An update on an indexed microbial culture collection and preservation of plant pathogens derived from Alberta crops. H. AHMED, Y. YANG AND J. FENG. The Alberta Plant Health Lab, Crop Diversification Centre North, 17 507 Fort Road NW, Edmonton, AB T5Y 6H3, Canada
Preservation and maintenance of plant pathogens in pure culture with known identities in a living and stable state is vital for pathological research, and is of critical importance when diagnosing new or emerging plant pathogens, as well as for confirming specific diseases to enable the development of appropriate control strategies. A bacterial and fungal culture collection revitalization initiative has been undertaken in Alberta. Diseased samples from different crops collected during field surveys and sent to diagnostic laboratories by agricultural fieldmen or researchers during 2016–2020 were the principal sources for pathogen isolation. Standard protocols for each pathogen were used for isolation and purification. The microbial cultures were identified based on morphological characteristics and DNA barcoding. Some cultures were obtained from other Plant Pathology laboratories, including the Crop Diversification Centre South, Brooks, AB, the Canadian Collection of Fungal Cultures, Ottawa, ON, the University of Alberta, Edmonton, AB, 20/20 Seed Labs, Nisku and Edmonton, AB, and SGS Biovision Seed Research Ltd., Sherwood Park, AB. These cultures were stored for short periods on Potato Dextrose Agar plates at 4°C, and for the long term in a Pro-Lab DiagnosticsTM MicrobankTM Bacterial and Fungal Preservation System, or in 16% glycerol at −80°C. Ninety-four percent of the fungal cultures and all of the bacterial cultures were collected locally by the APHL’s Provincial Diagnostics Lab. Currently, 303 cultures, including 68 fungal and 21 bacterial plant pathogenic species are conserved and included in a database. These isolates are available on request free of cost to researchers in Alberta.
Building Ug99 resistance in Canadian winter wheat germplasm by stacking up stem rust resistance genes Sr22 and Sr24. M. H. J. CRADDUCK, M. FRICK, R. J. GRAF AND A. LAROCHE. Lethbridge Research Centre, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB T1J 4B1, Canada
Wheat (Triticum aestivum L.) stem rust is caused by the pathogen Puccinia graminis. The deployment of effective resistance genes in cultivars since the 1950’s has mostly controlled this pathogen. However, a new race of stem rust known as Ug99, discovered in Uganda in 1998, defeats many common resistance genes including Sr31 and Sr38. Ug99 has since spread to thirteen countries in Africa and the Middle East and has evolved into a family of 13 different races with virulence to resistance genes that were previously effective against the original race. With the majority of the world’s wheat cultivars susceptible and the potential threat of further spread of Ug99, effective resistance must be developed. Pyramiding resistance genes using marker assisted selection (MAS) is an effective strategy to produce cultivars with durable resistance to Ug99 and other stem rust races. For this study, MAS was used to identify experimental winter wheat germplasm lines with two genes of interest, Sr22 and Sr24. A multiplex PCR was performed using primers specific for Sr22 and Sr24 to identify the presence of these resistance genes. Preliminary results from a stem rust nursery in Kenya have shown the effectiveness of our approach. Lines rated as more resistant were found to match the lines that carry resistance gene Sr22 and to a lesser extent Sr24. The ability to pyramid these genes in our germplasm could provide enhanced resistance to Ug99 variants in the future.
Development of mutant wheat populations under selection pressure for Fusarium head blight resistance. C. DOVELL, D. RYABOVA, A. BADEA, R. GRAF, H. RANDHAWA, D. SPANER, P. HUCL, M. A. HENRIQUEZ AND N. A. FOROUD. Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 5403 1st Avenue South, Lethbridge, AB T1J 4B1, Canada; (A.B.) Brandon Research and Development Centre, AAFC, 2701 Grand Valley Road, P.O. Box 1000A, Rural Route #3, MB R7A 5Y3, Canada; (D.S.) Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada; (P.H.) Crop Development Centre, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada; and (M.A.H.) Morden Research and Development Centre, AAFC, 101 Route 100 Unit 100, MB R6M 1Y5, Canada
Fusarium head blight (FHB) is a disease that affects wheat and barley, among other cereals, in Canada. We have been working with Canadian breeders to develop FHB-resistant doubled haploid (DH) populations using an in vitro selection approach. In this method, Microspores are isolated from F1 hybrids provided by wheat and barley breeders, and doubled haploid plants produced in the presence or absence of Fusarium mycotoxins. This approach will be modified to develop an ethyl methanesulfonate (EMS) mutant population while developing DH plants from spring wheat cultivars with poor or moderate FHB resistance, in the presence or absence of the in vitro selection (IVS) pressure. Plants will be screened for changes in disease response, and by comparing the control group with the IVS group, we will clearer picture on the efficacy of the in vitro selection approach. Furthermore, a TILLING approach can be employed to identify the mutations that have incurred, enabling the identification of candidate resistance and susceptibility genes. Finally, the EMS population can also be used directly in wheat-breeding programmes for cultivar development.
Effect of synthetic antimicrobial peptides on common fungal pathogens and application to field pea (Pisum sativum). A. J. HANNIG, S. CHATTERTON, D. J. BING AND R. K. GOYAL. Lacombe Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada; and (S.C.) Lethbridge Research and Development Centre, AAFC, 5403 1st Avenue South, Lethbridge, AB T1J 4B1, Canada
Plant antimicrobial peptides (AMP’s) are short amino acid chains (< 100 amino acids) which form a part of innate immunity in living organisms and display antimicrobial activity against a wide range of bacterial and fungal pathogens. AMP’s can be identified from transcriptomic and genomic data and be designed using computer softwares. Synthetic AMP’s can be produced which have similar or improved activity compared to natural AMP’s. In this study, the effect of eight AMP’s (PBL-1 to PBL-8) were tested on 20 fungal pathogens including pathogenic ascomycetes (Fusarium spp.), basidiomycetes (Rhizoctonia sp.) and oomycetes (Aphanomyces sp.). AMP’s were tested at concentrations of 1, 10 and 50 uM against pathogens under in vitro conditions. A rating scale of 0 (no growth inhibition) to 5 (complete growth inhibition) was used to rate the effect of AMP on germination of fungal spores and mycelial growth relative to controls. In summary, PBL-1 and PBL-5 ranked the highest in inhibition rating towards pathogens tested. The AMP’s having potent activity can be developed as bio-fungicides for foliar applications.
Impact of location and environmental factors on pulse, cereal and oilseed diseases in the Canadian prairies. M. HUBBARD, G. PENG, M. ENTZ, G. SEMACH, H. KUBOTA, B. TIDEMANN, F. LARNEY AND K. LIU. Swift Current Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 1 Airport Road, P.O. Box 1030, Swift Current, SK S9H 3X2, Canada; (P.G.) Saskatoon Research and Development Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (M.E.) Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; (G.S.) Beaverlodge Research Farm, AAFC, P.O. Box 29, Beaverlodge, AB T0H 0C0, Canada; (H.K., B.T.) Lacombe Research and Development Centre, AAFC, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada; and (F.L.) Lethbridge Research and Development Centre, AAFC, 5403 1st Avenue South, Lethbridge, AB T1J 4B1, Canada
Environmental and soil factors, including precipitation and soil pH, often affect the occurrence and severity of diseases in field crops. A multiple site-year experiment was established in the Canadian Prairies to assess the productivity, disease severity, pest incidence, soil health, resource use efficiency, environmental impact, and resilience of cropping systems. In 2018 and 2019, data on several important diseases of major field crops were collected at the seven sites in Alberta, Saskatchewan and Manitoba. Root rot of pulses, ascochyta blight of chickpea and pea, anthracnose of lentil, blackleg of canola and leaf spot diseases of cereals were assessed. Disease severity was generally low in both years, likely due to dry conditions. Overall disease did not differ between years. Disease severity varied significantly among sites, likely due to multiple site-specific characteristics. Precipitation and soil pH each explained a very small, but significant portion of overall disease severity. Root rot severity in pea or lentil, when considered separately, explained a small amount of yield variation. In contrast, pulse foliar diseases had a small, but significant impact on yield only when pea, lentil and chickpea were considered together. Blackleg was not significantly associated with canola yield. Surprisingly, higher severity of cereal leaf spot diseases or overall disease were significantly correlated with slightly increased yield, while explaining only a small portion of this parameter. Further exploration of site characteristics related to diseases, and relationships between disease and other agricultural and environmental parameters, especially in higher disease pressure years, is merited.
Detection and monitoring of Sclerotinia sclerotiorum ascospore levels in commercial canola fields during flowering. E. F. MCBAIN, T. K. TURKINGTON, S. E. STRELKOV AND J. FENG. Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada; (T.K.T.) Lacombe Research and Development Centre, Agriculture and Agri-Food Canada, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada; and (J.F.) Agriculture and Forestry, Plant and Bee Health Surveillance Section, Edmonton, AB T5Y 6H3, Canada
Sclerotinia sclerotiorum (Lib) de Bary (stem rot) is a common fungal disease of canola (Brassica napus L.) in western Canada. The incidence of stem rot has increased in recent years due to short crop rotations and increased canola acreage, highlighting the need for improved forecasting and management practices. The main objectives of this study were to: (1) refine the use of quantitative PCR analysis and spore traps to evaluate inoculum load before and during flowering for stem rot risk assessment, and (2) develop a better understanding of the relationship between environmental conditions, inoculum levels and final disease incidence and severity. In 2019, environmental data collected from four fields in the Fort Saskatchewan, Alberta, area included ambient and within crop temperature, relative humidity (RH), rainfall, and wind direction and speed. Sclerotinia sclerotiorum ascospore levels were monitored with a simple passive spore trap (‘Spornado’) and a rotorod sampler. One field was monitored more intensively, with five pairs of rotorod samplers and Spornados deployed to evaluate ascospore levels in different areas of the same field. Preliminary results indicated that a cool, wet summer, with a rain event occurring on at least two-thirds of days monitored, was favourable for disease development as reflected by increased disease incidence within monitored fields. Relative humidity within the crop canopy was higher relative to ambient RH. The Spornado and rotorod results showed moderate to high ascospore levels present at the start of flowering, followed by a second flush after 50% flower.
Identification of diverse chickpea pathogens in Montana State. S. MOPARTHI, M. E BURROWS AND K. MCPHEE. Department of Plant Sciences and Plant Pathology, Montana State University, P.O. Box 173 150, Bozeman, MT 59717–3150, USA
Montana is the leading producer of chickpeas in the United States. Diseases caused by fungal pathogens are major limiting factors for its production. The objective of this study was to survey the most prevalent and pathogenic species of chickpea crops in the State. A total of 53 chickpea fields from 10 counties were surveyed in June and July of 2020 to identify common foliar and root rot pathogens. Diseased chickpea plants were collected and fungal pathogens were isolated on potato dextrose agar (PDA). Pure cultures of isolates were obtained and identified to species level by amplifying part of translation elongation factor (EF1-α) gene (for Fusarium spp.), cytochrome oxidase (COI) gene (for Pythium spp.), glyceraldyhyde-3-phosphate dehydrogenase (G3PDH) gene (for Botrytis spp.), and entire internal transcribed spacer (ITS) region was amplified for other fungal species identification. Fusarium (95%) was the predominant fungal species recovered from the diseased chickpea roots, followed by Rhizoctonia (4%), and Pythium (1%). Among the Fusarium species, F. oxysporum (42%) was the predominant and widely distributed in all chickpea growing regions. Alternaria (76%) and Botrytis (20%) were the predominant fungal species from the foliage, followed by Ascochyta (4%). These preliminary results illustrate the pathogen diversity in the different chickpea producing regions of Montana. This information is essential for the management of diseases and breeding resistant chickpea varieties against economically important pathogens.
Efficacy of essential oils in managing Didymella rabiei, and evaluating their putative phytotoxicity. L. P. PARIKH AND M. E. BURROWS. Department of Plant Sciences and Plant Pathology, Montana State University, P.O. Box 173 150, Bozeman, MT 59717–3150, USA
Plant-derived essential oils (EOs) have potential antimicrobial benefits and can be incorporated in disease management practices as safer alternatives to synthetic fungicides. Previous studies identified in vitro inhibition of pathogens with palmarosa, oregano, clove, cinnamon, and thyme essential oils (EOs). In this study, five EOs were tested in vivo to control D. rabiei, and EO-seed treatments were evaluated for putative phytotoxicity on seed germination rate and root nodulation. Efficacy of EOs (diluted 1:1000) and fungicide Headline SC (0.29 lb ai/A) was evaluated in vivo on three chickpea varieties. Oregano (30–60%; SE 3.2–3.8) and thyme (30–45%; SE 3.1–3.8) oils significantly reduced disease severity compared to control (54–81%; SE 3.1–3.7) and were comparable to the fungicide (23–35%; SE 3.2–3.8) at P ≤ 0.05. EO-seed treatments (1:250 dilution in 0.01% water agar (WA)) and control (0.01% WA) were prepared for three varieties each of chickpea, pea, and lentil. Treatments were applied at 100 uL/g seeds. Seed germination rate was determined using a rolled paper towel assay (n = 200 seeds) and the experiment was conducted twice. Results indicated no phytotoxic effects of EO-seed treatments on seed germination rate (p > 0.05, n = 400 seeds). In a greenhouse assay, EO-treated seeds were inoculated with Rhizobium inoculant at 2.5 oz/100 lb chickpea seeds and 3.2 oz/100 lb pea/lentil seeds, and planted. Active root nodules were counted 28 days later, and the experiment was conducted thrice. No phytotoxic effect was observed on root nodulation (p > 0.05, n = 24 seedlings) in chickpea, pea, and lentil. EOs can be used in disease management of pulses and can minimize synthetic chemical use.
COVID staycation: A southern Alberta gravel road expedition to explore farmer responses to wheat diseases. B. L. PUCHALSKI AND B. J. PUCHALSKI. Paramoria Agri-Science, 216 23rd Street South, Lethbridge, AB T1J 3M6, Canada
COVID19 presented unique and difficult challenges to farmers during the 2020 growing season. The disease forced rural communities to isolate, yet farming activities continued. With limited external advice and assistance, how did farmers respond to emerging wheat diseases for the season? To identify farmer responses and disease patterns, we surveyed 166 cereal fields for foliar disease incidence and severity, using a modified Cobbs scale. The survey focused on spring, winter and durum wheat. The presence or absence of signs of fungicide application was recorded. A multivariant expert system which mapped disease risk in conjunction with the ability to apply fungicide using ground-based equipment was also created. Half of all fields did not have fungicides applied at the time of the survey. Sixty-one percent of those unsprayed fields exhibited disease incidence and severity below economic thresholds for applying fungicides. Surveyed fields demonstrated a spectrum of responses, with 20% of winter wheat fields at intermediate resistance and 33% of spring wheat fields being intermediate and susceptible to stripe rust. Considering the resistance of readily available varieties, this suggests that stripe rust resistance might be leaking. This same trend was not observed for tan spot of wheat. Despite a lack of resistance, tan spot struggled to capitalize to the same degree. Reliance on chemical control also presented difficulties. Multivariant analysis indicated that, for effective application of fungicides, the entire spray season might be decided in a window of only three days.
Developing a Brachypodium distachyon cell suspension culture pathosystem to study cellular defence responses. D. D. RYABOVA, G. PEÑA-FUNDORA AND N. A. FOROUD. (D.D.R., G.P.-F., N.A.F.) Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403–1st Avenue South, Lethbridge, AB T1J 4B1, Canada; and (G.P.-F.) University of Lethbridge, 4401 University Drive West, Lethbridge, AB T1K 6T5, Canada
A Brachypodium distachyon-21 (Bd21) cell suspension culture was established to study the processes of plant cellular responses to pathogens. Fusarium graminearum (Fg) is a wheat pathogen and it is known to infect different tissues of Brachypodium species, a model plant for cereals. The long-term objectives is establish high-throughput methods to study the Bd21 cell cultures Fg infection, Fusarium toxins and pathogen-associated molecular patterns (PAMPs), by monitoring reactive oxygen species (ROS) production and programmed cell death using multi-well formats. As a starting point, Bd21 cell suspensions were inoculated with Fg macroconidia, to observe their interactions. Spore germination and mycelium formation was observed at 22°C, 12–36 h after inoculation. Both wild-type strain Gz3639, and a GFP-labelled strain, were used to trace fungal structures. Germinated spores formed germ tubes, developed branching hyphae and some of the hyphae coincided in with Bd21 cells, but penetration of the host cells was not evident in most cases. The GFP strain developed fluorescent hyphae but did not form any structures inside Bd21 cells. Different techniques were employed to monitor the viability of plant cell culture, including MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and staining with vitality dyes (e.g. trypan blue, propidium iodide and fluorescein diacetate). Incubating Fg spores with Bd21 cells without shaking increased the chances to observe interaction events, but drastically dropped the viability of plant cells and also decreased fungal development.
Challenges in clubroot pathotype-specific molecular diagnostics. H. H. TSO, L. GALINDO-GONZÁLEZ, H. ASKARIAN, M. D. HOLTZ AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (M.D.H.) Field Crop Development Centre, Alberta Agriculture and Forestry, 5030–50 Street, Lacombe, AB T4L 1W8, Canada
Clubroot, caused by Plasmodiophora brassicae Wor., is an important soilborne disease of canola (Brassica napus L.). New pathotypes have recently emerged that can overcome host resistance, and the ability to distinguish pathotypes rapidly and reliably would be valuable for clubroot management efforts. To identify diagnostic polymorphic regions, we obtained variant genome information from 45 full genome P. brassicae isolates and aligned them with the original e3 P. brassicae reference genome published in 2015. Our preliminary computational analysis indicated numerous heterozygous positions when pathotype reads were mapped to the 2015 e3 genome assembly. When the reads were mapped to the most recent e3 genome published in 2019, the apparent heterozygosity was found to have resulted from the collapsing of reads corresponding to different genomic regions. To distinguish pathotypes, we developed an RNase H2-dependent PCR (rhPCR) assay with primers bearing a polymorphic ribonucleotide at the 3ʹ end. Although sensitive, this method produced residual unspecific amplification at times. The specificity of the rhPCR assay was improved with the incorporation of multiple SNPs expanding the primer region beyond the ribonucleotide. We also used SNaPshot, a single-base extension technology that fluorescently labels the discriminatory SNP, to compare its discriminatory power with rhPCR. Preliminary results showed a clear differentiation among pathotypes. While both technologies are useful for clubroot diagnostics, rhPCR is the most cost-effective and widely used molecular technique, and may be optimized into a quantitative assay. The main advantage of SNaPshot is the ability to distinguish all four alleles at the discriminatory SNP.
Identification of quantitative trait loci (QTL) associated with root rot caused by the root rot complex in field pea. L. WU, R. FREDUA-AGYEMAN, S. F. HWANG, K. F. CHANG, D. MCLAREN AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada; (K.F.C.) Alberta Agriculture and Forestry, Crop Diversification Centre North, 17 507 Fort Road NW, Edmonton, AB T5Y 6H3, Canada; and (D.M.) Brandon Research and Development Centre, Agriculture and Agri-Food Canada, 2701 Grand Valley Road, Brandon, MB R7A 5Y3, Canada
Root rot, caused by several soilborne pathogens including Fusarium spp., Aphanomyces euteiches, Pythium spp., Phytophthora spp. and Rhizoctonia solani, is a major threat to field pea production in western Canada. There are no completely effective strategies to manage the root rot complex, reflecting limitations in the effectiveness of cultural practices and fungicidal seed treatments, as well as a lack of completely resistant pea germplasm. The pea cultivar ‘00–2067ʹ is partially resistant to Aphanomyces root rot, with a major QTL on chromosome IV. In this study, the pea genotypes ‘00–2067ʹ and ‘Reward’ were screened for resistance to five isolates of Fusarium spp., one of Pythium spp., one of Phytophthora spp. and three isolates of Rhizoctonia spp. Two Fusarium spp. isolates (F4A and FG2) and one Rhizoctonia spp. isolate (CKP1) caused significant difference of root rot severity between ‘Reward’ and ‘00–2067ʹ. One hundred thirty-five individuals, derived from the cross ‘00–2067ʹ× ‘Reward’, were screened for resistance to F4A, FG2 and CKP1 in greenhouse experiments. Disease severity and plant height were evaluated as measures of resistance and agronomic traits, respectively. A significant genotypic effect and G × E interactions (P < 0.05) were detected with high heritability. The identification of quantitative trait loci associated with resistance to the three isolates using the 13.2 K SNP array and 222 SSR markers is in progress.
Effects of fall and spring lime applications on clubroot of canola. 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 [Plasmodiophora brassicae Wor.] is a major threat to sustainable canola (Brassica napus L.) production in western Canada. The clubroot pathogen prefers acidic soils, and hence, the application of lime to increase soil pH represents a possible management strategy. The finely ground Zero Grind limestone (ZG) and hydrated lime (HL) were applied in replicated field and greenhouse tub trials to evaluate their efficacy for clubroot control. The treatments included an untreated control (UTC), ZG applied at rates of 5 and 10 t/ha in fall or spring, spring applications of HL at 5 and 10 t/ha, and fall application of ZG combined with spring application of HL at 2.5 or 5 t/ha each. Two canola hybrids, ‘45H31ʹ (susceptible) and ‘CS2000ʹ (moderately resistant) were included in the tub trials and ‘45H31ʹ was used in the field trials. In the tub trials, the application of 5 t/ha fall ZG and 5 t/ha spring HD reduced clubroot severity the most on both cultivars, but did not result in significantly (p < 0.05) higher yields than any other treatment except for the UTC. In the field trials, the 10 t/ha fall ZG application and the combination of 5 t/ha fall ZG + 5 t/ha spring HD applications resulted in the lowest clubroot severity and highest yield at both sites. Significant reductions in clubroot incidence also were observed in all of the lime-treated plots or tubs, relative to the untreated controls. The data suggest that the application of lime products at different times holds potential as part of an integrated management strategy for clubroot of canola.