Estimation of viable resting spores of Plasmodiophora brassicae in a six-year crop rotation study using propidium monoazide-assisted PCR.
Al-Daoud, F., Robson, J., Pageau, D., Gossen, B.D., Dalton, J. A., and McDonald, M.R. 2017. Estimation of viable resting spores of Plasmodiophora brassicae in a six-year crop rotation study using propidium monoazide-assisted PCR. Can. J. Plant Pathol. 39: 114.
Resting spores of Plasmodiophora brassicae Woronin, the causal agent of clubroot in canola (Brassica napus L.) and other brassica crops, can remain viable in soil for many years. Quantitative PCR (qPCR) is used to quantify soil-borne resting spores, but it amplifies DNA from both viable and non-viable spores. However, pre-treatment with propidium monoazide (PMA) can suppress amplification of DNA from non-viable spores in subsequent qPCR (PMA-PCR). PMA penetrates non-viable cells, binds to the DNA when photo-activated, and prevents amplification. The objectives of this research were: 1) to develop a protocol for using PMA-PCR on soil samples, and 2) to compare qPCR and PMA-PCR analyses of soil samples from a six-year crop rotation field experiment at Normandin, Québec. A soil dilution technique, which involves simply mixing soil in water, retained up to 916-fold more spores than the standard sucrose solution-based techniques. This technique was used in conjunction with PMA-PCR to analyze the crop rotation soil samples. There were 2.0 x 106 ± 1.3 x 10^6 and 7.4 x 10^5 ± 2.6 x 10^5 spores g^-1 soil at year zero as estimated by qPCR and PMA-PCR, respectively. QPCR showed a quadratic decrease in spores over time with an 83% reduction in spore numbers after two years. PMA-PCR demonstrated a linear decrease in viable spores, with a 73% reduction in viable spores after three years. The half-life of spores at this site was estimated at 1.2 years by qPCR and 2.9 years by PMA-PCR. PMA-PCR is a useful technique to quantify viable resting spores, and reduce the need for resource-intensive bioassays.
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