Molecular Discrimination of Opposite Mating Type Haploids of Sporisorium scitamineum and Establishing Their Dimorphic Transitions During Interaction with Sugarcane

Sporisorium scitamineum , a biotrophic basidiomycete fungus, causes the ravaging sugarcane smut disease globally in almost all sugarcane growing countries. The fungus spans around three distinct developmental stages, viz., diploid teliospores, haploid sporidia and dikaryotic mycelia, during its life cycle. The yeast-like and non-pathogenic haploid sporidia represent two opposite mating types: MAT-1 and MAT-2 . Discrimination of these mating type haploids is essential for long-term preservation and maintenance of true-to-type isolate/pathotype, genetic investigations and to understand how the fusion of non-pathogenic, compatible mating type haploids turn into virulent dikaryotic mycelia. Hitherto, only the mating-based phenotyping assay is used for discrimination of opposite mating type haploids. In the present study, a quick and reliable PCR-based assay was developed for the first time for discriminating the distinct mating types ( MAT-1 and MAT-2 ) of the S. scitamineum by targeting the unique regions of bE / bW mating type genes. Among the different primer sets evaluated, two sets, viz., bE1F1/bE1-2R1 and bE2F2/bE1-2R1 that target bE mating type genes, precisely differentiated the MAT-1 and MAT-2 mating type haploids of two S. scitamineum isolates, Ss97009 (high virulent) and SsV89101 (less virulent). The pathogenicity of distinct developmental stages, viz., haploid sporidia, dikaryotic mycelia and teliospores, and the life cycle transitions of these two S. scitamineum isolates in planta were analyzed by phenotyping and microscopy, respectively. Quantitative real-time PCR was used to estimate the pathogen biomass of plants inoculated with individual haploid sporidia ( MAT-1 and MAT-2 ), mixture of haploid sporidia and teliospores of Ss97009 and SsV89101. These results altogether have substantiated the virulence nature of these two isolates and established the significance of fusion of opposite mating type haploids in causing smut disease in sugarcane. Overall, this simple and efficient PCR-based discrimination assay of haploids would serve as a valuable tool for genomic and functional studies of S. scitamineum.