Molecular pathotyping platforms for the clubroot pathogen, Plasmodiophora brassicae

Abstract

Clubroot, caused by the soilborne pathogen Plasmodiophora brassicae, is a threat to cruciferous crops worldwide and an important disease of canola (Brassica napus L.) in Canada. At present, pathotypes of P. brassicae are distinguished phenotypically based on their virulence patterns on host differential sets, including the systems of Williams, Somé et al., the European Clubroot Differential set, and most recently, the Canadian Clubroot Differential and the Sinitic Clubroot Differential sets. While these are frequently used because of their simplicity of application, they are time-consuming, labor-intensive, and can lack sensitivity. Early and preventative pathotype detection is imperative to maximize productivity and promote sustainable crop production. The decreased turnaround time and increased specificity of molecular pathotyping will be valuable for the development of integrated clubroot management strategies, and interest in molecular approaches to complement phenotypic bioassays is increasing. In this study, two rapid and sensitive molecular P. brassicae pathotyping assays were developed, the first using RNase H2-dependent PCR (rhPCR) technology, and the second using a modified single base extension technique known as SNaPshot. Both assays clearly distinguished between pathotype clusters. The results correlated fully with whole genome sequencing data in silico for all 38 single-spore isolates of P. brassicae tested. Additional isolates from pathotyped clubroot galls and from samples in a single-blind test were also identified correctly. The rhPCR assay generated differentiating electrophoretic bands without non-specific amplification. The SNaPshot assay was able to detect down to a 10% relative allelic proportion in template (pathotype) mixtures with both single-spore and field isolates. Collectively, the results demonstrated that the rhPCR-based and single base extension assays developed in this study may be used as fast and reliable diagnostic tools to detect and distinguish between P. brassicae pathotype clusters. The ability to identify pathotypes in a rapid manner will aid in clubroot diagnosis and surveillance activities, complementing traditional bioassays.