Quantitative real-time polymerase chain reaction for bacterial enumeration and allelic discrimination to differentiate Xanthomonas strains on citrus

Quantitative real-time polymerase chain reaction (QRT-PCR) was developed for identification and enumeration of bacteria in citrus plant samples infected with Xanthomonas axonopodis pvs. citri and citrumelo, the cause of citrus bacterial canker (CBC) and citrus bacterial spot (CBS), respectively. Three sets of primers based on the pathogenicity gene (Pth) in X. axonopodis pv. citri, a ribosomal gene in X. axonopodis pv. citrumelo, and the leucine-responsive regulatory protein (lrp) in both pathovars were combined with TaqMan probes and applied for specific strain detection and quantification. Calibration curves for bacterial abundance in plant samples obtained with the three primer-probe combinations were congruent with colony counts on plates of semiselective medium in most of the cases. However, apparent overestimation of bacterial cells by QRT-PCR indicated the presence of nonculturable or nonviable cells in some samples. In addition to quantification, the lrp primers and probes permitted differentiation by allelic discrimination of Xanthornonos strains infecting citrus tissues. This technique is based on the utilization of two probes that detect a single nucleotide difference in the target sequence between different strains and was validated with a collection of cultured Xanthomonas strains as well as tissue with CBC and CBS lesions. Allelic discrimination is demonstrated to be a more specific and sensitive protocol than previously developed PCR-based methods for strain identification and quantification.