Clubroot Resistant Canola Research Articles

A Hydroponic-Based Bioassay to Facilitate Plasmodiophora brassicae Phenotyping.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most devastating diseases affecting the canola/oilseed rape (Brassica napus) industry worldwide. Currently, the planting of clubroot-resistant (CR) cultivars is the most effective strategy used to restrict the spread and the economic losses linked to the disease. However, virulent P. brassicae isolates have been able to infect many of the currently available CR cultivars, and the options to manage the disease are becoming limited. Another challenge has been achieving consistency in evaluating host reactions to P. brassicae infection, with most bioassays conducted in soil and/or potting medium, which requires significant space and can be labor intensive. Visual scoring of clubroot symptom development can also be influenced by user bias. Here, we have developed a hydroponic bioassay using well-characterized P. brassicae single-spore isolates representative of clubroot virulence in Canada, as well as field isolates from three Canadian provinces in combination with canola inbred homozygous lines carrying resistance genetics representative of CR cultivars available to growers in Canada. To improve the efficiency and consistency of disease assessment, symptom severity scores were compared with clubroot evaluations based on the scanned root area. According to the results, this bioassay offers a reliable, less expensive, and reproducible option to evaluate P. brassicae virulence, as well as to identify which canola resistance profile(s) may be effective against particular isolates. This bioassay will contribute to the breeding of new CR canola cultivars and the identification of virulence genes in P. brassicae that could trigger resistance and that have been very elusive to this day.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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

BackgroundClubroot 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.ResultsBoth 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.ConclusionsThis 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.

Evaluation of Host Resistance, Hydrated Lime, and Weed Control to Manage Clubroot in Canola

Plasmodiophora brassicae Wor. is a soilborne parasite causing clubroot of canola (Brassica napus L.), a serious disease managed mostly by planting clubroot-resistant (CR) cultivars. Recently, new pathotypes of P. brassicae have emerged that overcome resistance, highlighting the need for a greater understanding of resistance stewardship and an integrated approach to clubroot management. Replicated field experiments were conducted in Edmonton, AB, in 2018 and 2019, to evaluate the effect of lime application and weed management on clubroot severity, crop yield and various growth parameters in clubroot-susceptible (CS) and CR canola cultivars. P. brassicae resting spore densities were also monitored by quantitative PCR for each treatment. When hydrated lime was applied to increase the soil pH from initial values of 5.2–5.5 to 7.2, clubroot severity decreased by 34–36% in the CS canola cultivar, while seed yield increased by 70–98%. The application of hydrated lime also resulted in resting spore densities that were 48–80% lower, relative to untreated controls, in plots where the CS cultivar was grown. Lime application or management of weeds did not significantly affect spore densities in plots with the CR cultivar. The results from the field trial suggest that the application of hydrated lime may be a useful strategy to manage clubroot, when used in combination with genetic resistance to reduce disease pressure.

Molecular evaluation of Plasmodiophora brassicae collections for the presence of divergent genetic pathogen populations before and after the release of clubroot resistant canola

Plasmodiophora brassicae Woronin, the cause of clubroot of crucifers, is now established across most of Alberta, Canada, and has become a major constraint in canola (Brassica napus L.) production. Disease control is achieved mainly by planting clubroot-resistant (CR) canola varieties. The first CR canola in Canada was released in 2009. In 2013, severe clubroot was detected on CR canola in several fields in Westlock County, Alberta, and found to be caused by a ‘new’ strain of P. brassicae termed ‘5X’. These strains have been shown to be genetically distinct from other P. brassicae strains in Alberta (‘population one’) and belong to a second population (‘population two’) of the pathogen. In this study, multiple molecular markers, specific for ‘population two’, were used to determine the occurrence of members of this population over time throughout central Alberta. Two hundred and twenty-four clubroot galls collected from 2005 to 2016 from non-CR canola varieties were examined. The presence of ‘population two’ DNA was detected at low levels in at least 47 of the galls, and it was the predominant population of P. brassicae in another 10 of the samples. These samples were found in Flagstaff County starting in 2008, Westlock County starting in 2009, the County of Vermillion River in 2011 and Red Deer County in 2014. Although relatively uncommon, members of this population were widespread, occurring at locations 168 km apart prior to the release of CR canola.

Pathotypes of Plasmodiophora brassicae collected from clubroot resistant canola (Brassica napus L.) cultivars in western Canada in 2017-2018

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important soilborne disease of canola (oilseed rape; Brassica napus L.). In Canada, clubroot management relies heavily on the planting of resistant cultivars, but since 2013, resistance has been broken in an increasing number of fields. Prior to the introduction of resistance, P. brassicae pathotype 3H, as defined on the Canadian Clubroot Differential (CCD) set, was predominant in Alberta. In testing of pathogen collections from 2014 to 2016, however, pathotype 3A was most common, indicating rapid shifts in the pathogen population. Up-to-date knowledge of pathotype composition is important for effective resistance breeding and stewardship. Therefore, clubbed roots were collected from clubroot-resistant canola crops from 166 fields in Alberta, Saskatchewan, and Manitoba in 2017 and 2018, with one isolate per field evaluated for pathotype designation on the CCD set and the differentials of Somé et al. Seventeen pathotypes were detected on the CCD set, including the previously reported pathotypes 3A, 3D, 3H, 5 L, 5X, 8E, 8N and 8P, plus the novel pathotypes 2C, 6D, 8D, 9A, 9B, 9C, 11A, 13A and 13B. Five pathotypes were identified on the hosts of Somé et al. including P1, P2, P3, P4 and P5, with P4 and P5 reported here from Canada for the first time. The majority of the isolates, representing 39 fields in 2017 and 92 fields in 2018, could overcome genetic resistance. The results suggest significant diversity in the virulence of P. brassicae populations and an increasing prevalence of resistance-breaking P. brassicae strains.

Development of molecular markers to identify distinct populations of Plasmodiophora brassicae

Clubroot, caused by Plasmodiophora brassicae, has become a serious production problem for canola (Brassica napus) on the Canadian prairies. Recently, clubroot-resistant (CR) canola cultivars have begun to show susceptibility to particular strains of P. brassicae. Using next generation sequencing data, a search was undertaken to identify genomic insertions unique to Canadian P. brassicae isolates belonging to two distinct populations. A total of 1353 candidate loci were detected that were present in Canadian P. brassicae isolates and were not homologous to sequences from any other species. Of these candidate loci, 600 appeared to be unique to isolates from the first population, which has been known to be present in Alberta for the longest period of time, while 85 appeared to be unique to isolates from the second population, in which virulence on CR canola cultivars was first detected. From these loci, 11 PCR markers were developed to identify P. brassicae isolates from these two populations. The primers did not amplify DNA from uninfected canola plants, soil samples, bacteria or fungal samples when tested. Assays Pb_pop1_52, Pb_pop1_60, Pb_pop1_255, Pb_pop1_327, Pb_pop1_595, Pb_pop1_673 were specific to population one, whereas primers Pb_pop2_2, Pb_pop2_26, Pb_pop2_33, Pb_pop2_57, and Pb_pop2_94 were specific to population two. The PCR assays were highly sensitive and could detect < 1 pg of target DNA. These methods should be useful for rapid diagnosis and identification of P. brassicae-infected canola plants.

Identification of lncRNAs in response to infection by Plasmodiophora brassicae in Brassica napus and development of lncRNA-based SSR markers.

Clubroot resistance in spring canola has been introgressed from different Brassica sources; however, molecular mechanism underlying this resistance, especially the involvement of long non-coding RNAs (lncRNAs), is yet to be understood. We identified 464 differentially expressed (DE) lncRNAs from the roots of clubroot-resistant canola, carrying resistance on chromosome BnaA03, and susceptible canola lines challenged with Plasmodiophora brassicae pathotype 3. Pathway enrichment analysis showed that most of the target genes regulated by these DE lncRNAs belonged to plant-pathogen interaction and hormone signaling, as well as primary and secondary metabolic pathways. Comparative analysis of these lncRNAs with 530 previously reported DE lncRNAs, identified using resistance located on BnaA08, detected 12 lncRNAs that showed a similar trend of upregulation in both types of resistant lines; these lncRNAs probably play a fundamental role in clubroot resistance. We identified SSR markers within 196 DE lncRNAs. Genotyping of two DH populations carrying resistance on BnaA03 identified a marker capable of detecting the resistance in 98% of the DH lines. To our knowledge, this is the first report of the identification of SSRs within lncRNAs responsive to P. brassicae infection, demonstrating the potential use of lncRNAs in the breeding of Brassica crops.

Virulence Spectrum of Single-Spore and Field Isolates of Plasmodiophora brassicae Able to Overcome Resistance in Canola (Brassica napus).

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important disease of canola (Brassica napus L.) that is managed mainly by planting clubroot-resistant (CR) cultivars. Field isolates of P. brassicae can be heterogeneous mixtures of various pathotypes, making assessments of the genetics of host-pathogen interactions challenging. Thirty-four single-spore isolates were obtained from nine field isolates of the pathogen collected from CR canola cultivars. The virulence patterns of the single-spore and field isolates were assessed on the 13 host genotypes of the Canadian Clubroot Differential (CCD) set, which includes the differentials of Williams and Somé et al. Indices of disease (IDs) severity of 25, 33, and 50% (±95% confidence interval) were compared as potential thresholds to distinguish between resistant and susceptible reactions, with an ID of 50% giving the most consistent responses for pathotype classification purposes. With this threshold, 13 pathotypes could be distinguished based on the CCD system, 7 on the differentials of Williams, and 3 on the hosts of Somé et al. The highest correlations were observed among virulence matrices generated using the three threshold IDs on the CCD set. Genetically homogeneous single-spore isolates gave a clearer profile of the P. brassicae pathotype structure. Novel pathotypes, not reported in Canada previously, were identified among the isolates. This large collection of single-spore isolates can serve as a reference in screening and breeding for clubroot resistance.

Changes in the range and virulence of Plasmodiophora brassicae across Canada

Plasmodiophora brassicae causes clubroot on Brassica vegetables and canola around the world. A diverse assemblage of new pathotypes have been identified on canola in the Canadian province of Alberta since 2012, but the pathotypes present in the rest of Canada have not been assessed in detail in recent years. Brassica vegetable and canola fields in Ontario were surveyed for clubroot in 2017–2019. The pathotype of isolates collected was determined using the Williams differential set. Clubroot samples or soil from six other Canadian provinces were also assessed. Clubroot appears to be spreading on canola in Ontario. Pathotype 6 was found only on vegetables and pathotype 2 was predominant on canola; pathotypes 2X and 3X were also identified on canola. The ‘X’ indicates collections that were virulent on the first generation of clubroot-resistant canola cultivars in Canada. Prior to 2012, the pathotypes in eastern Canada were 1, 2, 3, 4, 5, and 6 and the pathotypes in western Canada were 3, 5, and 8 with 6 found on vegetables in British Columbia. Additional virulent pathotypes 2X, 3X, and 5X were identified in eastern Canada and pathotype 9 was found in Manitoba. Two instances of rapid shifts in pathotype at research sites were demonstrated, one in Ontario (pathotype 6 to 2) and one in Quebec (pathotypes 2 and 5 to 2X and 5X).

Characterization of clubroot (Plasmodiophora brassicae) from canola (Brassica napus) in the Peace Country of Alberta, Canada

Clubroot of canola (Brassica napus L.), caused by Plasmodiophora brassicae Woronin, has spread to more than 3000 fields in Alberta, Canada, since it was first identified in 2003, but the disease had not been reported in the Peace Country Region in the northwest of the province. In 2017, 21 cases of clubroot were found in Big Lakes County, the southeastern-most district in this region. In 2018, an additional five cases were identified. Surveys of the infested crops in 2017 detected a clubroot disease index (DI) of < 10% in 15 fields and > 10% in the other six, while in 2018, the DI was < 5% in four fields and > 50% in one. Canola had been grown 3–6 times over the preceding 7–8 years in all fields for which rotation histories could be obtained. A subset of 23 P. brassicae field isolates was evaluated for pathotype classification on the hosts of the Canadian Clubroot Differential (CCD) set. Of the isolates collected in 2017, 11 were pathotype 3H, two each were pathotypes 5I and 8N, and three had novel virulence patterns designated 5A, 8A, and 8B. The five field isolates tested from 2018 included pathotypes 6C, 8A and 8 N, plus two isolates of the novel pathotype 8C. On the differential system of Somé et al., 17 isolates were classified as pathotype P2 and six as P3. As the CCD pathotype 8C is virulent on clubroot-resistant canola, integrated disease management strategies will be required for resistance stewardship in this region.

Effect of canola (Brassica napus) cultivar rotation on Plasmodiophora brassicae pathotype composition

In Canada, clubroot (Plasmodiophora brassicae) disease is managed mainly by planting clubroot resistant (CR) canola (Brassica napus). New pathotypes of P. brassicae have emerged recently, however, which are virulent on most CR canola cultivars. To understand the impact of cultivar rotation on pathotype abundance, greenhouse experiments were conducted in which different canola cultivar rotations were grown in a soil mix containing equal amounts of pathotypes 5X and 3, which are virulent and avirulent, respectively, on CR canola. The rotation treatments included: T1, the same susceptible cultivar planted over four cycles; T2, the same CR cultivar planted over four cycles; and T3, different CR cultivars planted in each cycle. Clubroot severity increased from cycles one to four in all treatments, with the exception of one CR cultivar in T3 that may carry a different source of resistance. Pathogen populations were recovered with a susceptible bait crop and pathotyped on the differentials of Williams plus a CR host (B. napus ‘Mendel’). The percentage of galls classified as pathotype 5X in T1 declined from 50% to 6.7% over the course of the experiment, while galls classified as pathotype 5X increased from 50% to 66.7% in both T2 and T3. Quantitative PCR analysis of the soil with pathotype 5X-specific primers generally confirmed an increase in 5X DNA. The results suggest that continuous planting of CR canola favours a rapid proliferation of virulent pathotypes of P. brassicae, as indicated by the increases in pathotype 5X observed in this study.

Suppression of Canola (Brassica napus) Resistance by Virulent Isolates of Plasmodiophora brassicae (Clubroot) During Primary Infection.

The planting of clubroot resistant (CR) canola (Brassica napus) is the most effective method to manage clubroot. Since 2013, many Plasmodiophora brassicae isolates capable of overcoming resistance have been detected, often in mixtures with avirulent isolates. To improve understanding of the effect of low concentrations of virulent isolates on host resistance, three CR canola cultivars (45H29, L135C, and L241C) were inoculated with pairs of isolates representing virulent/avirulent pathotypes (2*/2, 3*/3, and 5*/5) collected after or before the introduction of CR canola, respectively. Seven-day-old seedlings of each cultivar were incubated for 2 days in low concentrations (1 × 103 spores/ml) of the virulent isolates, followed by a second inoculation with a high concentration (1 × 107 spores/ml) of the avirulent isolates. Positive controls comprised seedlings inoculated with low concentrations of the virulent isolates followed by high concentrations of the virulent isolates (PC1) or only with high concentrations of virulent isolates (PC2). Negative controls comprised seedlings inoculated only with high concentrations of the avirulent isolates (NC1) or only with low concentrations of the virulent isolates (NC2). Clubroot severity was significantly higher in all nine experimental treatments (low virulent plus high avirulent) than in the negative control NC1 (high avirulent) but was lower in the experimental treatments than in the positive controls (PC1 and PC2). Low concentrations of virulent isolates alone (NC2) caused moderate clubroot. Disease severity correlated well with P. brassicae biomass in canola as determined by quantitative PCR analysis 28 to 35 days after inoculation. This study revealed that low concentrations of virulent isolates compromised canola resistance for infection by avirulent isolates.

Influence of resistant cultivars and crop intervals on clubroot of canola

Clubroot, caused by Plasmodiophora brassicae, is an important constraint on canola (Brassica napus) production in Canada. Rotations of clubroot-resistant (CR) canola cultivars in various sequences and planting intervals between canola with non-host crops and fallow periods were evaluated to determine their effects on clubroot severity and P. brassicae resting spore populations under field and micro-plot conditions. Under micro-plot conditions, the rotation sequences including CR canola, continuous fallow, and the non-host barley reduced gall weight by 63%–100% and clubroot severity by 34%–100% compared with continuous planting of susceptible canola. No visible clubroot symptoms developed following continuous fallow or the non-host crop. Under field conditions, clubroot severity was very high (78% disease index) in the continuous susceptible canola sequence. Most of the CR canola rotation sequences significantly reduced clubroot severity by 12%–23%, but continuous fallow, continuous barley, and alternating the CR canola cultivars ‘45H29’ or ‘73-47’ with ‘TC72429-10’ reduced clubroot severity by 32%–36%. A comparison of intervals between canola crops and four cropping sequences (continuous susceptible canola, alternating canola with barley or pea, a 2-yr non-host interval between canola crops, and a 3-yr non-host interval between canola crops) was conducted over 5 yr. A 2- or 3-yr non-host interval improved plant height, plant biomass, and seed yield, and reduced gall mass, P. brassicae propagules in the soil, and clubroot severity. A significant yield increase of more than 3600% was observed in a 3-yr non-host interval.

Plasmodiophora brassicae resting spore dynamics in clubroot resistant canola (Brassica napus) cropping systems

The soilborne pathogen Plasmodiophora brassicae, causal agent of clubroot of canola (Brassica napus), is difficult to manage due to the longevity of its resting spores, ability to produce large amounts of inoculum, and the lack of effective fungicides. The cropping of clubroot resistant (CR) canola cultivars is one of the few effective strategies for clubroot management. This study evaluated the impact of the cultivation of CR canola on P. brassicae resting spore concentrations in commercial cropping systems in Alberta, Canada. Soil was sampled pre‐seeding and post‐harvest at multiple georeferenced locations within 17 P. brassicae‐infested fields over periods of up to 4 years in length. Resting spore concentrations were measured by quantitative PCR analysis, with a subset of samples also evaluated in greenhouse bioassays with a susceptible host. The cultivation of CR canola in soil with quantifiable levels of P. brassicae DNA resulted in increased inoculum loads. There was a notable lag in the release of inoculum after harvest, and quantifiable P. brassicae inoculum peaked in the year following cultivation of CR canola. Rotations that included a ≥2‐year break from P. brassicae hosts resulted in significant declines in soil resting spore concentrations. A strong positive relationship was found between the bioassays and qPCR‐based estimates of soil infestation. Results suggest that CR canola should not be used to reduce soil inoculum loads, and crop rotations in P. brassicae infested fields should include breaks of at least 2 years away from B. napus, otherwise the risk of selecting for virulent pathotypes may increase.

Genotyping of Plasmodiophora brassicae reveals the presence of distinct populations

BackgroundPlasmodiophora brassicae is a soilborne pathogen of the family Brassicaceae and the causal agent of clubroot disease. In Canada, P. brassicae is now one of the most important constraints to canola (Brassica napus) production, and is managed mainly by the deployment of resistant cultivars. In recent years, however, new strains of the pathogen have emerged that are capable of overcoming host resistance, posing new challenges for disease management. Despite its economic significance, molecular studies of P. brassicae are rare, mainly because this microorganism cannot be cultured outside of its host.ResultsRestriction site-associated DNA sequencing (RADseq) was used to examine the genetic diversity within P. brassicae single-spore and field isolates collected from across Canada. The isolates included individuals that were either capable or incapable of causing disease on clubroot resistant canola cultivars. Over 8750 variants were identified through RADseq. Population analysis indicated that most isolates belonged to one of two distinct populations, corresponding with the ability of isolates to cause disease on resistant cultivars. Within each population, there were low levels of genetic diversity. One thousand and fifty of the genetic variants that distinguished the two populations were nonsynonymous, altering the coding sequences of genes.ConclusionThe application of RADseq revealed two distinct populations of P. brassicae in Canada, suggesting multiple introductions of the pathogen into the country. The genetic variation found here will be important for future research and monitoring of the pathogen.

Virulence and pathotype classification of Plasmodiophora brassicae populations collected from clubroot resistant canola (Brassica napus) in Canada

Clubroot, caused by Plasmodiophora brassicae Wor., is an important soilborne disease of canola (Brassica napus L.) in Canada that is managed mainly by planting clubroot-resistant (CR) cultivars. Populations of P. brassicae representing 106 fields in Alberta were obtained from galled roots of CR canola plants collected in 2014–2016 and characterized for virulence on seven CR canola cultivars. Sixty-one of these populations could overcome resistance in at least one CR cultivar and were evaluated further by inoculation on 13 Brassica hosts termed the Canadian Clubroot Differential (CCD) Set. The CCD Set included the differentials of Williams and Somé et al., selected hosts of the European Clubroot Differential Set, and the B. napus cultivars ‘Brutor’, ‘Mendel’, ‘Westar’ and ‘45H29’. Each unique virulence pattern on the CCD Set represented a distinct pathotype and was identified with a letter. Five reference isolates, obtained prior to the introduction of CR canola, also were assessed. A total of 17 pathotypes were detected using the CCD Set, compared with five pathotypes using the system of Williams and two with the system of Somé et al., suggesting that the CCD Set has a greater differentiating capacity. Pathotype A, a variant of pathotype 3 (as per Williams) which is able to overcome the resistance in CR B. napus, was predominant. The original pathotype 3, which is avirulent on CR canola, was classified as CCD pathotype H. An integrated strategy, combining other tools in addition to resistance, will be needed for the sustainable management of clubroot.

Metabolic cost of resistance in clubroot-resistant canola and napa cabbage

Genetic resistance is widely used to manage clubroot (Plasmodiophora brassicae) in canola (Brassica napus) and other brassica crops. Replicated field trials were conducted with three clubroot-resistant cultivars and one susceptible cultivar of either canola or napa cabbage (B. rapa) in southern Ontario in 2014 and 2015. These studies indicated that plant growth and development in resistant cultivars of both crops were often reduced at high concentrations of inoculum (resting spores) of the pathogen. In napa cabbage, leaf length was reduced by 31% in 2014 and 22% in 2015 at a site with high spore concentration relative to a site with lower concentration. In canola, height of resistant plants was reduced by 30%, biomass by 43%, and maturity was delayed at a site with high spore concentration relative to an adjacent site with lower spore concentration in 2014. However, there were no differences among sites in 2015. A growth room study demonstrated that plant height of a clubroot-resistant canola cultivar declined by 12–14% at high spore concentrations. Biomass of canola was also lower and maturity was delayed with increasing concentration of spores in some, but not all, field and laboratory studies. We suggest that the expression of resistance to clubroot in resistant cultivars can result in reduced plant growth and development in both B. rapa and B. napus, but that substantial reductions in plant growth and development occur only at high concentrations (> 106 g−1) of resting spores in soil.

The gene Cr811 is present exclusively in pathotype 5 and new emerged pathotypes of the clubroot pathogen Plasmodiophora brassicae

This study was conducted with the objective of finding genetic marker(s) to differentiate pathotypes of the clubroot pathogen Plasmodiophora brassicae. Ideally, such markers should be used as a diagnostic tool to identify or predict certain pathotypes. By using PCR with gene-specific primers, 117 non-housekeeping P. brassicae genes were investigated for their presence/absence in isolates representing pathotypes 2, 3, 5, 6 or 8 (Williams’ system), and in new strains of the pathogen recently identified from Alberta, Canada, which are highly virulent on clubroot resistant canola (Brassica napus) cultivars. One gene, named Cr811, was present in isolates of pathotype 5 and in a new strain of P. brassicae that also is classified as pathotype 5 on the system of Williams, but causes severe clubroot on resistant canola. Three additional newly identified virulent populations, similar to pathotype 3, also were tested and found to carry Cr811. In contrast, the Cr811 gene was absent in all isolates representing the original pathotypes 2, 3, 6 and 8. Quantitative real-time PCR analysis indicated greater copy numbers of Cr811 in the new virulent pathotype 3-like and pathotype 5-like populations versus the original pathotype 5, indicating a potential method to identify new pathotypes of P. brassicae before the breakdown of resistance could be detected in a canola crop. The association of Cr811 with infection by P. brassicae of previously resistant canola cultivars, as revealed by qPCR analysis, suggests that Cr811 plays an important role in the interaction between host resistance and P. brassicae pathogenicity.

Emergence of new virulence phenotypes of Plasmodiophora brassicae on canola (Brassica napus) in Alberta, Canada

Clubroot, caused by Plasmodiophora brassicae, is an important soilborne disease of canola (Brassica napus) in Alberta, Canada. Genetic resistance is the most effective clubroot management tool, and resistant cultivars are grown extensively in affected regions. In 2013, relatively severe symptoms of clubroot were observed in some fields of resistant canola. In greenhouse tests, four populations of P. brassicae from two of these fields caused significantly increased levels of clubroot on the cultivars from which they had been first recovered; these included three populations (L-G1, L-G2 and L-G3) recovered from the cultivar ‘L135C’, and one population (D-G3) recovered from ‘D3152’. Further testing showed that L-G1, L-G2 and L-G3 were highly virulent on a suite of six resistant canola cultivars (‘45H29’, ‘D3152’, ‘74–47CR’, ‘1960’, ‘L135C’ and ‘6056CR’) representing a cross-section of products available in Canada, while a seventh cultivar (‘9558c’) was moderately resistant to moderately susceptible. Bioassays of field soil with a dozen clubroot-resistant host genotypes confirmed that in most cases, resistance was no longer effective. Host responses to the population D-G3 were more variable, with most cultivars developing intermediate levels of disease. All four P. brassicae populations were classified as pathotypes 5, P3 and 16/6/8 on the differentials of Williams, Some et al., and the European Clubroot Differential set. The pathotype classifications, however, do not reflect the increased virulence of these populations on clubroot-resistant canola. The identification of new virulence phenotypes of P. brassicae capable of overcoming genetic resistance underscores the need for increased stewardship of resistance sources.

Adaptation to Brassica Host Genotypes by a Single-Spore Isolate and Population of Plasmodiophora brassicae (Clubroot).

Plasmodiophora brassicae, the cause of clubroot of crucifers, is an increasingly important pathogen of canola (Brassica napus) in Alberta, Canada. In response, clubroot-resistant canola genotypes are being deployed to help reduce yield losses. Two experiments were conducted to examine the effect on P. brassicae virulence of repeated exposure of a population and single-spore isolate of the pathogen to the same host. The first experiment examined changes in the index of disease over five cycles of infection on seven Brassica hosts (European Clubroot Differential [ECD] 02, ECD 04, ECD 05, ECD 15, '45H26', '45H29', and 08N823R). The second experiment tested the virulence of five cycled populations ('45H29', 08N823R, ECD 05, and ECD 15) and three cycled single-spore isolates ('45H29', 08N823R, and ECD05) on four resistant canola genotypes ('73-77', '73-67', VT-SD-09, and '9558C'). The results from these experiments clearly demonstrate the ability of both single-spore isolates and populations of P. brassicae to rapidly erode the resistance present in the two canola genotypes, '45H29' and 08N823R. Although the index of disease increased on these two genotypes, the four resistant canola genotypes remained resistant to all the cycled populations and single-spore isolates in the second experiment. These results underscore the importance of crop rotation in the management of clubroot in Alberta.