Development and evaluation of PCR-based diagnostic assays for the bacterial speck and bacterial spot pathogens of tomato

Bacterial speck and bacterial spot lesions can easily be confused with each other and with those formed by other tomato pathogens. To facilitate disease diagnosis, we developed and evaluated polymerase chain reaction (PCR)-based lesion assays using crude DNA extracts and primer sets COR1/2 (bacterial speck) and BSX1/2 (bacterial spot). All 29 pathogenic Pseudomonas syringae pv. tomato strains tested produced a 689-bp amplicon with COR1/2; 28 of the 37 geographically diverse bacterial spot-causing xanthomonad (BSX) strains that were tested generated the 579-bp BSX1/2 amplicon. The detection limit with plant samples was 30 to 50 CFU/reaction. In a 6-year study, the COR1/2 PCR assay diverged from the culture-based classical assay for only 3 of 70 bacterial speck lesion samples collected from Ontario greenhouses and tomato fields; the BSX1/2 assay was positive for 112 of the 124 confirmed bacterial spot lesions sampled. The majority (66%) of the BSX strains isolated from these lesions belonged to group D; the 12 strains that were BSX1/2-negative belonged to group C. Group D strains produced a 425-bp PCR product with crude DNA extracts but a 579-bp product with purified DNA; the former was identical to the latter except that it was missing 150 bp from the middle of the 579-bp sequence.