Pathogenicity and a TaqMan Real-Time PCR for Specific Detection of , a Bacterial Pathogen of Onions.

Bacterial diseases of onion are reported to cause significant economic losses. Brady, one of the pathogens causing the center rot on onions, has not yet been reported in Canada. We report the pathogenicity of on commercially available Canadian green onions (scallions). All -inoculated plants, irrespective of the inoculum concentration, exhibited typical leaf chlorotic discoloration on green onion leaves, which can reduce their marketability. Reisolation of from infected scallion tissues and reidentification by sequencing and phylogenetic analyses of the S gene suggest that the pathogen can survive in infected tissues 21 days after inoculation. This is the first report of as a potential pathogen of green onions. This study also reports the development and validation of a TaqMan real-time PCR assay targeting the S gene for reliable detection of in pure cultures and in planta. A 642-bp S gene fragment was targeted because it showed high nucleotide diversity and positively correlated with genome-based average nucleotide identity with respect to percent similarity index and identity of species. The assay specificity was validated using 61 bacterial and fungal strains. Under optimal conditions, the selected primers and FAM-labeled TaqMan probe were specific for the detection of nine reference strains by real-time PCR. The 52 strains of other spp ( = 25), non- spp. ( = 20), and fungi/oomycetes ( = 7) tested negative (no detectable fluorescence). Onion tissues spiked with , naturally infested onion bulbs, greenhouse infected green onion leaf samples, as well as an interlaboratory blind test were used to validate the assay specificity. The sensitivities of a 1-pg DNA concentration and 30 CFU are comparable to previously reported real-time PCR assays of other bacterial pathogens. The TaqMan real-time PCR assay developed in this study will facilitate reliable detection of and could be a useful tool for screening onion imports or exports for the presence of this pathogen.