A new inoculation method for spores of Plasmodiophora brassicae in roots of Brassica napus

Clubroot disease, caused by the biotrophic protist Plasmodiophora brassicae Woronin, is a significant disease affecting Brassicaceous crops worldwide, including canola (Brassica napus). Genetic resistance is the most effective and widely used control strategy to manage this disease. Effective disease management, through the deployment of clubroot resistance varieties, requires germplasm evaluation, usually carried out by directly injecting P. brassicae spores next to the roots of young seedlings of B. napus, planting into artificially inoculated soils, or by dipping roots in a spore suspension. The soil injection and soil inoculation methods require a large amount of inoculum, and the root-dipping method is labour-intensive and time-consuming, making effective and uniform large-scale inoculation experiments challenging to perform. This study describes a method for P. brassicae clubroot inoculation that involves coculturing young seedlings with a clubroot spore suspension for 24 hours prior to transplanting them into the soil. Radicle-emerged (1–2 days old) or cotyledon-emerged (2–3 days old) seedlings of the susceptible B. napus cultivar DH12075 were cocultured with the spore suspension of the Pb3H pathotype and evaluated for gall formation. This new inoculation method produced a high number of severely infected plants, similar to those obtained with root dipping method. Germinated seed inoculated at the cotyledon-emergence stage produced a slightly higher number of infected plants compared to those inoculated at the radicle-emergence stage. The coculturing method enables rapid, synchronized, and effective inoculation of B. napus with the clubroot pathogen, supporting large-scale resistance screening and crop management.