A molecular marker for the specific detection of new pathotype 5-like strains of Plasmodiophora brassicae in canola

Clubroot of crucifers, caused by Plasmodiophora brassicae, is managed in canola (Brassica napus) by the deployment of resistant cultivars. Recently, however, new strains of P.brassicae have been detected in Alberta, Canada, that can overcome this resistance. Some of these strains are classified as pathotype 5 on the differential system of Williams, but are distinguished by their ability to overcome host resistance. In order to expedite the identification of these new pathotype 5-like strains, three primer sets were developed based on the 18S-ITS region of the pathogen. With primers P5XF3 and P5XR3, a 127bp product was amplified from all new pathotype 5-like strains following optimized PCR analysis. A TaqMan probe-based quantitative assay was also developed. These protocols could be used to detect as little as 0.5pg P.brassicae DNA, and as few as 10(4)mL(-1) pathogen resting spores; infection of host tissues could be detected as soon as 4days after inoculation. The PCR and qPCR assays described in this study represent useful tools for the rapid and reliable diagnosis and quantification of new pathotype 5-like strains of P.brassicae.