Differentiation between Botryosphaeria dothidea and Neofusicoccum spp. based on a single nucleotide polymorphism in the ITS region

Fungi belonging to the Botryosphaeriaceae family are widespread pathogens of many angiosperms, causing disease on various high value crops. The most important members of the family for the Greek region and other Mediterranean countries are Botryosphaeria dothidea, Neofusicoccum hellenicum, Neofusicoccum mediterraneum and Neofusicoccum parvum. The frequently concurrent isolation of Botryosphaeriaceae species from the same host, as well as the extensive host range of B. dothidea, necessitate the development of rapid and reliable detection methods. This study presents a new and robust molecular diagnostic tool, in the form of a PCR method based on primers designed on an SNP (single nucleotide polymorphism) located in the ITS region (Internal Transcribed Region) of B. dothidea and Neofusicoccum species. SNP primers constructed with or without added mismatch nucleotides were combined with the same upstream universal primer to generate distinct amplicons. When evaluated in PCR assays, mismatched primers were found to have the highest differentiation capability. The potential for further development of SNP assays in order to differentiate between species is being evaluated.