European Clubroot Differential Set Research Articles

Pathotyping Systems and Pathotypes of Plasmodiophora brassicae-Navigating toward the Optimal Classification.

Plasmodiophora brassicae Woronin, an obligate biotrophic soil-borne pathogen, poses a significant threat to cruciferous crops worldwide by causing the devastating disease known as clubroot. Pathogenic variability in P. brassicae populations has been recognized since the 1930s based on its interactions with Brassica species. Over time, numerous sets of differential hosts have been developed and used worldwide to explore the pathogenic variability within P. brassicae populations. These sets encompass a range of systems, including the Williams system, the European Clubroot Differential set (ECD), the Brassica napus set, the Japanese Clubroot Differential Set, the Canadian Clubroot Differential Set (CCS), the Korean Clubroot Differential Set, and the Chinese Sinitic Clubroot Differential set (SCD). However, all existing systems possess both advantages as well as limitations regarding the detection of pathotypes from various Brassica species and their corresponding virulence pattern on Brassica genotypes. This comprehensive review aims to compare the main differential systems utilized in classifying P. brassicae pathotypes worldwide. Their strengths, limitations, and implications are evaluated, thereby enhancing our understanding of pathogenic variability.

Current and Future Pathotyping Platforms for Plasmodiophora brassicae in Canada.

Clubroot, caused by Plasmodiophora brassicae, is one of the most detrimental threats to crucifers worldwide and has emerged as an important disease of canola (Brassica napus) in Canada. At present, pathotypes are distinguished phenotypically by their virulence patterns on host differential sets, including the systems of Williams, Somé et al., the European Clubroot Differential set, and most recently the Canadian Clubroot Differential set and the Sinitic Clubroot Differential set. Although these are frequently used because of their simplicity of application, they are time-consuming, labor-intensive, and can lack sensitivity. Early, preventative pathotype detection is imperative to maximize productivity and promote sustainable crop production. The decreased turnaround time and increased sensitivity and specificity of genotypic pathotyping will be valuable for the development of integrated clubroot management plans, and interest in molecular techniques to complement phenotypic methods is increasing. This review provides a synopsis of current and future molecular pathotyping platforms for P. brassicae and aims to provide information on techniques that may be most suitable for the development of rapid, reliable, and cost-effective pathotyping assays.

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.

Variation in pathotypes and virulence of Plasmodiophora brassicae populations in Germany

Between 2012 and 2015, 49 new clubroot‐infested fields were identified in 12 German federal states. Clubroot disease incidence varied within these fields from 22% to 92%. Field information revealed that in 85% of fields, oilseed rape was grown in rotation once every 2 or 3 years. Frequency of OSR in the rotation was significantly correlated with the incidence and prevalence of clubroot disease. The disease was detected in fields with soil pH ranging from 5.1 to 8.3, and a significant negative correlation was found between soil pH and the disease incidence of infested fields. Furthermore, more cases of disease and severe incidences were observed in sandy loam and loamy sand as compared with other soil types. Pathotype classification of the 49 Plasmodiophora brassicae populations was conducted on two differential sets, the European Clubroot Differential set and the set of Somé (1996). Additionally, the degree of virulence of the collected isolates was analysed on the clubroot‐resistant oilseed rape cv. Mendel. The results showed variation in pathotype distribution in different regions in Germany. The majority of isolates according to Somé were pathotypes 1 and 3, respectively, with pathotypes 2 and 5 in the minority. Detailed classification according to Buczacki showed the dominance of 16/31/31, 16/14/30 and 16/14/31 populations among 20 distinct virulence patterns of collected isolates. From all populations tested for virulence on cv. Mendel, 15 isolates were found to be moderately or highly virulent. These virulent populations were not restricted to a small geographical area in the country.

Pathotypes of Plasmodiophora brassicae causing damage to oilseed rape in the Czech Republic and Poland

Winter oilseed rape (Brassica napus) is an important crop in the Czech Republic and Poland. Clubroot disease caused by the pathogen Plasmodiophora brassicae is a serious and still-growing problem for oilseed rape growers in both countries. The aim of this study was to evaluate the pathotype composition of P. brassicae populations from the Czech Republic and Poland, according to the three evaluation systems, and to determine soil inoculum loads for representative fields via traditional end-point PCR as well as quantitative PCR analysis. There were considerable differences between the populations of P. brassicae from both countries, and the number of pathotypes varied depending on the evaluation system and the threshold used to distinguish susceptible vs. resistant plant reactions. This is the first study comparing the effect of different thresholds. Using an index of disease (ID) of 25 % to distinguish susceptible vs. resistant reactions, there was a total of seven pathotypes identified based on the differentials of Williams, five with the system of Some et al., and 18 with the European Clubroot Differential (ECD) set. However, based on a threshold of 50 %, there were nine pathotypes according to the evaluation system by Williams, four based on the differentials of Some et al., and 15 with the ECD set. Changing of the thresholds led to the reclassification of some pathotypes. Several pathotypes were common in both countries. High amounts of pathogen DNA were found in many of the field soils analysed by quantitative PCR. There was a weak correlation between soil pH and infestation of P. brassicae for the Polish soils.

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.

Virulence and spread of Plasmodiophora brassicae [clubroot] in Alberta, Canada

Clubroot, caused by Plasmodiophora brassicae, has emerged as an important disease of canola (Brassica napus) in Alberta, Canada. Annual surveys from 2005 to 2008 revealed 405 clubroot-infested fields, mainly in central Alberta, but also in the southern part of the province. Within-field surveys suggested that the main mechanism of clubroot spread is through the movement of infested soil on farm machinery, as the frequency of infestation was highest at the field entrances and decreased sharply at distances of 150 and 300 m from the entrance. Characterization of P. brassicae populations from Alberta on the differentials of the European Clubroot Differential (ECD) series, P.H. Williams, and A. Somé et al. revealed that pathotype ECD –/15/12, 3, or P2, respectively, is predominant in the central part of the province, a finding consistent with earlier reports. However, novel virulence phenotypes were also observed. Two populations from southern Alberta were classified as pathotype 5 or P3 on the hosts of P.H. Williams and A. Somé et al., respectively, or as pathotypes ECD –/6/8 and –/4/0 on the ECD set, suggesting independent introductions of P. brassicae to this region or greater diversity than previously thought. Pathogen populations collected from infected canola plants from Quebec and Manitoba also yielded unique virulence patterns but were still virulent on most B. napus hosts, and an Ontario population induced significant disease only on a few of the vegetable differentials. Virulence levels overall were highest for the P. brassicae populations from central Alberta, which may help to explain the rapid increase in clubroot observed in this province.

Characterization of Plasmodiophora brassicae populations from Alberta, Canada

Clubroot, caused by Plasmodiophora brassicae, was identified in a number of canola (Brassica napus) fields in central Alberta in 2003. To characterize the virulence of the pathogen in the province, field populations from a number of locations in the Edmonton region were tested on the two most widely used sets of differential hosts, those of P.H. Williams and the European Clubroot Differential (ECD) series. Populations from British Columbia and Ontario were included for comparison. While the reaction of some hosts could be clearly defined as either resistant or susceptible, others showed intermediate disease index scores. If disease indices of 0%–49% and 50%–100% were regarded as resistant and susceptible, respectively, then populations from Alberta were classified as ECD 16/15/12 and 16/15/0 on the ECD set, or pathotypes 3 and 5, respectively, on the hosts of Williams. The population from British Columbia was classified as ECD 16/2/12 or pathotype 6, and the Ontario population was classified as ECD 16/0/14 or pathotype 6. The Alberta populations were more virulent on the B. napus hosts than those from other provinces, perhaps a reflection of their canola origin. In addition, 48 canola cultivars included in the 2004 Prairie Canola Variety Trials were screened for resistance to a local population of the pathogen, and all appeared to be highly susceptible. If clubroot were to become more widely established in western Canada, it could have a major negative impact on yields.

Production, characterization and interaction of single-spore isolates ofPlasmodiophora brassicae

Out of 164 plants of clubroot-susceptible Chinese cabbage inoculated with single resting spores ofPlasmodiophora brassicae, two plants developed clubroot symptoms. The two single-spore isolates (SSIs) extracted from these plants gave an identical reaction pattern on the European Clubroot Differential set (ECD) and seven doubled-haploid lines (DH-lines). Their reaction pattern differed from that of the original field isolate on four hosts: ECD hosts 06 and 07 were susceptible to the field isolate but resistant to both SSIs, while for DH-lines Bi and Pt the reverse was true. DH-line Pt was significantly less diseased by mixed inocula consisting of the field isolate and SSI-1 than by SSI-1 alone. It was concluded that the SSI-1 pathotype was a minor component of the field isolate, although it was isolated twice. The results also suggest that the alleviating effect of the field isolate in mixed inoculations with SSI-1 on DH-line Pt was due to induced resistance, rather than to competitive interactions.

A Simple Inoculation Method with a Single Resting Spore of Plasmodiophora brassicae.

Single inoculation method using a single resting spore of Plasmodiophora brassicae was developed. Single resting spore was obtained by placing a 0.5μl drop of spore suspension, which was adjusted to the concentration of 2000 spores per ml, on a piece of sterilized cover glass with a micropipette. The drop was checked under an inverted microscope to ensure that it exactly carried one spore alone. The cover glass carrying a single spore was placed on the surface of autoclaved soil in a pot. One- or two-day-old seedling of Chinese cabbage was inoculated by placing over the spore drop, covered with a small amount of soil, and grown in a growth chamber. The inoculation method could give an efficient infection. Pathogenicity of the single-spore isolates obtained was compared with their original populations to the host lines of European Clubroot Differential set, and was not always identical in those combinations.

Preliminary observations on variation in susceptibility to clubroot among collections of some wild crucifers

SUMMARYCollections of Capsella bursa‐pastoris and other common cruciferous weeds showed significant differences in susceptibility to populations of Plasmodiophora brassicae as defined on the European Clubroot Differential (ECD) set. Some evidence was obtained that selection for tolerance may occur within natural populations of C. bursa‐pastoris. Overall virulence of P. brassicae collections on the ECD set was not correlated with virulence to the weeds tested. The significance of wild hosts in studies of resistance to P. brassicae is discussed.