Science Edition: Genetics

Multiple genes and crop rotation enhance the durability of clubroot resistance

Cultivar resistance is the key to managing clubroot, which continues to spread on the Canadian prairies. Recently in Alberta, 17 “new” pathogen pathotypes were identified and all appeared virulent to previous resistant cultivars in the marketplace. This erosion of single-gene resistance is also being reported across western Canada, including a new variant of pathotype 3A in Manitoba. To address this challenge, researchers investigated the efficacy and durability of canola lines carrying single and multiple clubroot resistance (CR) genes against “new” and “old” pathotypes. Researchers also assessed resistance durability under heavy (Alberta) and lighter (Saskatchewan and Manitoba) infestation situations against the predominant pathotype 3H to better understand the risk of resistance erosion in recommending a CR canola cultivar in regions with different clubroot pathogen inoculum load on the Prairies.

For this three-year study, 20 canola-quality B. napus inbred and hybrid lines carrying single, double and triple CR genes were produced in collaboration with Nutrien Ag Solutions. Advanced RNA sequencing methods were used to compare single- and double-gene lines to determine the modes of action of the different genes. All of the genotypes selected were resistant to the predominant pathotype 3H and other ‘old’ pathotypes (2, 5, 6, 8), however resistance to new pathotypes was unknown.

This shows the size of galls for canola hybrid lines carrying the two resistance genes Rcr1 and CRB exposed to a population of pathotype 5X in five simulated generational cycles under controlled-environment conditions. In this experiment, lines with only one gene had much higher disease severity across all five generations.

Westar and 45H29 (resistant to old pathotypes), both susceptible to the newly identified pathotype 5X, were included as controls. These lines were assessed for resistance against three field populations of the pathotype 5X using inoculum from Stephen Strelkov’s lab at the University of Alberta and under simulated intensive canola-growing conditions. The lines went through five generational cycles of exposure, with all root galls recycled back into the growth media at the end of each generation. The experiment lasted about 18 months for each pathotype and repeated once.

Researchers found that using a variety with double CR genes located on two different chromosomes of the A genome, A8 (CRB) and A3 (Rcr1 or CRM), provided moderate resistance against all 5X populations, as well as the immunity to the old pathotypes 2, 3, 5, 6 and 8. The study also showed that in response to 5X infection, many genes involved in host immunity pathways were more strongly activated in lines carrying these two CR genes, relative to those controlled by either of the single CR genes alone. Although the resistance provided by stacking two CR genes with different modes of action only increased to a moderate level, this resistance to 5X proved to be quite robust and more durable than expected. Over the five generations of exposure to the inoculum, the clubroot galls were smaller and fewer, and the inoculum levels did not increase. In some cases, inoculum levels even went down slightly.

The study also confirmed that higher inoculum loads tend to accelerate the resistance erosion, especially for canola varieties carrying only a single CR gene. Therefore, for producers with fields of heavy clubroot infestation, an extended rotation is necessary to protect the performance and durability of clubroot resistance. Previous research by Peng shows that a two-year break between canola crops can potentially reduce the pathogen inoculum in the soil by up to 90 per cent relative to one-year break. Current study results also suggest that even in areas with low levels of clubroot infestation, such as Saskatchewan and Manitoba, using CR varieties is recommended to keep inoculum levels low and delay potential outbreak. Researchers plan to see if this successful strategy of stacking two CR genes to manage 5X also applies to other new virulent pathotypes in future research.

Key result: A three-year study showed that including two resistant genes with different modes of action in clubroot resistant canola cultivars is more durable than single resistance genes against the new virulent pathogen pathotype 5X. To improve durability of resistance, growers also need a break of at least two years between canola crops.

Project title, Principal investigator: “Enhancing the durability of clubroot resistance with multiple genes,” Gary Peng, Agriculture and Agri-Food Canada, Saskatoon SK

Funding: AAFC, WGRF, SaskCanola

‘New’ clubroot pathogen pathotypes were always present

Objectives of this project were to assess the genetic similarity among populations of Plasmodiophora 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. 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 in Saskatchewan, Manitoba and North Dakota. Other collections were obtained from the U.S. and China. In total, more than 80 collections are now available at Saskatoon.

Researchers developed a DNA extraction protocol for P. brassicae suitable for whole genome sequencing. Next-generation sequencing, high-speed computers and specialized software were used to compare the genetic similarity among strains of the pathogen at many spatial levels: field, region, province, country and continent. It showed that the new virulent pathotypes that have been identified in Alberta and across Canada were almost certainly always present in the pathogen population, and so were able to increase as soon as genetic resistance to clubroot was added to canola cultivars.

Key Results: Whole-genome sequencing showed that the new, virulent pathotypes causing the breakdown in clubroot resistance were almost certainly always present in the pathogen population, and increased rapidly (positive selection) when the first generation of resistance to clubroot was added to canola cultivars.

Project title, Principal investigator: “Using SNP markers to assess genetic variability of Plasmodiophora brassicae in Canada,” Bruce Gossen, AAFC Saskatoon

Funding: Alberta Canola, SaskCanola, Manitoba Canola Growers

Black mustard provides new source of disease resistance

Canola with genetic resistance remains the most important management strategy for blackleg and clubroot, however virulent pathogen populations have recently been reported that are able to overcome the resistance of canola cultivars for both diseases. In this four-year study, researchers studied black mustard (B. nigra), which is highly resistant to canola diseases, to identify unique and novel sources of clubroot and blackleg resistance genes and transfer those genes into B. napus breeding lines.

Researchers identified and genetically mapped a novel clubroot resistance gene highly resistant to pathotypes 3 and 5X, and other new pathotypes. They then successfully transferred this resistance into the B. napus (BC4) from the B. nigra. For blackleg, BC4 populations with resistance to two highly aggressive isolates collected in the Prairies were developed. The molecular markers and the new B. napus breeding lines will be made available to canola breeders for new cultivar development.

Key Results: Researchers successfully transferred both clubroot and blackleg resistance into Brassica napus canola breeding lines from a unique black mustard (B. nigra) line.

Project title, Principal investigator: “Introgression of disease resistance from Brassica nigra into canola using new-type Brassica napus,” Fengqun Yu, AAFC Saskatoon

Funding: SaskCanola, ADF

Screening B. napus lines uncovers better clubroot resistance

Recent emergence of new clubroot pathotypes and the erosion of clubroot resistance (CR) is of concern. The most efficient way to develop canola resistant to new pathotypes is to identify resistance in the species and then transfer resistance into elite canola breeding lines by intraspecific crosses.

The aim of this three-year project was to identify new sources of B. napus for resistance to the pathotype known as 5X, map clubroot resistance (CR) genes and develop markers tightly linked to the genes for use in marker-assisted breeding. A total of 845 B. napus lines collected worldwide were tested for resistance to pathotype 5X. In addition, a total of 53 B. napus with different levels of resistance to pathotype 5X were screened against three new pathotypes 3A, 2B, and 3D based on the Canadian Clubroot Differential (CCD) in 2017, and five B. napus lines highly resistant to the new pathotypes were identified. Three out of these five lines were found to be resistant to all of the new pathotypes used for this study. One of the best resistant lines has been distributed to canola seed companies for incorporation into elite canola breeding lines.

Key Results: More than ten Brassica napus lines highly resistant to the clubroot pathotype 5X, and five to pathotypes 3A, 2B, 3D and 5X were identified in the project. One of the best lines has been distributed to canola seed companies and several double haploid (DH) segregating populations are under development.

Project title, Principal investigator: “Identification and genetic mapping of Brassica napus for resistance to pathotype 5X of Plasmodiophora brassicae,” Fengqun Yu, AAFC Saskatoon

Funding: SaskCanola, Alberta Canola, Manitoba Canola Growers, Growing Forward 2