Stephen E. Strelkov

Characterization of Plasmodiophora brassicae populations from Alberta, Canada

Clubroot, caused by Plasmodiophora brassicae, was identified in a number of canola (Brassica napus) fields in central Alberta in 2003. To characterize the virulence of the pathogen in the province, field populations from a number of locations in the Edmonton region were tested on the two most widely used sets of differential hosts, those of P.H. Williams and the European Clubroot Differential (ECD) series. Populations from British Columbia and Ontario were included for comparison. While the reaction of some hosts could be clearly defined as either resistant or susceptible, others showed intermediate disease index scores. If disease indices of 0%–49% and 50%–100% were regarded as resistant and susceptible, respectively, then populations from Alberta were classified as ECD 16/15/12 and 16/15/0 on the ECD set, or pathotypes 3 and 5, respectively, on the hosts of Williams. The population from British Columbia was classified as ECD 16/2/12 or pathotype 6, and the Ontario population was classified as ECD 16/0/14 or pathotype 6. The Alberta populations were more virulent on the B. napus hosts than those from other provinces, perhaps a reflection of their canola origin. In addition, 48 canola cultivars included in the 2004 Prairie Canola Variety Trials were screened for resistance to a local population of the pathogen, and all appeared to be highly susceptible. If clubroot were to become more widely established in western Canada, it could have a major negative impact on yields.

Clubroot of cruciferous crops – new perspectives on an old disease†

Abstract Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is an economically important disease affecting plants in the family Brassicaceae worldwide. In Canada, it has mainly been a problem on cruciferous vegetables in the traditional production areas of Ontario, Quebec, British Columbia and the Atlantic Provinces. In the Prairie Provinces, clubroot has been reported sporadically in a few home gardens and commercial vegetable fields in Alberta and Manitoba over the past 80 years; however, this situation changed dramatically with the discovery of 12 infected canola (Brassica napus) fields near Edmonton, AB in 2003. Annual surveys carried out since 2003 have revealed that clubroot is a much more widespread and serious disease in Alberta than initially thought. By 2008, it had been detected in about 410 canola, mustard and vegetable fields in central and southern areas of the province. The Alberta Clubroot Management Plan was developed to guide farmers, agribusinesses, oil and gas companies, contractors, and the general public in adopting good growing practices or taking measures to prevent further spread of this disease. An unprecedented research effort is underway in western Canada to develop a better understanding of the biology and management of clubroot, especially in the canola production systems on the Prairies. These studies are broadly based and include activities such as the improvement of diagnostic tests, determination of P. brassicae pathotypes, investigation of modes of seed and soil transmission, evaluation of fungicide efficacy, soil amendments, biological control agents and equipment sanitation protocols, and modelling disease distribution and risk. Through these efforts, new information is being generated that will give the agricultural industry and other stakeholders some new perspectives on this old disease threat.

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/

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.

Diseases of Camelina sativa (false flax)

There is renewed interest in the crucifer Camelina sativa (false flax, camelina, gold of pleasure) as an alternative oilseed crop because of its potential value for food, feed, and industrial applications. This species is adapted to canola-growing areas in many regions of the world and is generally considered to be resistant to many diseases. A review of the literature indicates that C. sativa is highly resistant to alternaria black spot and blackleg of crucifers. Genotypes resistant to sclerotinia stem rot, brown girdling root rot, and downy mildew can be found among C. sativa accessions, raising the possibility of developing cultivars resistant to these diseases. However, C. sativa is susceptible to clubroot, white rust, and aster yellows disease. Until resistant cultivars or effective management practices have been developed, the susceptibility of C. sativa to these diseases will limit the cultivation of the crop in areas where these diseases are prevalent. Key words: false flax, alternaria black spot, sclerotinia stem rot, root rot, white rust, blackleg, downy mildew, clubroot, aster yellows, Brassicaceae. La cameline (Camelina sativa) est une espèce oléagineuse de plus en plus recherchée pour l’alimentation humaine et animale et pour des fins industrielles. L’espèce est adaptée aux zones de culture du Colza et de la Navette dans plusieurs régions du monde et est généralement considérée comme étant résistante aux maladies. Une recherche bibliographique a révélé que C. sativa est fortement résistante à l’alternariose et à la nécrose du collet. Certains génotypes de C. sativa sont résistants à la sclérotiniose, au rhizoctone commun ou au mildiou, ce qui permet d’entrevoir le développement de cultivars résistants à ces maladies. Camelina sativa est cependant sensible à la hernie des crucifères, l’albugine et la jaunisse de l’aster. La culture de C. sativa sera limitée dans les régions où ces maladies sont importantes. Mots-clés : cameline, alternariose, sclérotioniose, rhizoctone commun, albugine, nécrose du collet, mildiou, hernie, jaunisse de l’aster, Brassicaceae.

Detection and Measurement of Plasmodiophora brassicae

Clubroot, caused by Plasmodiophora brassicae, is one of the most important diseases of brassicas. Management of clubroot is difficult, and the best means of avoiding the disease include planting in areas where P. brassicae is not present and using plants and growing media free from pathogen inoculum. As P. brassicae is not culturable, its detection has traditionally relied on plant bioassays, which are time-consuming and require large amounts of glasshouse space. More recently, fluorescence microscopy, serology, and DNA-based methods have all been used to test soil, water, or plant samples for clubroot. The use of fluorescence microscopy to detect and count pathogen spores in the soil requires significant operator skill and is unlikely to serve as the basis for a routine diagnostic test. By contrast, serologic assays are inexpensive and amenable to high-throughput screening but need to be based on monoclonal antibodies because polyclonal antisera cannot be reproduced and are therefore of limited quantity. Several polymerase chain reaction (PCR)-based assays have also been developed; these are highly specific for P. brassicae and have been well-correlated with disease severity. As such, PCR-based diagnostic tests have been adopted to varying extents in Canada and Australia, but wide implementation has been restricted by sample processing costs. Efforts are underway to develop inexpensive serologic on-farm diagnostic kits and to improve quantification of pathogen inoculum levels through real-time PCR. Proper detection and quantification of P. brassicae will likely play an increasingly important role in the development of effective clubroot management strategies.

Minireview/ Minisynthèse The wheat/Pyrenophora tritici-repentis interaction: progress towards an understanding of tan spot disease †

Abstract Tan spot, caused by the fungus Pyrenophora tritici-repentis, is an important foliar disease of wheat. Eight races of P. tritici-repentis have been identified to date, based on their ability to cause necrosis and/or chlorosis on a set of wheat differential hosts. These symptoms result from the action of host-specific toxins, which are produced in different combinations by races of the fungus and mediate the interaction between host and pathogen. Ptr ToxA induces necrosis on sensitive wheat genotypes, and is a 13.2 kDa protein encoded by the ToxA gene. Ptr ToxB, encoded by the ToxB gene, is a 6.6 kDa, chlorosis-inducing protein. Ptr ToxC also induces chlorosis, but appears to be a non-proteinaceous, polar, non-ionic, low-molecular-mass molecule. In susceptible wheat, three dominant and independently inherited toxin-sensitivity genes have been identified, each controlling the reaction to a single toxin. Most studies have also found that sensitivity to the toxins and susceptibility to the producing races co-segregate, and appear to be controlled by the same genes. Therefore, the tan spot pathosystem conforms to the toxin model of host–pathogen interactions, in which the compatible interaction is the basis for specificity (as opposed to the incompatible interaction in the classical gene-for-gene model). It is likely that P. tritici-repentis produces additional toxins or pathogenicity factors, but these remain to be identified.

Clubroot in the Canadian canola crop: 10 years into the outbreak

Abstract 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.