Black rot, a disease of significance affecting vegetable Brassica crops, is primarily caused by the bacterium Xanthomonas campestris pv. campestris (Xcc). When the disease spreads extensively in the field, it can lead to substantial yield losses, particularly under favorable environmental conditions. Controlling the spread of this disease is challenging, and the primary approach involves utilizing resistant cultivars or disease-free seeds. Among the various methods available for identifying different Xcc races, Polymerase Chain Reaction (PCR)-based molecular markers have proven to be highly reliable. To date, the PCR method has successfully identified Xcc races 1 to 7. In this study, molecular markers were developed for races 4 and 9 through the sequencing and alignment of the whole genome sequences of Xcc races, closely related Xanthomonas campestris (Xc) pathovars, and two Xanthomonas species. These designed markers were subsequently validated by PCR with bacterial genomic DNA samples from Xcc races and 7 other bacteria. The results indicated successful amplification only for race 4 and race 9, yielding amplicon sizes of 1080 bp and 830 bp, respectively, while the other strains failed to amplify. Furthermore, the amplicons from races 4 and 9 were cloned and sequenced, confirming that both races exhibited matching sequences after alignment. Consequently, the molecular marker method offers a rapid and efficient means of differentiating between Xcc races 4 and 9 within a few hours, presenting itself as a viable alternative to conventional methods that rely on the use of differential cultivars of Brassicaceae for identifying Xcc races.

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