Multiplex PCR for specific identification and determination of mating type in Togninia minima (anamorphic Phaeoacremonium aleophilum), a causal agent of esca disease of grapevine

Abstract

Togninia minima is one of the fungi involved in esca disease of grapevine, worldwide. It has a biallelic heterothallic mating system. A multiplex PCR test was developed that can detect the species as well as the mating type. A T. minima-specific primer set, with expected amplicon size of 500 bp, was designed based on β-tubulin gene sequences. A previously designed degenerate primer set (NcHMG1 and NcHMG2) was successfully used to amplify a fragment of approximately 300 bp from the Mat1-2 gene of T. minima. The obtained sequence showed substantial homology to the Mat1-2 gene sequences of other related ascomycetes. A more specific primer set, with expected amplicon size of 230 bp, was designed based on the same Mat1-2 gene sequence. The specificity of the new primer set was verified on DNA extracted from a set of Phaeoacremonium and other fungal species frequently occurring on grapevine. Both primer sets were combined in a multiplex PCR for the simultaneous identification and determination of mating types of T. minima. A 500 bp amplicon was obtained from all available T. minima isolates and none from the other Phaeoacremonium spp. A 230 bp amplicon confirmed T. minima isolates that have the Mat1-2 allele. The species-specific β-tubulin-based primer set served as an internal control to confirm that the PCR reaction with the mating type primer set had worked properly. The efficacy of the multiplex test was evaluated on 31 isolates of T. minima from different vineyards in the Azarshahr region (East Azerbaijan province, Iran). Isolates of both mating types were found from the sampled areas; however, Mat1-2 isolates were more frequent than Mat1-1 isolates (19:12). This multiplex PCR assay developed can facilitate rapid screening of mating types in populations of T. minima.