A Long-Amplicon Viability-qPCR Test for Quantifying Living Pathogens that Cause Bacterial Spot in Tomato Seed

Bacterial spot is one of the most serious diseases of tomato. It is caused by four species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated or infected seed can be a major source of inoculum for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. Current seed testing protocols rely mainly on plating the seed extract and conventional PCR; however, the plating method cannot detect viable but nonculturable cells, and the conventional PCR assay has limited capability to differentiate DNA extracted from viable or dead bacterial cells. To improve the sensitivity and specificity of the tomato seed testing method for bacterial spot pathogens, a long-amplicon quantitative PCR (qPCR) assay coupled with propidium monoazide (PMA-qPCR) was developed to quantify selectively the four pathogenic Xanthomonas species in tomato seed. The optimized PMA-qPCR procedure was evaluated on pure bacterial suspensions, bacteria-spiked seed extracts, and seed extracts of inoculated and naturally infected seed. A crude DNA extraction protocol also was developed, and PMA-qPCR with crude bacterial DNA extracts resulted in accurate quantification of 10⁴ to 10⁸ CFU/ml of viable bacteria when mixed with dead cells at concentrations as high as 10⁷ CFU/ml in the seed extracts. With DNA purified from concentrated seed extracts, the PMA-qPCR assay was able to detect DNA of the target pathogens in seed samples spiked with ≥75 CFU/ml (about 0.5 CFU/seed) of the viable pathogens. Latent class analysis of the inoculated and naturally infected seed samples showed that the PMA-qPCR assay had greater sensitivity than plating the seed extracts on the semiselective modified Tween Medium B and CKTM media for all four target species. Being much faster and more sensitive than dilution plating, the PMA-qPCR assay has potential to be used as a standalone tool or in combination with the plating method to improve tomato seed testing and advance the production of clean seed.