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.     

Effect of susceptible and resistant canola plants on Plasmodiophora brassicae resting spore populations in the soil

Clubroot, caused by Plasmodiophora brassicae, has become a serious threat to canola (Brassica napus) production in western Canada. Experiments were conducted to assess the effect of growing resistant and susceptible canola genotypes on P. brassicae soil resting spore populations under greenhouse, mini‐plot and field conditions. One crop of susceptible canola contributed 1·4 × 10⁸ spores mL^?1 soil in mini‐plot experiments, and 1 × 10¹⁰ spores g^?1 gall under field conditions. Repeated cropping of susceptible canola resulted in greater gall mass compared to resistant canola lines. It also resulted in reduced plant height, increased clubroot severity in susceptible canola, and increased numbers of resting spores in the soil mix.     

Assessment of the impact of resistant and susceptible canola on Plasmodiophora brassicae inoculum potential

The impact on clubroot severity of growing susceptible canola or mixtures of resistant and susceptible canola genotypes was examined. Bioassays revealed greater clubroot severity and incidence, and reduced plant height, where 100% of a susceptible cultivar had been grown. A higher proportion of susceptible plants within a resistant canola crop increased root hair and secondary infections. Regression analysis of root hair infection and the amount of Plasmodiophora brassicae DNA (as determined by quantitative PCR) revealed strong linear relationships between the two parameters. The linear relationships between root hair infection and P. brassicae DNA were stronger for the resistant cultivar than for the susceptible cultivar when regression analysis was conducted by cultivar over the sampling dates. In conclusion, the cropping of a resistant cultivar reduced clubroot severity, while the presence of susceptible volunteer canola increased inoculum potential. Quantitative PCR was a reliable tool for the quantification of root hair infection.     

Potential biological control of clubroot on canola and crucifer vegetable crops

Clubroot caused by Plasmodiophora brassicae is an emerging threat to canola (Brassica napus) production in western Canada, and a serious disease on crucifer vegetable crops in eastern Canada. In this study, seven biological control agents and two fungicides were evaluated as soil drenches or seed treatments for control of clubroot. Under growth cabinet conditions, a soil‐drench application of formulated biocontrol agents Bacillus subtilis and Gliocladium catenulatum reduced clubroot severity by more than 80% relative to pathogen‐inoculated controls on a highly susceptible canola cultivar. This efficacy was similar to that of the fungicides fluazinam and cyazofamid. Under high disease pressure in greenhouse conditions, the biocontrol agents were less effective than the fungicides. Additionally, all of the treatments delivered as a seed coating were less effective than the soil drench. In field trials conducted in 2009, different treatments consisting of a commercial formulation of B. subtilis, G. catenulatum, fluazinam or cyazofamid were applied as an in‐furrow drench at 500 L ha^?1 water volume to one susceptible and one resistant cultivar at two sites seeded to canola in Alberta and one site of Chinese cabbage in Ontario. There was no substantial impact on the susceptible canola cultivar, but all of the treatments reduced clubroot on the susceptible cultivar of Chinese cabbage, lowering disease severity by 54–84%. There was a period of 4 weeks without rain after the canola was seeded, which likely contributed to the low treatment efficacy on canola. Under growth cabinet conditions, fluazinam and B. subtilis products became substantially less effective after 2 weeks in a dry soil, but cyazofamid retained its efficacy for at least 4 weeks.     

Virulence and inoculum density‐dependent interactions between clubroot resistant canola (Brassica napus) and Plasmodiophora brassicae

To mitigate the impact and dissemination of clubroot in western Canada, canola (Brassica napus) producers have relied on clubroot resistance traits. However, in 2013 and 2014, new strains of the clubroot pathogen, Plasmodiophora brassicae, emerged that are virulent on most clubroot‐resistant (CR) canola genotypes. Novel strains of the pathogen were inoculated onto two susceptible canola cultivars, one resistant line and six CR cultivars. Although all cultivars/lines showed a susceptible response to inoculation with the new strains of P. brassicae, the severity of disease reaction, root hair infection rates and the amount of P. brassicae DNA present in each canola genotype varied depending on the strain. In addition, the effect of inoculum density on disease severity and gall formation was recorded for one of these new strains on a universally susceptible Chinese cabbage cultivar and one susceptible and 10 resistant canola genotypes. Although root galls were observed at an inoculum density of 10³ spores per mL of soil, clear differentiation of susceptible and resistant reactions among canola cultivars/lines was not observed until the inoculum density reached 10⁵ spores mL^?1. At a spore density of 10⁶ spores mL^?1 and above, all cultivars/lines developed susceptible reactions, although there was some differentiation in the degree of reaction. This study shows the potential to develop a unique disease profile for emergent clubroot pathotypes and shows a useful range of spore densities at which to study new P. brassicae strains.     

Influence of cultivar resistance and inoculum density on root hair infection of canola (Brassica napus) by Plasmodiophora brassicae

The impact of cultivar resistance and inoculum density on the incidence of primary infection of canola root hairs by Plasmodiophora brassicae, the causal agent of clubroot, was assessed by microscopy. The incidence of root hair infection in both a resistant and a susceptible cultivar increased with increasing inoculum density, but was two‐ to threefold higher in the susceptible cultivar; the relationship between root hair infection and inoculum density was also substantially stronger and more consistent in the susceptible cultivar. In the susceptible cultivar, the root hair infection rate peaked between 6 and 8 days after sowing and then declined. In the resistant cultivar, it increased over the 14‐day duration of each study. It appears that examination of root hair infection by microscopy in a bait crop of susceptible canola could serve as a useful tool for estimating P. brassicae inoculum levels in soil. In a separate trial, the relationship between inoculum density and clubroot severity, plant growth parameters, and seed yield was assessed under greenhouse conditions. Inoculum density in the susceptible genotype was strongly and positively correlated with clubroot severity and negatively correlated with plant height and seed yield. In addition, a single cropping cycle of the susceptible cultivar contributed significantly higher levels of resting spores to the soil in a greenhouse test than did a cycle of the resistant cultivar, as assessed by quantitative PCR and microscope analysis.     

Efficacy of Vapam fumigant against clubroot (Plasmodiophora brassicae) of canola

Clubroot, caused by Plasmodiophora brassicae, has become a serious threat to canola (Brassica napus) production in western Canada. Experiments were conducted under greenhouse and field conditions to assess the effect of Vapam fumigant (dithiocarbamate; sodium N‐methyldithiocarbamate) on primary and secondary infection by P. brassicae, clubroot severity, and growth parameters in canola. Preliminary trials showed a 12–16‐fold reduction in primary and secondary infection and clubroot severity at all of the Vapam application rates (0·4–1·6 mL L^?1 soil) assessed. Vapam was also found to be effective in reducing clubroot severity and improving seed yield of canola under field conditions. Application of Vapam at soil moisture levels in the range of 10–30% (v:v) had a large effect on both disease severity and infection rates and plant growth parameters. The results suggest that Vapam can effectively reduce clubroot severity and may be useful for the treatment of transplant propagation beds in brassica vegetable production, and for the containment of small, localized clubroot infestations in commercial canola crops.     

Reducing the build‐up of Plasmodiophora brassicae inoculum by early management of oilseed rape volunteers

Clubroot of oilseed rape (OSR), caused by Plasmodiophora brassicae, is a disease of increasing economic importance worldwide. Previous studies indicated that OSR volunteers, Brassica crops and weeds play a critical role in the predisposition of the disease. To determine the effect of timing of foliar application of the herbicide glyphosate or mechanical destruction of OSR volunteers in reduction of clubroot severity and resting spore production, a series of studies was conducted under controlled conditions with a susceptible OSR cultivar and an isolate of P. brassicae. Plants were inoculated by injecting a spore suspension beside the root hairs at growth stage 11–12 (BBCH scale) and were terminated at 7 (early) or 21 (late) days post‐inoculation (dpi). Under controlled conditions, the first symptoms on roots were observed as early as 7 dpi. The early application of glyphosate as well as early mechanical destruction resulted in significant (P ≤ 0.05) reduction in the development of clubroot symptoms, root fresh weight and the number of resting spores?g root. Furthermore, the effect of volunteer management on clubroot severity in the succeeding OSR was studied by inoculating plants with the resting spores obtained from treated clubbed roots. Inoculated OSR exhibited root clubs similar to the initial symptoms after 35 dpi. Plants that were inoculated with spore suspension from early treated roots resulted in significant reductions in clubroot incidence and severity. Conversely, plants inoculated with the spore suspension from the late treated roots displayed levels of clubroot similar to the plants inoculated with the spore solutions of positive controls.     

Potential loss of clubroot resistance genes from donor parent Brassica rapa subsp. rapifera (ECD 04) during doubled haploid production

Clubroot resistance derived from the oilseed rape/canola Brassica napus ‘Mendel’ has been overcome in some fields in Alberta, Canada, by the emergence of ‘new’ strains of the protist Plasmodiophora brassicae. Resistance to the pathogen was assessed in 112 doubled haploid (DH) lines, derived from B. rapa subsp. rapifera (European clubroot differential (ECD) 04). The lines were evaluated against five single‐spore isolates representing the ‘old’ pathotypes 2, 3, 5, 6 and 8, and 15 field populations representing new strains of P. brassicae. The disease severity index (ID%) data revealed that none of the DH lines were resistant or moderately resistant to the new pathotype 5X (field populations L‐G1, L‐G2, L‐G3) and D‐G3, while 3–42% were resistant or moderately resistant to the other 11 new strains. Using the mean ID induced by the old pathotype 3 (approx. 13.5%) as the baseline, clubroot severity increased by 300–600% when inoculated with the new pathotypes. A significant finding of this study was the fact that ECD 04 showed absolute resistance to all of the old and new P. brassicae strains while the B. napus ‘Mendel’, although resistant to all of the old pathotypes, was resistant to only about 50% of the new strains. Similarly, all of the selected clubroot‐resistant commercial canola cultivars evaluated in this study were susceptible to 87% of the new P. brassicae strains. The molecular data revealed that the breakdown of clubroot resistance in Mendel and the canola cultivars was in part due to the non‐inheritance of the Crr1 gene on the A08 chromosome from ECD 04.     

Effects of temperature on infection and subsequent development of clubroot under controlled conditions

Controlled‐environment studies were conducted on two Brassica crops (canola, Brassica napus; and Shanghai pak choi, B. rapa subsp. chinensis var. communis) to examine the effects of temperature on infection and subsequent development of clubroot caused by Plasmodiophora brassicae. In the first experiment, canola seedlings were grown in infested soil for 3 weeks at 14–26°C to assess the impact on primary and secondary infection and transferred to 20°C for 3 weeks to assess symptom development under uniform conditions, or started at 20°C for 3 weeks and then placed at the treatment temperatures for the final 3 weeks to assess the impact of temperature on symptom development. A second experiment examined a wider range of temperatures (10–30°C). Similar experiments were also conducted on Shanghai pak choi. The studies demonstrated that clubroot severity was affected by temperature during both infection and vegetative development of the crop. Both early and late in crop development, little or no clubroot developed at temperatures at or below 17°C, and development was slower above 26°C than at 23–26°C for both crops throughout the study. In canola, the high levels of inoculum used in the study resulted in a high incidence of clubroot irrespective of temperature, but in pak choi incidence showed the same pattern as severity. This is the first study to demonstrate under controlled conditions that temperature during vegetative growth of the crop affects symptom development of clubroot.     

Comparison of techniques for estimation of resting spores of Plasmodiophora brassicae in soil

Clubroot (Plasmodiophora brassicae) is an important disease of canola (Brassica napus) and other brassica crops. Accurate estimation of inoculum load in soil is important for evaluating producer risk in planting a susceptible crop, but also for evaluation of management practices such as crop rotation. This study compared five molecular techniques for estimating P. brassicae resting spores in soil: quantitative polymerase chain reaction (qPCR), competitive positive internal control PCR (CPIC-PCR), propidium monoazide PCR (PMA-PCR), droplet digital PCR (ddPCR) and loop-mediated isothermal DNA amplification (LAMP). For ddPCR and LAMP, calibrations were developed using spiked soil samples. The comparison was carried out using soil samples collected from a long-term rotation study at Normandin, Québec, with replicated plots representing 0-, 1-, 2-, 3-, 5- and 6-year breaks following susceptible canola infested with clubroot. CPIC-PCR and ddPCR provided repeatable estimates of resting spore numbers in soil compared with estimates from qPCR or LAMP alone. CPIC-PCR provided the most robust measurement of spore concentration, especially in the 2 years following a crop of susceptible canola, because it corrected for effects of PCR inhibitors. PMA-PCR demonstrated that a large proportion of the DNA of P. brassicae detected in soil after the susceptible canola crop was derived from spores that were immature or otherwise not viable. Each assay provided a similar pattern of spore concentration in soil, which supported the conclusion of a previous study at this site that resting spore numbers declined rapidly in the first 2 years after a susceptible crop, but much more slowly subsequently.     

Direct evidence of surface infestation of seeds and tubers by Plasmodiophora brassicae and quantification of spore loads

Using quantitative PCR, DNA of Plasmodiophora brassicae, the causal agent of clubroot, was detected and quantified on canola, pea and wheat seeds, as well as on potato tubers, all harvested from clubroot‐infested fields in Alberta, Canada. Quantifiable levels of infestation were found on seven of the 46 samples analysed, and ranged from <1·0 × 10³ to 3·4 × 10⁴ resting spores per 10 g seeds; the vast majority (80–100%) of resting spores on these samples were viable, as determined by Evan’s blue vital staining. However, the levels of infestation found were generally lower than that required to cause consistent clubroot symptoms in greenhouse plant bioassays. While the occurrence of P. brassicae resting spores on seeds and tubers harvested from clubroot‐infested fields suggests that seedborne dissemination of this pathogen is possible, practices such as commercial seed cleaning may be sufficient to effectively mitigate this risk.     

Suppression of clubroot by Clonostachys rosea via antibiosis and induced host resistance

The mechanism of the biofungicide Prestop^® (Clonostachys rosea) was investigated for control of clubroot (Plasmodiophora brassicae) on canola. The key product components were partitioned and assessed for their effect on pathogen resting spores, root hair infection (RHI) and disease development using light microscopy, quantitative PCR and different application treatments during infection. The whole product of Prestop was consistently more effective than the C. rosea conidial suspension or product filtrate alone in reducing RHI and clubroot development. This biofungicide showed little effect on germination or viability of resting spores. Two‐application treatments at seeding and 7–14 days after seeding achieved greater clubroot control than a single application of the biofungicide at either seeding or post‐seeding stage. This may indicate the need to maintain a high biofungicide dose in the soil during primary and secondary infection. This biocontrol fungus colonized the rhizosphere and interior of canola roots extensively, and possibly induced plant resistance based on up‐regulation of the genes that are involved in jasmonic acid (BnOPR2), ethylene (BnACO) and phenylpropanoid (BnOPCL, BnCCR) biosynthetic pathways. It is concluded that the biofungicide Prestop suppressed clubroot on canola at least via root colonization and induced systemic resistance (ISR), and the latter may be through the modulation of phenylpropanoid and jasmonic acid/ethylene metabolic pathways elicited by the fungus.     

Identification of Brassica accessions resistant to ‘old’ and ‘new’ pathotypes of Plasmodiophora brassicae from Canada

Genetic resistance is the main tool used to manage clubroot of canola (Brassica napus) in Canada. However, the emergence of new virulent strains of the clubroot pathogen, Plasmodiophora brassicae, has complicated canola breeding efforts. In this study, 386 Brassica accessions were screened against five single-spore isolates (represented by pathotypes 2F, 3H, 5I, 6M and 8N on the Canadian Clubroot Differential Set) and 17 field isolates (represented by 12 unique pathotypes: 2B, 3A, 3D, 3O, 5C, 5G, 5K, 5L, 5X, 8E, 8J and 8P) of P. brassicae to identify resistance sources effective against these strains. The results showed that one B. rapa accession (CDCNFG-046, mean index of disease (ID) = 3.3%) and two B. nigra accessions (CDCNFG-263, mean ID = 3.1%; and CDCNFG-262, mean ID = 4.7%) possessed excellent resistance to all 22 of the isolates evaluated. Fifty other accessions showed differential clubroot reactions (resistant, moderately resistant or susceptible), including 27 (one B. napus, two B. rapa, four B. oleracea and 20 B. nigra) accessions that were each resistant to 8–21 P. brassicae isolates, but developed mean IDs in the range of 5.3–29.6%. The remaining 23 accessions (two B. napus, one B. rapa, five B. oleracea and 15 B. nigra) were each resistant to 3–13 isolates, but developed mean IDs in the range of 30.3–47.0%. The three accessions that showed absolute resistance and the 50 accessions that showed differential clubroot reactions could be used to breed for resistance to the new P. brassicae strains.     

Flooding affects the development of Plasmodiophora brassicae in Arabidopsis roots during the secondary phase of infection

Clubroot, a disease of Brassicaceae species, is caused by the soilborne pathogen Plasmodiophora brassicae. High soil water content was previously described to favour the motility of zoospores and their penetration into root cells. In this study, the effect of irrigation regimes on clubroot development during the post‐invasive secondary phase of infection was investigated. Three irrigation regimes (low, standard, high) were tested on two Arabidopsis accessions, Col‐0 (susceptible) and Bur‐0, a partially resistant line. In Col‐0, clubroot symptoms and resting spore content were higher under the ‘low irrigation’ regime than the other two regimes, thus enhancing the phenotypic contrast between the two Arabidopsis accessions. Clubroot severity under high and low irrigation regimes was evaluated in near‐isogenic lines derived from a Col‐0 ×  Bur‐0 cross, to assess the effect of soil moisture on the expression of each of four quantitative trait loci (QTL) controlling partial resistance. The presence of the Bur‐0 allele at the QTL PbAt5.2 resulted in reduced severity only under low irrigation, whereas the Bur‐0 allele at QTL PbAt5.1 was associated with partial resistance only under high irrigation. QTL PbAt4 reduced the number of resting spores in infected roots, but was not associated with reduced clubroot symptoms. The results indicated that soil moisture could have consequences for the secondary phase of clubroot development, depending on plant genotype. Future genetic studies may benefit from using combinations of watering conditions during the secondary stage of infection, thus opening up the possibility of identifying genetic factors expressed under specific environmental conditions.     

Nitrogen supply exerts a major/minor switch between two QTLs controlling Plasmodiophora brassicae spore content in rapeseed

Clubroot, caused by Plasmodiophora brassicae, is a worldwide disease affecting Brassica. Until now, the detection of genetic factors (QTLs) implicated in clubroot resistance has been based on estimates of disease index. However, as the amount of resting spores released in soil after club disintegration influences clubroot epidemics and resistance‐breaking dynamics, its genetic control may deserve specific attention. In a previous report, it was shown that nitrogen fertilization modulated quantitative partial resistance toward clubroot symptom development in rapeseed. The present work aimed to identify genetic factors involved in the control of resting spore production and to assess their regulation by nitrogen supply. A flow cytometer method was adapted for rapidly estimating resting spore content in a large series of samples. Linkage analysis was conducted to detect QTLs implicated in resting spore production in a Brassica napus doubled haploid progeny from the cross Darmor‐bzh × Yudal. DH lines inoculated with the P. brassicae isolate eH were grown under low‐ and high‐nitrogen supply. Under low‐nitrogen conditions, resting spore production was reduced compared to high‐nitrogen conditions, regardless of genotypes. Genetic architecture controlling resting spore production and clubroot symptom development was similar. Under high‐nitrogen conditions, resting spore production was controlled by one major QTL (C09a) and a few small‐effect QTLs. By contrast, two major QTLs (C02 and C09a) controlled resting spore production under low‐nitrogen conditions. This work highlighted a large see‐saw effect between the relative contribution of the C09a QTL (high effect under high‐nitrogen conditions) and the C02 QTL (high effect under low‐nitrogen conditions), with possible implications in resistance breeding.     

Root hair infection by Plasmodiophora brassicae in clubrootresistant and susceptible genotypes of Brassica oleracea,B. rapa and B. napus

The pathogenesis of clubroot, a disease of cruciferous crops caused by the fungusPlasmodiophora brassicae, starts with infection of the root hairs. This process was studied in 13 accessions ofBrassica oleracea, B. napus and B. rapa with varying levels of plant resitance toP. brassicae. Seedlings were grown in a mineral solution, inoculated with resting spores ofP. brassicae, and the number of plasmodia developing in root hairs was recorded. When compared with the standard susceptible cultivar Septa, both higher and lower resistance to root hair infection was found in the accessions of the differentBrassica species. No complete resistance to root hair infection was found. Over the accessions studied, there was no correlation between the plant resistance estimated from greenhouse tests and the resistance to root hair infection of seedlings. The resistance of all accessions must at least partly be caused by other mechanisms which operate after the root hair plasmodia are formed.     

Effect of temperature on primary infection by Plasmodiophora brassicae and initiation of clubroot symptoms

A study was conducted to assess the effect of temperature on infection and development of Plasmodiophora brassicae in root hairs of Shanghai pak choi (Brassica rapa subsp. chinensis) and on initiation of clubroot symptoms. Ten‐day‐old seedlings were grown in liquid‐sand culture, inoculated with resting spores and maintained in growth cabinets at 10, 15, 20, 25 and 30°C. Seedlings were harvested at 2‐day intervals, starting 2 days after inoculation (DAI) and continuing until swelling of the tap root was observed (maximum 28 days). Roots were assessed for root hair infection (RHI), stage of development of infection (primary plasmodia, zoosporangia, release of zoospores, secondary plasmodia), symptom development, and for clubroot severity at 24 DAI. Temperature affected every stage of clubroot development; RHI was highest and visual symptoms initiated earliest at 25°C, intermediate at 20 and 30°C, and lowest and latest at 15 and 10°C. Root hair infection was observed at every temperature, but clubroot symptoms developed only above 15°C. A substantial delay in the development of the pathogen was observed at 10 and 15°C. No symptoms were observed at 28 DAI in plants grown at 10°C. Swelling of the tap root was visible at 28 DAI in plants at 15°C, 14 DAI at 20 and 30°C, and 10 DAI at 25°C. These results support and explain the observation in companion studies that cool temperatures result in slower development of clubroot symptoms in brassica crops.     

Effect of soil type,organic matter content,bulk density and saturation on clubroot severity and biofungicide efficacy

Growth room experiments were conducted to assess the interaction of soil type, biofungicides, soil compaction and pathotype/host on infection and symptom development caused by Plasmodiophora brassicae, the cause of clubroot on Brassica spp. In two initial experiments, four soil types (peat soil, mineral soil, non‐calcareous sand, soil‐less mix), two biofungicides (Bacillus subtilis, Clonostachys rosea), and two pathotypes (3 and 6, Williams’ differential set) were assessed. Differences in clubroot severity associated with soil type were unexpectedly small and variable. Prestop (C. rosea) was often more effective than Serenade (B. subtilis) at reducing clubroot levels on peat and mineral soils, but less effective than Serenade on sand. Inoculation with pathotype 3 often resulted in a slightly higher mean severity than pathotype 6. The interaction of soil type × biofungicide was similar on both canola (B. napus) and Shanghai pak choy (B. rapa subsp. chinensis), whether the soil was kept saturated or allowed to drain after inoculation. The impact of soil type on biofungicide efficacy might explain, in part, why biofungicides are more effective in one location than another. The observation that clubroot severity in soil‐less mix was affected by compaction led to an investigation of soil bulk density. Severity was higher in soil‐less mix that was more compacted than in the initial experiments, and was lower in peat and mineral soils when soil bulk density was reduced by adding soil‐less mix. In this study, soil bulk density had a larger impact on clubroot than soil type, organic matter or pathotype.     

Mapping of clubroot (Plasmodiophora brassicae) resistance in canola (Brassica napus)

Clubroot disease, caused by Plasmodiophora brassicae, has become a major problem in the production of cruciferous crops worldwide. In this study, a population of 121 doubled haploid (DH) lines derived from a crossing between a resistant and a susceptible canola (Brassica napus) genotype was subjected to phenotypic and genotypic studies to determine the inheritance and location of the resistance gene(s). After inoculation with pathotype 3 of P. brassicae, the lines showed a 1:1 segregation ratio for resistance, indicating that resistance in this population is controlled by a single gene. Fifteen PCR‐based markers that were known to be linked to clubroot resistance (CR) genes were screened against genomic DNA from parents and resistant and susceptible bulks. Marker GC1680, linked to the CR gene CRa, exhibited polymorphism between the parents and between the resistant and susceptible bulks. CRa target primers were used to amplify fragments from the two parents and the resultant sequences were compared. A high degree of sequence similarity was found between the parents in the nucleotide binding site domain of CRa. In contrast, sequence polymorphisms were detected in the leucine‐rich repeat (LRR) domain. One pair of primers that amplify a band from the LRR region of the resistant parent but not the susceptible parent was used to screen the DH population. Amplicons were obtained from 60 of the 61 resistant lines and two of the 60 susceptible lines; thus, three recombinants were found. Based on these results, a resistance locus linked to CRa was found.