John F. Leslie

The fusarium laboratory manual

Foreword Preface 1. Introduction Techniques and Methods 2. Media - Recipes and Preparation 2.1 Media for Growing and Identifying Fusarium 2.2 Supplementary Identification Media 2.3 Media for Isolating Fusarium 2.4 Media for the Preparation of Natural Inocula 2.5 Synthetic and Semi-synthetic Media 2.6 Media for Sexual Crosses 2.7 Sterilization of Media and Materials 3. Techniques for Recovering Fusarium 3.1 Collecting strategy(ies) 3.2 Isolation Techniques - Plants 3.3 Isolation Techniques - Soil 3.4 Isolation Techniques - Spore Trapping and Air Sampling 3.5 Seed Disinfestation 4. Techniques for Growing and Maintaining Fusarium 4.1 Vegetative Propagation 4.2 Preparing Cultures for Identification 4.3 Single Spore Subcultures 4.4 Mutagenesis 4.5 Culture Preservation 5. Vegetative Compatibility Groups (VCGs) 5.1 History of and Genetic Basis Underlying Vegetative Compatibility 5.2 Overall Strategy for Determining if Strains are Vegetatively Compatible 5.3 Recovering and Identifying nit Mutants 5.4 Typical Pairing Protocols 5.5 Common Trouble Spots - HSI, crn, and NitMs 5.6 Characterizing a Population with VCGs 6. Fertility Concepts 6.1 Heterothallic, Homothallic and Pseudohomothallic 6.2 Mating Type 6.3 Population Effects of Mating Type 6.4 Male, Female, and Hermaphrodite 6.5 Crossing Protocols 6.6 Developing Female-Fertile Tester Strains 6.7 Species Identification Through Sexual Crosses 7. Nucleic Acid Analyses 7.1 DNA Extraction and Purification 7.2 PCR - Mating-Type Alleles 7.3 Amplified Fragment Length Polymorphisms (AFLPs) 7.4 Sequence Analysis and Sequenced Loci 7.5 Genetic Maps Taxonomy and Identification of Fusarium 8. A Brief History of Fusarium Taxonomy 9. Species Concepts in Fusarium 9.1 Generic Problems in Speciation in Fusarium 9.2 Morphological Species Concepts 9.3 Biological Species Concepts 9.4 Phylogenetic Species Concepts 9.5 How Many Strains Make a Species? 9.6 Species Names 9.7 Subspecific Terminology 9.8 A Species Concept for Fusarium 10. Teleomorphs of Fusarium 10.1 Taxonomy of Teleomorphs 10.2 General Teleomorph Characters 10.3 Sexual Development and Differentiation 10.4 Spore Killer 10.5 Anamorph-Teleomorph Connections 11. Practical Approaches to Identification 11.1 Overall Identification Strategy 11.2 The Diseased Plant and Its Geographic Origin 11.3 Native and Agricultural Populations 11.4 Culture Preparation 11.5 The Essence of Morphological Identifications 11.6 Beyond Morphology - Sexual Cross Fertility 11.7 Beyond Morphology - Molecular Diagnostics 11.8 The Special Case of Fusarium oxysporum 11.9 Differences Between Temperate and Tropical Regions 11.10 Conclusions Species Descriptions 12. Morphological Characters 12.1 Macroconidia 12.2 Microconidia 12.3 Chlamydospores 12.4 Other Characters 12.5 Secondary Characters 13. Species Descriptions F. acuminatum F. acutatum F. andiyazi F. anthophilum F. armeniacum F. avenaceum F. aywerte F. babinda F. begoniae F. beomiforme F. brevicatenulatum F. bulbicola F. camptoceras F. chlamydosporum F. circinatum F. compactum F. concentricum F. crookwellense (F. cerealis) F. culmorum F. decemcellulare F. denticulatum F. dimerum F. dlamini F. equiseti F. foetens F. fujikuroi F. globosum F. graminearum F. guttiforme F. heterosporum F. hostae F. konzum F. lactis F. lateritium F. longipes F. mangiferae F. merismoides F. miscanthi F. musarum F. napiforme F. nelsonii F. nisikadoi F. nurragi F. nygamai F. oxysporum F. phyllophilum F. poae F. polyphialidicum F. proliferatum F. pseudoanthophilum F. pseudocircinatum F. pseudograminearum F. pseudonygamai F. ramigenum F. redolens F. sacchari F. sambucinum F. scirpi F. semitectum (F. incarnatum) F. solani F. sporotrichioides F. sterilihyphosum F. subglutinans F. succisae F. thapsinum F. torulosum F. tricinctum F. udum F. venenatum F. verticillioides References Index

Sexual Recombination in Gibberella zeae

ABSTRACT We developed a method for inducing sexual outcrosses in the homothallic Ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum). Strains were marked with different nitrate nonutilizing (nit) mutations, and vegetative compatibility groups served as additional markers in some crosses. Strains with complementary nit mutations were cocultured on carrot agar plates. Ascospores from individual perithecia were plated on a minimal medium (MM) containing nitrate as the sole nitrogen source. Crosses between different nit mutants segregated in expected ratios (3:1 nit(-):nit(+)) from heterozygous perithecia. Analysis of vegetative compatibility groups of progeny of two crosses indicated two and three vegetative incompatibility (vic) genes segregating, respectively. For rapid testing of sexual recombination between nit mutants, perithecia were inverted over MM to deposit actively discharged ascospores. Development of proto-trophic wild-type colonies was taken as evidence of sexual recombination. Strains of G. zeae group 2 from Japan, Nepal, and South Africa, and from Indiana, Kansas, and Ohio in the United States were sexually interfertile. Four group 1 strains were not interfertile among themselves or with seven group 2 strains. Attempts to cross G. zeae with representatives of F. acuminatum, F. avenaceum, F. culmorum, F. crookwellense, F. oxysporum, and three mating populations of G. fujikuroi were not successful.

A Genetic Map of Gibberella fujikuroi Mating Population A (Fusarium moniliforme)

We constructed a recombination-based map of the fungal plant pathogen Gibberella fujikuroi mating population A (asexual stage Fusarium moniliforme). The map is based on the segregation of 142 restriction fragment length polymorphism (RFLP) markers, two auxotrophic genes (arg1, nic1), mating type (matA+/matA-), female sterility (ste1), spore-killer (Sk), and a gene governing the production of the mycotoxin fumonisin B1 (fum1) among 121 random ascospore progeny from a single cross. We identified 12 linkage groups corresponding to the 12 chromosome-sized DNAs previously observed in contour-clamped homogeneous electric field (CHEF) gels. Linkage groups and chromosomes were correlated via Southern blots between appropriate RFLP markers and the CHEF gels. Eleven of the 12 chromosomes are meiotically stable, but the 12th (and smallest) is subject to deletions in 3% (4/121) of the progeny. Positive chiasma interference occurred on five of the 12 chromosomes, and nine of the 12 chromosomes averaged more than one crossover per chromosome. The average kb/cM ratio in this cross is approximately 32.

PCR-based identification of MAT-1 and MAT-2 in the Gibberella fujikuroi species complex.

ABSTRACT All sexually fertile strains in the Gibberella fujikuroi species complex are heterothallic, with individual mating types conferred by the broadly conserved ascomycete idiomorphsMAT-1 and MAT-2. We sequenced both alleles from all eight mating populations, developed a multiplex PCR technique to distinguish these idiomorphs, and tested it with representative strains from all eight biological species and 22 additional species or phylogenetic lineages from this species complex. In most cases, either an ∼800-bp fragment from MAT-2 or an ∼200-bp fragment from MAT-1 is amplified. The amplified fragments cosegregate with mating type, as defined by sexual cross-fertility, in a cross of Fusarium moniliforme (Fusarium verticillioides). Neither of the primer pairs amplify fragments from Fusarium species such as Fusarium graminearum, Fusarium pseudograminearum, andFusarium culmorum, which have, or are expected to have,Gibberella sexual stages but are thought to be relatively distant from the species in the G. fujikuroi species complex. Our results suggest that MAT allele sequences are useful indicators of phylogenetic relatedness in these and otherFusarium species.

Population differentiation and recombination in wheat scab populations of Gibberella zeae from the United States

In limited previous studies of the Ascomycete fungus Gibberella zeae in North America, the populations examined were genetically and phenotypically diverse and could be viewed as subsamples of a larger population. Our objective in this study was to test the hypothesis that a homogeneous, randomly mating population of G. zeae is contiguous throughout the central and eastern United States across a span of several years. We analysed presence/absence alleles based on amplified fragment length polymorphisms (AFLPs) at 30 loci, 24 of which are defined genetically on a linkage map of G. zeae, from > 500 isolates in eight field populations from seven states collected during the 1998, 1999 and 2000 cropping seasons. All these strains had AFLP profiles similar to those of standard isolates of G. zeae phylogenetic lineage 7. All the populations are genetically similar, have high genotypic diversity and little or no detectable genetic disequilibrium, and show evidence of extensive interpopulation genetic exchange. Allele frequencies in some of the populations examined are not statistically different from one another, but others are. Thus, the populations examined are not mere subsamples from a single, large, randomly mating population. Geographic distance and genetic distance between populations are correlated significantly. The observed differences are relatively small, however, indicating that while genetic isolation by distance may occur, genetic exchange has occurred at a relatively high frequency among US populations of G. zeae. We think that these differences reflect the time required for the alleles to diffuse across the distances that separate them, because relatively little linkage disequilibrium is detected either in the population as a whole or in any of the individual subpopulations.

Diversity of Epidemic Populations of Gibberella zeae from Small Quadrats in Kansas and North Dakota.

ABSTRACT Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight (FHB) of wheat and barley and has been responsible for several billion dollars of losses in the United States since the early 1990s. We isolated G. zeae from the top, middle, and bottom positions of wheat spikes collected from 0.25-m(2) quadrats during severe FHB epidemics in a single Kansas (KS) field (1993) and in a single North Dakota (ND) field (1994). Three amplified fragment length polymorphism (AFLP) primer pairs were used to resolve 94 polymorphic loci from 253 isolates. Members of a subset of 26 isolates also were tested for vegetative compatibility groups (VCGs). Both methods indicated high levels of genotypic variability and identified the same sets of isolates as probable clones. The mean number of AFLP multilocus haplotypes per head was approximately 1.8 in each population, but this value probably underestimates the true mean due to the small number of samples taken from each head. Isolates with the same AFLP haplotype often were recovered from different positions in a single head, but only rarely were such apparently clonal isolates recovered from more than one head within a quadrat, a pattern that is consistent with a genetically diverse initial inoculum and limited secondary spread. The KS and ND samples had no common AFLP haplotypes. All G. zeae isolates had high AFLP fingerprint similarity (>70%, unweighted pair group method with arithmetic means similarity) to reference isolates of G. zeae lineage 7. The genetic identity between the KS and ND populations was >99% and the estimated effective migration rate was high (Nm approximately 70). Tests for linkage disequilibrium provide little evidence for nonrandom associations between loci. Our results suggest that these populations are parts of a single, panmictic population that experiences frequent recombination. Our results also suggest that a variety of population sampling designs may be satisfactory for assessing diversity in this fungus.

FUSARIUM THAPSINUM (GIBBERELLA THAPSINA) : A NEW SPECIES IN SECTION LISEOLA FROM SORGHUM

A group of Fusarium strains first distin- guished by the production of a diffusing yellow pig- ment is now described as a separate species, Fusarium thapsinum. The teleomorph, Gibberella thapsina, can be formed under laboratory conditions by crossing strains of opposite mating type on carrot agar. Fusar- ium thapsinum was recovered from banana, maize, peanut and sorghum in Egypt, South Africa, the Phil- ippines, Thailand, and nine states in the United States. Members of this species are morphologically similar to Fusarium moniliforme (Gibberella fujikuroi mating population A), but the two groups are repro- ductively isolated and can be distinguished by other characters such as mycotoxins produced, isozyme polymorphism, electrophoretic karyotype, benomyl sensitivity, and differences in the sequence of the in- ternally transcribed spacer (ITS) region of the ribo-

Genetic Diversity and Fitness of Fusarium graminearum Populations from Rice in Korea

ABSTRACT Fusarium graminearum is an important fungal pathogen of cereal crops and produces mycotoxins, such as the trichothecenes nivalenol and deoxynivalenol. This species may be subdivided into a series of genetic lineages or phylogenetic species. We identified strains of F. graminearum from the Republic of Korea to lineage, tested their ability to produce nivalenol and deoxynivalenol, and determined the genetic composition and structure of the populations from which they were recovered. Based on amplified fragment length polymorphism (AFLP), PCR genotyping, and chemical analyses of trichothecenes, all 249 isolates from southern provinces belonged to lineage 6, with 241 having the nivalenol genotype and 8 having the deoxynivalenol genotype. In the eastern Korea province, we recovered 84 lineage 6 isolates with the nivalenol genotype and 23 lineage 7 isolates with the deoxynivalenol genotype. Among 333 lineage 6 isolates, 36% of the AFLP bands were polymorphic, and there were 270 multilocus haplotypes. Genetic identity among populations was high (>0.972), and genotype diversity was low (30 to 58%). To test the adaptation of lineage 6 to rice, conidial mixtures of strains from lineages 3, 6, and 7 were inoculated onto rice plants and then recovered from the rice grains produced. Strains representing lineages 6 and 7 were recovered from inoculated spikelets at similar frequencies that were much higher than those for the strain representing lineage 3. Abundant perithecia were produced on rice straw, and 247 single-ascospore isolates were recovered from 247 perithecia. Perithecia representing lineage 6 (87%) were the most common, followed by those representing lineage 7 (13%), with perithecia representing lineage 3 not detected. These results suggest that F. graminearum lineage 6 may have a host preference for rice and that it may be more fit in a rice agroecosystem than are the other lineages present in Korea.

Toxicity, pathogenicity, and genetic differentiation of five species of Fusarium from sorghum and millet.

ABSTRACT Fusarium isolates recovered from sorghum and millet are commonly identified as F. moniliforme, but with the recognition of new species in this group, the strains given this name are being re-evaluated. We analyzed five strains each from five Fusarium species (F. andiyazi, F. nygamai, F. pseudonygamai, F. thapsinum, and F. verticillioides) often associated with sorghum and millet for their ability to produce fumonisin and moniliformin, their toxicity to ducklings, and their ability to cause disease on sorghum seedlings in vitro. These species can be distinguished with isozymes (fumarase, NADP-dependent isocitrate dehydrogenase, and malate dehydrogenase) and with banding patterns resulting from amplified fragment length polymorphisms. Two species, F. pseudonygamai and F. thapsinum, produced high levels of moniliformin, but little or no fumonisins, and were consistently highly toxigenic in the duckling tests. Two species, F. verticillioides and F. nygamai, produced high levels of fumonisins, but little or no moniliformin, and also were toxigenic in the duckling tests. F. andiyazi produced little or no toxin and was the least toxigenic in the duckling tests. In sorghum seedling pathogenicity tests, F. thapsinum was the most virulent followed by F. andiyazi, then F. verticillioides, and finally F. nygamai and F. pseudonygamai, which were similar to each other. Thus, these five species, which would once have all been called F. moniliforme, differ sufficiently in terms of plant pathogenicity and toxin production profile, that their previous misidentification could explain inconsistencies in the literature and differences observed by researchers who thought they were all working with the same fungal species.

One fungus, one name

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.