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 gerani- ums (Pelargonium spp.), both causing bacterial wilt and leaf spot. Asymptomatic infections are important reser- voirs 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 charac- terized PCR primer pair for Xcp that ampli®es a region of 200 bp. In addition, we designed a new primer pair speci®c for Rs that ampli®es a region of 822 bp. With these two primer pairs, we could detect either or both pathogens. As geranium tissue extracts frequently con- tain 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 `ampli®cation competenceʹ pri- mers, targeting a portion of the geranium 18 s rRNA gene, and generating a 494-bp ampli®cation product that con®rms ampli®cation 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 con- ®rms ampli®cation competence for each geranium sample.