A duplex-PCR method for species- and pathovar-level identification and detection of the quarantine plant pathogen Xanthomonas arboricola pv. pruni

A PCR-based method was developed for the stone fruit quarantine pathogen Xanthomonas arboricola pv. pruni (Xap), which provides rapid, sensitive and specific in planta detection and isolate identification. Primers specific for Xap were identified using random amplified polymorphic DNA (RAPD). Simplex PCR with these primers had a limit of detection per PCR reaction of approximately 10 CFU for isolate cultures and 50 CFU for plant material when used on tenfold dilutions of isolate culture or genomic DNA extracted from spiked samples, respectively. The primers were adapted as a high-throughput single-step screening based on a digoxigenin-labeled DNA probe assay with a detection limit of 4 × 102 CFU from isolate cultures. A duplex-PCR method was designed that includes the pathovar-level with species-level primers based on species-specific regions of the quinate metabolic gene qumA, increasing diagnostic confidence and offering the first molecular test for all X. arboricola pathovars.