Development of simple and quick DNA release protocol for conventional PCR based detection of Xanthomonas axonopodis pv. punicae causing bacterial blight in pomegranate

Abstract An economical, simple, rapid, and culture independent method was developed for routine analyses and detection of Xanthomonas axonopodis pv. punicae (Xap) that causes bacterial blight in pomegranate. Five DNA release buffers (B1-B5) were optimized for extracting bacterial genomic DNA (gDNA) directly from (a)symptomatic pomegranate leaves followed by conventional polymerase chain reaction (PCR)-based detection of Xap. B1, B3, and B4 were found suitable to release gDNA, which was subjected to PCR using universal primers for 16S rRNA and rpsL genes, and pathogen specific xopQ primers. DNA released from B1 and B4 successfully produced amplicons of expected sizes. Additional analyses found that DNA released using B4 buffer was stable up to 45 days at −20 °C/−80 °C and 35 days at 4 °C and 8-800 pg DNA could be detected by the PCR-based assay. B4 was further validated for versatility by extracting DNA of Xanthomonas spp. causing citrus canker and sorghum shoot stripe disease.