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.