First report of Apple bitter rot caused by Colletotrichum godetiae in Ontario.

Bitter rot is an emerging disease of apple (Malus domestica) fruit in Ontario in part due to changing weather conditions. The disease was mostly documented in warm and humid regions such as the southern USA, and Central and South America. Thirteen Ontario orchards in the fall of 2019 and 15 in 2020 were scouted for bitter rot based on their previous history of the disease. 100 fruit were collected from ten asymptomatic trees per cultivar and two susceptible cultivars, 'Empire' and 'Ambrosia' were scouted per orchard. If an orchard did not have either one of these cultivars, 'Honeycrisp' or 'Gala' were used. The fruit was stored at 4-5 oC for five months and then left at 22 oC for two weeks and assessed thereafter for bitter rot symptoms. Monoconidial cultures of Colletrotrichum spp. were established from the symptomatic fruit using potato dextrose agar media with antibiotics at 22 oC 14-hour light cycles. The fungal isolates were divided into two groups based on colony morphology observations seven days after culturing. The first group showed light grey, cottony, aerial mycelium on top and a pink to dark red color on the reverse of the plate. The second group showed light to dark grey, cottony mycelium on top and dark green colonies on the reverse of the plate. It is difficult to identify Colletrotricuhum species solely based on morphology, therefore the representative isolates from each group were used for multilocus gene sequencing and species identification. Genomic DNA was extracted using the Qiagen DNeasy Plant Mini Kit according to the manufacturer's protocols. The ITS region was amplified and sequenced using the primers ITS-1F (Grades & Bruns 1993) and ITS-4 (White et al. 1990). Primers T1 and T2 (O'Donnell & Cigelnik 1997) were used to amplify and sequence the 750 bp region of the TUB gene. Primers GDF1 and GDR1 (Guerber et. al. 2003) were used to amplify a 280 bp region of the GADPH gene. Following an NCBI nucleotide blast search, the isolates from group 1 were identified as C. fioriniae.The ITS sequences of group 2 isolates were matched 100% to the Colletotrichum godetiae type strain CBS133.44 and they were 99% matched to the closest species C. Johnstonii CBS128532. The TUB sequences matched 100% identity to 20 sequences belonging to C. godetiae, 99.59 % identity to C. godetiae type strain CBS133.44, and 97.75% to C. Johnstonii CBS128532. The GADPH sequences matched with 100% identity to C. godetiae ON241087.1 or MT816329.1 and 99-99.5% identity to the type strain CBS133.44 and 98.61-99% identity to CBS128532. Based on the blast results the group 2 isolates were identified as C. godetiae and their sequences were submitted to GenBank with ID OP702962 for ITS and OP972240 and OP972241 for GADPH and OP972242 for the TUB gene. Out of 50 isolates collected in this work, 94% belonged to group 1 and 6% belonged to group 2. Koch's postulates were performed on selected isolates by artificial inoculation of 5 healthy detached fruits of the cultivar, 'Empire.' Fruit surfaces were wiped with 70% ethanol, dried, wounded with a sterile needle, and then inoculated with a 10 µl of spore suspension containing 1x10^6 spores /ml. Inoculated fruits were incubated in a humid chamber at 22°C in dark. Symptoms started to appear at 5 days post-inoculation and looked like small brown circular lesions which developed orange spore masses as they grew into larger lesions. The non-inoculated control fruit did not develop lesions. Fungal colonies were established from the spores collected from the inoculated fruit and found to have identical morphological characteristics to the original isolates. C. godetiae has previously been reported to cause bitter rot in apples from various countries in Europe (Baroncelli et al. 2014; Munda, 2014; Wenneker et al. 2016). C. fioriniae has previously been reported as the dominant species causing bitter rot in apples although other species including C. chrysophilum and C. noveboracense have also been reported as causal agents from the Eastern USA (Khodadadi et al. 2020). To the best of our knowledge, this is the first report of Colletotrichum godetiae causing the bitter rot of apples in Ontario, Canada.

Characterization of a virulence factor in Plasmodiophora brassicae, with molecular markers for identification.

Symptom severity on differential host lines is currently used to characterize and identify pathotypes of Plasmodiophora brassicae, which is an obligate, soil-borne chromist pathogen that causes clubroot disease on canola (Brassica napus) and other brassica crops. This process is slow, variable and resource intensive; development of molecular markers could make identification of important pathotypes faster and more consistent for deployment of cultivars with pathotype-specific resistance. In the current study, a variant of gene 9171 was identified in the whole-genome sequences of only the highly virulent pathotypes of P. brassicae from around the world, including the new cohort of virulent pathotypes in Canada; its presence was confirmed using three KASP marker pairs. The gene was not present in the initial cohort of pathotypes identified in Canada. The putative structure, domains, and gene ontogeny of the protein product of gene 9171 were assessed using on-line software resources. Structural analysis of the putative protein produced by gene 9171 indicated that it was localized in the cytosol, and likely involved in cellular processes and catalytic activity. Identification of gene 9171 represents a potentially useful step toward molecular identification of the pathotypes of P. brassicae.

Influence of plant species, mycorrhizal inoculant, and soil phosphorus level on arbuscular mycorrhizal communities in onion and carrot roots.

Arbuscular mycorrhizal fungi (AMF) are ancient and ecologically important symbionts that colonize plant roots. These symbionts assist in the uptake of water and nutrients, particularly phosphorus, from the soil. This important role has led to the development of AMF inoculants for use as biofertilizers in agriculture. Commercial mycorrhizal inoculants are increasingly popular to produce onion and carrot, but their specific effects on native mycorrhizal communities under field conditions are not known. Furthermore, adequate availability of nutrients in soils, specifically phosphorus, can reduce the diversity and abundance of AMF communities in the roots. The type of crop grown can also influence the composition of AMF communities colonizing the plant roots. This study aimed to investigate how AMF inoculants, soil phosphorus levels, and plant species influence the diversity of AMF communities that colonize the roots of onion and carrot plants. Field trials were conducted on high organic matter (muck) soil in the Holland Marsh, Ontario, Canada. The treatments included AMF-coated seeds (three to five propagules of Rhizophagus irregularis per seed) and non-treated onion and carrot seeds grown in soil with low (~46 ppm) and high (~78 ppm) phosphorus levels. The mycorrhizal communities colonizing the onion and carrot roots were identified by Illumina sequencing. Five genera, Diversispora, Claroideoglomus, Funneliformis, Rhizophagus, and Glomus, were identified in roots of both plant species. AMF communities colonizing carrot roots were more diverse and richer than those colonizing onion roots. Diversispora and Funneliformis had a 1.3-fold and 2.9-fold greater abundance, respectively, in onion roots compared to carrots. Claroideoglomus was 1.4-fold more abundant in carrot roots than in onions. Inoculation with R. irregularis increased the abundance and richness of Rhizophagus in AMF communities of onion roots but not in carrot roots. The soil phosphorus level had no effect on the richness and diversity of AMF in the roots of either crop. In summary, AMF inoculant and soil phosphorus levels influenced the composition of AMF communities colonizing the roots of onion and carrot plants, but the effects varied between plant species.

Stemphylium Leaf Blight: A Re-Emerging Threat to Onion Production in Eastern North America.

Stemphylium leaf blight (SLB), caused by Stemphylium vesicarium, is a foliar disease of onion worldwide, and has recently become an important disease in the northeastern United States and Ontario, Canada. The symptoms begin as small, tan to brown lesions on the leaves that can progress to defoliate plants. Crop loss occurs through reduced photosynthetic area, resulting in smaller, lower-quality bulbs. Leaf necrosis caused by SLB also can compromise bulb storage, as green leaves are required for the uptake of sprout inhibitors applied prior to harvest. The pathogen can overwinter on infested onion residue and infected volunteer plants. Asymptomatic weedy hosts near onion fields may also be a source of inoculum. Production of ascospores of the teleomorph (Pleospora allii) peaks in early spring in northeastern North America, often before the crop is planted, and declines rapidly as daily mean air temperatures rise. Conidia are usually present throughout the growing season. Application of fungicides is a standard practice for management of the complex of fungi that can cause foliar diseases of onion in this region. Recent assessments have shown that populations of S. vesicarium in New York and Ontario are resistant to at least three single-site mode-of-action fungicides. Three disease prediction systems have been developed and evaluated that may enable growers to reduce the frequency and/or number of fungicide applications, but the loss of efficacious fungicides due to resistance development within S. vesicarium populations threatens sustainability. The lack of commercially acceptable onion cultivars with sufficient resistance to reduce the number of fungicides for SLB also limits the ability to manage SLB effectively. Integrated disease management strategies for SLB are essential to maintain profitable, sustainable onion production across eastern North America.

Risk assessment of secondary metabolites produced by fungi in the genus Stemphylium.

The fungal genus Stemphylium (phylum Ascomycota, teleomorph Pleospora) includes plant pathogenic, endophytic, and saprophytic species with worldwide distributions. Stemphylium spp. produce prodigious numbers of airborne spores, so are a human health concern as allergens. Some species also produce secondary metabolites, such as glucosides, ferric chelates, aromatic polyketides, and others, that function as toxins that damage plants and other fungal species. Some of these compounds also exhibit a low level of mammalian toxicity. The high production of airborne spores by this genus can result in a high incidence of human exposure. Concern about toxin production appears to be the reason that Stemphylium vesicarium, which is a pathogen of several vegetable crops, was classified in Canada as a potential risk of harm to humans for many years. A detailed assessment of the risk of exposure was provided to the relevant regulatory body, the Public Health Agency of Canada, which then determined that Stemphylium spp. in nature or under laboratory conditions posed little to no risk to humans or animals, and the species was re-assigned as a basic (level 1) risk agent.

Entomopathogenic nematodes for control of carrot weevil: efficacy and longevity in muck and mineral soils.

BACKGROUND: Carrot weevil is an important pest throughout carrot-producing regions in Canada. Pesticides to control carrot weevil adults require application when the majority of adults have emerged and often this occurs after oviposition has already commenced and damage will be realized. One alternative to conventional pesticides are entomopathogenic nematodes. We studied four commercially available entomopathogenic nematode products (Steinernema feltiae, S. carpocapsae, S. kraussei and Heterorhabditis bacteriophora) for efficacy against carrot weevil in Nova Scotia and Ontario carrot fields in 2017 and 2018. The longevity and infectivity of the products in fine sandy loam soil (Ontario) and sandy loam soil (Nova Scotia) were evaluated using Galleria mellonella larvae. RESULTS: In Nova Scotia soils, only S. kraussei when applied twice, showed some efficacy to reduce damage from carrot weevil in 2017. In Ontario, an early application of H. bacteriophora and S. feltiae significantly reduced the percentage of carrots with weevil damage in 2018. Longevity and infectivity of S. carpocapsae and S. feltiae (against G. mellonella) was obtained up to 6 weeks post application in Nova Scotia in 2017. Similarly, S. feltiae showed infectivity up to 9 weeks post application in Ontario and Nova Scotia in 2018. CONCLUSION: Entomopathogenic products showed an ability to survive and remain infective for up to 9 weeks in soils without irrigation. Timing of application to effect control of carrot weevil requires further study. The influence of soil moisture on the longevity and infectivity of these products is discussed. © 2021 Her Majesty the Queen in Right of Canada International Pest Management Science © 2021 Society of Chemical Industry. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

The Role of Ascospores and Conidia, in Relation to Weather Variables, in the Epidemiology of Stemphylium Leaf Blight of Onion.

Stemphylium leaf blight (SLB), caused by Stemphylium vesicarium, is an important foliar disease of onion in northeastern North America. The pathogen produces conidia and ascospores, but the relative contributions of these spore types to epidemics in onion is not known. Determining the abundance of ascospores and conidia during the growing season could contribute to a disease forecasting model. Airborne ascospores and conidia of S. vesicarium were trapped during the growing season of 2015 and 2016 at an onion trial in southern Ontario, Canada, using a Burkard 7-day volumetric sampler. Meteorological data were recorded hourly. Ascospore numbers peaked before the crop was planted and declined rapidly with time and at daily mean air temperatures >15°C. Conidia were present throughout the growing season and appear to be closely related to the development of SLB on onion. Daily spore concentrations were variable, but 59 to 73% of ascospores and approximately 60% of conidia were captured between 0600 and 1200 h. Spore concentrations increased 24 to 72 h after rainfall and precipitation and leaf wetness duration were consistently and positively associated with increases in numbers of conidia and subsequent SLB incidence. The first symptoms of SLB coincided with high numbers of conidia, rainfall, leaf wetness duration ≥8 h, and days with average daily temperature ≥18°C. The number of airborne ascospores was very low by the time SLB symptoms were observed. Ascospores may initiate infection on alternative hosts in early spring, while conidia are the most important inoculum for the epidemic on onions.

Oviposition, Feeding Preferences and Distribution of Delia Species (Diptera: Anthomyiidae) in Eastern Canadian Onions.

Delia antiqua, Delia platura and Delia florilega are three root maggot species commonly considered pests in Eastern Canadian onions. The onion maggot, D. antiqua, is considered the primary root maggot pest in onion but it remains unclear whether the other two species are also causing damage. In order to develop updated management strategies for root maggot, we tested adult oviposition and feeding preference by Delia larvae on four growth stages of onion using bioassays and we determined the Delia species composition across the four major onion growing regions in eastern Canada. Delia species oviposit readily on onion at the 5-7 true leaf growth stage but damage on onions is not statistically different between Delia species in our zero-inflated models. The four eastern Canadian onion growing regions have different proportions of Delia species. Southern Ontario and Quebec were the only two regions where Delia antiqua was collected. The highest average numbers of Delia spp. were caught in Quebec and Nova Scotia. Our study shows that timing is important in implementation of management strategies for root maggot in Eastern Canadian onions.

Whole-genome DNA similarity and population structure of Plasmodiophora brassicae strains from Canada.

BACKGROUND: Clubroot is an important disease of brassica crops world-wide. The causal agent, Plasmodiophora brassicae, has been present in Canada for over a century but was first identified on canola (Brassica napus) in Alberta, Canada in 2003. Genetic resistance to clubroot in an adapted canola cultivar has been available since 2009, but resistance breakdown was detected in 2013 and new pathotypes are increasing rapidly. Information on genetic similarity among pathogen populations across Canada could be useful in estimating the genetic variation in pathogen populations, predicting the effect of subsequent selection pressure on changes in the pathogen population over time, and even in identifying the origin of the initial pathogen introduction to canola in Alberta. RESULTS: The genomic sequences of 43 strains (34 field collections, 9 single-spore isolates) of P. brassicae from Canada, the United States, and China clustered into five clades based on SNP similarity. The strains from Canada separated into four clades, with two containing mostly strains from the Prairies (provinces of Alberta, Saskatchewan, and Manitoba) and two that were mostly from the rest of Canada or the USA. Several strains from China formed a separate clade. More than one pathotype and host were present in all four Canadian clades. The initial pathotypes from canola on the Prairies clustered separately from the pathotypes on canola that could overcome resistance to the initial pathotypes. Similarly, at one site in central Canada where resistance had broken down, about half of the genes differed (based on SNPs) between strains before and after the breakdown. CONCLUSION: Clustering based on genome-wide DNA sequencing demonstrated that the initial pathotypes on canola on the Prairies clustered separately from the new virulent pathotypes on the Prairies. Analysis indicated that these 'new' pathotypes were likely present in the pathogen population at very low frequency, maintained through balancing selection, and increased rapidly in response to selection from repeated exposure to host resistance.

Evaluation of the TOM-CAST Forecasting Model in Asparagus for Management of Stemphylium Leaf Spot in Ontario, Canada.

In the last 5 years, asparagus acreage in Canada has increased by over 25%. Stemphylium leaf spot, caused by Stemphylium vesicarium, has emerged as the predominant foliar pathogen of asparagus. Typically, contact fungicides are applied every 14 days; however, regardless of the number of applications, growers are not achieving adequate control of the disease. The TOM-CAST forecasting model is used widely in Michigan asparagus fields, but it has never been assessed for suitability in Ontario or in the popular cultivar, Guelph Millennium. Six field trials were conducted in 2012 and 2013 to evaluate the TOM-CAST forecasting model in two asparagus cultivars. The fungicides chlorothalonil or azoxystrobin/difenoconazole were applied according to the forecasting model or on a 14-day interval. The effectiveness of the forecasting model differed between sites and cultivars. Even though TOM-CAST is used in all cultivars in Michigan, TOM-CAST was not effective on Guelph Millennium. In the cultivar Jersey Giant, however, TOM-CAST with a 20 disease severity value spray interval improved control of Stemphylium leaf spot without increasing the number of sprays, compared with a 14-day treatment. The results in Guelph Millennium differed between sites. At one site, TOM-CAST maintained similar levels of Stemphylium leaf spot, but increased the number of applications, compared with a 14-day application interval. Of more concern, none of the fungicide treatments differed greatly from the untreated control at the other site. Our results show that forecasting models need to be validated locally in asparagus cultivars relevant to production today.

The Impact of the Carrot Rust Fly and Carrot Weevil Integrated Pest Management Program on the Ground-Dwelling Beetle Complex in Commercial Carrot Fields at the Holland Marsh, Ontario, Canada.

Carrot rust fly (CRF), Psila rosae (Fabricius, 1794) (Psilidae: Diptera) and carrot weevil (CW), Listronotus oregonensis (Le Conte, 1857) (Curculionidae: Coleoptera) are economic pests of carrot; larval tunneling on roots results in direct damage rendering the carrot unmarketable. The Holland Marsh in Ontario, Canada, is a major carrot production area. The ground-dwelling beetle (Coleoptera) fauna in commercial carrot fields in this region has not been described. In 2015 and 2016, eight commercial carrot fields were surveyed using pitfall traps to determine abundance and diversity of the ground-dwelling beetle complex. Research sites, which were used to evaluate the effectiveness of an existing integrated pest management (IPM) program, were also surveyed to determine the impacts of the IPM program on the natural enemy diversity, compared to insecticide-free sites. In total, 50 taxa and 4,127 individual ground-dwelling beetles were identified over the course of the 2 y. Known natural enemies of CRF and CW were identified and recovered in abundance. The abundance and diversity of ground-dwelling beetles among the commercial carrot fields varied greatly in 2015 and 2016 but was similar on research sites sprayed according to the IPM program compared to insecticide-free sites in both years. The importance of this research to promote conservation biological control through the naturalization of nonagricultural areas is discussed.

Proteomic Profiles of Adipose and Liver Tissues from an Animal Model of Metabolic Syndrome Fed Purple Vegetables.

Metabolic Syndrome (MetS) is a complex disorder that predisposes an individual to Cardiovascular Diseases and type 2 Diabetes Mellitus. Proteomics and bioinformatics have proven to be an effective tool to study complex diseases and mechanisms of action of nutrients. We previously showed that substitution of the majority of carbohydrate in a high fat diet by purple potatoes (PP) or purple carrots (PC) improved insulin sensitivity and hypertension in an animal model of MetS (obese Zucker rats) compared to a control sucrose-rich diet. In the current study, we used TMT 10plex mass tag combined with LC-MS/MS technique to study proteomic modulation in the liver (n = 3 samples/diet) and adipose tissue (n = 3 samples/diet) of high fat diet-fed rats with or without substituting sucrose for purple vegetables, followed by functional enrichment analysis, in an attempt to elucidate potential molecular mechanisms responsible for the phenotypic changes seen with purple vegetable feeding. Protein folding, lipid metabolism and cholesterol efflux were identified as the main modulated biological themes in adipose tissue, whereas lipid metabolism, carbohydrate metabolism and oxidative stress were the main modulated themes in liver. We propose that enhanced protein folding, increased cholesterol efflux and higher free fatty acid (FFA) re-esterification are mechanisms by which PP and PC positively modulate MetS pathologies in adipose tissue, whereas, decreased de novo lipogenesis, oxidative stress and FFA uptake, are responsible for the beneficial effects in liver. In conclusion, we provide molecular evidence for the reported metabolic health benefits of purple carrots and potatoes and validate that these vegetables are good choices to replace other simple carbohydrate sources for better metabolic health.

The Effect of Anthocyanin-Rich Purple Vegetable Diets on Metabolic Syndrome in Obese Zucker Rats.

Consumption of highly colored fruits and vegetables rich in anthocyanins has been associated with numerous health benefits. Purple carrots (PC) and purple potatoes (PP) have higher anthocyanin concentrations and higher biological activities compared with less pigmented cultivars. We hypothesized that substitution of the majority of carbohydrate in a high fat diet with PP or PC, for 8 weeks, would improve insulin resistance and hypertension, major components of metabolic syndrome, compared with orange carrots (OC), white potatoes (WP) or a control, high fat, sucrose-rich diet (HFD) in obese Zucker rats. After 8 weeks of feeding, intraperitoneal glucose tolerance test, intraperitoneal insulin tolerance test (ipITT), and invasive hemodynamic tests were performed. The PP group had better glucose tolerance compared with the WP and the HFD groups and higher insulin sensitivity as measured by the ipITT and homeostatic model assessment of insulin resistance (P = .018) compared with the HFD without having any effect on blood pressure. The PC reduced left ventricular pressure compared with both the HFD (P = .01) and the OC (P = .049) groups and reduced systolic and diastolic blood pressures compared with the HFD group (P = .01 and <.0001, respectively) without having any effect on glucose homeostasis. The PC animals consumed more and were more obese than other groups, possibly obscuring any benefit of this vegetable on glucose tolerance. The bioactives in the vegetables responsible for blood pressure and glucose homeostasis could be different, and their effects could be independent of each other. The specific bioactives of each vegetable and their molecular targets remain to be identified. Nonetheless, incorporation of purple vegetables in functional food products may provide metabolic/cardiovascular benefits in the background of a high-fat diet that promotes obesity.

Propidium Monoazide Improves Quantification of Resting Spores of Plasmodiophora brassicae with qPCR.

Plasmodiophora brassicae, which causes clubroot of Brassica crops, persists in soil as long-lived resting spores. Quantitative polymerase chain reaction (qPCR) analysis is often used to quantify resting spores but does not distinguish between DNA of viable and nonviable spores. The impact of pretreating spores with propidium monoazide (PMA), which inhibits amplification of DNA from nonviable microorganisms, was assessed in several experiments. Spore suspensions from immature and mature clubs were heat treated; then, PMA-PCR analyses and bioassays were performed to assess spore viability. Prior to heat treatment, assessments comparing PMA-PCR to qPCR for mature spores were similar, indicating that most of these spores were viable. However, only a small proportion (<26%) of immature spores were amplified in PMA-PCR. Bioassays demonstrated that clubroot severity was much higher in plants inoculated with mature spores than with immature spores. Heat treatment produced little or no change in estimates of mature spores from qPCR but spore estimates from PMA-PCR and clubroot severity in bioassays were both substantially reduced. Estimates of spore concentration with PMA-PCR were less consistent for immature spores. To facilitate use of PMA-PCR on infested soil, a protocol for extracting spores from soil was developed that provided higher extraction efficiency than the standard methods.

Identification of Genome-Wide Variants and Discovery of Variants Associated with Brassica rapa Clubroot Resistance Gene Rcr1 through Bulked Segregant RNA Sequencing.

Clubroot, caused by Plasmodiophora brassicae, is an important disease on Brassica species worldwide. A clubroot resistance gene, Rcr1, with efficacy against pathotype 3 of P. brassicae, was previously mapped to chromosome A03 of B. rapa in pak choy cultivar "Flower Nabana". In the current study, resistance to pathotypes 2, 5 and 6 was shown to be associated with Rcr1 region on chromosome A03. Bulked segregant RNA sequencing was performed and short read sequences were assembled into 10 chromosomes of the B. rapa reference genome v1.5. For the resistant (R) bulks, a total of 351.8 million (M) sequences, 30,836.5 million bases (Mb) in length, produced 120-fold coverage of the reference genome. For the susceptible (S) bulks, 322.9 M sequences, 28,216.6 Mb in length, produced 109-fold coverage. In total, 776.2 K single nucleotide polymorphisms (SNPs) and 122.2 K insertion / deletion (InDels) in R bulks and 762.8 K SNPs and 118.7 K InDels in S bulks were identified; each chromosome had about 87% SNPs and 13% InDels, with 78% monomorphic and 22% polymorphic variants between the R and S bulks. Polymorphic variants on each chromosome were usually below 23%, but made up 34% of the variants on chromosome A03. There were 35 genes annotated in the Rcr1 target region and variants were identified in 21 genes. The numbers of poly variants differed significantly among the genes. Four out of them encode Toll-Interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat proteins; Bra019409 and Bra019410 harbored the higher numbers of polymorphic variants, which indicates that they are more likely candidates of Rcr1. Fourteen SNP markers in the target region were genotyped using the Kompetitive Allele Specific PCR method and were confirmed to associate with Rcr1. Selected SNP markers were analyzed with 26 recombinants obtained from a segregating population consisting of 1587 plants, indicating that they were completely linked to Rcr1. Nine SNP markers were used for marker-assisted introgression of Rcr1 into B. napus canola from B. rapa, with 100% accuracy in this study.

A Multiplex qPCR Assay for Detection and Quantification of Plasmodiophora brassicae in Soil.

Various physical and chemical factors in soil can inhibit the detection and quantification of soilborne plant pathogens using quantitative polymerase chain reaction (qPCR) assays. A multiplexed TaqMan qPCR assay, including a competitive internal positive control (CIPC), was developed to identify and (where necessary) compensate for inhibition in the quantification of resting spores of Plasmodiophora brassicae from soil. The CIPC amplicon was developed by modifying a sequence coding for green fluorescent protein so that it could be amplified with P. brassicae-specific primers. Addition of CIPC at 5 fg/µl to the singleplex qPCR assay designed to quantify P. brassicae genomic DNA did not reduce the sensitivity, specificity, or reproducibility of the assay. Each of the soil samples, either artificially inoculated or naturally infested with P. brassicae, exhibited no amplification of the CIPC. When the samples were diluted and reassessed, the quantification cycle of the CIPC relative to the control (water only) was delayed in each sample. The magnitude of the delay was used to adjust the estimate of resting spore concentration. The corrected concentration estimates were significantly higher than the unadjusted estimate, which indicated the presence of DNA inhibitors in samples even after dilution. The only exception was a mineral soil sample inoculated with a low concentration (103 spores/g) of resting spores. The assay was optimized for use on a range of soil types. A sample of 0.25 g for mineral soil and 0.10 g for high-organic-matter soil was optimum for recovery of DNA of P. brassicae. The assay represents an improvement over existing assays for estimating resting spore concentration in infested fields.

The Role of Primary and Secondary Infection in Host Response to Plasmodiophora brassicae.

The disease cycle of Plasmodiophora brassicae consists of a primary phase in root hairs followed by a secondary phase in the root cortex and adjacent tissues. However, the role of root hair infection in subsequent cortical infection and development of P. brassicae is not well understood. To examine the role of the primary and secondary stages separately, inoculation studies with resting spores (source of primary zoospores) and secondary zoospores of a virulent and avirulent pathotype were conducted on canola (Brassica napus). The size of secondary zoospores and number of nuclei were also examined. The zoospores were larger (≈9.6 to 14.4 µm) than in previous reports and all were uninucleate. Inoculation with secondary zoospores alone produced both primary and secondary infection, even with the avirulent pathotype. No symptoms developed from inoculation with avirulent primary zoospores but tiny, bead-shaped clubs developed from inoculation with avirulent secondary zoospores. Inoculation with virulent secondary zoospores alone resulted in lower disease severity than inoculation with virulent resting spores alone. The results indicate that recognition of infection by the host and initiation of a response (induction or suppression of resistance) occurs during primary infection, although recognition can also occur during cortical infection and development.

Reaction of Lines of the Rapid Cycling Brassica Collection and Arabidopsis thaliana to Four Pathotypes of Plasmodiophora brassicae.

The clubroot reaction of five Rapid Cycling Brassica Collection (RCBC) lines (Brassica carinata, B. juncea, B. napus, B. oleracea, and B. rapa) and 84 lines of Arabidopsis thaliana to pathotypes 2, 3, 5, and 6 of Plasmodiophora brassicae (as classified on William's system) was assessed. Also, the reaction of the Arabidopsis lines to a single-spore isolate of each of pathotypes 3 and 6 was compared with that of a field isolate. Seedlings were inoculated with resting spores of P. brassicae, maintained at 25 and 20°C (day and night, respectively), and assessed for clubroot incidence and severity at 6 weeks after inoculation. Several lines of A. thaliana and RCBC exhibited a differential response to pathotype but none of the lines were immune. Among the RCBC lines, B. napus was resistant to all of the pathotypes; B. oleracea was resistant to pathotypes 2, 3, and 5; B. carinata and B. rapa were resistant to pathotypes 2 and 5; and B. juncea was susceptible to pathotypes 5 and 6 and had an intermediate response to pathotypes 2 and 3. Line Ct-1 of A. thaliana was highly resistant to pathotype 2, Pu2-23 was highly resistant to pathotype 5, and Ws-2 and Sorbo were highly resistant to pathotype 6. These results indicate that the lines of RCBC and A. thaliana have potential for use as model crops for a wide range of studies on clubroot, and could be used to differentiate these four pathotypes of P. brassicae. The reaction of the RCBC lines to pathotype 6 was highly correlated with response under field conditions but the reaction to the single-spore isolates of pathotypes 3 and 6 was not strongly correlated with reaction to the field collections in the Arabidopsis lines.

Effect of temperature on cortical infection by Plasmodiophora brassicae and clubroot severity.

A study was conducted to assess the effect of temperature on infection and development of Plasmodiophora brassicae in the root cortex of Shanghai pak choy (Brassica rapa subsp. chinensis) and on subsequent clubroot severity. Ten-day-old seedlings were grown individually, inoculated with resting spores, and maintained in growth cabinets at 10, 15, 20, 25, and 30?C. Seedlings were harvested at 2-day intervals, starting 8 days after inoculation (DAI) and continuing until 42 DAI. Roots were assessed at 4-day intervals for the incidence of cortical infection and stage of infection (young plasmodia, mature plasmodia, and resting spores), at 2-day intervals for symptom development and clubroot severity, and at 8-day intervals for the number of spores per gram of gall. Temperature affected every stage of clubroot development. Cortical infection was highest and symptoms were observed earliest at 25?C, intermediate at 20 and 30?C, and lowest and latest at 15?C. No cortical infection or symptoms were observed at 42 DAI in plants grown at 10?C. A substantial delay in the development of the pathogen was observed at 15?C. Resting spores were first observed at 38 DAI in plants at 15?C, 26 DAI at 20 and 30?C, and 22 DAI at 25?C. The yield of resting spores from galls was higher in galls that developed at 20 to 30?C than those that developed at 15?C over 42 days of assessment. These results support the observation in companion studies that cool temperatures result in slower development of clubroot symptoms in brassica crops, and demonstrate that the temperature has a consistent pattern of effect throughout the life cycle of the pathogen.

Management of Botrytis Leaf Blight of Onion: The Québec Experience of 20 Years of Continual Improvement.

Botrytis leaf blight (BLB) of onion (Allium cepa) is caused by Botrytis squamosa. The disease has been reported on onion crops in several of the onion production areas of the world including North and South America, Europe, Asia, and Australia, although it is not a problem in arid production regions such as the western United States. In eastern Canada, the disease is generally present every year and is especially severe on cultivars of yellow globe onion. The pathogen biology and disease epidemiology have been intensively researched. Over the last few decades, in the organic soil area of Quebec, extensive research effort has been devoted to the development and evaluation of predictive models and disease management strategies. There has been an active integrated pest management program for onions since the early 1980s, and scouting for disease has played a major role in disease management. In this article, the story of BLB management in eastern Canada over a period of two decades is summarized.