PCR-RFLP analysis of Botryosphaeriaceae species isolated from the Aegean and Mediterranean vineyards

The classical identification of Botryosphaeriaceae fungi, causing dieback and local dead arms in many plant species, is very difficult and time-consuming. Some of the molecular tools, such as PCR using species-specific primed, RFLP (Restricted Fragment Length Polymorphism) or gene sequencing are necessary for the fast identification of species. This study aims to contribute for the fast identification of some fungal species belonging Botryosphaeriaceae family reported up to now in Turkey vineyards. DNA was extracted from the 25 isolates obtained from the Aegean and Mediterranean Regions, and 4 different species (Botryosphaeria dothidea, Diplodia seriata, Lasiodiplodia theobromae, and Neofusicoccum parvum) and β-tubulin (TUB2) and ITS region (specific for Botryosphaeriaceae genus) on genomic DNA was amplified by polymerase chain reactions. This region was digested by five different restriction endonuclease enzymes (AluI, BsaHI, MboI, RsaI, TaqI) and band profiles were analyzed on an agarose gel (2%). According to the results of the analysis, the region, amplified with genus-specific primers, was not suitable for RFLP analysis. On the other hand, to discriminate these species, it was revealed that the β-tubulin gene region should be digested at least two different restriction enzymes. The enzyme BsaHI generated two different band sizes for B. dothidea and D. seriata (250 and 700 bp for the first; 400 and 600 bp for the second one) digesting β-tubulin gene of these species but this enzyme could not generate any discriminative bands for L. theobromae and N. parvum. However the enzyme TaqI could not discriminate B. dothidea and D. seriata but digest β-tubulin gene of L. theobromae and N. parvum at two points and it generated three different bands (for L. theobromae: 200-400-800 bp and for N. parvum: 200-400-650 bp)