A Multiplex Real-Time PCR Assay Differentiates Four Xanthomonas Species Associated with Bacterial Spot of Tomato

Bacterial spot of tomato, a major problem in many tomato production areas, is caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri. In order to detect and identify the bacterial spot pathogens, we evaluated a region of hrpB operon as a source for primers and probes for real-time polymerase chain reaction (PCR). A 420-bp fragment of the hrpB7 gene was amplified by PCR from 75 strains representing the four species. The PCR products were sequenced and phylogenetic analysis revealed that hrpB7 is highly conserved within each species, with a single-nucleotide polymorphism (SNP) among the X. vesicatoria strains. X. euvesicatoria and X. perforans varied by two SNP. Four probes and two primer sets were designed to target the four bacterial spot pathogens based on their hrpB7 gene sequences. In order to simultaneously detect the four bacterial spot pathogens, the four probes and two primer sets were optimized for a multiplex real-time TaqMan PCR assay. The optimized multiplex assay was determined to be highly specific to the four bacterial spot pathogens. Because the optimized multiplex assay facilitated the identification of each bacterial spot pathogen from pure cultures and infected plant tissue, it holds great potential as a diagnostic tool.