Propidium Monoazide Improves Quantification of Resting Spores of Plasmodiophora brassicae with qPCR

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 quantify resting spores but does not distinguish between DNA of viable and nonviable spores. The impact of pretreating spores with propidium monoazide (PMA), which inhibits amplification of DNA from nonviable 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 for mature spores were similar, indicating that most of these spores were viable. However, only a small proportion (<26%) of immature spores were amplified in PMA-PCR. 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 less consistent for immature spores. To facilitate use of PMA-PCR on infested soil, a protocol for extracting spores from soil was developed that provided higher extraction efficiency than the standard methods.

Comparison of techniques for estimation of resting spores of Plasmodiophora brassicae in soil

“…Most of the spores that remained after a 2‐year break were viable and probably highly resilient. The CIPC‐PCR and 0 PMA‐PCR treatments were functionally very similar, so the difference observed between the results was unexpected, not observed in previous assessments (Al‐Daoud et al ., ) and not explained.…”

“…The protocol followed the method of Al‐Daoud et al . (). Briefly, 2 g of air‐dried soil from each soil bulk were diluted in 100 mL sterile deionized water and mixed in a blender at maximum speed for 2 min.…”

“…To address this issue, several chemicals have been identified that enter nonviable cells, bind to DNA (similar to a vital stain), and inhibit DNA amplification during PCR. A method using one of these chemicals, propidium monoazide (PMA), has been developed to provide estimates of viable spores of P. brassicae (Al‐Daoud et al ., ).…”

“…Initial studies of spore concentration were based on visual counts, using a high‐powered microscope, of spores extracted from soil. However, the spores are small and lack distinctive structures or ornamentation, so assessment was time‐consuming and highly variable (Al‐Daoud et al ., ).…”

Rapid and equipment‐free detection of Phytophthora capsici using lateral flow strip‐based recombinase polymerase amplification assay

“…Molecular detection of pathogens enables early detection of plant diseases and is increasingly applied in agriculture. Various methods based on polymerase chain reaction (PCR) have been developed to diagnose plant diseases, including conventional PCR, nested PCR, multiplex PCR and real-time PCR (Al-Daoud et al 2017;Lau and Botella 2017). However, these methods rely on specialized equipment, which limits their utility in the field.…”