Cochliobolus miyabeanus species-specific identification using nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes associated with virulence to rice

Cochliobolus miyabeanus causes brown spot disease of rice. For necrotrophic Cochliobolus spp., some small molecule products of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are known pathogenicity and/or virulence factors, often acting in a host-specific manner. Previous whole genome analyses across Cochliobolus species identified 11 NRPS- and 21 PKS-encoding genes in C. miyabeanus. First purpose of the current study was to examine the effect of deletion of ten of the discontinuously distributed genes (corresponding to JGI protein IDs 4446, 5802, 6546, 7015, 9064, 41693, 41753, 83551, 98843 and 107726) on virulence of C. miyabeanus to rice. A second purpose, based on results of the first, was to develop a PCR method to specifically identify C. miyabeanus and to distinguish it from other closely related Cochliobolus spp. We show that deletion of seven of the genes resulted in mutants that were reduced in virulence compared to the wild-type strain WK1C. Wild-type developed large dark brown necrotic lesions surrounded by chlorotic halos, while the mutants produced smaller, light brown spots with chlorosis. These results suggest that the products of these genes contribute to brown spot disease. In addition, we identified PKS and NPS SNPs that discriminate between C. miyabeanus and other species of Cochliobolus.