Multiplex PCR for simultaneous identification of Ralstonia solanacearum and Xanthomonas perforans

Ralstonia solanacearum is a causative agent of bacterial wilt in many economically important crops, and Xanthomonas perforans is the causal organism of bacterial spot, one of the most important diseases of vegetables. A multiplex PCR protocol has been developed for the simultaneous, specific and rapid identification of R. solanacearum and X. perforans in plant materials. Species-specific primers RS-F-759 and RS-R-760 for R. solanacearum, RST2 and RST3 for X. perforans were used for identification of both pathogens at primer concentrations of 1:4 by optimization of multiplex PCR at annealing temperature of about 61 ± 1 °C. With these primer sets, specific amplification of 281- and 840-bp PCR products was obtained for R. solanacearum and X. perforans, respectively. The multiplex PCR assay was validated with susceptible plants mechanically inoculated with both the pathogens; specific PCR products confirmed the presence of R. solanacearum and X. perforans. The multiplex PCR is valuable in identification as well as primary screening of cultivars of both pathogens. The present study is a rapid and easy method for early identification of pathogens from asymptomatic and symptomatic plant materials.