Estimates of viable resting spores of Plasmodiophora brassicae using propidium monoazide and qPCR.
Gossen, B.D., Al-Daoud, F., and McDonald, M.R. 2016. Estimates of viable resting spores of Plasmodiophora brassicae using propidium monoazide and qPCR. Phytopathology 106 (S4): 41.
Plasmodiophora brassicae [clubroot of Brassicas] persists in soil as long-lived resting spores (RS). Quantitative polymerase chain reaction (qPCR) estimates of spore concentration do not distinguish between viable and non-viable RS. Propidium monoazide (PMA) penetrates non-viable cells and binds to DNA when exposed to high-intensity light, and so inhibits amplification with qPCR. The impact of pre-treating RS with PMA and light prior to qPCR (PMA-PCR) was assessed. Spore suspensions from 6-wk-old (immature) and 9-wk-old (mature) clubs were heat-treated, and concentrations of RS were estimated using qPCR and PMA-PCR. Spore viability was assessed using bioassays on canola seedlings. Prior to heat treatment, comparison of estimates from qPCR and PMA-PCR indicated that all mature RS were viable, but ≥ 74% of immature RS were not viable. This was consistent with substantially higher clubroot severity in plants inoculated with mature RS than with immature RS. Heat treatment produced little or no change in estimates of mature RS with qPCR, but substantially reduced both estimates of spore concentration from PMA-PCR and clubroot severity in bioassays. For immature RS, PMA-PCR did not consistently demonstrate a heat treatment effect. We conclude that PMA improves quantification of viable RS with qPCR.
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