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.
Clubroot of canola (Brassica napus, B. rapa), caused by the obligate parasite Plasmodiophora brassicae, is a soilborne disease that is responsible for severe crop losses in Alberta, Canada (Tewari et al., 2005; Strelkov et al., 2006; Hwang et al., 2011a,b). Once in a field, the pathogen builds up rapidly in the presence of susceptible crops and persists in the soil for more than 15 years (Wallenhammar, 1996). Therefore, crop rotations based on standard agronomic recommendations (canola grown only one year in four) may not significantly reduce clubroot inoculum levels (Strelkov et al., 2006). Even substantially longer rotations may not be effective because several common and endemic weed species, such as horseradish (Armoracia rusticana), white mustard (B. hirta), wild mustard (B. kaber), camelina/false flax (Camelina sativa), shepherd’s purse (Capsella bursa-pastoris) and stinkweed (Thlaspi arvense) are susceptible hosts (Howard et al., 2010). There is increasing concern that P. brassicae could spread across western Canada.
A range of alternative management strategies is being assessed for their usefulness in canola production, including the manipulation of seeding dates (Gossen et al., 2009) and biological control (Peng et al., 2011). The impact of bait crops (Kroll et al., 1983; Ikegami, 1985; Murakami et al., 2001) on soil resting spore populations and subsequent clubroot severity is also being examined under greenhouse and field conditions. Resting spore populations and subsequent clubroot severity were often slightly reduced following two cycles of cruciferous crops, but the impact was generally small (Ahmed et al., 2011). Bait crops had no effect on clubroot severity at two commercial field sites where populations of resting spores were high (1 × 106 spores per g of soil), suggesting that they are unlikely to become an important component of an IPM programme for clubroot of canola (Ahmed et al., 2011).
Effective clubroot management in canola will largely depend on utilization of cultivar resistance and the reduction of viable resting spore populations in the soil. Clubroot-resistant canola cultivars have recently become available for commercial production in Canada (Strelkov et al., 2011). However, data are needed on the effects of seeding resistant canola genotypes on resting spore populations in the soil of infested fields. Non-host or resistant crops grown prior to a susceptible host reportedly reduced clubroot disease severity (MacFarlane, 1952; Ikegami, 1985). Similarly, colonization and club formation were significantly reduced when susceptible and resistant radish (Raphanus sativus) cultivars were grown together, in comparison with treatments in which only susceptible cultivars were grown (Kroll et al., 1984). Knowledge of the impact of susceptible host genotypes on clubroot severity, and of their potential interaction with resistant hosts is particularly important in a canola cropping system, because susceptible volunteer plants will continue to be present in infested fields for many years, even when a resistant canola cultivar is sown. In addition, there will continue to be off-types in seed lots, gene segregation and incomplete resistance to some pathotypes, even within a resistant cultivar, as well as the presence of susceptible cruciferous weeds. The occurrence of large numbers of susceptible plants may offset the benefits of cropping a resistant cultivar. Furthermore, the deployment of resistant cultivars may create selection pressure on the pathogen to co-evolve and overcome resistance (Hwang et al., 2011a).
The objectives of this study were: (i) to evaluate the effect(s) that different proportions of susceptible canola plants (i.e. ‘volunteers’) within a stand of a resistant cultivar have on P. brassicae resting spore populations in the soil; and (ii) to examine the relationship between a bioassay for root hair infection and qPCR analysis of clubroot infection in resistant and susceptible canola genotypes.
Materials and methods
Impact of volunteer (susceptible) canola on inoculum potential
The hybrid canola cultivars 45H29 (clubroot-resistant) and 45H26 (clubroot-susceptible, Pioneer Hi-Bred) were grown together to simulate various proportions of susceptible plants (volunteer canola or Brassica weeds) growing within a stand of a clubroot-resistant canola cultivar. Seed of the susceptible and resistant cultivars were mixed together in ratios of 1:0, 3:1, 1:1, 1:3 and 0:1, respectively, and then four rows of 25 seeds each were planted in each experimental unit under greenhouse conditions (20 ± 2°C day/16 ± 2°C night, 16 h photoperiod). Each experimental unit consisted of a plastic tub (35 × 23 × 13·5 cm depth, with drainage holes) filled with soilless mix consisting of peat moss, coarse perlite, gypsum, dolomitic limestone and starter nutrient (Sunshine Mix 4; pH 6·5; SUN GRO®; Horticulture Canada Ltd). The soilless mix was inoculated with resting spores of P. brassicae isolate SACAN-ss1, which was isolated by Xue et al. (2008) and characterized as pathotype 3 on the differentials of Williams (1966). The pathogen was maintained as frozen root galls, and the resting spores were extracted as previously described (Strelkov et al., 2006). The spore concentration was adjusted to 1 × 106 spores mL−1 of soilless mix. After seeding, each tub was watered to saturation (water pH 6·5) and placed in a water-filled tray to maintain high soil moisture for the first 2 weeks, after which the plants were watered using an overhead sprinkler once each day for 4 weeks. At 6 weeks after seeding, the plants were pulled, the roots were washed under tap water, and data on plant height, root weight, and clubroot incidence and severity were assessed for 50 randomly chosen plants from each replication of a treatment, in order to provide an estimate of inoculum potential. Clubroot severity was assessed on a 0–3 scale, where: 0 = no galls, 1 = a few small galls (small galls on less than one-third of the roots), 2 = moderate galling (small to medium galls on one-third to two-thirds of the roots), and 3 = severe galling (medium to large galls on more than two-thirds of the roots) (Kuginuki et al., 1999). Severity ratings for each experimental unit were converted to an index of disease (ID) using the following formula (Strelkov et al., 2006):
where: n0, n1, n2 and n3 are the number of plants in each class, 0, 1, 2 and 3 are the symptom severity classes, and N is the total number of plants. After recording the data, all of the roots representing a replication were pooled and macerated in a blender, and the slurry was filtered through eight layers of cheesecloth to separate the resting spores of P. brassicae from the plant material. The resting spores from each tub were returned to the growth substrate in that tub and mixed thoroughly into the substrate. The substrate was then sown with 100% clubroot susceptible canola (cv. 45H26). Post-seeding agronomic practices followed the procedure described earlier. Six weeks after seeding, data on plant height, clubroot incidence and severity were recorded. The experiment was laid out in a randomized complete block design (RCBD) with six replications, and the study was repeated once.
In a second experiment, the effect of comparatively low proportions (0, 3, 5, 7 and 10%) of susceptible plants (cv. 45H26) in a stand of a resistant canola cultivar (cv. 71–45, Monsanto) was assessed. The experimental design, growing conditions, and agronomic practices were the same as in the initial experiment, except that the seeds of the resistant and susceptible cultivars were not mixed together. Instead, seeds of the susceptible cultivar were distributed at random in the four rows of 25 seeds, and marked with plastic tags to identify where seeds of the susceptible cultivar were sown. After 6 weeks, plant height, clubroot incidence and severity were recorded separately for plants of the resistant and susceptible cultivars. All of the roots of the susceptible plants (maximum of 3–10 roots, depending on the proportion of susceptible plants in each treatment) were collected. To make the number of roots equal (25 roots) for each replication, an additional 15–22 roots were randomly chosen from the resistant plants.
These 25 canola roots were macerated and filtered as described previously. The volume of the suspension for each tub was adjusted to 250 mL and the number of resting spores was estimated with a haemocytometer. The resting spores were returned to each tub, which was then planted with seed of the susceptible cultivar. Ten days after seeding, five seedlings per tub were uprooted and the roots were washed gently and then evaluated microscopically for root hair infection.
A 1·5 cm long segment of the taproot at 2–3 cm below the hypocotyl (Murakami et al., 2000) was cut from two seedlings per replication and mounted on a slide with 5% (v/v) glycerine in water. The incidence of root hair infection in the mounted specimens was then examined with a compound microscope at × 100 magnification. A root hair was regarded as infected when a primary plasmodium could be discerned within it. At least 100 root hairs were examined per sample (Hwang et al., 2011a). In addition to root hair infection, secondary plasmodia were also counted below the epidermis and in the cortex of the roots, which were rated on a 1–5 scale, where 1 = few secondary plasmodia and 5 = profuse secondary plasmodia. The remaining plants were allowed to grow to maturity. Plant height and clubroot severity were assessed at the flowering stage. The study was repeated once.
The homogeneity of variance of all the data sets was confirmed using normal probability plots, with the data from each study then examined by analysis of variance (GLM procedure; SAS Inc.). Analysis of variance revealed that there was no main effect of repetition, so the data from the two repetitions of each experiment were combined for subsequent analyses. Means of treatments were separated (where appropriate) using a protected LSD test at P ≤0·05.
Comparison of the bioassay and qPCR analysis
A trial to compare assessments of clubroot infection using a standard greenhouse bioassay and a quantitative PCR-based protocol was conducted under greenhouse conditions. Clubroot galls stored at −20°C were macerated in a blender and filtered through eight layers of cheesecloth. The concentration of resting spores in the ground inoculum was estimated with a haemocytometer. The inoculum was added to a soilless potting mix to produce a spore concentration of 1 × 106 spores mL−1 of soilless mix as described above.
The experiment was designed as a randomized complete block with five replicates. The main plot treatments were sampling dates (4, 6, 8 and 10 days after sowing) and the subplot treatments were hybrid canola cultivars [resistant cultivars 45H29 (Pioneer Hi-Bred) and 73-77RR (Monsanto); susceptible cultivars 34-65RR (Monsanto), 45H26 and 45H73 (Pioneer Hi-Bred)]. Each experimental unit consisted of a 9 cm diameter (450 mL) cup in which 10 canola seeds had been sown into soilless mix (Sunshine Mix 4; SUN GRO Horticulture). After seeding, the cups were placed in water-filled trays to ensure high soil moisture. At each sampling date, all the seedlings per replicate cup were uprooted and washed gently with tap water. Immediately after harvest, half of the plants were fixed in FAA (5 mL of formalin, 5 mL of acetic acid, 90 mL of 50% ethanol), and maintained at room temperature (20 ± 2°C) until needed (Hwang et al., 2011b). The remainder of the seedlings were stored at −20°C for qPCR analysis. Root hair infection was assessed using the protocol described above.
A parallel experiment was conducted with inoculated and non-inoculated seedlings as the main plot treatments and canola cultivars as subplots. The plants were grown for 6 weeks, and plant height, root weight and clubroot severity (0–3 scale, converted to ID) were assessed. Both experiments were conducted twice.
For qPCR analysis, roots of each sample were gently washed under tap water. Prior to DNA extraction, fresh root samples were washed with sterilized water and dried with a sterilized paper towel, and 200 mg subsamples were used for DNA extraction with a Fast DNA Spin Kit (Qbiogene Inc.) as per the manufacturer’s instructions. Quantitative PCR analysis was performed on undiluted DNA extracted from the root samples. Two P. brassicae-specific primer and TaqMan probe sets were in the qPCR: RC1F (5′-TCCCATATCCAACCCCATGT-3′), RC1R (5′-TCGGCTAGGATGGTTCGAAA-3′), RP1P (5′-6FAM-AACCGGTGACGTGCGG-MGBNFQ-3′) and RC2F (5′-TAATCGCCCTGGGTATGGTA-3′), RC2R (5′-CGCGATTCGACGTAGGACT-3′), RP2P (5′-6FAM-ACAACGGACAAGGATGTT-MGBNFQ-3′), as previously reported (Hwang et al., 2011a). All qPCR amplifications were conducted using a StepOnePlus Real Time PCR System (Applied Biosystems) in a 20 μL volume containing 1·0 μL of template DNA solution, 10 μm of each primer, 2 μm of MGB-probe and 10 μL TaqMan® Fast Universal PCR Master Mix (Applied Biosystems). All reactions were run at 95°C for 20 s, followed by 40 cycles at 95°C for 1 s and 60°C for 20 s.
The homogeneity of variance of each data set was examined using a normal probability plot. Analysis of variance (GLM procedure; SAS) indicated a cultivar × sampling date interaction for both root hair infection and the amount of P. brassicae DNA in infected roots, so the data were analysed separately by sampling date. Means comparison within each sampling date was performed using a LSMEAN t-test. Regression analysis was used to quantify the relationship between root hair infection and the amount of P. brassicae DNA in the infected roots. For this purpose, DNA and root hair infection data for each sampling date were pooled across canola cultivars. In addition, these data were also pooled for each cultivar across the sampling dates.
In the trial to examine the effect of cultivar resistance on clubroot severity and plant growth parameters, the mean data on plant height and root mass of inoculated versus control treatments were compared within a cultivar, and ID was compared among cultivars using a LSMEAN t-test. For each experiment, differences are significant at P ≤0·05 unless otherwise noted.
Impact of volunteer (susceptible) canola on inoculum potential
In the study with higher proportions of susceptible canola, plant height and clubroot levels (incidence and ID) increased with increasing proportions of susceptible plants in the mixed populations of resistant and susceptible canola cultivars (Fig. 1a). Disease severity and disease incidence were significantly greater, and plant height was lower, in soil where 100% susceptible plants had been grown after the mixed populations (Fig. 1b).
The study with lower proportions of susceptible canola indicated that the height of both resistant and susceptible canola plants was reduced relative to the control when inoculated with the clubroot pathogen. The susceptible canola was normally shorter than the resistant canola, even in the absence of inoculation (Table 1). There was no effect of treatment on clubroot severity in the susceptible cultivar grown in any proportion with the resistant cultivar (Table 2). As expected, the total resting spore count in the clubroot galls was greater after growth of a higher proportion of susceptible plants. Root hair infection in the successively grown susceptible cultivar also rose in treatments with a greater proportion of susceptible plants. Rates of secondary infection below the epidermis and in the cortex, as estimated by the number of secondary plasmodia, were also greater in the treatments in which a higher proportion of susceptible plants were grown (Table 2).
Table 1. Effect of ‘volunteer’ canola (proportion of crop that is susceptible) on plant height of resistant and susceptible canola plants grown under greenhouse conditions
Susceptible plants (%)
Plant height (cm)a
aMeans followed by the same letter for inoculated versus non-inoculated within a cultivar category do not differ at P ≤0·05 based on the LSMEAN t-test.
Table 2. Effect of ‘volunteer’ canola (proportion of crop that is susceptible) on resting spore loads and on root hair colonization and secondary plasmodium formation on a subsequent susceptible canola crop under greenhouse conditions
Susceptible plants (%)
Resting sporeb (x 109)
Root hair colonizationc (%)
aMeans in a column followed by the same letter do not differ at P ≤0·05 based on a LSMEAN t-test.
bAdditional resting spores contributed to the soilless mix after growing different proportions of volunteer canola, estimated using a haemocytomer. Data are the means of six replications.
cRoot hair colonization (formation of primary plasmodium) was estimated 10 days after seeding susceptible canola (cv. 45H26) after growing different proportions of the same susceptible canola cultivar as a volunteer with resistant canola Monsanto cv. 71-45, and incorporation of resting spores from the galls formed on the volunteer canola 6 weeks after the first cycle of seeding.
dThe secondary plasmodium was estimated using a qualitative scale 1–5 where 1 = few and 5 = profuse secondary plasmodia inside the root epidermis and cortex.
ID%, (index of disease %).
Comparison of bioassay and qPCR analysis
The analysis of variance indicated significant sampling date effects, cultivar effects, and a sampling × cultivar interaction for root hair infection and the amount of DNA extracted from clubroot-infected root hairs. The proportion of variance explained by sampling date, canola cultivar, and sampling × cultivar interaction was 35, 62 and 3% for root hair infection, and 85, 12 and 3% for DNA extraction, respectively. Root hair infection was always significantly higher in the susceptible canola cultivars (45H26, 34-65RR and 45H73) than in the resistant cultivars (45H29 and 73-77RR) at all sampling dates (Fig. 2a). The trend was similar for the DNA extracted from the root samples with the exception of one time point; at 4 days, no significant differences in the amount of DNA were observed (Fig. 2b). Root hair infection and P. brassicae DNA concentration increased over time. However, increases in root hair infection and the amount of DNA extracted were greater in the susceptible cultivars relative to the resistant cultivars (Fig. 2a,b).
Regression analysis of root hair infection and the amount of pathogen DNA revealed a strong linear relationship between these two variables at all sampling dates. The r2 values for the regressions were 80, 82, 90 and 99% at 4, 6, 8 and 10 days, respectively (Fig. 3). When regression analysis of root hair infection and P. brassicae DNA was conducted for each cultivar over the sampling dates, the linear relationships for the resistant cultivars (45H29, r2 = 93%; and 73-77RR, r2 = 81%) was strongest (Fig. 4). In contrast, the linear relationships between root hair infection and the amount of pathogen DNA in the susceptible cultivars was somewhat lower, with r2 values of 71, 73 and 76% for 45H73, 34-64RR and 45H26, respectively (Fig. 4). Moreover, in the resistant cultivars, the increase in root hair infection and amount of pathogen DNA rose slowly and gradually and stabilized at 8 and 10 days. In the susceptible cultivars, the increase in both root hair infection and amount of DNA rose sharply in two steps, first at 4–6 days and then at 8–10 days (Fig. 4). Regression of the pooled data over the sampling dates and cultivars revealed a weak linear relationship between root hair infection and amount of DNA in P. brassicae infected roots (r2 = 64%) (Fig. 5).
Results from the experiment to assess the effect of resistance on plant growth parameters and clubroot severity indicated that the height of both resistant and susceptible plants was reduced after inoculation with the pathogen, although this reduction was not significant for the susceptible cultivar 45H26. The greatest reduction in plant height was observed for the susceptible cultivars 45H73 (58%) and 34-65RR (37%). In both the resistant and susceptible canola cultivars, the root mass increased after inoculation with P. brassicae. However, in the susceptible cultivars, this increase was significantly higher than in the resistant cultivars. As expected, ID in the resistant cultivars was lower than in the susceptible cultivars. Among the susceptible cultivars, ID was greater on 34-65RR and 45H73 than on 45H26 (Table 3).
Table 3. Effect of resistance on plant height, root mass and disease severity of canola grown in infested soilless mix under greenhouse conditions
Plant heightb (cm)
Root mass (g)
aCvs 45H29 (Pioneer Hi-Bred) and 73-77RR (Monsanto) are clubroot-resistant canola cultivars; 45H26, 45H73 (Pioneer Hi-Bred) and 34-65RR (Monsanto) are clubroot-susceptible cultivars.
bMeans followed by the same letter for inoculated versus non-inoculated within a cultivar do not differ significantly by LSMEAN t-test at P ≤0·05. Data are the mean of five replications and two trials of the experiment.
ID%, (index of disease %).
Resting spores of P. brassicae persist in soil for long periods of time. In western Canada, the only viable option for clubroot management in canola is the cropping of resistant cultivars. However, the benefits of this strategy may be offset if not all of the plants sown in a resistant crop are actually resistant, for instance through the presence of off-types, volunteers and susceptible weeds. Earlier studies revealed that colonization and club formation were significantly reduced when susceptible and resistant radish (Raphanus sativas) cultivars were grown together, relative to the treatments using only susceptible plants (Kroll et al., 1984). Yamagishi et al. (1986) reported that resistant crucifers reduced the numbers of residual resting spores after continuous cultivation over 4 years, although such consistent reductions in P. brassicae populations has not been achieved under Canadian field conditions.
In the current study, the impact of different proportions of susceptible canola plants within a resistant crop on clubroot disease severity in successive susceptible crops was examined. The cropping of mixtures of susceptible and resistant cultivars reduced clubroot severity, relative to the cropping of only susceptible cultivars, in the subsequent susceptible canola crop. This suggests that volunteer canola plays a significant role in the persistence of resting spores in infested soil. However, no empirical data are available on the number of resting spores contributed by volunteers in infested fields where resistant canola is grown. Moreover, while resting spore populations were not directly quantified via microscopic analysis in the present study, previous research clearly showed that both root hair infection and clubroot severity increase with increased inoculum density (Hwang et al., 2011a,b). In this context, it is reasonable to assume that reductions in root hair infection or disease severity are reflective of a reduced resting spore population in the soil or potting mixture. Therefore, the increased rates of root hair infection observed in the greenhouse study when a greater proportion of susceptible canola plants were included in the crop suggest a higher number of resting spores. These findings reinforce the importance of rogueing susceptible volunteers from infested fields, even if the field is not going to be sown to canola, in order to prevent the build up of pathogen inoculum. Clubroot severity increased in the subsequent crop when higher proportions of susceptible plants were grown in the preceding crop population, indicating that susceptible volunteers (and weeds) contribute to the populations of viable resting spores in infested fields.
Previously the influence of cultivar resistance on root hair infection in resistant and susceptible cultivars was studied, and a qPCR protocol was developed to estimate P. brassicae populations in the soil (Hwang et al., 2011a). However, in that study only two cultivars (one resistant and one susceptible) were compared, over a range of inoculum densities. In contrast, in the current study, root hair infection in two resistant and three susceptible canola cultivars was compared, but at a single inoculum density. The results revealed that root hair infection and ID were greater, while plant height was reduced, in the susceptible cultivars relative to the resistant cultivars. Root hair infection and the amount of P. brassicae DNA increased over time. However, the increases in infection and pathogen DNA were greater in the susceptible cultivars than the resistant cultivars. Moreover, there were strong linear relationships between percentage root infection and the amount of P. brassicae DNA in the root hairs, when data from individual sampling dates over all five cultivars were considered. The strongest linear relationships between root hair infection and quantity of DNA were observed when individual cultivars versus sampling dates were compared. The slow increase in the amount of pathogen DNA and root hair infection observed in the resistant cultivars, and the sharp increases observed at 4–6 and 8–10 days in the susceptible cultivars, suggest that secondary infection and disease development in the latter proceeded quickly relative to the former. It is evident that successful management of P. brassicae through the deployment of genetically resistant cultivars will require careful control of susceptible weeds and canola volunteers, in order to maximize the impact of resistant cultivars on resting spore levels in the soil.
The authors thank the Canola Agronomic Research Program (Alberta Canola Producers Commission, Manitoba Canola Growers Association, SaskCanola and the Canola Council of Canada), the Alberta Crop Industry development Fund (ACIDF) Program, the Clubroot Risk Mitigation Initiative (CRMI) through Agriculture and Agri-Food Canada and the Canola Council of Canada for financial support, and Pioneer and Monsanto for supplying the canola seed.