Establishment and Application of Rapid Quantitative Detection of Viable Plasmodiophora brassicae by PMAxx-qPCR Method

Plasmodiophora brassicae is an obligate endoparasite that causes clubroot disease, which is the most devastating soil-borne disease in brassica crops. The propidium monoazide xx (PMAxx) could selectively bind to the chromosomal DNA of dead spores and therefore block DNA amplification by real-time fluorescent quantitative PCR (qPCR). In the present study, a strategy involving a PMAxx pre-treatment followed by the qPCR (PMAxx-qPCR) assay was developed for quantifying viable spores of P. brassicae, so as to provide a basis for early detection and prevention measurement of cloobroot disease.

PMA and PMAxx with concentrations of 0, 5, 10, 20, 40 and 60 µmol·L^-1 were prepared, respectively, and were used to pre-treat P. brassicae prior to DNA extraction, followed by qPCR. The inhibitory effects of PMA and PMAxx on DNA amplification of P. brassicae dead spores were compared, and the optimal nucleic acid dye and concentration to distinguish between live and dead spores were determined. The illumination time was set as 0, 2, 5, 10, 15 and 20 min, respectively, and the optimal exposure time was optimized to establish a PMAxx-qPCR assay for selectively detection of viable spores of P. brassicae. The mixed suspensions with different ratios of dead and viable spores (0, 0.01%, 0.1%, 1%, 10%, 25%, 50%, 75% and 100% viable spores) were prepared to determine the suitability of PMAxx-qPCR assay for distinguishing viable and dead spores. The assay was also applied to quantitative detection of viable spores of P. brassicae in 25 field soil samples.

PMAxx showed a better discrimination effect than PMA on the viable and dead spores of P. brassicae. When the concentration of P. brassicae was 1×10⁸ spores/mL, the optimal PMAxx concentration and light exposure time were 4 μmol·L^-1 and 10 min, respectively. The amplification of dead spores could be inhibited effectively, and only the DNA of living spores was targeted for selective amplification. For pre-defined ratio of viable spores, there was a good linear relationship between the lg of the P. brassicae DNA concentration assessed by PMAxx-qPCR and the theoretical viability (R²=0.992). For soil samples, viable P. brassicae was quantified in 11 of 25 samples, with infestation levels of approximately 32.35-6.97×10³ fg·g^-1.

The established method could quantitatively detect the viable spores of P. brassicae, with advantages of rapid, efficiency and sensitivity, which could be useful for avoiding the inability of qPCR method to distinguish between viable and nonviable spores. Application of the assay may potentially improve P. brassicae control and disease management.