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.     

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.     

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.     

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.     

Effect of host and non‐host crops on Plasmodiophora brassicae resting spore concentrations and clubroot of canola

Plasmodiophora brassicae, causal agent of clubroot of crucifers, poses a serious threat to Canadian canola production. The effects of fallow (F) periods and bait crops (clubroot‐susceptible canola (B) and perennial ryegrass (R)) on clubroot severity and P. brassicae resting spore populations were evaluated in five sequences: R–B, B–R, R–F, B–F and F–F. Both host and non‐host bait crops reduced clubroot severity in a subsequent crop of a susceptible canola cultivar compared with fallow. Resting spore and P. brassicae DNA concentrations decreased in all treatments, but were lowest for the R–B and B–R bait crop sequences. In addition, two studies were conducted in mini‐plots under field conditions to assess the effect of rotation of susceptible or resistant canola cultivars on clubroot severity and P. brassicae resting spore populations. One study included three crops of susceptible canola compared with a 2‐year break of oat–pea, barley–pea, wheat–wheat or fallow–fallow. The other study assessed three crops of resistant canola, two crops of resistant canola with a 1‐year break, one crop of resistant canola and a 2‐year break, and a 3‐year break with barley followed by a susceptible canola. The rotations that included non‐host crops of barley, pea or oat reduced clubroot severity and resting spore concentrations, and increased yield, compared with continuous cropping of either resistant or susceptible canola. Growing of a susceptible canola cultivar contributed 23–250‐fold greater gall mass compared with resistant cultivars.     

Reduction of clubroot (Plasmodiophora brassicae) formation in Arabidopsis thaliana after treatment with prohexadione‐calcium,an inhibitor of oxoglutaric acid‐dependent dioxygenases

Recently, flavonoids were shown to modulate the outcome of clubroot development in Arabidopsis thaliana after infection with the obligate biotrophic pathogen Plasmodiophora brassicae. Therefore, the development of clubroot disease was investigated in Arabidopsis after treatment with prohexadione‐calcium (ProCa), an inhibitor of ascorbic acid/2‐oxoglutaric acid‐dependent dioxygenases such as flavanone‐3‐hydroxylase. The treatment resulted in a reduction of the flavonols quercetin and kaempferol in clubroots, whereas the precursor naringenin highly accumulated. The root system of ProCa‐treated plants was better developed although galls were still visible. Thus, ProCa treatment resulted in reduced gall size. Flavonoids are thought to inhibit polar auxin transport by modulating auxin efflux carriers. It was investigated whether the auxin response might change as a consequence of the accumulation of naringenin in ProCa‐treated plants. In the areas of gall development an auxin response was indicated by the auxin‐responsive promoter DR5 coupled to the reporter β‐glucuronidase (GUS), whereas very little staining was found in healthy root parts. No differences in GUS activity were found between P. brassicae‐infected and ProCa‐treated plants, and plants only infected with P. brassicae, indicating that the effect of ProCa treatment on clubroot reduction is not via changes in auxin responses. As ProCa is also an inhibitor of late steps in gibberellin biosynthesis, a specific gibberellin biosynthesis inhibitor, chlormequatchloride (CCC), was tested on club development. However, CCC did not reduce disease symptoms, indicating that the observed reduced gall development was not because of gibberellin biosynthesis inhibition by ProCa.     

Sporulation of Pyrenopeziza brassicae (light leaf spot) on oilseed rape (Brassica napus) leaves inoculated with ascospores or conidia at different temperatures and wetness durations

In controlled environment experiments, sporulation of Pyrenopeziza brassicae was observed on leaves of oilseed rape inoculated with ascospores or conidia at temperatures from 8 to 20°C at all leaf wetness durations from 6 to 72 h, except after 6 h leaf wetness duration at 8°C. The shortest times from inoculation to first observed sporulation ( l ₀), for both ascospore and conidial inoculum, were 11–12 days at 16°C after 48 h wetness duration. For both ascospore and conidial inoculum (48 h wetness duration), the number of conidia produced per cm² leaf area with sporulation was seven to eight times less at 20°C than at 8, 12 or 16°C. Values of Gompertz parameters c (maximum percentage leaf area with sporulation), r (maximum rate of increase in percentage leaf area with sporulation) and l ₃₇ (days from inoculation to 37% of maximum sporulation), estimated by fitting the equation to the observed data, were linearly related to values predicted by inserting temperature and wetness duration treatment values into existing equations. The observed data were fitted better by logistic equations than by Gompertz equations (which overestimated at low temperatures). For both ascospore and conidial inoculum, the latent period derived from the logistic equation (days from inoculation to 50% of maximum sporulation, l ₅₀) of P. brassicae was generally shortest at 16°C, and increased as temperature increased to 20°C or decreased to 8°C. Minimum numbers of spores needed to produce sporulation on leaves were ≈25 ascospores per leaf and ≈700 conidia per leaf, at 16°C after 48 h leaf wetness duration.     

Effects of R gene‐mediated resistance in Brassica napus (oilseed rape) on asexual and sexual sporulation of Pyrenopeziza brassicae (light leaf spot)

The phenotype of the R gene‐mediated resistance derived from oilseed rape (Brassica napus) cv. Imola against the light leaf spot plant pathogen, Pyrenopeziza brassicae, was characterized. Using a doubled haploid B. napus mapping population that segregated for resistance against P. brassicae, development of visual symptoms was characterized and symptomless growth was followed using quantitative PCR and scanning electron microscopy on leaves of resistant/susceptible lines inoculated with suspensions of P. brassicae conidia. Initially, in controlled‐environment experiments, growth of P. brassicae was unaffected; then from 8 days post‐inoculation (dpi) some epidermal cells collapsed (‘black flecking’) in green living tissue of cv. Imola and from 13 to 36 dpi there was no increase in the amount of P. brassicae DNA and no asexual sporulation (acervuli/pustules). By contrast, during this period there was a 300‐fold increase in P. brassicae DNA and extensive asexual sporulation in leaves of the susceptible cv. Apex. However, when leaf tissue senesced, the amount of P. brassicae DNA increased rapidly in the resistant but not in the susceptible cultivar and sexual sporulation (apothecia) was abundant on senescent tissues of both. These results were consistent with observations from both controlled condition and field experiments with lines from the mapping population that segregated for this resistance. Analysis of results of both controlled‐environment and field experiments suggested that the resistance was mediated by a single R gene located on chromosome A1.     

A nested‐PCR method for rapid detection of Sclerotinia sclerotiorum on petals of oilseed rape (Brassica napus)

This study established a quick and accurate method to detect petal infection of oilseed rape (Brassica napus) by Sclerotinia sclerotiorum using a nested‐PCR technique. DNA samples were extracted from each petal using a microwave method, followed by two rounds of PCR amplification. The first‐round PCR amplification was performed using the universal fungal primer pair ITS4/ITS5, and the second‐round amplification with a specific primer pair XJJ21/XJJ222, which was designed using the single‐nucleotide polymorphisms among nuclear rDNA ITS sequences of Sclerotinia spp., Botrytis spp. and other selected fungi. The established technique is rapid and inexpensive, and has a high degree of specificity and sensitivity. This assay can distinguish Sclerotinia spp. from other fungi, including Botrytis cinerea, a closely related and frequent cohabitant on oilseed rape petals, and can detect 50 fg genomic DNA, five ascospores of S. sclerotiorumin vitro or 50 ascospores of S. sclerotiorum on one petal in approximately 6 h, even in the presence of a high background of oilseed rape DNA. This technique was successfully applied in detecting natural petal infections.     

Effects of cultivar resistance and fungicide application on stem canker of oilseed rape (Brassica napus) and potential interseasonal transmission of Leptosphaeria spp. inoculum

Phoma stem canker is a damaging disease of oilseed rape (Brassica napus) that causes annual yield losses to UK oilseed rape growers worth approximately £100 million, despite the use of fungicides. In the UK, oilseed rape is sown in August/September and harvested in the following July. The disease epidemics are initiated by ascospores released from Leptosphaeria spp. pseudothecia (ascocarps) on stem stubble in the autumn/winter. Control of this disease is reliant on the use of cultivars with “field resistance” and azole fungicides. This study investigated the effects of cultivar resistance and application of the fungicide prothioconazole on the severity of stem canker before harvest and the subsequent production of pseudothecia on the infected stubble under natural conditions in the 2017/2018, 2018/2019, and 2019/2020 cropping seasons. The application of prothioconazole and cultivar resistance decreased the severity of phoma stem canker before harvest, and the subsequent production of Leptosphaeria spp. pseudothecia on stubble in terms of pseudothecial density. Results showed that stems with less severe stem cankers produced fewer mature pseudothecia of Leptosphaeria spp. on the infected stubble. This investigation suggests that the most sustainable and effective integrated control strategy for phoma stem canker in seasons with low quantities of inoculum is to use cultivars with medium or good field resistance and apply only one spray of prothioconazole when required.     

A review of the relationship between primary and secondary dormancy,with reference to the volunteer crop weed oilseed rape (Brassica napus)

Fresh seeds of oilseed rape (Brassica napus) are reported to be nondormant and nonphotoblastic. However, a portion of the seeds can be induced into a light‐requiring state (secondary dormancy) for germination and also exhibit dormancy cycling. Thus, if seeds become buried in the soil they can form a persistent seedbank and become a serious volunteer weed in succeeding crops. The capacity of nondormant seeds of B. napus to be induced into secondary dormancy is contrary to results of studies on fresh nondormant seeds of some other species. A reanalysis of published and unpublished data shows that fresh seeds of this species have some degree of primary dormancy and that there is a significant relationship between primary dormancy and the capacity to enter secondary dormancy. However, most germination tests on B. napus have not been done in enough detail to detect primary dormancy (or not) in fresh seeds of this species. The usefulness of information on the relationship between primary dormancy and the capacity of the seeds to enter secondary dormancy is discussed in relation to management of weedy volunteers of this species.     

The interactions between piperonyl butoxide and E4, a resistance‐associated esterase from the peach‐potato aphid,Myzus persicae Sulzer (Hemiptera: Aphididae)

BACKGROUND: It has been reported previously that piperonyl butoxide (PBO) can inhibit both P450 and esterase activity. Although the method by which PBO combines with cytochrome P450 has been identified, the way in which it acts as an esterase inhibitor has not been established. This paper characterises the interactions between PBO and the resistance‐associated esterase in Myzus persicae, E4. RESULTS: After incubation with PBO/analogues, hydrolysis of 1‐naphthyl acetate by E4 is increased, but sequestration of azamethiphos is reduced. Rudimentary in silico modelling suggests PBO docks at the lip of the aromatic gorge. CONCLUSIONS: PBO binds with E4 to accelerate small substrates to the active‐site triad, while acting as a blockade to larger, insecticidal molecules. Structure–activity studies with analogues of PBO also reveal the essential chemical moieties present in the molecule. © 2012 Society of Chemical Industry