Dataset Information

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.

SUBMITTER: Tso HH

PROVIDER: S-EPMC9250251 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Clubroot, caused by Plasmodiophora brassicae, is one of the most important diseases of canola (Brassica napus) in Canada. Disease management relies heavily on planting clubroot resistant (CR) cultivars, but in recent years, new resistance-breaking pathotypes of P. brassicae have emerged. Current efforts against the disease are concentrated in developing host resistance using traditional genetic breeding, omics and molecular biology. However, because of its obligate biotrophic nature, limited resources have been dedicated to investigating molecular mechanisms of pathogenic infection. We previously performed a transcriptomic study with the cultivar resistance-breaking pathotype 5X on two B. napus hosts presenting contrasting resistance/susceptibility, where we evaluated the mechanisms of host response. Since cultivar-pathotype interactions are very specific, and pathotype 5X is one of the most relevant resistance-breaking pathotypes in Canada, in this study, we analyze the expression of genes encoding putative secreted proteins from this pathotype, predicted using a bioinformatics pipeline, protein modeling and orthologous comparisons with effectors from other pathosystems. While host responses were found to differ markedly in our previous study, many common effectors are found in the pathogen while infecting both hosts, and the gene response among biological pathogen replicates seems more consistent in the effectors associated with the susceptible interaction, especially at 21 days after inoculation. The predicted effectors indicate the predominance of proteins with interacting domains (e.g., ankyrin), and genes bearing kinase and NUDIX domains, but also proteins with protective action against reactive oxygen species from the host. Many of these genes confirm previous predictions from other clubroot studies. A benzoic acid/SA methyltransferase (BSMT), which methylates SA to render it inactive, showed high levels of expression in the interactions with both hosts. Interestingly, our data indicate that E3 ubiquitin proteasome elements are also potentially involved in pathogenesis. Finally, a gene with similarity to indole-3-acetaldehyde dehydrogenase is a promising candidate effector because of its involvement in indole acetic acid synthesis, since auxin is one of the major players in clubroot development.