Exploitation of the Xanthomonas Repetitive Intergenic Consensus Sequence for the Development of Efficient SCAR Markers for Detection of Xanthomonas citri pv. punicae in Field Samples of Bacterial Blight-infected Pomegranates

Bacterial blight in pomegranates (Xanthomonas citri pv. punicae (Xcp)), has become a severe concern in recent years. Hence, a molecular analysis of selected Xcp isolates and field samples from different locations was conducted. PCR amplification of the Xanthomonas Repetitive Intergenic Consensus (XRIC) box yielded 216 bp and 159 bp size bands in all Xcp as well as X. citri pv. citri (Xcc) isolates as well as infected field samples. The 16S rRNA gene and XRIC (216 bp) Xcp amplicons shared the highest sequence similarity with various Xcp and Xcc accessions. Two Xcp-specific SCAR primers were designed from in silico PCR simulation studies. SCAR Xcp1-20 and Xcp133-152 primers amplified a 152 bp band in all Xcp isolates (except one) as well as in infected pomegranate samples, while Xcc isolates yielded a 371 bp band. SCAR XcpF/R amplified a 200 bp band in all infected pomegranate samples as well as seven of eight Xcp isolates, while the Xcp Pune-Daund isolate, along with both Xcc isolates, amplified a 350 bp band. KM-gyrB primers amplified a 491 bp band in all Xcp isolates; and a 375 bp band in both Xcc isolates, as well as in 12 of 20 plant samples, with two yielding other-sized bands. Xac-rpf-specific primers amplified an expected 581bp band in all Xcp and Xcc isolates, as well as 14 out of 20 field samples. It can be concluded that the XRIC box amplification and sequencing method enables the rapid identification of various Xanthomonas species. Additionally, both SCAR primers developed can be directly used to identify the bacterial blight-infected field samples, facilitating rapid Xcp detection.