Poster: Biology & Disease Mgmt: Oomycetes
Estimates of viable resting spores of Plasmodiophora brassicae using propidium monoazide and qPCR
B. GOSSEN (1), F. Al-Daoud (2), M. McDonald (2) (1) Agriculture and Agri-Food Canada, Canada; (2) University of Guelph, Canada
Plasmodiophora brassicae [clubroot of Brassica spp.] persists in soil as long-lived resting spores. Quantitative polymerase chain reaction (qPCR) estimates of spore concentration do not distinguish between viable and non-viable spores. 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 resting spores 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 spores 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 resting spores were viable, but ≥ 74% of immature spores were not viable. This was consistent with substantially higher clubroot severity in plants inoculated with mature resting spores than immature spores. Heat treatment produced little or no change in estimates of mature spores with qPCR, but substantially reduced both estimates of spore concentration from PMA-PCR and clubroot severity in bioassays. For immature spores, PMA-PCR did not consistently demonstrate a heat treatment effect. We conclude that PMA allows for estimation of viable resting spores using qPCR.