Quantification of Viable Cells of Pv. in Tomato Seed Using Propidium Monoazide and a Real-Time PCR Assay

pv. is a seedborne pathogen that causes bacterial speck disease in tomato. pv. is typically detected in tomato seed using quantitative real-time PCR (qPCR) but the inability of qPCR to distinguish between viable and nonviable cells might lead to an overestimation of viable pv. cells. In the present study, a strategy involving a propidium monoazide (PMA) pretreatment followed by a qPCR (PMA-qPCR) assay was developed for quantifying viable pv. cells in contaminated tomato seed. PMA could selectively bind to the chromosomal DNA of dead bacterial cells and, therefore, block DNA amplification of qPCR. The primer pair Pst3F/Pst3R was designed based on gene to specifically amplify and quantify pv. by qPCR. The PMA pretreatment protocol was optimized for selectively detecting viable pv. cells, and the optimal PMA concentration and light exposure time were 10 μmol liter and 10 min, respectively. In the sensitivity test, the detection limit of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated tomato seed was 10 CFU ml and 11.86 CFU g, respectively. For naturally contaminated tomato seed, viable pv. cells were quantified in 6 of the 19 samples, with infestation levels of approximately 10 to 10 CFU g. The results indicated that the PMA-qPCR assay is a suitable tool for quantifying viable pv. cells in tomato seed, which could be useful for avoiding the potential risks of primary inoculum sources from contaminated seed.