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Hwang S.-F.,Crop Diversification Center North | Strelkov S.E.,University of Alberta | Feng J.,Crop Diversification Center North | Gossen B.D.,Agriculture and Agri Food Canada | Howard R.J.,Crop Diversification Center South
Molecular Plant Pathology | Year: 2012

Plasmodiophora brassicae causes clubroot disease in cruciferous plants, and is an emerging threat to Canadian canola (Brassica napus) production. This review focuses on recent studies into the pathogenic diversity of P. brassicae populations, mechanisms of pathogenesis and resistance, and the development of diagnostic tests for pathogen detection and quantification. Taxonomy: Plasmodiophora brassicae is a soil-borne, obligate parasite within the class Phytomyxea (plasmodiophorids) of the protist supergroup Rhizaria. Disease symptoms: Clubroot development is characterized by the formation of club-shaped galls on the roots of affected plants. Above-ground symptoms include wilting, stunting, yellowing and premature senescence. Disease cycle: Plasmodiophora brassicae first infects the root hairs, producing motile zoospores that invade the cortical tissue. Secondary plasmodia form within the root cortex and, by triggering the expression of genes involved in the production of auxins, cytokinins and other plant growth regulators, divert a substantial proportion of plant resources into hypertrophic growth of the root tissues, resulting in the formation of galls. The secondary plasmodia are cleaved into millions of resting spores and the root galls quickly disintegrate, releasing long-lived resting spores into the soil. A serine protease, PRO1, has been shown to trigger resting spore germination. Physiological specialization: Physiological specialization occurs in populations of P. brassicae, and various host differential sets, consisting of different collections of Brassica genotypes, are used to distinguish among pathotypes of the parasite. Detection and quantification: As P. brassicae cannot be cultured, bioassays with bait plants were traditionally used to detect the pathogen in the soil. More recent innovations for the detection and quantification of P. brassicae include the use of antibodies, quantitative polymerase chain reaction (qPCR) and qPCR in conjunction with signature fatty acid analysis, all of which are more sensitive than bioassays. Resistance in canola: Clubroot-resistant canola hybrids, recently introduced into the Canadian market, represent an important new tool for clubroot management in this crop. Genetic resistance must be carefully managed, however, as it has been quickly overcome in other regions. At least three resistance genes and one or two quantitative trait loci are involved in conferring resistance to P. brassicae. Root hair infection still occurs in resistant cultivars, but secondary plasmodia often remain immature and unable to produce resting spores. Fewer cell wall breakages occur in resistant hosts, and spread of the plasmodium through cortical tissue is restricted. More information on the genetics of clubroot resistance in canola is needed to ensure more effective resistance stewardship. © 2011 The Authors. Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd.


Hwang S.-F.,Crop Diversification Center North | Howard R.J.,Crop Diversification Center South | Strelkov S.E.,University of Alberta | Gossen B.D.,Agriculture and Agri Food Canada | Peng G.,Agriculture and Agri Food Canada
Canadian Journal of Plant Pathology | Year: 2014

Clubroot, caused by Plasmodiophora brassicae, has emerged as a serious disease threatening the canola production industry in western Canada. This review summarizes results from studies, conducted since 2007, on the development of effective strategies for the management of clubroot in canola. Several options have been proposed for the control of this disease in infested fields, including liming the soil to increase soil pH, crop rotation with non-hosts and bait crops, manipulating the sowing date, sanitization of farm equipment, and the deployment of resistant cultivars, all aimed at reducing the severity of infection. Research began by assessing existing clubroot treatments, originally developed for the cole crop vegetable industry, for their applicability to canola production systems. Although these treatments provide good levels of clubroot reduction for the intensive production of short-season brassica vegetables, most are not economically feasible for the large-scale production of canola, which requires protection over a greater field acreage. Genetic resistance to P. brassicae has been shown to be a practical option for the management of clubroot on canola, but resistance stewardship, coupled with crop rotation and appropriate cultural practices, will be required to maintain the performance and durability of genetic resistance. Pathogen resting spores can be disseminated on infested soil carried on both machinery and seed. Efforts to minimize spread of the pathogen between canola fields have focused largely on the sanitization of field equipment and seed. © 2014 © 2014 The Canadian Phytopathological Society.


Rennie D.C.,University of Alberta | Manolii V.P.,University of Alberta | Cao T.,University of Alberta | Hwang S.F.,Crop Diversification Center North | And 2 more authors.
Plant Pathology | Year: 2011

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 <1·0×10 3 to 3·4×10 4 resting spores per 10g 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. © 2011 The Authors. Plant Pathology © 2011 BSPP.


Strelkov S.E.,University of Alberta | Hwang S.-F.,University of Alberta | Hwang S.-F.,Crop Diversification Center North
Canadian Journal of Plant Pathology | Year: 2014

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is an important soilborne disease of the Brassicaceae. In Canada, clubroot has long been a problem in cruciferous vegetables, but was not reported on the Prairie canola (Brassica napus) crop until 2003, when 12 clubroot-infested fields were identified in central Alberta. Continued surveillance has shown that the disease is spreading, and at least 1064 fields in the province had confirmed clubroot infestations as of 2012. While the outbreak is still mainly confined to central Alberta, isolated clubroot infestations and the presence of viable P. brassicae inoculum have been confirmed in southern Alberta, Saskatchewan and Manitoba. Dissemination of the parasite appears to be predominantly through the movement of infested soil on farm and other machinery, although secondary mechanisms of spread, such as by wind and water erosion and soil contamination of seeds, also have been implicated. Numerous strains of P. brassicae occur in Canada, but pathotype 3 or 16/15/12, as classified on the differentials of Williams or the European Clubroot Differential series, predominates on canola. Given the significant economic value of Canadian canola, the emergence of clubroot on this crop has caused major concern and led to the initiation of a large, coordinated research effort aimed at understanding and managing this disease. This review summarizes the extent and nature of the clubroot outbreak in the Canadian canola crop, 10 years after it began, and provides a context for ongoing research and management efforts. © 2014 © 2014 The Canadian Phytopathological Society.


Feng J.,Crop Diversification Center North | Hwang S.-F.,Crop Diversification Center North | Strelkov S.E.,University of Alberta
Phytopathology | Year: 2013

A protocol for genetic transformation of the obligate parasite Plasmodiophora brassicae, causal agent of clubroot of crucifers, was developed. In this protocol, protoplast preparation was superseded with lithium acetate treatment and the selection step was omitted. In two independent experiments, germinating resting spores of P. brassicae were transformed by two fungal expression vectors containing either a green fluorescent protein (gfp) gene or a hygromycin resistance (hph) gene. Putative transformants were produced from both transformations, with ≈50% of the obtained galls containing resting spores from which transforming DNA could be detected by polymerase chain reaction (PCR). PCR, quantitative PCR (qPCR), and genome walking conducted on selected transformants indicated that the transforming DNA was intergraded into the P. brassicae genome. Transcript of hph but not gfp was detected by reverse-transcription qPCR from selected transformants. From all galls produced by transformants, no GFP activity could be identified. Verified transformants were inoculated on canola and new galls were generated. PCR and qPCR analyses based on these galls indicated that transforming DNA was still resident in P. brassicae. This is the first report on genetic transformation of P. brassicae. The information and data generated from this study will facilitate research in multiple areas of the clubroot pathosystem. © 2013 The American Phytopathological Society.


Gossen B.D.,Agriculture and Agri Food Canada | Mcdonald M.R.,University of Guelph | Hwang S.-F.,Crop Diversification Center North | Strelkov S.E.,University of Alberta | Peng G.,Agriculture and Agri Food Canada
Canadian Journal of Plant Pathology | Year: 2013

Clubroot of canola was identified for the first time on the Canadian prairies in 2003, and is spreading rapidly across the region. Although clubroot has been studied extensively on vegetable Brassica crops, it was not clear initially how much of the information would be directly transferable from the intensive production of vegetable crops to the extensive production practices used for canola. This review examines similarities and differences between clubroot development and management on vegetable crops and canola. One important difference was that clubroot generally has a larger economic impact on canola, which is harvested for seed, than on vegetables, especially those where early vegetative growth is the marketable component. Also, clubroot has spread within the production area more quickly than was expected based on vegetable production. This occurs in large part because the resting spores are readily moved within and between fields on the heavy machinery used for canola production, but movement of spores by wind and water is also being assessed. Interestingly, crop rotation to reduce yield losses may be a more viable approach for canola than in vegetable production. Resistance to clubroot is the most consistent and economically viable approach to clubroot management in canola, but several lines of evidence indicate that this resistance may not be durable. Fortunately, the large acreage of canola production in Canada ensures that new sources of resistance will be developed and deployed as existing sources are eroded. Pathogen development and cultural control are very similar on vegetables and canola; bait crops and soil amendments are generally not commercially viable in either system; and biocontrol has a limited potential at this time. Manipulation of seeding date, application of fungicide, and soil fumigation generally have more potential for use in vegetable production than for canola. Identification of approaches that reduce disease pressure in clubroot-infested fields and increase the durability and diversity of genes for clubroot resistance represent important lines of future research. © 2013 The Canadian Phytopathological Society.


Feng J.,Crop Diversification Center North | Hwang S.-F.,Crop Diversification Center North | Strelkov S.E.,University of Alberta
Plant Pathology | Year: 2013

The early stages of infection of canola roots by the clubroot pathogen Plasmodiophora brassicae were investigated. Inoculation with 1×105resting sporesmL-1 resulted in primary (root hair) infection at 12h after inoculation (hai). Secondary (cortical) infection began to be observed at 72hai. When inoculated onto plants at a concentration of 1×104mL-1, secondary zoospores produced primary infections similar to those obtained with resting spores at a concentration of 1×105mL-1. Secondary zoospores caused secondary infections earlier than resting spores. When the plants were inoculated with 1×107resting sporesmL-1, 2days after being challenged with 1×104 or 1×105resting sporesmL-1, secondary infections were observed on the very next day, which was earlier than the secondary infections resulting from inoculation with 1×107resting sporesmL-1 alone and more severe than those produced by inoculation with 1×104 or 1×105resting sporesmL-1 alone. Compared with the single inoculations, secondary infections on plants that had received both inoculations remained at higher levels throughout a 7-day time course. These data indicate that primary zoospores can directly cause secondary infection when the host is under primary infection, helping to understand the relationship and relative importance of the two infection stages of P. brassicae. © 2012 BSPP.


Feng J.,Crop Diversification Center North | Xiao Q.,University of Alberta | Hwang S.-F.,Crop Diversification Center North | Strelkov S.E.,University of Alberta | Gossen B.D.,Agriculture and Agri Food Canada
European Journal of Plant Pathology | Year: 2012

Clubroot, caused by Plasmodiophora brassicae, has two infection stages (primary and secondary). Although primary infection occurs in many plant species, secondary infection only continues to completion in susceptible hosts. As part of a larger study of clubroot pathogenesis, secondary zoospores collected from infected root hairs of canola and ryegrass were inoculated onto healthy roots of both plant species. The treatments consisted of all possible combinations of the two plant species and the two sources of inoculum. At 5 days after inoculation, levels of root hair infection were similar and in a range of 50-68% on roots in all of the treatments. Secondary infection was also observed from all of the treatments, with approximately 50% on canola and 40% on ryegrass. The proportion of secondary infection and the number of secondary plasmodia were higher in canola inoculated with zoospores from canola than in ryegrass inoculated with zoospores from ryegrass, with the other combinations intermediate. At 35 days after inoculation, typical clubs developed on 14% of the canola plants inoculated with secondary zoospores from canola, and tiny clubs developed on 16% of the canola plants inoculated with zoospores from ryegrass. Secondary infection occurred in about one-third of ryegrass plants but no clubs developed, regardless of inoculum source. These results indicate that resistance to secondary infection in ryegrass is induced during primary infection. This is the first report that secondary zoospores produced on a nonhost can infect a host and reconfirms that secondary infection can occur in a nonhost. © 2011 KNPV.


Feng J.,Crop Diversification Center North | Hwang S.-F.,Crop Diversification Center North | Strelkov S.E.,University of Alberta
Canadian Journal of Plant Pathology | Year: 2012

The infection of plants by pathogenic microbes and the subsequent establishment of disease involve substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. Clubroot of canola (Brassica napus), caused by the obligate parasite Plasmodiophora brassicae, has considerable economic impact but has not been characterized extensively at the molecular genetic level. Here we have used suppression subtractive hybridization (SSH) and expressed sequence tag (EST) analysis to investigate gene expression during the early stages of colonization of canola roots by P. brassicae. A cDNA library was constructed by SSH which consisted of 797 clones that represented 439 unigenes. Thirty-two of these genes were demonstrated to be of a P. brassicae origin, and of these, 24 had not been previously reported. The remaining 407 genes, which were of a canola origin, were subjected to gene ontology and in silico analyses. Real-time PCR analysis of ten P. brassicae and seven canola genes indicated that seven of the former and five of the latter were upregulated at 7 days after infection, suggesting the importance of these genes in pathogenesis or host resistance. © 2012 Copyright 2012 The Canadian Phytopathological Society.


Feng J.,Crop Diversification Center North | Zhang H.,Chinese Academy of Agricultural Sciences | Strelkov S.E.,University of Alberta | Hwang S.-F.,Crop Diversification Center North
PLoS ONE | Year: 2014

Leptosphaeria maculans is a fungal pathogen causing blackleg in canola. Its virulence has been attributed, among other factors, to the activity of hydrolytic cell wall degrading enzymes (CWDEs). Studies on the pathogenicity function of CWDEs in plant pathogenic fungi have been difficult due to gene redundancy. In microorganisms many CWDE genes are repressed by glucose and derepressed by the function of the sucrose non-fermenting protein kinase 1 gene (SNF1). To address the molecular function of SNF1 in L. maculans, the ortholog of SNF1 (LmSNF1) was cloned and functionally characterized using a gene knockout strategy. Growth of the LmSNF1 knockout strains was severely disrupted, as was sporulation, spore germination and the ability to attach on the plant surface. When inoculated on canola cotyledons, the LmSNF1 knockout strains could not cause any symptoms, indicating the loss of pathogenicity. The expression of 11 selected CWDE genes and a pathogenicity gene (LopB) was significantly down-regulated in the LmSNF1 knockout strains. In conclusion, knockout of LmSNF1 prevents L. maculans from properly derepressing the production of CWDEs, compromises the utilization of certain carbon sources, and impairs fungal pathogenicity on canola. © 2014 Feng et al.

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