Rhizoctonia leguminicola, which causes fungal blackpatch disease of legumes and other plants, produces slaframine and swainsonine that are largely responsible for causing salivation, lacrimation, frequent urination, and diarrhea in grazing animals including cattle, sheep, and horses. The original identification of R. leguminicola was based only on morphological characters of the fungal mycelia in cultures because of the lack of fungal genetic markers. Recent investigations suggested that R. leguminicola does not belong to genus Rhizoctonia and is instead a member of the ascomycetes, necessitating an accurate reclassification. The objective of this study was to use both genetic and morphological characters of R. leguminicola to find taxonomic placement of this pathogen within ascomycetes. Internal transcribed spacer region (ITS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) encoding gene were amplified from R. leguminicola isolates by PCR using universal primers and sequencing. Rhizoctonia leguminicola ITS and gpd sequences were aligned with other fungal sequences of close relatives, and phylogenetic trees were constructed using neighbor-joining and parsimony analyses. Rhizoctonia leguminicola isolates were clustered within a clade that contains several genera of ascomycetes belonging to the class dothideomycetes. We suggest that the fungus is misidentified in the genus Rhizoctonia and propose its reclassification in a new genus within the phylum Ascomycota.

The fungus Slafractonia leguminicola, the causal agent of blackpatch disease of legumes produces two mycotoxins slaframine and swainsonine, causing slobbers' symptoms and locoism of grazing animals, respectively. The genetics of this important fungus is poorly understood. This work aimed to develop a genetic transformation system and evaluate the efficacy of RNA interference (RNAi) in S. leguminicola. In this study, S. leguminicola was transformed using a PEG-mediated method with a fungal construct that carries a hygromycin resistance cassette. To assess the use of RNAi, a silencing construct pSilentPKS1-AS was constructed which includes inverted repeat transgenes of the polyketide synthase gene (pks1) that is involved in melanin biosynthesis. Transformation of S. leguminicola with the IRT pks1 vector decreased pks1 transcripts levels 82-92% in knockdown mutants when compared with the wild type and was accompanied with a reduction in melanin and swainsonine production. These results demonstrate that RNAi can be a useful tool for studying gene function in S. leguminicola.