A NEW ACCURATE GENOTYPING HRM METHOD FOR ALTERNARIA SPECIES RELATED TO FRUIT ROT DISEASES OF APPLE AND POMEGRANATE

Alternaria core rot and Alternaria black heart rot of apple and pomegranate fruit, respectively, are major pre- and postharvest diseases worldwide. However, it is very difficult to differentiate the rot related Alternaria species in the Alternaria complex as they are not always correlate to species-groups based upon morphological characteristics and due to the limited genetic variation these species exhibit among each other. Therefore, it is crucial to exploit novel assays towards the accurate identification and differentiation of these Alternaria species. We have developed, a real-time PCR assay [using species specific primers targeting the endopolygalacturonase ( EndoPG ) gene] combined with a high-resolution melting (HRM) curve analysis for discrimination of the 14 single nucleotide polymorphisms (SNPs)-based Alternaria haplotypes, which were assigned based on the aligned sequence profiles of 138 Alternaria spp. strains previously isolated from apple and pomegranate rotted fruit. This analysis specifically generated 14 unique HRM curve haplotype profiles among the Alternaria complex species tested. The results showed that HRM curve analysis allows the rapid and adequate identification and genotyping of the three Alternaria species ( A. alternata, A. tenuissima and A. arborescens ) responsible mostly for the apple and pomegranate fruit rot diseases.