Sheau-Fang Hwang

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.

Plasmodiophora brassicae: a review of an emerging pathogen of the Canadian canola (Brassica napus) crop

UNLABELLED 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. USEFUL WEBSITES http://www.canolacouncil.org/clubroot/resources.aspx, http://tu-dresden.de/die_tu_dresden/fakultaeten/fakultaet_mathematik_und_naturwissenschaften/fachrichtung_biologie/botanik/pflanzenphysiologie/clubroot, http://www.ohio.edu/people/braselto/plasmos/

Evidence that the Biofungicide Serenade (Bacillus subtilis) Suppresses Clubroot on Canola via Antibiosis and Induced Host Resistance

This study investigated how the timing of application of the biofungicide Serenade (Bacillus subtilis QST713) or it components (product filtrate and bacterial cell suspension) influenced infection of canola by Plasmodiophora brassicae under controlled conditions. The biofungicide and its components were applied as a soil drench at 5% concentration (vol/vol or equivalent CFU) to a planting mix infested with P. brassicae at seeding or at transplanting 7 or 14 days after seeding (DAS) to target primary and secondary zoospores of P. brassicae. Quantitative polymerase chain reaction (qPCR) was used to assess root colonization by B. subtilis as well as P. brassicae. The biofungicide was consistently more effective than the individual components in reducing infection by P. brassicae. Two applications were more effective than one, with the biofungicide suppressing infection completely and the individual components reducing clubroot severity by 62 to 83%. The biofungicide also reduced genomic DNA of P. brassicae in canola roots by 26 to 99% at 7 and 14 DAS, and the qPCR results were strongly correlated with root hair infection (%) assessed at the same time (r = 0.84 to 0.95). qPCR was also used to quantify the transcript activity of nine host-defense-related genes in inoculated plants treated with Serenade at 14 DAS for potential induced resistance. Genes encoding the jasmonic acid (BnOPR2), ethylene (BnACO), and phenylpropanoid (BnOPCL and BnCCR) pathways were upregulated by 2.2- to 23-fold in plants treated with the biofungicide relative to control plants. This induced defense response was translocated to the foliage (determined based on the inhibition of infection by Leptosphaeria maculans). It is possible that antibiosis and induced resistance are involved in clubroot suppression by Serenade. Activity against the infection from both primary and secondary zoospores of P. brassicae may be required for maximum efficacy against clubroot.

Crop rotation, cultivar resistance, and fungicides/biofungicides for managing clubroot (Plasmodiophora brassicae) on canola

Abstract Select biofungicides and fungicides, used alone or with cultivar resistance or crop rotation, were assessed for their potential in integrated management of clubroot disease. The synthetic fungicides pentachloronitrobenzene, fluazinam and cyazofamid showed activities against Plasmodiophora brassicae. The biofungicides Serenade® and Prestop® also suppressed the disease on canola via antibiosis and induced host resistance under controlled-environment conditions. Granular and seed-treatment formulations were developed to facilitate the delivery of biofungicide in field trials. Where P. brassicae resting spore populations were large in the soil, neither biofungicides nor synthetic fungicides were sufficiently effective when applied in the seed furrow. They occasionally reduced clubroot severity on Chinese cabbage. More than 5000 soil microbial isolates indigenous to the Canadian prairies were screened for potential clubroot control, but none showed consistent efficacy. Resistant cultivars reduced clubroot severity and canola yield losses significantly. A 2-year break from canola reduced P. brassicae resting spore concentrations by 90% relative to growing continuous canola or only a 1-year break in heavily infested field plots. This 2-year break alleviated disease impact on plant growth and development in a susceptible canola cultivar. Despite the substantial inoculum reduction after 2 years, the levels were still too high to obtain commercially acceptable yields in a susceptible cultivar. In a resistant cultivar, >2-year breaks increased yields by up to 25% relative to continuous growing of canola. A 2-year interval with non-hosts between canola crops, together with use of resistant cultivars, is recommended to reduce the inoculum load of P. brassicae in soil and achieve maximum yields of canola.

Effect of temperature, seeding date, fungicide seed treatment and inoculation with Fusarium avenaceum on seedling survival, root rot severity and yield of lentil.

Early seeding of lentil is necessary on the northern prairies to ensure that the crop has time to mature. However, planting into cold soils in spring results in slow germination, which may predispose seedlings to infection by soil-borne pathogens. In a controlled-environment study of the impact of temperature on infection of lentil seedlings (cv. Eston) byFusarium avenaceum, root rot symptoms were most severe at warm temperatures (20° to 27.5 °C) and declined in warmer or cooler soils. Field plots were seeded on three dates, which were spaced about 2 wk apart between early May and early June in four station years. Seedling emergence was most consistent and seed yield was highest in three of four sites at the second seeding date. Emergence and yield were substantially lower for the latest seeding date. Inoculation with F. avenaceum reduced establishment and seed yield. Seed treatment with Crown (thiabendazole and carbathiin) improved seedling survival, reduced root rot severity and increased seed yield rel...

Sensitivity of field populations of Ascochyta rabiei to chlorothalonil, mancozeb and pyraclostrobin fungicides and effect of strobilurin fungicides on the progress of ascochyta blight of chickpea

Chickpea production faces a major challenge from ascochyta blight (Ascochyta rabiei), a devastating disease that can cause total crop loss. To assess the effect of repeated fungicide application on disease progress, strobilurin fungicides, primarily alternating pyraclostrobin and azoxystrobin treatments, were applied up to five times per year in each of 2 yr. A single application or two early applications reduced blight severity. A third application resulted in additional benefits in 1 of 2 yr, but additional applications did not reduce severity further. To monitor for fungicide tolerance in populations of A. rabiei, 66 single- spore isolates were collected and grown on growth media amended with chlorothalonil, mancozeb, or pyraclostrobin. Insensitivity to one or more of the fungicides was detected in 49 (74%) of the isolates. Based on the effect on conidial germination, insensitivity to pyraclostrobin or chlorothalonil was observed in 26 of 37 isolates (70%). Repeated fungicide application may be selecti...

Molecular characterization of a serine protease Pro1 from Plasmodiophora brassicae that stimulates resting spore germination

Clubroot, caused by Plasmodiophora brassicae, is one of the most serious diseases of cultivated cruciferous crops in the world. However, the basis for pathogenicity in P. brassicae is not well understood. In this study, a serine protease gene (PRO1) was cloned from P. brassicae and its molecular characteristics were investigated. Southern analysis and specific polymerase chain reaction (PCR) amplification indicated that PRO1 is a single-copy gene present in a broad range of P. brassicae pathotypes. Northern analysis revealed that the expression of PRO1 was induced during plant infection, and that the quantity of transcript fluctuated according to the stage of pathogenesis. Amino acid sequence analysis suggested that the encoded protein (Pro1) belongs to the S28 family of proteases, with a predicted signal peptide and a theoretical molecular mass of 49.4 kDa. The open reading frame (ORF) of PRO1 was transferred into Pichia pastoris and Pro1 was heterologously produced. Pro1 showed proteolytic activity on skimmed milk and N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, and the activity could be inhibited by serine protease inhibitors and the chelating agent ethylenediaminetetraacetic acid. The optimal temperature of Pro1 was 25 degrees C, and it exhibited high activity at pH 6.0-6.4. These values coincide with the temperature and pH conditions favourable for P. brassicae resting spore germination in the field. When Pro1 was used to treat canola root exudates, it enhanced the stimulating effect of the root exudates on P. brassicae resting spore germination, indicating that Pro1 may play a role during clubroot pathogenesis by stimulating resting spore germination through its proteolytic activity.

Management of clubroot (Plasmodiophora brassicae) on canola (Brassica napus) in western Canada

Abstract 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.

An inexpensive method for extraction of genomic DNA from fungal mycelia

Abstract A rapid and efficient protocol for the extraction of genomic DNA from plant pathogenic fungi was developed. Key features of the protocol include the SDS-assisted lysis of fungal mycelium with inclusion of a glass bead to help break hyphal walls, followed by isopropanol precipitation of the DNA. The protocol was used to extract genomic DNA from a collection of 26 fungal species, representing many important plant pathogens. Yield of DNA ranged from 2.1–4.9 μg per 20 mg of mycelium or 0.4–0.6 μg per 20 mg of spores. The DNA was of sufficient purity to be digested by restriction enzymes, to serve as a template in the PCR-amplification of genomic fragments as large as 4.9 kb, and to be used in dot-blot hybridization for the detection of multiple- and single-copy genes.