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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 In this protocol, protoplast preparation was superseded with lithium acetate treatment and the selection step was omitted.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.

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.

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 | 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.

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.

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