We demonstrate that native grass species from coastal and geothermal habitats require symbiotic fungal endophytes for salt and heat tolerance, respectively. Symbiotically conferred stress tolerance is a habitat-specific phenomenon with geothermal endophytes conferring heat but not salt tolerance, and coastal endophytes conferring salt but not heat tolerance. The same fungal species isolated from plants in habitats devoid of salt or heat stress did not confer these stress tolerances. Moreover, fungal endophytes from agricultural crops conferred disease resistance and not salt or heat tolerance. We define habitat-specific, symbiotically-conferred stress tolerance as habitat-adapted symbiosis and hypothesize that it is responsible for the establishment of plants in high-stress habitats. The agricultural, coastal and geothermal plant endophytes also colonized tomato (a model eudicot) and conferred disease, salt and heat tolerance, respectively. In addition, the coastal plant endophyte colonized rice (a model monocot) and conferred salt tolerance. These endophytes have a broad host range encompassing both monocots and eudicots. Interestingly, the endophytes also conferred drought tolerance to plants regardless of the habitat of origin. Abiotic stress tolerance correlated either with a decrease in water consumption or reactive oxygen sensitivity/generation but not to increased osmolyte production. The ability of fungal endophytes to confer stress tolerance to plants may provide a novel strategy for mitigating the impacts of global climate change on agricultural and native plant communities.

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Stress tolerance in plants via habitat-adapted symbiosis

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.

https://science.sciencemag.org/content/sci/317/5843/1400.full.pdf

The Fusarium graminearum Genome Reveals a Link Between Localized Polymorphism and Pathogen Specialization

Members of the species-rich Fusarium solani species complex (FSSC) are responsible for approximately two-thirds all fusarioses of humans and other animals. In addition, many economically important phytopathogenic species are nested within this complex. Due to their increasing clinical relevance and because most of the human pathogenic and plant pathogenic FSSC lack Latin binomials, we have extended the multilocus haplotype nomenclatural system introduced in a previous study (D. C. Chang, G. B. Grant, K. O9Donnell, K. A. Wannemuehler, J. Noble-Wang, C. Y. Rao, L. M. Jacobson, C. S. Crowell, R. S. Sneed, F. M. T. Lewis, J. K. Schaffzin, M. A. Kainer, C. A. Genese, E. C. Alfonso, D. B. Jones, A. Srinivasan, S. K. Fridkin, and B. J. Park, JAMA 296:953-963, 2006) to all 34 species within the medically important FSSC clade 3 to facilitate global epidemiological studies. The typing scheme is based on polymorphisms in portions of the following three genes: the internal transcribed spacer region and domains D1 plus D2 of the nuclear large-subunit rRNA, the translation elongation factor 1 alpha gene ( EF - 1 α), and the second largest subunit of RNA polymerase II gene ( RPB2 ). Of the 251 isolates subjected to multilocus DNA sequence typing, 191 sequence types were differentiated, and these were distributed among three strongly supported clades designated 1, 2, and 3. All of the mycosis-associated isolates were restricted to FSSC clade 3, as previously reported (N. Zhang, K. O9Donnell, D. A. Sutton, F. A Nalim, R. C. Summerbell, A. A. Padhye, and D. M. Geiser, J. Clin. Microbiol. 44:2186-2190, 2006), and these represent at least 20 phylogenetically distinct species. Analyses of the combined DNA sequence data by use of two separate phylogenetic methods yielded the most robust hypothesis of evolutionary relationships and genetic diversity within the FSSC to date. The in vitro activities of 10 antifungals tested against 19 isolates representing 18 species that span the breadth of the FSSC phylogeny show that members of this complex are broadly resistant to these drugs.

Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the fusarium solani species complex

The fungus Fusarium fujikuroi causes “bakanae” disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen.

/pdf/deciphering-the-cryptic-genome-genome-wide-analyses-of-the-20083t1bpx.pdf

Deciphering the cryptic genome : genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites

Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated with human or animal mycoses encountered in clinical microbiology laboratories. The database comprises partial sequences from three nuclear genes: translation elongation factor 1 (EF-1), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2). These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium. Phylogenetic analyses of the combined data set reveal that, with the exception of two monotypic lineages, all clinically relevant fusaria are nested in one of eight variously sized and strongly supported species complexes. The monophyletic lineages have been named informally to facilitate communication of an isolate’s clade membership and genetic diversity. To identify isolates to the species included within the database, partial DNA sequence data from one or more of the three genes can be used as a BLAST query against the database which is Web accessible at FUSARIUM-ID (http://isolate.fusariumdb.org) and the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center (http://www.cbs.knaw.nl/fusarium). Alternatively, isolates can be identified via phylogenetic analysis by adding sequences of unknowns to the DNA sequence alignment, which can be downloaded from the two aforementioned websites. The utility of this database should increase significantly as members of the clinical microbiology community deposit in internationally accessible culture collections (e.g., CBS-KNAW or the Fusarium Research Center) cultures of novel mycosis-associated fusaria, along with associated, corrected sequence chromatograms and data, so that the sequence results can be verified and isolates are made available for future study.

/pdf/internet-accessible-dna-sequence-database-for-identifying-j6e4nnd6go.pdf

Internet-Accessible DNA Sequence Database for Identifying Fusaria from Human and Animal Infections

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

John F. Leslie

Papers

The Fusarium Laboratory Manual