Debunking the Myth of Fusarium poae T-2/HT-2 Toxin Production.

Fusarium poae is commonly detected in field surveys of Fusarium head blight (FHB) of cereal crops and can produce a range of trichothecene mycotoxins. Although experimentally validated reports of F. poae strains producing T-2/HT-2 trichothecenes are rare, F. poae is frequently generalized in the literature as a producer of T-2/HT-2 toxins due to a single study from 2004 in which T-2/HT-2 toxins were detected at low levels from six out of forty-nine F. poae strains examined. To validate/substantiate the observations reported from the 2004 study, the producing strains were acquired and phylogenetically confirmed to be correctly assigned as F. poae; however, no evidence of T-2/HT-2 toxin production was observed from axenic cultures. Moreover, no evidence for a TRI16 ortholog, encoding a key acyltransferase shown to be necessary for T-2 toxin production in other Fusarium species, was observed in any of the de novo assembled genomes of the F. poae strains. Our findings corroborate multiple field-based and in vitro studies on FHB-associated Fusarium populations which also do not support the production of T-2/HT-2 toxins with F. poae and therefore conclude that F. poae should not be generalized as a T-2/HT-2 toxin producing species of Fusarium.

Chromosome-level draft genome sequences of three isolates of the toxigenic fungus Claviceps purpurea showing structural rearrangements.

The whole genomes of three Claviceps purpurea strains were sequenced using Oxford Nanopore Technologies' MinION and assembled into complete, chromosome-level assemblies. The C. purpurea genome consists of eight conserved chromosomes, with evidence of inter-chromosomal structural rearrangements between strains.

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as "C16" are likely associated with pathogenicity. Presented here are the results of CRISPR-Cas9 gene-editing experiments disrupting the polyketide synthase and terpene synthase genes associated with the C16 biosynthetic gene cluster in F. graminearum. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products of the C16 biosynthetic gene cluster as the decalin-containing diterpenoid pyrones, FDDP-D and FDDP-E. Both decalin-containing diterpenoid pyrones were confirmed to be produced in wheat heads challenged with F. graminearum in growth chamber trials. The extent to which the F. graminearum C16 biosynthetic gene cluster is dispersed within the genus Fusarium is discussed along with a proposed role of the FDDPs as pathogen virulence factors.

Evolution of the Ergot Alkaloid Biosynthetic Gene Cluster Results in Divergent Mycotoxin Profiles in Claviceps purpurea Sclerotia.

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea.

Four phylogenetic species of ergot from Canada and their characteristics in morphology, alkaloid production, and pathogenicity.

Four ergot species (Claviceps ripicola, C. quebecensis, C. perihumidiphila, and C. occidentalis) were recognized based on analyses of DNA sequences from multiple loci, including two housekeeping genes, RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (TEF1-α), and a single-copy ergot alkaloid synthesis gene (easE) encoding chanoclavine I synthase oxidoreductase. Morphological features, ergot alkaloid production, and pathogenicity on five common cereal crops of each species were evaluated and presented in taxonomic descriptions. A synoptic key was also provided for identification.

Phylogeny of Canadian ergot fungi and a detection assay by real-time polymerase chain reaction.

The ergot disease of cereals has become increasingly important in agricultural areas of Canada since 1999. Generally, this disease is considered to be caused by Claviceps purpurea, but the taxonomy of Claviceps from these areas has not been well studied. The objectives of this study were (i) to determine the phylogenetic lineages (phylogenetic species) present in agricultural areas of Canada and (ii) to develop a molecular assay that can separate the lineages on crops from other lineages. Genetic diversity of Claviceps collected from agriculture areas in Canada were investigated using multilocus sequence typing. The loci sequenced include nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), partial fragments of translation elongation factor 1-α (TEF1), RNA polymerase II second largest subunit (RPB2), ß-tubulin (tubB), and two ergot alkaloid synthesis genes (easA, easE). Based on individual locus and concatenated alignments, phylogenetic analyses revealed seven lineages within the premolecular concept of C. purpurea, of which five corresponded with undescribed species (G2b and G4-7). Although lineages G2-7 had narrow host ranges, lineage G1 (= C. purpurea s.s.) had a broad host range that overlapped with other lineages. A molecular diagnostic quantitative polymerase chain reaction (qPCR) assay was developed and validated with 185 samples from a wide range of host plants and geographic origins, including 10 phylogenetic species in C. sect. Claviceps, 8 in C. sect. Pusillae, 1 in C. sect. Citrinae, and 1-2 species from Alternaria, Fusarium, and Penicillium. The assay can detect lineage G1 at a concentration of 7.5 pg/µL and distinguish it from other Claviceps species and lineages. This facilitates disease management by detecting the inocula from nonagriculture host plants.