The fusarium laboratory manual

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MANUAL DE IDENTIFICACION DE FUSARIUS

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The fusarium laboratory manual

  1. 1. launa M yrotarobaL m ui r a s u F e h T
  2. 2. kcolluB ennazuS yb shpargotohP lleremmuS .A tterB dna eilseL .F nhoJThe Fusarium Laboratory Manual
  3. 3. John F. Leslie is a professor in the Department of Plant Pathology at Kansas State University, Manhattan.Brett A. Summerell is Director of Science and Public Programs at the Royal Botanic Gardens – Sydney (Australia).They have been organizing and teaching Fusarium Laboratory Workshops annually since 2000 and began the work onthis manual in 2002 in Sydney under the sponsorship of the Australian-American Fulbright Commission.©2006 Blackwell PublishingAll rights reservedBlackwell Publishing Professional2121 State Avenue, Ames, Iowa 50014, USAOrders: 1-800-862-6657Office: 1-515-292-0140Fax: 1-515-292-3348Web site: www.blackwellprofessional.comBlackwell Publishing Ltd9600 Garsington Road, Oxford OX4 2DQ, UKTel.: +44 (0)1865 776868Blackwell Publishing Asia550 Swanston Street, Carlton, Victoria 3053, AustraliaTel.: +61 (0)3 8359 1011Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, isgranted by Blackwell Publishing, provided that the base fee of $.10 per copy is paid directly to the Copyright ClearanceCenter, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy licenseby CCC, a separate system of payments has been arranged. The fee codes for users of the Transactional Reporting Ser-vice are ISBN-13: 978-0-8138-1919-8; ISBN-10: 0-8138-1919-9/2006 $.10.Produced directly from camera-ready copy provided by the authors.Contribution no. 06-163-B from the Kansas Agricultural Experiment Station, Manhattan.First edition, 2006Library of Congress Cataloging-in-Publication DataLeslie, John F., 1953- Fusarium laboratory manual / John F. Leslie and Brett A. Summerell. p. cm. ISBN-13: 978-0-8138-1919-8 (alk. paper) ISBN-10: 0-8138-1919-9 (alk. paper)1. Fusarium-Laboratory manuals. I. Summerell, Brett A. II. Title. SB741.F9L47 2006 632.4677-dc22 2006004759The last digit is the print number: 9 8 7 6 5 4 3 2 1
  4. 4. Contents FOREWORD …………………………………………………………………………………………………… ix PREFACE ……………………………………………………………………………………………………… xi1. INTRODUCTION ……………………………………………………………………………………………….. 1Techniques and Methods…………………………………………………………………………………… 32. MEDIA – RECIPES AND PREPARATION ………………………………………………………………………... 5 2.1 Media for Growing and Identifying Fusarium ……………………………………………………….. 5 2.2 Supplementary Identification Media …………………………………………………………………. 7 2.3 Media for Isolating Fusarium ………………………………………………………………………… 7 2.4 Media for the Preparation of Natural Inocula ………………………………………………………… 9 2.5 Synthetic and Semi-synthetic Media …………………………………………………………………. 10 2.6 Media for Sexual Crosses …………………………………………………………………………….. 12 2.7 Sterilization of Media and Materials ………………………………………………………………….. 133. TECHNIQUES FOR RECOVERING FUSARIUM …………………………………………………………………… 15 3.1 Collecting strategy …...……………………………………………………………………………….. 15 3.2 Isolation Techniques – Plants ………………………………………………………………………… 16 3.3 Isolation Techniques – Soil …………………………………………………………………………… 18 3.4 Isolation Techniques – Spore Trapping and Air Sampling …………………………………………… 19 3.5 Seed Disinfestation …………………………………………………………………………………… 194. TECHNIQUES FOR GROWING AND MAINTAINING FUSARIUM ………………………………………………….. 21 4.1 Vegetative Propagation ……………………………………………………………………………….. 21 4.2 Preparing Cultures for Identification …………………………………………………………………. 24 4.3 Single-spore Subcultures ……………………………………………………………………………... 25 4.4 Mutagenesis …………………………………………………………………………………………... 26 4.5 Culture Preservation …………………………………………………………………………………... 285. VEGETATIVE COMPATIBILITY GROUPS (VCGS) ……………………………………………………………… 31 5.1 History of and Genetic Basis Underlying Vegetative Compatibility …………………………………. 32 5.2 Overall Strategy for Determining if Strains are Vegetatively Compatible …………………………… 34 5.3 Recovering and Identifying nit Mutants ……………………………………………………………… 35 5.4 Typical Pairing Protocols …………………………………………………………………………….. 38 5.5 Common Trouble Spots – HSI, crn, and NitMs ……………………………………………………… 40 5.6 Characterizing a Population with VCGs ……...………………………………………………………. 426. FERTILITY CONCEPTS ..………………………………………………………………………………………. 45 6.1 Heterothallic, Homothallic and Pseudohomothallic ………………………………………………….. 45 6.2 Mating Type …………………………………………………………………………………………... 47 6.3 Population Effects of Mating Type …………………………………………………………………… 50 6.4 Male, Female, and Hermaphrodite ……………………………………………………………………. 50 6.5 Crossing Protocols ……………………………………………………………………………………. 52 v
  5. 5. vi Contents 6.6 Developing Female-Fertile Tester Strains ……………………………………………………………. 54 6.7 Species Identification Through Sexual Crosses ………………………………………………………. 547. NUCLEIC ACID ANALYSES ……………………………………………………………………………………. 57 7.1 DNA Extraction and Purification ……………………………………………………………………… 57 7.2 PCR – Mating-Type Alleles …………………………………………………………………………... 63 7.3 Amplified Fragment Length Polymorphisms (AFLPs) ………………………………………………. 64 7.4 Sequence Analysis and Sequenced Loci ...……………………………………………………………. 69 7.5 Genetic Maps …………………………………………………………………………………………. 74Taxonomy and Identification of Fusarium………………………………………………….. 798. A BRIEF HISTORY OF FUSARIUM TAXONOMY ………………………………………………………………… 819. SPECIES CONCEPTS IN FUSARIUM ……………………………………………………………………………... 87 9.1 Generic Problems in Speciation in Fusarium ………………………………………………………… 88 9.2 Morphological Species Concepts ……………………………………………………………………... 89 9.3 Biological Species Concepts ………………………………………………………………………….. 90 9.4 Phylogenetic Species Concepts ……………………………………………………………………….. 91 9.5 How Many Strains Make a Species? …………………………………………………………………. 92 9.6 Species Names ………………………………………………………………………………………... 93 9.7 Subspecific Terminology .…………………………………………………………………………….. 94 9.8 A Species Concept for Fusarium ……………………………………………………………………... 9510. TELEOMORPHS OF FUSARIUM …………………………………………………………………………………. 97 10.1 Taxonomy of Teleomorphs …………………………………………………………………………… 97 10.2 General Teleomorph Characters ……………………………………………………………………… 97 10.3 Sexual Development and Differentiation …………………………………………………………….. 98 10.4 Spore Killer …………………………………………………………………………………………… 99 10.5 Anamorph-Teleomorph Connections …………………………………………………………………. 10011. PRACTICAL APPROACHES TO IDENTIFICATION ………………………………………………………………... 101 11.1 Overall Identification Strategy ………………………………………………………………………... 101 11.2 The Diseased Plant and Its Geographic Origin ……………………………………………………….. 102 11.3 Native and Agricultural Populations ………………………………………………………………….. 102 11.4 Culture Preparation …………………………………………………………………………………… 105 11.5 The Essence of Morphological Identifications ……………………………………………………….. 105 11.6 Beyond Morphology – Sexual Cross-Fertility ………………………………………………………... 108 11.7 Beyond Morphology – Molecular Diagnostics ……………………………………………………….. 108 11.8 The Special Case of Fusarium oxysporum …………………………………………………………… 109 11.9 Differences Between Temperate and Tropical Regions ……………………………………………… 109 11.10 Conclusions …………………………………………………………………………………………… 110Species Descriptions ....................................................................................................................................... 11112. MORPHOLOGICAL CHARACTERS ……………………………………………………………………………… 113 12.1 Macroconidia …………………………………………………………………………………………. 113 12.2 Microconidia ………………………………………………………………………………………….. 115
  6. 6. Contents vii 12.3 Chlamydospores ………………………………………………………………………………………. 116 12.4 Other Characters ……………………………………………………………………………………… 117 12.5 Secondary Characters ………………………………………………………………………………… 11713. SPECIES DESCRIPTIONS ………………………………………………………………………………………. 121 F. acuminatum ………………………………………………………………………………………... 122 F. acutatum …………………………………………………………………………………………… 124 F. andiyazi ……………………………………………………………………………………………. 126 F. anthophilum ……………………………………………………………………………………….. 128 F. armeniacum ……………………………………………………………………………………….. 130 F. avenaceum ………………………………………………………………………………………… 132 F. aywerte …………………………………………………………………………………………….. 134 F. babinda …………………………………………………………………………………………….. 136 F. begoniae …………………………………………………………………………………………… 138 F. beomiforme ………………………………………………………………………………………… 140 F. brevicatenulatum …………………………………………………………………………………... 142 F. bulbicola …………………………………………………………………………………………… 144 F. camptoceras ………………………………………………………………………………………... 146 F. chlamydosporum …………………………………………………………………………………… 148 F. circinatum ………………………………………………………………………………………….. 150 F. compactum …………………………………………………………………………………………. 152 F. concentricum ………………………………………………………………………………………. 154 F. crookwellense (F. cerealis) ………………………………………………………………………... 156 F. culmorum …………………………………………………………………………………………... 158 F. decemcellulare ……………………………………………………………………………………... 160 F. denticulatum ……………………………………………………………………………………….. 162 F. dimerum ……………………………………………………………………………………………. 164 F. dlamini ……………………………………………………………………………………………... 166 F. equiseti ……………………………………………………………………………………………... 168 F. foetens ……………………………………………………………………………………………… 170 F. fujikuroi ……………………………………………………………………………………………. 172 F. globosum …………………………………………………………………………………………... 174 F. graminearum ………………………………………………………………………………………. 176 F. guttiforme ………………………………………………………………………………………….. 180 F. heterosporum ………………………………………………………………………………………. 182 F. hostae ………………………………………………………………………………………………. 184 F. konzum ……………………………………………………………………………………………... 186 F. lactis ……………………………………………………………………………………………….. 188 F. lateritium …………………………………………………………………………………………... 190 F. longipes ……………………………………………………………………………………………. 192 F. mangiferae …………………………………………………………………………………………. 194 F. merismoides ………………………………………………………………………………………... 196 F. miscanthi …………………………………………………………………………………………… 198 F. musarum …………………………………………………………………………………………… 200 F. napiforme …………………………………………………………………………………………... 202 F. nelsonii …………………………………………………………………………………………….. 204 F. nisikadoi …………………………………………………………………………………………… 206 F. nurragi …………………………………………………………………………………………….. 208 F. nygamai ……………………………………………………………………………………………. 210 F. oxysporum …….……………………………………………………………………………………. 212 F. phyllophilum ……………………………………………………………………………………….. 219
  7. 7. viii Contents F. poae ………………………………………………………………………………………………... 220 F. polyphialidicum …………………………………………………………………………………… 222 F. proliferatum …….………………………………………………………………………………….. 224 F. pseudoanthophilum ………………………………………………………………………………... 228 F. pseudocircinatum ………………………………………………………………………………….. 230 F. pseudograminearum …….…………………………………………………………………………. 232 F. pseudonygamai ……………………………………………………………………………………. 234 F. ramigenum ….……………………………………………………………………………………… 236 F. redolens ……………………………………………………………………………………………. 238 F. sacchari ……………………………………………………………………………………………. 240 F. sambucinum ………………………………………………………………………………………... 242 F. scirpi ……………………………………………………………………………………………….. 246 F. semitectum (F. incarnatum) ……………………………………………………………………….. 248 F. solani …....…………….……...……………………………………………………………………. 250 F. sporotrichioides ……....……………………………………………………………………………. 256 F. sterilihyphosum …………………………………………………………………………………….. 258 F. subglutinans ………………………………………………………………………………………... 260 F. succisae ….…………………………………………………………………………………………. 262 F. thapsinum ……….…………………………………………………………………………………. 264 F. torulosum …….…………………………………………………………………………………….. 266 F. tricinctum …….…………………………………………………………………………………….. 268 F. udum ……………………………………………………………………………………………….. 270 F. venenatum ………………………………………………………………………………………….. 272 F. verticillioides …….……..……..……..….…………………………………………………………. 274 REFERENCES ………………………………………………………………………………………………….. 279 INDEX ………………………………………………………………………………………………………… 369
  8. 8. ForewordIn 1753, Linnaeus placed all the fungi in the genus species, considering that over 19,000 abstracts are avail-Chaos. In the case of the genus Fusarium things re- able in Biological Abstracts for Fusarium with betweenmained more or less chaotic until 1935 when Wollenwe- 5000 and 6000 for F. oxysporum alone. This kind of in-ber & Reinking created some order in their monumental formation is provided for the first time for many of themonograph, Die Fusarien (2359). The morphological 70 Fusarium species, particularly those described afterdifferences that they used to differentiate between 65 1983.species, 55 varieties and 22 forms were sometimes very Many mycologists and plant pathologists who mustsmall and variable. Subsequently two schools of Fusa- identify isolates of Fusarium are extremely concernedrium taxonomists developed – the “splitters” mainly in about the proliferation of Fusarium species that are diffi-Europe and the “lumpers” in the USA where Snyder and cult, if not impossible, to identify morphologically. OnHansen reduced the genus to nine species in 1945. Dur- the one hand they realize that many Fusarium taxa areing the 1980s, collaboration between Fusarium taxono- heterogeneous and that in many cases the apparent lim-mists from Europe, the USA, Australia and South Africa its of morphology have been reached for separating spe-resulted in a remarkable level of agreement between the cies. In short we have run out of morphological charac-taxonomic treatments of Gerlach & Nirenberg in Ger- ters before we have run out of species that need to bemany, Nelson, Toussoun & Marasas in the USA, and separated. On the other hand they are alarmed that newBurgess & Summerell in Australia. This relative uni- species are being described based on molecular dataformity shifted drastically again during the 1990s with from one or no more than a few isolates without due re-the application of the phylogenetic species concept to gard for the biology, pathology, toxicology and ecologyDNA sequences of diagnostic genes. The resulting pleth- of the taxa. The success or failure of implementing thisora of new species of Fusarium often cannot be distin-guished morphologically and the genus Fusarium might “new” Fusarium taxonomy will depend on the successfulappear to some to be heading for chaos again. integration of morphological characters, measures of Thus, the publication of this Fusarium Laboratory cross fertility and DNA sequence data into a speciesManual by John Leslie and Brett Summerell is most concept based on the whole fungus. The methods andtimely because it integrates the morphological, biological descriptions in this book provide a firm foundation fromand phylogenetic species concepts. The Fusarium Labo- which such discussions and descriptions can begin.ratory Manual includes detailed chapters on Techniques The Fusarium Laboratory Manual is a milestone inand Methods as well as on approaches to Taxonomy and the study of the genus Fusarium and will help bridge theIdentification of Fusarium followed by Species Descrip- gap between morphological and phylogenetic taxonomy.tions of 70 Fusarium species, arranged alphabetically It will be used by everybody dealing with Fusarium infrom F. acuminatum to F. verticillioides. Each species the Third Millennium.description contains photographs and descriptions of keymorphological characters, together with information on W.F.O. Marasassexual stage, taxonomy, pathology, ecology, genetics and PROMEC Unitmolecular biology. A comprehensive literature review is Medical Research Councilgiven for each species, including references to the most Tygerbergrecent publications. This is a formidable undertaking for 70 South Africa ix
  9. 9. PrefaceLaboratory workshops, in which researchers spend a Thanks are due to many for their efforts both withweek with experts in the field to learn to identify various the running of the workshops and the materials that haveFusarium species, are an established part of the Fusarium gone into this volume. First and foremost of these is Suz-community. The best known of these were run by the late anne Bullock, who is responsible for the numerous pho-Dr. Paul Nelson at Pennsylvania State University in the tomicrographs in this manual and whose efforts should1980s and 1990s. It was at one of these workshops where make the morphological species descriptions accessibleone of us (jfl) was first introduced not only to Fusarium even to those who have never before worked with theseas a taxonomic object, but also to a cohort of other be- fungi. Second to Kurt Zeller, Amgad Saleh, Jim Jur- genson, and Brook van Scoyoc, for their help in develop-ginning Fusarium researchers, e.g., Tom Gordon, Anne ing many of the protocols included in this volume andDesjardins, Jim Correll, and Marian Beremand, who distilling them to a form that is (hopefully) easily under-along with the instructors of the class, T. A. Toussoun, stood by those who have not used them before. Finally toW. F. O. Marasas, Carol Windels, Lester Burgess and all of those who have helped run the various workshops,Paul Nelson, have influenced much of the research in but especially Amy Beyers, Anita Kesler, Ingelin Leslie,Fusarium over the last 20 years. Brook van Scoyoc, and Celest McGowan, who have de- This manual springs from the tradition of Fusarium veloped the protocols and the timing for both the scien-Laboratory Workshops and is in some ways a successor tific, fiscal, and social aspects of the workshops that theto both the manual of Nelson et al. (1551) and that of rest of us have followed with such great success.Burgess et al. (279), which had their roots in these teach- Portions of this book were written while one of using and outreach efforts. However, our goal was to take (jfl) was on sabbatical leave at the University of Sydneythis tradition beyond morphological species descriptions and the Royal Botanic Gardens and Domain Trustto include additional techniques and identification proc- (RBGDT) and sponsored by the Australian-Americanesses that are widely used in the Fusarium research Fulbright Foundation and the RBGDT. We thank thecommunity. We also wanted to include some of the evo- American Phytopathological Society for permission tolutionary biology and population genetic thinking that reprint Table 11-1 and Figures 4-2 and 11-1, the Ameri-has begun to inform the understanding of agriculturally can Society for Microbiology for permission to reprintimportant fungal pathogens. The need for as many differ- Figure 7-2, and the Genetics Society of America for per-ent techniques to be used when examining these fungi mission to reprint Figure 7-3. Portions of Chapter 9 arehas never been clearer. In combination with the two re- based on Leslie et al. (1235), and portions of Chapter 11cent volumes from APS Press (468, 2083), this volume are based on Summerell et al. (2087). Both the work-presents a relatively current introduction to the genus shops and the manual have benefited from financial sup-Fusarium, the toxins these fungi produce and the diseases port from both our home institutions, and from INT-they can cause. SORMIL (the International Sorghum and Millet Collabo- Much of the material in this manual has been used rative Research Support Program, based at the Universityby participants who attended workshops held at Kansas of Nebraska, Lincoln, Nebraska), IITA (the InternationalState University (2000, 2001, 2003 and 2005), the Uni- Institute of Tropical Agriculture, Ibadan, Nigeria), andversity of Sydney (2002), and the University of Pretoria ABRS (the Australian Biological Resource Study).(2004). The material included in this manual is meant to Much of the book has been evaluated by others in-serve as the basis for future workshops, as well as a guide cluding Walter A. J. de Milliano, Anne Desjardins,to the field for those who are looking for a reference, a Stanley Freeman, David Geiser, Cristiano Lima, Antoniodescription of a common species, or a technique. The Logrieco, W. F. O. Marasas, Randy Ploetz, Amgadcontent also has been influenced by the presentations at Saleh, Baharuddin Salleh, Brenda Wingfield, and Kurtthese workshops of the other instructors – Lester Bur- Zeller. Their eyes have found errors and omissions thatgess, David Geiser, Antonio Logrieco, W. F. O. Marasas, we would have missed and their suggestions have onlyJohn Rheeder, Baharuddin Salleh, Keith Seifert, Carol improved the book’s accuracy, utility and readability. Er-Windels, Brenda Wingfield, and Kurt A. Zeller. Their rors that remain, however, are solely our own, and cor-thoughts on what is important, and what is not, were rections and suggestions for topics to be considered forpassed through our filters as we developed this manual. inclusion in future versions will be gratefully received. xi
  10. 10. xii Preface A final, and perhaps the greatest note of gratitude is July 2005to our wives, Ingelin and Liz, and families for their en- John F. Lesliecouragement, for sharing their homes and hospitality as Department of Plant Pathologyearly and final versions of the manuscript were first put Kansas State Universityto paper and then finally put to rest, and for tolerating our Manhattan, Kansas, USAinterest in this group of fungi, when they might have hadother places they would like for us to have focused in- Brett A. Summerellstead. They know better than we do that the idea of a Royal Botanic Gardens and Domain Trustbook is grand and the finished product is even better, but Mrs. Macquaries Roadthat the time in between is not always one of great joy. Sydney, New South Wales, Australia
  11. 11. The Fusarium Laboratory Manual John F. Leslie, Brett A. Summerell Copyright © 2006 Blackwell Publishing 1 IntroductionThe genus Fusarium was introduced by Link in 1809 the region. At the same time, the causal agent of Fusa-(1263), and is now approaching its third century as a ge- rium head scab can be used in commercial fermentationsnus that contains many plant-pathogenic fungi. The to produce a precursor for one of the most widely usedmembers of this genus can incite directly diseases in commercial cattle growth promotants (858). Recent prob-plants, humans, and domesticated animals, e.g., Boon- lems caused by strains of Fusarium which may havepasart et al. (221), Goldschmied et al. (718), Krcmery et originated from endophytes or pathogens of native Gos-al. (1136), Martino et al. (1373), Rabodonirina et al. sypium species (2286), are threatening the future of the(1760), Rebell (1783) and Vismer et al. (2263). The mor- cotton industry in Australia while simultaneously demon-tality rate for human patients with systemic Fusarium strating the relatedness of native and agricultural popula-infections is > 70% (1136), and HIV-infected patients are tions and suggesting new avenues for understanding howsusceptible to such Fusarium infections as well (562, these fungi evolve.778, 1486). In addition, Fusarium spp. produce an in- Many plants have at least one Fusarium-associatedtriguing array of secondary metabolites that are associ- disease. A recent perusal of the plant disease list main-ated with plant disease, as well as with cancer and other tained by the American Phytopathological Society (www.growth defects in humans and domesticated animals. apsnet.org/online/common/search.asp) revealed that overSome of these secondary metabolites are used commer- 81 of the 101 economically important plants on the listcially either directly or as the starting material for chemi- had at least one associated Fusarium disease. As thesecal syntheses of plant and animal growth promoters in fungi also may grow as apparently symptomless endo-both first world and third world settings (858, 1976, phytes under many conditions, the claim that, “If it is2157). Allegations of the use of mycotoxins produced by green, there is some Fusarium that can grow on it, in it,some of these fungi as biological weapons also have been or with it” probably is not too far removed from the truth.made (855, 1454, 1833). Two of these compounds, T-2 The types of diseases induced are quite varied as is theirand diacetoxyscirpenol (DAS) are on the USDA/CDC severity, and may include root or stem rots, cankers,select agent list and require special permits for study in wilts, fruit or seed rots, and leaf diseases. Thus identify-the United States (www.cdc.gov/od/sap/docs/saist.pdf). ing the Fusarium strain present in a diseased plant sam-Naturally occurring outbreaks of Fusarium toxicoses di- ple, usually to species and sometimes further, has beenrectly affecting humans have occurred historically, e.g., and remains an important task in many plant diagnosticAthens in the 5th century B.C. (1922), and in the Soviet laboratories.Union during World War II (666, 982). Thus, Fusarium The need to identify strains and to attach names tohas always been a visible genus with many strains, spe- them is as strong, or stronger, in Fusarium than it is incies and metabolites of an importance that transcends just any fungal genus. That these names have a value thatscience or agriculture. transcends the science from which they spring means that As a social phenomenon, Fusarium plant diseases these names need to be assigned and changed with carehave had several major impacts. One was the near devas- and caution. Depending on the era and the identificationtation of the commercial banana industry in the 1960s by scheme being followed (see Chapter 8), the number ofpanama wilt caused by Fusarium oxysporum f. sp. Fusarium species could range from as few as nine to wellcubense (1706). The recent losses of several billion dol- over a thousand. Since the 1980s the number of recog-lars (2342) by many wheat and barley farmers to Fusa- nized species has increased gradually, with the number ofrium head scab in the upper Midwest of the United States recognized species now > 80, of which 70 are describedhas shifted cropping strategies and bankrupted farmers in and illustrated in this text. If preliminary work from cur- 1
  12. 12. 2 Chapter 1rent molecular studies stands, then this number should a name to a culture, the apparent nomenclatural turmoilincrease, perhaps dramatically, during the coming years. can be confusing and perhaps even threatening. Fortu-As a genus, Fusarium lacks a large number of morpho- nately, many of the most important pathogens can be di-logical characters that can be used to easily differentiate agnosed, with some experience, on the basis of morpho-species, and it is not uncommon to “run out of characters logical characters. Even isolates not identified to a singlebefore you run out of species.” This problem has left nu- species can be assigned to a group of usually closely re-merous species definitions less-than-well defined, has lated species. Accurate morphological characterizationscomplicated problems of identifying the “real” Fusarium require that the fungus be subcultured and handled withxxx, and has resulted in spirited debates between indi- at least some care. Growing the fungus on media appro-viduals with quite different views of what a species is or priate for the morphological features to develop takesshould be. Although many morphological species con- time, sometimes several weeks, and means that a carefulcepts have changed, many have remained stable, even diagnosis often is a slow process. As molecular studieswhen challenged with genetic and molecular criteria. progress and the definition of common and important The relatively large amount of work done on the species solidify, the development of molecular diagnos-morphological taxonomy of these fungi means that as a tics for many species also should be possible. These di-genus, Fusarium often has served as testing ground for agnostics should be much faster than the present morpho-new speciation concepts in fungi. The use of molecular logical diagnoses, as at most limited culturing of the fun-approaches to differentiate species has been tried with a gus is required. The molecular diagnostics will need to benumber of strains usually considered problematic, i.e. not carefully evaluated on a broad range of species as well asclearly fitting within a given species, but not clearly dis- strains within the species to accurately define their diag-tinguishable from it either. Studies of phylogenetic line- nostic abilities and limitations. For laboratories that cur-ages based on multiple-gene genealogies, and the group- rently lack and are unable to acquire molecular expertise,ing patterns resulting from studies with amplified frag- morphological species definitions will remain the rule.ment length polymorphisms (AFLPs) both provide new Such scientists should seek partnerships with researchersmeans of evaluating relatedness. As these techniques are with access to molecular technologies to confirm criticalindependent of one another, each can be used to test the findings and conclusions, and to ensure that morphologi-hypothesis of distinctness generated by the other, to look cal similarities are not disguising significant genetic dif-for continuous gradations between representative types, ferences.and to identify putative inter-specific hybrids. Studies This book is designed as a laboratory manual andbased on biological species concepts also have been used guide to techniques and species likely to be encountered.to delimit some species, most commonly within the Gib- It is neither a mycological monograph (a number of spe-berella fujikuroi species complex, while also testing hy- cies are missing) nor a comprehensive summary of workpotheses of sameness/distinctness based on both molecu- that has been done with various Fusarium spp. (there arelar and morphological characters. Subspecific differences > 19,000 citations to Fusarium in Biological Abstractscould be indicative of “evolution in action”, suggesting alone). It does contain recipes for many commonly usedthat these fungi are not evolutionary fossils, but entities media, techniques that will work for common field andthat continue to change in response to both manmade and molecular applications, species descriptions of the mostnaturally occurring selective pressures and for the eluci- common species, and our thoughts on what a Fusariumdation of evolutionary mechanisms. Thus Fusarium has species is and how the field might be evolving. We hopebeen and remains an important genus for the testing and that you will find this manual a useful place to begin adevelopment of species concepts in fungi. serious study of a particular species or a Fusarium- For a researcher or diagnostician not interested in induced disease, or to find a quick answer regardingstate-of-the-art taxonomy and wanting primarily to attach these most interesting and important fungi.
  13. 13. The Fusarium Laboratory Manual John F. Leslie, Brett A. Summerell Copyright © 2006 Blackwell PublishingTechniques and Methods
  14. 14. The Fusarium Laboratory Manual John F. Leslie, Brett A. Summerell Copyright © 2006 Blackwell Publishing 2 Media – Recipes and Preparation 2.1 Media for Growing and Identifying Fusarium ..................... 5 2.2 Supplementary Identification Media ..................................... 7 2.3 Media for Isolating Fusarium .……………………………… 7 2.4 Media for the Preparation of Natural Inocula ..…………… 9 2.5 Synthetic and Semi-synthetic Media ………………………. 10 2.6 Media for Sexual Crosses …………………………………… 12 2.7 Sterilization of Media and Materials ………………………. 132.1 Media for Growing and Identifying dia for use in Petri dishes are made, autoclaved, and then Fusarium dispensed into sterile Petri dishes after autoclaving. Me- dia containing agar should be made in a flask that isVarious agar media have been used as standards on twice the volume of the medium being made, e.g., onewhich to grow cultures for the identification of Fusarium liter of medium in a 2-L flask, with a 2-L flask generallyspecies. Fusarium species have notoriously variable phe- the largest convenient size. Cooling the agar to ~50°Cnotypes when cultured on different agar media, e.g., Bur- before it is dispensed is essential if a heat-sensitive solu-gess et al. (280). Morphological features that are com- tion, e.g., an antibiotic, is to be added, and reduces themon in cultures grown on some media may be absent or amount of bubbles on the medium surface and the con-altered when the same strain is cultured on another me- densate on the dish lid in all cases. The most commonlydium. The basic principle is to use the same medium as used Petri dishes are those with a 100 mm or 60 mmthat used by the authors of the identification guide to nominal diameter. One liter of medium usually sufficeswhich comparisons are being made. Different media may to fill 40-50 of the larger dishes (20-25 ml medium/dish),have the same common name, e.g., V-8 juice agar or po- or 80-100 of the smaller dishes (10-12 ml medium/dish).tato dextrose agar, but vary in their composition. Thus, a If the medium is for sexual crosses, then more medium isstandard recipe needs to be followed in preparing media dispensed per dish than if the medium is for a vegetativeto be used for species characterization. Adding a deter- analysis (see section 2.6).gent to the media, e.g., Triton X-100 or tergitol, can re- The same media used in Petri dishes also can be usedstrict colony size and make processes such as colony in agar slants, but media that are viscous, e.g., carrot agarenumeration or replica plating simpler (1746, 2230). (see section 2.6) or oatmeal agar, generally cannot beCarnation Leaf-piece Agar (CLA), Spezieller Nährstof- used in slants since even the slowest decompression cy-farmer Agar (SNA), and Potato Dextrose Agar (PDA) are cle of most autoclaves results in the plugs capping thethe standard media used in the identification of Fusarium tubes being blown off or in the media boiling over thespecies in this volume. tops of the tubes. A medium to be dispensed into slants is Media usually are dispensed into Petri dishes or usually made up, melted, and dispensed into the slantsslants, sometimes termed an agar slope, in a test tube for with a repeating syringe before the medium is auto-media with agar, or in flasks for media without agar. Me- claved. It is very important to ensure good mixing and 5
  15. 15. 6 Chapter 2complete melting of the agar before dispensing the me- are macroconidia formed from more isolated phialides.dium; otherwise, some slants may receive too much agar Microconidia are more common on hyphae growing onand others too little, which can make the slants difficult the agar, often away from the leaf pieces. The mode ofto work with. The most commonly used test tube sizes formation of microconidia, i.e. monophialides or polyphi-for slants are 10×75, 13×100, and 16×150 mm nominal alides, the presence of false heads or chains of microco-sizes, which receive 1.25, 2.5 and 6.0 ml of medium/slant nidia, and the presence of chlamydospores can be deter-respectively. After the medium has been added, the tubes mined by direct examination with a compound micro-are stoppered with cotton or foam plugs, or covered with scope (100×) when strains are grown on small (60 mmcommercially available plastic or metal caps. When auto- diameter) plates of CLA. CLA also is suitable for theclaving slants it is very important to use a slow decom- production of large numbers of conidia for experimentalpression cycle to prevent the medium from boiling, as work, although many researchers also use the modifiedplugs wetted by boiling media render those cultures more Czapek-Dox medium (see section 2.5), and can be pre-susceptible to contamination. In practice, plugs wetted by pared for this purpose in large flat bottles or larger Petrimedia should be discarded and replaced with dry sterile plates, usually 150 mm nominal diameter.plugs (made by autoclaving some tubes that contain no Gibberella zeae (anamorph – Fusarium graminea-medium). After autoclaving, the tubes must be placed at a rum), homothallic strains of Haemanectria haemato-slant before they harden to provide additional surface cocca (anamorph – Fusarium solani), and appropriatelyarea for culture growth. To get the media at a slant, the mixed cultures of heterothallic Fusarium species form10×75 and 13×100 mm tubes usually are left in the racks perithecia on CLA if incubated under light (see sectionin which they were autoclaved, and the entire rack of 6.5). Older carnation leaves may give better results thantubes is slanted at one time. The larger slants, 16×150 younger carnation leaves for perithecia formation (2154).mm size, may be slanted in the rack, but this often results For genetic investigations, most researchers presently usein variation in the amount of slanting that actually occurs. carrot agar or V-8 juice agar (see section 2.6) to produceA more uniform slant for these tubes is obtained by plac- perithecia from which ascospores are collected.ing them individually on a slanting board to harden. Spezieller Nährstoffarmer Agar (SNA). SNA is aCarnation Leaf-Piece Agar (CLA). CLA is a natural weak nutrient agar used for the identification and main-substrate medium (625, 2017) prepared by aseptically tenance of strains of Fusarium and Cylindrocarponplacing sterile carnation leaf pieces, 3-5 mm², into a Petri (1566). SNA is prepared by autoclaving, in 1 L of dis-dish and adding sterile 2% water agar (20 g agar in 1 L of tilled H2O:H2O). Usually one carnation leaf piece is added per 2 ml KH2PO4 1 gof medium. In 60 mm diameter Petri dishes, 5-6 pieces KNO3 1 gper dish and in 100 mm diameter dishes, 10-12. MgSO4•7H2O 0.5 g The carnation leaf pieces are prepared from fresh KCl 0.5 gcarnation leaves free from fungicide or insecticide resi- Glucose 0.2 gdue. Immediately after collection, the leaves are cut into Sucrose 0.2 g5-8 mm² pieces (they shrink when they are dried) and Agar 20 gdried in a forced-air oven (~70°C) for 3-4 hours until Placing 1-2 pieces of sterile filter paper (Whatman 1), №brittle. Leaf pieces also can be dried in a microwave approximately 1 cm², on the agar surface after the me-oven; the time required varies by oven. The dried leaf dium has gelled can increase sporulation.pieces are packaged in aluminum or polycarbonate con- Culture degeneration, which is common on manytainers and sterilized by gamma irradiation (2.5 mega- synthetic media, usually does not occur on SNA. Therads). Sterilized leaf pieces can be stored dry at room medium promotes sporulation and good conidiogeneoustemperature for up to 12 months before use. cell development; however, because sporodochia forma- Most species of Fusarium sporulate on CLA in 6-10 tion is limited, macroconidial morphology is not as uni-days. Cultures grown on CLA produce macroconidia that form or as reliable as seen with CLA. Cultures grown onare more uniform in size and form, than do cultures SNA often are of value for examining microconidia, asgrown on carbohydrate-rich media such as PDA or this medium supports the formation of a range of differ-Czapek-Dox. Macroconidia form primarily in sporodo- ent microconidia. In some cases microconidia formed onchia, which usually develop on the leaf pieces. Macroco- SNA will differ morphologically from those seen onnidia formed in sporodochia are preferred for identifica- CLA. Many strains also form chlamydospores more read-tion as they are more consistent in shape and length than ily on SNA than they do on CLA, so for species in which
  16. 16. Media 7microconidial morphology or the presence of chlamy- tion series. The small amount of agar retards the sedi-dospores are important identification criteria, cultures mentation rates of fungal propagules. The water is heatedfrom both CLA and SNA should be examined. SNA is until the agar dissolves, and the heated medium distrib-transparent, so cultures can be viewed directly with a uted into flasks, or, more commonly, McCartney bottles.compound microscope (up to 100×) or small agar blocks Bottles are capped loosely during sterilization and thencan be mounted on a slide with a drop of water and cov- tightened after sterilization and cooling to room tempera-ered with a cover slip for observation at higher magnifi- ture.cations. Soil Agar (SA). Chlamydospore formation is enhancedPotato Dextrose Agar (PDA). PDA is a carbohydrate- on SA (1094), making this medium useful for the identi-rich medium that contains 20 g dextrose, 20 g agar, and fication of some species of Fusarium. SA is prepared bythe broth from 250 g white potatoes made up to 1 L with placing 250-500 g of sieved dry soil into a flask andtap water. The potatoes are unpeeled, washed, diced and bringing the total volume to 1 L with tap water and auto-boiled until soft (actual time varies with the size of the claving for 15 min. After the first autoclaving, add 15 gpotato). Filter the boiled potatoes through a single layer of agar and autoclave for a second 15 min. The amountof cheesecloth, which leaves some sediment in the broth. of soil used varies with soil type. Abundant chlamy-Commercially available preparations of PDA, e.g., Difco, dospore formation by various species has been observedBBL, etc., are often more convenient and usually are as on SA prepared with 250 g black clay soil, but other soilsuitable for identification purposes as home-made prepa- types also have been used successfully. While pouringrations. plates, the autoclaved medium should be regularly mixed Conidia formed on PDA are not as consistent in ei- or swirled to ensure even distribution of the solids to allther size or shape as those formed on CLA or SNA, and plates.thus are much less reliable for use for identification pur- KCl Agar. When 4-8 g/L KCl is added to WA or toposes. However, colony morphology, pigmentation and CLA the fungal cultures produce more and longer micro-growth rates of cultures of most Fusarium species on conidial chains (626). The chains also are easier to seePDA are reasonably consistent if the medium is prepared since there is less moisture on the agar surface and fewerin a consistent manner, and if the cultures are initiated moisture droplets in the aerial mycelium.from standard inocula and incubated under standard con-ditions. These colony characteristics often are useful sec- 2.3 Media for Isolating Fusariumondary criteria for identification. PDA is used by some researchers for the isolation of A number of media have been developed for the specificFusarium species. We do not recommend this medium isolation of Fusarium spp. Those with the longest historyfor this purpose, as many saprophytic fungi and bacteria of use are PPA and Komada’s, with Komada’s usuallyalso can grow on the medium and interfere with the re- preferred when the target is F. oxysporum, and PPA pre-covery of the Fusarium present. If PDA is used for the ferred for most other uses. MGA and RbGu media arerecovery of fungi from plant material, then the concentra- variations on PPA that have been widely used by sometion of potato and dextrose should be reduced by 50- researchers. MGA may become more prominent as the75%, and broad-spectrum antibiotics (see PPA Medium availability of PCNB decreases. New media are beingin section 2.3) included to inhibit bacterial growth. continuously developed to meet particular needs, e.g., SSM and SFA, or with formulations that are purportedly2.2 Supplementary Identification Media more selective for Fusarium species than were earlier media, e.g., the medium of Vujanovic et al. (2271) whichWater Agar (WA). WA (2%) consists of 20 g agar in 1 contains myclobutanil as the selective agent.L of distilled H2O. This medium is recommended for ger- Peptone PCNB Agar (PPA or Nash-Snyder Medium).minating conidia used to initiate Fusarium cultures (see The original medium was described by Nash & Snydersections 4.1-4.3). As hyphal growth is sparse on this me- (1534), with modifications by Nelson et al. (1551). PPAdium, it is suitable for growing cultures from which indi- is composed of a sugar-free basal medium supplementedvidual hyphal tips are taken to initiate new colonies (see with antibiotics and fungicides. The base medium con-section 4.3). In some instances, the sparse growth on WA tains:facilitates the isolation of Fusarium species from plantmaterial, particularly roots. Peptone 15 g WA (0.05%) consists of 0.5 g agar in 1 L of distilled KH2PO4 1 gH2O. WA (0.5%) is used in the preparation of soil dilu- MgSO4•7H2O 0.5 g
  17. 17. 8 Chapter 2 PCNB (Pentachloronitrobenzene) 750 mg PCNB is usually added as 1 g of Terrachlor, which con- Agar 20 g tains 75% PCNB (w/w). The pH is adjusted to 3.8 ± 0.2 H2O to 1 L with 10% phosphoric acid, if necessary, prior to auto-PCNB is usually added as 1 g of Terrachlor, which con- claving. The basal medium is autoclaved and cooled totains 75% PCNB (w/w). The pH should be adjusted to ~50°C before adding filter-sterilized supplemental stock5.5-6.5, if necessary. The streptomycin stock solution is 5 solutions. The streptomycin stock solution is 5 g of strep-g of streptomycin in 100 ml distilled H2O, and is used at tomycin in 100 ml distilled H2O, and is used at the rate ofthe rate of 20 ml/L of medium. The neomycin stock solu- 6 ml/L of medium. The Oxgall stock solution contains 5tion is 1 g of neomycin sulfate in 100 ml distilled H2O, g Oxgall and 10 g Na2B4O7•10H2O in distilled H2O, andand is used at the rate of 12 ml/L of PPA. Streptomycin is is used at the rate of 10 ml/L of medium.effective against Gram-negative bacteria, and neomycin Colonies of F. oxysporum are distinctly pigmentedagainst Gram-positive bacteria. on this medium, and usually separable from other Fusa- The medium may be poured into plates onto which rium species on this basis (272). However, there issoil or diseased plant material is placed, or dispensed into enough overlap with some related species, e.g., those invials into which the material is placed directly. Vials can the G. fujikuroi species complex, that simple counts ofbe particularly useful if samples are taken immediately colonies with different morphologies need not be an ac-from field material and then returned to the lab for proc- curate measure of the F. oxysporum levels in the sample.essing, and as a means to avoid problems associated with Growth of other Fusarium species may be suppressed bythe importation of diseased plant material that are usually the medium, and this medium often is not a good choicenot encountered with cultures. If plates are being used in for the recovery of Fusarium communities that containsoil dilutions (see section 3.3), then it is best if they are species other than F. oxysporum.allowed to sit for several days to dry before use. This Malachite Green Agar (MGA). MGA was developeddrying time allows the water in the soil suspensions to be in 1997 by Castellá et al. (326) as an alternative to PPAabsorbed more quickly into the agar gel. or Komada’s medium, and has performed well in some PPA is highly inhibitory to most other fungi and bac- comparison studies with other media (232). This mediumteria but allows slow growth of Fusarium. It enables the contains:selective isolation of Fusarium species from soil dilu- Peptone 15 gtions and from rotting plant material that often is infested KH2PO4 1 gwith other fast-growing microbes. Most species of Fusa- MgSO4•7H2O 0.5 grium do not form distinctive colonies on PPA. Often, Malachite green oxalate 2.5 mgsporulation is poor and conidial morphology abnormal. Agar 20 gThis means that colonies usually must be subcultured be- H2O to 1 Lfore a definitive identification can be made. Although thesporulation is poor, there may be enough spores so that a After autoclaving, streptomycin and chloramphenicol aresubculture originating from a single spore can be gener- added as for PPA. This medium has not been used asated from colonies growing on PPA. Cultures should not widely as has PPA or Komada’s. The only difference be-be left on PPA for more than 2-4 weeks. The sole nutri- tween this medium and PPA is that the PCNB is replacedent source in the medium is peptone, and its breakdown by 2.5 ppm malachite green. This medium probably willgenerates relatively high levels of ammonia that eventu- become more important as the availability of PCNB (dueally kill the colonies. to its carcinogenicity) declines. MGA also is reported to be more inhibitory of common contaminants, e.g., Asper-Komada’s Medium. Komada’s medium was developed gillus spp. and Penicillium spp., without reducing thefor the selective isolation of F. oxysporum from soil number of colonies of Fusarium spp. recovered than is(1115). The basal medium contains: PPA. The malachite green level in the medium is impor- D-Galactose 20 g tant, as the level of malachite green (15-50 ppm) used in L-Asparagine 2 g the Singh-Nene medium (1991), from which this formu- KH2PO4 1 g lation was derived, prevents germination of many Fusa- KCl 0.5 g rium spores. MgSO4•7H2O 0.5 g Another variant on PPA (712) is to replace the PCNB (Pentachloronitrobenzene) 750 mg PCNB with 1 g/L 2-benzoxazolinone, an antimicrobial Fe3Na EDTA 10 mg compound produced by many maize lines. This medium Distilled H2O to 1 L is particularly effective for selecting for maize pathogens
  18. 18. Media 9such as F. graminearum, F. subglutinans, and F. verticil- in selectivity to both PPA and Komada’s medium butlioides. with the advantage that colony morphology is somewhatSelective Fusarium Agar (SFA). SFA was developed easier to distinguish on this medium.for the selective isolation of Fusarium species from soil Specific Screening Media (SSM). SSM was developeddebris. SFA is a modified Czapek-Dox medium (see sec- for the selective isolation of Fusarium pseudograminea-tion 2.5) that contains antimicrobial agents (2151). rum and other fungal pathogens from the crown region of Glucose (Dextrose) 20 g wheat plants (2077). SSM contains 10 g dextrose, 20 g KH2PO4 0.5 g agar, and the broth from 125 g white potatoes made up to NaNO3 2 g 1 L with tap water. The potatoes are unpeeled, washed, MgSO4•7H2O 0.5 g diced, and boiled until soft. Filter the boiled potatoes Yeast extract 1 g through a single layer of cheesecloth, which leaves some 1% FeSO4•7H2O (aqueous) 1 ml sediment in the broth. Commercially available prepara- Agar 20 g tions of PDA, e.g., Difco, BBL, etc., may be used at half H2O to 1 L of the recommended strength, but must be supplementedThe basal medium is autoclaved and allowed to cool to with an additional 10 g/L agar. After the basal medium~50°C before adding filter-sterilized antibiotic stock so- has been autoclaved and cooled to ~50°C supplementallutions. The streptomycin and neomycin stocks are the stock solutions are added. The streptomycin stock solu-same as those for PPA, and are used at the same rate – tion is 5 g of streptomycin in 100 ml distilled H2O, and isstreptomycin stock is 5 g of streptomycin in 100 ml dis- used at the rate of 3 ml/L of medium. The neomycintilled H2O, and used at the rate of 20 ml/L of SFA, and stock solution is 1 g of neomycin sulfate in 100 ml dis-the neomycin stock is 1 g of neomycin sulfate in 100 ml tilled H2O, and is used at the rate of 6 ml/L of SSM. Di-distilled H2O, and used at the rate of 12 ml/L of SFA. chloran is prepared as 50 mg of dichloran (or 100 mg Al-Dichloran (2,6-dichloro-4-nitroanaline) is prepared as 50 lisan or Botran) in 100 ml ethanol, and used at the rate ofmg of dichloran (or 100 mg Allisan or Botran) in 100 ml 13 ml/L of SSM.ethanol, and used at the rate of 13 ml/L SFA. If dichloran SSM permits the formation of distinctive colonies ofis not available, then PCNB, as used in PPA – 750 mg or F. pseudograminearum, and suppresses the growth of1 g Terrachlor – can be incorporated into the medium mucoraceous fungi and Trichoderma species. This me-prior to autoclaving. dium is particularly well-suited for studies of crown rot SFA permits the slow growth of Fusarium species of wheat in which F. pseudograminearum is known tofrom plant roots and soil debris, and is less inhibitory form a morphologically distinguishable colony on thethan PPA to most fungi. Colonies of different species de- medium. In such studies, e.g., Burgess et al. (268), theveloping from a single root fragment or piece of debris medium is prepared in large trays to enable a very largeare easier to differentiate on SFA than on PPA. Although number of samples to be processed rapidly.SFA was developed for isolation of Fusarium from soildebris, it is not suitable for the isolation of Fusarium spe- 2.4 Media for the Preparation of Naturalcies from soil dilutions. InoculaRose Bengal-Glycerine-Urea Medium (RbGU). This Chaff-Grain Medium. Inoculum suitable for addition tomedium was developed by van Wyk et al. (2224) for iso- soil in pathogenicity tests can be prepared by using colo-lations from both soil and plant debris. nized chaff-grain as a substrate (we have successfully Glycerol 10 g used wheat, barley and oats, but expect that maize, sor- Urea 1 g ghum and rice also would work). Cereal chaff and grain L-Alanine 0.5 g are mixed together in an approximately 5:1 ratio (often a PCNB (Pentachloronitrobenzene) 1 g commercial horse feed will suffice). Rose Bengal 0.5 g To a 2-L beaker, add approximately 500 ml of the Agar 15 g chaff-grain mixture, and then add tap water to approxi- H2O to 1 L mately the 1-L level. Mix thoroughly to release any airThe basal medium is autoclaved and allowed to cool to bubbles and to wet the entire mixture; add additional wa-~50°C before adding a filter-sterilized streptomycin solu- ter to bring the total volume back to 1 L, if necessary.tion that is the same as that used for PPA – 5 g of strep- Place the beaker at 5°C for at least overnight, but nottomycin in 100 ml distilled H2O. The medium is similar more than 24 hours, to leach phenolic compounds. After
  19. 19. 10 Chapter 2leaching is complete, cover the mouth of the beaker with 2.5 Synthetic and Semi-synthetic Mediaa layer of cheesecloth or Miracloth, and invert the beakeron a drainboard to drain the water. After 5-10 min, wrap Synthetic and semi-synthetic media often are used forthe chaff-grain mixture in the cheesecloth and squeeze studying physiological characters, for isolating DNA, anduntil no more water can be released. for genetic studies. The media in this section are all based The drained mixture is distributed into glass jars or on the Czapek-Dox medium recipe, as modified by Cor-Erlenmeyer flasks, which are filled to a depth of ap- rell et al. (402). The basal medium contains:proximately 5 cm. The containers are sealed with a large KH2PO4 1 gcotton wool plug and autoclaved for 15 minutes on each MgSO4•7 H2O 0.5 gof two successive days. Containers are inoculated with a KCl 0.5 gconidial or mycelial suspension ( 105 cfu/ml) at the rate ≥ Agar (Bacto) 20 gof 2 ml of the fungal suspension per 250 ml chaff-grain Trace element solution 0.2 mlmixture. Distilled H2O to 1 L The inoculated material is shaken vigorously to It is critical to use distilled water in this medium to pre-evenly distribute the inoculum throughout the medium. vent unintentional contamination of the medium withInoculated material is incubated at 25°C until the mate- salts that may adversely affect growth characteristics orrial is completely colonized. The length of the incubation trace element levels. Agar should be at least of bacterio-depends on the rate of growth for the fungus. Usually 7- logical grade. Higher levels of agar purity occasionally14 days of incubation is sufficient. These cultures should are warranted, e.g., see nitrite medium below, but are notnot be maintained for more than 21 days, as viability be- necessary for routine work. Lower grades of agar maygins to decrease. Cultures should be shaken daily for the have trace amounts of vitamins, nucleic acids, and/orfirst 3-4 days to encourage more rapid and uniform colo- amino acids that may make it difficult to score auxotro-nization of the substrate. Once the substrate is completely phic mutations clearly. For work with liquid cultures, thecolonized, it is removed from the container and air dried agar is omitted completely.at room temperature (20-25°C) overnight (perhaps The trace element solution supplies critical nutrientssomewhat longer if the relative humidity is high). When required for some enzyme activities, e.g., Mo is essentialdry, then material is crushed to the required size (usually for nitrate reductase activity, and should not be omitted.particles pass through a 2 mm aperture sieve) for addition Note that all Fusarium species require iron, zinc, manga-to soil. The dried crushed substrate may be stored for up nese, copper, molybdenum and boron for proper vegeta-to 12 months at 2-5°C. tive growth and sporulation, but that excessive amounts In pot cultures or disease nurseries, the inoculum of- of these trace elements may be inhibitory (1310). Theten is mixed with soil at a rate of 1-2% of the final total trace element solution contains:volume before planting. In other cases, e.g., Liddell et al. Citric acid 5 g(1255), the inoculum is placed as a layer above the seed ZnSO4•6H2O 5 gand then covered with soil, and the seedlings grow Fe(NH4)2(SO4)2•6H2O 1 gthrough the inoculum layer. Infection may occur at this CuSO4•5H2O 250 mgtime, or it may be delayed by withholding moisture from MnSO4 50 mgthe layer that contains the inoculum. H3BO3 (Boric Acid) 50 mg This inoculum is particularly appropriate for species Na2MoO4•2H2O 50 mgthat do not form chlamydospores, but that persist in soil Distilled water 95 mlas hyphae in plant residues. This procedure has been used This recipe makes a nominal 100 ml. The citric acidwith numerous Fusarium species, e.g., Liddell (1253) should be added to the water first and dissolved com-and Liddell et al. (1255), including F. crookwellense, F. pletely to prevent other salts from precipitating. The solu-culmorum, F. oxysporum, F. pseudograminearum, F. so- tion may be filter-sterilized, although this is not neces-lani, and F. verticillioides. It also is suitable for studies sary. More commonly this solution is stored in a cappedwith a number of fungal genera, e.g., Summerell et al. bottle unsterilized at 4°C indefinitely following the addi-(2081), including Cylindrocarpon, Pythium, Rhizoctonia, tion of approximately 1 ml of CHCl3 to the stock solu-and Sclerotium. tion. The CHCl3 forms a small ball at the bottom of the container and it should be possible to detect its presence by smelling the solution in the bottle. If during storage the smell disappears or the ball of CHCl3 shrinks signifi-
  20. 20. Media 11cantly or is no longer visible, then the stock solution is Inositol 4 gprobably no longer saturated with CHCl3, and additional Ca pantothenate 200 mgCHCl3 should be added. A monthly check usually suf- Choline•Cl 200 mgfices. Thiamine 100 mg Additional ingredients are added to the basal me- Pyridoxine 75 mgdium (see below), depending upon the experimental pro- Nicotinamide 75 mgtocol, prior to autoclaving and use. Adding a drop or two Ascorbic acid 50 mgof vegetable-dye based food coloring, available at the lo- Riboflavin 30 mgcal grocery store, to media before autoclaving allows p-aminobenzoic acid 5 mgmedia types to be distinguished based on color rather Folic acid 5 mgthan by relying on stripes on the side of a plate or differ- Biotin 5 mgences in the plugs for slants to make these distinctions. 50:50 ethanol:H2O to 1 LThe food coloring does not affect the morphology or The vitamin solution may be filter-sterilized, althoughgrowth rates of Fusarium spp. this is not necessary. More commonly though, this solu- tion is stored indefinitely in the dark (dark bottle or bottleMinimal Medium (MM). This medium is the standard wrapped in aluminum foil) in a tightly capped bottle un-Czapek-Dox formulation as modified by Correll et al. sterilized at 4°C following the addition of 1-2 ml of(402). This medium is made by adding 2 g/L NaNO3 and CHCl3 to the stock solution. The CHCl3 forms a small30 g/L sucrose to the basal medium prior to autoclaving. ball at the bottom of the container and it should be possi-Most Fusarium species grow well vegetatively on this ble to detect its presence by smelling the solution in themedium, but sporulation characters vary, which makes it bottle. If during storage the smell disappears or the ballunsuitable for routine taxonomic studies. As with PDA, of CHCl3 shrinks significantly or is no longer visible,the high level of sugar can cause problems with character then additional CHCl3 should be added to maintain adegeneration in some strains. In studies of vegetative saturated solution. A monthly check usually suffices.compatibility, this medium functions as the NO3 test me- Storage in the dark is important as some of the vitaminsdium. This medium can be used to identify strains with decompose when exposed to light. Choline and panto-nutritional auxotrophies (such strains will grow poorly if thentic acid must be added as salts to maintain pH and toat all), and is suitable as a liquid medium for studies of increase solubility. Nicotinic acid is added as nicotina-enzyme activity and for the isolation of nucleic acids. mide for the same reason. Some researchers add addi-Complete medium (CM). This medium is a semi- tional vitamins to this mixture, but we have not observedsynthetic version of the Czapek’s-Dox medium that we any cases in which such additions have significantly af-routinely use in our laboratories. To the basal medium fected experimental results.the following additions are made prior to autoclaving: Virtually all Fusarium species grow well vegeta- tively on CM, but sporulation characters vary, which Sucrose 30 g makes it unsuitable for growing cultures for routine taxo- NaNO3 2 g nomic studies. As with PDA, the high level of sugar can N-Z Amine 2.5 g cause problems with character degeneration in some Yeast extract 1 g strains. The availability of fixed nitrogen means that nit Vitamin stock solution 10 ml mutants (see section 5.3) can revert without a noticeableN-Z Amine is an amino acid digest that is used as a change in phenotype. nit mutants isolated from sectors onsource of amino acids and fixed nitrogen. Other hydro- chlorate-containing medium usually must be grown onlytic protein digests can be substituted for N-Z Amine at CM for preservation or to obtain large numbers of spores,a similar level of supplementation. We routinely use but in general their culture on this medium should be asBacto (Difco, Detroit, Michigan) Yeast Extract, but other limited as possible. Auxotrophic mutants that requireyeast extracts should perform similarly. Yeast extracts amino acids not represented in casein usually growvary somewhat from lot to lot. This variation can be par- poorly on CM, as they rely solely on the amino acids inticularly important for methionine and adenine auxo- the yeast extract for growth. Arginine is almost alwaystrophs, and samples from several lots should be tested to limiting, and, if required, must be added as a separatemake sure that an adequate level of the supplement is supplement. Virtually all other Fusarium strains, bothpresent before embarking on extensive studies of such prototrophs and auxotrophs, grow well on this medium.mutants with this medium. The vitamin stock solution is Chlorate Medium. This medium was designed for thecomposed of: recovery of nitrate non-utilizing (nit) mutants for use in
  21. 21. 12 Chapter 2vegetative compatibility tests (402, 1745). To the basal cal agar in nitrite medium with Noble agar will make itmedium the following additions are made prior to auto- easier to score the growth on this medium.claving: Media to Restrict Radial Growth of Colonies. Mini- Sucrose 30 g mal medium + tergitol and sorbose (MMTS) and Com- NaNO3 2 g plete medium + tergitol and sorbose (CMTS) were modi- L-asparagine 1.6 g fied by Bowden & Leslie (230) from the original recipe KClO3 15 g of Puhalla & Spieth (1746) to restrict radial growth ofA common level of chlorate in the medium is 1.5% (the colonies on agar plates, and to enable the isolation oflevel reflected in the recipe). This level is usually the numerous colonies from a single plate following a selec-minimum chlorate level that is effective for generating tive treatment, e.g., a mutagenesis experiment. Thesenit mutant sectors. This recipe has numerous variants. If media are made by substituting 30 g/L sorbose for the 30too few NitM sectors are recovered, then changing the g/L sucrose in the minimal or complete recipes givenfixed nitrogen source from asparagine to proline may above, and adding 0.5 ml/L tergitol (type NP-10). Ifhelp (1090). If colonies seem to grow slowly across the colonies in a more “pelleted” form are desired from aplate without generating sectors, then omitting the aspar- liquid culture, then similar changes can be made to theagine often helps to restrict colony growth sufficiently to liquid media (1216). The sorbose level used can rangepermit the identification of nit sectors. The chlorate level from 2-6% (20-60 g/L), and tergitol from 0.05-0.2% (0.5-also can be increased, with 2-3% chlorate commonly 2 ml/L). Either compound can be used alone, but a higherused in many labs. The maximum chlorate level is 6% concentration of the single compound is required to ob-(60 g KClO3/L). Plates with high levels of chlorate dry tain the same net effect on colony diameter. In someeasily and have a feathery pattern of crystal formation in cases a small amount of other sugars, e.g., 0.2% dextrosethe medium. Such plates should be discarded. A final al- and 0.2% fructose, also may be added to the medium toternative is to drop the pH of the medium, but not so far support initial growth and spore germination. Both inter-as to prevent the agar from gelling. Conditions developed specific and intra-specific variation for sensitivity to ter-for generating nit mutants in Fusarium often, but not al- gitol and sorbose are known, and the level of sorboseways, work with other ascomycete fungi e.g., Colleto- and/or tergitol used in the medium may need to be ad-trichum (250), Aspergillus (156, 557), and Verticillium justed to reflect this variation.(1126). The media developed for studies in Aspergilluscan be particularly useful for strains that sector relatively 2.6 Media for Sexual Crossespoorly on the chlorate media described here. Obtaining a sexual stage is essential for analyses of bio-Phenotyping Medium. Phenotyping medium was de- logical species in some heterothallic species. Many spe-veloped by Correll et al. (402) to distinguish the muta- cies will form perithecia and mature ascospores on CLA.tions that affect different portions of the nitrate assimila- Some investigators also have used cultures grown ontion pathway (see section 5.3). There are three forms of various natural substrates, e.g., rice straw (905) or mul-phenotyping medium that are named according to the ni- berry twigs (469) on water agar or PDA, to obtain thetrogen source that is added to basal medium before auto- sexual stage. For studies of development or for geneticclaving: ammonium (1.6 g/L ammonium tartrate), hy- studies, however, more perithecia with a larger numberpoxanthine (0.2 g/L hypoxanthine), and nitrite (0.5 g/L of ascospores usually are required. Two media com-NaNO2). All contain 30 g/L sucrose as the carbon source. monly are used for this purpose, carrot agar and V-8 juiceThe ammonium medium is the control. Strains that will agar. Fertility on either medium occasionally can be in-not grow on this medium have another nutritional re- creased by placing a piece of sterile filter paperquirement that needs to be diagnosed. Hypoxanthine me- (Whatman . 1) over a portion of the plate prior to in- №dium is used to differentiate NitM mutants, as these mu- oculation, and perhaps impregenating it with a drop ortants are unable to utilize hypoxanthine as a sole nitrogen two of linoleic acid (535).source due to a defective molybdenum co-factor that is Carrot Agar. Carrot agar was developed for fertilityshared by nitrate reductase and purine dehydrogenase studies of Fusarium species with a Gibberella sexual(1376). Nitrite medium can be problematic. Nitrite is po- stage (1090). The medium is prepared by washing, peel-tentially toxic to the cells. If they are supplied with too ing and dicing 400 g of fresh carrots. Place the carrots inmuch NO2 they will not grow due to NO2 toxicity, but if a flask in 400 ml of water and autoclave for 20 minutes.there is not enough NO2 then they will not grow due to N Blend the carrots in a blender until the mixture appearsinsufficiency. In some cases, replacing the bacteriologi- smooth and with no large lumps. Add an additional 500

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