Protocol: rhPCR and SNaPshot assays to distinguish Plasmodiophora brassicae pathotype clusters

Abstract Background Clubroot of canola (Brassica napus), caused by the soilborne pathogen Plasmodiophora brassicae, has become a serious threat to canola production in Canada. The deployment of clubroot-resistant (CR) cultivars is the most commonly used management strategy; however, the widespread cultivation of CR canola has resulted in the emergence of new pathotypes of P. brassicae capable of overcoming resistance. Several host differential sets have been reported for pathotype identification, but such testing is time-consuming, labor-intensive, and based on phenotypic classifications. The development of rapid and objective methods that allow for efficient, cost-effective and convenient pathotyping would enable testing of a much larger number of samples in shorter times. The aim of this study was to develop two pathotyping assays, an RNase H2-dependent PCR (rhPCR) assay and a SNaPshot assay, which could quickly differentiate P. brassicae pathotypes. Results Both assays clearly distinguished between pathotype clusters in a collection of 38 single-spore isolates of P. brassicae. Additional isolates pathotyped from clubbed roots and samples from blind testing also were correctly clustered. The rhPCR assay generated clearly differentiating electrophoretic bands without non-specific amplification. The SNaPshot assay was able to detect down to a 10% relative allelic proportion in a 10:90 template mixture with both single-spore isolates and field isolates when evaluated in a relative abundance test. Conclusions This study describes the development of two rapid and sensitive technologies for P. brassicae pathotyping. The high-throughput potential and accuracy of both assays makes them promising as SNP-based pathotype identification tools for clubroot diagnostics. rhPCR is a highly sensitive approach that can be optimized into a quantitative assay, while the main advantages of SNaPshot are its ability to multiplex samples and alleles in a single reaction and the detection of up to four allelic variants per target site.