Molecular marker development for specific amplification of Xanthomonas campestris pv. campestris race 8 causing black rot disease in Brassica crops

Black rot of Brassica vegetables is primarily attributed to the bacterium Xanthomonas campestris pv. campestris (Xcc). Given its global significance, there is a pressing need for rapid, efficient and specific methods to identify specific Xcc races instead of using differential cultivars, which is time-consuming and labor-intensive. Molecular markers have been developed to specifically detect Xcc races 1–7 and 9, but not for other races. In this study, we developed a molecular marker for Xcc race 8 by sequencing race 8 and aligning the sequence with available genomic sequences of Xcc from the National Center for Biotechnology Information (NCBI) database. The PCR primer set that we designed was validated using a PCR with genomic DNA for Xcc races, Xc pathovars, Xanthomonas species, and other bacteria to differentiate race 8, yielding an amplicon of 352 bp for race 8 and 1405 bp for races 1–7 and 9 and two other Xc pathovars, but none for the other samples. Cloning and sequencing of specific DNA fragments from race 8 provided additional proof of the marker’s specificity. The race-8-specific primer set provides a quick, simple inexpensive and highly reliable tool to identify Xcc race 8 and differentiate it from other Xcc races.