Hierarchical genetic variation of Botryosphaeriaceae species associated with decline and dieback of grapevine in south-eastern Australia

Background and Aims: Species of Botryosphaeriaceae have been reported in association with various grapevine diseases, such as trunk diseases and fruit rots worldwide. Their distribution, however, has not been thoroughly studied at the genetic level. The aim of this study was to evaluate genetic variation within four species of Botryosphaeriaceae isolated from south-eastern Australian vineyards at several geographical scales. Methods and Results: Fungal isolates were taken using a hierarchical methodology. A range of polymerase chain reaction (PCR) primers, including a selected set of six inter-simple sequence repeat primers, the intron splice junction R1 primer and the M13 core sequence microsatellite primer, were selected after preliminary screening. A total of 171 isolates of the Botryosphaeriaceae was assessed, comprising 127 Diplodia seriata, 23 Neofusicoccum parvum, 18 Botryosphaeria dothidea and 3 Lasiodiplodia theobromae isolates from three closely located regions. From these, 280 highly reproducible polymorphic bands were detected across all isolates following PCR. Intra-and inter-specific differences were visualised as a dendrogram and a principle coordinates plot. Cluster analysis with the integrated locus matrix separated the isolates into four distinguishable groups according to species (cophenetic correlation = 0.97). Conclusions: Our study indicates that genetic differentiation by region was evident within D. seriata, and there was variation in genotype between vineyards in several instances. Isolates of N. parvum showed greater homogeneity between vineyards and regions, while B. dothidea showed homogeneity between regions from which they were isolated. Significance of the Study: The degree of variation in populations of Botryosphaeriaceae in vineyards indicated in this study can be helpful in the future for controlling the spread of the pathogen in Australian vineyards and can be used to guide sampling in future surveys and population studies of the genus. The unique banding patterns of each species detected in this study can be utilised in the future for designing species-specific primers, which would enable in-field real-time detection of Botryosphaeriaceae without destructive sampling.