Differentiating viable/non-viable and mature/immature Plasmodiophora brassicae resting spores using propidium monoazide-assisted qPCR.

Soil-borne resting spores (RS) of Plasmodiophora brassicae Woronin, the causal agent of clubroot of canola (Brassica napus L.) and other Brassicas, can remain viable for years. Soil spore load can be quantified using quantitative polymerase chain reaction (qPCR), but qPCR amplifies DNA from both viable and non-viable RS. Propidium monoazide (PMA) has been used in conjunction with qPCR (PMA-PCR) to prevent amplification of DNA from non-viable microorganisms. The objective of this study was to assess the potential of PMA-PCR to differentiate RS based on viability and maturity. Relatively immature and mature RS were isolated from younger and older clubs, respectively, and heat-treated at 80° C to produce a mixture of viable and non-viable spores. The spores were then treated with PMA (40 120 µM) followed by qPCR analysis. PMA-PCR analysis indicated that almost all of the non-heat-treated mature RS were viable whereas only 26% of non-heat-treated immature RS were viable, when compared to qPCR without PMA. After exposing mature and immature RS to the heat treatment, PMA-PCR detected a decrease of up to 98% in viable RS, when compared to qPCR without PMA. Bioassays using a susceptible canola cultivar confirmed that non-heat-treated and mature RS produced more clubroot than heat-treated or immature RS. These data indicate that PMA-PCR can be used to differentiate viable from non-viable and mature from immature RS.