Pushing the Limits: Multiplex Digital PCR for the Detection of Latent Infection by <i>Ralstonia solanacearum</i> Race 3 Biovar 2 in <i>Pelargonium</i> spp. Cuttings

Ralstonia solanacearum race 3 biovar 2 (R3Bv2; phylotype IIB, sequevars 1 and 2) is a bacterial plant pathogen internationally recognized as a quarantine pest due to its ability to cause potato brown rot and wilt in cool-temperate climates. It has recurrently been introduced to the United States through the importation of Pelargonium spp. (geranium) cuttings, where it causes latent infections that often harbor low pathogen titers which challenge the limits of detection by real time PCR. Here, we developed a multiplex digital PCR (dPCR) assay that incorporates four sets of primers and probes to allow for improved confidence in the detection of a low titer infection and for discrimination of the two sub-populations that comprise the R3Bv2 lineage (i.e. IIB-1 or IIB-2). Validation experiments indicated that the multiplex dPCR assay was approximately a magnitude of order more sensitive than real-time PCR, with a limit of detection corresponding to 0.3 genome equivalent copies per microliter in a plant DNA extraction. It also displayed 100% accuracy with a comprehensive panel of pure culture DNA (N = 139) spanning the diversity of the R. solanacearum species complex and related pathogens. Finally, the ability of the method to confirm R. solanacearum R3Bv2 in both naturally and artificially infected geranium stem cuttings with high real time PCR cycle quotient values was demonstrated. We expect that the multiplex dPCR assay developed here will strengthen agricultural biosecurity by improving confidence in the detection of low titer, latent infections by R. solanacearum R3Bv2 that challenge traditional diagnostic techniques.