Simultaneous PCR detection of the two major bacterial pathogens of geranium

Xanthomonas campestris pv. pelargonii (Xcp) and Ralstonia solanacearum (Rs) are the two most important bacterial pathogens of commercially cultivated geraniums (Pelargonium spp.), both causing bacterial wilt and leaf spot. Asymptomatic infections are important reservoirs of infections in commercial growing facilities. Our objective was to design a multiplex PCR (Polymerase Chain Reaction) assay to detect infection by either or both of these pathogens. We used a previously characterized PCR primer pair for Xcp that amplifies a region of 200 bp. In addition. we designed a new primer pair specific for Rs that amplifies a region of 822 bp With these two primer pairs. we could detect either or both pathogens. As geranium tissue extracts frequently contain inhibitors of the PCR process. a negative PCR Could result from either an accurate indication that the plant was pathogen-free or from a false negative assay. We therefore designed 'amplification competence' primers. targeting a portion of the geranium 18 s rRNA gene. and generating a 494-bp amplification product that confirms amplification competence and validates a negative assay result. Thus. the triple primer pair multiplex PCR screens for the two most important bacterial pathogens of geraniums simultaneously confirms amplification competence for each geranium sample.