A high‐throughput turbidimetric method for quantitative preparation of Plasmodiophora brassicae inoculum for bioassays

Abstract

Clubroot caused by Plasmodiophora brassicae is one of the major diseases on cruciferous crops. This disease has been a problem all over the world and led to serious economic losses in cruciferous crop production. The improvement of clubroot management is dependent on the effectiveness of bioassays on pathogenicity or plant resistance.P. brassicae resting spore inoculum prepared from clubroot tissues was widely used in bioassays. Traditionally, resting spore concentration was measured by visual counting with a haemocytometer; however, this method is time-consuming, labour intensive and with poor repeatability due to the tiny size of resting spores. In this study, we established a turbidimetric method to measure P. brassicae resting spore concentration of the inoculum. A regression curve was generated by plotting optical density of gradiently diluted resting spore suspension against the corresponding resting spore concentrations, which resulted in a logarithmic regression equation. This method was validated and proved to be robust and effective in determining resting spore concentration of different samples, and was further confirmed by bioassay of infection and clubroot development. In conclusion, we established a standard protocol to prepare P. brassicae inoculum and provided an effective method for resting spore concentration determination. The results of this study can be widely used in research activities on clubroot sustainable management when an effective assessment of P. brassicae biomass is needed.