Brett A. Summerell

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

Species and ecological diversity within the Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales)

The genus Cladosporium is one of the largest genera of dematiaceous hyphomycetes, and is characterised by a coronate scar structure, conidia in acropetal chains and Davidiella teleomorphs. Based on morphology and DNA phylogeny, the species complexes of C. herbarum and C. sphaerospermum have been resolved, resulting in the elucidation of numerous new taxa. In the present study, more than 200 isolates belonging to the C. cladosporioides complex were examined and phylogenetically analysed on the basis of DNA sequences of the nuclear ribosomal RNA gene operon, including the internal transcribed spacer regions ITS1 and ITS2, the 5.8S nrDNA, as well as partial actin and translation elongation factor 1-α gene sequences. For the saprobic, widely distributed species Cladosporium cladosporioides, both a neotype and epitype are designated in order to specify a well established circumscription and concept of this species. Cladosporium tenuissimum and C. oxysporum, two saprobes abundant in the tropics, are epitypified and shown to be allied to, but distinct from C. cladosporioides. Twenty-two species are newly described on the basis of phylogenetic characters and cryptic morphological differences. The most important phenotypic characters for distinguishing species within the C. cladosporioides complex, which represents a monophyletic subclade within the genus, are shape, width, length, septation and surface ornamentation of conidia and conidiophores; length and branching patterns of conidial chains and hyphal shape, width and arrangement. Many of the treated species, e.g., C. acalyphae, C. angustisporum, C. australiense, C. basiinflatum, C. chalastosporoides, C. colocasiae, C. cucumerinum, C. exasperatum, C. exile, C. flabelliforme, C. gamsianum, and C. globisporum are currently known only from specific hosts, or have a restricted geographical distribution. A key to all species recognised within the C. cladosporioides complex is provided.

Unravelling Mycosphaerella: do you believe in genera?

Many fungal genera have been defined based on single characters considered to be informative at the generic level. In addition, many unrelated taxa have been aggregated in genera because they shared apparently similar morphological characters arising from adaptation to similar niches and convergent evolution. This problem is aptly illustrated in Mycosphaerella. In its broadest definition, this genus of mainly leaf infecting fungi incorporates more than 30 form genera that share similar phenotypic characters mostly associated with structures produced on plant tissue or in culture. DNA sequence data derived from the LSU gene in the present study distinguish several clades and families in what has hitherto been considered to represent the Mycosphaerellaceae. In some cases, these clades represent recognisable monophyletic lineages linked to well circumscribed anamorphs. This association is complicated, however, by the fact that morphologically similar form genera are scattered throughout the order (Capnodiales), and for some species more than one morph is expressed depending on cultural conditions and media employed for cultivation. The present study shows that Mycosphaerella s.s. should best be limited to taxa with Ramularia anamorphs, with other well defined clades in the Mycosphaerellaceae representing Cercospora, Cercosporella, Dothistroma, Lecanosticta, Phaeophleospora, Polythrincium, Pseudocercospora, Ramulispora, Septoria and Sonderhenia. The genus Teratosphaeria accommodates taxa with Kirramyces anamorphs, while other clades supported in the Teratosphaeriaceae include Baudoinea, Capnobotryella, Devriesia, Penidiella, Phaeothecoidea, Readeriella, Staninwardia and Stenella. The genus Schizothyrium with Zygophiala anamorphs is supported as belonging to the Schizothyriaceae, while Dissoconium and Ramichloridium appear to represent a distinct family. Several clades remain unresolved due to limited sampling. Mycosphaerella, which has hitherto been used as a term of convenience to describe ascomycetes with solitary ascomata, bitunicate asci and 1-septate ascospores, represents numerous genera and several families yet to be defined in future studies.

One stop shop: backbones trees for important phytopathogenic genera: I (2014)

Many fungi are pathogenic on plants and cause significant damage in agriculture and forestry. They are also part of the natural ecosystem and may play a role in regulating plant numbers/density. Morphological identification and analysis of plant pathogenic fungi, while important, is often hampered by the scarcity of discriminatory taxonomic characters and the endophytic or inconspicuous nature of these fungi. Molecular (DNA sequence) data for plant pathogenic fungi have emerged as key information for diagnostic and classification studies, although hampered in part by non-standard laboratory practices and analytical methods. To facilitate current and future research, this study provides phylogenetic synopses for 25 groups of plant pathogenic fungi in the Ascomycota, Basidiomycota, Mucormycotina (Fungi), and Oomycota, using recent molecular data, up-to-date names, and the latest taxonomic insights. Lineage-specific laboratory protocols together with advice on their application, as well as general observations, are also provided. We hope to maintain updated backbone trees of these fungal lineages over time and to publish them jointly as new data emerge. Researchers of plant pathogenic fungi not covered by the present study are invited to join this future effort. Bipolaris, Botryosphaeriaceae, Botryosphaeria, Botrytis, Choanephora, Colletotrichum, Curvularia, Diaporthe, Diplodia, Dothiorella, Fusarium, Gilbertella, Lasiodiplodia, Mucor, Neofusicoccum, Pestalotiopsis, Phyllosticta, Phytophthora, Puccinia, Pyrenophora, Pythium, Rhizopus, Stagonosporopsis, Ustilago and Verticillium are dealt with in this paper.

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.

One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use.

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.

Myrtaceae, a cache of fungal biodiversity

Twenty-six species of microfungi are treated, the majority of which are associated with leaf spots of Corymbia, Eucalyptus and Syzygium spp. (Myrtaceae). The treated species include three new genera, Bagadiella, Foliocryphia and Pseudoramichloridium, 20 new species and one new combination. Novelties on Eucalyptus include: Antennariella placitae, Bagadiella lunata, Cladoriella rubrigena, C. paleospora, Cyphellophora eucalypti, Elsinoë eucalypticola, Foliocryphia eucalypti, Leptoxyphium madagascariense, Neofabraea eucalypti, Polyscytalum algarvense, Quambalaria simpsonii, Selenophoma australiensis, Sphaceloma tectificae, Strelitziana australiensis and Zeloasperisporium eucalyptorum. Stylaspergillus synanamorphs are reported for two species of Parasympodiella, P. eucalypti sp. nov. and P. elongata, while Blastacervulus eucalypti, Minimedusa obcoronata and Sydowia eucalypti are described from culture. Furthermore, Penidiella corymbia and Pseudoramichloridium henryi are newly described on Corymbia, Pseudocercospora palleobrunnea on Syzygium and Rachicladosporium americanum on leaf litter. To facilitate species identification, as well as determine phylogenetic relationships, DNA sequence data were generated from the internal transcribed spacers (ITS1, 5.8S nrDNA, ITS2) and the 28S nrDNA (LSU) regions of all taxa studied.

Biogeography and phylogeography of Fusarium: a review

Fusarium is a large, complex genus that causes a wide variety of plant diseases, produces a number of mycotoxins and is becoming increasingly recognized as a significant human pathogen. These fungi occur in ecosystems in all parts of the globe, which makes them useful as a model to better understand biogeographic processes affecting the distribution of fungi. Here we review the information available on the biogeography of different species and clades of Fusarium and some of the likely processes affecting dispersal and speciation.

Gibberella konza (Fusarium konzum) sp. nov. from prairie grasses, a new species in the Gibberella fujikuroi species complex

The Gibberella fujikuroi species complex (Fusarium section Liseola and allied taxa) is composed of an increasingly large number of morphological, biological and phylogenetic species. Most of the known species in this group have been isolated from agricultural ecosystems or have been described from a small number of isolates. We sampled Fusarium communities from native prairie grasses in Kansas and recovered a large number of isolates that superficially resemble F. anthophilum. We used a combination of morphological, biological and molecular characters to describe a new species, Gibberella konza (Gibberella fujikuroi mating population I [MP-I]), from native prairie grasses in Kansas. Although female fertility for field isolates of this species appears to be low, G. konza is heterothallic, and we developed reliably female fertile mating population tester strains for this species. The F. konzum anamorph is differentiated from F. anthophilum and from other Fusarium species in section Liseola by mating compatibility, morphology, AFLP fingerprint profile and differences in β-tubulin DNA sequence.

Introducing the Consolidated Species Concept to resolve species in the Teratosphaeriaceae

The Teratosphaeriaceae represents a recently established family that includes numerous saprobic, extremophilic, human opportunistic, and plant pathogenic fungi. Partial DNA sequence data of the 28S rRNA and RPB2 genes strongly support a separation of the Mycosphaerellaceae from the Teratosphaeriaceae, and also provide support for the Extremaceae and Neodevriesiaceae, two novel families including many extremophilic fungi that occur on a diversity of substrates. In addition, a multi-locus DNA sequence dataset was generated (ITS, LSU, Btub, Act, RPB2, EF-1α and Cal) to distinguish taxa in Mycosphaerella and Teratosphaeria associated with leaf disease of Eucalyptus, leading to the introduction of 23 novel genera, five species and 48 new combinations. Species are distinguished based on a polyphasic approach, combining morphological, ecological and phylogenetic species concepts, named here as the Consolidated Species Concept (CSC). From the DNA sequence data generated, we show that each one of the five coding genes tested, reliably identify most of the species present in this dataset (except species of Pseudocercospora). The ITS gene serves as a primary barcode locus as it is easily generated and has the most extensive dataset available, while either Btub, EF-1α or RPB2 provide a useful secondary barcode locus.