Does application of Trichoderma gamsii T6085 for control of Fusarium head blight alter the microbiome of wheat?
S. ALUKUMBURA, W. G. D. FERNANDO, S. SARROCCO, A. BIGI, G. VANNACCI AND M. G. BAKKER
Department of Plant Science, University of Manitoba, 66 Dafoe Road, Winnipeg, MB R3T 2 N2, Canada; (M.B.) Department of Microbiology, 45 Chancellor’s Circle, Winnipeg, MB R3T 2 N2, Canada; and (S.S., A.B., G.V.) Department of Agriculture, Food and Environment, University of Pisa, Lungarno Pacinotti 43, 6126 Pisa, Italy
Fusarium head blight (FHB), caused mainly by Fusarium graminearum, is a major constraint to wheat production worldwide. Trichoderma gamsii has shown promise as a potential biocontrol agent to manage FHB in wheat. However, the impact of T. gamsii application on the wheat microbiome is not known. The objective of this study is to evaluate the impact of T. gamsii application on bacterial and fungal communities associated with different wheat tissues. T. gamsii was applied on soil, crop residues at the beginning of stem elongation of wheat plants, and wheat spikes at anthesis. Wheat spikes, kernels and residue from the previous year’s wheat crop were collected at different time points for microbiome profiling and quantification of F. graminearum and T. gamsii densities. Densities of T. gamsii and F. graminearum were considerably high in residue while their detection rates in spikes and kernels were lower. The application of T. gamsii did not change the F. graminearum density. Despite the limited impacts of T. gamsii application, there were clear differences in microbial community structure over time. In crop residue, the F. graminearum density had decreased over time while in wheat spikes, it had increased over time. The crop residue had the highest microbial diversity and comparatively different microbial community structure. In wheat spikes and kernels, the predominant bacterial genera were Pseudomonas, Pantoea, and Enterobacter and the predominant fungal genera were Alternaria and Fusarium. These results indicate a minor impact of T. gamsii application on the microbiome associated with wheat tissues.
Potential use of vegetative indices to detect and estimate disease severities of Monilinia and Botrytis blight on wild blueberry fields
K. E. ANKU, D. C. PERCIVAL AND M. VANKOUGHNETT
Department of Plant, Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University, 50 Pictou Road, P.O. Box 550, Truro, NS B2N 5E3, Canada; and (M.V.) Applied Environmental/Agriculture Research Team, Nova Scotia Community College, 50 Elliot Road, Lawrencetown, NS BOS 1MO, Canada
Monilinia blight (MB) and Botrytis blossom blight (BB) diseases are endemic to most wild blueberry fields and affect both foliage and flowers. The severity of these diseases depends on the conditions present on the field. Present disease monitoring depends on the use of intensive sampling, using line transect among population structure. Therefore, the objective of this study is to assess the potential of using vegetative indices (VI’s) to detect and assess the severities of Monilinia blight and Botrytis blight disease on wild blueberry plants. Three patches each of Monilinia, Botrytis blight and healthy plant treatments were identified, and disease progression monitored. Disease sampling were grouped into three forms: low (1–30%), moderate (30–70%), and severe (70–100%) disease damage. Assessment of plant damage was conducted using VI’s and data analyzed using analysis of variance. Results indicated that there were significant differences (p < 0.05) between the VI’s computed for the healthy, Monilinia and botrytis blight diseased patch. At each damage level, healthy plants showed the highest significance, with Botrytis blight being significantly different from Monilinia blight. A discriminant accuracy of 94.4% showed that, normalized difference vegetative index (NDVI) can be used to differentiate between healthy and diseased plants. Green leaf index (GLI) gave an accuracy of 83.3% with enhanced NDVI (ENDVI) giving 72.2% accuracy. NDVI performed better than the other VI’s in the determination of disease severity. These results have illustrated the potential to use VI’s to discriminate healthy and disease plants and to assess disease pressures on wild blueberry fields.
In vitro screening of different forest tree species residues for their antifungal/antibacterial activity against strawberry pathogens
BARRO, M. DELISLE-HOUDE AND R. J. TWEDDELL
Département de phytologie, Université Laval, Québec, QC G1V 0A6, Canada
Large amounts of lignocellulosic wastes are generated every year from timber and forestry operations and from urban forestry and greening worldwide. Although they represent a promising source of bioactive molecules, lignocellulosic wastes are poorly valorized. In this study, crude extracts prepared from wastes (barks, needles, or leaves) of different species of forest trees were tested for their antifungal/antibacterial activity against three pathogens affecting strawberry (Fragaria × ananassa Duch.): Botrytis cinerea Pers., Colletotrichum acutatum J.H. Simmonds, and Xanthomonas fragariae Kennedy & King. Minimal inhibitory concentrations (MICs) of the crude extracts were determined using standard procedure. The strongest antifungal activity against B. cinerea was observed with white spruce (Picea glauca (Moench) Voss) needle extract while Canada yew (Taxus canadensis Marsh.) needle extract and sugar maple (Acer saccharum Marsh.) leaf extract showed the strongest antifungal activity against C. acutatum. Sugar maple leaf extract with a MIC value of 0.78 mg/mL showed the strongest antibacterial activity against X. fragariae. The efficacy of these extracts to control the pathogens in vivo will be investigated in future work.
Genetic diversity of Wheat streak mosaic virus in the wheat growing region of western Canada
H. BENNYPAUL, I. ABDULLAHI, M. W. HARDING AND R. ABOUKHADDOUR
Centre for Plant Health, Canadian Food Inspection Agency, 8801 East Saanich Road, North Saanich, BC V8L 1H3, Canada; (M.W.H.) Alberta Agriculture and Forestry, Crop Diversification Centre South, 301 Horticultural Station Road East, Brooks, AB T1R 1E6, Canada; and (R.A.) Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403 1 Avenue South, Lethbridge, AB T1J 4B1, Canada
Wheat streak mosaic virus (WSMV) is a positive-sense, single-stranded, monopartite RNA virus and is the type member of the genus Tritimovirus in the family Potyviridae. WSMV is known for causing damage to wheat in western Canada, the most recent outbreak occurred in southern Alberta and Saskatchewan in 2016–17. Understanding the genetic diversity of a pathogen is critical in developing management options. WSMV isolates have been divided into four clades (A-D) based on nucleotide sequence of the coat protein (CP) gene. Clade A contains isolates from Mexico, clade B from Europe and Russia, clade C from Iran, and clade D from the U.S., Turkey, Australia, and Argentina. The objective of this study was to explore genetic diversity of WSMV isolates collected from the wheat growing regions of western Canada, primarily from southern Alberta, during 2016–2017. Phylogenetic analysis of 16 isolates and the isolates available in GenBank was performed using the 1043-bp sequence corresponding to nucleotide positions 8192 to 9234 of the Sidney 81 strain coat protein gene using neighbour-joining analysis with bootstrapping support. Nine isolates clustered in clade B while five in clade D. Two isolates that did not cluster in known WSMV clades showed evidence of recombination. Among the local isolates investigated in this study, more polymorphic sites, parsimony informative sites, and increased diversity were observed in clade D isolates than clade B, suggesting more recent establishment of the virus in the latter. The observed diversity of WSMV could pose challenges to breeding durable resistance in wheat.
Disease dynamics of root rot complex in field pea
K. BISCAGLIA-HORVATH AND S. CHATTERTON
Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403 1 Avenue South, Lethbridge, AB T1J 4B1, Canada; and (K.B.) Department of Biology, University of Lethbridge, 4401 University Drive West, Lethbridge, AB T1K 3M4, Canada
Pea root rot is a disease complex composed of multiple biotic and abiotic stressors resulting in decay of the root system. Pathogens contributing to root rot in field peas include Aphanomyces euteiches and various Fusarium spp. However, little is understood of the nature of the interspecific interactions between pathogens and disease severity, as well as environmental influence on these interactions. The objective of our study was to simulate multiple infection scenarios in a greenhouse experiment to (i) characterize the nature and effect of pathogen interactions on disease severity and (ii) determine effect of multiple pathogens on pathogen biomass and host colonization patterns using quantitative PCR (qPCR). ‘CDC Meadow’ was grown in soil inoculated with varying concentrations of Fusarium avenaceum, Fusarium redolens and Aphanomyces euteiches. Four weeks post seeding, disease severity of roots was rated using a visual scale and fresh biomass measurements were taken. DNA was isolated from the tap and lateral roots for pending qPCR analysis. Results showed a positive correlation between pathogen load and disease severity in the case of all three pathogens. The presence of F. redolens, a weak pathogen, in the mixture resulted in a lower disease severity compared to single inoculations in some trials, but this was not consistent across all repeated trials. These results highlight that, even under controlled conditions, it is difficult to ascertain the nature of multiple pathogen interactions. This emphasizes the challenge of understanding pea root rot development under field conditions where more than three pathogens are often present.
Potato disease and insect pests in Manitoba, 2020
V. BISHT
Crop Industry Branch, Manitoba Agriculture and Resource Development, 65–3rd Avenue NE, Carman, MB R0G 0J0, Canada
Potato is a high-value crop with significant disease and insect pest risks. The 2020 cropping season had a cool-moist start, slowing emergence; and partly responsible for high incidence of blackleg disease and poor emergence in some fields. The growing season was generally dry with extended warm periods; dry conditions continued to harvest. There were periods of consecutive days with ‘>28°C days with >20°C nights’, which impacted potato yields and processing quality. Incidence of foliar diseases was lower than normal, but Verticillium wilt and black dot [Colletotrichum coccodes] diseases were extensive. Though Phytophthora infestans spores were trapped at one site in MB, there was no late blight disease reported. Colorado potato beetles continue to be problematic in select areas, possibly indicating resistance to the insecticides being used. Total aphid numbers trapped were significantly lower than normal. However, Green Peach Aphid numbers were higher, and risk for higher PVY in seed potato crops. Potato Mop Top Virus infection was noted in four of seven samples tested, ranging from 1.6 to 18.6% incidence. European corn borer injury was report and did not warrant insecticide applications. Broad mites (Polyphagotarsonemus latus) were reported on potato for the first time in Canada. Rains and warm conditions in August maybe partly responsible for tuber infections by Pythium leak and Pink rot diseases in poorly drained spots in fields. A field trial comparing ‘Direct Planting’ of potato in canola stubble vs ‘Traditional Planting’ after cultivation showed no differences in yield or tuber quality, including surface diseases.
Highlights from the 2020 Integrated Pest Management Programme for muck vegetable crops in the Holland/Bradford Marsh
T. BLAUEL AND M. R. MCDONALD
University of Guelph, Department of Plant Agriculture, Ontario Crops Research Centre – Bradford, 1125 Woodchoppers Lane, King, ON L7B 0E9, Canada
The Ontario Crops Research Centre – Bradford provides Integrated Pest Management services to vegetable growers in the Holland/Bradford Marsh. The main objective of the programme is to provide growers with information about the risk of the major insect pests and diseases, so they can take timely action as needed. This is accomplished by: scouting growers’ fields for diseases, weeds, and insect pests, providing disease and insect forecasting information, identifying and diagnosing diseases, insect pests and weeds, and implementing roto-rod spore traps to identify spores of vegetable crop pathogens. In 2020, 56 commercial onion, carrot, celery and potato fields were scouted twice a week for 21 growers. The forecasting models BOTCAST (for botrytis leaf blight of onion), DOWNCAST (for onion downy mildew), BREMCAST (for lettuce downy mildew), BSPCAST (for Stemphylium leaf blight of onion) and TOMCAST (for general leaf blights), along with models for white rot of onion and Sclerotinia white mould of carrot, were used to predict disease development. A harvest sample of 100 carrots and onions per field were assessed for below ground symptoms of diseases and insect damage. The forecasting models were generally useful for predicting blights and downy mildew development. Onion downy mildew spores were detected, and two fungicide applications were recommended in August. No downy mildew developed on onion in the Holland Marsh. Cavity spot and nematode/Pythium spp. forking were present in all scouted carrot fields. Stemphylium leaf blight (100% of fields) and pink root (96% of fields) were the most common onion diseases.
Nematicide efficacy for control of stem and bulb nematode (Ditylenchus dipsaci) in garlic (Allium sativum)
T. BLAUEL, K. VANDER KOOI AND M. R. MCDONALD
University of Guelph, Department of Plant Agriculture, Ontario Crops Research Centre – Bradford, 1125 Woodchoppers Lane, King, ON L7B 0E9, Canada
Stem and bulb nematode (Ditylenchus dipsaci, SBN) is one of the main pathogens of garlic in Ontario. The Ontario garlic industry is growing, and tools are needed to manage this nematode as there are currently no nematicides registered for control. The objective of this research was to identify nematicides that provide effective control of stem and bulb nematode. The nematicides tested were Velum Prime (fluopyram), Agri-Mek (abamectin), and two organic products, Promax (thyme oil) and Rhizovital 42 (Bacillus amyloliquefaciens). A total of three trials were conducted, one in 2018/19 (mineral soil) and two in 2019/20 (high organic matter soil and mineral soil). Nematicides were applied either as a seed (clove) soak prior to planting in the fall, or a drench over the seed at planting. Garlic treated with fluopyram applied as a soak or drench generally had the lowest incidence and severity of SBN damage at harvest. In 2020, the soak was most effective. As a result, percent marketability of garlic was higher in the fluopyram treatments and populations of SBN in these cloves were lower. In 2018/19, abamectin applied as a soak also provided good control of stem and bulb nematode. Thyme oil reduced severity in one of the three trials and the Bacillus product had no effect. The garlic industry needs effective tools to manage SBN. Fluopyram applied as a soak or drench is a strong candidate for registration. Research is continuing to evaluate other chemical and biological nematicides that may have efficacy in controlling this nematode.
Effect of clubroot [Plasmodiophora brassicae] on yield of canola (Brassica napus)
BOTERO-RAMÍREZ, S. F. HWANG AND S. E. STRELKOV
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada
Clubroot, caused by Plasmodiophora brassicae, is a major disease of canola (Brassica napus). The aim of this study was to evaluate the effect of clubroot development on the yield of canola under field and greenhouse conditions. Three canola hybrids, ‘45H31ʹ (susceptible), ‘45H29ʹ (1st generation resistance) and ‘CS2000ʹ (2nd-generation resistance), were inoculated with different quantities of P. brassicae inoculum and monitored for yield, pods per plant and 1000-grain weight. The field experiment was conducted over 2 years in biosecure clubroot nurseries inoculated with pathotype 5X or a mix of pathotypes 5X and 3 H at 5 × 108, 5 × 106 and 5 × 104 resting spores per plant. In the greenhouse experiment, plants were inoculated with pathotypes 5X, 3 H or a mixture of both at 1 × 103, 1 × 104 and 1 × 106 resting spores per plant. In both the field and greenhouse, clubroot incidence and disease severity index (DSI) increased along with inoculum density; the highest levels of disease were observed in the susceptible hybrid, while the lowest were found in ‘45H29.’ Yield, pods per plant and 1000-grain weight decreased as the DSI increased in all hybrids. Yield was affected by DSI and canola hybrid, but not by pathotype. Regression analysis indicated that under greenhouse conditions, an increment of 1% in the DSI resulted in a decrease of 0.49% in yield; under field conditions, this percentage was reduced to 0.26%. While the rate of yield reduction was similar among hybrids, overall yield losses were lower in the clubroot resistant hosts, since clubroot was less severe.
Efficacy of Bacillus pumilus and Bacillus subtilis to control grey mould on tomato and cucumber plants grown in greenhouse
M. BOUCHARD-ROCHETTE, T. T. A. NGUYEN, R. NAASZ, H. ANTOUN AND R. J. TWEDDELL
Département de phytologie, Université Laval, Québec, QC G1V 0A6, Canada; (R.N.) Premier Tech, 1 avenue Premier, Rivière-du-Loup, QC G5R 6C1, Canada; and (H.A.) Centre de recherche et d’innovation sur les végétaux, Université Laval, Québec, QC G1V 0A6, Canada
Gray mould [Botrytis cinerea Pers.] is one of the most damaging diseases affecting horticultural crops. Recent work reported the efficacy of Bacillus pumilus Meyer and Gottheil strain PTB180 and Bacillus subtilis (Ehrenberg) Cohn strain PTB185 to repress in vitro B. cinerea mycelial growth on detached tomato (Solanum lycopersicum L.) leaves and cucumber (Cucumis sativus L.) leaf discs. The objective of the study was to evaluate their efficacy to control grey mould on tomato and cucumber plants grown in greenhouse. Tomato and cucumber plants were sprayed two times (48 h interval) with either water (control), a suspension (1 × 107 colony-forming units/mL) of strain PTB180, PTB185, a mix (1:1, v:v) of both strains, or Rhapsody® (B. subtilis strain QST 713). Three days following the second application of the treatments, each plant was sprayed with a suspension (1 × 106 conidia/mL) of B. cinerea. On days 7, 10, and 14 post-inoculation of the pathogen, incidence and severity of the disease were measured. Foliar applications of PTB180, PTB185, and mix (1:1) of both strains were shown to significantly reduce grey mould incidence and severity on tomato and cucumber plants as compared to the control. The efficacy of the strains PTB180 and PTB185 was compared with that of the Rhapsody®.
Effect of biofumigation and fumigation on population density of root-lesion nematodes, Verticillium dahliae and potato yield in New Brunswick
D. CHEN, L.-P. COMEAU, T. DIXON, C. GOYER, K. NAHAR, M. ISLAM AND B. ZEBARTH
Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, 850, Lincoln Road, Fredericton, NB E3B 4Z7, Canada; (T.D.) McCain Foods (Canada), 8800 Main Street, Florenceville-Bristol, NB E7L 1B2,Canada; and (M.I.) Potatoes New Brunswick, 777 Everard H Daigle Boul, Grand Falls, NB E3Z 3 C7, Canada
Potato early dying disease complex (PED) is an endemic and a major yield-limiting factor for potato production in New Brunswick (NB). PED is primarily caused by Verticillium dahliae, and is exacerbated by the root-lesion nematode, Pratylenchus penetrans. In NB, PED management is mainly done through a 2-year potato-cereal crop rotation, but is ineffective despite cereals being a less favourable host. Soil fumigation with either metam sodium or chloropicrin can be used to control PED, but is not commonly practiced in Canada. Chloropicrin was demonstrated to reduce visual PED severity in some observational trials in NB, but its effect on pathogen population density and potato yield was not well studied. Biofumigation using mustard crops, as an alternative to chemical fumigation, showed potential in managing PED and other soil-borne pests, but its effectiveness needs to be evaluated under potato production systems of NB. Two trials were conducted to compare the effect of biofumigation and chloropicrin fumigation on PED management in a 2-year-rotation production system in commercial fields in NB from 2017 to 2020. The treatments included (1) spring barley representing conventional management, (2) spring barley plus fall chloropicrin fumigation, and (3) two crops of brown mustard as biofumigation. Biofumigation and fumigation significantly increased tuber yield by 10% and 15% of marketable yield, respectively, in trial one, and 3% and 19% of marketable yield, respectively, in trial two. Results indicate that both biofumigation and fumigation have a potential to suppress PED pathogens and increase potato yield in fields with high-PED pathogen pressure.
Identification and characterization of novel powdery mildew resistance genes in flax
V. CLEMIS, M. ZAIDI, S. CLOUTIER AND B. FOFANA
Charlottetown Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; and (S.C.) Ottawa Research and Development Centre, AAFC, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
Powdery mildew (PM), caused by Podosphaera lini, is a common fungal disease that can lead to reduced flax seed quality and yield. Currently however, the causative resistant genes are not well known and characterized. The objective of this study is to identify the flax genomic regions associated with PM resistance for a detailed identification and characterization of PM resistance genes. A recombinant inbred line (RIL) flax population derived from a cross between Linda (moderately resistant) and Norman (susceptible) was used for PM phenotyping, genotyping and a Genome Wide Association (GWAS) mapping. PM phenotypic data showed a normal distribution, indicating a quantitative inheritance of the PM resistance trait and the GWAS identified significant SNPs in three genomic regions on chromosomes 1, 9, and 14. These data, along with the progress achieved in the candidate genes characterization, were presented and discussed.
Assessing fungicide efficacy for improved management of the wild blueberry foliar disease complex in eastern Canada
W. CORNEL AND D. C. PERCIVAL
Department of Plant, Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University, 50 Pictou Road, P.O. Box, 550, Truro, NS B2N 5E3, Canada
The wild blueberry foliar disease complex, composed of blueberry leaf rust [Thekospora minima], Sphaerulina leaf spot [Sphaerulina vaccinia] and Valdensinia leaf spot [Valdensinia heterodoxa], significantly reduce yields when left unmanaged in the vegetative year of production. The once widely used chlorothalonil is no longer being used by many growers due to its deregistration in the European Union. Therefore, the objective of this study was to evaluate registered and future fungicide options for their impact on foliar diseases, leaf number and floral bud number. Field trials conducted in Collingwood Corner and Londonderry (Nova Scotia) consisted of ten treatments: untreated, mefentrifluconazole, mefentrifluconazole + fluxapyroxad, mefentrifluconazole + pydiflumetofen, pydiflumetofen, pydiflumetofen + difenoconazole, prothioconazole, fluopyram + prothioconazole, prothioconazole + benzovindiflupyr, and prothioconazole + pydiflumetofen. Mefentrifluconazole and pydiflumetofen are currently not registered for use on wild blueberries in Canada but, can are expected to be available within the next few years. Disease pressure was significantly higher at the Londonderry location. At both locations prothioconazole + benzovindiflupyr treatments recorded the highest leaf number, with prothioconazole + benzovindiflupyr and prothioconazole + pydiflumetofen treatments resulting in lowest disease incidence. At the Londonderry location, the untreated treatment had the lowest floral bud number. This study demonstrates that the current industry standard tank mix of prothioconazole + benzovindiflupyr offers the greatest control of foliar diseases. Furthermore, this study has also shown that pydiflumetofen combined with prothioconazole can be considered part of the foliar disease management strategy once pydiflumetofen is registered for use on wild blueberries in Canada.
Canadian prairie canola yield trends from 2011 to 2020
J. E. J. CORNELSEN AND N. W. W. ORT
Canola Council of Canada, 400–167 Lombard Avenue, Winnipeg, MB R3B 0T6, Canada
The average Canadian prairie canola (Brassica napus L.) yield has increased from 1867 to 2268 kg ha−1 from 2011 to 2020. This has been made possible in part because of an increase in genetic resistance to yield limiting diseases, the development and adoption of pod shatter tolerant cultivars, and the optimization of agronomic management practices. Developing genetic resistance to yield limiting diseases, like blackleg or clubroot, is a continuous effort because of their active shifts in pathogen virulence. Plant breeders and pathologists have and continue to work in collaboration to provide genetics that are resistant to current strains. The availability of resistant sources has strengthened integrated pest management to reduce the incidence and severity of diseases. The adoption of pod shatter tolerant cultivars has optimized canola production for direct harvesting, provided versatility in harvesting logistics, and has increased crop resilience to adverse environmental conditions. Agronomic management practices have been enhanced by adopting target seeding rates based on seed size, suitable fertilizer application practices, appropriate crop sequence, consulting established pest thresholds, and by reducing grain losses during harvest. As well, environmental conditions have affected overall disease development, yield, and the management practices deployed. The optimization of the aforementioned within our control has led to increased canola production and profitability, long-term positive environmental sustainability, and ultimately, the continued competitiveness of the Canadian canola industry. Ongoing research, innovation, and collaboration among farmers, agronomists, and researchers is necessary for current canola production levels to be maintained and increased.
Comparison of inoculation techniques for Verticillium longisporum on canola
J. CUI, S. F. HWANG AND S. E. STRELKOV
Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB T6G 2P5, Canada
Verticillium stripe, caused by Verticillium longisporum (VL), is an emerging soilborne disease of canola (Brassica napus) in Canada. Improved inoculation protocols will facilitate study of this disease and its management. Two inoculation techniques, a root-dip method at two inoculum concentrations and a grain inoculation method at four inoculum concentrations, were compared with the canola cultivars ‘45H31ʹ, ‘CS2000ʹ and ‘Westar’ under greenhouse conditions. Symptoms of Verticillium stripe appeared at early growth stages following root dip inoculation, resulting in seedling mortalities of 13%, 34%, and 33%, respectively, for ‘CS2000ʹ, ‘45H31ʹ and ‘Westar’ at 35 days post-inoculation. Plants inoculated by the root dip method incurred much greater mortality at the high vs. low inoculum concentration. In the surviving adult plants, ‘45H31ʹ became strongly resistant while ‘CS2000ʹ became more susceptible to VL. The grain inoculation method did not cause early stage mortality, although Verticillium stripe severity at the adult stage was significantly different between control and high inoculum concentrations for all cultivars. In addition, dry weight and plant height decreased as inoculum concentration increased. Inoculation of 1-week-old, 2-week-old, and 3-week-old seedlings of ‘CS2000ʹ and ‘Westar’ was also compared by the root-dip method. Symptoms were more severe when VL was inoculated at the later growth stages, perhaps because infected seedlings died following early inoculation. It seems that the timing and method of inoculation with VL will need to be carefully considered with respect to the objectives of a particular study.
Yield losses in canola caused by Verticillium longisporum
J. CUI, S. F. HWANG AND S. E. STRELKOV
Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB T6G 2P5, Canada
Verticillium stripe, a novel disease of canola (Brassica napus) caused by Verticillium longisporium, was first reported on the Canadian Prairies in 2014. The increasing prevalence of this soilborne disease has sparked concern among growers, since the potential for yield losses could be high. The relationship between Verticillium stripe severity and yield was evaluated at two infested field sites near Edmonton, Alberta, in 2020, using the canola hybrids ‘45H31ʹ and ‘CS2000ʹ. The disease severity was assessed on a 0–4 scale developed for this study, based on symptoms on the main stem and the amount of premature pod senescence, where 0 = no disease, 1 = discolouration with dark unilateral stripe on main stem, 2 = less than 25% of microsclerotia colonized on stem cortex, 3 = up to 75% of main stem colonized by microsclerotia, and 4 = entirely necrotic. Both canola hybrids developed symptoms of Verticillium stripe. Regression analysis indicated that seed yield declined with increasing disease severity. Seed yield per plant decreased by 12% to 75% and 17% to 86% in ‘45H31ʹ and ‘CS2000ʹ, respectively, as disease severity increased from 1 to 4. A greater percentage yield loss was observed on ‘CS2000ʹ vs. ‘45H31ʹ. At a disease severity of 3, yield losses in both cultivars exceeded 60% at one location. These preliminary results suggest that Verticillium stripe can cause very significant yield losses at high disease severity levels. The field trials are being repeated in 2021.
Alternative products for management of Cercospora leaf spot [Cercospora beticola Sacc.] of sugarbeet (Beta vulgaris L. spp. vulgaris)
C. DERVARIC, L. E. HANSON, M. R. MCDONALD AND C. TRUEMAN
Department of Plant Agriculture, University of Guelph, Ridgetown Campus, 120 Main Street East, Ridgetown, ON N0P 2C0, Canada; (L.E.H.) USDA-ARS, 612 Wilson Road, East Lansing, MI 48824, USA; and (M.R.M.) Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada
Cercospora beticola Sacc., causal agent of Cercospora leaf spot (CLS), is one of the most serious foliar pathogens of sugarbeets (Beta vulgaris spp. vulgaris). Resistance to FRAC Group 1 and 11 fungicides and increasing insensitivity to Group 3 fungicides challenge CLS management in Ontario. The active ingredient mancozeb, has been an important fungicide in standard spray programmes, but has been re-evaluated with fewer applications permitted due to safety concerns. Evaluation of alternative fungicides is needed to identify mancozeb replacements. Potassium bicarbonate and phosphites showed potential in managing CLS of sugarbeets in a 2019 field trial. These alternative fungicides and the registered fungicides mancozeb, copper octanoate, Bacillus amyloliquefaciens, and copper hydroxide, with and without canola oil spray adjuvant, were evaluated in a field experiment at Ridgetown, Ontario in 2020. Applications occurred on a 7 to 10-day calendar schedule. CLS intensity was moderate; disease severity in the non-treated control was 27% on the final assessment date. The area under the disease progress curve (AUDPC) was lower in the mancozeb, copper hydroxide, and copper hydroxide + canola oil treatments than the non-treated control. Phytotoxicity was observed in plots treated with copper hydroxide and copper hydroxide + canola oil. No differences were found among treatments for the percentage sugar recovered, beet yield, or sugar purity, possibly because of the relatively low disease pressure. This trial will be repeated in 2021 to confirm the results. Concurrent research on fungicide programmes integrating these new tools is expected to provide growers with more options to combat CLS.
Improving biosurveillance in the genomics era: using large-scale genome comparisons with machine learning to predict fungal phytopathogenic lifestyles
E. N. DORT, E. LAYNE, N. FEAU, M. BLANCHETTE AND R. C. HAMELIN
Department of Forest & Conservation Sciences, Faculty of Forestry, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada; and (E.L., M.B.) School of Computer Science, Faculty of Science, McGill University, 3480 University Street, Montréal, QC H3A 0E9, Canada
Fungal phytopathogens employ a diverse array of trophic modes, or lifestyles, that influence host--pathogen interactions and give plant pathologists important information on how a fungus is causing disease, how it might spread through an ecosystem, and ultimately, how best to approach disease mitigation. While current biosurveillance strategies enable regulatory agencies to identify known pathogens, they fail to monitor unknown or taxonomically unclassified pathogens, and they cannot determine the specific biological traits and lifestyles that facilitate disease outbreaks. We are demonstrating a genomics-based approach to address these challenges. Our research tested the hypothesis that there are genomic signatures associated with fungal phytopathogenic lifestyles that can be harnessed to develop more effective biosurveillance approaches. We compiled a database containing lifestyle and biological trait information for 537 fungal species, spanning the Ascomycota and Basidiomycota phyla, from the fungal genome portal MycoCosm. Using the gene annotation data from a subset of 387 species with published genomes, we performed both principal component analyses (PCAs) and machine learning to determine whether there were patterns associated with specific lifestyles or biological traits. While there were few discernable patterns beyond phylogeny from the PCAs, our novel machine learning approach, DendroNet, was able to separate the signals from phylogeny and gene content and successfully predict the lifestyles of species from their genomes with AUROC (area under the receiver operating characteristic curve) scores ranging from 0.84 to 1.00. These results have important implications for the future of phytopathogen biosurveillance as well as the development of more effective disease mitigation strategies.
Transcriptomic profiling of the host—pathogen interaction in tan spot of wheat
C. ESCOBAR-GIL, L. GALINDO-GONZALEZ, A. AKHAVAN AND S. E. STRELKOV
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada
Tan spot, caused by the necrotrophic fungus Pyrenophora tritici-repentis, is a foliar disease that affects wheat (Triticum aestivum and Triticum turgidum) by decreasing the kernel weight and number of kernels per spike. In recent years, tan spot has become one of the most detrimental diseases of wheat in Canada. Research on this pathosystem has focused on understanding the genetic basis of host resistance, yet important aspects of the plant-pathogen interaction remain unanswered. Using high-throughput RNA-sequencing, we have identified differences between isolates of P. tritici-repentis during saprophytic vs. parasitic growth. Differences were also identified in planta, at 12, 36 and 72 h after inoculation, between isolates producing the necrotrophic effectors ToxA and ToxB. The number of reads recovered indicated an increase in the ToxA-producer, while minor changes in read numbers were found for the ToxB producer. Preliminary analysis showed that the expression patterns correctly clustered the biological replicates according to each treatment, validating the experiment. For the ToxA-producing isolate, the number of differentially expressed genes decreased over time, in contrast with the ToxB-producing isolate, which presented a constant pattern of expression. This study will provide genomic-based resources to improve understanding of virulence mechanisms in P. tritici-repentis and identify patterns of molecular interactions between the host and pathogen. The knowledge can be applied to the development of improved tan spot management programmes, by identifying genes involved in virulence and potential targets in the host. Additionally, through comparative genomics with other necrotrophic fungal pathogens, these genes can provide insights into pathogenicity-related processes.
Temporal dynamics of the virome composition and transcriptional response of three grapevine cultivars
M. L. Fall, V. J. JAVRAN, P. LEMOYNE, G. S. MARTINS, D. XU, AND A. POURSALAVATI. (M.L.F., V.J.J., P.L., G.S.M., D.X., A.P.)
Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada; and (G.S.M.) Centre SÈVE, Département de biologie, Université de Sherbrooke, 2500 De L’Université Boulevard, Sherbrooke, QC J1K 2R1, Canada
Grapevine can be affected simultaneously by several pathogens, with a complex interaction that has largely been understudied. Among these pathogens, viruses represent a widespread class of pathogens that interact differentially with grapevine cultivars. In a recent study, we revealed a diverse virome (all the viruses and viroids) of a leafroll-infected interspecific hybrid grapevine cultivar and compared it to the virome of V. vinifera cultivar. However, co-occurrence analysis revealed that the presence of grapevine leafroll-associated virus species was randomly associated with the development of virus-like symptoms. To understand what drives the expression of symptoms, we used RNA sequencing analysis to profile the virome and transcriptome of grapevine leaves collected at two different times during the growing season (early August and late September). For cultivars such as Vidal, a total of 7047 genes were identified as differentially expressed genes (DEGs) from August to September, of which 2465 were upregulated and 4582 were downregulated. Further analysis revealed that many of these DEGs are involved in RNA-silencing pathway. For example, endoribonucleases dicer homolog 1 and 2 were significantly overexpressed. All asymptomatic grapevine plants showed viral-like symptoms in late September. The virome also showed a shift in terms of diversity and abundance, and the titre of the economically important viruses decreased from August to September, except in one case.
Cucumber powdery mildew detection using non-georeferenced multispectral images
C. I. FERNANDEZ, B. LEBLON, A. HADDADI, K. WANG AND J. WANG
Faculty of Forestry and Environmental Management, University of New Brunswick, 2 Bailey Drive, Fredericton, NB E3B 5A3, Canada; (A.H., K.W.) A&L Canada Laboratories Inc. 2136 Jetstream Road, London, ON N5V 3P5, Canada; and (J.W.) Department of Geography, University of Western Ontario, 1151 Richmond Street, London, ON N5G 2V4, Canada
Powdery mildew, which is caused by the fungus Podosphaera xanthii, is a major disease in cucumber greenhouses and may lead to yield losses between 30% to 50% of the total production This study evaluated three geometric transformations in an image registration method applied to non-georeferenced multispectral images acquired at close range over greenhouse cucumber plants with a Micasense® RedEdge camera. The detection of matching points was performed using SURF features, and outliers matching points were removed using the MSAC algorithm. For each geometric transformation (affine, similarity, and projective), we mapped the matching points of the blue, green, red, and NIR band images into the red-edge band space and computed the root mean square error (RMSE in pixel) to estimate the accuracy of each transformation. Then, registered band images were used to create an aligned RGB image and different vegetation indices. Using a pixel-by-pixel approach, a fine Gaussian support vector machine was trained to classify healthy and infected pixels. Our results achieved an RMSE of less than one pixel with the similarity and affine transformations and of less than two pixels with the projective transformation, whatever the band image. We determined that the best transformation was the affine transformation because it produces RMSEs of less than one pixel and having a Gaussian distribution. The classification using the RGB images presented a precision and F1 Score of 82 and 88%, respectively. Nevertheless, Cucumber powdery mildew detection is feasible using the five registered bands, reaching a precision and F1 Score of 97 and 98%, respectively.
How oomycete community structure responds to crop rotation and tillage
C. GAHAGAN, S. ARIS-BROSOU, M. J. MORRISON AND W. CHEN
Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (A.C.G., S.A.B.) Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6N5, Canada
Soil-borne oomycete pathogens cause devastating crop losses; thus, it is critical to find means of controlling them with standard agronomic practices. Past research has revealed little about how oomycete community structure changes in response to management strategies such as tillage and crop-rotation. Furthermore, despite their economic importance as serious pathogens, such as species from genera Pythium and Phytophthora, oomycete communities are still underrepresented in large soil microbiome studies. To address these knowledge gaps, 224 composite soil samples were collected from a split-plot tillage and rotation experiment over a 3-year period. An amplicon sequencing strategy was employed to assess oomycete community changes. To improve detection specificity, novel oomycete-specific primers targeting two DNA markers, the internal transcribed spacer 1 (ITS1) of ribosomal DNA and the ribosomal protein S10 gene (rps10), were used. This led us to develop a high-quality reference database (refDB) for the ITS1 region and to obtain a rps10 refDB from http://oomycetedb.cgrb.oregonstate.edu/. While both markers had a good coverage of Pythium which was most prevalent in soils, the rps10 marker recovered higher Phytophthora diversity as well as Aphanomyces which was not detected by the ITS1 marker. Preliminary analyses suggested that no-till increased oomycete diversity, while rotation effects were more complex. This study will shine a light on how common agricultural practices could help in managing and mitigating these important soil phytopathogens.
Using molecular biology and omics to manage clubroot of canola
L. GALINDO-GONZÁLEZ, Q. ZHOU, H. TSO, H. ASKARIAN, V. MANOLII, T. LOCKE, S. F. HWANG AND S. E. STRELKOV
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (T.L.) Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada
Clubroot, caused by Plasmodiophora brassicae, is one of the most important diseases of canola (Brassica napus) in Canada. Disease management relies heavily on planting clubroot resistant (CR) cultivars, but in recent years, new resistance-breaking pathotypes of P. brassicae have emerged. While efforts to develop new CR varieties using traditional breeding are underway, omics and molecular biology technologies can help to search for novel sources of resistance, improve understanding of host-pathogen interactions, and facilitate diagnosis. We performed two transcriptomic studies with two cultivar resistance-breaking pathotypes (3A and 5X) on B. napus hosts presenting contrasting resistance/susceptibility. The interaction with pathotype 5X showed a clear salicylic acid (SA)-mediated immunity response, especially in the resistant genotype. Inoculation with pathotype 3A showed that SA and ethylene were important as part of the defence mechanism of the resistant cultivar. As expected, susceptible hosts showed features of early cell growth modification. The P. brassicae reads from each interaction were used to predict putative effectors, indicating a predominance of effectors with interacting domains (e.g., ankyrin), supporting previous studies. Genes like benzoic acid/SA methyltransferase (BSMT), which methylates SA to render it inactive, showed higher levels of expression in the compatible interactions. We also sequenced 45 P. brassicae single spore and field isolates using Illumina short-reads, with reference-based mapping conducted to identify polymorphic sites that could be targeted for molecular pathotyping. Isolate clusters corresponding to pathotypes 5X and 3 H could be distinguished by rhPCR and SNapShot. These molecular diagnostics methods show promise for distinguishing additional pathotypes for proactive clubroot management.
Evaluation of plant-pollinator ecosystem health and rapid detection of plant and bee pathogens by high-throughput sequencing of European honey bee (Apis mellifera)
D. W. GLADISH, M. E. ROTT, M. M. GUARNA, S. F. PERNAL, J. F. GRIFFITHS AND G. J. BILODEAU
Canadian Food Inspection Agency (CFIA), 3851 Fallowfield Road, Ottawa, ON K2H 8P9, Canada; (M.E.R.) CFIA, 8801 East Saanich Road, North Saanich, BC V8L 1H3, Canada; (M.M.G., S.F.P.) Agriculture and Agri-Food Canada (AAFC), 1 Research Road, Beaverlodge, AB T0H 0C0, Canada; (J.F.G.) AAFC, 4902 Victoria Avenue North, Vineland Station, ON L0R 2E0, Canada
The European honey bee, Apis mellifera, is Canada’s most prevalent managed pollinator. Honey bee pollination services are valued up to 5.5 billion dollars annually in Canada. Being associated with microorganisms in their environment, they have the potential to interact with and acquire pathogens. As a result, they can serve as vectors, transporting bee pathogens back to their hive or transferring pathogens between individual plants. In our proof-of-concept experiment, we exploited this bee-pathogen relationship to develop a new way to rapidly detect pathogens in the environment and assess the health of plant-pollinator systems. High-throughput sequencing (HTS) of the conserved ITS1 and 16S regions in samples from pollen collected in hive-mounted pollen traps, hive-stored pollen, and bee foragers was used to categorize fungi and bacteria, respectively. Over 19 million reads per run were generated from 44 samples. The QIIME2 pipeline was used to do quality control, produce amplicon sequence variants, and conduct taxonomic classification. Using this method, we created community pathogen profiles for apples and blueberries from British Columbia and Ontario. Identified plant pathogens include fungi Monilinia spp. and Podosphaera leucotricha. Bee pathogens include the fungus responsible for chalkbrood disease, Ascosphaera apis, as well as the bacteria Melissococcus plutonius, the causative agent of European foulbrood disease, and its secondary invader Paenibacillus alvei. With this pilot project, we seek to determine the capacity of this approach to evaluate plant-pollinator ecosystem health, identify limitations, and set up a foundation from which future screening programmes may be built.
Boron (B) as an IPM tool for clubroot? Identification and potential utilization of B-insensitive lines of Brassica napus
B. D. GOSSEN, A. MCLEAN AND M. R. MCDONALD
Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; and (A.M, M.R.M.) University of Guelph, Department of Plant Agriculture, 50 Stone Road East, Guelph, ON N1G 2W1, Canada
Clubroot [Plasmodiophora brassica Wor.] severity can be suppressed by application of boron (B), but excess B produces phytotoxicity in canola (Brassica napus). Some lines of B. rapa are relatively insensitive to B, but sensitivity in B. napus had not been assessed. Assessment of 150 accessions of B. napus and B. rapa treated with 0, 8 and 16 kg/ha of B under controlled conditions identified several lines that were relatively insensitive to B. Lines with sufficient seed and germination (88 accessions) were planted at a site in Ontario, Canada where there was a natural infestation of clubroot. The trial was arranged in a replicated split-plot design, where the main plot treatments were a low level (8 kg/ha) of B applied versus a non-treated control. Application of B reduced clubroot only slightly in the nine most sensitive lines, but had a larger impact on severity in the 10 least sensitive lines. Insensitive lines generally had lower severity than the sensitive lines, even in the control. Five of the most insensitive lines were assessed in combination with clubroot inoculation under controlled conditions. Clubroot severity was much more strongly reduced by high rates of B applied to the insensitive lines relative to the B-sensitive control. This indicated that lines of B. napus were available that were much less sensitive to applied B than standard canola cultivars in Canada. It also indicated that application of high rates of B applied to B-insensitive lines could dramatically reduce clubroot severity, regardless of pathotype. B-insensitive cultivars might ultimately be used to facilitate canola production in clubroot-infested fields, by growing a B-insensitive cultivar and applying high levels of B to the clubroot patches.
Antimicrobial peptides display a potent activity against common fungal pathogens: A promising supplement in plant disease management
J. HANNIG, S. CHATTERTON, M. TETORYA, D. SHAH AND R. K. GOYAL
Lacombe Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada; (S.C.) Lethbridge Research and Development Centre, (AAFC), 5403–1 Avenue South, Lethbridge, AB T1J 4B1, Canada; and (M.T., D.S.) Donald Danforth Plant Science Center, St. Louis, MO 63132, USA
Plant disease management relies heavily on fungicides, especially where germplasm resistance is either weak or associated with unfavourable agronomic traits. Persistence application of fungicides often leads to insensitivity requiring a higher concentration for effectiveness, and a breakdown of protection needs a new formulation of fungicides. The evidences are emerging that fungicides negatively affect some of the beneficial microbial activity in soil, and the consequences of chemical residues in food and feed are yet to be fully determined. For climate preservation, there is a necessity to explore environmental-friendly alternatives. Antimicrobial peptides (AMPs) are an important component of innate immunity in living organisms that confers protection against biotic stresses. AMPs are short molecules (< 100 amino acids) which are highly potent at low concentrations against a wide range of pathogens. In this study, we assessed the antimicrobial activity of both the natural and synthetic AMPs against important fungal and oomycetes pathogens. An inhibitory concentration to reduce the pathogen growth by 50% (IC50) and a minimum inhibitory concentration (MIC) for each peptide were determined through absorbance and resazurin-based cell viability assays. A direct incubation of AMPs with the pathogens resulted in the latter’s reduced growth and the antimicrobial effect of an AMP was visible in multiple pathogens. A variation in an AMP’s IC50 value suggested a different level of pathogen susceptibility. The AMPs with low IC50 values (<10 uM) hold a great promise to develop them as biopesticides for foliar applications. Among the peptides tested, PD 30.1 and PN43 showed IC50 values less than 0.5 uM against F. graminearum. The other peptides displayed high toxicity against F. culmnorum. A work is in progress to evaluate in plantae effectiveness of AMPs.
A deep dive into the game of hide and seek in the Brassica napus-Leptosphaeria maculans pathosystem: combined transcriptome and proteome analysis
S. HUANG, F. LIU, Z. ZOU, W. G. D. FERNANDO AND G. PENG
Department of Plant Science, University of Manitoba, 66 Dafoe Road, Winnipeg, MB R3T 2N2, Canada; and (G.P.) Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada
Blackleg, caused primarily by the hemibiotrophic fungus Leptosphaeria maculans (Desmaz.) Ces. & De Not., is one of the most economically important diseases in many canola/oilseed rape (Brassica napus L.) growing regions in the world that often causes significant yield losses. Genetic resistance has been proven effective in mitigating blackleg infection at different growth stages (seedling and adult plant). Seedling resistance generally follows the gene-for-gene theory, with the exception that the recognition of avirulence genes AvrLm3 and AvrLm9 by the respective resistance genes Rlm3 and Rlm9 is masked by the presence of AvrLm4-7, known as the ‘game of hide and seek’. Recent evidence has shown that there is no direct interaction between AvrLm4-7 and AvrLm3 or AvrLm9, or between Rlm9 and AvrLm9 or AvrLm4-7, suggesting AvrLm4-7 may conform to the guard or decoy models and interact with unknown targets in the host that represses the recognition of AvrLm3-Rlm3 and AvrLm9-Rlm9. To reveal molecular networks underlying the masking effect, we combined RNA sequencing (NovaSeq 6000 System, Illumina) and proteomic (TMT-based technology) studies on B. napus 02–22-2-1 (Rlm3 carrier) seedlings at 3-, 7- and 11-days post inoculation (dpi) and Goéland (Rlm9 carrier) seedlings at 3 and 7 dpi with L. maculans isolates carrying avrLm4-7-AvrLm3-AvrLm9 and AvrLm4-7-AvrLm3-AvrLm9, respectively. Hundreds to thousands of genes were identified concurrently or specifically in these typical and masked interactions, in addition to a number of differentially expressed proteins. The further downstream analysis in this multi-omics study will be instrumental to pinpoint key players in the ‘game of hide and seek’ for this pathosystem and possibly illuminating similar interactions in other pathosystems.
Extreme resistance to potato virus A in potato cultivar Barbara is independently conferred by Ra and Rysto
W. HUANG, B. NIE, Z. TU, C. LI, A. MURPHY, M. SINGH, B. SONG, S. ZHANG, C. XIE AND X. NIE
Key Laboratory of Horticultural Plant Biology, Huazhong Agricultural University, Wuhan 430 070, China; (A.M., X.N.) Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Fredericton, NB E3B 4Z7, Canada; and (M.S.) Agricultural Certification Services, Fredericton, NB E3B 8B7, Canada
Potato virus A (PVA) and potato virus Y (PVY), both belonging to the genus Potyvirus (family Potyviridae), are two of the most economically important viruses of potato worldwide. Host resistance offers the most effective means for the control and management of the viruses. In this study, 20 potato clones (cultivars) were screened for their resistance against PVA and PVY by mechanical and/or graft inoculation assay. Six clones, including Barbara, Jizhangshu 8, Longshu 7, Longshu 8, M6, and Solara, were found to be extremely resistant to both PVA and PVY; three clones (AC142, Eshu 3, and Shepody) were deemed to be extremely resistant to PVA but susceptible to PVY. To further reveal the inheritance of the extreme resistance (ER) against PVA, a tetraploid F1 population of Barbara × F58050 and a tetraploid BC1 population of BF145 (a PVA-resistant but PVY-susceptible progeny of Barbara × F58050) × F58050 were obtained and analyzed. Based on the phenotype of all progenies, two independent loci were found to control ER against PVA in Barbara: one (i.e., Ry) confers ER to both PVA and PVY, and the other (i.e., Ra) confers ER to PVA only. The deduced genotype of Barbara is RyryryryRararara.
Pathogenicity of Fusarium graminearum and F. poae causing fusarium head blight on barley under controlled conditions
K. HUDSON, L. J. HARRIS, D. P. OVERY, A. XUE, A. FOSTER, AND R. KHANAL
Ottawa Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (A.F.) Charlottetown Research and Development Centre, AAFC, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada
Fusarium head blight (FHB) is one of the most damaging diseases of barley. FHB is caused by a species complex of Fusaria, of which Fusarium graminearum is the species of greatest concern for barley in Canada. Field surveys showed that two or more Fusarium species often co-exist within the same field or grain sample and F. poae has been reported as another dominant species in barley in eastern Canada. The aim of this study was to determine the pathogenicity of F. graminearum, F. poae and a co-inoculation of both species causing FHB in barley under controlled conditions. Spray inoculation was performed on two susceptible barley genotypes at 10 to 14 days after heading. FHB severities were rated on a scale of 0–9 at 4, 7, 14, 21, and 28 days after inoculation. There was a significant difference in FHB severity between F. graminearum and F. poae, with F. graminearum being more pathogenic species. Fusarium poae was less pathogenic and not statistically different from the control treatment (inoculated with deionized water only). When heads were co-inoculated with the mixture of both Fusarium species, the resulting FHB severity was significantly lower than that caused by F. graminearum alone, indicating that the presence of F. poae may reduce the pathogenicity of F. graminearum in causing FHB.
Genotyping by sequencing analysis of Fusarium poae field populations affecting small grain cereals in Canada
M. N. ISLAM, T. MOURITA, A. O. OGHENEKARO, H. R. KUTCHER, L. J. HARRIS, D. P. OVERY, T. K. TURKINGTON, W. XU AND X. WANG
Morden Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 101 Route 100, Morden, MB R6M 1Y5, Canada; (A.O.O.) Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; (H.R.K.) Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada; (L.J.H., D.P.O.) Ottawa Research and Development Centre, AAFC, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (T.K.T.) Lacombe Research and Development Centre, AAFC, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada
Fusarium head blight (FHB) is an important disease threatening the production of small grain cereals in Canada, reducing the yield and quality of crops. Multiple species within the genus Fusarium cause mycotoxin contamination in FHB disease. F. poae, F. graminearum, and F. sporotrichioides are the most common species associated with FHB. In recent years F. poae has become the predominant Fusarium species in commercial oat and barley fields in western Canada. Nevertheless, very little information is available on pathogenesis and genetic structure of this pathogen. To better understand the evolutionary processes affecting pathogen, the restriction site-associated DNA and Illumina shotgun sequencing were used for population genomics analyses using 198 Canadian F. poae isolate divided into 12 populations based on geographical origins and hosts specificity. The final filtered dataset included 2234 SNPs with a maximum of 2% missing data per SNP. The partitioning of the genetic variance within and among field populations using AMOVA revealed that 99.1% of the total genetic variance was within field populations, and only 0.9% was among populations. The corresponding overall Fixation index was 0.003, while pairwise differentiation between populations ranged from 0 to 0.033. These results indicate a high degree of gene flow and the individual populations in Canada being the parts of a single meta-population. We are currently utilizing these SNPs to study the structure of F. poae field populations in Canada, determine the extent of linkage disequilibrium, and identify hot spots in the F. poae genome that may contain candidate genes under strong selection.
Levels of mycotoxins in barley grains as infected by Fusarium head blight species in Manitoba, Canada from 2017 to 2020
M. NARZUL, M. TABASSUM, S. SURA, K. ANNE, D. KAMINSKI AND X. WANG
Morden Research and Development Centre, Agriculture and Agri-Food Canada, 101 Route 100, Morden, MB R6M 1Y5, Canada; and (K.A., D.K.) Manitoba Agriculture and Resource Development, P.O. Box 1041, 106–2nd Street SW, MB R0G 0J0, Canada
FHB can cause dramatic yield loss, lower grade, and contaminate small cereal grains (e.g., barley, oat, wheat) with fungal toxins (mycotoxin). This disease can savage crop and be responsible for up to $1.5 billion in income loss every year in Canada. Mycotoxins in barley have resulted in a growing concern of human and animal health hazards as recent increase of FHB in barley. We surveyed 180 barley fields over the four consecutive (2017–2020) seasons in Manitoba. Fusarium poae (FP), F. graminearum (FG), F. sporotrichioides (FS), F. equiseti (FE), and F. avenaceum (FA) were isolated, and multiplex PCR confirmed the presence of these pathogens in barley grains. FP and FG were predominant in barley grains in all 4 years. FP was detected in 84%, 65.9%, 61.7%, and 55.2%, compared to 34%, 56.8%, 40.4%, and 43.4% of barley fields infected with FG from 2017 to 2020, respectively. DNA concentration of FP (7.5, 2.87, 1.1 pg/ng) and FG (3.1, 1.1, 1.4 pg/ng) were also quantified using RT-qPCR from 2017 to 2019. Both PCR analyses indicate that last 4-year, the FP was detected as the most dominating species over FG. NIV appears to be the most common mycotoxin, was detected in 94% (mean 216 ppb, maximum 3252 ppb) and 66% (mean 412 ppb, maximum 2377 ppb) of barley fields from 2018 to 2019, respectively. DON was detected in 26% (mean 56 ppb, maximum 1488 ppb) and 24% (mean 65 ppb, maximum 2051 ppb) of barley fields. Results will help researchers to investigate potential management strategies to suppress barley-FHB in Manitoba.
Bioevaluation of seaweed extracts on the plant growth and resistance of tropical vegetables
J. JAYARAJ, O. ALI AND A. RAMSUBHAG
Department of Life Sciences, The University of the West Indies, St. Augustine, Trinidad and Tobago
The bioefficacy of extract preparations from two Caribbean seaweed species (Sargassum filipendula and Acathophora spicifera) were evaluated in sweet pepper and tomato plants. Foliar spray application of algal extracts have significantly improved the growth and yield of plants under greenhouse and field conditions. The treated plants recorded higher chlorophyl contents and growth parameters. Gene expression analyses revealed the upregulated expression of genes involved in auxin (IAA), gibberellin (Ga2Ox), cytokinin (IPT) synthesis and the genes involved in flowering regulation (SFT, SP, J, AN, FA and CO). The treated plants were resistant to pathogen infections. This was noticeable through reduced disease severity levels by, Xanthomonas campestris pv. vesicatoria and Alternaria solani pathogens in tomato and sweet pepper plants under both greenhouse and field conditions. Elevated levels of defence-related enzymes including phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, chitinase, and β-1,3-glucanase, as well as enhanced levels of total phenolic compounds were observed in the seaweed extract treated plants. The PR1a, PINII and ETR-1 genes were upregulated which is indicative of upregulation of SA/JA pathways. Significantly higher expression levels of multiple defence, stress responsive and growth-related genes was revealed in treated plants by RNA sequencing. A significant increase in colonization of microbes in the rhizosphere of plants was also noticed in treated plants. Multiple priming effects thus evoked by the seaweed extracts should have yielded in overall improved growth and productivity of plants and their enhanced resistance levels to pathogen infections.
Fusarium graminearum infection of differentially resistant barley cultivars reflect growth and deoxynivalenol production during malting
JAYATHISSA, M. BAKKER, D. FERNANDO, J. R. TUCKER AND A. BADEA
Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; (D.F.) Department of Plant Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; and (A.B., J.T.) Brandon Research and Development Centre, Agriculture and Agri-Food Canada, 2701 Grand Valley Road, Brandon, MB R7A 5Y3, Canada
Fusarium head blight (FHB) of barley causes significant losses for the malting and brewing industry; however, there has been insufficient attention towards understanding fungal growth and deoxynivalenol mycotoxin production during the malting process. Barley varieties: Newdale (moderately resistant to moderately susceptible to FHB) and AAC Goldman (moderately resistant to FHB) were inoculated with single strain conidial suspensions of each of seven different Fusarium graminearum strains (plus a non-inoculated control). In 2019, FHB severity was higher in Newdale (9%, by visual assessment) than in AAC Goldman (3%), and similar differences were observed for the fungal load (940.6 vs. 423.7 Fusarium: barley gene abundance ratio) and deoxynivalenol content (3.2 vs. 2.8 ppm via ELISA). We micro-malted this barley and found that cultivar differences in Fusarium-related variables persisted after malting; fungal load in the final malt was 17.03 in Newdale vs. 12.14 in AAC Goldman, and deoxynivalenol content was 3.8 vs. 1.4 ppm. Deoxynivalenol content increased in Newdale through the malting process but was reduced in AAC Goldman. Differences among cultivars were again evident in 2020: FHB severity was 38% in Newdale vs. 21% in AAC Goldman, the fungal load was 9.7 vs. 4.4, and deoxynivalenol content was 1.2 vs. 0.7 ppm. Toxin production varied among the seven pathogen strains as it was higher for 3-acetyl-deoxynivalenol compared to 15-acetyl-deoxynivalenol isolates. The outcome of this work will help to develop novel management strategies that could improve malt quality.
Use of high-throughput sequencing for the identification of plant pathogens
T. JEANNE, R. HOGUE, A. DIONNE, A. DROIT, C. BEAUPARLANT, J. D’ASTOUS-PAGÉ AND W. CHEN
Institut de Recherche et de Développement en Agroenvironnement, 2700 rue Einstein, Québec G1P 3W8, Canada; (A.D.) Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec (MAPAQ), 2700 rue Einstein, Québec G1P 3W8, Canada; (A.D., C.B.) Centre de recherche du CHUL, 2705, boulevard Laurier, Québec G1V 4G2, Canada; and (W.C.) Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
Field and vegetable crops are susceptible to many diseases which are caused by pathogenic organisms. These organisms co-exist on affected tissues, which complicates the diagnostic process. The decision-making process of disease diagnosis may involve the use of multiple microbiological, immunological and molecular assays, which can be time consuming, labour intensive and costly. The advent of high-throughput sequencing (HTS) techniques makes it possible to accurately identify most pathogenic organisms in a single analysis. By coupling available taxonomic information and using the latest sequencing data filtration strategies, it is now possible to assess unique genetic variants that can precisely identify pathogenic organisms. We evaluated the identification accuracy potential by HTS on nearly 500 diagnosed cases received at the Laboratoire d’expertise et de diagnostic en phytoprotection (MAPAQ) and at the Centre de recherche sur les grains (CEROM). These cases represented diverse crops (crucifers, cucurbits, potatoes, field crops and tomato) and pathogens. Amplicon-based HTS was used to assess pathogenic groups by targeting respective DNA regions, including the fungal Internal transcribed spacer ITS, bacterial and archaeal 16S rDNA gene, eukaryota18S rDNA and Fusarium Elongation factor 1-alpha (EF1α) regions. The HTS approach is based on an adapted sequence processing strategy, involving the use of public and custom-curated reference sequence databases. The HTS-based diagnostics results showed a good agreement with the diagnostic approaches used by LEDP for several pathogenic genera including Fusarium. The HTS data will be stored in a relational database and analyzed to define interpretation thresholds. A web application is to be developed to facilitate visualization of diagnosis based on HTS data.
Molecular diagnostics for bronze leaf disease affecting Canadian poplar and aspen trees
M. L. KALISCHUK, C. P. WIJEKOON, R. J. HOWARD AND L. M. KAWCHUK
Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; (C.P.W.) Canadian Centre for Agri-Food Research in Health and Medicine, 351 Taché Avenue, Winnipeg, MB R2H 2A6, Canada; (L.M.K.) Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB T1J 4B1, Canada; and (R.J.H.) RJH Ag Research Solutions Ltd., P.O. Box 1456, Brooks, AB T1R 1C3, Canada
Bronze leaf is a destructive disease that affects ‘Tower’ poplar (Populus × canescens Smith), Swedish columnar aspen (P. tremula L.), and Trembling Aspen (P. tremuloides Michx) and can cause economic losses to the nursery crop, processed wood industries and property owners where poplar and aspen trees are often used in amenity and shelterbelt plantings. Apioplagiostoma populi Barr (anamorph: Discula sp.) is the casual agent of bronze leaf disease; however, confirmation of the causal pathogen is challenging as it relies on observation of disease symptoms and morphological characteristics of A. populi when fruiting bodies form on infected leaves. Affected tissues develop a conspicuous, bronze-coloured pigment that becomes pronounced in late summer. The symptoms reduce the aesthetic and commercial value of infected trees and dieback can lead to premature death. In this study, we characterized the bronze leaf disease morphology in Canada and identified pathogen-specific sequences for sensitive diagnostics that indicate systemic infection of vascular tissues. Leaf samples from symptomatic trees were collected from British Columbia, Alberta and Manitoba and examined for perithecia to microscopically characterize A. populi asci and ascospores. Disease-associated β-tubulin 2, translation elongation factor-1- γ chain, and the internal transcribed spacer (ITS) 5.8S region of the nuclear ribosomal DNA sequences were isolated from perithecia and symptomatic tree samples. Morphological and molecular biological data were used to characterize the relationship and epidemiology of A. populi to develop a rapid diagnostic test to facilitate reliable, early detection and thereby help prevent and/or reduce aesthetic and economic losses from this devastating disease.
Fusarium head blight species diversity and deoxynivalenol (DON) levels in western Canadian producer wheat fields
S. K. KANNANGARA, P. BULLOCK, S. WALKOWIAK AND W. G. D. FERNANDO
Department of Plant Science, University of Manitoba, 222 Agriculture Building, 66 Dafoe Road, Winnipeg, MB R3T 2N2, Canada; (P.B.) Department of Soil Science, University of Manitoba, 362 Ellis Building, 13 Freedman Crescent, Winnipeg, MB R3T 2N2, Canada; (S.W.) Grain Research Laboratory, Canadian Grain Commission, 196 Innovation Drive, Winnipeg, MB R3T 6C5, Canada; and (W.G.D.F.) Department of Plant Science, University of Manitoba, 222 Agriculture Building, 66 Dafoe Road, Winnipeg, MB R3T 2N2, Canada
Fusarium head blight (FHB) is a devastating disease in western Canada that affects both the quantity and qualitity of wheat grain, mainly due to mycotoxin contamination (deoxynivalenol-DON and nivalenol-NIV). Fusarium graminearum is the principal causative agent associated with FHB of wheat in western Canada. Wheat spikes were collected in 2020 from an area not treated with fungicide in 65 participating farmers’ fields in western Canada to evaluate the presence of Fusarium species and their chemotypes. Inoculum availability was measured by setting up spore traps in each field from flowering until the soft dough stage. Both grain and chaff from spring wheat were subjected to mycotoxin testing by high sensitive 5/5 Vomitoxin kits from Neogen and fungal species and chemotype testing by DNA-based methods (PCR). The initial DON testing showed that the 2020 growing season experienced mild levels of FHB toxin contamination. In spring wheat, the highest mean DON content was recorded from Manitoba samples (0.3 ppm in grain and 2.12 ppm in chaff), while both Saskatchewan and Alberta samples contained average DON levels <0.2 ppm in grain and <1.00 ppm in the chaff. The chaff contained a significantly higher DON level than the grain, suggesting that chaff removal during harvest can reduce the impact of DON on the grain. The total genomic DNA was extracted from 24 Fusarium damaged kernels and ~0.5 g of chaff from each spring wheat sample. Different Fusarium species, including F. graminearum and their chemotype-producing genes, will be identified by 24 species-specific PCR assays and presented.
CRISPER/Cas9 mediated gene editing to create a Leptosphaeria maculans isolate carrying a single avirulent gene – AvrLepR1
S. K. KANNANGARA AND W. G. D. FERNANDO
Department of Plant Science, University of Manitoba, 222 Agriculture Building, 66 Dafoe Road, Winnipeg, MB R3T 2N2, Canada
Blackleg disease in canola, caused by Leptosphaeria maculans, is a major constraint to yield and trade. Although there is much research done on the interaction and identification of resistance, there is a significant limitation in characterizing the genes. A growing concern among blackleg researchers is that resistance (R) genes are not correctly identified as not all research labs use a standard set of well-characterized isolates. Further, the same R-gene may be determined by two different labs and named as two different genes. Therefore, the objective of the research is to develop universally acceptable isolates of L. maculans with single avirulent (Avr) genes that can be used for the identification of R genes and novel R genes. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 mediated gene editing is a promising tool in mutating Avr genes. We aim to create an isolate carrying only AvrLepR1 by knocking off the AvrLm5 gene of L. maculans isolate, D3. The CRISPR/Cas9 system was delivered to the L. maculans genome through the vector sgAvrlm5_pKHT332 via Agrobacterium-mediated transformation. The vector, sgAvrlm5_pKHT332, was created by combining a sgRNA, which was designed to bind AvrLm5 gene at 192 nucleotide position, and a section of pFC334 plasmid containing Aspergillus nidulans tef1 promoter, Cas9 gene and A. nidulans tef1 terminator via In-Fusion cloning. Antibiotic-resistant isolates were selected, and the mutant will be identified by sequencing the respective portion of the target gene. A successfully mutated AvrLepR1_avrlm5 isolate will be utilized for rapid phenotypic identification of the LepR1 gene in canola.
Elimination of zebra chip in Canada
L. KAWCHUK, D. JOHNSON, S. MEERS, Q. XIA AND J. LYNN
Lethbridge Research and Development Centre, Agriculture and Agriculture-Food Canada, 5403–1 Avenue South, Lethbridge, AB T1J 4B1, Canada; (D.J., Q.X.) Department of Geography and Environment, University of Lethbridge, 4401 University Drive West, Lethbridge, AB T1K 3M4, Canada; and (S.M.) Mayland Consulting, 1412 17A Street NE, Calgary, AB T2E 4V5, Canada
Zebra chip is a potentially devastating disease of solanaceous crops caused by ‘Candidatus Liberibacter solanacearum’ (Lso), a phloem-limited gram-negative bacterium that is transmitted by the potato-tomato psyllid (TPP) Bactericera cockerelli (Šulc). Originating in Mexico, the disease has become more prevalent and migrated northward through the US over several decades. To determine occurrence and epidemiology in Canada, TPP were collected from thousands of yellow sticky cards, and hundreds of samples from plants that exhibited putative disease symptoms, were collected during 2013 to 2020 from commercial farms. The TPP vector was observed primarily in AB, with fewer found in SK and MB. Under 100 TPP were observed annually except in 2017. Total nucleic acid was isolated from psyllids or plant samples and the cytochrome oxidase I or 16S ribosomal DNA was amplified with TPP and Lso specific PCR primers, respectively. All samples were negative for Lso except four TPP and a single post-harvest potato tuber recovered from southeastern Alberta in 2017. The Lso-positive potato tuber produced symptomatic plants with the proliferation of axillary shoots and abnormal tubers. BLAST analysis of the TPP and plant Lso showed central and western TPP haplotype and an identical match to Lso haplotype A isolates from the United States. Detection of Lso in Canada indicates the pathogen may be spreading to new areas, possibly due to favourable environmental conditions, and industry should continue to monitor movement of the vector and disease to minimize losses.
Natural antisense transcripts and stress response in Ustilago maydis
M. K. LARIVIERE AND B. J. SAVILLE
Environmental and Life Sciences Graduate Program, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada; and (B.J.S.) Forensic Science Program, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada
The climate is changing at an accelerated rate, raising concern about global food security. While advances are being made to increase crop yields, the threat of evolving pathogens must also be considered. Fungal pathogens and their host plants are evolving together, but as the climate changes, fungal pathogens are adapting at an accelerated rate compared to their hosts. We hypothesize that RNA-mediated mechanisms enhance fungal adaptation to stress. We are using the model fungal pathogen Ustilago maydis to investigate the role that natural antisense transcripts (NATs) play in modulating the stress response. NATs are a subset of non-coding RNAs which have regions of their sequence complementary to an mRNA. An RNA-seq comparison of three smut fungi revealed that 349 of the 2617 NATs found in U. maydis are conserved among all three smut species. The conservation of NATs among these smut species suggests that they have important functional roles. Genes complementary to these conserved NATs were investigated to identify those with previously documented roles in stress response. Among these genes, the levels of complementary NATs were assessed by reverse transcription-PCR, using RNA isolated from haploid U. maydis cells exposed to various stresses. Seven NATs with altered expression levels were identified and will be further characterized. Future experiments will assess whether expression of these NATs alters complementary mRNA stability, stress responses, or pathogenesis by the fungus. Identifying and describing these RNA-mediated responses to environmental stress may provide new tools for combating the increasing prevalence and severity of fungal diseases.
Characterization and developing effective screening methods of bacterial blight of highbush blueberry in the Lower Mainland of British Columbia
S. R. LATCHMAN AND R. R. BURLAKOTI
Agassiz Research and Development Centre, Agriculture and Agri-Food Canada, 6947 Hwy#7, Agassiz, BC V0M 1A0, Canada
The Lower Mainland of BC is a major region for the cultivation of highbush blueberry in Canada. Bacterial blight caused by Pseudomonas syringae (Ps) is an endemic disease of highbush blueberry in BC and causes significant damage in young plants. The objectives of this study were: (i) to characterize isolates of Ps and assess their virulence; and (ii) to develop reliable screening methods for pathogenicity test and disease resistance. About 200 isolates of Ps were collected in 2019 from suspected bacterial blight-infected plant samples. Of these, 51 representative isolates were used for characterization and virulence diversity. Isolates were confirmed as Ps by growing them on the Ps-selective media KBC (King’s B media with boric acid, cephalexin, and cycloheximide), and further characterized using the LOPAT test (levan, oxidase, pectolytic activity, arginine dihydrolase, and tobacco hypersensitivity reaction). Pathogenicity and virulence of these isolates were performed using blueberry leaf disc and detached leaf assays. About 61% of the tested isolates (n = 6) were confirmed as Ps and were pathogenic. Six pathogenic isolates were further evaluated by inoculating flower buds of two varieties ‘Chandler’ and ‘Duke’. All six isolates caused bacterial blight on flower buds of both varieties. In addition, six screening methods were compared by inoculating Ps in intact blueberry plants and three methods (wounding stem apex, removing leaf and wounding stem, and wounding leaf with carborundum) performed better than the remaining of screening methods.
Assessment of western redcedar for susceptibility to root and butt rot diseases by controlled inoculation
J.-J. LIU, K. PELLOW, M. CRUICKSHANK, A. ZAMANY, I. LEAL AND C. FILIPESCU
Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada; and (M.C., C.F.) Canadian Wood Fibre Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
Root and butt rot diseases are one of the most damaging groups of forest diseases. Western redcedar (WRC; Thuja plicata Donn) has the highest incidence of butt rot resulting in large economic losses in one of Canada’s most valuable tree species. The infection process and resistance of WRC to root and butt rot fungi are largely unknown; consequently, this presents a high risk for the WRC industry. To evaluate susceptibility of WRC to the diseases under greenhouse conditions, we developed two artificial inoculation methods using a block and stick to deliver inoculum to roots or an inoculum dowel plug directly inserted into the stem. Nine pathogenic fungi, including Armillaria ostoyae, Coniferiporia weirii, Phellinus (Porodaedalea) pini, Heterobasidion occidentale, Postia balsamea, Postia sericeomollis, Perenniporia subacida, Obba rivulosa, and Hypholoma fasiculare were used to inoculate 2-year-old seedlings. Symptoms of infection were assessed at 18–20 months post inoculations. Symptoms of infection were indicated by discoloured wood caused by fungal decay inside stem base cross-sections, or externally at the inoculation sites. Visual infection rates were 60% for H. occidentale and P. subacida, 30% for C. weirii, 20% for A. ostoyae, 10% for P. balsamea, and no obvious infection for the other four fungi. Furthermore, infected seedlings had stem height and stem diameter about 10% smaller than the controls, adding to the decay impacts. Infection assessment demonstrated that inoculation methods developed here were effective in measuring disease susceptibility, and could be used to screen WRC resistance to root and butt rot diseases in future breeding programmes.
Effects of two bacterial inoculants on the physiology and transcription of osmoprotectant-related genes in four generations of alfalfa breeding lines
S. LUNDELL AND B. BILIGETU
Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
Soil salinity is a malady inhibiting crop growth on 397 million ha of land worldwide, and 3.75 million ha in Western Canada. Planting deep rooted perennial forages such as alfalfa in salt affected land can act both as a means of land remediation by lowering the salt-laden groundwater table, and as a supplemental revenue stream for producers. Increased production of osmoprotectants is one means of raising the salinity tolerance of a plant. Mutualistic soil bacteria as well as selection for salt tolerant plant genotypes have both been demonstrated to be mechanisms capable of increasing the production of anti-stress-related compounds in plant tissues. This experiment aims to explore whether two strains of soil bacteria (Ensifer meliloti, Halomonas maura) can increase concentrations of osmoprotectants in alfalfa, whether salt-adapted genotypes from a multi-generation breeding programme targeting salinity tolerance produce greater concentrations of osmoprotectants, and whether soil bacteria can interact with salt-adapted genotypes to produce more osmoprotectants compared to earlier generations in 0, 8, and 16 ds/m soils. If increased levels of osmoprotectants are found among treatments, an rt-qPCR study will be conducted to deduce the cause(s) of increased osmoprotectant production among treatments. This study will aid in adapting alfalfa cultivars to growth on saline soil thereby producing a revenue stream for producers from marginal agricultural land.
Identification and diagnosis of plant pathogenic nematodes
M. MADANI
Formerly a post-doctoral at the Canadian Food Inspection Agency, Charlottetown, PE Canada; and University of Manitoba, Winnipeg, MB Canada
DNA-based technology has proved its usefulness for the identification and diagnosis of plant pathogens including species and subspecies of plant pathogenic nematodes. Traditionally, agarose gel electrophoresis has been the method of choice for separation of DNA bands. However, this can be a challenging step especially when dealing with the limited amount of starting materials used for loading on the gel, or when comparing amplicons sized less than 50 bp. The newly introduced Capillary Gel Electrophoresis (CGE) with the capability of running 1–2 µL of PCR (Polymerase Chain Reaction) product can separate amplicons with a few bp difference in length. The second approach is performing melting curve analysis as an additional step at the end of the real-time PCR cycles. Similar to CGE the generated results are visualized in the computer monitor that excludes the time-consuming agarose gel preparation and staining. The third technique is Loop-Mediated Isothermal Amplification (LAMP) which is a PCR-free-based amplification of the target DNA of pathogen. Preparation of a cocktail reaction carried out in a closed eppendorf which after incubation at a certain temperature will enable us to interpret the results based on the colour changes by the naked eye. The fourth approach is Integrative Analysis, by collective data derived from microscopy and host preference together with phylogenetic and DNA analysis of more than one molecular marker. This will generate robust and precise results on the diagnosis and species identification of the target pathogen, especially those closely related, enabling planning the proper control management strategy.
Top ten plant parasitic nematodes of economic importance, threatening prairie crops
M. MADANI
Formerly a post-doctoral at the Canadian Food Inspection Agency, Charlottetown, PE Canada; and University of Manitoba, Winnipeg, MB Canada
More than 4000 species of plant parasitic nematodes have been described worldwide, responsible for crop losses between 8.8 to 14.6%, equal to 100 to 157 billion USD annually, showing their potential threat to food security. The Canadian prairies with more than 32 Mha of suitable land for agriculture, and 4 M tones of pulses, and the main potato producer in Canada could potentially be subjected to several plant-parasitic nematodes. Vicinity with the North Dakota and Montana increasing this risk. Following is a short review on the most important species: 1) Soybean cyst nematode Heterodera glycines, on soybean, with the annual damage of $1 billion to USA agriculture. 2) Stem nematodes Ditylenchus spp., in Pulses and especially in fababean and pea, is an aggressive species with the economic threshold level of 0.8 eggs/g of soil, and complete crop failure at 64 eggs/g of soil in Europe. 3) Root-Knot Nematodes (Meloidogyne spp.), a cosmopolitan species and the capability for producing complex disease and synergism with other pathogens. 4) Root Lesion Nematode (Pratylenchus spp.), is a serious pathogen in field crops and fruit trees, with tolerance limit of 0.03 specimens/cm3 of soil, and yield losses of 58% at 2 specimens/cm3 of soil in chickpea in Europe and the USA. 5–6). Cereal Cyst Nematode, Heterodera trifolii and H. avenae, in cereals and grains, with crop losses of $4 million in Europe and $70 million in Australia. 7–8) Potato Cyst Nematodes Globodera pallida and G. rostocheinsis, with the damage threshold of one to two eggs/g of soil. 9) Stubby Root Nematode Thrichodorus spp., and 10) Spiral nematodes Helicotylenchus spp. in corn fields. Planning ahead regarding identification and control of plant parasitic nematodes is an essential component of sustainable crop production in Canadian agriculture.
Weather-based models for forecasting and managing Fusarium head blight risk in western Canadian cereal production
T. T. MATENGU, P. BULLOCK, M. S. MKHABELA, F. ZVOMUYA, M. A. HENRIQUEZ, T. OJO, R. PICARD, R. AVILA AND M. HARDING
Department of Soil Science, University of Manitoba, 13 Freedman Crescent, Winnipeg, MB R3T 2N2, Canada; (M.A.H.) Morden Research and Development Centre, Agriculture and Agri-Food Canada, 101 Route 100, Morden, MB R6M 1Y5, Canada; (T.O.) Soil and Ag Weather Surveillance, Manitoba Agriculture and Resource Development, 450 Broadway, Winnipeg, MB R3C 0V8, Canada; (R.P.) Crop Production Extension, Manitoba Agriculture and Resource Development, 279 Carlton Street, Somerset, MB R0G 2L0, Canada; (R.A.) Ministry of Agriculture, Government of Saskatchewan, 329–3085 Albert Street, Regina, SK S4S 0B1, Canada; and (M.H.) Plant and Bee Health Surveillance, Crop Diversification Centre South, Alberta Agriculture and Forestry, 301 Horticultural Station Road East, Brooks, AB T1R 1E6, Canada
Predicting the risk of Fusarium head blight (FHB) disease occurrence in cereal crops is critical for determining the need for and timing of fungicide sprays. Existing models for predicting FHB risk developed many years ago may no longer be suitable for the current Fusarium species complex that has evolved in Canada. Therefore, this study aims to develop and validate weather-based risk models around flowering for predicting FHB index (FHBi), Fusarium damaged kernels (FDK), and deoxynivalenol (DON) in spring wheat, winter wheat, barley, and durum across three Canadian Prairie provinces. Data collected from 15 sites in western Canada in 2019 and 2020 were used to classify an epidemic at a 5% FHBi (all crops), or 1 mg kg−1 DON (all crops) or 0.2, 0.3, 0.8, and 2% FDK thresholds for barley, spring wheat, winter wheat, and durum, respectively, to develop weather-based models for Fusarium epidemics. Kendall correlation and stepwise logistic regression analysis identified suitable combinations of temperature (temp), relative humidity (RH), precipitation (prec), and solar radiation (SR) at 4, 7, 10, 14 days pre-anthesis, and 3 days pre to 3 days post-anthesis for predicting FHB risk. The weather variables chosen across crop types for the FHBi models were RH, temp, and prec, and for the FDK and DON models RH was selected. Prediction accuracy of the models ranged from 74.6 to 80.6, 76.5 to 78.1 and 78.3 to 79.3% for FHBi, FDK, and DON, respectively. Fusarium head blight pressure was low in 2019 and 2020, most likely due to drier than normal weather conditions, which were unfavourable for the disease. The models will be used to power an interactive, online digital viewer and provide early warning of potential FHBi, FDK, and DON epidemics in prairie cereal crops.
Compost amendments affect plant performance and crop quality without affecting disease severity in a long-established crown gall-diseased vineyard
P. MCGONIGAL, T. M. VOEGEL, C. DOUGLAS AND L. M. NELSON
University of British Columbia, Okanagan Campus, 1177 Research Road, Kelowna, BC V1V 1 V7, Canada; and (C.D.) Quails’ Gate Estate Winery, 3303 Boucherie Road, Kelowna, BC V1Z 2 H3, Canada
Crown gall disease of grapevine has severe detrimental effects on grapevine performance and crop quality. The disease is induced by the soil-dwelling bacterium Allorhizobium vitis, which can be introduced into vineyard soil via infected planting material. Soil-borne A. vitis infections are enhanced by the presence of plant-parasitic nematodes because they induce root wounds through which the bacterium can enter the vine and establish systemic infection. Compost application has previously reduced pathogenic nematode populations in soil. Therefore, we hypothesized that compost amendments would reduce the severity of A. vitis infection by reducing soil nematode populations. In this study, three composts were applied in-row in a long-established vineyard infested with both plant-parasitic nematodes and A. vitis in the spring of 2019 and 2020. Bulk soil was collected in the spring and fall of both years to quantify A. vitis per gram of soil. Visual ratings of disease severity, plant performance, and crop quality measurements were also taken throughout 2019 and 2020. There were no significant differences in the concentration of A. vitis in bulk soil, nor in visual ratings of disease severity between treatments. Average berry weights (g; p = 0.023), berry titratable acidity (g/L; p = 0.040), and leaf greenness (p = 0.033) were affected by the application of compost treatments after 2 years. Our data indicate that berry tartness and sourness, berry weights, and overall plant vigour increased following compost application. Increased plant vigour may be detrimental as it can reduce grape production and make vines more susceptible to disease.
Screening for potential biological control agents against Allorhizobium vitis, the causal agent of crown gall disease of grapevine
P. MCGONIGAL, T. M. VOEGEL, D. VOLLMAN AND L. M. NELSON
University of British Columbia, Okanagan Campus, 1177 Research Road, Kelowna, BC V1V 1 V7, Canada
Crown gall disease of grapevine is an economically important disease in cold-climate grape growing regions around the world. The disease is characterized by its severe detrimental effect on grapevine performance and crop quality. Partial or complete vine death is common, especially in young vines. The causal agent of grapevine crown gall is the soil-dwelling bacterium Allorhizobium vitis, which can be introduced into vineyard soil via infected planting material. Current cultural management practices are often not effective and there are currently no commercial biocontrol products available. In this study, endophytic and rhizosphere bacteria collected from escaped vines (vines resistant to disease symptoms) in crown gall-diseased vineyards in British Columbia and Ontario were screened for inhibitory activity against several strains of A. vitis. The inhibitory screening assay employed consisted of four separate screens, each included to reduce the number of potential bacterial isolates to be used in subsequent greenhouse assays. Two separate media were included as antibiosis may be culture medium dependent. Five bacterial isolates that showed consistent inhibitory activity against A. vitis across all screens were identified as potential biological control agents. Three isolates were classified as Bacillus spp. and two were classified as Pseudomonas spp. Species within these genera have previously shown biological control activity against several plant pathogens. Simultaneous inoculation of A. vitis and each of the potential biological control agents identified in this study at a 1:1 ratio did not result in gall reduction compared to untreated controls. Further studies are currently being conducted.
Validation of prediction models for sclerotinia stem rot [Sclerotinia sclerotiorum] in soybean (Glycine max) in Quebec
C. MORIER-GXOYIYA, T. COPLEY, G. BOURGEOIS AND V. GRAVEL
Department of Plant Science, Faculty of Agricultural and Environmental Sciences, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Québec H9X 3V9, Canada; (T.C.) Centre de recherche sur les grains (CÉROM), Inc., 740 chemin Trudeau, St-Mathieu-de-Beloeil, Québec J3G 0E2, Canada; and (G.B.) Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada
Sclerotinia stem rot (SSR) in soybean (Glycine max (L.) Merr) is a disease caused by the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary which leads to yield losses for farmers in many regions, including Quebec. The disease is commonly kept at bay by chemical fungicides sprayed during the crop’s vulnerable growth stages or using tolerant cultivars. However, whether pesticides are needed depends on the risk of disease outbreak, which is largely influenced by environmental conditions prevailing prior to and during the soybean flowering period. Unnecessary or improperly timed fungicide applications are costly not only economically, but also environmentally. To evaluate the risk of SSR disease development, researchers have created multiple SSR prediction models. In 2019 and in 2020, pre-conditioned S. sclerotiorum sclerotia were placed in commercial and research soybean plots throughout Quebec. Plots were scouted for the presence of apothecia and SSR disease development. Observations were used to evaluate the ability of previously published bioclimatic models to predict the formation of S. sclerotiorum apothecia under Quebec’s agro-environmental conditions through receiver operating characteristic (ROC) curve analysis. Upon validation, models were customized using the data collected in Quebec to improve their predictive ability in the local context. The most accurate published models used maximum temperature alone or maximum temperature, wind speed and relative humidity, with an accuracy of 80.0% and 75.9% respectively at a probability threshold of 40%. These results suggest that published models provide a reliable basis for the development of an adapted SSR forecasting system for Quebec soybean growers.
Comparing extraction methods to quantify Aphanomyces euteiches inoculum levels in field soils
C. K. MORRISON, S. BANNIZA AND S. CHATTERTON
Department of Plant Sciences, University of Saskatchewan, Room 4D36, Agriculture Building, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada; and (S.C.) Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB T1J 4B1, Canada
Root rot of pea and lentil caused by the oomycete pathogen Aphanomyces euteiches is a serious threat to pea and lentil production in Canada and elsewhere around the world. Accurate detection and quantification of the inoculum of this pathogen, in the form of long-lived oospores, is a necessary tool for growers as control of this disease is limited to avoiding peas or other hosts in a field until inoculum load is reduced sufficiently. Standard commercial kit DNA extractions coupled with quantitative polymerase chain reaction amplification (qPCR) can be used to quantify this pathogen in soil samples but can provide inconsistent results for some soil types. To improve upon existing methods, multiple extraction methods were performed followed by standard qPCR to assess detection limits, precision, and accuracy. Methods include a sugar-based centrifugation oospore extraction with subsequent DNA extraction, standard commercial kit DNA extraction coupled with pre-extraction steps including grinding, bead-beating and sonication of soil samples, and greenhouse bioassays. Both naturally infested field soil and spiked soil were used. Pre-extraction steps involving grinding and bead-beating slightly increased extraction efficiency, likely by helping to separate the oospores from the soil matrix. Extraction efficiency from naturally infested field soils remained low, but the alternative extraction methods using substantially larger volumes of soils (two orders of magnitude) than the 250 mg required for the commercial kit may be needed in soils where oospore counts are low. Comparing these methods in different soil types will be important to determine whether methods are reproducible across soil types.
Competition between Plasmopara viticola clade aestivalis and clade riparia: who can win the aggressiveness battle?
R. A. MOUAFO-TCHINDA, P.-O. HEBERT, M. L. FALL, C. BEAULIEU AND O. CARISSE
Centre SÈVE, Département de biologie, Université de Sherbrooke, 2500 De L’Université Boulevard, Sherbrooke, QC J1K 2R1, Canada; and (P.-O.H., M.L.F., O.C.) Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada
Since the evidence of the existence of two clades of Plasmopara viticola (clades riparia and aestivalis) in eastern Canada, it has been reported that epidemics caused by clade riparia start 2 to 3 weeks before those caused by clade aestivalis, however, clade aestivalis was more aggressive than clade riparia. The objective of this work was to study the competition between the clades riparia and aestivalis, and to compare the aggressiveness of both clades in mono- and co-infection situations. Solutions of sporangia from both clades were co-inoculated in six percentage combinations on leaf discs. Subsequently, the progression of sporangia production expressed as sporangia percentages (SP) was estimated after eight cycles of infection-sporulation. The aggressiveness of clades in mono-infection situations on leaf discs was then compared to that in co-infection situations. The results show that the percentage of sporangia produced by clade aestivalis increases with the infection-sporulation cycle, while that produced by clade riparia decreases. The aggressiveness of the clades riparia and aestivalis in co-infection situations were different from those in mono-infection situations and were strongly influenced by the SP of the clades in competition. These results suggest that P. viticola clade aestivalis is more competitive than clade riparia and that the management of downy mildew in eastern Canada should consider the percentages of each clade present in the vineyard. Even though these results raise new questions, they should be considered for the management of grape downy mildew.
Potential role of root neutral lipids in mediating forage soybean acclimation to cultivation on acidic soil in boreal climate
M. NADEEM, O. A. ADIGUN, C. F. MANFUL, T. H. PHAM, R. THOMAS AND M. CHEEMA
School of Science and the Environment, Memorial University of Newfoundland and Labrador, 20 University Drive, Corner Brook, NL A2H 5 G4, Canada
Crop plants are known to adapt to their environment to produce desirable harvest by modulating their membrane lipids. Conversely, less is known concerning storage lipid’s role in successful plant adaptation and acclimation to abiotic stressor in the growth environment. Triacylglycerol (TAG) and diacylglycerol (DAG) are the main storage lipids and limited information suggest that their metabolism appears to be important in plant response to different abiotic stresses such as phosphorus or nitrogen starvation. To improve our understanding of neutral lipids contributions to successful plant acclimation to environmental stress we assessed TAG and DAG metabolism in soybean roots exposed to varying soil pH (6.8, 6.0 and 5.1) and their possible relationship with plant agronomic performance and adaptation during cultivation in boreal climate. A field study was conducted in Newfoundland where root membrane lipids were determined at pod formation stage using ultra high-performance liquid chromatography linked to hydrophilic interaction chromatography coupled to heated electrospray ionization accurate mass tandem mass spectrometry. We observed a significant decrease in total neutral lipids across the soil pH continuum {from neutral soil pH (79.93 ± 0.60 nmol%) to acidic soil pH − 5.1 (70.60 ± 0.39 nmol%)} whereas a concomitant increase was noticed in forage biomass production. Soybean plants produced significantly higher biomass of 3.59 ± 0.13 Mg ha−1 at pH 5.1, (3.55 ± 0.07 Mg ha−1 at pH 6 compared to lower biomass (2.18 ± 0.23 Mg ha−1) at pH 6.8 which suggests that neutral lipids remodulation appears to assist forage soybeans plants to adapt and attain higher forage biomass at low soil pH in boreal climate.
Potato mop-top virus, an emerging challenge to potato industry
X. NIE, M. SINGH, J. LAVOIE, V. BISHT, M. SHUKLA, A. CREELMAN AND M. LAI
Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Fredericton, NB E3B 4Z7, Canada; (M.SINGH) Agricultural Certification Services, Fredericton, NB E3B 8B7, Canada; (J.L.) New Brunswick Department of Agriculture, Aquaculture and Fisheries, Potato Development Centre, Wicklow, NB E7L 3S4, Canada; and (V.B.) Manitoba Agriculture, P.O. Box 1149, #65 – 3rd Avenue NE, Carman, MB R0G 0J0, Canada
Potato mop-top virus (PMTV, genus Pomovirus, family Virgaviridae) is a soil-borne virus that causes a severe tuber disease called spraing, which is characterized by internal necrotic arcs or rings in infected potato tubers. The disease can result in significant economic losses to the potato industry when disease incidence becomes sufficiently high. Since its first discovery in North America in 2003, the virus has been reported in many potato production areas in USA and Canada. To unveil the occurrence of PMTV in Canada and to develop management strategies for the virus as well as other tuber necrotic viruses including tobacco rattle virus, alfalfa mosaic virus and potato virus Y strain NTN, a 5-year study has been underway. An ongoing annual survey of potato tubers from Manitoba and New Brunswick suggests that PMTV is the primary virus responsible for virus-caused tuber necrotic diseases in both provinces. To facilitate the identification of PMTV-infested fields, a real-time PCR-based high-resolution DNA melting (HRM) assay was developed for the simultaneous detection of PMTV and its protist vector, Spongospora subterranea f.sp. subterranea (Sss) in soil and for the identification of PMTV- and/or Sss-infested fields. Indeed, by using this method, several fields have been determined to be PMTV-infested, and one of them is being used for the trial of potato cultivar sensitivity to PMTV-induced spraing disease.
Altering nitrogen management to improve winter wheat yields and nitrogen efficiency in the Northern Great Plains
J. L. OWENS, E. RAHMANI, X. HAO, L. M. HALL, K. COLES, C. HOLZAPFEL AND B. L. BERES
Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403 1st Avenue South Lethbridge, AB T1J 4B1, Canada; (L.M.H.) University of Alberta Department of Agricultural, Food, and Nutritional Science, 410 Ag/Forestry Building, Edmonton, AB T6G 2P5, Canada; (K.C.) Farming Smarter, 211 034 Hwy 512, Lethbridge County, AB T1J 5N9, Canada; and (C.H.) Indian Head Agricultural Research Foundation, #1 Government Road, Indian Head, SK S0G 2 K0, Canada
Optimizing nitrogen management for winter wheat crops can maximize agronomic performance and reduce greenhouse gas emissions, but these factors are rarely considered together. We conducted two experiments at seven sites throughout the Canadian Prairies over three to four crop cycles. We measured grain yield and protein at all sites, and greenhouse gas emissions from one site (Lethbridge, Alberta). Experiment I applied: urea, urea+eNtrench® (nitrification inhibitor), SuperU® (urease and nitrification inhibitor), and Environmentally Smart Nitrogen® (ESN®), a polymer-coated urea. All N was applied: 100% side-banded at planting, 30% side-banded at planting and 70% broadcast in-crop in late fall, and 30% side-banded at planting and 70% broadcast in-crop in early spring. Experiment II applied: urea ammonium nitrate (UAN), UAN+eNtrench®, UAN+Agrotain Ultra® (urease inhibitor), UAN+Agrotain Plus® (urease and nitrification inhibitors), and a 50–50 mix of urea and ESN®, with all N applied 50% side-banded at planting and 50% broadcast in-crop in early spring. All N banded at planting and split applying in spring produced similar yields and protein, but grain yields and protein were 4.2% and 1.8% when N was split applied in late fall compared to all N banded at planting (p ≤ 0.05). Including urease or urease and nitrification inhibitors also incrementally improved yields and N uptake. Greenhouse gas emissions scaled per unit grain yield, grain-N, and total N uptake did not vary significantly between treatments showing improvements in winter wheat agronomic performance did not equate to substantial reductions in greenhouse gases linked to climate change.
Is less virulent Leptosphaeria biglobosa robust enough to protect canola from highly virulent L. maculans?
K. R. E. PADMATHILAKE AND W. G. D. FERNANDO
Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Leptosphaeria biglobosa is a less virulent pathogen that causes blackleg disease in canola (Brassica napus). Previous literature has shown the inoculation of less virulent L. biglobosa can boost the host resistance of canola against the highly virulent L. maculans. This study focused to study the effectiveness of L. biglobosa as a biocontrol agent against L. maculans in depth at morphological and transcriptomic levels. The in-situ development of two Leptosphaeria species inoculated at different timepoints was assessed using fluorescent-protein-tagged isolates: GFP-tagged L. maculans and DsRed-tagged L. biglobosa. The control of L. maculans mycelia with pre inoculated and co-inoculated L biglobosa was clear in fluorescence protein tagged mycelial observations under confocal microscope. The quantification of each pathogen done by qPCR and host defence-related real-time gene expressions confirmed the potential of L. biglobosa in the management of blackleg disease. The study shows how the less virulent species L. biglobosa protect the canola crop from the invasion of the highly virulent L. maculans in canola. Morphological and Transcriptomic data are being evaluated, and will be presented at the conference.
Does trophic switch of Leptosphaeria maculans happen under both incompatible and compatible interactions with canola (Brassica napus)?
K. R. E. PADMATHILAKE, Z. ZOU AND W. G. D. FERNANDO
Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Canola (Brassica napus) is a 29.6-billion-dollar crop in Canada. Leptosphaeria maculans causes blackleg, is one of the most economically significant diseases of canola. Since the breakdown of resistance in B. napus genotype grown in Canada, it is pivotal to introduce new resistant genotypes. This study focused to evaluate the potential of B. napus genotype, ‘01–23-2-1ʹ which carries Rlm7 to be the next candidate gene to be introduced. 01–23-2-1 line was inoculated with L. maculans isolate (UMAvr7) carrying an avirulence gene AvrLm7, and the CRISPR/Cas9 knockout AvrLm7 mutant (umavr7) of the same isolate to make incompatible and compatible interactions, respectively. The susceptible Westar was used as the control targeting key time points in these interactions and analyzed by dual RNA-seq. L. maculans is a hemibiotrophic fungus which starts its life cycle as a biotroph and turns into a necrotroph later. Out of phytohormone composition, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play vital roles in plant-pathogen interactions and trophic switch of the pathogen. Genes related to host SA metabolism, phytohormone considered against biotrophs, upregulated early stage of the incompatible interaction compared to the compatible interactions. In contrast, genes of JA and ET metabolism, which are considered to be high against necrotrophs, were significantly higher in later stages of compatible interactions. However, the genes of JA and ET metabolism were several times lower at later time points in incompatible interaction suggesting the L. maculans incompatible interaction remain under control at 7- and 11- dpi and the pathogen remains more as a biotroph without switching into necrotrophic stage.
Development of a consistent greenhouse inoculation method for Fusarium avenaceum for screening germplasm of Pisum sativum
S. PANDIT, S. CHATTERTON AND C. VUCUREVICH
University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada; and (S.C., C.V.) Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403–1 Avenue South Lethbridge, AB T1J 4B1, Canada
Fusarium avenaceum is the most dominant pathogen in the pea (Pisum sativum) root rot complex in the prairies. Management of fusarium root rot (FRR) is challenging as fully resistant cultivars are absent and control options are limited to crop rotation. To develop a screening system for breeding purposes, three experiments were conducted. In the first experiment, disease severity (DS) of two cultivars (‘CDC Meadow’ and ‘Carmen’) in response to seed soak inoculation by three F. avenaceum isolates at four concentrations (1000, 5000, 10 000, 50 000 macroconidia/mL water) was measured. Results indicated that 10,000 macroconidia/mL was the best inoculum dose to achieve moderate disease severity. ‘CDC Meadow’ had significantly higher DS at all inoculum concentrations compared to ‘Carmen’. In the second experiment, 13 pea germplasm lines were screened in a Fusarium disease nursery, and in a greenhouse using the seed soak method. Germplasm lines ‘K-2ʹ, ‘5005ʹ, and ‘5004ʹ had significantly lower DS in the 2019 field nursery while none of the lines performed differently from the susceptible control in 2020 field or greenhouse trials. Due to inconsistent results between greenhouse and field screening, four different inoculation methods were then compared in two cultivars (‘CDC Meadow’ and ‘CDC Dakota’). The inoculation methods were: 1) seed soaking; 2) pipetting inoculum to seed at planting; 3) pipetting inoculum to lower stem and roots 2 weeks after planting; or 4) soaking roottrainers in inoculum 2 weeks after planting. Root soaking resulted in consistent FRR development in both cultivars, and will be tested further on more germplasm lines.
Can new sainfoin cultivars be profitably used for mixed stands with alfalfa and grass?
H. P. POUDEL AND S. N. ACHARYA
Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403–1 Avenue South, Lethbridge, AB T1J 4B1, Canada
Sainfoin is a highly nutritious, non-bloating leguminous forage crop of temperate regions. Despite non-bloating properties, its use as pasture was limited because of lower dry matter yield (DMY) and its lack of persistence in a mixed pasture. The paradigm then shifted when new sainfoin cultivars planted in mixtures with alfalfa reduced bloat incidences in ruminants by 98%. However, the yield potential of these sainfoin cultivars is still undervalued. In the study described here, we determined the yield potential of these newly developed sainfoin cultivars and their sister populations in the primary mixture with alfalfa cv. Longview or orchardgrass cv. Kayak. The experiments were established by drilling the two crops in alternate rows or cross-drilling them under irrigated and non-irrigated conditions in Lethbridge, AB in 2013. Forage mass and its composition by species were observed for 3 consecutive years after the establishment year. Monoculture orchardgrass produced the least DMY among all combinations of sainfoin-orchardgrass mixtures under both irrigated and non-irrigated conditions and both planting schemes consistently over all 3 years. The DMY of monoculture alfalfa was less than its mixture with new sainfoin cultivars under irrigated conditions, but this did not happen consistently under non-irrigated conditions. Under non-irrigated conditions, alfalfa-orchardgrass yielded significantly greater than sainfoin-orchardgrass mixed stands. The percentage of DMY contribution of sainfoin in alfalfa-sainfoin mixtures decreased, yet it increased in sainfoin-orchardgrass mixtures across harvest time in both growing conditions. We conclude that new sainfoin cultivars with alfalfa have the potential to produce bloat-free pasture without compromising the biomass yield when supplemented with irrigation.
Effectiveness of biofungicides to control several fungal diseases in vineyards
C. PROVOST, O. CARISSE AND A. LEFEBVRE
Centre de recherche agroalimentaire de Mirabel, 9850 Belle-Rivière, Mirabel, Québec J7N 2X8, Canada; and (O.C., A.L.) Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada
Canadian growers are under constant pressure to reduce their use of pesticides. However, to ensure the industry’s competitiveness, it is essential to secure yields by improving disease management. Winegrowers are threatened by several leaves and berry diseases, including bunch rot [Botrytis cinerea], downy mildew [Plasmopara viticola], powdery mildew [Erysiphe necator], and black rot [Guignardia bidwellii]. Therefore, preventing and delaying the development of these diseases with minimal use of fungicides is a daily challenge for winegrowers and advisers. The control of these diseases is based on the use of several control methods, including synthetic fungicides. However, the resistance to these products is high, which results in a loss of effectiveness. In this context, it becomes essential to encourage the use of biofungicides. However, we must first assess their effectiveness and determine the best use strategy to provide winegrowers with precise information that will allow them to integrate these products into their disease control management. The main objective of this project was to evaluate the effectiveness of biofungicides approved for the control of grapevine diseases and propose application strategies within an integrated pest management framework. Within the framework of this project, some biological fungicides allowed good disease control, such as Botector, Oxidate, Actinovate, and Double nickel55. Significant reductions in disease were observed compared to treatment with water, but disease levels remained high, demonstrating that the use of these products as unique method of control was not enough for acceptable disease control. These results support the importance of developing an integrated approach based on best cultural practices, scouting, and risk estimation.
Impact of cluster leaf removal on grape disease pressure for cold-hardy hybrid cultivars under climatic conditions of eastern Canada
C. PROVOST, A. LEFEBVRE AND O. CARISSE
Centre de recherche agroalimentaire de Mirabel, 9850 Belle-Rivière, Mirabel, Québec J7N 2X8, Canada; and (A.L., O.C.) Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada
There is a general agreement among scientists and grape specialists that proper canopy (CM) and fruit zone management (FZM) are essential practices to obtain good quality grapes. Fruit zone management involves leaf removal around the clusters. The ultimate objective of both CM and FZM is to improve grape aroma, flavour and pigment profiles, favour earlier maturity and reduce diseases. Practices of fruit zone management were evaluated for their effect on disease management during summer 2019 and 2020. The practices were leafing around the cluster zone on: 1) one side of the row at nouaison; 2) two sides of the row at nouaison; 3) one side of the row at veraison; 4) two sides of the row at veraison; 5) no leafing (control). Downy mildew, powdery mildew, and botrytis bunch rot were assessed weekly on leaves and at harvest on clusters. Regardless of the treatment, the effect of fruit zone management practices was small but significant. For both timing of leaf removal, nouaison and veraison, lower disease severity was observed when leaves were removed on both sides of rows. Overall, lowers disease severity was observed when leaves were removed at nouaison as compare with veraison. The difference in disease severity may be explained by lower humidity and better fungicide penetration in the canopy in sub-plots where leaves around the clusters were removed on both sides of rows at veraison. The removal of leaves from the fruiting area promotes the penetration of fungicides during a localized treatment but also of general coverage.
Assessment of compatibility of biofungicides in the development of a strategy for the control of diseases in vineyards
C. PROVOST, A.-A. DURAND, P. CONSTANT AND C. GUERTIN
Centre de recherche agroalimentaire de Mirabel, 9850 Belle-Rivière, Mirabel, Québec J7N 2X8, Canada; and (A.A.D., P.C., C.G.) Institut National de Recherche Scientifique- Centre Armand-Frappier Santé Biotechnologie, 531 boulevard des Prairies, Laval, Québec H7V 1B7, Canada
Several leaf and berry diseases are present in vineyards and cause significant economic losses. In recent years, several biofungicides have been registered to control these diseases. However, the results in the field are sometimes inconsistent. Moreover, some aspects of their use are not well known, such as the compatibility of the different fungicides or the best strategy for using them. A better knowledge of their mode of action and the possible effects on each other is necessary to prevent interference with a previous application and avoid unnecessary application. This project aims to determine the compatibility of lower risk fungicides (including biofungicides) and understand their respective effects to propose an optimal use strategy to fight against grapevine diseases. The first part was carried out in the laboratory to understand the functioning of biofungicides and establish the compatibility of the products. The results show that the population of the active ingredient of Double Nickel 55 and Serenade Opti decreases rapidly after the application, whereas Botector microbial population increase up to 6 days following application, before decreasing. Oxidate 2.0 fungicide significantly affects all three biofungicides and causes significant mortality of the active ingredient, unlike Cosavet DF, Copper 53 W, and Milstop, which have little effect on microorganisms from biofungicides. Secondly, field trials will be carried out during the 2021 and 2022 seasons to evaluate different application strategies based on the results observed in the laboratory.
Cannabis inflorescence yield and cannabinoid concentration are not improved with long-term exposure to UV radiation
V. RODRIGUEZ-MORRISON, D. LLEWELLYN AND Y. ZHENG
School of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada
It is commonly believed that exposing Cannabis sativa L. (cannabis) plants to ultraviolet (UV) radiation can enhance Δ9-tetrahydrocannabinol (Δ9-THC) concentrations in female inflorescences. However, a lack of published scientific studies has left knowledge-gaps in the effects of UV on cannabis. In this study we investigated the effects of UV exposure level on photosynthesis, growth, inflorescence yield, and secondary metabolite composition of two indoor-grown cannabis cultivars: ‘Low Tide’ (LT) and ‘Breaking Wave’ (BW). After growing vegetatively for 2 weeks under a canopy-level photosynthetic photon flux density (PPFD) of ≈225 μmol·m–2·s–1 in an 18-h light/6-h dark photoperiod, plants were grown for 9 weeks in a 12-h light/12-h dark ‘flowering’ photoperiod under a canopy-level PPFD of ≈400 µmol·m–2·s–1 and 3.5 h·d–1 of supplemental UV radiation with UV photon flux densities (UV-PFD) ranging from 0.01 to 0.8 μmol·m–2·s–1 provided by light-emitting diodes (LEDs) with a peak wavelength of 287 nm. The severity of UV-induced morphology and physiology symptoms worsened as UV exposure level increased. Dry inflorescence yield decreased with increasing UV exposure level in LT, but not in BW. In LT, total equivalent Δ9-THC and total equivalent cannabidiol (CBD) concentrations decreased with increasing UV exposure level, whereas there were no UV treatment effects on total equivalent concentrations of individual cannabinoids in BW. The potential for using UV to enhance cannabis quality must still be confirmed before it can be used as a production tool for modern, indoor-grown cannabis cultivars.
A hydroponics-based high-throughput screening system for clubroot disease indexing
R. SALIH AND E. PÉREZ-LÓPEZ. (R.S., E.P.-L.)
Department of Plant Sciences, FSAA, 2425, rue de l’Agriculture, Université Laval, Québec G1V 0A6, Canada; and (E.P.-L.) Centre de recherche et d’innovation sur les végétaux (CRIV), 2480, boulevard Hochelaga, Université Laval, Québec G1V 0A6, Canada
In recent years, clubroot has become one of the most devastating diseases affecting canola worldwide. Control of clubroot disease has proven to be very difficult to achieve. To this day, the best alternative is the use of resistant genotypes, but after 1 or 2 years of using certain clubroot resistant canola, resistance has been broken by different P. brassicae pathotypes. A very important step to identify which canola variety is the best for each field is the identification of the different pathotypes and how the plants respond to them. Pathotyping rely on the determination of disease index (DI) through a very laborious observational and subjective process that would benefit of digitalization. Selecting the appropriate growing conditions of the sample population is essential in selecting the suitable digital software. P. brassicae infection is conditioned by the transition of resting spores into motile spores, zoospores, which are chemotactically attracted by canola roots under water-saturated conditions. Therefore, a hydroponic system could provide the ideal condition to initiate and observe infections. Here we present the preliminary results of a hydroponics-based high-throughput screening system for clubroot disease indexing. We found that after 21 days post inoculation of Westar canola with P. brassicae field isolate pathotype 3 H, 100% of the plants showed large galls and the ratio shoot/root was significatively lower in the infected plants than in the mock-inoculated control. In this poster we will also present what are the next steps in our project to increase the efficiency of the system.
Metalaxyl sensitivity of Phytophthora spp. associated with red raspberry in British Columbia
S. SAPKOTA AND R. R. BURLAKOTI
Agassiz Research and Development Centre, Agriculture and Agri-Food Canada, 6947 Hwy#7, Agassiz, BC V0M 1A0, Canada
Root rot and wilting complex (RRWC), caused by Phytophthora spp., is the most devastating disease of red raspberry in British Columbia (BC). Metalaxyl is the most commonly used fungicide to manage the disease in the province. P. rubi and P. gonapodyides were isolated from red raspberry samples showing symptoms of RRWC. The metalaxyl sensitivity of strains of Phytophthora spp. (12 strains of P. rubi and six strains of P. gonapodyides) isolated from diverse raspberry farms of BC were evaluated in vitro using the radial growth method. Metalaxyl was amended to 20% clarified V8 agar to yield final concentrations of 0, 0.001, 0.01, 0.1, 1, 10 µg mL−1. Each strain was tested in triplicate for treatments and experiments were repeated twice independently. Fifty percent effective concentration (EC50) values and dose response curves were determined for each strain. Results showed that ~ 83% of P. rubi and 100% of P. gonapodyides strains were highly sensitive to metalaxyl with mean EC50 values of 0.0576 (range from 0.0375 to 0.0738) for P. rubi and 0.0542 (range from 0.0244 to 0.0758) for P. gonapodyides. A detailed sensitivity study of a large number of Phytophthora isolates is in progress.
Unravelling RNA helicases in Ustilago maydis and their role during teliospore dormancy and germination
M. SETO AND B. J. SAVILLE
Environmental and Life Sciences Graduate Program, Trent University, 1600 West Bank Drive, Peterborough, ON K9L 0 G2, Canada; and (B.J.S.) Forensic Science Program, Trent University, 2140 East Bank Drive, DNA Building, Peterborough, ON K9L 0 G2, Canada
The emergence of fungal diseases is a major threat to sustainable crop production. Fungal pathogens can persist in the environment by forming resistant spores. As such, spore dormancy, dispersal, and germination are integral to the spread of fungal diseases. We use Ustilago maydis D.C. Corda as a model to study molecular aspects of teliospore germination. Teliospores are thick-walled, dormant, diploid cells that germinate and complete meiosis to initiate new rounds of infection. Early fungal spore studies indicated that all the necessary components required for germination, including preformed messenger RNAs (mRNAs), are stored in the spore during dormancy. We hypothesize that some stored mRNAs are stabilized in the dormant teliospore through double-stranded RNA (dsRNA) formation and are unwound by RNA helicases during germination to be made available for translation. RNA helicases are highly conserved enzymes that are capable of forming RNA clamps, unwinding RNA-RNA duplexes, and displacing proteins. We identified all RNA helicases in U. maydis and determined, by RNA seq, that five RNA helicases had elevated transcript levels in dormant teliospores and that these levels decreased during germination. Deletion strains were created, and their phenotypes were assessed to gain insight into the function of two RNA helicases. The information gained from this study enhances our understanding of fungal spore germination, which may lead to the development of novel disease control measures.
Sporulation potential of Phytophthora ramorum EU2 lineage on selected Canadian broadleaf and conifer tree species
S. F. SHAMOUN, G. SUMAMPONG, A. ISLAM AND M. ELLIOTT
Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada; and (M.E.)Washington State University, Puyallup Research and Extension Centre, 2606 West Pioneer, Puyallup, WA 98371–4998, USA
The invasive alien pathogen Phytophthora ramorum (Oomycetes) causes sudden oak death (SOD) in forests and Ramorum blight in nurseries, resulting in mortality of several forest species in western North America and Europe. Our previous investigation results based on the assessment of 33 Canadian trees, shrubs and landscape plants which were reported in the 7th SOD Science and Management Proceedings 2020, showed a large variation in aggressiveness and sporulation potential among the evaluated hosts. Since the sporulation potential of P. ramorum drives the epidemic of SOD, the objective of this study was to determine the most efficient inoculum reservoir of an isolate from the P. ramorum EU2 lineage on a selected sub-set of 11 tree species including red alder (Alnus rubra), bigleaf maple (Acer macrophyllum), California bay laurel (Umbellularia californica), sugar maple (Acer saccharum), vine maple (Acer circinatum), Himalayan blackberry shrub (Rubus armeniacus), western hemlock (Tsuga heterophylla), western larch (Larix occidentalis), white spruce (Picea glauca), Sitka spruce (Picea sitchensis), and Japanese larch (Larix kaempferi). Detached leaves/needles of five conifer and six broadleaf species were inoculated with P. ramorum EU2 lineage mycelia isolated from a stream bait near an infected larch plantation in Scotland, UK. Of the 11 plants tested, red alder (Alnus rubra) had sporulation potential comparable to California bay laurel (Umbellularia californica) which drives the SOD epidemic in California forests. Sitka spruce (Picea sitchensis) and sugar maple (Acer saccharum) had moderate levels of sporulation. These results confirm the potential threat of the EU2 lineage of P. ramorum to Canadian flora.
Preharvest UV treatment a potential ecological approach for the control of Xanthomonas campestris pv. vitians induces key genes associated with homoeostasis, growth, and defence in lettuce
SIDIBÉ, M. T. CHARLES, O. NICOLAS AND C. BEAULIEU. (A.S., M.T.C., C.B.)
Department of Biology, Université de Sherbrooke, 2500 Boulevard de l’Université, Sherbrooke, QC J1K 2R1, Canada; (A.S., M.T.C.) Saint‐Jean‐sur‐Richelieu Research and Development Centre, Agriculture and Agri‐Food Canada (AAFC), 430 Boulevard Gouin, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada; and (O.N.) Harrow Research and Development Centre, AAFC, 2585 County Road 20, Harrow, ON N0R 1 G0, Canada
Lettuce (Lactuca sativa L.) is one of the most widely consumed leafy vegetables in the world. Its susceptibility to bacterial leaf spot (BLS) caused by Xanthomonas campestris pv. vitians (Xcv) can result in 100% yield losses. Despite the excessive and repeated use of pesticides, control of this disease remains difficult. Consumers, concerned about human health and environmental protection, denounce the presence of residues on horticultural products and the abusive use of pesticides in agriculture. The search for new and effective biological approaches is needed to improve the control of BLS. In the present study, a series of independent trials were conducted to evaluate the repeated use of a hormetic dose of 0.4 kJ/m2 of UV-C radiation to control Xcv and to assess the impact of this treatment on lettuce yield. This study showed a 30–50% reduction in lettuce susceptibility to BLS as a function of the number of cycles of UV-C treatment without negative effects on agronomic characteristics and with an increase in total mineral concentrations, shelf life, and dry mass. Data revealed differential expression of genes associated with homoeostasis, growth, and defence. We conclude that UV-C hormesis, under the conditions described in the present study, is an effective eustress that will not interfere with normal growth or the ability of plants to defend themselves against other potential stressors. The knowledge gained from this work could promote pre-harvest UV-C hormesis as an environmentally friendly approach in the implementation of an integrated pest management programme.
Fungal pathogen emergence: an Ustilago maydis x Sporisorium reilianum model
E. R. M STORFIE AND B. J. SAVILLE. (B.J.S.)
Environmental and Life Sciences Graduate Program, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada; and (E.R.M.S., B.J.S.) Forensic Science Program, Trent University, 2140 East Bank Drive, Peterborough, ON K9L 0 G2, Canada
The emergence of fungal hybrid pathogens threatens sustainable crop production worldwide. To investigate hybridization, the related smut fungi, Ustilago maydis and Sporisorium reilianum, were selected, because they infect a common host (Zea mays), can hybridize, and there are tools available for their analysis. Hybrid dikaryons demonstrated filamentous growth on plates, similar to U. maydis dikaryons, but showed reduced virulence and colonization of Z. mays compared to both parental dikaryons. To understand the hybrid’s limited pathogenic development, select virulence gene transcript levels were assessed during pathogenic time courses, using RT-PCR and/or RT-qPCR with species-specific primers. These genes included transcription factors, cell signalling proteins, and effectors characterized in U. maydis, as well as their orthologs in S. reilianum. During the hybrid infection, these virulence genes had altered transcript levels relative to the parental dikaryon infections. Genes involved in late development were either downregulated or not detected in the hybrid, suggesting that the hybrid’s pathogenic development had stalled. To determine if hybrid pathogenesis could be altered, characterized U. maydis virulence genes were constitutively expressed in the U. maydis nucleus of the hybrid. Little impact was observed in hybrids with increased expression of tin2, pit2, and cce1 effectors, which are known to modify host response. However, increased expression of rbf1 and hdp2, transcription factors that are known to regulate early-stage specific pathogenic development, increased the hybrid’s ability to infect and induce symptoms, including the rare induction of small leaf tumours. These results establish a base for investigating molecular aspects of fungal hybrid pathogen emergence.
Fungicide sensitivity of Stemphylium vesicarium in Ontario
S. M. STRICKER, E. MCFAUL, B. D. GOSSEN AND M. R. MCDONALD
Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (B.D.G.) Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada
Stemphylium leaf blight (SLB), caused by Stemphylium vesicarium (Wallr.) E. G. Simmons, has become an important disease of onion in Ontario, Canada and north-eastern USA in recent years. Commercial cultivars are highly susceptible, so growers apply foliar fungicides at 10–14-day intervals to manage the disease. However, the efficacy of several fungicides has declined over time. Isolates of S. vesicarium collected in southern Ontario from onion, asparagus, and leek in 2012–2020 were assessed for sensitivity to the technical grade active ingredients of commonly used fungicides. Two isolates collected from oat in Saskatchewan in 1995 provided an historical baseline. The oat isolates were sensitive to azoxystrobin (FRAC 11), fluopyram (FRAC 7) and difenoconazole (FRAC 3), but insensitive to pyrimethanil (FRAC 9). Sensitivity to azoxystrobin was present in isolates collected from asparagus in 2012, but the population trended towards insensitivity by 2016, with 97% insensitive in 2020. Isolates from 2012 were sensitive to fluopyram, but 37% of the 95 isolates collected in 2018 and 2019 and 100% of 30 isolates in 2020 were insensitive. Most isolates (99%) were sensitive to difenoconazole based on a mycelial growth assessment, but conidial germination was not inhibited even a high rate (100 ppm). For pyrimethanil, 94% of isolates from 2018 and 2019 were insensitive. These active ingredients, which have been and are being used on onion in Ontario, no longer provide effective SLB suppression. Identification of alternative management strategies is required.
Genetic stability analysis of stem solidness and grain yield in spring wheat
M. SUBEDI, H. A. CÁRCAMO, J. J. KNODEL, D. K. WEAVER, R. D. CUTHBERT, C. J. POZNIAK, K. T. NILSEN AND B. L. BERES
Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 5403–1st Avenue South, Lethbridge, AB T1J 4B1, Canada; (J.J.K.) Extension Entomology, 210 Walster Hall, P.O. Box 6050, North Dakota State University, Fargo, ND 58108–6050, USA; (D.K.W.) Department of Land Resources and Environmental Sciences, 334 Leon Johnson Hall, Montana State University, Bozeman, MT 59971, USA; (R.D.C.) Swift Current Research and Development Centre, AAFC, P.O. Box 1030, 1 Airport Road, Swift Current, SK S9H 3X2, Canada; (C.J.P.) Crop Development Centre, Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada; and (K.T.N.) Brandon Research and Development Centre, AAFC, P.O. Box 1000A, 2701 Grand Valley Road, Brandon, MB R7A 5Y3, Canada
The wheat stem sawfly, Cephus cinctus Norton (Hymenoptera: Cephidae), is a major pest of wheat (Triticum aestivum L.) in the northern Great Plains where it is a constant threat in Montana and is resurging in Alberta, Saskatchewan and North Dakota. Adoption of solid-stemmed cultivars is an important management tool for wheat growers; however, the inconsistent pith expression first noted with the release of ‘Rescue’ has been repeatedly observed in modern cultivars in Canada. Given the extensive hectares planted to solid-stemmed wheat cultivars during an outbreak, identification of cultivars that display stable stem solidness and grain yield across a wide range of environments where stem sawfly infestations occur is desirable. We assessed eight solid-stemmed and two hollow-stemmed spring wheat genotypes grown across diverse environments for their response in stem solidness and yield using multiple statistical models. Study sites included southern Alberta and Saskatchewan, Montana and North Dakota. Most models agreed that the genotypes, ‘Choteau’ ‘BW925ʹ and ‘Mott’ consistently displayed high and stable stem solidness concomitant with high grain yield. ‘Choteau’ and ‘BW925ʹ also consistently met or exceeded the desired threshold of a 3.75/5 pith rating for optimum resistance whereas, ‘Mott’ developed optimal pith at a specific (early) phenological stage when resistance to WSS infestation is critical. The identified stable and ideal genotypes would be useful to enhance germplasm development, production and adoption of spring wheat cultivars in the WSS prone regions.
Delineating the SAR pathway in common hexaploid wheat
S. SUN, R. LINNING, Y. ZHANG, C. HERRFURTH, I. FEUSSNER AND G. BAKKEREN. (S.S., Y.Z., G.B.)
The University of British Columbia, Department of Botany, 6270 University Blvd, Vancouver, BC V6T 1Z4, Canada; (S.S., R.L., G.B.) Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97 South, Summerland, BC V0H 1Z0, Canada; and (C.H., I.F.) Georg-August University, Plant Biochemistry, Goettingen, D-37077, Germany
In response to biotic stressors, plants mobilize metabolic compounds in defence. Local threats can quickly lead to a robust, whole-plant immune response, or systemic acquired resistance (SAR). A L-lysine catabolic pathway was recently characterized in Arabidopsis, where the metabolites Pipecolic acid (Pip) and N-hydroxylated pipecolic acid (NHP), produced by the reductase SAR-Deficient 4 (AtSARD4) and Flavin-monooxygenase 1 (AtFMO1), respectively, are crucial for proper SAR establishment. While recent studies implicate NHP in pathogen defence across angiosperms, the functional characterization of SARD4 and FMO1, and their participation in SAR in common hexaploid wheat (Triticum aestivum), is lacking. Here, we employed a reverse-genetics approach to identify functional orthologs of AtSARD4 and AtFMO1 in wheat. Gene and protein homology searches and transcriptome analyses yielded 48 TaFMO1 and 3 TaSARD4 candidates. Representatives were selected and two TaFMO1 candidates were successfully expressed in a SAR-deficient Arabidopsis fmo1 deletion mutant. These transgenic TaFMO1 Arabidopsis lines revealed a potential partial recovery in SAR to oomycete Hyaloperonospora arabidopsidis, indicating possible functional complementation. Similarly, TaSARD4-expressing transgenic Δsard4 Arabidopsis lines are being generated. Preliminary trials of wheat supplemented with 1 mM Pip and 10 mM NHP significantly enhanced wheat resistance to two fungal pathogens: Blumeria graminis f.sp. tritici (powdery mildew) and Puccinia triticina (wheat leaf rust). Evaluating levels of Pip and NHP in the transgenic Arabidopsis lines and in wheat when challenged by pathogens is in-progress using HPLC-MS. We are exploiting the vast knowledge emanating from Arabidopsis research to draw parallels with wheat, as to improve disease resistance in a major crop plant.
Fusarium head blight (FHB) and deoxynivalenol (DON) index for Ontario commercially grown winter wheat
L. TAMBURIC-ILINCIC AND E. SPARRY
University of Guelph, Ridgetown Campus, 120 Main Street East, Ridgetown, ON N0P2C0, Canada; and (E.S.) C&M Seeds, 6150 5th Line, Palmerston, ON 0 G2P0, Canada
Fusarium head blight (FHB) is a serious disease of wheat and deoxynivalenol (DON) is the most common mycotoxin produced by Fusarium graminearum (FG). All wheat commercially grown in Ontario is entered in the Performance Trials and tested for agronomic traits and for FHB resistance in nurseries inoculated with FG and mist irrigated. The FHB nursery at Ridgetown, Ontario was established in 1996. FHB index and DON level information from 2007 is available on www.gocereals.ca. The lowest (4.4%) and highest (50.2%) mean FHB index was recorded in 2018 and 2013, respectively, while the lowest (0.2 ppm) and highest (22.1 ppm) mean DON level was recorded in 2020 and 2013, respectively. Wheat cultivars are grouped based on FHB visual symptoms and DON level, using historical and the most recent data. Categories include moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS). The Ontario Cereal Crop Committee (OCCC) checks are: MR (‘Marker’, ‘AC Morley’ and ‘Ava’), MS (‘CM614ʹ, ‘25 R46ʹ, ‘Princeton’ and ‘Priesley‘), S/HS (‘DS572SRW’, ‘25R40ʹ and ‘AC Sampson‘). During FHB epidemic years in Ontario, high DON level was recorded in S/HS cultivars with some cultivars differing in category for FHB index and DON level. For example, ‘UGRC Ring’ is in MS category for FHB symptoms, and in MR category for DON level. The OCCC activities facilitates screening of new cultivars, with increased level of FHB resistance and higher yield. Importantly to encourage cultivation of the most promising new cultivars, yield, agronomic and FHB/DON information is available to growers in Ontario.
A single base extension assay for pathotyping clubroot [Plasmodiophora brassicae] of canola
H. H. TSO, L. GALINDO-GONZÁLEZ AND S. E. STRELKOV
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada
Clubroot, caused by the soilborne pathogen Plasmodiophora brassicae, is one of the most detrimental diseases of canola (Brassica napus) in Canada. Several host differential sets have been developed for pathotype identification, including the Canadian Clubroot Differential (CCD) set. However, pathotyping based on the reaction of host differentials is time-consuming, labour-intensive, and requires biosecure greenhouse facilities. To facilitate rapid pathotype identification, a highly specific assay was developed to detect clusters corresponding to P. brassicae pathotypes 3 H and 5X. This assay is based on SNaPshot, a single base extension (SBE) reaction. Primers are designed upstream of the discriminating polymorphic base and extended by one base with fluorescently labelled dideoxynucleotides (ddNTPs) when the respective template is in the mix. A conventional polymerase chain reaction (PCR) primer pair was designed to produce the 304 base pair amplicon to be used as template for the SBE reaction, and a SNaPshot primer was designed for pathotyping. The SNaPshot primer was optimized on individual templates from single-spore isolates providing the expected base resolution, and then assessed for its limit of detection in template proportions of 10:90, 20:80, 30:70, 40:60, and 50:50. Additional testing was carried out on DNA extracted from canola root galls collected from the field to evaluate the sensitivity of the assay against field isolates. The assay was able to detect a 10% relative allelic proportion in a 10:90 template mixture with both single-spore isolates and field isolates. The application of this technique appears promising for large-scale SNP pathotyping to produce highly sensitive results.
Modern breeding strategies assist development of disease-resistant, two-row malting barley adapted for production in western Canada
J. R. TUCKER, C. W. HIEBERT, W. XU, A. BADEA AND W. G. D. FERNANDO
Brandon Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 2701 Grand Valley Road, Brandon, MB R7A 5Y3 Canada; (J.R.T., W.G.D.F.) Department of Plant Science, University of Manitoba, 66 Dafoe Road, Winnipeg, MB R3T 2N2 Canada; and (C.W.H., W.X.) Morden Research and Development Centre, AAFC, 101 Route 100 Unit 100, Morden, MB R6M 1Y5, Canada
Barley (Hordeum vulgare L.) is a versatile crop used primarily for animal feed or malt production, and is of major economic importance to western Canadian farmers. However, barley is a common host to numerous plant pathogens, which impose constraints on production that can affect both yield and grain quality. Plant breeding is a successful approach for disease management in barley, which minimizes reliance on pesticides, helps Canada’s approach for IPM and sustainability. Molecular marker assisted selection for rpg4/Rpg5 gene complex has supported development of elite germplasm with resistance to virulent races (Ug99) of stem rust (Puccinia graminis f. sp. tritici). Advances in Genomic sciences have supported new approaches that exploit genome-wide markers for genomic prediction and enhancement of resistance to fusarium head blight (Fusarium graminrearum) and low deoxynivalenol accumulation. With the recent release of the first reference genome of a Canadian malting cultivar ‘AAC Synergy’, it will be possible to investigate unique foliar disease resistance mechanisms for further use in developing improved germplasm. Accelerated development of disease-resistant cultivars will help maintain competitiveness of barley in face of evolving pathogens.
Integration of cover crop programmes in organic soils to improve soil health in the Holland/Bradford Marsh
K. VANDER KOOI, D. VAN DYK, T. BLAUEL AND M. R. MCDONALD
University of Guelph, Department of Plant Agriculture, Ontario Crops Research Centre – Bradford, 1125 Woodchoppers Lane, King, ON L7B 0E9, Canada; and (D.V.) Ontario Ministry of Agriculture Food and Rural Affairs, 1 Stone Road West, Guelph, ON N1G 2W1, Canada
The Bradford-Holland Marsh has high organic matter soil (45–80%) that is ideal for vegetable production. The two main crops, onions and carrots, are harvested from August to November and there is a need for cover crops to protect the soil after harvest. The objectives of this project were to identify cover crops to follow onion and carrot harvest and to investigate biofumigant crops to improve soil health and reduce soilborne plant pathogens. Onions and carrots were mechanically transplanted or direct seeded, grown to maturity and mechanically harvested. Cover crops were seeded following harvest for both crops. Diakon radish, barley, a Nitro Mix, fumigant mustards Caliente 199 and Caliente Rojo were used. Soil analysis was conducted before and after biofumigant crops to assess the effects on plant pathogens. Soil coverage and biomass of the cover crops was measured. All species of cover crops that followed onions grew well. Canopy coverage was good for all except Caliente Rojo. In carrots, interseeded oats, radish and forage pea grew under and in-between carrot rows. Barley, seeded prior to, or drilled following carrot harvest, was also able to establish before freezing in late fall. In the biofumigant trial, all crops had good canopy coverage except for Caliente 199. A DNA multiscan showed few differences in the levels of soilborne plant pathogens between the two fumigation crops. Research is continuing to investigate full season cover crops on organic soils and identify good fall cover crops for use after onions and carrots.
Impact of blackleg [Leptosphaeria maculans] on canola yield
Y. WANG, S. E. STRELKOV AND S. F. HWANG
Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB T6G2P5, Canada
Blackleg, caused by Leptosphaeria maculans, is an economically important disease of canola (Brassica napus) worldwide. Severe epidemics can result in yield losses of 30–50% and total crop failure can occur when susceptible canola cultivars are grown. In western Canada, blackleg have been widely observed, while growers plant blackleg resistant cultivars to manage the disease. In order to establish the relationship between blackleg severity and the yield of blackleg-resistant canola hybrids, field experiments were conducted over 2 years in four canola hybrids, ‘1950 RR’, ‘45H29ʹ, ‘45H31ʹ and ‘CS2000ʹ. Adult plants were assessed for blackleg disease on a 0 to 5 scale, where 0 indicates a completely healthy plant and 5 indicates plant death. The average disease severity on the inoculated canola ‘1950 RR’, ‘45H29ʹ, ‘45H31ʹ and ‘CS2000ʹ over 2 years was 1.4, 1.6, 1.9 and 1.1, respectively. Seed yield in non-inoculated plots were higher than inoculated plots for all four canola hybrids. Regression analysis indicated that there were polynomial relationships between blackleg severity and pod number and seed yield. Plants with a blackleg severity of 0 had a slightly lower seed yield loss than plants with a severity of 1 for ‘1950RR’, ‘45H29ʹ and ‘CS2000ʹ, while as disease severity increased to 2–5, yields began to decline. Seed yield decreased as disease severity increased from 0 to 5 for ‘45H31ʹ. The results suggest the potential for significant losses in blackleg-resistant canola hybrids, and integrated disease management strategies should be employed to mitigate the impact of L. maculans on canola.
Novel mutations in the CYP51 gene in a global collection of Pyrenophora tritici-repentis (tan spot of wheat)
B. WEI, M. HAFEZ, R. GOURLIE, S. E. STRELKOV AND R. ABOUKHADDOUR. (B.W., M.H., R.G., R.A.)
Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403 1 Avenue South, Lethbridge, AB T1J 4B1, Canada; and (B.W., S.E.S.) Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada
Sterol demethylation inhibitors (DMIs) are a group of site-specific fungicides applied to control fungal plant pathogens. The target site of all DMIs is CYP51, belongs to the cytochrome P450 monooxygenase superfamily, an essential enzyme for the biosynthesis of fungal sterols. Mutations in CYP51 are the most common mechanism by which fungi evolve tolerance to DMIs. In this study, mutations in the CYP51 gene were explored in a global collection of 36 isolates of Pyrenophora tritici-repentis, the pathogen causing tan spot of wheat. The mutations at the DNA and protein levels were determined. The results showed that CYP51 occurred as two gene copies in the tested isolates, the first copy was most common and found in 33 isolates and the second copy was only found in three isolates with over 60% similarity. Four different haplotypes of CYP51 were identified (H1- H4). Haplotype H1 was the most common and found in 30 of the isolates. In contrast, the two non-pathogenic isolates and a weakly virulent race 5 isolate from Canada all carried different haplotypes. Eight non-synonymous mutations were identified, but none were identical to previously reported mutations. The non-pathogenic isolates were collected from grasses and likely were not subjected fungicide application. Further work to estimate the tolerance to DMIs in relation to these CYP51 mutations is P. tritici-repentis is needed.
Effects of low temperature stress on physiological and biochemical processes of silage-corn genotypes
J. WU, M. NADEEM, D. SVESHNIKOV, R. THOMAS, L. GALAGEDARA AND M. CHEEMA
School of Science and the Environment, Memorial University of Newfoundland, Corner Brook, NL A2H 5G4, Canada
Low temperature stress hampers seedling establishment, growth and productivity of crops. However, low temperature stress tolerance varies within crop species and genotypes. In this study, we conducted a growth chamber experiment to determine the effects of low temperature regime on the seedling establishment, physiological and biochemical parameters of three silage corn genotypes. Experimental treatments were five temperature regimes including 25°C (control), 20°C, 15°C, 10°C and 5°C and three genotypes (Yukon-R, DKC26-28RIB, A4177G3-RIB) with different genetic traits. The experiment was set up in a completely randomized design in split-plot arrangement with three replications. Low temperature stress was imposed at the third leaf stage for 5 days, and then physiological and biochemical parameters were recorded. Results depicted that silage-corn vegetative growth, photosynthetic system and redox homoeostasis were affected when seedlings were exposed to 15°C or lower temperatures. Silage-corn genotypes Yukon-R and DKC26-28RIB expressed significantly (p < 0.05) higher agronomic performance than A4177G3-RIB, particularly root development. Similarly, Yukon-R and DKC26-28RIB showed minimum stress-induced malonaldehyde and hydrogen peroxide content. However, photosynthesis rate and photochemical efficiency (Fv/Fm) were higher in Yukon-R and DKC26-28RIB compared to A4177G3-RIB under low temperature conditions. Taken together, our data suggested that Yukon-R and DKC26-28RIB have better adaptation and low temperature stress tolerance, which could be considered as the candidate genotypes to be grown in boreal climate. The outcome of this research will further enhance our insights in understanding low temperature stress tolerance mechanisms of silage-corn and designing future field experiments to enhance forage production in boreal climates.
Identification of quantitative trait loci associated with partial resistance to Fusarium root rot and wilt caused by Fusarium graminearum in field pea
L. F. WU, R. FREDUA-AGYEMAN, S. F. HWANG AND S. E. STRELKOV
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada
Fusarium root rot, caused by a complex of Fusarium spp., is a major disease affecting field pea production. While the development of genetic resistance represents one of the most promising approaches for root rot management, no sources of complete resistance have been identified. The aim of this study was to detect quantitative trait loci (QTL) conferring partial resistance to root rot and wilting caused by Fusarium graminearum, a major component of this pathogen complex. An F8 recombinant inbred line (RIL) population consisting of 129 individuals derived from the cross ‘Reward’ (root rot susceptible) × ‘00–2067ʹ (root rot tolerant) was inoculated with F. graminearum under various greenhouse conditions. Plants were assessed for root rot and wilt severity in the fourth week after inoculation. The F8 population was genotyped using 13.2K single nucleotide polymorphisms (SNPs) and 212 simple sequence repeat (SSR) markers evenly distributed on seven pea chromosomes. A significant genotypic effect (P < 0.05) and high heritability (92.1%) indicated that a high proportion of the genetic variance was transmitted to the progeny. Seven QTL for root rot severity were detected on Linkage Group (LG) I, III, IV and V, including four stable QTL identified in more than one environment. The two largest stable QTL, Fg-Ps3.1 and Fg-Ps3.2, were located on LG III and explained 6.56–12.75% and 8.18–13.24% of the phenotypic variance, respectively. The flanking markers may contribute to marker-assisted selection in breeding programmes for partial resistance to F. graminearum.
Virulence structure of wheat powdery mildew population and effective resistance genes for Ontario in 2018 and 2019
G. XUE, S. LIM, Y. CHEN, G. HUMPHREYS, W. CAO, J. MENZIES, C. COWGER, H. LI AND M. SERAJAZARI
Ottawa Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; (J.M.) Morden Research and Development Centre, AAFC, 101 Route 100, Morden MB R6M 1Y5, Canada; (C.C.) USDA-ARS, Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695, USA; (H.L.) National Engineering Laboratory for Crop Molecular Breeding, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100 081, China; and (M.S.) Plant Agriculture, University of Guelph, Guelph, ON N1G 2W1, Canada
Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a major disease of wheat (Triticum aestivum) in Ontario, which can cause up to 20% yield loss. The development of resistant commercial wheat cultivars is the most economical means of controlling this disease, but only if the resistance genes used are incompatible with the virulence phenotypes present in the pathogen population. The virulence structure of Bgt in Ontario was examined in 2018 and 2019. Of the 42 single colony isolates collected in Ontario greenhouses and commercial fields, 40 virulence phenotypes, assigned as VP1 to VP40, were identified on a set of 24 single-gene differential genotypes. Of the 24 resistance genes possessed by the differential genotypes, eight genes, namely, Pm1a, Pm1b, Pm1c, Pm12, Pm16, Pm21, Pm37, and MlAG12 were effective against all of the Bgt isolates. Four genes, Pm3d, Pm29, Pm34, and NCAG13, were mostly effective, with resistance reactions to more than 80% of the isolates. No significant differences in the numbers of virulence genes per isolate were found between the 2 years or between the greenhouse and field origins. The virulence frequencies of Bgt isolates for these effective and most effective genes were not significantly affected by the year of collection or their origin, suggesting that the Bgt population is relatively stable. The effective genes identified in this study may be deployed singly or used for gene pyramiding to develop powdery mildew-resistant cultivars in Ontario.
The regulation of intrinsic signalling in Brassica napus defending against Leptosphaeria maculans
C. YANG AND W. G. D. FERNANDO
Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Plants are able to trigger multiple signalling pathways to cope with pathogenic invasion. Gene-for-gene interaction, one of the effective mechanisms, is triggered by the interaction between pathogenic Avr effectors from the pathogen and plant R proteins. Plant tissues induce distinct activities when they have, or have no HR, and those differences help to find out the factors in efficient defence against plant pathogens. An excellent pathosystem to investigate this is the Brassica napus – Leptosphaeria maculans pathosystem. Three cultivars of B. napus, Westar, Surpass400 and 01–23-2-1 that are susceptible, intermediate-resistant and resistant respectively, were inoculated by two L. maculans isolates (HCRT75 8–1 (Genotype: avrLm1, AvrLm2, avrLm3, avrLm4, AvrLmJ1-5, AvrLm7, AvrLm6, avrLm9, AvrLm11, avrLepR1 and AvrLepR2) and HCRT77 7–2 (Genotype: AvrLm1, avrLm2, avrLm3, AvrLm4, AvrLmJ1-5, AvrLm7, AvrLm6, avrLm9, AvrLm11, avrLepR1 and avrLepR2)) to cause three distinct levels of severity: susceptible, intermediate and resistant. Expression studies using RT-qPCR, histochemical assays (such as trypan blue staining) were applied on the cotyledons of those cultivars to search the differences in defence response from those cultivars (with distinct severities). Histochemical assays suggested that the intermediate and resistant genotypes (i.e. Surpass400 and 01–23-2-1) displayed earlier H2O2 accumulation and cell death on the cotyledons. Transcriptional analysis (RT – qPCR) also observed that the genes related to salicylic acid (SA) and ROS were activated from the two genotypes as early as 3 and 5 dpi. The results indicated that the early activation of SA/ROS signalling is one of the crucial components for B. napus to defend against L. maculans.
Effect of Brassica napus seedling age at time of inoculation on fusarium wilt severity
C. X. YANG, S. F. HWANG, R. FREDUA-AGYEMAN AND S. E. STRELKOV
Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB T6G 2P5, Canada
Fusarium wilt is a widespread vascular fungal disease caused by the soilborne pathogen Fusarium oxysporum. In Canada, fusarium wilt was first reported on canola (oilseed rape; Brassica napus) in 1999 in northeast Alberta, becoming more severe after 2002. At present, fusarium wilt is managed mainly by the deployment of resistant cultivars, but has the potential to re-emerge as a destructive disease of canola on the Prairies. The objective of this study was to evaluate the effect of seedling age at the time of inoculation on fusarium wilt severity. Seedlings of the B. napus cultivars ‘Westar’ and ‘Mendel’ were inoculated at different stages (7, 14 and 21 days old) under greenhouse conditions. Fusarium wilt severity was assessed on a 0–5 scale, where: 0 = no symptoms, 1 = mild chlorosis of one leaf, 2 = moderate chlorosis and stunting of one to two leaves; 3 = severe chlorosis (> 50% of the plant), some necrosis and moderate stunting and wilting, 4 = severe necrosis, wilting and stunting of the whole plant, and5 = dead plant. The disease severity data were analyzed using a mixed-effect linear model with estimated marginal means, which showed that seedlings inoculated at 21 days old developed the highest disease severity, while those inoculated at 7 days had the lowest disease severity for both cultivars. These results suggest that fusarium wilt is more severe when older seedlings are inoculated with F. oxyporum.
Greenhouse and field evaluation of amisulbrom for clubroot management
Z. YU, S. F. HWANG AND S. E. STRELKOV
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada
Clubroot, caused by Plasmodiophora brassicae, is an important constraint to Canadian canola (Brassica napus) production. The fungicide amisulbrom, a quinone inside inhibitor (QiI), was evaluated for effectiveness as a clubroot management tool. Liquid (GWN10440, 1000 g active ingredient (ai) ha−1) and fertilizer formulations (GWN10750, 700 g ai ha−1; GWN10751, 1000 g ai ha−1; GWN10752, 1500 g ai ha−1) of amisulbrom were compared on clubroot susceptible and moderately resistant canola hybrids under greenhouse conditions, and in field trials conducted in Edmonton in 2019 and 2020. No significant differences were observed among the amisulbrom treatments in the field in 2019, possibly due to excessive raining. In 2020, treatment with amisulbrom resulted in significant reductions in the clubroot disease severity index (DSI) in the field; the best control (nearly 60% reduction in DSI) was obtained with GWN10752 and GWN10440. Under greenhouse conditions, the treatments were tested at low (1 × 105 resting spores g−1 soil) and high (1 × 107 resting spores g−1 soil) inoculum pressure. Clubroot severity decreased with increasing amisulbrom rate for all fertilizer formulations, with a DSI < 20% observed in both cultivars following treatment with GWN10752 at the low inoculum level. The greatest reductions in DSI were achieved with the liquid formulation GWN10440 at both the low and high spore concentrations. While both liquid and fertilizer formulations of amisulbrom show promise as part of an integrated clubroot management strategy, the fertilizer form may be more suitable for Canadian canola growers given the costs of equipment, time and labour.
Applications of deep learning methods to genomic prediction in breeding wheat for fusarium head blight resistance
L. ZHAO AND W. G. D. FERNANDO
Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Fusarium head blight (FHB) is a major wheat disease resulting in reduced yield and mycotoxin-contaminated grain. Fusarium graminearum, by producing several trichothecene mycotoxins, is considered as the major aetiological agent to wheat FHB. Wheat resistance to FHB is a quantitative trait which is controlled by many unique quantitative trait locus [QTLs; (Bai et al. (2018) CJPP 40:336–346)]. Such a complex trait presents a huge breeding challenge with traditional marker-assisted selection (MAS) which could only employ a limited number of markers. To overcome the limitation of MAS, the genomic prediction (GP)-based breeding strategy could include all available markers to facilitate germplasm screening and increase the accuracy in predicting phenotype. However, GP frequently faces statistical challenges given the unbalanced number between markers and plant individuals. Traditional GP linear models such as genomic best linear unbiased prediction (GBLUP), Bayesian GP family, or least absolute shrinkage and selection operator (LASSO) have made considerable achievements in GP but still hard to fully exploit hidden non-additive information from markers. In contrast, deep learning (DL) models for GP could include all non-additive effects and are capable of extracting complex features to make predictions. This is an interesting and promising strategy for polyploid plants. However, this method has not been widely applied in plant disease resistance identification, especially for wheat FHB. In this presentation, we will review the application of DL methods such as Convolutional neural network (CNN) and Transformers in conducting crop GP. With these experiences, a general DL-based strategy is proposed for selecting wheat germplasm with FHB resistance.
Identification of Arabidopsis Phospholipase A knock-out mutants with increased susceptibility to Plasmodiophora brassicae
Q. ZHOU, K. JAYAWARDHANE, G. CHEN, S. F. HWANG AND S. E. STRELKOV
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada
Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most significant disease threats to canola (Brassica napus) production in Canada. The identification of novel genes that contribute to clubroot resistance is important for the sustainable management of this disease, as they may be used in the development of resistant canola cultivars. Phospholipase As (PLAs) play important roles in plant defence signalling and stress tolerance, and thus are attractive targets for crop breeding. However, since canola is an allopolyploid and has multiple copies of each PLA gene, it is time-consuming to test the functions of PLAs directly in this crop. In contrast, the model plant Arabidopsis thaliana has a simpler genetic background and only one copy of each PLA. Therefore, it would be reasonable and faster to validate the potential utility of PLA genes in Arabidopsis first. In this study, we identified seven homozygous T-DNA insertion pla knockout Arabidopsis mutants, and tested their performance following inoculation with P. brassicae. Five mutants (pla1-IIα, pla1-Iγ3, pla1-III, spla2-α, pla-IIIβ, pla-IIIδ) developed more severe clubroot than the wide-type, suggesting increased susceptibility to P. brassicae. The homologs of these AtPLAs in B. napus were identified through blast tools and phylogenic analysis. BnPLAs expression profiles were subsequently examined in transcriptomic datasets generated from canola infected by P. brassicae, and promising candidates will be selected for further characterization in canola.
How do single and combined genes work in resistant breeding to northern corn leaf blight?
X. ZHU, L. M. REID, K. K. JINDA, T. WOLDMARIAM, J. WU, A. KEBEDE, C. VOLOACA, A. TENUTA AND D. HOOKER
Ottawa Research and Developmental Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; (A.T.) Ontario Ministry of Agriculture, Food and Rural Affairs, P.O. Box 400, Ridgetown, ON N0P 2C0, Canada; and (D.H.) Department of Plant Agriculture, University of Guelph, Ridgetown Campus, Ridgetown, ON N0P 2C0, Canada
Breeding resistant corn (Zea mays L.) is the best way to control northern corn leaf blight. From 2006 to 2014, Htm1 and Htn1 were introgressed into susceptible inbred CO388, Ht1, Ht2, Ht3, Htm1, and Htn1 (Ht(s) were introgressed into polygenic (PG) resistant inbred CO428 and got PGHt(s). Both CO388 and CO428 families crossed with A619 family (A619, A619 Ht1, A619Ht2, and A619Ht3) and other seven testers, including T3 with partial resistance. From 2015 to 2017, three experiments were designed: to assess single and combined gene effects to three disease and four yield traits under artificial inoculation; to assess their effects under different inoculation times; and to find higher Yield and stability crosses and inbreds for corn production. Ht1, Ht2 and Ht3 reduced number of lesions per leaf (NLPL), Lesion size (LS), and percent leaf area affected (PLAA) >30%, Htm1 and Htn1 > 20%. If both parents with resistant genes, (Htm1, Htn1)/(Ht1, Ht2, Ht3, Partial) and PGHt(s)/Ht(s) reduced NLPL, LS, and PLAA >50% and >39%, respectively. All single genes increased Yield, Kernel moisture, kernel number per ear and 100-kernel weight. Yield increased 7.5%, 21.1%, and 30.1% for females (Htm1 and Htn1), males (Ht1, Ht2, Ht3, and Partial), and their crosses, respectively. Additive action played a major role in most cases could be used to predict PLAA and yield. Linear regressions were found between four yield traits and PLAA. CO388 × A619-related resistant crosses had yield advantage >12% under twice inoculations. Three resistant crosses and four inbreds could be used in corn production.
Functional analysis of exopolygalacturonase genes in Verticillium dahliae during interaction with potato
X. ZHU, M. SAYARI AND F. DAAYF
Faculty of Agricultural and Food Sciences, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Verticillium dahliae is one of the most destructive plant pathogens, with the capability of infecting plant species in more than 200 families worldwide. Previous subtractive hybridization/cDNA-AFLP revealed higher level of expression of an exopolygalacturonase (ExoPG) gene (VDAG_03463) in highly than in weakly aggressive V. dahliae isolates. We aimed to check the effect of this gene in the pathogeneicity of V. dahliae. ExoPG transcripts increased dramatically in the highly aggressive isolate compared to the weakly aggressive one when treated with potato leaf or stem extracts. We used a split-marker technique to knock-out this gene in V. dahliae and generated six mutants. No changes in the pathogenicity or ExoPG activity was observed in the mutants compared to the wildtype. To investigated more, we identified eight polygalacturonase/pectinase-related genes in the genome of V. dahliae namely PGA, PGB, PGC, PGD, PEC1, PEC2, PEC3 and PEC4. Expression of PGA dramatically increased in the Δexopg mutant treated with different potato extracts, compared to the wild type. Except for PGA gene, similar expression trends of the above-mentioned genes were observed in the Δexopg mutant and weakly aggressive isolate when treated with different potato extracts. This indicates that other genes/pathways compensate the activity of the knocked out ExoPG gene in the Δexopg mutant. This is the first report of protoplast-based split marker method for transformation of V. dahliae.
Role of NOX genes in Verticillium dahliae’s pathogenicity
X. ZHU, M. SAYARI, M. R. ISLAM AND F. DAAYF
Faculty of Agricultural and Food Sciences, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; and (M.R.I.) Department of Plant Pathology, Faculty of Agriculture, Bangladesh Agricultural University, Bangladesh
NADPH oxidase (Nox) genes are crucial for the production Reactive Oxygen Species (ROS) and are important for many fungi’s pathogenicity. Genome investigation of Verticillium dahliae revealed the existence of three Nox-related genes namely NoxA, NoxB, and NoxC. Here we investigated the role of these genes in the pathogenicity of V. dahliae. In vitro expression of NoxA, NoxB, and NoxC showed significantly higher expression of NoxB in the highly than the weakly aggressive isolate of V. dahliae after treatment with different potato extracts. More transcripts of NoxA were detected in the weakly than the highly aggressive isolate in response to leaf and stem extracts. Significantly higher expression of NoxC was observed in the highly aggressive isolate treated with stem extracts. Single gene disruption mutants were generated for each of these genes to better understanding of the role of these genes in the pathogenicity of the highly aggressive isolates. Knocking out of both noxa and noxb genes, significantly decreased pathogenicity of the fungus, indicating the essential roles of these genes for V. dahliae against its hosts. The penetration ability of both noxa and noxb knock-out strains was drastically reduced, compared to the wild type. Gene complementation restored the pathogenicity as well as penetration capability of the fungus in both noxa and noxb mutants. Our data revealed that NoxA and NoxB genes play important roles in the pathogenicity of V. dahliae. These findings shed more light on the biology of V. dahliae and the mechanisms of its pathogenicity.