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.