Conventional and Real-Time PCR-Based Assay for Detecting Pathogenic Alternaria brassicae in Cruciferous Seed

Abstract

Alternaria brassicae is an important seedborne pathogenic fungus responsible for the black spot disease of crucifers. Sanitary control of commercial seed is necessary to limit the spread of this pathogen. Current detection methods, based on culture and morphological identification of the fungus, are time consuming, laborious, and not always reliable. Therefore, a polymerase chain reaction (PCR)-based assay was developed with A. brassicae-specific primers designed on the basis of the sequence of two clustered genes potentially involved in pathogenicity. Two sets of primers were selected for conventional and real-time PCR, respectively. In both cases, A. brassicae was specifically detected using DNA extracted from seed. The real-time PCR-based method presented here can be automated easily and preliminary results indicate that it is efficient for quantitative estimation of seed infection.