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

Clubroot [Plasmodiophora brassicae] is an important disease of canola and other brassica crops. Accurate estimation of inoculum load in soil is important for evaluating producer risk in planting a susceptible crop, but also for evaluation of management practices such as crop rotation. This study compared five molecular techniques for estimating resting spores of this pathogen in soil: quantitative polymerase chain reaction (qPCR), competitive positive internal control PCR (CPIC-PCR), propidium monoazide PCR (PMA-PCR), digital droplet PCR (ddPCR) and loop-mediated isothermal DNA amplification (LAMP). For ddPCR and LAMP, calibrations were developed using soil that had been inoculated with a known number of resting spores. The comparison was carried out using soil samples collected from a long-term rotation study at Normandin, QC, with replicated plots representing 0-, 1-, 2-, 3-, 5- and 6-year break following susceptible canola infested with clubroot. CPIC-PCR and ddPCR provided repeatable estimates of resting spore numbers in soil compared with estimates from qPCR or LAMP alone. Each assay provided a similar pattern of spore concentration in soil, which supported the conclusion of a previous study at this site that resting spore numbers declined rapidly in the first 2 years after a susceptible crop, but much more slowly after that. Estimates of spore concentration from CPIC-PCR were very useful because they were corrected for the effects of PCR inhibitors. This was especially important in the 2 years following a crop of susceptible canola. PMA-PCR demonstrated that a large proportion of the DNA of the pathogen detected in the spring after a susceptible canola was from spores that were immature or not viable.