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

Clubroot ( Plasmodiophora brassicae ) is an important disease of canola ( Brassica napus ) 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 P. brassicae resting spores in soil: quantitative polymerase chain reaction ( qPCR ), competitive positive internal control PCR ( CPIC ‐ PCR ), propidium monoazide PCR ( PMA ‐ PCR ), droplet digital PCR (dd PCR ) and loop‐mediated isothermal DNA amplification ( LAMP ). For dd PCR and LAMP , calibrations were developed using spiked soil samples. The comparison was carried out using soil samples collected from a long‐term rotation study at Normandin, Québec, with replicated plots representing 0‐, 1‐, 2‐, 3‐, 5‐ and 6‐year breaks following susceptible canola infested with clubroot. CPIC ‐ PCR and dd PCR provided repeatable estimates of resting spore numbers in soil compared with estimates from qPCR or LAMP alone. CPIC ‐ PCR provided the most robust measurement of spore concentration, especially in the 2 years following a crop of susceptible canola, because it corrected for effects of PCR inhibitors. PMA ‐ PCR demonstrated that a large proportion of the DNA of P. brassicae detected in soil after the susceptible canola crop was derived from spores that were immature or otherwise not viable. 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 subsequently.