Clubroot Management Research Articles

Optimizing Clubroot Management and the Role of Canola Cultivar Mixtures.

The sustainable cultivation of canola is under threat from clubroot disease (Plasmodiophora brassicae). The pathogen's resting spores can survive in the soil for extended periods, complicating disease management. Therefore, effective clubroot control requires a combination of tactics that provide multiple layers of protection. Management strategies have focused on pathogen avoidance and reducing disease levels in infested fields. The sanitation of machinery and field equipment remains the most effective method for preventing the pathogen's introduction into non-infested fields. For disease reduction, crop rotation, liming, chemical control, and host resistance are commonly employed, with the use of clubroot-resistant cultivars being the most effective to date. However, resistance breakdown has been observed within four years of the introduction of new cultivars, jeopardizing the long-term effectiveness of this approach. A promising yet underexplored strategy is the use of cultivar mixtures. This approach leverages mechanisms such as the dilution effect, the barrier effect, induced resistance, disruptive selection, and the compensatory effect to control the disease. Cultivar mixtures have the potential to reduce the impact of clubroot on canola production while preserving pathogen population structure, thereby minimizing the likelihood of resistance breakdown. Given its potential, further research into cultivar mixtures as a management strategy for clubroot disease is warranted.

Biocontrol potential of cell-free supernatant of Paenibacillus chitinolyticus against Plasmodiophora brassicae in two important Brassica species

Plasmodiophora brassicae is a serious threat to Brassica crops worldwide, resulting in substantial economic losses for growers. Biocontrol with chitinolytic bacteria producing chitinase is gaining attention as a natural alternative to chemicals. This approach is favored due to the essential role chitinases play in protecting against chitin-containing pathogens. Given that chitin is a major component in the resting spores of P. brassicae and plays a crucial role during pathogenesis, it is probable that Paenibacillus chitinolyticus, producing a high level of chitinase, could suppress P. brassicae by targeting chitin in a critical stage of this pathogen’s life cycle. Our research aimed to evaluate the effect of various applications of P. chitinolyticus on clubroot suppression in two economically important Brassica species: Chinese cabbage and rapeseed. The effectiveness of the cell-free supernatant (CFS) of an endemic strain of P. chitinolyticus from the Czech Republic at five different time points was studied in the greenhouse by measuring the disease severity index. Early application of P. chitinolyticus decreased the disease index significantly within both plant species resulting in increased shoot dry weight. In conclusion, the CFS of P.chitinolyticus has significant antagonistic activity against clubroot in Chinese cabbage and rapeseed in the early developmental stages of clubroot occurrence and holds the potential as a biofertilizer as well as bioprotectant agent in clubroot management of P. brassicae.

Integrated Management of Clubroot in Zhejiang Province, China

Clubroot, caused by Plasmodiophora brassicae, is a destructive soil-borne disease significantly harming global Brassica crop production. This study employed the Williams and European Clubroot Differential (ECD) and Williams systems to identify the pathotypes of P. brassicae collected from Hangzhou City, Yuhang District and Quzhou City, Kaihua County in Zhejiang Province. Greenhouse and field trials were conducted to evaluate the effects of plastic film covering and four chemical agents on the growth parameters and clubroot severity of the Chinese cabbage cultivar ‘Granaat’. Potential treatment mechanisms on clubroot were explored through a qPCR analysis of the resting spore density and pH measurement of the soil. Furthermore, treatment with 1-napthaleneacetic acid (NAA), a synthetic auxin, was also evaluated for its potential role in suppressing clubroot. The results indicate that the pathotypes of P. brassicae in the two districts were P1, ECD20/31/12, and P3, ECD20/15/4. While an individual application of plastic film covering could not effectively control clubroot, calcium cyanamid, dazomet and ammonium bicarbonate demonstrated significant efficacy in its management. These three agents significantly reduced the resting spore density in the soil, with calcium cyanamid and ammonium bicarbonate also increasing soil alkalinity. Additionally, ammonium bicarbonate promotes lateral root development in ‘Granaat,’ helping infected plants access adequate water and nutrients. However, NAA exhibited no efficacy in clubroot control. Therefore, sustained lateral root development is crucial for effectively resisting P. brassicae invasion. Considering application costs and environmental friendliness, we propose the field application of ammonium bicarbonate as the optimal method for clubroot disease management in Zhejiang Province.

Evaluation of Amisulbrom Products for the Management of Clubroot of Canola (Brassica napus).

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola (Brassica napus). Amisulbrom, a quinone inside inhibitor (QiI), was evaluated for its effectiveness in clubroot management in Alberta, Canada. Resting spores of P. brassicae were treated in vitro with 0, 0.01, 0.1, 1, and 10% (w/v) amisulbrom to determine its effect on spore germination and viability. Amisulbrom inhibited resting spore germination by up to 79% and reduced viable spores by 31% relative to the control. Applications of a liquid solution (AL1000, 1000 g active ingredient (ai) ha-1) and granular formulations (AF700, 700 g ai ha-1; AF1000, 1000 g ai ha-1; AF1500, 1500 g ai ha-1) of amisulbrom were tested on the canola cultivars '45H31' (clubroot-susceptible) and 'CS2000' (moderately resistant) under greenhouse conditions and in field experiments in 2019 and 2020. In the greenhouse, the treatments were evaluated at inoculum concentrations of 1 × 105 or 1 × 107 resting spores g-1 soil. A trend of decreasing clubroot severity with an increasing amisulbrom rate was observed. At the lower spore concentration, treatment with AF1500 resulted in a clubroot disease severity index (DSI) <20% for both cultivars, while the lowest DSI under both low and high spore concentrations was obtained with AL1000. The field results indicated a significant reduction in DSI, with varied effects of rates and liquid vs. granular formulations. The greatest reductions (up to 58.3%) in DSI were obtained with AF1500 and AL1000 in 2020. These findings suggest that amisulbrom holds promise as part of an integrated clubroot management approach.

Effect of Mulching on Soil Temperatures and Its Impact on Plasmodiophora brassicae and Clubroot.

Clubroot caused by Plasmodiophora brassicae is a serious soilborne disease on cruciferous crops worldwide. Agricultural practice is a preferable clubroot management strategy because of its low investment requirement and environmental safety. Among the agricultural practices, solarization has been widely applied in the integrated management of other soilborne diseases. However, only few reports exist on the effect of solarization on clubroot management. In this study, we measured the effect of plastic mulching on soil temperature at different depths and on clubroot incidence and severity under greenhouse and field conditions. The pathogen density in the soil after solarization was measured by quantitative PCR analysis. Results indicated that the mulching treatment increased soil temperature especially in the soil layer ranges of 0 to 20 cm. Solarization with mulching also effectively reduced the incidence and severity of clubroot in the greenhouse assay and the field trial by decreasing the P. brassicae population in the soil. This study suggested that solarization with mulching can impair clubroot development and thus contribute to the sustainable management of clubroot.

Characterization of Plasmodiophora brassicae pathotypes from western Canada in 2019–2020

Clubroot, caused by Plasmodiophora brassicae, is a major soilborne disease of canola (Brassica napus) on the Canadian Prairies. Most canola cultivars carry what is now referred to as ‘first-generation’ resistance, which represents the most widely used clubroot management tool. Unfortunately, new pathotypes of P. brassicae have emerged that can overcome this resistance. By 2018, 36 unique pathotypes of P. brassicae had been identified on the Prairies based on their virulence on the Canadian Clubroot Differential (CCD) set, with pathotypes 3A, 3D and 3 H being predominant. Since the virulence of P. brassicae populations can shift rapidly in response to host selection pressure, continued pathotype monitoring is important for resistance stewardship. Clubbed roots were collected from 133 canola crops (126 in Alberta, 6 in Saskatchewan, 1 in Manitoba) in 2019 and 127 crops (113 in Alberta, 13 in Saskatchewan, 1 in Manitoba) in 2020, and evaluated for their CCD pathotype designations. Twenty-five unique pathotypes were identified across the sampled crops over the two years of the study; these included seven novel pathotypes, six of which could overcome first-generation resistance (3C, 8 G, 8I, 9D, 9E and 9F). While pathotypes 3A, 3D and 3 H continued to be identified frequently in 2019 and 2020, several others, most notably pathotype 8E (virulent on hosts with first-generation resistance), had also become more common by 2020. The results indicate the continued emergence of new P. brassicae pathotypes on canola.

INTEGRATED APPROACH FOR CLUBROOT MANAGEMENT IN COLE CROPS: A REVIEW

Clubroot is a severe cosmopolitan disease of cole crops. This disease is caused by Plasmodiophora brassicae which is a soil-borne pathogen. This disease is emerging as one of the biggest threats to cole crops. This pathogen was first detected in Europe. Several experiments have been conducted to understand the nature of the pathogen but the complexity of the pathogen is not properly understood yet. Due to this reason, the management of clubroot has been quite a challenge to the researchers. Given the lack of knowledge about the pathogen, farmers, and plant pathologists around the globe are struggling to control the disease. Integrated approaches are being introduced for better management of the disease. The integrated approach includes various physical, chemical, cultural, and biological methods that are combined to overcome the problem.

First Report of Plasmodiophora brassicae causing clubroot on Thlaspi arvense L. in Sichuan Province of China.

Pennycress (Thlaspi arvense L.), an annual herb of the mustard family Brassicacae, is native to Eurasia and now widely distributed throughout temperate North America. This species is currently being developed as a medicinal herb used to treat nephritis in China and an oilseed crop for biofuel production (Roque et al.2012). In November 2020, stunt and wilt symptoms were observed on above ground parts and swollen club-shaped galls were observed on the roots of T. arvense in most of the Chinese cabbage growing area in Kangding (30°03'"N,102°02'"E), Sichuan Province of China. The average disease incidence of swollen roots on T. arvense was 91.2% ( n=80). To identify the causal agent of this disease, the swollen roots of T. arvense were collected, crushed and observed under microscope (Fei et al.2017). Abundant resting spores were found in the root galls, which were spherical and 2.0 to 3.1 μm in diameter with an average length of 2.7 nm (n=100). The healthy roots and the root galls of T. arvense plants were further evaluated by PCR with P. brassicae-specific primers TC2F/TC2R (Cao et al. 2007). The results showed that a DNA fragment with an estimated size of 520 bp, as expected for that of P. brassicae, was consistently amplified in diseased roots, No PCR amplification occurred in the healthy roots with the TC2F/TC2R primers. Blast analysis of the 520 bp segment (GenBankMZ040496) showed the highest identity with the sequence of small subunit ribosomal RNA gene of P. brassicae (GenBankMH762161, 97.7%, E value=0). These results confirmed that the pathogen in the galled roots of T. arvense was P. brassicae. The pathogenicity of isolated P. brassicae was tested on both T. arvense and Chinese cabbage (B. campestris ssp. pekinensis). Resting spores were isolated from the diseased roots (Castlkbury et al. 1994) of T. arvense and suspended in Hoagland's solution to the final concentration of 1 × 107 spores per milliliter. Fifteen plastic pots (10 cm bottom diameter, 16 cm upper diameter, 13 cm high) were filled with soil (1 kg per pot) that was sterilized twice with high-temperature (121℃), high pressure (19 PSI) for 1.5 hours with a time interval of 2 days between. Inoculated pots received 100 mL spore suspension each. Fifteen control pots with sterilized field soil were treated with 100 mL Hoagland's solution each. Seeds of T. arvense and B. campestris were pre-germinated at 20°C on moist filter paper for 7 days and transplanted into the pots, five seedlings each and five pots per treatment. The pots were maintained in a greenhouse with 16 hours photoperiod at 24°C/16°C day/night temperature. After 7 weeks, plants in each pot were uprooted and the roots cleaned in running water and inspected for clubroot symptoms. Plants of T. arvense and Chinese cabbage in pots inoculated with resting spores showed clubroot symptoms while no disease symptoms were observed on any control plants. The disease incidence rate was 95.4% on T. arvense and 81.2% on B. campestris. Therefore, it was confirmed that P. brassicae could cause clubroot disease on T. arvense. To our knowledge, this is the first published report of clubroot disease on T. arvense in China. This finding is helpful for the management of clubroot on herbs and plants of biological origin in the cruciferous family.

Soil fumigation with Vapam (metam sodium) to control clubroot (Plasmodiophora brassicae) of canola (Brassica napus)

Clubroot, a damaging disease of canola ( Brassica napus L.) caused by the soilborne parasite Plasmodiophora brassicae Woronin, is spreading across Alberta and other provinces of western Canada. The movement of infested soil on field machinery is the main mechanism of dispersal, with clubroot generally occurring first as localized patches near field entrances. In this study, the soil fumigant Vapam (metam sodium) was evaluated as a management option for foci of P. brassicae infestation. Replicated experiments at two field sites in central Alberta showed reductions in clubroot severity ranging from 9% to 51% following treatment with varying rates of Vapam. Decreases in clubroot severity of up to 28% were observed in the year following Vapam treatment, indicating some potential residual effects and (or) a reduction in the amount of inoculum returned to the soil in the previous year. While Vapam shows some promise as a clubroot management tool, an integrated approach will be required for the sustainable management of this disease on canola.

Grasses and field crops reduce the concentration of resting spores of Plasmodiophora brassicae in soil under controlled conditions

AbstractClubroot, caused by Plasmodiophora brassicae, dramatically reduces yields of brassica crops, and once present in a field, can persist in the soil for many years via long‐lived resting spores. Some nonhost crops can stimulate the germination of resting spores, which reduces clubroot inoculum because the pathogen cannot complete its life cycle in the absence of a living host. The effect of selected grasses and other field crops on the concentrations of spores in soil was assessed in two replicated and repeated short‐duration (8‐week) studies under controlled conditions. The spore concentration declined precipitously in the bare soil control relative to the initial spore concentration over the course of both studies. Cultivars of meadow bromegrass, smooth bromegrass and one cultivar of perennial ryegrass reduced the spore concentration compared to the bare soil control in both repetitions of one study, while two cultivars of perennial ryegrass did not reduce the spore concentration. In a second study, spring wheat, perennial ryegrass, barley and field pea reduced spores compared to the bare soil control, but soybean did not. Our hypothesis that larger root systems would produce larger reductions in spore numbers was not supported; there was no correlation between root weight and reduction in spore concentration. We conclude that perennial grasses, spring cereals and field pea can contribute to the reduction of clubroot spores in soil. The advantage of grass crops over annual crops for clubroot management is that movement of infested soil is minimized over the entire duration of the grass stand.

Fine Mapping of Clubroot Resistance Loci CRA8.1 and Candidate Gene Analysis in Chinese Cabbage (Brassica rapa L.).

Clubroot is caused by Plasmodiophora brassicae, which threatens Brassicaceae crop production worldwide. In recent years, there has been an outbreak and rapid spread of clubroot in many major cruciferous crop-producing areas of China. In this study, we identified a cabbage material DingWen (DW) with different resistant capabilities from Huashuang5R (H5R) and Huayouza62R of Brassica napus, which are currently used as the main resistant cultivars for clubroot management in China. We used a next-generation sequencing-based bulked segregant analysis approach, combined with genetic mapping to identify clubroot-resistant (CR) genes from F1 population generated from a cross between the DW (CR) and HZSX (clubroot susceptible). The CR locus of DW (named CRA8.1) was mapped to a region between markers A08-4346 and A08-4853, which contains two different loci CRA8.1a and CRA8.1b after fine mapping. The CRA8.1b loci contain a fragment of 395 kb between markers A08-4624 and A08-4853 on A08 chromosome, and it is responsible for the resistance to PbZj and PbXm isolates. However, together with CRA8.1a, corresponding to a 765-kb region between markers A08-4346 and A08-4624, then it can confer resistance to PbXm+. Finally, through expression analysis between resistant and susceptible materials, two genes encoding TIR-NBS-LRR proteins (BraA08g039211E and BraA08g039212E) and one gene encoding an RLP protein (BraA08g039193E) were identified to be the most likely CR candidates for the peculiar resistance in DW.

A Loop-Mediated Isothermal DNA Amplification (LAMP) Assay for Detection of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is a serious threat to cruciferous crops around the world. The resting spores of P. brassicae are a primary source of infection and can survive in soil for many years. Detection of resting spores in soil is essential for forecasting clubroot prevalence. Detection of P. brassicae has been relying on plant bioassays or PCR-based methods. The loop-mediated isothermal DNA amplification (LAMP) is a promising approach for microorganism detection with the advantage of high sensitivity, accuracy, and convenience in viewing. In this study, we developed a LAMP assay for detection of P. brassicae in soil, roots, and seeds. This method can detect P. brassicae at a minimal amount of 1 fg of plasmid DNA or 10 resting spores in the soil. Compared with conventional PCR, the LAMP was more sensitive in detection of P. brassicae at the lower levels in soil samples. In conclusion, we elaborated a sensitive, accurate, and easy-to-use LAMP assay to detect P. brassicae, which will facilitate sustainable clubroot management and planning.

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.

Identification of Arabidopsis Phospholipase A Mutants With Increased Susceptibility to Plasmodiophora brassicae.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most devastating diseases of canola (Brassica napus) in Canada. The identification of novel genes that contribute to clubroot resistance is important for the sustainable management of clubroot, as these genes may be used in the development of resistant canola cultivars. Phospholipase As (PLAs) play important roles in plant defense signaling and stress tolerance, and thus are attractive targets for crop breeding. However, since canola is an allopolyploid and has multiple copies of each PLA gene, it is time-consuming to test the functions of PLAs directly in this crop. In contrast, the model plant Arabidopsis thaliana has a simpler genetic background and only one copy of each PLA. Therefore, it would be reasonable and faster to validate the potential utility of PLA genes in Arabidopsis first. In this study, we identified seven homozygous atpla knockout/knockdown mutants of Arabidopsis, and tested their performance following inoculation with P. brassicae. Four mutants (pla1-iiα, pla1-iγ3, pla1-iii, ppla-iiiβ, ppla-iiiδ) developed more severe clubroot than the wild-type, suggesting increased susceptibility to P. brassicae. The homologs of these Arabidopsis PLAs (AtPLAs) in B. napus (BnPLAs) were identified through Blast searches and phylogenic analysis. Expression of the BnPLAs was subsequently examined in transcriptomic datasets generated from canola infected by P. brassicae, and promising candidates for further characterization identified.

A high‐throughput turbidimetric method for quantitative preparation of Plasmodiophora brassicae inoculum for bioassays

AbstractClubroot caused by Plasmodiophora brassicae is one of the major diseases on cruciferous crops. This disease has been a problem all over the world and led to serious economic losses in cruciferous crop production. The improvement of clubroot management is dependent on the effectiveness of bioassays on pathogenicity or plant resistance. P. brassicae resting spore inoculum prepared from clubroot tissues was widely used in bioassays. Traditionally, resting spore concentration was measured by visual counting with a haemocytometer; however, this method is time‐consuming, labour intensive and with poor repeatability due to the tiny size of resting spores. In this study, we established a turbidimetric method to measure P. brassicae resting spore concentration of the inoculum. A regression curve was generated by plotting optical density of gradiently diluted resting spore suspension against the corresponding resting spore concentrations, which resulted in a logarithmic regression equation. This method was validated and proved to be robust and effective in determining resting spore concentration of different samples, and was further confirmed by bioassay of infection and clubroot development. In conclusion, we established a standard protocol to prepare P. brassicae inoculum and provided an effective method for resting spore concentration determination. The results of this study can be widely used in research activities on clubroot sustainable management when an effective assessment of P. brassicae biomass is needed.

Plasmodiophora brassicae in Its Environment: Effects of Temperature and Light on Resting Spore Survival in Soil.

Clubroot caused by Plasmodiophora brassicae is an important disease on cruciferous crops worldwide. Management of clubroot is challenging, largely because of the millions of resting spores produced within an infected root that can survive dormant in the soil for many years. This study was conducted to investigate some of the environmental conditions that may affect the survival of resting spores in the soil. Soil samples containing clubroot resting spores (1 × 107 spores/g soil) were stored at various temperatures for 2 years. Additionally, other samples were buried in soil or kept on the soil surface in the field. The content of P. brassicae DNA and the numbers of viable spores in the samples were assessed by quantitative PCR (qPCR) and pathogenicity bioassays, respectively. The results indicated that 4°C, 20°C, and being buried in the soil were more conductive conditions for spore survival than -20°C, 30°C, and at the soil surface. Most (99.99%) of the spores kept on the soil surface were nonviable, suggesting a negative effect of light on spore viability. Additional experiments confirmed the negative effect of ultraviolet light on spore viability because spores receiving 2 and 3 h ultraviolet light exhibited lower disease potential and contained less DNA content than the nontreated control. Finally, this work confirmed that DNA-based quantification methods such as qPCR can be poor predictors of P. brassicae disease potential because of the presence and persistence of DNA from dead spores.

Evaluation of lime products for clubroot (Plasmodiophora brassicae) management in canola (Brassica napus) cropping systems

Clubroot, caused by Plasmodiophora brassicae, is an important soilborne disease of canola (Brassica napus) in Alberta, Canada. As clubroot prefers acidic soils, the application of lime to increase soil pH may reduce disease. Replicated field plot experiments were conducted to evaluate the efficacy of hydrated lime for clubroot management. In 2017, moderate (11.4 t ha−1) to high rates (12.7 t ha−1) of hydrated lime reduced the index of disease (ID) by 35–91% in a susceptible canola cultivar 8 weeks after seeding. In contrast, no effect of lime treatment was observed in the 2018 field trials, due to several environmental factors. A greenhouse study compared the efficacy of hydrated lime and limestone in reducing ID in susceptible and resistant canola cultivars, at different application rates and inoculum concentrations. In treatments that did not receive lime, ID was very high (92–100%) in the susceptible and low (9–13%) in the resistant canola. The application of hydrated lime at 4.7, 8.1, 11.4 and 14.8 t ha−1 eliminated visible symptoms in both cultivars, whereas limestone reduced the ID only at the two lowest inoculum concentrations. Root tissues from the same study were analyzed by quantitative PCR to measure P. brassicae proliferation in planta. Inoculum concentration and the type and rate of lime significantly affected the amount of P. brassicae DNA present in the root tissue. Repeated trials with less virulent inoculum indicated similar trends. Hydrated lime may represent a more effective tool than limestone to manage P. brassicae on canola in highly infested soils.

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.

A New Identification Method Reveals the Resistance of an Extensive-Source Radish Collection to Plasmodiophora brassicae Race 4

Raphanus sativus, an important cruciferous vegetable, has been increasingly affected by clubroot disease. Establishing a stable and accurate resistance identification method for screening resistant germplasms is urgently needed in radish. In this study, the influence of inoculum concentration, inoculation methods, and pH of the substrate on disease occurrence was studied. The result showed that the disease index (DI) was highest at 2 × 108 spores/mL, the efficiency of two-stage combined inoculation methods was higher than others, and pH 6.5 was favorable for the infection of P. brassicae. By using this new method, DIs of 349 radish germplasms varying from 0.00 to 97.04, presented significantly different levels of resistance. Analysis showed that 85.06% germplasms from China were susceptible to P. brassicae, whilst 28 accessions were resistant and mainly distributed in east, southwest, northwest, and south-central China. Most of the exotic germplasms were resistant. Repeated experiments verified the stability and reliability of the method and the identity of germplasm resistance. In total, 13 immune, 5 highly resistant and 21 resistant radish accessions were identified. This study provides an original clubroot-tolerance evaluation technology and valuable materials for the development of broad-spectrum resistant varieties for sustainable clubroot management in radish and other cruciferous crops.

Identification of resistance loci against new pathotypes of Plasmodiophora brassicae in Brassica napus based on genome-wide association mapping

Genetic resistance is a successful strategy for management of clubroot (Plasmodiophora brassicae) of brassica crops, but resistance can break down quickly. Identification of novel sources of resistance is especially important when new pathotypes arise. In the current study, the reaction of 177 accessions of Brassica napus to four new, virulent pathotypes of P. brassicae was assessed. Each accession was genotyped using genotyping by sequencing to identify and map novel sources of clubroot resistance using mixed linear model (MLM) analysis. The majority of accessions were highly susceptible (70–100 DSI), but a few accessions exhibited strong resistance (0–20 DSI) to pathotypes 5X (21 accessions), 3A (8), 2B (7), and 3D (15), based on the Canadian Clubroot Differential system. In total, 301,753 SNPs were mapped to 19 chromosomes. Population structure analysis indicated that the 177 accessions belong to seven major populations. SNPs were associated with resistance to each pathotype using MLM. In total, 13 important SNP loci were identified, with 9 SNPs mapped to the A-genome and 4 to the C-genome. The SNPs were associated with resistance to pathotypes 5X (2 SNPs), 3A (4), 2B (5) and 3D (6). A Blast search of 1.6 Mb upstream and downstream from each SNP identified 13 disease-resistance genes or domains. The distance between a SNP locus and the nearest resistance gene ranged from 0.04 to 0.74 Mb. The resistant lines and SNP markers identified in this study can be used to breed for resistance to the most prevalent new pathotypes of P. brassicae in Canada.