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 a fungus namely Claviceps purpurea, but the taxonomy of this fungus and its relatives from these areas has not been well studied. The objectives of this study were: (i) to determine the phylogenetic lineages present in agricultural areas of Canada. A lineage represents a group within which all the individuals are more closely related to each other than to other individual outside the group. 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 multi-locus sequence typing. The loci sequenced include nuc rDNA ITS1-5.8S-ITS2 (ITS), partial fragments of translation elongation factor 1- (TEF1), RNA polymerase II second largest subunit (RPB2), β-tubulin (tubB), and two ergot alkaloid synthesis genes (easA, easE). Based on individual locus and concatenated alignments, phylogenetic analyses revealed seven lineages within the pre-molecular 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 qPCR assay was developed and validated with 185 samples from a wide range of host plants and geographic origins, including ten phylogenetic species in C. sect. Claviceps, eight in C. sect. Pusillae, one in C. sect. Citrinae, and one to two 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 non-agriculture host plants.