Tiesen Cao

Isolation and Variation in Virulence of Single-Spore Isolates of Plasmodiophora brassicae from Canada

Clubroot of crucifers, caused by Plasmodiophora brassicae, is emerging as an important disease of canola (Brassica napus) in Alberta, Canada. Populations of the pathogen often consist of a mixture of different pathotypes. Therefore, a simple and efficient method to isolate single resting spores of P. brassicae was developed, based on serial dilution of spore suspensions. The virulence of 24 single-spore isolates, representing five populations of the pathogen from Alberta, Ontario, and British Columbia, was characterized on the differentials of Williams and Somé et al. Symptoms were rated 6 weeks after inoculation and Fishers least significant difference (P < 0.05) was used to differentiate resistant from susceptible host reactions. The pathotype composition of P. brassicae in Canada appeared more diverse when single-spore isolates were examined rather than populations of the pathogen. In Alberta, at least three and possibly four pathotypes were identified among the 14 isolates tested, whereas a maximum of only two pathotypes had been reported previously when populations of the pathogen were examined. Pathotype 3 or P2, as classified on the differentials of Williams and Somé et al., respectively, was found to be predominant in the province. The occurrence of other pathotypes at lower frequencies suggests that caution should be used in any breeding strategy, because rare pathotypes of P. brassicae may quickly become predominant if susceptible host genotypes are continuously grown.

Virulence and spread of Plasmodiophora brassicae [clubroot] in Alberta, Canada

Clubroot, caused by Plasmodiophora brassicae, has emerged as an important disease of canola (Brassica napus) in Alberta, Canada. Annual surveys from 2005 to 2008 revealed 405 clubroot-infested fields, mainly in central Alberta, but also in the southern part of the province. Within-field surveys suggested that the main mechanism of clubroot spread is through the movement of infested soil on farm machinery, as the frequency of infestation was highest at the field entrances and decreased sharply at distances of 150 and 300 m from the entrance. Characterization of P. brassicae populations from Alberta on the differentials of the European Clubroot Differential (ECD) series, P.H. Williams, and A. Somé et al. revealed that pathotype ECD –/15/12, 3, or P2, respectively, is predominant in the central part of the province, a finding consistent with earlier reports. However, novel virulence phenotypes were also observed. Two populations from southern Alberta were classified as pathotype 5 or P3 on the hosts of P.H. Williams and A. Somé et al., respectively, or as pathotypes ECD –/6/8 and –/4/0 on the ECD set, suggesting independent introductions of P. brassicae to this region or greater diversity than previously thought. Pathogen populations collected from infected canola plants from Quebec and Manitoba also yielded unique virulence patterns but were still virulent on most B. napus hosts, and an Ontario population induced significant disease only on a few of the vegetable differentials. Virulence levels overall were highest for the P. brassicae populations from central Alberta, which may help to explain the rapid increase in clubroot observed in this province.

Molecular Detection of Plasmodiophora brassicae, Causal Agent of Clubroot of Crucifers, in Plant and Soil

Clubroot of crucifers, caused by Plasmodiophora brassicae, recently has been identified in canola (Brassica napus) fields in Alberta, Canada. An effective strategy for managing the disease is to avoid planting cruciferous crops in P. brassicae-infested soil, because the pathogen produces resting spores that can remain infectious for many years. A simple, one-step polymerase chain reaction (PCR) protocol was developed to detect the pathogen in plant and soil samples. The primers TC1F and TC1R, based on a P. brassicae partial 18S ribosomal RNA (rRNA) gene sequence from GenBank, yielded a 548-bp product in the optimized PCR. A second pair of primers, TC2F and TC2R, which amplified a fragment of the 18S and internal transcribed spacer (ITS) 1 regions of the rDNA repeat, also was tested and produced a 519-bp product. Neither set of primers amplified any DNA fragment from noninfected plant hosts, noninfested soil, or common soil fungi and bacteria tested in this study. Quantities of 100 fg or less of total P. brassicae DNA, or 1 × 103 resting spores per gram of soil, could be detected consistently using these primers and PCR protocol, corresponding to an index of disease of 11% or lower when the soil was bioassayed. The protocol also enabled detection of P. brassicae in symptomless root tissue 3 days after inoculation with the pathogen. Therefore, the PCR assay described in this study could provide a reliable diagnosis for routine detection of P. brassicae in plant and soil materials in a specific and rapid manner.

A proteomic evaluation of Pyrenophora tritici-repentis, causal agent of tan spot of wheat, reveals major differences between virulent and avirulent isolates.

Pyrenophora tritici‐repentis causes tan spot, an important foliar disease of wheat. The fungus produces multiple host‐specific toxins, including Ptr ToxB, a chlorosis‐inducing protein encoded by the ToxB gene. A homolog of ToxB is also found in avirulent isolates of the fungus. In order to improve understanding of the role of this homolog and evaluate the general pathogenic ability of P. tritici‐repentis, we compared the proteomes of avirulent race 4 and virulent race 5 isolates of the pathogen. Western blotting analysis revealed the presence of Ptr ToxB in spore germination and culture fluids of race 5 but not race 4. A comprehensive proteome‐level comparison by 2‐DE indicated 133 differentially abundant proteins in the secretome (29 proteins) and mycelium (104 proteins) of races 4 and 5, of which 63 were identified by MS/MS. A number of the proteins found to be up‐regulated in race 5 have been implicated in microbial virulence in other pathosystems, and included the secreted enzymes α‐mannosidase and exo‐β‐1,3‐glucanase, heat‐shock and BiP proteins, and various metabolic enzymes. These proteome‐level differences suggest a reduced general pathogenic ability in race 4 of P. tritici‐repentis, irrespective of toxin production. Such differences may reflect an adaptation to a saprophytic habit.

Emergence of new virulence phenotypes of Plasmodiophora brassicae on canola (Brassica napus) in Alberta, Canada

Clubroot, caused by Plasmodiophora brassicae, is an important soilborne disease of canola (Brassica napus) in Alberta, Canada. Genetic resistance is the most effective clubroot management tool, and resistant cultivars are grown extensively in affected regions. In 2013, relatively severe symptoms of clubroot were observed in some fields of resistant canola. In greenhouse tests, four populations of P. brassicae from two of these fields caused significantly increased levels of clubroot on the cultivars from which they had been first recovered; these included three populations (L-G1, L-G2 and L-G3) recovered from the cultivar ‘L135C’, and one population (D-G3) recovered from ‘D3152’. Further testing showed that L-G1, L-G2 and L-G3 were highly virulent on a suite of six resistant canola cultivars (‘45H29’, ‘D3152’, ‘74–47CR’, ‘1960’, ‘L135C’ and ‘6056CR’) representing a cross-section of products available in Canada, while a seventh cultivar (‘9558c’) was moderately resistant to moderately susceptible. Bioassays of field soil with a dozen clubroot-resistant host genotypes confirmed that in most cases, resistance was no longer effective. Host responses to the population D-G3 were more variable, with most cultivars developing intermediate levels of disease. All four P. brassicae populations were classified as pathotypes 5, P3 and 16/6/8 on the differentials of Williams, Somé et al., and the European Clubroot Differential set. The pathotype classifications, however, do not reflect the increased virulence of these populations on clubroot-resistant canola. The identification of new virulence phenotypes of P. brassicae capable of overcoming genetic resistance underscores the need for increased stewardship of resistance sources.

Interaction of pH and temperature affect infection and symptom development of Plasmodiophora brassicae in canola

Abstract The occurrence of clubroot on canola (Brassica napus), caused by the soilborne protozoan Plasmodiophora brassicae, in western Canada is currently centred in a region of slightly acidic soils near Edmonton, AB. Warm temperatures and slightly acidic conditions are known to favour the development of clubroot. The current study was conducted as part of a larger project to assess the risk that P. brassicae will spread to other areas in the prairie region, e.g., where soil pH is neutral or alkaline. The interaction of temperature (10, 15, 20, 25, 30 °C) and pH (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) on root hair infection (RHI) and clubroot symptom development in canola roots was studied under optimum moisture conditions and moderate (1–5 × 106 resting spores per seedling) inoculum levels under controlled conditions. The highest levels of RHI (max = 61%) and clubroot incidence and severity (max. = 100%) developed at pH 5.0–6.5 × 20–25 °C. Clubroot levels were intermediate at pH 7.0–8.0 × 20–25 °C, and very low at 10 and 15 °C, regardless of pH. Surveys of clubroot-infested canola fields in Alberta demonstrated that there was only a weak correlation between soil pH and clubroot level (r = −0.30 for incidence and r = −0.33 for severity, based on 267 fields). The absence of a strong correlation supports the results of the controlled environment study. We conclude that moderate levels of clubroot can develop at pH levels well above its pH optimum when temperature and moisture are suitable. This may be the underlying cause of failures in clubroot control that occasionally occur in infested vegetable fields treated with lime. This study indicates that there is a substantial risk that moderate levels of clubroot will develop in regions where soils are neutral or slightly alkaline if other conditions (temperature, moisture, inoculum load) are favourable for disease development.

Histopathology of internal fruit rot of sweet pepper caused by Fusarium lactis

Abstract The etiology of internal fruit rot of greenhouse-grown sweet pepper, caused by the fungus Fusarium lactis, was investigated during anthesis and fruit development using stereo, fluorescence and scanning electron microscopy. Proliferation of fungal mycelium was observed on the surface of the stigma one day after inoculation (DAI). Mycelial growth was also visible in the transmitting tissues of the style and inside the ovary at 5 and 6 DAI. When externally symptomless sweet pepper fruits were sectioned 45 DAI, typical internal fruit rot symptoms were observed. Culturing of hyphal-like strands and ovules from inoculated flowers on potato dextrose agar (PDA) confirmed the presence of F. lactis. Similarly, symptomless seeds from infected fruit also yielded colonies of the fungus on PDA. These findings support the hypothesis that internal fruit rot of greenhouse sweet pepper caused by F. lactis is initiated through the infection of the stigma and style during anthesis. Moreover, symptomless seed infection may contribute to disease spread between greenhouses, although further work is needed to understand how F. lactis becomes established and spreads from infected seeds to flowers.

Spread of Plasmodiophora brassicae on canola in Canada, 2003–2014: Old pathogen, new home

Abstract Clubroot caused by Plasmodiophora brassicae has been reported at sites across North America on brassica vegetables for more than 50 years. However, it had not been reported on canola (Brassica napus) on the Canadian prairies until the initial discovery of a cluster of 12 infested fields near Edmonton AB in 2003. The purpose of this review is to consolidate and summarize the data on the spread of P. brassicae on canola in Canada since 2003, to compare this pattern of distribution with observations from an infested site in Ontario, and to draw inferences about the relative importance of short- and long-distance transmission of the pathogen on clubroot distribution in the prairie region. Over the last decade, P. brassicae has spread across central Alberta, with the leading edge of the epidemic moving at about 20 km per year, resulting in more than 1850 fields confirmed infested. DNA of the pathogen has also been detected from soil collected at sites across Saskatchewan and Manitoba, and very slight clubroot symptoms have been observed at isolated sites across the prairies. Transport of resting spores in soil carried on farm equipment has been shown to be an important mechanism of short-distance dissemination in this region. Dispersal of resting spores with wind-borne soil may also have an important role in both short- and long-distance dissemination. Dispersal on seed does not appear to be an important factor in clubroot spread. In contrast to the rapid spread observed in Alberta, P. brassicae is spreading very slowly, if at all, at the site in Ontario. This likely reflects the relatively small size and strength of the inoculum source and the absence of susceptible hosts nearby at the site in Ontario, relative to the thousands of hectares of heavily infested fields that provide a large, strong inoculum source in central Alberta.

Host–parasite interactions in clubroot of crucifers

Abstract Clubroot, caused by Plasmodiophora brassicae, is an important root disease of crucifers worldwide. In this review, the molecular aspects of clubroot pathogenesis and resistance are discussed. Topics covered include recent studies on the processes associated with infection by primary and secondary pathogen zoospores, examination of the expression patterns of P. brassicae genes at different stages of infection, and the concurrent identification of candidate genes for functional studies. Although the whole genome sequence of P. brassicae is not yet available, molecular studies of the pathogen are nonetheless moving into the genomics era. Transcriptomics and functional analyses of individual genes involved in the P. brassicae/Brassica interaction have recently been conducted to complement studies on the excellent model host, Arabidopsis thaliana. Two resistance genes have been cloned and their molecular functions illustrated, supporting suggestions that the clubroot pathosystem follows the gene-for-gene model. Although much about the pathogenesis of clubroot remains unknown, significant advances have been made in recent years, which may facilitate the identification of targets for agro-chemical development and resistance breeding.

Virulence and pathotype classification of Plasmodiophora brassicae populations collected from clubroot resistant canola (Brassica napus) in Canada

Abstract Clubroot, caused by Plasmodiophora brassicae Wor., is an important soilborne disease of canola (Brassica napus L.) in Canada that is managed mainly by planting clubroot-resistant (CR) cultivars. Populations of P. brassicae representing 106 fields in Alberta were obtained from galled roots of CR canola plants collected in 2014–2016 and characterized for virulence on seven CR canola cultivars. Sixty-one of these populations could overcome resistance in at least one CR cultivar and were evaluated further by inoculation on 13 Brassica hosts termed the Canadian Clubroot Differential (CCD) Set. The CCD Set included the differentials of Williams and Somé et al., selected hosts of the European Clubroot Differential Set, and the B. napus cultivars ‘Brutor’, ‘Mendel’, ‘Westar’ and ‘45H29’. Each unique virulence pattern on the CCD Set represented a distinct pathotype and was identified with a letter. Five reference isolates, obtained prior to the introduction of CR canola, also were assessed. A total of 17 pathotypes were detected using the CCD Set, compared with five pathotypes using the system of Williams and two with the system of Somé et al., suggesting that the CCD Set has a greater differentiating capacity. Pathotype A, a variant of pathotype 3 (as per Williams) which is able to overcome the resistance in CR B. napus, was predominant. The original pathotype 3, which is avirulent on CR canola, was classified as CCD pathotype H. An integrated strategy, combining other tools in addition to resistance, will be needed for the sustainable management of clubroot.