Duplicate of 38857 - Propidium monoazide improves quantification of viable resting spores of Plasmodiophora brassicae with qPCR.

Citation

Al-Daoud, F., Gossen, B.D., and McDonald, M.R. 2017. Propidium monoazide improves quantification of viable resting spores of Plasmodiophora brassicae with qPCR. Plant Dis. 101: 442–447. http://dx.doi.org/10.1094/PDIS-05016-0715-RE.

Plain language summary

Plasmodiophora brassicae, which causes clubroot of Brassica crops, persists in soil as long-lived resting spores. The molecular test that is often used to determine the concentration of resting spores does not distinguish between viable (infectious) and non-viable spores. Treating spores with propidium monoazide (PMA) to exclude non-viable spores from these assessments was assessed. Spores from immature and mature clubs were heat-treated, and then their viability was assessed using the molecular test with and without PMA and compared with the results of bioassays. The bioassays showed that, prior to heat treatment, most of the spores from mature clubs were infectious, only a small proportion of spores from immature clubs were infectious, and that heat treatment killed most of the spores. The heat treatments produced little or no change in estimates of mature spores using the assay without PMA, but spore estimates from the PMA treatment were substantially reduced. This demonstrated that treatment with PMA improved the accuracy of the molecular assessment. To facilitate using PMA-PCR on soil, a simple protocol for extracting spores from soil was developed that provided higher extraction efficiency than the standard sucrose-based protocols.

Abstract

Plasmodiophora brassicae, which causes clubroot of Brassica crops, persists in soil as long-lived resting spores. Quantitative polymerase chain reaction (qPCR) analysis is often used to determine the concentration of resting spores, but this approach does not distinguish between DNA of viable and non-viable spores. The impact of pretreating spores with propidium monoazide (PMA), which inhibits amplification of DNA from non-viable microorganisms, was assessed in several experiments. Spore suspensions from immature and mature clubs were heat-treated, then PMA-PCR analyses and bioassays were performed to assess spore viability. Prior to heat treatment, assessments comparing PMA-PCR to qPCR indicated that the majority of mature spores were amplified in PMA-PCR, indicating that most of these spores were viable. However, only a small proportion (<26%) of immature spores were amplified. Bioassays demonstrated that clubroot severity was much higher in plants inoculated with mature spores than with immature spores. Heat treatment produced little or no change in estimates of mature spores from qPCR, but spore estimates from PMA-PCR and clubroot severity in bioassays were both substantially reduced. Estimates of spore concentration with PMA-PCR were much less consistent for immature spores. To facilitate using PMA-PCR on soil, a simple protocol for extracting spores from soil was developed that provided higher extraction efficiency than the standard sucrose-based protocols.

Publication date

2017-02-01