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Propidium monoazide-assisted PCR may quantify viable Plasmodiophora brassicae resting spores to the exclusion of non-viable resting spores.

Al-Daoud, F., Deora, A., Robson, J., Gossen, B.D., and McDonald, M.R. 2016. Propidium monoazide-assisted PCR may quantify viable Plasmodiophora brassicae resting spores to the exclusion of non-viable resting spores. Can. J. Plant Pathol. 38: 113.

Abstract

Clubroot of canola (Brassica napus L.) caused by Plasmodiophora brassicae Woronin is a major threat to canola production in the Canadian prairies and worldwide. This is partly due to the ability of P. brassicae resting spores to remain viable in soil for years. qPCR can be used to quantify soil-borne spores, however it amplifies DNA from viable and non-viable spores. Propidium monoazide (PMA) has been used in conjunction with qPCR (PMA-PCR) to prevent amplification of DNA from non-viable microorganisms. PMA is thought to penetrate damaged cells, and intercalate into DNA thereby inhibiting its amplification. The objective of this study was to assess the potential for using PMA-PCR to quantify viable spores of P. brassicae while excluding non-viable spores. Naturally-infested muck soil and resting spores isolated from clubbed roots of cabbage (B. oleracea var. capitata) were heat treated (80 °C for 10 min) to produce a mixture of viable and non-viable spores. Extracted spores were then treated with different PMA concentrations (40-120 µM) followed by qPCR analysis. Heat treatment did not affect spore numbers obtained from qPCR with no PMA. In contrast, PMA-PCR detected a reduction in the number of resting spores after heat treatment: 641-fold (99%) for spores extracted from clubs and 5-fold (79%) for spores extracted from soil. This study indicates that PMA-PCR may quantify viable resting spores of P. brassicae while excluding non-viable resting spores. Current research is comparing different manual extraction protocols for use with PMA-PCR, and comparing PMA-PCR with different vital stains.

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