Gossen: Using SNP Markers to Assess Genetic Variability of Plasmodiophora brassicae in Canada

Date: April 2018
Term: 2 years
Status: Completed
Researcher(s): Bruce Gossen, F. Yu, Agriculture and Agri-Food Canada, Saskatoon SK, M. R. McDonald, University of Guelph, S.E. Strelkov, University of Alberta
SaskCanola Investment: n/a
Total Project Cost: n/a
Funding Partners: n/a

Project Summary

Clubroot caused by Plasmodiophora brassicae has spread across central Alberta and to isolated fields in southern Alberta, Saskatchewan, Manitoba and North Dakota since it was first identified on canola in 2003. Researchers conducted a two-year project to attempt to trace the dispersal of the pathogen back to its source. In the study, researchers identified breakdown of genetic resistance to clubroot in canola outside of Alberta, and demonstrated that a similar breakdown is occurring wherever canola is grown in Canada, e.g., ON, QC, and even PEI. These studies will permit comparison of the genetic similarity among new pathotypes from widely different geographic locations.

Clubroot caused by Plasmodiophora brassicae has spread across central Alberta and to isolated fields in southern Alberta, Saskatchewan, Manitoba and North Dakota since it was first identified on canola (Brassica napus) in 2003. The pathogen had only been reported previously on canola in North America from Quebec. The source of the clubroot outbreak on canola has never been determined.

Researchers at Agriculture and Agri-Food Canada (AAFC) in Saskatoon conducted a two-year project (2016-17) to attempt to trace the dispersal of the pathogen back to its source. The objectives of the project were to use whole-genome sequencing to assess the genetic similarity among populations of P. brassicae from various collections and to examine pathogen variability at various levels, from a single clubbed root, to a field, to a region and then among regions. These analyses will be used to determine whether the pathogen populations in Alberta, Saskatchewan, Manitoba and North Dakota represent one or more separate introductions.

The predominant pathotype in central Alberta is pathotype 3, but pathotype 5 is predominant in southern Alberta. It will also be used to compare the similarity of new collections (known as pathotype X) that are virulent on previously resistant canola cultivars with one another, with the original population(s) in Alberta, and populations at other sites in Canada to identify the origin of these isolates. Quantification of the genetic variability of P. brassicae from across Canada may also provide insights into the durability of resistance by providing a basis for comparison against variability estimates reported from other parts of the world.

For the project, researchers collected isolates of P. brassicae from across Canada, including several examples of breakdown of genetic resistance from Alberta and from other sites across Canada, as well as from recent infestations identified in Saskatchewan, Manitoba and North Dakota. Other collections were obtained from Wisconsin, California and even China. The project experienced delays in accessing collections from Alberta, so not all of the new X pathotypes were available for comparison. In total, more than 80 collections are now available at Saskatoon. Containment facilities at the Saskatoon Research Centre were used for studies of unique or foreign collections of the pathogen, and some of the work with local collections was conducted at the University of Guelph.

Researchers assessed several DNA extraction protocols to develop material for submission for sequencing. The DNA extraction protocols and kits that are normally used for molecular techniques, such as PCR, did not produce sufficient quantities of DNA for whole-genome sequencing. However, an older extraction technique, CTAB, was identified that generally produced adequate quantities of DNA from dried clubs. Researchers validated this approach for production of DNA libraries and developed a DNA extraction protocol for P. brassicae suitable for whole genome sequencing.

Clubroots of inoculated Brassica rapa var. Chinensis seedlings after 6 weeks

A total of 52 isolates/collections were submitted to the NRC facility of PBI Saskatoon. These collections were compared to collections and single-spore isolates from Alberta using entire genetic sequencing of pathogen DNA. Twenty of the isolates/collections examined in an initial screening produced callus from infested roots of B. rapa (which does not generally produce callus). Callus infested with P. brassicae appeared to be an excellent source of DNA for the study. Microscopic analysis of callus tissue demonstrated that all of components of the pathogen's life cycle were present in the infested callus cells, including resting spores, sporangia, zoospores, and plasmodia.

The breakdown of genetic resistance to clubroot in Alberta has been well characterized. In the current study, researchers identified breakdown of genetic resistance to clubroot in canola outside of Alberta. This demonstrated that a similar breakdown is occurring wherever canola is grown in Canada, e.g., ON, QC, and even PEI. These studies will permit comparison of the genetic similarity among new pathotypes from widely different geographic locations.

Researchers also identified that dual cultures of P. brassicae with host callus cells were effective for DNA sequencing. Once the data has been analyzed, which will take several months using high-speed computers and specialized software, it will be possible to compare the genetic similarity among isolates at many spacial levels: field, region, province, country and continent. This information will be shared with provincial extension specialists and directly with producers on clubroot.ca and through a close partnership with the Canola Council of Canada.

Two weeks old dual callus culture of Brassica rapa

var. Chinensis and plasmodiophora brassicae

Scientific publications.

Al-Daoud, F., Gossen, B.D., and McDonald, M.R. 2018. A shift in the pathotype of Plasmodiophora brassicae at a site in Ontario. Can. J. Plant Pathol. 40:. (in press).

Sedaghatkish, A. Al-Daoud, F. Lee, S.H., Zwiazek, J.J., Gossen, B.D., and McDonald, M.R. 2018. The effect of selected mycorrhizae fungi on clubroot of canola. Can. J. Plant Pathol. 40:. (in press).

Al-Daoud, F., Gossen, B.D., and McDonald, M.R. 2016. Change in virulence of Plasmodiophora brassicae at a site in Ontario. Can. J. Plant Pathol. 38: 541.

Yu, F., Zhang, X., Huang, Z., Song, T., Chu, M., Falk, K.C., Gossen, B.D., Deora, A., McDonald, M.R., and Peng, G. 2016. SNP markers for clubroot resistance gene CR01 based on RNA sequencing. Can. J. Plant Pathol. 38: 133.

Full Report PDF: n/a