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MANUAL DE IDENTIFICACION DE HONGOS

MANUAL DE IDENTIFICACION DE HONGOS

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    Los hongos y deterioro de los alimentos Los hongos y deterioro de los alimentos Document Transcript

    • Fungi and Food Spoilage
    • John I. Pitt l Ailsa D. HockingFungi and Food Spoilage13
    • John I. Pitt Ailsa D. HockingHonorary Research Fellow Honorary Research FellowCSIRO Food and Nutritional Sciences CSIRO Food and Nutritional SciencesNorth Ryde, NSW 2113 North Ryde, NSW 2113Australia AustraliaJohn.Pitt@csiro.au Ailsa.Hocking@csiro.auISBN 978-0-387-92206-5 e-ISBN 978-0-387-92207-2DOI 10.1007/978-0-387-92207-2Springer Dordrecht Heidelberg London New YorkLibrary of Congress Control Number: 2009920217# Springer ScienceþBusiness Media, LLC 2009All rights reserved. This work may not be translated or copied in whole or in part without the writtenpermission of the publisher (Springer ScienceþBusiness Media, LLC, 233 Spring Street, New York,NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use inconnection with any form of information storage and retrieval, electronic adaptation, computersoftware, or by similar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if theyare not identified as such, is not to be taken as an expression of opinion as to whether or not they aresubject to proprietary rights.Printed on acid-free paperSpringer is part of Springer ScienceþBusiness Media (www.springer.com)
    • Preface to the Third EditionIn contrast to the second edition, the third edition of ‘‘Fungi and Food Spoilage’’is evolutionary rather than revolutionary. The second edition was intended tocover almost all of the species likely to be encountered in mainstream foodsupplies, and only a few additional species have been included in this new edition.The third edition represents primarily an updating – of taxonomy, physiology,mycotoxin production and ecology. Changes in taxonomy reflect the impact thatmolecular methods have had on our understanding of classification but, it mustbe said, have not radically altered the overall picture. The improvements in theunderstanding of the physiology of food spoilage fungi have been relativelysmall, reflecting perhaps the lack of emphasis on physiology in modern micro-biological science. Much remains to be understood about the specificity ofparticular fungi for particular substrates, of the influence of water activity onthe growth of many of the species treated, and even on such basic parameters ascardinal temperatures for growth and the influence of pH and preservatives.Since 1997, a great deal has been learnt about the specificity of mycotoxinproduction and in which commodities and products-specific mycotoxins arelikely to occur. Changes in our understanding of the ecology of the includedspecies are also in most cases evolutionary. A great number of papers have beenpublished on the ecology of foodborne fungi in the past few years, but with fewexceptions the basic ecology of the included species remains. Recent changes in our understanding of foodborne fungi include the realisa-tion that Aspergillus carbonarius is a major source of ochratoxin A in the worldfood supply, that A. westerdijkiae and not A. ochraceus is the other commonAspergillus species making this toxin and that these species are responsible forochratoxin A in foods outside the cool temperate regions, where Penicilliumverrucosum is the important species. In recent years a number of new specieshave been found to be capable of producing aflatoxin, but the fact remains thatmost aflatoxin in the global food supply is produced by A. flavus and A. para-siticus. The taxonomy of Fusarium species is still undergoing major revision.However, the renaming of Fusarium moniliforme as F. verticillioides is the onlychange of importance here. Recent publications have improved our understand-ing of species – mycotoxin relationships within Fusarium. v
    • vi Preface to the Third Edition Among the colleagues who helped us to prepare this edition, we wish toparticularly thank Dr Anne-Laure Markovina, now of the University of Sydney,who assisted in literature searches and some cultural and photographic work,and Mr N.J. Charley who has continued his excellent work of curating the FRRculture collection, on which so much of the descriptive work in this book is based.
    • Preface to the First EditionThis book is designed as a laboratory guide for the food microbiologist to assistin the isolation and identification of common foodborne fungi. We emphasise thefungi which cause food spoilage, but also devote space to the fungi commonlyencountered in foods at harvest, and in the food factory. As far as possible, wehave kept the text simple, although the need for clarity in the descriptions hasnecessitated the use of some specialised mycological terms. The identification keys have been designed for use by microbiologist with littleor no prior knowledge of mycology. For identification to genus level, they arebased primarily on the cultural and physiological characteristics of fungi grownunder a standard set of conditions. The microscopic features of the various fungibecome more important when identifying isolates at the species level. Nearly allof the species treated have been illustrated with colony photographs, togetherwith photomicrographs or line drawings. The photomicrographs were takenusing a Zeiss WL microscope fitted with Nomarski interference contrast optics.We are indebted to Mr W. Rushton and Ms L. Burton, who printed the manyhundreds of photographs used to make up the figures in this book. We also wish to express our appreciation to Dr D.L. Hawksworth, Dr A.H.S.Onions and Dr B.C. Sutton of the Commonwealth Mycological Institute, Kew,Surrey, UK, Professor P.E. Nelson and the staff of the Fusarium ResearchCenter, University of Pennsylvania, USA and Dr L.W. Burgess of the Universityof Sydney, who generously provided facilities, cultures and advice on some of thegenera studied. vii
    • Preface to the Second EditionIn planning for the second edition of ‘‘Fungi and Food Spoilage’’, we decidedthat the book would benefit from a larger format, which would permit improvedillustrations, and from some expansion of the text, in both numbers of speciestreated and overall scope. These aims have been realised. The Crown Quarto sizehas allowed us to include substantially larger, clearer illustrations. Many newphotographs and photomicrographs have been added, the latter taken using aZeiss Axioscop microscope fitted with Nomarski differential interference con-trast optics. We have taken the opportunity to include more than 40 additionalspecies descriptions, to add a new section on mycotoxin production for eachspecies and to update and upgrade all of the text. Since the first edition, changes in the climate for stabilising fungal nomencla-ture have resulted in development of a list of ‘‘Names in Current Use’’ for someimportant genera, including Aspergillus and Penicillium. Names of species usedin the second edition are taken from that list, which was given special status bythe International Botanical Congress, Tokyo, 1994. Names used in this editionhave priority over any other names for a particular species. Publication of a list of‘‘Authors of Fungal Names’’ (P.M. Kirk and A.E. Ansell, Index of Fungi,Supplement: 1–95, 1992) has also stabilised names of authorities for all fungalspecies. Abbreviations of authors’ names used in this edition conform to thoserecommended by Kirk and Ansell. Some progress in standardisation of methodsand media has also been made, primarily through the efforts of the InternationalCommission on Food Mycology. The first edition included some 400 references. When we began revisionarywork, we felt that the number of references in the area of food mycology hadprobably doubled or increased by perhaps 150% during the intervening years. Infact, this second edition includes over 1900 references, almost a five-fold increaseover the 1985 edition! This provides a clear indication that interest in, and studyof, food mycology has greatly increased in recent years. Modern referencingsystems have enabled us to expand information from tropical sources, especiallyin Asia and Africa, but we are conscious of the fact that treatment of fungi foundin foods on a worldwide basis remains rather incomplete. We gratefully acknowledge support and assistance from colleagues who havecontributed to this new edition. Ms J.C. Eyles formatted and printed the camera ix
    • x Preface to the Second Editionready copy, Ms C. Heenan collated, arranged and formatted the illustrations andMr N.J. Charley looked after the culture collection, culture growth and colonyphotography. Without this level of support, the book would not have beencompleted.
    • Contents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 The Ecology of Fungal Food Spoilage . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.1 Water Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.2 Hydrogen Ion Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.3 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.4 Gas Tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.5 Consistency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.6 Nutrient Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.7 Specific Solute Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.8 Preservatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.9 Conclusions: Food Preservation . . . . . . . . . . . . . . . . . . . . . . . . 93 Naming and Classifying Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.1 Taxonomy and Nomenclature: Biosystematics . . . . . . . . . . . . . 11 3.2 Hierarchical Naming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.3 Zygomycotina. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.4 Ascomycotina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 3.5 Basidiomycotina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 3.6 The Ascomycete – Conidial Fungus Connection . . . . . . . . . . . . 15 3.7 Dual Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 3.8 Practical Classification of Fungi . . . . . . . . . . . . . . . . . . . . . . . . 164 Methods for Isolation, Enumeration and Identification . . . . . . . . . . . . 19 4.1 Sampling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4.2 Enumeration Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4.2.1 Direct Plating. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 4.2.2 Dilution Plating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 4.2.3 Incubation Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . 22 4.3 Sampling Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 4.4 Air Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.5 Isolation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.5.1 Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.5.2 Moulds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 4.5.3 Short Term Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 4.6 Choosing a Suitable Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 4.6.1 General Purpose Enumeration Media. . . . . . . . . . . . . . . 26 4.6.2 Selective Isolation Media. . . . . . . . . . . . . . . . . . . . . . . . . 27 4.6.3 Techniques for Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 4.6.4 Techniques for Heat-Resistant Fungi . . . . . . . . . . . . . . . 32 4.6.5 Other Plating Techniques . . . . . . . . . . . . . . . . . . . . . . . . 33 4.7 Estimation of Fungal Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.7.1 Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.7.2 Ergosterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4.7.3 Impedimetry and Conductimetry . . . . . . . . . . . . . . . . . . 37 xi
    • xii Contents 4.7.4 Adenosine Triphosphate (ATP) . . . . . . . . . . . . . . . . . 37 4.7.5 Fungal Volatiles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 4.7.6 Immunological Techniques . . . . . . . . . . . . . . . . . . . . . 38 4.7.7 Molecular Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 4.8 Identification Media and Methods. . . . . . . . . . . . . . . . . . . . . . 41 4.8.1 Standard Methodology . . . . . . . . . . . . . . . . . . . . . . . . 41 4.8.2 Plating Regimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 4.8.3 Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 4.8.4 Additional Media and Methods . . . . . . . . . . . . . . . . . 42 4.8.5 Identification of Fusarium Species. . . . . . . . . . . . . . . . 43 4.8.6 Yeasts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 4.9 Examination of Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 4.9.1 Colony Diameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 4.9.2 Colony Characters. . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 4.9.3 Preparation of Wet Mounts for Microscopy. . . . . . . . 46 4.9.4 Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4.9.5 Microscopes and Microscopy . . . . . . . . . . . . . . . . . . . 47 4.10 Preservation of Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4.10.1 Lyophilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4.10.2 Other Storage Techniques . . . . . . . . . . . . . . . . . . . . . . 49 4.11 Housekeeping in the Mycological Laboratory. . . . . . . . . . . . . 50 4.11.1 Culture Mites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 4.11.2 Problem Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 4.11.3 Pathogens and Laboratory Safety . . . . . . . . . . . . . . . . 51 5 Primary Keys and Miscellaneous Fungi . . . . . . . . . . . . . . . . . . . . . . . . 53 5.1 The General Key . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 5.1.1 Notes on the General Key . . . . . . . . . . . . . . . . . . . . . . 55 5.2 Miscellaneous Fungi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 5.3 Genus Acremonium Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 5.4 Genus Alternaria Nees: Fr.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 5.5 Genus Arthrinium Kunze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 5.6 Genus Aureobasidium Viala and G. Boyer . . . . . . . . . . . . . . . . 65 5.7 Genus Bipolaris Shoemaker . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 5.8 Genus Botrytis P. Micheli: Fr. . . . . . . . . . . . . . . . . . . . . . . . . . 68 5.9 Genus Chaetomium Kunze . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 5.10 Genus Chrysonilia Arx . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 5.11 Genus Cladosporium Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 5.12 Genus Colletotrichum Corda . . . . . . . . . . . . . . . . . . . . . . . . . . 81 5.13 Genus Curvularia Boedijn. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 5.14 Genus Drechslera S. Ito . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 5.15 Genus Endomyces Reess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 5.16 Genus Epicoccum Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 5.17 Genus Fusarium Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 5.18 Genus Geotrichum Link: Fr.. . . . . . . . . . . . . . . . . . . . . . . . . . . 122 5.19 Genus Hyphopichia Arx and van der Walt. . . . . . . . . . . . . . . . 124 5.20 Genus Lasiodiplodia Ellis and Everh. . . . . . . . . . . . . . . . . . . . . 125 5.21 Genus Monascus Tiegh. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 5.22 Genus Moniliella Stolk and Dakin . . . . . . . . . . . . . . . . . . . . . . 129 5.23 Genus Nigrospora Zimm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
    • Contents xiii 5.24 Genus Pestalotiopsis Steyaert . . . . . . . . . . . . . . . . . . . . . . . . . . 133 5.25 Genus Phoma Sacc. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 5.26 Genus Stemphylium Wallr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 5.27 Genus Trichoconiella B.L. Jain. . . . . . . . . . . . . . . . . . . . . . . . . 137 5.28 Genus Trichoderma Pers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 5.29 Genus Trichothecium Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 5.30 Genus Ulocladium Preuss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 6 Zygomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6.1 Genus Absidia Tiegh. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 6.2 Genus Cunninghamella Matr. . . . . . . . . . . . . . . . . . . . . . . . . . . 149 6.3 Genus Mucor P. Micheli: Fr. . . . . . . . . . . . . . . . . . . . . . . . . . . 151 6.4 Genus Rhizomucor (Lucet and Costantin) Vuill. . . . . . . . . . . . 157 6.5 Genus Rhizopus Ehrenb.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 6.6 Genus Syncephalastrum J. Schrot. . . . . . . . . . . . . . . . . . . . . . . ¨ 165 6.7 Genus Thamnidium Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 7 Penicillium and Related Genera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 7.1 Genus Byssochlamys Westling . . . . . . . . . . . . . . . . . . . . . . . . . 170 7.2 Genus Eupenicillium F. Ludw. . . . . . . . . . . . . . . . . . . . . . . . . . 175 7.3 Genus Geosmithia Pitt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 7.4 Genus Paecilomyces Bainier . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 7.5 Genus Scopulariopsis Bainier . . . . . . . . . . . . . . . . . . . . . . . . . . 187 7.6 Genus Talaromyces C.R. Benj.. . . . . . . . . . . . . . . . . . . . . . . . . 188 7.7 Genus Penicillium Link. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 7.7.1 Penicillium subgenus Aspergilloides Dierckx . . . . . . . . . 196 7.7.2 Penicillium subgenus Furcatum Pitt. . . . . . . . . . . . . . . . 207 7.7.3 Penicillium subgenus Penicillium . . . . . . . . . . . . . . . . . . 223 7.7.4 Penicillium subgenus Biverticillium Dierckx . . . . . . . . . 263 8 Aspergillus and Related Teleomorphs. . . . . . . . . . . . . . . . . . . . . . . . . . 275 8.1 Genus Emericella Berk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 8.2 Genus Eurotium Link: Fr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 8.3 Genus Neosartorya Malloch and Cain . . . . . . . . . . . . . . . . . . . 292 8.4 Genus Aspergillus Fr.: Fr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 9 Xerophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 9.1 Genus Basipetospora G.T. Cole and W.B. Kendr. . . . . . . . . . . 340 9.2 Genus Chrysosporium Corda . . . . . . . . . . . . . . . . . . . . . . . . . . 342 9.3 Genus Eremascus Eidam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 9.4 Genus Polypaecilum G. Sm. . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 9.5 Genus Wallemia Johan-Olsen. . . . . . . . . . . . . . . . . . . . . . . . . . 350 9.6 Genus Xeromyces L.R. Fraser . . . . . . . . . . . . . . . . . . . . . . . . . 353 10 Yeasts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 11 Fresh and Perishable Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 11.1 Spoilage of Living, Fresh Foods . . . . . . . . . . . . . . . . . . . . . . . 383 11.2 Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 11.2.1 Citrus Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384 11.2.2 Pome Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 11.2.3 Stone Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
    • xiv Contents 11.2.4 Tomatoes and other Solanaceous Fruit. . . . . . . . . . . 387 11.2.5 Melons and other Cucurbits . . . . . . . . . . . . . . . . . . . 387 11.2.6 Grapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 11.2.7 Berries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 11.2.8 Figs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 11.2.9 Tropical Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 11.3 Vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 11.3.1 Peas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 11.3.2 Beans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 11.3.3 Onions and Garlic . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 11.3.4 Potatoes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 11.3.5 Roots and Tubers . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 11.3.6 Yams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 11.3.7 Cassava . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 11.3.8 Leafy and other Green Vegetables . . . . . . . . . . . . . . 393 11.4 Dairy Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 11.5 Meats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 11.6 Cereals, Nuts and Oilseeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 11.6.1 Wheat, Barley and Oats. . . . . . . . . . . . . . . . . . . . . . . 395 11.6.2 Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396 11.6.3 Maize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396 11.6.4 Soybeans and Mung Beans . . . . . . . . . . . . . . . . . . . . 397 11.6.5 Other Beans and Pulses . . . . . . . . . . . . . . . . . . . . . . . 398 11.6.6 Sunflower Seed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 11.6.7 Sorghum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 11.6.8 Peanuts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 11.6.9 Cashews and Brazil Nuts . . . . . . . . . . . . . . . . . . . . . . 399 11.6.10 Almonds, Hazelnuts, Walnuts and Pecans . . . . . . . . 399 11.6.11 Pistachios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 11.6.12 Copra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 40112 Spoilage of Stored, Processed and Preserved Foods . . . . . . . . . . . . . . . 12.1 Low Water Activity Foods: Dried Foods . . . . . . . . . . . . . . . . 401 12.1.1 Cereals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402 12.1.2 Flour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 12.1.3 Pasta. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 12.1.4 Bakery Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 12.1.5 Maize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404 12.1.6 Soybeans, Mung Beans, other Beans and Chickpeas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405 12.1.7 Nuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406 12.1.8 Peanuts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406 12.1.9 Hazelnuts, Walnuts, Pecans and Almonds . . . . . . . . 407 12.1.10 Pistachio Nuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408 12.1.11 Other Nuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409 12.1.12 Coconut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409 12.1.13 Spices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 12.1.14 Coffee Beans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 12.1.15 Cocoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 12.1.16 Dried Meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
    • Contents xv 12.2 Low Water Activity Foods: Concentrated Foods . . . . . . . . . . 412 12.2.1 Jams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412 12.2.2 Dried Fruit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413 12.2.3 Fruit Cakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414 12.2.4 Confectionery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 12.2.5 Fruit Concentrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 12.2.6 Honey and Syrups . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416 12.3 Low Water Activity Foods: Salt Foods . . . . . . . . . . . . . . . . . . 416 12.4 Intermediate Moisture Foods: Processed Meats . . . . . . . . . . . 417 12.5 Heat Processed Acid Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . 418 12.6 Preserved Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418 12.7 Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419 Media Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 Glossary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
    • Chapter 1IntroductionFrom the time when primitive man began to culti- remains an enormous problem throughout the world.vate crops and store food, spoilage fungi have Figures are difficult to obtain. However, even given ademanded their tithe. Fuzzes, powders and slimes dry climate and advanced technology, losses of foodof white or black, green, orange, red and brown to fungal spoilage in Australia must be in excess ofhave silently invaded – acidifying, fermenting, dis- $10,000,000 per annum: losses in humid tropical cli-colouring and disintegrating, rendering nutritious mates and countries with less highly developed tech-commodities unpalatable or unsafe. nology remain staggering. An estimate of 5–10% of Until recently, fungi have generally been all food production is not unrealistic. Research intoregarded as causing only unaesthetic spoilage of fungal food spoilage and its prevention is clearly anfood, despite the fact that Claviceps purpurea was urgent necessity: lacking in spectacular appeal, it is,linked to human disease more than 200 years ago, however, often neglected. A further point, of the high-and the acute toxicity of macrofungi has long been est significance, needs emphasis here. Research on theknown. Japanese scientists recognised the toxic nat- fungi which cause food spoilage, and the mycotoxinsure of yellow rice 100 years ago, but 70 years elapsed they produce, can only be carried out effectively ifbefore its fungal cause was confirmed. Alimentary based on accurate identification of the microorgan-toxic aleukia killed many thousands of people in the isms responsible. Taxonomy and nomenclature (sys-USSR in 1944–1947; although fungal toxicity was tematics) make up the vital root system of all the treessuspected by 1950, the causal agent, T-2 toxin, was of biological science.not clearly recognised for another 25 years. The prevention of fungal food spoilage as an art Forgacs and Carll (1952), in a prophetic article, is old, but as a discipline, young. Drying, the oldestwarned of the danger from common spoilage fungi, method of food preservation, has been practiced forbut it was not until 1960, when the famous "Turkey millennia and is still the most common, effectiveX" disease killed 100,000 turkey poults in Great and cheap technique for preserving food. OnlyBritain, and various other disasters followed in rapid recently have we been able to identify with certaintysuccession, that the Western world became aware that the fungal species which cause spoilage of driedcommon spoilage moulds could produce significant foods. Prediction of their responses to a given envir-toxins. Since 1960 a seemingly endless stream of toxi- onment, specified by physico-chemical parametersgenic fungi and potentially toxic compounds has been such as water activity, temperature, pH and oxygendiscovered. On these grounds alone, the statement tension, even now is often uncertain. Within historic‘‘It’s only a mould’’ is no longer acceptable to food times, newer methods of food preservation havemicrobiologist, health inspector or consumer. The been introduced – salting, curing, canning, refrig-demand for accurate identification and characterisa- eration, freezing, the use of preservatives, irradia-tion of food spoilage fungi has become urgent. tion and most recently, high hydrostatic pressure. In the flurry of research into mycotoxins, however, Freezing excepted, each new technique hasit must not be forgotten that food spoilage as such selected for one or more fungal species resistant toJ.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_1, 1Ó Springer ScienceþBusiness Media, LLC 2009
    • 2 1 Introductionthe process applied. As examples we can take Poly- given on known habitats and sources, physiology,paecilum pisce on salt fish, Xeromyces bisporus on heat resistance, etc., together with a selective biblio-fruit cake, Cladosporium herbarum on refrigerated graphy. Accurate information on mycotoxin pro-meat, Zygosaccharomyces bailii in preserved juices, duction is also included.Z. rouxii in jams and fruit concentrates, Aspergillus As far as possible, the precise terminology forflavus on peanuts, Eurotium chevalieri on hazel nuts, fungal structures used by the pure mycologist andPenicillium roqueforti on cheeses, Byssochlamys indeed most necessary for him has been avoided infulva in acid canned foods . . . the list of quite specific these chapters. Some concepts and terms are offood – fungus associations is extensive. The study of course essential: these have been introduced assuch associations is one of the more important needed and are listed in a glossary.branches of the young discipline, food mycology. The taxonomic sections of this book are designed This book sets out to document current knowl- to facilitate identification of food spoilage and com-edge on the interaction of foods and fungi, in the mon food contaminant fungi. A standardised plat-context of spoilage and toxicity, not food produc- ing regimen is used, originally developed for thetion or biotechnology. Four aspects are examined. identification of Penicillium species (Pitt, 1979b)First, ecology: what factors in foods select for par- and extended here to other genera relevant to theticular kinds of fungi? A chapter is devoted to the food industry. Under this regimen, cultures arephysical and chemical parameters which influence incubated for 1 week at 5, 25 and 378C on a singlethe growth of fungi in foods. Second, methodology: standard medium and at 258C on two others. Inhow do we isolated fungi from foods? What are the conjunction with the appropriate keys, this systembest media to use? How do we go about identifying will enable identification of most foodborne fungifood spoilage fungi? Third, the commodity: what to species level in just 7 days. For a few kinds offungi are usually associated with a particular food? fungi, notably yeasts and xerophiles, subsequentHere ecological factors interact to produce a more growth under other more specialised conditionsor less specific habitat. Major classes of foods and will be necessary.their associated spoilage fungi are described. Finally, this book is dedicated to the general foodFinally, the fungus: what fungus is that? In a series microbiologist. May it help to restore equilibrium andof chapters, the main food spoilage moulds and assist in continued employment, when the qualityyeasts are described and keyed, together with others assurance manager demands: ‘‘What is it?’’ . . . ‘‘Howcommonly associated with food but not noted for did it get in?’’ . . . ‘‘What does it do?’’ . . . ‘‘How do wespoilage. Where possible, further information is get rid of it?’’ . . . and, worst of all . . . ‘‘Is it toxic?’’
    • Chapter 2The Ecology of Fungal Food SpoilageFood is not commonly regarded as an ecosystem, fresh foods is limited to particular species. Such spe-perhaps on the basis that it is not a ‘‘natural’’ sys- cific relationships between fresh food and fungus aretem. Nevertheless an ecosystem it is and an impor- discussed in Chapter 11 and under particular species.tant one, because food plants and the fungi that Other kinds of foods are moribund, dormant orcolonise their fruiting parts (seeds and fruit) have nonliving, and the factors which govern spoilage arebeen co-evolving for millennia. The seed and nut physical and chemical. There are eight principal factors:caches of rodents have provided a niche for the (l) water activity;development of storage fungi. Fallen fruit, as they (2) hydrogen ion concentration;go through the cycle of decay and desiccation, have (3) temperature – of both processing and storage;provided substrate for a range of fungi. Humans (4) gas tension, specifically of oxygen and carbonhave aided and abetted the development of food dioxide;spoilage fungi through their vast and varied food (5) consistency;stores. It can be argued, indeed, some rapidly evol- (6) nutrient status;ving organisms, such as haploid asexual fungi, are (7) specific solute effects; andmoving into niches created by man’s exploitation of (8) preservatives.certain plants as food. Food by its very nature is expected to be Each will be discussed in turn below.nutritious: therefore, food is a rich habitat formicroorganisms, in contrast with the great naturalsystems, soil, water and plants. Given the rightphysico-chemical conditions, only the most fasti- 2.1 Water Activitydious microorganisms are incapable of growth infoods, so that factors other than nutrients usually Water availability in foods is most readily measuredselect for particular types of microbial populations. as water activity. Water activity (aw), is a physico- Perhaps the most important of these factors chemical concept, introduced to microbiologists byrelates to the biological state of the food. Living Scott (1957), who showed that aw effectively quan-foods, particularly fresh fruits, vegetables, and also tified the relationship between moisture in foodsgrains and nuts before harvest, possess powerful and the ability of microorganisms to grow on them.defence mechanisms against microbial invasion. Water activity is defined as a ratio:The study of the spoilage of such fresh foods ismore properly a branch of plant pathology than aw ¼ p=po ;food microbiology. The overriding factor determin-ing spoilage of a fresh, living food is the ability of where p is the partial pressure of water vapour in thespecific microorganisms to overcome defence test material and po is the saturation vapour pres-mechanisms. Generally speaking, then, spoilage of sure of pure water under the same conditions.J.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_2, 3Ó Springer ScienceþBusiness Media, LLC 2009
    • 4 2 The Ecology of Fungal Food Spoilage Water activity is numerically equal to grow below 0.95 aw. A few halophilic algae and bac-equilibrium relative humidity (ERH) expressed as teria can grow in saturated sodium chloride (0.75 aw),a decimal. If a sample of food is held at constant but are confined to salty environments. Ascomycetoustemperature in a sealed enclosure until the water in fungi and conidial fungi of ascomycetous origin com-the sample equilibrates with the water vapour in the prise most of the organisms capable of growth belowenclosed air space (Fig. 2.1a), then 0.9 aw. Fungi capable of growth at low aw, in the presence of extraordinarily high solute concentrations aw ðfoodÞ ¼ ERH ðairÞ=100: both inside and out, must be ranked as among the most highly evolved organisms on earth. Even among the Conversely, if the ERH of the air is controlled in fungi, this evolutionary path must have been of thea suitable way, as by a saturated salt solution, at utmost complexity: the ability to grow at low aw isequilibrium the aw of the food will be numerically confined to only a handful of genera (Pitt, 1975).equal to the generated ERH (Fig. 2.1b). In this way, The degree of tolerance to low aw is most simplyaw can be experimentally controlled, and the rela- expressed in terms of the minimum aw at whichtion of aw to moisture (the sorption isotherm) can be germination and growth can occur. Fungi able tostudied. For further information on water activity, grow at low aw are termed xerophiles: one widelyits measurement and significance in foods see used definition is that a xerophile is a fungus able toDuckworth (1975); Pitt (1975); Troller and Chris- grow below 0.85 aw under at least one set of envir-tian (1978); Rockland and Beuchat (1987). onmental conditions (Pitt, 1975). Xerophilic fungi In many practical situations, aw is the dominant will be discussed in detail in Chapter 9.environmental factor governing food stability or spoi- Information about the water relations of manylage. A knowledge of fungal water relations will then fungi remains fragmentary, but where it is known itenable prediction both of the shelf life of foods and of has been included in later chapters.potential spoilage fungi. Although the water relationsof many fungi will be considered individually in laterchapters, it is pertinent here to provide an overview. Like all other organisms, fungi are profoundly 2.2 Hydrogen Ion Concentrationaffected by the availability of water. On the aw scale,life as we know it exists over the range 0.9999þ to 0.60 At high water activities, fungi compete with bacteria(Table 2.1). Growth of animals is virtually confined to as food spoilers. Here pH plays the decisive role.1.0–0.99 aw; the permanent wilt point of mesophytic Bacteria flourish near neutral pH and fungi cannotplants is near 0.98 aw; and most microorganisms cannot compete unless some other factor, such as low waterFig. 2.1 The concept ofwater activity (aw) (a) therelationship between aw andequilibrium relativehumidity (ERH); (b) onemethod of controlling aw bymeans of a saturated saltsolution, which generates aspecific ERH at a specificconstant temperature
    • 2.3 Temperature 5Table 2.1 Water activity and microbial water relations in perspectiveaaw Perspective Foods Moulds Yeasts1.00 Blood, plant wilt point, seawater Vegetables meat, milk fruit0.95 Most bacteria Bread Basidiomycetes Basidiomycetes Most soil fungi0.90 Ham Mucorales Fusarium Most ascomycetes0.85 Staphylococcus aureus Dry salami Rhizopus, Cladosporium Zygosaccharomyces rouxii (salt)0.80 Aspergillus flavus Xerophilic Penicillia Zygosaccharomyces bailii0.75 Salt lake Jams Xerophilic Aspergilli Debaryomyces hansenii Halophiles Salt fish Wallemia Fruit cake Eurotium0.70 Confectionery Chrysosporium Dried fruit Eurotium halophilicum Dry grains0.65 Xeromyces bisporus Zygosaccharomyces rouxii (sugar)0.60 DNA disordereda Modified from data of J.I. Pitt as reported by Brown (1974). Water activities shown for microorganisms approximateminima for growth reported in the literature.activity or a preservative, renders the environment As noted above, heat-resistant fungal spores mayhostile to the bacteria. As pH is reduced below about survive pasteurising processes given to acid foods.5, growth of bacteria becomes progressively less Apart from a few important species, little informa-likely. Lactic acid bacteria are exceptional, as they tion exists on the heat resistance of fungi. Much ofremain competitive with fungi in some foods down to the information that does exist must be interpretedabout pH 3.5. Most fungi are little affected by pH with care, as heating menstrua and conditions canover a broad range, commonly 3–8 (Wheeler et al., vary markedly, and these may profoundly affect1991). Some conidial fungi are capable of growth heat resistance. High levels of sugars are generallydown to pH 2, and yeasts down to pH 1.5. However, protective (Beuchat and Toledo, 1977). Low pHas pH moves away from the optimum, usually about and preservatives increase the effect of heatpH 5, the effect of other growth limiting factors may (Beuchat, 1981a, b; Rajashekhara et al., 2000) andbecome apparent when superimposed on pH. also hinder resuscitation of damaged cells (BeuchatFigure 2.2 is an impression of the combined influence and Jones, 1978).of pH and aw on microbial growth: few accurate data Ascospores of filamentous fungi are more heatpoints exist and the diagram is schematic. resistant than conidia (Pitt and Christian, 1970; For heat-processed foods, pH 4.5 is of course cri- Table 2.2). Although not strictly comparable, datatical: heat processing to destroy the spores of Clostri- of Put et al. (1976) indicate that the heat resistancedium botulinum also destroys all fungal spores. In acid of yeast ascospores and vegetative cells is of thepacks, below pH 4.5, less severe processes may permit same order as that of fungal conidia.survival of heat-resistant fungal spores (Section 2.3). Among the ascomycetous fungi, Byssochlamys species are notorious for spoiling heat processed fruit products (Olliver and Rendle, 1934; Richardson, 1965). The heat resistance of B. fulva2.3 Temperature ascospores varies markedly with isolate and heating conditions (Beuchat and Rice, 1979): a D valueThe influence of temperature in food preservation between 1 and 12 min at 908C (Bayne and Michener,and spoilage has two separate facets: temperatures 1979) and a z value of 6–7C8 (King et al., 1969) areduring processing and those existing during storage. practical working figures. The heat resistance of
    • 6 2 The Ecology of Fungal Food SpoilageFig. 2.2 A schematic diagram showing the combined influence of water activity and pH on microbial growthB. nivea ascospores is marginally lower (Beuchat role. Food frozen to –108C or below appears to beand Rice, 1979; Kotzekidou, 1997a). microbiologically stable, despite some reports of Ascospores of Neosartorya fischeri have a similar fungal growth at lower temperatures. The lowestheat resistance to those of Byssochlamys fulva, but temperatures for fungal growth are in the range –7 tohave been reported less frequently as a cause of food 08C, for species of Fusarium, Cladosporium, Penicilliumspoilage. Heat resistant fungi are discussed further and Thamnidium (Pitt and Hocking, 1997). Nonsterilein Chapter 4. food stored at ca. 58C in domestic refrigerators, Food products may be stored at ambient tem- where conditions of high humidity prevail, willperatures, in which case prevention of spoilage eventually be spoiled by fungi of these genera. Atrelies on other parameters, or under refrigeration, high aw and neutral pH, psychrophilic bacteria maywhere temperature is expected to play a preservative also be important (mostly Pseudomonas species).Table 2.2 Comparative heat resistance of ascospores and conidiaa Survivors (%)Fungus Spore type Initial viable count/ml 508C 608C 708CEurotium amstelodami Ascospores 5.0 Â 102 93 85 3 Conidia 7.3 Â 102 107 0.3 0Eurotium chevalieri Ascospores 1.0 Â 103 103 62 21 Conidia 8.9 Â 102 128 0.1 0Xeromyces bisporus Ascospores 1.0 Â 103 93 30 0.3Aspergillus candidus Conidia 3.8 Â 102 102 0 0Wallemia sebi Conidia 7.1 Â 102 42 0 0a Heated at temperatures shown for 10 min. Data from Pitt and Christian (1970).
    • 2.4 Gas Tension 7 Thermophilic fungi, i.e. those which grow only at oxygen concentration, at least up to 90% carbonhigh temperatures, are rarely of significance in food dioxide (Yates et al., 1967). Both Byssochlamysspoilage. If overheating of commodities occurs, nivea and B. fulva were capable of growth in atmo-however, in situations such as damp grain, thermo- spheres containing 20, 40 or 60% carbon dioxidephiles can be a very serious problem. with less than 0.5% oxygen, but inhibition increased Thermotolerant fungi, i.e. species able to grow at with increasing carbon dioxide concentrationboth moderate and high temperatures, are of much (Taniwaki et al., 2001a). Byssochlamys fulva isgreater significance. Aspergillus flavus and A. niger, capable of growth in 0.27% oxygen, but not in itsable to grow between ca. 8 and 458C, are among the total absence (King et al., 1969). It is also capable ofmost destructive moulds known. fermentation in fruit products, but presumably only if some oxygen is present. At least some species of Mucor, Rhizopus and Fusarium are able to grow and ferment in bottled2.4 Gas Tension liquid products and sometimes cause fermentative spoilage. Growth under these conditions may beFood spoilage moulds, like almost all other filamen- yeast-like. Species of Mucor, Rhizopus and Amylo-tous fungi, have an absolute requirement for myces used as starter cultures in Asian fermentedoxygen. However, many species appear to be effi- foods can grow under anaerobic conditions,cient oxygen scavengers, so that the total amount of demonstrated by growth in an anaerobe jar with aoxygen available, rather than the oxygen tension, hydrogen and carbon dioxide generator (Hesseltinedetermines growth. The concentration of oxygen et al., 1985). Other authors have reported growthdissolved in the substrate has a much greater influ- under anaerobic conditions of such fungi as Mucorence on fungal growth than atmospheric oxygen species, Absidia spinosa, Geotrichum candidum,tension (Miller and Golding, 1949). For example, Fusarium oxysporum and F. solani (Stotzky andPenicillium expansum grows virtually normally in Goos, 1965; Curtis, 1969; Taniwaki, 1995). The2.1% oxygen over its entire temperature range yeast-like fungus Moniliella acetoabutans can cause(Golding, 1945), and many other common food fermentative spoilage under totally anaerobic con-spoilage fungi are inhibited only slightly when ditions (Stolk and Dakin, 1966).grown in nitrogen atmospheres containing approxi- As a generalisation, however, it is still correct tomately 1.0% oxygen (Hocking, 1990). Paecilomyces state that most food spoilage problems due to fila-variotii produced normal colonies at 258C under mentous fungi occur under aerobic conditions, or at650 mm of vacuum (Pitt, unpublished). least where oxygen tension is appreciable, due to Most food spoilage moulds appear to be sensitive leakage or diffusion through packaging.to high levels of carbon dioxide, although there are In contrast, Saccharomyces species, Zygosac-notable exceptions. When maintained in an atmo- charomyces species and other fermentative yeastssphere of 80% carbon dioxide and 4.2% oxygen, are capable of growth in the complete absence ofPenicillium roqueforti still grew at 30% of the rate in oxygen. Indeed, S. cerevisiae and Z. bailii can con-air (Golding, 1945), provided that the temperature tinue fermentation under several atmospheres pres-was above 208C. In 40% CO2 and 1% O2, P. roque- sure of carbon dioxide. This property of S.forti grew at almost 90% of the rate in air (Taniwaki cerevisiae has been harnessed by mankind for hiset al., 2001a). Xeromyces bisporus has been reported own purposes, in the manufacture of bread andto grow in similar levels of carbon dioxide (Dallyn many kinds of fermented beverages. Z. bailii, onand Everton, 1969). the other hand, is notorious for its ability to con- Byssochlamys species appear to be particularly tinue fermenting at reduced water activities in thetolerant of conditions of reduced oxygen and/or presence of high levels of preservatives. Fermenta-elevated carbon dioxide. Growth of Byssochlamys tion of juices and fruit concentrates may continuenivea was little affected by replacement of nitrogen until carbon dioxide pressure causes container dis-in air by carbon dioxide, and growth in carbon tortion or explosion. The closely related speciesdioxide–air mixtures was proportional only to Zygosaccharomyces rouxii is a xerophile and causes
    • 8 2 The Ecology of Fungal Food Spoilagespoilage of low-moisture liquid or packaged pro- Some xerophilic fungi are known to be moreducts such as fruit concentrates, jams and dried demanding. Ormerod (1967) showed that growthfruit. The difference in oxygen requirements of Wallemia sebi was strongly stimulated by proline.between moulds and fermentative yeasts is one of Xerophilic Chrysosporium species and Xeromycesthe main factors determining the kind of spoilage a bisporus also require complex nutrients, but theparticular commodity will undergo. factors involved have not been defined (Pitt, 1975). Yeasts are often fastidious. Many are unable to assimilate nitrate or complex carbohydrates; a few, Zygosaccharomyces bailii being an example, cannot2.5 Consistency grow with sucrose as a sole source of carbon. Some require vitamins. These factors limit to some extentConsistency, like gas tension, exerts considerable the kinds of foods susceptible to spoilage by yeasts.influence over the kind of spoilage to which a food A further point on nutrients in foods is worthis susceptible. Generally speaking, yeasts cause making here. Certain foods (or nonfoods) lackmore obvious spoilage in liquid products, because nutrients essential for the growth of spoilage fungi.single celled microorganisms are able to disperse Addition of nutrient, for whatever reason, can turnmore readily in liquids. Moreover, a liquid substrate a safe product into a costly failure.is more likely to give rise to anaerobic conditions Two cases from our own experience illustrate thisand fermentation is more readily seen in liquids. In point, both involving spoilage by the preservative-contrast, filamentous fungi are assisted by a firm resistant yeast Zygosaccharomyces bailii. In the first,substrate, and ready access to oxygen. a highly acceptable (and nutritious) carbonated bev- The foregoing is not intended to suggest that yeasts erage containing 25% fruit juice was eventually forcedcannot spoil solid products nor moulds liquids: merely from the Australian market because it was impracticalthat all other factors being equal, fermentative yeasts to prepare it free of occasional Z. bailii cells. Effectivehave a competitive advantage in liquids and cause levels of preservative could not be added legally andmore obvious spoilage under these conditions. pasteurisation damaged its flavour. Substitution of the fruit juice with artificial flavour and colour removed the nitrogen source for the yeast. A spoilage free product resulted, at the cost of any nutritional2.6 Nutrient Status value and a great reduction in consumer acceptance. The other case concerned a popular water-iceAs noted in the preamble to this chapter, the nutri- confection, designed for home freezing. This con-ent status of most foods is adequate for the growth fection contained sucrose as a sweetener and a pre-of any spoilage microorganism. Generally speaking, servative effective against yeasts utilising sucrose.however, it appears that fungal metabolism is best One production season the manufacturer decided,suited to substrates high in carbohydrates, whereas for consumer appeal, to add glucose to thebacteria are more likely to spoil proteinaceous formulation. The glucose provided a carbon sourcefoods. Lactobacilli are an exception. for Zygosaccharomyces bailii, and as a result several Most common mould species appear to be able months production, valued at hundreds of thou-to assimilate any food-derived carbon source with sands of dollars, was lost due to fermentativethe exception of hydrocarbons and highly con- spoilage.densed polymers such as cellulose and lignin. Mostmoulds are equally indifferent to nitrogen source,using nitrate, ammonium ions or organic nitrogensources with equal ease. Some species achieve only 2.7 Specific Solute Effectslimited growth if amino acids or proteins must pro-vide both carbon and nitrogen. A few isolates As stated earlier, microbial growth underclassified in Penicillium subgen. Biverticillium are conditions of reduced water availability is mostunable to utilise nitrate (Pitt, 1979b). satisfactorily described in terms of aw. However,
    • 2.9 Conclusions: Food Preservation 9the particular solutes present in foods can exert The use of chemical preservatives in foods is lim-additional effects on the growth of fungi. Scott ited by law in most countries to relatively low levels(1957) reported that Eurotium (Aspergillus) amste- and to specific foods. A few fungal species possesslodami grew 50% faster at its optimal aw (0.96) when mechanisms of resistance to weak acid preservatives,aw was controlled by glucose rather than magne- the most notable being Zygosaccharomyces bailii.sium chloride, sodium chloride or glycerol. Pitt This yeast is capable of growth and fermentation inand Hocking (1977) showed a similar effect for fruit-based cordials of pH 2.9–3, of 458C Brix andEurotium chevalieri and reported that the extreme containing 800 mg/L of benzoic acid (Pitt andxerophiles Chrysosporium fastidium and Xeromyces Hocking, 1997). The yeast-like fungus Moniliellabisporus grew poorly if at all in media containing acetoabutans can grow in the presence of 4% aceticsodium chloride as the major solute. In contrast Pitt acid and survive in 10% (Pitt and Hocking, 1997).and Hocking (1977) and Hocking and Pitt (1979) Of the filamentous fungi, Penicillium roquefortishowed that germination and growth of several appears to be especially resistant to weak acid pre-species of Aspergillus and Penicillium was little servatives and this property has been suggested as aaffected when medium aw was controlled with useful aid to isolation and identification (Engel andglucose–fructose, glycerol or sodium chloride. Teuber, 1978). Zygosaccharomyces rouxii, the second most xer-ophilic organism known, has been reported to growdown to 0.62 aw in fructose (von Schelhorn, 1950).Its minimum aw for growth in sodium chloride is 2.9 Conclusions: Food Preservationreportedly much higher, 0.85 aw (Onishi, 1963). Some fungi are halophilic, being well adapted to It is evident from the above discussion that thesalty environments such as salted fish. Basipetospora growth of fungi in a particular food is governedhalophila and Polypaecilum pisce grow more rapidly largely by a series of physical and chemical para-in media containing NaCl as controlling solute meters, and definition of these can assist greatly in(Andrews and Pitt, 1987; Wheeler et al., 1988c). assessing the food’s stability. The situation in prac-Such fungi have been called halophilic xerophiles to tice is made more complex by the fact that suchdistinguish them from obligately halophilic bacteria. factors frequently do not act independently, but synergistically. If two or more of the factors out- lined above act simultaneously, the food may be2.8 Preservatives safer than expected. This has been described by Leistner and Rodel (1976) as the ‘‘hurdle concept’’. ¨Obviously, preservatives for use in foods must be This concept has been evaluated carefully for somesafe for human consumption. Under this constraint, commodities such as fermented sausages and is nowfood technologists in most countries are limited to widely exploited in the production of shelf stablethe use of weak acid preservatives: benzoic, sorbic, bakery goods and acid sauces.nitrous, sulphurous, acetic and propionic acids – or, For most fungi, knowledge remains meagreless commonly, their esters. In the concentrations about the influence of the eight parameters dis-permitted by most food laws, these acids are useful cussed here on germination and growth. However,only at pH levels up to their pKa plus one pH unit, sufficient information is now available that somebecause to be effective they must be present as the rationale for spoilage of specific commodities byundissociated acid. For studies of the mechanism of certain fungi can be attempted, especially whereaction of weak acid preservatives see Warth (1977, one or two parameters are of overriding impor-1991); Brul and Coote (1999); Stratford and Anslow tance. This topic is considered in later chapters(1998) and Stratford and Lambert (1999). devoted to particular commodities.
    • Chapter 3Naming and Classifying FungiAs with other living organisms, the name applied to correctly naming plants, algae and fungi. It isany fungus is a binomial, a capitalised genus name amended every 6 years by special sessions at eachfollowed by a lower case species name, both written International Botanical Congress and is repub-in italics or underlined. The classification of organ- lished thereafter. The 17th version of the ICBNisms in genera and species was a concept introduced (the Vienna code) is the most recently publishedby Linneaus in 1753 and it is the keystone of biolo- (McNeill et al., 2006). The ICBN impinges onlygical science. It is as fundamental to the biologist as indirectly on the work of the practicing mycologistArabic decimal numeration is to the mathematician. or microbiologist. It is nevertheless of vital impor-Here the analogy ends: the concept of ‘‘base 10’’ is tance to the orderly naming of all plant life; torigorous; the concept of a species, fundamental as it ignore the ICBN is to invite chaos.is, is subjective and dependent on the knowledge Where confusion arises over the correct name forand concepts of the biologist who described it. a botanical species – a constant source of irritation to the nontaxonomist – it stems usually from one of three causes: indecision by, or disagreement among,3.1 Taxonomy and Nomenclature: taxonomists on what constitutes a particular spe- Biosystematics cies; incorrect application of the provisions of the ICBN; or ignorance of earlier literature.Once biologists began to describe species and to To return to our example, when species x andassemble them into genera, questions about their species y are seen to be the same, x has priorityrelationships began to arise: is species x described because it was published earlier; y becomes a syno-by Jones in 1883 the same as species y described by nym of x. Important synonyms are often listed afterSmith in 1942? Does species z, clearly distinct from x a name to aid the user of a taxonomy, and thisand y in some characters, belong to the same genus? procedure has been followed here.The study of these relationships is termed taxon- Through ignorance, the same species name may beomy. Modern taxonomy is based on sound scientific used more than once, for example, Penicillium thomiiprinciples, but still involves subjective judgment. Maire 1915 and P. thomii K.M. Zalessky 1927. The When the decision is made that species x and species name P. thomii has been given to two quite differenty are the same, however, the taxonomist must follow fungi. Clearly P. thomii Maire has priority; the laterclearly established procedures in deciding which name name is not valid. To avoid ambiguity, correct practicemust be used (‘‘has priority’’). The application of these in scientific publication is to cite the author of a speciesprocedures is termed nomenclature and, for fungi, at first mention, and before any formal description.plants and algae, is governed by the International The ICBN provides rules to govern change ofCode of Botanical Nomenclature (ICBN). genus name also. In our example, if species z is The ICBN is a relatively complex document of transferred to the genus to which species x and yabout 70 Articles dealing with all aspects of belong, it retains its species name but takes the newJ.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_3, 11Ó Springer ScienceþBusiness Media, LLC 2009
    • 12 3 Naming and Classifying Fungigenus name. The original author of the name z is connection between the Ascomycetes, the fungiplaced in brackets after the species name, followed by that produce sexual spores in sacks, and the Deu-the name of the author who transferred it to the teromycetes, where spores are always asexual, hascorrect genus. For example, Citromyces glaber been known for a long time. However, molecularWehmer 1893 became Penicillium glabrum (Wehmer) taxonomy has provided the fundamental assuranceWestling 1893 on transfer to Penicillium by Westling needed to make this change. From the point of viewin 1911. Note the use of Latinised names: glaber of the food mycologist, this is a mixed blessing. The(masculine) became glabrum (neuter) to agree with demands of the molecular systematists may yetthe gender of the genus to which it was transferred. make the taxonomy of foodborne fungi even more Further points on the use of the ICBN arise from complicated. The taxonomic system used here isthis example. P. glabrum retains its date of original believed to be both practical and in line with thepublication, and therefore takes priority over current ‘‘best practice’’ of the nomenclaturalists.P. frequentans Westling 1911 if the two species are The hierarchical subdivisions in Kingdom Fungicombined. When Raper and Thom (1949) combined of interest in the present context are shown below,the two species, a taxonomically correct decision, they using as examples three genera and species impor-retained the name P. frequentans, which was nomen- tant in food spoilage:claturally incorrect, causing confusion whenSubramanian (1971) and Pitt (1979b) took up the cor- Kingdom Fungi Fungi Fungirect name. It is worth pointing out that the confusion in Subkingdom Zygomycotina Ascomycotina Basidiomycotinathis and similar situations arose from Raper and Class Zygomycetes Plectomycetes Wallemiomycetes Order Mucorales Eurotiales WallemialesThom’s action in ignoring the provisions of the Family Mucoraceae Trichocomaceae WallemiaceaeICBN, not from that of later taxonomists who cor- Genus Rhizopus Eurotium Wallemia Species stolonifer chevalieri sebirectly interpreted it. Variety intermedius3.2 Hierarchical Naming Note that names of genera, species and varieties are italicised or underlined, while higher taxonomicA given biological entity, or taxon in modern terminol- ranks are not.ogy, can be given a hierarchy of names: a cluster of related Three subkingdoms of the kingdom Fungi includespecies is grouped in a genus, of related genera in families, genera of significance in food spoilage. As indicated inof families in orders, orders in classes, and classes in the examples above, these are Zygomycotina, Asco-subkingdoms. Similarly a species can be divided into mycotina and (much less commonly) Basidiomyco-smaller entities: subspecies, varieties and formae speciales tina. Fungi from each of these subkingdoms have(a term usually reserved for plant pathogens). quite distinct properties, shared with other genera In most modern classifications, the fungi are and species from the same subkingdom. Unlikeranked, like plants and animals, as a separate king- other texts, this book will not rely on initial recogni-dom. Traditionally, fungi have been divided into tion of a correct subkingdom before identification ofseveral subkingdoms, based on spore type and genus and species can be undertaken. Nevertheless,some environmental considerations. Modern mole- identification of the subkingdom can provide valuablecular methods have revolutionised this. Fungi have information about a fungus, so the principal proper-been shown to be more closely related to animals ties of these three subkingdoms are described below.than plants, where traditional taxonomy has alwaysplaced them. Some of the so-called ‘‘lower fungi’’have been shown not to be fungi of all (thoughmycologists will no doubt continue to study them). 3.3 Zygomycotina The most important change from the point ofview of the food mycologist is the demise of the Most fungi within the subkingdom Zygomycotinasubkingdom Deuteromycotina, and its absorption belong to the class Zygomycetes. Fungi in this class(almost entirely) into the Ascomycotina. The possess three distinctive properties:
    • 3.4 Ascomycotina 131. Rapid growth. Most isolates grow very rapidly, this, growth of fungi in this subkingdom is usually often filling a Petri dish of malt extract agar with slower than that of Zygomycetes, although there are loose mycelium in 2–4 days. some exceptions.2. Nonseptate mycelium. Actively growing mycelia Fungi in the subkingdom Ascomycotina, loosely are without septa (cross walls) and are essentially called ‘‘ascomycetes’’, characteristically produce unobstructed. This allows rapid movement of cell their reproductive structures, ascospores, within a contents, termed ‘‘protoplasmic streaming’’, sac called the ascus (plural, asci, Fig. 3.2a, b). In which can be seen readily by transmitted light most fungi, nuclei normally exist in the haploid under the binocular microscope. In wet mounts state. At one point in the ascomycete life cycle, the absence of septa is usually obvious (Fig. 3.1a). diploid nuclei are produced by nuclear fusion,3. Reproduction by sporangiospores. The reproduc- which may or may not be preceded by fusion of tive structure characteristic of Zygomycetes is the two mycelia. These nuclei undergo meiosis within sporangiospore, an asexually produced spore which the ascus, followed by a single mitotic division and in genera of interest here is usually produced inside a then differentiation into eight haploid ascospores. sac, the sporangium, on the end of a long specialised In most genera relevant to this work, asci can be hypha. Sporangiospores are produced very rapidly. recognised in stained wet mounts by their shape, which is spherical to ellipsoidal and smoothly From the food spoilage point of view, the out- rounded; size, which is generally 8–15 mm in dia-standing properties of Zygomycetes are very rapid meter; and the presence when maturity approachesgrowth, especially in fresh foods of high water activ- of eight ascospores tightly packed within their walls.ity; inability to grow at low water activities (no At maturity asci often rupture to release the ascos-Zygomycetes are xerophiles); and lack of resistance pores, which are thick walled, highly refractile, andto heat and chemical treatments. From the food often strikingly ornamented (Fig. 3.2c, d).safety point of view, Zygomycetes have rarely been Two other characteristics of asci are significant:reported to produce mycotoxins. generally they mature slowly, after incubation for 10 days or more at 258C, and they are usually borne within a larger, macroscopic body, the general term for which3.4 Ascomycotina is ascocarp. Genera of interest here usually produce asci and ascospores within a spherical, smooth-walledThe subkingdom Ascomycotina is distinguished body, the cleistothecium (Fig. 3.3a), or a body withfrom Zygomycotina by a number of fundamental hyphal walls, the gymnothecium (Fig. 3.3b).characters, the most conspicuous being the produc- Ascospores are highly condensed, refractiletion of septate mycelium (Fig. 3.1b). Consequent on spores, which are often resistant to heat, pressureFig. 3.1 (a) Nonseptate mycelium of Syncephalastrum racemosum; (b) septate mycelium of Fusarium equiseti
    • 14 3 Naming and Classifying FungiFig. 3.2 Asci and ascocarps: (a) asci of Talaromyces species; (b) asci of Byssochlamys fulva; (c) ascospores of Eupenicilliumalutaceum; (d) ascospores of Neosartorya quadricincta. Bars ¼ 5 mmand chemicals. Almost all xerophilic fungi are exist for specific kinds of conidia. Along the evolu-ascomycetes. tionary process, some Ascomycetes with well- Besides their sexual spores, ascospores, ascomy- developed asexual stages lost the ability to producecetes commonly produce asexual spores. Formed ascospores, and rely entirely on conidia forafter mitotic nuclear division, these spores are dispersal.borne singly or in chains, in most genera of interest Conidia, and the specialised hyphae from whichhere from more or less specialised hyphal structures. they are borne, are astonishingly diverse in appear-The general term for this type of spore is conidium ance. The size, shape and ornamentation of conidia(plural, conidia), but other more specialised terms and the complexity of the structures producing themFig. 3.3 (a) Cleistothecia of Eupenicillium; (b) gymnothecia of Talaromyces. SEM. Bars = 50 mm
    • 3.7 Dual Nomenclature 15provide the basis for classification of Ascomycetes anamorph, has a separate name, this strictly speak-that no longer produce the ascosporic (sexual) stage. ing applies to the conidial state. It should be used Lacking ascospores, conidial fungi are not usually only when the ascomycete state is absent, or to referheat resistant, but conidia may be quite resistant to specifically to the conidial state if the ascomycete ischemicals. Some conidial fungi are xerophilic. present. However, the reader is warned that some anamorphic names are, and will continue to be, in common use for holomorphic fungi. Under the Articles of the ICBN, a generic name3.5 Basidiomycotina originally given to an anamorphic or conidial fungus cannot be used for a teleomorphic or ascomycetousThe Subkingdom Basidiomycotina includes mush- fungus. For example, the name Penicillium, originallyrooms, puffballs and the plant pathogenic rusts and given to an anamorphic fungus with no known tele-smuts. Until recently it was not considered of any omorph, cannot be used for the teleomorphs laterinterest to the food mycologist. However, molecular found to be produced by other Penicillium species.studies indicate that the small brown species Such teleomorphs are classified in the genera Eupeni-Wallemia sebi, long a curiosity because of its lack cillium or Talaromyces, depending on whether ascos-of resemblance to any other fungus, is a basidiomy- pores are produced in cleistothecia or gymnothecia.cete. It has no obvious phylogenetic affinity with Correct species names for the ascomycetous andany other genus and has now been classified in its conidial states of a single holomorphic fungus may orown order, Wallemiales (Zalar et al., 2005). Only may not be the same, depending both on the circum-one other species of foodborne fungi, Trichosporo- stance in which the names were originally given, andnoides nigrescens Hocking and Pitt (1981), has a on later synonymy. For example, Eupenicilliumknown affinity with this subkingdom. ochrosalmoneum Scott and Stolk and Penicillium ochrosalmoneum Udagawa refer to the teleomorph and anamorph of a single fungus. Udagawa (1959) described the anamorph; the teleomorph was later3.6 The Ascomycete – Conidial Fungus found, in the same isolate, by Scott and Stolk (1967). Connection On the other hand, the anamorph of Eupenicil- lium cinnamopurpureum Scott and Stolk (1967) isIt was established more than a century ago that many Penicillium phoeniceum van Beyma (1933), with P.fungal species carry the genetic information to pro- cinnamopurpureum Abe ex Udagawa (1959) as aduce both ascospores and conidia. These two kinds of synonym. Scott and Stolk (1967) found a teleo-spores are produced by different mechanisms and morph in Udagawa’s P. cinnamopurpureum; Pitthave different functions, so they are not always (1979b) later showed that this species was a syno-formed simultaneously. Not surprisingly, mycologists nym of the earlier P. phoeniceum. E. cinnamopurpur-sometimes have given different generic and species eum, the first name applied to the teleomorph, isnames to a single fungus producing both an ascospo- unaffected by this change in the anamorph name. Inric and a conidial state. The usage of these names passing, note that ‘‘Abe ex Udagawa’’ indicatesunder the ICBN depends on the circumstances under invalid (incomplete) publication of this species bywhich they were originally given. Some of these cir- Abe, with validation later by Udagawa. The speciescumstances are discussed briefly below. dates from the year of validation. The ascomycete state, now usually referred to asthe teleomorph, is regarded by nomenclaturalists asthe more important reproductive state, and thename applied to the teleomorph should be used 3.7 Dual Nomenclaturewhen the ascomycete state is present. If the conidialstate is also in evidence, the fungus is now a holo- An important point here is that some isolates ofmorph and is still correctly known by the teleo- Penicillium phoeniceum regularly produce the teleo-morph name. If the conidial state, known as the morphic state Eupenicillium cinnamopurpureum, while
    • 16 3 Naming and Classifying Fungiothers, taxonomically indistinguishable, fail to produce principal factors influencing food spoilage bya teleomorph at all. Because of this, it is essential to have fungi are physico-chemical and have already beena separate name for teleomorph and anamorph. The outlined in Chapter 2. The point being made here issystem of two names for a single fungus, known as dual that food spoilage fungi are classified in just a fewnomenclature, has a place in the classification of fungi orders and a relative handful of genera. For thisdespite its apparent complexity. In the descriptions in reason there is much to be said for food mycologistslater chapters, fungi for which both teleomorphs and avoiding the use of a traditional, hierarchical classi-anamorphs are known have both names listed. As noted fication as outlined above and employing a lessabove, if both states are found in a particular isolate, the formal approach to the identification of the fungiteleomorph name is the more appropriate: to use that of interest to them.given to the anamorph is not incorrect, but this name is In the present work, this pragmatic approach hasmore sensibly applied to the conidial state only. been followed as far as possible: Dual nomenclature would be relatively simple if  The use of specialised terms has been kept to athe relationship between anamorph and teleomorph minimum, while being cognisant of the need forwas always one to one. This is not the case. As has clarity of expression.already been mentioned, species classified in Peni-  Hierarchical classification has been avoided ascillium may produce teleomorphs in two genera, far as possible, consistent with retaining a logicalEupenicillium and Talaromyces. On the other hand approach to the presentation of fungi which areTalaromyces produces anamorphs in two genera, related or of similar appearance.Penicillium and Paecilomyces. Aspergillus is the  Identification procedures used have beenanamorph of eight or ten teleomorphic genera. designed to be simple and comprehensible,Most teleomorph–anamorph relationships encoun- avoiding the use of specialised equipment or pro-tered in food mycology belong to the genera men- cedures unavailable in the routine laboratory. Totioned here. These relationships will be described this end, identification of nearly all specieswhere necessary under these particular genera. included in this work is based entirely on inocu- lation of a single series of Petri dishes, incubation under carefully standardised conditions and3.8 Practical Classification of Fungi examination by traditional light microscopy.  A standard plating regimen has been used for the initial examination of all isolates (except yeasts),Fungi are classified in a vast array of orders, so that identification procedures can be carriedfamilies, genera and species. Among natural organ- out without foreknowledge of genus or evenisms, the numbers of taxa of fungi are rivalled only subkingdom.by those of the flowering plants and insects. Esti-  Cultural characters, which can be broadlymates of fungal species range as high as 1.5 million; defined as the application of microbiologicalonly 5% of this number have so far been described techniques to mycology, have been used(Hawksworth, 1991). throughout. Many fungi are highly specialised. Some willgrow only in particular environments such as soil The use of cultural characters has long beenor water; many are obligate parasites and require a implicit in the study of fungi in pure culture onspecific host, such as a particular plant species, and artificial substrates, especially in such genera aswill not grow in artificial culture; many grow only in Aspergillus and Penicillium, genera of paramountassociation with plant roots. From the point of view importance in food spoilage. In Penicillium, culturalof the food microbiologist, these kinds of fungi are characters have been used as taxonomic criteriairrelevant. In one sense, most fungi which spoil since the turn of the 20th century, but have assumedfoods are also highly specialised, their speciality greater importance through the work of Pitt (1973,being the ability to obtain nutrients from, and 1979b), who used the measurement of colony dia-hence grow on, dead, dormant or moribund plant meters, following incubation under standardisedmaterial more or less regardless of source. The conditions, as a taxonomic criterion. The use of
    • 3.8 Practical Classification of Fungi 17pure culture techniques and growth data in fungal and adds unnecessary complexity to the task of thetaxonomy is now widespread. nonspecialist confronted with a range of fungal Food microbiologists, the primary audience for genera.this book, are familiar with cultural techniques and The approach used here has been to examinethe use of a wide range of media and varied incuba- every isolate (excluding yeasts) by a single system:tion conditions, so the authors make no apology for inoculation onto a standard set of Petri dishes andthe taxonomic approach used in the present work. examination of them culturally and microscopicallyThis approach is a logical extension of the system after 7 days incubation. Most of the genera andused by the first author in Penicillium taxonomy species included in this book can be identified imme-and which has been found to have a much broader diately, at that point. Only in exceptional cases hasapplicability. it been found necessary to reinoculate isolates onto In the field of mycology, different genera have a further set of media in order to complete identifi-been studied by many different people of varied cation. The exceptional fungi are first the xero-backgrounds and for different reasons. Conse- philes, many of which grow poorly if at all on thequently, keys and descriptions have been based on standard media, and, second, genera such as Fusar-a wide variety of media, often traditional formula- ium and Trichoderma, in which some species cannottions incorporating all sorts of natural products. readily be differentiated on the standard regimen.This heterogeneity makes comparisons difficult Details of the techniques used are given in Chapter 4.
    • Chapter 4Methods for Isolation, Enumeration and IdentificationThis chapter describes techniques and media suitable Florida (2007). Papers from the third and fourthfor the enumeration, isolation and identification workshops were published in the Internationalof fungi from foods. Some techniques are similar to Journal of Food Microbiology and the proceedingsthose used in food bacteriology; others have been of the fifth (Samsø) workshop were publisheddeveloped to meet the particular needs of food as ‘‘Advances in Food Mycology’’ (Hocking et al.,mycology. Most of the media have been specifically 2006a).formulated for foodborne fungi. The approach taken The methodology described below is based onhere is designed to provide a systematic basis for the recommendations from ICFM and representsstudy of food mycology. current thinking within the food mycology com- In 1984 a group of about 30 of the world’s fore- munity. However, no formal endorsement frommost scientists in food mycology met in Boston, ICFM is implied.Massachusetts, USA, to hear and discuss a widerange of presentations that explored many aspectsof methodology in food mycology. Agreement wasreached on broad issues and areas requiring further 4.1 Samplingwork pinpointed. The proceedings were publishedas ‘‘Methods for the Mycological Examination of It must be emphasised at the outset that results fromFood’’ (King et al., 1986). At a second workshop, mycological assays of foods are only as good as theheld in Baarn, the Netherlands, in 1990, results of samples used. However, sampling is beyond thea number of collaborative studies on media and scope of this text. Excellent treatises on samplingmethods were presented and some standardised plans for food bacteriological purposes have beenprotocols developed. The proceedings, published as produced by the International Commission on‘‘Modern Methods in Food Mycology’’ (Samson Microbiological Specifications for Foods (ICMSF,et al., 1992), provided a comprehensive overview 1986, 2002) and are generally applicable to foodof current thinking in this field. mycology. The working group which organised those twoworkshops was then formalised as the InternationalCommission on Food Mycology (ICFM), a com-mission under the auspices of the Mycology Divi- 4.2 Enumeration Techniquession of the International Union of MicrobiologicalSocieties (IUMS). ICFM is dedicated to interna- Quantification of the growth of filamentous fungitional standardisation of methods in food myco- is more difficult than for bacteria or yeasts. Vegeta-logy. Subsequent ICFM workshops were held in tive growth consists of hyphae, which are notCopenhagen, Denmark (1994), Uppsala, Sweden readily detached from the substrate and which(1998), Samsø, Denmark (2003) and Key West, survive blending poorly. When sporulation occurs,J.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_4, 19Ó Springer ScienceþBusiness Media, LLC 2009
    • 20 4 Methods for Isolation, Enumeration and Identificationvery high numbers of spores may be produced, The standard protocol recommended by the ICFMcausing sharp rises in viable counts, often without (Hocking et al., 2006a, p. 344) is given below, withany great increase in biomass. amplification where necessary. The estimation of fungal growth or biomass is Surface disinfection. Surface disinfection is car-not easy, because no primary standard exists (such ried out by immersing particles in a chlorine solu-as cell numbers used for yeasts and bacteria). tion. Household chlorine bleach, nominally 4–5%Although techniques for quantifying biomass have active chlorine, is effective. Dilute the chlorine 1 toimproved in recent years, most food laboratories 10 with water before use, to provide an approximatelycontinue to rely on viable counting (dilution plat- 0.4% solution. Immerse particles for 2 min, stirringing) for detecting and quantifying fungal growth in occasionally, then drain the chlorine. Chlorine solu-foods. tions are rapidly denatured by organic matter, so it As well as dilution plating, a second standard is important to use a surplus of chlorine solutionmethod, known as direct plating, has been devel- (10 times the volume of the particles) and to use theoped for estimating fungal numbers and growth in solution only once.foods. Both methods are described in detail below. This process is conveniently carried out inTechniques for biomass estimation will be discussed 250–500 ml beakers. Place 50 or more particles inlater. the beaker and add chlorine. To dislodge air bubbles, immediately stir with a pair of forceps, leaving them in the solution, and preferably cover the beaker with a watch glass. The watch glass simplifies decanting4.2.1 Direct Plating of the chlorine, and the forceps, disinfected by the chlorine, may be used to plate the particles.Contributions at the international workshops men- Studies in our laboratory have shown that thetioned above emphasised the use of direct plating as treatment outlined here may be inadequate underthe preferred method for detecting, enumerating some conditions. In commodities such as peanutsand isolating fungi from particulate foods such as or maize where high levels of Aspergillus flavus orgrains and nuts. In direct plating, food particles are Penicillium species may be present, surface disinfec-placed directly on solidified agar media. In most tion may be difficult. Here 2 min immersion in 70%situations, particles should be surface disinfected ethanol followed by 2 min in 0.4% chlorine isbefore plating, as this removes the inevitable surface recommended.contamination arising from dust and other sources Rinsing. After the chlorine is poured off, particlesand permits recovery of the fungi actually growing may be rinsed once with sterile water. Use a 1 minin the particles. This process provides an effective treatment, with stirring, then pour the water off.measure of inherent mycological quality and permits Again a watch glass should cover the beaker duringassessment of the potential presence of mycotoxins this period, and the sterile forceps should be usedas well. for stirring. It is not clear whether rinsing fulfils Surface disinfection should be omitted only where any essential function. Early direct plating regimenssurface contaminants become part of the down- used agents such as mercuric chloride for disinfec-stream mycoflora, for example, in grain intended tion, and rinsing was essential to remove such toxicfor flour manufacture. Even here, surface disinfec- materials before they penetrated the particles tootion before direct plating provides the most realistic deeply. However, chlorine is effectively denaturedappraisal of actual grain quality. by the particles and is believed to penetrate very Results from direct plating analyses are expressed little. In our opinion, the rinsing step can be omittedas percentage infection of particles. The technique without loss of efficacy of the treatment, with sav-provides no direct indication of the extent of fungal ings in time and materials, and reduced risk ofinvasion in individual particles. However, it is reason- recontamination from the air.able to assume that a high percentage infection is Plating. After disinfection and the optional rinse,correlated with extensive invasion in the particles particles should be plated onto solidified agar, atand a higher risk of mycotoxin occurrence. the rate of 6–20 particles per plate, depending on
    • 4.2 Enumeration Techniques 21particle size. Use the disinfected forceps. Plating Diluents. The recommended diluent is aqueousshould be carried out immediately: keep the watch 0.1% peptone (Hocking et al., 2006a, p. 344), sui-glass on the beaker if this is not possible. table for both filamentous fungi and yeasts. Saline Incubation. Incubate plates upright, under nor- solutions, phosphate buffer or distilled water are nomal circumstances for 5 days at 258C. See more longer recommended by ICFM as they may be dele-detailed notes below. terious to yeasts (Mian et al., 1997). The addition of Examination. After incubation, examine plates a wetting agent such as polysorbate 80 (Tween 80)visually, count the numbers of infected particles may be desirable for some products, but the naturaland express results as a percentage. Differential wetting ability of the peptone is usually adequate.counting of various genera is often possible. Correct Special diluents may be necessary in some cir-choice of media, a stereomicroscope and experience cumstances. If yeasts are to be enumerated fromwill all assist in this process. dried products or juice concentrates, the diluent should also contain 20–30% sucrose, glucose or glycerol, as the cells may be injured or be susceptible to osmotic shock.4.2.2 Dilution Plating Dilution. Serial dilutions of fungi are carried out by the same procedures as those used in bacteriol-Dilution plating is the appropriate method for ogy, and the recommended dilution rate is 1:10mycological analysis of liquid or powdered foods. (=1+9). Fungal spores sediment more quicklyIt is also suitable for grains intended for flour man- than bacteria, so it is important to draw aliquotsufacture and other situations where total fungal for dilution or plating as soon as possible, prefer-contamination is relevant. ably within 1 min (Beuchat, 1992). Sample preparation. The two most common Plating. Spread (surface) plating is recom-methods of sample preparation for dilution plating mended. When pour plates are used, fungi developare stomaching and blending: stomaching is recom- more slowly from beneath the agar surface and maymended by ICFM (King et al., 1986). The Colworth be obscured by faster growing colonies from surfaceStomacher (Sharpe and Jackson, 1972), or equiva- spores. Hence spread plating allows more uniformlent equipment (e.g. BagMixer1, Interscience, Saint colony development, improves the accuracy of enu- `Nom La Breteche, France), is a very effective device meration of the colonies and makes subsequent iso-for dispersing and separating fungi from finely lation of pure cultures easier.divided materials such as flour and spices, and soft The optimum inoculum for surface plating isfoods such as cheeses and meats, and its use is 0.1 ml. Best results will be obtained if plates arestrongly recommended. Treatment time in the sto- dried slightly before use. After adding the inoculum,macher should be 2 min. Harder or particulate foods spread it evenly over the agar surface with a sterilesuch as grains, nuts or dried foods, like dried bent glass rod (‘‘hockey stick’’). Sterilise the rod byvegetables, should be soaked before stomaching. flaming it with ethanol before use. A plate spinner isSoaking times from 30 to 60 min are generally suffi- a useful accessory. It is usually possible to enumer-cient, but for extremely hard particles such as dried ate plates with up to 150 colonies, but if a highlegumes, soaking for up to 3 h may be required. proportion of rapidly growing fungi are present,Comminution in a Waring Blender or similar the maximum number which can be distinguishedmachine is a suitable alternative for these types of with any accuracy will be lower. Because of thissamples and may give a more satisfactory homoge- restriction on maximum numbers, it may be neces-nate. Blending times should not exceed 60 sec, as sary on occasion to accept counts from plates withlonger treatments may fragment mycelium into as few as 10–15 colonies. Clearly, such limitationslengths too short to be viable or overheat the on numbers per plate and the overgrowth of slowhomogenate. colonies will result in higher counting errors than The sample size used should be as large as possi- are usually achieved with bacteria or yeasts.ble. If a stomacher or BagMixer 400 is used, a Enumerating yeasts is less difficult. In the absencesample size of 10–40 g is suitable. of filamentous fungi, from 30 to 300 colonies per
    • 22 4 Methods for Isolation, Enumeration and Identificationplate can be counted and errors will be comparable The techniques are based on those described bywith those to be expected in bacterial enumeration. Langvad (1980) for studying the fungal flora of Incubation. The standard incubation conditions leaves.are 258C for 5 days. However, other conditions may If samples are particulate, or can be cut up, sterilebe more suitable in some circumstances (see notes forceps can be used to press pieces of a suitable sizebelow). (up to about 10 mm2) onto a suitable medium in a Reporting results. As in bacteriology, results Petri dish. The sample is then removed, leaving anfrom dilution plating are expressed as viable counts impression, and any spores or mycelium transferredper gram of sample. Note that such results are not will form colonies within a few days. This techniquedirectly comparable with those obtained from direct is known as press plating.plating and may not offer a direct indication of the For packaging materials such as cardboard, anextent of fungal growth. alternative method is to cut a piece which will fit in a standard Petri dish. The dish is prepared by adding a sterile filter paper moistened with 10% glycerol and then placing a bent glass rod on it as a separa-4.2.3 Incubation Conditions tor. After adding the sample, a thin layer of an appropriate agar medium is poured over its surface.As noted above, the standard incubation conditions To reduce evaporation, the dish should be sealedspecified by ICFM are 258C for 5 days (Hocking with Parafilm or a similar material or placed in aet al., 2006a). Undoubtedly, 258C is the most suita- polyethylene bag before incubation at 258C for able temperature for routine work in temperate to few days. If contamination levels are not too heavy,subtropical environments. Few if any common the number and types of moulds present can befungi are sensitive to this temperature, even those effectively estimated by this method. Colonies maywhich grow readily under refrigeration. Higher tem- be subcultured for identification.peratures are unacceptable in the temperate zone: For sampling walls or other surfaces, or for non-308C is close to the upper limit for growth of some destructive sampling, impressions may be takenimportant Penicillium species. In tropical regions, using adhesive tape. Carefully handled, tape comingincubation at 308C is recommended as a more rea- from the roll will be virtually sterile. Press a shortlistic temperature for enumerating fungi from com- length of tape firmly onto the surface to be sampled,modities stored at ambient temperatures. In cool adhesive side down, then transfer it, still with thetemperate regions such as Europe, 228C may be a same side down, onto a suitable growth medium.more suitable incubation temperature. After 1–2 days incubation at 258C, the tape may be When used for fungi, Petri dishes should be removed to allow development and sporulation ofstored upright. The principal reason is that some colonies.common fungi can shed large numbers of spores Surface sampling techniques for assessment ofduring handling, which in an inverted dish will be sanitation in food production and processing areastransferred to the lid. Reinversion of the Petri dish are discussed by Evancho et al. (2001). Surfaces mayfor inspection or removal of the lid may liberate be sampled by using sterile swabs or by agar contactspores into the air or onto benches and cause serious methods. The swab method involves rubbing acontamination problems. moistened sterile cotton swab over the test surface and placing the swab in a dilution bottle to be sub- sequently diluted and plated on appropriate media. Agar contact plates, also known as RODAC (repli-4.3 Sampling Surfaces cate organism direct agar contact) plates, are restricted to use on smooth or semi-smooth flatMethods are outlined below for directly sampling surfaces. An alternative to agar contact plates isthe mycoflora of surfaces of commodities such as the agar slice technique, where agar is filled into afruits, meats, cheeses, salamis and dried fish and syringe-like apparatus or into an artificial sausagealso packaging materials, machinery and walls. casing. The solidified agar can be pushed out onto
    • 4.5 Isolation Techniques 23the surface to be sampled, then the portion making Hycon RCS and RCS High Flow centrifugal aircontact sliced off with a sterile scalpel or wire and samplers (Biotest, Solihull, UK) and the MAS-100placed in a Petri dish for incubation. air sampler (Merck, Darmstadt, Germany) are more convenient, as they are small and readily por- table. The Biotest RCS Plus sampler was reported to give comparable results to larger and more4.4 Air Sampling sophisticated machines, but its sampling efficiency gradually decreased for particle sizes below 4 mmAir sampling in the food processing environment is (Benbough et al., 1993).discussed by Evancho et al. (2001). The simplestmethod of air sampling is by sedimentation or settleplates. A Petri dish containing an appropriate agarmedium is exposed to the atmosphere for a fixed 4.5 Isolation Techniquesperiod of time (usually 15–60 min), then closed andincubated at 258C (Samson et al., 2004a). This The term ‘‘isolation’’ is used here in its strict sense:method can be useful in food production areas, as the preparation of a pure culture, free from anyit gives a direct indication of the number and types contamination and ready for identification.of fungi likely to come into contact with exposedproduct. However, the settle plate technique lacksprecision, and volumetric air sampling is a muchmore reliable indicator of air quality. 4.5.1 Yeasts A number of air sampling devices are commer-cially available. Of these, the Anderson sampler Streaking techniques commonly used for bacterial(either the two-stage or six-stage model; Anderson purification are equally suitable for the isolation ofInstruments Inc, Atlanta, GA, USA) is probably yeasts (Fig. 4.1). A method widely used by yeastthe best, giving accurate and consistent results specialists is to disperse a portion of a colony in 2–(Buttner and Stetzenbach, 1993). However, the 3 ml of sterile water, then streak a single loop of thisAnderson sampler is expensive and requires mains suspension over the whole surface of a plate, mov-power or a large battery for operation. In factory ing the loop slowly down from top to bottom whilesituations, the dry cell battery-operated Biotest simultaneously moving it rapidly across the plateFig. 4.1 Petri dishes of yeasts growing on malt extract agar, showing a streaking method suitable for producing isolatedcolonies
    • 24 4 Methods for Isolation, Enumeration and Identificationfrom side to side. After suitable incubation, well- visible – and inoculate a single point on a plate orseparated single colonies should appear in the lower slant.half of the plate (Fig. 4.1). The same procedure can be applied to mixed If all of these single colonies appear to be of cultures arising from direct plating or surface sam-similar size and appearance (taking into account pling techniques. It is advisable to keep notes on thethe effect of crowding), the culture may be judged appearance of the colony area sampled, as this willto be pure. Microscopic checks of some single colo- give an indication of whether the culture whichnies are also desirable. Disperse a needle point of grows up is the same as that intended to be isolated.cells from a colony in a drop of water, add a cover It is generally easy to isolate rapidly growingslip and examine by bright field illumination at fungi from those which grow more slowly. The out-about 400Â. Cell outlines will be clearly visible. ermost hyphal tips are usually free of contamina-Note that, unlike those of bacteria, yeast cell sizes tion. The reverse process is often much more diffi-often vary considerably in a pure preparation. Pur- cult. The isolation of slowly growing fungi in theity is indicated not so much by uniformity of cell size presence of rapidly growing ‘‘weeds’’ often requireswithin a preparation as by similarity in microscopic skill, patience and ingenuity. It is desirable to watchcell appearance from colony to colony. When a the point inocula daily over several days at least,culture is considered to be pure, streak it onto an because a particular stage in the life cycle may giveappropriate slant. some advantage. The slow colony may germinate more rapidly, or have a sector accessible to a needle, or spore more freely. The use of higher or lower incubation temperatures, or media of low aw or4.5.2 Moulds low nutrient status, or the addition of dichloran or rose bengal (see below) may all be of value in thisStreaking techniques are ineffective for filamentous process.fungi and are not recommended. Isolation depends Freeing fungi from bacteria, long considered toon picking a small sample of hyphae or spores – be a very difficult procedure, has been greatly sim-judged to be pure by eye, by hand lens or preferably plified in recent years with the advent of mediaunder the stereomicroscope – and placing this sam- containing antibiotics. With use of the mediaple on a fresh plate as a point inoculum. Purity is recommended in the next section, bacterial contam-subsequently judged by uniformity in appearance of ination of cultures should be a rare event.the colony which forms after incubation. The When a pure colony is obtained, it should beappearance of a mixed culture depends on the inoculated onto a slant of an appropriate mediumgrowth rates of the fungi present. If rates are and incubated until ready for identification. Again,diverse, a mixed culture is often indicated by a moulds should always be inoculated at a singleclump of dense hyphae at the inoculum point, sur- point, preferably near the centre of the slant. Thisrounded by looser wefts of spreading hyphae. With permits the best colony development and sporula-fungi of approximately equal growth rates, mixtures tion in most fungi.are often indicated by colonies with sectoringgrowth: sectors will show differences in mycelial,spore or reverse colours or in radial growth rates. The simplest starting place for isolating fungi is 4.5.3 Short Term Storagean enumeration plate with well-separated colonies.Use a needle of platinum or nichrome, preferably For short to medium-term storage, fungi are usuallycut to a chisel point with a pair of pliers, or a steel stored on slants, most commonly in McCartney orsewing needle. Sterilise it by heating, then plunge Universal bottles which have the advantages ofthe tip into cold agar and leave until cool – with being free standing and having caps that can benichrome or steel this will require several seconds. sealed to retard drying during storage. During incu-With the tip of the cold, wet needle pick off a few bation and until colonies are fully mature, however,spores or a tuft of mycelium – just enough to be caps must be kept loose on slants in bottles. Moulds
    • 4.6 Choosing a Suitable Medium 25require free access to oxygen for typical growth and The most important division in types of enu-sporulation. Oxygen starvation during growth will meration media lies between those suitable forat best lead to retarded sporulation or at worst high water activity foods, such as eggs, meat, vege-death of the culture. tables and dairy products, and those suited to the Long-term preservation of fungi is dealt with enumeration of fungi in dried foods. The mostlater in this chapter. suitable media for dried foods depend on the type of food, the major categories being foods low in soluble solids such as cereals, high sugar foods4.6 Choosing a Suitable Medium such as confectionery and dried fruit, and salt foods. A second consideration lies in whether theFood laboratories often rely on a single all-purpose primary interest is in moulds, or yeasts, or both;medium to produce a ‘‘yeast and mould’’ count in and a third concerns the presence or absence ofeverything from raw material to finished product. preservatives. Finally, media are available forBut just as the food bacteriologist uses selective specific mycotoxigenic fungi, notably Aspergillusmedia for particular purposes, so too food mycolo- flavus and related species and Penicillium verruco-gists are developing a range of media suited to spe- sum plus P. viridicatum.cific applications. It is plainly unrealistic to expect a An overview of media considered most suitablesingle medium to answer all questions about mould for particular purposes is given in Table 4.1. Theand yeast contamination in all foods. The fungi that table is derived from Pitt and Hocking (1997),spoil meats or fresh vegetables are not the same as together with recent recommendations fromthose that grow on dried fish. Although often used ICFM (Samson et al., 1992; Hocking et al., 2006a).for this purpose, very dilute media such as potatodextrose agar are of little or no value for enumerat-ing fungi from dried foods.Table 4.1 Recommended media for fungal detection, enumeration and isolationaType of food Selecting for Medium RemarksFresh foods: milk and milk Moulds DRBC Blend (where necessary) products, fruit, cheese, sea Yeasts TGY, MEA, OGY and dilution plate foods General DRBCFreshly harvested grains, nuts General DRBC Direct plate Dematiaceous Hyphomycetes DRBC, CZID Direct plate Fusarium CZID Direct plate Yeasts TGY, MEA, OGY Dilution plateFruit juices, fresh Yeasts TGY, MEA, OGY Dilution plateFruit juices, preserved Preservative resistant yeasts TGYA, malt acetic Dilution plate agarFruit juices, to be pasteurised, or Heat resistant moulds PDA, MEA Special protocol pasteurised productsFruit juice concentrates Xerophilic yeasts MY50G Special diluentsDried foods in general General DG18 Direct plateStored cereals, nuts General DG18 Direct plate Dematiaceous Hyphomycetes DRBC, CZID Direct plate Fusarium CZID Direct plate
    • 26 4 Methods for Isolation, Enumeration and IdentificationTable 4.1 Recommended media for fungal detection, enumeration and isolationa (continued)Type of food Selecting for Medium RemarksGrain for milling into flour General DG18 Stomach or blend and dilution plateDried fruit, confectionery, Xerophilic moulds and yeasts MY50G Direct plate chocolate, etc. Fastidious xerophiles MY50G Direct plate – in presence of Eurotium spp. MY70GF Direct plateSalt foods, e.g. salt fish General DG18 Direct plate or press plate Halophilic xerophiles MY5-12, MY10-12 Direct plate or press plateGeneral Fungi producing aflatoxins AFPA Direct or dilution plateGeneral Fungi producing ochratoxins DRYS Direct or dilution platea For medium acronyms, see Section 4.6.4.6.1 General Purpose Enumeration for the inhibition of bacteria. Such media allow Media better recovery of moribund and sensitive fungi than the acidified media commonly used in theTo be effective, a general purpose enumeration past. For many years rose bengal has been addedmedium must fulfil several requirements (Pitt, to media to slow colony spread, while more recently1986). As these are sometimes overlooked, they are 2,6-dichloro-4-nitroaniline (dichloran) has beenlisted here: added to inhibit rapidly spreading moulds. Many common spoilage fungi, Aspergillus and Penicillium to inhibit bacterial growth completely, without species in particular, develop better on media with affecting growth of foodborne fungi (filamen- adequate nutrients. Low nutrient media of very high tous or yeasts); aw, such as potato dextrose agar, have lost favour to be nutritionally adequate and support the because they are selective against some species in growth of fastidious fungi; these genera. to suppress the growth of rapidly spreading The media described below are considered to be fungi, especially the Mucorales, but not to pre- the most satisfactory general purpose enumeration vent their growth entirely, so they too can be media available at this time (Hocking et al., 2006a). enumerated; Formulations are given in the Media Appendix. to slow radial growth of all fungi, to permit Dichloran rose bengal chloramphenicol (DRBC) counting of a reasonable number of colonies agar. DRBC (King et al., 1979, Pitt and Hocking, per plate, without inhibiting spore germination; 1997) is recommended for both moulds and yeasts. to promote growth of relevant fungi; and It is particularly suited to fresh and high aw foods to suppress growth of soil fungi or others gener- (Hocking et al., 1992). This medium contains both ally irrelevant in food spoilage. rose bengal (25 mg/kg) and dichloran (2 mg/kg), Fulfilling the above requirements necessitates the which restrict colony spreading without affectinguse of potent inhibitory compounds, and there is spore germination unduly. Compact coloniessometimes a fine line between inhibition of undesir- allow crowded plates to be counted more accu-able microorganisms and suppression of growth of rately. This combination of inhibitors also effec-those being sought. Modern fungal enumeration tively restricts the rampant growth of most of themedia rely on the use of antibiotics at neutral pH common mucoraceous fungi such as Rhizopus and
    • 4.6 Choosing a Suitable Medium 27Fig. 4.2 Petri dishes of (a) DRBC and (b) RBC showing effective control of Rhizopus growth by rose bengal and dichloran inDRBCMucor (Fig. 4.2), although it does not completely identification. Eurotium species are usually identi-control some other troublesome genera such as fied on Czapek yeast extract agar with 20% sucroseTrichoderma. (CY20S), described later in this chapter. In routine use, it is recommended that DRBC Other general purpose media. Under circumstancesplates be incubated away from light at 258C for where rapidly spreading moulds do not cause pro-5 days. blems, two alternative general purpose enumeration Dichloran 18% glycerol (DG18) agar. Hocking media are satisfactory. These are rose bengal chlor-and Pitt (1980) developed DG18 to be selective for amphenicol agar (RBC; Jarvis, 1973), from whichxerophilic fungi from low moisture foods such as DRBC was developed, and oxytetracycline glucosestored grains, nuts, flour and spices. DG18 was yeast extract agar (OGY; Mossel et al., 1970). OGYdesigned for enumeration of a range of nonfasti- has been found to be very suitable for yeasts in thedious xerophilic fungi and yeasts. However, it has absence of moulds (Andrews, 1992a).been shown since that it supports growth of the Most of the media discussed above are availablecommon Aspergillus, Penicillium and Fusarium spe- in ready to use dehydrated form from media suppli-cies, as well as most yeasts, and many other com- ers such as Oxoid, Difco, BBL, etc.mon foodborne fungi. DG18 can now be described as a general purposemedium with emphasis on enumeration of fungi fromdried foods. DG18 is also recognised to be a very 4.6.2 Selective Isolation Mediauseful medium for enumeration of airborne fungi(Wu et al., 2000; Samson et al., 2004a). It is an effec- Although considerable progress has been made intive inhibitor of Mucoraceous fungi, and bacteria are the past 20 years, the formulation of selective mediatotally suppressed. However, growth of Eurotium for foodborne fungi still requires a great deal ofspecies (previously known as ‘‘the Aspergillus glaucus research. The availability of effective media cangroup’’) is still somewhat too rapid, and colonies may greatly simplify the isolation and identification ofhave diffuse margins. The addition of detergent to significant food spoilage and mycotoxigenic fungi.DG18 has been reported to be an improvement in Most attention has been paid so far to the require-this respect (Beuchat and de Daza, 1992). ments of xerophilic fungi because of their failure to Although DG18 is a satisfactory isolation med- develop on standard high aw media. For mycotoxi-ium for Eurotium species, it is not suitable for their genic fungi, satisfactory media exist only for
    • 28 4 Methods for Isolation, Enumeration and IdentificationAspergillus flavus and closely related species, Penicil- Coconut Cream agar (CCA) to detect aflatoxin pro-lium verrucosum and P. viridicatum, and the genus duction in Aspergillus flavus and A. parasiticus. CCAFusarium. These media are considered below. can be made using any brand of commercial canned Media for Aspergillus flavus and related species. coconut cream (available from Asian food stores inBothast and Fennell (1974) developed Aspergillus many places). Dilute 50:50 with water, add agardifferential medium after finding that Aspergillus (1.5%) and autoclave. Inoculate solidified plates withflavus and A. parasiticus produced conspicuous, up to four colonies (picked with a wet needle) anddiagnostic orange yellow colours in the colony incubate at 308C for 5–7 days. Examine, reversereverses in the presence of an appropriate nitrogen upmost, under long wave length UV light. Coloniessource and ferric salts. Few other fungi produced a producing aflatoxins will fluoresce bluish white orsimilar colouration. It was later shown that these white, especially in the centres. Ignore fluorescencespecies produce aspergillic acid or noraspergillic from the Petri dish itself. Use an uninoculated coconutacid, which react with ferric ammonium citrate to cream agar plate as a control (Dyer and McCammon,form the colour complex (Assante et al., 1981). 1994). Plates inoculated with known nontoxigenic andHowever, development of the orange yellow reverse toxigenic strains are also useful controls. A. parasiticuspigment is not indicative of aflatoxin production. isolates almost always produce aflatoxins. This medium was reformulated by Pitt et al. Media for fungi producing ochratoxin A. Although(1983) to produce Aspergillus flavus and parasiticus ochratoxin A was first described from Aspergillusagar (AFPA). As well as more effective concentra- ochraceus, recent molecular studies indicate that A.tions of the active ingredients, AFPA contains westerdijkiae is the major ochratoxin A producingdichloran and chloramphenicol to inhibit spreading species in Aspergillus Section Circumdati (Frisvadfungi and bacteria, respectively. et al., 2004). Aspergillus carbonarius is also an impor- When incubated at 308C for 42–48 h, colonies of tant source of ochratoxin A, particularly in grapeAspergillus flavus, A. parasiticus and A. nomius are products (Abarca et al., 2004; Leong et al., 2006adistinguished by bright orange yellow reverse col- and references therein). However, there are no selec-ours. Only A. niger can be a source of error: it grows tive or indicative media for these fungi. When DG18 isas rapidly as A. flavus and sometimes produces a used as the isolation medium, selection of coloniesyellow, but not orange, reverse. After 48 h A. niger with light brown (ochre) or black sporulation is acolonies begin production of their diagnostic black good starting point for detecting A. westerdjikiae oror dark brown heads, which provide a ready dis- A. carbonarius respectively. Coconut cream agartinction from A. flavus. Prolonged incubation of (Dyer and McCammon, 1994) can also be used toAFPA, beyond 4 days, is not recommended because screen for ochratoxin production in A. carbonariusA. ochraceus and closely related species may also and species in Aspergillus Section Circumdati (Heenanproduce a yellow reverse after this time. AFPA is et al., 1998). Plates are best incubated at 258C ratherrecommended for the detection and enumeration of than 308C for potentially ochratoxigenic species ofpotentially aflatoxigenic fungi in nuts, maize, spices Aspergillus.and other commodities. Its advantages include Penicillium verrucosum is the most importantrapidity, as 48 h incubation is usually sufficient, ochratoxin a producer in the genus Penicilliumspecificity, and simplicity, as little skill is required (Pitt, 1987), although P. nordicum can also producein interpreting results. In consequence, it can be a this mycotoxin (Frisvad and Samson, 2004). Fris-simple, routine guide to possible aflatoxin contam- vad (1983) developed Dichloran rose bengal yeastination (Pitt, 1984). This medium is also very effec- extract sucrose agar (DRYS) for selective enumera-tive for enumerating A. flavus in soils, where levels tion of P. verrucosum. P. nordicum, P. viridicatumdown to 5 spores per gram can be detected. Where and P. aurantiogriseum are also selected by DRYS.soils carry a heavy bacterial load, doubling the P. verrucosum and P. nordicum produce ochratoxinquantity of chloramphenicol or adding other anti- A, P. verrucosum also produces citrinin, while thebiotics may be of value. latter two species produce xanthomegnin and vio- Detection of aflatoxin production using Coconut mellein. According to Frisvad (1983), P. verrucosumCream Agar. Dyer and McCammon (1994) developed colonies on DRYS have a violet brown reverse, and
    • 4.6 Choosing a Suitable Medium 29the latter two species produce yellow colonies with a Bragulat et al. (2004) compared the efficacy of MGAyellow reverse. The incubation regimen recommended 2.5 medium with DCPA, CZID and several otherby Frisvad (1983) is 7–8 days at 208C. Subsequently, Fusarium media, using pure cultures of twelve FusariumFrisvad et al. (1992) developed dichloran yeast extract species commonly found in foods as well as naturallysucrose 18% glycerol agar (DYSG) on which P. ver- contaminated samples. They reported that there wasrucosum produces a red brown reverse. Because of its no statistically significant difference in colony counts oflower aw, DYSG inhibits rapidly growing fungi such the Fusarium spp. tested, but that colonies on MGA 2.5as Rhizopus and Mucor more effectively than were smaller than the other media. MGA 2.5 didDRYES. In a study of 76 samples of wheat, rye and not allow growth of other fungi such as Zygomycetesbarley, Lund and Frisvad (2003) found that DYSG and yeasts from naturally contaminated samples, thuswas more effective than DRYES for screening grain providing better selectivity than the other media.samples for the presence of P. verrucosum and poten- Media for dematiaceous Hyphomycetes. Althoughtial ochratoxin A contamination. designed primarily for Fusarium isolation, CZID has Media for Fusarium species. Dichloran chloram- been found in our laboratory to be very useful forphenicol peptone agar (DCPA; Andrews and Pitt, isolating dematiaceous Hyphomycetes, provided1986) can be used to isolate Fusarium species from that iprodione is added at half the usual concentra-grains and other substrates. The medium was devel- tion, as full strength iprodione tends to restrictoped from Nash-Snyder medium (Nash and Snyder, colony diameters too severely. Alternaria, Bipolaris,1962) a medium for enumeration of Fusarium species Curvularia, Stemphylium and Ulocladium species willfrom soils. DCPA uses a low level of dichloran as a grow and sporulate well when incubated for 5 dayssubstitute for the high level of pentachloronitroben- at 258C with a 12 h photoperiod. Drechslera specieszene (PCNB), and chloramphenicol rather than the will grow but will not sporulate on this medium.antibiotic mixture used by Nash and Snyder (1962). DRBC is also of value for isolating these fungi, When Fusarium species are dominant, DCPA is but some species do not sporulate readily on it.effective for their isolation from grains, animal feeds DCPA, originally developed for isolation ofand soil. However, DCPA has been found to be less Fusarium, may also be used as an isolation mediumeffective in mixed populations. DCPA is a very useful for the dematiaceous Hyphomycete genera men-medium for the identification of Fusarium species tioned above. Alternaria, Curvularia and relatedbecause it often induces the abundant formation of genera usually grow rapidly and sporulate well onmacroconidia (Hocking and Andrews, 1987). DCPA. However, fungi should not be maintained A much more stringent medium, effective for iso- or stored on DCPA for more than two weeks, aslation of most Fusarium species occurring in foods, is ammonia is produced by aging cultures.Czapek iprodione dichloran agar (CZID; Abildgren Media for xerophilic fungi. Xerophilic fungi are ofet al., 1987). As well as dichloran, this medium con- great importance in food spoilage, and hence mediatains the fungicide iprodione. CZID is highly selec- and techniques for their enumeration and isolationtive for Fusarium species, and is probably the best have received much attention. Xerophiles range frommedium for Fusarium enumeration and isolation. those which grow readily on normal media andCZID is suitable for isolation of Fusarium species which are only marginally xerophilic, such as manyby direct plating of surface disinfected grains and Aspergillus and Penicillium species, to those, such asother commodities, or dilution plating of homoge- Xeromyces bisporus, which will not grow at all onneous samples such as flour. Some questions remain normal (high aw) media. It is not surprising that noconcerning whether CZID may be too selective and single medium is suitable for quantitative estimationnot support growth of all foodborne Fusarium spe- of all xerophilic fungi found causing food spoilage.cies. However, the common species all grow well. As noted earlier, DG18 was developed as a general ´ Castella et al. (1997) developed a Fusarium selective medium for xerophiles, and remains the medium ofmedium (MGA 2.5) using 2.5 mg/L malachite green as choice for this purpose. DG18 should be used in anythe selective agent. They reported that MGA 2.5 was general examination of the mycoflora of dried foods.more selective than Nash-Snyder medium as it did not Media for extreme xerophiles. Fungi discussedallow development of colonies of other fungal genera. here include Xeromyces bisporus, xerophilic
    • 30 4 Methods for Isolation, Enumeration and IdentificationChrysosporium species, Eremascus species, Eurotium The most satisfactory medium is malt extract yeasthalophilicum and the halophilic xerophiles Polypae- extract 70% glucose fructose agar (MY70GF).cilum pisce and Basipetospora halophila. These MY70GF is of similar composition to MY50G,extreme xerophiles grow slowly if at all on DG18 except that it contains equal parts of glucose andand are quickly overgrown by rapidly spreading fructose to prevent crystallisation of the medium atxerophiles such as common Eurotium species. Such the concentration used (70% w/w). It is made in aspecies require special media and techniques. similar manner to MY50G. Since the early 1950s, studies on extremely Growth of even the extreme xerophiles onxerophilic fungi have been carried out in our labora- MY70GF is extremely slow, and plates should betory (Scott, 1957). A very wide variety of media incubated for at least 4 weeks at 258C. Once growthhave been used for their cultivation. The most effec- is apparent, pick off small portions of colonies andtive of these, suitable for all except the halophilic transfer them to MY50G, to allow more rapidxerophiles, is malt extract yeast extract 50% glucose growth and sporulation.agar (MY50G; Pitt and Hocking, 1997). Media for halophilic xerophiles. Some xerophilic Isolation techniques for extreme xerophiles. As fungi from salted foods such as salt fish grow morewell as special media, extreme xerophiles require rapidly on media containing NaCl and hence arespecial techniques for isolation. If a low aw com- correctly termed halophilic xerophiles. Malt extractmodity such as ground spice, dried fruit, fruit cake, yeast extract 5% salt 12% glucose agar (MY5-12)confectionery or dried fish shows signs of white and malt extract yeast extract 10% salt 12% glucosemould growth, it is likely that the fungus responsi- agar (MY10-12) are suitable for these fungi. Tech-ble is an extreme xerophile. Fungi of this kind are niques for enumeration and isolation are similar tousually sensitive to diluents of high aw and often those described above for other extreme xerophiles.cannot be isolated by dilution plating. For directplating, a convenient technique is to place smallpieces of sample, without surface disinfection, 4.6.3 Techniques for Yeastsonto a rich, low aw medium, such as MY50G. Alter-natively, examine the food under the stereomicro- The simplest enumeration and growth medium forscope, which will often provide useful information most food spoilage yeasts is malt extract agarin any case, and use an inoculating needle to pick off (MEA). Although originally introduced as a growthpieces of mycelium or spores from the surface of the medium for moulds, its rich nutritional statusspoiled food. Place these pieces (three to six per makes it very suitable for yeasts, and its relativelyplate) directly onto MY50G. Colonies should low pH (usually near 5.0) reduces the possibility ofdevelop after 1–3 weeks incubation at 258C. Quite bacterial contamination.frequently extreme xerophiles will be present in pure More recently, tryptone glucose yeast extractculture and can be readily isolated. Examination agar (TGY) has been recommended for enumera-with the stereomicroscope will usually indicate tion of yeasts (Hocking et al., 1992, 2006a). Due to awhether this is the case. Selection of growth which higher glucose concentration (10%) and higher pHappears to cover the range of types seen will assist (5.5–6.0), this medium is more effective in recoveryisolation of the principal fungi present. of stressed yeast cells, and colony development is Direct sampling by the press plating technique usually faster than on MEA. However, its higherdescribed earlier in this chapter can be useful for pH means that an antibiotic should be incorporatedisolating xerophiles. for enumeration of yeasts from food samples which Sometimes extreme xerophiles are accompanied may contain bacteria. Recommended antibioticsby Eurotium species, also capable of growth at very are chloramphenicol or oxytetracycline at the con-low aw. The latter are identifiable under the stereo- centrations used in DRBC and OGY.microscope by their Aspergillus heads. Under these Both MEA and TGY are suitable for enumera-circumstances, isolation of extreme xerophiles is tion of yeasts in products such as fruit juices, fruitmore difficult. A medium of sufficiently low aw to purees and yoghurt, where moulds are usually pre-slow the growth of Eurotium species is necessary. sent only in low numbers.
    • 4.6 Choosing a Suitable Medium 31 If large numbers of moulds are present, often the and replace it with sterile water. Leave the cap loose,case with solid products such as cheese, a general incubate at room temperature or 308C and watch forpurpose enumeration medium such as DRBC or evidence of fermentation. Shaking the container dailyDG18 should be used. will help to detect gases resulting from fermentation. For circumstances where yeast enumeration Classical enrichment techniques used in bacterio-remains difficult on media such as DRBC, a valu- logy can also be used for yeasts. TGY broth has beenable technique is that of de Jong and Put (1980), used very successfully in our laboratory for enrichmentwhich involves a 3-day anaerobic incubation. They of low numbers of yeasts in liquid products. Add 10 mlrecommended using pour plates or streak plates of of product to 90 ml TGY broth and incubate at 258Cmycophil agar (BBL Microbiological Systems, for 3–4 days, or 308C for 2–3 days. Look for signs ofCockeysville, Maryland, USA), but malt extract fermentation and streak out onto TGY agar.agar should be equally suitable. Antibiotics may Detection of preservative resistant yeasts. A fewbe added if necessary. Plates are incubated at 258C species of yeasts are able to grow in products contain-for 3 days in an anaerobic jar, then taken out and ing preservatives such as sorbic, benzoic and aceticincubated aerobically for another 2 days at 258C. As acids or sulphur dioxide. The most important of thesemany spoilage yeasts are able to grow anaerobically, is Zygosaccharomyces bailii. The simplest and mostcolonies will commence development without the effective way to screen for preservative resistant yeastspossibility of overgrowth by moulds. This technique is to spread or streak product onto plates of maltis unlikely to be suited to the enumeration of yeasts acetic agar (MAA), which is MEA with 0.5% aceticon surfaces of fresh fruit or vegetables, as yeasts acid added (Pitt and Richardson, 1973) or TGY withwhich colonise these surfaces are often strict aerobes. 0.5% acetic acid added (TGYA, Hocking, 1996). Enrichment for yeasts in liquid products. In liquid MAA and TGYA are made by adding glacialfood products, low numbers of yeasts may be diffi- (16 N) acetic acid to melted and tempered basalcult to detect, but may have a serious potential to medium to give a final concentration of 0.5%. Mixcause spoilage. Enrichment techniques are the only and pour immediately. These media cannot be heldsatisfactory way of monitoring products in these molten for long periods or remelted because of theircircumstances. low pH (approximately 3.2 and 3.8 for MAA and For products or raw materials free from sus- TGYA, respectively). The acetic acid does not needpended solids and of low viscosity, standard mem- sterilisation before use.brane filtration techniques are a satisfactory MAA and TGYA are suitable media for moni-method for detecting low numbers of yeasts. The toring raw materials, process lines and productsfilter can be placed directly onto a suitable medium containing preservatives for resistant yeasts. Theysuch as MEA or TGY and staining can be carried are also effective for testing previously isolatedout subsequently. Centrifugation may also be used, yeasts for preservative resistance.but has the disadvantage that only relatively small Erickson (1993) developed a selective medium forvolumes of product can be screened. Zygosaccharomyces bailii. Zygosaccharomyces bailii If products or raw materials are viscous, of low medium (ZBM) is based on Sabouraud dextrose agaraw, or contain pulps and cannot be filtered efficiently, amended with fructose, NaCl, tryptone, yeast extractother enrichment techniques are needed. In many and trypan blue dye, then acetic acid (0.5%) andcases, the best enrichment medium is the product potassium sorbate (0.01%) are added to make theitself, diluted 1:1 with sterile water. A 1:1 dilution medium selective. It is designed as a plating mediumincreases the aw of juice concentrates or honey to a for detection of Z. bailii in acidified ingredients inlevel which will allow growth of potential spoilage conjunction with hydrophobic grid membrane filtra-yeasts without causing a lethal osmotic shock to the tion (Erickson, 1993).cells. If the product contains preservative, dilution When compared with MAA and TGYA inwill lower the concentration and permit cells to grow. an interlaboratory study, ZBM was found to be highly To detect low numbers of spoilage yeasts in cor- selective for Zygosaccharomyces bailii, to the exclu-dials, fruit juice concentrates and similar materials, sion of other important preservative resistant yeastssimply decant half the product from the container such as Schizosaccharomyces pombe and Pichia
    • 32 4 Methods for Isolation, Enumeration and Identificationmembranaefaciens. In addition, recovery of Z. bailii system compared with 10% in TGY. Yeasts whichcells sublethally injured by lyophilisation was signi- are unable to grow in the presence of acetic acid orficantly lower on ZBM than on TGYA or MAA other weak acid preservatives (sorbic or benzoic(Hocking, 1996). Its high selectivity and complex for- acids and their salts) are not detected by this method.mulation make ZBM unsuitable for routine laboratoryuse as a medium for detection of preservative resistantyeasts. Enrichment of preservative resistant yeasts. A 4.6.4 Techniques for Heat-Resistanttechnique capable of detecting yeast numbers as low Fungias 1 cfu/ml within 4 days has been developed in ourlaboratory (Hocking et al., 1996). This method is parti- Heat resistant spoilage fungi, such as Byssochlamys,cularly suitable for the detection of Zygosaccharomyces Talaromyces, Neosartorya and Eupenicillium speciesbailii in raw materials or finished product, but can also can be selectively isolated from fruit juices, pulpsbe used for the detection of Schizosaccharomyces and concentrates by laboratory pasteurisation usingpombe, Pichia membranaefaciens and other species of various methods (Beuchat and Rice, 1979; Hockingpreservative resistant yeasts. The method involves a 2- and Pitt, 1984; Beuchat and Pitt, 2001; Houbrakento 3-day enrichment step followed by a plating step and Samson, 2006). Three methods are describedwith a further 2 days of incubation. Triplicate 20 ml here: a plating method based on that of Murdock andtryptone glucose yeast extract (TGY) broths containing Hatcher (1978), a direct incubation method and a fil-0.5% acetic acid (TGYA) are each inoculated with 1 g tration method for liquid samples such as liquid sugar.or 1 ml of product and the broths incubated at 308C. Plating method. If the sample to be tested is moreAfter incubation for 48 and 72 h, 0.1 ml from each concentrated than 358Brix, it should first be dilutedbroth is spread plated onto TGY agar containing 0.5% 1:1 with 0.1% peptone or similar diluent. For veryacetic acid and the plates incubated at 308C. acid juices such as passionfruit, normally about pH The detection time of the method is shortened by 2.0, the pH should be adjusted to 3.5–4.0. Two 50 mlincubating broths and plates at 308C rather than the samples are taken for examination. Erlenmeyertraditional temperature of 258C, as the optimum flasks (250 ml) or polyethylene Stomacher bagsgrowth temperature for Zygosaccharomyces bailii may be used as heat penetration into these contain-is 30–328C (Jermini and Schmidt-Lorenz, 1987b). ers will be rapid. If using Stomacher bags, the topsThe sensitivity of the method is greatly increased by should be heat sealed. If a heat sealer is not avail-using triplicate broths instead of single or duplicate able, the tops may be folded over and secured with abroths and by spread plating 0.1 ml from each broth clip and should not be fully immersed. The twoinstead of streaking a loopful onto TGYA agar. samples are heated in a closed water bath at 808C This method has been used to detect low num- for 30 min, then rapidly cooled. Each 50 ml samplebers of cells of Zygosaccharomyces bailii in experi- is then mixed with an equal volume of doublementally inoculated cordial syrup, mayonnaise, strength MEA distributed over four 150 mm Petrisalad dressing and barbecue sauce and other preser- dishes. The Petri dishes are loosely sealed in a plasticvative-resistant yeasts such as Schizosaccharomyces bag to prevent drying and incubated at 308C for uppombe, Pichia membranaefaciens and some to 30 days. Plates are examined weekly for growth.preservative resistant strains of Saccharomyces Most moulds will produce visible colonies withincerevisiae. Yeasts of intermediate preservative 10 days, but incubation for up to 30 days will allowresistance (e.g. Debaryomyces hansenii, Candida for the possible presence of badly heat damagedkrusei and Torulaspora delbrueckii) can also be spores, which may germinate very slowly. This longdetected by this method. Better recoveries were incubation time also allows most moulds to matureobtained using TGY than a malt extract agar and and sporulate, aiding their identification.broth system, possibly due to the fact that the pH of The main problem associated with this techniqueTGY broth + 0.5% acetic acid is 3.8, compared with is the possibility of aerial contamination of the platespH 3.2 for MEA + 0.5% acetic acid, and there is a with common mould spores, which will give falselower concentration of glucose (2%) in the ME positive results. The growth of green Penicillium
    • 4.6 Choosing a Suitable Medium 33colonies, or colonies of common Aspergillus species antibiotics to each plate, mix the agar and sample well,such as A. flavus and A. niger, is a clear indication of then let the plates solidify. Incubate at 308C for up tocontamination as these fungi are not heat resistant. 30 days, examining weekly. Count colonies and reportTo minimise this problem, plates should be poured count per 100 g. This method was developed by BCNin clean, still air or a Class 2 biohazard cabinet if Research Laboratories, Rockford, Tennessee, USA.possible. If a product contains large numbers of heatresistant bacterial spores (e.g. Bacillus species), anti-biotics can be added to the agar. The addition of 4.6.5 Other Plating Techniqueschloramphenicol (100 mg/l of medium) will preventthe growth of these bacteria. Three other techniques which were developed Direct incubation method. A more direct method for counting bacteria have been applied to fungalused for screening fruit pulps and other semisolid enumeration.materials avoids the problems of aerial contamina- Spiral plate count. Zipkes et al. (1981) evaluatedtion. Place approximately 30 ml of pulp in each of the application of the spiral plate procedure to thethree or more flat bottles such as 100 ml medicine enumeration of yeasts and moulds. They comparedflats. Heat the bottles in the upright position for this procedure with the traditional pour plate and30 min at 808C and cool, as described previously. streak plate methods and found that spiral platingThe bottles of pulp can then be incubated directly, gave a higher overall recovery and lower replicatewithout opening and without the addition of agar. plating errors than the other two methods. TheThey should be incubated flat, allowing as large a medium they used was potato dextrose agar, butsurface area as possible, for up to 30 days at 308C. the technique should be no less efficient using theAny mould colonies which develop will need to be media recommended here. Automation of spiralsubcultured onto a suitable medium for identification. plate counting was studied by Manninen et al.If containers such as Roux bottles are available, larger (1991). They compared counts of pure bacterial,samples can be examined by this technique, but heat- yeast and mould cultures using standard platinging times must be increased. Bottle contents should methods and spiral plate counts determinedreach at least 758C for 20 min when checked by a both manually and with a laser colony detector.thermometer suspended near the centre of the pulp. They concluded that counts were not significantly For further details of the above methods see different except where large colonies (10–15 mmHocking and Pitt (1984) or Beuchat and Pitt (2001). diameter) of Rhizopus oligosporus were enumerated. Filtration method for liquid sugars. This method Other Rhizopus and Mucor species are also likely topermits the detection of very low numbers of cells in interfere with this method unless a suitable platingclear liquids such as liquid sugar. Sample size should medium (such as DRBC) is used. Alonso-Callejabe at least 100 g, taken after vigorously shaking the et al. (2002) found that the spiral plate techniquecontainer from which the sample is drawn. Add compared well with standard plate counting on100 ml diluent (0.1% aqueous peptone) to 2 Â 50 g OGYE agar for enumeration of yeasts and mouldssamples and mix well to dissolve. Filter both samples in goat’s milk cheeses; however, Garcı´ a-Armestosequentially through the same sterile 0.45 mm mem- et al. (2002) found the method unsuitable forbrane filter. After both samples have passed through enumeration of yeasts in raw ewe’s milk.the filter, rinse the interior of the funnel with 3 Â 20– Hydrophobic grid membrane filters. Membrane30 ml volumes of sterile diluent. Remove the filter filters overprinted with a square hydrophobic gridfrom the filter holder using sterile forceps and place have been developed for rapid enumeration ofit in a sterile bottle or Stomacher bag. Add 10 ml bacteria. The hydrophobic grid membrane filter TMdiluent to the bottle or bag containing the filter and (HGMF) system is marketed as ISO-Grid byplace in a water bath at 758C for 30 min. Ensure the Neogen (Lansing, MI, USA). The HGMF ‘‘count’’sample is submerged in the water bath (weigh down if is determined by a most probable number (MPN)necessary). Cool rapidly to room temperature, shake calculation. Brodsky et al. (1982) applied thewell, then divide the 10 ml of diluent between three HGMF technique to counting yeasts and mouldsPetri dishes. Add a generous portion of MEA with in foods. They compared it with spread plating on
    • 34 4 Methods for Isolation, Enumeration and Identificationpotato dextrose agar amended with antibiotics and 4.7 Estimation of Fungal Biomassfound that the HGMF technique produced highercounts in 2 days than the traditional method did A deficiency in all of the enumeration techniquesafter 5 days. The HGMF method could find use for which rely on culturing fungi is that the result is atevaluating the quality of raw materials before incor- best poorly correlated with growth or biomass.poration into a product. For example, Erickson Biomass is usually regarded as the fundamental(1993) has proposed a method using HGMF in measure of fungal growth in biotechnology, but itconjunction with a selective medium for detection is not easy to quantify under the conditions exist-of the preservative resistant yeast Zygosaccharo- ing in foods. Mycelial dry weight is most com-myces bailii in acidified ingredients. The HGMF monly used as a biomass estimate, but its relation-method was collaboratively trialled by 20 labora- ship to mycelial wet weight and to metabolismtories using 6 naturally contaminated foods. varies widely in foods, due to the great influenceAlthough there were some differences between of aw on both of the latter parameters. Fungicounts obtained by HGMF and the reference growing at reduced aw can be expected to bemethod (5 day pour plate), the differences were more dense than at high aw due to increased con-not significant, and the HGMF method was centrations of internal solutes, though this isadopted by the AOAC International (Entis, 1996). exceptionally difficult to measure experimentally.Spangenberg and Ingham (2000) found that the The question of a satisfactory fundamental mea-HGMF method gave equivalent results to DRBC sure of fungal biomass remains unanswered.for enumeration of yeasts and moulds in grated low Despite these basic problems, several chemicalmoisture Mozzarella cheese. and biochemical techniques are available to esti- Use of HGMF requires special holders for the mate the extent of fungal growth in commodities.square membrane filters, and an automated counting These techniques rely either on some unique com-system is necessary to take full advantage of the ponent of the fungus that is not found in othermethod. The number of colonies on an HGMF can microorganisms or foods or on immunological orbe counted visually, but it is a relatively slow process. molecular techniques. Some are still in the develop- TM Petrifilm Yeast and Mould. Petrifilm YM (3 M mental phase: the most important ones areCompany, St. Paul, MN, USA) is a proprietary sys- described briefly here.tem for enumerating fungi on a layer of mediumenclosed in a plastic film, which eliminates the use ofPetri dishes. A collaborative study carried out by theAOAC (Knight et al., 1997) concluded that Petrifilm 4.7.1 ChitinYM gave comparable results with the BAM method(USFDA, 1992) for yeast and mould counts in five Chitin is a polymer of N-acetyl-D-glucosamine anddifferent food types inoculated with a cocktail of sev- is a major constituent of the walls of fungal sporeseral species of Aspergillus and Penicillium and two and mycelium. It also occurs in the exoskeleton ofspecies of yeast. Consequently, Petrifilm YM was insects but is not present in bacteria or in foods.adopted first action by AOAC International. Other Hence the chitin content of a food or raw materialworkers (Beuchat et al., 1990, 2007; Taniwaki et al., can provide an estimate of fungal contamination.2001b; Ferrati et al., 2005) have shown that Petrifilm Chitin is most effectively assayed by the methodYM performed satisfactorily compared with conven- of Ride and Drysdale (1972). Alkaline hydrolysis oftional cultural methods when tested on a range of the food sample at 1308C causes partial depolymer-foods. However, Petrifilm was not particularly effec- isation of chitin to produce chitosan. Treatmenttive in inhibiting the growth of spreading moulds such with nitrous acid then causes partial solubilisationas Rhizopus and Mucor. In addition, subculturing and deamination of glucosamine residues to pro-colonies for identification was more difficult from duce 2,5-anhydromannose, which is estimated col-Petrifilm YM than from traditional Petri dishes. Pet- orimetrically using 3-methyl-2-benzothiazolonerifilm YM has a high aw and we do not recommend it hydrazone hydrochloride as the principal reagent.for analysis of foods of less than 0.95 aw. Alkaline hydrolysis is more readily accomplished at
    • 4.7 Estimation of Fungal Biomass 351218C in an autoclave (Jarvis, 1977). Improved assay 4.7.2 Ergosterolsensitivity was achieved by derivatisation of glucosa-mine and other products with o-phthalaldehyde, Ergosterol is the major sterol produced by fungi, butseparation by high performance liquid chromatogra- at most is a minor component of plant sterols (Weete,phy and detection of fluorescent compounds with 1974). Ergosterol occurs as a component of fungal cella spectrofluorimeter (Lin and Cousin, 1985). Ekblad membranes, so is inherently likely to be correlatedand Nasholm (1996) also described an HPLC method with hyphal growth and biomass. It is therefore awhich measured fluorescence of a 9-fluorenylmethyl- good candidate as a chemical for measuring fungalchloroformate derivative of glucosamine. The growth in foods and raw materials. Methodology forchitin assay remains rather complex and slow, usually estimating ergosterol in cereals was developed by Seitzrequiring about 5 h. et al. (1977, 1979). Samples are blended with metha- A number of studies have indicated that the nol, saponified with strong alkali, extracted with pet-chitin assay is a valuable technique for estimating roleum ether and fractionated by high pressure liquidthe extent of fungal invasion in foods such as maize chromatography. Ergosterol is detected by ultravioletand soybeans (Donald and Mirocha, 1977), wheat absorption, optimally at 282 nm, a wavelength at(Nandi, 1978) and barley (Whipps and Lewis, 1980) which other sterols exhibit little or no absorbance.to estimate mycorrhizal fungi and fungal pathogens For refinements to this methodology, see Newellin plant material and soil (Ekblad and Nasholm, et al. (1988). Liquid chromatography or gas chroma-1996; Ekblad et al., 1998; Penman et al., 2000; tography with mass spectrometric detection (LC-MS;Singh, 2005) and measure wood rotting fungi GC-MS) have also been used to estimate ergosterol(Nilsson and Bjurman, 1998). Particular attention (Headley et al., 2002; Dong et al., 2006; Varga et al.,has been paid to the possibility of developing the 2006). GC-MS in combination with non-chitin assay as a replacement for the Howard mould discriminating flash pyrolysis (Py-GC-MS) providescount for tomato products (Jarvis, 1977; Bishop the advantages of little sample preparation and smallet al., 1982; Cousin et al., 1984). sample size (Parsi and Gorecki, 2006). The chitin assay has some shortcomings and has The ergosterol assay provides a useful methodbeen severely criticised by some authors (e.g. for quantifying fungal growth, so numerous studiesSharma et al., 1977). The relationship between dry have assessed the relationship between fungalweight and chitin content varies at least twofold for growth and ergosterol production, using both liquiddifferent food spoilage fungi (Cousin et al., 1984; and solid substrates.Lin and Cousin, 1985; Cousin, 1996). Some foods Using liquid cultures, Zill et al. (1988) showed acontain naturally occurring amino sugars such as correlation between ergosterol production, mycelialglucosamine and galactosamine, which should be wet weight and mycelial protein in Fusariumremoved by acetone extraction prior to hydrolysis graminearum. Matcham et al. (1985) reported that(Whipps and Lewis, 1980). Products from rot-free ergosterol correlated better with mycelial dry weighttomatoes give positive glucosamine assays even than chitin or laccase, a polyphenol oxidase. Varia-after acetone extraction (Cousin et al., 1984) and tion exists, however. Torres et al. (1992) reported thatchitin content does not increase proportionally with Aspergillus ochraceus grown in liquid culture showedfungal growth (Sharma et al., 1977). Insect contam- a threefold increase in ergosterol concentration inination of grain samples has been reported to relation to mycelial dry weight as the culture agedproduce grossly misleading results (Sharma et al., from 2 to 26 days. Other reports (Matcham et al.,1977), but the presence of fruit flies in tomato- 1985; Newell et al., 1987; Seitz et al., 1979) havebased products was less serious (Lin and Cousin, indicated smaller variations, only one to twofold.1985). Materials such as stored grains frequently Marfleet et al. (1991) showed that fungal biomasscontain insect fragments and need to be checked and ergosterol levels were correlated for three repre-before chitin assays are attempted. Because of these sentative fungal species over a range of aw on soliddifficulties, the use of chitin as a chemical assay for substrates but not in liquid media. Nout et al. (1987)fungi in foods has largely been superseded by the showed that the ergosterol content of Rhizopusergosterol assay. oligosporus varied widely, from 2 to 24 mg/mg
    • 36 4 Methods for Isolation, Enumeration and Identificationmycelial mass, and varied with substrate, aeration better on DG18 (r = 0.77) than on MEA (r = 0.69).and growth phase. The ergosterol content was low Ergosterol levels of food grade wheat ranged fromduring the rapid growth phase but tended to increase, 2.4 to 2.8 mg/g dry weight, samples from field trialsat times sharply, as growth slowed. Taniwaki (1995) (of unspecified quality) from 3.0 to 5.6 mg/g and feeddemonstrated that in fungi growing in atmospheres grains from 8 to 15 mg/g dry weight.low in O2 and with elevated CO2 levels, the ergosterol After an extensive survey of ergosterol levels incontent of hyphae was significantly reduced. In Danish crops, Hansen and Pedersen (1991) con-atmospheres containing 60% CO2, ergosterol con- cluded that the normal levels of ergosterol in barleytent per unit of hyphal length was up to six times less were 7.6 – 2.8, wheat for bread making 5.0 – 1.5, ryethan in air. Growth medium also affected ergosterol for bread making 6.8 – 2.2, peas 2.2 – 2.7 and rape-concentrations: on average, seven foodborne fungal seed 2.4 – 1.3 mg/g dry weight. Ochratoxin A inspecies grown on PDA produced more than twice as barley correlated well with ergosterol content andmuch ergosterol per unit hyphal length as when reached significant levels when ergosterol increasedgrown on CYA (Taniwaki, 1995; Taniwaki et al., to 25 mg/g dry weight. However, aflatoxin B1 became2006). However, there was a reasonable correlation detectable in cottonseed meal when ergosterolbetween ergosterol and mycelium dry weight for reached only 4 mg/g. ‘‘Burned’’ rapeseed, a measureseven of the eight species tested. Eurotium chevalieri of quality, became significant when ergosterolwas the exception: this species produced little ergos- reached 1.4 mg/g dry weight. Lamper et al. (2000)terol and appeared to produce several other sterols found that ergosterol content correlated well (r =(Taniwaki et al., 2006). Marı´ n et al. (2005) examined 0.87) with deoxynivalenol levels in wheat inoculated16 species of food spoilage fungi and concluded that with Fusarium graminearum or F. culmorum. Moraesergosterol content and colony diameters were better et al. (2003) found that there was good correlationcorrelated to fungal biomass than fungal counts were. between mould counts and ergosterol content ofMarı´ n et al. (2008) showed that, for 14 common food Brazilian maize (r = 0.94) but a poor correlation (rspoilage fungi, correlation coefficients between ergos- = 0.4) between ergosterol and aflatoxin content.terol and colony diameters were sufficiently significant Pietri et al. (2004) found significant correlationover a range of aw values (0.95–0.85), pHs (5–7) and between ergosterol content of Italian maize and thepotassium sorbate concentrations (0.5–1.5%) for major mycotoxins, fumonisin B1 (1995 crop) or zear-both parameters to be useful in growth modelling. alenone and deoxynivalenol (1996 crop). Ergosterol Quantifying ergosterol production in foods has content correlated strongly with fat acidity valuesproved more difficult. Seitz et al. (1977) showed a and germination ability of stored canola (Pronykgood correlation between damage in rice grains and et al., 2006). These authors also noted that Penicil-their ergosterol content, between ergosterol in lium and Aspergillus species contributed more towheat and rainfall during the growing season and ergosterol than Eurotium species. Ergosterol levelsbetween ergosterol content and fungal invasion in in sound canola were between 1.46 and 1.67 mg/g,several sorghum hybrids. Matcham et al. (1985) whereas levels above 2 mg/g indicated significantreported good correlations between linear extension levels of spoilage (Pronyk et al., 2006).of Agaricus bisporus grown on rice grains and chitin, Karaca and Nas (2006) examined ergosterol con-ergosterol and laccase production. Ergosterol con- tent of dried figs and found good correlation (r =tent correlated with colony counts of fungi on wheat 0.92) between aflatoxin and ergosterol in reject figsgrains at 0.95 aw but not at 0.85 aw (Tothill et al., which were fluorescent, but no significant correla-1992). Using a stereomicroscope for visual exami- tion with patulin content. Kadakal et al. (2005)nation, they concluded that sound grain contained found good correlation (r = 0.98) between ergos-up to 6 mg/g ergosterol, microscopically mouldy terol and patulin in apple juice and that both patulingrain 7.5–10 mg/g and visibly mouldy grain more (r = 0.99) and ergosterol (r = 0.99) were linearlythan 10 mg/g ergosterol. From studies on ergosterol related to the proportion of decayed apples usedlevels, colony counts and mould growth in a variety to make the juice. Ergosterol has been used toof grain samples, Schnurer and Jonsson (1992) con- ¨ assess mould growth in cheese with variable resultscluded that ergosterol correlated with colony counts (Pecchini, 1997; Taniwaki et al., 2001a).
    • 4.7 Estimation of Fungal Biomass 37 Ergosterol content has also been investigated as influence of product variability and induced higheran indicator of the mycological status of tomato conductance changes (Owens et al., 1992). An impe-products. Battilani et al. (1996) found a significant dimetric method for detection of heat resistant fungicorrelation between ergosterol, Howard mould in fruit juices was described by Nielsen (1992). Thecount (HMC) and fungal growth, but with a high detection limit in artificially contaminated juiceslevel of uncertainty. Kadakal et al. (2004) found a was one Neosartorya ascospore per millilitre,linear relationship between degree of decay in detectable in 100 h. Huang et al. (2003) reportedtomato pulp and HMC (r = 0.97) and ergosterol an impedance method for detection of bacteria and(r = 0.96) and concluded that ergosterol has the fungi in bottled water which shortened the detectionpotential to be used in quality assessment of toma- time from 5 days to 27.1 h for fungi and from 48 totoes. Sio et al. (2000) described an improved method 11.3 h for bacteria.for extraction of ergosterol from tomato products. Although impedimetry and conductimetry pro- Other applications of ergosterol as a measure- mised to be effective rapid methods when usedment of fungal biomass include estimation of under well-defined conditions for a specific purposemould spores in indoor air and aerosols (Miller with a particular kind of food, the methodologyand Young, 1997; Robine et al., 2005; Lau et al., does not appear to have been broadly taken up for2006), estimation of wood decay by fungi (Eikenes food mycology applications.et al., 2005) and estimation of fungi in soil and wet-lands (e.g. Headley et al., 2002; Zhao et al., 2005). The ergosterol assay is reported to have a high 4.7.4 Adenosine Triphosphate (ATP)sensitivity and, in contrast to the chitin assay,requires only 1 h for completion (Seitz et al., 1979). ATP has also been suggested as a measure of micro-Despite its limitations, it appears to be a useful indi- bial biomass because bioluminescence techniquescator of fungal invasion of foods and to hold promise provide a very sensitive assay (Jarvis et al., 1983).as a routine technique for quality control purposes. Provided that background levels of ATP in plant or other cells are very low, or that microorganisms can be effectively separated from such other materials,4.7.3 Impedimetry and Conductimetry the method has some potential as a microbial assay. A good correlation was shown between ATP pro-Metabolites produced by growth of microorgan- duction and viable counts of six species of psychro-isms in liquid media alter the medium’s impedance trophic yeasts grown in pure culture (Patel andand conductance. The use of changes in these prop- Williams, 1985). The effective detection of low levelserties as a measure of bacterial growth was sug- of yeasts in carbonated beverages by ATP has alsogested by Hadley and Senyk (1975) and was first been reported (LaRocco et al., 1985). However, liv-applied to yeasts by Evans (1982) and to moulds by ing plant cells contain high levels of ATP and fungiJarvis et al. (1983). Most of the subsequent work on are often very difficult to separate from food materi-fungi has been carried out with yeasts, but the als. Moreover, extraction of molecules from fungalmethodology is often applicable to moulds also. cells is notoriously difficult, so this potential may be During the 1980s there were a number of studies difficult to realise in food mycology. The most wide-aimed at optimising media for inducing detectable spread application of ATP bioluminescence in theand reproducible changes in either conductance or food industry is for monitoring hygiene of surfacescapacitance during fungal growth (see Pitt and in food production facilities (Easter, 2007).Hocking, 1997). Watson-Craik et al. (1989, 1990)studied 27 mould species on a wide range of bothcommercially available and specially preparedmedia and concluded that conductance and capaci- 4.7.5 Fungal Volatilestance were both medium and species specific. Theuse of media high in ammonium ions and glucose, Methods for detection and characterisation of fun-with added yeast extract and peptone, decreased the gal volatiles are finding increasing applications in
    • 38 4 Methods for Isolation, Enumeration and Identificationfood mycology, for the detection of fungal dete- of volatile compound production as an indicator ofrioration of grain and other food products, for the mould deterioration in grains has been extensivelyidentification of particular fungi and for assessing assessed and reviewed (Kaminski and Wasowicz,potential mycotoxin contamination. Over the last 1991; Schnurer et al., 1999; Magan and Evans, 2000; ¨two decades, there has been rapid growth in the Paolesse et al., 2006; Balasubramanian et al., 2007).development of gas sensor arrays. Gas sensors are Fungal volatiles can be used to detect potentialchemical sensors that produce an electronic signal mycotoxin contamination, to discriminate betweenwhich is used as input into a pattern recognition fungal species (Sunesson et al., 1995; Keshri et al.,system in order to recognise different volatiles and 1998) and even between toxigenic and non toxigenicodours. This integrated system of gas sensor and strains of particular fungi (Sahgal et al., 2007).pattern recognition is often called an ‘‘electronic Karlshøj et al. (2007a) used an electronic nose tonose or e-nose’’ (Gardner and Bartlett, 1994; Schal- differentiate between closely related Penicilliumler et al., 1998). Arrays comprising equipment to species (P. camemberti, P. nordicum, P. paneum,collect headspace volatiles, analysis by GC-MS or P. carneum, P. roqueforti and P. expansum) fromother sensitive analytical methods, coupled with cheese. Volatile profiles can be used to predictcomputer analysis are also commonly used. mould spoilage in bakery products (Vinaixa et al., Deterioration of stored grain results from a com- 2004; Marı´ n et al., 2007a) and to detect and diffe-bination of chemical and biological changes, with rentiate between toxigenic and non toxigenicchanges due to fungal growth often predominant. P. verrucosum strains in bakery products (NeedhamDeterioration is marked by off-odour development, and Magan, 2003). Volatile profiles have also beenloss of germinability, caking, rancidity and some- used to differentiate between toxigenic and nontimes mycotoxin development (Abramson et al., toxigenic Fusarium strains (Keshri and Magan,1980). Fungi produce volatile chemicals during 2000; Demyttenaere et al., 2004), to identify myco-growth and particular chemicals may be associated toxins (aflatoxins, ochratoxin A and deoxynivale-with grain deterioration (Kaminski et al., 1974, nol) in durum wheat (Tognon et al., 2005) and to1975). Sinha et al. (1988) monitored production of detect and quantify ochratoxin A and deoxynivale-3-methyl-1-butanol, 1-octen-3-ol and 3-octanone in nol in barley (Olsson et al., 2002). This technologystored grain and found that the presence of these has also been applied to predict the presence ofvolatiles usually correlated with seed infection by P. expansum and patulin in apple products (KarlshøjAlternaria alternata, Eurotium repens, Aspergillus et al., 2007b) and to detect and discriminate diseasesversicolor and several Penicillium species. Octenol of potato tubers (Kushalappa et al., 2002) and stem-and octanone in particular seemed to be associated end rot and anthracnose in mangoes (Moalemiyanwith deterioration in grain due to fungal growth. et al., 2006). Electronic nose technology has alsoAdamek et al. (1992) identified methylfuran, been used for early detection of moulds in libraries2-methylpropanol and 3-methylbutanol as the and archives (Pinzari et al., 2004).most important metabolites from Eurotium amste-lodami, Aspergillus flavus, Penicillium cyclopiumand Fusarium culmorum growing on wheat. Thevolatiles produced by several Penicillium species 4.7.6 Immunological Techniqueswere also studied. Borjesson et al. (1989) collected ¨volatiles produced by several food spoilage fungi Cell wall proteins of fungi produce antigens, whichgrown in pure culture on wheat grain and found that can be detected by immunological methods. Somesome compounds, especially 3-methyl-1-butanol, were antigens are derived from components common to aproduced early in fungal growth and could be used as wide range of fungi, and hence are indicative ofan early warning of potential deterioration, before general fungal growth, while others are genus orfungal growth became visible. The production of vola- even species specific. A variety of methods havetile metabolites by Penicillium species in grain corre- been developed to take advantage of these fungallated well with carbon dioxide production and ergos- antigens, although none is in widespread commer-terol formation (Borjesson et al., 1990, 1992). The use ¨ cial use.
    • 4.7 Estimation of Fungal Biomass 39 Enzyme-linked immunosorbent assay (ELISA). (amount of ochratoxin A, r ¼ 0.93; percentageThe preparation of antigens from three common infection r ¼ 0.89) reported by Lu et al. (1995)foodborne fungi (Penicillium aurantiogriseum, but poorer correlation with colony forming unitsMucor racemosus and Fusarium oxysporum) was (r ¼ 68) and glucosamine content (r ¼ 0.64).described by Notermans and Heuvelman (1985). Anand and Rati (2006) did not attempt to correlatePreparation of immunoglobulin antibodies against their ELISA with external measurements of fungalthese antigens was followed by development of an biomass, but used an internal comparison methodELISA assay. Fungi were detected in both unheated using freeze dried mycelium. Immunological detec-and heat processed foods by this method. Antigens tion of aflatoxigenic fungi has also been the subject ofwere relatively genus specific: the M. racemosus several studies, using antibodies raised to A. flavus orantigens reacted with other Mucor and Rhizopus A. parasiticus (Candlish et al., 1997; Tsai and Yu,species, and the Penicillium antigen reacted with 1997, 1999; Yong and Cousin, 2001). Tsai and Yuthe Aspergillus species tested. It was subsequently (1999) reported good correlation between viableshown that the Penicillium antigen reacted with 43 count and ELISA measurements, depending onof 45 Penicillium species tested and that antigen substrate, with correlation coefficients varying fromproduction correlated with mycelial weight and 0.96 for wheat to 0.86 for peanuts in artificiallywas unaffected by culture conditions, medium, tem- inoculated samples.perature and aw (Notermans et al., 1986). The Peni- ELISA tests for Botrytis and Monascus in foodscillium antigen also reacted with Aspergillus flavus (Cousin et al., 1990) and for the detection in rice ofand the level of antigen correlated with aflatoxin Penicillium islandicum (Dewey et al., 1990) andproduction (Notermans et al., 1986). Humicola lanuginosa (Dewey et al., 1992) have ELISA techniques have also been studied as a been described. ELISA tests have also been appliedpotential replacement for the Howard mould count. to detection of yeasts in dairy products (GarciaAntigens from tomato moulds (Alternaria alternata, et al., 2004) and fruit juice (Yoshida et al., 1996),Geotrichum candidum and Rhizopus stolonifer) were moulds in paprika powder (Kisko et al., 1998) andused to produce an ELISA test sensitive to 1 mg/g of moulds in agricultural commodities (Park et al.,mould in tomato. A correlation was observed between 2003) with varying success. Linfield et al. (1995)antigen formation and mould added to tomato puree, reported that polyclonal antibodies raised againstwhile background interference was very low (Lin Botrytis allii were useful in early detection of necket al., 1986). The method was tested against a broader rot of onions caused by this pathogen.range of foods, with encouraging results (Lin and Immunochemical detection of fungi in food andCousin, 1987). Robertson and Patel (1989) improved feeds has been reviewed by Li et al. (2000).the sensitivity of the method for tomato paste by using Latex agglutination. A different approach to thea polyclonal antibody against Botrytis cinerea, Mucor immunological detection of fungi in foods has beenpiriformis and Fusarium solani in addition to the three the coating of latex beads with antibodies and detec-species used by Lin et al. (1986). tion of agglutination of the beads in the presence of ELISA based methodology has been reported for antigens (Kamphuis et al., 1989; Notermans andthe detection of Fusarium species in corn (Meirelles Kamphuis, 1992; Stynen et al., 1992). It was foundet al., 2006), cornmeal (Iyer and Cousin, 2003) and that 0.8 mm latex beads coated with antibodiesgrain (Rohde and Rabenstein, 2005). Correlations from the extracellular polysaccharide produced bywith other measures of fungal growth (ergosterol, Penicillium digitatum specifically detected Aspergilluscolony count, mycotoxin levels) were variable. and Penicillium species (Kamphuis et al., 1989). Aspergillus species have also been examined as Detection limits were as low as 5–10 ng/ml of thetargets for immunological detection because of their purified antigen. Commercially produced latex agglu-importance in mycotoxin contamination. ELISA tination tests were of value for screening the mycolo-detection of A. ochraceus in wheat (Lu et al., gical quality of grains and processed foods (Braendlin1995), coffee powder, chilli powders and poultry and Cox, 1992; van der Horst et al., 1992), althoughfeed (Anand and Rati, 2006) has been investigated, one kit performed poorly in detection of mould inwith reasonable correction with other parameters tomato products (van der Horst et al., 1992).
    • 40 4 Methods for Isolation, Enumeration and Identification Schwabe et al. (1992) compared the latex aggluti- cerevisiae, Candida albicans, C. guilliermondii,nation assay with ergosterol production for detection C. lusitaniae, C. tropicalis, Schizosaccharomycesof Penicillium, Aspergillus and Fusarium species in japonicus and Sch. pombe) (Broad Institute,pure culture. They concluded that the two methods 2008). Penicillium species are noticeably absentwere comparable for Penicillium and Aspergillus but from the list, although the genome of the patho-that ergosterol was more sensitive for Fusarium. In genic species P. marneffei has recently beenfood samples, both the latex agglutination test and sequenced (JCVI, 2008).ergosterol were effective means of detecting mould There have been rapid advances in molecular meth-growth, but no clear correlation existed in values ods for the detection of mycotoxigenic fungi. Probesobtained by the two methods. Kesari et al. (2004) have been developed that target genes in the myco-used a latex agglutination test to detect teliospores of toxin biosynthetic or regulatory pathways for manyKarnal bunt (Tilletia indica) in single grains of wheat species of Aspergillus, Penicillium and Fusarium,and were able to detect a few as 750 teliospores, which enabling these fungi to be detected and/or quantifiedthey reported as suitable for single seed analysis. in grains, grapes, apples, coffee and other food Fluorescent antibody techniques. Fluorescent anti- matrices (Edwards et al., 2002; Paterson, 2006; Gei-body techniques have also been used directly for the sen, 2007; Niessen, 2007a, b; Rossi et al., 2007).detection of mould in foods. Warnock (1971) detected Molecular methods have also been developedPenicillium aurantiogriseum in barley by this method, for the detection of spoilage fungi, particularlywhile Robertson et al. (1988) used antisera from five yeasts, e.g. Brettanomyces/Dekkera in grapes andfungi to visualise moulds and simplify their detection wine (Phister and Mills, 2003; Agnolucci et al.,in the Howard mould count technique. 2007; Hayashi et al., 2007), Hanseniaspora in wine (Phister et al., 2007); Zygosaccharomyces bailii in wine and fruit juices (Rawsthorne and Phister, 2006) and Kluyveromyces marxianus in4.7.7 Molecular Methods yoghurt (Mayoral et al., 2006). More general tech- niques have been described for detection of yeastsThe first techniques for detecting DNA sequences in juices (Casey and Dobson, 2004; Ros-Chumillasusing specific probes were devised more than 30 et al., 2007), milk, yoghurt and cheese (Cocolinyears ago (Southern, 1975). With the development et al., 2002; Bleve et al., 2003; Garcı´ a et al., 2004;of the polymerase chain reaction (PCR), gene clon- Lopandic et al., 2006; Gente et al., 2007), sour-ing and oligonucleotide synthesis, DNA sequences dough fermentation (Meroth et al., 2003), tablecan now be prepared in large quantities for use in olives (Arroyo-Lopez et al., 2006) and vacuumprobes. Depending on its role in the genome, DNA packaged ham (Sanz et al., 2005). PCR basedmay be specific at almost any taxonomic level. methods have also been described for detection ofMolecular methods can be used in both detection moulds in orange juice (Wan et al., 2006), Alter-and identification of fungi, and real-time PCR naria in cereal grains (Zur et al., 2002) and Botrytismethods even allow quantitative detection. This in onion seeds (Walcott et al., 2004).field of research continues to develop very rapidly, As gene sequencing has become more readilyand significant advances may be expected. available and cheaper, along with freely accessible Producing a broadly based or even strain specific databases such as GenBank (http://www.ncbi.nlm.-DNA probe is theoretically possible for any organ- nih.gov/Genbank/), sequence analysis and compar-ism. Full genome sequences are available for a ison is becoming a routine method for identificationnumber of important foodborne fungi, including of yeasts and filamentous fungi. For yeasts, theseveral Aspergillus species (A. nidulans, A. fumiga- most common target region is the D1/D2 domaintus, A. oryzae, A. flavus, A. niger, A. terreus, A. of the 26S rDNA (Kurtzman et al., 2003), but theclavatus and Neosartorya fischeri), Botrytis cinerea, internal transcribed spacer (ITS) region of riboso-Chaetomium globosum, Fusarium graminearum, mal DNA (rDNA) may also be used (van derF. oxysporum, F. verticillioides, Rhizopus oryzae Vossen et al., 2003; Pulvirenti and Giudici, 2003;and several yeast species (Saccharomyces Solieri et al., 2006; Villa-Carvajal et al., 2006).
    • 4.8 Identification Media and Methods 41 Sequencing of the ITS region along with ‘‘house- 4.8.2 Plating Regimenkeeping genes’’ such as calmodulin, b-tubulin andelongation factor 1-a is now commonly applied for As noted above, cultures for identification arepurposes of identification and phylogenetic analysis grown on three media and at three temperatures.of important food spoilage and mycotoxigenic fungi Maximum efficiency in time, incubator space andin the genera Aspergillus (Varga, 2006; Geiser et al., materials is achieved by inoculating two cultures on2007; Peterson, 2008), Penicillium (Peterson, 2004, a single Petri dish of G25N, and at 5 and 378C, as2006; Samson et al., 2004b; Wang and Zhuang, shown in Fig. 4.3. Cultures are rarely mutually2007; Serra et al., 2008) and Fusarium (O’Donnell inhibitory under these conditions, although over-et al., 2004; Scott and Chakraborty, 2006; Leslie growth of one culture by another is occasionallyet al., 2007). a problem at 378C. Standard sized Petri dishes Despite the apparent power of molecular techni- (90–100 mm) are used, except that at 58C, smallerques, they need to be applied with some caution, plates (50–60 mm in diameter) can be an advantage.particularly when comparing DNA sequences with The smaller size is easier to examine under the lowthose in the publicly available databases to identify power microscope. All plates are incubated for ayeast or mould isolates. Correct identification relies standard time of 7 days.on the database sequences having the correct name Plates incubated at 378C should be enclosed inattached to them by the depositor, which is not polyethylene bags to prevent evaporation and dry-always the case. If the identification makes sense, ing of the medium. Unless the humidity is very low,the percent homology is 98% or greater and the plates at 258C will not dry excessively in 7 days. If nonumber of base pairs on which the homology is 58C incubator is available, use a polyethylene foodscored is high, then the answer is probably correct. container or insulated box in a household refrigera- tor. The box should be equipped with a thermo- meter, and its location moved by trial and error until a place with a 58C average temperature is4.8 Identification Media and Methods located. Temperatures at 5 and 378C should ideally be –0.58C and be checked frequently; at 258C, –28C4.8.1 Standard Methodology control is adequate (Okuda, 1994).The identification keys in this book are based primar-ily on the standardised procedure described for theidentification of Penicillium species by Pitt (1979b). 4.8.3 InoculationCultures are grown for 7 days on three standardmedia at 258C, and on one of these at 5 and 378C As shown in Fig. 4.3, Petri dishes of CYA and MEAalso. The three Media are Czapek yeast extract agar for incubation at 258C are inoculated with a single(CYA; Pitt, 1973) used at all three temperatures; malt culture at three points, equidistant from the centreextract agar (MEA; Raper and Thom, 1949) and 25% and the edge of the plate and from each other. Platesglycerol nitrate agar (G25N; Pitt, 1973). Their formu- of the other media are inoculated with two pointslae are given in the Media Appendix. Preparation time per culture, as illustrated.of CYA and G25N is reduced by the use of Czapek With some fungi, especially Penicillium andconcentrate (Pitt, 1973), which is added to the media Aspergillus, it is important to minimise coloniesat the rate of 1% of the aqueous portion. from stray spores. The most satisfactory technique As media ingredients have become more purified is to inoculate plates with spores suspended in semi-in recent years, difficulties with extent and colour of solid agar (Pitt, 1979b). Dispense 0.2–0.4 ml ofsporulation on CYA have been encountered, espe- melted agar (0.2%) and detergent (0.05%), such ascially with some Penicillium species. To overcome polysorbitan 80 (Tween 80), in small vials and ster-this problem, Czapek concentrate has been reformu- ilise. To use, add a needle point of spores and myce-lated (Pitt, 2000) by the inclusion of traces of zinc and lium to a vial and mix slightly. Then, before flamingcopper (Smith, 1949) (see Media Appendix). the needle, use it to stab inoculate the 58C plate;
    • 42 4 Methods for Isolation, Enumeration and IdentificationFig. 4.3 Schematic of regimen used for culturing fungal isolates for identificationresidual spores on the needle make a good inocu- Penicillia are very common in foods, and manylum. Next, take a sterile loop, mix the vial contents produce mycotoxins, so correct identification isthoroughly and inoculate the standard plates. Used often critical. Species within subgenus Penicilliumvials can be sterilised by steaming and reused several fall into two groups: those with an affinity for pro-times before being washed or discarded. teinaceous foods and those which grow more vigor- ously in foods high in carbohydrate. Frisvad (1981, 1985) introduced creatine sucrose agar (CREA) to permit differentiation between these two groups.4.8.4 Additional Media and Methods Creatine (as the sole nitrogen source) permitted growth of the former group while inhibiting theThe plating regimen outlined above can be used to latter. Incorporation of bromocresol purple enabledidentify most of the fungi described in subsequent visualisation of pH changes, either acid or alkaline,chapters of this book. Some exceptions exist, depending on the creatine or sucrose metabolism ofbecause certain genera either grow poorly or fail to a particular species. However, discriminationsporulate on the standard media. As noted earlier in between positive and negative responses was notthis chapter, fastidious xerophiles are identified on always clear, and the main tabulation of speciesMY50G agar. Eurotium species, traditionally iden- reactions to CREA (Table 1 in Frisvad, 1985) wastified on Czapek agar with 20% sucrose, are identi- difficult to interpret. Frisvad (1993) subsequentlyfied here on Czapek yeast extract agar with 20% produced a number of variations of CREA, includ-sucrose (CY20S; see Appendix). Trichoderma ing acid and neutral pH formulations and substitu-species are best identified on potato dextrose tion of sucrose with fructose or lactose. Althoughagar (PDA) after a relatively short incubation time the merits of each formulation were discussed, no(3–4 days), as the structures tend to autolyse as firm recommendation resulted from this exercise.cultures mature. Pitt (1993a) modified Frisvad’s strongly alkaline Penicillium subgenus Penicillium. Many species CREA medium by studying several sucrose andclassified in subgenus Penicillium are morphologi- creatine concentrations over a wide pH range. Thecally similar, and identification using traditional result was neutral creatine sucrose agar (CSN), amorphological techniques remains difficult. These medium producing eight different reactions among
    • 4.8 Identification Media and Methods 43the 20 Penicillium subgenus Penicillium species Single sporing. The technique for preparing sin-tested. When included in the normal plating regi- gle spore cultures is as follows (Nelson et al., 1983;men for identification of Penicillium cultures from Leslie and Summerell, 2006). Pour about 10 ml ofthe subgenus Penicillium, CSN provides a very use- 2% water agar into unscratched glass or plasticful aid to distinguishing between these difficult and Petri dishes and allow to dry, either by holding theclosely related species. See Chapter 7 for details of plates at room temperature for several days or byuse of CSN, its reactions and interpretation. Its placing them inverted in an oven at 37–458C forformulation is given in the Media Appendix. about 30 min. Prepare a suspension of conidia in Dematiaceous Hyphomycetes. The natural habi- a 10 ml sterile water blank so that it containstats for many dematiaceous Hyphomycetes are 1–10 spores per low-power (10Â) microscope fieldplants or plant material, so finding suitable labo- when a drop from a 3 mm loop is examined on a slide.ratory media and conditions to induce typical With experience, this concentration can be gaugedsporulation can be difficult. Alternaria, Curvularia, simply by observing the turbidity of the suspension.Stemphylium and Ulocladium species are best iden- Pour the suspension of spores onto a dried water agartified from dichloran chloramphenicol malt extract plate, drain off the excess liquid and incubate in anagar (DCMA; Andrews, 1992b) plates incubated inclined position at 20–258C for 18–20 h.for 7 days at 258C under lights. Conidial character- After incubation, open the Petri dish, shake offistics of Bipolaris species vary with type of medium. any accumulated moisture droplets and examineSpecies described here are best identified from tap under a stereomicroscope using transmitted light.water agar (TWA) containing a natural substrate The germinating conidia should be visible undersuch as sterilised wheat or millet seeds, or wheat 25Â magnification. A dissecting needle with a flat-straw. Drechslera species will grow well on DCMA tened end and sharpened edges is used to cut outbut sporulation is poor. The best sporulation is small squares of agar containing single, germinat-achieved with V-8 juice (V-8 J) agar or TWA with ing conidia. These single conidia are then trans-one of the natural substrates mentioned above, ferred on the agar blocks to the desired growthincubated at 258C for 7–10 days under lights with medium.a 12 h photoperiod. Light/dark periodicity is impor- If the original culture is contaminated with bac-tant as Drechslera cultures require light to produce teria, a drop of 25% lactic acid may be added to theconidiophores and darkness to produce conidia, water blank. Allow this acidified spore suspensionpossibly due to light inactivation of flavine neces- to stand for 10 min before pouring onto a watersary for conidial formation (Knan, 1971; Platt et al., agar plate. Germination of acid-treated Fusarium1977). conidia may be delayed by 24 h or more. For the identification of Trichoderma and Fusar- Media. Two media have been used in this bookium species, potato dextrose agar (PDA) is used. for the identification of Fusarium isolates: potatoFusarium species also require additional methods dextrose agar (PDA) for colony characteristics andand media as outlined below. colour and dichloran chloramphenicol peptone agar (DCPA) for the development of diagnostic macro-, micro- and chlamydoconidia. A third medium, carnation leaf agar (CLA), is4.8.5 Identification of Fusarium Species recommended by some Fusarium specialists for Fusarium cultivation and identification (NelsonFusarium isolates exhibit unusually high variability et al., 1983; Leslie and Summerell, 2006). CLA isin colony morphology and also may deteriorate an excellent medium, on which most Fusarium spe-rapidly in culture. Thus, they should be identified cies readily produce their diagnostic macroconidia.as soon as possible after initial isolation with a Production of macroconidia on DCPA is usuallyminimum of subculturing to avoid deterioration. It comparable with that on CLA, but microconidiais common practice to prepare cultures of Fusaria and chlamydoconidia are often more plentiful onfrom single spores for growth on identification CLA due to greater production of aerial hyphae.media, as this reduces both of these problems. DCPA is used in the present work rather than CLA,
    • 44 4 Methods for Isolation, Enumeration and Identificationhowever, because dried, gamma-irradiated carna- have been published in the literature, and bothtion leaves are difficult to obtain in many localities. automated and manual yeast identification systems Inoculation and incubation. For identification by are now commercially available.the methods used in this book, single spore cultures ´ Deak and Beuchat (1987) published a simplifiedof Fusarium isolates should be prepared on agar identification key (SIK) which included 215 speciesblocks as outlined above, inoculated, one per of foodborne yeasts. They subsequently modifiedplate, onto two plates each of PDA and DCPA their system, restricting it to the 76 species mostand incubated at 258C for 7 days. Individual plates frequently occurring in foods (SIM), and reportedmay be used for each medium, or alternatively that it was much more successful than the API 20Cdivided plates may be used, with one medium on ´ system (BioMerieux, Marcy-l’Etoile, France) foreach half of the plate. Illumination during incuba- ´ identification (Deak, 1992). The SIM uses onlytion is essential for the production of macroconidia. two Petri dishes and three test tubes to examineThe light source may be diffuse daylight (not direct each strain for ability to assimilate 10 carbonsunlight) or light from a bank of fluorescent tubes. sources, fermentation of glucose, assimilation ofA photoperiod of 12 h per day is normally used. nitrate and splitting of urea. These biochemicalAlternating temperatures of 20 and 258C have been tests are supplemented by morphological observa-recommended (Nelson et al., 1983; Leslie and Sum- tions. SIM separates the yeasts into six groups by amerell, 2006) but are not essential. dichotomous key utilising the results of five key A simple light bank may be constructed from a tests. Further tests are used to differentiate thestandard 40 watt fluorescent fixture with two cool yeasts in these six groups using secondary dichoto-white tubes, suspended 0.5–l m above the labora- mous keys.tory bench or shelf supporting the cultures. The A dichotomous key to 25 common species ofaddition of a black light tube (e.g. Philips TL foodborne yeasts was published by Smith and40 W/80 RS F40BLB) is also desirable and in Yarrow (1995), who used 17 biochemical andsome cases essential to induce macroconidial or physiological tests to distinguish the species.chlamydoconidial production. Of the commercial systems available, the most widely used for foodborne yeasts are the Biolog, which is an automated system, and the BioMerieux ´ ID32C yeast identification strips which can be4.8.6 Yeasts read manually or automatically using the ATB sys- ´ tem. BioMerieux also markets the fully automatedYeast identification systems. Identification of food- VITEK 2 system, based on a card containingborne yeasts remains a difficult task, as colony 64 tests. However, the database comprises onlycharacteristics and microscopic morphology are of 46 clinically important species (Aubertine et al., 2006)limited value. Generally it has been necessary to use and so has limited application to foodborne yeasts.biochemical and physiological tests such as fermen- The Biolog (Biolog Inc., Hayward, CA, USA;tation of carbohydrates, assimilation patterns for a http://www.biolog.com/microID.html) utilises arange of carbon and nitrogen sources and growth at yeast identification test panel (YT MicroPlateTM)various temperatures. Details of these methods and consisting of a matrix of 8 Â 12 wells. The first threemedia may be found in Kurtzman and Fell (1998), rows contain 35 carbon source oxidation tests usingKurtzman et al. (2003) or Barnett et al. (2000). tetrazolium violet as an indicator of oxidation. TheMolecular methods (see below) are being used next five rows contain carbon assimilation testsincreasingly in yeast identification, replacing time which are scored turbidimetrically against a nega-consuming biochemical testing. tive control panel containing only water. The last Identification using systems based on biochem- row contains two carbon sources and tests forical and physiological testing is complex and time the co-utilisation of various carbon sources withconsuming. However, a number of attempts have D-xylose. The hardware (Biolog MicroStationbeen made to assist those who wish to persevere Reader) consists of an automated plate readerwith yeast identification. Several simplified systems coupled with a computer, which interprets the
    • 4.9 Examination of Cultures 45results and compares them with the resident data- Even using the available systems, identificationbase which currently includes 267 species. Manual of yeasts still requires some specialist knowledgeinterpretation of the Biolog plates is not recom- and interpretation and remains time consuming.mended. This system has been designed with the Our experience indicates that no more thanfood industry in mind, and the database contains 12 species of spoilage yeasts are of real concern inall the common foodborne yeasts, unlike other foods. It is possible to differentiate these few speciessystems which are usually aimed at the clinical by relatively simple techniques, i.e. colony andmarket. microscopic morphology, growth on the standard ´ The ATB ID32C system (BioMerieux, Marcy- media used for filamentous fungi, growth on otherl’Etoile, France) is an automated system utilising media which test for preservative resistance, ability ´the BioMerieux ID32C yeast identification strips. to use nitrate as a nitrogen source and adaptation toThese strips contain 30 assimilation tests plus a high NaCl concentrations. Details of these techni-positive (glucose) and a negative control well, all ques are given in Chapter 10.of which are inoculated with a yeast suspension ofspecified density. The strips are incubated at 308Cfor 48–72 h. As with the Biolog, the ATB automatedsystem consists of a plate reader attached to a 4.9 Examination of Culturescomputer. The database associated with the ATBsystem contains 63 yeast species. As noted above, all cultures for identification ´ The BioMerieux ID32C strips can be read manu- should first be grown on the standard regimenally, and the results enable identification of yeasts described earlier. After 7 days incubation, the fol-using published keys or computer identification lowing examination should be carried out and thenprogrammes such as that of Barnett et al. (1996). the general key to fungi in Chapter 5 should be used.This system may be used with reasonable success, That key will be of assistance even in the event ofparticularly when the test results from the ID32C cultures failing to grow under one or more of thestrips are supplemented with extra tests (glucose standard conditions.fermentation, urease production, nitrate utilisation,growth in 0.5 and 1% acetic acid and in 50 and 60%glucose, growth at 378C, production of pseudohy-phae, ascospore formation and morphological 4.9.1 Colony Diametersobservations) to give a more comprehensive basefor the identification. Growth in 0.5 and 1% acetic Measure the diameters of macroscopic colonies inacid indicates preservative resistance, and growth in millimetres from the reverse side (Fig. 4.4). Micro-50 and 60% glucose gives an indication of ability to scopic growth or germination at 58C is assessed bygrow at reduced aw. Both these parameters are low power microscopy (60–100Â), by putting theimportant in determining a yeast’s ability to cause 58C Petri dish on the microscope stage and examin-spoilage in particular products. ing by bright field transmitted light. Growth at 378C Identification using DNA sequencing is is assessed macroscopically only; germination ofincreasingly becoming the method of choice, as spores at 378C is an unreliable character.extensive databases such as GenBank are freelyavailable for identification purposes. The 600–650 nucleotide D1/D2 region of the large subunit(26S) ribosomal DNA is the most widely targeted 4.9.2 Colony Characterssection of the genome, and sometimes the ITSregion may also be used (Kurtzman et al., 2003). Colony appearance can be judged by eye or with aSequencing of the D1/D2 region, along with some hand lens, but examination is more effective if asupplementary physiological and biochemical stereomicroscope is used. Magnifications in thetests, is the identification method currently used range of 5–25Â are the most useful. Charactersin our laboratory. such as type and location of sporing structures and
    • 46 4 Methods for Isolation, Enumeration and IdentificationFig. 4.4 Technique for measuring colony diameters by transmitted lightextent of sporulation are best gauged with the buried in the agar, e.g. pycnidia, take a sample ofstereomicroscope. Reflected light is usually more these with a small piece of the agar. Float the cuteffective than transmitted light. colony sample from the needle onto a slide with the To determine colony colours, examine colonies aid of a drop of 70% ethanol. It may be necessary toby daylight or by daylight-type fluorescent light. In tease out the specimen with the needle and the cor-some genera, reference to a colour dictionary is ner of a cover slip (square cover slips are best).helpful. The Methuen ‘‘Handbook of Colour’’ Fungal specimens may be highly hydrophobic: the(Kornerup and Wanscher, 1978) has been used in ethanol helps to wet the preparation, minimising thethis work and is highly recommended. amount of entrapped air. When most of the ethanol has evaporated, add a drop of lactic acid (for phase or interference contrast optics) or lactofuchsin stain (see below) for bright field. Add a cover slip; if4.9.3 Preparation of Wet Mounts for necessary remove excess liquid from the prepara- Microscopy tion by gently blotting with facial tissue or similar absorbent paper. The preparation is now ready forFungi should always be examined microscopically examination.as wet mounts rather than fixed and stained likebacteria. To prepare a wet mount, use an inoculat-ing needle to cut out a small portion of the colonywhich includes sporing structures. Examination 4.9.4 Stainingwith the stereomicroscope can be an invaluable aidhere. For freely sporing fungi with little mycelium, A wide variety of stains are in use for mycologicalcut a piece of colony near the edge, where fruiting work. However, most are time consuming to pre-structures are young and spore numbers not exces- pare, or to use, or are slow to act, because fungalsive. Take structures which may enclose spores, i.e. walls and spores are highly resistant to stains. By farcleistothecia, from near colony centres, where the the most effective stain for use in food mycology isprobability of mature spores is highest. If the only lactofuchsin (Carmichael, 1955), which suffers fromdifferentiated parts of the colony appear to be none of these faults. It consists of 0.1% acid fuchsin
    • 4.9 Examination of Cultures 47dissolved in lactic acid of 85% or higher purity. equipped with an eyepiece micrometer, which isYoung actively growing fungal structures are pre- essential for measuring dimensions of spores andferentially coloured bright pink, so sporing struc- sporogenous structures.tures can usually be readily distinguished against a In the examination of fungal mounts, it is stressedbackground of older mycelium. Cleistothecial initi- that it is most important to use low power opticsals, developing asci and maturing ascospores are before succumbing to the temptation to use oil immer-also seen more readily in preparations stained with sion. The principal reason is that fungal preparationslactofuchsin. usually remain as small clumps and do not disperse as Like most other mycological stains, lactofuchsin bacteria do. Only under low power is the search foris corrosive. Take care to clean it off microscope the optimal area of the slide for the observation ofparts or skin! Be especially careful of the objective fruiting structures likely to be rewarded. Once a sui-faces, because lactic acid will slowly corrode the table area is located under the 10Â or 16Â objective,relatively soft glass used in lenses. move to the 40Â. This should be the lens most used; microscope optics are such that only the finest details of ornamentation can be observed more effectively4.9.5 Microscopes and Microscopy under oil immersion than at this magnification. Aligning the microscope. Correct alignment of the microscope is essential, so that its resolution is asChoice of microscope. Fungal identifications inevi- high as possible and it can be used for long periodstably involve microscopy. A high quality, binocular without discomfort. An incorrectly aligned micro-compound microscope is essential for serious myco- scope will lead to poor observation, discomfort,logical work. Bright field, phase contrast and inter- fatigue, headache and eye strain. A person of nor-ference contrast optics are all suitable. If bright field mal visual acuity should be able to use a correctlyoptics are used, preparations should be stained. aligned instrument throughout a whole workingPhase contrast optics avoid the need for staining, day without discomfort. The steps to correctlyalthough as explained above, staining has merit for align a microscope are given below. They shouldcertain structures in any case. Resolution under be read in conjunction with the microscope manu-phase contrast sometimes suffers from excessive facturer’s instructions.halo effects because fungal structures are highlyrefractile. Experience suggests that bright field 1. Mount a slide on the stage and bring it intooptics are more satisfactory than phase contrast approximate focus. If a prepared slide is notfor examining most fungi. Interference contrast is available, a slide marked with a marking pen orsuperior to both bright field and phase contrast and ink line is a satisfactory substitute.is strongly recommended. The very thin optical sec- 2. Close the microscope’s field diaphragm (the onetion cut by this system provides higher resolution of at the microscope base nearer the light source).dense structures and the relatively low contrast is The image of the diaphragm opening should nowrestful to use. be visible in the microscope field. If it is, first focus The microscope should be equipped with at least it with the condenser focusing knob and thenfour objectives: 6Â for examining Petri dishes under centre it in the field with the condenser centringlow power (for germination, etc.); 10Â or 16Â for screws. If the diaphragm opening cannot be seen,searching fields for sporing structures; 40Â for first rack the condenser up and down and watchexamining structural details and measuring stipes, to see if the opening becomes visible; if it does not,fruiting structures and large conidia; and 100Â oil rack the condenser to its highest position and thenimmersion for observation of details of spore slowly open the field diaphragm until the openingattachment, surface texture, ornamentation of comes into view. Centre the diaphragm approxi-hyphae and spores and fine measurements. Oculars mately and proceed as above.should be 10Â or 12.5Â and may with advantage be 3. For bright field optics, the condenser diaphragmwide field and have a high eye point suitable for use should be adjusted each time the objective powerwith spectacles. One should be a focusing ocular, is changed. Remove one ocular; close the
    • 48 4 Methods for Isolation, Enumeration and Identification condenser diaphragm so that the field seen down Always check the settings on the microscope the open tube is about two-thirds its maximum before use and after making measurements with size. With phase contrast and interference con- the micrometer. It is very easy to upset the ocular trast systems, this adjustment is less critical. alignment when measuring. The preceding steps align the microscope itself and should be checked frequently. If optimal illumination is desired, each step should be car- ried out for each new slide and each objective 4.10 Preservation of Fungi change. As a routine habit, the whole process should take only a few seconds. Many fungi are stable in culture and can be subcul- If the available microscope is not equipped tured repeatedly without apparent change or dete- with a built-in light source, a field diaphragm rioration. Others, especially Fusarium species and and a fully centring condenser, it is unlikely to other plant pathogens and some mycotoxigenic spe- be satisfactory for the identification of small cies, will degenerate rapidly after only a few transfers. spored fungi such as Penicillium species. For stable isolates that are used routinely in the The following steps are designed to align the laboratory, storage on agar slopes is satisfactory. observer with the microscope, compensating for Many freely sporing fungi will survive for several individual differences in sight. Provided settings months, and sometimes much longer, when stored at on the microscope are remembered, these steps 1–48C on a medium such as CYA. One of the hazards need be carried out only occasionally, to check of storage at these temperatures is contamination by that visual acuity has not altered. Different set- psychrophilic Penicillium or Cladosporium species. tings will be needed for an individual with and Storage at freezer temperatures (–18 to –208C) without spectacles or contact lenses. prevents such contamination, but some fungi do not4. Assuming the microscope is binocular, pull the survive well under these conditions. oculars out to their greatest distance apart and Storage at room temperatures, at 108C or above, then, while watching a focused field, move them for long periods is not advisable because of the gradually together until a single circular field is likelihood of invasion by culture mites (see below). seen without strain or head movement. Note the distance on the scale between the oculars; this is the individual’s interpupillary distance. Repeat this operation two or three times until satisfied 4.10.1 Lyophilisation that the correct distance has been found. This distance should always remain the same and be For unstable cultures, and indeed for the long term similar on any microscope. storage of any food spoilage fungi, freeze drying or5. Under the 40Â or 100Â objective, locate a tiny, lyophilisation is probably the best method of pre- readily recognised point on the slide and focus on servation. Many commercial systems are now avail- it. Take a piece of white card and place it between able for carrying out this process. the focusing ocular and the corresponding eye. A satisfactory menstruum for lyophilisation of Leave the eye open. Now focus the tiny point most fungi is l.5Â normal strength reconstituted with the other eye, carefully, with the microscope nonfat milk powder (15% in distilled water). For fine focus. Next, transfer the white card to the fungi with hydrophobic conidia, such as Aspergillus other ocular and, using the focusing collar beneath and Penicillium, a small amount of detergent the ocular, refocus the tiny point. Remove the card (0.05%) such as polysorbitan 80 (Tween 80) should and note the setting on the scale. Repeat until be added to the milk. Dispense the milk in 10 ml satisfied the correct setting has been found. lots in small tubes or 12.5 ml (0.5 oz) McCartney6. On some microscopes, the eyepiece micrometer bottles, and sterilise by steaming for 20 min on three can be focused independently. Use the focusing sys- successive days (the Tyndallisation process). The tem on the ocular itself to focus the micrometer. milk must be stored at 208C or above between Note the setting on the scale on the side of the ocular. steamings, to permit bacterial spores to germinate.
    • 4.10 Preservation of Fungi 49Occasionally bacterial spores will survive this pro- cryovials. An 80% solution of glycerol remains vis-cess: it is advisable to store the milk at room tem- cous at –808C, which enables cultures to be removedperature for some days after Tyndallisation. Any from the freezer for subculture without the need forbottles which show clotting or other breakdown defrosting.should be autoclaved and discarded. Autoclaving at For smaller laboratories that do not have access1218C, even for 10 min or less, is not recommended, to lyophilisation, liquid nitrogen or ultra-low tem-as browning will occur and browning compounds are perature storage, some simple techniques exist thatknown to be inhibitory to microorganisms. can be used to maintain fungal cultures over rela- Most common spoilage fungi survive lyophilisa- tively long periods (e.g. 1–10 years) without thetion well. However, in our experience and that of need for subculturing.others (Mikata et al., 1983; Smith and Onions, Water storage. A simple and inexpensive method1994), some yeasts, plant pathogens and xerophiles of fungal culture preservation is storage of agardo not. Storage of lyophilised ampoules at refrig- blocks in water (Smith and Onions, 1994). Smalleration temperatures (0–48C) is recommended, but agar blocks (7–10 mm2) are cut from the growingroom temperature is probably satisfactory provided margin of a young fungal colony and placed insunlight is avoided and temperatures do not exceed sterile water in a bottle such as a Bijou bottle308C. Some laboratories routinely store lyophilised (6.25 ml or 0.25 oz McCartney bottle). The rubbercultures at –18 to –208C. lined cap is screwed down and the bottles stored in a Strains being maintained for a particular trait or cool room (1–108C). Cultures may be revived byutilised for a specific purpose such as system testing removal of a block and placing it on a suitableor metabolite production should always be lyophi- growth medium. Using this method, cultures havelised. Continued subculturing often leads to dete- been reported to remain viable and retain theirrioration or loss of the desired character. The ability characteristics for up to 7 years (Boeswinkel, 1976;of a strain to produce a particular mycotoxin, for Smith and Onions, 1994).example, may decrease with each transfer. Isolates Some yeasts may be maintained by storing asshould be lyophilised as soon as possible after pri- suspensions in water (Kirsop, 1984). Growth frommary isolation to prevent degeneration. a late logarithmic slant culture is suspended in ster- ile distilled water and transferred to a sterile con- tainer so that 90% of the volume is filled with the suspension. Containers are stored at room tempera-4.10.2 Other Storage Techniques ture. Survival of some Candida, Saccharomyces, Cryptococcus, Rhodotorula and Schizosaccharo-A variety of systems other than lyophilisation have myces species for up to 4 years has been reportedbeen proposed for long term storage of fungi. Of (Kirsop, 1984).these, liquid nitrogen storage has found most accep- Silica gel storage. Many fungal cultures may betance with major culture collections. This type of maintained for long periods (often more than 10storage appears to be superior to any other for plant years) by drying spore suspensions onto silica gelpathogens and fungi which will not sporulate in (Smith and Onions, 1983; Smith, 1984). Thispure culture. However, liquid nitrogen systems are method is not suitable for mycelial cultures, butexpensive to establish and maintain and are only can be used with some success for yeasts (Kirsop,suitable for large collections. 1984). As silica gel liberates heat when moistened, Freezer units which run at very low temperatures the technique depends on keeping the cultures cool(–808C or below) are available and are well suited to enough to avoid damaging the spores during pre-the needs of culture collections. In our laboratory paration. Medium grain plain (non-indicating)we routinely store cultures at –808C, using glycerol silica gel of 6–22 mesh is placed in suitable glass(60–80%) as a cryoprotectant. Spore suspensions bottles (Bijou or McCartney bottles) to a depth ofare prepared by taking conidia or ascospores from about 1 cm and sterilised, either by dry heat ata freely sporulating sector of the colony, dispersing 1808C for 2–3 h or by autoclaving at 1218C forthem in the glycerol then freezing in screw capped 15 min. Autoclaved silica gel must be thoroughly
    • 50 4 Methods for Isolation, Enumeration and Identificationdried in an oven before use. Bottles are precooled by If for any reason fungal spore concentrations doplacing in a tray of ice or refrigerating for 24 h, then build up in a laboratory and cause an unacceptabletransferring to an ice tray for inoculation. level of contamination, the air should be purified. Suspensions of fungal spores or yeasts are pre- The simplest technique is to spray the air through-pared in sterile skimmed milk (as for lyophilisation, out the laboratory with an aerosol before it is closedabove) and the suspension added to the cooled silica in the evening. Any aerosol spray, such as a roomgel to wet three-quarters of it. The bottles and gel deodoriser or air freshener, is effective. Aerosolare allowed to dry at room temperature for 10–14 droplets entrain fungal spores very efficiently anddays with caps slightly loosened. Caps are then carry them to the floor.screwed down and the bottles stored at 48C (storage A more drastic and effective treatment in cases ofat room temperature is also satisfactory) in air-tight severe contamination is to spray a solution of 2%containers over indicating silica gel to absorb any thymol in ethanol around the room and close it for amoisture. weekend. The spray is rather pungent, and while not Cultures are revived by shaking a few crystals of harmful to humans, it effectively kills fungal sporessilica gel onto a suitable growth medium (broth and mites (see below). Do not leave cultures onculture may be better for yeasts). Survival varies benches before fumigation.according to species or even strain, but survival ofyeasts for up to 5 years (Kirsop, 1984) and fungi formore than 10 years (Smith and Onions, 1983) hasbeen reported. 4.11.1 Culture Mites A major hazard in growing and maintaining fungal cultures is the culture mite. Many species of mites4.11 Housekeeping in the Mycological live on fungal hyphae as their main or sole diet in Laboratory nature and find culture collections an idyllic envir- onment. Mites crawl from culture to culture, con-Like any other microbiological laboratory, a myco- taminating them with fungi and bacteria as they gological laboratory should be kept in a clean con- or, given long enough, may eat them out entirely.dition. Discard unwanted cultures regularly and Mites are very small (0.05–0.15 mm long),dispose of them by steaming or autoclaving. Wipe usually just visible to the observant naked eye.bench tops regularly with ethanol (70–95%). Floors They are arachnoids, related to spiders, and her-should be wet mopped or polished only with maphroditic. Each mite leaves a trail of eggs aboutmachines equipped with efficient vacuum cleaners half adult size as it goes. Eggs hatch within 24 h andand dust filters. Where possible store food and plant reach adulthood within 2 or 3 days. The damage anmaterials away from the laboratory. Open Petri unchecked mite plague can do to a fungal culturedishes carefully. Use small inocula on wet needles. collection has to be experienced to be believed.Transport Petri dishes to the stereomicroscope The most common sources of mites are plantstage before removing lids. Do not bump cultures material, soil, contaminated fungal cultures andduring transport. mouldy foodstuffs. Mites can also be carried on Contrary to popular belief, a well-run mycologi- large dust particles. Building work near a labora-cal laboratory is not a source of contamination to tory almost always induces a mite infestation.bacteriological laboratories. The air in a mycologi- The avoidance of losses due to mites requirescal laboratory should not carry a significant popu- constant vigilance. Always watch for telltale signs,lation of fungal spores. The reverse problem can such as contaminants growing around the edges of aoccur, however, because bacteria multiply more Petri dish, a ‘‘moth-eaten’’ appearance to coloniesrapidly than do fungi. Bacterial spores are often or ‘‘tracks’’ of bacterial colonies across agar. Exam-present in food laboratories, readily infect fungal ination of suspect material or cultures under theplates and can rapidly outgrow and inhibit fungal stereomicroscope will readily reveal the presencemycelia, especially at 378C. of mites and mite eggs.
    • 4.11 Housekeeping in the Mycological Laboratory 51 Adult mites are rapidly killed by freezing, and Rhizopus stolonifer is a ubiquitous fungus in manymite eggs will only survive 48–72 h at –208C. Cul- kinds of foods. It grows rampantly at 258C, filling atures contaminated by mites can often be recovered Petri dish with sparse, dark mycelium in 2 days. Itby freezing for 48 h, then subculturing from unin- produces barely macroscopic aerial fruiting structuresfected portions of the culture with the aid of the which are at first white, then become black. Givenstereomicroscope. Suspect food and other samples seven undisturbed days, it sheds dry, black sporesbeing brought into the laboratory should also be outside the Petri dish, providing an effective inoculumfrozen for 24 h to destroy mites before enumeration for a continuous chain of future contamination. Onceor subculturing is carried out. such an infection occurs, it is essential to carefully Infestation by mites can be minimised by good place the contaminated plate in a suitable containerhousekeeping, i.e. by avoiding accumulation of dust such as a plastic bag before transporting it to steameror old cultures in the laboratory. It is also good or autoclave. Then use 70% ethanol to clean areas onpractice to handle and store food and plant samples which the plate had been incubated or placed. At dailywell away from areas where fungi are inoculated intervals, carefully examine all plates inoculated sub-and incubated. sequently, discarding any which show Rhizopus con- To control a mite infestation, remove all con- tamination, until infection ceases. Spraying the airtaminated material, including cultures. Freeze with aerosols or thymol in ethanol will assist. UnlikePetri dishes and culture tubes which must be recov- A. fumigatus, R. stolonifer is not pathogenic.ered; autoclave, steam or add alcohol to all others. The third problem fungus, Chrysanilia sitophila,Clean benches thoroughly with sodium hypochlor- is known as ‘‘the red bread mould’’. It used to beite (household bleach) or 70% ethanol. Incubators common, but due to changes in manufacturingcan be disinfested with aerosol insecticides. practice, it is now encountered less frequently, either in the bakery or in the laboratory. Like Rhizopus stolonifer, it grows with great rapidity at 258C. It4.11.2 Problem Fungi forms a thin, pink mycelial growth across a Petri dish, clearly following the oxygen gradient which leads to the open air. It will force its way unerringlyThere are three fungal invaders which should be between dish and lid and, once outside, will producewatched for carefully in a food mycology laboratory: masses of pink spores, which are quickly shed andAspergillus fumigatus, Rhizopus stolonifer and Chryso- build up around the base of the Petri dish. Decon-nilia sitophila. The first is a human pathogen; the others tamination relies on the same techniques as forcan cause a contamination chain which is difficult to Rhizopus: C. sitophila is probably the more difficultbreak. fungus to eradicate. Again, apart from its nuisance Aspergillus fumigatus may cause invasive asper- value in the laboratory, it is harmless.gillosis in the lungs or serious allergenic responses insome individuals. It is sound practice to immedi-ately kill and discard cultures of this fungus as soonas it is recognised. On no account should it be used 4.11.3 Pathogens and Laboratory Safetyfor experimental studies in food spoilage or biode-terioration without precautions to prevent dissemi- While it must be said that any fungus which isnation of spores. The morphology of A. fumigatus is capable of growth at 378C is a potential mammaliandescribed in detail in Chapter 8, but it is readily pathogen, the physiology of the healthy human isrecognisable in the unopened Petri dish: highly resistant to nearly all of the fungi encoun- colonies are low, dull blue and broadly spread- tered in the food laboratory. Nevertheless, fungi ing, with a velvety texture; which can grow at 378C should be treated with growth is very rapid at 378C, covering a Petri caution. In particular, the habit of sniffing cultures dish in 2 days; is to be avoided wherever possible. It is true that long columns of blue conidia are readily seen odours produced by fungi have been used quite under the stereomicroscope. frequently as taxonomic criteria, especially in older
    • 52 4 Methods for Isolation, Enumeration and Identificationpublications, but their subjective and ephemeral A. fumigatus and also grow prolifically at 378C. Othernature makes them of little value for this purpose, Aspergillus species, particularly A. flavus, A. niger andand the risks involved are serious. Some labora- A. terreus, have been isolated from human specimenstories regard fungal volatiles as such a serious risk from time to time. For more details, see de Hoog et al.that cultures such as Penicillium and Aspergillus (2000). These species appear to be mainly opportunistsspecies are handled in a biohazard cabinet. Many and pose little threat to healthy people. Careful hand-types of fungal spores are allergenic or carry myco- ling and good housekeeping are all that are required.toxins. Inhalation of fungal spores should be However, immunocompromised individuals areavoided as far as possible. in a different category. It is increasingly evident Of the fungi described in this book, only the Asper- that such people have little resistance to fungalgilli normally pose any direct threat to health. Asper- infection. Persons suspected to be immunocompro-gillus fumigatus has already been mentioned, and care mised, regardless of the cause, should not work inshould also be taken when handling cultures of a mycology laboratory nor indeed be permittedNeosartorya species, which are closely related to knowingly to enter one.
    • Chapter 5Primary Keys and Miscellaneous FungiPrinciples underlying fungal classification have become astonishingly complex in recent years; for-been outlined in Chapter 3, including a brief over- tunately most of it is not essential for the recogni-view of the relevant divisions of the Kingdom Fungi tion of the genera discussed in this text. Termsand their principal methods of reproduction. Some which are important in the keys which follow arefurther detailed information is necessary in this described below.chapter to assist in the use of the keys which follow. A fundamental division within the asexual fungi Ascomycetes. As discussed in Chapter 3, Asco- separates genera which form conidia aerially,mycetes produce ascospores in asci (Fig. 3.2). One grouped in the class Hyphomycetes, from those ingenus, Byssochlamys, produces asci which are unen- which conidia are borne in some sort of envelopingclosed; all other genera of relevance here produce body, the class Coelomycetes.asci in some kind of fruiting body, or ascocarp. The Hyphomycetes. Fungi have developed seeminglytwo kinds of ascocarp commonly seen in food spoi- endless ways of extruding or cutting off conidia,lage fungi, the cleistothecium and the gymnothe- solitarily or in chains, from fertile cells which them-cium, have been described and illustrated in selves may be borne solitarily or aggregated intoChapter 3 (Fig. 3.3). Both types of ascocarp are more or less ordered structures. Hyphomycete tax-usually pale or brightly coloured, not dark, and onomy attempts to thread a way through this maze.release ascospores by rupturing irregularly. Of gen- In general, type and degree of aggregation of theera relevant here, cleistothecia are produced by fertile cells, and type of conidium, provides the basisEmericella, Eurotium, Eupenicillium, Monascus and for generic classification, while details of these char-Neosartorya, and gymnothecia by Talaromyces. acters and of spore size, shape and ornamentation A third class of ascocarp, less commonly encoun- are more commonly used to distinguish species.tered in foodborne fungi, is the perithecium. Peri- Features of conidia used in the keys in this workthecia have cellular walls like cleistothecia, but are are length, septation, ornamentation and colour,distinguished by the presence of an apical pore or particularly whether walls are light or dark. Theostiole through which asci or ascospores are liber- method of conidium formation (ontogeny) is sel-ated; also asci are long and clavate with ascospores dom emphasised here, because terminology is com-arranged linearly within them. In the one perithecial plex and distinctions may not be obvious. The prin-genus of interest here, Chaetomium, the perithecia cipal point to note is the disposition of conidia: theyare black and have stout hyphae attached to the may be borne solitarily, i.e. just one conidium perwalls (Fig. 5.10). point of production; singly, i.e. successively from a Conidial fungi. The strictly conidial fungi, also single point, but unattached to each other; or inknown as Fungi imperfecti or Deuteromycetes or chains. Solitary conidia are borne on a relativelyanamorphic fungi, possess an amazing variety of broad base and usually adhere to the fertile cell.ways of producing conidia. Terminology for struc- Conidia formed in chains are usually extrudedtures bearing conidia and for conidia themselves has from a small cell of determinate length, often aJ.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_5, 53Ó Springer ScienceþBusiness Media, LLC 2009
    • 54 5 Primary Keys and Miscellaneous Fungiphialide, which in most genera narrows to a distinct or less spherical body with one or more poresneck. Conidia borne singly may be extruded in this (ostioles) through which conidia are released, andsame manner, or be borne by extrusion from a pore the acervulus, a flat body from which conidia arein a hypha or fertile cell, or be cut off by hyphal released by lifting or rupturing of a lid. The majorityfragmentation. of Coelomycetes are pathogens on plants and many Phialidic Hyphomycetes. Hyphomycetes may have not been studied in pure culture. In consequence,produce phialides solitarily (the genus Acremonium) their taxonomy is difficult and genera and species areor in loosely ordered structures (Trichoderma) or often poorly delimited. For a complete account ofhighly ordered structures (Aspergillus, Penicillium Coelomycete taxonomy see Sutton (1980).and related genera). Genera of interest here with Yeasts. Yeasts are fungi which have developedless ordered phialidic structures can mostly be differ- the ability to reproduce by forming single vegetativeentiated by macroscopic characters, e.g. colony dia- cells by budding or, in a few species, by fission, in ameters and colours. However, differentiating genera manner similar to bacteria. Like bacteria, andwith highly ordered phialidic structures will necessi- unlike fungal spores, such cells are metabolicallytate careful microscopic examination. Phialides in active and may in turn reproduce by budding (orAspergillus, Penicillium and related genera are some- fission). Yeast cells may survive for long periodstimes borne directly on a stalk or stipe which arises both in culture and in nature. In consequencefrom a hypha; in other species, however, the phia- many yeasts produce true spores rarely or not at all.lides are borne from supporting cells, termed metulae Yeasts are readily distinguished from filamen-(sing. metula) and in some species the metulae may in tous fungi on the agar plate by their soft-texturedturn be supported by other cells, termed branches or colonies and limited growth. They are usually alsorami (sing. ramus). The whole structure, including readily distinguished from bacteria by their raisedthe stipe, is called a conidiophore. and often hemispherical colonies, white or pink In Aspergillus, stipes are always robust, with thick colours and lack of ‘‘bacterial’’ odour. If in doubt,walls and usually without septa; the stipe terminates in make a simple wet mount of a colony in water ora more or less spherical swelling, the vesicle, which lactofuchsin, add a cover slip and examine with thebears phialides, or metulae and phialides, over most oil immersion lens. Yeast cells are larger than bac-of its surface. In Aspergillus, phialides (and metulae) teria, measuring at least 3 Â 2 mm and are nonuni-are always produced simultaneously, and this feature form in size. If the culture is not too old, some cellscan readily be recognised by examining young devel- will usually show developing buds.oping conidiophores (Fig. 8.1a). Similar structures, Yeasts cannot be classified solely by morphologi-though smaller, are produced by some Penicillium cal features or growth on the standard media, and sospecies: these are clearly distinguished from Aspergillus are considered in a separate chapter (Chapter 10).species by stipes which are septate and by phialideswhich are produced over a period of time (successively;Fig. 8.2b). Most Penicillium species, and those of 5.1 The General Keyrelated genera, do not produce phialides on vesicles,but in a cluster directly on a stipe, or on metulae and/or The taxonomic terms discussed above will enablerami. The fruiting structure in Penicillium and related the use of the general and miscellaneous keys whichgenera is termed a penicillus, while that in Aspergillus follow, although some other taxonomic terms may(for want of a better term) is called a head (or more be introduced in discussions of particular genera. It isrecently, an aspergillum, Klich, 2002). emphasised that these keys are designed for use on Coelomycetes. As noted earlier, Coelomycetes isolates which have been incubated for 7 days onproduce conidia within an enveloping body, termed the standard plating regimen outlined in Chapter 4.a conidioma (pl. conidiomata). In Petri dish culture, Colony diameters should be measured in millimetresconidiomata are produced on or just under the agar from the reverse side by transmitted light. The gen-surface and are macroscopically visible, usually eral key has been designed to be as simple as possiblebeing 100–500 mm in diameter. Two kinds of con- and suitable for routine use, but it should be read inidioma are important here: the pycnidium, a more conjunction with the notes below it.
    • 5.1 The General Key 55 General key to food spoilage fungi1 No growth on any standard medium in 7 days Chapter 9 – ‘‘Xerophilic fungi’’ Growth on one or more standard media 22 (1) Colonies yeasts, either recognisably so on isolation or in culture, i.e. colonies soft, not exceeding 10 mm diam on any standard medium Chapter 10 – ‘‘Yeasts’’ Growth filamentous, exceeding 10 mm diam on one or more standard media 33 (2) Growth on CYA and/or MEA faster than on G25N 4 Growth on G25N faster than on CYA and MEA Chapter 9 – ‘‘Xerophilic fungi’’4 (3) Hyphae frequently and conspicuously septate 5 Hyphae lacking septa or septa rare Chapter 6 – ‘‘Zygomycetes’’5 (4) No mature spores present in 7 days 6 Mature spores present in 7 days 96 (5) Immature fruiting structures of some kind present 7 No fruiting structures (or spores) detectable by low- power microscopy or wet mounts from CYA or MEA See section on ‘‘Miscellaneous fungi’’ below7 (6) Colonies and fruiting structures white or brightly coloured 8 Colonies or fruiting structures dark Continue incubation; when spores mature, refer to section on ‘‘Miscellaneous fungi’’ below8 (7) Colonies and fruiting structures white Chapter 8 – ‘‘Aspergillus and its teleomorphs’’ Colonies or fruiting structures brightly coloured Chapter 7 – ‘‘Penicillium and related genera’’9 (5) Spores (conidia) less than 10 mm long, borne in chains on clustered fertile cells (phialides), on well-defined stipes 10 Spores (conidia) of various sizes, borne singly or solitarily, or if borne in chains, then chains not in aggregates See section on ‘‘Miscellaneous fungi’’ below10 (9) Phialides or metulae and phialides borne on more or less spherical swellings on the stipe apices 11 Phialides borne on penicilli, i.e. on unswollen stipes with or without intervening metulae and rami Chapter 7 – ‘‘Penicillium and related genera’’11 (10) Conidia blue or green, phialides produced successively on vesicles, vesicles less than 10 mm diam, stipes usually septate Chapter 7 – ‘‘Penicillium and related genera’’ Conidia variously coloured, phialides and/or metulae produced simultaneously on vesicles, vesicles larger than 10 mm diam, stipes nonseptate Chapter 8 – ‘‘Aspergillus and its teleomorphs’’5.1.1 Notes on the General Key isolates are usually white or rarely golden brown. If the original culture used as inoculum is colouredCouplet 1. No growth on any standard medium indi- other than pure white or golden brown, it is prob-cates an extreme xerophile, i.e. Xeromyces bisporus or ably nonviable.a Chrysosporium species, or a nonviable culture. Next Couplet 2. Yeasts are usually readily distinguishedinoculate culture onto MY50G for 7 days at 258C. If by slow growth, soft, easily sampled colonies, smallgrowth occurs, enter the key in Chapter 9, ‘‘Xerophi- spherical to ellipsoidal cells, often of variable sizelic Fungi’’; no growth on MY50G indicates a non- and shape and by reproduction by budding. Seeviable culture. Chrysosporium and Xeromyces Chapter 10, ‘‘Yeasts’’ for identification procedures.
    • 56 5 Primary Keys and Miscellaneous Fungi Couplet 3. The ability to grow more rapidly on A finely drawn glass needle will sometimes be ofG25N than on CYA or MEA indicates a xerophile assistance in making mounts from delicate conidial(at least for keying purposes here). Check the key in structures on the colony.Chapter 9, ‘‘Xerophilic Fungi’’. Some isolates can be Nearly all dark fruiting structures encounteredidentified from the standard plates, while others will mature at 258C within 2 weeks. Light does notwill require growth on CY20S or MY50G for usually influence this process. When mature sporesidentification. are formed, refer to the following section. Couplet 4. The absence of septa in young, grow-ing hyphae indicates an isolate belongs to subking-dom Zygomycotina, discussed here in Chapter 6 5.2 Miscellaneous Fungi‘‘Zygomycetes’’. Couplets 5, 6. Some isolates from a variety offungal genera will not produce spores on the stan- In this section are considered the genera which dodard media in 7 days. Continue to incubate such not logically fit into some larger grouping consid-cultures, preferably in diffuse daylight such as a ered elsewhere. Some are important in specific foodlaboratory window sill, at temperatures near 258C. spoilage problems, others are found in particularAlso inoculate such cultures onto two or three habitats such as cereals, while still others representplates of DCMA and incubate these at 258C or the aerially dispersed fungal flora found as ubiqui-thereabouts in darkness and in diffuse daylight or, tous contaminants or saprophytes. As will be seen,if possible, under fluorescent illumination (see they are a very heterogeneous collection.Chapter 4). After 1–2 weeks, check again for spores Most fungi significant in food spoilage or foodor fruiting bodies. If such structures are not seen, contamination and not treated in other chapters arethe isolate is unlikely to be significant in foods. included here. It is inevitable, though, that occasional Apparently asporogenous cultures should also isolates from foods will not belong to the generabe checked with a stereomicroscope while scraping considered in this section. The key has not beenup a sector of the colony with a needle. Fruiting designed to take account of this, as it would be abodies submerged in the agar will sometimes practical impossibility. So when an isolate appears tobecome visible with this technique. key out satisfactorily, it must be checked against the Couplet 7. Some isolates which produce white or description to confirm the identification. Some iso-brightly coloured fruiting bodies also produce very lates will of course belong to a recognisable genus,sparse aerial conidial structures which are easily but not the species described; in that case the refer-overlooked. Check such cultures carefully with the ences indicated will provide further information.stereomicroscope; if conidial structures are found, The miscellaneous fungal genera are consideredmake a wet mount and reenter the key at Couplet 5. in alphabetical order following the key. Key to miscellaneous genera1 Colonies on CYA and MEA not exceeding 60 mm diam in 7 days 2 Colonies on CYA or MEA exceeding 60 mm diam in 7 days 122 (1) Conidia borne within a fruiting body on or beneath the agar surface Phoma Conidia borne from aerial or surface hyphae 33 (2) Mycelium and conidia hyaline or brightly coloured 4 Mycelium and/or conidia dark coloured 114 (3) Conidia with a single lateral septum Trichothecium Conidia nonseptate or with more than one septum 55 (4) Conidia borne from gradually tapering fertile cells (phialides) 6 Conidia borne directly on hyphae or by budding or hyphal fragmentation 7
    • 5.2 Miscellaneous Fungi 576 (5) Colonies exceeding 50 mm diam on CYA Fusarium Colonies not exceeding 50 mm diam on CYA Acremonium7 (5) Colonies exceeding 45 mm diam on MEA; conidia borne solely by the breakup of hyphae to form arthroconidia Geotrichum Colonies not exceeding 40 mm diam on MEA; conidia not exclusively arthroconidia 88 (7) Conidia exceeding 12 mm long; developing cleistothecia, fist-like on arm-like stalks, also present Monascus Conidia not exceeding 12 mm long; no evidence of cleistothecial development 99 (8) Conidia yeast-like, borne on spicules (small projections) from hyphae, or by budding; conidia 5 mm or less long Endomyces Some conidia yeast-like, borne by budding; hyphal fragments, arthroconidia and/or chlamydoconidia also present; conidia >5 mm long 1010 (9) Budding hyphal fragments (10–50 mm long) present Hyphopichia Hyphal fragments absent; or if present, not budding Moniliella11 (3) Colonies low, mucoid and yeast-like, becoming grey to black in both obverse and reverse Aureobasidium Colonies dry and velutinous, obverse green, reverse olive or deep blue black Cladosporium12 (1) Spores borne within an enclosed fruiting body on or under the agar surface 13 Spores borne from aerial or surface hyphae 1713 (12) Spores consistently less than 15 mm long 14 The larger or all spores more than 15 mm long 1514 (13) Fruiting bodies perithecia with stout, black hyphae attached to the walls Chaetomium Fruiting bodies pycnidia, without attached hyphae Phoma15 (13) Fruiting bodies roughly spherical (pycnidia) Lasiodiplodia Fruiting bodies flat (acervuli) 1616 (15) Conidia hyaline or brightly coloured, nonseptate, without terminal appendages Colletotrichum Conidia dark, with three or four septa and with spike-like, sometimes branched, terminal appendages Pestalotiopsis17 (12) Colonies and conidia hyaline or brightly coloured 18 Colonies and/or conidia dark coloured 2218 (17) Colonies with grey or green areas 19 Colonies white, orange, pink or purple 2019 (18) Colonies green Trichoderma Colonies grey Botrytis20 (18) Colonies low and persistently white Geotrichum Colonies floccose, white or becoming brightly coloured 2121 (20) Colonies predominantly orange, orange conidia shed profusely around the Petri dish rim Chrysonilia Colonies white, pink or purple, sporulation on MEA weak or absent, better on DCPA under lights Fusarium22 (17) Conidia consistently less than 15 mm long 23 Conidia frequently exceeding 15 mm long 25
    • 58 5 Primary Keys and Miscellaneous Fungi23 (22) Conidiophores long, branched, apically swollen, bearing closely packed pale brown conidia Botrytis Conidiophores short or ill-defined, dark brown or black conidia borne irregularly 2424 (23) Conidia dark brown, often with a lighter coloured band around the periphery Arthrinium Conidia uniformly jet black Nigrospora25 (22) Conidia approximately spherical Epicoccum Conidia elongate 2626 (25) Conidia with transverse septa (or thick walls between cells) only 27 Larger conidia with both transverse and longitudinal septa or irregularly septate 3027 (26) Conidia clavate (club shaped), often with long hyphal appendages at the apices; found almost exclusively on rice Trichoconiella Conidia cylindroidal, ellipsoidal or an elongate ‘‘D’’ shape; source more general 2828 (27) Conidia cylindroidal, with parallel sides except at the terminal cells Drechslera Conidia fusoid, narrowing from the central cells to the terminal cells, often bent or ‘‘D’’ shaped 2929 (28) Conidia not exceeding 40 mm long Curvularia Conidia usually exceeding 40 mm long Bipolaris30 (26) Conidia clavate (club shaped) Alternaria Conidia spherical to roughly ellipsoidal or short cylindrical 3131 (30) Conidia often tapering towards the base and sometimes pointed, i.e. pyriform or apiculate Ulocladium Conidia spherical to short cylindrical, not tapering from base to apex, with rounded ends Stemphylium5.3 Genus Acremonium Link Acremonium strictum W. Gams Fig. 5.1 Cephalosporium acremonium (name of uncertain application; noCommonly referred to as Cephalosporium Corda in valid authority).pre-1970 literature, Acremonium is a large and variedgenus characterised by the production of small, hyaline, Colonies on CYA 20–30 mm diam, white or orangesingle-celled conidia borne singly, i.e. successively but to pink, dense to floccose or funiculose; reversenot connected to each other, from solitary phialides. pale or with orange to pink tones. Colonies on A variety of species have been recorded from foods MEA 13–20 mm diam, similar to those on CYAfrom time to time. One species, A. strictum, described or of slimy texture. Colonies on G25N <5 mmhere, is of relatively common occurrence. In this spe- diam, usually 1–2 mm, of white mycelium. Some-cies, the phialides gradually taper to the apices without times growth at 58C. No growth at 378C.basal thickening or formation of a distinct neck, and Conidia borne successively but singly from theconidia aggregate in balls of slime. Under the stereo- apices of long, solitary, usually unbranched phia-microscope, the slime balls look like large single lides, aggregating in a slime ball at the phialide tip,spores, but their true nature becomes evident in wet cylindrical to ellipsoidal, hyaline, 3–6 Â 1–2 mm,mounts. A. strictum appears mainly in the earlier food smooth walled.literature under the name C. acremonium. As noted by Distinctive features. See genus preamble.Domsch et al. (1980), this name has been used for a Taxonomy. As noted above, Acremonium strictumvariety of species, so that reports on physiology and is the correct name for the fungus commonly calledoccurrence are unreliable (Fig. 5.1). C. acremonium in the earlier food literature (Gams, 1971).
    • 5.3 Genus Acremonium 59Fig. 5.1 Acremonium strictum (a) colonies on CYA and MEA, 7 d, 258C ; (b, c) phialides, bar = 10 mm; (d) conidia, bar = 5 mm Physiology. Mycelial growth and sporulation of isolated from black bean seeds in Argentina (Cas-Acremonium strictum were found to be enhanced by tillo et al., 2004) and raw cork (Quercus suber) inthe addition of mannitol to MEA (Teixeira et al., Spain (Alvarez-Rodriguez et al., 2002). Other2005). records, including those of Acremonium spp. or the Mycotoxins. A. strictum and other Acremonium earlier name Cephalosporium acremonium, includespecies encountered in foods are not known to wheat, barley and rice, bananas showing crown rot,produce mycotoxins. fresh vegetables, peanuts, pecans, hazelnuts and wal- Ecology. The main source of Acremonium stric- nuts, soybeans, frozen meat, salami and biltong (Pitttum is maize (Pitt et al., 1993; Pitt and Hocking, and Hocking, 1997).1997; Williams et al., 1992). It has recently been References. Gams (1971); Domsch et al. (1980).
    • 60 5 Primary Keys and Miscellaneous Fungi5.4 Genus Alternaria Nees: Fr. Trichoconiella forms conidia similar to those of Alternaria, but with transverse septa only. ThisAlternaria produces large brown conidia with both genus is associated with rice.longitudinal and transverse septa, borne from Emory Simmons has spent a lifetime workinginconspicuous conidiophores, and with a distinct with Alternaria and related genera and has recentlyconical narrowing or ‘‘beak’’ at the apical end. revised the taxonomy of Alternaria comprehen-These conidia are often formed in chains. Two sively (Simmons, 2007). The simple notions ofother genera, Stemphylium Wallroth and Ulocla- earlier authors, i.e. that Alternaria alternata isdium Preuss, produce similarly septate conidia, but everywhere and that specific plants were colonisedare uncommon in foods. Those of Ulocladium are by specific pathogens, have been painstakinglyroughly ellipsoidal, usually somewhat narrower at replaced by a much more robust taxonomy. Forthe base, and at most have small beaks. Both Alter- example, Alternaria from citrus fruits may be Alter-naria and Ulocladium produce new conidia by lat- naria citri, but may also be one of 20 other species.eral growth of the conidiophore from near the apex The monograph by Simmons (2007) includes 276or through the beak area. Stemphylium conidia species and is an essential laboratory manual forlook like those of Ulocladium, but new conidia anyone who wishes to identify Alternaria speciesnever form from a beak. New conidia are produced from a range of sources. Alternaria is not an easythrough the pore from which a conidium has genus for the inexperienced, however. Treatmentalready been produced. For a more detailed here is confined to three common species only,account of the distinctions among these three gen- with cereals as their major source.era, see Simmons (1967). Key to included species1. Conidia produced in unbranched chains on DCMA Alternaria tenuissima (see A. alternata) Conidia produced in branched clusters on DCMA 22. Conidia in unbroken chains, i.e. narrow hyphal elements between spores lacking; species occurring on wheat and many other substrates A. alternata Primary conidia producing secondary conidia, i.e. conidia often separated by short hyphal lengths; species usually occurring on wheat A. infectoriaAlternaria alternata (Fr.) Keissl. Fig. 5.2 chains of up to 10 units, and then septating bothAlternaria tenuis Nees laterally and longitudinally, with up to six transverse and two to three longitudinal or oblique septa,Colonies on CYA and MEA 50–70 mm diam, or usually of clavate or pyriform shape overall, taperingcovering the whole Petri dish, plane, of deeply floc- towards the apices, forming a short beak, in culturecose off-white to grey brown mycelium; reverse brown usually 20–40 Â 8–12 mm, with walls smooth to con-to nearly black. Colonies on G25N 10–15 mm diam, spicuously roughened.low and dense, olive brown or grey; reverse brown Distinctive features. A. alternata is the most com-to almost black. At 58C, at least microcolonies, mon saprophytic Alternaria species, occurring on aoften colonies up to 4 mm diam. Usually no growth wide variety of sources, in contrast to other speciesat 378C, occasionally colonies up to 10 mm diam, considered here. On DCMA, conidia are producedsimilar in appearance to those at 258C, or white. more or less uniformly above the agar surface, in Colonies on DCMA 60–70 mm diam or covering quite lengthy chains. The larger conidia have boththe whole Petri dish, plane, floccose but not sparse, longitudinal and transverse septa.mycelium grey to dark grey; reverse grey to dark Taxonomy. The first edition of this book notedgrey or black, less commonly bluish black. that with acceptance of the 1821 starting point date Conidia blown out from the apices of undistin- for Alternaria nomenclature, A. alternata slowlyguished conidiophores as short, irregularly branched became accepted as the correct name for this
    • 5.4 Genus Alternaria 61 a b cFig. 5.2 Alternaria alternata (a) colonies on PDA and DCMA, 7 d, 258C ; (b) conidia in situ, bar ¼ 50 mm; (c) conidia, bar ¼ 20 mmspecies. With the reversion to be the 1753 starting (v/v) in N2, with growth rates being proportionalpoint date, the correct name again became A. tenuis. to oxygen concentration (Follstad, 1966; WellsHowever, publications by Fries, 1821–1832, were and Uota, 1970). The presence of the volatilelater given special status under the ICBN, so compounds 3-methyl-1-butanol, 1-octen-3-ol and 3-A. alternata is once more the correct name. Further octanone in stored wheat grains is usually correlatedchanges appear unlikely. with infection by A. alternata, amongst other fungi Physiology. Optimum growth of Alternaria alter- (Sinha et al., 1988).nata is near 258C with minima variously reported as – Mycotoxins. Alternaria alternata produces sev-5 to 6.58C and maxima near 368C (Hasija, 1970; eral toxins, of which the most important is tenua-Domsch et al., 1980). The minimum aw for growth zonic acid (TA) (Logrieco et al., 2003). Tenuzoicat 258C is 0.86 (Hocking et al., 1994). Optimal acid is toxic to a wide range of plants and animals,growth occurs at pH 4–5.4, and the pH range for particularly mice, chick embryos and chickensgrowth is 2.7–8.0 (Hasija, 1970). A. alternata is able (Logrieco et al., 2003). Sorghum grains containingto grow in oxygen concentrations as low as 0.25% high levels of TA were associated with the human
    • 62 5 Primary Keys and Miscellaneous Fungihaematological disorder known as onyalay quoted in Pitt and Hocking, 1997), but also in(Bottalico and Logrieco, 1998). Less toxic com- rice, rapeseed, sunflower seed (Pozzi et al., 2005)pounds include alternariol (AOH) and alternariol and sorghum (Fakhrunnisa et al., 2006). In Aus-monomethyl ether (AME), altenuene (AE) and tralia and Southeast Asia, A. alternata has causedaltertoxin I, II and III (Logrieco et al., 2003). severe damage, spoilage and mycotoxin productionAOH and AME are usually found in combination in wheat and sorghum (Pitt et al., 1994; Webleyand have some teratogenic and fetotoxic effects in et al., 1997 and our unpublished observations).pregnant mice (Bottalico and Logrieco, 1998). The This species has been recorded quite frequentlyproduction of one or more of these toxins has been from nuts, including peanuts, hazelnuts and pecansreported in tomatoes (Bottalico and Logrieco, (see Pitt and Hocking, 1997). Other sources include1998), citrus fruits (Logrieco et al., 1990a), capsi- soybeans, cold stored and frozen meat, biltong,cum and melons (Bottalico and Logrieco, 2001), bottled water and spices (Pitt and Hocking, 1997;cereal grains (Logrieco et al., 1990b; Webley et al., Cabral and Fernadez Pinto, 2002). This species has1997), rapeseed and olives (Logrieco et al., 2003), and occasionally been associated with gushing in beerapples (Robiglio and Lopez, 1995; Tournas and (Niessen et al., 1992).Stack, 2001). Maximum production of alternariol, its Closely related species. Alternaria tenuissimamonomethyl ether and altenuene occurred at 258C (Kunze) Wiltshire differs from A. alternata by theand 0.98 aw (Magan et al., 1984). However, the opti- formation of longer conidia (up to 60 mm) inmum for tenuazonic acid production was reported as unbranched chains (Simmons, 2007). The metabo-0.90 aw at 258C (Etcheverry et al., 1994). Altertoxins I, lite profiles of A. alternata and A. tenuissima areII and III are produced by many Alternaria species, very similar. In a recent study, it was found thathowever, A. alternata is the most important producer. most A. tenuissima isolates examined producedAltertoxins have received much attention due to their alternariol, alternariol monomethyl ether and tenu-mutagenic activity, particularly that of altertoxin III, zoic acid, whereas three of the A. alternata isolateswhose mutagenicity is approximately ten times lower produced altenuene, alternariol, alternariol mono-than that of aflatoxin B1 (Bottalico and Logrieco, methyl ether and altertoxin I (Andersen et al, 2002).1998). Andersen and Frisvad (2004) reported A. tenuissima Ecology. Under the names Alternaria alternata and from mouldy tomatoes. In our experience this spe-A. tenuis, this species has been reported from a very cies is of common occurrence in cereals.wide range of foods. However, the very broad species References. Simmons (1967, 2007); Ellis (1971);concept used by many authors is not accepted by Domsch et al. (1980).Simmons (2007). The following reports undoubtedlyinclude a number of other species. It is a major patho-gen of fresh tomatoes (Harwig et al., 1979; Zitter and Alternaria infectoria E.G. Simmons Fig. 5.3Wien, 1984; Pose et al., 2004), though other Alter-naria species are probably involved here too. Spoilage Colonies on CYA and MEA 60 mm diam or more,of eggplants and peppers (Snowdon, 1991), apples on CYA plane, low to moderately deep, mycelium(Combrink et al., 1985) and bananas stored under dark grey to almost black, sometimes with a palermodified atmospheres (Wade et al., 1993) have been grey floccose overlay; reverse bluish black; on MEAreported. Damage may occur also to a wide variety of moderately deep to deep, dense to floccose, of darkfresh vegetables including beans, cauliflowers, grey mycelium sometimes overlaid with lighter greycucumbers, melons, peas and potatoes (Webb and hyphae; reverse bluish black. On G25N, coloniesMundt, 1978; Snowdon, 1991). A. alternata has 8–15 mm diam, of grey or greenish grey mycelium;been recorded frequently from a wide range of cer- reverse pinkish, grey or black. At 58C, at least ger-eals, particularly wheat (Pitt et al., 1998b; Clear et al., mination, usually colonies of 2–5 mm diam formed.2005, Fakhrunnisa et al., 2006; Lugauskas et al., At 378C, colonies 5–25 mm diam, low to deep, grey,2006 and references quoted in Pitt and Hocking, reverse dark.1997), barley (Andersen et al., 1996; Fakhrunnisa Colonies on DCMA 60 mm or more diam,et al., 2006; Medina et al., 2006 and references usually with wide margins, low to subsurface,
    • 5.4 Genus Alternaria 63 a b cFig. 5.3 Alternaria infectoria (a) colonies on PDA and DCMA, 7 d, 258C ; (b) conidia, bar ¼ 50 mm; (c) conidia, bar ¼ 20 mmcentrally usually sparse and floccose, aerial myce- irregularly septate, walls and septa thick and darklium white to pale olive brown; reverse pale brown, brown, and with walls sometimes conspicuouslybrown or olive brown, sometimes darker in annular roughened.or irregular patches. Distinctive features. After growth on DCMA, Conidia on DCMA observed under the stereo- Alternaria infectoria is readily distinguished frommicroscope characteristically formed in short, irre- A. alternata and other Alternaria species under thegularly branched chains, forming clusters, with pri- stereomicroscope. A. infectoria forms clusters ofmary conidia borne from knobby hyphae, usually conidia borne in irregularly branching chains that,bearing secondary conidia borne from the tips, at unlike those of A. alternata, have short hyphal seg-the end of short (30–100 mm) hyphal lengths; con- ments between the conidia. Colonies on DCMAidia characteristically clavate (clubshaped) 20–50 Â have low, wide margins, are less floccose than7–14 mm, with up to 7 transverse septa and 1 or those of A. alternata and (apart from conidia)more longitudinal septa as well, or sometimes have paler colours.
    • 64 5 Primary Keys and Miscellaneous Fungi Taxonomy. This species was commonly known A. infectoria more likely reflects failure by mostas the Alternaria state of Pleospora infectoria until investigators to differentiate Alternaria species,Simmons (1986) provided a binomial name, rather than true rarity. It seems probable that thisA. infectoria. He showed that its teleomorph does species may be confined to small grains.not belong in Pleospora, as had been widely References. Simmons (1986, 2007).accepted, and erected a new genus, Lewia Barr andSimmons, for it (Simmons, 1986). Simmons (1986)reported that Alternaria species related to A. infec- 5.5 Genus Arthrinium Kunzetoria have very rarely been seen to produce a tele-omorph; however, on several occasions we haveseen Lewia teleomorphs in isolates of A. infectoria Arthrinium, in earlier literature more commonlygrown on autoclaved wheat grains. known by its synonym Papularia, is not a common Physiology. No physiological studies on this spe- genus in foods, but has occasionally caused spoi-cies have been reported. lage. Arthrinium produces relatively large, dark- Mycotoxins. Alternaria infectoria is chemically walled (but not black) conidia borne solitarily,very different from other Alternaria species (Ander- both terminally and laterally, on short, narrow con-sen et al., 2002). A. infectoria does not produce any idiophores. Two species are treated here, A. phaeos-of the known Alternaria metabolites (Andersen and permum and the Arthrinium state of Apiospora mon-Thrane, 1996) and shares only a few metabolites tagnei, distinguished from each other by conidialwith other Alternaria species (Andersen et al., size.2002). We have confirmed the absence of any toxinproduction by this species (unpublished). Arthrinium phaeospermum (Corda) Ecology. Alternaria infectoria is most commonly M.B. Ellis Fig. 5.4associated with grains, having been reported from Papularia sphaerosperma (Pers.) Hohn. ¨small grains in the United States (Bruce et al., 1984),Australian wheat (Pitt et al., 1998b), Danish barley Colonies on CYA and MEA covering the whole Petri(Andersen et al., 1996) and Norwegian grains dish, mycelium low or floccose, coloured white or(Kosiak et al., 2004). The lack of other reports of grey, sometimes with conspicuous areas of pink,Fig. 5.4 Arthrinium phaeospermum (a) colony on CYA, 7 d, 258C; (b) conidia, bar ¼ 10 mm
    • 5.6 Genus Aureobasidium 65darkening in age; reverse yellow or brown. Colonies on sweet potatoes (Ravichandran and Sullia, 1983).on G25N 10–18 mm diam, of white mycelium. Some- We isolated A. phaeospermum at low levels fromtimes germination at 58C. No growth at 378C. paddy rice, mung beans, soybeans, black beans and Reproduction by solitary conidia, blown out cashews in Thailand (Pitt et al., 1993).from the ends of, or from denticles on the sides of, Closely related species. The Arthrinium state ofshort, narrow, sometimes sinuous conidiophores, Apiospora montagnei Sacc., also known as Papulariathemselves borne in clusters from mother cells on arundinis (Corda) Fr., is similar to A. phaeospermum innatural substrates, but often singly in culture; con- all characters examined, except for the production ofidia circular in plan view but elliptical from the side, smaller conidia, 6–8 mm long. Data sheets at the Inter-8–12 Â 5–7 mm, dark brown, smooth walled, often national Mycological Institute, Egham, Surrey, UK,with a narrow, hyaline band around the longest record its isolation from white flour and molasses; itperiphery. has also been reported from wheat (Pelhate, 1968), Distinctive features. The conidium of Arthrinium barley (Flannigan, 1969) and cashews (Pitt et al.,is distinctive: solitary, dark-walled, circular in plan 1993).but elliptical from the side, and often with a hyaline References. Ellis (1971); Domsch et al. (1980).peripheral band. Cultivation on DCPA and underlights may assist sporulation. In the present context,Arthrinium is distinguished from Nigrospora by the 5.6 Genus Aureobasidium Vialalatter’s production of jet black conidia entirely and G. Boyerdevoid of ornamentation. Physiology. Conidia of Arthrinium appear to be Growth of Aureobasidium isolates is at first yeast-like,highly heat resistant. In apple juice, conidia sur- but, while remaining very low and mucoid, coloniesvived a pasteurising process of 888C for 1.5 min, spread rapidly and turn black in patches. Microsco-and in water, heating at 1058C for 2.5 min (Anon, pically, hyphae as well as budding yeast-like cells are1967). No experimental details, i.e., numbers heated present; the latter are actually conidia. The conidiaor come-up time, were reported, however, and a are borne from small denticles (minute projections)decimal reduction time cannot be calculated. Even directly from the hyphal walls or from short lateralso, survival of a heat treatment at 888C indicates protrusions on the hyphae; characteristically 2–4 den-that A. phaeospermum is uncommonly heat resistant ticles on one cell will produce conidia synchronously.for a conidial fungus. Optimum germination of There are more than 10 species (Hermanides-Nijhof,A. phaeospermum occurs when the conidia are sus- 1977), but only one, Aureobasidium pullulans, is ofpended in sterile saline solution at 208C for 15 min importance in foods (Fig. 5.5).before plating onto 2% malt extract agar (Agut andCalvo, 2004). The authors also noted that the via-bility of Arthrinium conidia was low, making this Aureobasidium pullulans (de Bary)genus relatively rare in the environment. G. Arnaud Fig. 5.5 Mycotoxins. Arthrinium species are not known to Dematium pullulans de Baryproduce mycotoxins, however, reports of secondary Pullularia pullulans (de Bary) Berkhoutmetabolites with antimicrobial properties have beenfound for some Arthrinium species (quoted in Agut Colonies on CYA and MEA 25–35 mm diam, lowand Calvo, 2004). and mucoid, faintly pink, becoming grey to black in Ecology. Arthrinium phaeospermum (reported as areas at 7–10 days; reverse in similar colours. Colo-Papularia sphaerosperma) has been recorded as a nies on G25N 10–12 mm diam, similar to those oncause of spoilage of pasteurised apple juice (Anon., CYA. At 58C, microcolonies to colonies up to 3 mm1967). It has also been reported on barley, wheat flour, diam. No growth at 378C.rice, pecans and airline meals (see Pitt and Hocking, Conidia borne on denticles directly from hyphae1997). It was isolated from Morelo cherries (Olszak, or sometimes small lateral protrusions; conidia1994) and Iranian barley (Asgari et al., 2004). yeast-like, primary ones borne from the denticles,A. phaeospermum has also been recorded as a pathogen usually measuring 10–16 Â 3–6 mm, and secondary
    • 66 5 Primary Keys and Miscellaneous Fungi a b cFig. 5.5 Aureobasidium pullulans (a) colonies on CYA and MEA, 7 d, 258C; (b, c) conidia, bar ¼ 10 mmones by budding from the primaries, 7–10 Â 3–5 in low temperature habitats, suggests that somemm, not adhering to each other, smooth walled. strains grow down to –58C (Michener and Elliott, Distinctive features. Within the present context, 1964). It is of common occurrence in salterns (Buti-Aureobasidium pullulans is readily recognised by its nar et al., 2005a), so is likely to be a xerophile. Heatdistinctive low, mucoid, white then pink to black resistance is very low (Skou, 1969). The ability of A.colonies and yeast-like conidia. Several other genera pullulans to grow on sugars used in yeast taxonomyhave a similar appearance (Hermanides-Nijhof, has been reported by de Hoog and Yurlova (1994)1977; Beh, 2007) but they rarely occur in foods. and Beh (2007). Taxonomy. Earlier literature discusses this spe- Ecology. A ubiquitous saprophyte from all sortscies under the names Dematium pullulans and of moist and decaying environments, AureobasidiumPullularia pullulans. pullulans has been reported from a very wide range of Mycotoxins. Mycotoxins are not known to be foods, but only rarely as a cause of spoilage. Itsproduced. prevalence in frozen foods is noteworthy, being the Physiology. The temperature range for growth of predominant mould isolated from blueberry, appleAureobasidium pullulans has been reported as and cherry pies by Kuehn and Gunderson (1963). It2–358C, with an optimum of 258C (Skou, 1969), was the most frequently isolated mould in Canadianbut some earlier data, together with its abundance icewine must, but played no role in fermentating
    • 5.7 Genus Bipolaris 67must (Subden et al., 2003). The surface of healthy Bipolaris maydis (Nishik. and C. Miyake)grapes is predominately colonised by A. pullulans Shoemaker Fig. 5.6(Fleet, 2003a; Prakichaiwattana et al., 2004; Beh,2007). A. pullulans has been reported in yoghurt in Colonies on CYA and MEA 45–55 mm diam, plane,a survey of a Slovakian dairy plant (Pieckova et al., deeply floccose, of light to dark grey mycelium;2002) and has been associated with the spoilage of reverse dark brown to dark blue black. On G25N,cold stored meat and cheese (see Pitt and Hocking, colonies 2–6 mm diam. At 58C, germination. At 378C,1997). A. pullulans has been recorded in a variety of colonies 5–15 mm diam, usually low, dense andfresh foods and commodities including fresh vegeta- wrinkled, of grey mycelium, reverse deep blue black.bles, cabbage, strawberries, grapes, citrus and pas- Colonies on DCMA 30–40 mm diam, plane, ofteurised orange juice (see Pitt and Hocking, 1997). sparse to dense, floccose grey mycelium, reverseOther records include shrimp, green olives, barley, brown, grey or black.wheat and flour, oats and nuts (see Pitt and Hocking, Conidia borne singly from nodes on knobby or1997). geniculate mid to dark brown hyphae, usually References. Hermanides-Nijhof (1977); Domsch slightly curved, (40–)50–80 Â 12–16 mm, with sixet al. (1980); de Hoog and Yurlova (1994). to nine inconspicuous septa and with smooth, dark, thick walls. Distinctive features. Large conidia, often slightly curved, of fusoid shape and with several5.7 Genus Bipolaris Shoemaker thick transverse septa are characteristic of this genus. Conidial curvature and shape distinguishIn separating unrelated species from Helminthos- it from Drechslera, while conidial length and theporium, Shoemaker (1959) revised the genus presence of more than three, thick septa allow dis-Drechslera S. Ito and erected Bipolaris. The fact tinction from Curvularia. The species treated herethat species of Bipolaris (in the sense of Shoe- is common on maize.maker, 1959) were associated with two distinct Taxonomy. Some species of Bipolaris produceteleomorph types, and that correlated with mor- teleomorphs, classified in the genus Cochliobolus.phological differences, led to segregation of Exser- Physiology. Species of Bipolaris sporulate signif-ohilum from Bipolaris by Leonard and Suggs icantly better when grown on substrates containing(1974). These genera produce long, large conidia cellulose (Pratt, 2006).with transverse septa only; the septa are thick and Mycotoxins. No significant mycotoxins arequite different from those in e.g. Trichoconiella. In known.Drechslera, conidia are cylindrical and germinate Ecology. We have isolated Bipolaris maydisat any cell, while conidia of Bipolaris and Exser- from 1% of Thai maize kernels, and less fre-ohilum are fusoid, i.e. gradually narrowing, and quently from Thai sorghum and mung bean sam-conidia germinate only from the end cells (bipolar ples (Pitt et al., 1993, 1994). It was also found atgermination). In Bipolaris, germ tubes develop similar levels in commodities from Indonesia androughly along the line of the conidium axis, while the Philippines (Pitt et al., 1998a). Other recordsin Exserohilum, the germ tube is often offset from of Bipolaris in foods are rare. However, referencesthe axis and develops at an angle (Alcorn, 1983). to Drechslera species in foods often have referredExserohilum species are apparently rare in foods, to taxa correctly classified in Bipolaris, e.g. in rice,although E. rostratum (Drechsler) K.J. Leonard B. oryzae (Breda de Haan) Shoemaker; in barley,and Suggs has been reported from sorghum B. australiensis (M.B. Ellis) Tsuda and Uemaya,(Usha et al., 1994). B. maydis and B. setariae (Sawada) Shoemaker; in The literature on these genera is voluminous and peanuts, B. spicifera (Bainier) Subram.; in corian-complex. The most useful publications are by der seed, B. bicolor (Mitra) Shoemaker; and inAlcorn (1983) and Sivanesan (1987). One common avocados, B. setariae (see Pitt and Hocking,species of Bipolaris, B. maydis, is treated here 1997).(Fig. 5.6). References. Alcorn (1983); Sivanesan (1987).
    • 68 5 Primary Keys and Miscellaneous Fungi a b cFig. 5.6 Bipolaris maydis (a) colonies on MEA and DCMA, 7 d, 258C; (b, c) conidiophores and conidia, bars ¼ 10 mm5.8 Genus Botrytis P. Micheli: Fr. is known as Botrytis cinerea, although it is probable that this name includes a group of related speciesBotrytis is a common genus in the temperate zones, rather than a single well defined taxon (Coley-Smithwhere it occurs mainly as a pathogen on a variety of et al., 1980).plant crops. Vegetables and small berry fruits areparticularly susceptible. Invasion may occur beforematurity or postharvest, both in transport and in Botrytis cinerea Pers. Fig. 5.7storage. Onions and other Allium species and grapesare the most susceptible crops. In the latter, it is Colonies on CYA and MEA covering the wholenotable that the disease is sometimes encouraged. Petri dish, floccose, growth sometimes patchy orGrapes affected by Botrytis, in this circumstance irregular, mycelium white, becoming grey to darkcalled ‘‘the noble rot’’, are used in the production grey as conidiogenesis proceeds; reverse pale toof certain high quality sweet wines in France, Ger- grey. On G25N, colonies 10–18 mm diam, irregularmany, Australia and other countries. in outline, floccose centrally or in patches, becom- Botrytis species are characterised by the produc- ing grey; reverse grey. At 58C, colonies up to 5 mmtion of conidia on pegs from spherical swellings. diam produced, low and sparse. No growth atThe most commonly encountered species in foods 378C.
    • 5.8 Genus Botrytis 69Fig. 5.7 Botrytis cinerea (a) colony on CYA, 7 d, 258C; (b) conidiophores and conidia, bar = 25 mm Ecology. Botrytis cinerea is a virulent pathogen, Conidiophores borne from aerial hyphae, stipes the cause of rots in many kinds of fresh fruits (Snow-of indeterminate length, each bearing terminally an don, 1990). It is the most important cause of disease inirregular cluster of short branches, 10–30 mm long, grapes, both before harvest and in storage and thewith swollen spherical apices, 8–10 mm diam; con- most common cause of spoilage in berries such asidia borne singly from these apices on denticles strawberries, blueberries, raspberries and blackberries(small pegs), ellipsoidal, 8–12 mm long, smooth (Snowdon, 1990; Tournas and Katsoudas, 2005), andwalled, not released at maturity. also causes large losses in apples and pears, tomatoes, Distinctive features. Solitary conidia borne on stone fruits and kiwi fruit (see Pitt and Hocking,denticles from terminally swollen, short branches 1997). A gel-like turbidity can be caused in raspberryare characteristic of Botrytis. juice, making filtration difficult (Will et al., 1992). For Physiology. Growth has been reported at 0.93 aw a review of many aspects of occurrence, pathogenicity(Snow, 1949) and at 0.90 aw (Jarvis, 1977). Lahlali and control, see Elad et al. (2004).et al. (2007) observed no growth of Botrytis cinerea Although vegetable spoilage is perhaps lessat 0.93 aw in the presence of NaCl or at 0.89 aw in the important, the range of crops affected is verypresence of nonionic solute. Reported growth large, and includes asparagus, beans, cabbages, car-temperatures are rather variable, with minima rots, celery, melons and potatoes (Snowdon, 1991;from –2 to 58C or even 128C, maxima 28–358C, Lugauskas et al., 2005; Tournas, 2005). It has alsoand optima of 22–258C (Domsch et al., 1980). been isolated from a wide variety of dried or pro-B. cinerea will grow from pH 2–8 (Jarvis, 1977) and cessed foods, but here its role is uncertain and it isin O2 concentrations down to 1% (Follstad, 1966). probably present only as a contaminant.The effect of low temperatures and controlled atmo- Other important species. Several other species ofsphere storage on the growth of B. cinerea on suscep- Botrytis are commonly occurring pathogens. Oftible fruits and vegetables has been reported (Reyes, interest here are B. allii Munn, B. byssoides Walker1990). B. cinerea produces pectinases (polygalactur- and the Botrytis state of Sclerotinia porri vanonase) and the cell wall degrading enzymes cellulase Beyma, all of which cause spoilage of onions andand xylanase (El-Habbaa, 2003). leeks (Ellis, 1971; Snowdon, 1990). Mycotoxins. Mycotoxin production has not been Botrytis allii grows optimally at 20–258C, with areported – fortunately, in view of this species role in minimum below 58C and a maximum near 358C. Thespeciality wine production. optimum aw for growth is in excess of 0.99. Growth
    • 70 5 Primary Keys and Miscellaneous Fungioccurs down to 0.96 aw in media containing NaCl conidiomata (pycnidia); ascospores in Chaetomiumand 0.93 aw in media containing KCl or sucrose, or are often freed from the ascus before being dis-on onion leaves (Alderman and Lacy, 1984). charged singly from the perithecium, and hence References. Ellis (1971); Coley-Smith et al. could be mistaken for conidia. Chaetomium is readily(1980); Domsch et al. (1980); Elad et al. (2004). distinguished from Phoma by the presence of stout black or dark coloured hyphae attached to the peri- thecial walls, which are visible in culture under the low power microscope.5.9 Genus Chaetomium Kunze Chaetomium species are notable as producers of cellulases and so commonly occur on wood andChaetomium is the only genus of Ascomycetes which paper products. They are relatively common inproduces black ascocarps and which is commonly some kinds of foods. C. globosum is the most fre-encountered in foods. The ascocarps in Chaetomium quently encountered species and is described here asare perithecia, similar in appearance to cleistothecia, representative of the genus. In addition descriptionsbut having an opening (ostiole) through which the of C. brasiliense and C. funicola, both quite commonascospores are released. There is a superficial resem- in tropical commodities, are provided here.blance to the genus Phoma, which produces black Key to Chaetomium species included here1 Colonies on CYA at 378C much larger than at 258C C. brasiliense Colonies on CYA at 378C smaller than at 258C or absent 22 (1) Colonies on CYA and MEA at 258C less than 50 mm diam, ascospores 5–7 mm long C. funicola Colonies on CYA and MEA at 258C usually covering the whole Petri dish, ascospores 8–10 mm long C. globosumChaetomium brasiliense Bat. & subspheroidal to broadly ellipsoidal, 6–8 mm long,Pontual Fig. 5.8 brown, with smooth walls. Conidia not produced. Distinctive features. Unlike the other Chaetomium species considered here, C. brasiliense grows much more rapidly at 378C than 258C, and perithecia areColonies on CYA and MEA 25–35 mm diam, on formed in much greater numbers at the higher tem-CYA low to moderately deep and dense, of white to perature. Perithecia are black, but under the stereo-grey mycelium; reverse sometimes pale, more com- microscope appear surrounded by grey hyphae.monly dark grey to almost black; on MEA low and Ascospores are smaller than those of C. globosum.sparse, dark grey, with some paler grey aerial Physiology. No studies on physiology are known tohyphae; reverse dark grey. No growth on G25N or us. However, the description above indicates that thisgermination at 58C. Colonies at 378C covering the species has a high temperature optimum for growth.whole Petri dish, 65 mm or more diam, general Mycotoxins. Significant mycotoxins are notappearance similar to those on CYA at 258C; known to be produced.reverse very dark grey to greyish black (Fig. 5.8). Ecology. We have isolated this species not infre- Reproductive structures perithecia not observed on quently from tropical sources. From Thailand, 1% ofCYA or MEA at 258C, but on CYA at 378C abundant, mung beans, black beans, paddy rice grains andforming a deep, contiguous layer on the agar surface, cashew kernels, and 7–10% of soy bean and sorghummost readily observed at colony margins, especially samples, but <1% of individual seeds, contained C.between colonies, surrounded by radiating long, stout brasiliense (Pitt et al., 1993, 1994). Similar figures werehyphae, straight at first but at maturity curling into obtained from Indonesian commodities (Pitt et al.,long corkscrew shapes, 75–100 mm diam, maturing in 1998a). In samples from the Philippines, this species7 days at colony centres; asci not seen, ascospores was found in maize, peanuts, soybeans, mung beans,
    • 5.9 Genus Chaetomium 71Fig. 5.8 Chaetomium brasiliense (a) colony on CYA, 7 d, 258C; (b) perithecium in situ, bar ¼ 25 mm; (c) asci, bar ¼ 10 mm; (d)ascospores, bar ¼ 5 mmblack pepper, paddy and milled rice, all near 1% of Perithecia borne in a layer on the agar surface,total particles examined (our unpublished data). 120–160 mm diam, surrounded by almost straight, Reference. Von Arx et al. (1986). dark walled, rough to spiky hyphae; asci maturing in 14 days, breaking down immediately, ascosporesChaetomium funicola Cooke Fig. 5.9 ellipsoidal, 5–7 Â 3–5 mm, grey or brown, sometimes with one or two internal oil droplets visible, oftenColonies on CYA 20–25 mm diam, plane, of low collapsing in age, smooth walled.and sparse white mycelium, sometimes pale grey Distinctive features. Chaetomium funicola growscentrally, enveloping abundant developing perithe- only slowly at 258C and usually not at 378C. Ascos-cia, reverse pale or greyish brown. Colonies on pores are smaller than those of C. globosum.MEA 35–40 mm diam, plane, sparse, low to some- Physiology. No studies are known to us.what floccose, mycelium white or grey, enveloping Domsch et al. (1980) indicated that perithecialdeveloping perithecia; reverse pale to greyish olive production is maximal at about 208C, indicatingor pale brown. On G25N, usually no growth. At that this species is better adapted to low than high58C, no germination. At 378C, usually no growth. temperatures.
    • 72 5 Primary Keys and Miscellaneous FungiFig. 5.9 Chaetomium funicola (a) colonies on CYA and MEA, 7 d, 258C; (b) perithecium in situ, bar ¼ 50 mm; (c) ascospores,bar ¼ 5 mm Mycotoxins. Mycotoxin production has not been Chaetomium globosum Kunze Fig. 5.10reported. Ecology. Despite its low temperature optimum, Colonies on CYA and MEA covering the whole Petriwe repeatedly isolated Chaetomium funicola from dish, on CYA low and sparse, of scanty white myce-tropical commodities. It was present in 23% of Indo- lium and conspicuous though usually sparse blacknesian soybean samples, ranging up to 50% infection perithecia, ca. 0.2 mm diam; on MEA growth morein some samples, and in 3% of beans overall (our dense but still low, coloured grey or greenish blackunpublished data). Lower levels of infection (1% or from hyphae enveloping abundant perithecia;more overall) were observed in peanuts, cashews, reverse on both media usually brown. On G25N,copra, mung beans and sorghum from Thailand colonies less than 5 mm diam produced. No growth(Pitt et al., 1993, 1994), in mung beans and maize at 58C. At 378C, colonies usually 20–30 mm diam, offrom Indonesia, and paddy rice from the Philippines white mycelium.(Pitt et al., 1998a and our unpublished data). Reproductive structures perithecia, black, References. Domsch et al. (1980); von Arx et al. 150–200 mm diam, with numerous stout, dark hyphae(1986). appended; ascospores produced after 1–2 weeks,
    • 5.10 Genus Chrysonilia 73Fig. 5.10 Chaetomium globosum (a) colony on CYA, 7 d, 258C; (b) perithecium in situ, bar ¼ 100 mm; (c) ascospores, bar ¼5 mmspheroidal, broadly ellipsoidal or apiculate, com- frequently from tropical commodities: from 12 tomonly 8–10 mm long, smooth walled. Conidia not 17% of maize samples, 14 to 32% of mung beangenerally produced. samples and 33 to 35% of soybean samples from Distinctive features. In contrast to Chaetomium Thailand, Indonesia and the Philippines, with over-brasiliense, C. globosum grows much more rapidly all infection rates of 1–4% of all particles examined.at 258C than 378C. Ascospores are larger than those Relatively high infection levels were also encoun-of C. funicola. tered in Thai cashews, copra and paddy rice. Kemiri Physiology. Chapman and Fergus (1975) reported (candle) nuts from Indonesia were highly infected:that Chaetomium globosum ascospores germinated 37% of samples, up to 40% in infected samples andfrom a minimum temperature of 4–108C to a max- 9% of nuts overall (Pitt et al., 1998a). This speciesimum of 388C, and most rapidly at 24–388C. They has not been reported to cause food spoilage,also reported germination over the whole pH range although it has recently been implicated in causingtested, 3.5–7. Heat resistance was low: 1% of ascos- disease in pears in Egypt (Ismail and Abdalla, 2005).pores survived 10 min at 558C, but none survived at References. Domsch et al. (1980); von Arx et al.578C for a similar period. Ahammed et al. (2005) (1986).observed maximum sporulation at 288C and pH 5.0on a medium containing sucrose and thiamine. C.globosum can grow down to 0.94 aw in soil (Kouyeas,1964). 5.10 Genus Chrysonilia Arx Mycotoxins. Chaetomium globosum produceschaetoglobsins A and C, moderately toxic com- Chrysonilia was erected by von Arx (1981a) topounds reported to be produced in building materi- accommodate Monilia sitophila, for many yearsals (Fogle et al., 2007). Their relevance to foods is known as ‘‘the red bread mould’’. Chrysonilia sito-unknown. phila is characterised by very rapid growth and the Ecology. This species has been isolated from a production of conidia in chains, cut off from thevariety of commodities, particularly wheat, barley, apices of undifferentiated hyphae. This species isrice, beans and soybeans. Chaetomium globosum has readily recognisable in Petri dish culture: sparse,also been recorded in nuts (see Pitt and Hocking, white to pink mycelium spreads rapidly across the1997) and spices (Mandeel, 2005). We isolated it dish and up the walls, then within 3 days sheds
    • 74 5 Primary Keys and Miscellaneous FungiFig. 5.11 Chrysonilia sitophila (a) colony on CYA, 7 d, 258C; (b) conidia, bar ¼ 10 mmenormous numbers of orange conidia outside the low, dense and mucoid. No growth at 58C. At 378C,dish. This attribute makes it a particularly trouble- colonies covering the whole Petri dish, similar tosome laboratory contaminant. Cultivation in Petri at 258C.dishes is therefore not recommended: observation Reproductive structures arthroconidia, cut off inof these characters in a culture tube is sufficient for succession from the apices of branching hyphae;positive identification. conidia at maturity variable in size and shape, sphe- The teleomorph of this species is Neurospora rical to ellipsoidal, pale orange, 6–15 mm diam, withsitophila, a well-known Ascomycete which has thin smooth walls.been of great value in genetic studies. It is not Distinctive features. See remarks above.found in foods, however. Physiology. According to Panasenko (1967), Chrysonilia sitophila is capable of growth down to 0.88–0.90 aw.Chrysonilia sitophila (Mont.) Arx Fig. 5.11 Mycotoxins. No mycotoxins have been recordedMonilia sitophila (Mont.) Sacc. for this species. Ecology. For long a common sight in bakeriesTeleomorph: Neurospora sitophila Shear and B.O. and on bread, Chrysonilia sitophila is less commonlyDodge encountered now, but can still be a great source of trouble as a persistent contaminant in laboratoriesColonies in 28 ml McCartney bottles or test tubes (see Chapter 4). It has sometimes been reported fromdistinguished by pale pink floccose growth, filling other foods: pastries, hazelnuts, beans and meat pro-the entire bottle, then turning salmon, first at the ducts (see Pitt and Hocking, 1997). We isolated itbottle neck or tube plug and subsequently through- frequently from Thai cassava, where it infected 36%out, as sporulation occurs. of all pieces examined, and Philippine peanuts, where On CYA and MEA, colonies covering the whole it infected up to 90% of nuts in some samples, andPetri dish, mycelium pink, reaching the lid in tufts was present in 1% of nuts overall. It was also presentor patches and all around the rim, producing vast in 1% of all Indonesian peanuts, sorghum and maizenumbers of salmon conidia at or near the rim and grains examined (Pitt et al., 1998a). Infection rates insometimes for several millimetres beyond it, and maize and peanuts ranged up to 40% in some sam-shedding them profusely; reverse salmon or pink. ples (our unpublished data). We also found it atOn G25N, colonies up to 30 mm diam produced, lower levels in Thai soybeans, black beans and
    • 5.11 Genus Cladosporium 75cashews (Pitt et al., 1994). Recently, C. sitophila was stereomicroscope. They disintegrate partially orreported in the mycoflora of cork slabs (Oliveira totally in wet mounts, leaving masses of conidia,et al., 2003; Pereira et al., 2006). which may show buds or bud scars. In shape, but Reference. Von Arx (1981a). not colour, the conidia often resemble yeast cells; however, walls are thick, coloured olive and often roughened.5.11 Genus Cladosporium Link Cladosporium species occur as pathogens on fresh fruit and vegetables (Snowdon, 1990, 1991)Cladosporium is a very commonly isolated genus and may cause spoilage of strawberries (Benekeand is often dominant in studies of airborne micro- et al., 1954; Dennis et al., 1979) or tomatoes (Har-flora. Species occur both as saprophytes and as wig et al., 1979). On other foods, Cladosporiumplant pathogens. Conidia of Cladosporium species species usually occur as contaminants rather thanare particularly well adapted to aerial dispersal, as spoilage fungi. However, all common speciesbeing small, dry, heavily pigmented and apparently grow at temperatures near 08C, and Cladosporiahighly resistant to sunlight. The genus is highly have been reported to cause spoilage of chilleddiverse, and includes many species not yet charac- meats, cheese and other refrigerated commoditiesterised (Crous et al., 2007). from time to time (see Pitt and Hocking, 1997). In culture, Cladosporium is readily recognised. A major taxonomic revision of Cladosporium, itsColonies are 15–40 mm in diameter, low, dense teleomorphs and related genera has recently beenand velvety, and coloured olive. Colony reverses published (Crous et al., 2007). In overall terms, theon CYA are often a deep iridescent blue black, taxonomy of the common species treated here hasuncommon among slowly growing genera. been unaffected. The most frequently reported Reproductive structures are fragile, tree-like con- saprophytic species is Cladosporium herbarum, butidiophores, which branch irregularly by budding C. cladosporioides and C. sphaerospermum arefrom the youngest cells. The structures can be seen also ubiquitous. In addition, C. macrocarpum isby examination of colonies under the described here. Key to Cladosporium species included here1 Single celled conidia small, less than 4 mm wide 2 Single celled conidia often exceeding 4 mm wide 32 (1) Single celled conidia ellipsoidal or apiculate (lemon-shaped) C. cladosporioides A high proportion (greater than 40%) of single-celled conidia roughly spherical or pyriform C. sphaerospermum3 (1) Conidia 4–6 mm wide C. herbarum Conidia commonly exceeding 6 mm wide C. macrocarpumCladosporium cladosporioides (Fresen.) Conidiophores in situ dendritic (tree-like), clos-G.A. de Vries Fig. 5.12 ely packed, with stipes bearing branching structures of acropetally produced cells, all functioning as conidia at maturity and separating in liquid mounts;Colonies on CYA and MEA 25–40 mm diam, low conidia heavy walled, pale olive brown, larger onesand dense, lightly wrinkled or plane, surface veluti- non- or singly septate, 10–30 Â 2–5 mm, smoothnous or lightly floccose; conidia abundant, coloured walled, smaller ones nonseptate, ellipsoidal to api-olive; reverse bluish grey. Colonies on G25N 5–12 mm culate, 3–7 Â 2–4 mm, with walls smooth to finelydiam, plane, sometimes centrally raised, velutinous, roughened.coloured as on CYA; reverse bluish black. At 58C, Distinctive features. Cladosporium cladospor-colonies usually 1–2 mm diam, occasionally only ger- ioides produces smaller conidia than C. herbarummination occurring. No growth at 378C. and C. macrocarpum; unlike C. sphaerospermum,
    • 76 5 Primary Keys and Miscellaneous FungiFig. 5.12 Cladosporium cladosporoides (a) colonies on CYA and MEA, 7 d, 258C; (b) fruiting structures in situ, bar = 50 mm;(c) conidiophores, bar = 5 mm; (d) conidia, bar = 5 mmthe majority of conidia are ellipsoidal. Growth on Ecology. Cladosporium cladosporioides has beenCYA and MEA is much faster than that of C. isolated from a very wide variety of foods, includingsphaerospermum. wheat and flour, barley, rice, sorghum, fresh and Physiology. Gill and Lowry (1982) reported a frozen meat and fresh vegetables (see Pitt andminimum growth temperature of –58C for Clados- Hocking, 1997). It occurred on 16% of 74 driedporium cladosporioides; the maximum is near 328C fish samples from Indonesia (Wheeler et al., 1986),(Domsch et al., 1980). It is capable of growth down and was common on Egyptian walnuts and hazel-to 0.86 aw at 258C (Hocking et al., 1994) and is nuts (Abdel-Hafez and Saber, 1993). It has alsorelatively resistant to microwave heating (Dragoni been found in peanuts (El-Magraby and El-et al., 1990). The minimum inhibitory concentration Maraghy, 1988).of sorbic acid is 160 mg/kg at pH 5 (Skirdal and Cladosporium cladosporioides is not as common aEklund, 1993). cause of spoilage in fresh fruit as C. herbarum, but Mycotoxins. This species is not known to pro- can cause rots of raspberries and melons andduce mycotoxins. sooty discolouration of bananas (Snowdon,
    • 5.11 Genus Cladosporium 771990). Because of its psychrophilic nature, 14% of mung bean samples from Indonesia (PittC. cladosporioides causes spoilage of refrigerated et al., 1998a and our unpublished data).foods such as cheese (Northolt et al., 1980) and References. De Vries (1952); Ellis (1971);meat (Gill et al., 1981). It was the dominant fungus Domsch et al. (1980).causing losses in vacuum packed Australian cheeseblocks (Hocking and Faedo, 1992). Cladosporium cladosporioides is also very com- Cladosporium herbarum (Pers.) Link Fig. 5.13mon in major Southeast Asian commodities: we Colonies on CYA and MEA 18–32 mm diam, veluti-isolated this species from 33% of mung bean, 14% nous to lightly floccose, plane or slightly wrinkled,of peanut, 13% of soybean and 10% of maize sam- coloured olive; reverse olive grey to dark greenishples from the Philippines and 21% of soybeans and grey. Colonies on G25N 5–10 mm diam, low andFig. 5.13 Cladosporium herbarum (a) colonies on CYA and MEA, 7 d, 258C; (b) fruiting structures in situ, bar ¼ 50 mm;(c) conidiophores, bar ¼ 5 mm; (d) conidia, bar = 5 mm
    • 78 5 Primary Keys and Miscellaneous Fungisparse to deep and dense, coloured as on CYA or paler. (Ribeiro and Dias, 2005) and pawpaw (EcherenwaAt 58C, colonies usually 1–2 mm diam; occasionally and Umechuruba, 2004). Its common occurrence ononly germination. No growth at 378C. fresh apples can lead to contamination of apple juice Conidiophores in situ sparsely and irregularly and high acid fruit based products. As a psychrophile,branched dendritic structures, borne on long, dark C. herbarum can cause ‘‘black spot’’ spoilage of meat instipes, separating in fluid mounts; conidia ellipsoi- cool stores (Gill et al., 1981), of cheese during ripeningdal to cylindroidal, extremities sometimes irregular (Gueguen, 1988) and in storage under vacuum (Hock-due to bud scars, usually nonseptate but larger ing and Faedo, 1992). It has been isolated from freshcells with one to two septa, commonly 8–15(–20) Â and frozen meat and processed meat products (see Pitt4–6 mm, pale brown, with densely roughened walls. and Hocking, 1997). C. herbarum has also been iso- Distinctive features. The conidia of Cladosporium lated from eggs, peanuts, hazelnuts, walnuts, cereals,herbarum are 4–6 mm wide, with distinctly rough- chickpeas, soybeans and frozen fruit pastries (see Pittened walls; conidia of C. macrocarpum are 6–9 mm and Hocking, 1997; Kuehn and Gunderson, 1963;wide and have finely roughened walls. Arya, 2004). Growth on dough carrying machinery Taxonomy. It was concluded by Dugan and (Gradel and Muller, 1985) and on wine corks (Daly ¨Roberts (1994) that Cladosporium herbarum repre- et al., 1984) has been reported.sented part of a continuum with C. macrocarpum, References. Dugan and Roberts (1994); Ho et al.while Ho et al. (1999) considered that C. macrocar- (1999); Schubert et al. (2007).pum was a variety of C. herbarum. However, in amajor study, Schubert et al. (2007) have kept thesetwo species. They indicated that C. herbarum as Cladosporium macrocarpum Preuss Fig. 5.14commonly used includes several other species, butthe concept of C. herbarum used here is still valid. Colonies on CYA 18–25 mm diam, velutinous toThe teleomorph of C. herbarum is Davidiella tassi- lightly floccose, plane or slightly wrinkled, colouredana (De. Not.) Crous and U. Braun (Schubert et al., olive to dark olive; reverse bluish grey. Colonies on2007). This is not usually seen in pure culture. MEA 16–24 mm diam, low to floccose, pale olive to Physiology. Growth of this species has been olive; reverse olive grey to dark olive grey. Coloniesreported down to 0.88 aw (Snow, 1949) and down on G25N 3–8 mm diam, low and sparse to deep andto –108C (Joffe, 1962). The maximum growth tem- dense, coloured as on MEA or paler. At 58C, colo-perature is 28–328C (Domsch et al., 1980). This nies usually 1–2 mm diam; occasionally germinationspecies can grow and sporulate in an atmosphere only or no growth observed. No growth at 378C.containing 0.25% oxygen (Follstad, 1966). Conidiophores as seen under the stereomicro- Mycotoxins. No mycotoxins are known to be scope long and dark, with small clusters of conidiaproduced. borne in short, irregularly branched chains; conidia Ecology. Because Cladosporium herbarum has been borne from irregularly located pores on geniculatethe best known Cladosporium name for a long time, it (‘‘knee-like’’) swellings, ellipsoidal or short cylin-seems likely that some reports of this species really refer droidal, with rounded ends, (10–)15–20 Â 6–9 mm,to other Cladosporia, especially C. cladosporioides, in usually aseptate, with finely roughened brownour experience the more common species in foods. For walls.example, Waghray et al. (1988) reported C. herbarum Distinctive features. Colonies are similar to thoseas common on Indian rice, but according to Pitt et al. of Cladosporium herbarum, but of somewhat paler(1994, 1998a and unpublished), the common Clados- colour. Conidia are considerably larger than thoseporium species on Southeast Asian rice is C. cladospor- of C. herbarum or of the other Cladosporium speciesioides. In contrast to C. cladosporioides, C. herbarum described here.causes spoilage of fresh fruits and vegetables: rots in Taxonomy. From an examination of 32 isolatesfresh yams (Dioscorea sp.; Adeniji, 1970a), stored from cherries in Washington state, USA, Duganapples (Snowdon, 1990; Kalafatoglu and Karapinar, and Roberts (1994) concluded that Cladosporium1991), stone fruit, tomatoes, melons, grapes (Snowdon, macrocarpum and C. herbarum represented part of1990, 1991; Camili and Benato, 2005), passionfruit a continuum and that C. macrocarpum was a
    • 5.11 Genus Cladosporium 79Fig. 5.14 Cladosporium macrocarpum (a) colonies on CYA and MEA, 7 d, 258C; (b) fruiting structures in situ, bar ¼ 50 mm;(c) conidiophores, bar ¼ 5 mm; (d) conidia, bar ¼ 5 mmsynonym. Ho et al. (1999) considered that C. macro- latter as one of the principal fungi found in a largecarpum was a variety of C. herbarum. However, in a survey. Dugan and Roberts (1994) reported fre-major study, Schubert et al. (2007) kept the two quent occurrence on Bing cherries in the Unitedspecies separate. States (see Taxonomy). It occurs on hazelnuts and Physiology. No reports on the physiology of this walnuts in Egypt (Abdel-Hafez and Saber, 1993).species have been located. Properties should be We have isolated this species at a low frequencysimilar to those of Cladosporium herbarum, to from cashews in Thailand (Pitt et al., 1993) andwhich it is closely related. mung beans in the Philippines (our unpublished Mycotoxins. No mycotoxins have been reported. data). It was also reported on fresh asparagus Ecology. Cladosporium macrocarpum is uncom- (Kadau et al., 2005).mon in foods. Abdel-Kader et al. (1979) and Pet- References. Dugan and Roberts (1994); Ho et al.ters et al. (1988) both reported it from barley, the (1999); Schubert et al. (2007).
    • 80 5 Primary Keys and Miscellaneous FungiCladosporium sphaerospermum Penz. Fig. 5.15 all functioning as conidia at maturity, and separating in liquid mounts; conidia heavy walled, pale oliveColonies on CYA 18–25 mm diam, low and dense, brown, larger ones 10–30 Â 2–4 mm, only occasion-usually plane, surface velutinous; conidia abundant, ally septate, smooth walled, smaller ones nonseptate,coloured olive to dark olive; reverse bluish grey. subspheroidal, ogival or apiculate, 4–6(–10) Â 3–4Colonies on MEA 12–20 mm diam, of similar mm, with walls smooth to definitely roughened.appearance to those on CYA, sometimes lighter in Distinctive features. Similar to Cladosporium cla-colour. Colonies on G25N 8–12 mm diam, plane or dosporioides in most respects, C. sphaerospermum iscentrally raised, velutinous, coloured as on CYA; distinguished by slower growth on CYA and MEAreverse almost black. At 58C sometimes germina- and by production of a high proportion (40% plus)tion. No growth at 378C. of more or less spherical conidia. Conidiophores under the stereomicroscope den- Taxonomy. Several new species very similar todritic (tree-like), closely packed, with stipes bearing Cladosporium sphaerospermum were described bybranching structures of acropetally produced cells, Zalar et al. (2007), but none was of common occur-Fig. 5.15 Cladosporium sphaerospermum (a) colonies on CYA and MEA, 7 d, 258C; (b) fruiting structures in situ, bar ¼ 50 mm;(c,d) conidiophores and conidia (slide culture), bar ¼ 10 mm; (e) conidia, bar ¼ 5 mm
    • 5.12 Genus Colletotrichum 81rence. The concept of C. sphaerospermum as used the opinion of Sutton (1980), C. gloeosporioides ashere remains unchanged. currently accepted is an agglomerate of several spe- Physiology. Cladosporium sphaerospermum has cies with a similar appearance. The species descrip-an optimum aw for growth near 0.97 at 258C. This tion given below is taken from a limited number ofspecies is a xerophile, able to germinate and grow isolates and undoubtedly does not cover the fullslowly at 0.815 aw (Hocking et al., 1994). No other range of variation.physiological studies have been reported. Mycotoxins. No mycotoxins are known to be Colletotrichum gloeosporioididesproduced. (Penz.) Sacc. Fig. 5.16 Ecology. Although less common than Cladospor-ium cladosporioides, C. sphaerospermum has been Teleomorph: Glomerella cingulata (Stonemason)isolated from a wide range of foods, including barley Spauld. & Schrenk.and peanuts in Egypt, US pecan nuts, Europeanmeat products, spoiled cheese in Europe and Austra- On CYA and MEA, colonies 60 mm diam or more,lia, stored apple fruit in Turkey (see Pitt and Hock- often covering the whole Petri dish, with a denseing, 1997) and carbonated soft drinks in Nigeria basal layer of hyphae and conidial fruiting bodies(Odunfa, 1987) and Argentina (Ancasi et al., 2006) . (acervuli) overlaid by areas of floccose white,We have isolated this species at low frequency from orange or grey mycelium; acervuli up to 500 mmpeanuts from Thailand (Pitt et al., 1993), rice and long, pale, grey or orange; reverse with pale greysoybeans from the Philippines, and maize and mung or orange areas. On G25N, colonies 2–5 mm diam,beans from Indonesia (Pitt et al., 1998a). pale or black. At 58C, no growth to germination. References. Zalar et al. (2007). No growth at 378C. Reproductive structures flat, lidded acervuli, opening irregularly, containing a single closely packed layer of phialides, of irregular dimensions;5.12 Genus Colletotrichum Corda conidia borne singly, cylindroidal, with rounded ends, nonseptate, 12–18 Â 3–3.5 mm, hyaline andColletotrichum is a genus with many species, wide- smooth walled.spread as plant pathogens. In this genus, conidia are Distinctive features. In the present context,borne inside an acervulus, a structure with a more or Colletotrichum gloeosporioides is distinguished byless closed lid which eventually ruptures. Acervuli producing conidia in acervuli and by pathogenicityare readily seen on the agar surface with the unaided on tropical fruits; conidia are aseptate cylinderseye or by low power microscope. Conidia of Colle- with rounded ends, 12–18 mm long.totrichum are single celled and hyaline or brightly Taxonomy. This species as currently delimited iscoloured. They may be cylindrical or pointed, probably too broadly defined (Sutton, 1980). Thestraight or curved. teleomorph of Colletotrichum gloeosporioides, Glo- A number of Colletotrichum species cause spoilage merella cingulata (Stonemason) Spauld. andof fresh fruits and vegetables. Examples include C. Schrenk., is not found in foods.acutatum J.H. Simmonds on strawberries, C. circans Physiology. Colletotrichum gloeosporioides was(Berk.) Voglino on leeks and onions, C. coccodes insensitive to storage in 10–13% CO2 in the pre-(Wallr.) S. Hughes on potatoes, tomatoes and egg- sence of 3–7% O2 (Wade et al., 1993).plants, C. higginsianum Sacc. on cabbage and other Mycotoxins. Mycotoxins are not known to becucurbits, C. lindemuthianum (Sacc. and H. Magn.) produced.Broome and Cavara on beans and C. musae (Berk. Ecology. According to Sutton (1980), the Inter-and Curtis) Arx on bananas (Snowdon, 1990, 1991). national Mycological Institute Herbarium has One species, Colletotrichum gloeosporioides, a records of Colletotrichum gloeosporioides from 470common cause of spoilage of fruits, especially different host genera – a remarkable host range,from the tropics, is treated here as an example of even taking into account that several biological spe-this rather ill-defined though important genus. In cies may be involved.
    • 82 5 Primary Keys and Miscellaneous FungiFig. 5.16 Colletotrichum gloeosporioides (a) colonies on CYA and MEA, 7 d, 258C; (b) immature conidiophores, bar = 10 mm;(c) conidia, bar = 5 mm Colletotrichum gloeosporioides and closely and Segall, 1963; Hall and Scott, 1977). Gammarelated species cause anthracnoses of tropical fruits, radiation can be used to control C. gloeosporioidesincluding avocados, bananas, pawpaws (papayas) in pawpaws (papayas) (Cia et al., 2007).and mangoes, and of temperate fruits, including Colletotrichum species were isolated from 40% ofapples, stone fruits and grapes. Colletotrichum spe- paddy rice samples examined from Thailand (Pitt et al.,cies cause damage to bananas (C. musae), strawber- 1994). C. dematium was found at low levels in severalries (C. acutatum), grapefruit, tomatoes and other Southeast Asian commodities, including soybeans,fruits (Snowdon, 1990; Pitt and Hocking, 1997). cowpeas, peanuts and black pepper (Pitt et al., 1998a). Anthracnoses on fruit are dark, relatively dry, References. Von Arx (1957); Mordue (1971);shrunken skin blemishes which expand rapidly as Sutton (1980); Gunnell and Gubler (1992).the fruit ripens. Except in advanced stages, theblemishes are only skin deep, and the fruit is edible,if unsightly. Advanced lesions may develop pinkish 5.13 Genus Curvularia Boedijnmasses of conidia. Control in bananas is generallypossible with benzimidazole or similar fungicides; in In Curvularia, conidia are long and ellipsoidal, withmangoes, pawpaws and avocados, hot water dips, three to four transverse septa. As the name implies,with or without fungicide, can be beneficial (Smoot conidia are often curved due to an asymmetrically
    • 5.13 Genus Curvularia 83swollen central cell. Most species are plant patho- Curvularia lunata; Curvularia pallescens is describedgens. The species most common in foods is here as a second foodborne species. Key to Curvularia species included here1 Mycelium on CYA and MEA grey to dark grey; reverse often blue black; conidia with central cells dark-walled C. lunata Mycelium on CYA and MEA pale to mid grey; reverse brown; all cells in conidia with uniform wall colour C. pallescensCurvularia lunata (Wakker) Boedijn Fig. 5.17 conidiophores, elongate, smooth walled, with three septa, almost always curved at an asymmetric cellTeleomorph: Cochliobolus lunatus R.R. Nelson & third from the base, 16–25(–30) Â 8–14 mm, end cellsF.A. Hassis pale brown, central cells darker. Distinctive features. Curvularia lunata is the speciesOn CYA and MEA, colonies at least 60 mm diam, of Curvularia commonly isolated from foods. It isoften covering the whole Petri dish, usually deep, mod- distinguished from C. pallescens by darker colonies,erately dense and floccose, mycelium off-white to grey, a blue black reverse, and nonuniform pigmentation inoften approaching black, in age sometimes developing conidial walls. Conidial production usually occurs onorange or salmon coloured areas; reverse usually grey MEA, but culturing on DCMA and/or under lightsto bluish black, sometimes with areas of salmon. On may assist recognition if MEA plates are sterile.G25N, colonies 5–15 mm diam, low and dense, grey to Taxonomy. When grown in pairs, some isolatesblack with reverse similar. At 58C, usually germination. of this species mate to produce an ascomycetousAt 378C, colonies (5–)20–40 mm diam, of similar state, Cochliobolus lunatus R.R. Nelson and F.A.appearance to those at 258C. Haasis. This state is not encountered in agar culture Colonies on DCMA 55–65 mm diam or covering of single isolates.the whole Petri dish, plane, sparse, velutinous, Physiology. Curvularia lunata was able to germi-reverse brown or dark grey. nate at 0.86 aw, but only grew down to 0.89 aw at Conidia, best seen in mounts from growth close 258C (Hocking et al., 1994).to the agar surface on MEA or on DCMA, borne Mycotoxins. No reliable reports of mycotoxinfrom pores along the sides of short knobby production by this species are known to us.Fig. 5.17 Curvularia lunata (a) colony on CYA, 7 d, 258C; (b, c) conidiophore and conidia, bar = 10 mm
    • 84 5 Primary Keys and Miscellaneous Fungi Ecology. As Curvularia lunata is primarily an diam, similar to those on CYA at 258C, sometimesinvader of monocotyledon plants (Domsch et al., less floccose, reverse dark blue black. Colonies on1980), the most common food sources are cereals: DCMA 55–65 mm diam, plane, sparse, floccose, espe-records include rice, barley, wheat, maize and sor- cially at colony junctions, reverse brown or dark grey.ghum (Pitt and Hocking, 1997; Mtisi and McLaren, Conidia are similar to those of C. lunata.2003; Echemendia, 2005, Fakhrunnisa et al., 2006). The variety is distinguished from Curvularia lunataIt has been reported as one cause of spoilage of fresh var. lunata by a more floccose growth habit and fastertomatoes in Nigeria (Muhammad et al., 2004) and growth at 378C. It occurs in similar foods and com-has also been found on litchi fruit (Wells et al., 1981), modities to C. lunata, the highest level we havesoybeans (Ahammed et al., 2006), hazelnuts and wal- encountered being 2% of all Thai rice grains and 1%nuts (Abdel-Hafez and Saber, 1993), peanuts and of all Thai sorghum grains examined (Pitt et al., 1994).spices (see Pitt and Hocking, 1997). References. Ellis (1971); Domsch et al. (1980); In Thailand, Curvularia lunata is a major invader Sivanesan (1987).of sorghum: we isolated it from 56% of samples and8% of all grains examined. It was also prevalent in Curvularia pallescens Boedijn Fig. 5.18paddy rice samples, where 28% of samples and 3%of all grains examined were infected (Pitt et al., Colonies on CYA 50–65 mm diam, on MEA cover-1994). It was found in 9–10% of paddy rice samples ing the whole Petri dish, plane, of low to floccosefrom Indonesia and the Philippines (Pitt et al., mycelium, pale grey to mid grey, reverse brown to1998a and unpublished data). dark brown. On G25N, colonies 3–6 mm diam, Additional variety. Curvularia lunata var. aeria brown or grey. At 58C, usually germination. At(Bat.) M.B. Ellis is a floccose taxon accepted as a 378C, colonies 40–50 mm diam, of floccose greyvariety of C. lunata. Colonies of C. lunata var. aeria mycelium, sometimes brown soluble pigment,on CYA 50–65 mm diam, plane, floccose, of pale to reverse dark brown.mid grey mycelium, reverse deep blue black. Colonies On DCMA, colonies 50–65 mm diam, plane,on MEA covering the whole Petri dish, plane, deeply with pale brownish grey to mid brown mycelium,floccose, especially so at colony to colony and colony reverse reddish brown to dark brown.to Petri dish junctions, mycelium white to orange grey, Conidia borne from pores in geniculate hyphae,reverse yellow brown, dark brown or blue black. ellipsoidal, with three septa, sometimes inconspicu-Colonies on G25N 4–6 mm diam, of dark grey myce- ous, almost straight along one side but with thelium. No growth at 58C. At 378C, colonies 50–65 mm eccentric swelling of the penultimate cell character-Fig. 5.18 Curvularia pallescens (a) colony on MEA, 7 d, 258C; (b) fruiting structures in situ, bar = 50 mm; (c) conidia, bar = 10 mm
    • 5.14 Genus Drechslera 85istic of the genus, 18–25 Â 9–12 mm, with walls pale to mid grey mycelium, sometimes lightlysmooth, pale to mid brown, and with all cells in sporing in upper mycelial layers; exudate andeach conidium of a similar colour. soluble pigment absent; reverse dark grey to Distinctive features. This species lacks the abrupt blue black. Colonies on MEA covering thecurvature in the conidia observed in other foodborne Petri dish, similar to on CYA but sporulationspecies; conidia are relatively light in colour and char- usually absent; reverse brownish grey to bluishacterised by an even colour in all cells. Colony reverses black. Colonies on G25N 4–8 mm diam, ofon CYA and MEA are brown, not blue black. sparse, pale grey mycelium. At 58C, sometimes Physiology. Like Curvularia lunata, C. pallescens germination. At 378C, no growth.was able to germinate at 0.86 aw, but only grew Colonies on DCMA when grown under lightsdown to 0.89 aw at 258C (Hocking et al., 1994). 45–70+ mm diam, similar to those on CYA, Mycotoxins. This species is not known to pro- sometimes lightly sporing; reverse pale to darkduce mycotoxins. grey. Ecology. The major known source of Curvularia Conidiophores 150–250(–400) mm long, straightpallescens is sorghum (Pitt et al., 1994; Ishrat et al., or sinuous, sometimes knobby terminally, bearing2005). However, it has also been recorded on rice solitary conidia; conidia cylindroidal, commonly(Pitt et al., 1994; Gutierrez et al., 2002) and causing 70–120 Â 14–17 mm, with five to seven septa,rots in melons (Sharma et al., 2002). brown, smooth walled. References. Ellis (1971); Sivanesan (1987). Distinctive features. See genus preamble. Taxonomy. The teleomorph of this species is the Ascomycete Pyrenophora tritici-repentis (Diedicke)5.14 Genus Drechslera S. Ito Drechsler. This is not usually seen in pure culture; sometimes immature perithecia may be formedShoemaker (1959) revised the genus Drechslera, pro- (Sivanesan, 1987).vided a clear separation from Helminthosporium, and Physiology. No physiological studies on Drech-erected Bipolaris. Leonard and Suggs (1974) segre- slera species are known to us. In common with mostgated Exserohilum from Bipolaris. These three genera other plant borne dematiaceous Hyphomycetes,produce long, large conidia with transverse septa, growth is unlikely to occur below about 0.90 aw.which are much thicker than those in e.g. Trichoco- Species appear to be mesophilic.niella. In Drechslera, conidia are cylindrical and ger- Mycotoxins. No mycotoxins are known to beminate at any cell, while in Bipolaris and Exserohi- produced.lum, conidia narrow gradually and germinate only Ecology. Drechslera tritici-repentis is one cause offrom the terminal cells. ‘‘pink tip’’ in wheat. We have isolated it from 56% Drechslera species are serious pathogens on cer- of 344 surface disinfected North American wheateal plants, and a number of species have been samples, where it was present at up to 27% inrecorded to occur on grains from time to time. infected samples and 2% of all grains examinedHowever, they appear to have little or no role in (unpublished data). Occurrence in Australianfood spoilage. Species are mostly distinguished by wheat was much lower (5% of samples; unpublisheddifferences in spore size; one, Drechslera tritici- data) and it was not isolated from Southeast Asianrepentis, is treated here as an example. commodities (Pitt et al., 1994, 1998a). Several other Drechslera species have beenDrechslera tritici-repentis (Diedicke) reported from grains and seeds. However,Shoemaker Fig. 5.19 most records examined refer to species now included in Bipolaris or, less commonly, Exser-Teleomorph: Pyrenophora tritici-repentis (Die- ohilum. Only D. sorokiniana (Sacc.) Shoemaker,dicke) Drechsler from German durum wheat (Nirenberg et al., 1995) relates with certainty to a DrechsleraColonies on CYA 55–70+ mm diam, sometimes species.covering the Petri dish, of low to deep, floccose, References. Alcorn (1983); Sivanesan (1987).
    • 86 5 Primary Keys and Miscellaneous FungiFig. 5.19 Drechslera tritici-repentis (a) colony on MEA, 7 d, 258C; (b) colonies on tap water agar plus millet seed and on V-8juice, 7 d, 258C; (c) conidia in situ, bar = 50 mm; (d) conidia, bar = 10 mm5.15 Genus Endomyces Reess and spreading on agar plates, their decision is fol- lowed here. The conidial state consists of yeast-likeEndomyces is a genus of yeast-like Ascomycetes, cells borne on spicules or small projections. Theindeed often classified with the yeasts, in Sacchar- Ascomycete state occurs sometimes in culture: theomycopsis (Kurtzman and Fell, 1998; Barnett et al., production of unenclosed, evanescent asci is sugges-2000). However, Yamada et al. (1996) considered tive of a relationship with the true yeasts. The onlythat differences in DNA from the ITS region are species common in foods is Endomyces fibuliger. Seesufficient to consider Endomyces as a genus separate also Hyphopichia, which appears to be a closelyfrom Saccharomycopsis. As growth is filamentous related genus.
    • 5.15 Genus Endomyces 87Fig. 5.20 Endomyces fibuliger (a) colonies on CYA and MEA, 7 d, 258C; (b) conidiophores and conidia (slide culture), bar =10 mm; (c) adherent ascospores, bar = 5 mmEndomyces fibuliger Lindner Fig. 5.20 tight clusters of 2–4, ellipsoidal, with a longitudinalEndomycopsis fibuliger (Lindner) Dekker flange, offset from the spore axis to give a ‘‘bowlerSaccharomycopsis fibuligeria (Lindner) Klocker ¨ hat’’ appearance, 7–8 mm long, including the flange. Distinctive features. This species produces white,Colonies on CYA 18–30 mm diam and on MEA powdery, filamentous colonies which bear conidia15–25 mm diam, of low and sparse to moderately from spicules on vegetative hyphae, and sometimesdense white to pale grey mycelium, sometimes cen- also by budding (yeast-like cells).trally umbonate; reverse pale, off-white or very Taxonomy. This species has been described underpale yellow. Colonies on G25N 8–15 mm diam, both Endomycopsis and Saccharomycopsis; in thesimilar to those on CYA. No germination at 58C. latter case the epithet is spelled ‘‘fibuligera’’. AsAt 378C, colonies usually 5–20 mm diam, similar to explained above, we consider E. fibuliger to be theat 258C; reverse off-white to dull yellow brown. valid name. More recent taxonomies list this species Conidia borne from spicules (small projections) under Saccharomycopsis fibuligera (Lindner)along the length of undifferentiated hyphae, yeast- Klocker (Kurtzman and Fell, 1998; Barnett et al., ¨like, spherical, ellipsoidal or pyriform, 3–6 mm long, 2000) without taxonomic justification.with thin, smooth walls. Ascospores sometimes pro- This species and Hyphopichia burtonii have aduced in cultures on CYA or MEA, asci evanescent, close resemblance in culture. Both grow at more orascospores observed singly or more commonly in less the same rates and have similar habitats. The
    • 88 5 Primary Keys and Miscellaneous Fungifragmenting of fertile hyphae in H. burtonii is the brown, or sometimes reddish or greenish, in freshonly feature which provides a characteristic differ- isolates enveloping or surmounted by brown blackence in morphology. clusters of conidia, sometimes dominating colony Physiology. The physiology of Endomyces fibuliger appearance; clear to red brown exudate sometimeshas not been studied in any detail. A minimum growth produced; reverse usually orange brown to black ortemperature around 58C has been reported (Spicher, with pink, red or green areas. On MEA colonies1986b). E. fibuliger on wheat and rye bread was less generally similar to on CYA, sometimes with aaffected by reduced O2 levels than other spoilage different colour combination, or occasionally withmoulds and its growth could not be prevented by mod- surface slimy. On G25N colonies 3–10 mm diam,ified atmosphere packaging (Suhr and Nielsen, 2005). low to deep, yellow to dark brown; reverse similar, Mycotoxins. This type of fungus is unlikely to sometimes with yellow soluble pigment. At 58C,produce toxic compounds. response variable, no growth to colonies up to Ecology. This species is not uncommon in cereals 8 mm diam formed. No growth at 378C.and cereal products, especially packaged bread Conidia borne solitarily on short conidiophores,(Spicher, 1984a, 1985). In Europe, along with Hypho- usually in dense clusters, spherical with a broad,pichia burtonii, it is known as ‘‘chalky mould’’ of bread tapering, truncate base; brown, irregularly septate(Spicher, 1986b). It produces powerful amylases when mature, commonly 15–25 (–30) mm diam, with(Gogoi et al., 1987; Manilal et al., 1991). Endomyces rough walls obscuring numerous septa.fibuliger (Saccharomycopsis fibuligera) was found in According to Schol-Schwarz (1959), conidia onfermented potato pulp used in an Indonesian starter stems of sterile lupins measure 7–65 Â 6–54 mm. AsRagi tape (Abe et al., 2004) and in ‘‘Marcha’’, a tradi- has been observed with other genera, conidia ontional amylolytic starter used to produce sweet-sour agar media are much less variable in size and oftenalcoholic drinks in the Himalayan regions of India, smaller than on natural substrates.Nepal, Bhutan and Tibet (Tsuyoshi et al., 2005). Distinctive features. See the genus preamble. References. Von Arx (1981b); Barnett et al. Taxonomy. Kilpatrick and Chilvers (1981) exam-(1990; 2000). ined the variability of 2000 isolates of Epicoccum and concluded that all belonged to a single, geneti- cally variable species. A study of the 5.8S and ITS5.16 Genus Epicoccum Link regions of DNA confirmed this (Wang and Guo, 2004). In early literature this species was usuallyEpicoccum is a Hyphomycete genus, a saprophyte or known as E. nigrum Link. With the gradual accep-secondary invader of senescent plant tissue. It is char- tance of the 1821 starting date (See Chapter 3), thisacterised by the production of masses of large, sphe- name was in time replaced by E. purpurascens.rical, stalked, irregularly septate conidia, borne on Now, with the reversion to the 1753 starting date,rapidly growing multicoloured colonies. Epicoccum E. nigrum is again the correct name.is very widely distributed in the air, in soil and on Physiology. According to Kilpatrick and Chilversdecaying vegetation, one particular source being dry- (1981), maximum growth rates vary widely among iso-ing grass (Kilpatrick and Chilvers, 1981). Its ubiquity lates of this species. However, optimal temperatures forin the environment means it is commonly found on growth are usually 20–258C, with a maximum at 30–foods but it is an uncommon cause of spoilage. There 358C and a minimum below 58C. The optimum wateris a single species, Epicoccum nigrum. potential for growth is –20 bars (0.98 aw) and minimum –120 bars (0.91 aw) (Kilpatrick and Chilvers, 1981). Mycotoxins. Mycotoxin production has not beenEpicoccum nigrum Link Fig. 5.21 recorded.Epicoccum purpurascens Ehrenb. ex Schltdl. Ecology. As noted in the genus preamble, Epi- coccum is common in the general environment andColonies on CYA 60 mm diam or more, often hence readily finds its way onto foods such ascovering the whole Petri dish, low and dense or cereals and nuts. It has been reported to causefuniculose or floccose; mycelium orange brown, spoilage of cantaloupes, in which it produces a
    • 5.17 Genus Fusarium 89Fig. 5.21 Epicoccum nigrum (a) colonies on CYA and MEA, 7 d, 258C; (b) conidiophores and conidia, bar ¼ 10 mm;(c) conidia, bar ¼ 10 mmred discolouration (Snowdon, 1991), and as a Epicoccum nigrum was isolated from 29% ofpathogen on cucumbers, tomatoes, apples and soybean samples and 7% of cashew samples inpears (Bruton et al., 1993). Among other fungi, Thailand, with up to 12% infection in infected soy-E. nigrum can produce core rot of apples (Com- bean samples, 15% in cashews and 1% infectionbrink et al., 1985). It has been associated with overall in both (Pitt et al., 1994).spoilage of pecans and is sometimes present in References. Schol-Schwarz (1959); Domsch et al.barley after harvest and during malting, but is (1980); Kilpatrick and Chilvers (1981).not a cause of gushing (see Pitt and Hocking,1997; Niessen et al., 1991; Hudec, 2007). E. nigrumhas been reported on fresh vegetables, nuts and 5.17 Genus Fusarium Linkcereals. Other records include rice, wheat, maizeand wheat; gourds and muskmelons, pecans, pea- The character which defines the genus Fusarium isnuts, frozen and cured meats and biltong (see Pitt the production of septate, fusiform to crescent-and Hocking, 1997; Gros et al., 2003). shaped conidia, termed macroconidia, with a foot-
    • 90 5 Primary Keys and Miscellaneous Fungishaped basal cell and a more or less beaked apical Taxonomy. A bewildering array of taxonomiccell. Macroconidia may be produced in discrete systems have been applied to Fusarium, rangingpustules, called sporodochia, or in confluent, slimy from the milestone work of Wollenweber andmasses, known as pionnotes. Mounts from these Reinking (1935), which distinguished 65 species, toareas, which are usually cream, salmon pink or the simplification by Snyder and Hansen, whoorange, will reveal masses of these spores. However, accepted only 9 species in a series of papers in thethey are rarely widely distributed over the colony 1940s. The interrelationships of the principal taxo-and frequently are entirely absent. A light bank is nomic schemes for Fusarium are discussed by Boothinvaluable for promoting formation of macroconi- (1971), Nelson et al. (1983) and more recently bydia in Fusarium cultures. Leslie and Summerell (2006). In recent years, Fusar- Many species of Fusarium also produce smaller ium taxonomy and nomenclature has undergoneone to two celled conidia, microconidia, of various many changes, with molecular methods elucidatingshapes. Chlamydoconidia, either terminal or inter- the existence of cryptic species within some of thecalary, are characteristic of some species also. broader species concepts. In addition, many newFusarium colonies are usually fast growing and con- species have been described, often from naturalsist of felty aerial mycelium which may be pale, or ecosystems rather that economically importantbrightly coloured in shades of pink, red, violet or crops (see Leslie and Summerell, 2006). The nomen-brown. clature of Fusarium species described in this work Fusarium species are renowned for their role as follows that of Leslie and Summerell (2006) which isplant pathogens, causing a wide range of diseases based on Nelson et al. (1983) which, in turn, is basedsuch as vascular wilts, root and stem rots, pre- and on Wollenweber and Reinking (1935). Reference ispost-emergence blight and many others. Leslie and made to the names used by Nelson et al. (1983)Summerell (2006) include 70 species in their labora- where they differ from those adopted by Leslie andtory manual but the taxonomy of Fusarium is still Summerell (2006).undergoing change, with biological and phyloge- Mycotoxins. Fusarium is one of the three majornetic criteria as well as morphological concepts fungal genera producing toxins. The most wide-used to delineate species (Leslie and Summerell, spread Fusarium mycotoxins are the trichothecenes,2006). Fusarium species are widely distributed in a family of sesquiterpenes. More than 50 such com-soils, particularly cultivated soils, and are active in pounds are known to be produced by species in thisthe decomposition of cellulosic plant materials. genus. Some are highly toxic: none appears to beThey are a major cause of storage rots of fruits benign. Trichothecenes are often produced as mix-and vegetables and are commonly associated with tures even under pure culture conditions and arecereals and pulses, which they usually invade before very difficult to separate, so the toxicity of manyharvest. De Nijs et al. (1996) have published a com- compounds remains uncertain. The most importantpendium of the occurrence of more than 60 Fusar- are deoxynivalenol (and sometimes nivalenol), pro-ium species in raw materials, foods and feeds but, in duced by F. graminearum, F. culmorum and relatedour experience, a relatively small number of species species. Zearalenone, not a true mycotoxin, reallyare important in food spoilage. an oestrogen analogue produced by fungi, is formed A number of Fusarium species have teleomorphs by the same species that make deoxynivalenol. T-2belonging to the genera Nectria Fr., Calonectria De toxin, produced primarily by Fusarium sporotri-Not. and Gibberella Sacc. These teleomorphs are chioides, caused the deaths of many people andperithecial Ascomycetes, and are generally not pro- animals during the 20th century, but is of uncom-duced in culture. However, fertile strains of F. solani mon occurrence now. Some Fusarium species pro-sometimes form reddish brown perithecia on PDA duce other types of toxic compounds. The mostand F. graminearum usually produces dark purple important of these are the fumonisins, producedperithecia on Carnation Leaf Agar. These teleo- by F. verticillioides, F. proliferatum and severalmorphs are known as Nectria haematococca Berk. other species. Fumonisins are sphingosine analo-and Broome and Gibberella zeae (Schwein.) Petch, gues, interfering with membrane function in manrespectively (Booth, 1971). and animals (Miller and Trenholm, 1994). Other
    • 5.17 Genus Fusarium 91less toxic or less important compounds, such as Pigs are especially sensitive to zearalenone. Signsmoniliformin and fusaric acid, are discussed briefly of hyperoestrogenism generally appear at dietaryunder the appropriate species. levels > 1 mg/kg, but can occur at lower levels Deoxynivalenol and nivalenol. Like all trichothe- sometimes. In prepubertal gilts, clinical signscene mycotoxins, deoxynivalenol (DON) and niva- include vulval swelling, uterine enlargement andlenol are inhibitors of protein synthesis (Feinberg mammary development. Mature sows can developand MacLaughlin, 1989). Nivalenol is much more ovarian atrophy, constant oestrus and pseudopreg-toxic than DON, but is produced in much lower nancy (Hagler et al., 2001). Prepubertal male pigsquantities in grains, and is considered a less signifi- can undergo a feminising effect with mammarycant mycotoxin (Miller et al., 2001). DON is espe- development, decreased testicular size and loss ofcially toxic to pigs, where, at quite low intake levels libido, but mature boars are resistant (Hagler et al.,(< 5 mg/kg in feed) it causes vomiting, feed refusal 2001).and reduced weight gain due to neurotoxic effects. It was once claimed that humans had shownCattle and poultry are resistant to reasonable levels similar signs in Puerto Rico, but an FDA investiga-(> 5 mg/kg in feed) of DON (Miller et al., 2001). tion failed to confirm this (Goodman et al., 1987). In humans, high doses of DON may cause T-2 toxin. T-2 toxin is the cause of alimentaryabdominal pains, dizziness, headache, nausea, toxic aleukia (ATA) a devastating disease whichvomiting and other effects. Cases of such acute occurred in the USSR during and after Secondpoisoning are rare, but have been recorded from World War, in times of extreme food shortageIndia (Bhat et al., 1989), Japan (Udagawa, 1988) resulting in consumption of overwintered cerealsand several outbreaks in China (JECFA, 2001). (Joffe, 1978; Beardall and Miller, 1994). ManyDON is also immunosuppressive, and because of people, probably hundreds of thousands, died as aits widespread occurrence in grains, is undoubtedly result (Marasas et al., 1984). ATA was characterisedresponsible for susceptibility to bacterial and viral by leucopoenia, bleeding from nose, throat anddiseases in man and animals (Miller et al., 2001). gums, haemorrhagic rash, exhaustion of the bone The most comprehensive study of DON marrow and fever. Vomiting, nausea, diarrhoea andincluding occurrence, toxicity, analytical meth- abdominal pain also usually occurred. Decrease inods and intake levels is that by JECFA (2001). immunological functions led to susceptibility toThey concluded that DON is not significantly bacterial and viral diseases, and often death (Joffe,carcinogenic, mutagenic or teratogenic (JECFA, 1978; Beardall and Miller, 1994). Haemorrhagic2001). The Committee set a provisional tolerable syndrome in cattle, pigs and poultry in the Uniteddaily intake of 1 mg/kg weight per day. They were States in the 1960s was also probably due to T-2unable to estimate the level of DON in foods toxin (Desjardins, 2006).below which any acute effect in humans would T-2 toxin is produced by F. sporotrichioides and,occur. less commonly, F. poae (Desjardins, 2006). It Zearalenone. Zearalonone is one of the five most appears to be produced only under cold conditions,significant mycotoxins (Miller, 1995). It is not a true and fortunately is now uncommon. The occurrence,mycotoxin, being a nonsteroidal oestrogen pro- toxicity and biology of T-2 toxin were examined induced by a fungus. It is not acutely toxic and has detail by JECFA (2001). They established that T-2not been associated with any fatal disorder in ani- (and its metabolite HT-2) were immunotoxic andmals or humans. However, it has caused oestrogenic haemotoxic compounds in several animal speciessyndromes in pigs, and perhaps in human adoles- after short-term intake. Long-term effects couldcents as well. Zearlaenone is produced by the same not be evaluated. T-2 was at most weakly genotoxic.Fusarium species that produce deoxynivalenol, i.e. In the absence of long-term studies, T-2 was notF. graminearum and related species, especially classifiable as to carcinogenicity (JECFA, 2001).F. culmorum, F. equiseti and F. crookwellense. The Fumonisins. Fumonisins are produced by F.association of these fungi with cereal crops is world- verticillioides (formerly known as F. moniliforme),wide, as is the production of zearalenone (Marasas by the closely related species F. proliferatum,et al., 1984). uncommonly by F. subglutinans and F. oxysporum,
    • 92 5 Primary Keys and Miscellaneous Fungiand about 10 other species not treated here. The with neural tube defects such as spinal bifida in afirst two named species are by far the most impor- population along the Texas–Mexican border (Des-tant. F. proliferatum is one cause of maize ear rot in jardins, 2006). For the human population, a provi-Europe and is a pathogen on a variety of other sional maximum tolerable daily intake (PMTDI) of 2crops. F. verticillioides is endemic in maize and mg/kg body weight per day was established byfound wherever that crop is grown. This species JECFA (2001).grows well at higher temperatures, and ear rot and Determining which particular Fusarium speciesfumonisin accumulation are associated with produces which mycotoxins has not been easy.drought, insect stress and growing hybrids outside Unstable taxonomy and misidentification havetheir areas of adaptation (Miller, 2001, 2008). combined to cloud the picture. In a major contribu- Fumonisins are analogues of sphingosines, essen- tion, Marasas et al. (1984) examined several hun-tial components of cell membranes. Fumonisins have dred Fusarium isolates, identifying them accordingbeen reported to interfere specifically with dihydro- to Nelson et al. (1983) and then assessing mycotoxinceramide, an essential enzyme in the biosynthesis of production. Sections on mycotoxins in the speciessphingosine and related compounds (Wang et al., descriptions which follow are based on Marasas1991). Fumonisins have been found to be biologi- et al. (1984), Desjardins (2006) and Leslie andcally active in many animal species, but horses and Summerell (2006). Claims concerning mycotoxinpigs are the most affected. In horses, fumonisins production by particular species in more recentare responsible for leucoencephalomalacia, a rapidly references should be treated with some caution.progressing disease that causes tissue liquefaction in Cultural instability. Many Fusarium species areequine brains. It has been known in the United States notorious for their instability in culture. Isolates offor more than 100 years that feeding maize to horses some species will degenerate quickly, often afteris a dangerous practice (Desjardins, 2006): it was not only one or two transfers. For this reason, it isuntil 1988 that the reason became apparent (Gelder- important to identify Fusaria as soon as possibleblom et al., 1988; Marasas et al., 1988). For horses, after primary isolation. Pure cultures for identifica-consumption of feed containing > 10 mg/kg fumo- tion are traditionally started from a single germi-nisin B1 in the diet (equivalent to 0.2 mg/kg body nated spore, as the mass transfer of Fusaria appearsweight per day) was associated with increased risk of to increase the rate of deterioration of strains indeveloping this disease (JECFA, 2001). In pigs, culture.fumonisins cause pulmonary oedema, due to left Identification procedures. Identification of Fusar-ventricle heart failure (JECFA, 2001), while in ium isolates is often difficult, but the task can berats the primary effect is to cause liver cancer made easier by observing a few basic rules:(Gelderblom et al., 1991), but they also cause pro-  identify cultures as soon as possible after primarygrammed cell death (apoptosis; Tolleson et al., 1996). isolation;It is unusual for a single toxin to have such diverse  always grow cultures for identification from sin-effects in different animal species, and the reasons for gle germinated conidia;that remain unknown (Desjardins, 2006).  use standardised media and incubation In humans, fumonisins and Fusarium verticil- conditions;lioides are associated with oesophageal cancer.  use a light bank (Chapter 4) whenever possible.Extensive studies in areas of low and high maizeconsumption in South Africa have established this Diagnostic features. The main characters usedconnection (Rheeder et al., 1992; JECFA, 2001). to distinguish species of Fusarium are (1) the sizeThis disease is also prevalent in areas of China and and shape of the macroconidia; (2) the presenceoccurs at significantly higher levels than background or absence of microconidia; (3) the manner inalso in parts of Iran, northern Italy, Kenya and a which microconidia are produced; (4) the type ofsmall area of the southern USA (JECFA, 2001). In phialide on which microconidia are produced; (5)all of those areas consumption of maize and maize the presence or absence of chlamydoconidia; andproducts is very high (JECFA, 2001). There is also (6) the colours and morphology of colonies onincreasing evidence that fumonisins are associated PDA.
    • 5.17 Genus Fusarium 93 The morphology of macroconidia is a principal compound microscope. Some species produce micro-diagnostic feature for Fusarium species. Macroco- conidia in false heads (small, mucoid, adherent ballsnidia generally have at least three septa, with a of conidia) and others produce them singly. In somedifferentiated apical cell which may be pointed, species, microconidia are produced on phialides withrounded, hooked or filamentous, and a basal cell only one pore, and these are termed monophialides,which may be foot-shaped, with a distinct heel, or but a few species produce phialides with more thanjust slightly notched. Fusarium macroconidia gen- one pore (polyphialides). Species which produce poly-erally exhibit some degree of curvature, the convex phialides usually produce monophialides as well.and concave sides being referred to as the dorsal and Descriptions. Descriptions of the microscopic fea-ventral sides, respectively. Although some macro- tures of species in this book are based on structuresconidia are usually produced in the aerial mycelium, formed in cultures grown on DCPA at 258C, with athe shape and size of those in sporodochia are more 12 h photoperiod, under a light bank consisting ofregular and are used for identification purposes two cool white fluorescent tubes and one black lightwhere possible. tube (see Chapter 4). Cultures should be examined at Microconidia are usually produced in the aerial 7 days, then at 10 days and 14 days if sporulation ismycelium and their shape can be very important in poor. Aerial mycelium often develops better after 10Fusarium identification. Most species which pro- days incubation on DCPA and chlamydoconidiumduce microconidia form only a single type, the production is more reliable in older cultures.most common shape being ellipsoidal to clavate. Descriptions of the colony characteristics are takenHowever, F. poae produces spherical to apiculate from cultures grown on PDA at 258C for 7 days, alsomicroconidia and F. sporotrichioides produces a vari- with a 12 h photoperiod. Additional information mayety of shapes: ellipsoidal, pyriform and spherical. be gained by recording the growth rates of colonies onThe method of production of microconidia and the PDA at 258C and 308C after 3 days (Burgess et al.,types of phialides on which they are borne are also 1994). Growth rates at 308C on PDA are particularlyuseful diagnostic criteria. F. verticillioides produces helpful in distinguishing isolates of Fusarium avena-its microconidia in long, delicate, dry chains, which ceum from those of Fusarium acuminatum.are best observed by using the 10Â objective of the Key to Fusarium species included here1 Microconidia abundant 2 Microconidia rare or absent 92 (1) Colonies on PDA with mycelium and/or reverse coloured greyish rose or burgundy 3 Colonies on PDA in shades of cream, pale salmon or violet 53 (2) Microconidia spherical to apiculate, borne singly on monophialides F. poae Microconidia ellipsoidal, clavate, fusiform and/or pyriform, borne on polyphialides or both polyphialides and monophialides 44 (3) Microconidia clavate only, produced profusely, giving colonies on PDA a powdery appearance F. chlamydosporum Microconidia various shapes: clavate, pyriform and spindle shaped (check PDA cultures also) F. sporotrichioides5 (2) Microconidia produced in long or short chains (some false heads may also be present) 6 Microconidia produced singly or in false heads 76 (5) Microconidia produced from monophialides only F. verticillioides Monophialides and polyphialides both present F. proliferatum
    • 94 5 Primary Keys and Miscellaneous Fungi7 (5) Colonies cream or bluish, sporodochia cream F. solani Colonies pale salmon or violet, sporodochia salmon 88 (7) Microconidia borne on short, stout monophialides; chlamydoconidia usually produced F. oxysporum Microconidia borne on polyphialides and slender monophialides; chlamydoconidia not produced F. subglutinans9 (1) Colonies cream, pale salmon or brown 10 Colonies greyish rose to burgundy 1110 (9) Macroconidia cigar- or spindle-shaped, produced in the aerial mycelium F. semitectum Macroconidia obviously curved, produced in sporodochia F. equiseti11 (9) Macroconidia robust, ventral side straight; aerial mycelium tan to brown 12 Macroconidia delicate and slender, slightly or definitely curved; aerial mycelium white or pinkish 1312 (11) Macroconidia short and stout, up to 7 mm wide F. culmorum (See F. graminearum) Macroconidia longer and narrower, maximum width 5.5 mm F. graminearum13 (11) Macroconidia with elongated basal cell and long, whip-like apical cell F. longipes Macroconidia with basal and apical cells not obviously elongated 1414 (13) Macroconidia delicate and needle-like, with sides almost parallel F. avenaceum Macroconidia with slight to definite curvature F. acuminatumFusarium acuminatum Ellis & Everh. Fig. 5.22 the widest point often one third of the distance from the base, giving a ‘‘bottom-heavy’’ appearance;Telemorph: Gibberella acuminata Wollenw. microconidia produced sparsely by some isolates; chlamydoconidia produced, relatively slowly.Colonies on CYA 40–50 mm diam, of dense, felty Distinctive features. Ruby to dark ruby reversemycelium, white to greyish rose or greyish magenta; colours on PDA, and relatively slender, slightlyreverse uniformly pale or with areas of greyish rose. curved macroconidia, usually with five septa, areColonies on MEA 45–65 mm diam, yellow brown the distinctive features of Fusarium acuminatum.centrally, greyish rose at the margins; reverse deep However, unless chlamydoconidia are present, thisbrownish yellow to brownish orange, occasionally species can be confused with F. avenaceum (seepale. Colonies on G25N 9–15 mm diam. At 58C, below). F. armeniacum is very closely related (Bur-colonies 7–12 mm diam. No growth at 378C. gess and Summerell, 2000). On PDA, colonies usually covering the whole Taxonomy. Perithecia of Gibberella acuminata Wol-Petri dish, of dense to floccose white to pale salmon lenw., the teleomorph of F. acuminatum, are formed inmycelium, sometimes greyish rose at the margins; the laboratory when opposite mating types are inocu-reverse dark ruby centrally, greyish ruby at the lated onto sterile wheat straws (Booth, 1971). Isolatesmargins. On DCPA, colonies sparse, of floccose to of F. acuminatum show considerable variability in cul-funiculose white to pale salmon mycelium; reverse ture, and this variability, correlated with secondarypale or with brownish red annular rings. metabolite production (Logrieco et al., 1992), has Macroconidia relatively slender, usually with been shown to be due to the fact that two speciesfive septa, but three and four septa not uncommon, were included within F. acuminatum. F. armeniacumwith a long, tapering apical cell and foot-shaped (Forbes et al.) L.W. Burgess and Summerell, previouslybasal cell, distinctly but not highly curved, with described as F. acuminatum var. armeniacum G.A.
    • 5.17 Genus Fusarium 95Fig. 5.22 Fusarium acuminatum (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) macroconidia, bar = 10 mmForbes et al. has been elevated to species level (Burgess Logrieco et al. (1992) divided the isolates into threeand Summerell, 2000) being distinguished on morpho- categories, (a) enniatin B and/or moniliforminlogical grounds, isoenzyme patterns, molecular mar- producers (probably F. acuminatum sensu stricto),kers and mycotoxin profiles. Macroconidia of F. arme- (b) T-2, HT-2 and/or neosolaniol producersniacum are produced in distinct apricot coloured (probably F. armeniacum) and (c) nontoxigenic.sporodochia. F. acuminatum sensu stricto produces moniliformin Physiology. Some isolates of Fusarium acumina- (Chelkowski et al., 1990; Logrieco et al., 1992) andtum have antioxidant enzyme activity (Kayali and enniatins (Logrieco et al., 1992; Kononeko et al.,Tarhan, 2005). 1993; Desjardins, 2006; Leslie and Summerell, 2006) Mycotoxins. Most of the mycotoxin production as well as some other minor toxins (Desjardins,reported from Fusarium acuminatum, particularly 2006).the production of trichothecene toxins, is probably Ecology. Fusarium acuminatum has been isolatedmore correctly due to F. armeniacum (Desjardins, from a wide variety of plants throughout the world.2006; Leslie and Summerell, 2006). Isolates produ- Although some isolates may cause severe root rot incing type A trichothecenes including T-2 toxin, particular legume species (Leslie and Summerell,HT-2 toxin and diacetoxyscirpenol (see Pitt and - 2006), F. acuminatum is generally regarded as a sapro-Hocking, 1997) are most likely to be F. armeniacum. phyte. It has been reported to cause rot in pumpkinsIn a survey of 25 isolates of F. acuminatum (Elmer, 1996), is one cause of rot in stored potatoesfrom different sources and geographic locations, (Theron and Holz, 1990) and is weakly pathogenic in
    • 96 5 Primary Keys and Miscellaneous Fungi ´bananas (Jimenez et al., 1993). It is quite common in mycelium coloured white, very pale rose or deeperpoor quality wheat from cool temperate zones (Mills greyish rose; reverse varying from pale to paleand Wallace, 1979; Abramson et al., 1987). It has been yellow or with areas of greyish rose or sometimesisolated from developing peanut pods (Barnes, 1971), uniformly deep burgundy. Colonies on MEA 45–barley (Abdel-Kader et al., 1979) and, in our labora- 55 mm diam, low to moderately deep, of opentory, from rain damaged sorghum and soybeans. The floccose to funiculose mycelium, coloured white,incidence of F. acuminatum in tropical commodities pale rose or greyish rose, sometimes brown cen-was low (Pitt et al., 1993, 1994). trally; reverse brownish orange, sometimes paler References. Domsch et al. (1980), under G. acu- centrally or at the margins. Colonies on G25N 9–minata; Nelson et al. (1983); Desjardins (2006); 15 mm diam. At 58C, colonies 10–12 mm diam.Leslie and Summerell (2006). No growth at 378C. On PDA, colonies moderately deep to deep, of denseFusarium avenaceum (Fr.) Sacc. Fig. 5.23 mycelium coloured white, pale salmon or sometimes dark brownish red, with central masses of reddishTeleomorph: Gibberella avenacea R.J. Cooke orange sporodochia, sometimes surrounded by an outer ring of paler sporodochia; reverse greyish red,Colonies on CYA covering the whole Petri dish, with darker annular rings, paler towards the margins.moderately deep to deep, of open, floccose On DCPA, colonies deep, of moderately dense white to pale salmon mycelium with a central mass of orange toFig. 5.23 Fusarium avenaceum (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) macroconidia, bar = 10 mm
    • 5.17 Genus Fusarium 97salmon sporodochia, often surrounded by concentric Ecology. Fusarium avenaceum has a worldwiderings of sporodochia; reverse pale. distribution wherever crops are grown, but is relatively Macroconidia long, slender, with four to seven uncommon in food commodities. It is a majorsepta, thin-walled, straight or slightly curved, with a component of Fusarium head blight in cereals in Eur-tapering apical cell and a notched or foot-shaped ope, the US Pacific Northwest region and Canadabasal cell; microconidia produced sparsely by some (Desjardins, 2006). Logrieco et al. (2002) identified F.isolates; chlamydoconidia absent. avenaceum as a component of Fusarium ear rot of Distinctive features. Fusarium avenaceum is dis- maize in Europe. It has been reported from barleytinguished by thin walled, needle-like macroconi- (Flannigan, 1969; Petters et al., 1988; Stenwig anddia and by the absence of chlamydoconidia. Liven, 1988) where it may inhibit germination of malt-Despite the fact that F. avenaceum and F. acumi- ing grains (Hudec, 2007), but is of minor importance innatum are not considered by Fusarium taxonomists gushing of beer (Niessen et al., 1992). Other reportedto be closely related, these two species can be diffi- sources are sorghum (Onyike and Nelson, 1992), pea-cult to distinguish, as isolates with macroconidia nuts (Joffe, 1969), pigeon peas (Maximay et al., 1992)of intermediate form are not uncommon. Colony and, in our laboratory, triticale. F. avenaceum has beendiameters on PDA at 308C after 3 days can be a reported to cause spoilage of cool-stored broccoli (Mer-useful differentiating feature: under these condi- cier et al., 1991), dry rot of stored carrots in Italytions colonies of F. avenaceum are usually 8– (Marziano et al., 1992) and dry rot of rutabaga (swede15 mm diam, whereas those of F. acuminatum are turnip) in Canada (Peters et al., 2007). It has occasion-15–28 mm diam (Burgess et al., 1994). Isolates of F. ally caused spoilage of apples, pears, asparagus, toma-avenaceum show an unusually broad range of col- toes, eggplant and potatoes (Snowdon, 1990, 1991) andours on PDA and also have a very broad host has been reported as a postharvest pathogen of stone-range. However, extensive genetic analysis has fruit in New Zealand (Hartill and Broadhurst, 1989).shown no bases from splitting the species, and References. Domsch et al. (1980), as G. avenacea;pathogenicity tests on single strains have con- Nelson et al. (1983); Leslie and Summerell (2006).firmed the broad host range (Nalim, 2004). Taxonomy. The teleomorph of Fusarium avena- Fusarium chlamydosporum Wollenw. &ceum is Gibberella avenacea R.J. Cook. It is not Reinking Fig. 5.24usually seen in culture on the media used here. Fusarium fusarioides (Gonz. Frag. & Cif.) Booth Physiology. The optimum growth temperature forFusarium avenaceum is 358C, the minimum near –38C Colonies on CYA covering the whole Petri dish, ofand the maximum 318C (Domsch et al., 1980). The low to moderately deep floccose mycelium, colouredminimum aw for growth is approximately 0.90 at 258C white to pale rosy pink, often with surface appearing(Magan and Lacey, 1984c), and the pH optimum powdery due to production of microconidia; reverseranges between 5.4 and 6.7 (Domsch et al., 1980). pale to greyish rose or brownish red. Colonies on Mycotoxins. This species has been reported to pro- MEA 55–70 mm diam, of low, moderately denseduce a variety of trichothecene and other mycotoxins. mycelium in shades of yellow brown, or greyish roseHowever, Nelson et al. (1983) regarded only reports of to greyish ruby, paler at the margins; reverse deepmoniliformin production as accurate. Later reports yellow brown to orange brown. Colonies on G25Nhave confirmed this (Chelkowski et al., 1990; Abbas 15–20 mm diam. At 58C, colonies 1–2 mm diam. Atet al., 1991; Bosch and Mirocha, 1992). Reports of 378C, colonies 5–15 mm diam.production of fusarin C (Farber and Sanders, 1986; On PDA, colonies of felty mycelium, colouredThrane, 1988; Leonov et al., 1993) and enniatins pale salmon, sometimes browner, or with patches of(Blais et al., 1992; Kononeko et al., 1993) also appear greyish red, often with a powdery appearance fromto be reliable (Desjardins, 2006; Leslie and Summerell, profuse microconidial production; reverse deep vio-2006). Production of any trichothecene toxin has not let brown to dark ruby, paler at the margins. Onbeen confirmed, and Fusarium avenaceum does not DCPA, colonies of sparse, floccose, pale salmoncarry the tri5 gene which is essential for trichothecene mycelium, often powdery with microconidia, show-production (Tan and Niessen, 2003). ing poorly defined annulations; macroconidia
    • 98 5 Primary Keys and Miscellaneous FungiFig. 5.24 Fusarium chlamydosporum (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) polyphialides, bar = 10 mm; (d)macroconidia, bar = 10 mm; (e) microconidia, bar = 10 mmoccasionally produced near the colony centres in Taxonomy. Fusarium chlamydosporum has prior-salmon sporodochia; reverse pale. ity over F. fusarioides as the correct name for this Macroconidia often rare, relatively short and species (Domsch et al., 1980; Nelson et al., 1983;stout, usually with three to five septa, slightly Leslie and Summerell, 2006).curved; microconidia produced abundantly from Physiology. This species has minimum, optimumpolyphialides in the aerial mycelium, with zero to and maximum temperatures for growth of 5, 27 andtwo septa, fusiform to slightly clavate. Chlamydo- 378C (Seemuller, 1968). ¨conidia usually abundant in older cultures, pro- Mycotoxins. Production of type A trichothecenesduced singly, in pairs or in clumps. (including T-2 toxin, HT-2 toxin, monoacetoxyscir- Distinctive features. The presence of abundant penol, neosolaniol and iso-neosolaniol) by Fusar-fusiform microconidia borne on polyphialides is ium chlamydosporum was reported by Park andthe most outstanding feature of Fusarium chlamy- Chu (1993); however, subsequent studies havedosporum. Also colonies on PDA have dark violet found no evidence of trichothecene production inbrown to dark ruby reverse colours. this species (Desjardins, 2006). Moniliformin is the
    • 5.17 Genus Fusarium 99major mycotoxin produced by F. chlamydosporum Fusarium culmorum (W.G. Smith) Sacc.(Marasas et al., 1984; Desjardins, 2006). Fig. 5.25 Ecology. Fusarium chlamydosporum is mainly aninhabitant of soils in warmer climates (Domsch Colonies on CYA covering the whole Petri dish, ofet al., 1980; Leslie and Summerell, 2006), and is dense felty mycelium, often with a floccose overlay,not regarded as a plant pathogen or spoilage fun- sometimes reaching the Petri dish rim, pale red togus. However, it is commonly isolated from grains pastel red; reverse pastel red to deep red. Coloniesin drier areas, particularly in the Middle East, on MEA 60 mm or more diam, floccose, in age oftensouthern Europe, central Asia and Australia (Leslie reaching the Petri dish lid, pale red to pastel red,and Summerell, 2006), and has also been isolated commonly with a greyish orange to yellowish brownfrom pearl millet (Wilson et al., 1993; Jurjevic et al., overlay; reverse brown to reddish brown. Colonies2007), pecans (Huang and Hanlin, 1975) and sor- on G25N usually 5–10 mm diam, mycelium orangeghum (Rabie et al., 1975; Onyike and Nelson, 1992). white, reverse yellow to orange. At 58C, germina-A low incidence of F. chlamydosporum was found in tion. No growth at 378C.peanuts from both Indonesia and the Philippines On PDA, colonies covering the whole Petri dish,(Pitt et al., 1998a) and from mung beans and sor- of dense to floccose mycelium, pale red and paleghum in Thailand (Pitt et al., 1994). Involvement in yellow brown; reverse red to deep red. On DCPA,dry rot of potatoes has also been reported (Somani, colonies 50–65 mm diam, of sparse mycelium,2004; Esfahani, 2006). orange to pinkish white, bearing abundant macro- References. Domsch et al. (1980); Nelson et al. conidia in orange sporodochia; reverse dull orange(1983); Leslie and Summerell (2006). brown.Fig. 5.25 Fusarium culmorum (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) macroconidia, bar = 10 mm
    • 100 5 Primary Keys and Miscellaneous Fungi Macroconidia relatively short, wide and only Chandler et al., 2003; Jennings et al., 2004) andslightly curved, with four to five septa, 30–45 mm zearalenone (Bakan et al., 2001; Hestbjerg et al.,long, with rounded or sometimes papillate apical 2002; Llorens et al., 2004a; Brinkmeyer et al.,cells; basal cells with a slight to definite notch, some- 2005). Moniliformin production was reported bytimes papillate. Microconidia not produced. Chla- Scott et al. (1987) but was not detected in 42 isolatesmydoconidia sometimes formed, in conidia, or of F. culmorum from Canada by Abramson et al.intercalary in the hyphae, singly or in chains, 9–14 (2001). Reports of production of type A trichothe-mm diam, smooth walled. cenes (T-2 toxin, HT-2 toxin) have not been sub- Distinctive features. Short, stout macroconidia stantiated (Leslie and Summerell, 2006).are the prime feature distinguishing Fusarium cul- The existence of two chemotypes of Fusariummorum from most other species. F. culmorum may culmorum, those that produce deoxynivalenol andbe confused with F. crookwellense L.W. Burgess those that produce nivalenol (Miller et al., 1991),et al., but macroconidia of the latter species have a has been confirmed by molecular studies. Withindistinctly foot-shaped basal cell, whereas those of F. the trichothecene gene cluster, isolates possessingculmorum are shorter and stouter, and the basal cell the Tri7 and Tri13 genes produce nivalenol andis not distinctly foot-shaped. related compounds, whereas sequences in the Tri3, Taxonomy. No teleomorph is known for this Tri5 and Tri6 genes are associated with deoxyniva-species. lenol production (Chandler et al., 2003; Jennings Physiology. Fusarium culmorum has been et al., 2004; Quarta et al., 2005, 2006). Of 55 Eur-reported to be psychrotrophic, growing down to opean isolates examined by Quarta et al. (2005), 1108C, with an optimum at 218C and a maximum of were the nivalenol chemotype, and the remainderonly 318C (Arsvoll, 1975); however, Magan and were deoxynivalenol producers. Jennings et al.Lacey (1984c) reported growth at 358C. The mini- (2004) examined 153 isolates from England andmum aw for growth is 0.87 at 20–258C and pH 6.5: at Wales, and found that the DON chemotype waspH 4.0, growth did not occur below 0.90 aw (Magan dominant over the NIV chemotype (59% vs 41%,and Lacey, 1984a). A strain of F. culmorum produced respectively). Lauren et al. (1992) examined 45 iso-deoxynivalenol optimally at 258C, but only between lates of F. culmorum from New Zealand soil and0.995 and 0.97–0.96 aw (Hope and Magan, 2003; pasture and found none produced deoxynivalenolHope et al., 2005). At 158C, deoxynivalenol was or its monoacetyl isomers. Two chemotypes wereproduced in lower concentrations later in the growth identified, one producing diacetylnivalenol withcycle, but over a slightly greater aw range (0.995 to culmorin as the major metabolite accounted for0.95–0.94 aw). The dynamics of nivalenol production 95% of isolates, while the other chemotype pro-for this strain was similar (Hope and Magan, 2003). duced diacetoxyscirpenol.Zearalenone production by F. culmorum was Ecology. This species has a worldwide distributionreported to be optimal above 258C (Bottalico et al., in soil and as a pathogen of cereals and other hosts,1982). F. culmorum is very tolerant of low O2 tensions with a higher incidence in temperate climates(Magan and Lacey, 1984b). Radiation resistance of (Domsch et al., 1980; Nelson et al., 1983; Leslie andF. culmorum was relatively high: up to 0.8 kGy were Summerell, 2006). It is an important component of theneeded for a tenfold reduction in spore numbers on cohort of Fusarium species that cause head blight ofgrain, and up to 1.39 kGy on media (O’Neill et al., wheat and associated cereal crops in Europe, Canada,1991). China and other areas with cool weather during the Mycotoxins. Fusarium culmorum produces a growing season (Desjardins, 2006). In wheat it causesvariety of mycotoxins: indeed the list in our files is extensive internal damage to the grain, and reductionsof more than 40 compounds. However, the most in flour yield and baking quality (Meyer et al., 1986).important toxins confirmed to be produced by this F. culmorum was reported as the dominant Fusariumspecies (Nelson et al., 1983; Marasas et al., 1984; species on barley in Europe (see Pitt and Hocking,Desjardins, 2006; Leslie and Summerell, 2006) are 1997). It also occurs in triticale (Perkowski et al.,deoxynivalenol, nivalenol and their derivatives 1988). F. culmorum has been identified as a compo-(Abramson et al., 2001; Hestbjerg et al., 2002; nent of Fusarium ear rot of maize in Europe (Logrieco
    • 5.17 Genus Fusarium 101et al., 2002). F. culmorum was one cause of crown rot covering the whole Petri dish, of open, floccosein bananas (Wade et al., 1993), and is a minor cause of white to pale brown mycelium; reverse pale, orspoilage of apples and pears (Snowdon, 1990). sometimes showing areas of pale greyish red. Colo- References. Domsch et al. (1980); Nelson et al. nies on G25N 12–20 mm diam. At 58C, colonies of 1–(1983); Desjardins (2006); Leslie and Summerell 4 mm diam produced. At 378C, usually no growth,(2006). although in isolates from the tropics, colonies up to 35 diam Produced.Fusarium equiseti (Corda) Sacc. Fig. 5.26 On PDA, colonies of dense to floccose mycelium, white to pale salmon, becoming brown with age, withTeleomorph: Gibberella intricans Wollenw. a central mass of orange to brown sporodochia, some- times surrounded by poorly defined sporodochialColonies on CYA filling the whole Petri dish, often rings; reverse pale salmon, often with a brown centralto the lid, of dense to floccose white mycelium; area and brown flecks. On DCPA, colony appearancereverse pale or pale salmon. Colonies on MEA usually dominated by salmon, orange or brownishFig. 5.26 Fusarium equiseti (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) macroconidia, bar ¼ 10 mm;(d) chlamydoconidia, bar ¼ 10 mm
    • 102 5 Primary Keys and Miscellaneous Fungisporodochia centrally and in poor to well defined Ecology. A cosmopolitan soil fungus, Fusariumconcentric rings; mycelium low and sparse, coloured equiseti, has a distribution extending from Alaska towhite or pale salmon; reverse pale. the tropics (Domsch et al., 1980). It has been isolated Macroconidia distinctly curved, often with a from a variety of plants, particularly cereals, where it‘‘hunchbacked’’ appearance, with five to seven septa, may cause stem and root rots (Leslie and Summerell,ranging from relatively short in some isolates to very 2006). F. equiseti has been identified as a minor com-long in others, with a wide range of sizes often present ponent of maize ear rot in Europe, and head blight ofin a single isolate, basal cell distinctly foot-shaped, wheat and associated grains in Europe and Northoften with an elongated heel, apical cell elongated American (Desjardins, 2006). It has been reportedand curved, in long spores becoming filamentous; from various cereal grains including wheat, maize,chlamydoconidia usually produced abundantly, in barley, rye and rice (see Pitt and Hocking, 1997; Pittchains or clumps; microconidia absent. et al., 1998a) and also from peanuts, walnuts, soy- Distinctive features. Fusarium equiseti produces beans, cowpeas, sorghum, oilseeds and herbs (seedistinctly curved macroconidia which are often Pitt and Hocking, 1997). It has been isolated fromelongate, especially in the basal and apical cells. tomatoes (Okoli and Erinle, 1989) and capsicumsColonies on PDA are floccose, usually with at (Adisa, 1983; Hashmi and Ghaffar, 1991), and hasleast a central mass of orange sporodochia, and been reported as contributing to soft rot of bell pep-with reverse pale salmon, often flecked with pers in India (Shukla and Sharma, 2000) and crownbrown. Chlamydoconidia are usually abundant; ´ rot of bananas (Wallbridge, 1981; Jimenez et al.,microconidia are not produced. 1993). It has also been implicated as a cause of rots Taxonomy. The teleomorph of Fusarium equiseti of cucurbit fruits in contact with soil (Burgess et al.,is Gibberella intricans Wollenw. However, there are 1994), dry rots of potato tubers (El-Hassan et al.,few records of its occurrence in nature (Booth, 2004) and soft rot of pumpkins (Elmer, 1996).1971). F. equiseti has caused spoilage of UHT processed Physiology. Fusarium equiseti grows strongly at fruit juices, due to its ability to grow in very low O2308C (Burgess et al., 1994) but, from observations in tensions (Hocking, 1990).our laboratory, most isolates grow poorly or not at References. Domsch et al. (1980) as Gibberellaall at 378C. The minimum aw for growth has been intricans; Nelson et al. (1983); Desjardins (2006);reported to be 0.92 aw (Chen, 1966). Growth Leslie and Summerell (2006).occurred at pH 3.3 but not 2.4, and was still rapidat pH 10.4 (Wheeler et al., 1991). Tripathi et al. Fusarium graminearum Schwabe Fig. 5.27(1999) reported growth between pH 2.0 and 11.00.Growth in N2 with <1% O2 was 90% of that in air Teleomorph: Gibberella zeae (Schwabe) Petch(Hocking, 1990). Mycotoxins. Fusarium equiseti has been reliably Colonies on CYA filling the whole Petri dish, oftenreported to produce a number of mycotoxins. Tri- to the lid, of dense, floccose mycelium colouredchothecene production in F. equiseti is variable, greyish rose, greyish yellow or paler; reverse usuallywith both type A trichothecenes (diacetoxyscirpenol orange red to greyish ruby, though sometimes paleand related compounds, T-2 toxin, HT-2 toxin and brownish pink. Colonies on MEA filling the wholeneosolaniol) and type B trichothecenes (deoxyniva- Petri dish, often reaching the lid at the margins atlenol, 15-acetyldeoxynivalenol, fusarenone-X and least, of dense to openly floccose mycelium, innivalenol) reliably reported (see Pitt and Hocking, shades of greyish rose and greyish yellow to golden1997; Langseth et al., 1999; Hestbjerg et al., 2002; brown; reverse orange brown to yellowish brown,Kosiak et al., 2005; Desjardins, 2006). Zearalenone, sometimes paler at the margins. Colonies on G25Nmoniliformin, beauvericin, fusarochromanone and 20–30 mm diam, occasionally more. At 58C, colo-related compounds have also been reported (see Pitt nies of 5–12 mm diam produced. No growth atand Hocking, 1997; Logrieco et al., 1998a; Desjar- 378C.dins, 2006), but production of butenolide is ques- On PDA, colonies filling the whole Petri dish, oftionable (Desjardins, 2006). dense, floccose mycelium coloured olive brown,
    • 5.17 Genus Fusarium 103Fig. 5.27 Fusarium graminearum (a) colonies on PDA and DCPA, 7 d, 258C; (b) Gibberella zeae perithecium and ascospores,bar ¼ 25 mm; (c) macroconidia, bar ¼ 10 mmyellowish brown, reddish brown or pale salmon, or dense to floccose greyish rose to golden brownin combinations of those colours; sometimes with a mycelium and dark ruby reverse. Macroconidiacentral mass of red brown to orange sporodochia; are relatively straight and thick-walled, with areverse ruby to dark ruby centrally, sometimes foot-shaped basal cell. Microconidia are notviolet brown. On DCPA, colony appearance domi- produced.nated by salmon to orange sporodochia in con- Taxonomy. The teleomorph of Fusarium grami-centric rings, overlaid by sparse, floccose, pale sal- nearum is Gibberella zeae (Schwabe) Petch. Francismon mycelium. and Burgess (1977) described two populations of F. Macroconidia usually with five septa, sometimes graminearum, which they termed Group 1 andless, thick walled, straight to moderately curved, Group 2. Group 2 isolates, the real F. graminearum,with the ventral surface almost straight and a are responsible for head scab of wheat and alsosmoothly arched dorsal surface; basal cell distinctly cause destructive ear rot of maize (Burgess et al.,foot-shaped, apical cell tapered; chlamydoconidia 1981; Desjardins, 2006; Leslie and Summerell,formed tardily in some isolates; microconidia 2006). Group 1 isolates, which cause root andabsent. crown rot of wheat, were given species status and Distinctive features. On PDA, colonies of Fusar- described as Fusarium pseudograminearum O’Don-ium graminearum are usually highly coloured, with nell and T. Aoki (teleomorph Gibberella coronicola
    • 104 5 Primary Keys and Miscellaneous FungiT. Aoki and O’Donnell) by Aoki and O’Donnell et al., 1983; Logrieco et al., 1988; Miller et al., 1991).(1999a, b). F. graminearum is homothallic and North American strains are predominantly chemo-forms perithecia readily in nature and in culture type IB (Abbas et al., 1989b; Mirocha et al., 1989b;on Carnation Leaf Agar (Leslie and Summerell, Miller et al., 1991; Abramson et al., 1993; Desjardins,2006), whereas F. pseudograminearum is heterothal- 2006). This chemotype also predominates in thelic and does not. O’Donnell et al. (2004) proposed Ukraine (Leonov et al., 1990) and New Zealandthat the ‘‘F. graminearum clade’’ be split into nine (Lauren et al., 1992). Chemotype IA predominatesdifferent species, based on geographical and mole- in China (Miller et al., 1991; Zhang et al., 2007b),cular data; however, the morphological distinctions Argentina (Ramirez et al., 2006b) and Italy (Logriecobetween these species are small. This proposal has et al., 1988) while in Japan (Sugiura et al., 1990) andnot been taken up by all Fusarium researchers Poland (Visconti et al., 1990) both chemotypes IA(Leslie and Summerell, 2006). The complete genome and IB are commonly isolated. Chemotype II occursof F. graminearum has been sequenced and is avail- only rarely in North America (Miller et al., 1991) butable (Broad Institute, 2003). is more common elsewhere, including New Zealand Physiology. The optimal temperature for growth (Lauren et al., 1992), Australia (Blaney and Dod-of Fusarium graminearum is between 24 and 268C on man, 1988, 2002; Tan et al., 2004), Japan (Sugiuraboth liquid and solid media at pH 6.7–7.2 (Booth, et al., 1990), Korea (Lee et al., 1986; Kim et al.,1971), and 258C on irradiated wheat grains (Ramirez 1993), Italy (Logrieco et al., 1988) and South Africaet al., 2006a). The minimum aw for growth is close to (Sydenham et al., 1991).0.90 at 15–258C (Cuero et al., 1987; Ramirez et al., Ramirez et al. (2006a) reported that deoxyniva-2006a). The minimum pH for growth is temperature- lenol production occurred most rapidly at 258C, butdependent, near 3.0 at 25 and 378C, and 2.4 at 308C. the maximum amount was produced at 308C. TheThe maximum is near 9.5 at 378C, but greater than aw range for deoxynivalenol production was 0.95–10.2 at the lower temperatures (Wheeler et al., 1991). 0.995 aw. Llorens et al. (2004b) reported that 288C Mycotoxins. Fusarium graminearum produces was optimal for deoxynivalenol production, butnumerous mycotoxins: almost 50 toxic compounds nivalenol and 3-acetyldeoxynivalenol were pro-have been reported. The most important economic- duced optimally at 20 and 158C, respectively. Zear-ally are type B trichothecenes – deoxynivalenol alenone production was optimal at 208C (Llorens(DON) and its derivatives 3-acetyldeoxynivalenol et al., 2004a).(3-AcDON) and 15-acetyldeoxynivalenol (15- Other toxins produced by Fusarium graminearumAcDON), nivalenol (NIV) and its derivatives and include butenolide, culmorin, sambucinol, calonec-zearalenone (see Marasas et al., 1984; Pitt and Hock- trin and related compounds, fusarins and a numbering, 1997; Desjardins, 2006; Leslie and Summerell, of other minor metabolites (Desjardins, 2006).2006). See Desjardins (2006) for a comprehensive Ecology. Fusarium graminearum is primarily areview of trichothecene mycotoxins. Other toxins pathogen of gramineous plants, particularlyreported include aurofusarin, culmorins, fusarin C wheat, in which it causes crown rot and head scab.and steroids (see Pitt and Hocking, 1997; Leslie and Reports have come from Australia, Canada, theSummerell, 2006). Type A trichothecenes, including United States, Japan, Korea, South Africa andT-2 toxin, HT-2 toxin, diacetoxyscirpenol and neoso- many European countries (see Pitt and Hocking,laniol, have been reported from some isolates. How- 1997; Desjardins, 2006; Leslie and Summerell,ever, production of these toxins by F. graminearum was 2006). F. graminearum also causes cob rot andqueried by Nelson et al. (1983) and remains in doubt. stalk rot of maize in many countries leading to Based on production of the different trichothe- DON and zearalenone formation in maize (see Pittcenes, Fusarium graminearum has been divided into and Hocking, 1997; Leslie and Summerell, 2006).two chemotypes, chemotype I for deoxynivalenol Occurrence of F. graminearum in barley has beenproducers and chemotype II for nivalenol produ- less frequent, but it is believed to be one cause ofcers. Strains producing 3-acetyldeoxynivalenol gushing in beer (Niessen et al., 1992). Other sourcesare classified as chemotype IA and those producing include sugar beets (Bosch and Mirocha, 1992),15-acetyldeoxynivalenol as chemotype IB (Ichinoe soybeans (Clear et al., 1989; Jacobsen et al., 1995),
    • 5.17 Genus Fusarium 105sorghum (Onyike and Nelson, 1992) and triticale different species, F. venenatum Nirenburg (O’Donnell(Perkowski et al., 1988). F. graminearum has been et al., 1998). For reviews of this topic see Edwardsreported to cause dry rot in potatoes (Ali et al., (1993); Trinci (1994) and Wiebe (2004).2005) and postharvest rots in pumpkins (Elmer, References. Domsch et al. (1980) (as Gibberella1996). Crown rot of bananas may also be due to F. zeae); Nelson et al. (1983); Desjardins (2006); Leslie ´graminearum (Wallbridge, 1981; Jimenez et al., and Summerell (2006).1993). Quorn, a mycoprotein produced for human con- Fusarium longipes Wollenw. &sumption in the United Kingdom, is produced from a Reinking Fig. 5.28strain of Fusarium which was originally identified asF. graminearum. This strain, which does not produce Colonies on CYA and MEA covering the wholeany trichothecene toxins, has since been assigned to a Petri dish, plane, deep and floccose or sometimesFig. 5.28 Fusarium longipes (a) colonies on PDA and DCPA, 7 d, 258C; (b) macroconidia, bar ¼ 10 mm; (c) chlamydoconidia,bar ¼ 10 mm
    • 106 5 Primary Keys and Miscellaneous Fungifuniculose, mycelium pale orange to pale red or dull Fusarium oxysporum Schltdl. Fig. 5.29reddish, reverse on CYA brownish orange to lightbrown, on MEA deep red to brownish red. On G25N, Colonies on CYA 50–70 mm diam, sometimescolonies 15–22 mm diam, of floccose, pale orange covering the whole Petri dish, moderately deep, ofmycelium, reverse in similar colours. No germination floccose white to greyish mycelium; reverse pale toat 58C. At 378C, colonies of variable size, up to 65 mm pale greenish grey. Colonies on MEA 65–70 mmdiam, of white to very pale pink mycelium, reverse diam, often covering the whole Petri dish, of floc-dull yellow to pinkish brown. cose white to pale greyish magenta mycelium; On PDA, colonies covering the whole Petri dish, reverse greyish magenta to dark purple, often palersimilar to those on CYA, reverse dull red to dark at the margins. Colonies on G25N 12–16 mm diam,red. On DCPA, colonies 55–65 mm diam, sparse, occasionally larger. At 58C, germination to forma-mycelium pale pink, showing abundant orange tion of microcolonies. At 378C, no growth, or colo-sporodochia containing macroconidia, reverse dull nies up to 5 mm diam formed.yellow to brown. On PDA, colonies of white, pale salmon or pale Macroconidia on DCPA strongly curved, 50–80 mauve mycelium, sometimes dense and floccose, 4–6 mm, with four to six septa; apical cells usually sometimes low, often with salmon sporodochia in awith a long papilla or a long slender whip-like end; central mass, and sometimes in one or two poorlybasal cells with a deep notch and long ‘‘foot’’. Micro- defined concentric rings also; reverse pale salmon,conidia not produced. Chlamydoconidia produced often mauvish centrally, sometimes dark magenta.by some isolates, in chains or small clusters, spherical On DCPA, colonies low, sometimes with concentricto ellipsoidal or irregular in shape, 7–12 mm wide, rings of sparse, floccose aerial mycelium, powderywith thin smooth walls, becoming rough in age. with microconidia on the agar surface, or sometimes Distinctive features. Pale orange to pale red uniformly low with little aerial mycelium and thenmycelium on CYA and MEA, fast growth at 378C with appearance dominated by surface macroconidia.and very long, slender, strongly curved macroconi- Macroconidia only slightly curved, usually withdia are the main distinguishing features of Fusarium three septa, occasionally more, thin walled, withlongipes. notched or foot shaped basal cells and short, some- Taxonomy. No teleomorph is known for this times hooked, apical cells; microconidia abundant,species. fusiform to kidney-shaped, produced in false heads Physiology. No studies on the physiology of this from short, stout monophialides. Chlamydoconidiaspecies are known to us. produced singly or in pairs. Mycotoxins. This appears to be a nontoxigenic Distinctive features. Fusarium oxysporum pro-species (Wing et al., 1993; Nelson et al., 1994), duces abundant fusiform to kidney-shaped micro-although Logrieco et al. (1998a) reported beauver- conidia in false heads from short, stout, flask-icin production by one isolate. shaped phialides in the aerial mycelium. The col- Ecology. Fusarium longipes principally comes from ony reverse on PDA is usually mauve, violet ortropical soils (Burgess et al., 1994; Sangalang et al., greyish magenta.1995). It has been reported from peanuts from West Taxonomy. Many plant pathogenic races exist inJava (Dharmaputra and Retnowati, 1996), and we Fusarium oxysporum; these are called formae spe-isolated it from 13% of Indonesian peanut samples, ciales (abbreviated f. sp.). Recent DNA sequenceat up to 16% of kernels in infected samples and 1% of analyses have indicated that some of these are poly-all kernels examined (Pitt et al., 1998a). It was also phyletic (Summerell et al., 2003). The most impor-found at low levels in Indonesian maize and cowpeas, tant of these are discussed by Booth (1971) andThai maize, mung beans, cassava and sesame seed, Leslie and Summerell (2006). No teleomorph isand paddy rice from the Philippines (Pitt et al., 1993, known.1998a). F. longipes has also been reported from cereals Physiology. Domsch et al. (1980) reported anin Iran (Zare and Ershad, 1997). optimum growth temperature between 25 and 308C References. Nelson et al. (1983); Leslie and for Fusarium oxysporum, with a minimum aboveSummerell (2006). 58C, and a maximum at or below 378C. The
    • 5.17 Genus Fusarium 107Fig. 5.29 Fusarium oxysporum (a) colonies on PDA and DCPA, 7 d, 258C; (b) phialides, bar ¼ 10 mm; (c) macroconidia andmicroconidia, bar ¼ 10 mmoptimum pH for growth is 7.7 with the wide range of not after exposure to 658C for 6 s in 208 Brix sucrosepH 2.2–9.0 being tolerated (Domsch et al., 1980). solution at pH 4.2. F. oxysporum was more resistantThe minimum aw for growth is 0.89 at 208C, after a to dry heat, with sporadic survival detected aftergermination time of 2 months (Schneider, 1954). exposure to 1408C for 60 s (our unpublished data).Growth in N2 with <1% O2 was 97% of that in air Mycotoxins. Fusarium oxysporum has been widely(Hocking, 1990). Some growth occurred in 99% reported to produce moniliformin (Marasas et al.,CO2, with only trace amounts of O2 and N2 present 1979; Abbas et al., 1989b, 1991; Mirocha et al.,(Hocking, 1990). Atmospheres of 20–40% CO2 with 1989a; Chelkowski et al., 1990) and zearalenone5 or 1% O2 reduced colony growth rate of F. oxy- (Per, 1995; see also Pitt and Hocking, 1997),sporum by 40–50%, but ergosterol content was although production of the latter compound appearsreduced by 80–90% under these conditions (Tani- to be in doubt as F. oxysporum does not possess thewaki et al., 2001a). F. oxysporum chlamydoconidia tri5 gene required for trichothecene biosynthesisdo not exhibit exception heat resistance, despite its (Tan and Niessen, 2003). F. oxysporum isolatesability to cause spoilage in beverages (see below). In reported to produce wortmannin (Abbas and Miro-experiments in our laboratory, survival was recorded cha, 1988; Abbas et al., 1989a, 1992; Gunther et al.,from mixed suspensions of microconidia and chla- 1989) have been re-examined and classified as F.mydoconidia after heating at 638C for up to 48 s, but torulosum (Thrane, 2001). Other mycotoxins reported
    • 108 5 Primary Keys and Miscellaneous Fungito be produced by F. oxysporum are beauvericin References. Booth (1971); Domsch et al. (1980);(Logrieco et al., 1998a; Moretti et al., 2002), type B Nelson et al. (1981, 1983); Leslie and Summerellfumonisins (Abbas et al., 1995; Proctor et al., 2004) (2006).and type C fumonisins (Seo et al., 1996). Ecology. Fusarium oxysporum is a serious wilt Fusarium poae (Peck) Wollenw. Fig. 5.30pathogen of many crop plants, including sweetpotatoes, cabbage and other crucifers, cucumbers Colonies on CYA filling the whole Petri dish, deep,and melons, oil and date palms, tomatoes, peas, of moderately dense white to pale rose mycelium;soybeans and cowpeas, clovers, cotton and a variety reverse unevenly coloured, pale to rose red or deepof others (Booth, 1971; Nelson et al., 1981) and is red. Colonies on MEA filling the whole Petri dish,geographically widespread (Sangalang et al., 1995; deep, of sparse, pale rose mycelium; reverse brown-Leslie and Summerell, 2006). Considering these ish orange or paler. Colonies on G25N 8–12 mmfacts, its common occurrence in foods is not unex- diam. At 58C, germination to colonies up to 2 mmpected. It occurs in cereals, including maize in the diam formed. No growth at 378C.United States, Australia, Turkey, South Africa and On PDA, colonies moderately deep, of floccose,the Philippines; rice in India, Egypt and Indonesia; pale salmon to pale rose mycelium, darker centrally,barley in Egypt and sorghum from Africa (see Pitt reverse varying from pale salmon at the margins toand Hocking, 1997). It is widespread on nuts: pea- greyish ruby centrally, or entirely dark ruby to darknuts, pecans, hazelnuts and walnuts (see Pitt and magenta. On DCPA, colonies moderately deep, ofHocking, 1997). floccose pale salmon mycelium in poorly defined This species is one cause of crown rot of bananas concentric rings; sporodochia rarely present; ´(Wallbridge, 1981; Jimenez et al., 1993; Boruah reverse pale or, in highly pigmented isolates, withet al., 2004), and also occurs as a minor pathogen annular brownish red rings.on citrus, pome fruits, tomatoes, melons (Snowdon, Macroconidia usually sparsely produced, varying1990) and pineapples (Biswal et al., 2007). Other in shape, mostly with three septa, occasionally more,sources include coriander seeds (Hashmi and slightly curved, with a foot-shaped basal cell. Micro-Ghaffar, 1991), cacao beans (Ribeiro et al., 1986), conidia abundant, spherical, often with a distinctnavy beans (Tseng et al., 1996), adzuki and mung papilla, occasionally also lemon-shaped, aseptate orbeans (Tseng and Tu, 1997), garlic (Rath and with one septum, produced in the aerial myceliumMohanty, 1986), capsicums (Hashmi and Thrane, from short flask-shaped phialides on compact1990), truffles (Bokhary et al., 1990), asparagus branched stipes, often appearing like bunches of grapes(Weber et al., 2006), cheese (Hocking and Faedo, when examined in situ under the low power1992; Lund et al., 1995) and potatoes (Vrany et al., microscope.1989; Kim and Lee, 1994; Venter and Steyn, 1998). Distinctive features. The abundant production ofF. oxysporum has been implicated as a cause of gill spherical microconidia borne from flask-shapedblackening disease in Kumma prawns (Penaeus phialides on compact, branched stipes distinguishesjaponicus) (Souheil et al., 1999). Fusarium poae from other species. It also has a Due to its ability to grow in low O2 tensions, distinctive odour, described as ‘‘fruity’’ or ‘‘sweet’’Fusarium oxysporum has caused spoilage of UHT- (Leslie and Summerell, 2006).processed fruit juices (Hocking, 1990). This problem Taxonomy. A group of cultures morphologi-became widespread from 2001 onwards, and our cally similar to Fusarium poae, referred to as ‘‘pow-laboratory investigated several spoilage incidents dery’’ F. poae, have recently been assigned to aoccurring in Australia and New Zealand in fruit, separate species Fusarium langsethiae Torp anddairy- and soy-based aseptically filled beverages. Nirenburg (Torp and Nirenburg, 2004). This spe-We had anecdotal evidence of similar problems in cies has been isolated from oats, wheat and barleyEurope, Asia and North America. F. oxysporum in cooler areas of Europe and Scandinavia. F.appeared to be colonising the aseptic area of the langsethiae, which does not have the characteristicpackaging equipment, and once established, was sweet odour of F. poae, is also distinguished by itsvery difficult to eliminate. metabolic profile (Thrane et al., 2004) and by
    • 5.17 Genus Fusarium 109Fig. 5.30 Fusarium poae (a) colonies on PDA and DCPA, 7 d, 258C; (b) macroconidia, bar ¼ 10 mm; (c) microconidia, bar ¼10 mm; (d) phialides, bar ¼ 10 mmmolecular techniques (Schmidt et al., 2004). It that it was perhaps responsible for Alimentaryappears to be more closely related to F. sporotri- Toxic Aleukia due to the production of T-2 toxinchioides than F. poae (Niessen et al., 2004). have largely been discounted, as F. sporotrichioides, Physiology. The optimum temperature for the major producer of that toxin, was also com-growth of Fusarium poae is near 258C, with a mini- monly present during that disease syndrome, andmum near 2.58C and a maximum of 358C (Kvash- in the haemorrhagic diseases that occurred in cattle,nina, 1976; Torp and Nirenburg, 2004). The mini- pigs and poultry in the United States in the 1960smum aw for growth is near 0.90 between 17 and (Marasas et al., 1984). However, it has been con-258C (Magan and Lacey, 1984c). cluded more recently that certain strains of F. poae Mycotoxins. Reports of toxin production by can indeed produce T-2 toxin (De Nijs et al., 1996;Fusarium poae have been very variable. Reports Langseth et al., 1999; Thrane et al., 2004).
    • 110 5 Primary Keys and Miscellaneous Fungi Most strains of Fusarium poae produce nivalenol, On PDA, colonies covering the whole Petri dish,diacetoxyscirpenol, monoacetoxyscirpenols, fusare- similar to on MEA, reverse light orange to greyishnone-X and scirpentriol (Thrane et al., 2004), with red. On DCPA, colonies 45–55 mm diam, of palesome strains also producing beauvericin and ennia- orange mycelium, plane, floccose, similar in texturetins (Thrane et al., 2004; Chelkowski et al., 2007). to on CYA, orange sporodochia usually evident onLogrieco et al. (1998a) also reported beauvericin pro- the agar surface, reverse orange brown.duction by F. poae. T-2 toxin is more often produced Macroconidia on DCPA slightly curved, withby F. sporotrichioides and F. langsethiae (Thrane three to four septa, 40–50 Â 3.0–4.0 mm, roundedet al., 2004; Desjardins, 2006). or papillate at both ends, or sometimes with a notch Ecology. Fusarium poae mainly occurs in tempe- at the basal end, with thin, smooth walls. Microco-rate regions, where it is found on woody seedlings or nidia borne in chains and false heads from mono-herbaceous and gramineous hosts (Booth, 1971; phialides or polyphialides, ellipsoidal or bean-Leslie and Summerell, 2006). From foodstuffs, it shaped, commonly 8–12 Â 2.5–4.0 mm and nonsep-has been reported most commonly from grains, tate, occasionally longer and with one or moreespecially wheat, from France, Canada, Japan, septa, with thin smooth walls. ChlamydoconidiaEthiopia, Hungary, Poland, Germany and Norway not produced.(see Pitt and Hocking, 1997; Birzele et al., 2002; Distinctive features. Similar to Fusarium verti-Bottalico and Perrone, 2002; Ioos et al., 2004; cillioides in most respects, F. proliferatum is distin-Kosiak et al., 2003, 2004). It has been implicated guished by the formation of microconidia fromas a cause of panicle blight of oats in Poland polyphialides. Microconidial chains are usually(Mielniczuk, 2001), head blight of oats in Canada shorter in F. proliferaturm than F. verticillioides.(Tekauz et al., 2004) and as a pathogen of barley in Freshly isolated cultures form abundant macroco-Argentina (Barreto et al., 2004). It has been associated nidia in sporodochia, but this feature is often lostwith maize in cooler climates, where it may be after repeated transfer (Leslie and Summerell,involved in ear rot (see Pitt and Hocking, 1997; Log- 2006).rieco et al., 2002; Chelkowski, et al., 2007). F. poae has Taxonomy. Morphologically, Fusarium prolifer-also been recorded from soybeans in the United States atum is indistinguishable from F. fujikuroi Niren-(Abbas and Bosch, 1990), heart rot of sugar cane in burg. These two species can be differentiated usingSouth Africa, rice in Australia and decayed stored mating tests or by DNA sequencing. F. fujikuroi,citrus fruit in Georgia, CIS (Booth, 1971). which is associated mainly with rice, does not pro- References. Booth (1971); Domsch et al. (1980); duce fumonisins, whereas most isolates of F. prolif-Nelson et al. (1983); Desjardins (2006); Leslie and eratum do.Summerell (2006). Physiology. The optimum pH and temperature for growth of Fusarium proliferatum are 5.5 and 258C,Fusarium proliferatum (Matsush.) Nirenberg respectively, with growth occurring at 5 and 378C, Fig. 5.31 but not 408C on maize meal (Marı´ n et al., 1996). The minimum aw for germination reported by Marı´ nTeleomorph: Gibberella intermedia (Kuhlman) et al. (1996) was 0.88, with no germination occurringSamuels at 0.85 aw after 40 days. However, Samapundo et al. (2005a) reported a minimum aw of 0.87 for growth ofColonies on CYA covering the whole Petri dish, F. proliferatum on maize, with optimum growthplane, floccose, of pale orange mycelium, reverse occurring at 308C at 0.97 aw, the highest aw tested. F.light orange to pastel red. Colonies on MEA similar proliferatum was able to grow on irradiated maizeto those on CYA but much less dense, mycelium in kernels at 0.98 and 0.93 aw in atmospheres containingsimilar colours, reverse orange, greyish red or violet up to 60% CO2 with 20% O2, although the lag timebrown. On G25N, germination or colonies up to was longer and the growth rate slower than the con-5 mm diam. At 58C, no germination. At 378C, colo- trols (Samapundo et al., 2007a). Reduction of head-nies 5–20 mm diam, of pale orange mycelium, space oxygen from 20 to 2% (without elevated CO2)reverse usually orange. had no effect on growth of F. proliferatum, but
    • 5.17 Genus Fusarium 111Fig. 5.31 Fusarium proliferatum (a) colonies on PDA and DCPA, 7 d, 258C; (b) phialides bearing microconidia in chains andfalse heads in situ, bar ¼ 50 mm; (c) macroconidia and microconidia, bar ¼ 10 mm; (d) polyphialides, bar ¼ 10 mm; (e)monophialides, bar ¼ 10 mmvacuum packaging and the incorporation of oxygen hydroxyanisole (BHA) can also inhibit growth andscavenging sachets prevented growth (Samapundo fumonisin production by F. proliferatum under someet al., 2007b). Application of 1% ammonium bicarbo- conditions (Etcheverry et al., 2002).nate to maize at aw values between 0.99 and 0.92 Fumonisin B1 production was inhibited atprevents growth and fumonisin production by F. headspace CO2 concentrations of 40, 30 and 10%proliferatum and was proposed as a possible treat- at 0.98, 0.95 and 0.93, respectively (Samapundoment for stored maize (Samapundo et al., 2007c). et al., 2007a). When headspace oxygen was con-The antioxidants propylparaben and butylated trolled, optimum fumonisin production occurred
    • 112 5 Primary Keys and Miscellaneous Fungiwith 20% O2 at 0.98 aw, but at 0.95 and 0.93 aw, Fusarium semitectum Berk. & Ravenel Fig. 5.32more fumonisin was produced with 10% head- Fusarium incarnatum (Desm.) Sacc.space O2 (Samapundo et al., 2007b). Marı´ n et al. Fusarium pallidoroseum (Cooke) Sacc.(1995) reported fumonisin production was higherat 25 than at 308C, and highest at the highest aw Colonies on CYA filling the whole Petri dish, some-tested (0.97), with low levels still produced at 0.92 times to the lid at the edges, of dense, floccose, white,aw. Samapundo et al. (2005b) found that there was pale salmon or pale brown mycelium, sometimes pow-little difference in fumonisin production at 25 and dery from macroconidia produced in the aerial myce-308C, but lower amounts were produced at 158C, lium; pale salmon sporodochia occasionally producedirrespective of aw value (between 0.97 and 0.92). at the inoculation points; reverse pale salmon, yellow-Conversely, Melcion et al. (1998) observed max- ish, greyish yellow or with brown patches. Colonies onimal fumonisin production at 158C on maize at MEA filling the whole Petri dish, of floccose or funi-very high aw. culose white to pale greyish yellow mycelium; reverse Mycotoxins. Fusarium proliferatum is a major pale, sometimes greyish orange centrally or in patches.producer of fumonisins B1, B2 and B3 (Ross et al., Colonies on G25N 15–20 mm diam. At 58C, usually1990; see also Pitt and Hocking, 1997; Desjardins, only microcolonies produced. Usually no growth at2006), and indeed strains of F. proliferatum are 378C, but occasionally colonies of 2 mm diam formed.among the highest fumonisin producers (Leslie et al., On PDA, colonies of dense, floccose mycelium,2004). Other mycotoxins produced include monilifor- coloured pale salmon, brown or yellow, sometimesmin, beauvericin, enniatins and fusarin (Marasas with a powdery appearance from macroconidia inet al., 1986; Desjardins et al., 2000a; see also Desjar- the aerial mycelium; reverse pale salmon, oftendins, 2006). brownish centrally. On DCPA, colonies in similar Ecology. Reporting of Fusarium proliferatum in colours to those on PDA, but less dense, aerialfoods and feeds has increased sharply with the dis- mycelium often powdery with macroconidia, andcovery of fumonisins and that this species is an occasionally a central area of salmon to orangeimportant source of these toxins. It has been sporodochia; reverse pale.shown to colonise maize plants throughout the Macroconidia of two types produced: the first inworld, and is increasingly important in maize ear the aerial mycelium, often from polyphialides, cigar-rot in Europe (see Bacon and Nelson, 1994; Desjar- or spindle-shaped, straight or slightly curved, withdins, 2006; Leslie and Summerell, 2006). It was four to five septa, and with poorly differentiatedreported from sorghum in Nigeria by Onyike and basal and apical cells; the second in sporodochia,Nelson (1992) and in pine nuts in Spain (Marı´ n more curved, with a foot-shaped basal cell andet al., 2007b). We have encountered this species in curved apical cell. Chlamydoconidia usually pro-maize, mung beans, paddy rice and occasionally duced; microconidia not produced.sorghum in Southeast Asia (Pitt et al., 1998a). F. Distinctive features. The production of spindle-proliferatum has also been reported as a cause of or cigar-shaped macroconidia from polyphialides inblack point in wheat (Conner et al., 1996; Desjar- the aerial mycelium is the most distinctive feature ofdins et al., 2007). It is a pathogen of bananas (Jime-´ Fusarium semitectum. These macroconidia can benez et al., 1993), onions (Toit et al., 2003; Stankovic observed in situ with the low power microscope,et al., 2007), garlic (Dugan et al., 2003, 2007; Stan- and are often arranged in pairs in ‘‘V’’ shapes,kovic et al., 2007) and asparagus (Elmer, 2000; resembling rabbit ears.Gossman et al., 2005; Corpas-Hervias et al., 2006; Taxonomy. Type material of Fusarium semitectum,Weber et al., 2006; Liu et al., 2007). Fumonisins examined by Booth and Sutton (1984), was found tohave been reported in infected garlic (Seefelder be a Colletotrichum species, C. musae. However, typeet al., 2002) and asparagus (Logrieco et al., 1998b; material of F. pallidoroseum (Cooke) Sacc., whichSeefelder et al., 2002; Weber et al., 2006; Liu et al., they also examined, contained polyphialides and2007). macroconidia typical of F. semitectum. Subsequently, References. Nelson et al. (1983); Desjardins Nirenberg (1990) pointed out that the species epithet(2006); Leslie and Summerell (2006). incarnatum predates pallidoroseum by about 30
    • 5.17 Genus Fusarium 113Fig. 5.32 Fusarium semitectum (a) colonies on PDA and DCPA, 7 d, 258C; (b) phialides bearing macroconidia in aerialmycelium in situ, bar ¼ 50 mm; (c) phialides, bar ¼ 10 mm; (d) macroconidia, bar ¼ 10 mmyears, so if the name were to be changed, it should Marin et al., 1996; Shukla and Sharma, 2000). Cher-be F. incarnatum. However, both Desjardins (2006) ian (2007) reported maximum rotting of bananas byand Leslie and Summerell (2006) agree that F. semi- F. semitectum occurred at 308C.tectum is really a species complex, with possibly five Mycotoxins. Confusion still exists over the myco-intra-specific groups (Abd-Elsalam et al., 2003), toxins produced by Fusarium semitectum (Desjar-and until such time as it is thoroughly examined dins, 2006). Zearalenone production has beenboth morphologically and phylogenetically, the reported (see Marasas et al., 1984; Pitt and Hock-name F. semitectum should continue to be used. ing, 1997, Desjardins, 2006, Leslie and Summerell, Physiology. The temperature range for growth of 2006). Production of T-2 toxin has also beenFusarium semitectum is 38C to ca. 378C, with the reported, but is unsubstantiated (Marasas et al.,optimum near 258C (Kakker and Mehrotra, 1971; 1984; Desjardins, 2006). Japanese isolates of F.
    • 114 5 Primary Keys and Miscellaneous Fungisemitectum have been reported to produce diacetox- examined; Pitt et al., 1994) and Indonesia (60% ofyscirpenol, nivalenol, fusarenone-X and neosola- samples, up to 60% of grains in infected samplesniol (Suzuki et al., 1980, 1981). Diacetoxyscirpenol and 7% of all grains; Pitt et al., 1998a). It was theproduction has also been reported by Molto et al. most common fungus in Thai mung beans (55% of(1997). samples; up to 76% infection in infected samples, Ecology. Fusarium semitectum is widespread in and 15% infection overall). It was also common intropical and subtropical countries, although reports Thai black beans (6% of all beans examined), sor-from foods are relatively uncommon. It has been ghum (4% of all grains) and soybeans (2% of allfound at low levels in maize in Italy (Logrieco et al., beans; Pitt et al., 1994). Generally similar figures1995) and the USA (Katta et al., 1995), and we have were found for infection in rice, soybeans, blackfound high levels of F. semitectum in Thai maize beans and mung beans in Indonesia and the Philip-(45% of samples, up to 22% infected grains in pines (Pitt et al., 1998a). Indeed, F. semitectum wasinfected samples and 4% of all kernels examined; by far the most common fungal invader of beansPitt et al., 1993). Incidence in Indonesian maize was that we encountered in Southeast Asia.similar, but levels in Philippine maize lower (Pitt References. Domsch et al. (1980); Nelson et al.et al., 1998a). F. semitectum is one of the dominant (1983); Desjardins (2006); Leslie and Summerellfungi of pearl millet (Wilson, 2002; Jurjevic et al., (2006).2007) and is common in peanuts (Joffe, 1969; Gil-man, 1969; Oyeniran, 1980; Pitt et al., 1993; Dhar- Fusarium solani (Mart.) Sacc. Fig. 5.33maputra and Retnowati, 1996). It has also beenreported from rice (Desjardins et al., 2000a) and Teleomorph: Haemanectria haematococca (Berk. &sorghum (Onyike and Nelson, 1992; Usha et al., Broome) Samuels & Nirenberg1994; Shabbir and Rajasab, 2004). It causes storagerots of various fruits (Snowdon, 1990), especially Colonies on CYA 60–65 mm diam, low, of moder-crown rot of bananas (see Pitt and Hocking, 1997; ately dense white mycelium, often covered in veryVesonder et al., 1995; Marin et al., 1996; Cherian, fine droplets of clear exudate; a central, cream spore2007) and soft rot of bell peppers (Shukla and mass sometimes present; reverse pale, sometimesSharma, 2000; Sharma and Shukla, 2003). It has with bluish or greenish areas. Colonies on MEAalso been reported to cause postharvest spoilage of 50–60 mm diam, low to moderately deep, of sparse,tomatoes (Muniz et al. 2003; Chaturbhuj and Rai, often slightly funiculose, white to pale violet myce-2005), passionfruit (Muniz et al., 2003), mushrooms lium; reverse pale, sometimes bluish grey centrally.(Kang et al., 2002) and disease in walnuts (Belisario Colonies on G25N 3–8 mm diam. No growth atet al., 1999). This species has also been isolated from 58C. At 378C, either no growth, or colonies up topigeon peas (Maximay et al., 1992), soybeans (Vaa- 10 mm diam formed.monde et al., 1987; and our unpublished data), black On PDA, colonies low to moderately deep, ofgram (Goyal and Jain, 1998), black beans (Castillo white to cream mycelium in concentric rings, oftenet al., 2004), coriander (Hashmi and Thrane, 1990; alternating with rings of cream or bluish grey spor-Hashmi and Ghaffar, 1991) and sunflower seeds odochia; in some isolates the teleomorph produced,(Shahnaz and Ghaffar, 1991), though with few of dark orange perithecia scattered over the centralreports in each case. Earlier reviews indicate isola- area of the colony; reverse pale or with areas oftions from citrus fruits, tomatoes, melons, cucum- turquoise grey or pale violet brown. On DCPA,bers and potatoes (Booth, 1971; Domsch et al., colonies low, of sparse, white mycelium in annular1980). rings, with a central mass of cream sporodochia; In our experience, Fusarium semitectum is much reverse pale to pale yellow brown.more common in tropical commodities than the Macroconidia abundant, stout, thick walled,above citations would indicate. This species appears with three to four, or less commonly five septa,to be endemic in paddy rice from Thailand (in 94% straight, parallel sided for most of the length, apicalof samples examined, up to 44% of grains infected cells blunt and rounded, basal cells either rounded,in infected samples and a very high 20% of all grains notched or sometimes distinctly foot-shaped.
    • 5.17 Genus Fusarium 115Fig. 5.33 Fusarium solani (a) colonies on PDA and DCPA, 7 d, 258C; (b) phialides bearing macroconidia in false heads in situ,bar ¼ 50 mm; (c) phialides, bar ¼ 10 mm; (d) microconidia, bar ¼ 10 mm; (e) macroconidia, bar ¼ 10 mmMicroconidia usually abundant, ellipsoidal, fusi- F. oxysporum. However, phialides in F. solani areform or kidney-shaped, produced in false heads much longer than those of F. oxysporum.on very long, straight phialides. Chlamydoconidia Taxonomy. The teleomorph of Fusarium solani isproduced singly or in pairs. Haemanectria haematococca (Berk. and Broome) Distinctive features. Fusarium solani is the only Samuels and Nirenberg, which produces orangeFusarium species which produces cream or bluish brown perithecia. However, only about 1% ofrather than salmon or orange sporodochia. The F. solani isolates produce the teleomorph in culturemacroconidia are stout and straight or only slightly (Booth, 1983). Leslie and Summerell (2006) believecurved. Microconidia are produced in false that there are a number of biological species withinheads (mucoid balls) on very long, slender phialides. F. solani and that further work is required to clarifyThis species is morphologically very similar to the taxonomy and phylogeny of this group.
    • 116 5 Primary Keys and Miscellaneous Fungi Physiology. Domsch et al. (1980) noted that var- Fusarium solani can attack keratin, and has beenious authors have reported the optimum growth isolated from human fingernails and eyes (Domschtemperature for Fusarium solani to be between 27 et al., 1980; de Hoog et al., 2000; Leslie and Summerell,and 318C, with strong growth at 378C. However, 2006). It can be a serious pathogen of crustaceans,our observations indicate only weak growth at this attacking and destroying the chitinous exoskeletontemperature, and Chaturvedi et al. (2003) reported (Fisher et al., 1978; Gonzalez, 1995; Le et al., 2005)optimum growth at 258C. Schneider (1954) reported and has also been reported to cause disease in seagrowth of F. solani down to 0.90 aw after a germina- ´ turtles (Cabanes et al., 1997; Castella et al., 1999a; de ˜tion time of 8 weeks at 208C. Hoog et al., 2000). Mycotoxins. Mouldy sweet potatoes (Ipomoea References. Domsch et al. (1980); Nelson et al.batatas) or extracts from them have been shown to (1983); Desjardins (2006); Leslie and Summerell (2006).be toxic to a variety of animals, including cattle (Wil-son et al., 1970; Doupnik et al., 1971), chickens Fusarium sporotrichioides Sherb. Fig. 5.34(Doupnik et al., 1971; Peckham et al., 1972) andmice (Boyd and Wilson, 1972). The toxic principles Colonies on CYA filling the whole Petri dish,are believed to be furanoterpenoids, ipomeanols and deep, of floccose white to pale pink and brownipomeanine (Nelson et al., 1983, 1994; Mawhinney mycelium; reverse pale salmon centrally, brownishet al., 2008). These compounds are not mycotoxins, at the margins. Colonies on MEA similar to thosebut result from fungal metabolism of phytoalexins on CYA except more highly coloured; reverse violetproduced by the plant (see Leslie and Summerell, brown centrally, paler at the margins. Colonies on2006). Fusarium solani has been reported to produce G25N 12–15 mm diam. At 58C, colonies 5–10 mmfusaric acid (Bacon et al., 1996) and the immunosup- diam. No growth at 378C.pressive compound cyclosporin (Sugiura et al., 1999). On PDA, colonies of dense, floccose, salmon Ecology. A cosmopolitan soil fungus, Fusarium to pink mycelium, sometimes with a central masssolani, has frequently been isolated from subterra- of orange sporodochia; reverse greyish rose to bur-nean crops such as potatoes, sweet potatoes, yams gundy, occasionally paler. On DCPA, colonies ofand peanuts (see Pitt and Hocking, 1997; Ismail, floccose pale salmon mycelium in concentric rings;2001; Peters et al., 2008). Hot water dipping orange sporodochia sometimes present on the agar(57.58C, 20–30 min) can reduce the incidence of surface; reverse pale.storage rots of potatoes caused by F. solani (Ran- Macroconidia abundant, moderately curved,ganna et al., 1998). F. solani may also invade a wide with three to five septa and with a curved, pointedvariety of other vegetable crops (Snowdon, 1991), apical cell and a notched or foot-shaped basal cell.especially cucurbits including muskmelons (Cham- Microconidia abundant, produced from polyphia-paco et al., 1993) and squash (Assawah and Al- lides in the aerial mycelium, fusiform, broadly ellip-Zarari, 1984), and legumes such as beans, soybeans soidal or pyriform, the latter often produced onlyand peas (see Pitt and Hocking, 1997; Tseng and Tu, on PDA, often with a papilla at the base. Chlamy-1997). Other sources are diverse, including small doconidia formed abundantly, singly or in chains orgrains, where it may be part of the cohort of Fusar- clumps, as cultures age.ium species causing head blight (Bottalico and Per- Distinctive features. On PDA, Fusarium sporotri-rone, 2002), maize, sorghum, bananas, guavas, cas- chioides has a similar appearance to F. poae,sava, sugar beets, capsicums, garlic, and seeds of F. chlamydosporum and F. tricinctum. The produc-sunflower, sesame and coriander (see Pitt and tion of both fusiform and pyriform microconidiaHocking, 1997). We have isolated F. solani from from polyphialides distinguishes F. sporotrichioidesbeer, shortening flakes, chilled water lines in a bev- from these closely related species. Pyriform microco-erage production plant, the inside of a juice filling nidia are produced more commonly, and sometimesmachine, and an air conditioning duct. exclusively, on cultures on PDA. Colony reverses on This species was widespread in Indonesian crops, PDA are always greyish rose or burgundy.especially beans, usually at about 1% of all seeds Physiology. Domsch et al. (1980) gave the optimumexamined, in paddy rice, maize, peanuts and pepper growth temperature of Fusarium sporotrichioides as(Pitt et al., 1998a). 22.5–27.58C, with a maximum of 358C. Joffe (1962)
    • 5.17 Genus Fusarium 117Fig. 5.34 Fusarium sporotrichioides (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) polyphialides, bar ¼ 10 mm;(d) microconidia, bar ¼ 10 mm; (e) macroconidia, bar ¼ 10 mmreported the growth of toxigenic isolates of this species Abramson et al., 2004; see also Pitt and Hocking,down to –28C. Schneider (1954) reported 0.88 aw as the 1997; Desjardins, 2006; Leslie and Summerell,minimum for growth, after 8 weeks incubation at 208C. 2006). F. sporotrichioides has been used to elucidate Mycotoxins. Fusarium sporotrichioides is the biochemistry and genetics of trichothecene bio-regarded as the major cause of alimentary toxic synthesis (Desjardins et al., 1993), with the firstaleukia (ATA) a devastating disease which occurred trichothecene biosynthetic gene, trichodienein the USSR during and after World War II, in synthase (TRI5) cloned from F. sporotrichioides intimes of extreme food shortage resulting in con- 1989 (see Desjardins, 2006; Leslie and Summerell,sumption of overwintered cereals (Joffe, 1978). 2006). Early reports of deoxynivalenol and nivale-Hundreds of thousands of people died from T-2 nol production have not been confirmed and may betoxin produced by this species and F. poae (Marasas due to misidentification of isolates (Desjardins,et al., 1984). F. sporotrichioides also produces a 2006). Other mycotoxins produced by F. sporotri-number of T-2 toxin derivatives, including HT-2, chioides include aurofusarin, beauvericin and occa-T-2 triol and T-2 tetraol, and biosynthetic inter- sionally enniatins, but not nivalenol, fusarenone-Xmediates such as neosolaniol, diacetoxyscirpenol or fumonisins (Thrane et al., 2004; Desjardins,and 15-monoacetoxyscirpenol (Thrane et al., 2004; 2006).
    • 118 5 Primary Keys and Miscellaneous Fungi Fusarium sporotrichioides has been implicated spore mass; reverse pale, salmon or yellowish. Colo-as the cause of mouldy corn toxicosis (haemorrha- nies on MEA less dense than on CYA, of white togic syndrome) in cattle, pigs and poultry in the pale salmon mycelium, sometimes powdery withUSA and elsewhere, fescue foot in cattle feeding microconidia; reverse pale yellow, salmon or violeton winter pastures in the USA, Australia and grey. Colonies on G25N 5–12 mm diam, occasion-New Zealand, akakabi-byo (scabby grain intoxica- ally larger. At 58C, germination to microcolonytion) and bean hulls poisoning in animals in Japan formation. No growth at 378C.(Marasas et al., 1984; Wu et al., 1997). On PDA, colonies low to moderately deep, of Ramakrishna et al. (1996) reported that T-2 floccose to funiculose mycelium coloured white,toxin formation by F. sporotrichioides on barley pale salmon, pale pink or mauve, sometimes pow-grain occurred optimally at 208C and toxin produc- dery with microconidia, reverse violet grey to deeption was stimulated by the presence of Aspergillus violet centrally, paler at the margins, or uniformlyflavus or Penicillium verrucosum. pale. On DCPA, colonies low at the margins, floc- Ecology. While not so commonly isolated as some cose centrally, of pale salmon mycelium, sometimesother species described here, Fusarium sporotrichioides overlying a thin layer of pale orange macroconidiais important because of its toxicity. It occurs more on the agar surface; reverse pale.commonly in cool climates and, in foods, is almost Macroconidia slightly curved to almost straight,entirely confined to grains. Historically, this species with three to five septa and thin, delicate walls, nar-has been associated with rye in Europe (Joffe, 1978; row, tapered apical cells and foot-shaped basal cells.Matossian, 1989). More recently, it has been reported Microconidia abundant, fusiform or broadly ellip-most frequently from wheat, in Japan, Spain, Hungary, soidal, aseptate or with a single septum, produced inPoland, Canada, Ethiopia (see Pitt and Hocking, 1997) false heads from polyphialides and also from simpleand Finland and Russia (Yli-Mattila et al., 2004). Other phialides. Chlamydoconidia not produced.grain sources include maize (Logrieco et al., 1995, 2002; Distinctive features. The production of microco-Adejumo et al., 2007; and see Pitt and Hocking, 1997), nidia in false heads from polyphialides and thebarley (Yli-Mattila et al., 2004; Bourdages et al., 2006), absence of chlamydoconidia distinguish Fusariumsorghum, wild rice (Nyvall et al., 1999) and cereal grains subglutinans from otherwise similar species such asin general (see Pitt and Hocking, 1997; Langseth et al., F. verticillioides and F. oxysporum.1998; Kosiak et al., 2003). This species has also been Taxonomy. Fusarium subglutinans was raised toisolated from peanuts (Austwick and Ayerst, 1963), species status by Nelson et al. (1983), previouslysugar beets (Bosch and Mirocha, 1992) and in our having been regarded as a variety of F. moniliformelaboratory from Australian soybeans. or F. sacchari. The teleomorph of F. subglutinans is References. Domsch et al. (1980); Nelson et al. Gibberella subglutinans (Edwards) P.E. Nelson et al.(1983); Desjardins (2006); Leslie and Summerell (2006). Recent phylogenetic studies indicate that there may be at least two cryptic species within F. subglutinans (Steenkamp et al., 2002; Desjardins et al., 2006).Fusarium subglutinans (Wollenw. & Reinking) Physiology. Martelleto et al. (1998) reportedP.E. Nelson et al. Fig. 5.35 growth of Fusarium subglutinans between 10 andFusarium moniliforme J. Sheld. var. subglutinans Wollenw. &Reinking 308C, with 258C being optimal. Castella et al.´Fusarium sacchari (Butler) W. Gams var. subglutinans Wollenw. (1999b) found growth of F. subglutinans was opti-& Reinking mal between 20 and 258C on maize cultures, but on rice, growth was best at 158C. Highest productionTeleomorph: Gibberella subglutinans (Edwards) of fusaproliferin was at 208C on maize after 6 weeksP.E. Nelson et al. ´ (Castella et al., 1999b). Mycotoxins. Usually regarded as one of the lessColonies on CYA covering the whole Petri dish, toxic of major Fusarium species, F. subglutinans has,of dense white to very pale salmon mycelium, in however, been reported to produce moniliformindegenerate cultures of low and sparse funiculose as its major toxin (see Pitt and Hocking, 1997;white mycelium; sometimes with a central orange Kostecki et al., 1999; Sewram et al., 1999; Logrieco
    • 5.17 Genus Fusarium 119Fig. 5.35 Fusarium subglutinans (a) colonies on PDA and DCPA, 7 d, 258C; (b) polyphialides, bar ¼ 10 mm; (c) macroconidiaamd microconidia, bar ¼ 10 mmet al., 2002; Desjardins, 2006; Desjardins et al., et al., 1995), Canada (Neish et al., 1983), the Car-2006; Leslie and Summerell, 2006; Sørensen et al., ibbean (Julian et al., 1995), Peru (Logrieco et al.,2007). Production of fumonisin B1 has also been 1993a), Argentina (Torres et al., 2001; Reynosoreported (Visconti and Doko, 1994), but levels are et al., 2004, 2006), Mexico (Desjardins et al.,generally low (Reynoso et al., 2004). Other toxins 2000b; Morales-Rodriguez et al., 2007), Southreported are beauvericin and fusaproliferin Africa (Marasas et al., 1978, 1979; Rheeder et al.,(Moretti et al., 1995; Logrieco et al., 1998a; 1995), Zimbabwe (Mubatanhema et al., 1999), ´Castella et al., 1999b; Kostecki et al., 1999; Shep- Indonesia (Pitt et al., 1998a), Australia (Burgesshard et al., 1999; Torres et al., 2001; Reynoso et al., et al., 1981; Blaney et al., 1986) and New Zealand2004). (Hussein et al., 1991, 2002). F. subglutinans has also Ecology. Closely related to Fusarium verticil- been isolated in Mexico from teosintes, the nearestlioides, F. subglutinans appears to have a similar wild relatives of maize (Desjardins et al., 2000b).host range, and a worldwide distribution. Its F. subglutinans has been reported from wheat inmajor food habitat is maize. It has been isolated Europe (Bottalico and Perrone, 2002; Ioos et al.,from Europe (Italy, Logrieco et al., 1995; Austria, 2004; Cosic et al., 2007), South Africa (BoshoffLew et al., 1991; Poland, Logrieco et al., 1993b), the et al., 1998), Tanzania (van Dyk, 2004), EgyptUnited States (Abbas et al., 1988, 1989b; Katta (Gherbawy et al., 2006) and Iran (Moosawi-Jorf
    • 120 5 Primary Keys and Miscellaneous Fungiet al., 2007), but it does not appear to play an active Fusarium verticillioides (Sacc.) Nirenbergrole in head blight. Fig. 5.36 Fusarium subglutinans is a pathogen of pineap- Fusarium moniliforme J. Sheld. (in part)ples (Bolkan et al., 1979; Rohrbach and Taniguchi, ´1984, Matos et al., 2000), bananas (Jimenez et al., Teleomorph: Gibberella moniliformis Wineland1993, 1997; Wade et al., 1993; Vesonder et al., 1995)and capsicums (bell peppers) (Hashmi and Thrane, Colonies on CYA usually covering the whole1990; Utkhede and Mather, 2004). Other sources Petri dish, low at the margins to moderately deephave been sorghum (Onyike and Nelson, 1992), centrally, of floccose to funiculose white mycelium;sugar beets (Bosch and Mirocha, 1992), traditional reverse pale, or in shades of pale salmon or violet.African vegetables (van der Walt et al., 2006), toma- Colonies on MEA usually covering the whole Petritoes and lemons (Muniz et al., 2003), and black dish, low to moderately deep centrally, of white orpepper (Pitt et al., 1998a). pale salmon funiculose mycelium, sometimes pow- References. Booth (1971); Nelson et al. (1983); dery with microconidia; reverse of some isolatesDesjardins (2006); Leslie and Summerell (2006). pale, of others violet or greyish magenta. ColoniesFig. 5.36 Fusarium verticillioides (a) colonies on PDA and DCPA, 7 d, 258C; (b) phialides bearing chains of microconidia, bar¼ 50 mm; (c) phialides, bar ¼ 10 mm; (d) macro- and microconidia, bar ¼ 10 mm
    • 5.17 Genus Fusarium 121on G25N 5–12 mm diam. At 58C, either germina- from maize and a few other sources (Leslie andtion or no growth. At 378C, colonies of 4–10 mm Summerell, 2006). Morphologically similar speciesdiam produced. within the Gibberella fujikeroi complex, associated On PDA, colonies of low, densely funiculose with different plant hosts and different mating types,mycelium coloured white to pale salmon, usually have been described: F. thapsinum from sorghum,powdery with chains of microconidia; reverse vary- F. sacchari from sugar cane, F. mangiferae froming from isolate to isolate, pale salmon, greyish mango and F. fujikuroi from rice, and these namesviolet, brownish violet or deep violet; dark blue have now been taken up by Leslie and Summerellsclerotia produced by some isolates. On DCPA, (2006).colonies low, of thin, funiculose white to pale sal- Physiology. The maximum temperature formon mycelium in concentric rings, powdery with growth of Fusarium verticillioides has been reportedmicroconidia, and sometimes alternating with to be 32–378C, the minimum as 2.5–58C, and therings of macroconidia produced on the agar surface; optimum near 258C (Nirenberg, 1976). The mini-reverse pale. mum aw for growth was 0.87 at 258C, after a Macroconidia usually long and slender, almost 4-month germination time (Armolik and Dickson,straight, thin walled, with foot-shaped basal cells; 1956). Reduction of headspace oxygen from 20 tomicroconidia fusiform to clavate, produced from 2% had no effect on growth of F. verticillioides, butlong monophialides, forming chains readily seen vacuum packaging with oxygen absorbing sachetsin situ under the low power microscope (6Â to completely inhibited growth (Samapundo et al.,10Â); chlamydoconidia not produced. 2007b). Fumonisins B1 and B2 were produced Distinctive features. The production of microco- down to at least 0.92 aw (Marı´ n et al., 1995) butnidia in long chains from relatively long phialides were not detected at 0.89–0.91 aw on irradiateddistinguishes Fusarium verticillioides from most other maize grains (Marı´ n et al., 1999). Maximum fumo-common Fusarium species. F. proliferatum is very nisin production occurred with 15% oxygen atsimilar to in appearance to F. verticillioides but is 0.976 aw, but with 5% O2, 0.93 was the optimumdistinguished from it by the production of polyphia- aw (Samapundo et al., 2007b).lides bearing microconidia in short chains. Mycotoxins. The major mycotoxin produced by Taxonomy. In 1877, Saccardo described Oospora Fusarium verticillioides is fumonisin B1, the cause ofverticillioides from maize in Italy. In 1904, John leucoencephalomalacia in horses, pulmonarySheldon described Fusarium moniliforme from oedema in pigs and liver cancer in rats. It is amaize that was associated with animal toxicosis in possible cause of human oesophageal cancerthe United States (see Desjardins, 2006), and for (Gelderblom et al., 1988; Sydenham et al., 1990;most of the twentieth century, the epithet F. mon- Shephard et al., 2007) and neural tube defectsiliforme was used for this fungus. From an extensive (Desjardins, 2006). For a comprehensive accountstudy of the group of Fusarium species which of fumonisins, see Marasas (2001) and Desjardinsincluded F. moniliforme, Nirenberg (1976) con- (2006). Isolates of F. verticillioides from maize fromcluded that this species should be called F. verticil- many countries have been shown to produce fumo-lioides, but at the time, this proposal was not gen- nisins B1 and B2 (Bezuidenhout et al., 1988; Laurenterally accepted. However, Seifert et al. (2003), et al., 1989; and see Pitt and Hocking, 1997; Desjar-representing the International Society for Plant dins, 2006). This species also commonly producesPathology and the International Committee on the fusarins, fusaric acid and naphthoquinones, but notTaxonomy of Fungi (ISPP/ICTF) Subcommittee beauvarin, moniliformin or fusiproliferin (see Pitton Fusarium Systematics, proposed that the name and Hocking, 1997; Desjardins, 2006; Leslie andF. verticillioides be applied to the fumonisin produ- Summerell, 2006). Reports of zearalenone, diace-cing fungus on maize, on the grounds that the name toxyscirpenol and deoxynivalenol production byF. moniliforme represented an unacceptably broad F. verticillioides were discounted by Nelson et al.species concept, and that F. verticillioides was undis- (1983). Recently identified isolates of F. verticil-putably the older name, which therefore had prior- lioides from banana rots (Mirete et al., 2004;ity. This name is now generally accepted for isolates Moretti et al., 2004) produce moniliformin but not
    • 122 5 Primary Keys and Miscellaneous Fungifumonisins (Patino et al., 2006). These isolates need ˜ to mating population A (G. moniliformis), they arefurther characterisation. genetically distinct from F. verticillioides isolates Ecology. Fusarium verticillioides is widespread in from maize, produce moniliformin and are unable tothe tropics and humid temperate areas of the world produce fumonisins (Moretti et al., 2004; Mirete et al.,(Leslie and Summerell, 2006). This species is an 2004; Patino et al., 2006). ˜endemic pathogen of maize, causing both stalk rot In light of recent taxonomic changes, many of theand cob rot which can be present at all stages of above reports of Fusarium moniliforme occurrence mayplant development. It has been isolated from maize need to be verified.kernels in every major location where it has been References. Nirenberg (1976); Domsch et al.sought, including the United States, Canada, (1980); Nelson et al. (1983) (both under F. monili-Mexico, Honduras, Ecuador (Pacin et al., 2002), forme); Desjardins (2006); Leslie and SummerellPeru and Argentina; from Iran (Ghiasian et al., (2006).2004; 2006), India, China, Nepal (Desjardins andBusman, 2006) and Taiwan; from Europe (Logriecoet al., 2002), and from Australia, South Africaand other countries on the African subcontinent 5.18 Genus Geotrichum Link: Fr.(Adejumo et al., 2007; Bigirwa et al., 2007) (seealso Pitt and Hocking, 1997; Desjardins, 2006; For a long time, the genus Geotrichum has beenLeslie and Summerell, 2006). Incidence in indivi- distinguished by its method of reproduction,dual maize kernels, where reported, has almost which is exclusively the formation of arthroconi-always been very high, e.g. 100% in Sardinia (Bot- dia from vegetative hyphae. Arthroconidia aretalico et al., 1995), 54% in mainland Italy (Logrieco cylindrical spores formed by septation of vegeta-et al., 1995), 97% in Thailand (Pitt et al., 1993), tive hyphae into short segments, which separate at92% in the Philippines and 73% in Indonesia (Pitt maturity. Young colonies sometimes appearet al., 1998a). yeast-like on CYA, but soon spread rapidly. Fusarium verticillioides (as F. moniliforme in ear- Recently, the definition of Geotrichum has beenlier literature) has been reported as a pathogen of other broadened to include some species which alsoGraminae, particularly sorghum (Leslie et al., 2005), produce blastospores (yeast-like cells), but theserice (Desjardins et al., 2000a) and millet (Leslie et al., are not of interest here. The most recent taxon-2005) (see also Pitt and Hocking, 1997; Desjardins, omy is that of de Hoog et al. (1986), in which 232006). It has been reported from several types of species are described, together with two teleo-nuts, i.e. hazelnuts, pecans, peanuts and kola nuts morphs, Dipodascus and Galactomyces. Only one(see Pitt and Hocking, 1997). F. verticillioides has species, Geotrichum candidum, is significant inalso been reported from oilseeds: sunflower, amar- foods (Fig. 5.37).anth, and soybeans; also spices, including coriander,fenugreek, cardamom and pepper (see Pitt and Hock-ing, 1997). It has also been found in mung beans (Pitt Geotrichum candidum Link: Fr. Fig. 5.37et al., 1994), biltong (van der Riet, 1976) and cheeses Oidium lactis Fresen.(Northolt et al., 1980). Oospora lactis (Fresen.) Sacc. Fusarium verticillioides (or F. moniliforme) hasbeen reported to cause storage rot of oranges and Teleomorph: Galactomyces geotrichum (E.E. Butlerother citrus (Snowdon, 1990), garlic (Gargi and Roy, & L.J. Peterson) Redhead & Malloch1988), yams (Ogundana, 1972), pineapples (Lim,1983), bananas (Chorin and Rotem, 1961; Jimenez ´ Colonies on CYA of variable size, 20–45 mmet al., 1993; Vesonder et al., 1995), asparagus (Snow- diam, very low and quite sparse, plane, of whitedon, 1991) and dry rot of passion fruit (Lutchmeah, mycelium, often leathery and difficult to dissect1993). The molecular and chemical profiles of isolates with a needle; reverse pale. Colonies on MEA 50–of F. verticillioides that are pathogenic on bananas 65 mm diam, similar to on CYA but of softer,have been examined. Whilst banana isolates belong yeast-like texture. On G25N, no growth to
    • 5.18 Genus Geotrichum 123Fig. 5.37 Geotrichum candidum (a) colonies on MEA, 7 d, 258C; (b) arthroconidia, bar ¼ 10 mmgermination. At 58C, no germination to colonies Mycotoxins. This species is not known to pro-up to 4 mm diam, of dense white mycelium. At duce any toxic compounds.378C, usually no growth, occasionally sparse colo- Ecology. Geotrichum candidum is a significantnies up to 10 mm diam formed. pathogen of citrus fruits during postharvest storage, Conidiophores undifferentiated hyphae, at causing sour rots (Butler et al., 1965; Hall and Scott,maturity fragmenting almost entirely to form 1977; Snowdon, 1990). It occurs in fruit weakened byarthroconidia; arthroconidia hyaline, cylindrical, over maturity and long storage. Lemons and grape-sometimes developing rounded ends and thick- fruit are particularly susceptible, but a variety of otherened walls, commonly 5–8 Â 2–5 mm, smooth fruit can also be affected (Butler, 1960). Initial infec-walled. tion is mainly through injuries. The primary control Distinctive features. See genus description. measure is preventative, and lies in choosing sound, Taxonomy. Some isolates of Geotrichum candi- young fruit for long term storage (Hall and Scott,dum have been reported to form a teleomorph, 1977). As G. candidum grows poorly below 108C,Galactomyces geotrichum, which consists of single cold storage can assist in control (Rippon, 1980,ascospores within solitary asci borne along fertile Plaza et al., 2003).hyphae (de Hoog et al., 1986). Geotrichum candidum causes spoilage of toma- Physiology. Geotrichum candidum is restricted to toes (Okoli and Erinle, 1989; Snowdon, 1991),habitats of high water availability, its minimum aw dried capsicums (Atanda et al., 1990) and sapodillasfor growth being 0.90 (Heintzeler, 1939). Growth (Kusum and Geeta, 1990) in tropical countries. Aoccurred at 0.95 but not at 0.90 aw (Plaza et al., wide variety of vegetables, including carrots,2003). Optimal growth temperatures are 25–308C cucumbers, onions, peas and potatoes are also sus-(Plaza et al., 2003), with maxima of 35–388C ceptible (Snowdon, 1991).(Domsch et al., 1980). The conidia have a very Geotrichum candidum has long been a problem inlow heat resistance, with a D value of 30–40 min the canning and freezing industries. Known asat 528C (Beuchat, 1981b). Miller and Golding ‘‘machinery mould’’ (Eisenberg and Cichowicz,(1949) reported that G. candidum was able to 1977), it is a frequent contaminant of processinggrow at very low oxygen tensions, but not lines, and consequently of products such as frozenanaerobically. foods (Pitt and Hocking, 1997). Standard methods
    • 124 5 Primary Keys and Miscellaneous Fungihave been established for estimating G. candidum in Hyphopichia burtonii (Boidin et al.) Arx &food processing machinery, by physical counting van der Walt Fig. 5.38rather than microbiological techniques (Cichowicz Pichia burtonii Boidin et al.and Eisenberg, 1974). Improvements in sanitation Endomycopsis burtonii (Boidin et al.) Lodder & Kregerprocedures have reduced this problem in recent Endomycopsis chodatii Wick.years. Kure et al. (2004) concluded that air was the Anamorph: Candida chodatii (Nechitsche) Berkhoutmajor source of G. candidum contamination in Cladosporium chodatii (Nechitsche) Sacc.cheese factories in Norway. Trichosporon behrendii Lodder & Kreger This mould is a very common problem in raw Trichosporon variabile (Lindner) Delitschmilk in Europe especially when it is used in themanufacture of soft, fresh cheeses such as quarg Conidia borne from spicules (small projections)or Roblochon (see Pitt and Hocking, 1997; along the length of undifferentiated hyphae, yeast-Lopandic et al., 2006). Contamination of sur- like, ellipsoidal, pyriform or near cylindroidal, 2.5–face-ripened cheeses such as Brie and Camembert 5.0 Â 1.5–2.5 mm, with thin, smooth walls; hyphalcan also be a problem (Pitt and Hocking, 1997). fragments also evident, 10–50 Â 2.5–3.0 mm, stillHowever, recent surveys indicate that G. candi- functioning as fertile hyphae, producing conidiadum may play an important role in the ripening (blastospores) from lateral spicules and terminalof many soft and semi-hard cheeses as well as buds. Ascospores not observed in pure culture,contributing desirable cheese flavours (Boutrou two mating strains needed; ascospores hat-shaped,and Gueguen, 2005; Boutrou et al., 2006; Ghosh 1–4 per ascus.et al., 2006). Distinctive features. This species produces white, Geotrichum candidum has been isolated from a powdery, filamentous colonies which bear conidiavariety of other foods, including meats, raw and from spicules on vegetative hyphae, and also byParma hams, hard cheeses and traditional fermen- budding (yeast-like cells). It is distinguished fromted foods, and Nigerian alcoholic beverages (see Pitt E. fibuliger by the fragmentation of hyphae into shortand Hocking, 1997). Low levels were found in lengths; these can also act as reproductive structures.maize, peanuts, sorghum, soybeans and black rice Taxonomy. This species and Endomyces fibuligerin Thailand (Pitt et al., 1993, 1994). It is of quite have rarely been placed in the same genus by taxo-common occurrence in barley during malting (see nomists, but in culture the resemblance is unmistak-Pitt and Hocking, 1997). able. Both grow at more or less the same rates and References. von Arx (1977); de Hoog et al. have similar habitats. One (E. fibuliger) is homothal-(1986). lic and the other heterothallic; however, the frag- menting of fertile hyphae in Hyphopichia burtonii is the only obvious difference in morphology. The teleomorph of Hyphopichia burtonii has been5.19 Genus Hyphopichia Arx and van der seen only in the laboratory, after mixing of two mat- Walt ing strains. However, the teleomorph name is used here, both because it has become accepted in theHyphopichia is a genus of yeast-like fungi, probably recent literature and because there are several con-closely related to Endomyces, from which it differs fusing anamorph names, at both genus and speciesby the production of hyphal fragments which pro- level. Some recent taxonomies have included thisduce yeast-like conidia by budding from spicules. species in the yeasts, as Pichia burtonii Boidin et al.There is a single species, H. burtonii. (Kurtzman and Fell, 1998; Barnett et al., 2000). Colonies on CYA 22–25 mm diam and on MEA Physiology. Hyphopichia burtonii grows more25–30 mm diam, of low and sparse to moderately rapidly at 308C than 208C (Ramakrishna et al.,dense white to pale grey mycelium, sometimes cen- 1993). The minimum is >58C and growth occurs attrally umbonate; reverse pale. Colonies on G25N 378C. Growth occurs at least down to 0.90 aw on10–12 mm diam, similar to those on CYA. No barley grains (Ramakrishna et al., 1993).germination at 58C. At 378C, colonies 5–10 mm Mycotoxins. This type of fungus is unlikely todiam, similar to at 258C; reverse pale. produce toxic compounds.
    • 5.20 Genus Lasiodiplodia 125Fig. 5.38 Hyphopichia burtonii (a) colonies on CYA and MEA, 7 d, 258C; (b) vegetative cells, bar = 10 mm; (c,d) vegetativecells, bar = 5 mm Ecology. Hyphopichia burtonii is not uncommon characterised by distinctive large, striate conidia, asin cereals and cereal products, especially packaged described below.bread (Spicher, 1984a, 1985). In Europe, along withEndomyces fibuliger, it is known as ‘‘chalky mould’’ Lasiodiplodia theobromae (Pat.) Griffon(Spicher, 1986b). It is also part of the flora of Parma & Maubl. Fig. 5.39hams during ripening (Simoncini et al., 2007). Botryodiplodia theobromae Pat. Reference. Barnett et al. (1990). Teleomorph: Botryosphaeria rhodina (Berk. & Curt.) Arx5.20 Genus Lasiodiplodia Ellis and Everh. Colonies on CYA and MEA usually filling the whole Petri dish with a loose to moderately denseLasiodiplodia is the correct generic name for the weft of light to dark grey hyphae, adhering to thefungus previously known as Botryodiplodia theobro- Petri dish lid; pycnidia borne beneath the agarmae Pat. (Sutton, 1980). Both genus and species are surface (visible from the reverse), and sometimes
    • 126 5 Primary Keys and Miscellaneous FungiFig. 5.39 Lasiodiplodia theobromae (a) colonies on CYA, 7 d, 258C; (b) conidia, bar = 10 mmat the base–lid interface or on the lid itself; reverse Taxonomy. A molecular study using a combinedpale, black near pycnidia, uniformly grey-black analysis of the ITS region and the translation elon-elsewhere. On G25N, colonies 30–50 mm diam, gation factor 1-a indicated that Lasiodiplodia theo-occasionally more, low and spreading, with sparse bromae as currently understood includes three spe-white aerial mycelium; reverse pale or grey. No cies, distinguishable by differences in the size ofgrowth at 58C. At 378C, colonies greater than their conidia (Alves et al., 2008).50 mm diam, of low, dense mycelium, coloured This species is sometimes known by its teleo-grey or deep red; red brown soluble pigment and morph name, Botryosphaeria rhodina, but thisoccasionally exudate produced; reverse deep red state is not usually seen in culture.brown to almost black. Physiology. Alasoadura (1970) reported cardinal Reproductive bodies on CYA pycnidia, grey- temperatures for Lasiodiplodia theobromae as mini-black and roughly spherical, 200–400 mm diam, mum 158C, optimum 288C and maximum 408C.but on other media reportedly up to 5 mm diam This is difficult to reconcile with statements by(Punithalingam, 1976), or forming a stroma Uduebo (1974) that ‘‘The vegetative growth at(Alasoadura, 1970), easily ruptured, of dark 378C is scanty’’, or that this species does not growbrown to black pseudoparenchymatous cells; con- at 358C on potato dextrose agar (Adeniji, 1970b).idia at 7 days ellipsoidal, 15–20 Â 9–12 mm, with Isolates of this species which we have studied grewsmooth, brownish walls and little ornamentation, rapidly at 378C, and our observations are consistentat maturity (18–)20–30 Â 10–15 mm, with a trans- with the statement that distribution of L. theobro-verse septum and ornamented by longitudinal mae is mainly confined to the area between 408Nstriations. and 408S (Punithalingam, 1976). Several references Distinctive features. Lasiodiplodia theobromae is suggest that germination and growth of L. theobro-distinguished in culture by fast growth on G25N mae is confined to high water activities: vigorousand at 378C: on G25N growth is sparse and usually growth observed on G25N in this study is at var-colourless, but at 378C it is dense, with mycelium iance with this.grey and/or red. Microscopically, this species is Mycotoxins. Mycotoxin production has not beendistinguished by large ellipsoidal conidia which reported.are borne in black pycnidia, and are ornamented Ecology. Punithalingam (1976) aptly describedwith a transverse septum and longitudinal Lasiodiplodia theobromae as ‘‘an unspecialisedstriations. virulent rot pathogen causing numerous diseases:
    • 5.21 Genus Monascus 127dieback, root rot, rot or decay of various fruits and with fermented foods; the third, M. ruber Tiegh., isstorage rot of yams.’’ Spoilage by Lasiodiplodia of uncommon in fermented foods but of widespreadavocados, bananas, grapes, guavas, mangoes, okra, occurrence elsewhere, and it sometimes causes foodpapayas, passionfruit, traditional Nigerian foods spoilage. Only M. ruber is treated here. For informa-and yams has been reported (see Pitt and Hocking, tion on the other species, see Hawksworth and Pitt1997). Control measures for fruits are well documen- (1983). Further species have been described fromted by Punithalingam (1976). Iraqi soils (Cannon et al., 1995) but these are not Lasiodiplodia theobromae has also been reported relevant to foods.from pecans, peanuts, wheat and soybeans (see Pittand Hocking, 1997). In our experience, it is of com- Monascus ruber Tiegh. Fig. 5.40mon occurrence in many tropical food commod-ities. We isolated it from 58% of Thai maize samples, Colonies on CYA 20–32 mm diam, occasionally onlyat up to 46% of kernels in infected samples and in 15 mm, plane, sparse, surface texture floccose to7% of all kernels examined (Pitt et al., 1993). It was deeply floccose; mycelium white at first, then becom-also common in Thai peanuts, mung beans and sor- ing pale brown as cleistothecia and aleurioconidiaghum, with 30–33% of samples showing infection develop, in age sometimes becoming dark brown;(Pitt et al., 1993, 1994). Indonesian crops were brown soluble pigment sometimes produced; reverseequally affected, particularly peanuts, where 47% sometimes uncoloured, usually brown to dark brownof samples were positive, with up to 64% of kernels near sepia. Colonies on MEA 30–42 mm diam, planeaffected in those samples, and overall 9% of the more and sparse, sometimes with little aerial growth butthan 12,000 peanut kernels examined were infected usually with deeply floccose mycelium, white at thewith this species. More than 25% of maize, talo bean, margins, becoming brown to orange brown as cleis-velvet bean, black soybean and cowpea samples also tothecia and aleurioconidia develop; orange or brownshowed infection (Pitt et al., 1998a). soluble pigment sometimes produced; reverse pale at References. Punithalingam (1976); Domsch et al. the margins, khaki or brownish orange at the centres.(1980), under Botryosphaeria. Colonies on G25N usually 16–20 mm diam, some- times only 10 mm, plane, sparse and floccose; myce- lium white; reverse uncoloured or brown. No growth at 58C. At 378C, colonies 12–30 mm diam, low and5.21 Genus Monascus Tiegh. sparse, coloured as on CYA at 258C, or with reverse deeper brown and sometimes with a reddish tinge.Monascus is a genus of Ascomycetes characterised Cleistothecia spherical, 30–60 mm diam, borne as aby the production of colourless to pale brown cleis- hyphal knot from a well-defined stalk, with cellulartothecia and aleurioconidia. Each cleistothecium is walls, becoming brown during maturation; ascosporesborne from a knot of hyphae on a well defined stalk, ellipsoidal, hyaline, 5–7 Â 4.0–4.5 mm, smooth walled.in 7 day old cultures resembling a clenched fist on a Aleurioconidia sometimes borne on pedicels from thenarrow forearm. Aleurioconidia occur singly or in sides of hyphae, but more commonly terminally, some-short chains. Asci break down rapidly so that, when times borne singly but more often in chains of up toascospores are mature, the impression under the 10 cells long, spherical to pyriform, often rounding atmicroscope is of a sac filled with a mass of ellipsoi- maturity, 10–14 mm diam or 10–18 Â 8–14 mm, withdal, smooth walled, refractile spores. thick, smooth, brown walls. Chlamydoconidia and Species of Monascus are best known for their role arthroconidia produced by most isolates also.in the fermentative production of Oriental foods, of Distinctive features. The cleistothecium producedwhich red rice (ang-kak), rice wine and kaoliang by Monascus is distinctive, being borne as a fist-likebrandy are the best known (Hesseltine, 1965; Lin, hyphal knot on an arm-like stalk. M. ruber is dis-1975). Three species were accepted in the most recent tinguished from other Monascus species by rela-revision of the genus (Hawksworth and Pitt, 1983): tively rapid growth, especially on MEA, and bytwo, M. purpureus Went and M. pilosus K. Saito ex brown pigmentation in the walls of cleistotheciaD. Hawksw. & Pitt, are associated almost exclusively and aleurioconidia.
    • 128 5 Primary Keys and Miscellaneous FungiFig. 5.40 Monascus ruber (a) colonies on CYA and MEA, 7 d, 258C; (b) cleistothecia, bar = 10 mm; (c) aleurioconidia, bar =10 mm; (d) ascospores, bar = 5 mm Taxonomy. Sequencing of the ITS region and in a solution of NaCl at 0.92 aw (Panagou et al., 2003).part of the b-tubulin gene indicate that Monascus Growth of M. ruber was little affected over the pHruber and M. pilosus should be considered as a single range 3.0–5.0 (Panagou et al., 2003) and was able tospecies (Park et al., 2004a). grow down to pH 2.2 at 308C in high aw (Panagou Physiology. The cardinal temperatures reported et al., 2005). The combined effect of temperaturefor Monascus ruber are minimum 15–188C; optimum (25–358C), water activity (0.999–0.92 aw) and pH358C and maximum near 458C (Manandhar and Api- (2–6.8) was studied on gradient plates by Panagounis, 1971; Panagou et al., 2003). M. ruber has been et al. (2005), and also modelled against several equa-isolated from relatively concentrated substrates (such tions (Panagou et al., 2003, 2005, 2007).as high moisture prunes, ca. 0.85 aw) and is probably Ascospores of Monascus ruber are heat resistant.weakly xerophilic. However, an isolate failed to grow D80 in citrate buffer (pH 4) was 2.1 min. NaCl
    • 5.22 Genus Moniliella 129provided some protection: D80 values were 0.88 min prunes (Hawksworth and Pitt, 1983). It was isolatedin 5.6% brine, but 1.04 min in 10.5% brine. z values quite frequently from Indonesian dried fishover the temperature range 70–808C varied from 7.4 (Wheeler et al., 1986). Other sources includeto 7.9 C8 under the conditions used (Panagou et al., mayonnaise (Muys et al., 1966a), bread (Spicher2002). They concluded that a heat process of 5 min and Isfort, 1988), table olives (Panagou et al.,at 808C would assist the olive industry in producing 2002), and minced meat, soft cheese, fruit sauce,a stable product. spices, honey, cacao beans, soy beans, peas, palm Pigmentation has received a great deal of atten- kernels, maize and various animal feeds, and silagetion, having been used to distinguish a number of (data sheets, International Mycological Institute,species utilised in Oriental fermentations. However, Egham, Surrey; Hawksworth and Pitt, 1983).Carels and Shepherd (1977) showed that red pig- References. Domsch et al. (1980); Hawksworthments form during growth near neutral pH values, and Pitt (1983).whereas cultures become orange if the pH of thefermenting food becomes strongly acid. Pigmentstability is affected by pH, temperature, light, oxy- 5.22 Genus Moniliella Stolk and Dakingen and water activity (Dufosse et al., 2005). Theproduction of pigments by Monascus species hasbeen reviewed by Miyake et al. (2008). Moniliella is a genus of yeast-like fungi, charac- Mycotoxins. Monascus ruber (and M. purpureus) terised by the production of budding cells, thinproduce citrinin (Blanc et al., 1995a, b) both in sub- walled arthroconidia and, in one species, by rela-merged and solid state culture. This is of importance tively large chlamydoconidia (up to 3Â hyphal dia-as red pigments produced by Monascus species are meter). In some respects it is similar to Hyphopichia,used as food colourants. Recent studies indicate that but conidia are larger, and the hyphal fragmentshigh pigment production can be achieved without bearing conidia on spicules, characteristic ofcitrinin synthesis by incorporating histidine in the Hyphopichia, are absent. The latest taxonomy (degrowth media (Hajjaj et al., 2000; Xu et al., 2003). Hoog, 1979) accepts two species, M. suaveolens andWang et al. (2005) surveyed type cultures of 23 spe- M. acetoabutans. M. suaveolens is found in buttercies of Monascus (most now regarded as synonyms) and margarine, and other substrates rich in oil,for their ability to synthesise citrinin and found that while M. acetoabutans is of interest because of itsall, including M. ruber, produced citrinin. resistance to acetic acid and hence potential ability Ecology. A widespread species, Monascus ruber to spoil mayonnaise and other products preservedhas caused spoilage of high moisture Australian with acetic acid. Key to Moniliella species included here1 Spherical chlamydoconidia produced, 8–12 mm diam, with thick, brown walls; growth on malt acetic agar M. acetoabutans Thick walled chlamydoconidia not produced; no growth on malt acetic agar M. suaveolensMoniliella acetoabutans Stolk & Dakin hyphal tips, and chlamydoconidia, in intercalary or Fig. 5.41 terminal positions on hyphae, solitary or in short chains; budding conidia ellipsoidal, arthroconidiaColonies on CYA and MEA 22–30 mm diam, cylindroidal, both 5–9 mm long, chlamydoconidia sphe-deeply floccose, especially on CYA, mycelium rical, 8–12 mm diam, with thick brown walls.pure white; reverse yellow brown. Colonies on Distinctive features. Moniliella acetoabutans isG25N 5–10 mm diam, deep but often mucoid, morphologically distinguished by its three types ofuncoloured. No growth at 58C. Usually no growth conidia: budding conidia like a hyaline Cladospor-at 378C, occasionally colonies up to 5 mm diam. ium; arthroconidia like Geotrichum, but not so Conidia of three types produced, budding cells from extensive; and relatively large, brown walled chla-hyphal extremities, arthroconidia by differentiation of mydoconidia. In culture, the definitive test for M.
    • 130 5 Primary Keys and Miscellaneous FungiFig. 5.41 Moniliella acetoabutans (a) colonies on CYA and MEA, 7 d, 258C; (b,c) conidiophores producing budding cells(slide culture), bar = 10 mm; (d) arthroconidia, bar = 10 mm; (e) chlamydoconidia, bar = 10 mmacetoabutans is its ability to produce quite rapidly Ecology. Dakin (Stolk and Dakin, 1966) isolatedgrowing white colonies on malt acetic agar – or even Moniliella acetoabutans from spoiling sweet fruiton MEA containing 2% acetic acid. sauce, and then a variety of other similar products. Physiology. This species appears to be unique in its It also caused fermentative spoilage of a large pro-tolerance of acetic acid and other weak acid preserva- duction run of mayonnaise in Australia in 1971. Thetives. In our laboratory, Moniliella acetoabutans has source was wooden vinegar tanks, where the fungusbeen able to grow in MEA with 4% added acetic acid, was surviving in 10% acetic acid, then growing onto our knowledge a unique property. This species is the wood as the tank level lowered and the acetic acidcapable of fermentative growth, like a true yeast, and became diluted by evaporation. Data sheets from thealso appears to be highly tolerant of acid pH. International Mycological Institute, Egham, Surrey, Mycotoxins. Toxic compounds are not produced. record isolations from various acetic acid preserves.
    • 5.23 Genus Nigrospora 131 Moniliella acetoabutans infections fortunately appear Taxonomy. The taxonomy of Moniliella suaveolensto be rare. They may be overcome either by holding is complicated by the fact that the two varieties acceptedvinegar or acetic acid in stainless steel tanks, or by by de Hoog (1979) culturally bear little resemblance topasteurising this ingredient before addition to product. each other. One (M. suaveolens var. suaveolens) produces References. Stolk and Dakin (1966); Dakin and low, spreading colonies, which remain white; the otherStolk (1968); de Hoog (1979). (M. suaveolens var. nigra) forms compact, dense colo- nies, which rapidly become olive. Their incorporationMoniliella suaveolens (Lindner) Arx Fig. 5.42 into a single species by de Hoog (1979) was due toCladosporium suaveolens (Lindner) Delitsch similarities in conidial production and conidia.Cladosporium butyri O.S. Jensen Physiology. No physiological studies are known to us.Colonies on CYA and MEA highly variable in Mycotoxins. Mycotoxins are not produced.character, 15–40 mm diam, either white, floccose, Ecology. This species has caused spoilage of mar-sparse and persistently white, or low, dense, veluti- garine in Europe (Muys et al., 1966a, b; Bours andnous and olive, lightly to heavily sporing; reverse Mossel, 1973) and Australia (Hocking and Pitt,colourless or olive. Colonies on G25N 3–8 mm unpublished). It has been reported as one of thediam, of white mycelium. At 58C, sometimes germi- causes of chalky bread in Europe (Spicher, 1985,nation. At 378C, no growth. 1986b; Spicher and Isfort, 1987). It has occasionally Conidiophores undifferentiated, bearing conidia in been isolated from cheese (Chabalier et al., 1997;short, sometimes branched, acropetal chains (the Torkar and Vengust, 2008).youngest spore at the end), breaking up in liquid Reference. de Hoog (1979).mounts; conidia subspheroidal or ellipsoidal to cylin-droidal, or somewhat irregular, nonseptate, when ellip-soidal 9–13 Â 7–10 mm, when cylindroidal commonly 5.23 Genus Nigrospora Zimm.15–20 Â 5–7 mm, with smooth, slightly thickened walls. Distinctive features. Unlike Moniliella acetoabu- Characterised by the production of relatively large,tans, this species does not make chlamydoconidia. It solitary, jet black, smooth-walled, oblate conidia,is not preservative resistant, and is normally asso- Nigrospora occurs in nature mainly as a plant andciated with oils and oil-based foods. seed pathogen, but is found also in air, soil andFig. 5.42 Moniliella suaveolens (a) colonies on MEA, 7 d, 258C; (b,c) vegetative cells (slide culture), bar = 10 mm
    • 132 5 Primary Keys and Miscellaneous Fungiwater. Two species are of significance in foods: Taxonomy. Nigrospora oryzae produces an asco-Nigrospora oryzae and Nigrospora spherica. mycete teleomorph, Khuskia oryzae, which is found only as a pathogen on certain plants. Physiology. Optimum growth for Nigrospora oryzae has been recorded at 0.995 aw and 258C. N.Nigrospora oryzae (Berk. & Broome) Petch oryzae sporulated at 0.98 and 0.995 aw but not at Fig. 5.43 lower aw values (0.95, 0.90 or 0.85) at 258C (Sem- pere and Santamarina, 2006). Inability to grow atTeleomorph: Khuskia oryzae H.J. Huds. low or high temperatures has been noted above. Mycotoxins. Mycotoxin production has not beenColonies on CYA and MEA covering the whole reported for Nigrospora species.Petri dish, low to moderately deep, dense to floc- Ecology. As a plant pathogen on cereal crops, thecose, mycelium flesh coloured or pale orange to widespread distribution of Nigrospora oryzae in cerealpure grey; black conidia conspicuous at low magni- grains is to be expected. It has been reported from barleyfications; reverse pale, greyish orange or grey to (Fakhrunnisa et al., 2006), wheat, other leguminous anddeep bluish grey. Colonies on G25N usually 10– cereal foods, pecans and various health foods (see Pitt15 mm diam, white or mucoid. No growth at 5 and Hocking, 1997). It also causes a storage disease ofor 378C. apples in India (Khanna and Chandra, 1975). Conidiophores borne from aerial hyphae, short, We isolated Nigrospora oryzae frequently in surveysdark walled, bearing conidia in isolation or in clus- of Southeast Asian food commodities (Pitt et al., 1993,ters from groups of irregular cells; conidia solitary, 1994, 1998a and unpublished). The highest level ofjet black, oblate, sometimes collapsing, mostly occurrence was on paddy rice, where it was isolated12–15 mm long, with smooth, featureless walls, from an average of 40% of samples, with up to 68%remaining attached or (on natural substrates) vio- of infected kernels in infected samples and in 10% oflently discharged. more than 7,000 grains examined from Thailand, Indo- Distinctive features. See genus description. nesia and the Philippines. This species was isolated fromNigrospora is clearly distinguished from Arthrinium, high percentages of samples (25% or more) of maize,which it superficially resembles, by its jet black sorghum, soybeans, cashews and copra from Thailand,conidia which lack surface features or markings. with overall infection rates of 2–5%. In Indonesia,Fig. 5.43 Nigrospora oryzae (a) colonies on MEA, 7 d, 258C; (b) conidia, bar = 10 mm
    • 5.24 Genus Pestalotiopsis 133kemiri nuts showed infection at 5% overall, while maize, conidia with three or four transverse septa and spikysorghum, soybeans, kidney beans, cowpeas and talo appendages from one or both ends. Neither genus isbeans were all infected at the rate of 1% or more of all encountered frequently in foods other than cereals orseeds examined (Pitt et al., 1998a). Maize, mung beans nuts, where they can be spoilage agents. In our experi-and black pepper from the Philippines showed similar ence, direct plating of cereals will frequently detect Pes-levels of infection (our unpublished data). talotiopsis, but dilution plating techniques are ineffective. Additional species. Nigrospora spherica (Sacc.) The name Pestalotia De Not. has frequently beenE.W. Mason is similar in culture to N. oryzae, but incorrectly applied to foodborne fungi which shouldproduces larger conidia, mostly 15–18 mm long. have been identified as Pestalotiopsis or, less fre-This species is associated with two diseases of bana- quently, Truncatella. In his comprehensive revision,nas, crown rot (Wallbridge, 1981) and squirter (Hall Steyaert (1949) confined Pestalotia to a single spe-and Scott, 1977; Snowdon, 1990), and Nigrospora cies, not found in foods. Sutton (1980) accepted arot of apples (Snowdon, 1990). It has also been single species in Pestalotiopsis, P. guepinii. However,recorded from wheat, peanuts, pecans, maize and many new species have been described since, oftenbiltong (see Pitt and Hocking, 1997). based on host specificity. Molecular studies on ITS Although less common in Southeast Asian food and 5.8S DNA have shown that host specificity is notcommodities than Nigrospora oryzae, we isolated a sound basis for species delimitation in Pestalotiop-N. spherica from kemiri nuts, maize, peanuts, milled sis (Jeewon et al., 2004).rice, cowpeas and black soybeans in Indonesia (Pittet al., 1998a), and from paddy rice, maize, soybeansand black pepper in the Philippines (our unpublished Pestalotiopsis guepinii (Desm.) Steyeartdata). Fig. 5.44 References. Ellis (1971); Domsch et al. (1980). Colonies on CYA and MEA growing rapidly, covering the whole Petri dish, plane and floccose;5.24 Genus Pestalotiopsis Steyaert mycelium usually white, sometimes off-white to pale brown; reverse pale or in similar colours toPestalotiopsis and the closely related genus Truncatella the mycelium. Colonies on G25N 10–16 mm diam,Steyaert (see below) are characterised by the formation of low, white mycelium. Sometimes germination orof black acervuli containing relatively large fusiform growth at 58C. Usually no growth at 378C.Fig. 5.44 Pestalotiopsis guepinii (a) colonies on CYA, 7 d, 258C; (b) conidia, bar = 10 mm
    • 134 5 Primary Keys and Miscellaneous Fungi Conidia produced in flat, black acervuli, borne just one or more small ostioles (orifices), are producedbeneath the agar surface, opening irregularly at matur- beneath the agar surface, and exude the conidia inity, filled with a dense layer of conidia; conidia fusiform, slime. Several Phoma species have been reportedfive celled (four septate), 20–28 Â 6–9 mm, the central 3 from foods, but until recently the taxonomy of thecells brown, 15–20 mm long, the apical and basal cells genus was uncertain, so identifications to specieshyaline, the basal one with a single usually unbranched were often unreliable. A new monograph has clar-spike-like appendage and the apical one with two or ified the taxonomy of the genus (Boerema et al.,more simple or branched spiky appendages. 2004). This book provides keys and descriptions of Distinctive features. Pestalotiopsis shares with Trun- 223 species and subspecies in 10 sections, with acatella the production in subsurface acervuli of relatively great deal of other information including methodslarge fusiform conidia with appendages. In Pestalotiop- for identifying species in natural habitats. A singlesis, conidia have four septa, in Truncatella, three. species, Phoma sorghina, is described here as repre- Physiology. Optimum sporulation of Pestalotiop- sentative of the genus.sis guepinii was achieved on media containing 3%sucrose. In addition, reduced nitrogen concentration Phoma sorghina (Sacc.) Boerema Fig. 5.45also favoured sporulation (Ebenezer et al., 2002). Mycotoxins. Mycotoxin production has not been Colonies on CYA and MEA variable, usuallyreported. 50–55 mm diam, of dense to floccose grey green or Ecology. There are few records under the name olive mycelium, with characteristic white to salmonPestalotiopsis in the food literature. Most relate to pink tinges; reverse salmon or reddish. Colonies onfruit spoilage: Pestalotiopsis sp. from crown rot of G25N 8–10 mm diam, of sparse brown mycelium.bananas and rots in litchis, canker in guavas, and No growth at 5 or 378C.tarry rots in pomegranates (Snowdon, 1990). Most Pycnidia produced abundantly on MEA, justreports of Pestalotia species, which probably refer beneath the agar surface, 300–400 mm diam, withto Pestalotiopsis, have come from pecans. Other one or more inconspicuous ostioles exuding conidiaPestalotia records include wheat, rice, almonds in a slimy matrix; conidia cylindroidal, 4–5 Â 2–2.5and hazelnuts (see Pitt and Hocking, 1997). mm, hyaline, with thin, smooth walls. We isolated Pestalotiopsis guepinii at low levels Distinctive features. Phoma species producefrom Thai copra, maize, black beans and red beans black subsurface pycnidia which exude small(Pitt et al., 1993, 1994). cylindrical conidia in slime. These features are suffi- Additional genus. Truncatella Steyaert produces cient in the present context; however, see Suttoncolonies similar to those of Pestalotiopsis, and pro- (1980) for a range of other similar genera.duces similar conidiomata. Conidia are fusiform Taxonomy. Taxonomic problems in Phomawith three septa, and measure 15–20 Â 6–8 mm; the have been addressed by Kovics et al. (2005) andtwo median cells are brown and 11–14 mm long; a molecular taxonomy has recently been con-apical and basal cells are hyaline; and the basal cell structed (Aveskamp et al., 2006) for the 223 spe-is without appendages. Appendages from the apical cies and subspecies accepted by Boerema et al.cell are variable in number and branching. (2004). No records of Truncatella in foods were found, Physiology. Sporulation by Phoma species maybut it is probable that some references to Pestalotia be stimulated by growth on DCPA or PDA. Carna-include Truncatella species. tion Leaf Agar, developed for Fusarium species, Reference. Sutton (1980). induces sporulation in most Phoma isolates. Mycotoxins. Phoma sorghina has been reported to produce tenuazonic acid (Shephard et al., 1991)5.25 Genus Phoma Sacc. and P. lingam the first naturally occurring epimo- nothiodiocoperazine, named sirodesmin H (PedrasPhoma is a large and variable genus, characterised et al., 1988). No studies have been reported on theby the production of small, single celled conidia in significance, if any, of production of these toxins bypycnidia. The pycnidia in Phoma are black, have these species.
    • 5.25 Genus Phoma 135Fig. 5.45 Phoma sorghina (a) colonies on CYA and MEA, 7 d, 258C; (b) pycnidium, bar = 10 mm; (c) conidia, bar = 5 mm Ecology. Phoma sorghina is the principal Phoma raw milk, spoiled cheese, sunflower seed, corianderspecies found in grain sorghum. Other Phomas, seed, infant cereal food, biltong and a variety ofusually unidentified to species, have been reported Japanese foods (see Pitt and Hocking, 1997).from cereals, including barley, wheat, maize and Phoma species (unidentified) were common inrice (see Pitt and Hocking, 1997; Do Amaral et al., paddy rice from Thailand (in 56% of samples, up to2005; Perello and Moreno, 2005). Phomas, some- 44% of grains in infected samples and 8% of all grainstimes identified to species, have been reported from examined) (Pitt et al., 1994). Similar levels occurred ina wide variety of fruits and vegetables (Snowdon, sorghum from the Philippines and black soybeans1990, 1991). Important are black rot of papayas due from Indonesia (Pitt et al., 1998a and our unpublishedto P. caricae-papayae (Tarr) Punith., black rot of data). Lower but significant levels (1–2% infectionmelons due to Phoma cucurbitacearum (Fr.) Sacc. overall) were found in sorghum, mung beans and(Snowdon, 1990), crown rot of bananas caused by soybeans from Thailand, paddy rice and soybeansP. sorghina and Phoma rot of sugar beets due to from the Philippines and paddy rice, mung beans,P. betae A.B. Frank. Other records have come from soybeans and talo beans from Indonesia (Pitt et al.,pecans, peanuts, apples, potatoes, fresh vegetables, 1994, 1998a and our unpublished data).
    • 136 5 Primary Keys and Miscellaneous Fungi Although they occur in a variety of foods, Phoma Conidiophores undifferentiated, of variablespecies are most likely to cause spoilage in weather- length, with slightly swollen (up to 7 mm) tipsdamaged cereals. bearing conidia successively, blown out from a References. Boerema et al. (2004). pore as a hyaline cell, then septating both long- itudinally and transversely, becoming thick- walled and detaching with maturity; the smaller5.26 Genus Stemphylium Wallr. conidia more or less spherical, with one to two septa, the larger ones at maturity usually shortLike Alternaria and Ulocladium, Stemphylium pro- cylindrical, with rounded ends and straight orduces conidia with both longitudinal and transverse irregularly swollen sides, with two to four trans-septa. Stemphylium conidia are more or less ellip- verse and one to two longitudinal septa, some-soidal to short cylindroidal, like those of Ulocla- times near spherical or irregular in shape, overalldium. Whereas those of Ulocladium are narrower ranging in size from 13 mm diam up to 30–32 Âat the base than the apex, and can be pointed at 16–20 mm, with brown walls, slightly roughenedone end, to give a pyriform or apiculate shape, those or with short spines, or occasionally with largerof Stemphylium have rounded ends and are usually dark warts.symmetrical longitudinally. For further details con- Distinctive features. As noted above, Stemphy-cerning the differences among these three genera, lium is distinguished by dark conidia with bothsee the section dealing with Alternaria in this chap- transverse and longitudinal septa, with endster, or Simmons (1967). In an extensive molecular rounded, symmetrical from end to end. S. botryo-study of Alternaria, Stemphylium and related genera sum conidia characteristically are slightly con-(Ulocladium, Embellisia and Nimbya), Pryor and stricted in the middle.Gilbertson (2000) and Pryor and Bigelow (2003) Taxonomy. The teleomorphs of this and otherprovided strong molecular evidence that the genus Stemphylium species are in the Ascomycete genusStemphylium is distinct from these other genera. Pleospora (Simmons, 1969, 1985). They are not Species of Stemphylium occur commonly as weak often seen in pure culture in the Petri dish. Metabo-parasites or saprophytes on a variety of plants and lite profiles have been used to distinguish severalplant materials. The taxonomy of the genus has Stemphylium species, including S. botryosumrecently been revised: molecular methods showed (Andersen et al., 1995).good agreement with existing morphological taxon- Physiology. Potato dextrose agar supportedomy (Camara et al., 2002). One species is treated ˆ abundant mycelial growth, whereas conidiogenesishere, S. botryosum (Fig. 5.46). was highest on Sabouraud maltose agar (Susuri and Doga-Gashi, 2003).Stemphylium botryosum Wallr. Fig. 5.46 Mycotoxins. This species has been reported to produce a toxin called stemphol, but mamma-Teleomorph: Pleospora tarda E.G. Simmons lian toxicity was not indicated (Solfrizzo et al., 1994).Colonies on CYA 50–65 mm diam, of low to some- Ecology. This and other Stemphylium specieswhat floccose, rather sparse mycelium, olive to olive have been reported as pathogens on mangoesbrown; reverse olive grey to dark grey. Colonies on (Johnson et al., 1990), squash (Assawah and Zarari,MEA 50–70 mm diam, low to floccose, in the latter 1984), sweet potatoes (Ravichandran and Sullia,case sometimes with areas of white or pale yellow 1983), asparagus and lettuce (Snowdon, 1991),mycelium, elsewhere pale to dark grey; reverse light tomatoes (Dodds et al., 1991; Muniz et al., 2003)to dark grey, sometimes pale yellow or pinkish. Colo- and peppers (Muniz et al., 2003). Stemphylium spe-nies on G25N 6–15 mm diam, pale to dark grey. At cies have also been isolated from malting barley58C, usually germination. At 378C, no growth. (Sepitkova and Jesenska, 1986), lucerne seeds (Sus- Colonies on DCMA 35–45 mm diam, low to uri and Doga-Gashi, 2003) and soybeans (oursomewhat floccose, sparse, grey to dark grey in unpublished data).areas; reverse in similar colours. References. Simmons (1967, 1969, 1985).
    • 5.27 Genus Trichoconiella 137Fig. 5.46 Stemphylium botryosum (a) colonies on CYA and MEA, 7 d, 258C; (b) conidiophores and conidia in situ, bar = 50mm; (c,d) conidia, bar = 10 mm5.27 Genus Trichoconiella B.L. Jain sometimes with pale grey areas; yellow exudate sometimes produced; reverse pale or orange at theThis genus was established to accommodate the margins, centrally very dark brown to bluish black.fungus more commonly known as Alternaria pad- Colonies on MEA 45–55 mm diam, plane, low towickii (Ganguly) M.B. Ellis. Conidia of this species floccose, mycelium pale orange or grey; reverse paleproduce only transverse septa, excluding it from or pinkish at the margins, overall bluish black. NoAlternaria (Jain, 1975). growth on G25N or at 58C. At 378C, colonies up to 5 mm diam formed, or no growth.Trichoconiella padwickii (Ganguly) B.L. Jain Colonies on DCMA 35–55 mm diam, similar in Fig. 5.47 appearance to on MEA, but mycelium mid to dark grey; reverse grey, dark grey or bluish black.Alternaria padwickii (Ganguly) M.B. Ellis Conidia best observed on colonies grown on sterile wheat grains, borne singly from long hyphae,Colonies on CYA 30–45 mm diam, plane, dense to with five to six transverse septa, 40–55 Â 9–11 mmfloccose, mycelium yellowish grey to light orange, between the tip and last septum, with the central
    • 138 5 Primary Keys and Miscellaneous FungiFig. 5.47 Trichoconiella padwickii (a) colonies on CYA and MEA, 7 d, 258C; (b,c) conidia, bar = 10 mmcells larger, giving a narrowly ellipsoidal shape, and Physiology. No reports of physiological studieswith smooth, brown walls; hyphae sometimes are known to us.remaining attached to the conidium and then giving Mycotoxins. Mycotoxins are not known to bethe impression of long appendages. produced. Distinctive features. This genus superficially Ecology. This species (by either generic name)resembles Alternaria, but produces conidia with has rarely if ever been reported from the foodtransverse septa only. Long, whip-like hyphal literature. However, we have found Trichoconiellaappendages are also distinctive. Trichoconiella padwickii, reported as Alternaria padwickii, to bepadwickii appears to be almost exclusively asso- endemic in rice from Southeast Asia. It was pre-ciated with rice. sent in 9 of 18 samples of Thai paddy rice, with up
    • 5.28 Genus Trichoderma 139to 60% of grains in individual samples showing in the literature on Trichoderma species should beinfection, and was found in 12% of all individual treated with caution, as green-spored Trichodermagrains examined (Pitt et al., 1994). The incidence isolates have often been called T. viride regardless ofin paddy rice from Indonesia (71% of samples, the texture of their conidial walls.19% of all particles) and the Philippines (72% of Because most Trichoderma isolates have similarsamples, 23% of all particles) was even higher. morphology, taxonomy proved very difficult untilIndividual rice samples from Indonesia had up the advent of molecular methods. The use ofto 69% of particles infected by T. padwickii, sequences from multiple gene loci has resulted in awhile incidence was up to 90% of grains in sam- large increase in species numbers, from 9 in 1970 toples from the Philippines (Pitt et al., 1998a and now over 80 (Samuels, 2006). Most species haveour unpublished data). been shown to have teleomorphs in Hypocrea, Ellis (1971) reported Trichoconiella padwickii (as though few are found in laboratory cultureA. padwickii) from rice grains from Egypt, India, (Samuels, 2006). The taxonomy of Trichoderma isMalaysia, Nigeria, Pakistan and Sabah, indicating still evolving: see Samuels (2006) for a clear over-that this species is very widespread. Occurrence on view of current understanding.crops other than rice has rarely been reported. The most commonly isolated Trichoderma spe- References. Ellis (1971); Jain (1975); Domsch cies is Trichoderma harzianum Rifai (Bisset, 1991a;et al. (1980). Samuels, 2006). This is the species we have encoun- tered most frequently from foods, and it is probable that most foodborne Trichoderma isolates belong in T. harzianum.5.28 Genus Trichoderma Pers. Care should be exercised in handling Tricho- derma isolates in the laboratory. Conidia areIn this ubiquitous genus, reproduction is by small small, are shed and dispersed readily, and contami-single-celled conidia produced from phialides which nant colonies grow rapidly. Many Trichodermasare arranged in irregular verticils, with the subterm- produce powerful chitinases and cellulases and, ininal phialides borne more or less at right angles to time, can overrun and destroy other culturesthe stipe. Colonies are low and spread rapidly. completely.Mycelial growth is loose textured and characteristi-cally develops irregularly, often appearing in tuftsor isolated patches. Conidia, green in the common Trichoderma harzianum Rifai Fig. 5.48species, sometimes develop only after exposure tolight. Colonies on CYA and MEA generally covering the Trichoderma species have usually been consid- whole Petri dish, often irregular in outline or withered to be soil fungi, but the genus is now also isolated tufts evident, of white to yellow mycelium,known to include plant symbionts and fungal with bright to dull yellow green conidia developingpathogens. Some are also of increasing importance over the whole surface or in patches or tufts; reverseas human pathogens. Interest in the genus has pale or yellowish. Colonies on G25N less than 5 mmincreased sharply in recent years, as some species diam, with growth weak. At 58C, usually no growth,have been proposed as biocontrol agents against occasionally germination. No growth at 378C.plant pathogenic fungi. Conidiophores consisting of highly branched Speciation in Trichoderma has proved to be structures, with a stipe bearing branches and theexceptionally difficult. The most common species branches rebranching, all approximately at rightname in the literature, Trichoderma viride Pers., angles, to form a pyramidal shape, with each branchhas often been incorrectly used (Rifai, 1969) and it bearing phialides irregularly; phialides ampulli-is now considered to be a rare species (Jaklitsch form, commonly 5–7 Â 3.0–3.5 mm, larger whenet al., 2006). Conidia produced by T. viride have borne apically, bearing conidia singly, not in chains;rough walls, whereas the majority of Trichoderma conidia often adhering in small clusters, spheroidal,isolates produce conidia with smooth walls. Reports subspheroidal, or sometimes broadly ellipsoidal,
    • 140 5 Primary Keys and Miscellaneous FungiFig. 5.48 Trichoderma harzianum (a) colonies on MEA, 7 d, 258C; (b) conidiophores, bar = 10 mm; ( c) conidia, bar = 5 mm2.5–3.2(–4.0) mm diam or in length, smooth walled. parts of the world (Domsch et al., 1980). It hasSolitary aleurioconidia also formed by some iso- been reported from rotting tubers of cassavalates, spherical to broadly ellipsoidal, 6–8 mm diam. (Ekundayo and Daniel, 1973) and as a cause of Distinctive features. Trichoderma harzianum pro- spoilage in apples (Penrose et al., 1984). It hasduces conidiophores which are compactly branched been reported from salmon and peas (data sheets,in a pyramidal shape, the base being highly International Mycological Institute, Egham, Sur-branched, and the apex usually bearing a solitary rey). We found T. harzianum, at levels up to 1%phialide. Conidia are smooth-walled, nearly spheri- of all grains or nuts examined, in maize fromcal and less than 3.5 mm long. Thailand and Indonesia and sorghum from Indo- Taxonomy. The teleomorph of Trichoderma har- nesia. It was encountered at low levels in Philip-zianum is Hypocrea lixii. This is not seen in labora- pine maize, Thai peanuts and Indonesian cowpeastory culture (Samuels, 2006). (Pitt et al., 1993, 1994, 1998a). T. viride was not Physiology. Because of the confusion in the encountered at all.literature over the identity of Trichoderma iso- Trichoderma harzianum is no doubt widely dis-lates, it is possible that much of the information tributed in foods, but it has often been reported asreported for T. viride actually is based on studies T. viride.on T. harzianum. Domsch et al. (1980) reported References. Bisset (1984, 1991a, b; Samuels,the optimum growth temperature for T. harzia- 2006).num as approximately 308C, with a maximumnear 368C. Our observations indicate a minimumgrowth temperature at or slightly above 58C. Theminimum aw for growth is 0.91 at 258C (Griffin, 5.29 Genus Trichothecium Link1963). Mycotoxins. Mycotoxin production by Tricho- Trichothecium is a distinctive genus with a singlederma harzianum has not been reported. common species, T. roseum, characterised by Ecology. Trichoderma harzianum is frequently sparse, pinkish colonies and conidia formed in aisolated from cultivated and forest soils in all unique V-formation on long stipes. T. roseum is a
    • 5.29 Genus Trichothecium 141Fig. 5.49 Trichothecium roseum (a) colonies on CYA and MEA, 7 d, 258C; (b) conidiophores and conidia in situ, bar = 25mm; ( c) conidia, bar = 10 mmcommon saprophyte in damp and decaying habitats Conidiophores long, simple hyphae, bearingand is sometimes a weak pathogen of both plants conidia at the tip successively, each formed as aand animals. blown-out cell below the previous one, offset from the hyphal axis and adhering loosely to form char-Trichothecium roseum (Pers.) Link Fig. 5.49 acteristic short, V-shaped chains; conidia approxi- mately ellipsoidal to pyriform, with a single trans-Colonies on CYA and MEA 50–60 mm diam, low verse septum, 16–20 Â 8–12 mm, and with thin,and often sparse, characteristically coloured smooth walls.orange pink near salmon; reverse similarly Distinctive features. See genus preamble.coloured, or less intense, or brownish. Colonies Physiology. Domsch et al. (1980) reportedon G25N barely macroscopic, 1–2 mm diam at growth temperatures for Trichothecium roseum asmost. At 58C, no germination to germination. No minimum 158C, optimum 258C and maximumgrowth at 378C. 358C. Our data on growth at low temperature is at
    • 142 5 Primary Keys and Miscellaneous Fungivariance with this, indicating a minimum growth been maintained by Simmons (2007), moleculartemperature of 58C or near. Growth occurs down analyses have indicated that Ulocladium cannotto 0.90 aw (Snow, 1949). be separated from Alternaria (Pryor and Gilbertson, Mycotoxins. Trichothecium roseum has been ´ 2000; de Hoog and Horre, 2002; Pryor and Bigelow,reported to produce trichothecene toxins, including 2003).trichothecin, trichothecolon and 12,13-epoxy-4- Ulocladium botrytis is treated here as representa-(1-oxobut-2-enyloxy)-trichothec-9-ene (Konishi tive of the genus.et al., 2003). Some of these toxins have been foundin grapes and wheat in measurable quantities, but asspoilage or heavy growth of T. roseum is rare, theyare unlikely to be significant in human health. How- Ulocladium botrytis Preuss Fig. 5.50ever, their presence at quite low levels in grapemusts has been reported to inhibit wine yeasts Colonies on CYA and MEA 60 mm or more diam,(Flesch et al., 1986). plane, dense to somewhat floccose, dark olive to Ecology. As a ubiquitous and readily recognised very dark grey, reverse blue black. On G25N, colo-saprophyte, Trichothecium roseum has been isolated nies 6–10 mm diam, of dense olive to dark greyfrom a variety of foods. Cereals are a common mycelium, reverse blue black. At 58C germinationsource, including barley, wheat, maize, sorghum to formation of microcolonies. At 378C, coloniesand paddy rice (see Pitt and Hocking, 1997). Other 15–20 mm diam, olive grey, reverse grey brown orimportant sources include apples (Valletrisco and blue black.Niola, 1983) and grapes (see Pitt and Hocking, On DCMA, colonies 30–40 mm diam, similar to1997; Serra et al., 2005; Blancard et al., 2006). This those on CYA.species has caused spoilage of a wide variety of Conidia borne singly from nodes or pores onother fruits and vegetables from time to time, but knobby or geniculate dark conidiophores, on CYAis usually only a minor pathogen (Snowdon, 1990, and DCMA variable in size and shape, often1991). It has also been isolated from meat products, roughly spherical, 9–12 mm diam, occasionally sin-cheese, beans, hazelnuts, pecans and pistachios (see gle celled, usually with four or more septa, but alsoPitt and Hocking, 1997). We isolated T. roseum characteristically ellipsoidal, 15–25 Â 9–10 mm,from sorghum, cashews, paddy and milled rice, pea- and with lateral and longitudinal or irregularnuts, maize and soybeans in Southeast Asia, but septa, usually with only a small basal protrusionalways at a low incidence (Pitt et al., 1998a). In but occasionally stalked and resembling Alter-our experience, levels of T. roseum in foods other naria, dark brown, with thick, often very roughthan fruits are usually low, and spoilage walls.exceptional. Distinctive features. As noted above, the genus Reference. Domsch et al. (1980). Ulocladium is distinguished from Alternaria and Stemphylium by conidial shape. Conidia of Ulocla- dium are obovoid, i.e. narrower at the base than at the apex. U. botrytis is the only species at all com-5.30 Genus Ulocladium Preuss mon in foods. Physiology. No physiological studies on Ulocla-Ulocladium, in common with Alternaria and dium species are known to us.Stemphylium, produces conidia with both longitu- Mycotoxins. No mycotoxins are known to bedinal and transverse septa. Those of Ulocladium are produced. None of 52 Ulocladium isolates producednarrower at the base than the apex, and can be altertoxins or tenuazonic acid, toxic metabolitespointed at one end, to give a pyriform or apiculate produced by many Alternaria species (Andersenshape, while those of Stemphylium have rounded and Hollensted, 2008).ends and are usually symmetrical longitudinally. Ecology. Ulocladium species are infrequentlyConidia of Alternaria are definitely club-shaped found in foods. They have been isolated from hazel-(Simmons, 1967). Although this distinction has nuts and walnuts, pistachios, peanuts and barley
    • 5.30 Genus Ulocladium 143Fig. 5.50 Ulocladium botrytis (a) colonies on CYA and MEA, 7 d, 258C; (b) conidiophores and conidia in situ, bar = 50 mm;(c) conidia, bar = 10 mm(see Pitt and Hocking, 1997). U. chartarum can beans, maize, peanuts and soybeans (Pitt et al.,cause rots in melons (Snowdon, 1990). We isolated 1993, 1994, 1998a).Ulocladium species, at low levels, from cashews, red References. Simmons (1967).
    • Chapter 6ZygomycetesZygomycetes (currently the phylum Zygomycota) can be grown in the laboratory, see O’Donnellare relatively primitive fungi characterised by the (1979).production of solitary spores, zygospores, as their The genera of Zygomycetes commonly found inteleomorph. Zygomycetes of significance here are foods are confined to a single order, Mucorales.characterised by hyphae with few if any cross walls Order Mucorales. The order Mucorales is mono-(septa): the hyphae are essentially unobstructed phyletic (having a single evolutionary origin) withtubes. Absence of septa facilitates rapid transloca- all genera closely related genetically (White et al.,tion of nutrients and organelles such as mitochon- 2006). However, classification within the order isdria and nuclei between sites of growth, nutrient less certain, as molecular studies have indicatedadsorption and spore formation. In consequence, poor correlation with traditional morphologicalZygomycetes are also characterised by rapidity of taxonomy (O’Donnell et al., 2001). All of thegrowth. Many species are able to fill a Petri dish families and many of the genera within the orderwith loosely packed mycelium and to produce cannot be sustained using molecular data.mature spores within 2 days of inoculation. Family Mucoraceae. At the same time, O’Don- Zygospores are large (usually greater than 30 mm nell et al. (2001) consider that all of the generadiam), dark walled, distinctive bodies (Fig. 6.1) on included here fit within modern concepts of thewhich Zygomycetes rely for long-term survival. For- family Mucoraceae, which is now being broadenedmation of zygospores by the majority of species to include many of the other previously recognisedencountered in food spoilage requires mating by families (White et al., 2006). As the task of reorder-two strains, so zygospores are not commonly ing the genera within this family remains incom-observed in the pure cultures used for identification. plete, the generic taxonomy provided here is basedA few species such as Rhizopus sexualis do produce on traditional systems. Comments on the taxo-zygospores in pure culture, and this is a valuable nomic status of each genus is included under thattaxonomic aid. Perhaps because they are physically name as appropriate.difficult to separate from other more abundant spore Although some species within this familytypes, little is known about the physiological proper- produce zygospores as a sexual state in culture,ties of zygospores, such as resistance to heat and traditional classification has been based on asexualchemicals. (anamorphic) reproduction. Asexual reproduction Zygomycetes embrace a wide variety of fungi, in the order Mucorales is primarily by sporangios-with diverse habitats. Almost all require high pores, which are typically borne within sporangia.water activities for growth. In damp situations, Sporangia (Fig. 6.2a) are closed sacs, borne ontheir rapid growth habit provides a selective advan- stipes (stalks). Stipes are often termed sporangio-tage over most, more advanced fungi with septate phores, although this name is more appropriatelyhyphae. Many are found on dung or as insect patho- applied to the whole fruiting structure in line withgens. For a guide to genera of Zygomycetes which conventional terminology in other asexual fungi.J.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_6, 145Ó Springer ScienceþBusiness Media, LLC 2009
    • 146 6 Zygomycetes In one genus, Syncephalastrum, sporangiospores are borne in cylindrical merosporangia in a radial array on the columella surface (Fig. 6.2d). Under low magnifications, these structures have some resemblance to the fruiting structures of Aspergillus. In another, Thamnidium, sporangiospores are borne both in normal sporangia and in small sporangioles, formed in clusters (Fig. 6.2e), often on the same stipe as a sporangium. In Cunninghamella, sporan-Fig. 6.1 Zygospore of Rhizopus sexualis, bar ¼ 50 mm gioles are also borne in clusters, on spicules, but never produce sporangiospores. Here the sporan-Stipes may be borne singly or in clusters from fertile giole itself acts as the reproductive unit.hyphae, may be branched or unbranched or may Some species in the Mucoraceae also producegrow out from beneath a terminal sporangium to chlamydoconidia as a second type of anamorph.produce a succession of sporangia (sympodial These are cylindrical to spherical cells with rela-branching, Fig. 6.2b). tively thick walls formed in hyphae and stipes, Sporangia appear under the low-power micro- sometimes in great numbers (Fig. 6.2f). They prob-scope as small (less than l mm diam) brown to ably function as a resting stage, more resistant toblack spheres distributed on long stipes throughout light, heat and desiccation than sporangiospores,the aerial mycelium. Sporangial walls disintegrate in and are probably analogous to similar structuresage, releasing the sporangiospores. formed by Hyphomycetes. In microscopic mounts from 7 day old cultures, Identifying genera in Mucoraceae. One theme ofintact sporangia are rare: the structures mostly seen this book is to provide a standardised system forat stipe apices are columellae (Fig. 6.2c), formed within identification of food spoilage fungi. Where possi-the sporangia and remaining intact in age. The man- ble, identification is achieved after 7 days incuba-ner in which columellae collapse after sporangial dis- tion, by a single macroscopic and microscopicintegration provides useful taxonomic information. inspection of a standard set of Petri dishes. TheFig. 6.2 Anamorphic reproductive structures in genera of Mucorales: (a) sporangium; (b) sympodial branching;(c) columellae; (d) merosporangium; (e) sporangioles; (f) chlamydoconidia
    • 6 Zygomycetes 147keys to Mucoraceae hereunder are accordingly secretion of mucous material. Rhizoids arebased on this system. short root-like structures produced by Rhizopus To the experienced mycologist it will be obvious at the base of each sporangiophore (Fig. 6.3c).that because they grow so rapidly, isolates of Mucor species do not produce rhizoids, whereasMucoraceae can be identified much earlier than Absidia and Rhizomucor species produce them7 days. However, a second theme of this book is irregularly.the facilitation of fungal identifications by the bac- As the name implies, Mucor produces sporan-teriologist who does not instinctively recognise a giospores in a layer of mucous which causes themMucor from a Monascus, so the standard 7 day to adhere to the colony when disturbed or pickedschedule has been maintained here. There is an with a needle. Absidia and Rhizomucor are similar,unexpected bonus: the characteristic shapes of colu- but Rhizopus produces dry spores.mellae collapsing in age simplifies recognition of If a Mucor culture fills the Petri dish the lid can besome genera (Fig. 6.3). removed with minimal disturbance to the culture; Significant genera. Seven genera in the family however, the rhizoids of Rhizopus adhere to the lidMucoraceae are treated here: Absidia Tiegh., and part of the culture will detach with it. Beware!Cunninghamella Matr., Mucor P. Micheli: Fr., Rhizo- Carry out this operation away from your inoculat-mucor (Lucet and Costantin) Vuill., Rhizopus ing area. Dry spores, which are aerially dispersedEhrenb., Syncephalastrum Schroter and Thamnidium ¨ when plates are opened, and very rapid growthLink. These genera are differentiated primarily by the make Rhizopus species a serious source of labora-morphology of their sporangiophores and sporangia. tory contamination. Rhizoids, mucous and contamination. Other use- The seven genera of Mucoraceae considered hereful features for distinguishing some of these are keyed below and then treated in alphabeticalgenera are the production of rhizoids and the order.Fig. 6.3 Collapse of columellae in age: (a) little collapse (Mucor, Rhizomucor); (b) funnel shapes (Absidia); (c) umbrella shapes(Rhizopus)
    • 148 6 Zygomycetes Key to genera of the family Mucoraceae included here1 All reproductive cells borne on spicules around vesicles 2 Some or all reproductive cells sporangiospores, borne within sporangia (Fig. 6.2a, b, c) 32 (1) Reproductive cells sporangiospores, borne in cylindrical sacs (merosporangia; Fig. 6.2d) (at low magnification reminiscent of Aspergillus) Syncephalastrum Reproductive cells single-celled spherical sporangioles Cunninghamella3 (1) As well as sporangia, clusters of smaller sacs (sporangioles, Fig. 6.2e) present Thamnidium Only sporangia, or columellae derived from sporangia, present 44 (3) Columellae retaining approximately spherical shape after sporangiospore discharge (Fig. 6.3a), sporangiospore walls smooth or spiny 5 Columellae collapsing to form funnel or umbrella shapes, sporangiospore walls smooth or striate 65 (4) Sporangiospores rarely exceeding 5 mm in long axis Rhizomucor Sporangiospores commonly exceeding 5 mm in long axis Mucor6 (4) Columellae collapsing inwardly from the apex to form a funnel shape (Fig. 6.3b), sporangiospore walls smooth Absidia Columellae collapsing outwardly to form an umbrella shape (Fig. 6.3c), sporangiospore walls striate Rhizopus6.1 Genus Absidia Tiegh. columella is abrupt, and the columella is wholly within the sporangial wall. In age, columellae ofThe genus Absidia produces a distinctive type of Absidia frequently collapse inward from the apexcolumella which widens gradually at the junction to form funnel-shaped structures.with the stipe, outside the circumference of the Absidia species form rhizoids, irregular root-likesporangium (Fig. 6.4b). In other Zygomycete gen- outgrowths at the bases of the stipes, but these areera considered here, the junction of stipe and less conspicuous and less regular than in RhizopusFig. 6.4 Absidia corymbifera (a) colonies on CYA, 7 days, 258C; (b) sporangia and columellae, bar ¼ 25 mm; (c) sporangios-pores, bar ¼ 10 mm
    • 6.2 Genus Cunninghamella 149species. Only one Absidia species, Absidia corymbi- recorded as a pathogen of peaches (Singh and Pra-fera, is at all common in foods. Several recent shar, 1988). A. corymbifera is probably widespreadauthors have concluded that Absidia is polyphyletic in the tropics: Oyeniran (1980) recorded isolations(evolutionarily diverse) based on molecular studies from cocoa, palm kernels and maize. We have(e.g. Voigt et al., 1999; O’Donnell et al., 2001; found it, always at low levels, in sorghum andKwasna et al., 2006) and that A. corymbifera is not mung beans from Thailand (Pitt et al., 1993,closely related to most other species in the genus and 1994); in peanuts, kemiri nuts, milled rice and cor-would be better classified in Rhizomucor. However, iander from Indonesia (Pitt et al., 1998a); and pea-this name remains in common use. nuts, paddy, milled rice, soybeans and black pepper from the Philippines (our unpublished data).Absidia corymbifera (Cohn) Sacc. & References. Ellis and Hesseltine (1966); NottebrockTrotter Fig. 6.4 et al. (1974); de Hoog et al. (2000).Absidia ramosa J.J. Ellis & Hesselt.On CYA, colonies covering the Petri dish, low and 6.2 Genus Cunninghamella Matr.sparse, white to pale brown or grey; reverse colour-less. On MEA, colonies filling the whole Petri dishwith deep floccose mycelium, coloured mid-grey This genus is unusual in that sporangiophores giveby sporangia; reverse pale. On G25N, colonies rise to sporangioles (small sporangia) which do not10–30 mm diam, sparse and floccose, coloured as differentiate into sporangiospores but themselveson MEA. No growth at 58C. At 378C, colonies cov- act as the conidial stage. The sporangioles areering the whole Petri dish, similar to those at 258C. borne on spicules (spikes) from vesicles; the vesicles Sporangiophores borne from aerial hyphae, stipes are borne terminally or irregularly on the sporan-sometimes irregularly branched; sporangia hyaline, giophores. A recent monograph by Zheng and Chen15–50 mm diam, appearing pyriform due to external (2001) lists 15 species, and a molecular study of 12 ofconical columellae; columellae pyriform 10–30 mm these showed good agreement with traditional spe-diam, sometimes with small projections on the apices ciation (Liu et al., 2001). Zheng and Chen (2001)or with collarettes above the base, in age often col- report that a great deal of literature confusion existslapsing inward from the apex to form funnel-shaped over the names of species, in particular with thestructures; sporangiospores hyaline, broadly ellipsoi- common species Cunninghamella bertholletiae anddal to spheroidal, 3–6 mm long, smooth walled. C. elegans. These two species are distinguishable by Distinctive features. See genus description. small morphological differences and by the fact that Physiology. Evans (1971) recorded growth tem- C. bertholletiae grows to 428C or more, while theperatures for Absidia corymbifera as minimum 148C, maximum growth temperature for C. elegans ismaximum 508C and optimum near 408C. This spe- usually 358C. Although the name C. elegans iscies is able to germinate and grow down to 0.88 aw most commonly seen in the food literature, our(Hocking and Miscamble, 1995). Growth in nitrogen experience indicates that C. bertholletiae is the(<1% O2) was similar to that in air (Hocking, 1990). most common species, along with C. echinulata. Mycotoxins. Mycotoxin production has not been C. bertholletiae is described below: C. echinulatareported. differs by the production of sporangioles with Ecology. Absidia corymbifera is a weak human conspicuously spiny walls (Zheng and Chen, 2001).and animal pathogen, with a very wide host rangeand capable of infecting many body organs (de Cunninghamella bertholletiae Stadel Fig. 6.5Hoog et al., 2000). From foods, most isolationshave been from wheat, barley, malted barley and Colonies on CYA and MEA covering or filling thecereal products such as flour and bran. It has also whole Petri dish, sparse, mycelium off-whitebeen found in meat products and biltong, cassava, to beige, reverse colourless to pale yellow. Onhazelnuts and sunflower seeds and, as A. ramosa, in G25N, ranging from no growth to colonies 15 mmpecans (see Pitt and Hocking, 1997). It has been diam, of sparse, translucent mycelium. At 58C, no
    • 150 6 ZygomycetesFig. 6.5 Cunninghamella bertholletiae (a) colonies on CYA and MEA, 7 days, 258C; (b) developing sporangioles in situ,bar ¼ 50 mm; (c, d) vesicles and mature sporangioles, bar ¼ 10 mmgermination. At 378C, colonies similar to those on mating types from the same species are grownCYA at 258C, mycelium off-white to grey, reverse together.pale yellow to brown. Physiology. Cunninghamella bertholletiae grows Sporangiophores long, bearing solitary vesicles up to 42–458C. Growth in soil is confined to above –terminally and in irregular verticils subterminally or 90 bars (ca. 0.93 aw) (Kouyeas, 1964).along the length; sporangioles borne from spicules, Mycotoxins. Mycotoxins are not produced.breaking at maturity, spherical to ellipsoidal, 7–11 mm Ecology. In common with other Cunninghamelladiam or 9–13 mm long, with smooth to finely species, C. bertholletiae mostly occurs in tropicalroughened walls. and subtropical soils (Zheng and Chen, 2001). Zygospores not commonly seen in pure culture, C. echinulata was a cause of rotting in kola nutsfide Zheng and Chen (2001) irregularly ellipsoidal to (Adebajo, 1994). We have seen C. bertholletiae atspherical, brownish, 20–45 mm diam, with rough low levels in maize from the Philippines andwalls. peanuts from Indonesia (Pitt et al., 1998a and Distinctive features. See genus description. unpublished). Taxonomy. Most Cunninghamella species are References. O’Donnell (1979); Domsch et al.heterothallic, producing zygospores only when two (1980).
    • 6.3 Genus Mucor 1516.3 Genus Mucor P. Micheli: Fr. which are smooth or spiny, but not striate. This is in contrast to Rhizopus species, which commonly pro-As traditionally described, Mucor is a very common duce sporangiospores with striate walls.and widespread genus in nature, occurring in soils, Some Mucor species are able to grow and weaklydecaying vegetation, dung and many other moist habi- ferment under anaerobic conditions and occasionallytats where rapidly growing fungi have an advantage. cause spoilage of beverages in this manner. Growth isRecent molecular studies (Voigt et al., 1999; O’Don- yeast-like in appearance (Fig. 6.6), although individualnell et al., 2001; Kwasna et al., 2006) have indicated cells are much too large to be mistaken for a true yeast.that Mucor includes at least two phylogenetically dis- Inoculation of such cells onto aerobic media producestinct groups. However, the species included here, inso- normal growth. Yeast-like growth has also beenfar as they have been examined, appear to fall into the reported to occur when Mucor and some related generamajor Mucor clade, so the traditional description is grow in the presence of high sodium chloride concentra-used here. Unlike those of Absidia, sporangia of tions (Tresner and Hayes, 1971). At least 20 species ofMucor have columellae borne wholly within the spor- Mucor have been reported from foods. Five appear toangial wall; the columellae collapse irregularly, if at all, be most significant: Mucor circinelloides, M. hiemalis,in age (Fig. 6.3a). Unlike Rhizopus species, Mucors do M. piriformis, M. plumbeus and M. racemosus. Thesenot produce rhizoids. In species of interest here, spor- species are keyed and described below.angiospores are longer than 5 mm and have walls General reference. Schipper (1978a). Key to Mucor species included here1 Strong growth at 378C M. circinelloides No growth at 378C 22 (1) Columellae frequently with small irregular projections on the apices; sporangiospores with minute spines M. plumbeus Columellae without apical projections; sporangiospores smooth walled 33 (2) Columellae 50–100 mm diam; growth on G25N weak or absent M. piriformis Columellae usually less than 50 mm diam; colonies on G25N greater than 10 mm diam 44 (3) Chlamydoconidia abundant, often dominating microscopic appearance M. racemosus Chlamydoconidia present in low numbers or absent M. hiemalisMucor circinelloides Tiegh. Fig. 6.7 relatively low and sparse, appearing pale grey or yellowish; reverse uncoloured. On MEA, coloniesOn CYA, colonies 60 mm diam or more, often filling the whole Petri dish, in colours similar tospreading across the whole Petri dish, but growth those on CYA. On G25N, colonies 15–25 mm diam, low and relatively dense, golden yellow in both obverse and reverse. At 58C, colonies 4–10 mm diam, low and sparse. At 378C, colonies 20–40 mm diam, sparse and floccose, with colours more brown than at 258C. Sporangiophores borne from aerial hyphae, stipes commonly branched, often sympodially, sporangia spherical, 25–50 mm diam, sometimes up to 80 mm; columellae roughly spherical, up to 50 mm diam; sporangiospores hyaline, ellipsoidal, mostly 4.5–7 mm long, smooth walled. Chlamydoconidia uncom- mon, spherical, cylindrical or rather irregular, up toFig. 6.6 M. plumbeus showing yeast-like growth, in liquidculture under anaerobic conditions, bar ¼ 25 mm 15 mm diam. Zygospores not formed in pure culture.
    • 152 6 ZygomycetesFig. 6.7 Mucor circinelloides (a) colonies on CYA, 7 days, 258C; (b) columellae; (c) sporangia; (d) chlamydoconidia;(e) sporangiospores; (f) sporangium; (g) sporangiospores, all bars ¼ 10 mm Distinctive features. Mucor circinelloides has numer- Ecology. This species has been reported fre-ous characters in common with M. hiemalis. Unlike quently as an animal pathogen and occasionally asM. hiemalis, M. circinelloides grows well at 378C a human pathogen (de Hoog et al., 2000). It can(20 mm or more in 7 days), grows weakly at 58C (less cause spoilage in cheese and yams (see Pitt andthan 10 mm vs. greater than 20 mm in 7 days) and Hocking, 1997) and can be pathogenic on mangoescommonly produces sympodially branched stipes. (Johnson et al., 1990) and peaches (Restuccia et al., Taxonomy. DNA sequence data from the ITS1 2006). It has been isolated from meat, hazelnuts andand 2 regions indicate that Mucor circinelloides is walnuts and also from maize, mung beans, soybeanscorrectly placed in the genus Mucor (Kwasna et al., and barley (Pitt et al., 1993, 1994, 1998a; see Pitt and2006). Hocking, 1997). Physiology. Tresner and Hayes (1971) reported References. Schipper (1976); Domsch et al.that Mucor circinelloides grew in media containing (1980).15% (w/v) NaCl (=0.90 aw) but not 20% (=0.86aw). On a medium with glucose as controlling Mucor hiemalis Wehmer Fig. 6.8solute, this species germinated and grew down to0.90 aw (Hocking and Miscamble, 1995). On CYA, colonies spreading across, and sometimes Mycotoxins. This species is not known to pro- filling, the whole Petri dish, growth relativelyduce mycotoxins.
    • 6.3 Genus Mucor 153Fig. 6.8 Mucor hiemalis (a) colonies on CYA, 7 days, 258C; (b) sporangia and columellae, bar ¼ 25 mm; (c) sporangiospores,bar ¼ 10 mmsparse, greyish; reverse pale. On MEA, colonies Ecology. Mucor hiemalis has been reported as afilling the whole Petri dish, relatively dense, greyish rare cause of cutaneous mycosis (de Hoog et al.,to distinctly yellow; reverse yellow to golden yellow. 2000). This species causes rots in guavas (Ito et al.,On G25N, colonies 10–15 mm diam, moderately 1979), carrots and cassava (Snowdon, 1991). It hasdense, bright yellow in both obverse and reverse. been reported from spoilage of yoghurt due toAt 58C, colonies 20–30 mm diam, low and sparse. inward collapse of containers (Foschino et al.,No growth at 378C. 1993) and cheese (Hayaloglu and Kirbag, 2007). It Sporangiophores borne aerially, stipes generally has also been reported from fresh vegetablesunbranched, less commonly sympodially branched; (Lugauskas et al., 2005), from chestnuts (Jerminisporangia up to 60 mm diam; columellae ellipsoidal, et al., 2006), hazelnuts and soybeans (see Pitt and15–30 mm diam; sporangiospores hyaline, narrowly Hocking, 1997), from wheat based fast foods into broadly ellipsoidal or reniform (kidney shaped), Nigeria (Fapohunda and Ogundero, 1990), from5–11 mm long, smooth walled. Chlamydoconidia preprepared airline food in Egypt (Saudi and Man-uncommon, spherical to cylindrical or irregular, sour, 1990) and from chocolate confectionery inup to 15 mm diam. Zygospores not formed in pure Italy (Dragoni et al., 1989). We have isolated it atculture. low levels from Thai and Indonesian maize and Distinctive features. Mucor hiemalis is similar to paddy rice (Pitt et al., 1993, 1998a).M. circinelloides, but grows more rapidly at 58C and Reference. Schipper (1973).does not grow at 378C. Moreover, it produces largersporangiospores which are sometimes reniform andusually has unbranched stipes. Mucor piriformis A. Fisch. Fig. 6.9 Taxonomy. DNA sequence data from the ITS1and 2 regions indicate that Mucor hiemalis is correctly On CYA, colonies low and sparse, spreading acrossplaced in the genus Mucor (Kwasna et al., 2006). the Petri dish or discrete; mycelium colourless, over- Physiology. Growth of some Mucor hiemalis iso- all colour buff from sporangia; reverse pale. Onlates occurs at temperatures below 08C (Joffe, 1962). MEA, colonies 35–60 mm diam, coloured grey or Mycotoxins. Mycotoxin production has not been brownish; reverse pale. On G25N, colonies less thanreported. 7 mm diam, or growth absent. At 58C, colonies
    • 154 6 ZygomycetesFig. 6.9 Mucor piriformis (a) colonies on MEA, 7 days, 258C; (b) sporangia, bar ¼ 25 mm; (c) columellae, bar ¼ 25 mm;(d) sporangiospores, bar ¼ 10 mm; (e) developing sporangium, bar ¼ 25 mm; (f) sporangiospores, bar ¼ 10 mm30–45 mm diam, low and sparse, or with some aerial 258C is often contorted and highly branched. Spor-hyphae. No growth at 378C. angia, columellae and many sporangiospores are Sporangiophores on CYA borne from surface larger than those of other common Mucor species.hyphae, stipes short, broad, often sympodially Physiology. Mucor piriformis is a psychrophile.branched and with encrusted walls; sporangia up Cardinal temperatures are minimum, near 08C,to 150 mm diam, with spinulose walls; columellae optimum 20–218C, maximum 268C (Michailidesspheroidal to short cylindroidal, 25–80(–100) mm and Spotts, 1990). Mycelium and sporangiosporesdiam or in length, the larger ones collapsing irregu- were inactivated by heating to 43–46 and 52–558C,larly; sporangiospores hyaline, spherical to broadly respectively (Michailides and Ogawa, 1989).ellipsoidal, 6–12(–20) mm diam or in long axis, Mycotoxins. Mycotoxin production has not beensmooth walled. Chlamydoconidia uncommon; reported.zygospores not formed in pure culture. Ecology. Mucor piriformis is a destructive patho- Distinctive features. Mucor piriformis grows gen of fresh strawberries (Snowdon, 1990; Pitt andrapidly on CYA at 58C, but relatively poorly Hocking, 1997) and causes rotting of cold storedunder the standard 258C incubation conditions. pears, apples and tomatoes (see Pitt and Hocking,This is especially noticeable on G25N, where 1997), plums (Borve and Vangdal, 2007) and yamsgrowth is weak or absent. Mycelium on CYA at (Amusa and Baiyewa, 1999; Iwata, 2006). A variety of
    • 6.3 Genus Mucor 155treatments for control of pear spoilage have been Mucor plumbeus Bonord. Fig. 6.10proposed: dips in hot water (Michailides and Mucor spinosus Tiegh.Ogawa, 1989), a hot water pressure process (Spottset al., 2006), salt and surfactant solutions (Spotts On CYA and MEA, colonies at least 50 mm diam,and Cervantes, 1989), prestorage drench in thia- low to deep, often spreading across the Petri dish;bendazole (Lennox et al., 2004) or treatment of mycelium colourless, overall colour pale to deepwash water with chlorine dioxide or peracetic grey from sporangia; reverse colourless. Coloniesacid (Roberts and Reymond, 1994; Mari et al., on G25N 20–35 mm diam, low, moderately dense,2003). This species has recently been reported as a white to pale yellow brown; reverse pale. At 58C,dominant species in the mycoflora of cassava and colonies 8–15 mm diam, low and sparse. No growthyam chips (Gnonlonfin et al., 2008). at 378C. References. Schipper (1975); Michailides and Sporangiophores borne from surface or aerialSpotts (1990). hyphae, stipes unbranched or branched sympodially,Fig. 6.10 Mucor plumbeus (a) colonies on CYA, 7 days, 258C; (b) sporangia, bar ¼ 25 mm; (c) columellae, bar ¼ 25 mm;(d) chlamydoconidia, bar ¼ 25 mm; (e) sporangiospores, bar ¼ 10 mm; (f) sporangia, bar ¼ 25 mm; (g) sporangiospores,bar ¼ 10 mm
    • 156 6 Zygomycetessporangia dark greyish brown, up to 80 mm diam, and has been observed to cause anaerobic spoilage ofwith spiny walls; columellae pyriform to ellipsoidal apple juice in our laboratory. Meat (Gros et al., 2003;or short cylindroidal, up to 50 Â 30 mm, often with Pitt and Hocking, 1997), nuts and cereals are otherirregular projections at the apices; sporangiospores commodities from which this species, and its synonymbrown, spheroidal, commonly 7–8(–12) mm diam, M. spinosus, have been reported (see Pitt and Hock-with walls rough or minutely spiny. Chlamydoconidia ing, 1997). It was isolated at low levels from blackuncommon; zygospores not formed in pure culture. rice in Thailand, soybeans in the Philippines and Distinctive features. Mucor plumbeus is distin- coriander in Indonesia (Pitt et al., 1994, 1998a).guished by its grey colour, columellae with irregular Reference. Schipper (1976).apical projections and brown sporangiospores withrough or spiny walls. Mucor racemosus Fresen. Fig. 6.11 Taxonomy. DNA sequence data from the ITS1 and2 regions indicate that Mucor plumbeus is correctly On CYA and MEA, colonies spreading across theplaced in the genus Mucor (Kwasna et al., 2006). Petri dish, low to moderately deep; mycelium col- Physiology. Panasenko (1967) reported growth ourless, overall colour light to mid brown fromof Mucor plumbeus from 4 or 5 to 358C, with an sporangia and chlamydoconidia; reverse lightoptimum of 20–258C. The minimum aw for growth brown. On G25N, colonies 25–40 mm diam, low,was reported to be 0.93 by Snow (1949). Growth in moderately dense, similar in colour to those onN2 (<1% O2) was 80% of that in air; some growth CYA. At 58C, colonies 12–25 mm diam, low andoccurred in an atmosphere of !97% CO2, with only sparse. No growth at 378C.trace amounts of O2 (Hocking, 1990). As measured by Sporangiophores borne from surface or aerialcolony diameters, growth on cheddar cheese in an mycelium, stipes branched sympodially or irregularly,atmosphere of 20% CO2 and 5% O2 was 50% of that sporangia up to 80 mm diam, light brown, within air, and growth in 20% CO2 and 1% O2, 40% CO2 encrusted walls; columellae ellipsoidal to pyriform,and 5% O2 and 40% CO2 and 1% O2 was 40, 50 and up to 40 mm long; sporangiospores hyaline to pale30% of that in air, respectively (Taniwaki et al., 2001a). brown, broadly ellipsoidal to subspheroidal, com- Mycotoxins. Mycotoxins are not known to be monly 5–8 mm diam, smooth walled. Chlamydoconi-produced. dia and arthroconidia formed abundantly, 5–20 mm or Ecology. Mucor plumbeus has been reported to more in diam or long axis. Zygospores not formed inspoil cheese (Northolt et al., 1980; Devoyod, 1988) pure culture.Fig. 6.11 Mucor racemosus (a) colonies on CYA, 7 days, 258C; (b) columellae, bar ¼ 25 mm; (c) chlamydoconidia, bar ¼ 25mm; (d) sporangiospores, bar ¼ 10 mm
    • 6.4 Genus Rhizomucor 157 Distinctive features. Mucor racemosus is similar 6.4 Genus Rhizomucor (Lucet andin many respects to M. plumbeus. M. racemosus Costantin) Vuill.differs by faster growth on G25N, brown not greycolony colouration, the much greater abundance of Schipper (1978b) revived the genus Rhizomucor for threechlamydoconidia, smooth sporangiospores and the species previously accepted in Mucor, but distinguishedabsence of irregular projections on columellae. by the production of stolons and by their thermophilic Taxonomy. DNA sequence data from the ITS1 nature. Molecular studies (Voigt et al., 1999) supportand 2 regions indicate that Mucor racemosus is cor- this concept. Rhizomucor species are significant in foodsrectly placed in the genus Mucor (Kwasna et al., 2006). mainly in tropical regions. The most common species in Physiology. Mucor racemosus grows between –3 foods is Rhizomucor pusillus; R. miehei is also mentioned.or –48C and 30–358C, with an optimum of 20–258C(Panasenko, 1967). The minimum aw for growth is Rhizomucor pusillus (Lindt) Schipper Fig. 6.120.92 (Panasenko, 1967). Mucor pusillus Lindt Mycotoxins. No mycotoxins are produced. Ecology. Mucor racemosus is responsible for a On CYA, colonies sometimes 25–35 mm diam, ofspongy soft rot of cool stored sweet potatoes, pota- colourless to white floccose mycelium surmountedtoes and citrus (Chupp and Sherf, 1960). It has been by brown sporangia, or sometimes covering thereported, along with M. hiemalis, from spoilage of whole Petri dish and then similar to that on MEA.yoghurt due to inward collapse of containers On MEA, colonies covering the whole Petri dish,(Foschino et al., 1993) and as a cause of spoilage of low and relatively sparse, pale to mid-grey; reversecheese (Devoyod, 1988; and in our laboratory) and pale, yellowish or greenish grey. On G25N, growthcheesecake (Piskorska-Pliszczynska and Borkowska- sparse, not exceeding 5 mm diam, or absent. NoOpacka, 1984). It has also been found contaminating growth at 58C. At 378C, colonies similar to thosecheese (Kivanc, 1992; Lund et al., 1995; Cantoni et al., ¸ at 258C on MEA, but more dense, brown to dark2003), salami (Cantoni et al., 2007), raisins (Youssef grey.et al., 2000), frozen and processed meats, salted horse Sporangiophores borne from surface hyphae,meat, fermenting cacao beans, maize, barley, soy- stipes sometimes appearing unbranched, but usuallybeans and paddy rice (see Pitt and Hocking, 1997). extensively and irregularly branched; poorly formed Reference. Schipper (1976). rhizoids sometimes apparent but not adjacent to theFig. 6.12 Rhizomucor pusillus (a) colonies on CYA, 7 days, 258C; (b) columellae, bar ¼ 25 mm; (c) sporangia, bar ¼ 25 mm;(d) sporangiospores, bar ¼ 10 mm
    • 158 6 Zygomycetesstipe bases, a distinction from Rhizopus; sporangia to 50(–60) mm diam with spiny walls, with columel-spherical, brown or grey, 40–60(–80) mm diam; colu- lae rarely larger than 30 mm diam.mellae spherical, ellipsoidal or pyriform, 20–45 mm Reference. Schipper (1978b).diam, sometimes collapsing irregularly (similar toMucor species); sporangiospores hyaline, sphericalto broadly ellipsoidal, 3–4 mm diam, smooth walled. 6.5 Genus Rhizopus Ehrenb.Zygospores produced by occasional isolates, black,broadly ellipsoidal, 60–70 mm diam. The genus Rhizopus needs little introduction to food Distinctive features. Rhizomucor species are most microbiologists, because R. stolonifer is among thereadily distinguished by rapid growth at 378C and most obvious moulds encountered on Petri dishesby their sporangiospores, which are small (less than inoculated with food materials. Coarse, rampant5 mm diam) and smooth walled. growth and rapidly maturing spores make this spe- Physiology. Rhizomucor pusillus is a thermophile. cies a source of endless contamination to the unw-Growth has been reported at temperatures as high ary laboratory worker. Other Rhizopus species areas 608C (Crisan, 1973). Optimum growth conditions less common in foods and cause less nuisance.are 37–428C, with a lower limit of 208C (Panasenko, Rhizopus species, especially R. oligosporus, have1967; Evans, 1971). been used for millennia to modify basic foods in the Mycotoxins. Mycotoxin production is unknown. Orient. The subject of fermented foods is outside the Ecology. Rhizomucor pusillus is a frequent agent of scope of this book: for further information see, forbovine mycotic abortion and a rare cause of human example, Hesseltine (1965), Gray (1970), Beuchatzygomycosis (de Hoog et al., 2000). This species (1987) or Rombouts and Nout (1995).appears to be of widespread though uncommon Rhizopus is distinguished from other genera inoccurrence in foods. Reported sources include cereals the order Mucorales by the formation of rhizoids(Lugauskas et al., 2006), kola nuts (Adebajo and (short root-like appendages), which are conspicu-Popoola, 2003), various spices (Mandeel, 2005), olives ous at the base of the sporangiophores; by columel-(Roussos et al., 2006), meat products, pecans, hazel- lae which often collapse into umbrella shapes in age;nuts and walnuts, sunflower seeds and various tropi- and by dry sporangiospores with striate walls. Thecal products (see Pitt and Hocking, 1997). It is used in most recent complete taxonomic treatment is bycheese manufacture in Japan (Koizumi, 2001). We Zheng et al. (2007a) who accepted 10 species. Mole-isolated R. pusillus from mung beans in Thailand cular phylogenetic analyses have recently been car-and kemiri nuts in Indonesia (Pitt et al., 1994, 1998a). ried out by Abe et al. (2006) and Liou et al. (2007), Additional species. Rhizomucor miehei (Cooney with good agreement with the morphological con-and R. Emers.) Schipper (synonym M. miehei Coo- clusions by Zheng et al. (2007a). Five species areney and R. Emers.) has occasionally been isolated considered here and keyed below: R. microsporus,from foods (Kuthubutheen, 1979; Ogundero, 1981). R. oligosporus, R. oryzae, R. sexualis and R. stolonifer.It is similar in most characters to R. pusillus, includ- Because of its ubiquitous nature, R. stolonifer ising thermophily. R. miehei produces sporangia up described first. Key to Rhizopus species included here1 Abundant zygospores present on CYA and MEA R. sexualis Zygospores not produced 22 (1) Rapid growth at 378C; spores commonly less than 8 mm diam 3 Growth at 378C weak or absent; spores commonly 8–20 mm diam R. stolonifer3 (2) Columellae up to 100 mm diam; spores 5–8 mm long R. oryzae Columellae not more than 80 mm diam; spores less than 5 mm long 44 (3) Columellae up to 75 mm diam; spores less than 4 mm long, with spinose walls; source food fermentations and fermented foods R. oligosporus Columellae less than 40 mm diam; spores 3–5 mm long, with finely striate walls; source other types of foods R. microsporus
    • 6.5 Genus Rhizopus 159Rhizopus stolonifer (Ehrenb.: Fr.) with maturity. Sporangiospores are large, with stri-Lindner Fig. 6.13 ate walls. In contrast to that at 258C, growth at 5Rhizopus nigricans Ehrenb. and 378C is weak or absent. Taxonomy. Twenty nine isolates identified asOn CYA, colonies covering the whole Petri dish, Rhizopus stolonifer or possibly related species were phy-sometimes low and sparse, with black sporangia ´ ¨ logenetically analysed by RAPD analysis (Vagvolgyionly at the margins, sometimes filling the whole et al., 2004) and ca. 30 different isolates by Liou et al.Petri dish and then similar to those on MEA; reverse (2007) using DNA sequences from the D1/D2 ofpale. On MEA, colonies filling the whole Petri dish rDNA. These analyses divided R. stolonifer and somewith floccose white mycelium bearing conspicuous varieties into two or four clades. In each case, the iso-sporangia, at first white, then with maturation lates commonly seen in foods remained in R. stolonifer.rapidly becoming black, either distributed uniformly Physiology. Rhizopus stolonifer has beenor concentrated at dish peripheries; reverse unco- reported to grow from 4.5 or 58C up to 308C (Schip-loured. On G25N, colonies similar to that on MEA per, 1984) or 35–378C (Pierson, 1966, and ourbut less dense. At 58C, spores barely germinating. At observations), with an optimum near 258C. How-378C, usually no growth, sometimes colonies up to ever, Zheng et al. (2007a), who tested 36 isolates of15 mm diam, very thin and sparse. R. stolonifer, reported maximum growth tempera- Sporangiophores borne in groups of three to tures of only 30–328C. This species germinatedfive from clusters of rhizoids, stipes unbranched, down to 0.84 aw at 258C, but growth was veryrobust and up to 3 mm long, with brown walls; slow, and was absent at 308C (Hocking and Mis-sporangia 100–350 mm diam, usually spherical; camble, 1995). It produced the fastest mycelialcolumellae roughly spherical, up to 200 mm diam, growth we have recorded: at 258C and an aw inin age often collapsing downwards and outwards to excess of 0.99, the radial growth rate reachedproduce umbrella shapes; sporangiospores commonly 2 mm/h (i.e. nearly 5 cm/day). Like some other8–20 mm in long axis, pale brownish, with striate walls. species in the Mucorales, R. stolonifer can grow Distinctive features. Rhizopus stolonifer is distin- under anaerobic conditions (Stotzky and Goos,guished by its habit, with coarse hyphae and ram- 1965); however, an atmosphere of 80% O2 + 20%pant growth at 258C, and by sporangia which are CO2 halved its growth rate at 258C after 40 h (Hoo-white when first formed but which change to black gerwerf et al., 2002).Fig. 6.13 Rhizopus stolonifer (a) colonies on CYA, 7 days, 258C; (b) sporangiophores with rhizoids and collapsed columellae,bar ¼ 25 mm; (c) sporangiospores, bar ¼ 10 mm
    • 160 6 Zygomycetes Mycotoxins. Rhizopus stolonifer (a single isolate 2005), cheese (Hayaloglu et al., 2007) and meattested) was moderately toxic to ducklings (Rabie products (see Pitt and Hocking, 1997).et al., 1985). The possible toxins involved have not R. stolonifer is a cosmopolitan fungus, particularlybeen elucidated. in tropical and subtropical regions, in almost all Ecology. Rhizopus stolonifer is by far the most kinds of fresh, moist or partially dried foods.commonly occurring species of the order Mucorales References. Schipper (1984); Zheng et al. (2007a).in foods. It causes destructive rots of a wide range offruits (Snowdon, 1990, 1991). The most importantare fresh berries, especially strawberries (Beneke Rhizopus microsporus Tiegh. Fig. 6.14et al., 1954; Dennis et al., 1979; Harris and Dennis,1980), and stone fruits of all kinds (Snowdon, 1990; On CYA and MEA, colonies covering or filling theSingh and Shukla, 2005). Tomatoes, eggplant and whole Petri dish, of fine, pale grey mycelium,capsicums are also seriously affected in some coun- becoming dark grey as sporangia mature; reversetries (Kassim, 1987; Snowdon, 1991). These diseases pale to dull yellow. On G25N, germination tooccur postharvest and are commonly known as microcolony formation. No growth at 58C. At‘‘transit rot’’. Whole cases of fruit can decay in just 378C, colonies filling the whole Petri dish, similara few days. Spoilage is usually initiated through to those on CYA or MEA at 258C.damage and then spreads by contact. Control Sporangiophores borne singly or in groups ofusually relies on preharvest or postharvest sprays two to three from clusters of poorly formed rhi-or dips of fungicides. Thiabendazole (Benlate) or zoids, stipes unbranched, relatively short, com-2,6-dichloro-4-nitroaniline (Dichloran or Botran) monly 200–500 mm long, with brown walls; sporan-provides reasonable control (Snowdon, 1990, 1991 gia black, 100–200 mm diam, usually spherical;and see Pitt and Hocking, 1997). Other treatments columellae 20–35 mm diam, spherical, some remain-suggested include hot water dips and irradiation ing so in age, others collapsing downwards, to form(Nicoue et al., 2004), ultraviolet light (Pan et al., umbrella shapes; sporangiospores ellipsoidal, 3–52004) and chlorine dipping (Sabaa-Srur et al., mm long, with thin, striate walls.1993). Control of postharvest rot has been satisfac- Distinctive features. Rhizopus microsporus differstorily achieved with applications of yeasts such as from R. stolonifer by shorter stipes and smallerMetschnikowia fructicola (Karabulut et al., 2004) columellae, by the absence of white immature spor-and Cryptococcus laurentii (Zhang et al., 2007a). angia and by strong growth at 378C. It is distin-Transgenic tomatoes with reduced polygalacturo- guished from R. oryzae by production of smallernase activity appear to have a higher resistance to columellae and smaller spores. R. oligosporus istransit rot (Sanders et al., 1992). very similar to R. microsporus, but produces larger Pectic enzymes of Rhizopus stolonifer survive the columellae and smooth to spinose spores.canning process normally applied to fruit. If even a Taxonomy. Sporangia up to 80 mm and spores upsmall proportion of infected fruit is processed, these to 6.5(–7.5) mm long were described by Schipperenzymes can cause softening and spoilage of canned and Stalpers (1984). This species was divided intoapricots (Harper et al., 1972). six varieties by Zheng et al. (2007a), one of which, Many types of vegetables are also susceptible to Rhizopus oligosporus, is considered here to be aspoilage by Rhizopus stolonifer (Snowdon, 1991). separate species. Other varieties are uncommon orBeans and peas, carrots, sweet potatoes, yams and unknown from foods.cassava (see Pitt and Hocking, 1997) are all highly Physiology. Rhizopus microsporus grows up tosusceptible. 46–488C (Schipper and Stalpers, 1984; Zheng Isolation of this species has been reported et al., 2007a). The lower limit for growth is 0.90 awfrom many other food sources, including wheat (Hocking and Miscamble, 1995).and barley (Lugauskas et al., 2006), soybeans Mycotoxins. Wilson et al. (1984) were the first to(Tariq et al., 2005), peanuts (Gachomo et al., report the production of rhizonin A, a nonspecific2004), hazelnuts (Senser, 1979), pecans and bran hepatotoxin, by Rhizopus microsporus. However, a(see Pitt and Hocking, 1997), spices (Mandeel, recent study demonstrated that the toxin was not
    • 6.5 Genus Rhizopus 161Fig. 6.14 Rhizopus microsporus (a) colonies on CYA, 7 days, 258C; (b) sporangiophores with rhizoids, bar ¼ 25 mm;(c) sporangiospores, bar ¼ 10 mmactually synthesised by the fungus, but by a bacter- Rhizopus oligosporus Saito Fig. 6.15ial symbiont belonging to the genus Burkholderia(Partida-Martinez et al., 2007). This finding is of Colonies on CYA 50–60 mm diam, not usuallyinterest as species of Rhizopus are frequently used in covering the whole Petri dish, low, plane and sparse,the food industry for the preparation of fermented with ill-defined margins, pale brown, sporangiafoods such as tempe and sufu. sparsely produced, brown, sometimes enclosed in Ecology. Rhizopus microsporus causes human clear to brown droplets; reverse uncoloured to palezygomycoses, particularly cutaneous and gastroin- brown. Colonies on MEA covering the whole Petritestinal infections (de Hoog et al., 2000). This spe- dish, sometimes reaching, and adhering to, the lid,cies appears to be uncommon in foods from tem- coloured dark grey to black, sporangia abundant,perature zones; however, we isolated it quite black; reverse uncoloured. On G25N, sometimesfrequently from tropical commodities. It was germination. At 58C, no germination. At 378C,found in 18% of maize samples from the Philippines colonies covering the whole Petri dish in a low,and from 3% of all kernels examined. It was also very sparse, often cobwebby growth; reversepresent, at lower numbers, in peanuts, paddy rice, uncoloured.soybeans and black pepper from the Philippines, Sporangiophores best observed on MEA, stipesand maize, peanuts, paddy and milled rice, soy- 150–400 mm long, usually with well developed shortbeans, mung beans and coriander from Indonesia rhizoids at or near the base, terminating in dark(Pitt, 1998a and our unpublished data). sporangia, 80–120 mm diam, at 7 days usually bro- Reference. Schipper and Stalpers (1984); Zheng ken with spores dispersed; columellae persistent,et al, (2007a). often split, but remaining spheroidal to pyriform,
    • 162 6 ZygomycetesFig. 6.15 Rhizopus oligosporus (a) colonies on MEA, 7 days, 258C; (b) sporangiospores, bar ¼ 10 mm; (c) developingsporangia, bar ¼ 25 mm; (d) sporangia, bar ¼ 25 mm25–75 mm diam or long; sporangiospores spherical probably confined to, food fermentations. Its deriva-to subspheroidal, 3.0–3.5 mm diam, with thin, finely tion from R. microsporus seems likely, as the two taxaspinose walls. share many properties. Using a detailed light and Distinctive features. In common with Rhizopus scanning electron microscopy study, Jennessen et al.microsporus, R. oligosporus differs from R. stolonifer (2008) have illustrated criteria for separating R. oligos-by shorter stipes and smaller columellae, by the porus from related species. Sporangiospores of R. oli-absence of white immature sporangia and by gosporus are often larger and less regularly shapedstrong growth at 378C. It is distinguished from R. than those of other species. This is likely to be theoryzae by production of smaller columellae and result of domestication (Jennessen et al., 2008).smaller spores. R. oligosporus produces larger Physiology. Maximum growth temperature ofcolumellae than isolates of R. microsporus and 13 isolates was 45–498C (Zheng et al., 2007a).forms smooth to spinose spores. Mycotoxins. No mycotoxins are known to be Taxonomy. Schipper and Stalpers (1984) reduced produced. Weak toxicity to ducklings was reportedRhizopus oligosporus to the status of a variety of by Rabie et al. (1985). However, only a single isolateR. microsporus, and this was maintained by Zheng was tested.et al. (2007a). However, this taxon deserves species Ecology. Rhizopus oligosporus is used in food fer-status as a domesticated species used in, and mentations, the most notable product being tempeh,
    • 6.5 Genus Rhizopus 163which is produced in Southeast Asian countries, 30–60 mm diam, or occasionally filling the wholeespecially in Indonesia. Cooked, dehulled soybeans Petri dish, relatively low and sparse. No growth atare soaked for 2–3 days, then inoculated from a 58C. Colonies at 378C covering and sometimes fill-previous batch or, more commonly now, with a star- ing the Petri dish, similar to that on CYA or moreter culture, usually R. oligosporus. Fermentation sparse.takes place under ambient (tropical) conditions, pre- Sporangiophores borne in clusters of one to threeferably with air temperatures of 25–288C for 36–48 h from rhizoids, with stipes up to 1500 mm long,(Hesseltine, 1965, 1991; Ko and Hesseltine, 1979; usually unbranched; sporangia spherical, up toBeuchat, 1987; Nout and Rombouts, 1990). Feng 150 mm diam, white at first then becoming greyishet al. (2007) reported the production of volatiles by black at maturity; columellae usually spherical, upR. oligosporus during the fermentation of soybean to 100 mm diam, pale brown, in age often collapsingand barley tempeh. R. oligospor