Detection of xanthomonas citri subsp. Citri A^*, A^W and x. fuscans subsp. aurantifolii B, C using PCR and real-time PCR

Citrus bacterial canker (CBC) is an economically important disease in many citrus producing regions, and several canker-causing pathotypes have been characterized within the genus Xanthomonas pathogens. In view of the potential for economic losses and the quarantine significance of the pathogens, rapid and reliable pathogen diagnostic methods are needed for controlling this disease. In this study, we report novel molecular markers for Xanthomonas strain-specific detection by polymerase chain reaction (PCR). The primer sets XcAsF/XcAswR, XaBF/ XaBR, and XaCF1/XaCR1 specifically differentiated Xanthomonas citri subsp. citri A^* and A^w, X. fuscans subsp. aurantifolii B and X. f. subsp. aurantifolii C strains, respectively. In addition, real-time PCR (qPCR) assays were conducted to analyze the sensitivity, specificity and efficiency of the designed primers by estimating the minimum quantity of bacterial cells required for detection. With qPCR analysis, it was possible to detect a range of one to 1 × 10⁷ bacterial cell(s), which will be helpful to detect the species at pre-symptom development stage of quarantine period of the plant. PCR/primer efficiency was also demonstrated by using raw bacterial cells, which will considerably reduce time and cost needed for genomic DNA isolation. The identified molecular markers and qPCR method will be useful for routine diagnosis of Xanthomonas species, which will improve the citrus canker disease management.