis, Minter
and Caine 1980) and Euepixylon Fuisting (Whalley 1976, Laessoe and Spooner
1994).

123

 124

Figs 9-14. Nodulisporium anamorph of Biscogniauxia nummularia (Bull.:Fr.) O. Kuntze and Daldinia
eschscholtzii (Ehrenb.) Rehm.
Scale bar = 10 um. Figs 9, 10, 13. B. nummularia conidiogenous cells and conidia. Figs. 11, 12, 14. D.
eschscholtzii. Fig. 12. Young conidium developing (arrowed). Fig. 14. Succession scar on conidiogenous
cell marking the former point of attachment of a conidium (arrowed). Figs. 9-14. Cryo SEM micrographs.

Figs 15-16. Geniculosporium anamorph of Nemania serpens (Pers.:Fr.) Gray. Scale bar = 10 um. Cryo
SEM micrographs.

Geniculosporium Chesters & Greenhalgh (Figs 15-16) is associated with

Leprieuria Laessoe, Rogers & Whalley (Samuels and Muller 1980) Nemania S.F.
Gray, many species of Rosellinia Ces. & De Not. (Greenhalgh and Chesters 1968,
Martin 1967, Jong and Rogers 1972, Petrini and Muller 1986, Petrini 1992)
Phylacia Lev. (Rodrigues and Samuels 1989), Anthostomella Sacc. (Francis,
Minterand Caine 1980) and Euepixylon Fuisting (Whalley 1976, Laessoe and
Spooner 1994). If Nodulisporium can be seen as the main anamorphic type in the
family it should be noted that there are variations on the theme and various authors
have selected Periconiella Sacc., Virgariella S.J. Hughes or Rhinocladiella Nannf.
as more appropriate depending on subtlety of branching and degree of complexity of
the conidiophores. Thus Petrini and Muller (1986) elected to place anamorphic
states of Biscogniauxia in Periconiella but Gonzalez and Rogers (1992) preferred
Nodulisporium. Jong and Davies (1974) selected the similar Sporothrix Hektoen &
C.F. Perkins as the most appropriate form genus to accommodate Phaeosporis
melasperma (Nyl.) Clem. (=Areolospora terrophila S.C. Jong & E.E. Davies.

125

 An interesting and unusual variation is shown by D. occidentalis Child where the

anamorph, although essentially a Nodulisporium, produces its conidia
holoblastically where each scar apparently represents the former production site of a
single conidium. Thus the conidiogenous cells are annellated and do not exhibit the
usual sympodial proliferation typical for a Nodulisporium (Petrini and Muller,
1996). A similar observation has been made for X. longipes Nits. (Rogers 1983). To
what extent these variations are significant in relation to systematic arrangements in
the Xylariaceae is unknown but it should be noted that even the distinction between
Nodulisporium and Geniculosporium has been questioned (Cole 1971, Kendrick and
Carmichael 1973). However the clear association of Geniculosporium with
Nemania, Rosellinia and a number of other closely related genera provides powerful
reason for maintaining separate status for these two genera. The Dematophora Hart.
state associated with Rosellinia necatrix Prill. can be considered as a synematious
form of Geniculosporium and therefore still in harmony with the anamorphs
produced by most other species of Rosellinia (Teixeira de Sousa and Whalley 1991).
However a fundamental question raised by Cole (1973) which still requires
attention concerns his comment that "it is often difficult to be sure whether a
conidiophore or conidiogenous cell has proliferated sympodially when only the
mature state is examined". According to Cole " in Nodulisporium hinnuleum
(Preuss) G. Smith conidia are produced in a basipetal succession, clustering about
the apex of the conidiogenous cell, which does not itself change in length during the
process of conidium formation". This is in conflict with the usual interpretation of
Nodulisporium anamorphs in the Xylariaceae where the conidia are produced by
continuous acropetal succession with the youngest conidium produced at the apices
of a succession of growing points produced by a sympodially proliferating
conidiogenous cell (Greenhalgh and Chesters 1968).
Although the overall pattern of anamorphic type in the Xylariaceae can be
viewed as one of consistency there are some notable exceptions. As Ju, Gonzalez
and Rogers (1993) stated "one of the hallmarks of taxa of family Xylariaceae is the
type of hyphomycetous anamorph that features dry amerosporous conidia produced
holoblastically from sympodially proliferating conidiogenous cells". Their discovery
of three Xylariaceous taxa [Lopadostoma turgidum (Pers.:Fr.) Traverso;
Creosphaeria sassafras (Schwein.:Fr.) Ju, Gonzalez & J.D. Rogers and a fungus
close to Nummularia viridis Theiss.] that, "in culture, produce more or less slimy
scolecosporous conidia holoblastically from sympodially proliferating or
sympodially and percurrently proliferating conidiogenous cells" (Ju et al. 1993) is
an interesting development. It leads them to conclude "that these fungi represent a
heretofore unrecognized evolutionary line in the Xylariaceae" ... and... "the
relationships among these three taxa are presently unknown" (Ju et al. 1993).
Although these undoubtedly represent a major departure from the 'norm' for the
family there are other curiosities. Padixonia C.V. Subraman. (Fig. 20) is unique to
Xylaria furcata Fr., one of several species associated with termite nests
(Subramanian 1972) and Acanthodochium Samuels, Rogers and Nagasawa (Fig. 19)
has to date been reserved for anamorphs of Collodiscula japonica Hino & Katumoto
and Astrocystis Berk. & Broome (Samuels, Rogers and Nagasawa 1987, Ju and
Rogers 1990).

126

Figs 17-20. Conidiogenous cells and conidia of Fig.17. Hadrotrichum (after Petrini and Candoussau), Fig.
18. Lindquistia (after Subramanian and Chandrashekara), Fig. 19. Acanthodochium (after Samuels et al.),
Fig. 20. Padixonia (after Subramanian).

Other isolated anamorphic forms which deserve consideration include

Hadrotrichum Fuckel which was the chosen form genus for the anamorph of
Hypoxylon deustum (Hoffm.:Fr.) Grev. (= Kretzschmaria deusta (Hoffm.:Fr.)
P.M.D. Martin (Petrini & Muller, 1986) and the Xylocoremium flabelliforme
(Schwein.:Fr.) J.D. Rogers anamorph of Xylaria cubensis (Mont.) Fr., X. allantoidea
(Berk.) Fr. and allies (Callan and Rogers 1990). We draw attention to Hadrotrichum
pyrenaicum Petrini & Candoussau and the statement that "Hadrotrichum
pyrenaicum has probably been overlooked, or confused, for a long time with
immature forms of Hypoxylon spp., although a microscopical examination leaves no
doubt as to its identity. No connection with any known species of Hypoxylon could
be established even though the presence of Nodulisporium - like conidiophores and
conidia in pure culture indicate a possible link" (Petrini and Candoussau 1983).
Later Petrini and Muller (1986) connected H. pyrenaicum with Hypoxylon.

127

moravicum Pouzar. It is interesting to note that Hypoxylon fraxinophillum Pouzar

(=H. argillaceum Berk.) is also restricted to Fraxinus (Pouzar 1972) and it develops
conidiophores, which as illustrated by Greenhalgh & Chesters (1968), are
remarkably similar to those of H. pyrenaicum. It certainly would not be unexpected
to find similar anamorphs in these closely related taxa but the presence of a
Hadrotrichum anamorph in Hypoxylon deustum as recorded by Petrini and Muller
(1986) is much more difficult to explain in terms of teleomorph relationships. It is
generally agreed that Hypoxylon deustum has little in common with Hypoxylon s. str.
and it is usually referred to Ustulina Tul. & C. Tul. or to Kretzschmaria Fr. if the
proposal to synonomize these genera is accepted (Laessoe, 1994).
Regardless of argument over the finer details of assignment to hyphomycetous
genera the value of the anamorph in developing systematic arrangement within the
Xylariaceae must not be undervalued. As stated by Rodrigues and Samuels (1989) in
reference to Phylacia "Anamorphs can be useful in determining relationships of
fungi" and "the anamorphs that we observed are species of Geniculosporium (sensu
Petrini and Petrini 1985) and are wholly consistent with anamorphs that have
previously been described for the family Xylariaceae". In a similar vein Callan &
Rogers (1990) stated that "Xylaria Hill ex Schrank is increasingly recognized as a
diverse and complicated assemblage of taxa. It has become apparent that the biology
and systematics of Xylaria and its allies can be understood only after consideration
of the holomorphs".

Table 1. Teleomorph-anamorph connections in the Xylariaceae

TELEOMORPH ANAMORPHS
Anthostomella Sacc. Nodulisporium & Virgariella
Astrocystis Berk. & Broome Acanthodochium
Biscogniauxia Kuntze Geniculosporium, Periconiella &
Nodulisporium
Calceomyces Udagawa & Ueda Nodulisporium
Camillea Fr. Xylocladium
Collodiscula * I.Hino & Katum. Acanthodochium
Daldinia Ces. & De Not. Nodulisporium
Entonaema A.Moller Nodulisporium
Euepixylon * Fuisting Geniculosporium
Hypoxylon Bull. Nodulisporium, Virgariella
&Rhinocladiella
Induratia Samuels, E.Mull. & O.Petrini Nodulisporium
Leprieuria Laessoe, J.D.Rogers & Whalley. Geniculosporium
Nemania * S.F.Gray Geniculosporium
Obolarina * Pouzar ? Rhinocladiella
Phaeosporis Clem. Sporothrix
Phylacia Lev. Geniculosporium
Podosordaria Ellis & Holw. Lindquistia
Poronia Willd. Lindquistia
Rhopalostroma D.Hawks. Nodulisporium
Rosellinia De Not. Geniculosporium, Dematophora
& Nodulisporium
Thamnomyces Ehrenb. Nodulisporium
Theumenella Penz. & Sacc. Nodulisporium
Xylaria Hill ex Schrank Xylocoremium
* not recognised by Eriksson & Hawksworth ( 1993 ) .

The value of cultural studies in the Xylariaceae is clearly proven: at their least
the anamorphs provide a range of new characters for taxonomic purposes but their
128

outstanding contribution lies in the confirmation of connections with known

teleomorphs and the subsequent development of more natural groupings. Currently
accepted anamorph teleomorph connections in the Xylariaceae are listed in Table 1;
others will undoubtedly follow.

Acknowlegments
We wish to thank the British Mycological Society for permission to reproduce
Figures 1-8 from Mycological Research.

References
Callan B. and Rogers J.D. 1990. Teleomorph-anamorph connections and correlations in some Xylaria
species. Mycotaxon 36, 343-369.
Chesters C.G.C. and Greenhalgh G.N. 1964. Geniculosporium serpens gen. et sp. nov., the imperfect state
of Hypoxylon serpens. Transactions of the British Mycological Society 47, 393-401.
Cole G.T. 1971. The Sympodula and the Sympodioconidium. In Taxonomy of Fungi Imperfecti Kendrick
W.B. ed., University of Toronto Press, Toronto. Canada, pp. 141-159.
Eriksson O. and Hawksworth D.L. 1993. Outline of the ascomycetes - 1993. Systema Ascomycetum 12,
51-257.
Francis S.M., Minter D.W. and Caine T.S. 1980. Three newspecies of Anthostomella. Transactions of the
British Mycological Society 75, 201-206.
Gonzalez F.S.M. and Rogers J.D. 1993. Biscogniauxia and Camillea in Mexico. Mycotaxon 47, 229-258.
Gowan S.P. and Vilgalys R. 1991. Ribosomal length polymorphisms within populations of Xylaria
magnoliae (Ascomycotina). American Journal of Botany 78, 160-1607.
Greenhalgh G.N. and Chesters C.G.C. 1968. Conidiophore morphology in some British members of the
Xylariaceae. Transactions of the British Mycological Society 51, 57-82.
Hawksworth D.L. and Whalley A.J.S. 1985. A new species of Rhopalostroma with a Nodulisporium
anamorph from Thailand. Transactions of the British Mycological Society 84, 560-562.
Jong S.C. and Davies E.E. (1974). Areolospora, a new humicolous genus in the Xylariaceae. Norwegian
Journal of Botany 21, 23-30
Jong S.C. and Rogers J.D. 1972. Illustrations and descriptions of conidial states of some Hypoxylon
species. Washington Agricultural Experiment Station Technical Bulletin 71, 1-51.
Ju Y.-M. and Rogers J.D. 1990. Astrocystis reconsidered. Mycologia 82, 42-349.
Ju Y.-M., Gonzalez F.S.M. and Rogers J.D. 1993. Three xylariaceous fungi with scolecosporous conidia.
Mycotaxon 47, 219-228.
Kendrick W.B. and Carmichael J.W. 1973. Hypomycetes. In The Fungi: an Advanced Treatise., Volume
4A, A taxonomic Review with Keys: Ascomycetes and Fungi Imperfecti. Ainsworth G.C., Sparrow
F.K. and Sussman A.S.eds., Academic Press, New York, pp. 323-509.
Laessoe T. 1994. Index Ascomycetum. 1. Xylariaceae. Systema Ascomycetum (in press).
Laessoe, T., Rogers, J.D. and Whalley, A.J.S. (1989). Camillea, Jongiella and light-spored species of
Hypoxylon. Mycological Research 93, 121-155.
Laessoe T. and Spooner B.M. 1994. Rosellinia & Astrocystis (Xylariaceae): new species and generic
concepts. Kew Bulletin 49, 1-70.
Martin P. 1967. Studies in the Xylariaceae: 1. New and old concepts. Journal of South African Botany 33,
205-240.
Petrini L.E. 1992. Rosellinia species of the temperate zones. Sydowia 44, 169-281.
Petrini L.E. and Muller E. 1986. Untersuchungen uber die Gattung Hypoxylon (Ascomycetes,
Xylariaceae) und verwandte Pilze. Mycologia Helvetica 1, 501-639.
Petrini L.E. and Petrini O. 1985. Xylariaceous fungi as endophytes. Sydowia 38, 216-234.
Petrini O. and Candoussau F. 1983. Hadrotrichium pyrenaicum nov. sp., a new deuteromycete from the
Pyrenees (France). Mycotaxon 18, 91-95.
Pouzar Z. 1972. Hypoxylon fraxinophilum spec. nov. and H. moravicum spec. nov., two interesting
species found onFraxinus angustifolia. Ceska Mykologie 26, 129-17.
Rodrigues K.F. and Samuels G.J. 1989. Studies in the genusPhylacia (Xylariaceae). Memoirs of the New
York Botanical Garden 49, 290-297.
Rogers J.D. 1983. Xylaria bulbosa, Xylaria curta and Xylaria longipes in Continental United States.
Mycologia 75, 457-467.
Rogers, J.D. 1985. Anamorphs of Xylaria: Taxonomic considerations. Sydowia 38, 255-262.
Rogers J.D. 1993. Teleomorph, Anamorph, and Holomorph considerations in the Xylariaceae. In The
Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics. Reynolds

129

 D.R. and Taylor J.W. eds., CAB International: Wallingford, UK, pp. 179-182.
Rogers, J.D. and Laessoe T. 1992. Podosordaria ingii sp. nov. and its Lindquistia anamorph. Mycotaxon
44, 435-443.
Samuels G.J. and Muller E. 1980. Life history studies of Brazilian Ascomycetes 8. Thamnomyces
chordalis (anam.: Nodulisporium) and Camillea bacillum (anam.: Geniculosporium) with notes on
taxonomy of the Xylariaceae. Sydowia 33, 274-281.
Samuels G.J., Rogers J.D. and Nagasawa E. 1987. Studies in the Amphisphaeriaceae (sensu lato) 1.
Collodiscula japonica and its anamorph, Acanthodochium collodisculae. Mycotaxon 28, 453-459.
Stiers D.L., Rogers J.D. and Russell D.W. 1973. Conidial state of Poronia punctata. Canadian Journal of
Botany 51, 481-484.
Subramanian C.V. (1972). Padixonia, a new genus of hyphomycetes. Current Science 41, 282-283.
Subramanian C.V. and Chandrashekara, K.V. 1977. Lindquistia, a new hyphomycete genus. Boletin de la
Sociedad Argentina de Botanica 18, 145-151.
Teixeira de Sousa A.J. and Whalley A.J.S. 1991. Induction of mature stromata in Rosellinia necatrix and
its taxonomic implications. Sydowia 43, 281-290.
Whalley A.J.S. 1976. Notes on the conidial state of Hypoxylon udum. Transactions of the British
Mycological Society 67, 515-517.
Whalley A.J.S. 1985. The Xylariaceae: some ecological considerations. Sydowia 38, 369-382.
Whalley A.J.S. 1993. Tropical Xylariaceae: their distribution and ecological characteristics. In Aspects of
Tropical Mycology. Isaac S., Frankland J.C., Watling R. & Whalley A.J.S. eds., Cambridge
University Press: Cambridge, UK, pp. 103-119.

________

130

ANAMORPHS AND THE CLASSIFICATION OF XYLARIACEOUS FUNGI

KATLEEN VAN DER GUCHT

Laboratorium Plantkunde, Vakgroep Morfologie, Systematiek & Ecologie,

Universiteit Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgi.

Introduction
As Rogers (1985) pointed out Xylaria Hill ex Schrank is a complex and difficult
genus. Several mycologists (Fries 1851, Miller 1942, Dennis 1961, 1970, 1974, Joly
1968, Martin 1970, Bertault 1984) have tried to organize the genus into well-defined
groups, based upon stromatal characteristics. Though these efforts have some merit,
most of the proposed schemes are difficult to use consistently and the groupings
tend to be artificial due to variability and often ambiguous nature of some of the
major characters employed.

Classifiction
Rogers (1985) was the first to propose a subdivision of the genus Xylaria based
upon anamorphic characteristics. The division is primarily based on two aspects of
Xylaria anamorphs: the place of the anamorph in the life cycle, and the morphology
of the conidiogenous structure. He recognizes 8 groups divided upon 4 sections (see
table 1).

Table 1. The subdivision of Xylaria into four sections based on anamorph characteristics (Rogers 1985).

sections characteristic features

Section 1 Conidia produced from palisade of conidiogenous cells over entire young
telemorphic stromata or at least not limited to specialized appendages.
Conidiogenesis holoblastic. Conidia produced in more or less sympodial
sequence, seceding individually and passively.
This section contains 5 groups: Xylaria polymorpha group, X. hypoxylon group, X.
multiplex group, X. pyramidata group and the X. pedunculata group.
Section 2 Conidia produced on special and localized peg- or hair-shaped appendages on
young teleomorphic stromata. Conidiogenesis apparently holoblastic. Conidia
apparently seceding individually and passively.
This section is represented by the X. comosa group.
Section 3 Conidia produced on special anamorphic stromata or conidiomata which usually
are produced earlier in the year than teleomorphic stromata. Teleomorphic
stromata never bear conidia. Conidiogenesis holoblastic. Conidia produced in
more or less sympodial sequence, seceding individually and passively.
This section is represented by the X. cubensis group.
Section 4 Conidia produced on young teleomorphic stromata. Conidiogenesis apparently
holoblastic. Conidia produced in tandem, seceding forcibly.
This section is represented by the X. furcata group.

The division proposed by Rogers (1985) has special merit, since the recognized
sections may represent more natural assemblages of Xylaria species. However, as
the author pointed out many more anamorphic data must be obtained and correlated
with teleomorphic data to fill in gaps and thereby ameliorate and/or alter the
proposed system.
The subdivision proposed here, which became apparent through my study of
anamorphic and teleomorphic features of Papua New Guinean material, is inspired

131

on the work of Rogers (1985), but differs in the grouping of some of the species.
These differences can largely be explained by the fact that I have been able to study
the anamorphic state (including the anamorph as found in nature) of a number of
species for which there were no previous data.
Based on the Papua New Guinean material 5 groups could be recognized.

1. Xylaria anisopleura group

Members of this group are X. anisopleura (Mont.) Fr., X. schweinitzii Berk. &
M.A. Curtis, and, judging from the teleomorphic features, X. scruposa (Fr.) Fr.
- Teleomorphic features: This group is well characterised by the (usually) large
teleomorphic stromata with a roughened stromatal surface due to wrinkles, warts
and tomentum, with greyish to brownish scales when young, becoming blackish to
black at maturity; ostioles umbilicate, appearing as small hemispherical black discs;
apical apparatus rectangular, constricted sub-apically; ascospores fairly large, over
15 m long, usually with a short, straight to curving germ slit, obliquely oriented.
- Anamorph in nature: The anamorph as found in nature is formed on stromata
growing side by side with mature teleomorphic stromata. These stromata are, except
for being smaller, similar to the teleomorphic stromata (e.g. clavate to spathulate for
X. anisopleura (Mont.) Fr., and cylindric-fusoid for X. schweinitzii Berk. & M.A.
Curtis).
Most probably, the anamorph in this group develops on young immature
teleomorphic stromata, iIn contrast to the taxa of the X. cubensis group (see further)
where the anamorph develops on stromata distinct from the teleomorphic stromata.
- Cultural characteristics: The colony colours are white, grey and black, and the
mycelium is characterised by the presence of coiled hyphae. The conidia are
produced upon cylindrical stromata and are ellipsoid. In most instances, the cultures
remained sterile, or the material did not germinate at all.
This group represents part of the X. polymorpha group as recognized by Rogers
(1985) (see further).

2. Xylaria feejeensis group

Members of this group are X. feejeensis (Berk.) Fr. and X. luteostromata Lloyd.
- Teleomorphic features: Characteristic for this group are the slender stromata,
usually less than 5 mm diam., with a surface finely and reticulately cracked into
small, angular closely spaced scales so as to outline the individual perithecia;
ostioles finely papillate in the centre of a dome-shaped to hemispherical black disc;
apical apparatus more or less quadrate; ascospores rather small (up to 11 m long)
with a straight germ slit running almost full spore length.
- Anamorph in nature: unknown.
- Cultural characteristics: Colonies appressed, white to grey (usually with a reddish
to orange shine). The mycelium is characterised by the presence of highly coiled
hyphae. Conidia are produced upon cylindrical stromata or on small rudimentary
stromata. Conidia subglobose, obovoid to cylindrical.
Rogers (1985) placed X. feejeensis (Berk.) Fr. together with X. anisopleura
(Mont.) Fr. and X. schweinitzii Berk. & M.A. Curtis in the X. polymorpha group.
Though cultural characteristics indicate that the X. feejeensis group and the X.
anisopleura group may be closely related, it is my opinion that based on the above
mentioned differences in teleomorphic and anamorphic features warrent their
classification in two distinct groups.

132

3. Xylaria apiculata group

Members of this group are X apiculata Cooke, X. palmicola G. Winter and X.
mellisii (Berk.) Cooke.
- Teleomorphic features: Stromata with cylindric-conical fertile parts, on narrow
hirsute stipes, and attenuated acute sterile apices; surface with peeling or flaking
greyish to brownish outer layer, the remnants persisting as conspicuous plates or
shreds in some species or not noticeably persisting in other species; ostioles
umbilicate, usually obscure, or slightly raised; ascospores over 10 m long with a
straight or slightly spiralling germ slit, from less than to almost full spore length.
- Anamorph in nature: formed on filiform apiculi or branches, white to grey, of
immature teleomorphic stromata. Conidia obovoid, small, 3.5-5.5x1-2.5 m.
- Cultural characteristics (after Rogers and Samuels 1986, Rogers et al. 1988).
Colonies velvety, white, becoming grey to black (with a greenish shine). Slender
cylindrical stromata are usually formed, less than 2 mm diam., covered with dark
hyphae and with an acute white tip. Conidia rarely forming in culture, arising from
loosely dispersed, aerial hyphae along the white upper part of the stroma.
Rogers (1985) recognized a X. multiplex group with the following members: X.
apiculata Cooke, X. arbuscula Sacc. (= X. mellisii (Berk.) Cooke), X. multiplex
(Kunze) Fr. and X. schreuderiana van der Bijl. I agree on the grouping of X.
apiculata Cooke and X. arbuscula Sacc. but according to my observations, X.
multiplex (Kunze) Fr. belongs to a group clearly distinct from the here proposed X.
apiculata group (see further).

4. Xylaria grammica group

Members of this group are X. grammica (Mont.) Fr., X. papulis Lloyd, X.
multiplex (Kunze) Fr. and X. cf. pallida Berk. & Cooke.
- Teleomorphic features: Immature stromata brightly coloured, white or yellow. The
brightly coloured surface layer is worn off in mature stromata. Surface of mature
stromata smooth, with remnants of the bright-coloured surface layer, or
characterized by an outer layer which splits longitudinally around the ostioles,
exposing the underlying black surface. Ostioles punctiform and located in the black
stripes, or finely papillate, often surrounded by an annular disc; ascospores
inaequilaterally ellipsoid with a straight germ slit running almost full spore length.
- Anamorph in nature: formed on wart-like structures developed upon immature
bright coloured teleomorphic stromata.
- Cultural characteristics: Colonies at first brightly coloured (e.g. light yellow), later
darkening from centre outwards, finally becoming black with a greenish shine.
Mycelium characterized by the presence of thick walled, dark brown hyphae, with
short protuberances, breaking down into segments. Cylindrical stromata covered
with villose black hyphae are formed, but they usually remain sterile, or the
conidiogenous structures are produced on wart-like structures upon the cylindrical
stromata. Anamorph usually formed on small tufts or subglobose stromata in the
centre of the colony. Conidia elongated ellipsoid.
This is a conspicuous group due to the bright colouration of the colonies as well
as of the immature stromata, the production of conidia on wart-like structures, and
the shape of the conidia. This group was not previously recognized.

133

5. Xylaria cubensis group

Members of this group are X. cubensis (Mont.) Fr., X. allantoidea (Berk.) Fr. and
X. poitei (Lv.) Fr. This group corresponds to the X. cubensis group as described by
Rogers (1985).
- Teleomorphic features: Stromata brown, bronze or copper-coloured, becoming
blackened with age; surface smooth and plane; ostioles papillate; ascospores
inaequilaterally ellipsoid, germ slit inconspicuous or conspicuous, straight, running
almost full spore length.
- Anamorph in nature: produced on special anamorphic stromata which develop
earlier in the year than the teleomorphic stromata.
- Cultural features: Colonies orange-white. Anamorph formed on cylindrical to
flabelliform stromata. Conidiogenous cells long cylindrical. Conidia obovoid.

Conclusion
It was not possible to place all the Xylaria taxa, found in Papua New Guinea, in
one of the above circumscribed groups, especially due to the lack of observations on
the anamorphic state.
It is obvious that much more study needs to be done before one can come to a
more natural subdivision within the genus Xylaria. In my opinion, however, the
subdivision as proposed by Rogers (1985) together with the scheme presented
above, though both far for complete and in need of further corroboration, provide an
indication that the combination of anamorphic and teleomorphic features can lead to
a less artificial grouping, and may thus provide insight into the evolution of the large
and complex genus Xylaria.
It is also admitted that a subdivision of a complex genus like Xylaria should
ideally be carried out in the frame-work of a worldwide revision rather than being
based on a study of the funga 3 of one region. However, I have judged it worthwhile
to propose the scheme obtained, such that it can be verified and complemented
through studies on taxa from other regions.

References
Barr M.E. 1983. The ascomycete connection. Mycologia 75: 1-13.
Bertault R. 1984. Xylaires d'Europe et d'Afrique du Nord. Bull. Soc. Mycol. France 100: 139-175.
Dennis R.W.G. 1961. Xylarioideae and Thamnomycetoideae of Congo. Bull. Jard. Bot. tat 31: 109-154.
Dennis R.W.G. 1970. Fungus flora of Venezuela and adjacent countries. Kew Bull., Addit. Ser. 3: 1-531.
Dennis R.W.G. 1974. Xylariaceae from Papua and New Guinea. Bull. Mens Soc. Linn. Lyon, num. spc.
43: 127-138.
Fries N. 1851. Novae symbolae mycologicae. Nova Acta Regiae Soc. Sci. Upsal. ser. 3 1: 17-136.
Joly P. 1968. Elments de la flore mycologique du Viet-Nam, Troisime contribution: A propos de
quelques Xylarias. Rev. Mycol. Paris 33: 155-207.
Martin P. 1970. Studies in the Xylariaceae: VIII. Xylaria and its allies. J. S. African Bot. 36: 73-138.
Miller J.H. 1942. South African Xylariaceae. Bothalia 4: 251-272.
Rogers, J.D. 1985. Anamorphs of Xylaria: Taxonomic considerations. Sydowia 38: 255-262.
Rogers J.D., Callan, B.E., Rossman, A.Y.and Samuels, G.J. 1988. Xylaria (Sphaeriales, Xylariaceae)
from Cerro de le Neblina, Venezuela. Mycotaxon 31: 103-153.
Rogers, J.D. and Samuels, G.J. 1986. Ascomycetes of New Zealand. 8. Xylaria. New Zealand J. Bot. 24:
615-650.

134

 First occurrence in nature of the teleomorph of Cochliobolus

R. RAEMAEKERS* AND J. COOSEMANS**

*Belgian Administration for Development Cooperation (BADC),

**Faculty of Agricultural and Applied Biological Sciences, K.U. Leuven

Introduction
The genus Cochliobolus was erected by Drechsler (1934) to include those fungi
with teleomorphs producing asci with helically coiled filiform ascospores.
Teleomorphs of Cochliobolus are rare in nature (Sivanesan 1987). The teleomorph
Cochliobolus sativus (Ito & Kurib.) Drechsler ex Dastur (anamorph Bipolaris
sorokiniana (Sacc.) Shoem., syn. Helminthosporium sativum Pammel, King &
Bakke) syn. Drechslera sorokiniana (Sacc.) Subram. & Jain, has not been recorded
in the field (Sivanesan 1987, personal comm. 1990, Tinline and Dickson 1958).
Sivanesan (1987) reports that several Cochliobolus spp. were produced since
1927 by pairing opposite mating strains of single spore isolates at temperatures
above 20 C on agar media prepared with plant material. Cochliobolus sativus was
produced in the laboratory and described by Ito and Kuribayashi, and by Tinline
(1951) and Tinlin and Dickson (1958).
This paper describes the occurrence in nature of C. sativus in Zambia following
the introduction of rainfed wheat, on which annually epidemics of Bipolaris
sorokiniana diseases occur (Raemaekers 1991, Raemaekers et al. 1991). The
naturally occurring teleomorph is compared with the teleomorph produced by Ito &
Kuribayashi and by Tinline (1951) in the laboratory.

Materials and methods

Rainfed wheat or summer wheat research was re-introduced by the Department
of Agriculture (Zambia) in the late 1970s. The research described was carried out at
Mount Makulu Research Station (MMRS) near Lusaka, where wheat plots were
seeded early January and harvested in May. At this and at other locations in the
country, B. sorokiniana diseases (spot blotch, head blight, stem break and black
point) were destructive (Raemaekers 1991).
The weather conditions during the period from harvest to seeding of the new
crop (autumn to summer) when the teleomorph develops are summarised (Table 1).
To examine the above-ground crop residues from autumn till summer, wheat
culms with and without ears were collected after plot-combining and taken to an
area (garden) protected from bushfires. There, the straw was spread on a net of
chickenwire (1 x 8m) suspended 20cm above soil level in an area without shade. It
was exposed to normal weather conditions, similar to those occurring in the adjacent
fields. This procedure started in 1985 but due to circumstances it had to be repeated
annually till 1989-90. Straw from the Mbala area, 1,000km north of Lusaka, was
collected after harvest and exposed to the weather in the same way. Partially
decomposed straw, used as mulch under coffee trees in the same area, was also
collected.
Selected parts were examined at regular intervals by stereoscope and fruiting
bodies were carefully removed, often from in between a dense mat of conidia and
conidiophores. They were cleaned following a method described by Tinline (1951).
A cleaned fruiting body would then be mounted in a drop of sterile distilled water
and squashed for microscopic examination.
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Table 1. Weather conditions from autumn to summer at MMRS1

month mean mean mean % rainfall

temp. max temp. min temp. R.H. Sunshine hrs/day mm
C C C
May 17.3 24.5 11.3 57 9.1 5
June 16.1 23.3 9.8 55 8.5 0
July 16.0 23.2 9.2 51 9.0 0
August 18.3 25.7 11.9 44 9.1 0
September 22.0 29.4 16.4 37 9.5 1
October 24.8 31.8 18.4 40 8.8 15
November 22.7 29.0 17.9 59 6.6 97
December 21.1 26.3 17.2 74 5.7 222
January 21.0 26.3 17.4 75 4.9 166
1MMRS: Mount Makulu Central Agricultural Research Station, Chilanga, Zambia. (Meteorological

Department, Lusaka, 1975).

Asci and single spores were placed on PDA with bactericide. Single spores were
separated under the stereoscope into a small drop of sterile distilled water and
transferred to the agar. Petri dishes were sealed and kept at room temperature in the
laboratory where they were exposed to daylight.
Plants of the susceptible wheat variety 'Loerie' were grown in pots in the
greenhouse for pathogenicity tests. At heading, the bottom leaves were removed and
the two top leaves were kept for inoculation. In the laboratory, these plants were
atomised with distilled water. A drop of sterile distilled water was placed a few cm
away from the leaf base either on the flag leaf or the penultimate leaf. The following
treatments were compared with a control: single B. sorokiniana conidium from
wheat straw, single conidium from monoascosporic isolate, single ascospore, groups
of ascospores, ascus. Spores were collected with fine pointed tweezers under the
stereoscope and transferred to the drop of water on the leaf. The plants were covered
with plastic bags after being atomised with distilled water. The pots were placed on
a bench in a tray with water. The bags were removed 24 hours later. The maximum
and minimum temperatures in the laboratory were 29 and 22C respectively during
the period 15 to 30 March 1990.

Results
Ascocarps developed in abundance towards the end of the year on the straw; this
occurred annually during the years of observations. Most ascocarps were observed
on dark brown discoloured straw and ears, the discolouration being the result of
severe infection during crop life. Conidia and conidiophores were also present in
abundance on this dry material. The ascigerous stage was present predominantly on
the upper part of the plant including glumes, rachis, peduncle, top internode and
nodes, but not on the lower internodes. It was not observed on the infected grains in
the glumes. Most of the leaves were disintegrated by the time the ascocarps started
to develop; fruiting bodies did develop in and underneath leaf sheaths.
Mature fruiting bodies started to develop in December. By March-April, most
ostioles were open and such ascocarps were found to be empty upon examination. A
few immature (nonseptate) spores were observed by mid-December. By late
December the first septate ascospores were noticed. The first observation of such a
spore was on 24.12.85. By mid-January, pseudothecia with immature and mature
asci were present. One hundred and four fruiting bodies were examined mid-
136

January 1986. Only 15% of those contained asci (with mature and immature
ascospores). In February and March 1990, only one third of all asci examined in
many fruiting bodies contained ascospores. The other asci were either empty or
filled to various degrees with granular protoplasma. Pseudothecia always contained
a mixture of asci with and without ascospores. Ascocarps developed on the Mbala
straw, but less frequent than on the more infected MMRS straw. A few ascocarps
with mature ascospores were found on the partially decomposed straw (mulch).
Globose superficial pseudothecia were smooth and black, occasionally with
setae. Most of them developed amidst concentrations of conidia and conidiophores
from the infection during crop growth. Often they were held to the straw by strips of
disintegrating epidermis through which they had grown. They were usually scattered
on the wheat tissue. Occasionally they developed closely together in groups of 4 to
6. Most ascocarps had a short subcylindrical ostiolar beak. Several pseudothecia
were observed without beak; one fruiting body had two beaks. Upon contact with
water on a glass slide, or after being crushed by a needle if not opening
spontaneously, the mature fruiting bodies released their content of asci and
paraphyses together. Asci in all stages of development appeared.
The number of asci per ascocarp varied between 1 and 39 with a mean of 16.
One ascocarp with 50 asci was observed. The number of asci with spores varied
between 1 and 9 per ascocarp. The number of ascospores per ascus varied from 1 to
8, with a mean of 5. Similar to descriptions by Drechsler (1934) and Tinline (1951),
the shape of the asci was subcylindrical to clavate, slightly curved and short-
stipitate.
When the ascus was filled with spores, they were always coiled in a tight helix,
the typical characteristic of the genus Cochliobolus. Coiled spores discharged
simultaneously through the ascus apex which ruptured circumscissally. The
ascospores uncoiled gradually in water. Asci contained any number of spores from
one to eight. When an ascus contained one ascospore only, this one was bent or
positioned irregularly within the ascus. Nonseptate spores were hyaline; mature
spores somewhat olivaceous. They were long, filiform and with many septa.
Constrictions at the septa were visible, especially at or prior to germination. Mature
ascospores broke up easily into segments which germinated individually.
Single septated ascospores quickly germinated on PDA and after 2 to 3 hours the
developing germtube was visible. Nonseptate ascospores did not germinate. Spores
not liberated from within the ascus germinated in situ through the ascus wall on
PDA.

Table 2. Comparison of measurements of C. sativus naturally occurring versus laboratory produced

teleomorph (units in m)

C. sativus in nature C. sativus in laboratory1

mean Tinline 1951 Ito
ascocarp width 240 - 660 (440)2 310 - 460 370 - 530
ascocarp height 180 - 576 (383) 270 - 420 340 - 470
beak width 50 - 144 (85) 90 - 140 80 - 110
beak length 40 - 90 (69) 66 - 190 90 - 150
ascus length 43 - 259 (162) 160 - 255 110 - 220
ascus width 10 - 39 (27) 27 - 42 32 - 45
ascospore length 113 - 420 (220) 182 - 343 160 - 360
ascospore width 7 - 15 (9) 5 - 10 6 - 9
number of septa 3 - 11 (7) 6 - 14 6 - 13

137

 Table 2 compares the measurements of ascocarps, asci and ascospores of C.

sativus occurring in nature with those recorded by Ito and Kurib (Tinline 1951) of
artificially produced teleomorphs made by mating compatible fertile strains under
controlled laboratory conditions.
In general, the observations show the species to have a wider range of
measurements than previously recorded. Some ascocarps found in nature were
smaller while others were larger than those produced in the laboratory. Longer beaks
were recorded on the laboratory produced teleomorphs. The ascus measurements
include both mature and immature asci. Immature asci varied in length between 50
and 250 m and in width between 10 and 38 m.The length of mature asci
fluctuated between 43 and 246 m. In the range of 50 to 110 m only immature asci
were observed. Most ascus lengths fell within the range covered by Tinline's and
Ito's, but the asci were somewhat narrower than theirs. Most measurements of
ascospore length and width were within the range observed by both authors. Some
were wider and shorter; longer ascospores were observed as well. Nonseptate
ascospores measured 134-276 m by 5-10 m. The maxima of 14 and 13 septa as
observed by Tinline and Ito & Kuribayashi respectively were not observed.
Monoascosporic cultures yielded Bipolaris sorokiniana with typical brown
conidia and dark mycelium on PDA, identical to cultures from single conidia
directly transferred from straw to the agar medium.
In the pathogenicity tests, all inoculations produced tiny brown specks within 48
hours. Within 4 days such a speck developed into a light brown elliptical lesion with
a dark centre and a yellow halo. Six days after inoculations, small parts of the
lesions were plated on PDA and B. sorokiniana was recovered.

Discussion
This is the first report of abundant formation in nature and annual re-occurrence
in spring of the teleomorph of Cochliobolus sativus. A reference sample was
deposited in 1990 at the International Mycological Institute under IMI-number
34112 and the fungus was confirmed as C. sativus (Sivanesan, personal
communication). Moist chamber tests and plating of infected plant parts on agar
media never yielded the teleomorph during the period 1974-90.
Tinline (1951) considered the slight differences between his measurements and
those by Ito and Kurib. to be due to variant strains of the fungus, and environmental
and cultural differences. The differences observed here between the laboratory
produced and naturally occurring C. sativus may be attributable to different
conditions during development in 'controlled environment' in Canada and Japan and
'normal weather conditions in Zambia.
The occurrence of asci with fewer than 8 ascospores, also observed by others
(Drechsler 1934, Nelson 1964, Tinline 1951), is attributed to the chromosomal
behaviour in the developing ascus (Hrushovetz 1956). This may also explain why
many asci were either empty or partially filled with granular protoplasma, at a time
when most asci were expected to contain mature ascospores.
The presence of the perfect stage in central and northern Zambia at locations
1,000km apart and the severity of B. sorokiniana infections in rainfed wheat
experiments in central Zimbabwe (Raemaekers 1987) are an indication that the
teleomorph could develop abundantly in a large region of southern Africa, if a
susceptible crop such as summer wheat is introduced.
Tinline and Dickson (1958) reported that the formation and maturation of C.

138

sativus was greatly affected by temperature, requiring more precise environmental

conditions than the vegetative stage. Sexually the fungus developed only on natural
media between 16 and 24C with the optimum at 20C, whereas asexually it grew
on natural and synthetic media between 4 and 36C with optimum growth at 28C.
Shoemaker (1955) investigated the temperature range and observed that the
formation of male and female reproductive organs in C. sativus took place at 24 and
20C respectively. Beyond these temperatures no fertilisation took place and only
protothecia developed. Similar temperatures as those at which the teleomorph was
produced in the laboratory occur in Zambia during spring and early summer.
C. sativus and other Cochliobolus species are heterothallic (Nelson 1957, Tinline
1951). Opposite mating strains are randomly distributed in nature, and may even
occur on the same lesion (Nelson 1957). The dense concentration of Bipolaris
sorokiniana on wheat straw in Zambia therefore is thought to contain both mating
strains.
What the implications are of finding the complete life cycle of Cochliobolus
sativus in nature in Zambia is not yet clear. It was demonstrated that it can serve as
primary inoculum along with conidia from debris. Its role will be better understood
when rainfed wheat production expands and wheat-wheat rotations are introduced.
Production of more virulent C. sativus strains is not excluded.

References
Drechsler C. 1934. Phytopathological and taxonomic aspects of Ophiobolus, Pyrenophora,
Helminthosporium, and a new genus, Cochliobolus. Phytopathology 24: 953-983.
Hrushovetz S.B. 1956. Cytological studies of ascus development in Cochliobolus sativus. Can. J. Bot. 34:
641-651.
Meteorological Department. 1975. Climatological summaries for Zambia. Periods ending December
1970. Meteorological Department Lusaka. Government Printer, Lusaka, Zambia. 59 pp.
Nelson R.R. 1957. Heterothallism in Helminthosporium maydis. Phytopathology 47: 191-192.
Nelson R.R. 1964. The perfect stage of Helminthosporium cynodontis. Mycologia 56: 64-69.
Raemaekers R. 1987. Annual report 1986. Rainfed and irrigated wheat pathology-breeding. Belgian
Development Cooperation. Mount Makulu Research Station, Zambia. 138 pp.
Raemaekers R. 1991. Contribution to the epidemiology of Bipolaris sorokiniana diseases and the
development of rainfed wheat, a new crop in Zambia. Ph.D. thesis nr. 205. Dissertationes de
Agricultura. Faculteit der Landbouwwetenschappen, Katholieke Universiteit Leuven. 136 pp.
Raemaekers R.H., Nawa I.N., Chipili J. and Sakala A. 1991. Revised checklist of plant diseases in
Zambia. Agricultural Editions nr.18, General Administration for Development Cooperation, Brussels.
119 pp.
Shoemaker R.A. 1955. Biology, Cytology, and Taxonomy of Cochliobolus sativus. Can. J. Bot. 33: 562-
576.
Sivanesan A. 1987. Graminicolous species of Bipolaris, Curvularia, Drechslera, Exserohilum and their
teleomorphs. C.A.B. International Mycological Institute (CMI). 261 pp.
Tinline R.D. 1951. Studies on the perfect stage of Helminthosporium sativum. Can. J. Bot. 29: 467-478.
Tinline R.D. and Dickson J.G. (1958). Cochliobolus sativus. I. Perithecial development and the in-
heritance of spore color and mating type. Mycologia 50(5): 697-706.

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139

 PYXIDIOPHORA, A POSSIBLE TELEOMORPH OF PLEUROCATERA

ACICULARIS

GNTER R.W. ARNOLD

Friedrich-Schiller Universitt Jena, Pilzkulturensammlung, Freiherr-Vom-Stein-Allee 2, D-9925,

Germany

Abstract. In old cultures of the recently re-discovered hyphomycete Pleurocatena acicularis mature
perithecia were found which doubtless are the teleomorph of this fungus and belongs to the genus
Pyxidiophora. Until now a connection between Pyxidiophora and Pleurocatena was unknown.

Introduction
Establishing connections between anamorphs and teleomorphs, or to find a
teleomorph of an existing anamorph is one of the tasks even of the modern
mycology. There are various methods and techniques to gain positive results, as was
pointed out by Kendrick (1979). The factor of a lucky chance should not be
forgotten. It was indeed one lucky chance which led us to a surprising possible
connection. This "possible" should be underlined, because the result until now was
impossible to repeat. The fact is that in our laboratory the teleomorph of
Pleurocatena acicularis was discovered.

Material and Methods

A pure culture of the hyphomycete Pleurocatena acicularis was obtained from
conidia from the natural host and subcultured.
The fungus was isolated from a minute colony on Hymenochaete tabacina,
covered partially with Hypocreopsis lichenoides, collected by Bjrn Brandt on Salix
aurita, Germany, Mecklenburg-Vorpommern, between Bad Slze and Triebsees,
15.03.1992, and identified by G.R.W. Arnold (A 92/39.2 = MW i 3147). The
fungus, surely, is mycophilic.
The growth in liquid Czapek medium is almost zero. Subcultures were obtained
on malt-agar (MA), oat-meal-agar (OMA) and synthetic nutrient agar (SNA) after
Nirenberg, incubated in the incubator-room at 21 1C and a relative humidity of
76%. The growth of the fungus was checked at great intervals (4-6 weeks).

Results
P. acicularis proved to be a highly difficult fungus. The mycelial growth was, in
general on all three employed media, slow and scanty; the best growth was observed
on MA, the poorest on SNA. A thin, whitish superficial mycelium was developed.
Conidiation, characteristic for P. acicularis (Fig. 1), was satisfaying on MA, not so
good on OMA, and rare on SNA. But after a period of four months small darkish
points were detected with naked eye, which under the dissecting microscope were
identified as ascomata! A contamination of Pleurocatena with an ascomycete was to
be excluded. Therefore, the observed ascomycete had to be connected with
Pleurocatena acicularis. The perithecia were scattered over the elder parts of the
colony which at this time covered almost completely the surface of the agar slant in a
10cm Petri dish; they were superficial, comparatively simple, slender and elongated
piriforme, almost translucent, with a fimbriate neck (Fig. 2). Asci were not seen. The
ascospores were very peculiar and characteristic: long guttuliforme, with a broader

140

apex and an attenuated basal part, thinwalled, hyaline, two-to multicelled. In some
cases a dark eye-spot could be observed in the upper part of the ascospore (Fig. 3).

Fig.1. Pleurocatena acicularis: conidiophores and conidia

Until now, the existence of a teleomorph-connection of Pleurocatena acicularis

was unknown. On the basis of tho described characters, it is proposed to place this
ascomycetous fungus in the genus Pyxidiophora, tentatively. It certainly differs from
the described species of Pyxidiophora which have anamorphs belonging to the
genera Chalara and Thaxteriola (Lunquist 1980, Blackwell et al.1986).

Conclusions
The formation of a teleomorph of the recently rediscovered and cultivated
hyphomycete Pleurocatena acicularis (Arnold 1995) was an unexpected surprise.
Attempts have to be made to improve the conditions for growth of the fungus, to
repeat the production of perithecia based on mono-conidial and mono-ascosporic
cultures, to study the physiology of nutrition, biochemistry, a possible antibiotic
activity of the fungus, and its ontogeny and cytology.

141

Fig.2. Pyxidiophora teleomorph of Pleurocatena acicularis: three stages of perithecia

Fig.3. Pyxidiophora teleomorph of Pleurocatena acicularis: ascospores of different kinds,

and"Hullhyphen" of the earlier stages of perithecial development

References
Arnold G.R.W. 1995. Pleurocatena acicularis, ein seltener, wiederentdeckter Hyphomyzet. Feddes
Repertorium.
Blackwell M. Perry T.J., Bridges J.R. and Moser J.C. 1986. A new species of Pyxidiophora and its
Thaxteriola anamorph. Mycologia 78: 605-612,.
Kendrick W.B, Samuels G.J., Webster J. and Luttresll E.S. 1979. Techniques for establishment
connections between Anamorph and Teleomorph. In The Whole Fungus, Kendrick, B. ed., National
Museum of Natural Sciences, National Museums of Canada, Ottawa, and The Kananaskis Foundation
2: 635-651.
Lundquist N. 1980. On the genus Pyxidiophora sensu lato (Pyrenomycetes). Botaniska Notiser 133: 121-
144.

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142

 ILLUSTRATIONS OF THE CULTURAL FEATURES OF GYRODONTIUM BOVEANUM

(MONTAGNE) MAAS GEESTERANUS
CONY DECOCK* AND ANXIOUS J. MASUKA**

* Mycothque de l'Universit catholique de Louvain. Louvain-la-Neuve, Belgium

** Forest Research Center,P.O. Box HG 595,Highlands, Harare, Zimbabwe

Abstract. Main cultural features of Gyrodontium boveanum (Montagne) Maas

Geesteranus are illustrated on the basis of two cultures which recently became
available to the authors. One was obtained from a specimen of Gyrodontium
boveanum (Montagne) Maas Geesteranus collected by the authors in the small wet
forest surrounding the Victoria Falls (Zimbabwe), inside an empty dead trunk. The
second was kindly provided by the IFO, Osaka, Japan, under the name Gyrodontium
versicolor (Berkeley & Broome) Maas Geesteranus.

In culture on Malt Agar 2% or on Cherry decoction agar at 25C, both strains

produce arthroconidia. Those arthroconidia rapidly become thick walled, turn
yellow brown and round off, soon disjoining and forming a brown powdery layer at
the colony surface. Fresh cultures obtained from teeth produced fruitbodies in Petri
dishes but, after several subculturing, new fruitbody formation were no more
observed. Thin strands are also formed.

143

 Several authors have reported the occurrence of yellow brown thick walled
spores in the flesh of Gyrodontium basidiocarp, sometimes forming a dense brown
powdery layer (Reid 1963, Maas Geesteranus 1964). Although they interpreted such
spores either as chlamydospores or as basidiospores, owing to their close
resemblance to true basidiospores, both authors were doubtful about their origin.
Study of pure culture allowed to determinate the nature of these spores.

References
Reid D.A. 1963. New or interesting records of Australasian Basidiomycetes: V, Kew Bull. 17: 267-308.
Maas Geesteranus R.A. 1964. Notes on Hydnums II, Persoonia 3: 155-192.
________

THE LACTARIUS GYMNOCARPUS COMPLEX IN TROPICAL AFRICA

A. VERBEKEN

University of Ghent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

Abstract. The Lactarius gymnocarpus group has been studied as part of a

revision of the genus Lactarius in tropical Africa.

Fig. Collecting sites of Lactarius gymnocarpus () and L. longisporus () and other Lactarius species
().

Detailed study of morphological and anatomical characters combined with

ecological data of 99 collections of so called or close to Lactarius gymnocarpus
Heim maily from African miombo woodland lead us to recognize 6 new species to
Lactarius gymnocarpus. They are L. pseudogymnocarpus, L. gymnosporus, L.
gymnocarpoides, L. luteopus, L. medusae and L. flammans. The diagnostic
differences are illustrated by drawings, photographs and distributions maps.
_______

144

 SCORIAS SPONGIOSA (SCHW.)FRIES FROM KENYA

R. MIBEY

University of Nairobi, Department of Botany, P.O. Box 30197 Nairobi, Kenya

Abstract. This paper describes the "sooty mould" fungus, Scorias spongiosa
(Schw.) Fries from Kenya usually found associated with scale insects of the genus
Coccus. The fungus is found mainly during dry weather conditions.

______

AN INTERESTING SPECIES OF PUCCINIA ON GENTIANA

EVANGELIA KAPSANAKI-GOTSI AND I. VOVOU

University of Athens, Department of Biology, Section of Ecology and Systematics, Panepistimiopolis, 157
84 Athens, Greece

Abstract. An interesting specimen of Puccinia living on Gentiana cruciata L.

has been collected near Vikos gorge, at Ipiros, Greece. A detailed study of its
morphological characteristics, revealed some remarkable differences from the
known species of Puccinia on Gentiana. It seems to be closely related to Puccinia
gentianae (F. Strauss) Rohl., which is widespread in the northern hemisphere, but
unknown from Greece. A comparative study of our specimen with authentic material
of P. gentianae is necessary, for the determination of its exact taxonomic position.
It is the first time that the genus Gentiana is reported from Greece, as a host of a
rust fungus.
________

CULTURAL CHARACTERISTICS AND THEIR TAXONOMIC

POTENTIAL IN LENTINELLUS SPECIES

ZAPI GONOU-ZAGOU AND MARIA PANTIDOU

University of Athens, Department of Biology, Section of Ecology and Systematics, Panepistimiopolis, 157
84 Athens, Greece

Abstract. Mycelial cultures of three species of the genus Lentinellus Karsten

have been studied. The cultural, macroscopical and microscopical, as well as
cytological characters of the species and their ability to produce polyphenol oxidases
are presented. The aim of this study is not only to give the culture features of the
three species but also to get additional viewpoints for a better understanding of the

145

taxonomic relationships within the genus Lentinellus. Thus there is a taxonomic

approach in which the study of cultures is intended to contribute to the evaluation of
fungal relationships.
_______

RESPONSES TO THE THERMAL STRESS IN THE INTERSTERILITY

GROUPS OF GANODERMA AUSTRALE IN TAIWAN
ZUEI-CHING CHEN

Department of Botany, National Taiwan University, Taipei, Taiwan 10764, R.O.C.

Abstract. The diversity of responses to thermal stress of two "intersterility

groups" of Ganoderma australe (Fr.) Pat. in Taiwan was studied and its impact on
the migration of tropical fungi to the temperate region in Asia was discussed. The
intersterility Group 1 (isolate TAI-01) is a tropical strain distributed in the lowland
area of Taiwan and has a higher optimum growth temperature range i.e., 28-32C
than that of Group 2. The tropical strain responded to 40C heat shock treatment by
synthesizing seven heat shock proteins with mol. wt. of 13.5 -98 kDa. The
intersterility Group 2 (isolate TAI-05) is distributed in the high land area of 1.650 m
altitude of Taiwan and has an optimum growth temperature range of 24-28C but
showed no response to heat shock. Instead, it responded to cold shock by
synthesizing a cold shock protein of 41 kDa. Evidently the Group 2 of G. australe in
Taiwan has been reproductively isolated from Group 1 and has adapted to the
habitat of the temperate climate and is a temperate strain. The migration of tropical
fungi into the temperate zone requires a speciation process and an induction of cold-
shock proteins. The subtropical island Taiwan can provide an excellent bridge for
the floristic migration between the Torrid and the Temperate species of fungi in
Asia.
______

146

 MOLECULAR TAXONOMY
___________

18S RRNA AND THE FUNGAL TREE: EVOLUTIONARY

RELATIONSHIPS AMONG ASCOMYCETES, BASIDIOMYCETES,
ZYGOMYCETES, AND CHYTRIDIOMYCETES.

Yves VAN DE PEER1, Grgoire L. HENNEBERT2, Rupert DE WACHTER1

1Departement Biochemie, Universiteit Antwerpen (UIA), Universiteitsplein 1, B-2610 Antwerpen,.

2Mycothque de l'Universit Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium

Introduction
A valuable and reliable classification of organisms should ideally be based on
their natural evolutionary relationships. Although morphology is an inevitable tool
for the description of a species, drawing conclusions about its genealogy from
morphological or biochemical characteristics is far more difficult. This is especially
true for prokaryotes, but also for eukaryotes with simple morphology, like yeasts.
But even for organisms with a more pronounced morphology, there can be
misinterpretation of the data due to convergent evolution and disagreement about
phenotypic characteristics being ancestral or derived.
However, the evolutionary history of an organism is recorded in its genes and the
development of sequence analysis techniques has allowed reconstruct biological
evolution by comparing the structure of genes or gene-products in different species.
One of the molecules exceedingly suited for this purpose is the small ribosomal
subunit RNA (further abbreviated as SSU rRNA) or 18S rRNA (in eukaryotes). SSU
rRNA, present in all living organisms, as well as in organelles like plastids and
mitochondria, has a conserved structure and an average chain length of about 1800
nucleotides in eukaryotes. As a consequence of its popularity in the construction of
evolutionary trees, over 3000 complete (or nearly complete) sequences have been
determined. They are recorded in our SSU rRNA database, which is made available
to the scientific community (Neefs et al. 1993). About 700 of these sequences are
from eukaryotes and about 165 of these are from "true" fungi, covering a wide range
of different genera and families.

Results
Figure 1 shows an evolutionary tree based on all SSU rRNA sequences known to
date of ascomycetes, basidiomycetes, zygomycetes, and chytridiomycetes. Only one
rRNA sequence is included if several are determined for the same species, usually
by different authors. Dissimilarity, being the fraction of substitutions between two
sequences, was converted into evolutionary distance by using the equation of Jukes
and Cantor (1969), which corrects for multiple mutations per site. The tree topology
was inferred by the neighbour-joining method of Saitou and Nei (1987). The
complete alignment was used for tree construction. Confidence values for individual
branches were determined by a bootstrap analysis in which 100 bootstrap trees were
generated from resampled data (Felsenstein 1985). Distance calculation, tree
construction and bootstrap analysis were performed with the software package
TREECON (Van de Peer and De Wachter 1993).

147

 148

Fig.1. Evolutionary tree based on 145 SSU rRNA sequences of chytridiomycetes, zygomycetes,
ascomycetes, and basidiomycetes. Zea mays and Homo sapiens were included as outgroup organisms.
The distance between two organisms or groups of organisms, measured in substitutions per nucleotide, is
obtained by summing the lengths of the connecting branches along the horizontal axis, using the scale on
top. Bootstrap percentages higher than 50% are placed alongside the node considered.

Phylogeny of Chytridiomycetes and Zygomycetes

In the tree of Fig. 1, the chytridiomycete Blastocladiella emersonii and the
zygomycete Mucor racemosus form the first lines of divergence within the fungi.
Next there is a splitting of, on the one hand, Ascomycetes and Basidiomycetes, and
on the other hand, the remaining Zygomycetes and Chytridiomycetes. Thus, based
on this result, the Chytridiomycetes as well as the Zygomycetes seem polyphyletic.
However, since both Blastocladiella and Mucor form long branches because of a
higher evolutionary rate, their divergence should be interpreted with caution. It is a
known phenomenon that long branches can be pulled to some extent towards the
root of the tree (Olsen 1988), because of an underestimation in the Jukes-Cantor
conversion of dissimilarity into evolutionary distance (Golding 1983). Furthermore,
until now, only one representative is sequenced for the Blastocladiales and for the
Mucorales and it is possible that addition of more representatives of these orders
may change and stabilize their respective position in the tree. Blastocladiella and
Mucor not considered, Zygomycetes and Chytridiomycetes are clustered together
and seem to form a monophyletic lineage, supported at a fairly high bootstrap level
(80%). In this tree, the zygomycete Endogone pisiformis is clustered with the
Chytridiomycetes, as also noticed by Bruns et al. (1992). However, its position is
unstable (only supported in 51% of the bootstrap samples) and depends on the
composition of the dataset.

Phylogeny of Ascomycetes
Basidiomycetes and Ascomycetes clearly form two separate monophyletic
groups based on SSU rRNA data (Bruns et al. 1992, Van de Peer et al. 1992,
Wilmotte et al. 1993, this study). Furthermore they are sister groups supported at a
high bootstrap level (96%). The Ascomycetes are subdivided into two major
lineages: the Euascomycetes and the Hemiascomycetes. Within the Euascomycetes,
three clearly separated groups can be distinguished, viz. Plectomycetes,
Loculoascomycetes, and Pyrenomycetes, while the Discomycetes are divided over
two different lineages. Overall, this subdivision of the Euascomycetes agrees well
with classical taxonomic views.
The Hemiascomycete cluster consists of yeasts and yeast-like fungi, most of
which were formerly classified as Deuteromycetes. Based on SSU rRNA it is clear
that some of these yeast genera like Candida and Pichia, are heterogenous and it
would be meaningful to reconsider their taxonomy. A detailed discussion of many of
the species included in both the Euascomycete and the Hemiascomycete cluster is
given in Wilmotte et al. (1993).
Within the Ascomycetes, a third cluster, although not supported by bootstrap
analysis, is formed by the genera Neolecta, Pneumocystis, Taphrina,
Schizosaccharomyces, Protomyces, and Saitoella. The exact taxonomic position of
these species is not clear and deserves further attention.

Phylogeny of Basidiomycetes
Within the Basidiomycetes, three main lineages can be discerned, two leading to

149

the Ustomycetes and one leading to the true Basidiomycetes, two taxa created by
Moore (1972) on the basis of differences in septal ultrastructure and life cycle.
According to this tree and some previously published trees based on SSU rRNA
(e.g. Wilmotte et al. 1993), the Ustomycetes seem to be paraphyletic, although this
finding is not supported at a high level by bootstrap analysis.

Acknowledgements
This work was supported by the Federal Office for Scientific, Cultural and Technical Matters of the
Belgian State, by the Programme on Interuniversity Poles of Attraction (contract 23) of the Belgian State,
and by the F.K.F.O.

References
Bruns T.D., Vilgalys R., Barns S.M., Gonzales D., Hibbett D.S., Lane D.J., Simon L., Stickel S., Szaro
T.M., Weisburg W.G. and Sogin M.L. 1992. Evolutionary relationships within the fungi: analysis of
nuclear small subunit rRNA sequences. Mol. Phyl. Evol. 1:231-241.
Felsenstein J. 1985 Confidence limits on phylogenies: an a pproach using the bootstrap. Evolution 39:
783-791.
Golding G.B. 1983. Estimates of DNA and protein sequence divergence: an examination of some
assumptions. Mol. Biol. Evol. 1: 125-142.
Jukes T.H. and Cantor C.R. 1969. Evolution of protein molecules. In: Mammalian Protein Metabolism,
Munro H.N. ed., Academic Press, New York pp 21-132.
Moore R.T. 1972. Ustomycota, a new division of higher fungi. Antonie v. Leeuwenhoek 38: 567-584.
Neefs J.-M., Van de Peer Y., De Rijk P., Chapelle S. and De Wachter R. 1993 Compilation of small
ribosomal subunit RNA structures. Nucl. Acids Res. 21: 3025-3049.
Olsen G.J. 1988. Phylogenetic analysis using ribosomal RNA. Methods in Enzymology 164: 793-812.
Saitou N. and Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic
trees. Mol. Biol. Evol. 4: 406-425.
Van de Peer Y., Hendriks L., Goris A., Neefs J.-M., Vancanneyt M., Kersters K., Berny J.-F., Hennebert
G.L. and De Wachter, R. 1992 Evolution of basidiomycetous yeasts as deduced from small ribosomal
RNA sequences. System. Appl. Microbiol. 15: 250-258.
Van de Peer Y.and De Wachter R. 1993. TREECON: a software package for the construction and
drawing of evolutionary trees. Comput. Applic. Biosci. 9: 177-182.
Wilmotte A., Van de Peer Y., Goris A., Chapelle S., De Baere R., Nelissen B., Neefs J.-M., Hennebert
G.L. and De Wachter R. 1993. Evolutionary relationships among higher fungi inferred from small
ribosomal subunit RNA sequence analysis. System. Appl. Microbiol. 16: 436-444.

______

150

 A RAPID MOLECULAR TECHNIQUE TO DISTINGUISH

CYLINDROCLADIUM SPECIES

P.W. CROUS1, A. KORF2 AND W.H. VAN ZYL2

Departments of 1Plant Pathology and 2Microbiology, University of Stellenbosch, South Africa

Introduction
In a recent monograph of Cylindrocladium Morgan, Crous and Wingfield (1994)
recognized 22 species and 2 varieties. Four of these species, namely C. clavatum
Hodges & May, C. pteridis Wolf, C. gracile (Bugn.) Boesewinkel and C.
hawksworthii Peerally are known to have 1-septate conidia and thin-walled stipe
extensions terminating in a clavate vesicle. C. hawksworthii can easily be
distinguished in having curved conidia, whereas those of C. clavatum, C. gracile and
C. pteridis are straight. The latter three species have similar temperature
requirements for growth, and are primarily distinguished on cultural characteristics
and dimensions of conidia and stipe extensions. These species represent a range in
size of conidia and stipe extensions from the smaller C. clavatum and C. gracile to
the larger C. pteridis (Crous and Wingfield 1994). Calonectria teleomorphs have
been described for C. pteridis, C. gracile and recently also for C. clavatum (Crous et
al. 1993b, El-Gholl et al. 1993). However, an examination of the type strains of
Cylindrocladium clavatum and Calonectria clavata Alfieri et al. found them to be
morphologically distinct.
Complementing alpha and beta taxonomy, several molecular techniques have
recently been employed in Cylindrocladium, ranging from total protein and isozyme
banding patterns to DNA restriction fragment length polymorphisms (RFLPs)
(Crous et al. 1993a, 1993c). These techniques proved to be time consuming, and in
the case of proteins, often influenced by host and geographical variation. It has been
shown, however, that DNA RFLPs can clearly distinguish variation between and
among species, and therefore substantially enhance attempts to allocate isolates in
Cylindrocladium (Crous et al. 1993a). In the Hypocreales, ribosomal DNA (rDNA)
restriction fragment length polymorphisms (RFLPs) have been successfully used as
a rapid technique to demonstrate species-specific differences in Fusarium Link
(Lodolo et al. 1992). The aim of the present study, therefore, was to use this
technique to establish variation among the four Cylindrocladium species discussed
above, and to determine the correct taxonomic position of the type strain of
Calonectria clavata.

Material and Methods

Morphological characterization
The following type and verified strains of Cylindrocladium and Calonectria spp.
were studied: Cylindrocladium clavatum (PPRI 3994 = CPC 328), (ATCC 22833, ex
type strain); Calonectria clavata (078-1261 = ATCC 66388 and 078-1543 = ATCC
66389, ex type strains); Cylindrocladium gracile (PC 551197, ex type strain);
Calonectria gracilis Crous, Wingfieldand Alfenas (PPRI 4176 = AR 2677, ex type
strain), Cylindrocladium hawksworthii (MUCL 30866, ex type strain);
Cylindrocladium pteridis (PPRI 4157 = UFV 43).

151

 Single-conidial isolates were cultured on 2 % malt extract agar (MEA) (Oxoid),

plated onto carnation-leaf agar (CLA), incubated at 25 C under near-ultraviolet
light, and examined after 7 d. Only material occurring on carnation leaves was
examined. Mounts were prepared in lactophenol cotton blue, and measurements
made at 1000 x magnification.

Chromosomal DNA isolation

Single-conidial isolates were grown on MEA, and plugs of seven-day-old
cultures transferred into 500 ml Erlenmeyer flasks containing 100 ml glucose-yeast
extract broth (Biolab). Cultures were incubated for 7-14 d in the dark at 25 C until
sufficient growth occurred. Mycelia were harvested by filtration (Whatman No. 1
filter paper), the mycelial mat immersed into liquid nitrogen and chromosomal DNA
isolated. Chromosomal DNA was subsequently redissolved in 200 l TE buffer (pH
8.0).

Restriction enzyme analysis and Southern hybridization

Chromosomal DNA (ca. 5 g) of each isolate was subjected to restriction
digestion with EcoRI, HindIII and XhoI for 3 h respectively, according to the
recommendation of the suppliers (Boehringer Mannheim). The DNA was separated
on horisontal 0.8 % agarose gels and transferred to Hybond-N nylon membranes
(Amersham) according to standard procedures. The Neurospora crassa rDNA was
purified from plasmid pMF2 (Russell et al. 1984) as a 6.3 kb PstI fragment and
labelled with [-32P]dATP (Amersham). The Southern hybridizations and
stringency washes were performed according to the method of Sambrook et al.
(1989).
Results
Morphological characterization
All the species examined in the present study produced stipe extensions
terminating in clavate vesicles. Based on conidium morphology on CLA, two groups
could be distinguished. Conidia of C. clavatum, C. gracile and C. pteridis were
straight, whereas those of C. hawksworthii and Calonectria clavata were curved.
Conidia of Cylindrocladium clavatum were 38-52 x 4-6 m, overlapping with the
slightly larger conidia of Cylindrocladium gracile (PC 551197), which were 40-56 x
3.5-5 m. Conidia of Calonectria gracilis were 40-65 x 4-5 m, overlapping to
some degree with the lower range of Calonectria pteridis, which were 62-121 x 5-6
m. The same trend was also observed for their ascospores, those of C. gracilis
being 1- septate, (27-)36.5(-50) x (4-)5(-6) m, and those of C. pteridis 1(-3)-
septate, (30)-51.5(-75) x (4.5)5.5(-7) m. Of the two species with curved conidia,
those of C. hawksworthii (42-76 x 4-4.5 m) were similar in length, but slightly
narrower than those of Calonectria clavata (50-80 x 5-6 m).

Restriction enzyme analysis and Southern hybridization

Using the Neurospora crassa rDNA probe five distinct EcoRI, XhoI and HindIII
restriction patterns were obtained for the different strains (Figs. 1-3). The South
African collection of Cylindrocladium clavatum (CPC 328) was similar to that of the
type culture of C. clavatum (ATCC 22833). However, the type culture of C. gracile
(PC 551197) could not be distinguished from that of C. clavatum. The two
heterothallic isolates of Calonectria clavata were similar to each other, but distinct
from that of Cylindrocladium clavatum, and all other species studied. The profile of

152

Calonectria gracile (AR 2677) was distinct from Cylindrocladium gracile (PC
551197), and closer to that of Cylindrocladium pteridis.

Discussion
Type and verified strains of four Cylindrocladium species were investigated in
this study. Three of these have been associated with Calonectria teleomorphs, and
type strains of the latter were also included. Comparisons were done on the basis of
morphology, culture characteristics and nuclear DNA polymorphisms.

Cylindrocladium hawksworthii
The species is known from two collections made in Mauritius (Peerally 1991). It
is distinguished from other species in Cylindrocladium in having primarily clavate
vesicles, and curved 1-septate conidia. The phenomenon of curved macroconidia in
Cylindrocladium is known from C. curvatum Boedijn and Reitsma (1950) with
sphaeropedunculate vesicles, and C. variabile Crous et al. (1993a), with
sphaeropedunculate to ellipsoidal or clavate vesicles and (1-)3(-4)-septate conidia.
The present study also found the two heterothallic strains of Calonectria clavata to
have prominently curved macroconidia. The latter strains could, however, be
distinguished from Cylindrocladium hawksworthii by their larger conidium
dimensions and septation, as well as distinct rDNA restriction patterns.

Cylindroladium clavatum and Cylindrocladium gracile

C. clavatum was described by Hodges and May (1972) with conidia being 37.5-
48 x 3.5-5.5 m. Bugnicourt (1939) described Cylindrocarpon gracile with conidial
dimensions as being 24-48 x 2.5-4 m. Boesewinkel (1982) transferred
Cylindrocarpon gracile to Cylindrocladium, and retained it as a separate species
because of its longer stipe extensions and narrowly clavate vesicles.
Results obtained with rDNA restriction patterns for EcoRI, XhoI and HindIII in
the present study clearly indicate, however, that the type culture of Cylindrocladium
gracile (PC 551197) is indistinguishable from that of C. clavatum (ATCC 22833).
The similarity in morphology and rDNA restriction pattern suggests therefore that
the two species are synonymous. Because gracile is the older epithet, C. gracile
Bugnicourt is to be accepted as the earlier name for this species.

Calonectria pteridis (anam. Cylindrocladium pteridis) and Calonectria gracilis

Of the 1-septate species with clavate vesicles, C. pteridis has the largest
macroconidia, and frequently also forms curved microconidia in culture. In the
present study curved microconidia have also been observed for Calonectria clavata.
Similar to C. clavata, C. pteridis has also been found to be heterothallic, with
successful matings producing the teleomorph in culture (Crous et al. 1993b).
A homothallic strain with conidial dimensions of 40-65 x 4-5 m was recently
obtained from Brazil. Because these dimensions overlapped considerably more with
that of C. gracile than C. pteridis, and C. pteridis was known to be heterothallic with
larger, 1(-3)-septate ascospores, the isolate was described as the Calonectria
teleomorph of Cylindrocladium gracile (Crous et al. 1993b). As stated above, the
present study found Cylindrocladium gracile to be distinct from Calonectria gracile,
and synonymous with Cylindrocladium clavatum.
The rDNA restriction patterns obtained with EcoRI and HindIII showed only
minor differences between Calonectria gracile and Calonectria pteridis. These two

153

isolates could, however, easily be distinguished with the restriction enzyme XhoI.
Based on the differences in the banding patterns, as well as conidium and ascospore
dimensions, it would appear that Calonectria gracilis represents a distinct species
between Cylindrocladium clavatum and Cylindrocladium pteridis. We are, however,
of the opinion that additional isolates of Calonectria pteridis will have to be studied
to suitably resolve the validity of Calonectria gracilis.

Fig.1. rDNA hybridization patterns for EcoRI-digested nDNA of strains of Cylindrocladium and
Calonectria species. Lane 1: Cylindrocladium clavatum PPRI 3994. Lane 2: Calonectria gracilis PPRI
4176. Lane 3: C. pteridis PPRI 4157. Lane 4: Cylindrocladium gracile PC 551197. Lanes 5 and 6:
Calonectria clavata ATCC 66388 and 66289. Lane 7: Cylindrocladium hawksworthii MUCL 30866.
Lane 8: C. clavatum ATCC 22833. Size markers are lambda DNA digested with EcoRI and HindIII.
Fig.2. rDNA hybridization patterns for XhoI-digested nDNA of strains of Cylindrocladium and
Calonectria species. Details as in Fig. 1.
Fig.3. rDNA hybridization patterns for HindIII-digested nDNA of strains of Cylindrocladium and
Calonectria species. Details as in Fig. 1

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Calonectria clavata
The two heterothallic strains of Calonectria clavata are morphologically distinct
from all presently described species of Cylindrocladium, C. clavatum included.
Furthermore, their rDNA restriction patterns (Figs. 1-3) show these two isolates to
be similar, but distinct from all other species investigated. These results therefore
suggest that these isolates represent a presently undescribed species of
Cylindrocladium.
Conclusion
The present study has found rDNA RFLPs to be effective in distinguishing
among morphologically similar species of Cylindrocladium. Our results further
suggest that this is also an excellent technique for validating anamorph teleomorph
relationships where the two states have been described from separate collections.
With the recently completed monograph of Cylindrocladium, results obtained using
this technique can now be integrated with alpha and beta taxonomic criteria. This
approach would help to determine the morphological and genetic range of species
occurring in morphologically similar complexes.

Acknowledgments
We thank Dr. N.E. El-Gholl (Division of Plant Industry, Florida, USA), Dr. J. Mouchacca (Laboratoire de
Cryptogamie, Paris, France), Pr A.C. Alfenas (Department of Plant Pathology, Viosa, MG, Brazil) and
the curators of MUCL and ATCC for providing cultures examined in this study. Financial support for this
study was provided in the form of a rolling grant from the Foundation for Research Development to the
first and junior author.

References
Boedijn K.B.and Reitsma J. 1950. Notes on the genus Cylindrocladium. Reinwardtia 1: 51-60
Boesewinkel H.J. 1982. Heterogeneity within Cylindrocladium and its teleomorphs. Trans. Br. mycol.
Soc. 78: 553-556
Bugnicourt F. 1939 Les Fusarium et Cylindrocarpon de l'Indochine. Encycl. Mycol. 11: 1-206
Crous P.W., Wingfield, M. J. 1994. A monograph of Cylindrocladium, including anamorphs of
Calonectria. Mycotaxon 51: 341-435
Crous P.W., Janse B.J.H., Victor D., Marais G.F.and Alfenas A.C. 1993a. Molecular characterization of
Cylindrocladium spp. with three-septate conidia and ovoid-like vesicles. System. Appl. Microbiol. 16:
266- 273.
Crous P.W., Wingfield M.J.and Alfenas A.C. 1993b. Additions to Calonectria. Mycotaxon 46: 217-234.
Crous P.W., Wingfield M.J.and Alfenas A.C. 1993c. Cylindrocladium parasiticum sp. nov., a new name
for C. crotalariae. Mycol. Res. 97: 889-896.
El-Gholl N.E., Alfieri S.A.and Barnard E.L. 1993. Description and pathogenicity of Calonectria clavata
sp. nov. Mycotaxon 48: 201-216.
Hodges C.S.and May L.C. 1972. A root disease of pine, Araucaria, and Eucalyptus in Brazil caused by a
new species of Cylindrocladium. Phytopathology 62: 898-901.
Lodolo E.J., Van Zyl W.H.and Rabie C.J. 1992. A rapid molecular technique to distinguish Fusarium
species. Mycol. Res. 97: 345-346.
Peerally A. 1991. Cylindrocladium hawksworthii sp. nov. pathogenic to water-lilies in Mauritius.
Mycotaxon 40: 367-376.
Russell P.J., Wagner S., Rodland K.D., Feinbaum R.L., Russel J.P., Bret-Harte M.S., Free S.J.and
Metzenberg R.L. 1984. Organization of the ribosomal ribonucleic acid genes in various wild-type
strains and wild-collected strains of Neurospora. Mol. Gen. Genet.196: 275-282.
Sambrook J., Fritsch E.F.and Maniatis T. 1989. Molecular cloning: a laboratory manual. 2nd ed. Cold
Spring Harbor/NY, Cold Spring Harbor Laboratory Press.

(The present study forms part of a paper submitted to System. Appl. Microbiol.)
________

155

 IDENTIFICATION OF BASIDIOMYCETOUS YEASTS USING WHOLE-

CELL PROTEIN ELECTROPHORESIS

M. VANCANNEYT1, G. L. HENNEBERT2 AND K. KERSTERS1

1
Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium;
2
Mycothque de lUniversit Catholique de Louvain, Place Croix du Sud 3, B-1348 Louvain-la-Neuve,
Belgium

Introduction
There is a continuing need for simple and reliable laboratory procedures to
differentiate yeast species. With an increasing number of species it becomes
obvious that the conventional diagnostic tests frequently provide insufficient
phenotypic characterization to permit unambiguous assignment at the species level.
In this study the use of one-dimensional sodium dodecyl sulphate-polyacrylamide
gel electrophoresis of whole-cell proteins (SDS-PAGE) as a possible method for
classification and identification of basidiomycetous yeasts is investigated.

Materiel and methods

Approximately 240 different strains representing at least 70 different species in
the basidiomycetous genera Cystofilobasidium, Cryptococcus, Filobasidium,
Filobasidiella, Kondoa, Leucosporidium, Mrakia, Rhodosporidium, Rhodotorula
and Tremella were studied. Cells were cultivated in liquid cultures and harvested in
the stable growth phase. The methods used for preparation and electrophoresis of
the SDS-protein extracts were those described previously (Vancanneyt et al.
1992b). Protein patterns were scanned and digitized. Numerical analysis was
performed using the Pearson product moment correlation coefficient and UPGMA
clustering.

Results
Except for the genus Mrakia, all species of the teleomorphic genera
Cystofilobasidium, Filobasidium, Filobasidiella, Kondoa, Leucosporidium, Mrakia
and Rhodosporidium constituted separate protein electrophoretic clusters
(Vancanneyt et al. 1992a). The species of the genus Mrakia (M. frigida, M. gelida,
M. nivalis and M. stokesii) showed highly similar protein patterns, suggesting that
these four species may be synonymous. Strains of two varieties of Filobasidiella
neoformans, F. neoformans var. neoformans and F. neoformans var. bacillispora
could not be differentiated, neither by protein electrophoresis, visually, or by
numerical analysis.
A study of 107 Rhodosporidium and Rhodotorula strains revealed after
numerical analysis of protein electrophoregrams eighteen clusters (Vancanneyt et
al. 1992b). Only the type strain was included for the following ten species:
Rhodotorula armeniaca, Rt. auriculariae, Rt. bacarum, Rt. bogoriensis, Rt.
diffluens, Rt. hordea, Rt. hylophila, Rt. ingeniosa, Rt. muscorum and Rt. philyla. All
these type strains demonstrated a qualitatively well-characterized protein profile,
which might indicate that each of these species occupies a unique and separate
taxonomic position within the genus Rhodotorula. Homogeneous and separate
protein clusters were found for strains of the species Rt. fragaria, Rt. javanica, Rt.
lactosa, Rt. pustula and Rt. sonckii. Intraspecific heterogeneity was demonstrated

156

within the following seven Rhodotorula species: Rt. acheniorum, Rt. araucariae, Rt.
aurantiaca, Rt. foliorum, Rt. glutinis, Rt. graminis and Rt. minuta. One or more
strains of the anamorphic species Rt. glutinis grouped with strains of Rs.
sphaerocarpum, Rs. toruloides, Rs. diobovatum strains, Rs. kratochvilovae and Rt.
mucilaginosa strains. For Rt. graminis, the type strain grouped with Rs. diobovatum
strains and most of the strains investigated grouped with Rs. paludigenum strains.

Table 1: DNA base composition and type of coenzyme Q within protein electrophoretic groups of
Rhodotorula and Rhodosporidium strains.

Protein Species Number of strains mol %(G+C) Coenzyme Q

group

1 Rt. glutinis 2 59.6-60.7 Q10

Rt. mucilaginosa 15
2 Rt. glutinis 1 62.0-63.0 Q10
Rs. sphaerocarpum 4
3 Rt. glutinis 1 59.9-60.0 Q9
Rt. sp. 1
Rs. toruloides 6
4 Rt javanica 2 ND Q9
5 Rt. graminis 4 62.5-63.0 Q10
Rs. paludigenum 4
6 Rt. pustula 2 ND ND
7 Rt. aurantiaca 1 61.3 Q10
Rt. graminis 1
8 Rt. glutinis 7 66.4-67.2 Q10
Rt. graminis 1
Rs. diobovatum 5
9 Rt. glutinis 2 64.2-65.0 Q10
Rs. kratochvilovae 1
10 Rt. fragariae 2 ND Q10
11 Rt. acheniorum 2 52.8 Q10
12 Rs. dacryoidum 3 ND ND
13 Rt. aurantiaca 2 58.4-59.3 Q10
14 Rt. aurantiaca 2 55.1 Q10
15 Rt. minuta 10 48.6-51.0 Q10
16 Rt. lactosa 2 56.6 Q9
17 Rt. sonckii 2 ND Q10
18 Rt. aurantiaca 2 57.6-57.9 Q9

ND: not determined

A study of 78 Cryptococcus strains, 3 Filobasidium strains and 10 Tremella

reference strains revealed after numerical comparison eighteen protein
electrophoretic clusters (Vancanneyt et al. 1994). Only the type strain was included
for the following Cryptococcus species: Cr. curiosus, Cr. dimennae, Cr. flavus, Cr.
heveanensis, Cr. magnus, Cr. marinus, Cr. skinneri and Cr. tsukubaensis. These
type strains showed a well-characterized protein fingerprint. Homogeneous and
separate protein clusters were found for strains of the species Cr. amylolentus, Cr.
curvatus, Cr. gastricus and Cr. macerans. A significant protein electrophoretic
heterogeneity was observed within the species Cr. albidus, Cr. humicola, Cr.
laurentii and Cr. luteolus. Highly similar protein patterns were observed for some
strains of Cryptococcus albidus and the investigated strains of Cr. kuetzingii, for the

157

type strain of Cr. elinovii and the studied strains of Cr. terreus and for a strain of
Cr. luteolus and two strains of Cr. laurentii. Concerning the reference strains of the
genus Tremella, T. aurantia, T. brasiliensis and T. globospora showed a unique
protein pattern. A highly similar protein fingerprint was found between one strain of
Cr. Laurentii and T. foliacea. A qualitatively and quantitatively highly similar
protein pattern was found for respectively T64. coalescens and T. mesenterica, T.
fuciformis and T. samoensis and T. encephala and T. subanomala.
The taxonomic validity of the obtained groupings was evaluated using other
chemotaxonomical criteria such as the determination of the DNA base composition
and the ubiquinone type (Table 1 and 2; Vancanneyt et al. 1992a, b, 1994). These
data confirmed the protein electrophoretic heterogeneity within several anamorphic
Rhodotorula and Cryptococcus species and indicated a possible close relationship
between strains of different species.

Table 2: DNA base composition and type of coenzyme Q within protein electrophoretic groups of
Cryptococcus, Filobasidium and Tremella strains.

Protein Species Number of strains mol %(G+C) Coenzyme Q

group

1 Cr. curvatus 6 56.7-56.9 Q10

2 Cr. amylolentus 2 ND Q9
3 Cr. laurentii 2 54.7-55.2 Q10
Cr. luteolus 1
4 Cr. laurentii 3 57.2-58.1 ND
5 Cr. elinovii 1 54.7-55.0 Q10
Cr. terreus 3
6 Cr. albidus var. aerius 1 53.7-54.2 Q10
Cr. albidus var. albidus 1
7 Cr. albidus var aerius 2 49.8-50.1 ND
8 Cr. albidus var. albidus 7 51.3-53.0 Q10
Cr. kuetzingii 4
9 Cr. albidus var. albidus 4 49.1-49.6 Q10
10 T. encephala 1 47.6-47.7 Q10
T. subanomala 1
11 T. fuciformis 1 55.9-56.1 Q10
T. samoensis 1
12 Cr. albidus var. albidus 3 52.8-53.5 Q10
13 Cr. ater 2 51.3-52.0 Q10
F. foliforme 3
14 Cr. humicolus 2 57.4 ND
15 Cr. gastricus 2 ND ND
16 Cr. macerans 4 ND ND
17 T. coalescens 1 46.4-47.2 ND
T. mesenterica 1
18 Cr. humicolus 8 61.1-62.3 ND

ND: not determined

158

 Conclusion
We conclude that SDS-PAGE of cellular proteins allows a rapid and reliable
grouping of a large number of yeast strains. The majority of the species are
delineated as separate clusters. The technique is successful in delineating
anamorph/teleomorph relations, in revealing synonymy between species and in
demonstrating taxonomic heterogeneity within several species. Moreover, the
possibility of computerized processing of the patterns suggests a method useful for
analytical characterization of yeasts. Identification of new isolates becomes an easy
routine procedure once a database of reference protein electrophoregrams is
constructed.

References:
Vancanneyt M., Coopman R., Tytgat R., Berny J.-F., Hennebert G.L. and Kersters K. 1992a. A
taxonomic study of the basidiomycetous yeast genera Rhodosporidium and Rhodotorula based on
whole-cell protein patterns, DNA base compositions and coenzyme Q types. J. Gen Appl. Microbiol.
38: 363-372.
Vancanneyt M., Van Lerberge, E., Berny J.-F., Hennebert G.L. and Kersters K. 1992b. The application of
whole-cell protein electrophoresis for the classification and identification of basidiomycetous yeast
species. Antonie van Leeuwenhoek 61: 69-78.
Vancanneyt M., Coopman R., Tytgat R., Hennebert G.L. and Kersters K. 1994. Whole-cell protein
patterns, DNA base composition and coenzyme Q types in the yeast genus Cryptococcus Ktzing
and related taxa. Syst. Appl. Microbiol. 17: 65-75.
______

159

 PHYTOPATHOGENIC FILAMENTOUS (ASHBYA, EREMOTHECIUM)

AND DIMORPHIC FUNGI (NEMATOSPORA) WITH FALCATE
ASCOSPORES AS NEW MEMBERS WITHIN THE
SACCHAROMYCETACEAE.

PRILLINGER1, H., MESSNER1, R., BREITENBACH2, M., BRIZA2, P., STAUDACHER3, E., MOLN R1, O.,
WEIGANG4, F., LOPANDIC1, K., IBL5, M., HIMMLER1, G.
1
Univ. Bodenkultur, Inst. f. Angew. Mikrobiol.; Nudorfer Lnde 11, A-1190 Wien; 2 Univ. Salzburg,
Inst. f. Genetik u. Allgemeine Biologie; Hellbrunnerstr. 34, A-5020 Salzburg; 3 Univ. Bodenkultur, Inst. f.
Chemie; Gregor Mendel Str. 33, A-1180 Wien; 4 Hewlett-Packard Ges. m. b. H.; Lieblgasse 1, A-1222
Wien; 5 Codon Genetic Systems Ges. m.b.H.; Colloredogasse 29/13 A-1180 Wien.

Introduction
Although geneticists and molecular biologists commonly separate yeasts and
filamentous fungi, Meyen, 1838 has already acknowledged in the name
Saccharomyces the fact that yeasts are fungi (van der Walt 1987). Wickerham
(1951, 1952) concluded that the classification of yeasts will be incorrect as long as
the related filamentous forms had been studied thoroughly by persons who knew
these fungi as well as yeasts.
In the present work we investigated the phylogenetic relationship between the
saprophytic and predominantly unicellular yeast genera Saccharomyces and
Kluyveromyces and the plant pathogenic, filamentous fungi Ashbya gossypii and
Eremothecium ashbyi. Dimorphic yeast, Nematospora coryli, parasitic on hazelnuts
and soybeans was included in our investigations. To characterize yeasts and
filamentous fungi from these genera on the molecular level we have chosen the cell
wall monosaccharide composition, the presence of DL-dityrosine-containing
macromolecules from ascospore walls (Briza et al. 1990), the ubiquinone spectra,
and the DNA sequence information from the small and large ribosomal subunit
genes as well as from the rapidly evolving ITS1 and ITS2 regions.

Material and Methods

Cell wall sugars/Ubiquinone-system. The qualitative and quantitative
monosaccharide pattern of purified yeast and hyphal cell walls and ubiquinone
spectra were determined as described in Prillinger et al. (1993) and Messner et al.
(1994a). Dityrosine analysis was performed according to Briza et al. (1994).
Analysis of ribosomal DNA-sequences: Sequences were obtained by direct,
automatic sequencing of PCR products (Codon Genetic Systems, Vienna; Messner
et al. 1994b).

Results and Discussion

Ascospore morphology and the presence of hyphal growth were considered to
separate the genera Ashbya, Eremothecium, and Nematospora at least on the family
level (Metschnikowiaceae: von Arx and van der Walt 1987) from other yeast genera
belonging to the Endomycetales. The ascospores of Saccharomyces species are
smooth and spherical, in Kluyveromyces they are reniform. Falcate ascospores, often
whiplike at one end and occasionally septate are known in Nematospora coryli,
Ashbya gossypii and Eremothecium ashbyi (von Arx and van der Walt 1987).
Kurtzman (1993), Kurtzman and Robnett (1994), and Prillinger et al. (1990,
1993) presented molecular evidence that the Endomycetales except the
Schizosaccharomycetaceae are homogenous. The Schizosaccharomycetaceae were
160

included in the new order Schizosaccharomycetales. According to Kurtzman &

Robnett (1994) and Prillinger et al. (1993) representatives of the Endomycetales like
Ashbya, Eremothecium, Kluyveromyces, Nematospora, and Saccharomyces
investigated in this study are primitive fungi ancestral to filamentous Asco- and
Basidiomycetes.
To clarify the phylogenetic relationship between the saprophytic unicellular
species of Kluyveromyces and Saccharomyces on the one hand and the
phytopathogenic dimorphic (Nematospora) or filamentous Ashbya or Eremothecium
species on the other hand we used a polyphasic molecular approach. In the present
paper we have investigated:
1. The qualitative and quantitative monosaccharide composition of purified yeast
and fungal cell walls after hydrolysis with trifluoroacetic acid.
2. The presence of dityrosine in the cell walls of ascospores.
3. The major component of the ubiquinone system.
4. The DNA sequence of six regions of the ribosomal RNA genes.
The cell wall carbohydrate pattern of the phytopathogenic filamentous fungi A.
gossypii and E. ashbyi comes close to the characteristic mannose-glucose pattern of
several Saccharomyces and Kluyveromyces species (Prillinger et al. 1990). Whereas
the proportion of mannose appeared to be commonly higher in the Kluyveromyces
and Saccharomyces yeast strains as well as in N. coryli, glucose dominates in the
filamentous strains of A. gossypii and E. ashbyi.
As far as ascospores could be observed on different sporulation media (Kreger-
van Rij 1984), dityrosine was detected in all species of Saccharomyces,
Kluyveromyces, and the two filamentous fungi A. gossypii and E. ashbyi. Dityrosine
was absent in sporulating cultures from Nadsonia fulvescens (Ubiquinone Q-6),
Debaryomyces hansenii (Q-9), Pichia farinosa (Q-9), and Schizosaccharomyces
pombe (Q-10).
As already shown by Yamada et al. (1977, 1987), the ubiquinone Q-6 was
present in all species of the genera Kluyveromyces and Saccharomyces as well as in
A. gossypii and E. ashbyi. Ubiquinone Q-5 was found in the type strain of
Nematospora coryli. According to Yaada et al. (1987) there is an additional strain of
N. coryli where ubiquinone Q-6 was found. Conspecificity was established for both
strains based on partial base sequences of 18S and 26S ribosomal DNA (Yamada
and Nagahama 1991).
For molecular phylogeny based on DNA sequences three different targets were
chosen. Regions of 800 to 900 bases length of the genes for the small and large
ribosomal subunit as well as both the complete internal transcribed spacers ITS1 and
ITS2 had been sequenced (Messner et al. 1994b).
Separate phylogenetical computation of these three types of ribosomal sequences
results in the clustering of the Saccharomyces species, the Kluyveromyces species,
E. ashbyi, N. coryli, and A. gossypii for all regions investigated. As illustrated by
Fig. 1 deriving from ITS1 and ITS2 data, the genera Saccharomyces and
Kluyveromyces are well defined by 82 and 98 % bootstrap confidence, respectively.
E. ashbyi, N. coryli and A. gossypii are clustered here at the species level, and the
separation of these three subclades from Candida by tree topology and a bootstrap
value of 65 % is quite good for sequences of the rapidly evolving ITS regions.

161

Fig. 1. Cladogram based on both internal transcribed spacer regions ITS1 and ITS2. An alignment of 655
bases in length was computed under maximum likelihood hypothesis. Bootstrap confidence values
derived from 600 repeats.

Our data corroborate the work of Kurtzman and Robnett (1994), who found by
ribosomal sequences a close relationship between Ashbya, Eremothecium, and
Nematospora together with Holleya tight enough to be congeneric. The genus
Metschnikowia, however, appears to be only distantly related according Kurzmann
and Robnett (1994).

Conclusions
1. Phenotypic criteria like ascospore shape and ornamentation or presence or
absence of hyphae are in most cases unreliable for definition of families in the
Endomycetales.
2. Based on a similar cell wall carbohydrate composition, the presence of dityrosine
in endospores, which are formed by free cell formation, and a high degree of
ribosomal DNA sequence similarity especially with respect to the ITS1 and ITS2
regions we redefine the family Saccharomycetaceae Winter to include unicellular
saprophytic fungi like the genera Kluyveromyces and Saccharomyces, as well as
dimorphic or filamentous parasitic fungi like species of the genera Ashbya,
Eremothecium, Holleya, and Nematospora.
3. Our data suggest that unicellular ascomycetous yeasts like Kluyveromyces and
Saccharomyces have evolved from filamentous plant pathogens with ontogenetic
saprophytic yeast stages. The lack of the respective parasitic filamentous forms can
be explained by an extinction of the specific hosts.
4. Molecular characteristics like cell wall sugars or ribosomal DNA sequence
information are reliable tools to trace ascomycetous yeasts back to a polykaryotic
coenocytic ("siphonal") ancestor (Prillinger 1987).

Acknowledgements
For kindly providing type strains we are indebted to Dr. C. P. Kurtzman (Peoria, U.S.A.), Prof. U. Stahl
and K. Scheide (T.U. Berlin). Supported by grants from the "Jubilumsfonds der sterreichischen
Nationalbank" and FWF (project: PO9255-Bio).

162

 References
Arx J.A. von, and van der Walt J.P. 1987. Ophiostomales and Endomycetales. Stud. Mycol. 30: 167-176.
Briza P., Ellinger A., Winkler G. and Breitenbach M. 1990. Characterization of a DL-dityrosine
containing macromolecule from yeast ascospore walls. J. Biol. Chem. 265: 15118-15123.
Briza P., Eckerstorfer M. and Breitenbach M. 1994. The sporulation-specific enzymes encoded by DIT1
and DIT2 genes catalyze a two-step reaction leading to a soluble LL-dityrosine-containing precursor of
the yeast spore wall. Proc. Natl. Acad. Sci. USA 91: 4524-4528.
Kreger-van Rij N.J.W. 1984. The yeasts - a taxonomic study. Elsevier Sci. Publ., Amsterdam, 1082 p..
Kurtzman C.P. 1993. Systematics of the ascomycetous yeasts assessed from ribosomal RNA sequence
divergence. Antonie van Leeuwenhoek 63: 165-174.
Kurtzman C. P. and Robnett C. J. 1994a. Orders and families of ascosporogenous yeasts and yeast-like
taxa compared from ribosomal RNA sequence similarities. In Ascomycete Systematics: Problems and
Perspectives in the Nineties. Hawksworth, D. L. ed., Plenum Press, New York, USA pp 249-260.
Messner R., Prillinger H., Altmann F., Lopandic K., Wimmer K., Molner O. and Weigang F. 1994a.
Molecular characterization and application of random amplified polymorphic DNA analysis of Mrakia
and Sterigmatomyces species.Int. J. Syst. Bacteriol. 44: 694-703.
Messmer R., Prillinger H., Ibl L. and Himmler G. 1995 Sequences of ribosomal genes and internal
transcribed spacers. GenBank acc. N UO9327. Mol. Evol. Biol. 4:406-425.
Messner R., Prillinger H., Ibl M. and Himmler G. 1995. Sequences of ribosomal genes and internal
transcribed spacers move three plant parasitic fungi, Eremothecium ashbyi, Ashbya gossypii, and
Nematospora coryli, towards Saccharomyces cerevisiae. J. Gen. Appl. Microbiol. 41: 31-42.
Meyen J. 1838. Wiegmann Arch. Naturgesch. 4: 1-186.
Prillinger H., 1987. Yeasts and anastomoses: their occurrence and implications for the phylogeny of
Eumycota. In Evolutionary biology of the fungi. Rayner A.D.M. et al. eds., Cambridge Uiv. Press pp.
355-377
Prillinger H., Drfler C., Laaser G., Eckerlein B. and Lehle L. 1990. Ein Beitrag zur Systematik und
Entwicklungsbiologie hherer Pilze: Hefe-Typen der Basidiomyceten. Teil I. Schizosaccharomycetales,
Protomyces-Typ. Z. Mykol. 56: 219-250.
PrillingerH., Oberwinkler F., Umile C., Tlachac K., Bauer R., Drfler C. and Taufratzhofer E. 1993.
Analysis of cell wall carbohydrates (neutral sugars) from asco- and basidiomycetous yeasts with and
without derivatization. J. Gen. Appl. Microbiol. 39: 1-34.
van der Walt J.P. 1987. The yeasts: a conspectus. In The expanding realm of yeast-like fungi. De Hoog
G.S., Smith M.T. and Weijman A.C.M. eds. Stud. Mycol. 30: 19-31.
Wickerham L.J. 1951. Taxonomy of yeasts. 1. Techniques of classification. 2.
A classification of the genus Hansenula. Tech. Bull. 102: 56 p., U.S. Dept. Agric. Washington D.C.
Wickerham L.J. 1952. Recent advances in the taxonomy of yeasts. Annu. Rev. Microbiol. 6: 317-332.
Yamada Y.and Nagahama T. 1991. The molecular phylogenyof the ascomycete yeast genus Holleya
Yamada based on the partial sequence of 18S and 26S ribosomal RNAs. J. Gen. Appl. Microbiol. 37:
199-206.
Yamada Y., Nojiri M., Matsuyama M. and Kondo K., 1977. Coenzyme Q system in the classification of
the ascosporogenous yeast genera Debaryomyces, Saccharomyces, Kluyveromyces and Endomycopces.
J. Gen. Appl. Microbiol. 22: 325-337.
Yamada Y., Banno I., Arx J.A. von, and van der Walt J.P. 1987. Taxonomic significance of the coenzyme
Q system in yeasts and yeast-like fungi. In The expanding realm of yeast-like fungi. De Hoog G.S.,
Smith M.T. and Weijman A.C.M. eds. Stud. Mycol. 30: 299-308.

______

163

 GENETIC ORGANIZATION OF THE YEAST YARROWIA LIPOLYTICA

Serge CASAREGOLA, Huu Vang NGUYEN, Chantal FEYNEROL, Monique DIEZ & Claude GAILLARDIN

Collection de Levures d'Intrt Biotechnologique (CLIB)

Laboratoire de Gntique Molculaire et Cellulaire, INRA-CNRS,
Centre de Biotechnologies Agro-Industrielles, Institut National Agronomique,
78450 Thiverval-Grignon, France

Introduction
Yarrowia lipolytica (otherwise known as Candida lipolytica and
Saccharomycopsis lipolytica) is a dimorphic yeast which forms both yeast-like cells
and true mycelium. It can grow on hydocarbons and has been used for the production
of single-cell protein and citric acid as well as various other various metabolites. In
addition, Y. lipolytica is able to secrete naturally a number of enzymes in large
amount. These characteristics make this yeast an organism with high biotechnogical
potential.
Although sexuality in this yeast was discovered more than 20 years ago, genetic
studies were hampered by unusual features including low mating frequencies, low
fertility of hybrids, irregular meiotic segregation and mitotic haploidization. Despite
the development of programs to generate numerous mutants and to improve mating
procedures and tetrad analysis (Gaillardin et al. 1973, Ogrydziak et al. 1978), the
construction of a genetic map did not allow the detection of any centromere-linked
genes and the number of chromosomes in this yeast.
Recent studies in our laboratory (Naumova et al. 1993) have defined, using Pulse
Field Gel Electrophoresis (PFGE), the existence of three groups of electrophoretic
karyotypes amongst 27 natural isolates. Moreover, within these groups an important
variability of the number of chromosomes (from 3 to 6 varying in size from 2 to 6
Mb) was observed as well as an important chromosomal-length polymorphism.
Hybridization studies with few cloned genes to these karyotypes revealed that these
markers were not linked to specific chromosomes when strains were compared. This
result is in agreement with the absence of centromere linkage observed with tetrad
analysis during the establishment of the genetic map (Ogrydziak et al. 1982). Taken
together, these results could account for the genetic anomalies displayed by this
species.
In a different approach based on the separation of chromosomes by PFGE and
the assignement of cloned genes to these chromosomes, we set up the construction of
a physical map of the genome of Y. lipolytica, in order to facilitate genetic studies
and to gain better insights on the unusual genetic characteristics of this yeast.
Material and Methods
Strains used in this study

E150 lab. strain, from the crossing W29 x CBS6124-2 CLIB 122
E129 lab. strain, from the crossing W29 x CBS 6124-2 CLIB 121
H222 lab. strain from H. Weber (Germany) CLIB 80
CX161-1B lab. strain from D.M. Ogrydziak (U.S.A.) CLIB 153
W29 wild type, ATCC 20460 CLIB 89
CBS 6124-1 monosporous segregant from the wild type CBS 6124 CLIB 77
CBS 6124-2 monosporous segregant from the wild type CBS 6124 CLIB 78
B204-12D lab. strain from G. Barth (Swizerland)

164

Preparation of chromosomal DNA:

This was essentially performed as described by Naumova et al. (1993).

Chromosome separation
Chromosomes were separated in 0.8% agarose gels run in 0.5xTAE buffer at
12C in a Bio-Rad CHEF-DRII. Electrophoresis was carried out at 50V for 48hrs
with a switching time of 2400s, 43V for 70hrs with a switching time of 3000s and
40V for 47.7hrs with a switching time of 3300s.

Southern blotting and DNA/DNA hybridization

DNA from agarose was depurinated, denaturated and alkali-transferred overnight
to GeneSceen membranes. The membranes were then neutralized.
DNA used as probe was directly 32P-dCTP labelled with the random priming method
(MegaPrime, Amersham) in low-melting agarose or after purification from agarose
gel with the GeneClean kit. Hybridizations were performed at 65 C in 3xSSPE with
Denhardt and dextransulfate. The final wash was in 40mM Na-phosphate/1% SDS at
65 C.

Results and Discussion

Important efforts were devoted to the improvement of Y. lipolytica chromosomes
separation by PFGE and to the transfer of DNA to membranes in Southern blotting.
For this purpose, a laboratory strain, E150, was chosen and we were able to show
that this strain possesses at least 5 chromosomes whose sizes vary from 2.6 to 4.9
Mb using the chromosomes of Schizosaccharomyces pombe as size markers. The
size of the Y. lipolytica genome is thus estimated at 18.1 Mb.
36 genes, mostly cloned in the Laboratoire de Gntique Molculaire et
Cellulaire, others kindly provided by foreign laboratories, were used as probes in
hybridization experiments to the PFGE separations. This allowed the obtention of 5
linkage groups comprising:
- 2 markers on Chrom. I
- 6 markers on Chrom. II
- 12 markers on Chrom. III
- 7 markers on Chrom. IV
- 11 markers on Chrom. V.
It must be pointed at that:
(1) 3 centromeres were used as probes defining the chromosomes I, III and IV. This
allows for the first time, a certain number of markers to be linked to centromeres.
(2) rDNA clusters were shown, unlike other yeasts, to be present on 4 chromosomes
out of 5. It is remarkable that, unlike other yeasts too, Y. lipolytica possesses at least
2 classes of rDNA units (Fournier et al. 1986). The rDNA clusters being highly
variable in size, their presence on most of the chromosomes of the strain tested could
explain the chromosome-length polymorphism observed in different Y. lipolytica
strains.
In order to validate the construction of a physical map of the genome, we tested
the distribution of the already assigned markers in the E150 strain to a number of
widely used wild type and laboratory strains.
First, electrophoretic karyotypes of 7 different isolates were obtained. This
allowed us to confirm the results obtained by Naumova et al. (1993) on the

165

important variability of the genome structure of realy related Y. lipolytica isolates.

Then, a pool of genes (Bio6, His1, Pho2, PGK) known to hybridize to the E150
chromosome III was used as probes to hybridize to the chromosome separations of
the 7 isolates. Preliminary results indicate that the linkage previously obtained with
the 4 genes in E150 was not conserved in these strains, including in the parental
strains of E150. Indeed, whereas only one signal was observed on the chromosome
III of E150, hybridization signals were scattered over the separations of the other
strains.
Surprisingly, in the same test performed with a marker of the E150 chromosome
I, the Ryl2 gene, a single signal on the smallest chromosome of each separation was
observed, indicating that this chromosome did not display important length
polymorphism or gross rearrangement from strain to strain. This is to be linked to the
fact that the chromosome I is not carrying rDNA clusters unlike the other
chromosomes.
These observations lead us to incriminate homologous recombination between
the rDNA clusters on most of the chromosomes to explain the chromosomal
rearrangements and the specific genetic features of Y. lipolytica. This hypothesis is
being tested through the study of the conservation of linkage groups at meiosis and a
better characterization of the location of the rDNA clusters in respect with the
assigned markers.

Acknowledgements
We are indebted to Dr. Ph. Fournier who initiated this work, set up the PFGE conditions and showed
constant interest and support. We would like to thank all our colleagues for kindly providing us with the
cloned genes. This work was supported by the Insititut National de la Recherche Agronomique and by an
EEC grant (BIOT-CT91-0267 DSCN).

References
Gaillardin C.M., Charoy V. and Heslot H.. 1973. A study of copulation, sporulation and meiotic
separation in Candida lipolytica. Arch. Microbiol. 92: 69
Fournier P., Gaillardin C.M., Persuy M.A., Klootwijk J. and van Heerikhuizen H. 1986. Heterogeneity in
the ribosomal family of the yeast Yarrowia lipolytica: genomic organization and separation studies.
Gene 42: 273
Naumova E., Naumova G., Fournier P., Nguyen H.V. and Gaillardin C.M. 1993. Chromosomal
polymorphism of the yeast Yarrowia lipolytica and related species: electrophoretic karyotyping and
hybridization with cloned genes. Current Genet. 23(5-6): 450-454.
Ogrydziak K.D., Bassel D., Contopoulou J.R. and Mortimer R.K. 1978. Development. of. genetic
techniques and the genetic map of the yeast. Saccharomycopsis lipolytica. Molec. Gen. Genet. 163:
229-239.
Ogrydziak K.D., Basel J. and Mortimer R.K. 1982 Development of the genetic map of the yeast
Saccharomycopsis lipolytica. Molec. Gen. Genet. 188: 179

_________

166

 TAXONOMIC REVISION OF SACCHAROMYCES SENSU STRICTO

STRAINS OF NCAIM

J. TORNAI-LEHOCZKI., D. DLAUCHY., G. PETER

National Collection of Agricultural and Industrial Microorganisms, Budapest, H-1118, Somloi ut 14-16,
Hungary

Introduction
In the last decades, classification of species in the Saccharomyces sensu stricto
group have been a hot issue (Lodder 1970, Kreger-van Rij 1984, Barnett 1992).
Using conventional identification tests for the determination of phenotypic
characters delimitation of species within the group cannot be done satisfactorily. On
the basis of nDNA/nDNA reassociation studies, recently four species have been
reestablished, namely Saccharromyces cerevisiae, S. paradoxus, S. bayanus, and S.
pastorianus (Vaughan-Martini and Martini 1987, 1989, 1993). In 1990, Rodrigues
de Sousa et al. investigated the fructose proton symport activity in species of
Saccharomyces sensu stricto group. They reported that four strains of S. pastorianus
and five strains of S. bayanus tested showed fructose proton symport activity
whereas five strains of S. cerevisiae and three strains of S. paradoxus did not. They
suggested that active fructose transport is a reliable phenotypic character that
correlates with species delimitation based on the genotype.
The aim of our study was to check the reliability of the active fructose proton
symport in differentiating 72 species of Saccharomyces sensu stricto, using yeast
strains isolated from Hungarian wine and beer.

Material and Methods

Yeast strains
The type strains of Saccharomyces sensu stricto species and some of their
synonyms and 72 wine and brewer yeast strains identified earlier with species
belonging to Saccharomyces sensu stricto were studied. Thirteen type strains were
obtained from the Industrial Yeast Collection of the Dipartimento di Biologia
Vegetale, Universita di Perugia, Italy (DBVPG) and the Culture Collection Unit,
Fermentation Section, Northern Utilization Research Branch, U.S. Department of
Agriculture, Peoria, Illinois, USA (NRRL). The other strains were obtained from the
National Collection of Agricultural and Industrial Microorganism, Budapest,
Hungary (NCAIM).
Yeast strains were characterized and identified according to methods and keys of
Kreger-van Rij (1984) and Barnett et al. (1990).

Measurement of fructose transport

Detection of active fructose transport system was performed according to the
method of Rodrigues de Sousa et al. (1990). Yeast strains were propagated in a
liquid mineral medium (van Uden 1967) with 0.5% (w/v) fructose (Merck). Proton
symport activity was tested by recording the alkalinization of an aqueous cell
suspension after addition of fructose to a final concentration of 10 mmol l-1 using a
RADELKIS OP-211/1, (Hungary) pH meter (precision 0.05 pH) with OP-0808P
pH sensitive combination glass electrode, and registered with a Potentiometric OH-
814/1 type recorder (RADELKIS, Hungary).

167

Table 1. Active fructose transport of yeast strains belonging to Saccharomyces sensu stricto group

--
Species Collection Isolation Fructose
original epithet numbers source H+ symport
--
Saccharomyces cerevisiae
Sacch. cerevisiaeT DBVPG 6173 beer -
Sacch. cerevisiae NCAIM Y.00201 wine -
Sacch. cerevisiae NCAIM Y.00204 wine -
Sacch. cerevisiae NCAIM Y.00205 wine -
Sacch. cerevisiae NCAIM Y.00206 wine -
Sacch. cerevisiae NCAIM Y.00222 wine -
Sacch. cerevisiae NCAIM Y.00223 wine -
Sacch. cerevisiae NCAIM Y.00225 wine -
Sacch. cerevisiae NCAIM Y.00227 wine -
Sacch. cerevisiae NCAIM Y.00228 wine -
Sacch. cerevisiae NCAIM Y.00235 wine -
Sacch. cerevisiae NCAIM Y.00237 wine -
Sacch. cerevisiae NCAIM Y.00238 wine -
Sacch. cerevisiae NCAIM Y.00248 wine -
Sacch. cerevisiae NCAIM Y.00249 wine -
Sacch. cerevisiae NCAIM Y.00252 wine -
Sacch. cerevisiae NCAIM Y.00254 wine -
Sacch. cerevisiae NCAIM Y.00255 wine -
Sacch. cerevisiae NCAIM Y.00256 wine -
Sacch. cerevisiae NCAIM Y.00259 wine -
Sacch. cerevisiae NCAIM Y.00261 wine -
Sacch. cerevisiae NCAIM Y.00294 wine -
Sacch. cerevisiae NCAIM Y.00295 wine -
Sacch. cerevisiae NCAIM Y.00297 wine -
Sacch. cerevisiae NCAIM Y.00299 wine -
Sacch. cerevisiae NCAIM Y.00301 wine -
Sacch. cerevisiae NCAIM Y.00303 wine -
Sacch. cerevisiae NCAIM Y.00304 wine -
Sacch. cerevisiae NCAIM Y.00305 wine -
Sacch. cerevisiae NCAIM Y.00307 wine -
Sacch. cerevisiae NCAIM Y.00309 wine -
Sacch. cerevisiae NCAIM Y.00312 wine -
Sacch. cerevisiae NCAIM Y.00368 wine -
Sacch. cerevisiae NCAIM Y.00370 wine -
Sacch. cerevisiae NCAIM Y.00378 wine -
Sacch. cerevisiae NCAIM Y.00417 wine -
Sacch. cerevisiae NCAIM Y.00418 wine -
Sacch. cerevisiae NCAIM Y.00419 wine -
Sacch. cerevisiae NCAIM Y.00420 wine -
Sacch. cerevisiae NCAIM Y.00424 wine -
Sacch. cerevisiae NCAIM Y.00425 wine -
Sacch. cerevisiae NCAIM Y.00426 wine -
Sacch. cerevisiae NCAIM Y.00427 wine -
Sacch. cerevisiae NCAIM Y.00429 wine -
Sacch. cerevisiae NCAIM Y.00431 wine -
Sacch. cerevisiae NCAIM Y.00442 wine -
Sacch. cerevisiae NCAIM Y.00445 wine -
Sacch. cerevisiae NCAIM Y.00768 wine +*
Sacch. cerevisiae NCAIM Y.00769 wine +*
Sacch. cheresiensis NCAIM Y.00817 wine -
Sacch. prostoserdovii NCAIM Y.00818 wine -
Sacch. beticus NCAIM Y.00819 wine -

168

Saccharomyces bayanus
Sacch. bayanusT DBVPG 6171 beer +
Sacch. globosusT NRRL-Y 12645 pear juice +
Sacch. heterogenicusT NRRL-Y 1354 apple juice +
Sacch. inusitatusT NRRL-Y 12648 beer +
Sacch. abuliensisT NRRL-Y 11845 Mesophylax adopersus +
Sacch. bayanus NCAIM Y.00500 wine -*
Sacch. bayanus NCAIM Y.00826 wine -*
Sacch. bayanus NCAIM Y.00827 wine -*
Sacch. bayanus NCAIM Y.00828 wine -*
Sacch. bayanus NCAIM Y.00829 wine -*
Sacch. bayanus NCAIM Y.00830 wine -*
Sacch. heterogenicus NCAIM Y.00831 wine -*

Saccharomyces pastorianus
Sacch. pastorianus NT DBVPG 6047 beer +
Sacch. carlsbergensis NCAIM Y.00820 beer +
Sacch. carlsbergensis NCAIM Y.00821 beer +
Sacch. carlsbergensis NCAIM Y.00822 beer +
Sacch. carlsbergensis NCAIM Y.00832 beer +
Sacch. carlsbergensis NCAIM Y.00833 beer +
Sacch. carlsbergensis NCAIM Y.00834 beer +
Sacch. carlsbergensis NCAIM Y.00770 beer -*
Sacch. carlsbergensis NCAIM Y.00835 beer -*
Sacch. uvarum NCAIM Y.00823 beer -*
Sacch. pastorianus NCAIM Y.00824 beer -*

Saccharomyces paradoxus
Sacch. paradoxusNT DBVPG 6411 -
Sacch. paradoxus NCAIM Y.00223 -
Sacch. paradoxus NCAIM Y.00399 -
Sacch. paradoxus NCAIM Y.00825 -
--
T = Type strain; NT = Neotype strain; *= Differing results. Strains have been examined for
nDNA/nDNA homology, see Table 2; NCAIM = National Collection of Agricultural and Industrial
Microorganisms; DBVPG = Industrial yeast collection of the Dipartimento di Biologia Vegetale,
Universit di Perugia, Italy; NRRL = ARS Culture Collection, Northern Regional Research Laboratory,
U.S. Department of Agriculture, Peoria, Illionis, USA

Table 2. Results of nDNA/nDNA homology %

--
Sacch. Sacch. Sacch.
cerevisiaeT pastorianusT bayanusT
DBVPG 6173 DBVPG 6047 DBVPG 6171
--
Sacch.cerevisiae NCAIM Y.00768 52 98 -
Sacch.cerevisiae NCAIM Y.00769 50 97 -
Sacch.carlsbergensis NCAIM Y.00770 96 50 -
Sacch.carlsbergensis NCAIM Y.00835 98 48 -
Sacch. uvarum NCAIM Y.00823 89 45 -
Sacch. pastorianus NCAIM Y.00824 94 42 -
Sacch. bayanus NCAIM Y.00826 96 - 18
Sacch. bayanus NCAIM Y.00827 94 - 19
Sacch. bayanus NCAIM Y.00828 90 - 17
Sacch. bayanus NCAIM Y.00829 88 - 22
Sacch. bayanus NCAIM Y.00830 89 - 20
Sacch. heterogenicus NCAIM Y.00831 90 - 12
--
T = Type strain; NCAIM = National Collection of Agricultural and Industrial Microorganisms
DBVPG = Industrial yeast collection, Dipartimento di Biologia Vegetale, Universita di Perugia,Italy

169

nDNA/nDNA reassociation studies

Extraction and purification of nDNA was accomplished by a combination of
procedures by Marmur (1961) and Price et al. (1978). nDNA/nDNA reassociation
was determined spectrophotometrically using a Gilford Response 2 instrument with
a thermoprogrammer, according to Kurtzman et al. (1980). nDNA/nDNA homology
% was calculated from the Cot curves (Britten and Kohne 1968) using the formula
of Seidler and Mandel (1971).

Results and Discussion

Presence or absence of active fructose transport in 72 yeast strains, which were
identified earlier as belonging to the Saccharomyces sensu stricto group and in eight
Saccharomyces type strains are summarized in Table 1. Results obtained with type
strains corresponded in all cases with those of Rodrigues de Sousa et al. (1990).
Thirteen out of 72 wine and brewer strains differed in active fructose transport
ability from that expected for the species identified earlier. These strains were
examined for deoxyribonucleic acid relatedness. Results of nDNA/nDNA
reassociation studies are summarized in Table 2.
Results of active fructose transport investigation in accordance with results of
nDNA/nDNA homology confirmed that species of Saccharomyces sensu stricto can
be divided into two groups: active fructose transport is present in S. bayanus and S.
pastorianus and active fructose transport is absent in S. cerevisiae and S. paradoxus
in agreement with finding of Rodrigues de Sousa et al. (1990). Vaughan-Martini and
Martini (1993) by testing 12 strains of the four species also confirmed that they can
be discriminated on the basis of this property. Molecular taxonomy has reformed
yeast classification and has become of prime importance in the differentiation of
species (Kurtzman 1987). While nDNA/nDNA reassociation method is an excellent
research tool, it is too expensive and sophisticated for routine yeast identification.
Active fructose transport can be determined easily and this phenotypic character
correlates with the molecular classification of species in the group of
Saccharomyces sensu stricto. Hence it is a useful method for the identification of
yeast strains belonging to Saccharomyces sensu stricto. Investigations are in
progress to find appropriate and reliable conventional physiological tests as
suggested by Vaughan-Martini and Martini (1993) to differentiate between pairs of
Saccharomyces species differentiated on the basis of the presence or absence of
active fructose transport.

References
Barnett J.A. 1992. The taxonomy of the yeast Saccharomyces Meyen ex Rees: a short review for non-
taxonomists. Yeast 8: 1-23.
Barnett J.A., Payne R.W. and Yarrow D. 1990. Yeasts: characteristics and identification. 2nd ed.,
Cambridge Univ. Press, Cambridge, pp. 594-602.
Britten R.J. and Kohne D.E. 1968. Repeated sequences in DNA. Science 161: 529-540.
Kreger-van Rij N.J.W. ed. 1984.The Yeasts. A Taxonomic Study. 3rd ed. Elsevier Science Publishers,
Amsterdam, pp.379-395.
Kurtzman C.P., Smiley M.J. and Johnson C.J. 1980. Emendation of the genus Issatchenkia Kudriavzev
and comparison of species by deoxyribonucleic acid reassociation, mating reaction and ascospore
ultrastructure. International Journal of Systematic Bacteriology 30: 503-513.
Kurtzman C.P. 1989. Molecular taxonomy. In The Yeasts. Rose A. H. and Harrison J. S. eds., 2nd ed.,
Academic Press, London, pp. 63-89.
Lodder J. ed. 1970. The Yeasts: A Taxonomic Study. North-Holland Publ. Co., Amsterdam, pp. 555-718.
Marmur J. 1961. A procedure for the isolation of DNA from microorganisms. Journal of Molecular
Biology 3: 208-218.

170

Price C.W., Fuson G.B. and Phaff H.J. 1978. Genome comparison in yeast systematics: delimitation of
species within the genera Schwanniomyces, Saccharomyces, Debaromyces, and Pichia.
Microbiological Reviews 42: 161-193.
Rodrigues de Sousa H., Spencer-Martins I. and van Uden N. 1990. Active fructose transport in
Saccharomyces sensu stricto. Taxonomic implications. Acta Varia 5:127-134.
Seidler R. J. and Mandel M. 1971. Quantitative aspects of deoxyribonucleic acid renaturation: Base
composition, state of chromosome replication, and polynucleotide homologies. Journal of
Bacteriology 106: 608-614.
van Uden N. 1967. Transport-limited fermentation and growth of Saccharomyces cerevisiae and its
competitive inhibition. Archives of Microbiology 58: 155-168.
Vaughan Martini A. and Martini A. 1989. Saccharomyces paradoxus comb. nov., a newly separated
species of the Saccharomyces sensu stricto complex based upon nDNA/nDNA homologies.
Systematic and Applied Microbiology 12: 119-122.
Vaughan Martini A. and Martini A. 1987. Three newly delimited species of Saccharomyces sensu stricto.
Antonie van Leeuwenhoek 53: 77-84.
Vaughan Martini A. and Martini A. 1993. A taxonomic key for the genus Saccharomyces. Systematic and
Applied Microbiology 16: 113-119.

________

171

 PHYLOGENIC RELATIONSHIPS WITHIN THE SACCHAROMYCES

CEREVISIAE COMPLEX SPECIES.

DILNORA E. GOULIAMOVA1 GRGOIRE L. HENNEBERT2

1Institute of Microbiology, Acad. G. Bonchev str. 26, 1113 Sofia, Bulgaria

2Mycothque de l'Universit Catholique de Louvain, Place Croix du Sud 3, B-1348 Louvain-la-Neuve,
Belgium

Introduction
Saccharomyces cerevisiae, as conceived by Kreger-van Rij (1984), is a large
species covering 75 differently named taxa (S. cerevisiae and 75 synonyms). That is
the taxon she called Saccharomyces cerevisiae sensu lato.
Vaughan-Martini and Martini (1987) demonstrated by DNA/DNA reassosiation
the weak relatedness between the type or neotype strains of three of the
synonymyzed taxa (S. pastorianus, S. bayanus, S. paradoxus) and the neotype strain
of S. cerevisiae, while relatedness between two of the synonimized species
themselves is also weak, as shown here.

S. cerevisiae 52% S. paradoxus

\ /
52% 20% 24%
/ \
S. pastorianus 72% S. bayanus

That means that Saccharomyces cerevisiae as a large species cannot stand. It is a

group of related but distinct species: three species are shown distinct from
Saccharomyces cerevisiae sensu stricto.
To be convincing those results needed confirmation. Kurtzman and Robnett
(1991) proved the non-similarity between the three synonymized species and S.
cerevisiae, using partial sequencing (300 bases) in two regions of 28S rRNA and o,e
region of 18S rRNA. But, they used to charactere S. pastorianus the type strain of S.
carbergensis and therefore were characterizing the later species instead.

Material and methods

The purpose of the present work is to confirm the Vaughan-Martini and Martini's
statement (1987) by using the proper type strains of the same four species names:
MUCL 31497, NT of S. cerevisiae, MUCL 31496, NT of S. pastorianus, MUCL
31495, T of S. baynus, MUCL 3149, NT of S. paradoxus.

rRNA extraction
Cells were grown at 25C in 100ml of DYP liquid medium (dextrose, yeast
extract, peptone) on rotary shaker at 200rpm during 16 hours. Celles were harvested
by centrifugation, washed with BPS buffer and freeze dried. Undergraded rRNA
was isolated according two procedures, one by Guadet et al. (1989) and the other by
guanidinium thiocyanate (Promega). Purity of rRNA samples was estimated from
spectrophotometric absorbance ratios 260/280=1.70-2.00 and their integrity by
denaturating agarose gel electrophoresis (Fig. 1).

172

Fig. 1 Electrophoretic test of rRNA

purity

rRNA sequencing
Two regions of 28 S RNA were
sequenced using specific
oligonucleotide primer (401), primer 5'-
GGTSSGTGTTSAAGASGG-3' (635)
and primer 5'-
TTGGAGASSTGSTGSGG-3' (1841),
totalizing 820 bases, following the
dideoxynucleotide chain termination
method as described by Lane et al.
(1985). Nucleotide fragments generated
in the chain elongation reactions were
separated on 8% acrylamide 8M urea
gels and visualized by autoradiography.
Fig. 2 presents sequences obtained from
the four strains using primer 401.
Alignment of the four sequences was
carried out by Clustal W 1.4 program
(Higgins and Sharp 1988).

Fig. 2 Sequences of 28S rRNA portion from 1, S. pastorianus, 2, S. bayanus, 3, S. cerevisiae, 4, S.

paradoxus

173

Phylogenetic tree
A phylogenetic tree depincting the four sequenced species has been produced
using the YVDP program (Van de Peer and De Wachter 1993) and including the
sequences of another strain of S. cerevisiae and of five other fungi available from Pr
De Wachter's data base, University of Antwerp. Evaluation of the branching
reliability has been obtained by bootstrapping.

Fig. 3.Phylogenetic tree constructed using YVDP software.

Results
The obtained tree (Fig. 3) does not differ from the one obtained by Kurtzman and
Robnett (1991) exepted by the fact that Saccharomyces cerevisiae in our tree seems
to be phylogenetically older than the derivated species. This might result from
sequencing a larger portion of rRNA but might also be due to the fact that the medial
branching of the cluster is poorly reliable with 55% bootstrap.
In both trees the four tested Saccharomyces species cluster together separately
from other fungal species. Saccharomyces pastorianus and S. bayanus cluster
closely together with high bootstrap valuie (100%) and confirm the DNA
relatedness of 72% as shown by Vaughan-Martini and Martini (1987). It also
confirms the weak DNA relatedness of 20% between S. cerevisiae and S. bayanus
and between S. paradoxus and S. pastorianus.
S. carlsbergensis sequenced by Kurtzman and Robnett (1985) falls into the same
position than S. pastorianus. But their synonymy needs demonstration.
It is noteworthy to observe the close relatedness between Candida albicans and
Saccharomyces cerevisiae, both hemiascomycetous species, and the segregation of
Pneumosystis carinii and Schizosaccharomyces pombe from the Hemiascomycetes,
as it has been shown by 18S rRNA sequencing. But the two latter taxa are certainly
not mutually related as shown by 57% bootstrap value.
The other yeast Cryptococcus neoformans, is here also confirmed to be very
distinct, in the same position as found in 18S rRNA trees where it culsters together
with Basidiomycetes.

174

 Conclusions
Our results based on the sequence of two portions of 28S rDNA, confirms the
distinction of Saccharomyces cerevisiae from the three first thought synonyms S.
paradoxus, S. pastorianus and S. bayanus and the close relatedness between S.
pastorianus and S. bayanus.
References
Guadet J., Julien J., Lafey JF. and Brygoo Y. A989. Phylogeny of some fusarium species, as determined
by large subunit rRNA sequence comparison. Mol. Biol. Evol. 6: 227-242.
Higgins D.J. and Sharp P.M. 1988. Clustal: a package for performing multiple sequence alignment on a
microcomputer. Gene 73: 237-244.
Kreger-van Rij N.J.W. ed. 1984 The Yeasts, a taxonomic study. 3rd edn. Elsevier Science Publ.
Amsterdam.
Kurtzman C. and Robnett C. 1991. Phylogenetic relationships among Saccharomyces,
Schizosaccharomyces, Debaryomyces and Schwanniomyces determined from partial ribosomal RNA
sequences. Yeast 7: 61-72.
Martini-Vaughan A. and Martini A. 1987. Three newly delimited species of Saccharomyces sensu stricto.
Antonie van Leewenhoek 53: 77-84.
Van de Peer Y. and De Wachter R. 1993. TREECON, a software package for the construction ans
drawing of evolutionary trees. Comput. Appl. Biosc. 9: 177-182.
________

WHAT ABOUT THE "SPECIES GROUP" CONCEPT IN ASPERGILLUS

FLAVUS ?

JOELLE DUPONT 1, MARIE-FRANCE ROQUEBERT1 AND EVELYNE GUEHO2

1Laboratoire de Cryptogamie, M.N.H.N., 12 Rue Buffon, 75005 Paris

2Unit de Mycologie, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris

Abstract. Whemer (1901) was the first to introduce the idea of "species groups"
to accommodate the great diversity and variability of Aspergilli.
This concept is the foundation of the actual taxonomy of the genus.
The flavus group contains 16 species (Christensen 1985) from yellow green to
brown in color. Some of them are morphologically well defined but others of
industrial importance, referred as the Koji molds, A. oryzae, A. sojae, A. tamarii are
difficult to differentiate from the toxinogenic and cosmopolitan species A. flavus and
A. parasiticus.
Numerous technics have been applied to find new characters: electrophoretic
mobility patterns of proteins and specific enzymes, pyrolysis profiles, DNA
homology, GC contents of DNA. We propose a view of the flavus group through the
comparison of sequences of the 5' end of 28S rRNA. Two points are clear and make
the group heterogeneous:
- the complete similarity of 10 "species", including the Koji molds and the
toxinogenic species,
- the split of A. zonatus and A. clavatoflavus together with the teleomorphic
Penicilliopsis.

175

 A COMPARATIVE STUDY OF ELECTROPHORETIC KARYOTYPES IN

THE YEAST GENERA TORULASPORA LINDNER AND
ZYGOSACCHAROMYCES BARKER

GUIDO CAPRIOTTI AND ANN VAUGHAN-MARTINI

Dipartimento di Biologia Vegetale, Sez. Microbiologia Applicata, Universita degli Studi di Perugia, Italy

Abstract. In the most recent edition of the monograph The Yeast, a taxonomic
study (Yarrow 1984) the genera Torulaspora and Zygosaccharomyces were formally
separated from Saccharomyces. These genera differ from Saccharomyces in having
a predominately haploid vegetative phase and in the mechanism of ascus formation:
conjugation of independent cells that can be preceded by the formation of specific
protuberances or by the union of a cell with its bud.
While the distinction between Torulaspora and Zygosaccharomyces is usually
straightforward, some problems have been encountered in the separation of species
within Zygosaccharomyces since several taxa are characterized by limited
assimilative and fermentative profiles. Since these genera has been clarified by
DNA/DNA reassociation experiments (Price et al. 1978, Kurtzman 1991), the
definition of electrophoretic karyotypes of all type strains of the two genera can be
useful as an aid for the identification of unknown strains.

References
Kurtzman C.P. 1991. DNA relatedness among species of the genus Zygosaccharomyces. Yeast 6: 213-
219.
Price C.W., Fuson G.B. and Phaff H.J. 1978. Genome comparison in yeast systematics. Delimitation of
the species within the genera Schwanniomyces, Saccharomyces, Debaryomyces and Pichia. Microbiol.
Rev. 42: 161-193.
Yarrow D. 1984. Saccharomyces Meyen ex Reess. In The yeasts, a taxonomic study. Kreger-van-Rij
N.J.W. ed. Amsterdam, Elsevier Science Publishers. pp.379-395.
______

176

 FUNGAL BIODIVERITY
BIODIVERSITY OF CYLINDROCLADIUM ON WATER-LILIES AT SSR
PAMPLEMOUSSES BOTANIC GARDEN IN MAURITIUS

A. PEERALLY

Abstract. An interesting case of fungal biodiversity within the same genus is described. Six species
of Cylindrocladium are reported to occur on water-lilies in three contiguous ponds in Mauritius. The
species are C. hawksworthii, recently descibed as a new species, C. quinqueseptatum, C. colhounii, C.
floridanum, C. camelliae and C. infestans, all of which are pathogenic. Of these species with the
exception of C. hawksworthii, the five others were previously observed to occur on tea in Mauritius.

Introduction
The taxonomy and pathology of the genus Cylindrocladium has fairly recently
been comprehensively reviewed (Peerally 1991a). In the present paper the
occurrence of six species of Cylindrocladium is reported on water-lilies at
Pamplemousses Botanic Garden in Mauritius.

Three species of water-lilies, Nelumbo nucifera Gaertn., Nymphaea lotus L. and

Victoria amazonica L. have, for many years, been known to be affected by a foliage
disease (Fig. 1a-c). These water-lilies grow on three contiguous ponds, two of which
contain N. nucifera and N. lotus, while the third larges pond bears N. lotus and the
spectacular V. amazonica, of significant attraction to visitors. The occurrence of
Cylindrocarpon quinqueseptatum Boedijn & Reitsma and of C. hawksworthii on N.
nucifera and N. lotus has already been described (Peerally 1991b), since the latter
pathogenen was reported as a new species.
The associatopn of Cylindrocarpon as a pathogen of tea had been the subject of
previous papers (Peerally 1973, 1974a-C, 1976a, b, 1991a), and the species reported
are:
C. colhounii Peerally
C. floridanum Sob. & Seymour
C. clavatum Hodges & May
C. camelliae Venkataaara & Venkata Ram
C. quiqueseptatum Boedijn & Reitsma
C. infestans (Boersew.) Peerally.

The occurrence of two or more species of Cylindrocladium on the same hosts has
been frequently reported in the literature. Thus significant mortality rates of
seedlings of Eucalytus grandis and E. terreticornis were due to a disease complex
which included C. quinqueseptatum, C. clavatum and C. ilicicola (Anon. 1982),
Mohanan and Sharma (1986) found several species of Cylindrocladium associated
with Eucalyptus spp. causing a complex damping off, stem canker and leaf and
shoot blight in Kerala, India.
Several papers in the literature have emphasized the disease enhancing effects of
the high moisture levels with Cylindrocladium diseases. Thus Barnard (1984)
reported extensive damage of E. grandis and E. robusta seedlings under very humid
conditions caused by overhead irrigation. Various workers (Petch 1921, Loos 1951,
Wabster 1954) reported that the disease incidence by C. theae on tea to be enhanced
by wet conditions and was checked with the cessation of rain.

177

 Material and method

The Cynlindrocladium species reported in this paper were all isoltad from
diseased leaves of three species of water-lilies. All six species were examined from
incubated diseased material and from colonies on PDA isolated from diseased leaves
after surface sterilistation. Artificially and naturally infected leaves (Fig. 2a) of
water-lilies were maintained in boxes with wet cotton to induce disease and
sporulation which occurred very easily after a few days at 22-27C.

Results
The occurrence of Cylindrocladium on the three species of water-lilies wa as
follows:
Host species Cylindrocladium species
Nelundo nucifera C. quinqueseptatum, C. hawksworthi
Nymphaea lotus C. quinqueseptatum, C. hawksworthii,
C. colhounii
Victoria amazonica C. colhounii, C. floridanum,
C. camelliae, C. infestans

Fig. 1. a. Necrotic spots on Nymphaea lotus leave. b. Spot on Nelumbo nucifera leaf. c. Spot on
Victoria amazonica leaf. d. Cylindrocladium hawksworthii (upper colony) and C. quinqueseptatum (lower
colony) on N. nucifera. e. C. hawksworthii conidial masses on N. nucifera.

178

 Spots on Nelundo nucifera are grey becoming brown to black and coalesce to
give large circular necrotic areas. The masses of conidiophores and conidia of C.
quinqueseptatum are generally less dense (Fig. 1d) than those of C. hawksworthii
(Fig. 1d, e) which look decidedly denser, more powdery and white.
Spots on Nymphaea lotus are initially water-soaked areas turning brown and later
black.
On Victoria amazonica (Fig. 1c) there are conspicuous water-soaked areas which
soften to produce a soft rot. It can be easily observed under a microscope that in
infected areas the host cells separate easily due to the dissolution of the middle
lamella. Areas of infected leaves of V. amazonica producing condidiophoes of C.
infestans and C. camelliae are clearly discernible.

Species description

Cylindrocladium colhounii Peerally (Fig. 2b)

Coniophores 308-413 m long, with strile appendage, much shorter without
appendage. Stipe narrower towards an apical slender vesicle 7.8-82.0 x 2.0-5.2 m.
Primary conidiophore branches aseptate, rarely 1-septate, 13-26 m long; secondary
branches aseptate, 7.8-18.2 m long; tertiary branches aseptate 7.8-10.4 m.
Phialides in groups of 2-4, occasionally arising directly from the stipe, 7.6-13.6 m
long. Conidia hyaline, cylindrical, straight, 3-septate, rarely 1- or 2-septate, never 4-
or 5-septate, 38.3-84.2 x 3.4-5.7 m, in average 66.0-5.0 m.

Cylindrocladium quinqueseptatum Boedijn & Reitsma (Fig.2c, d)

Conidiophores branches arising laterally from a stipe; primary branches aseptate
or 1-septate, 8.4-26.6 m long; secondary and tertiary branches aseptate, 11.5-15.6
m. Phialides hyaline, 13.0-15.6 m long. Conidia hyaline, cylindrical, straight, 1-
to 6-septate, usually 5-septate, old conidia in culture developing more than 6 septa,
59.8-104.6 x 5.2-7.0 m in vivo, 72.8-119.6 x 5.2-7.8 m in vitro. Sterile filament
terminating in a narrowly clavate vesicle 2.5-3.0 m wide.

Cylindrocladium hawksworthii Peerally (fig. 3)

Conidiophores erect, hyaline, septate, 250-500 m long. Stipe conspicuously
setate; primary conidiophore branches 1-septate or aseptate 12-80 m long;
secondary branches 1-septate or aseptate 12-45 m long; tertiary branches usually
aseptate 8-30 m long; quaterny branches usually aseptate 7-35 m long; sterile
appendage 160-280 m; phialides solitary or in small groups, sometimes arising
directly from the stipe, hyaline, aseptate, 7-13 m long; conidia hyaline, cylindrical,
curved, 1-septate, 36-62 x 3-5 m.

Cylindrocladium floridanum Sob. & Seymour (Fig. 4a-d)

Conidiophores banches arising lateraly from a stipe; primary and secondary
branches asteptate or 1-septate, 10.2-26 m long; tertiary branches aseptate, 7.8-10.2
m. Phialides hyaline, 7.8-15.6 m. Conidia hyaline, cylindrical, 1-septate, 36.4-
57.2 x 2.6-4.6 m. Sterile filmaments temenating in a globose vesicle, 8-18 m
diam. Secondary sterile filaments present in most isolates.

179

 Fig.2. Cylindrocladium colhounii. b. Conidiophore and conidia. Cylindrocladium quinqueseptatum.

a. spots on in vitro inoculated Nelundo nucifera leaf. c. Sterile appendate with vesicle. d. Conidia.
Fig. 3. Cylindrocladium hawksworthii. a-C. Sterile appendage with vesicle. d. Conidia.

Cylindrocladium camelliae Venkatara & Venkata Ram (Fig. 5c-g)

Conidiophores of two types: penicillate or verticillate. In penicillate stipe, sterile
filament usually present; primary branches aseptate, 12.0-28.2 m long; secondary
branches aseptate, 8.4-19.6 m; phialides navicular, hyaline, 9.2-18.6 m. In
verticillate stipe, sterile filament absent, phialides 16.8-31.2 m long. Conidia
hyaline cylindrical, 1-septate, 9.6-15.6 x 1.8-2.2 m. Sterile filament elongate,
aseptate, thick-walled, 60.0-110.6 m, apically vesiculate. Vesicle lanceolate or
ellipsoidal, sometimes lanceolate curved, thin walled, 4.0-5.2 m wide.
Chlamydospores present. Microconidia unfrequent in culture.

Cylindrocladium infestans (Boesew.) Peerally (Fig 5a, b)

Conidiophores of two types: penicillate or most often subverticillate. Penicillate
conidiophores 190-250 m long; primery branches 13-22 m long; secondary
branches 8-16 m long; phialides avicular 5-15 m. In verticillate conidiophores,
significantly phialides longer, 14-32 m long. Sterile filament 70-130 m long,
ending a a cylindrical or narrowly lanceolate vesicle 20-70 x 4-5 m.
Chlamydospores prsent. Microconidia unfrequent in culture.

180

 Fig. 4. Cylindrocladium floridanum. a, b. Conidiophores. c. Sterile appendage with vesicle. d.

Conidia.
Fig. 5. Cylindrocladium infestans. a. Penicillate conidiophore. d. vertcillate conidiophore.
Cylindrocladium camelliae. b-c. Penicillate conidiophores. e-g. Vesicles .

Discussion
Six species of Cylindrocladium pathogenic on water-lilies in Mauritius
are described from three contiguous ponds in Pamplemousses Botanic Garden. In
addition to the pathology of this disease complex it is interesting to highlight the
biodiversity and ecological aspects. Several workers like Peerally (1974b), Rattam
and Dhanda (1985) and Mohanan and Sharma (1986), have reported mixed
infections involving two or morespecies of Cylindrocladium. The foliage disease of
water-lilies in Mauritius associated with six species of Cylindrocladium is a
particularly stricking example of the biodiversity of a single fungus genus on three
species of water-lilies growing within a very small area. The fact that this disease on
water-lilies occurs in a particulaly humid habitat is quite characteristic of
Cylindrocladium, which has been constantly reported to thrive best under very
humid conditions in both artificial and natural growing conditions. Of the six species
occuring on water-lilies five are pathogenic to tea in Maritius in the central plateau,
about 300km away from the Pamplemousses Gargen. The sixth species, C.
hawksworthii, was described as a new species which has not so far been observed on
tea or any other host.

Acknowlegements
My appreciation is due to Mr V. Aumeer for technical assistance.

References

181

Anonymous 1982. Nursery diseases of Eucalyptus in Kerala. Evergree 8: 2-4. Div. of Pathology, Kerala
Forest Res. Inst., Peechi, India.
Barnard E.L. 1984. Occurrence, impact and fungicidal control of girdling stem cankers caused by
Cylindrocladium scoparium on Eucalyptus seedlings. Plant Disease 68: 471-473.
Loos C.A. 1951. Pathological problems. Tea Quartely 22: 27-30.
Mohanan C. and Sharma J.K. 1986. Epidemiology of Cylindrocladium diseases of Eucalyptus . In
Eucalyptis in India: Past, present and future. Proceedings of the National Seminar, Kerala Forest
Inst. India.
Peerally A. 1972a. A new disease on tea. Revue agricole et Sucrire de l'Ile Maurice 51: 115-117.
P eerally A. 1972b. A decline of tea bushes associated woth a root rot. Revue Agricole et Sucrire de l'Ile
Maurice 51:147-152.
Peerally A. 1973. Calonectria colhounii sp. nov., a common parasite of tea in Maritius. Trans. Brit.
mycol. Soc. 61: 89-93.
Peerally A. 1974a. Cylindrocladium clavatum. CMI Descriptions of Pathogenic Fungi and Bacteria 422.
Peerally A. 1974b. Calonectria quinqueseptata (conidial state: Cylindrocladium quinqueseptatum). CMI
Descriptions of Pathogenic Fungi and Bacteria 423.
Peerally A. 1974c. Cylindrocladium camelliae. CMI Descriptions of Pathogenic Fungi and Bacteria 428.
Peerally A. 1974d. An elucidation of certain diseases of tea caused by Calonectria spp. Revue Agricole et
Sucrire de l'Ile Maurice 53: 57-66.
Peerally A. 1991a. The classification and phytopathology of Cylindrocladium species. Mycotasxon 40:
323-366.
Peerally A. 1991b. Cylindrocladium hawksworthii sp. nov. pathogenic in water-lilies in Mauritius.
Mycotaxon 40: 367-376.
Petch T. 1921. Report of the Botanist/Mycologist. Tropical Agriculturist 57: 318-319.
Rattan G.S. and Dhanda R.S. 1985. Leaf blight and seedling diseases of Eucalyptus caused by
Cylindrocladium spp. in Punjab. Annals of Biology (India) 1: 184-188.
Webster B.N. 1854. Notes on pathological matters. Tea Quarterly 25: 17-19.

___________

182

 THE OCCURRENCE OF WOOD DECAYING FUNGI IN BELGIUM

HOUSES

CONY DECOCK AND GREGOIRE L. HENNEBERT

Mycothque de lUniversit catholique de Louvain (MUCL) Laboratoire de Mycologie Systmatique et

Applique, Place Croix du Sud 3, 1348 Louvain-la-Neuve, Belgium

Abstract. From 1990 to 1993, 602 cases of timber decay from anish houses have been analysed at
MUCL. 18 species of wood decaying fungi have been recorded. 11 species cause a brown rot and
represent more than 72% of the timber decay while 7 species cause white rot. Serpula lacrymans occurs
in 46% of the cases and is the more frequent species. After this, Donkioporia expansa, Coniophora
marmorata and Asterostroma ochroleucum are observed at a respective frequency of 11%, 7.3% and
6.3%. Noteworthy, Coniophora marmorata is here much more common than Coniophora puteana,
reputed in other countries as most frequent. Another species, rarely documented as a wood decaying
fungus in houses, is Asterostroma ochroleucum. The fungus is causing a white rot of wood and expands
easily on and in plasterwork and masonry in very wet areas.
Keywords. Timber decay, house fungi, Serpula lacrymans, Donkioporia expansa, Coniophora
marmorata, Asterostroma ochroleucum.

Introduction
Fungal decay in houses immediately suggests an attack by the true dry rot
fungus, Serpula lacrymans. Indeed, Serpula lacrymans is the most frequently
reported but also the most destructive fungi in all North Western Europe.
Nevertheless, other lignicolous basidiomycetes occur in buildings beside Serpula
lacrymans. For instance Coniophora puteana, the cellar fungus, is known as
frequent. But few informations are available over the presence and frequency of
others species than the true dry rot in buildings of central and western Europe. Koch
(1985) gives an overview of the principal wood-rot fungi occurring in Danish
buildings for years 1946-1983. More recently, Paajanen and Viitanen (1989) do the
same for Finland for the years 1978 to 1988.
From 1990 to 1993, 683 cases of timber decaying fungi were analysed by
MUCL. This paper presents a brief draft of the species encountered and their
frequency in Belgian housing with some comments on noteworthy species.

Situation in Belgium

Between 1990 and 1993, 683 timber decaying fungi were analysed at MUCL.
Most samples were sent or brought to the laboratory by private owners or repairing
companies. 92.4% of the samples were identified to the species level, doubtful cases
identified only to the genus level, representing 7.6%. 18 species belonging to 8
families have been recorded. Table 1 gives the list of species encountered, their
respective case number and frequency. Table 2 gives the families with their
respective species number, incidence and kind of rot (classification according to
Jlich 1981). 11 species cause a brown rot -or laccase negative rot- while 7 species
cause a white rot -or laccase positive rot. Incidence of brown rotter is much more
important than that of white rotter. Indeed, more than 72% of timber decay is by
brown rot against only 28% by white rot. Almost two third (62.7%) of the decay are
assigned to members of the Coniophoraceae represented by 3 genera and 5 species.

183

 50

45

40

35

30

25

20

15

10

0
S.l.
D.e.
C.m.
A.o.
C.sp.
C.r.
T.f.
A.x.
P.c.
C.p.
A.sp.
G.t.
P.v.
P.p.
S.b.
L.p.
S.h.
P.m.
P.o.
D.q.
S.l.: Serpula lacrymans D.e.: Donkioporia expansa C.m.: Coniophora marmorata A.o.:
Asterostroma ochroleucum C. sp.: Coniophora species C.r.: Coprinus radians T.f.: Trechispora
farinacea P.c.: Phellinus contiguus A.x.: Antrodia xantha C.p.: Coniophora puteana A.sp.:
Antrodia species G.t.: Gloeophyllum trabeum P.p.: Paxillus panuoides P.v.: Poria vaillantii S.b.:
Sistotrema brinkmanii L.p. Leucogyrophana pinastri S.h.: Serpula himantioides P.m.: Perenniporia
medulla-panis P.o.: Pleurotus ostreatus D.q.: Daedelea quercina.

Table 1. List of species seen during the studies and their respective number, frequency and kind of rot.

Species raw number Percentage Kind of rot

Serpula lacrymans 319 46.7 Brown
Donkioporia expansa 76 11.12 White
Coniophora marmorata 50 7.32 Brown
Asterostroma ochroleucum 43 6.29 White
Coniophora sp. 39 5.7 Brown
Coprinus radians 32 4.68 White
Trechispora farinacea 19 2.78 White
Antrodia xantha 16 2.34 Brown
Phellinus contiguus 15 2.19 White
Coniophora puteana 14 2.05 Brown
Antrodia sp. 13 1.90 Brown
Gloeophyllum trabeum 12 1.75 Brown
Antrodia vaillantii 10 1.46 Brown
Paxillus panuoides 8 1.17 Brown
Sistotrema brinkmanii 7 1.02 Brown
Leucogyrophana pinastri 3 0.44 Brown
Serpula himantioides 2 0.30 Brown
Perenniporia medulla-panis 2 0.30 White
Pleurotus ostreatus 2 0.30 White
Daedalea quercina 1 0.15 Brown
TOTAL 683 100

184

Table 2. List of families encountered with their respective frequency and kind of rot.

Fungal family Brown rot white rot Total

% incidence % incidence % incidence
species species species
Coniophoraceae 5 62.5 (427) - 0 (0) 5 62.5 (427)
Polyporaceae 4 7.6 (52) 2 11.4 (78) 6 19 (130)
Lachnocladiaceae - 0 (0) 1 6.3 (43) 1 6.3 (43)
Coprinaceae 0 0 (0) 1 4.7 (32) 1 4.7 (32)
Corticiaceae 1 1.0 (7) 1 2.8 (19) 2 3.8 (26)
Hymenochaetaceae - 0 (0) 1 2.2 (15) 1 2.2 (15)
Paxillaceae 1 1.2 (8) - 0 (0) 1 1.2 (8)
Pleurotaceae - 0 (0) 1 0.3 (1) 1 0.3 (1)
Total number of
11 72.3 (493) 7 27.7 (190) 18 100
species

100
S.l. C.m.
90
D.e. A.o.
80

70

60

50

40

30

20

10

0
90 91 92 93
years

Fig. 2. Frequency of the 4 principal species year by year.

Discussion
Before discussing the present data, we must advice that relative occurrence given
above are not necessarily representative of the real Belgian situation. In first hand,
we do not have information from all around Belgium. Most of the specimens
analysed originate from Brussels and Louvain-la-Neuve areas. In second hand, we
have not collected all the samples ourselves in a randomly way. Most were sent to
MUCL and we can not certify they were always collected randomly. It may occur
that a selection has been applied before. This is particularly obvious for Serpula
lacrymans. Indeed, information about the dry rot in house has been widely spread in
the public the last years in Belgium by means of media, books and vulgarisation
leaflets (Leclercq 1989, Rammeloo et al. 1989, 1992, Hennebert et al. 1990).
Peoples dealing with timber decay (owners, repair companies, architects and
insurance experts) may have acquired by this way a relatively good knowledge of
the true dry rot symptoms allowing them to identify Serpula lacrymans. Samples
sent to the laboratory might be only the most critical ones. This can alter the real
species occurrence, leading to some under-estimation of Serpula lacrymans and
consequently to some over-estimation of the others species.

185

Fig.3. Cubic rot or Brown rot due to Serpula lacrymans.

Fig.4. Micro-cubic rot due to Coniophora puteana.
Fig.5. Fibrous rot or White rot due to Donkioporia expansa.

186

The occurrence given above must therefore be regarded more as a general overview
rather than the real situation which could be slightly different. Nevertheless, for the
other species than S. lacrymans, the number of cases per year represents a good draft
of the situation. More than the real occurrence, the presence and the relative position
of each species are important.

1. Serpula lacrymans (Wulfen:Fries) Schroeter

Serpula lacrymans, the true dry rot fungus, as in all north western European
countries, is by far the most important species involved in timber decay. Almost
50% of the timber breakdown is attributed to that species. It is also the most
destructive fungi for timber.

Fig. 6. Hymenium of the fruitbody of Serpula lacrymans.

2. Donkioporia expansa (Desmazires) Kotlaba & Pouzar

Syn. Boletus expansus Desmazires
Poria expansa (Desmazires) H. Jahn
Phellinus cryptarum Karsten
Polyporus megaloporus Persoon
Phellinus megaloporus (Persoon) Heim
Poria megalopora (Persoon) Cooke

Donkioporia expansa (Desmazires) Kotlaba & Pouzar comes in second position

with an occurrence of 11% (76 cases in 4 years). This species causes a white rot
mainly of deciduous wood but can also be found on coniferous (Cartwright and
Findlay 1958, Findlay 1950, Buchwald 1986).

187

 According to Heineman (1979), the earliest Belgian record of this species dates
on 1854. This author considered this species rare in Belgium and cited only 5 more
records after the first one (Heineman 1979). Since then, in 1989, Guillitte (in
Rammeloo et al. 1989) noted a very weak occurrence of only 2 % (5 cases over 250
samples between 1985 and 1988). In 1992, the same author (in Rammeloo et al.
1992) mentioned 71 cases from 1985 to 1991 (for 749 wood decaying fungi analysed
in 7 years), thus 10 cases/year. In the present analysis one the 4 last years, 76 cases
have been identified with an average of 19 cases/year
D. expansa was also considered as scarce in others western European countries.
The species is known in Belgium since at least 1922 (as P. cryptarum) after the
destruction of the Versailles Palace woodwork (Mangin and Patouillard 1922).
There is no recent data available on the occurrence of the species in Belgium. In
England, Findlay (1950) reports the species in a dozen or more buildings. Jlich
(1984) reports the same as rare in CS, D, DDR, GB and NL. Ritter (1983) draws
attention on the species in a small report. As far as we know, Buchwald (1986) was
the first to demonstrate that D. expansa occurs much more frequently in Germany
than previously known. He reported an average of 15 to 20 cases/year while the
species was stated as seldom.
Nor Koch (1985) nor Paajanen and Viitanen (1989) do mention the species in
their survey of timber decaying fungi in North Europe. According to Ryvarden and
Gilbertson (1993) D. expansa is a Central and Southern species.
Buchwald (1986) draws the attention on the fact that D. expansa is not strictly
restricted to hardwood (oak and chestnut mainly) as stated previously but is also
able to decay softwood. Well, in the past, most records were made on oak or
chestnut wood (Cartwright and Findlay 1958, Findlay 1950), but it is well
demonstrated now that th species occurs also on coniferous wood. Numerous
collections are made on softwood now since oak wood is less used in modern
buildings.
For more literature concerning this species, see Mangin and Patouillard 1922
(under P. cryptarum), Cartwright and Findlay 1958 (under P. cryptarum), Domanski
and Orlicz 1967 (under Polyporus megaloporus Persoon), Drfelt and Sommer 1973
(under Poria expansa), Heineman 1979 (under D. expansa), Ritter 1983 (under D.
expansa), Buchwald 1986 (under D. expansa),

3. Coniophora marmorata Desmazires.

Coniophora marmorata Desmazires represent 7.9% of the specimens analysed
(40 cases over 520) while Coniophora puteana represent only 2.4% (12 cases). Up
to 1992, no mention of this species existed for Belgium. Ginns (1982), in its
monograph of the genus Coniophora cited only 9 specimens. Few data on its
occurrence in Europe are available. C. marmorata seems to be restricted to western,
central and southern Europe. Indeed, nor Koch (1985) nor Paajanen and Viitanen
(1989) mention the species in their survey. At the same, Hallenberg (1985) do not
mention the species for northern Europe. However Beech-Andersen (1992) mentions
the species in Denmark.

188

Fig. 7. fruitbody of Coniophora marmorata on wood.

Table 2. Main distinguishing features between C. marmorata and C. puteana(Ginns, 1982).

Speciies Spores Mycelium

C. marmorata subglobose dimitic with

to ovoid tan to pale yellow
dextrinoid skeletal hyphae
7-10 m x 4.5-7 (-8) m 2-7 m wide

C. puteana broadly monomitic

ellipsoid
non dextrinoid
9.5-14 x 6-7.5 m

4. Asterostroma ochroleucum Bresadola.

Asterostroma ochroleucum Bresadola represents 6.2% of samples analysed (43
cases over 683).
A. ochroleucum produces a white fibrous rot of both hardwood and coniferous
wood. It expands easily on and in plasterwork and masonry in very wet areas
forming whithish to ochraceous strands network which can be mistaken for S.
lacrymans strands with the naked eyes. It is easily recognised under the microscope
by its typical brown asterosetae. When fruitbodies is present, which occurs scarcely
in Belgian buildings, spores are globose to subglobose and strongly tuberculate (fig.
9) with amyloid tubercule.

189

 Fig.8. Mycelium et fruitbody (in centre) of Asterostroma ochroleuca on plasterwork.

Fig.9. Asterosetae and tuberculate spores of Asterostroma ochroleucum.

Few data are available on the occurrence of the species in building, in Europe.
Findlay (1950) found the species on several occasions in England while Koch,
according to Hallenberg (1985), mentioned it as quite common in Denmark.

190

However, she did not mention it in her survey of wood decay fungi in Danish houses
(Koch 1985).
This species is more important than previously known.

Conclusions
Like in all North and Western European countries, Serpula lacrymans is the first
cause of wood decay in Belgium. It is also the most destructive fungus. From the
data analysed, the species is involved in almost 50 % of the wood damage in
Belgium. And its occurrence is most probably under-estimated.
Donkioporia expansa is much more important than previously established. It has
to be looked for, not only on oak wood but also on coniferous wood.
Coniophora marmorata is also an important wood decayer and has been
certainly mistaken for C. puteana in the past.
Asterostroma ochroleucum is not uncommon in Belgian buildings and was
certainly overlooked till recently..

References
Beech-Andersen J. 1993. The dry rot fungus and other fungi in houses. Internat. Res. Group on Wood
Pres. IRG/WP 2389: 1-46.
Buchwald G. 1986. On Donkioporia expansa (Desm.) Kotlaba et Pouzar. Internat. Res. Group on Wood
Pres. IRG/WP1285: 1-9.
Cartwright K.S.G. and Findlay W.P.K. 1958. Decay of timber and its prevention. Her Majestys
Stationery Office, London 356 pp.
Domanski S. and A Orlicz. 1967. Polyporus megaloporus Pers. in the Family Polyporaceae s. str. Acta
Mycologica 3: 51-62.
Drfelt H. and B. Sommer 1973. Poria expansa (Desm.) Jahn in Botanischen Garten halle Gefunden.
Mykologisches Mitt. Bl. 17: 44-47.
Findlay W.P.K. 1950. A note on the fungi of less common occurrence in houses. Trans. Brit. Mycol. Soc.
34: 35-37.
Ginns J., 1982. A monograph of the genus Coniophora (Aphyllophorales, Basidiomycetes). Opera
Botanica 61: 1-61.
Hallenberg N. 1985. The Lachnocladiaceae and Coniophoraceae of North Europa. Ed. Fungiflora, Oslo.
96 pp.
Heineman P. 1979. Poria expansa, polypore peu connu ou mconnu en Belgique. Dumortiera 13: 1-2.
Hennebert G.L., Balon F. and Boulenger P. 1990. La mrule: science, technique et droit. Ed. Ciaco,
Bruxelles, 250 pp.
Jlich W. 1981. Higher taxa of basidiomycetes. Bibliotheca Mycologica 85, ed. J. Cramer. 485 pp.
Jlich W. 1984. Kleine Kyptogamenflora Bd. II. Ed. Fischer Verlag, Stuttgart: XXX pp.
Koch A.P. 1985. Wood decay in danish buildings. Internat. Res. Group on Wood Pres. IRG/WP 1261: 1-
9.
Mangin L. and Patouillard N. 1922. Sur la destruction des charpentes au Belgique de Versailles par le
Phellinus cryptarum Karst. C.R. Acad.Sci.Paris 175: 389-394.
Paajanen L. and Viitanen H. 1989. Deacy fungi in Finnish houses on the basis of inspected samples from
1978 to 1988. Internat. Res. Group on Wood Pres. IRG/WP 1401: 1-5.
Rammeloo J., O. Guillitte, G. Draye, M. Van Leemput, F. Deroy and S. Roland, 1989. La mrule et autres
champignons nuisibles dans les btiments : une approche multidisciplinaire. Jardin Botanique
National de Belgique : 55 pp.
Rammeloo J., Guillitte O., Draye G., Van Leemput M., Deroy F. and Roland S., 1992. La mrule et
autres champignons nuisibles dans les btiments: une approche multidisciplinaire, 2d d. Jardin
Botanique National de Belgique 55 pp.
Ritter G. 1983. Neufund von Donkioporia expansa. Boletus 7(1): 3-4.
______

191

 SYMBIOTIC FUNGI IN THE GALLERIES OF THE STRIPED BARK

BEETLE, TRYPODENDRON LINEATUM (COLEOPTERA, SCOLYTIDAE)

G. BABUDER AND F. POHLEVEN

Department of Wood Science and Technology, Biotechnical Faculty, University of Ljubljana, Slovenia

Abstract. Symbiotic fungi from the galleries of the striped bark beetle, Trypodendron lineatum (Ol.)
found in spruce logs lying at the timber storage yard of a woodprocessing industry in Ljubljana were
studied. In laboratory conditions the following fungi were isolated and determined: Trichoderma
harzianum Rifai, yeast Pichia anomala Hansen (Kurzman), blue-stain fungus Ceratocystis piceae
(Mnch) Bakshi (anam. Graphium) and a fungus closely similar to Ceratocystis araucariae (anamorph)
(in the following text designed as FsCa). Isolated fungi stained the wood but did not cause any major
weight loss of wood samples in laboratory conditions. The hyphae of symbiotic fungi primarily colonized
the parenchyma tissues of the sapwood, and also tracheids. Enzyme activity of fungi was studied by using
simple laboratory test methods. The presence of specific enzymes cooperating in wood decay was
established.

Introduction
The striped bark beetle, Trypodendron lineatum (Ol.) is considered to be one of
the most serious pest among ambrosia beetles in the northern hemisphere (Uusvaara
and Loyttyniemi 1975). Ambrosia beetles are coleopteran insects (Scolytidae and
Platypodidae) that indirectly utilize wood.
The damage is caused by the female beetle of T. lineatum tunneling in sapwood
and introducing into it spores of mutualistic fungi which then grow in the gallery.
Such fungi can spread and stain the wood near gallery or up to several centimeters
from the gallery. They convert wood components into the fungal growth which
serves as food for the beetle (Baker, 1963; Batra, 1963). T. lineatum attacks logs of
autumn and winter felled coniferous trees lying unpeeled, and also weakened or
dying trees (Anilla, 1975).
From the first reports of symbiosis between insects and fungi till the early 1960s,
the concept of one ambrosial fungus per one ambrosia beetle was emphasized. But
some subsequent studies confirmed the view that each ambrosia beetle is
symbiotically associated with more than one fungus (Baker 1963, Norris 1965).
Experimental results show that different species of bacteria, yeasts, yeastlike fungi
and ambrosial fungi compose the symbiotic community of microorganisms with the
ambrosia beetle. Norris (1979) used for the assemblage of symbiotic micro- and
macroorganisms (including the beetle) the term a multi-species complex or a
supraspecies. The activities of the microbial complex as a whole, not just those of
the ambrosial fungus, allow such beetles to indirectly utilize nutrient-poor substrates
as wood.
Nevertheless, many questions concerning the relationship and interaction
between symbiotic fungi and ambrosia beetles are still open. The following
problems are perhaps the most interesting for research in near future: isolation and
determination of fungi species associated with the ambrosia beetles in different
countries, antagonism and synergism among symbiotic fungi in vivo and in vitro, the
influence of fungi on wood decay and utilization of wood components, succession of
fungi in attacked wood etc.

192

 Materials and Methods

Isolation of fungi
Spruce logs containing galleries of active T. lineatum were identified by the
presence of the whitish bore-dust at the entrance holes in bark crevices and by
chiseling into galleries. 20cm-thick disks of the logs were removed in areas
containing the highest number of the entrance holes. These disks were then brought
to laboratory for dissection and isolation of associated fungi.
Isolations were made from the different parts of the galleries: entrance tunnels,
lateral mother tunnels, axial larval cradles, wood near tunnels and abandoned
tunnels. Samples were taken with sterile forceps and scalpel. Each sample was
brought on the surface of potato-dextrose agar (PDA).
Adult females were aseptically removed from the tunnels and allowed to walk on
agar surfaces, to transfer the spores of fungi to the medium.

Determination of decay capacity

Decay capacity of isolated fungi was determined by partly modified EN 113. In
laboratory conditions the wood samples of spruce (P. abies (L.) Karst.) and red pine
(P. sylvestris L.) were exposed to fungi activity on malt extract agar (MEA) in Kolle
flasks for 16 weeks. The decay of wood samples was determined by measuring
weight loss.

Enzyme activity
The presence of specific enzymes and their activity in the symbiotic fungi were
demonstrated by simple test methods. Cellulase activity was determined according
to Rautela and Cowling (1966), that of oxidase according to Bavendamm (1928) and
Jorgensen and Vejlby (1953). Activity of laccase, which is important for the splitting
of the phenol ring of the lignin complex, was determined according to Stalpers
(1978).

Results and discussion

From the different parts of the galleries of T. lineatum, four species of symbiotic
fungi were constantly isolated: Trichoderma harzianum Rifai, yeast Pichia anomala
(Hansen) Kurzman, blue-stain fungus Ceratocystis piceae (Mnch) Bakshi (anam.
Graphium) and a fungus closely similar to Ceratocystis araucariae (anam. 'FsCa')
(Table 1).
The ambrosial fungus of T. lineatum known as Ambrosiella ferruginea
(Mathiesen-Krik) Batra was not isolated. The four species of fungi were found
during the pupa, callow adult and adult beetle stage. Some other microorganisms
were isolated from either the beetles or their galleries, but not from both. They were
found at only one, or a very few locations on beetles or in galleries. Therefore, they
were not considered as symbionts and not determined.
Nevertheless, no report on the establishment of T. harzianum, P. anomale and
FsCa from the galleries of T. lineatum were found in the literature. Consequently,
this might be the first observation of the symbiosis of T. lineatum with these fungi. It
is known for a long time that C. piceae is an important member of the fungal
community of microbes associated with different ambrosia and bark beetles (Bakshi

193

1950, Wilson 1959, Krik 1971). Because of limited and extremely slow growth P
anomala was not used in further experiments.

Table 1: Symbiotic fungi isolated from the different parts of the galleries of Trypodendron lineatum (Ol.).

Symbiont Entrance tunnel Lateral mother tun Axial larval cr Wood near t Abandoned tunnel
T. harzianum + + + + +
P. anomala + + + - -
C. piceae + + + + +
FsCa + + + + +

The symbiotic fungi caused the stain of spruce and red pine wood after 16
weeks, but did not produce any major weight loss of wood samples. Weight loss
was found to be the highest in samples exposed to C. piceae, and medium weight
loss in samples exposed to FsCa which caused the most intensive wood staining.
The lowest weight loss was observed in samples exposed to T. harzianum
which discoloured the wood by forming masses of pigmented spores on the wood
surface (Table 2). C. piceae caused 11.4 % weight loss in American beech and 17.3
% in sugar maple after 3 months (Eslyn and Davidson, 1976). Fungus can act as
typical soft-rot and attack the S2 layer of tracheid walls (Levy, 1967).

Table 2: Weight loss of wood samples (%) produced by symbiotic fungi of Trypodendron lineatum (Ol.).

Symbiont cellulase oxidase laccase

T. harzianum + -
C. piceae ++ ++ +++
FsCa +++ - +++

a) mean values of weight loss of ten samples; b) standard deviation

Specific enzymes are needed for the fungi to penetrate the wood elements.
Cellulase was proved in T. harzianum, C. piceae and FsCa. Laccase activity was
observed in FsCa and C. piceae which produced also oxidase.T. harzianum did not
synthesize any of these enzymes (Table 3).

Table 3: Enzyme activity of symbiotic fungi of Trypodendron lineatum (Ol.). - = no activity; + = slight

Symbiont Red pine Spruce

xa SDb xa SDb
T. harzianum 0.05 0.002 0.07 0.002
C. piceae 2.21 0.012 1.02 0.009
FsCa 0.79 0.012 0.12 0.006

activity: ++ = medium activity; +++ = strong activity.

References
Annila E. 1975. Effect of felling date of trees on the attack density and flight activity of Trypodendron
lineatum (Oliv.) (Col., Scolytidae). Comm. Inst. For. Fenn. 86: 6, 28 p.
Baker J.M. 1963. Ambrosia beetles and their fungi, with particular reference to Platypus cylindrus Fab.
Sym. Soc. Gen. Microbiol. 13: 232-265.

194

Bakshi B.K. 1950. Fungi associated with ambrosia beetles in Great Britain. Trans. Brit. Mycol. Soc. 33:
111-120.
Batra L. R. 1963. Ecology of ambrosia fungi and their dissemination by beetles. Trans. Kansas Acad. Sci.
66: 213-236.
Bavendamm W. 1928. Uber das Vorkommen und den Nachweis von Oxisasen bei Holzzerstorenden
Pilzen. Zuschr. Pflanzenkrank. u. Pflanzenschutz 38: 257-276.
Eslyn W.E. and Davidson R.W. 1976. Some wood-staining fungi from pulpwood chips. Mem. N.Y. Bot.
Garden 28(1): 50-57.
Jorgensen E. and Vejlby K. 1953. A new polyphenol oxidase test. Physiologia Plantarum 6: 533-537.
Krik A. 1971. The relation beetwen blue-stain fungi and bark beetles. IRG/WP Bull.19, 6 p.
Levy J.F. 1967. Decay and degrade of wood by soft-rot fungi and other organisms. Inter. Pest Control
(Nov./Dec.): 28-34.
Norris D.M. 1965. The complex of fungi essential to the growth and development of Xyleborus sharpi in
wood. Mat. u.Organ. Beih. 1: 523-529.
Norris D.M. 1979. The mutualistic fungi of Xyleborini beetles. In Insect fungus symbiosis.Batra, L.R. ed.,
Allanheld, Osmun and Co, Montclair, pp.53-63.
Rautela G.S.and Cowling E.B. 1966. Simple cultural test for relative cellulolitic activity of fungi. App.
Microb.: 892-898.
Stalpers, J.A. 1978. Identification of wood-inhabiting Aphyllophorales in pure cultures. CBS Studies in
mycolog 16: 1-248.
Uusvaara O. and Loyttyniemi K. 1975. Effect of injury caused by the ambrosia beetle (Trypodendron
lineatum Oliv., Coleoptera, Scolytidae) on saw timber quality and value. Folia Forestalia 231: 1-14.
Wilson C.L. 1959. The Columbian timber beetle and associated fungi in white oak. For. Sci. 5: 114-127.
______

195

 CERCOSPOROID FUNGI ASSOCIATED WITH THE EYESPOT DISEASE

OF GRAMINICOLOUS HOSTS

G.F. CAMPBELL1, B. ROBBERTSE2, P.W. CROUS2, AND B.J.H. JANSE1

Departments of 1Microbiology and 2Plant Pathology, University of Stellenbosch, South Africa

Introduction
The fungus commonly associated with eyespot disease symptoms of wheat was
first described as Cercosporella herpotrichoides Fron (Sprague 1936). Based on the
unthickened, inconspicuous conidial scars, several Cercosporella species were
reallocated to the newly erected genus Pseudocercosporella Deighton (1973). Two
varieties of P. herpotrichoides (Fron.) Deighton were distinguished, namely P.
herpotrichoides var. herpotrichoides and P. herpotrichoides var. acuformis
Nirenberg (Nirenberg 1981). In addition, P. anguioides Nirenberg and P. aestiva
Nirenberg were also described from eyespot symptoms on cereals in Germany
(Nirenberg 1981). In a study of the various Mycosphaerella Johanson species and
their respective anamorphs (von Arx 1983), P. herpotrichoides was reallocated to
the genus Ramulispora Miura as R. herpotrichoides (Fron) Arx . This decision was
chiefly based on the fact that Ramulispora species had conidia forming lateral
branches. The genus was furthermore restricted to all species occurring on Poaceae.
Pseudocercosporella species were recognized as anamorphs of Mycosphaerella
(Corlett 1991). The description of the teleomorph of Ramulispora herpotrichoides as
Tapesia yallundae Wallwork & Spooner (1988) [0culimacula yallundae (Wallroth
& Spooner) Crous & W. Gams, 2003, ndlr], further supported its separation from
Pseudocercosporella.
New combinations for the varieties of R. herpotrichoides were subsequently
introduced as R. herpotrichoides var. herpotrichoides (Fig. 1) and var. acuformis
(Nirenberg) Boerema, Pieters & Hamers (1992). This variety was later reconsidered
at species level as R. acuformis (Nirenberg) Crous. Braun (1993) furthermore
reduced Pseudocercosporella. anguioides to an additional variety of R.
herpotrichoides. No reasons were given, however, for not retaining it as a separate
species in Ramumispora. Because of the uncertainty surrounding the generic
placement of Pseudocercosporella aestiva (Fig. 2), the latter species was not
introduced into Ramulispora.
Using isozyme and mitochondrial DNA (DNA) banding patterns, the three
varieties of Ramulispora herpotrichoides could clearly be distinguished from P.
aestiva (Julian & Lucas 1990, Priestly et al. 1992; Nicholson et al. 1993), which
appeared to be unrelated to R. herpotrichoides. The aim of the present study,
therefore, was to use scanning electron microscopy (SEM) and random amplified
polymorphic DNA (RAPDs) to investigate the generic and species status of the four
taxa discussed above.

Material and Methods

Scanning electron microscopy
Verified isolates of R. herpotrichoides var. herpotrichoides (CBS 494.80), var.
acuformis (CBS 495.80), var. anguioides (86.45.64C) and P. aestiva (CBS 497.80)
were used to compare their conidial morphology using SEM. Sporulation of isolates
were obtained after 2 wks on synthetic nutrient agar (SNA) (Nirenberg 1981) at
196

15C. Specimens for SEM were cut from colonized agar, fixed overnight in 3%
glutaraldehyde, and rinsed in 0.05 M Na Cacodylate buffer the next day. The
material was further fixed in 2% Osmium tetroxide in 0.05 M Na Cacodylate buffer,
rinsed in the latter buffer, and dehydrated in a series of alcohol concentrations.
Specimens were critical point dried, coated with gold and viewed with a Jeol JSM
6100 scanning electron microscope. Specimens were also viewed using kryo-
fixation.

DNA isolation
Colonized agar plugs from 14-d-old colonies grown on PDA were inoculated
into 200 ml liquid cultures (8 g/l yeast extract and 5 g/l glucose). Cultures were
incubated at 25C for 10 d on an orbital shaker and the mycelia harvested by
filtration (Whatman no.1 filter paper). Freeze-dried mycelia mixed with river sand
was ground to a fine powder with a mortar and pestle. Twenty ml lysis buffer (3%
[v/v] SDS; 1% [v/v] 2-mercaptoethanol; 50 mM Tris [pH 7.2] and 50 mM EDTA)
was added and the mixture incubated at 65C overnight. Cetyltrimethylammonium
bromide (CTAB) and 5 M NaCl were then added to a final concentration of 1%
[v/v] and 0.7 M respectively and incubated for 4 hrs at 65C. The mixture was
extracted with phenol/chloroform/isoamylalcohol (25:24:1) (PCI), and followed by
two extractions with chloroform/isoamylalcohol. The resulting aqueous fraction was
mixed with 7.5M NH4OAc and 0.54 volumes isopropanol and incubated at -20C
overnight to precipitate the nucleic acids. After centrifugation at 5000 rpm for 5
min, the pellet was washed with 70% [v/v] ethanol, vacuum dried and dissolved in
300 l TE buffer (10 mM Tris [pH 8.0] and 1 mM EDTA). The sample was treated
with Rnase A (10 mg/ml) and incubated at 37C for 90 min after which the DNA
was PCI extracted, precipitated with isopropanol in the presence of NH4Oac,
washed, vacuum dried and redissolved in 300 l TE buffer.

RAPD analysis
The 10-mers used as random primers were purchased from Operon Technologies
(Kit E, Operon Technologies Inc. Alameda CA94501, USA). Taq Polymerase
together with its 10X concentrated buffer (100 mM Tris-HCl [pH 8.3] [20C]; 15
mM MgCl2; 500 mM KCl) was supplied by Boehringer Mannheim (Boehringer
Mannheim, Johannesburg, South Africa). PCR reaction mixtures (50 l final
volumes) contained 25 ng genomic DNA, dATP, dCTP, dGTP and dTTP each at
200 M final concentration, 100 nM oligonucleotide primer, 1X Taq polymerase
buffer and 1 unit of Taq polymerase. One mM MgCl2 was also added. Each reaction
was overlaid with a 100 l of mineral oil to prevent evaporation. The random
sequence primers used were OPE-14 5TGCGGCTGAG and OPE-15
5ACGCACAACC. Amplifications were conducted in a Hybaid Omnigene (Hybaid
Ltd., Waldegrave Rd., Middlesex, UK) thermal cycler. Samples were subjected to
60 repeats of the following cycle: 1 min at 94C, 1 min at 36C and 2 min at 72C.
After the last cycle, a final extension step of 72C for 5 min was included, followed
by cooling to 4C until recovery of the samples. Electrophoresis of aliquots (15 l)
of amplification products was through 1.4% agarose gels using 1X TAE buffer.
Phage lamda DNA digested with EcoRI and HindIII was used as a standard.

197

 Results and Discussion

Generic placement
When von Arx (1983) separated the graminicolous Pseudocercosporella species
into Ramulispora, it was chiefly based on the hosts falling in the Poaceae, and the
conidia having the ability to form lateral branches.
All species of Pseudocercosporella studied by the present authors have been
found to have conidia arranged in dry masses. However, conidia of R. sorghi (Ell. &
Ev.) Olive & Lefebvre, the type species of Ramulispora, aggregate in slimy masses.
The same phenomenon has also been observed for R. herpotrichoides.
With the description of the teleomorph of R. herpotrichoides var.
herpotrichoides in Tapesia, additional support was given for the separation of these
anamorphs from Pseudocercosporella. Furthermore, based on the research of King
(1990), T. yallundae var. acuformis Boerema, R. Pieters & Hamers (1992) has also
recently been described as the teleomorph for R. herpotrichoides var. acuformis.and
later named Tapesia acuformis [Oculimacula acuformis (Boerema, R. Pieters &
Hamers) Crous & W. Gams, 2003; ndlr].
Other than Tapesia and Mycosphaerella teleomorphs, the formation of lateral
conidium branches is the chief morphological character proposed by von Arx (1983)
to separate Pseudocercosporella from Ramulispora. Von Arx (1983) clearly
illustrated lateral branches in Ramulispora sorghi and R. herpotrichoides as is
typically observed in other Cercosporoid genera such as Mycovellosiella Rangel
(Crous & Braun 1994). However, in a paper dealing with the morphology of R.
sorghi, Olive et al. (1946) presented several figures indicating that the lateral
branches in R. sorghi are in fact secondary conidia forming directly on the surface of
primary conidia. Chang and Tyler (1964) were the first to report microcyclic
conidiation in R. herpotrichoides var. herpotrichoides. Fernandez et al. (1991)
recently defined this process of conidiation in Cercospora kikuchii (Matsumoto &
Tomoyasu) M.W. Gardner. Although Chang and Tyler (1964) reported short
secondary conidiophores to be present, the latter was not observed in our study.
Secondary conidia were, however, found to develop directly on the surface of
primary conidia. Microcyclic conidiation, also known as iterative germination or
precocious sporulation was defined as the recapitulation of conidiation following
conidial germination without an intervening phase of mycelial growth.
The phenomenon observed for R. herpotrichoides and P. aestiva in the present
study was identical to that illustrated by Olive et al. (1946) for R. sorghi, and
distinct from microcyclic conidiation (sensu Fernandez et al. 1991), and lateral
branches (sensu Crous & Braun 1994). We conclude, therefore, that in Ramulispora
the formation of secondary conidia and not lateral branching or microcyclic
conidiation occurs. The process should therefore be seen as conidia forming directly
on the surface of the primary conidia, without the presence of an intervening germ
tube with conidiogenous locus.
From these data it is clearly shown that the three varieties discussed above are
presently best retained in Ramulispora, as they cannot be accommodated in
Pseudocercosporella. The generic placement of P. aestiva remains, however,
uncertain. The present study suggests that the possible collection of its teleomorph
and further molecular studies will prove necessary to resolve the generic position of
P. aestiva.

198

Species delimitation

Ramulispora herpotrichoides var. herpotrichoides & var. acuformis

Several morphological criteria are used to distinguish var. herpotrichoides and
var. acuformis. Conidia of var. herpotrichoides are curved, whereas those of var.
acuformis are straight (Nirenberg 1981). Our SEM study showed both var.
herpotrichoides and var. acuformis to have polyblastic conidiogenous cells (Fig. 3).
When cultured on PDA, var. herpotrichoides has fast growing colonies with smooth
margins, whereas R. herpotrichoides var. acuformis has slow growing colonies with
feathery margins (Lange-de la Camp 1966, Scott et al. 1975). The two varieties also
differ in their pathogenicity towards wheat and rye, with var. herpotrichoides being
more pathogenic to wheat than to rye, and var. acuformis being equally pathogenic
to wheat and rye (Lange-de la Camp 1966, Hollins et al. 1985). Furthermore, an
ultrastuctural study showed that the infection process on a wheat coleoptile was also
distinctly different for var. herpotrichoides and var. acuformis (Daniels et al. 1991).
Distinct mating populations have also recently been isolated for var. acuformis and
var. herpotrichoides in England (P. Dyer, personal communication). Molecular
markers including isozymes, restriction fragment length polymorphisms (RFLPs)
and RAPDs have successfully been used to distinguish var. herpotrichoides and var.
acuformis (Julian and Lucas, 1990; Nicholson et al. 1991, 1993, Nicholson and
Rezanoor 1994, Priestley et al. 1992, Thomas et al. 1992).
Using RAPDs, the present study supported the distinction made between var.
herpotrichoides and var. acuformis based on general morphology, cultural
characteristics, pathology, and molecular studies of other workers discussed above.
The F-values obtained with two primers in the present study indicated var.
herpotrichoides to have 50% similarity to var. acuformis. Using four primers, this
figure decreased to 35% (Fig. 5, Table 1). This low percentage similarity, together
with the other differences discussed above, suggests that these two varieties should
be seen as two separate species.

Ramulispora herpotrichoides var. anguioides

Nirenberg (1981) described var. anguioides as having grey- pink, velvety
colonies on PDA with even colony margins. Conidia were described as forming on
simple, seldomly branched conidiophores. Successful protoplast fusion between var.
herpotrichoides, var. acuformis and var. anguioides has recently been obtained by
Hocart & McNaughton (1994), indicating that these varieties are closely related.
Using isozyme electromorphs, var. anguioides has been shown to be distinct
from var. herpotrichoides, var. acuformis and P. aestiva (Julian and Lucas 1990,
Priestley et al. 1992). However, var. anguioides has been shown to have a certain
percentage of similarity with var. acuformis. Using RFLPs, Nicholson et al. (1993)
found that strong hybridization occurs between var. anguioides and var.
herpotrichoides, indicating that they may be closely related. However, DNA
electromorphs of the three varieties were distinct.
Using RAPDs, the present study found var. anguioides to be distinct from the
other two varieties and P. aestiva. F-values showed var. anguioides to have 17%,
9% and 8% similarity to var. herpotrichoides, var. acuformis and P. aestiva
respectively (Fig. 5, Table 1).

199

Pseudocercosporella aestiva:
Nirenberg (1981) described the conidiophores of var. anguioides as hyaline,
thin, polyblastic, simple, and seldomly branched. Results obtained in the present
study found all four taxa could have polyblastic conidiogenous cells. However, the
conidiogenous cells of var. herpotrichoides were frequently swollen and
ampulliform, whereas those of P. aestiva were mostly reduced (Fig. 4).
Isozyme and RFLPs have indicated little relationship between P. aestiva and the
three varieties of R. herpotrichoides (Julian and Lucas 1990, Nicholson et al. 1993,
Priestley et al. 1992).

Fig.1. Conidiophore of R. herpotrichoides var. herpotrichoides with ampulliform conidiogenous cells

giving rise to an aggregated, slimy mass of conidia.
Fig.2. Typical conidia of P. aestiva on SNA.
Fig.3. Conidium of R. herpotrichoides var. herpotrichoides attached to a polyblastic conidiogenous cell.
Fig.4. Older, swollen conidium of P. aestiva giving rise to a secondary conidium (arrow) via a
conspicuous conidiogenous locus (double arrow).

200

Fig.5. Polymorphic bands of amplified DNA of the three varieties of R. herpotrichoides and P. aestiva.
Lane 1: Phage lamda DNA digested with EcoRI (A) and HindIII (B) was used as a standard. Lane 2: R.
herpotrichoides var. herpotrichoides; Lane 3: R. herpotrichoides var. acuformis; Lane 3: R.
herpotrichoides var. anguioides; Lane 4: P. aestiva.

Table 1. Combined F-values (% similarity) following RAPD- analysis (2 primers) of the three varieties of
R. herpotrichoides and P. aestiva.

Using RAPDs, the present study supported these results, and also showed P.
aestiva to be distinct from the three varieties of R. herpotrichoides. F-values
between P.aestiva and var. herpotrichoides, var. acuformis and var. anguioides were
found to be 9%, 29% and 8% respectively, indicating little relationship (Fig. 4,
Table 1).

201

 In conclusion, the use of molecular techniques in Ramulispora is shown to

enhance the taxonomic importance of seemingly insignificant morphological
differences. Results obtained with SEM and RAPDs indicate that the four taxa
studied are distinct, and that P. aestiva is probably not a species of Ramulispora.
Furthermore, the low % percentage similarity obtained with RAPDs, as well as other
morphological differences suggest that the three varieties of R. herpotrichoides
should be treated as three separate species.

References
Arx, von, J.A. 1983. Mycosphaerella and its anamorphs. Proceedings of the Koninklijke Nederlandse
Akademie van Wettenschappen, Ser. C: Biological and Medical Sciences 86: 15-54.
Bateman G.L. 1988. Pseudocercosporella anguioides, a weakly pathogenic fungus associated with
eyespot in winter wheat at a site in England. Plant Pathology 37: 291-296.
Boerema G. H., Pieters R. and Hamers M.E.C. 1992. Check- list for scientific names of common parasitic
fungi. Supplement Series 2b (additions and corrections): Fungi on field crops: cereal and grasses.
Netherlands Journal of Plant Pathology 98: 1-32.
Braun. U. 1993. Studies on Ramularia and allied genera (VI). Nova Hedwigia 56: 423-454.
Chang E.P. and Tyler, L.J. 1964. Sporulation by Cercosporella herpotrichoides on artificial media.
Phytopathology 54: 729-735.
Corlett M. 1991. An annotated list of the published names in Mycosphaerella and Sphaerella. Mycologia
Memoir 18: 1-328.
Crous P.W. and Braun U. 1994. Cercospora species and similar fungi of South Africa. Mycological
Research 99:31-36.
Daniels A., Lucas J.A. and Peberdy J.F. 1991. Morphology and ultrastructure of W and R pathotypes of
Pseudocercosporella herpotrichoides on wheat seedlings. Mycological Research 95: 385-397.
Deighton F.C. 1973. Studies on Cercospora and its related genera. IV. Cercosporella Sacc.,
Pseudocercosporella gen. nov. and Pseudocercosporidium gen. nov. Mycological Papers 133: 1-62.
Fernandez F.A., Glawe D.A. and Sinclair J.B. 1991. Microcycle conidiation and nuclear behavior during
conidiogenesis in Cercospora kikuchii. Mycologia 83: 752-757.
Hocart M.J. and Mc Naughton J.E. 1994. Interspecific hybridisation between Pseudocercosporella
herpotrichoides and P. anguioides achieved through protoplast fusion. Mycological Research 98: 47-
56.
Hollins T.W., Scott P.R. and Paine J.R. 1985. Morphology, benomyl resistance and pathogenicity to
wheat and rye of isolates of Pseudocercosporella herpotrichoides. Plant Pathology 34: 369-379.
Julian A.M. and Lucas J.A. 1990. Isozyme polymorphisms in pathotypes of Pseudocercosporella
herpotrichoides and related species from cereals. Plant Pathology 39: 178- 190.
King A.C. 1990. First record of Tapesia yallundae as the teleomorph of Pseudocercosporella
herpotrichoides var. acuformis, and its occurrence in the field in the Federal Republic of Germany.
Plant Pathology 39: 44-49.
Lange-de la Camp M. 1966. Die Wirkungsweise von Cercosporella herpotrichoides Fron dem Erreger
der Halmbruchkrankheit des Getreides. II. Aggressiviteit des Erregers. Phytopathologische Zeitscrift
56: 155-190.
Nicholson P., Hollins T.W., Rezanoor H.N. and Anamthawat-Jonsson K. 1991. A comparison of cultural,
morphological and DNA markers for the classification of Pseudocercosporella herpotrichoides.
Plant Pathology 40: 584-594.
Nicholson P., Rezanoor H.N. and Hollins T.W. 1993. Classification of a world-wide collection of isolates
of Pseudocercosporella herpotrichoides by RFLP analysis of mitochondrial and ribosomal DNA and
host range. Plant Pathology 42: 58-66.
Nicholson P. and Rezanoor H.N. 1994. The use of random amplified polymorphic DNA to identify
pathotype and detect variation in Pseudocercosporella herpotrichoides. Mycological Research 98(1):
13-21.
Nirenber H.I. 1981. Differenzierung der Erreger der Halmbruchkrankheit. I. Morphologie. Zeitscrift fr
Pflanzenkrankheiten und Pflanzenschutz 88: 241-248.
Olive L.S., Lefebvre C.L. and Sherwini H.S. 1946. The fungus that causes sooty stripe of sorghum spp.
Phytopathology 36: 190-200.
Priestly R.A., Dewey F.M., Nicholson P. and Rezanoor H.N. 1992. Comparison of isoenzyme and DNA
markers for differentiating W-, R- and C-pathotypes of Pseudocercosporella herpotichoides. Plant
Pathology 41: 591-599.
Scott P.R., Hollins T.W. and Muir P. 1975. Pathogenicity of Cercosporella herpotrichoides to wheat,

202

 barley, oats and rye. Transactions of the British Mycological Society 65: 529-538.
Sprague R. 1936. Relative susceptibilty of certain species of gramineae to Cercosporella herpotrichoides.
Journal of Agricultural Research 53: 659-670.
Thomas D., Maraite H. and Boutry M. 1992. Identification of rye- and wheat-types of
Pseudocercosporella herpotrichoides. Journal of General Microbiology 138: 2305-2309.
Wallwork H. and Spooner B. 1988. Tapesia yallundae, the teleomorph of Pseudocercosporella
herpotrichoides. Transactions of the British Mycological Society 91: 703- 705.

______

DIVERSITY OF COLLETOTRICHUM GLOEOSPORIOIDES PATHOGENIC

TO STYLOSANTHES SP. IN TROPICAL AREAS

F. MUNAUT, N. HAMAIDE & H. MARAITE

Universit catholique de Louvain, Facult des Sciences agronomiques, Unit de Phytopathologie, Place
Croix du Sud 2 bote 3, B-1348 Louvain-la-Neuve, Belgium.

Abstract. Virulence spectra of Colletotrichum gloeosporioides strains isolated from Stylosanthes sp.
sampled in Africa and Australia were investigated on a set of Stylosanthes genotypes. Virulence spectra
as well as aggressiveness of strains were highly variable. The distinction of strains causing type A
(restricted lesions) and type B (extensive necrosis) is confirmed. Esterases extracted from both types of
strain showed isozymic variation. Polymorphism was observed between and within types ; the latter can
be clearly separated by specific bands. Strains isolated from Stylosanthes sp. presented esterase patterns
different from those of strains isolated from other tropical plants. By Random Amplified Polymorphic
DNA (RAPD) technique markers have been identified which separate strains of types A and B.
Polymorphism was also observed among strains of type B from various geographical origins.

Introduction
Stylosanthes guianensis is a tropical pasture legume very interesting for its
tolerance to dry conditions in tropical areas. Since 1970's, anthracnose caused by
Colletotrichum gloeosporioides Penz. is an important limiting factor in South
America (Grof et al. 1979), Africa (Clatworthy 1975) and Australia (O'Brien and
Pont 1977). Selection of resistant genotypes is commonly used to control
anthracnose, but strains able to partially or totally overcome the resistance appear
sometimes rapidly.
In order to understand the mechanisms of this pathogen-host coevolution and to
assist in the collection and selection of resistant Stylosanthes genotypes, the
diversity in pathogenicity, DNA and isozymic patterns of C. gloeosporioides strains
from various hosts and origins is investigated.

Material and methods

Pathogenicity test
Virulence spectra of C. gloeosporioides strains isolated from Stylosanthes
sampled in Africa and Australia (Table 1) were investigated on a set of S. guianensis
5 6
genotypes by inoculation of conidia suspensions (10 -10 conidia/ml) on first-leaf
stage plants. Plants were kept 48 h in the dark at 23 C before further incubation
under a photoperiod of 14 h. Symptom severity was quoted after eleven days.

203

Esterase assay
Esterases were extracted from mycelium and conidia grown in V8 liquid cultures
in a Tris buffer at pH 7.5, loaded on non-denaturing gel for electrophoresis and
stained with the a-naphtyl acetate/fast red TR salt method (Shaw and Prasad 1970).
DNA study
DNA was extracted by the CTAB method (Roger and Bendich 1985). The
Random Amplified Polymorphic DNA (RAPD) was developed to distinguish
strains, using primers from the Kit A of Operon Technologies Inc. and the
DynaZyme polymerase (Techgen International).

Table 1. Host, type, geographical origin of Colletotrichum gloeosporioides strains

1
Strain Host Type Location

HM 334 Stylosanthes sp. A Australia

HM 335 Stylosanthes sp. A Australia
HM 336 S. guianensis cv. Graham B Australia
HM 337 S. guianensis B Australia
HM 313 S. guianensis B Burundi
HM 312 S. guianensis B Zaire
HM 373 S. guianensis cv Cook B Zaire
HM 376 S. guianensis B Zaire
HM 502 S. guianensis cv. Cook B Zaire
HM 495 S. guianensis cv. CIAT 184 B Ivory Coast
HM 498 S. guianensis cv. CIAT 11366 B Ivory Coast
HM 514 S. guianensis cv. CIAT 184 B Ivory Coast
HM 515 S. guianensis cv. Cook B Ivory Coast
1
Type of symptom from which the strain was isolated : A, restricted clear spots with a brown margin ; B,
extensive dark brown necrosis.

Preliminary results and discussion

Pathogenicity
Two types of anthracnose, previously described by Irwin and Cameron (1978),
are distinguished on diseased samples and in inoculation tests. Symptoms caused on
the three genotypes inoculated with the strain of type A were characterized by a
clear spot surrounded by a dark margin. Susceptible genotypes infected with strains
of type B presented a general necrosis of plants, starting by dark brown spots
extending on the leaflets, causing their premature shedding as also on the stem.
Virulence spectra as well as aggressiveness of strains appeared highly variable (Fig.
1), symptoms induced ranging from a few small spots to death of plants.
Evaluation of the degree of necrosis from 1 (spots less than 1 mm in diameter) to
5 (more than 75 % necrosis of the leaf surface), is made by visual inspection of the
first leaf inoculated. For each strain the results are expressed by the mean indice of
the three leaflets of eighteen plants per genotype. A mean index is calculated for
each genotype.

Esterases
Isozymic variation was observed for esterases extracted from both types of
strain. Types could be clearly separated by specific bands. Polymorphism was also
observed among strains of type B. Strains isolated from various other tropical plants
204

presented esterases patterns different from the strains isolated from Stylosanthes sp.
Moreover, subcultures from sector appearing on PDA cultures of a C.
gloeosporioides strain isolated from banana, showed different isozymic patterns.

DNA polymorphism
RAPD permitted to identify markers which clearly separate type A and B.
Polymorphism was also observed among type B strains which could be grouped
according to geographical origin

4 HM 335
HM 373
SEVERITY INDICES

HM 502
3
HM 498
HM 514
2 MEAN

CIAT 10136 CIAT 184 SCHOFIELD

Figure 1. Anthracnose severity on the first leaf of three Stylosanthes guianensis genotypes, 11 days after
inoculation with five Colletotrichum gloeosporioides strains

Strains from Australia, Burundi and Zaire were closely related while those isolated
in Ivory Coast formed a second distinct group. Subcultures of strain HM 498 and
from sectors of the HM 515 presented DNA polymorphism.

Prospects
Study of coevolution of Colletotrichum gloeosporioides and Stylosanthes sp. is
planned in collaboration with the Universidad Nacional Autonoma Mexico and the
Laboratorium voor Gentechnologie (Katholieke Universiteit Leuven) in the frame of
a project coordinated by the International Board for Plant Genetic Resources
(IBPGR).

Acknowledgements
The authors are grateful to IBPGR and the Belgian Administration for Development Cooperation for
funding this research project.

References
Clatworthy J.N. 1975. Introduction and preliminary screening of pasture legumes at Marandellas,
Rhodesia. Proceedings of the Grassland Society of Southern Africa 10: 57-63.
Grof B.R., Schultze-Kraft R.and Muller F. 1979, Stylosanthes capitata Vog., some agronomic attributes,
and resistance to anthracnose (Colletotrichum gloeosporioides Penz.). Tropical grasslands 13: 28-37.
Irwijn J. A.G. and Cameron D.F. 1977. Two diseases in Stylosanthes spp. caused by Colletotrichum
gloeosporioides in Australia, and pathogenic specialization within one of the causal organisms.
Australian Journal of Agricultural Research 29: 305-317.
O'Brien R.G.and Pont W. 1977. Diseases of Stylosanthes in Queensland. Queensland Agricultural
Research 103: 126-128.

205

Rogers S.O.and Bendich A.J. 1985 Extraction of DNA from milligram amounts of fresh, herbarium and
mummified plant tissues. Plant Molecular Biology 5: 69-76.
Shaw R.and Prasad R. 1970. Starch gel electrophoresis.
A compilation of recipes. Biochemical Genetics 9: 297-320.
________

DIVERSITY IN NON-SPECIFIC FOLIAR PATHOGENS OF WHEAT

FROM NON-TRADITIONAL WARM AREAS

DI ZINNO T., DUVEILLER E.*, LONGREE H. AND MARAITE H.

Unit de Phytopathologie, Facult des Sciences Agronomiques, Universit Catholique de Louvain (UCL),
Place Croix du Sud, 2, Bte 3, 1348 Louvain-la-Neuve, Belgium.
*Centro Internacional de Mejoramiento de Maz y Trigo (CIMMYT), Lisboa 27, Apdo. Postal 6-641,
06600 Mexico D.F., Mexico.

Abstract. In order to characterize the variability of the non-specific foliar pathogens of wheat from warm
areas, a world-wide collection of strains is established through a collaboration between CIMMYT,
Mexico and UCL, Belgium. By isolation of leaf samples sent from non-traditional warm area (South
Asia, Africa, Latin America) in 1993 and 1994, we confirmed that Bipolaris sorokiniana is the most
important foliar pathogen followed by Drechslera tritici-repentis, representing respectively 75 and 17%
of the fungi isolated from leaf spots in 1993. This collection is the base for characterisation of the NSFP
diversity by pathogenicity tests on host differentials of wheat lines developed at CIMMYT, by an analysis
of the spectrum of toxins produced and by studies of genetic diversity with RAPD.

Introduction
Changes in cropping systems and progresses obtained in genetic resistance to
classical diseases like rust, led to the important development of the non-specific
foliar pathogens (NSFP) of wheat. With the support of the Belgian Agency of
Development Cooperation, CIMMYT, Mexico and UCL, Louvain-la-Neuve
collaborate to evaluate in warmer areas the sustainability of wheat production
related to cropping systems and to promote research on resistance to this
pathosystem.
In non-traditional warm wheat growing areas, helminthosporium blights, spot
blotch and tan spot caused respectively by Bipolaris sorokiniana (Sacc. in Sorok)
Shoem., teleomorph Cochliobolus sativus (Ito & Kurib.) Drechs. ex Dastur and
Drechslera tritici-repentis (Died.) Shoem., teleomorph Pyrenophora tritici-repentis
(Died.) Drechs., are considered as the most important foliar pathogens, the incidence
of other pathogens such as Alternaria triticina being poorly documented (various
authors in Saunders 1990).
Data concerning variability among strains are useful to assist selection of
resistant or tolerant cultivars. New lines being selected in "hot spot" fields where the
pathogen is naturally present at a high pressure, it is important to determine if
variability of pathogenicity among the strains occurring in these fields is
representative of variability found in other locations. Differences in virulence among
strains of B. sorokiniana of a same country were shown i.e. for Thailand by Hetzler
(1992) and for Brazil by Mehta (1981). Concerning D. tritici-repentis, different
pathotypes producing different type of lesions in correlation with toxin production
were described by Lamari and Bernier (1989).
A reference collection of pathogens from diverse origins is thus the first priority.
This will be achieved through a collaborative network gathering UCL, CIMMYT
and National Agricultural Research Systems from the warm wheat growing area.

206

 Material and methods

Foliar samples were collected during 1993 and 1994 principally in the South
Asian region (Bangladesh, Nepal and India) but also in South-Africa and Latin
America in farmer or trial fields in characteristic locations on a great range of wheat
varieties, some classical in these environments like Sonalika and Kanchan, some
new resistant lines like crosses with Agropyrum curvifolium.

Others
D. tritici-repentis
B. sorokiniana
100

80

60

40

20

0
Total Bangladesh Nepal India
warm

Figure 1: Distribution of the NSFP isolated from wheat in South Asian area in 1993: the number of strains
isolated was 77 for all warm areas, 30 for Bangladesh, 26 for Nepal and 6 for India.

Lesions on wheat leaves and stems are observed under stereomicroscope and
microscope to detect fungal fructifications.
After description, pieces of lesions are surface sterilized with NaOCl (0.5%
during 3 minutes), rinsed with three changes of sterile water and placed in Petri
dishes containing water agar (1.5%, streptomycin 150mg/l). After incubation in the
dark at 20C, the plates are placed under continuous light (15cm below a white and a
near-ultra-violet tube) at room temperature to develop conidiophores and then in the
dark at 20C to induce sporulation. Conidia are transferred to fresh water agar plates
until they germinate and then on V8-PDA plates. Monoconidial cultures are
conserved in V8 slants under oil.

Results
Symptoms of foliar blights range from little black points to complete chlorosis
and necrosis of the leaves, besides the characteristic symptoms of helminthosporium
leaf blights: oval to elongated, light brown to dark brown blotches with or without a
central dark spot.

The association of Bipolaris sorokiniana with the symptoms can be easily

identified by the help of a stereomicroscope by the presence of dark conidia and for
207

Drechslera tritici-repentis under a light microscope by the presence of conidia

showing the characteristic conical basal cell (Drechsler 1923). However if no
conidia are visible, seen the diversity of pathotype-genotype combinations and of
environmental conditions, a distinction between leaf symptoms of the species is
almost impossible without isolation.
In 1993, a majority of Bipolaris sorokiniana (75%) has been isolated from samples
with foliar blights from warm areas. The rest is made up of 17% of Drechslera
tritici-repentis and 8% of other fungi like Curvularia sp. and Alternaria sp (fig. 1).
This distribution is globally the same in the particular environments analysed
(Bangladesh, Nepal and India) and confirmed by the isolation of 1994.

Conclusion and prospects

This collection is the base for characterisation of the diversity of the non-specific
foliar pathogens of wheat in warm areas.
Four items will particularly interest us for this characterization:
- time course evolution of the NSFP distribution in warm area by the annual visit
and collect of samples in particular known locations and various cropping systems.
- variability in pathogenicity of the isolates from different location and year through
inoculation test on host differentials of wheat lines developed at CIMMYT.
- variability in the spectrum of toxins produced by the isolates: non-host-specific
Bipolaris toxins such as prehelminthosporal and prehelminthosporol (De Mayo et al.
1965); host-specific ones such as Drechslera toxins: necrosis and chlorosis inducing
toxins (Ballance et al. 1989, Brown and Hunger 1993)
- genetic diversity with RAPD.

References
Ballance G.M., Lamari L. and Bernier C.C. 1989. Purification and characterisation of a host-selective
necrosis toxin from Pyrenophora tritici-repentis. Physiological and Molecular Plant Pathology 35 :
203-213.
Brown D.A. and Hunger R.M. 1993. Production of a Chlorosis-Inducing, Host-Specific, Low-Molecular
Weight Toxin by Isolates of Pyrenophora tritici-repentis, Cause of Tan Spot of Wheat. Journal of
Phytopathology 137 : 221-232.
De Mayo P., William R.E. and Spencer E.Y. 1965. Terpenoids. VIII. The immediate precursor of
Helminthosporal and Helminthosporol. Canadian Journal of Chemistry 43 : 1357-1365.
Drechsler C. 1923. Some graminicolous species of Helminthosporium I. J. agric. Res., 24 : 641-739.
Hetzler J., 1992. Host-pathogen interaction in population of Bipolaris sorokiniana in the Warm Non-
Traditional Areas. Doctoral Dissertation, Institute of Plant pathology and Plant Protection,
Gttingen, 129 p.
Lamari L. and Bernier C.C. 1989. Wheat genotype that Virulence of isolates of Pyrenophora tritici-
repentis on 11 wheat cultivars and cytology of th differential host reactions. Canadian Journal of
Plant Pathology 11 : 284-290.
Mehta Y.R. 1981. Identification of races of Helminthosporium sativum of wheat in Brasil. Pesq. Agropec.
Bra. 16 : 331-336.
Saunders D.A. 1990. Wheat for the Nontraditional Warm Areas.UNDP/CIMMYT Iguau, Brazil, 549 pp.
______

208

 PATHOGENIC DIVERSITY IN MAGNAPORTHE GRISEA, THE RICE

BLAST PATHOGEN

J.B.BAHAMA, J.L.NOTTEGHEM* AND H.MARATE

Universit Catholique de Louvain, Facult des Sciences agronomiques, Unit de Phytopathologie, Place
Croix du Sud 2 bote 3, 1348 Louvain-la-neuve, Belgique
*Laboratoire de Phytopathologie CIRAD/IRAT , Av. du val de Montferrand B.P.5035, 34032 Montpellier
cedex 1, France

Abstract. Rice cultivation has been introduced in high elevation swamps of Burundi in 1980. Six years
after, the released cultivar, Yunnan3, was severely attacked by Magnaporthe grisea, the rice blast
pathogen. In order to identify resistant genotypes, race diversity encountered in this new rice growing
area was studied. The results discussed in this paper have been obtained by inoculating 25 isolates of M.
grisea on 27 rice cultivars including 13 Japanese differentials. All the known resistance genes are broken
down by Burundian isolates except Pi-ta2. A high diversity is thus established.

Introduction
Blast, caused by Magnaporthe grisea (Hebert) Barr (anamorph Pyricularia
oryzae Cavara), is one the most widespread disease of rice in tropics. All the rice
breeding programmes are aimed at controlling that disease by resistant cultivars.
As a preliminary to resistance breeding for blast, the pathogen variability has to
be well known. Virulence spectrum of isolates collected from rice growing areas has
to be determined.
In developping countries where rice is included in the subsistence crop system-a
low input one- vertical resistance, which is race specific, would be avoided since the
pathogen adapts to such cultivars very quickly. Moreover, when severe epidemics
break out, the farmers don't have means to afford fungicides. Hence, more efforts
must be done to select quantitative resistance which ensures durable protection and
more stable yield.
In Burundi, rice has been introduced in high elevations in 1980 with the release
of Yunnan 3. Six years after, in 1986, high damages were caused to this cultivar by
neck blast. Therefore, blast is taken into account in resistance breeding.
A collection of isolates was made up and a high diversity was observed by
inoculation on differentials and some reference cultivars from various rice growing
areas. Results of those tests are discussed below.

Symptom description
Magnaporthe grisea infects rice at all growth stages, resulting in two types of
symptoms: leaf blast and panicle blast.
The leaf spots are typically elliptical with usually grey or whitish center and a
brown or reddish-brown margin. The shape and colour vary depending upon the
degree of susceptibility of the cultivars and the environmental conditions. Highly
resistant cultivars develop minute brown specks of pin head size, while susceptible
ones growing in conducive conditions show no or very little brown margin.
Panicle blast can affect any part of the panicle but often its base, causing the
rotten neck also called neck rot. Panicles often fall over and grains are empty.
Panicle branches and glumes can also be attacked. This type of blast is the most
detrimental to yield.

209

 Material and methods

Twenty five isolates of M. grisea including 20 from Burundi (BD) and 5 from
Cameroon (CM28), Columbia (CL6), Ivory Coast (CD45), Korea (CR5) and
Philippines (PH11) were grown on rice flour agar medium (1 litre of water, 15g of
agar, 20g of rice flour) in Petri dishes at 28C; 60% of relative moisture and 12 h of
light per day. After 10 days of fungal growth, a spore suspension adjusted to 25,000
conidia/ml was injected between leaf sheaths with a hypodermic syringe.
Thirteen differentials carrying different resistance genes and 13 reference
cultivars were used in this experiment. Plants were grown in a greenhouse up to 4-5-
leaf stage in batches containing compost and watered with nutritive solution.
Seven days after inoculation, lesions were assessed using Notteghem's 6-class
scale where scores 1-3 correspond to resistant cultivars, 4-6 to susceptible ones.

Results
All the resistance genes carried by differentials are broken by Burundian isolates
2
except Pi-ta of Pi n4 (Table1). Isolates CL6, CM28 and CR5 from respectively
Columbia, Cameroon and Korea overcome Pi-ta2 gene. PH11, CL6, CM28, CR5
and CD45 are highly aggressive on IR841, IR8, L9 and Mafushi which are resistant
to Burundian isolates (Table2).
BD29, BD30, BD32, BD37 and BD38 attack Tetep which is widely used as
resistant donor in most of the rice breeding programmes. On IRAT13 only moderate
type lesions are observed. All the isolates severely infect Yunnan 3.

Discussion and Conclusion

There is a high pathogenic diversity in the M. grisea population from high
elevation swamps in Burundi despite the limited number of rice genotypes grown in
this environment. All the known resistance genes are broken down by Burundian
2
isolates except Pi-ta . The latter is, however, matched by isolates from other
geographic areas. In addition, all the isolates have overcome five genes or more.
Improving resistance strategies based on these genes separately or in combination
can thus not be effective for a long time. This suggest that in Burundian context,
2
cultivars carrying only major genes should be avoided even those carrying Pi-ta
since material exchange is not rigorously controlled.
More emphasize should be put on quantitative resistance which is often durable.
Cultivars with a good level of quantitative resistance such as IRAT13, L9 and
Mafushi should be used as parents. Promising progenies should be tested in a large
number of rice growing countries and tests would be repeated during several
seasons.
Recent molecular techniques especially Restriction Fragment Length
Polymorphism (RFLP), have enabled to map blast resistance genes conferring
quantitative resistance (Wang et al. 1993). Using that tool should help to find out
quantitatively resistant cultivars to broaden the resistance background.
Other workers (Notteghem 1993, Wang et al. 1993) propose combinating a
quantitative resistance with a qualitative one to face M. grisea variability.
To carry out this breeding, it is essential to understand the M. grisea population
structure. Appearance of new isolates and their spectrum should be monitored. DNA
markers identification has already proved its efficiency (Wolfe 1993).

210

 211

 Acknowledgement
The authors greatly thank the European Community and the Belgian Administration for Development
cooperation for their financial support.

References
Notteghem J.L. 1993. Durable resistance to rice blast disease. In Durability of Disease Resistance. Jacobs
Th., Parlevliet J.E. eds., Kluwer Academic Publishers. Dordrecht/Boston/London
Wang G.L., Mackill D.J., Bonmann J.M., McCouch S.R. and Nelson R.J. 1993. RFLP mapping of genes
conferring complete and partial resistance in a rice cultivar with durable resistance to blast. In
Durability of Disease Resistance. Jacobs Th. and Parlevliet J.E. eds., Kluwer Academic Publishers.
Dordrecht, Boston, London.
Wolfe M.S. 1993. Can the strategic use of disease resistant hosts protect their inherent durability? In
Durability of Disease Resistance. Jacobs Th. and Parlevliet J.E. eds., Kluwer Academic Publishers.
Dordrecht, Boston, London.
______

CHARACTERIZATION OF POLYMYXA GRAMINIS INVOLVED IN THE

TRANSMISSION OF PEANUT CLUMP VIRUS IN TROPICAL AREAS

A. LEGREVE1, B. VANPEE1, P. DELFOSSE2, D. V. R. REDDY2 & H. MARAITE1

1Unit de Phytopathologie, Universit catholique de Louvain, Facult des Sciences agronomiques, Place
Croix du Sud 2 bte 3, B-1348 Louvain-la-Neuve, Belgium .
2International Crops Research Institute for the Semi-Arid Tropics, Patancheru, Andhra Pradesh 502 324,
India.

Abstract. The plasmodiophoraceous fungus, Polymyxa graminis Ledingham, is involved in the

transmission of Peanut Clump Virus (PCV), a soil-borne virus widely distributed in India and in West
Africa. This fungus is known in temperate areas to be the vector of soil-borne viruses on barley, wheat
and oat. Ecological requirements of P. graminis isolates from various origins are possibly different.
Indian and African isolates indeed grow easily at 25-30C, whereas those from temperate areas are
favoured by temperatures between 15 and 20C. Host range of tropical isolates is apparently wider and
less specific than that of isolates from temperate areas. Indian P. graminis isolates grow on
monocotyledonous plants as well as on dicotyledonous plants, whereas in temperate areas, two fungal
species are listed as separate species according to the host specificity; P. graminis growing only on
graminaceous plants and Polymyxa betae Keskin on Chenopodiaceae. In order to clarify the taxonomic
position and precise the requirements of P. graminis associated with PCV transmission, pure strains
obtained from a single cystosorus are prepared from isolates of various origins. Host range, ecological
requirements, such as temperature optimum and pH, are studied. First results confirm differences in the
temperature optimum, the Indian strain growing best on sorghum between 20 and 30C compared to less
than 20C for the strains from northern origins on barley.

Introduction
Peanut clump is a soil-borne virus disease widely distributed in India and West
Africa. Infected plants are stunted conspicuously and have dark green leaflets.
Flowers can be produced, but any pods formed are poorly developed (Thouvenel et
al. 1976; Reddy et al. 1983). The virus is transmitted with seed from groundnut,
pearl millet and finger millet infected plants, but is also soil-transmitted by a
plasmodiophoraceous fungus, Polymyxa graminis Ledingham, an ubiquitous
obligate root parasite of graminaceous plants (Thouvenel et al., 1976; Thouvenel &
Fauquet 1981, Ratna et al. 1991). The resting spores of the fungus may contain the
virus and can survive in soils for many years. In specific conditions, spores
212

germinate and zoospores infect epidermic root cells of a host plant. The
plasmodium, the zoosporangium, producing secondary zoospores, and the
cystosorus, the survival stage of the fungus, are all formed in the infected cell.
In temperate areas, two fungal species are distinguished according to the host
specificity : P. graminis growing only on Poaceae (Ledingham 1939, Barr 1979,
Bastin et al. 1989, Adams 1990), and Polymyxa betae Keskin on Chenopodiaceae
and some related families (Barr 1979, Barr and Asher 1992). The two species are
known to be the vector of soil-borne viruses on their specific hosts. In India, P.
graminis isolates grow on monocotyledonous plants, such as pearl millet, sorghum
and wheat, as well as on different families of dicotyledonous plants including
Chenopodiaceae. According to this wider and less specific host range, the taxonomic
position of the P. graminis strains associated with peanut clump virus transmission
is questionable. Is it the same species as P. graminis found in temperate areas or is it
a third one of the same genus, able to infect and transmit virus(es) over a wider host
range? Is the host specificity a relevant criterion to separate species?
The in depth understanding of the P. graminis populations involved in the
transmission of the peanut clump virus and the acquisition of basic information for
development of an integrated control of PCV at small holdings level, are the main
objectives of the collaborative research project between Universit catholique de
Louvain - Unit de Phytopathologie and International Crops Research Institute for
the Semi-Arid Tropics.
The program started in March 1993 with the isolation of P. graminis from
various origins and the production of single cystosorus strains. The characterization
of the P. graminis populations involved in the transmission of the peanut clump
virus and the comparison with strains from temperate areas are in progress. The
results presented here concern a first experiment on the temperature requirements
for 5 strains from various origins.

Materials and methods

Single cystosorus cultures
P. graminis isolates were obtained by growing bait plants in infested soils from
various origins. Pieces of rootlets containing P. graminis cystosori were soaked in
sterile distilled water, cut in 1 or 2 mm fragments and homogenized in Omnimixer
Virtis at 25000 rpm. Few drops were spread on 2% water agar in Petri dishes. The
surface was carefully analysed with a Wild Macroscope at 40-100 x magnification to
detect single cystosorus.S ingle cystosori were taken with a micro spear and placed
adjacent to the roots of a 2-day-old pregerminated seedling planted in single tube on
sterile quartz sand. Hundred to 300 plants were each inoculated with a single
cystosorus for each isolates. The plants were watered with half-strength nutrient
solution and grown at 15-20C (night-day) or 25-30C according to the temperate or
tropical origin of the P. graminis isolates. After 2 or 3 months, each plant was
removed and checked under the microscope to detect the fungus. Infected roots were
dried and stored at room temperature.
Fungal multiplication was performed in an automatic immersion device. The
latter was constituted by PVC tubes suspended in a container, linked with a pump to
an independent nutrient solution tank. Host plants were planted in the tubes
containing sterile quartz sand inoculated with root extract infested with the P.
graminis strain. The basis of each tube was formed by a polyamid grid allowing
flowing in and out of nutrient solution and zoospores. Watering is programmed

213

during 6 hours each 12 hours. After 2 months, plants were removed and the roots
observed to ascertain the presence of the fungus. The strains are stored in dried root
fragments at room temperature.

Temperature requirement
Five single cystosorus strains were tested : strains I1-20 and I1-229 from India
multiplied in sorghum roots, and the strains B1 from Belgium, C1 from Canada and
F11 from France in barley roots.
Culture tubes filled with sterile quartz sand were inoculated with cystosori
suspensions of each strain (1000 cystosori/tube). The effect of temperature on the
development of temperate and tropical strains, respectively on barley or sorghum,
was tested by transplanting 2-day-old seedlings in inoculated sand and growing
them at the test temperatures, 10-15C, 15-20C, 20-25C, 25-30C or 30-35C
(night/12h-day/12h). Periodically, 4 plants grown at the various temperatures were
removed for each strain. The roots were coloured in blue lactophenol and observed
under the microscope for assessment of fungal infection.

First results
Up to now, six P. graminis strains have been obtained and multiplied: three
Indian strains on sorghum and isolated from a soil sampled at Patancheru in a field
infected by the peanut clump (strain I1-1, I1-20, I1-229), one Belgian strain on
barley and isolated from a soil sampled at Loupoigne in a field infected by the
barley yellow mosaic (strain B1), one Canadian strain on barley obtained from
barley roots grown on Ottawa soil in 1987 and send by Dr D. J. S. Barr (strain C1),
and one French strain on barley and isolated from a soil sampled in 1988 at
Carcassonne (strain F11).
Temperature requirements for P. graminis infection are different according to the
origin of the strains (Fig. 1). The two Indian strains have a narrow optimum
temperature around 25C. Below 20C and above 30C, any trace of infection was
not yet observed after six weeks. Strains from temperate areas are favoured by
cooler temperature: the strains B1 and C1 grew easily between 10 and 20C, and the
strain from the south of France grew only at 15-20C, 5 weeks after the inoculation.
Differences in infection capacity are also evident. Belgian strain B1 showed
100% infection on barley after 2 weeks, whereas minimum 4 weeks of incubation
are necessary to the strain C1 to reach this level. The 3 other strains seem to have a
lower infection capacity, especially the strains coming from India.

Discussion and prospects

The first results demonstrate differences in the temperature requirements of P.
graminis strains according to the origin, the Indian strains being able to develop at
temperatures inhibitory to the Belgian strain. The results with the Canadian and the
French strains suggest a continuous range of temperature optimum. Further
repetitions of these experiments will specify the differences in response at the
various growth stages of the fungus and the influence of the host in the observed
differences. In view to a better understanding of the P. graminis populations
involved in the transmission of the peanut clump virus, soil water potential and pH
requirements will also be tested. The host ranges including dicotyledonous plants
and weeds of the tropical strains will be analysed and compared to temperate P.
betae and P. graminis species. A morphological analyse of the various stages of the

214

fungus and a genomic analyse of the various strains on DNA fragments am plified
by PCR will be done to define the taxonomic position of the fungus involved in the
PCV transmission.

Acknowledgements
The authors are grateful to ICRISAT and the Belgian Administration for Development Cooperation for
funding this research project.

Indian strain I1-20 Indian strain I1-229

Percentage of infected roots

Percentage of infected roots

100 100

80 80

60 60
P-Z-C P-Z-C 20-25C
40 40
P P-Z P-Z P-Z 25-30C P-Z P-Z P-Z 20-25C
20 20
others others
0 0
1 2 3 4 5 6 7 1 2 3 4 5 6 7
Time (weeks) Time (weeks)

Belgian strain B1 Canadian strain C1

P-Z P-Z P-Z P-Z
Percentage of infected roots

10-15C P-Z P-Z P-Z 10-15C

Percentage of infected roots

100 100
P-Z P-Z P-Z-C P-Z-C 15-20C P-Z-C 15-20C
80 80 P P-Z-C

60 60
P
40 40
20-25C
P P P-Z P-Z
20 20
others others
0 0
1 2 3 4 5 6 7 1 2 3 4 5 6 7
Time (weeks) Time (weeks)

French strain F11

Percentage of infected roots

100

80 P-Z P-Z 15-20C

60
10-15C
40 15-20C
20-25C
P 25-30C
20 30-35C
others
0
1 2 3 4 5 6 7
Time (weeks)

Fig. 1 Comparison of temperature requirement of Indian P. graminis strains (strains I1-20 and I1-229 on
sorghum) and temperate strains (strains B1, C1 and F11 on barley). Fungal stages observed in the
roots: P = plasmodium, Z = zoosporangium and C = cystosorus.

215

 References
Adams M.J. 1990. Host range and transmission of barley viruses by isolates of Polymyxa graminis.
Proceedings of the first symposium of the international working group on plant viruses with fungal
vectors. Schriftenreine der Deutschen Phytomedizinischen geselleschaft 1:121-123. Ed. R. Konig.
Stuttgart - Ulmer.
Barr D.J.S. 1979. Morphology and host range of Polymyxa graminis, Polymyxa betae and Ligniera
pilorum from Ontario and some other areas. Canadian Journal of Plant Pathology 1: 85-94
Barr K.J. and Asher M.J.C. 1992. The host range of Polymyxa betae in Britain. Plant Pathology 41: 64-68
Bastin V., Boute C. and Maraite H. 1989. Inoculum potential and host range of Polymyxa graminis.
EPPO Bulletin 19: 541-546.
Ledingham G.A. 1939. Studies on Polymyxa graminis, n. gen. n. sp., a Plasmodiophoraceous root parasite
of wheat. Canadian Journal of Research C 17: 38-51.
Ratna A S., Rao A.S., Reddy A.S., Nolt B.L., Reddy D.V.R., Vijayalakshmi M. and McDonald D. 1991.
Studies on the transmission of Indian peanut clump virus disease by Polymyxa graminis. Ann. appl.
Biol. 118: 71-78.
Reddy D.V.R., Rajeshwari R., Iizuka N., Lesemann D.E., Nolt B.L.and Goto T. 1983. The occurrence of
Indian peanut clump, a soil-borne virus disease of groundnuts (Arachis hypogea) in India. Ann. appl.
Biol. 102: 305-310.
Thouvenel J.-C., Dollet M.and Fauquet C. 1976. Some properties of peanut clump, a newly discovered
virus. Ann. appl. Biol. 84: 311-320.
Thouvenel J.-C.and Fauquet C. 1981. Further properties of peanut clump virus and studies on its natural
transmission. Ann. Appl. Biol. 97: 99-107.
_______

216

 PRACTICAL MYCOLOGY
PRACTICAL ADVICE FOR COLLECTING WOOD-ROTTING FUNGI IN
THE TROPICS

ERAST PARMASTO

Institute of Zoology & Botany, 21 Vanemuise St., EE 2400 Tartu, Estonia

Number of new species of mainly wood-rotting fungi of Aphyllophorales is

increasing rapidly. Nevertheless, only a small part of the tropical territories has been
mycologically explored; thousands of species have not yet found and described.
Type specimens and other collections of Aphyllophorales collected earlier are in
many if not most cases sterile (without basidia and spores). Their original
consistency and colour have usually changed, fruit-bodies are sometimes badly
damaged by insects. Comparatively very small number of species has been
introduced to pure culture.
For developing modern taxonomy of this economically important group of fungi,
extensive collecting of specimens is needed. Some practical advises in addition to
the ones published in several handbooks are given below.
1. When sampling a specimen growing on wood, the tree species (or at least
genus) must be identified if possible: there are externally similar sibling species
specialized in several genera of wood-rotting fungi (Phellinus, Inonotus,
Peniophora, Corticium etc.). Sample also a piece of rotten wood (substrate) or
describe the rot type (white, brown, pocket rot...).
2. Describe the colour of all parts of the fresh basidiomata (fruit-bodies) as soon
as possible using standard colour charts (e. g., the rather cheap "Methuen Handbook
of Colour" by A. Kornerup & J.H. Wanscher). Note colour change after bruising
(handling) the basidioma.
3. Dry the specimens in the same day you collected them. When kept moist or
dried slowly, basidia will collapse and spores will be damaged or destroyed (by
bacteria?).
4. Separate a part of the big basidioma (or one of the basidiomata) to take a spore
print. This is extremely important! If the collected basidioma is dry, put it (in the
evening) for 10-30 min. into water. After some drying (basidioma must be moist but
not wet) put it on a piece of black and white paper collection number written on it.
Hymenophore (pores, lamellae, folds) must be facing downwards. Put the basidioma
with the paper strip below it for the night in a plastic bag to avoid desiccation. Next
morning you can usually get a good spore print; in several cases, sterile when
collected specimens have developed new basidia and began to sporulate. Dry the
basidioma used for taking a spore print, and mark it: it may be the best part of your
collection. The piece of paper with spore print must be dried promptly (with no
heating) and kept in a labelled envelope.
5. Spore prints when kept dry (if possible, in frigerator at 25 C) may be used
even after one or some weeks to inoculate suitable medium in your lab to get a pure
culture. In some species, spores are viable up to several months.
6. When studying herbarium specimens microscopically, use the spore print to
measure 25-50 randomly taken spores to calculate mean spore size and spore
length/width index Q.

217

 7. After returning from a collecting trip, disinfect your collections in a deep-

freezer. Keep them at -18 C (better: at -25 or even -30) 1-2 days, in big fruit
bodies of polypores - up to 3 days. If you put every disinfected specimen in a
minigrip plastic bag, further infection by insects is surely avoided. Use of poisonous
disinfectants must be avoided: some of these are almost useless (as is the frequently
used in tropical herbaria naphthalene), all are dangerous for health, and many of
them may disturb the use of specimens in molecular taxonomy studies later.
8. If you have found an interesting collection you consider to belong to a new
species, do not hurry describing it as a new one. About 70-75 % of "new" species are
based on only one collection nowadays. In many cases these are not new species but
"deviating" specimens of old ones of unknown variability. Keep in mind that
variation of size of spores, basidia, cystidia, pores etc. in one specimen does not give
any information on variability of the species. Avoid increasing taxonomic "noise,"
better continue collecting in other areas to find more specimens of your fungus.
______

218

 THE INFLUENCE OF PREPARATION ON ASCOSPORE

MEASUREMENTS

BELLIS KULLMAN, AIVO JAKOBSON, MART RAHI

Institute of Zoology and Botany of the Estonian Academy of Sciences,

21 Vanemuise St., EE2400 Tartu, Estonia

Abstract. The influence of various media (tap water (TW), 2% potassium hydroxide solution (KOH),
cotton blue solution in lactophenol (CB), polyvinyl lacto-phenol (PVLPh), detergent solution in water
(DE), fixative (FAA)) and the storage on the ascospore measurements of Byssonectria terrestris (Alb. &
Schw.:Fr.) Pfister, Neotiella vivida (Nyl.) Dennis & Rifai and Peziza domiciliana Cooke were compared.
The influence of the medium and the storage on spore dimensions of different species has the same trend
but its effect may be different. The comparison of spore dimensions taken from spore print, from
rehydrated herbarized material and from living material immediately or up to 45 days after preparation is
not justified.
Additional keywords. Ascomycetes, spore measurements, microscopy.

Introduction
In the systematics of Ascomycetes spore measurements as quantitative characters
are used for the delimitation of populations and species, for the study of geographic
speciation and for the construction of phylogenetic trees (Huhtinen 1989, Fogel
1992, Mls Raitviir 1974, Schumacher 1990, Wu 1993). The natural variability of
spore dimensions of a specimen may be considerable, and recorded variability
always includes measurement errors. These errors have two main causes: 1). errors
of the measurement procedure itself, e.g. due to the orientation of a spore in a
microscopic specimen (Rahi Kullman 1993); 2) spore shape distortions (shrinkage or
swelling) due to differences in the osmotic concentration (water potential) of the
spore content and the medium.
There is strong experimental evidence that spore dimensions depend on the type
and concentration of the fixative, medium as well on spore/specimen storage. Spore
shrinkage up to 26% in linear dimensions in the Melzer reagent (56% of the
calculated volume) has been reported (Baral 1992); these data indicate that one
should be extremely cautious in comparing spore measurement data when details of
the measurement procedure are incomplete or missing. Dealing with data on type
specimens this recommendation is essential but sometimes unrealistic.
Considering the huge amount of published data on spore dimensions it is of
interest to evaluate effects on spore dimensions due to: 1) type and storage of a
specimen (fresh, air dried herbarized fruitbody or spore print); 2) type of medium.

Material and methods

The influence of the following media on spore dimensions were evaluated: tap
water (TW); 2% potassium hydroxide solution (KOH); 0.2% cotton blue solution in
lactophenol (40%) (CB); polyvinyl lactophenol containing 56% of polyvinyl alcohol,
22% of lactic acid, 22% of phenol (by volume) (PVLPh); fixative water solution
containing 4% of formalin, 23% of ethanol, 1% of acetic acid (FAA); detergent
solution (some drops of Fotoflo 200 in 100ml 1% NH3 solution - DE). The effect of
the time of taking spore measurements (immediately or up to 45 days after
preparation) and the type of a microscopic specimen (made from spore print, fresh,
fixed or rehydrated herbarized fruitbody) on spore dimensions were also examined.
The species under study were: Byssonectria terrestris (Alb. & Schw.:Fr.) Pfister

219

(spore print from several fruitbodies of one specimen, 8 fresh and 17 herbarized
fruitbody), Peziza domiciliana Cooke (spore print from one fruitbody) and Neotiella
vivida (Nyl.) Dennis & Rifai (fixed fruitbody).
From each fruitbody/spore print 25 spores were measured with an ocular
micrometer (microscope "Amplival", immersion objective HI 100) with an accuracy
of 0,3 m. A database of spore dimensions was compiled. The mean spore length
(L) and width (W), L/W ratio (Q), standard deviation (s) and the coefficient of
variation (V) were computed for each sample.
In the analysis of variance "Statgraph" procedures were applied: One- and Two-
Way ANOVA for means and variances; Analysis of the Nested Design for
evaluating the effects of the medium and the fruitbody. The significance level 0.05
was applied throughout the analyses.

Results
The spores of Byssonectria terrestris from one specimen measured in CB were
examined. One-Way ANOVA revealed that a) samples from the same herbarized
fruitbody do not differ significantly (p > 0.3) and b) spore samples from the same
common spore print of several fruitbodies do not differ significantly (p > 0.9).
Variance of spore means is the smallest in spore print (Table 1. Fig.1 A) which may
serve as a "reference standard" in comparative study (Table 2).

The spore dimensions of a fixed Neotiella vivida fruitbody do not change

significantly in TW, while they diminish essentially in CB.

Nested Design ANOVA revealed significant temporal differences spore

dimensions measured on a fresh fruitbody and half a year later on the same dried
specimen (FFinTW and HFinTW; FFinTW and HFinCB) and also significant
difference spore dimensions measured in CB on a spore print and on the herbarized
specimen (SPinCB and HFinCB).

Table 1. Spore measurements (L, W, Q) of spore print (SP), fresh (FF) and herbarized (HF) fruitbodies of
one B. terrestris specimen; coefficients of variation (V , V ) and standard deviations (s , s ) of sample
L W L W
means (_, _); coefficients of variation of samples (V , V ) in different media (TW, CB). Samples from the
l w
same common spore print of several fruitbodies - *; samples from the same fruitbody -.; samples from
the several fruitbodies - .; N-number of samples (of size 25)

N L V s W V s V Vw Q
L L W W
*SPinCB 4 18.8 0.4 0.1 8.7 0.3 0.03 4-5 3-5 2.17
HFinCB 5 18.8 1 0.2 7.9 2 0.2 4-6 7-11 2.38
FFinTW 7 20.0 1 0.1 8.5 1 0.1 4-11 3-5 2.35
HFinCB 5 18.6 2 0.3 7.8 2 0.2 4-5 6-9 2.39
HFinTW 7 18.5 3 0.5 8.0 3 0.2 3-4 4-6 2.31

Tables 2-4. Mean spore measurements (L,W,Q) of spore samples (n=25) and their coefficients of variation
(V), standard deviations (s) in different media; differences of these measurements with respect to those
measured in CB (Tables 2, 3) or in FAA (Table 4) (%, m) and the significance level (p) of the
difference.

Table 2. Byssonectria. terrestris from the same common spore print of several fruitbodies.

220

 Media m s V m % p Q
CB L 18.8 0.8 4
W 8.7 0.4 4 2.17
PVLPh L 18.9 0.7 4 +0.1 0 0.64
W 8.5 0.4 4 -0.2 2 0.06 2.22
FAA L 18.7 0.6 3 -0.1 1 0.51
W 8.6 0.3 3 -0.2 1 0.32 2.18
KOH L 19.0 1.0 6 +0.2 1 0.28
W 8.9 0.5 6 +0.2 2 0.03 2.14
TW L 19.5 1.0 5 +0.6 3 0.00
W 9.1 0.4 4 +0.2 5 0.00 2.13
DE L 19.5 1.1 5 +0.7 4 0.00
W 9.5 0.6 7 +0.8 9 0.00 2.10

Table 3. Peziza domiciliana from the same common spore print of one fruitbody.

Media m s V m % p Q
CB L 14.8 0.7 4
W 9.0 0.7 8 1.65
PVLPh L 14.5 0.6 4 -0.3 2 0.07
W 8.2 0.4 5 -0.8 8 0.00 1.76
TW L 14.9 0.6 4 +0.1 1 0.89
W 9.6 0.5 5 +0.6 6 0.00 1.56

Table 4. Neotiella vivida from the one fixed fruitbody

Media m s V m % p Q
FAA L 23.8 1.2 5
W 13.1 0.6 4 1.8 3
TW L 23.6 0.9 4 -0.2 1 0.43
W 13.4 0.7 5 +0.3 2 0.10 1.76
CB L 21.8 0.9 4 -2.0 8 0.00
W 12.0 0.6 5 -1.1 8 0.00 1.82

Table 5. Analysis of Nested Designs. Variance components of the medium (ME%), fruitbody (FB%), and
the residual (Spore%) for spore length (L) and width (W) on the basis of the B. terrestris spore
measurements of fresh (FF) and herbarized (HF) fruitbodies and the spore print (SP) as compared in
different media CB, TW, TW_CB. If Femp > Fkr, the variance component is significant at a=0.05.

ME% Femp Fkr FB% Femp Fk Spore%

FFinTW and HfinTW L 53 63.0 > 4.8 4 3.5 > 1.8 43
W 40 25.0 > 4.8 9 5.6 > 1.8 51
FFinTW and HfinCB L 46 91.5 > 5.3 0 1.2 < 3.8 54
W 43 55.7 > 5.3 2 1.8 < 3.8 5
HFinTW and HFin CB L 2 1.3 < 5.3 21 7.9 > 3.8 77
W 2 1.5 < 5.3 14 5.1 > 3.8 84
HFinTW and HfinTW-CB L 5 1.7 < 5.3 34 15.0 > 3.8 61
W 8 2.7 < 5.3 23 7.5 > 3.8 69
SPinCB and HfinCB L 3 2.7 < 6.0 4 2.1 < 2.1 93
W 67 456 >> 6.0 0 0.4 < 2.1 33

The temporal changes in spore dimensions of Byssonectria terrestris are illustrated

221

in Fig. 2. Changes of spore width measured in spore print in PVLPh are mixed: a
small but significant decrease is followed by an increase. A significant decrease in
spore dimensions was observed in a microscopic specimen of a fruitbody and a spore
print mounted on PVLPh and CB respectively. Decrease is not significant up to the
6th day in spore print in CB.

Discussion and recommendations

1. Spores discharged from the ascus are significantly wider in comparison with
spores in fruitbodies (Table 5: SpinCB, HFinCB. Fig.1: L4>LE or LA>LB).
2. Spore dimensions decrease with the storage of a herbarized specimen. Spores in
fruitbodies measured a half year later are significantly smaller in comparison with
spores in fresh fruitbodies (Table 5: FFinTW and HFinTW; FFinTW and HFinCB.
Fig.1: E>C). Weekly drying does not cause any essential change (E and 6).
3. Preliminary rehydration of a herbarized specimen in TW increases significantly
the variance of means of spore dimensions in comparison with those measured on
spores introduced directly into CB (Table 1. Fig. 1: C,D - B). The means of spore
dimensions of fresh fruitbodies (Table 1. Fig.1: E) vary less than those of herbarized
fruitbodies.
4. Variance of the spore means is the smallest in spore print (Table 1. Fig.1: A)
which may serve as a "reference standard" in a comparative study. The spore
discharge as a pretreatment factor probably account for this homogeneity.
5. Considering effects on spore dimensions and variability (Table 2. Fig.1: A, 1-5)
on the wall structure, the media investigated can be divided as follows:
a) FAA, CB, PVLPh -impact on spore sizes and variability is minor. Preference
should be given to FAA;
b) TW - causes no wall disintegration, but impact on dimension variability is greater.
This may be caused by different spore viability (Stojanovic, Ristanovic and Jencic
1986);
c) KOH, DE - due to disintegrating effects on the spore wall the impact on
dimension variability is the greatest. The effects of KOH, TW and DE on spore
dimensions are significantly greater than that of CB.
6. The effect of various media on the length/width ratio (Q) differs, Q is maximal in
PVLPh and minimal in DE. Therefore, one must be extremely cautious when
comparing data on Q (Table 2).
7. The essential increase in TW is the same for spore measurements of Byssonectria
terrestris and Peziza domiciliana. Differences in its effect are probably due to
different spore wall rigidity (Table 2 and 3).
8. The fixative seems to preserve the measurement of both dry spores and those
taken from live fruitbodies. On the contrary in CB, it dehydrates spore in live
fruitbodies (see B. terrestris spore print, Table 2. and a fixed N. vivida fruitbody,
Table 4).
9. In microscopic specimens from fresh fruitbodies and spore print mounted in CB
and PVLPh temporal changes in spore dimensions may differ considerably. In
microscopic specimens of spores in PVLPh the distance between glass surfaces
decreases due to the medium loss by evaporation and spores may become
compressed and distorted. In microscopic specimens from fruitbody these effects do
not take place.
It is recommended to measure spores in CB in the spore print and in the herbarized
fruitbody during the first six days (Fig.2-4).

222

Fig.1. Mean spore length and width in one specimen of B. terrestris. SP - spore print, FF - fresh fruitbody,
HF -herbarized fruitbody. Points: 1 - in fixative (FAA), 2 - in polyvinyl lacto-phenol (PVLPh), 3 - in
potassium hydroxide solution (KOH), 4 - in tap water (TW), 5 - in detergent solution (DE), 6 - one week
storage of the fruitbody in TW, clusters A - spore samples of the same common spore print from different
fruitbodies in CB; B - samples from different fruitbodies in CB; B' - samples from the same fruitbodies in
CB; C - rehydrated samples in TW, D - rehydrated samples mounted in CB; E -samples from the fresh
fruitbody in TW.

Fig. 2. Temporal changes in spore dimensions in a microscopic specimen of B. terrestris. Spore print
mounted in polyvinyl lacto-phenol (A), fruitbody mounted in polyvinyl lacto-phenol (B) and spore print
mounted in cotton blue solution (C).

References
Baral H.O. 1992. Vital versus herbarium taxonomy: Morphological differences between living and dead
cells of Ascomycetes, and their taxonomical implications. Mycotaxon 44(2): 333-390.
Fogel R. 1992. Utility of spore length/width ratio in separating Geopora cooperi form Cooperi and G.
cooperi f. gilkeyae. Mycologia 84(1): 124-127.
Huhtinen S. 1989. A monograph of Hyaloscypha and allied genera. Karstenia 29(2): 45-252.
Rahi M. and Kullman B. 1993. Pitfalls in ascospore measurements. In Fungi and Lichenes in the Baltic
Region, Abstracts: 123, 12th International Conference on Mycology and Lichenology. Vilnius.

223

Mls T. and Raitviir A. 1974. Morphometrics and the taxonomiy of fungi. Tallinn (in Russian), pp. 159.
Schumacher T. 1990. The genus Scutellinia (Pyronemataceae). Opera Bot. 101: 1-107.
Stojanovic S., Ristanovic M., Jencic R. 1986. Influence of the age of cleistothecia Erysiphe graminis DC.
ex Merat f. sp. tritici em. Marshal on the morphologic properties and vitality of ascospores. 5th.
Jugoslovenski Simpozijum O. Zastiti Bilja. Struga (Yugoslavia). 22 25 Oct. 1985. Zastita bilja
37(176): 169-174.
Wu C-G. 1993. Clomales of Taiwan: iv. A monograph of Sclerocystis (Glomaceae). Mycotaxon 49: 327-
349.
______

MICROPLATE TECHNIQUE OF PHYSIOLOGICAL TESTS FOR YEAST

IDENTIFICATION
PIERE EVRARD AND GRGOIRE L. HENNEBERT

Mycothque de l'Universit Catholique de Louvain, Unit de Microbiologie, Facult des Sciences

Agronomiques, Place Croix du Sud 3, B-1348 Louvain-la-Neuve, Belgium

Abstract. Yeasts are important in various industrial processes as producers or as

contaminants. To identify yeasts quickly and reliably is crucial. Therefore exploring
fast, simple and inexpensive identification methods is necessary.

Fig. 1. Microplate 85 assimilative tests of one yeast strain. 2. Microplate grouping 12 fermentative tests of
8 strains in anaebiosis.

Fig. 3.Traditional optical reading of fermentative tests in tubes of one strain. 4. Automatic non-subjective
reading of microplates of assimilative and fermentative tests.

Traditionally, yeasts are identified by morphological characters and

physiological properties such as assimilation and fermentation tests according to the

224

procedure described by Van der Walt & Yarrow (1984) emended by Barnett et al.
(1990).
Physiological tests using tubes are time consuming and expensive. We propose a
new, fast and inexpensive technique for physiological tests using microplates. The
oxydative microplate includes 48 carbone sources and 9 nitrogen sources for
assimilation, 10 vitamines for minimum requirement, 3 cycloheximide and sodium
chloride concentrations for tolerance, and the urease, the arbutin splitting and the
starch formation tests. The fermentative microplate includes 12 carbon sources. The
microplates are read automatically on a microplate reader. The absorbency
measurements are transferred to a computer in a data matrix. A software has been
created and programmed to interpret the absorbency measurement data in terms of
positive, negative and weak results.

Together with morphological data, physiological data are then treated by the
yeast identification expert system ALLEV for identification (Robert et al. 1994).

References
Van der Walt & Yarrow 1984. In The Yeasts, a taxonomic study. NJW. Kreger-van Rij ed.Elsevier
Amsterdam.
BarnettJ.A. 1990. The Yeasts, characteristics and identification. 3d ed. Cambridge Univ. Pr.
Robert V., De Bien J.E., Buyck B. and Hennebert G.L. 1994. "ALLEV", a new program for computer-
assisted identification of yeasts. Taxon, 43 : 433-439.

______

CRYOPRESERVATION OF ASPERGILLUS REPENS

DAVID SMITH

International Mycological Institute, Bakeham Lane, Egham, Surrey TW20 9TY, United Kingdom

Abstract. Cryogenic light microscopy has been used to develop optimum

cooling protocols for fungi. The response to freezing and thawing of a fungus is
demonstrated well by Aspergillus repens on cooling at -30C min -1 to -30C. Ice
crystalization, solute crystalization and gas bubble formation cause injury to the
fungus cell. Complete evacuation of cytoplasm from the hyphae has been seen.
_______

225

 CULTIVATION OF THE FUNGI

ACTIVATED CHARCOAL-MEDIATED SPOROPHORE INITIATION IN
AGARICUS AND TRICHOLOMA
ABED PEERALLY

Faculty of Science, University of Mauritius, Reduit, Mauritius

Abstract. Agaricus bisporus is known not to produce sporophore primordia in aseptic conditions
unless activated charcoal is present. Using different techniques Agaricus bisporus, A. bitorquis and
Tricholoma spectabilis were grown aseptically on distilled water agar, polyethylene foam, malt extract
agar and soil with and without activated charcoal. Only A. bitorquis produced ill-defined primordia in the
form of mycelial aggregates on water agar and malt extract agar in the absence of activated charcoal. In
the presence of activated charcoal the three species produced primordia but only A. bitorquis and T.
spectabilis produced normal pins and sporophores. The best fruiting was obtained with A. bitorquis on
water agar treated with activated charcoal and with T. spectabilis on activated charcoal treated soil. It is
concluded that all three species produce similar fruiting inhibitory factors capable of being neutralised by
soil bacteria and activated charcoal.

Introduction
The usefulness of activated charcoal in stimulating the fruiting process of
Agaricus bisporus (Lange) Sing. was initially reported by Mader (1943). This
observation led Mader to conclude that volatiles were involved in the formation of
carpophores. The stimulating effects of soil bacteria in the natural initiation of
fruiting in A. bisporus was first observed by Eger (1961, 1972) and subsequently
reliable additional observation on the bacterial induction of fruiting was made by
Hayes et al. (1969), Hayes (1981) and Peerally (1978, 1981).
Eger (1961, 1972) also reported the ability of activated charcoal to replace the
effects of bacteria. Similar results were also reported by various other workers
(Long and Jacobs 1974, Convy 1976, Wood 1976, Peerally 1978, Stoller 1978).
That activated charcoal could also stilmulate fruiting in Agaricus bitorquis
(Sacc.) Quel. was shown by Peerally (1978). However Stoller (1978) observed that
on malt extract agar, activated charcoal and lignite separately had no effects on the
initiation of fruiting in A. bitorquis but when mixed, fruiting was profusely
stimulated.
It is generally believed that activated charcoal stimulates fruiting by removal of
one or more inhibitors present in the mycelium. To date it is still not understood
what precise role bacteria and activated charcoal play in carpophore initiation
Ingratta and Patrick (1986) suggested that the major stimulus in carpophore
initiation in A. bisporus might be related to the intensified microbial activity in the
casing layer which results in a reduced nutritional base in the casing and an increase
of fungistasis.
The present paper reports the fruiting response in A. bisporus (strain 133), A.
bitorquis (strain 1) and in Tricholoma spectabilis Peerally & Sutra (local strain 1)
under the influence of activated charcoal, in four different techniques.

Methods
Cultures were first obtaine on standard potato dewtrose agar (pH 7.5) and then
used to onoculate autoclaved wheat grain in conical flasks. Grain spawn of A
bisporus was incubated at 23C while that of A. bitorquis and T. spectabilis was kept
at 28C. The three spawns were fully invaded and ready after about three weeks.
226

 Plates of malt 2% agar (MEA) and double distilled water agar (DWA), both
adjusted to pH 7.5 with sodium hydroxide and calcium carbonate, were prepared and
after setting, the agar surface was thoroughly covered with a thin layer of activated
charcoal powder (about 0.7g per plate). Plates were inoculated with single grain of
spawn or with a small quantity of spawn.
Loam soil, pH adjusted to about pH 7.3 with calcium hydroxide, and
polyethylene foam (8cm diam, 4mm thick) were placed in separate Petri dishes.
Sterilisation of soil was achieved on three consecutive days for one hour. Plates with
foam were autoclaved one for one hour. After sterilisation, activated charcoal
powder was applied to the soil (0.3g or 1.0g per plate) and to the foam (1.0g per
plate). Plates were then inoculated on one side with a small quantity of grain spawn,
and sterile distilled water added.

Fig. 1. Agaricus bisporus a-c Primordia production.

Fig. 2. Agaricus bitorquis a. On loam + activated charcoal. b. On soil + 0.3g activated charcoal. c. On soil
+ 1g activated charcoal.

They were at least ten replicates per treatment including the untreated control
sets of plates. Experiments were repeated at least once.
Plates with A. bisporus were incubated for six days at 23C and then transferred
to an aerated room at 18-20C. Plates with A bitorquis and T. spectabilis were
incubated at 28C for six days and then transferred to 21-26C.
227

 Results
Activated charcoal treated plates and control plates were observed daily. The
state of mycelial growth and of fruiting initiation and development was studied over
a period of two to four weeks. No attempt was made to count the number of
primordia which for some treatments was indeed not feasible.
Colony growth in all untreated DWA plates was fluffy or slightly strandy.
Primordia were not produced in A. bisporus and T. specatabilis. Some of the DWA
plates with A. bitorquis produced carpophore initials but in a rather disorganized
pattern, in contrast to organized, synchronous and regular development at the
periphery of the colony obtained with activated charcoal. Those initials consisted of
fuzzy hyphal aggregates (Wood 1978). Smooth-surfaced primordia and pins as
produced in the presence of activated charcoal were never seen on untreated DWA.
Initials were formed from day 7 or 8 after inoculation.
In DWA plates treated with activated charcoal colony growth was conspicuously
adpressed with little aerial mycelium, in A. bisporus and A. bitorquis. Strandy
growth sometimes occurred in A. bisporus and A. bitorquis conspicuously (Fig. 2a).
In T. spectabilis strands were never observed, but instead isolated areas of fluffy
mycelium were frequently produced (Fig. 1) from which well developed smooth
primordia, pins and carpophores developed. Fully mature carpophores with stipe,
pileus and gills were best formed by A. bitorquis and were profuse throughout the
periphery of the colonies A. bisporus produced a few primordia (Fig. 3a-c) which
did not develop further.
On MEA, A. bisporus and T. spectabilis did not produce any primordia whether
activated charcoal was supplied or not. A. bitorquis produced a few mycelial
aggregates but nothing further in presence of activated charcoal.
On foam without activated charcoal, mycelial growth of the three strains was
very fluffy. In the presence of activated charcoal A. bitorquis produced very strandy
mycelium, primordia, pins and mature carpophores (Fig. 2a). A. bisporus formed
strandy mycelium but no primordia, while T. spectabilis grew to a limited extend
and formed some primordias and pins.
In autoclaved soil treated with activated charcoal A. bitorquis (Fig. 2b, c) and T.
spectabilis produced fluffy mycelial growth, primordia, pins and mature
carpophores.

Discussion
Only A. bitorquis was capable of producing initials in the form of fuzzy
myceliual aggregates on untreated DWA. In the presence of activated charcoal all
three species produced pimordia, but only A. bitorquis and T spectabilis formed well
developed smooth primordia, pins and carpophores, similar to those produced in
normal casing and in nature, respectively. Only T. spectabilis showed the peculiarity
of forming fluffy aerial mycelium capable of readily producing well developed pins
and carpophores.

228

Fig. 3. Tricholoma spectabilis. a-c. fruiting on activated charcoal.

Fig. 4. Tricholoma spectabilis. a, b. mycelium and primordial on non aseptic soil. c. sterile mycelium on
aseptic soil.

The fact that activated charcoal did not stimulated primordial on MEA supports
the results obtained by Stolla (1978)
San Antonio and Peerally (1978) working with several strains of A. bitorquis
concluded that there was no correlation between the formation of initials on agar
media and productivity under normal growing conditions. This might possibly be
attributed to the disactivation of activated charcoal by chemicals present in the malt
agar. It is, thus, conceivable that such a correlation might exist between formation of
primordias in the presence of activated charcoal and productivity of the strain.
The fact that all three species were capable of producing at least primordias in
the presence of activated charcoal is interesting. Bacteria are known to be necessary
for the formation of carpophores in A. bisporus, as well as for A. bitorquis and T.
spectabilis (Peerally unpublished) (Fig. 4a-c).
It must therefore be concluded that the same inhibitory factors are present in the
mycelium of these basidiomycetes and once neutralised or removed, fruiting takes
229

place. The induction of fruiting in widely different terricolous basidiomycetes by

bacteria and activated charcoal could well have the same mechanism but the nature
of this induction still remains to be elucidatedc.

Acknowlegement
My thanks are due to Mr V. Aumeer for technical assistance.

References
Couvy J. 1976. La fructification d'Agaricus bisporus en milieu aseptique: un modle experimental pour
l'tude des substances impliques dans l'initiation fructifre. Mushroom Science 9(1): 157-164.
Eger G. 1961. Untersuchungen ber die Funktion der Deckschicht bei der Fruchtkrperbildung des
Kultuurchampignons, Psalliota bispora Lge. Arch. Mikrobiol. 39: 313-334.
Eger G. 1972. Experiments and comments on the action of bacteria on sporophore initiation in Agaricus
bisporus. Mushroom Science 8: 719-725.
Hayes W.A., Randle P.E. and Last F.T. 1969. The nature of the microbiological stimulus affecting
sporophore formation in Agaricus bisporus (Lange) Sing. Annals of Applied Biology 64: 177-187.
Hayes W.A. 1981. Iterrelated studies of physical, chelmical and biological factors in casing soild and
relationships with productivity in commercial culture of A. bisporus (Lange) Pilat. Mushroom
Science 11 103-129.
Ingratta F.J. and Patrick Z.A. 1986. Influence of microorganisms and fungistasis in sporophore initiation
in Agaricus brunnescens. Proceedings International Symposium on the Scientific and Technical
Aspects of Cultivating Edible Fungi. The Penn. State Univ. Univ. Park, USA, pp. 27-40.
Long P.E. and Jacobs L. 1974. Aseptic fruiting of the cultivated mushroom, Agaricus bisporus. Trans.
Brit. mycol. Soc. 63: 99-107.
Mader E.O. 1943. Some factors inhibiting the fructification and production of the cultivated mushroom.
Phytoapthology 33: 1134-1145.
O'Donoghue Maguire D.C. and Ryan J.P. 1991. Influences of a wide range of bacteria, actinomycetes and
fungi on mycelial growth of Agaricus bisporus (Lange) Sing. and the special fruiting requirement of
A. bisporus. In Science and Cultivation of Edible Mushroom, Maher M. ed. Proc. 13th. Int. Congress,
Dukin, pp. 753-759.
Peerally A. 1978. Sporophore initiation in Agaricus bisporus and Agaricus bitorquis in relation to
bacteria and activated charcoal. Mushroom Science 10(1): 611-639.
Peerally A. 1981. A Petri plate agar technique for obtaining primordial in Agaricus bisporus (Lange)
Sing. Mushroom Science 11:153-158.
San Antonio J.P. and Peerally A. 1978. Growth, primordial formation and fruiting of twenty-two different
strains of Agaricus bitorquis. Mushroom Science 10(1): 587-594.
Stoller B.B. 1978. Synthetic casing for mushroom beds. Mushroom Science 10(2): 187-215.
Wood D.A. 1978. Studies on primordium initiation in Agaricus bisporus and A. bitorquis. Mushroom
Science 10(1): 565-585.

_______

IN VITRO CULTURE OF ENDOMYCORRHIZAL FUNGI: WHY THAT

INTEREST?
1 2 1 2
S. DECLERCK , T. DIOP , C. PLENCHETTE AND D.G. STRULLU .

1Station d'agronomie, INRA, B.V. 1540, 17 rue Sully, 21034 Dijon Cedex, France. 2Universit d'Angers,
2 Bd. Lavoisier, 49045 Angers Cedex, France.

Introduction
Vesicular arbuscular mycorrhizal (VAM) fungi are found naturally in almost all
tropical soils and under all terrestrial ecosystems (Sieverding 1991). Much of the
interest in VAM fungi relates to their role in plant growth and phytoprotection.

230

Taxonomy of these fungi has been largely based on spore characteristics (Morton,
1988). Nevertheless, accurate identification of VAM fungi in field soils is not easy
as the spores may be present in different stages of their development. Mixtures of
spores of different species could also lead to identification problems (Abbott and
Robson 1991). Taxonomy using biochemical and genetical characterizations has
also been hampered by the lack of powerful techniques of specific and sensitive
detection and obtention of DNA fragments in axenic conditions. Recently, the
upsurge of the polymerase chain reaction (PCR) technology has given a new
impetus in biomolecular research and has widespread applications in VAM fields as
diverse as phylogeny, evolutionary history and taxonomy (Simon 1993).
Mosse and Hepper (1975) were the first to propose an in vitro system based on a
dual culture of spores and excised roots. Mugnier and Mosse (1987) and Becard and
Fortin (1988) improved the system by using roots genetically transformed by
Agrobacterium rhizogenes. Diop et al. (1992) were able to follow the entire
vegetative cycle of Gigaspora margarita using Ri-TDNA transformed carrot roots.
Another method of investigation was proposed by Strullu and Romand (1986, 1987
and 1991) who developed a culture technique based on the intraradical phase of the
fungus i.e. the vesicles.
The continuous culture of VAM fungi means that the mycorrhizal fungi must be
indefinitively subcultured in order to maintain and increase its biomass. Becard and
Fortin (1988) achieved continuous cultures of G. margarita using the spores
produced in vitro by this VAM fungus. Strullu and Romand (1986) proposed a
method using mycorrhizal root as starting material and based on the successive
reassociation of mycorrhizal roots with non mycorrhizal root systems. This system
of in vitro subcultures has been applied successfully by these authors with Glomus
species.
The indefinite in vitro maintenance of VAM fungal species would make it
possible to maintain biodiversity through the creation of fungal libraries. Taxonomy
based on morphology and molecular biology (PCR) should also lead to the
characterization of these species which should become "reference species".
We propose an application of in vitro culture using the intraradical phase of the
VAM fungi, applied to banana in order to obtain appropriate strain for sustainable
agriculture.
Methodology
Sampling, trapping and isolation procedures
Infective VAM fungal propagules are collected in the field by soil and root
sampling (1)*. Soil samples are taken in the upper layer of the soil where most root
growth occurs. The indigenous VAM population present in soil and roots were then
multiplied and maintained on trap plants (Allium porum). The procedure of trap pot
culture is simple (2). Pots are filled with calcined clay (Terra Green) on top of wich
the field soil or root sample is placed. Another layer of Terra Green is placed above
the soil or root sample. A high mycotrophic plant with good growth and rooting e.g.
leek, is then planted in the pot. After several months, soil and root samples are taken
and screened for mycorrhizal infection. Soil and root material is then suspended in
water and after 10 - 15 s (for sedimentation of coarse sand) the suspension is
decanted over a series of soil sieves (2mm - 500 - 250 - 125 - 50 microns). Roots in
the top sieve are washed under tap water and transferred to a petri dish. Infected
roots are further separated from the noninfected roots under the stereomicroscope
(3). Infected roots are conserved at 4C. Spores (4) are isolated from the sieves and
231

used as starting material for monosporal culture on leek plants (4'). Spores are also
used for taxonomic description of the field isolates (4''). Collections of the VAM
isolates are maintained by using the sieved soil to establish pot culture (5).

ROOTS

(1) FIELD
SOIL

(2) TRAP POT CULTURE

(5) SIEVED SOIL (3) INFECTED ROOTS (4) SPORES

(4') TAXONOMY (4'') MONOSPORAL CULTURE

(6) DESINFECTION PROCESS

(7) ISOLATION OF SINGLE VESICLE (8) INFECTED ROOTS WITH VESICLES

OF THE SAME SPECIES

(9) GERMINATION ON MM
SRM

TOMATO ROOT CULTURE

(10) GERMINATION SEED

(13) DUAL CULTURE ON MM
(11) SUBCULTURE SRM
(12) CLONING

PRODUCTION OF SPORES AND INFECTED ROOTS

(17) CONTINUOUS (18) RHIZOSPHERE (15) FUNGAL (18) TAXONOMY (19) LARGE
CULTURE MODEL DEVELOPMENT SCALE
AND GROWTH PROD.

FUNGAL LIBRARY MORPHOLOGICAL GENETICAL

In vitro culture
Fungal inocula
Infected roots containing vesicles are desinfected using the procedure developed
by Strullu and Romand (1986) (6). The roots are surface sterilized succesivelly with
95 alcohol for 10 s, 6 % Ca hypochlorite for 2 min, 2 % chloramine T for 10 min
and 200 mg/l streptomycin - 100 mg/l gentamycin for 10 min. Roots are rinsed with
sterilized water after each sterilization step. Sterilized roots are conserved in
Streptomycin (200 mg/l) - gentamycin (100 mg/l) at 4C until ready for use.
Vesicles (7) are extracted from roots and germinated on Minimal (Becard and
Fortin, 1988) or on Strullu -Romand (1986) medium (9). Roots containing vesicles
of the same species (8) are cut in pieces of 0.5cm and also placed on the same
mediums as described above (9).
Root organ culture

232

 Initiation of root culture requires seed germination under axenic conditions.

Emerging radicles are then excised and cultured on White's medium (White 1943).
Tomato seeds are surface sterilized by soaking in 15 % hydrogene peroxide for 2 - 3
minutes. Seeds are then rinsed in sterile water and germinated (10) in the dark in
sterile Petri dishes on water - agar. When radicles are 2 cm long, they are excised
and transferred onto White's medium (White 1943) (11). Roots with the most
laterals are then chosen to initiate clonal culture (12).
Dual culture
Dual culture of root and fungus is established on Minimal medium (Becard and
Fortin 1988) or on Strullu - Romand medium (1986) (13). Pre-germinated single
vesicles or mycorrhizal root pieces, containing vesicles of the same species, showing
hyphal regrowth are used to inoculate the root system by placing them close to
emerging lateral roots. Extra and intra-radical development of the fungus is
eventually obtained depending on the fungus, the host and the environmental
conditions.
Conclusion
The root organ culture technique for VAM fungi has numerous advantages over
traditional inoculum production systems. It allows large scale production of
inoculum free of pathogens (14). It permits reproducible observations of all stages of
VAM fungi development (15) and can be used as a simplified rhizosphere model
(16) for ecological studies i.e. effect of heavy metal toxicity, competition with other
microorganisms, interactions with plant root pathogens, pesticides... The system is
also useful for determining symbiotic factors provided by the root which govern
fungal growth (CO-, root exudates...) (16). The continuous culture of VAM fungi
would make it possible to maintain biodiversity through the creation of fungal
libraries (17). Taxonomy based on morphology and molecular biology (PCR) should
also lead to the characterization of these species which should become "reference
species" (18).
Development of pure culture of endomycorrhizal fungi continues to represent
one of the most challenging goals in fungal biology. It is accepted that
understanding the mechanisms involved in the symbiotic process will lead to the
development of pure culture in the near future.

Acknowlegement
This work was supported by a grant of CEE - STD3 N 92104

References
Abbott L.K. and Robson A.D.. 1991. Factors influencing ther occurrence of vesicular-arbuscular
mycorrhizas. Agriculture, Ecosystems and Environment 35: 121-150.
Becard G. and Forti J.A.. 1988. Early events of vesicular-arbuscular mycorrhiza formation on Ri T-DNA
transformed roots. New Phytol. 108: 211-218.
Diop T.A., Becard G. and Piche Y.. 1992. Long - term in vitro culture of an Endomycorrhizal fungus,
Gigaspora margarita, on Ri T-DNA transformed roots of carrot. Symbiosis 12: 249-259.
Morton J.B.. 1988. Taxonomy of VA mycorrhizal fungi : classification, nomenclature, and identification.
Mycotaxon 32: 267-324.
Mosse B. and Hepper C.M.. 1975. Vesicular-arbuscular mycorrhizal infections in root organ cultures.
Physiol. Plant Pathol. 5: 215-223.
Mugnier J. and Mosse B. 1987. Vesicular-arbuscular mycorrhizal infections in transformed Ri T-DNA
roots grown axenically. Phytopathology 77: 1045-1050.

233

Sieverding E. 1991. Vesicular-Arbuscular Mycorrhiza Management in tropical Agrosystems. Deutsche

Gesellschaft fr Technische Zusammenarbeit (GTZ) Gmbh - 372 p., ISBN 3-88085-462-9.
Simon L., Bousquet J., Levesque C. and Lalonde (M. 1993. Origin and diversification of
endomycorrhizal fungi and coincidence with vascular land plants. Nature 363: 67 - 69.
Strullu D.G. and Romand C. 1986. Mthode d'obtention d'endomycorrhizes vsicules et arbuscules en
conditions axniques. Compt. Rend. Hebd. Sance Acad. Sci. 303: 245-250.
Strullu D.G. and Romand C. 1987. Culture axnique de vsicules isoles partir d'endomycorrhizes et r-
association in vitro des racines de tomate. Compt. Rend. Hebd. Sance Acad. Sci. 305: 15-19.
Strullu D.G., Romand C and Plenchette C. 1991. Axenic culture and encapsulation of the intraradical
forms of Glomus spp. World journal of Microbiology and Biotechnology 7: 292-297.
White P.R. 1943. A handbook of plant tissue culture. Cattel J., Lancaster, PA.
______

ENHANCEMENT OF CARPOPHORE PRODUCTION AND PROTEIN

CONTENT OF PLEUROTUS SPP. BY CULTIVATION ON GRASS HAY.
KANDANDA TSHINYANGU
Mycothque de l'Universit Catholique de Louvain, Place Croix du Sud 3 Bte 6, B-1348 Louvain-la-
Neuve, Belgium

Abstract. Seven strains of Pleurotus mushrooms (Pleurotus ostreatus var.

columbinus MUCL 28785, Pleurotus ostreatus var. columbinus MUCL 30495, P.
cornucopiae MUCL 31019, P. ostreatus var. ostreatus cv. Florida MUCL 31018, P.
pulmonarius MUCL 28784 and P. sajor-caju MUCL 31017) were cultivated on
grass hay and wheat straw without additives. The grass hay increases both
production of carpophore and protein content of all strains.
Stimulating factors of grass hay was identified by supplementing wheat straw
with selected and combined chemical compounds.That study was conducted with
Pleurotus ostreatus var. columbinus MUCL 28785 cultivated on all supplemented
susbstrates. Several compounds (alanine, asparagine, biotin, gibberellic acid,
glutamic acid, glycin, indole-acetic acid, indole-butyric acid, kinetin, manganese and
serine) have been able to enhance carpophore production. The main stimulating
factor of grass hay was the interaction between amino acids and manganese.
_______

IS THE RUST OF THE CREEPING THISTLE (CIRSIUM ARVENSE) THE

MICROSYMBIONT OF ITS VAM ?

HARTMUT HEILMANN
Birkenstrasse 10, 74592 Kirchberg/Jagst, Germany

Abstract. This paper investigates the causing factor of growth of the creeping
thistle (Cirsium arvense) as representative of a group of plants which really bears
the qualities of weeds as it can crop up anywhere and cannot be controlled by
normal soil cultivation methods. On the other hand they often do not endanger the
yield of the crop. Rumex crispus, Agropyron repens, Convolvulus arvense and
Equisetum arvense belong to this group of weeds. After use of pesticides and
fungicides the problem subsists. The failing control of the creeping thistle shows a
very unsatisfactory theory of its growth. Growing factor and sprouting are obviously
the same. The thistle has a VAM. Rhythm of growth and physiologic conditions of

234

the soil are similar to those of orchids. Sprouting from the seed does not show
cotyledons but a whole plant.
The invincibility of these weeds obviously rests on an assimilatoric contribution
by dissimilation in the soil.
Some Orchidaceae, Convolvulaceae and Scrophulariaceae posses VAM. In the
wild a regular vegetal association of the creeping thistle can be found with orchids
such as Epipactis helleborina and Platanthera bifolia, and Convolvulaceae such as
Convolvulus arvense. The question arises whether exists a similar symbiotic
contribution to thistle growth, also in agricultural systems.
The ecologic niche of orchids can be destroyed easily by stress on the soil
microbiology e.g. by action of fertilizers or other
chemicals, fungicides or pesticides. The creeping thistle
is also sensitive but temporally.
The best method of creeping thistle regulation are
legumes in crop rotation. Red clover (Trifolium
pratense) or alfalfa (Medicago sativa) obviously have
an antagonitic effect on the thistles. The action maybe
due to stem rot by Sclerotinia trifoliorum, but the
attacked patches in the field are very soon subjected
again to growth of thistles, Agrpyron repens and/or
Rumex crispus.
The root of Cirsium arvense can have a phase of
dormancy for many years, this means that roots survive
in soil but do not emerge. Also thousands of seeds are
waiting in the soil. Do both are waiting for the action of
a microsymbiont, like do species on Orchidaceae?
Indeed the author interprets the fact as the possible
existence of a comparable mycothrophy by VAM.
Creeping thistle is associated with a number of
fungi, foliar pathogens such as rusts Puccinia suaveolens or Albugo trapognonis,
Coelomycetes Septoria circii or Phyllosticta cirsii, stem fungi like Leptosphaeria or
Ophiobolus spp. or root attack by nematodes. But none of these organisms is known
to enhance growth of thistles. Then an unidentified VAM or endophytic fungus
might be responsible of the weed survival and might provide the key of its control.
_________

MORPHOLOGICAL STUDIES OF MYCELIA FROM

ECTOMYCORHIZIAN FUNGI CULTIVATED IN VITRO
J. JEANFILS, J.-J. CUVELIER
Universit Mons-Hainaut, Facult de Mdecine, Avenue du Champ de Mars 8, 7000 Mons

Abstract. Different ectomycorhizian fungi belonging at different genus were put

in culture from carpophore fragments. Morphological studies of mycelium growing
on different culture media were described. Chemical reactions obtained on in vitro
mycelium are compared with results obtained on carpophore.
_______

235

 FUNGAL BIOTECHNOLOGY
YEAST FUNGI AND ALCOHOL FROM BAMBOOS

FRANCIS S.S. MAGINGO

Applied Microbiology Unit, Department of Botany, University of Dar es Salaam, P.O. Box 35060
Dar es Salaam, Tanzania.

Introduction
The bamboo Oxytenanthera braunii is used by the local people in southwestern
Tanzania, to obtain an alcoholic beverage locally known as ulanzi (IBRN 1993).
This is attained through fermenting sugary exudates, from young, cut and bruised
shoots of the bamboo, which come out during the rainy season (Kigomo 1990). The
sugary exudates are made to flow into a collecting container known as mbeta (Fig.
1 & 2). Exudates from many mbeta are then pooled together in 20-liter plastic
containers, brought into a cool corner of a house and left to ferment.
The fermentation of the bamboo exudates is a wild and natural one, as no special
fermenting microorganisms are purposely inoculated into the bamboo sugary
exudates. The beverage, reaches its maximum alcohol amount within 24 hours.
Obtainage of an alcoholic beverage from bamboos, is practised only in the
southwestern areas of Tanzania, and no where else in the world.
So far, microorganisms involved in the fermentation of the bamboo sugary
exudates, have not yet been documented. This work was aimed at isolating
microorganisms involved in fermenting sugary exudates of the bamboo to alcohol;
and also to quantify amounts of alcohol produced.

Materials and methods

Slants of sterile medium in MacCartney bottles were brought to a bamboo
growing area in southwestern Tanzania, for initiating isolation of the fermenting
microorganisms. An initiation of isolation of microorganisms from plant exudates is
best done on the surface of agar-containing medium (Carmo-Sousa 1969). The agar
medium in the McCartney bottles was a Wickerham maintenance agar medium
(Phaff et al. 1978) containing yeast extract (0.3%), glucose (1.0%), malt extract
(0.3%), peptone (0.5%) and agar (2.0%).
While working in a kitchen at the bamboo growing area, a wire loop was
sterilized by heating it to red hot on a spirit lamp, and cooled in absolute alcohol. On
cooling to ambient temperature, the wire loop was used to take samples from sterile
membrane filters which had just been used to filter-sterilize fresh bamboo exudates
for other studies. The contents on the wire loop were striken on the slants in the
McCartney bottles. The bottles were then properly closed and brought to the
laboratory for purification and identification of the prevalent fermenting
microorganisms (Lodder 1970).
Several aliquots of fresh bamboo exudates 900 ml in volume, were hygienically
also collected in pre-sterilized 1 1iter bottles with butyl rubber stoppers and metallic
caps. Two sterile hypodermic needles were injected on each bottle through the
rubber stoppers, to release possible CO2 that could be produced during
transportation. The bottles were immediately placed in well protected ice-boxes and
transported to reach the laboratory in Dar es Salaam, the same day. In the laboratory,
the exudates were fermented naturally, at room temperature (28 1C).
236

Fig.1. A stand of bamboos, having many mbeta hung on various young, cut bamboo shoots
Fig.2. Close up of a yound, cut bamboo shoot with a mbeta hung on it for collection of exudates

Samples were taken from the fermenting set-ups on daily basis for determination
of types and amounts of alcohols in them on a Gas-Chromatograph (Magingo and
Gijzen 1989).

237

Fig.3. Asexual cells of the Saccharomyces sp. From bamboo exudates

Fig.4.Cells of the isolated Saccharomyces sp., some having ascospores

Results and Discussion

A Saccharomyces species (Fig 2-3) was the most prevalent microorganism
fermenting the bamboo sugary exudates. The isolated yeast could also ferment
glucose, galactose, sucrose and raffinose. The yeast isolated, could ferment 27%
w/w pure glucose, to produce and withstand 9.5% w/w ethanol.
In terms of alcohols, only ethanol was detected in the fermented bamboo
exudates taken from one village in Iringa, Tanzania. The alcohol production reached
6.5% w/w on average. It was also established that, at the sampling village, one stand
(clump) of the bamboo has an average of 15 young shoots, each actively producing
an average of 800g of the sugary exudates in one day. Such a clump, occupies an

238

average area 3m in diameter. It thus follows that one stand of the bamboos can
produce 12 Kg of the sugary exudates in a day which, on fermentation, will have
0.78 Kg or 1 liter of pure ethanol. If a 2m inter-stand space is allowed in a hectare
of the bamboo, there will be 400 stands of the bamboo in the hectare, producing a
total of 312 Kg of pure ethanol in a day. In other words, 400 liters of pure ethanol
can be produced by the bamboo, per hectare, per day. If such alcohol is distilled by
solar-powered equipment, it might be a cheap source of ethanol for various uses.
The present isolated yeast from bamboo exudates, could withstand 9.5% alcohol.
It is proposed here that, further work on the bamboo fermenting microorganisms,
could be directed at searching for other microbial strains or types in the bamboo
exudates, which can ferment higher concentrations of sugars, and/or those which can
withstand higher amounts of alcohols.

References
Carmo-Sousa L. do 1969. Distribution of yeasts in nature. In The Yeasts. Vol. 1 Biology of yeasts. Rose
A.H. and Harrison J.S. eds. Academic Press, London. pp. 79-l05.
IBRN Newsletter 1993. The bamboo wine. IDRC Bamboo/Rattan Network Newsletter 14:.2 IDRC-South
Asia Regional Office. New Delhi.
Kigomo B.N. 1990. Bamboo resources in the Eastern Africa Region. In Bamboos. Ramanuja Rao I.V.,
Gnanaharan R. and Sastry C. B. eds. Current Research. Proceedings of the International Bamboo
Workshop, Cochin, India 14-18 Nov. 1988. IDRC, Canada, pp. 22-28.
Lodder J. 1970. The Yeasts. A taxonomic study. North-Holland publishing Company, Amsterdam.
Magingo F.S. and Gijzen H.J. 1989. Assessment of wine quality and state of fermentation process of
Dodoma Wine Company and suggestions for improvement. Consultancy Report, Project funded by
Tanzania Commission for Science and Technology, and Delegation of European Communities in
Tanzania.
_______

GROWTH OF FUSIDIUM COCCINEUM ON SOLID MEDIA

HELEN E. SMITH & GLYN HOBBS

School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street,Liverpool L3 3AF

Abstract. Growth of Fusidium coccineum on both complex and defined agar media resulted in the
production of fusidic acid into the solid medium. Production of the antibiotic was found to be growth
associated in both cases with production ceasing on depletion of glucose. Conidiation did not occur in
any of the cultures examined indicating the absence of a temporal link between antibiotic production and
conidiation in this organism.

Introduction
Fusidic acid is a clinically important antibiotic that inhibits protein synthesis at
the level of translocation in both prokaryotes and eukaryotes (Tanaka et al. 1968). It
exhibits potent activity against gram-positive bacteria (Godtfredsen et al. 1966) and
is used extensively in the treatment of staphylococcal infections in either cream or
tablet form.
Fusidic acid is produced by conventional submerged fermentation using the
fungus Fusidium coccineum (Von Daehne et al. 1984). Despite the obvious clinical
importance of this metabolite little information has been published on the
physiology of the producing organism, for a review see Von Daehne et al. (1984).
This organism differentiates in both surface and submerged culture and a link
between conidiation of the fungus and fusidic acid production has been reported,

239

(Navashin et al. 1981). To study this relationship further we examined the growth of
the organism on agar solidified media.

Material and Methods

Organisms
Fusidium coccineum (CBS 197.55) was used throughout the study. This was
maintained by weekly subculture on YEPG agar and incubation at 28C.
Corynebacterium xerosis (NCTC.9755) was used in the bioassay protocol and was
maintained by subculturing on CX agar slopes with incubation at 37C.
Media
YEPG medium contained, per litre: 10g yeast extract (Difco), 20g glucose, 10g
mycological peptone (Difco), 20g agar. Liquid starter cultures, 50ml in a 250ml
conical flask, were prepared using the above medium without agar, followed by
inoculation with a 5mm diameter plug from a 7 day old YEPG plate. Cultures were
incubated in an orbital shaker (200 rpm), at 30C for 7 days. Aliquots (100l) of the
liquid cultures were used to inoculate plates of YEPG agar. Each plate was prepared
with a cellophane membrane overlay and confluent growth was produced by
spreading the inoculum across the surface of the cellophane. Plates were incubated
at 30C for various times as shown. Minimal medium contained, per litre final
concentration: 10g glucose, 3.36g sodium dihydrogen orthophosphate (dihydrate),
0.85g sodium nitrate, 0.75g potassium chloride, 0.64g sodium sulphate decahydrate,
0.26g magnesium chloride hexahydrate, 2.2mg calcium chloride, 24g sodium
molybdate dihydrate, 0.292g EDTA. Trace salts. 2.0mg zinc oxide, 27mg ferric
chloride hexahydrate, 10mg manganese chloride 0.85mg cupric chloride dihydrate,
2.4mg cobalt chloride hexahydrate, 0.3mg boric acid and 20g biotin. The pH of the
medium was adjusted to 6.8 with 1M NaOH prior to sterilization. The trace salts
solution was made up separately as a hundred fold concentrate, the pH was adjusted
to 2.0 with concentrated hydrochloric acid and sterilised at 121 C for 15min.
Glucose was also sterilised separately as a ten fold concentrate.
For the physiological experiments 20ml of medium was dispensed into each
plate and allowed to solidify prior to placing a pre-sterilised cellophane membrane
on the surface. Cultures were inoculated directly on the surface of the membrane and
spread with a glass spreader to achieve a confluent inoculum.

Analysis of samples
Biomass was determined by dry weight after removal of cell material from the
surface of the membrane with a razor blade. The biomass was placed on a Whatman
No1 filter and dried in a microwave oven for 15 min. on the "defrost" setting.
Samples were taken from beneath the membrane using a cork borer (5mm
diam.). The agar plugs were then subjected to a freeze squeeze protocol to provide
liquid medium samples using the method of Tautz & Renz (1983). Glucose was
measured using a glucose oxidase kit (Boehringer-Mannheim). Fusidic acid was
measured by bioassay using C. xerosis (Bywater 1975).

Results

240

Fig.1. Growth of F. coccineum on YEPG medium. (a) biomass, (b) glucose, (c) fusidic acid. Values
represent the mean of at least three determinations.

241

Fig.2. Growth of on minimal medium. (a) biomass, (b) glucose, (c) fusidic acid. Values represent the
mean of at least three determinations.

Growth of F. coccineum on complex medium was rapid with maximum biomass

levels being achieved within one day of inoculation (Fig 1a). The biomass accretion
coincided with the consumption of glucose from the medium (Fig

242

1b).Approximately half the available glucose was consumed during this initial
growth period. After 21 hours of growth, biomass levels declined possibly due to
nutrient limitation or diffusion limitation. Measurements of fusidic acid
concentrations in the medium revealed a biphasic production pattern, with an
exponential increase in antibiotic during the growth phase followed by a linear
accumulation after biomass accumulation had ceased (Fig 1c). Glucose levels
throughout the experiment were found to decrease, reaching an undetectable value
after 90 hours post-inoculation. The complete removal of glucose from the medium
resulted in the cessation of fusidic acid accumulation, suggesting an intimate link
between antibiotic production and glucose availability. The cultures were monitored
throughout for the appearance of morphological differentiation, particularly for the
appearance of conidia. At no time during the experiment were conidia observed
suggesting that under these conditions at least, no link between antibiotic production
and conidiation could be shown.
To further investigate the relationship between glucose availability, conidiation
and antibiotic production we developed a defined minimal medium that would
support growth and antibiotic production. Growth of the culture on minimal medium
was slower than for complex medium, reaching maximum biomass after 120 hours
(Fig 2a). Biomass accumulation ceased close to the point of glucose exhaustion
suggesting that growth in this culture was probably glucose limited (Fig 2b). Fusidic
acid production was growth linked under these conditions and as with the complex
medium the production of the metabolite ceased at the point when glucose
completely disappeared from the medium (Fig 2c). Examination of the cultures for
evidence of conidiation revealed a complete absence of conidia throughout the
experiment, providing further evidence for the lack of association between
conidiation and antibiotic production in this organism.

Discussion
Plate cultures provide a simple and convenient way of investigating the
connection between morphological differentiation and metabolite consumption and
production. The evidence provided herein suggests that conidiation and fusidic acid
production are physiologically separate entities. This data conflicts with earlier work
of Navashin et al.(1981) who describe a temporal link between conidiation and
antibiotic synthesis in F. coccineum. Furthermore, our work would also suggest that
the cells responsible for the production of the antibiotic are the hyphal form rather
than the conidia themselves. The kinetics of production of the antibiotic on solid
medium is not typical of a secondary metabolite with the greatest rate of production
occurring during the rapid growth phase. The question then arises as to whether
fusidic acid is an "overflow metabolite" of primary metabolism or a true secondary
metabolite.
Clearly fusidic acid production is dependent upon available glucose, an efficient
protocol for production therefore may be to use immobilised resting cells of the
fungus which could be fed continuously with glucose.

References

Bywater M.F. 1975. Fusidic Acid. Laboratory Methods in Antimicrobial Chemotherapy. 219-221.
Godtredsen W.O., Daehne W. von, Tybring L. and Vangedal S.1966. Relationship between structure and
antibacterial activity. Journal of Medical Chemistry 9:15-22.
Navashin S.M., Bartoshevich Y.E., Telesnina G.N. Zvjagilskaya R.A., Dimitriyeva S.V., Saykin Y.O.

243

 and Krakhmaleva I.N. 1981. Respiratory system of Fusidium coccineum and regulation of
biosynthesis of fusidic acid and sporogenesis. European Journal of Applied Microbiology and
Biotechnology 12:220-225
Tanaka N., Kinoshita T. and Masukawa H. 1968. Mechanism of protein synthesis inhibition by fusidic
acid and related antibiotics. Biochemical and Biophysical Research Communications 30:278-283.
Tautz D. and Renz M. 1983. An optimised freeze-squeeze method for the recovery of DNA fragments
from agarose gels. Analytical Biochemistry 132:14-19.
Von Daehne W., Jahnsen J., Kirk I., Larsen R. and Lorck H. 1984. Fusidic acid: Properties, biosynthesis
and fermentation. Drugs and the Pharmaceutical Sciences 22:427-450.

_______

STRAIN POLYMORPHISM IN PDR1, A TRANSCRIPTION REGULATOR

OF PLEIOTROPIC DRUG RESISTANCE GENES IN SACCHAROMYCES
CEREVISIAE.

E. CARVAJAL1, E. BALZIL2 AND A. GOFFEAU2

1 Instituto de Quimica Universidade Federal do Rio de Janeiro, Cidade Universitaria, IIha do Fundao,
CEP 21945, Rio de Janeiro, RJK, Brasil
2 Universite Catholique de Louvain, Unite de Biochimie Physiologique, Place Croix du Sud, Louvain-la-
Neuve.

Abstract. PDR1 is a Zn2 Cys6 transcription factor that regulates positively

PDR5 and probably other mutidrug pumps in Saccharomyces cerevisiae. (Balzi et
al. 1994).
The comparison of the PDR1 DNA sequence has been carried out on the
following seven so called wild type laboratory strains: IL 125-2B, D 286-2A, FM
11, GR 350, JG 200, FY 1679-28C, S288C.
Seven nucleotidic differences clearly distinguished the strains of european origin
(IL1252B, D 286-2A) from those of american origine (JG200, GR 350, FM11, FY
1679-28C, S288C). Four out of the seven differences resulted in aminoacid changes,
and three others were only at the nucleotide sequence. In all european strains, the
amino acid residues 411, 821 and 921 were found to be lysine, alanine and
isoleucine instead of glutamine threonine and threonine respectively in the american
strains. The fourth difference was observed in the polyasparagine strech beginning at
residue 1009: in the european strains five successive asparagine residues were
observed instead of ten asparagines in the american strains.
Thus the polymorphism is more pronounced in the C-terminal region of the protein.
In this family of transcription factors the C-terminus is usually involved in
transactivation of transcription whereas the N-terminus bears the zinc finger domain
which binds DNA on specific consensus motif. We will construct a set of a isogenic
strains carrying the different wild type alleles to investigate whether or not these
modifications affect the transactivation activity of PDR1.

Reference
Balzi E., Wang M., Leterme S., Van Dyck L. and Goffeau A. 1994. PDR5, a novel yeast multidrug
resistance conferring transporter controlled by the transcription regulator PDR1. J. Biol. Chem. 269:
2206-2214
______

244

 STRUCTURAL IDENTIFICATION AND BIOCHEMICAL MODE OF

ACTION OF A MYCOTOXINE ISOLATED FROM COCHLIOBOLUS
SATIVUS (MUCL 18528)

D. VILRET, P. GOBLET, J. HUTSCHEMACKERS, N. COSYNS, M. NOL AND M. BRIQUET

Universit Catholique de Louvain, Facult des Sciences Agronomiques, Unit de Biochimie

Physiologique, Place Croix du Sud 2/20, B-1348 Louvain-la-Neuve

Abstract. A mycotoxin extracted from the culture medium of Cochliobolus

sativus has been purified by chromatography (TLC, HPLC) and identified by
infrared, mass and NMR spectrometry. The chemical structure corresponds to the
"helminthosporol", a sesquiterpene discovered in 1965 by S. Tamura.
Our results on the biochemical activity show that this compound inhibits the
oxidative phosphorylation of plant mitochondria (ATP synthase) but does not inhibit
the ATP phosphohydrolase (ATPase) activity. In contrast the mitochondrial ATPase
activity of the yeast Saccharomyces cerevisiae is strongly inhibited by the toxin. The
plasma membrane ATPase activity of Saccharomyces cerevisiae is not inhibited by
helminthosporol.
_____

CHARACTERIZATION OF THE CARBOHYDRATE MOIETY ON

PROTEINS SECRETED FROM DIFFERENT FUNGI

M. MARAS1, W. LAROY1, W. FIERS1, G.L HENNEBERT 2 AND R. CONTRERAS1

1Laboratory of Molecular Biology, State University, Ledeganckstraat 35, B-9000 Ghent, Belgium.
2Mycothque MUCL, Universit Catholique de Louvain, Place Croix du Sud 3, B-1348 Louvain-la-
Neuve, Belgium.

Abstract. Many different yeasts and filamentous fungi are attractive candidates
for the production of secreted, therapeutically valuable proteins. Since the
glycosylation pathway in these lower eukaryots differs from that in mammalian
cells, oligosaccharides different from the complex, mammalian type are synthesized.
Fungi-type glycosylation, known to be mannan or (for certain species)
galactomannan forms an obstacle to application of heterologous proteins as
therapeutic agents. The latter proteins are rapidly cleared from the blood because of
recognition and binding of fungi-like oligosaccharides to sugar binding receptor
proteins (Summerfield et al., 1986). With the exception of Saccharomyces
cerevisiae, detailed knowledge about glycosyl structures is very poor. Yeasts are
known for their capacity to hyperglycosylate proteins. Filamentous fungi, on the
other hand, seem to synthesize smaller glycans (Berka, R.M. and Barnett, C.C,
1989). This resembles more the situation in mammalian cells.
With the aim to obtain more information about the diversity of oligosaccharide-
structures and about the possible existence of glycosyl structures more similar to the
mammalian system, we studied glycoproteins from a collection of yeasts and
filamentous fungi. Lectins were used to investigate the identity of terminal sugar
residues different from mannose. In a collection of yeasts, a few species seemed to
have terminal a-linked galactose residues: Binding of Bandeiraea simplicifolia I
245

lectin, highly specific for terminal a-linked galactose, to their secreted proteins, was
demonstrated. We are now investigating a collection of filamentous fungi, to
determine whether new glycosyl patterns can be found.

References
Summerfield J.A. and Tayler M.E. 1986. Mannose-binding proteins in human serum: identification of
mannose-specific immunoglobulins and a calcium-dependent lectin, of broader carbohydrate
specificity, secreted by hepatocytes Biochim. Biophys. Acta 883: 197-206.
Berka R.M. and Barnett C.C. 1989. The development of gene expression systems for filamentous fungi.
Biotech. Adv. 7: 127-154.
_______

SPECIFIC AND NON-SPECIFIC INTERACTIONS IN YEAST CELL

FLOCCULATION
P.B. DENGIS, L.R. NLISSEN, AND P.G. ROUXHET

Universit Catholique de Louvain, Unit de Chimie des Interfaces, Place Croix du Sud 2 Bte 18, B-1348
Louvain-la-Neuve.

Abstract. At the end of the fermentation in brewery, cell aggregates are formed
which accumulate on top of the fermented wort (top strains) or which sediment to
the bottom (bottom strains). The aim of this research is to understand better the
mechanisms which control the yeast aggregation, in particular the importance of
long range interactions (double layer interactions, van der Waals forces), short range
non-specific interactions (hydrophobic interactions, salt bridges) and specific
interactions (molecular recognition).
The yeast surface chemical composition is determined by X-Ray Photoelectron
Spectroscopy (XPS); the hydrophobicity is evaluated by contact angles and the
surface electrical properties are characterized by the zeta potential. The tendency of
the cells to form aggregates is determined by turbidimetric measurements after
controlled stirring and subsequent settling in defined solutions.
Top fermentation strains have been found to be more hydrophobic, to have a
lower phosphate surface concentration and a less negative zeta potential. A more
detailed comparison has been performed between one top and one bottom strain. For
both strains aggregation takes place only at the end of the exponential growth phase
and it requires the presence of calcium. The tendency of the top strain to form
aggregates is maximum between pH3 and 4.5, i.e. close to its isoelectric point of 4.
For the bottom strain the isoelectric point is much lower than the optimum
aggregation pH range: however the decrease of the phosphate surface concentration
during the culture is in accordance with an influence of electrostatic interactions. In
the adequate range of pH, flocculation of the bottom strain requires addition of about
0.1 mM Ca++ while the top strain flocculates without calcium addition. The two
strains exhibit also different responses with respect to the addition of organic
compounds in the solution. For the bottom strain, certain sugars provoke an
inhibition of flocculation or a redispersion of previously formed flocks; this is a
specific effect, occurring with 10 to 500 mM glucose, mannose and maltose, but not
with galactose, which points to the involvement of molecular recognition in cell
aggregation. Flocculation of the top strain, on the contrary, is not influenced by
these sugars; furthermore, addition of 5% ethanol broudens considerably the pH

246

range (2.5-5.5) in which flocculation takes place suggesting that solvation forces
play an important role.
_________

MODIFICATION OF SUPPORT SURFACE PROPERTIES TO

IMPROVE MICROORGANISM ADHESION

P.A. GERIN1, M.-N. BELLON-FONTAINE2, M. ASTHER3 AND P.G. ROUXHET1

1Unite de Chimie des Interfaces, UCL, Place Croix du Sud, 2/18, B-1348 Louvain-la-Neuve,
Belgium2Laboratoire de Gnie de l'Hygine et des Procds Alimentaires, INRA, 25 avenue de la
Rpublique, F-91300 Massy, France
3Laboratoire de Biotechnologie des Champignons Filamenteux, INRA, 163 avenue de Luminy, Case
Postale 929, F-13288 Marseille, France

Abstract. For immobilisation of fungi in bioreactor, a quick adhesion and

homogenous distribution of the spore inocculum on the support surface is desirable.
This is expected to lead to a more uniform growth and colonisation of the support.
Polycations and ferric ions were adsorbed on the surface of a flat polycarbonate
support, either as such, or pretreated by sulpho-chromic mixture. The pretreatment
grafted sulphate or sulphonate groups, which lowered the isoelectric point and
rendered the surface more negative. The adsorption of polycations and ferric ions
increased the zeta potential and isoelectric point of these surfaces. 5-10 atomic
layers of adsorbed polycations were found at the pretreated support surface; on the
non-pretreated supports, less than one atomic layer was adsorbed.
The supports were then submitted to a flow of Phanerochaete chrysosporium
spore suspension; these fungal spores have a hydrophobic and negatively charged
surface. A few spores adhered in a patchy way on the native supports. Adsorption of
polycations and ferric ions increased the amount of adhering spores and led to a
homogenous distribution. This might be due to the reduction of the electrostatic
repulsion between the spores and the support, which increased the chance for the
spores to come in contact with the surface and to adhere.
The spores adhering to the supports were later submitted to a flow of culture
medium. Neither adhesion on the support, nor the presence of adsorbed polycations
inhibited or delayed the swelling, the gerrnination and the growth of the spores.
_______

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 BACTERIOLOGY
GAS CHROMATOGRAPHIC ANALYSIS OF CELLULAR FATTY ACIDS
IN THE IDENTIFICATION OF MICROORGANISMS

P. WAUTHOZ, M. EL LIOUI AND J. DECALLONNE

Unit de Microbiologie, Facult des Sciences agronomiques, Universit catholique de Louvain, Place
Croix du Sud, 2, bte 12, B-1348 Louvain-la-Neuve, Belgium

Since Abel et al. (1963) demonstrated that fatty acid composition can be useful
to classify microorganisms into genera and species, various workers have
successfully used it to characterize, differentiate and classify bacteria.
As the cellular fatty acid profile is affected by a number of parameters, rigid
standardization of experimental conditions and techniques is required. The
extraction and derivation procedure proposed by Miller and Berger (1985) is
commonly used in bacterial analysis. It consists in the main following steps:
1. saponification (NaOH in MeOHaq, 100C, 30 min.)
2. methyation (HCI/MeOH, 80C, 10 min.)
3. extraction (hexane/ethylether, 10 min.)
4. base wash (dilute NaOH, 10 min.).
The advantage of this method are the low time consumption, the low volume of
reagents (each 1 to 2 ml) and the quantity of bacterial material require (about 40mg).
We applied this procedure to about 300 foodborne bacteria, grown under
standardized conditions. The reproducibility, from bacterial cultivation to the GC
analysis, was estimated at So=94.202.80 (n=84), where So is the overlap
coefficient (Bousfield et al. 1985). Inside a species, the So was always greater than
79.
The great diversity of bacterial fatty acids, including saturated, monounsaturated,
straight chain, branched-chain, 2- and 3-hydroxy and cyclopropane types, and the
variability of their relative proportions permit one to define arge groups of strains
(lactic acid bacteria, Enterobacteriaceae,...) and to separate closely related genera
(Micrococcus, Staphylococcus) (Table 1).

The same extraction and derivatization procedure was also applied to

characterise yeast strains. It should be noted that this procedure does not allow one
to separate fatty acids from long-chain hydrocarbons, which elute after fatty acids in
GC analysis.
Four stains of Saccharomyces (S. bayanus MUCL 31495; S. pastorianus MUCL
31496; S. cerevisiae MUCL 31497; S. paradoxus MUCL 31498) were investigated
after grown for 72 hours, at 25C, in dextrose 20g/l, yeast extract 5g/l, meat peptone
10g/l broth, on a rotary shaker at 160 rpm.
The profiles obtained were rather simple with a preponderance of saturated and
monounsaturated C16 and C18 acids (Table 2).
In addition, in certain genera (Bacillus, Pseudomonas), the profile So obtained
may be used as characteristics in speciation (Fig. 1).

248

 Table 1. So value calculated between different bacterial genera.

N 1 2 3 4 5 6 7 8 9
Gram -
Enterobacteriaceae
Escherichia coli ATCC 10536 1 -
Salmonella RL 2 92 -
Pseudomonas fluorescens UPB 3 71 75 -
408
Gram+
Bacillus subtilis ATCC 12711 4 3 3 4 -
Lactic acid bacteria
Lactobacillus plantarum DSM 5 57 55 46 4 -
20174
Leuconostoc mesenterodes 6 56 57 47 5 89 -
NCIB 3354
Lactococcus lactis LMG 6897 7 40 39 29 5 70 65 -
Pediococcus acidilactici NCIB 8 61 58 52 3 74 69 66 -
6990
Staphylococcus aureus MBLA 9 4 4 5 73 5 7 6 5 -
210

Fig.1. Dendrogram of Bacillus species based on the So values.

Table 2. Fatty acid composition of Saccharomyces strains (% total fatty acids)

Strain Fatty acid

12:0 14:0 16:0 18:0 14:1( 16:1 18:1 18:1

9c) (9c) (9c) (11c)
S. bayanus MUCL 31495 1,37 0,59 9,51 3,22 0,78 61,18 19,69 0,01
S. pastorianus MUCL 31496 1,56 0,70 2,42 0,24 2,32 77,25 14,28 0,20
S. cerevisiae MUCL 31497 2,33 0,81 3,32 0,74 3,64 63,04 23,50 2,26
S. paradoxus MUCL 31498 0,83 0,93 5,98 0,45 1,48 68,28 19,78 0,95

249


 The four strains had palmitoleic acid as the major component with an occurrence
of up to 76% of the total fatty acid. The proportion of oleic acid varied from 14 to
27% among strains. The palmitic acid is several folds more abundant than stearic
acid. Saturated and monounsaturated C12 and C14 were present in lower
proportions. Traces (< 1%) of 15:1, 15:0, 16:1 (11c) and 17:1 acids were observed.
No branched-chain and cyclopropane acids, which are common components of
certain bacterial lipids, appeared in the profiles.
The qualitative composition is in agreement with previous reports on
Saccharomyces strains (Augustyn and Kock 1989, Kock et al. 1986, Moss et al.
1982) and is highly similar to those found for other yeast genera (Moss et al. 1982,
Smit et al. 1987). Therefore, the relative simplicity of the fatty acid composition of
yeasts does not seem to offer as much aid in the taxonomy of the unicellular fungi as
it does for the bacteria. Betwee the four strains studied, So was greater than 82,
which is a higher value than for bacterial intra-species similarity. This does not
allow one to draw conclusions on the classification of these strains in one or
different species. Further experimental work is needed on more strains to evaluate
the ability of using quantitative differences to composition to characterise, classify
and identify yeast strains.

Acknowledgements
We thank Pr Hennebert, MUCL, for the yeast cultures. This work was supported by the Ministre de
l'Agriculture de la Region Wallone and by Requasud.

References
Abel K., 1963. Classification of microorganismsby analysis of chemical composition I. Feasibility of
utilizing gas chromatography. J. Bacteriol. 85: 1039-1044.
Augustyn O.P.H. and Kock L.F. 1989. Application of an adapted technique to differentiate between
strains of Saccharomyces cerevisiae. J. Microbiol. Methods. 10: 9-23.
Bousfield I.J., Smith G.L, Dando T.R. and Hobbs G. Total fatty scid profiles in the identification of
coryneform, nocardioform and some other bacteria. J. Gen. Microbiol. 129: 375-394.
Kock J.L.F., Cotrell M. and Lategan P.M. 1986. A rapid method to differentiate between five species of
the genusSaccharomyces. Appl. Microbiol. Biotechnol. 23: 499-501.
Miller L. and Berger T. 1985. Bacteria identification by gas chromatography of whole cell fatty acids.
Application Note 228-41, Hewlett Packard.
Moss C., Shinoda T and Samuels J W. 1982. Determination of cellular fatty acid compositions of various
yeasts by gas-liquid chromatography. J. Clin. Microbiol. 16: 1073-1079.
Smit E. et al. 1987. Dependence of culture age on the cellular long-chain fatty acid composition of three
basidiomycetes yeasts. Syst. Appli. Microbiol. 10: 38-41.
______

IDENTIFICATION OF LACTIC ACID BACTERIA USING RAPD-PCR.

NOL A. AND J. DECALLONNE

Unit de Microbiologie, Universit Catholique de Louvain-Facult des Sciences agronomiques, Place
Croix du Sud 2, BP 12, 1348 Louvain-La-Neuve - Belgium.

Abstract. Lactic acid bacteria play an essential role in food technology. They
improve the aroma and texture in food, and inhibit the growth of spoilage bacteria.
However, some strains of lactic bacteria are involved in food spoilage and few are
found even pathogens.
Therefore, studies of reliable, simple and time saving identification methods are
required for both basic research on lactic acid bacteria and their applications in
industrial food fermentations. These methods aim to distinguish genera and species.

250

 Identification methods of lactic acid bacteria are conventionally based on

morphological and biochemical criteria. Recently, they have highly developed by
using DNA based analysis, and particularly by the Polymerase Chain Reaction
(PCR) (Saki et al. 1986).
A recent innovation is amplification fragment length polymorphism analysis by
arbitrary primers (Welsh and Mac Clelland 1990). This strategy called RAPD
(Random Amplified Polymorphic DNA) involves the enzymatic amplification of
template DNA directed by one or more arbitrary oligonucleotide primers to produce
a characteristic spectrum of products, a proportion of which can be polymorphic.
The procedure is fast, independent of prior genetic and biochemical knowledge
of the organism tested.
The present work has been carried out as a contribution to this new method in the
field of lactic acid bacteria identification. We describe the use of RAPD as an
effective and rapid method to discriminate 12 Lactobacillus strains that were
previously characterized in our laboratory by gas chromatographic analysis of
cellular fatty acids (HRGC) (Wauthoz 1992).
Lactobacillus brevis NCIB 4617, NCIB 4036; Lactobacillus buchneri NCIB 8007, ENSI 9;
Lactobacillus casei casei ENSI; Lactobacillus confusus NCIB 4037;Lactobacillus fermentumDSM
20052, CIP 53163, EPI 7, EPI 11; Lactobacillus plantarum DSM 20174, CIP A169.

Lactobacillus strains were grown under standardized conditions in MRS medium

(Man, Rogosa and Sharpe) and total genomic DNA for RAPD was extracted from 3
ml of overnight culture following Cancilla (1992). RAPD was performed by the
method of Mazurier (1992) using ten 10-mer primers (kit OPC-01, Operon) and the
amplification products were separated according the size by agarose gel
electrophoresis.
Using the oligonucleotide OPC-01 (TTCGAGCCAG) as primer, specific DNA
fingerprints have been obtained for each of the studied strains (figure 1). The
number of DNA bands in these fingerprints ranged from 1 to about 12 and
differences in intensity were observed between these bands.
Although only a limited number of Lactobacillus strains were evaluated, these
data demonstrate the ability of RAPD to differentiate between Lactobacillus strains
that are not always discriminated by HRGC analysis and by conventional
microbiological methods.

References.
Cancilla M.R., Powell I.B., Hillier A.J. and Davidson B.E. 1992. Rapid genomic fingerprinting of
Lactococcus lactis strains by arbitrarly primed PCR with 32P and fluorescent labels. Appl. and Env.
Microbiol. 58: 1772-1775.
Mazurier S.I. and Wernars K. 1992. Typing of Listeria strains by random amplification of polymorphic
DNA. Res. Microbiol. 143: 499-505.
Saiki R.K., Gelfand D.H., Stoffel S., Scharff S.J., Higuchi R.G., Horn G.T., Mllis K.B. and Erlich H.A.
1986. Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase.
Science 239: 487-491.
Wauthoz P.1992. Caractrisation et identification de la flore bactrienne des aliments par analyse
chromatographique des acides gras cellulaires. PhD Thesis, Universit Catholique de Louvain,
UCL.
Welsch J. and Mac Clelland M. 1990. Fingerprinting genome using PCR with arbitrary primers. Nucleic
Acids Res. 18: 7213-7218.
______

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 252

 LAST LECTURE

5 December 1995

253

 254

 TAXONOMY AND NOMENCLATURE OF THE

FUNGI:
REASONS FOR A MYCOTHEQUE

RETROSPECTIVE 4

G. L. HENNEBERT

Introduction

Philibert Biourge, in 1895, then assistant of Professor Jean-Baptiste

Carnoy, studying the moulds of brewery malts, went to question the
diversity of the Penicillium isolates, all being in greenish tinges close to that
of Penicillium glaucum. Addressing the matter to Carnoy, he received this
answer: "Then, Biourge, do a monographic study of the genus!" (Biourge in
Glibert 1912, Hennebert 1979).
In 1956, Victor Estienne, my professor in plant pathology, observed with
similar perplexity that "authors describe distinct species of Botrytis on the
onion, Allium cepa" and asked me, "What are they, exactly? Are they not all
Botrytis cinerea? You should study them." It was his suggestion for a PhD
research, to solve a problem that he thought to be of host specificity:
"Compare them through cross-inoculations in host plants." But cross-
inoculations failed.
Professor N. Fabritius Buchwald (Fig.8), author of the Studies of
Sclerotiniaceae (Buchwald 1949) in the steps of Prof. Whetzel of Cornell
University, said to me in Copenhagen, "The matter is primarily taxonomical.
There are many names of Botrytis species in the literature. Many are not
Botrytis, some are, and among these, a number are host specific, others are
not. Differentiation of the species is first of all morphological. You should
compare fresh collections morphologically, from nature and in culture. You
should look for the perfect stage in nature and find out the correct name to
apply". His impulse, in 1957, launched me into the taxonomy and the
nomenclature of the fungi.
The literature from the oldest to the most recent includes more than 380
species names in Botrytis, but many have become synonymous to names of
taxa as distant as Phytophthora. The remaining names, often with old
descriptions in Latin, too short or lacking illustration, were investigated
through examination of the original or type material, when extant, from
diverse herbaria.
In the meantime, I was exploring woods, swamps, ponds, fields,
vineyards, orchards and vegetable plots, flower gardens, agricultural and

4
Last lecture delivered at UCL on December 5, 1995.
255

forest soils, even caves (Hennebert 1960b, 1971) collecting specimens, and
isolated Botrytis strains in pure culture.
Most of the cultures were conidial and sclerotial. I was disturbed finding
in some of them small globose conidia like Penicillium conidia, even in single
Botrytis conidium culture. I could not obtain their germination and
concluded that they were spermatia, first discovered by De Bary (1884) in
Botrytis and described by Istvanffi (1905) and Brierley (1918) (Hennebert,
1960a).
The multiplicity of forms in Botrytis cultures, conidial, sclerotial,
spermatial, expressed the pleomorphy found as a quite general character of
the fungi (Tulasne 1851). Fungal pleomorphy is a phenomenon that was
misunderstood and confused with polymorphism and 'transformism' by
Carnoy (1870) who described the transformation of a Mucor into a yeast that
turned to become a Penicillium (Hennebert 1979).
The forms observed in Botrytis were said to be asexual and "imperfect".
Guided by the works of Whetzel (1945), Raabe (1938) and Buchwald (1949,
1953), who observed and described the sexual state of Botrytis species, I
collected apothecia on sclerotia, together with conidiophores of Botrytis, in
the field under Allium ursinum, Ficaria verna and Caltha palustris,
representing three different species, Botryotinia globosa Buchwald, B.
ficariarum Hennebert and B. calthae Hennebert and Elliott (Fig. 1-3)
(Hennebert 1958, 1960a, Hennebert and Groves 1963). The apothecia, the
sexual, ascosporic form of reproduction, were said to be "perfect" states of
the asexual Botrytis states. Apothecia were also developing spontaneously in
a pure culture of Botrytis porri Buchwald from Allium porrum, confirming its
identity as Botryotinia porri (van Beyma) Whetzel (Fig. 4) (Hennebert 1960a,
1963b). The species is homothallic. But in heterothallic species to produce
apothecia needs crossing.
To each state of the fungi, imperfect state and perfect state, a distinct
Latin binomial name was given. This is heritage of the past when
mycologists considered each reproductive form a distinct species. The most
common species of the genus Botrytis, thus received the following names:
Sclerotium durum Pers.: Fr., described by Persoon (1794a) for the sclerotial
form, belonging to the genus Sclerotium Tode: Fries (Tode 1790).
Botrytis cinerea Pers.: Fr., described by Persoon (1797) for the conidial
form, renamed from the polynomial nomenclature of Micheli (1729).
Botryotinia fuckeliana (De Bary) Whetzel, described for the ascosporic
sexual ("perfect") form of reproduction, the apothecium. The apothecial state
was discovered on Sclerotium echinatum Fuckel, a synonym of S. durum Pers.:
Fr., by De Bary (1866), who named it Peziza fuckeliana De Bary in honour of
Fuckel, a name that Fuckel (1870) himself changed into Sclerotinia fuckeliana
(De Bary) Fuckel. The genus Sclerotinia was divided by Whetzel (1945) on
the basis of the organic connection to diverse asexual forms and Botryotinia
connected to the form Botrytis as established by De Bary (1864, 1884, 1887).

256

 The spermatial form of Botrytis cinerea, discovered in this species by De

Bary (1884), belongs to Myrioconium Sydow (1912) but has not been named
specifically. Spermatia, so-called by Tulasne (1856), are phialoconidia which
rise from both external and internal phialides on hyphae.

Fig. 1. Botryotinia ficariarum (Botrytis ficariarum anamorph) ; 2 and 3. Botryotinia calthae, apothecia
and culture ; 4. Botryotinia porri (sclerotium and apothecium in culture.

Several names for one fungus species appeared contrary to Linn's

principle and that of the International Code of Botanical Nomenclature, one
taxon - one name. In such cases the Code prescribes the predominance of the
name of the sexual form over the other names and tolerates however the use
of the other names. That peculiarity of the nomenclature of the pleomorphic
fungi struck me from the early days.

257

 The embryo of a mycotheque

Soon I had hundreds of Botrytis isolates in pure culture beside isolates of
many other fungi, an embryo of a fungus culture collection, a 'mycotheque'
(Hennebert 1968d). "But why bring nature in small tubes?" questioned a
relative of mine, long ago. Why a fungus culture collection? That is the
question I wish to answer today, through an overview of 35 years of
mycology.
Carnoy (1870) had already answered the question: "J'ai toujours cru que,
pour faire sortir de l'enfance la science mycologique, il fallait cultiver les
champignons, les cultiver toujours, les placer dans des milieux les plus
divers, et, le microscope en main, suivre pas pas leur dveloppement.
Lorsqu'on trouve une forme, au lieu de la dcrire immdiatement comme
une espce nouvelle, n'est-il pas bien plus naturel de se demander d'abord si
une culture mthodiquement varie la reproduirait toujours ou si elle ne
pourrait pas en tirer autre chose? Aussi on doit savoir gr tous ceux qui
s'adonnent aux tudes exprimentales sur le dveloppement et les
mtamorphoses de cette classe d'tres singuliers." Here was an early
promotion of the cultivation of the fungi in pure culture and of its use in
experimental mycology to help fungal taxonomy.
Why a mycotheque? With Carnoy, I answer: to help elaborate a more
natural taxonomy of the fungi, through demonstration and comparison in
pure culture of their morphology and pleomorphy, and correlatively to
resolve nomenclatural problems that pleomorphy may cause. Taxonomy
and nomenclature were, to me, the prime reasons for building a
mycotheque.
In those early days, I frequently visited the Centraalbureau voor
Schimmelcultures, a well organized mycotheque, successively directed by
Dr. Johanna Westerdijk, Dr A. van Beverwijk (Fig. 9) and Dr J.A. von Arx
(Fig. 10) and where Prof. Biourge deposited Penicillium type strains in 1929.
The Mycotheque developed from that of Prof. Biourge at the University
of Louvain, named MUCL, included 34,000 herbarium specimens and 24,000
living strains based upon them in 1994 5.

1. DEFINITIONS

Taxonomy and nomenclature are too often abhorred activities and thus
eliminated from training in many universities. However, I wish to let them
be seen more serenely.
Taxonomy and nomenclature are at the base of all activities of man who
observes, thinks and communicates. Observation leads to thought, thought
to concepts. Taxonomy is the result of observation and thought.
Nomenclature results from thought and concepts. Taxonomy and
nomenclature is at the base of all common knowledge.

5
MUCL preserves presently around 51.000 herbarium specimens and 35.000 living strains of
fungi.
258

 Observation of the diversity of things induces comparison and

distinction, and leads to elaboration of concepts and to formation of words.
If a concept is a category, the word is a noun; if it is an attribute, the word is
an epithet. Categories are genera, epithets refer to species. That is taxonomy.
Nouns are generic names, nouns plus epithets are species names. That is
nomenclature. Common knowledge is made of taxonomy and
nomenclature.

Fig. 5. Taxonomy and nomenclature in the old time (by an unknown mycologist).

The first approach of fungal biodiversity consists of taxonomy and

nomenclature. The names of fungal species are, in the binomial
nomenclature of Linn, a specific epithet combined with a generic name. For
the stability of nomenclature, a reference specimen of the species, designated
as type of its name and preserved under that name, guarantees the name
application.
Classification is the direct product of taxonomy, by creation of categories
of hierarchical rank: species, genera, families, orders, etc. When built on
different criteria, classifications differ. Different classifications constitute
different systems.
Identification is the recognition of an individual, a specimen, as
belonging to a known species through its close similarity with the type or
reference individual of the species, and application of the species name. If an
individual does not belong to any described species, it represents a new
species, perhaps a new genus, to be described, typified and named.

2. TAXONOMY

2A. Study of fungi starts in the field, by observation and collecting

The first step of any mycological study is the observation of the fungi in
the field. To teach this basic requirement, Dr Stanley J. Hughes, the first day

259

after my arrival in Canada for a two-year postdoctoral stay (1960-1962), took

me to the forest, in Gatineau Park, to collect microfungi, field shoes on feet,
knife and basket in hands (Fig. 5).
Professor Richard P. Korf, who I visited at Cornell University in 1961 for
the first time of many, was doing the same with his students: no mycology
in class or laboratory before a discovery of the fungi in the field (Fig. 6). The
bonnet of the car could serve as a bench to sort the collections. But take care:
some fresh polypores, like Ganoderma applanatum, exude drops of so active
enzymes that within one hour time you may have to let repaint the bonnet.
Nathalie Buffin, my assistant, repeatedly went collecting, whether
thermophilic fungi in hot springs from coal tips in the Borinage, South of
Belgium, or nematophagous Arthrobotrys species, or coprophilous or semi-
aquatic hyphomycetes.
Mycology starts in, and proceeds from the field. It is necessary in the
field to annotate the collected specimens with indication of the substrate,
host, habitat, locality, collecting date, collector's name, and any other
ecological information observable in the field. Necessary also is the
microscope examination and the drawing of the fungus mounted when
fresh, for, as I experienced, microscope slides are much better from fresh
than from dried material. Drawings, made at the same magnification, and a
written description on prepared format, help to attempt a first identification
and apply a name, even a provisional one. Isolation of the fungus shall be
made on different culture media, preferably by transfer of single spores,
conidia or mycelial threads. The fungus is then labelled and registered with
number and name. Finally, specimens are dried and preserved, together
with microscope slides and dried cultures, in properly sealed and labelled
envelopes in the herbarium (Hennebert 1968d).

2B. Building a mycotheque for experimental taxonomy

The herbarium is the first part of the mycotheque. Specimens are generally
disposed by name (genus and species), either taxonomically (classes, orders,
families) or alphabetically throughout. Both taxonomic and alphabetic
disposition are subjected to name changes, but the former is also dependent
on the classification. I adopted the alphabetical order. Packets are disposed
like cards in boxes, what proved more flexible and space-saving than
packets glued on herbarium sheets in folders, which I first used. Each set of
specimens of the same species is separate and headed by a color card with
the species name. Five suggestive colors were selected for species cards, grey
for Myxomycetes, blue for Oomycetes, violet for Zygomycetes, red for
Ascomycetes, brown for Basidiomycetes and green for Deuteromycetes,
providing information on the class of the fungi.

260

 Fig. .6. Dr Stanley Hughes on collecting fungi in the Gatineau Park,

near Ottawa, Canada, September 1960

Fig. 7 Dr Richard P. Korf at the end of a collecting trip with students

in a forest near Ithaka, N.Y., September 1961.

261

Fig. 8. Pr. Fabritius N. Buchwald, Copenhague, DK; 9. .Dr. Agatha van Beverwijk and 10. Pr. J.A. von
Arx, successive directors of CBS, Baarn, NL (photo 9 of a drawing by Dr. G.A. de Vries).

262

 The collection of living strains is the second part of the mycotheque. Living
cultures are disposed numerically and preserved in three different ways: on
agar slants in two or more culture media under mineral oil, some also fresh
and under water; lyophilized when sporulating; and cryopreserved at -
140C. Much research was carried out to select the optimal cooling speed
and the cryoprotectant medium in lyophilisation and cryopreservation
(Hennebert 1987a, 1987c, 1987d, Berny and Hennebert 1988, 1989a, 1989b,
1990a, 1991a).

Fig. 11. The database is a set of four linked data sets, specific names and data, strain data, morphonyms
and synonyms.

The register and the database make the third part of the mycotheque. All
fungi collected are registered with a unique serial number. The number is
applied to the specimen, the label, the slides, the drawing, the photographs,
the descriptions, the living and dried cultures, the computed data set and
the printed data sheet.
The numerical registration allows one to know the last number applied.
Beside the numerical register, an alphabetical card system records the names
and useful synonyms of the species with the numbers of the living strains
represented in the collection. From 1985 onwards, computerization of the
collection made the card system, not the handwritten register, obsolete.
Strain and species data are recorded in the database, following a
standardized scheme of data (Fig. 11) (Gams et al. 1988, Hennebert 1994a).
The database allows recording and retrieval of the data, management and
statistics of the mycotheque and the publication of the catalogues of strains
(Hennebert et al. 1989, 1992).

The iconotheque is the fourth part of the mycotheque. It contains all

drawings, descriptions and computerized datasheets of the collected fungi.
The drawings are made at 1700x (Fig. 12), sometimes also 680 x or 170 x

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magnification from natural material and, often only,

from culture material. The iconotheque includes
photographs as well.
An alphabetical index of the fungal names supported
by a mycological library makes a fifth part of the
mycotheque. This alphabetical index with
bibliographic reference was gradually compiled
from diverse indexes, from Streinz's Nomenclator
Fungorum (1862), Saccardo's indexes, Petrak's
indexes, to the most recent CAB Index of fungi. The
compilation greatly helped bibliographical research.

2C. Difficulty of interpreting old literature and tracing the type

Many names of fungi were created during the last two and half centuries.
The interpretation of the ancient authors before 1900 and of their taxonomy
remains uneasy. To interpret correctly old descriptions, it is necessary to
- understand Latin,
- be aware that fungi were described from nature, not in culture,
- take into account the tools of observation in current use at the time,
- know the precise meaning of descriptive terms and taxonomic criteria used
at the time,
- know the value of measurement units of the period,
- remember that the rules of nomenclature applied were those of Linn until
1867, with some possible deviations peculiar to certain authors, and those of
De Candolle and successors after 1867.

Tracing the type

In each case, tracing and carefully examining of the type, paratype or
authentic material preserved in the author's herbarium or other herbaria is a
requirement to make an enlightened interpretation of the description.
Sometimes the type material is to be found, far away in a lost box above a
herbarium cabinet, or covered with dust and mouse frass under the roof of a
long deceased mycologist's house.

Necessity of type examination

Dr S. J. Hughes proposed to interpret adequately a two-line description
like that of Wallroth's Balanium (Fig. 13) (Wallroth 1833). The type material
of was necessary and made possible an adequate description in modern
terms (Hughes and Hennebert 1961). At the same to justify the name
Oedemium didymum (Schmidt) Hughes as correct for Botrytis didyma Schmidt
1817, required looking through one century of mycology and finding up to 5
synonymous genera and 16 synonymous species (Fig. 15, 16). Among the
species

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 Fig. 13. Balanium stygium Wallroth; 14. Oedemium didymum, as O. tomentosum Corda after Corda
1837 and his type material; 15. Synonymy of Oedemium. didymum: five generic and seven specific
names; 16. Oedemium didymym (Schm.)Hughes.

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investigated, Oedemium atrum Corda was described and illustrated by Corda

(1828) while he was only 19 years old. Corda's data could be interpreted
only by examination of the type material, fortunately preserved in Prague.
The specimen was composed of dematious sterile fungal setae mixed with
globules that Corda (1833) recognized of heterogenous nature, being insect
frass containing Penicillium conidia, three elements described by Corda as
part of the fungus (Fig. 14). The decision to declare the name Oedemium
atrum a nomen confusum could not have been undertaken without an
examination of the type (Hughes and Hennebert 1963).

Iconotypes and neotypes

Dierckx (1901), student of Biourge, published in a short 6-page note, 22
new species of Penicillium that he had fully described and illustrated from
pure culture, in a handwritten thesis. Unfortunately, when Dierckx went
travelling in Indonesia, all his cultures were lost with the exception of P.
griseo-roseum. On his return, he attempted to recover the species. Not very
successful, he left notes and drawings to Biourge who succeeded to recover
20 of Dierckx 22 new species, and re-described them in his monograph of
Penicillium (1923). In 1979, during an exhibition commemorating Biourge's
microbiological carrier, a member of his family let us have a box from under
the roof of Biourge's house containing the three plates of Dierckx's drawings
(Fig. 17, 18, 19), but the handwritten thesis remains lost. The drawings are
now considered as type material (iconotypes), which support Biourge's
selected neotype cultures for Dierckx's species of Penicillium (Hennebert
1979, 1985).

Guidance of the protologue

If no original material can be traced, the originally published description,
illustration and comments about habitat, locality etc., constituting the
protologue, are the only data available to select a type. Any contemporary or
later interpretation of the description of the taxon, and any application of its
name by another author, should be regarded with caution.
Ostracoderma pulvinatum Fries, the only species of the genus Ostracoderma
Fries 1825 (Syst. Orb. Veg. 1:150), was collected in Sweden and later recognized
by Juel (1920). The fungus matched perfectly Fries's protologue of O.
pulvinatum 1829 (Syst. mycol. 3: 214, hereunder) for which no type is available

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Fig. 17. Orignal Dierckx' drawings of Penicillium species (1901): from left to right, 1st row, (first drawing
lost), P. candidofulvum Dx., P. aurantiobrunneum Dx.; 2d row, P. roseopurpureum Dx., P. citreoroseum
Dx., P. citreonigrum Dx.; 3d row, P. corylophilum Dx., P. carneoviolaceum Dx..

267

Fig. 18. Original Dierckx' drawings of Penicillium species (1901): from left to right, 1st row, P. duclauxii
Delacroix?, P. aeruginosum Dx., P. minioluteum Dx.; 2d row, P. congolense Dx., P. elongatum Dx., P.
glaucum Link?; 3d row, P. atroviride Dx., P. verrucosum Dx., P. griseobrunneum Dx..

268

Fig. 19. Original Dierckx' drawings of Penicillium species (1901): from left to right, 1st row, P.
brevicompactum Dx., P. biourgei Dx., P. aurantiocandidum Dx.; 2d row, P. brunneorubrum Dx., P.
griseofulvum Dx., P. aurantiogriseum Dx.; 3d row, P. hirsutum Dx., P. griseoroseum Dx..

269

 Juel considered the fungus conspecific of Hyphelia terrestris Fries 1849 and
therefore synonymized Ostracoderma with Hyphelia Fries 1825. But Hyphelia
Fr. is not available because of misapplication of his type species name
(Hughes 1958). In another hand Hughes (1958) synonymized
Chromelosporium Corda 1833 with Ostracoderma Fries 1829, but did not
consider the absence of a "crustose peridium" in the former, clearly indicated
in Fries's protologue of Ostracoderma pulvinatum, giving the fungus the
appearance of a Myxogaster ("Facile igitur crederes esse Myxogatrem") and also
observed by Juel. This feature forced the rejection of Hughes' synonymy,
although conidiogenesis in both genera is similar, and the distinction of
Chromelosporium and Ostracoderma (Hennebert 1973).

The type locality

In absence of any original, type or
authentic material of a fungus, but
guided by the published protologue,
one last resource is to go collecting the
fungus anew in the type locality. When
the locality is not destroyed, the fungus
can be found unless it is of rare
occurrence. I discovered Arachnophora
fagicola Hennebert (Fig. 26) from a few
conidiophores only, just enough to
prepare a microscope slide, on a cupule under a beach tree in Parc
d'Arenberg, Heverlee, Belgium (Fig. 20). Later wanting to provide sufficient
type material for herbarium deposit in view of publication, I had to examine
eight thousand beach cupules from the same locality to finally have eighteen
minute colonies of that new fungus to serve as a type (Hennebert 1963a).

Tracing the type host

The same conduct is to be followed when a living culture is wanted for
reconsideration of a rare species. Seaverinia geranii (Seaver and Horne)
Whetzel (1945) had been found on Geranium maculatum in localities of New
York and Wisconsin States five times in 50 years, successively by Seaver,
Davis, Whetzel, and Korf. Ten years after the last collection by Korf, in the
forest along the Cayuga Lake near
Ithaca, I went collecting on the same
spot and found the species (Fig. 21).
Fresh single ascospores could
cultivated and yielded the verrucose
conidial state Botrytis geranii Seaver.
After a proposal by Buchwald
(1949), I redisposed the conidial
state in the new form-genus,
Verrucobotrys Hennebert, as

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Verrucobotrys geranii (Hennebert 1973).

Ex-type cultures
Sometimes, natural type or authentic material is lost, either not deposited
in herbarium, or destroyed, but ex-type cultures are preserved in culture
collections. Should a taxonomist describes or re-describes a fungus from
culture, he should then deposit it in a dried state in herbarium. So did I with
Wardomyces columbinus (Fig. 37) and Asteromyces cruciatus (Fig. 27)
(Hennebert 1962), of Custingophora olivacea (Fig. 28) (Stolk and Hennebert
1968), of Lomentospora prolificans (Fig. 40) (Hennebert and Desai 1974), and
many others isolated from soil (Hennebert 1968a, 1968c, 1969). Conversely,
in order to spare type material in herbarium, it is recommended to deposit
living ex-type isolates in culture collections (Banno et al. 1993, 1994).

Tracing earlier names

Many taxonomists dare to describe and publish rapidly a species they
consider as new, taking the risk to see it synonymized soon after. To be
certain that the species has not yet been described or the name already used,
before publishing a new species or a new name, a full survey of mycological
literature and indexes is necessary.
A Wardomyces-like fungus isolated from soil in Belgium and considered a
new species was also isolated from soil in the Netherlands. After screening
the available literature for some times, I prepared a description of the new
species for publication, when I received a reprint from Subramanian
describing the fungus as Adoghamina ruchira Subramanian n. gen. n. sp. Two
weeks later I found Barron's description of the same fungus as Gilmaniella
humicola Barron n. gen. n. sp., preceding by a few weeks the publication by
Subramanian. The synonymy remained to be published (Hennebert 1968b).
Nathalie Buffin collected an interesting white arbuscular hyphomycete
bearing cylindrical conidia and forming white patches on semi-immersed
dead leaves at a pond in the Lauzelle Forest, Louvain-la-Neuve (Fig. 23). We
though the fungus to be undescribed. We composed a descriptive name,
Cylindrodendrum album. Checking for a possible earlier use of the name in
several indexes, we found that the name had been used
already by Bonorden (1851) (Fig. 22) which description and illustration were
matching our fungus exactly. There is no type material left of Bonorden's
species, but a search in other herbaria revealed that the fungus had been
collected twice in 120 years after Bonorden. Our collection was dsignated
neotype (Buffin and Hennebert 1984).

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Fig. 22. Cylindrodendrum album Bonord. original drawing; 23. Drawing made by N. Buffin from fresh
collection and culture.

Publishing rapidly
Even after searching for guarantee of the novelty, it is important to
publish rapidly. As very few journals were editing manuscripts with a delay
shorter than 6 months, Richard P. Korf and I decided to edit a new journal
offering only a short delay together with quality, designed to promote the
taxonomy of fungi and lichens (Hennebert and Korf 1974). Created in July
1974, Mycotaxon has published, in 1995, 67 volumes of around 540 pages
each, about 36000 pages in 24 years 6.

2D. Evolution of taxonomic criteria from morphology to morphogenesis

Taxonomy of fungi evolved greatly after Linn with development of tools

for a finer observation and characterization of life. New characteristics of
fungi are tested as taxonomic criteria. That evolution requires constant
adaptation by the taxonomist.
It does not mean that traditional criteria such as static descriptive
morphology are abandoned. To the contrary, they make the basis of present
taxonomy, but enriched by morphometry, morphogenesis, sporogenesis,
anatomy, physiology, biochemistry and genetics.
It is possible to distinguish four periods in the evolution of taxonomical
criteria: a first period of static morphology from field material, a second

6
Mycotaxon published its 100th volumes in 2007.
272

period enriched by morphogenesis in experimental mycology

(pleomorphism, sexuality, culturing), a third period aided by physiology
and biochemistry, and a fourth, the current period, revised by genetics and
phylogeny.
From Linn (1753), through Tode, Persoon, Link, Fries, Corda, Wallroth,
Fresenius, Fuckel, De Bary, R. & F. Tulasne, Saccardo and many others until
the 1850-1880s, taxonomic criteria in fungi were merely morphological and
static, based on the observation of natural material and evolved from
macromorphology to micromorphology with the development of the
microscope.
Then morphogenetic criteria were added owing repeated developmental
observations in nature and lately helped by use of aseptic culture on liquid
substrates by Pasteur (1866) and on solid substrates by Brefeld (1875, 1881)
Presumed sexually functioning organs in fungi were observed by
Ehrenberg (1820) in Zygomycetes, by Lveill (1837) who described the
hymenium of Basidiomycetes, by Tulasne (1856), who first described
"spermatia", and De Bary (1863) who observed the "carpogone" and the
"pollinode" in the Ascomycetes.
Brefeld (1872) observed sexual
fusion in Zygomycetes and other
fungi. Stahl (1877) was the first to
follow the fusion of spermatia with
the ascogonial hypha he called
"trichogyne" in Ascolichens.
Blakeslee (1904) made the fusion of
heterothallic partners in
Zygomycetes. These discoveries led
to the differentiation of sexual
sporogenesis from conidiogenesis.
Fresenius (1850) already
described the conidiogenesis of
Botrytis cinerea (Fig. 24). The development of several kinds of secondary
spores, or conidia, in addition to spores suspected to be sexual, led L.
Tulasne (1851) to define pleomorphism in fungi. Numerous examples of this
pleomorphism were illustrated in the Ascomycetes by L. Tulasne and C.
Tulasne in the Selecta Fungorum Carpologia (1861-1865) and in the
Basidiomycetes by De Seynes (1874) and in all groups of fungi by Brefeld
(1872-1912).
Availability of the fungi in living culture greatly favoured the
observation of pleomorphism and conidiogenesis and its use in taxonomy.
An early example of the advantage of cultures was the morpohogenetic
study of several Penicillium species made in vitro by Brefeld (1874).
More recent mycologists, Vuillemin (1910), Mason (1933), Hughes (1953)
and Tubaki (1958), defined and described different types of conidiogenesis,

273

and used them as criteria for generic distinction of asexual fungi. Most of
them were confirmed by electron microscopy (Samson and Cole 1970).

2E. Development of morphogenetic criteria: the unitary parameters of

conidiogenesis

The conidiogenetic system of Hughes (1953) and Tubaki (1958) guided

my descriptions and illustrations of the conidial fungi. On basis of
conidiogenesis, were excluded from the genus Botrytis, species with blastic
synchronous conidia but dissimilar from what was called the Botrytis cinerea-
type (Whetzel 1945), belonging to Chromelosporium, Haplotrichum, Mycotypha,
Nematogonium and Ostracoderma, species with sympodial blastic conidia
belonging to Beauveria, Calcarisporium, Cephalotrichum, Conoplea, Costantinella,
Hansfordia, Nodulisporium, Rhinocladiella, Rhinotrichum and Virgaria, species
with phialidic conidia referable to Verticillium, species with retrogressive
conidia as in Cladobotryum and species with arthric conidia which were
disposed in Geomyces (Hennebert 1960a).
At the 1st Kananaskis conference on the Taxonomy of the Fungi Imperfecti
(Kendrick 1971), advances were made in the understanding of
conidiogenesis. Several new terms proved to be useful: among them,
"holoblastic" and "holoarthric", "enteroblastic" and "enteroarthric", that I
proposed to characterize the ontogeny of the conidia in relation to the wall
of the conidiogenous cell. Also emphasis was given to the ontogeny of the
conidiophore itself, beside that of conidia (Pirozynski 1971). Considering the
conidiophore also, I segregated several form-genera among the Botrytis-like
species (Fig. 25). A first group of them is linked, or presumably so, to
operculate Discomycetes, order Pezizales, distinct from another group
linked to the inoperculate Discomycetes, order Helotiales, family
Sclerotiniaceae. In Pezizales, the new form-genus Dichobotrys Hennebert
(Fig. 25-5) was described for the conidial form of Lamprospora,
Sphaerosporella, and Trichophaea species, while two species of form-genus
Chromelosporium Corda are conidial states of Peziza species. Botrytis-like
form-genera presumably related to operculate discomycetes, Pulchromyces
Hennebert (Fig. 25-7) and Phymatotrichopsis Hennebert (Fig. 25-8) were
described as new, beside the "peridial hyphomycetes" Ostracoderma Fries
and Glischroderma Fuckel (Fig. 25-10) (Hennebert 1973, Hennebert and Korf
1975, Bellemre and Hennebert 1979, Sutton and Hennebert 1994, Korf 1994).
In Sclerotiniaceae, distinctive features of conidiomata were enhanced as
generic criteria to discriminate, beside Botrytis sensu stricto, three distinct
new form-genera, Streptobotrys Hennebert (Fig. 25-2) for the twisted
conidioma of Streptotinia, Amphobotrys Hennebert (Fig. 25-3) for the
dichotomous conidioma of one Botryotinia species and Verrucobotrys
Hennebert (Fig. 25-4) for its verrucose conidioma of Seaverinia (Hennebert
1973).

274

 Fig. 25. Diagrammatic illustrations of coniophomata of 1. Botrytis; 2. Streptobotrys; 3. Amphobotrys;

4. Verrucobotrys; 5. Dichobotrys; 6. Chromelosporium; 7. Pulchromyces; 8. Phymatotrichopsis; 9.
Ostracoderma; 10. Glischroderma.

.
However, the more conidial fungi were examined, the more intermediate
categories of conidiogenesis were discovered: Wardomyces and Asteromyces
species have solitary blastic or thalloblastic conidia born in basipetal
succession on the conidiophore (Hennebert 1962), Arachnophora fagicola (Fig.
26) has solitary blastic conidia borne successively trough percurrent
proliferation of the conidiophore (Hennebert 1963a), Sporoschismopsis
moravica (Fig. 29) produces successive arthric conidia borne in percurrently
successive phialides (Holubova-Jechova and Hennebert 1972).

275

Fig. 26. Arachnophora fagicola Hennebert; 27. Asteromyces cruciatus F.Me. Moreau ex Hennebert 28.
;Custingophora olivacea Stolk, Hennebert & Klopotek; 29. Sporoschismopsis moravica Hol.-Jech. &
Hennebert

I thus progressively developed a more detailed, yet still unsatisfactory,

scheme of conidiogenesis for my students (Hennebert 1982). Adressing the
need for a finer analysis of conidiogenesis, I introduced the concept of
unitary parameters acting together or successively in each specific
conidiogenesis, instead of describing categories or types of conidiogenesis
(Fig.30).

276

 Fig. 30. One of the plates figuring some of the parameters, the combination of whichh shall define a
conidiogenic pattern (Hennebert in Hawksworth 1994b)

The parameters are numerous and should be defined under a univocal

descriptive terminology. A first and tentative analysis of a non-exhaustive
number of unitary parameters of conidiogeneis was presented at the
Ascomycetes Conference in Paris in 1993 (Hawksworth 1994b). When
eventually fully refined, the unitary parameters should allow the
characterization of any conidial fungus and hopefully, when computerized,
serve as a basis for an expert identification system of conidial fungi
(Hennebert and Sutton 1994).

2F. Value of cultures in demonstrating pleomorphism and variations in

fungi

Like Biourge in 1892-93 who spent a year in both Pasteur and Hansen's
laboratories learning the single cell culture (Hanssen 1886), I spent two years

277

in Ottawa where Dr J. W. Groves taught me the single ascospore culture and

the spermatization technique.
Single spore culture has been a great help in the separation of close
species. I used single spore culture currently in Penicillium, and other genera
for isolation of strains. Mass conidial colonies often sectorize, showing
variants. Strains received for the collection, particularly ex-type strains, were
regularly checked for homogeneity from 50 single spore cultures. In an
attempt to rebuild the Biourge's Penicillium collection, type strains returned
from other collections were found to contain up to 4 variants. Indeed,
mutations occur during maintenance and preservation of ex-type cultures
and selection of the original strain has frequently been necessary. In an
experiment eight different variants were isolated from a homogeneous
parental strain of Penicillium expansum following exposure to diverse
preservation processes (Fig. 31) (Berny and Hennebert 1990a).

Fig. 31. Eight variants of Penicillium expansum wild strain (left) obtained from monospores
after preservation processes.

Cultures help to demonstrate pleomorphism, as well as variability and

mutability. Culture under diverse conditions allows the fungus to reveal
itself in more facets than expected. An example is the dimorphism of many
yeast and yeast-like fungi, even Saccharomyces cerevisiae, which turns
filamentous under nitrogen starvation. Moniliella pollinis (Hennebert and
Verachtert) De Hoog turned from arthric conidia to a sympodial budding
yeast depending on the shaking speed in liquid culture (Dooms et al. 1971).
Only through single spore culture, it could be demonstrated that spermatia
belong to Botrytis cinerea.
Analysis of conidiogenesis In culture helped to reduce the taxonomy of
the genus Botrytis to some thirty six species (Hennebert 1958, 1960a, 1963b,
1964a, 1966, 1969, 1973). Cultures also helped to demonstrate the sexual-
asexual connection. Through crossing single conidial isolates of Botrytis by
spermatization of sclerotia, apothecia of heterothallic Botryotinia ficariarum
and Botryotinia ranunculi could be obtained in pure culture (Hennebert and
Groves 1963). Homothallic species like Botryotinia porri can fruit easily.
Similar proof of pleomorphic connection In yeasts were also atteined
through rDNA sequencing from cultures.

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2 G. Classification of asexual fungi with sexual fungi

With or without cultures, another

way to infer taxonomic relationship of
conidial fungi in absence of a sexual
reproduction, is by analogy of their
morphology. In Chromelosporium three
species only, C. fulvum (Link: Fr.)
McGinty et al.[C. ollare (Pers.)
Hennebert] (Fig. 32), C. macrospermum
Hennebert and C. trachycarpum
Hennebert, are conidial states of Peziza
species, but the type species C.
ochraceum Corda and six other species
are only presumed to be states of
Operculate Discomycetes by analogy,
although they had often been
considered states of Basidiomycetes such as Hypochnus or Tomentella. At the
same Phymatotrichopsis omnivora (Duggar) Hennebert is considered by some
authors a state of Hydnum or Trechispora.
Ostracoderma Fr. and Glischroderma Fuck. are similarly grouped with
conidial states of Operculate Discomycetes. A peridium, absent in the others,
gives to Ostracoderma pulvinatum and Glischroderma cinctum Fuck. the
external pulvinate smooth appearance of the myxomycete Lycogala. A
closely related peridial fungus, Glischroderma cinctum Fuckel was described
by Fuckel (1870) in the Gasteromycetes, and distinguished from
Ostracoderma. Another 'peridial hyphomycete' with elliptical conidia, found
by Bresadola, was named Lycogala torrendii as if a Myxomycete. It was
renamed by Torrend (1913) Lycoperdellon torrendii and disposed in the
Gasteromycetes. Heim and Malenon (1933), Heim (1934, 1949), Dissing and
Lange (1962), Malenon (1960, 1964), Demoulin (1966) placed both genera in
the Gasteromycetes and families were erected for them. Zeller (1948) rightly
placed the fungi in the Deuteromycetes. Because of the analogy of the
conidiogenesis with Chromelosporium states of Peziza species, following Donk
(1962), I chose to dispose Lycoperdellon as synonym of Ostracoderma Fr. and
with Glischroderma among conidial states of Operculate Discomycetes.
Glischroderma cinctum and another undescribed species of Glischroderma
Fuckel, with a glutinous peridium, were found by Korf and his students in
the surroundings of Ithaca, NY. (Korf 1994).
The presumed position of Ostracoderma (= Lycoperdellon) and
Glischroderma amongst conidial forms of certified Operculate Discomycetes
(Hennebert 1973) was strongly supported by the presence of ascomycetous
septal pores demonstrated in SEM by Bronchart and Demoulin (1975).

279

Ultrastructure is indeed another way approach relationships between

conidial and sexual fungi and their phylogeny.

2H. Relative aid of additional criteria

Physiological characters
After the promotion of physiological characteristics in yeast taxonomy by
Guilliermond (1912), some additional characteristics, enzymatic activities,
requirements in growth factors and tolerance to environmental conditions,
were added. Those criteria are still in use (Berny and Hennebert, 1990b). In
1985, I initiated the miniaturization of all physiological tests of assimilation,
fermentation, enzymatic activity, requirements and tolerances, using 96 well
microplates read by optical densitometry. The method has greatly increased
test rapidity. It was presented at the 8th International Symposium on Yeasts
in Atlanta in 1992. Today, the microplate results, together with yeast
morphological and genetic data, are integrated in the computer-assisted
expert identification system BCCM/ALLEV-2 created by Vincent Robert at
MUCL (Robert et al. 1994, 1997) and presented again as a poster at the
MUCL centenary symposium (Evrard and Hennebert 1994) 7.

Biochemical characters
The development of analytical methods enhanced extensive biochemical
investigations on both filamentous and yeast-like fungi. However the new
criteria are so far applicable to sufficiently large fresh biomass of fungi from
culture, or nature. Their application to type material in herbaria is excluded.
That is a handicap of the biochemical approach.
Dominant Q co-enzymes have become a good generic criterium and a
good indicator of the generic homogeneity. Applied to Candida the genus
was found heterogeneous and composed of four distinct groups
(Vancanneyt et al. 1991b, 1994a).
Enzyme and protein profiles have provided taxonomic help at the species
level and lower ranks (De Bertoldi and Hennebert 1977, Berny and
Hennebert 1991b, Hennebert 1996b, Hennebert and Vancanneyt 1998).
Whole cell protein profiles proved to be useful in confirming by maximum
similarity the organic connection established otherwise between asexual and
sexual strains in both ascomycete and basidiomycete yeasts. The same
degree of similarity between unconnected anamorph and teleomorph
species led to presume of their close relationship, if not their ontogenic
identity (Vancanneyt et al. 1990, 1991a, b, c, 1992, 1994b).

Genomic characters
Genomic analysis for fungal taxonomy developed rapidly.
Polymorphism of fragmented DNA (RFLP) demonstrated the relation of

7
The System ALLEV later received further developments and world wide distribution as
BIOLOMIX by the CBS, the Netherlands.
280

produced mutants with the parent strain of Penicillium expansum (Cercel et

al., 1998a, 1998b). Ratios in guanidine and cytosine confirmed similarities
observed in protein profiles of yeasts (Vancanneyt et al. 1991c, 1994a).
Here also, genomic analysis is up to now applied only to living strains in
culture, less easily to dried type material or small nature collection. The
strains currently analysed are otherwise classically pre-identified.
Taxonomic conclusions inferred from genomic analysis might therefore
depend on the identifications of reference sequences. Since all names are
obligatory linked to type material preserved in a herbarium, it is highly
recommended that molecular taxonomists analyse type specimens and
authenticated ex-type strains. Neglecting this basic requirement would be
equivalent to ignoring 240 years of taxonomy and nomenclature
(Gouliamova and Hennebert 1994a, 1994b, 1995)).
With that in mind, sequencing of nuclear rDNA or RNA associated with
numerical analysis applied on ex-type strains and other reference strains
provided much valuable data for both taxonomy and phylogeny of
ascomycete and basidiomycete yeasts (Hendricks et al. 1989, 1991, 1992, Van
de Peer et al. 1992, Wilmotte et al. 1993, Gouliamova and Hennebert 1994a,
1994b). They allowed confirmation of the anamorph-teleomorph connection
in all cases tested and detection of presumed such connections in some other
cases. Sequencing part of 18S RNA and the region ITS1-5.8S rDNA-ITS2 of
ex-type or ex-neotype yeast strains, Saccharomyces cerevisiae and some
reputedly synonymous species were compared.. S. chevalieri and S.
ellipsoideus were found synonyms of S. cerevisiae, while S. pastorianus, S.
paradoxus, S. uvarum and S. bayanus were confirmed to be distinct species
(Gouliamova and Hennebert 1994b, 1998). Using the same ITS region,
specific distinctions and seveal new species were established among thirteen
unidentified strains of Lipomyces, otherwize detected by DNA reassociation
(Gouliamova and Hennebert 1995).

3. NOMENCLATURE

Nomenclature Is linked intimately to taxonomy, as language is linked to

concepts, and as locality names are to each destination box at the post office.

3A. Typification: the basis of nomenclature

Typification of names is one of the two pillars of botanical nomenclature

stability, the other being priority of names.
Automatic typification of a new name by the type of an earlier name
included in the protologue was the rule until the Montreal International
Code of Botanical Nomenclature (Lanjouw 1961).
Interpretation of cases differs whether names are applied to the same
taxon, or to two different taxa, one designated by the earlier name, the other
described under the new name.

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 When a recent name is published for a taxon while an earlier legitimate

and priorable name for the same taxon is cited as a synonym in the
protologue and should have been used, automatic typification applies and
the recent name is nomenclaturally superfluous and illegitimate. It was the
rule until 1966. If the author in some way excludes the type of the cited
earlier name, e.g., by designating a type of the new name for the same taxon,
the new name with its type should be accepted and automatic typification
should not apply. The result would be that the new name is legitimate and
taxonomic synonym of the earlier name. That was the proposal made by
Weresub and Hennebert (1963), accepted by the Edinburgh Code (Lanjouw
1966).
But often authors misinterpreted the description of an earlier species and
therefore misapplied its name to a different taxon. There is then a conflict
between the taxon represented by the earlier name cited as a synonym and
its type and the taxon as described. The question in interpreting such
mycological literature was: is the intention of the later author to be respected
or automatic typification applied?
In such cases of misapplication of an earlier legitimate name, the
Edinburgh Code (1966) maintained the automatic typification to be applied
"in all circumstances" (Art. 7), no regards to the intention of the later author.
The new name is then obligate synonym of the earlier one. That is the case of
Hyphelia Fr.
Persoon (1794b) had described Trichoderma roseum and deposited a
specimen on Populus bark in his herbarium in Leiden. Hoffmann (1795)
collected the same species, named it Trichoderma rosea Pers. and described
the conidia under the lens as oblong, phaseoliform and uniseptate. Later
Persoon (1801) listed his Trichoderma roseum Pers. with reference to Persoon
1794 and Hoffmann 1795, adding its occurrence on Salix bark based on
another specimen in his herbarium. Link (1809) erected the new genus
Trichothecium Link with uniseptate conidia on the basis of Trichoderma roseum
Pers.
Fries (1825) erected the new genus Hyphelia Fr., and included in the same
description the name of one species "Hyphelia rosea", based on Trichoderma
roseum Pers. which he explicitely propoposes as type, but commenting "not
Trichothecium Link". In the same work, Fries recognized the genus
Trichothecium Link, with the comment that it differs from Trichoderma roseum
Pers.
. Fries (1829) in the Systema mycologicum, already written at the time of
the publication of the new genus Hyphelia in 1825, indicated having seen a
Persoon specimen with reference to Persoon 1801. Hughes (1958) indeed
examined Persoon's three authentic specimens of Trichoderma roseum Pers. in
Leiden herbarium and recognized Trichothecium roseum (Pers.) Link: Fr Fries
might have had in hand a poor specimen from Persoon like the one
deposited in 1801 and misinterpreted Persoon's description, consequently
misapplying the name. Hyphelia Fr., based on a misapplication of the type

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species name Trichoderma rosea Pers. is therefore obligate synonym of

Trichothecium Link and therefore not available for the species Fries called
Hyphelia terrestris Fr. (Chromelosporium tuberculatum (Pers.) Hennebert)
(Hennebert 1973)

3B. Pleomorphy in fungi and nomenclature

Linn decided his taxonomy to be based on sexual reproductive

structures. In the introduction of the Philosophia botanica (1753) he classified
plant taxonomists according to their basic principles of classification and
qualified himself as "sexualist" (Fig. 33). Consequently, fungal sexuality and
pleomorphism being unknown at the time, any distinct form of
reproduction in fungi was considered a distinct species and named.

Fig. 33. Linn (1753) declares himself a "sexualist" taxonomist.

Corda (1839) described Echinobotryum parasitans as a parasite of Stysanus

caput-medusae ignoring that it was another form of spores of the same fungus
(Fig. 35). Even Saccardo (1882) and Penzig (1882) made the same 'mistake'
when accepting Echinobotryum citri as a parasite of Stysanus monilioides , later
corrected as Echinobotryum atrum(Fig. 36).
Tulasne (1851), who called pleomorphy the coexistence of several forms of
reproduction in fungi, demonstrated the organic connection between
ascosporic fungi and conidial fungi as the "perfect state" (which they
presumed to be sexual) and the "imperfect state or states" of the same

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species, as several conidial forms of reproduction may exist in the same

perfect species (Fig. 34). Number of pleomorphic fungi had therefore more
than one name. Tulasne (1851), in agreement with Linne's principle, asserted
the predominance of the name of the perfect sexual state upon the names of
the imperfect states, to designate the whole fungus (Hennebert 1971).

Fig. 34. Pleomorphy: Hypomyces rosellus and anamorph Cladobotryum dendroides (L.R. & C.
Tulasne, III, 1865)

However, vast amount of conidial species remained, and still remain,

without connection to a sexual form. Consequently, Fuckel (1870) classified
the fungi in two major groups, the Fungi Perfecti and the Fungi Imperfecti.
Saccardo (1889) named them Fungi Superiores and Fungi Inferiores, the latter
being "metagenetic forms" (formae metageneticae,) which he designated later
Deuteromycetae or Fungi secundarii (Saccardo 1899).
Although the Lois de la Nomenclature Botanique Art. 15 by De Candolle
(1867) reinforced the principle "one taxon - one name", Saccardo (1904) in his
De Diagnostica et nomenclatura mycologica, recommended to classify duly
demonstrated pleomorphic fungi by their appropriate names in Fungi
Perfecti and Fungi Imperfecti respectively. Saccardo was supporting a
tolerated use of the imperfect names beside the correct perfect name.

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 The matter, submitted to the International Botanical Congress of Vienna

in 1905, was left to the International Commission of Botanical Nomenclature
(Briquet 1905). The proposal of the Commission (Atkinson 1909) integrating
the positions of Tulasne and Saccardo, was adopted at the Botanical
Congress of Brussels in 1910, and became part of the Brussels International
Code of Botanical Nomenclature (Briquet 1912) as Art. 49 bis. Art. 49 bis was
the first rule of nomenclature for pleomorphic fungi. It recognized the
predominance of the name of the perfect state of Ascomycetes and
Basidiomycetes over the names of their imperfect states, and tolerated the
use of the existing names of imperfect states, to be classified in "form-
genera" and assessing them "only a temporary value". A later proposal made
by the British mycologists (Anonymous 1929) to extend the rule 49 bis to the
Zygomycetes and the Mastigomycetes (Phycomycetes), in order to make
coherent the entire nomenclature of the fungi, was defeated at the Botanical
Congress of Cambridge in 1930 (Briquet 1935, ICBN Art. 57).
The Stockholm Code (Lanjouw 1952), accepted for the first time, and
ruled, in Art. 69, the creation of new names for the imperfect states of
already named pleomorphic fungi, while the temporary value of such names
was forgotten. The rule still evolved further to a very complex Art. 59
adopted in Edinburgh Code (Lanjouw 1966) and remained unchanged until
1981. The rule intended to separate the nomenclature of the imperfect fungi
from that of the perfect fungi and to preserve the predominance and
stability of the names of perfect species, by subordinating taxa of imperfect
states of fungi as "form-taxa", and making invalid epithets transferred from
form-species to perfect species and valid but illegitimate perfect names not
typified by a perfect state in accordance with their protologue. In practice
this version of Art. 59, was "undoubtedly one of the thorniest articles of the
Code for mycologists to apply" (Korf 1982). It was "extraordinarily
complicated, so that in some instances, up to three separate conclusions
could be drawn regarding names to be used. There was a need for a drastic
reconsideration of some basic principles" (Hawksworth 1984).

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 Fig. 35-38. Pleoanamorphy in fungi. 35. Echinobotryum parasitans Corda on Stysanus caput-
medusae Corda after Corda , Prachtflora (1840); 36. Echinobotryum atrum Corda after Saccardo,
Fungi delineati (1878); 37. Wardomyces columbinus, MUCL 6959, from culture; 38. Mammaria
echinobotryoides, MUCL 7564, from culture.

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Fig. 39-42. Pleoanamorphic fungi. 39. Raffaelea ambrosiae Arx & Hennebert: 40. Lomentospora
prolificans Hennebert; 41. Chalaropsis punctulata Hennebert; 42. Isthmolongispora lanceata De Hoog &
Hennebert.

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3C. Revision of Art. 59 for the nomenclature of pleomorphic fungi

Such a reconsideration of basic principles of nomenclature I attempted to

offer at the First Conference of Kananaskis, Canada, on the Taxonomy of the
Fungi Imperfecti in 1969 (Kendrick 1971). In a paper on the Pleomorphism in
Fungi imperfecti, I analysed the fundamentals of fungal nomenclature of
pleomorphic fungi and 'pleoanamorphic' fungi (a new term at the time). The
matter was not easy to put into words. Supported by Richard P. Korf at
Cornell University, I formulated the concepts of "phase" versus "state",
"form" of a fungus, of "whole fungus" versus "perfect state" and "imperfect
states", of "botanical taxon" versus "form-taxon", and of "botanical
nomenclature" versus "anatomical nomenclature", in order to define
adequately the divergent options applied by fungal taxonomists in naming
pleomorphic fungi, to deduce the nomenclatural implications of each option
(Hennebert 1971).
A "sexual phase" and an "asexual phase" were distinguished in each
fungus, even when the sexual phase is not actually known by man, both
phases composing together the "whole fungus". The sexual phase is
represented by a unique sexual form of reproduction, while the asexual
phase is possibly represented by one or several asexual forms of
reproduction or by nothing else than resting organs or mycelium.
The Edinburgh Code (Lanjouw 1966) treats the fungi according two
distinct systems of nomenclature.
For the Mastigomycetes and Zygomycetes, the Code applies the
"botanical nomenclature", meaning that only one name is given to "the whole
fungus" as known, it being either sexual, or sexual and asexual, or only
asexual, in strict application of the Linnean principle "one taxon - one name",
based on what I call "botanical typification" governed by Art. 7: "The
nomenclatural type is not necessarily the most typical or representative element of a
taxon". An example is the genus Mucor that accommodates equally either
sporangiosporic species, or zygo-sporangiosporic species ('imperfect'
species), or even zygo-sporangio-blastosporic species, like Mucor troglophilus
Zalar et al. (1997), no matter what part of the fungus the type specimen
contains.
On the other hand, for the Ascomycetes, Basidiomycetes and
Deuteromycetes, the Brussels (Briquet 1912) and subsequent Codes accept
the use of several names for one particular fungus, one name for the sexual
phase, typified by sexual reproduction and representing the "taxon", one or
several names for the asexual phase typified by asexual reproduction and
which represent one or several "form-taxa". The rule prescribes the
predominance of the name of the sexual phase to designate the "taxon",
excluding from consideration the names of "form-taxa". That system of
nomenclature applies a different principle of typification than the rest of
botanical nomenclature. Governed by Art. 59, the type of Ascomycete and

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Basidiomycete names must be obligatorily the typical representative element

of the taxon, i.e. the sexual fructification, while the type of Deuteromycete
form-taxa is restricted to an asexual state, fructification or organ. In this
system, names are clearly anatomical names, names of an anatomical part of
the fungus. I called this system "anatomical nomenclature" with two distinct
name statuses based on what I call here "anatomical typification". Even though
the Code gives to names of sexual organs a botanical status, the resulting
nomenclature of Ascomycetes or Basidiomycetes is not truly "botanical", for
an obligation restricts typification, excluding from priority otherwise
potentially competing names of asexual states.
Some fungal taxonomists refusing to create names for asexual states of
pleomorphic fungi apply, to refer to them, an intermediate system called the
"cross-reference naming system", using only the name of the form-genus
associated to the sexual name (Hennebert 1971, 1983).
As a result of this analysis of the dual system of nomenclature, the First
Kananaskis Conference, in 1969, decided the creation of a committee for
reconsideration of Art. 59. This was formed as Subcommittee A of the
International Mycological Association Nomenclature Committee at the First
International Mycological Congress in Exeter, September 1971. The
Subcommittee was composed of 12 members, with Dr Luella Weresub as
Chairman.

3D. Anamorph, teleomorph, holomorph: new nomenclatural terms

In 1977, just playing with words in a conversation with Dr Luella

Weresub in Ottawa, I proposed some less 'imperfect' terms than the
anthropomorphic 'perfect' and 'imperfect', hopefully terms without value
judgements and possibly objective and unequivocal to designate the
anatomical parts of a fungus deserving a name. Urged on by Luella Weresub
who believed in those terms, we published the terms "anamorph",
"teleomorph" and "holomorph" to differentiate any fungal "morph" deserving a
name (Hennebert and Weresub 1977). I was inspired by the term
"anamorphosis" used by Donk (1960a, 1960b) following its first use by the
Vienna International Commission of Botanical Nomenclature (Briquet 1905),
and by the ICBNs of 1912, 1935 and 1947, for the asexual states of
pleomorphic fungi. The term was preferred to the term "forma
metagenetica" used by Saccardo (1889), and to the term "deuteroform"
proposed by Shear (1929) as a replacement for the German "Nebenfrucht".
The term "anamorph" was proposed for any asexual state of the
"anamorphosis" (the asexual phase), the term "teleomorph" for the unique
sexual state of the fungus and the term "holomorph" for the "whole fungus",
including even all unknown "morphs". Any asexual fungus (imperfect
fungus) is therefore an "anamorphic fungus", as lacking a teleomorph, either
by a gap in our knowledge or having lost the capacity for sexual
reproduction, irrespectively. The anamorphosis of a fungus can be

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represented by one or several anamorphs that deserve names. When several,

they were called "synanamorphs" by Hughes (1979). Fungi with
synanamorphs are said "pleoanamorphic" and raise problems of nomenclature
similar to those of pleomorphic fungi (Hennebert 1987c, 1991).
After several years of work, IMA Subcommittee elaborated a proposal for
the modification of Art. 59 recommended by the Second International
Mycological Congress in Tampa in 1977. In agreement with the proposal, the
Nomenclature Session of the International Botanical Congress in Sydney in
1979 voted in favour of the use of the new terms anamorph, teleomorph and
holomorph, in a new version of Art. 59. This, adopted by the Sydney Code
(Stafleu 1981), simplified the rule, but reinforced the segregation of
anamorphic nomenclature from teleomorphic (holomorphic) nomenclature
of Ascomycetes and Basidiomycetes, based on their respective "anatomical
typification". It clearly declared anamorphic names unpriorable in
holomorphic nomenclature in case the anamorphic fungi were ever found to
be sexual. It was the most conservative option, far from the true botanical
nomenclature requiring the suppression of Art. 59, what I considered the
final solution. New Art. 59 had however two advantages, to solve a serious
nomenclatural knot by a clear practical line and, overall, to stimulate
reconsideration of the basic principles of nomenclature of the fungi.
For plant pathologists, anamorphic names of plant pathogens often serve
to designate plant diseases. Similarly, in medical mycology, names of
mycoses commonly derive from the anamorphic name of the causal agent.
Both practices are regrettable since they link disease names to a not
infrequently changing reference basis. Using the practice as an argument in
favour of an anatomical nomenclature and the maintenance of the
Deuteromycetes is fallacious (De Vroey and Hennebert 1983). The true
reason in maintaining Art. 59 was the fear of the major disturbance that its
suppression and a return to unrestricted typification and absolute priority of
names would cause in the present usual fungal nomenclature.

3E. Anamorph-teleomorph correspondence

Anxious to nurture a trend toward integration of conidial fungi into the

taxonomy of Ascomycetes, Bellemre and Hennebert presented before the
Mycological Congress in Tampa in 1977 a compilation of the various
conidiogeneses known in the Discomycetes together with their connections
with teleomorphic genera (Hennebert and Bellemre 1977, 1979).
In 1979, the Second Conference of Kananaskis considered "The Whole
Fungus". Enlightened by the new terminology (Hennebert and Weresub
1979) and the new rules of nomenclature for pleomorphic fungi (Weresub
1979), a wider survey of anamorph-teleomorph connections in the
Ascomycetes and Basidiomycetes was elaborated (Kendrick 1979). The
survey showed the intricacy of the correspondences between anamorphic
and teleomorphic genera, making the dreamed integration of the

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Deuteromycetes into the Ascomycetes and Basidiomycetes an uneasy but

challenging puzzle.
This is possibly due to lack of refined taxonomic criteria for the
differentiation of genera at both sides. As far as anamorphs are concerned, a
more detailed analysis of the unitary parameters of conidiogenesis, from the
origin of the conidioma till the maturity of conidia, is needed. Hennebert
and Sutton (1994) made such an attempt, which is still incomplete. A similar
approach would presumably be helpful, too, in revision of teleomorphic
genera.
A new survey of conidiogenesis in Ascomycete families by Sutton and
Hennebert (1994) did not show any correspondence but, rather,
heterogeneity between taxonomies, suggesting that reciprocal amendments
might improve generic congruency. Using this approach, I obtained a strict
correspondence between anamorphic and teleomorphic species of
Melampsora (Hennebert 1964b). Also, through emphasis of characters
discriminating new genera in the Botrytis-like complex, the anamorphic
taxonomy could be appropriated to that of the correlated Discomycete
teleomorphs (Hennebert 1973, Sutton and Hennebert 1994).

3F. Nomenclature of pleoanamorphic fungi

Pleoanamorphic fungi (synanamorphoses) raise the same problems as

those of plemorphic fungi (teleomorphosis-anamorphosis). I studied a
number of pleoanamorphic fungi, beside Botrytis species: Ambrosiella
xyleborii and Raffaelea ambrosiae (Fig. 39) both with two kinds of conidia (von
Arx and Hennebert 1965), Chalaropsis punctulata (Fig. 41) with sympodial
thalloblastic conidia and Chalara phialidic thallic conidia (Hennebert 1967),
Spirosphaera species with bulbiferous conidia disintegrating into secondary
arthroconidia (Hennebert 1968a), Echinobotryum atrum (Fig. 35, 36),
Wardomyces columbinus (Fig. 37) and Mammaria echinobotryoides (Fig. 38) with
basipetally successive blastic conidia and phialidic or annelidic conidia
(Hennebert 1968c), Trichocladium and Thermomyces species with thallobastic
and philidic conidia (Hennebert 1971), Lomentospora prolificans (Fig. 40) with
sympodial conidia and chamydospores (Fig. 35) (Hennebert and Desai 1974)
and Isthmolongispora lanceata (Fig. 42) with sympodial conidia and its Zalerion
thalloblastic morph (De Hoog and Hennebert 1983). They raised the still
unanswered question of a monoanamorphic versus a pleoanamorphic
definition of anamorphic genera.
Here also, I distinguished three systems applied by mycologists to
pleoanamorphic fungi, from the botanical (pseudo-botanical) to the
anatomical system with the intermediate cross-reference system (Hennebert
1971, 1987b). The new Art 59 of the Sydney Code (Stafleu 1981), which
adopts the anatomical system for pleomorphic fungi, does not require the
dissection of the anamorphosis into synanamorphs with distinct names, as
long as the application of any anamorphic name corresponds to its type.

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Therefore the choice of a monoanamorphic or pleoanamorphic definition of

an anamorphic genus is free. It should be governed only by the perspective
of a more natural classification (Weresub 1979). Examples exist in the
botanical nomenclature of Entomophthorales where "one- state" and "three-
state" species are disposed of and named in different genera in a unique
taxonomy and nomenclature (Remaudire and Hennebert 1980).
To awaken consciousness to the nonsense of an anatomical system
pushed so far as to dissect the anamorphoses of fungi and thus to mupltiply
the names of synanamorphs, I took two extreme positions. Nathalie Buffin
and myself decided naming provocatively the same fungus by two names,
Cylindrodendrum album Bonorden and Cylindrocarpon hydrophilum Buffin and
Hennebert, after it produced distinct conidiophores and conidia, although of
the same phialidic conidiogenesis, under different culture conditions (Fig.
43) (Buffin and Hennebert 1984). The position actually conformed to
anatomical nomenclature of anamorphic fungi. It is clear however that
Cylindrocarpon hydrophilum, in a pseudo-botanical system, is a synonym of
Cylindrodendrum album.
In non-conformity with that system, we described, under a unique name,
Basifimbria spinosa Buffin and Hennebert, a fungus producing two kinds of
conidia on the same conidiophore, and indicated, ironically, where
eventually to cut the conidiophore with scissors to give the different parts
different names(Fig. 44) (Buffin and Hennebert 1985, Hennebert 1987b,
1991).

Fig. 43-44. Basifimbria spinosa Buffin & Hennebert. 43. Variability on the same culture medium.;
44. Variability or pleoanamorphy needing sectioning into pieces for three anamorphic names?

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Obviously, good sense must prevail. The fundamental aim of naming living
beings is indeed to make possible their recognition, inventory and
classification, by giving each of them a single name and not a multiplicity of
names (Hennebert 1991). I therefore prefer giving one name only to
pleoanamorphic fungi. So I proceeded in naming and describing Botrytis,
Chalaropsis, Wardomyces, Mammaria, Humicola species, using eventually a
cross-reference generic denotation for what I considered the "secondary"
conidiogenesis. Clearly the anatomical system of nomenclature, allowing a
micro-dissection of the fungi, might "encourage les amateurs de gloriole,
ceux qui se plaisent voir leur nom imprim" (De Candolle 1867). As
asserted by mycologists 90 years ago, "names given to other states" than the
teleomorph "have only a temporary value" (Briquet 1905), we should
remember that all anamorphic names coined for pleomorphic fungi are by
definition and typification of a subordinate status, some day in the future
destined to become obsolete and to disappear (Hennebert 1987b).

3G. Towards a more natural classification

My dream remains to see the day when taxonomy and nomenclature of

the Deuteromycotina (anamorphs and anamorphic fungi) shall be integrated
with those of the Ascomycotina and Basidiomycotina (holomorphs) with the
abandonment of names for anamorphs of teleomorphic fungi, in order to use
only one name per fungus, the nomen botanicum. This dream I share with
others. Already Weresub and Pirozynski (1979) declared "We trust that the
time will come when our grasp of the morphology, biochemistry and
genetics of all fungi will enable us to classify all anamorphic fungi
botanically" with correlatively the demise of the Deuteromycota. After a
critical analysis of the new Art 59 of the Sydney Code, I concluded: "The
morphological diversity, the ontogenetic plasticity of forms and the unity of
the chemotaxonomical characters expressed by the unique gene pool in the
same fungus demonstrate clearly enough the artificial nature of anatomical,
monoanamorphic taxa and the need for a more synthetic concept of the
species to approach a more natural taxonomy" (Hennebert 1991).
When in August 1992, Reynolds and Taylor (1993) convened a conference
at Newport, Oregon, on the theme of "The Fungal Holomorph", they had, as
objective, the integration of Deuteromycotina into the taxonomy of
Ascomycotina and Basidiomycotina. The trend was confirmed by a large
majority of mycologists at the Newport Conference (Hawksworth 1993). The
arguments brought in by the speakers were mostly biomolecular: the close
phylogenetic relatedness between many anamorphic and teleomorphic
fungi.
The push towards integration will stimulate interactive adjustment of
both taxonomies and consequently the mixing of their nomenclature so as to
reach a genus - form-genus, species - form-species correspondence. The

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integration should mark different steps. The minimal step is to distribute the
Deuteromycotina into two groups, the Ascodeuteromycotina and the
Basidiodeuteromycotina, like Cryptococcus was segregated from Candida. A
further step is to place those anamorphic genera near holomorphic genera in
orders and families to which they belong: Aspergillus and Penicillium in the
Eurotiales, Trichochomaceae, for instance. A next possible step would be to
integrate biologically connected anamorph and teleomorph genera in unique
genera, e.g., Monilia in Monilinia, Botrytis and Amphobotrys in Botryotinia;
such genera should include holomorphic species and anamorphic species
with their respective holomorphic or anamorphic names (Hennebert 1993).
In the integration process, what shall be the status of the anamorphic
names? Two options can be conceived. One option might reject Art. 59, to
anihilate anatomical typification in higher fungi; making any name of equal
and concurrent status would realize a truly botanical nomenclature. Even
were it applied from a future date, that option would create such a turn-over
of fungal nomenclature, that it shall not be acceptable. The other option, the
only feasible one, is to maintain the anatomical typification approved by Art.
59, which prescribes an obligate teleomorphic typification for holomorphic
names of Ascomycotina and Basidiomycotina and a restricted anamorphic
typification for anamorphic names of Asco- and Basidiodeuteromycotina,
making the latter names forever non-synonymous and non-priorable, in
other words "deprived of nomenclatural status by typification" in regard to
holomorphic names. In such an integrated classification, the names of
anamorphs of pleomorphic fungi will disappear by use of the holomorphic
names. The names of those anamorphic fungi still lacking any connection to
teleomorphs ("anamorphic holomorphs", according Reynolds 1993) should
remain treated as "pseudo-botanical" names (Hennebert 1991) for so long as
a related teleomorph remains undiscovered.
At the Newport Conference in August 1992 , the terms anamorph, and
teleomorph were disputed by Sutton (1993) and Hawksworth (1993), who
preferred the term "mitosporic" and "meiosporic" to qualify the fungus or
parts of the ascomycete or basidiomycete fungus, the term "mitospore"
replacing the 150 year-old term "conidium". After the matter was raised
again at the Conference on Ascomycete Systematics in Paris in 1993, Korf and
Hennebert (1993) answered the argument and emphasized the disastrous
situation that such deviations in terminology already foreseen by Weresub
and Hennebert (1979) would create. The terms anamorph and teleomorph
are not karyological but nomenclatural terms, which adequately serve the
nomenclature of higher fungi. Fungal spores are said meiotic or mitotic
depending on their direct parental nuclear division, the meiotic spores being
those spores containing one or more of the four meiotic nuclei. The
adjectives "meiosporic" cannot correctly qualify Ascomycotina and
Basidiomycotina, as spores of most Ascomycetes and numerous
Basidiomycetes are mitotic, containing none of the four nuclei of meiosis. In
prospect of the conceivable and hopefully predictable integration of the

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Deuteromycotina in the Ascomycotina and the Basidiomycotina, and as long

as the rule of anatomical typification is maintained in these fungi, the terms
anamorph and teleomorph will remain necessary.

CONCLUSION
Taxonomy and nomenclature of the fungi, reasons for a Mycotheque

Fungal taxonomy and fungal nomenclature are linked intimately. Both

will really progress towards a natural classification of the fungi if they are
based on an accurate and extensive knowledge of the fungi based on their
morphology and development. This requires repeated collections of fungi in
the field and their isolation in pure culture on various media and under
diverse conditions, and on their morphological and morphogenetic
description in pure culture in such media and under such conditions, using
a harmonized and unequivocal terminology, and finally the application of a
correct name in regard to the taxonomic literature and the rules of
nomenclature.
For such achievement, a mycotheque is the basic and necessary tool for
mycology. It includes a herbarium, culture collection, iconotheque, library,
data bases and expert systems. Surely, fungal systematics will not be
successfully completed without collaborative morphological, biological and
genomic studies. But, at the least, it should first be started with field work
and morphological descriptions. Genomic analysis is only an additional,
currently and unfortunately luxurious, approach.
The task for fungal taxonomists is urgent:, for the undiscovered
biodiversity of the fungi will not wait. Only 5 % of the mycoflora consisting
of a presumed 1,500,000 species is known. Much of that important and still
undiscovered diversity (more than 1,400,000 species) lies in tropical zones,
mainly in tropical forests (Hawksworth 1991, 1994). The actual destruction
rate of the tropical forests is such that that reservoir of biodiversity shall be
entirely burnt up within 100 years, by 2100 AD (Hennebert 1996a). At the
current rate of 14 new species published per day by the existing mycological
community, about 350 years of work by such a community will be needed if
the objectives remain what they are today. Spending manpower, time and
money in elaborating a genetic phylogeny, a pretended natural classification
of the fungi from a non-representative sample (5%) of the fungal world is
clearly an impossible vision. The goal is not to achieve the impossible, but
rather it is in the field we must spend our energies. Let's go to work.
Wonders are waiting for us before being all too soon incinerated.

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302

 INDEX OF FUNGAL NAMES

Species names in summaries, tables, plates and dendrograms are not indexed.
Names of families, orders and classes are not indexed.

Acanthodochium 126, 127

Agaricus 69, 72, 226
A. bisporus 226, 227, 228
A. bitorquis 226, 228
Adoghamina ruchira 271
Albugo trapogonensis 235
Alternaria 208
A. triticina 206,207
Amanita zambiana 69
A. muscaria 70
Amauroderma 67, 71
Ambrosiella 51
A. ferruginea 193
A. xyleborii 291
Ambrosyozima 41
Amphobotrys 274, 294
Anthosspomella 123, 125
Antrodia vaillantii 70
Arachnophora fagicola 270, 275, 276
Areolospora terrophila 125
Armillaria 67, 73, 74
Arthroascus 41
Ascoidea 41
Ashbya 41, 160, 161
A. gossypii 160, 161
Aspergillus 11, 20, 21, 114
A. clavatoflavus 175
A. flavus 175
A .niger 11, 20, 114
A. oryzae 175
A. parasiticus 175
A. repens 225
A. sojae 175
A. tamarii 175
A. zonatus 175
Asteromyces 275
A. cruciatus 275, 280
Asterostroma ochroleucum 189, 181
Astrocystis 126
Aureobasidium 41
Auricularia 72
Balanium 264
B. stygium 264, 265
303

Ballistosporomyces 43
Basidiobotrys 123
Beauveria 274
Besingtonia 43
Bipolaris sorokiniana 135, 138, 206, 207, 208
Biscogniauxia 123, 125
B. nummularia 125
Blastocladiella emersonii 149
Boletus edulis 70
Boletus expansus 187
Botryotinia 256, 274, 294
B. calthae 256, 257
B. globosa 256
B. ficariarum 256, 257, 278
B. fuckeliana 256
B. porri 256, 257
B. ranunculi 278
Botrytis 21, 255,293, 298
B. cinerea 255, 259, 273
B. didyma 264
B. geranii 270
B. porri 256
Brettanomyces 44
Broomeia congregate 72
Byssonectria terrestris 219, 220, 222
Calcarisporium 274
Calonectria 151, 152
C. clavata 151, 152, 153, 155
C. gracilis 151, 152
C. pteridis 153
Camillea 123
C. leprieurii 123
C tinctor 123
Candida 44, 148, 161, 294
C. albicans 44, 174
C. guilliermondii 45
C. lipolytica 164
C. tropicalis 45
Cantharellus 69
Cephalascus 40, 41
Cephalotrichum 274
Ceratocystis 41, 51
C. araucariae 193
C. piceae 193, 194
Cercospora kikuchii 198
Cercosporella 196
C. herpotrichoides 196
Chalara 291
Chalaropsis 293

304

 C. punctulata 287, 291

Chlorophyllum molybdites 69
Chromelosporium 270, 274
C. fulvum 279
C. macrospermum 279
C. ochraceum 279
C. ollare 279
C. trachycarpum 279
C. tuberculatum 283
Cladobotryum 274
C. dendroides 284
Cochliobolus 135
C. sativus 135, 139, 206 207, 245
Colletotrichum gloeosporioides 203, 204
Collodiscula japonica 126
Collybia aurea 72
Coniophora puteana 70, 183, 188, 191
Coniophora marmorata 188, 191
Conoplea 274
Cookeinia 72
Coriolopsis 73
C. polyzona 70
Corticium 217
Costantinella 274
Creosphaeria sassafras 126
Cryptococcus 43, 44, 156, 294
C. albidus 157
C. alylolentus 157
C. curiosus 157
C. curvatus 157
C. dimennae 157
C. elinovii 158
C. flavus 157
C. gastricus 157
C. heveanensis 157
C. humicola 157
C. kuetzingii 157
C. laurentii 157
C. luteolus 157
C. macerans 157
C. magnus 157
C. marinus 157
C. neoformans 174
C. skinneri 157
C. terreus 158
C. tsukubaensis 157
Cryphonectria parasitica 53
Curvularia 208
Custingophora olivacea 271, 276

305

Cylindrocarpon hydrophilum 296

Cylindrocladium 151, 152, 177, 181
C. camelliae 177, 180
C. clavatum 151, 152, 153, 177
C. colhounii 177, 179
C. floridanum 177, 179
C. gracile 151, 152, 153
C. hawksworthii 151, 152, 153, 178, 179
C. illicicola 177
C. infestans 177, 180
C. pteridis 151, 152, 153
C. quinqueseptatum 177, 179
C. theae 177
Cylindrodendrum album 275, 296
Cystofilobasidium 43, 156
Daldinia 72, 123
D. escholtzii 125
D. occidentalis 126
Debaryomyces 41, 161
D. hansenii 161
D. larala 42
Dekkera 44, 45
Dematophora 126
Dichobotrys 278
Dichomitus 70
Dipodascus 41
Donkioporia expansa 187
Drechslera sorokiniana 135, 209
Drechslera tritici-repentis 206, 207, 208
Echinobotryum atrum 287, 290, 295
Echinobotryum citri 287
Echinobotryum parasitans 287, 290
Endogone pisiformis 149
Epichloe 43
Eremascus 41
Eremothecium 41, 160, 161
E. ashbyii 160, 161
Erythrobasidium 43
Euepixylon 123, 125
Filobasidiella 156
F. neoformans var.neoformans 156
F. neoformans var.bacillispora 156
Filobasidum 156
Flavodon flavus 72
Fomitopsis widdringtoniae 67
Fusaroium oxysporum sp.f. cubense 27
Fusicoccum coccineum 239, 240, 242
Galactomyces 41
Ganoderma 67, 71, 118

306

 G. australe 146
G. applanatum 260
Geniculisporium 123, 125, 126
Geomyces 274
Gigaspora marginata 231
Gilmaniella humicola 271
Glischroderma 274, 279
G. cinctum 279
Gloeophyllum trabeum 70
Grammothele 70
Grammothelopsis 70
Graphium 193
Guillermondella 41
Gyrodontium boveanum 70, 143
Hadrotrichum 127
H. pyrenaicum 127
Hansfordia 274
Haplotrichum 274
Helminthosporium sativum 135
Holleya 41, 162
Hormoascus 41
Humicola 293
Hymenochaeta tabacina 140
Hyphelia 271, 282
H. rosea 282
H. terrestris 271, 283
Hypochnus 279
Hypocreopsis lichenoides 140
Hypomyces rosellus 284
Hypoxylon 72, 123, 127
H. argillaceum 128
H. deustum 127, 128
H. fraxinophilum 128
H. moravicum 128
H. pyrenaicum 127
Inonotus 217
Irpex 73
Isthmolongispora lanceata 287, 291
Kathistes 51
Kluyveromyces 160, 161
Kondoa 156
Kretzschmaria 128
K. deusta 127
Lactarius 69, 72, 144
L. flammans 144
L. gymnocarpoides 144
L. gymnocarpus 144
L. gymnosporus 144
L. kabansus 72

307

 L. longisporus 144
L. luteopus 144
L. medusae 144
L. pseudogymnocarpus 144
Lamprospora 274
Lentinellus 145
Lentinus cladopus 69
Leptosphaeria 235
Leprieuria 123, 125
Leucocoprinus 69
Leucosporidium 156
Linquistia 123, 127
Lomentospora prolificans 271, 287, 291
Lopadostoma turgidum 126
Lycogala 72
L. torrendii 287
Lycoperdellon 287
L. torrendii 287
Lypomyces 281
Macrolepiota 69, 72
Magnaporthe grisea 209, 210
Mammaria 293
M. echinobotryoides 286, 291
Megasporoporia 70
Melampsora 291
Metschnokowia 162
Monilia 294
Monilinia 294
Moniliella pollinis 278
Monochaeta 118
Mrakia 43, 156
M. frigida 156
M. gelida 156
M.. nivalis 156
M. stokesi 156
Mucor racemosus 149
Mucor troglophilus 288
Mycosphaerella 196, 198
Mycotypha 274
Mycovellosiella 198
Myrioconium 41, 257
Nadsonia fulvescens 161
Nemania 123, 125, 126
N. serpens 125
Nematogonium 274
Nematospora 41, 160, 161
N. coryli 160, 161
Neolecta 149
Neotiella vivida 220, 222, 224

308

Neurospora crassa 152

Nodulisporium 123, 125, 125, 127, 278
N. hinnuleum 126
Nummularia viridis 126
Oculimacula yallundae 196
Oculimacula acuformis 198
Oedemium atrum 265
Oe. didymum 264, 265
Oe. tomentosum 265
Ophioboulus 235
Ophiostoma 51
O. ulmi 53
Ostracoderma 266, 270, 274, 279
O. pulvinatum 266, 270, 279
Padixonia 126, 127
Penicilliopsis 175
Penicillium 11,15, 19, 20, 114, 255, 258, 265, 267-3, 278
P. aeruginosum 268
P. atroviride 268
P. aurantiobrunneum 267
P. aurantiocandidum 269
P. aurantiogriseum 269
P. biourgei 269
P. brefeldianum 21
P. brevicompactum 268
P. brunneorubrum 268
P. candidofulvum 268
P. carneoviolaceaum 267
P. citreonigrum 267
P. citreoroseum 267
P. chrysogenum 63
P. congolense 268
P. corylophilum 267
P. duclauxii 268
P. elongatum 268
P. expansum 278, 281
P. flexuosum 21
P. glaucum 255, 268
P. griseobrunneum 268
P. griseofulvum 11, 15, 20, 21, 114, 269
P. griseoroseum 19, 265, 269
P. hirsutum 269
P. minioluteum 269
P. roseopurpureum 267
P. verrucosum 268
Peniophora 217
Perenniporia mundula 69
Periconiella 125
Pestalotia 118

309

Peziza 274
P. domiciliana 220, 222
P. fuckeliana 256
Phaeosporis melasperma 125
Phanerochaete chrysospermum 247
Phellinus 217
P. rimosus 71
P. cryptarum 187
P. megaloporus 187
Phialophora 41
Phylacia 123, 125, 128
Phyllosticta circii 235
Phymatotrichopsis 274
P. omnivora 279
Phytophthora 53, 259
Ph. infestans 53
Pichia 40, 41, 42, 149
P. anomala 193
P. carsonii 41
P. etchellsii 41
P. farinosa 161
Pizolithus arhizus 117
Pleurocatera acicularis 140, 141
Pleurotus 234
P. cornucopiae 234
P. ostreatus 234
P. ostreatus var. columbinus 234
P. ostreatus cv.Florida 234
P. pulmonarius 234
P. sajor-caju 234
Pneumocystis 50, 96, 149
P. carinii 174
Podaxis pistillaris 72
Polymyxa betae 213
Polymyxa graminis 212, 213, 214
Polyporus squamosus 11
Polyporus tenuiculus 69
Poria expansa 187
Poria megalopora 187
Poronia 123
Porosordaria 123
Protomyces 50, 96, 149
Pseudocercosporella 196, 198
P. aestiva 196, 198, 199
P. anguioides 196
P. herpotrichoides var. herpotrichoides 196, 198
P. herpotrichoides var. acuformis 196, 198
Puccinia gentiana 145
P. suaveolens 235

310

Pulchromyces 274
Pycnoporus 73
Pyricularia oryzae 209, 210
Pyrenophora tritici-repentis 207
Pyxidiophora 51, 140, 141
Raffaelea ambrosiae 50, 287, 291
Ramulispora 196, 198
R. herpotrichoides var. herpotrichoides 196, 198
R. herpotrichoides var. acuformis 196, 198
R. herpotrichoides var. anguioides 199
R. sorghi 198
Rhizopus oryzae 117
Rhinocladiella 125, 274
Rhinotrichum 274
Rhodosporidium 40, 156
R. spaerocarpum 157
R. diobovatum 157
R. kratochvilovae 157
R. paludigenum 157
R. toruloides 40
Rhodotorula 43, 156
R. acheniorum 157
R. araucariae 157
R. armeniaca 156
R. aurantiaca 157
R. auriculariae 156
R. bacarum 156
R. bogoriensis 156
R. buffonii 44
R. diffluens 156
R. foliorum 157
R. fragaria 156
R. glutinis 157
R. graminis 157
R. hordea 156
R. hylophila 156
R. ingeniosa 156
R. javanica 156
R. lactosa 156
R. minuta 157
R. mucilaginosa 157
R. muscorum 156
R. philyla 156
R. pustula 156
R. sonckii 156
Rhopalostroma 123
Rosellinia 123, 125, 126
Russula 69, 72
Saccharomyces 40, 51, 160, 161, 167, 170, 172, 176, 238, 244-48, 281

311

 S. bayanus 167, 172, 174, 248, 281

S. carlsbergensis 174
S. cerevisiae 40, 167, 172, 174, 248
S. chevalieri 281
S. ellipsoideus 281
S. paradoxus 167, 172, 174, 248, 281
S. pastorianus 167, 172, 174, 248, 281
S. uvarum 248, 281
Saitoella 96, 149
Saturnospora 42
Schizosaccharomyces 41, 42, 50, 96, 149
S. pombe 161, 174
Saccharomycopsis lypolitica 164
Schwanniomyces 42
S. occidentalis 41
Sclerotium durum 256
Sclerotium echinatum 256
Scorias spongiosa 145
Seaverinia 274
S. geranii 270
Septoria circii 235
Serpula lacrymans 183
Sphaerosporella 274
Spirosphaera 291
Sporobolomyces 43
Sporoschismopsis moravica 275, 276
Sporothrix 125
Streptobotrys 274
Streptotinia 274
Stysanus caput-medusae 283, 286
Stysanus monilioides 283
Subbaromyces 51
Suillus cothurnatus 117
S. granulatus 70
Symbiotaphrina 51
Tapesia 196
T. yallundae 196
T. yallundae var. acuformis 198
T. acuformis 198
Taphrina 50, 95, 149
Termitomyces 69
Thermomyces 291
Torulaspora 176
Trametes 73
Trametes versicolor 70
Tremella 156
T. aurantia 158
T. brasiliensis 158
T. coalescens 158

312

 T. encephala 158
T. foliacea 158
T. fuciformis 158
T. globospora 158
T. mesenterica 158
T. samoensis 158
T. subanomala 158
Trichocladium 291
Trichoderma harzianum 193, 194
Trichoderma roseum 282
Tricholoma 226
T. spectabilis 226, 227, 228
Trichomonascus 41
Trichophaea 274
Trichothecium 282
T. roseum 282
Tseuchiyaea 43
Ustilago maydis 43
Ustulina 128
Verrucobotrys 270, 274
V. geranii 271
Verticillium 274
Virgaria 274
Virgariella 125
Wardomyces 275, 293
W. columbinus 271, 286, 291
Williopsis 42
Wyngea 42
W. robertsii 41
Xylaria 72, 114, 128, 131
X. allantoidea 127, 134
X. anisopleura 132
X. apiculata 133
X. arbuscula 133
X. cubensis 127, 132, 134
X. feejeensis 132
X. furcata 126, 131
X. grammica 133
X. longipes 126
X. luteostromata 132
X. magnoliae 95, 121
X. mellisii 133
X. multiplex 133
X. pallida 133
X. palmicola 133
X. papulis 133
X. poitei 134
X. polymorpha 21, 132
X. schreuderiana 133

313

 X. schweinitzii 132
X. scruposa 132
Xylocoremium flabelliforme 127
Xylocladium 123
Yarrowia lipolytica 164
Zygosaccharomyces 176

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314

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