Gary Peng

Detached and attached Arabidopsis leaf assays reveal distinctive defense responses against hemibiotrophic Colletotrichum spp.

The agriculturally important genus Colletotrichum is an emerging model pathogen for studying defense in Arabidopsis. During the process of screening for novel pathogenic Colletotrichum isolates on Arabidopsis, we found significant differences in defense responses between detached and attached leaf assays. A near-adapted isolate Colletotrichum linicola A1 could launch a typical infection only on detached, but not attached, Arabidopsis leaves. Remarkably, resistance gene-like locus RCH1-mediated resistance in intact plants also was compromised in detached leaves during the attacks with the virulent reference isolate C. higginsianum. The differences in symptom development between the detached leaf and intact plant assays were further confirmed on defense-defective mutants following inoculation with C. higginsianum, where the greatest inconsistency occurred on ethylene-insensitive mutants. In intact Arabidopsis plants, both the salicylic acid- and ethylene-dependent pathways were required for resistance to C. higginsianum and were associated with induced expression of pathogenesis-related genes PR1 and PDF1.2. In contrast, disease symptom development in detached leaves appeared to be uncoupled from these defense pathways and more closely associated with senescence: an observation substantiated by coordinated gene expression analysis and disease symptom development, and chemically and genetically mimicking senescence.

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.

Genetics and breeding for clubroot resistance in Canadian spring canola (Brassica napus L.)

Abstract Clubroot disease caused by Plasmodiophora brassicae Woronin is a concern to the canola (Brassica napus L.) growers in Canada. A crop management strategy that includes deployment of genetic resistance and appropriate cultural practices is needed for long-term management of this disease. Resistance to the P. brassicae pathotypes present in Canada has been found in the primary and secondary gene pools of spring B. napus canola. Some of these sources, such as winter canola ‘Mendel’, rutabaga and Pak Choi (Brassica rapa L.) ‘Flower Nabana’, were used in genetic studies and breeding for the development of clubroot-resistant canola cultivars. A dominant gene in ‘Mendel’ and ‘Flower Nabana’ was found to confer resistance to P. brassicae pathotype 3, while a simple or a complex genetic control of resistance was found in rutabaga. The clubroot resistance (CR) gene in ‘Flower Nabana’ was mapped to chromosome A3, and molecular markers linked to the CR gene were identified for use in marker-assisted breeding (MAB). Using the CR genes from ‘Mendel’ and rutabaga, several clubroot-resistant spring canola lines were developed. Often the CR genes of ‘Mendel’ and rutabaga conferring resistance to pathotype 3 also conferred resistance to other pathotypes of P. brassicae found in Canada, including pathotypes 5, 6 and 8. The CR gene of ‘Flower Nabana’ was introgressed into B. napus and B. rapa canola through MAB. Since single-gene controlled resistance can be eroded, other strategies such as pyramiding different CR genes into B. napus canola should be considered for durable resistance.

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.

Heteroconium chaetospira Induces Resistance to Clubroot via Upregulation of Host Genes Involved in Jasmonic Acid, Ethylene, and Auxin Biosynthesis

An endophytic fungus, Heteroconium chaetospira isolate BC2HB1 (Hc), suppressed clubroot (Plasmodiophora brassicae -Pb) on canola in growth-cabinet trials. Confocal microscopy demonstrated that Hc penetrated canola roots and colonized cortical tissues. Based on qPCR analysis, the amount of Hc DNA found in canola roots at 14 days after treatment was negatively correlated (r = 0.92, P<0.001) with the severity of clubroot at 5 weeks after treatment at a low (2×105 spores pot−1) but not high (2×105 spores pot−1) dose of pathogen inoculum. Transcript levels of nine B. napus (Bn) genes in roots treated with Hc plus Pb, Pb alone and a nontreated control were analyzed using qPCR supplemented with biochemical analysis for the activity of phenylalanine ammonia lyases (PAL). These genes encode enzymes involved in several biosynthetic pathways related potentially to plant defence. Hc plus Pb increased the activity of PAL but not that of the other two genes (BnCCR and BnOPCL) involved also in phenylpropanoid biosynthesis, relative to Pb inoculation alone. In contrast, expression of several genes involved in the jasmonic acid (BnOPR2), ethylene (BnACO), auxin (BnAAO1), and PR-2 protein (BnPR-2) biosynthesis were upregulated by 63, 48, 3, and 3 fold, respectively, by Hc plus Pb over Pb alone. This indicates that these genes may be involved in inducing resistance in canola by Hc against clubroot. The upregulation of BnAAO1 appears to be related to both pathogenesis of clubroot and induced defence mechanisms in canola roots. This is the first report on regulation of specific host genes involved in induced plant resistance by a non-mycorrhizal endophyte.

A comparison of clubroot development and management on canola and Brassica vegetables

Abstract 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.

Spray Retention on Green Foxtail (Setaria viridis) and its Effect on Weed Control Efficacy by Pyricularia setariae1

The importance of spray retention to the biocontrol of green foxtail with Pyricularia setariae was characterized using airbrush and broadcast sprayers at variable application volumes. Spray retention was determined by measuring amounts of a tracer dye solution on treated plants using fluorescence spectrophotometry. Depending on the droplet size, broadcast spraying at 1,000 to 2,000 L/ha produced a level of retention equivalent to that of airbrush spraying until runoff. The trend of P. setariae spore retention on green foxtail was similar to that of liquid retention. Broadcasting P. setariae at volumes producing equivalent spray retention to that of airbrush inoculation resulted in a similar level of weed control under greenhouse conditions. Reducing broadcast volume from 2,000 to 250 L/ha lowered biocontrol efficacy only slightly, when the concentration of P. setariae was increased proportionally to keep the applied fungal dose the same. A nozzle with a fine droplet spectrum (volume median diameter [VMD] 207 μm) had a significantly greater retention efficiency than a coarse spray (VMD 325 μm), but this retention difference was not translated into consistent enhancement of biocontrol efficacy. Higher retention increases may be necessary for more substantial improvement in biocontrol of green foxtail by P. setariae. Nomenclature: Green foxtail, Setaria viridis (L.) Beauv. #3 SETVI; Pyricularia setariae Nisikado [synonym Pyricularia grisea (Cooke) Sacc.]. Additional index words: Application volume, biocontrol, droplet size, mycoherbicide. Abbreviations: AAFC, Agriculture and Agri-Food Canada; AAV, actual application volume; VMD, volume median diameter.

Effect of environmental parameters on clubroot development and the risk of pathogen spread

Abstract Clubroot (Plasmodiophora brassicae) was initially discovered on canola (Brassica napus) in western Canada near Edmonton, Alberta in 2003. Since then, the disease has spread rapidly but is still most common and most destructive on the heavy, slightly acidic soils in the region where the first infested fields were identified. However, there was (and is) concern that the pathogen might continue to spread and become a constraint to canola production across the Canadian prairies. To assess the risk of continued spread, the effect of factors such as temperature and soil type, pH and micronutrients on clubroot development were examined. Temperatures below 17 °C were shown to slow or inhibit the development of P. brassicae at all stages of its life cycle. Alkaline pH also reduced infection and symptom development in both controlled environment and field situations, but alkaline pH did not eliminate clubroot when other conditions were conducive for infection. Differences in the concentration of boron and other micronutrients in soil were shown to affect clubroot development on canola, but the effect of these differences was unlikely to limit the development of clubroot in situations where inoculum concentration was high. Soil moisture, especially in the rhizosphere during primary and secondary infection, had an important impact on clubroot development, but is a difficult factor for most growers to manipulate. Soil type had a small effect on clubroot severity in controlled environment studies, but a strong interaction of soil type with soil moisture is extremely likely under field conditions. Assessment of the interaction of environmental factors affecting infection success with the mechanisms of pathogen dispersal indicated that clubroot on canola has the potential to spread across large portions of the Canadian prairies.

Sources of resistance to Plasmodiophora brassicae (clubroot) pathotypes virulent on canola

Abstract A collection of 955 Brassica accessions including B. rapa (718), B. napus (94), B. juncea (93), B. oleracea (30), B. carinata (12) and B. nigra (8) was screened against Plasmodiophora brassicae pathotype 3 (1 × 106 resting spores cc−1 growth medium), the predominant strain of the pathogen on canola in western Canada. A total of 35 accessions (mostly B. rapa) showed at least 50% reduced clubroot severity relative to a susceptible control, with 15 showing complete resistance (clubroot-free). Ten resistant accessions representing Brassica A-, B- and C-genome species were tested further using a 10-fold higher pathogen inoculum dose (1 × 107 resting spores cc−1 growth medium) and by testing them against the five pathotypes (2, 3, 5, 6 and 8) of P. brassicae found in Canada. One B. nigra, two B. oleracea and four B. rapa (oriental vegetable) accessions maintained a high level of resistance under the higher pathogen inoculum pressure, while one B. nigra and two B. rapa (turnip) accessions showed moderate resistance. Most of the selected clubroot-resistant accessions showed consistent resistance to each of the five P. brassicae pathotypes found in Canada, except for one B. nigra and two turnip accessions, which varied slightly against different pathotypes. Several promising sources of clubroot resistance were identified in this study that can be used to develop new canola germplasm with a diverse clubroot resistance background for potentially more durable clubroot resistance.