Degradome studies provide new insights into viroid pathogenicity. C. R. ADKAR PURUSHOTHAMA AND J.-P. PERREAULT. RNA Group/Groupe ARN, Département de Biochimie, Faculté de médecine et des sciences de la santé, Pavillon de Recherche Appliquée au Cancer, Université de Sherbrooke, 3201 rue Jean Mignault, Sherbrooke, QC J1E 4K8, Canada
Viroids are single stranded, non-coding RNA molecules that infect and cause diseases in several economically important plants. Though it is well established that the viroid derived small RNA (vd-sRNA) down-regulate endogenous mRNAs by RNA silencing mechanism, it is not known how exactly viroid infections can induce severe disease symptoms given the fact that a smaller number of vd-sRNA binding to the specific target mRNAs were recovered from the infected plants. Hence in the present study, the role of viroid derived small RNA in pathogenesis was studied using in silico and in cellulo experiments. More specifically, the genome of potato spindle tuber viroid was dissected in silico into 21-nucleotide fragments, which were then used to interrogate publicly available tomato transcriptome data sets using the WMD3 Web-based tool. The resulting putative target mRNA sequences with known functions were selected for further analysis. The effect of potato spindle tuber viroid on the putative target mRNA was analysed by RT-qPCR. The accumulation of viroid derived small RNAs in the viroid infected plants were verified by high-throughput sequencing, and the cleavage of putative target mRNA by Parallel Analysis of RNA Ends (PARE). Mapping of PARE sequences against endogenous mRNAs and subsequent comparison with control revealed the extensive degradation of endogenous mRNAs in viroid infected plants. This implies the possible involvement of secondary products of viroid infection in disease severity.
Estimation of viable resting spores of Plasmodiophora brassicae in a 6-year crop rotation study using propidium monoazide-assisted PCR. F. AL-DAOUD, J. ROBSON, D. PAGEAU, B. D. GOSSEN, J. A. DALTON AND M. R. MCDONALD. Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; (D.P.) Agriculture and Agri-Food Canada (AAFC), 1468 St-Cyrille Street, Normandin, QC G8M 4K3, Canada; and (B.D.G.) Saskatoon Research and Development Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada
Resting spores of Plasmodiophora brassicae Woronin, the causal agent of clubroot in canola (Brassica napus L.) and other brassica crops, can remain viable in soil for many years. Quantitative PCR (qPCR) is used to quantify soil-borne resting spores, but it amplifies DNA from both viable and non-viable spores. However, pre-treatment with propidium monoazide (PMA) can suppress amplification of DNA from non-viable spores in subsequent qPCR (PMA-PCR). PMA penetrates non-viable cells, binds to the DNA when photo-activated, and prevents amplification. The objectives of this research were: (1) to develop a protocol for using PMA-PCR on soil samples, and (2) to compare qPCR and PMA-PCR analyses of soil samples from a 6-year crop rotation field experiment at Normandin, Québec. A soil dilution technique, which involves simply mixing soil in water, retained up to 916-fold more spores than the standard sucrose solution-based techniques. This technique was used in conjunction with PMA-PCR to analyse the crop rotation soil samples. There were 2.0 × 106 ± 1.3 × 106 and 7.4 × 105 ± 2.6 × 105 spores g−1 soil at year zero as estimated by qPCR and PMA-PCR, respectively. QPCR showed a quadratic decrease in spores over time with an 83% reduction in spore numbers after 2 years. PMA-PCR demonstrated a linear decrease in viable spores, with a 73% reduction in viable spores after 3 years. The half-life of spores at this site was estimated at 1.2 years by qPCR and 2.9 years by PMA-PCR. PMA-PCR is a useful technique to quantify viable resting spores, and reduce the need for resource-intensive bioassays.
A detached fed-leaf bioassay to assess bacterial antagonists against Phytophthora infestans isolates. P. AUDY, N. FORAN, S. M. BOYETCHKO AND V. GRAVEL. Quebec Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), Quebec, QC G1V 2J3, Canada; (N.F., V.G.) Department of Plant Science, McGill University, Macdonald Campus, 21 111 Lakeshore Road, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada; and (S.M.B.) Saskatoon Research and Development Centre, AAFC, Saskatoon, SK S7N 0X2, Canada
Late blight of potato (Solanum tuberosum L.) caused by Phytophthora infestans (Mont.) de Bary, is the most devastating disease affecting potato, accounting for over US$6 billion worldwide each year due to production losses and prevention measures. Chemical fungicides are heavily used by potato producers to prevent and control the disease but public pressure has created a demand for environmentally friendly control products and a preference for pesticide-free foods are driving the exploration for biological control options. In this study, a detached fed-leaf bioassay was developed to assess the ability of some bacteria to control potato late blight under environmental conditions optimum for disease development. Forty-six bacterial strains were tested against a P. infestans US-8 isolate (A2 mating-type). Four treatments were used: pathogen alone, whole bacterial culture + pathogen, bacterial filtrate + pathogen and autoclaved bacteria culture + pathogen. The six leading bacterial agents were then evaluated for their abilities to impede P. infestans development (four A1 and A2 mating-type isolates) using the same detached fed-leaf assay. Data analysis revealed that the level of biocontrol differed greatly among the six bacterial strains. Results showed that bacterial antagonists could be an effective addition to current prevention methods against late blight and contribute to the reduction of synthetic fungicides in potato production.
Fusarium spp. complex infecting oat in Manitoba. M. BANIK, M. BEYENE AND X. WANG. Morden Research and Development Centre, Agriculture and Agri-Food Canada, 101 Route 100, Morden, MB R6M 1Y5, Canada
Oat is one of the most important cereal crops in western Canada. This crop has become desirable for human consumption due its high nutritional value. In recent years, Fusarium head blight (FHB) emerged to be one of the most serious diseases on oat, especially in the Canadian Prairies. Several Fusarium spp. can infect oat and produce different mycotoxins in contaminated grains which cause immunosuppression and various health issues when consumed by human and animals. Of special interest among these Fusarium mycotoxins are the trichothecenes (e.g. T-2 and HT-2 toxins produced by F. sporotrichioides Sherb.), deoxynivalenol (DON, mainly produced by F. graminearum Schwabe and F. culmorum Sacc.) as well as beauvericin and enniatin (mainly produced by F. poae (Peck) Wollenw.). In this study, we investigated Fusarium spp. infecting oat in Manitoba. Oat samples were collected from 36 commercial oat fields in 2015. Species identification was performed based on morphological characteristics and species-specific PCR. Fusarium biomass in contaminated grain was assessed by real-time qPCR using primer sets specific to F. poae, F. graminearum and F. sporotrichioides. Our results indicate that F. poae was the most common Fusarium spp. infecting oat in Manitoba in 2015, followed by F. graminearum and F. sporotrichioides. Most oat samples are contaminated by multiple Fusarium spp. and pathogen biomass mostly originated from F. poae, hence suggesting that the production and/or accumulation of diverse mycotoxins in oat should be taken into consideration due to the mixed Fusarium infection commonly present in field samples.
Development of a botanical pesticide for the control of powdery mildew: the challenge of performing fungicide efficacy trial on obligate biotrophic fungi. S. BEAUSEIGLE, P. H. ONTCHANGALT, A. BILLONG, S. KERNER, Y. RUDOLPH-BINETTE AND A. VIALLE. Biopterre-Bioproducts development center, 1642 rue de la ferme, La Pocatière, QC G0R 1Z0, Canada; (S.K.) iFact inc. 1117 rue Sainte-Catherine Ouest, suite 410, Montréal, QC H3B 1H9, Canada; and (Y.R.-B.) Arbressence inc., 77 rue Omer Desserres, suite 6A, Blainville, QC J7C 5N3, Canada
Powdery mildew fungi cause important economic losses on a wide range of ornamental and agricultural plants. Repetitive application of synthetic fungicides is currently the main practice for powdery mildew management in crops. However, there is a high potential for powdery mildew species to develop fungicide resistance. Botanical pesticides, such as plant extract-based biopesticides, offer a good alternative to conventional chemical pesticides since they are less likely to have resistance issues because of their broad and non-specific modes of actions. The objective of this project is to perform fungicide efficacy trials against powdery mildew species using Northern white cedar (Thuja occidentalis) hydrosol formulations as botanical fungicide. In order to proceed to the homologation of the product, the first step is to determine the fungicide efficacy among the range of powdery mildew species in vitro. However, the fact that powdery mildews are obligate biotrophs, coupled with the fact that there are multiple pathogen/host combinations, make any in vitro tests challenging to realize. We have developed a specific strategy to perform a pesticide efficacy trial on obligate biotrophic pathogens. First, we have narrowed down the number of pathogen/plant host combinations targeted in focusing on powdery mildew species occurring under controlled production systems such as greenhouse (vegetables) and growth chamber production (cannabis). Since there are a limited number of fungal strains available, a ‘fungal strains harvest campaign’ has been initiated throughout Canada. Lastly, we have developed a protocol to test the fungicide efficiency of obligate, biotrophic fungal species in vitro.
Survival and productivity in an environment of multiple stressors: responses to drought in interior Douglas-fir (Pseudotsuga menziesii var. glauca) seedlings. E. M. BECKER, M. G. CRUICKSHANK AND R. N. STURROCK. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
The interior Douglas fir (Fdi) variety, Pseudotsuga menziesii Mirb. Franco var. glauca, is a dominant and highly valued tree that grows more slowly and is better adapted to drought than the coastal variety of the species (var. menzies). These long-lived trees face selective pressures for both growth and stress tolerance and must be able to compete for resources and light as well as being able to withstand decades of exposure to multiple biotic and abiotic stressors. We are characterizing the survival and tolerance responses of Fdi families from four distinct seed breeding zones in B.C. to multiple stressors, including drought and fungal diseases. We are testing the hypothesis that growth and stress tolerance are linked, and obtaining insight into underlying mechanisms of responses to drought and diseases. Objectives of the present study were to analyse the survival of Fdi in an environment of drought, and to characterize the responses of Fdi grown in a water-limited environment, compared to in an ideal environment (=tolerance). In this study, trees from the driest breeding zones, SA and WKL, were those with the most growth in the driest water-limited treatment. Trees from these drier zones, however, had the highest risk of mortality by drought. This work provides a foundation for future genetic studies to associate phenotypic stress responses with their genetic components. The results of these experimental trials will increase our knowledge of conifer responses to multiple disease agents, and inform and guide specific breeding strategies for Douglas-fir trees.
Next-generation sequencing (NGS) using Ion Torrent technology for biosurveillance from spore and insect traps. G. J. BILODEAU, É. TREMBLAY AND J. A. BERUBE. Canadian Food Inspection Agency, Ottawa Plant Laboratory, Ottawa, ON K2H 8P9, Canada; and (J.A.B.) Natural Resources Canada, Laurentian Forestry Centre, P.O. Box 10380, Stn. Sainte-Foy Québec, QC G1V 4C7, Canada
Plant pathogenic fungal spores spread by wind, rain and through vectors (insects) can cause the introduction of exotic diseases and are responsible for the devastation of various plant species. New technologies such as metagenomics are now an option for quicker detection by avoiding time-consuming culturing methods. As a proof of concept of biosurveillance using this new technology, DNA was extracted from spore trap filtrates and insect trap preservation liquids from collections from 2013–2015. DNA was sequenced on the Ion Torrent PGM™ platform using fusion primers designed to multiplex three genic regions (350–400bp amplicons from 2 ribosomal DNA region ITS1 (fungi and oomycetes) and the mitochondrial DNA region ATP9-NAD9 (Phytophthora spp.)). Bioinformatic analyses were also done to process the millions of sequences generated, which were then compared with species-specific qPCR testing for multiple specific targets. We compared the NGS contents to qPCR data obtained for the detection of forest pathogen species, which led us to find several closely related species to our targeted Phytophthora spp. and forest fungi. The method we developed allows us to identify potential sources of entry into Canada and to access sample abundance, biodiversity and phylogeny in order to accelerate the identification process of current risk associated with these exotic plant pathogen species.
Update on Manitoba horticultural crops disease and insect pests in 2015. V. BISHT. Crop Industry Branch, Manitoba Agriculture, 65, 3rd Avenue NE, Carman, MB R0G 0J0, Canada
Potato and horticultural crops are high value and high input crops with significant disease and insect pest risks. Along with weather, pests are main issues for Manitoba. In 2015, late blight appeared late in season, in the first week of September, but still led to some storages having rot issues, leading them to be processed early. Scattered disease appeared in the west, central and southern parts of the province. The late blight strain was identified as US #23. A couple of fields of tomato crop also had significant late blight on fruits. Verticillium wilt appears to be an endemic issue in some fields. A GF2 project has been initiated to understand various factors involved in impacting the productivity in MB. European corn borer injury incidence was quite noticeable in some fields, where early infested plants had reduced tuber production. Preliminary results from a national PVY-strain distribution study suggest that from Manitoba overall PVY in fields was low (<1% in most fields), but most PVY strains were necrotic (PVYNTN being predominant). Carrot forking caused by Pythium spp. was found in some fields. Onion neckrot disease was significantly lower than 2 years ago; and may be attributed to dry harvest time. None of the fungicides tested gave consistent and effective control of neckrot. Cauliflower blackrot disease was a significant disease in a couple of fields. High incidence of blackleg and cabbage maggots on crucifer vegetables, including rutabaga was recorded. On raspberry, fireblight along with the Spotted Wing Drosophila (SWD) caused significant losses.
Molecular mapping of common bunt resistance in a ‘Vesper’ × ‘Lilian’ population. F. BOKORE, R. CUTHBERT, R. E. KNOX, C. POZNIAK, A. N’DIAYE, A. SHARPE AND Y. RUAN. Swift Current Research and Development Centre, Agriculture and Agri-Food Canada, P.O. Box 1030, Swift Current, SK S9 H 3X2, Canada; (C.P., A.N’D.) Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada; and (A.S.) National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada
Common bunt caused by Tilletia tritici (Bjerk.) Wint. and T. laevis Kühn is an economically important disease of wheat (Triticum aestivum L.) causing grain yield and quality losses. An effective way to control common bunt is the use of resistant varieties. The objective of this study was to identify and map quantitative trait loci (QTL) for common bunt resistance in the Canadian hard red spring wheat variety ‘Lillian’. A population of 280 doubled haploid lines, from the cross ‘Vesper’/‘Lillian’, and parents were inoculated with races L16 (T. laevis) and T19 (T. tritici). The lines were evaluated for common bunt incidence in the field near Swift Current, Canada in 2014 and 2015. The lines were genotyped with the Infinium iSelect 90 K wheat assay and a set of 1975 informative single nucleotide polymorphisms were used for QTL analysis. The bunt incidence scores of the lines ranged from 0 to 45% in 2014 and 1 to 50% in 2015. The susceptible check cultivar ‘Biggar’ scored 37.5% incidence in 2014 and 50% in 2015. ‘Lillian’ was rated at 5% incidence for both 2014 and 2015, and ‘Vesper’ at 30% in 2014 and 25% in 2015. ‘Lillian’ contributed QTL for common bunt resistance on chromosomes 3D, 5A and 7A, and ‘Vesper’ on 1D. The QTL explained from 4.8 to 7.7% of the variation in disease incidence. The 5A QTL expressed in both years, whereas the other QTL were confined to one season. The QTL have potential in breeding for bunt resistance in wheat.
Advances in elucidating the mode of action of mineral oil on reduction of aphid-mediated PVY transmission. S. BOQUEL, R. HEPAT AND X. NIE. Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada; and (S.B.) SIPRE – Comité Nord, Rue des Champs Potez, 62217 Achicourt, France
Mineral oil (MO) has been used in the management of non-persistent virus and more specifically Potato virus Y (PVY) for a long time. MO has been commercially available to Canadian seed potato growers for a few years and yet, its mode of action remains unknown. Deciphering the mode of action of mineral oil will allow refinement of the current recommendations given to growers, increasing its reliability in managing PVY. The aim of this study was to test the efficacy of a single or five MO sprays on PVY acquisition and inoculation by aphids under field conditions. Results showed no significant reduction of PVY acquisition in the field. However, a 39% and 44% reduction of PVY transmission was observed 15 days post-inoculation (dpi) with plants treated with a single or five sprays of mineral oil, respectively. Quantitation of PVY amplicons based on band intensity revealed that amongst plants successfully infected with PVY, plants treated with MO harbour less PVY than untreated plants. The percentage of strong intensity bands was reduced after one and five sprays as well as the percentage of medium intensity bands after five sprays of MO. Mineral oil was not effective in reducing PVY acquisition but provided a good control of PVY transmission. These results suggest that mineral oil could have reduced (i) the number of viral particles inoculated by the aphid while probing the treated plant, (ii) virus replication and/or accumulation in inoculated leaves between inoculation and 15 dpi. Experiments are ongoing to answer these questions.
The Biovigilante: Monitoring threats to plant health in an era of globalization and climate change. K. BRODERS. 307 University Avenue, Colorado State University, Fort Collins, CO 80523–1177, USA
The Anthropocene epoch is defined as the point when human activities started to have a significant global impact on Earth’s ecosystems. Certainly the phenomenon of globalization and changes in global climate patterns have resulted in an upheaval of how we view disease ecology, epidemiology, plant-microbe coevolution and the plasticity of the disease triangle in both forested and agricultural ecosystems. In order to sustain our ecosystem services and feed an ever-growing population we must adapt to this new reality as many of our pathogen nemeses have already done. The number of emerging pathogens has increased significantly over the course of the last two decades, and will probably continue to increase in the more globally interconnected future. I will provide four independent scenarios of plant pathogen emergence including: the introduction of an invasive forest pathogen; an endemic pine pathogen that has reached epidemic levels due to changes in regional climatic patterns; an endemic beetle vectored pathogen that has expanded its host range due to climate change and anthropogenic forces; and the combination of an introduction of a new, better adapted race of a crop pathogen followed by a rapid range expansion. Finally, based on these four examples, I provided a perspective on preparing for new emerging forest and crop pathogens in the future through the development of next-generation methods for monitoring emerging pathogens, as well as developing networks for rapidly communicating information regarding emerging pathogens.
Using biovigilance-based information for strategic and tactical disease management decisions. O. CARISSE. Centre de recherché et de developpement en horticulture, Agriculture et Agroalimentaire Canada, 430 Gouin Bvld., Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada
Biovigilance can be defined as the study of the unintentional effects of farming practices on pest populations and ecological services (biodiversity). Since the second half of the last century, farming and pest management practices have undergone considerable changes, which were driven by increasing mechanization, the intensive use of fertilizers and pesticides, and the genetic improvement of crops. These practices have exerted pressure on pest populations, which have adapted by, among other means, developing pesticide resistance and overcoming crop resistance. In addition, climate change and the movement of plant products between different areas or countries also influence the diversity of pest populations and their natural enemies. As a result, pest management decisions must take into account these changes in pest populations. Biovigilance-based information can be used for strategic (long-term) decisions about matters such as the type of production system, crop rotation, and host genetic selection for perennial crops. Similarly, knowledge on pathogen aggressiveness or fungicide resistance should be considered when making tactical (short-term) disease management decisions. During this presentation, we discussed how biovigilance information can help with making both types of disease management decisions. The discovery of new Plasmopara viticola (Berk. & Curtis) Berl. & De Toni ff. sp. (P. viticola f. sp. riparia, P. viticola f. sp. aestivalis and P. viticola f. sp. vinifera) and their significance will be used as a case study for strategic decisions. Fungicide resistance in Botrytis spp. populations will be used as a case study for tactical decisions. The ultimate objective of biovigilance-based disease management is to mitigate potential threats before they become a huge problem. Typically, this approach to disease management is forward-looking and requires a long-term commitment for research.
Differential proliferation of Plasmodiophora brassicae in Brassica napus cultivars. T. CAO, S. F. HWANG, I. FALAK AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (S.F.H.) Alberta Agriculture and Forestry, Crop Diversification Centre North, 17507 Fort Road, Edmonton, AB T5Y 6H3, Canada; and (I.F.) Pioneer Hi-Bred Production Ltd, 12111 Mississauga Road, Caledon, ON L7C 1X1, Canada
Clubroot is a devastating disease of canola (Brassica napus L.) in Alberta, Canada, and effective management relies mainly on genetic resistance. In order to improve understanding of host resistance, the proliferation of Plasmodiophora brassicae Woronin (Pb) within the roots of susceptible (45H26) and resistant (P2008-6) canola genotypes was monitored by quantitative PCR (qPCR) and histological analyses. Roots were sampled 1–44 days post-inoculation (dpi) with a single-spore isolate representing Pb pathotype 3. The concentration of pathogen DNA was significantly greater in 45H26 (4.68–1668.57 µg g−1) than in P2008-6 (2.91–538.60 µg g−1) starting at 4 dpi and at most of the subsequent time-points examined. Root hair infections were observed from 4 to 7 dpi in both hosts. Zoosporangia were found on P2008-6 at 10 dpi, and plasmodia were visible on 45H26 and P2008-6 starting from 11 and 12 dpi, respectively. From 11 to 30 dpi, plasmodia dramatically increased in both host genotypes, with more plasmodia in 45H26 than in P2008-6. At 37 and 44 dpi, more resting spores were visible in 45H26 than in P2008-6. While the extent of pathogen proliferation and the speed of its development were greater in the susceptible vs. the resistant genotype, Pb was able to complete its life-cycle and produce resting spores on the resistant host. This suggests that resistant canola genotypes may contribute to the build-up of Pb inoculum in the soil, although not to the same extent as susceptible varieties.
The Innate potato: from concept to commercialization. N. CHAMPOURET. Simplot, P.O. Box 27, Boise, ID 83707, USA
Simplot Innate® technology permits the selective improvement of positive traits or minimization of negative traits of widely used, conventional potato varieties without incorporation of foreign genes. The Innate® platform of genetic modification also leverages the utilization of disease resistance traits from wild potato species. Resistance genes can be combined in various desirable permutations to keep at bay a particular pathogen population or to enhance the natural resistance of a variety. The technology effectively accelerates genetic improvement of conventional varieties faster than is currently possible using traditional breeding methods. The second generation of Innate® potatoes will have reduced blackspot bruise, reduced asparagine (which reduces the potential for the formation of acrylamide), cold storage capability and foliar late blight resistance against common North American strains. Therefore, Innate® potatoes are genetically engineered with traits appealing to potato growers, packers, processors, retailers and consumers.
Occurrence of soybean root rot and associated pathogens in western Canada. K. F. CHANG, S. F. HWANG, H. U. AHMED, Q. ZHOU, H. FU, S. E. STRELKOV, R. L. CONNER, D. L. MCLAREN, M. W. HARDING AND G. D. TURNBULL. Crop Diversification Centre (CDC) North, Alberta Agriculture and Forestry (AAF), 17507 Fort Road, Edmonton, AB T5Y 6H3, Canada; (S.E.S.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (M.W.H.) CDC South, AAF, 301 Horticultural Station Road East, Brooks, AB T1R 1E6, Canada; (R.L.C.) Morden Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 101 Route 100, Morden, MB R6M 1Y5, Canada; and (D.L.M.) AAFC, 2701 Grand Valley Road, Brandon, MB R7A 5Y3, Canada
Soybean (Glycine max (L.) Merr.) has great potential as an alternative crop in western Canada. Root rot is a common constraint to soybean production worldwide. Surveys in 2015 determined the incidence, severity and causal organisms of root rot of soybean in 41 crops across nine counties in southern Alberta. A total of 100 root samples were collected per field. Root rot occurred at all locations, with an average incidence of 78% and a range of 33–100%. The average disease severity was 1.3 and ranged from 0.4 to 2.6 on a scale of 0–4. Species of Fusarium were most commonly isolated, followed by Pythium spp. and then Rhizoctonia solani Kühn. A total of 94 soil samples were collected from Alberta and Manitoba and seeded with the oomycete-susceptible pea cultivar ‘Midas’. Isolation from the infected roots on semi-selective media yielded 151 isolates with morphological characteristics of Pythium or Phytophthora species. In greenhouse assays, 85 of these isolates caused more than 50% reduction in seedling emergence. Twenty-one of these isolates were moderately virulent (50–69% emergence reduction), 26 were virulent (70–84% emergence reduction) and 38 isolates were highly virulent (85–100% emergence reduction). Most of the isolates caused severe disease on the host (ratings of 3.0–4.0), while 13 isolates caused more moderate symptoms (ratings of 2.1–2.9). Plant height decreased as disease severity increased. The identity of these isolates will be determined by examining their morphological and reproductive structures, along with the use of molecular diagnostic tools.
Oospore dose-response and spatial distribution of the pea root pathogen, Aphanomyces euteiches, in Saskatchewan soils. S. CHATTERTON, A. ERICKSON AND S. BANNIZA. Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403-1 Avenue South, Lethbridge, AB T1J 4B1; and (S.B.) Crop Development Centre, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
Aphanomyces root rot, caused by Aphanomyces euteiches Drechs., was first detected in pea fields in Saskatchewan and Alberta in 2012 and 2013, respectively, and can cause significant crop loss in both provinces. In order to determine oospore dose-response relationships, soils were collected from brown, dark brown and black soil zones in Saskatchewan, and sterilized or used as is (raw). Oospores were added to soils to provide final concentrations from 0–2000 oospores g−1 soil, A. euteiches DNA quantified using real-time PCR (qPCR), and then used to grow pea plants for root rot assessments. The threshold level for disease in raw soils was 100 oospores g−1 soil in all soil types, which was also the limit of detection using qPCR. For black and brown soil types, the threshold level for disease was 750 oospores g−1 soil in sterilized soils, demonstrating that other soil-borne microorganisms increase disease risk. To determine spatial distribution of oospores in the field, soils were collected from 0–20, 20–40 and 40–60 cm depths at 11 sites from three pea fields in each of the three soil zones in Saskatchewan. Peas were grown in each soil sample and rated for root rot. Disease levels were highest in the top 0–20 cm layer, and decreased with increasing depth. Horizontal distribution of A. euteiches varied between fields. Real-time PCR analysis to quantify A. euteiches DNA levels in these soil samples is underway. Results will be used to develop sampling protocols and soil tests to determine root rot risk.
Expression analysis of three NADPH oxidase genes in germination and infection of wheat leaf rust. M. Z. CHE, X. B. WANG, B. D. MCCALLUM, H. B. KHALIL, G. BAKKEREN AND B. J. SAVILLE. (M.Z.C., X.B.W., B.D.M.) Morden Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 101 Route 100, Unit 100, Morden, MB R6M 1Y5, Canada; (M.Z.C.) Department of Plant Pathology, China Agricultural University, Beijing, 100193, People’s Republic of China; (H.B.K., G.B.) Summerland Research and Development Centre, AAFC, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada; and (B.J.S.) Forensic Science Program Trent University, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada
Leaf rust [Puccinia triticina Eriks. (Pt)] of wheat (Triticum aestivum L.) is a widespread disease that causes significant reductions in grain yield and quality. Urediniospore germination and the infection of wheat plants are critical steps in the establishment of the pathogen. Reactive oxygen species (ROS) often plays an important role during host and pathogen interaction. However, very little is known about the role of these molecules in the pathogenesis of Pt. In this study, we identified two NADPH oxidase genes (PtnoxA and PtnoxB) and a regulatory gene (PtnoxR) which were potentially involved in the production of ROS from EST and RNAseq databases of Pt. We analysed the expression levels of three NADPH oxidase genes over time during spore germination and plant infection using RT-qPCR. Total RNA was extracted from urediniospore germ tubes at 6, 12 and 24 h after incubation (hai), and from inoculated wheat leaf tissues (Thatcher, leaf rust susceptible line) at 24, 48, 96, 144 h post-inoculation (hpi) and 8 days post-inoculation. During urediniospore germination, the expressions of PtnoxA and PtnoxR peaked at 24 hai whereas the highest level of PtnoxB transcripts was found at 6 hai. In comparison, the expression of three transcripts all peaked at 24 hpi during the infection on Thatcher and downregulated when Pt has successfully penetrated leaf-surface. This result indicates that PtnoxA, PtnoxB and PtnoxR are differentially regulated during urediniospore germination and plant infection. The production of ROS is important for the pathogenesis of Pt and needs to be carefully regulated during host plant infection.
Geographic atlas of mycotoxigenic fungi through metagenomic surveys of DNA barcodes using a novel taxonomic classification approach. W. CHEN, C. VISAGIE, M. LIU, K. SEIFERT, T. GRAEFENHAN, S. HAMBLETON AND C. A. LEVESQUE. Ottawa Research and Development Centre, Science and Technology Branch, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
Mycotoxins are toxic secondary metabolites produced by species of fungal genera such as Alternaria, Aspergillus, Claviceps, Fusarium and Penicillium. Species of one genus often produce different biosynthetic groups of mycotoxins, which have adverse health effects on humans and livestock. Some of these species are considered pathogens of agricultural/medical importance. For risk assessment and mycotoxin management, it is important to accurately identify these organisms. This is challenging for Metagenomic surveys using DNA barcodes. The internal transcribed spacer (ITS) of the rDNA region is the official fungal barcode, but often lacks sufficient discriminatory power at the species/subspecies ranks. Furthermore, existing algorithms for sequence classification are similarity- and composition-based, which are imprecise for taxonomic profiling below the genus level and for accommodating the inherent error rate of various Next Generation Sequencing (NGS) platforms. Our three objectives were: (1) using clades-specific oligonucleotides to facilitate and improve species/subspecies-level interpretation of metabarcodes; (2) developing a living geographic atlas of mycotoxin producing fungi in Canada; (3) discovering known and putatively unknown species and provide critical information to taxonomists for species discovery and reference database development. A total of 45 million ITS1 and ITS2 454-pyro-tagged sequences from air/rain samples and commodity seed wash samples collected during 2009–2013 were compared with the curated UNITE fungal ITS database. Preliminary results showed that ITS1 and ITS2 recovered different numbers of putative species (Operational Taxonomic Unit, OTU) at 97% similarity cutoff, with ITS2 recovering almost double the OTUs for Aspergillus, Penicillium and Claviceps.
Transcription factor Zfp1 and its role in Ustilago maydis pathogenesis. H. Y. K. CHEUNG, M. E. DONALDSON, K. L. SPENCE and B. J. SAVILLE. (M.E.D., B.J.S.) Environmental and Life Sciences Graduate Program, Trent University, Peterborough, ON K9J 7B8, Canada; (K.L.S.) Department of Population Medicine, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (M.E.D., B.J.S.) Forensic Science Program, Trent University, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada
The basidiomycete biotrophic plant pathogen, Ustilago maydis (DC.) Corda, alters the host (Zea mays) to favour fungal growth through the secretion of effectors. The function of effectors has been well studied, yet little is known about the control of their expression. We identified a U. maydis transcription factor linked to effector gene expression, zinc finger protein 1 (Zfp1). It contains a DNA binding Zn(II)2Cys6 binuclear cluster domain and is localized to the nucleus. Infection by ∆zfp1 (solopathogen deletion) strains is reduced. Pathogenesis, when it occurred, produced little or no anthocyanin and arrested at the leaf tumour stage. Complementation with wild-type zfp1 partially restored pathogenesis and fully complemented anthocyanin production. Putative Zfp1 target genes were identified by RNA-seq analysis. Of the 1870 genes with significantly altered transcript levels in ∆zfp1 infections, 111 code for predicted effectors, with a majority of the altered effectors down-regulated relative to wild-type infections. Analysis of the corn transcriptome in ∆zfp1 infections relative to wild-type infections revealed increased photosynthesis gene transcript levels, consistent with the maintenance of C4 photosynthesis observed in uninfected corn, as well as decreased transcript levels of genes encoding hormone metabolism, secondary metabolism and pathogenesis-related proteins. Microscopy investigation of ∆zfp1 in planta development showed minimal hyphal branching consistent with the host detecting and containing fungal growth. This plant response is consistent with a change in effector gene expression by U. maydis ∆zfp1 strains. The combined analyses support a role for Zfp1 as a transcription factor involved in pathogenesis through the control of effector gene expression.
What are Plant Canada and the Global Plant Council? D. ERRAMPALLI. Agriculture and Agri-Food Canada, 4902 Victoria Avenue North, Vineland Station, ON L0R 2E0, Canada
Plant Canada Federation of Canadian Plant Science Societies is an independent, not-for-profit umbrella organization of seven Canadian plant science societies including Canadian Phytopathological Society, Canadian Botanical Association, Canadian Society of Plant Biologists, Canadian Weed Science Society, Canadian Society of Agronomy, Canadian Society for Horticultural Science, Canadian Association for Plant Biotechnology. Plant Canada seeks to being together all those in research, education and training in plant science and related disciplines in Canada. The Global Plant Council is a coalition of national, regional and international societies representing plant, crop and agricultural and environmental sciences across the globe. The global plant council has 28 member societies from six continents. Plant Canada is the founding member of the Global Plant Council. Deena Errampalli, the president of Plant Canada, presented goals and achievements of both these organizations.
Management of apple scab in organic apple orchards. D. ERRAMPALLI, A. HALDAR, C. JACKSON, A. ZWIEP, F. BETANCOURT, L. KRZYWDZINSKI, M. PARCEY AND K. SCHNEIDER. Agriculture and Agri-Food Canada, 4902 Victoria Avenue North, Vineland Station, ON L0R 2E0, Canada
Apple scab caused by Venturia inaequalis (Cooke) Wint. occurs in many apple growing regions in the world, including Ontario, Canada and also causes economic losses. The conidia of V. inaequalis are spread to leaves and the developing fruit through rainfall and air. This study aims to examine the effect of organic fungicides against V. inaequalis in reducing the development of apple scab in ‘McIntosh’ and ‘Empire’ apples in the orchard. During the summers of 2014 and 2015, apples were sprayed with three organic fungicides, a water control and a chemical control (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide). The three organic fungicides used were Sulphur (Kumulus), a 0.25% Reynoutria sachalinensis (Regalia #1) and 0.25% pre bloom and 0.75% post bloom R. sachalinensis (Regalia#2). A disease rating scale was used to take observations of the presence of apple scab on ‘McIntosh’ leaves and fruits. Observations on apple scab disease were made over a period of 3 months in 2014 and in 2015 which showed a gradual increase in disease progression. Two-way ANOVA tests were also performed to determine any statistical differences between and among all five treatments. Results indicated that the Sulphur treatment was the most effective organic fungicide followed by R. sachalinensis in reducing apple scab disease in ‘McIntosh’ apple leaves and fruits.
Sequence-based identification of fungi from ginseng roots and soils. D. ERRAMPALLI, C. NICOL, A. HALDAR, M. PARCEY, K. I. SCHNEIDER AND S. WESTERVELD. Agriculture and Agri-Food Canada, 4902 Victoria Avenue North, Vineland Station, ON L0R 2E0, Canada; and (S.W.) Ontario Ministry of Agriculture, Food and Rural Affairs, 1283 Blueline Road, Simcoe, ON N3Y 4N5, Canada
Replant disease causes severe yield losses in ginseng. Detection and identification of pathogens are the first critical steps in developing disease control. The goal of this study was to determine whether sequence analysis of internal transcribed spacer region (ITS) can be used to detect and identify fungal pathogens in soils in which the ginseng was grown and symptomatic ginseng roots. Soils were collected from gardens (fields) with and without replant disease (control) in 2014. Ginseng roots with symptoms were collected from three ginseng gardens in Ontario. Fungi were isolated from soils and root samples on different selective media. Genomic DNA was extracted from each of the fungal isolates and the ITS region was amplified by PCR with ITS1 and ITS4 primers. The amplified DNA was sequenced and aligned against sequences in GenBank. Sequence based identification was carried out on 109 fungal isolates: (a) Fusarium oxysporum Schlecht. emend Snyder & Hans. (44%), Fusarium solani (Mart.) Sacc. (14.8%), Cladosporium cladosporoides (Fresen.) de Vries (16.0%), and Fusarium spp. (13%) were from 55 isolates from the soils in which ginseng was cultivated, (b) Bionectria spp./Clonostachys spp. (27.3%), Cylindrocarpon spp. (13.6%) and Mortierella spp. (13.6%) were from 22 isolates from the soils around wild ginseng plants, and (c) F. solani (46%), Epicoccum nigram Link (12.5%) and Pseudallescheria boydii (Shear) McGinnis et al./Scedosporium minutisporum (Gilgado et al.) Lackner & de Hoog (9.4%) were from 32 isolates from the symptomatic ginseng roots. This information is important for determining the complex that causes ginseng replant disease.
Effect of maleic hydrazide on stem rust seedling infection type. T. FETCH JR. Brandon Research and Development Centre, Agriculture and Agri-Food Canada, 2701 Grand Valley Road, Brandon, MB R7A 5Y3, Canada
Stem rust of wheat (Triticum aestivum), caused by Puccinia graminis f. sp. tritici Pers.:Pers./Eriks. & Henn., has recently inflicted substantial yield losses worldwide due to new races that have arisen. Races are typically characterized by their reaction (infection type, IT) on the first leaf of single-gene differential lines. Rating the IT is often hampered by extensive growth of subsequent leaves, and IT can be affected by shading. Maleic hydrazide (MH) is a growth inhibitor used by rust pathologists to stunt leaf elongation, but the effect of MH on stem rust IT is unknown. Seeds of 20 single-gene differential lines were planted in root-trainers, and 10 mL of MH (0, 100, 200, 300, 400 or 500 ppm) were applied at primary leaf tip emergence. Seedlings were inoculated 6 days after MH application with race QCCJB. Seedling ITs were assessed 14 days later, and first and second leaf lengths (four reps) were measured. The MH treatments did not significantly reduce the length of primary leaves (except Sr9d), but significantly reduced second leaf length, starting at 100 ppm for most lines. Treatment using MH generally increased pustule size and IT score, but did not greatly alter the IT response of genes Sr6, Sr9e, Sr30, Sr36, Sr38 or SrMcN. However, MH treatments substantially changed the IT from resistant to susceptible for genes Sr7b, Sr8a, Sr9a, Sr9b, Sr11, Sr24 and SrTmp, beginning at the 200 ppm concentration. Further work is needed to optimize the MH application level that will reduce the length of second leaves but not affect the IT response.
A new twist on antibiosis: Exposure to sub-inhibitory concentrations of antibiotics alters the transcriptome of plant pathogens. M. FILION. Université de Moncton, 18 Antonine-Maillet, Moncton, NB E1A 3E9, Canada
Antibiotic-producing rhizobacteria show promise for use as successful biocontrol agents against many plant pathogens affecting agricultural crops. Antibiosis, or the capacity to control plant pathogens using antagonistic microorganisms that produce antibiotics, usually involves a significant reduction in the pathogen’s population following exposure to lethal doses of antimicrobial compounds, leading to reduced disease pressure and symptoms. Although this classical definition of antibiosis has been shown to be the main biocontrol mechanism operating in many pathosystems, recent advances in transcriptomics depict an alternate mode of action for antibiosis. It is now increasingly believed that under natural rhizosphere conditions, mostly sub-lethal doses of antibiotics are produced by rhizobacteria and therefore pathogen populations are not necessarily altered when exposed to such low doses of antibiotics. Instead, exposition to sub-lethal doses of antibiotics may lead to targeted effects on the expression of key genes involved in pathogenesis, leading to reduced disease symptoms. In this presentation, the impact of the phenazine-1-carboxylic acid producer Pseudomonas fluorescens LBUM223 on the transcriptome of the plant pathogen Streptomyces scabies (ex Thaxter) Lambert & Loria causing common scab of potato was presented to illustrate this new mode of action for antibiosis.
DNA-barcoding the rusts – sampling herbarium specimens in the National Mycological Herbarium (DAOM). S. HAMBLETON, Q. EGGERTSON, S. WILSON, S. A. REDHEAD AND C. A. LEVESQUE. Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, K.W. Neatby Building, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
Plant pathologists, diagnosticians and regulatory agencies rely on access to comprehensive, accurate and curated reference DNA sequence databases. These data are essential for rapid and authoritative identification of plant pathogens in field samples, commodity imports/exports and environmental samples as represented by DNA sequences derived from next-generation sequencing (NGS) techniques. Herbarium collections are the most easily accessible source of obligate fungal pathogens and their hosts, with specimens often dating back to the 1800s and representing a broad diversity of hosts and geographic provenance. A processing pipeline was developed to minimize destructive sampling of these irreplaceable collections while maximizing DNA yield and facilitating high throughput. DNA-barcoding techniques were applied to both pathogen and host, targeting the ITS2 and rbcLa gene regions respectively, for over 1200 specimens of rust fungi (Pucciniales/Uredinales) preserved in DAOM. For specimen selection, from the more than 38 K available, we focused on species of quarantine concern as listed by various countries, those not yet represented in public sequence databases, those occurring on agricultural hosts, and alternate hosts for species with complex life cycles. Our success rate ranged from 51–72% (rusts, depending on genus) and 60% (hosts), and the oldest specimen successfully sequenced for both was collected in 1889. We now have consensus sequences representing 88% (23/26) of genera and 73% (242/330) of species sampled. The primary impacts will be to increase the number of publicly available and authoritatively identified reference DNA barcodes and reduce the risk of misinterpreting DNA signatures of innocuous species with those of regulated species.
DNA-barcoding the Powdery Mildews – sampling herbarium specimens in the National Mycological Herbarium (DAOM). S. HAMBLETON, Q. EGGERTSON, C. A. LEVESQUE, W. CHEN, T. BARASUBIYE, S. A. REDHEAD AND M. LIU. Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, K.W. Neatby Building, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
The obligate plant pathogens known as Powdery Mildews (Erysiphales) comprise a morphologically diverse group of fungi producing conspicuous symptoms of white mycelium covering mainly leaves. These fungi infect a wide range of plants, causing premature leaf fall, hypertrophy and deformation, and consequently significant economic losses in agricultural, horticultural and viticulture industries. Since the description of the first powdery mildew by Linnaeus in 1753, a comprehensive system for species identification gradually developed based on various biological characters and more recently, incorporating molecular characters. Although using DNA sequences is an efficient and effective approach for diagnoses by regulatory departments and pest diagnostic agencies, considerable data gaps still exist for DNA-based identifications and species complexes remain unresolved. In a metagenomics study of environmental samples, only 3% OTUs matched to the genera Podosphaera and Erysiphe could be identified to species. To fill gaps in reference sequences, 305 historical specimens of 149 species in 15 genera from DAOM were sampled to amplify ITS region for the pathogens and rbcLa for the hosts, using published primers from various sources. The preliminary data set generated included 105 ITS sequences of 35 species and 63 partial rbcLa of 15 species. Based on analyses supplemented with data from GenBank, the identifications of 82 DAOM specimens were revised in light of the new phylogeny-based classifications. No primer set was found to be universally specific for all species sampled and some also amplified non-target fungal contaminants co-occurring on the specimens. Primer design and optimization is a priority for this fungal group.
Protecting Canada’s forests using next generation genomic biosurveillance. R. C. HAMELIN. The University of British Columbia, 2329 West Mall, Vancouver, BC V6T 1Z4, Canada; and Institut de Biologie Intégrative et des Systèmes, Université Laval, Quebec, QC G1V 0A6, Canada
The world’s forests face unprecedented threats from invasive insects and pathogens that can cause large irreversible damage to the environment. This threatens Canada’s capacity to provide long-term fibre supply and ecosystem services that range from carbon storage, nutrient cycling, water and air purification, soil preservation and maintenance of wildlife habitat. The number of new introductions and interceptions of invasive alien forest pathogens is escalating at an alarming rate and the movement of goods and people is responsible for most of the documented incursions. The key to reduce the likelihood of invasive alien pathogen introductions is via vigilant biosurveillance combined with rapid and accurate detection. This leads to increased preparedness and early interventions that help prevent establishment. The TAIGA (Tree Aggressor Identification using Genomes Approaches) team has developed a pipeline to generate and analyse genome sequences and identify genome regions that are unique and can be translated into detection tools and regions that are highly variable and can be developed into monitoring tools. Real-time genome sequencing is promising to become part of routine diagnoses and could help predict disease outcome and model transmission risks to inform forest managers about prevention. Genome-wide population sequencing data will generate global databases that will provide accurate identification of forest enemies and reveal genomic patterns that can identify sources and novel variants and can inform outbreak management. This genomic biosurveillance pipeline has the potential to generate transformative changes to address the challenges of biosurveillance of invasive alien forest pathogens.
Fusarium graminearum chemotypes from infected winter wheat crops in Manitoba. M. A. HENRIQUEZ, B. D. MCCALLUM, M. F. BELMONTE, C. A. MCCARTNEY, T. OUELLET, F. M. YOU AND H. S. RANDHAWA. Morden Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 101 Route 100, Morden, MB R6M 1Y5, Canada; (M.F.B.) Department of Biological Sciences, University of Manitoba, 50 Sifton Road, Winnipeg, MB R3T 2N2, Canada; (T.O.) Ottawa Research and Development Centre, AAFC, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (H.S.R.) Lethbridge Research and Development Centre, AAFC, 5403 – 1st Avenue South, PO Box 3000, Lethbridge, AB T1J 4B1, Canada
Fusarium head blight (FHB) is mainly caused by various Fusarium spp., of which the most important is Fusarium graminearum Schwabe. FHB is considered as the most serious disease affecting wheat (Triticum aestivum L.), causing significant yield losses worldwide. Yield loss results mainly by reduction in grain quality due to shrivelled and/or discoloured kernels, which are referred to as Fusarium-damaged kernels (FDK) and contamination of grain with the trichothecene mycotoxin deoxynivalenol (DON). Fusarium graminearum frequently produces one of three sets of trichothecene metabolites: (i) deoxynivalenol and 3-acetyldeoxynivalenol (3-ADON chemotype), (ii) deoxynivalenol and 15-acetyldeoxynivalenol (15-ADON chemotype), or (iii) nivalenol and its acetylated derivatives (NIV chemotype). The objective of this research was to create a culture collection of F. graminearum species from infected winter wheat and to identify the most prevalent chemotypes in Manitoba. Spikes collected in 2015 from commercial winter wheat crops were processed for pathogen isolation and identification in the laboratory. Ninety-two monosporic cultures of Fusarium isolates from 32 fields were sequenced using the nuclear ribosomal intergenic spacer (IGS) to confirm their species identity. A multiplex-PCR was used to identify chemotype-specific nucleotide variation among a panel of F. graminearum isolates and it showed that 46 isolates were of 3-ADON chemotype and 25 isolates were of 15-ADON chemotype. Fusarium graminearum isolates from different geographic areas in Manitoba and representing different chemotypes will be tested in autumn/winter 2016/17 for their pathogenicity on winter wheat varieties. The most aggressive F. graminearum isolate will be selected for further transcriptome studies.
In vitro molecular interaction between the helper component-proteinase of Potato virus Y and cuticle proteins of potato aphid. R. HEPAT, S. BOQUEL AND X. NIE. Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada; and (S.B.) SIPRE – Comité Nord, Rue des Champs Potez, 62217 Achicourt, France
Potato virus Y (PVY) is transmitted by aphids in a non-persistent manner. Upon acquisition, the virus is retained within the stylets of aphids. When the viruliferous aphids probe on a plant shortly after the acquisition, the virus is released from the stylets into plant cells. For better management of PVY, a study was undertaken to identify aphid proteins that may be involved in the virus-vector interactions. Cuticle proteins (CuPs), which are a major component of the insect cuticle, were examined for in vitro binding to the PVY helper component-proteinase (HC-Pro) and purified PVY virions. Proteins in 8 M urea extracts from Macrosiphum euphorbiae and Myzus persicae were separated by 12% SDS-PAGE, electroblotted onto membranes, and incubated with cuticular protein-specific antibody for detection/identification of CuPs. Several CuPs ranging from 17 KD to 70 KD were detected. To test whether the CuPs interact with HC-Pro and PVY virion, the blotted protein extracts were overlaid with HC-Pro alone or in combination with purified PVY virion, and then incubated with HC-Pro-specific or PVY-specific antibodies. In blots overlaid with HC-Pro and incubated with HC-Pro antibody, CuPs-like proteins were detected; and in blots overlaid with HC-Pro+PVY and incubated with PVY antibody, similar CuPs-like proteins were also detected. Nevertheless, no bands were detected by PVY antibody in blots overlaid with PVY alone or HC-Pro alone. Together, these results demonstrate that HC-Pro acts as a bridge for PVY virion and aphid CuPs. Molecular identification of CuPs that interact with HC-Pro is underway.
An assessment of the genetic basis for resistance to stem rust race TRTTF in Canadian hexaploid wheat cultivars. C. W. HIEBERT, M. N. ROUSE, J. NIRMALA, C. A. MCCARTNEY, M. T. KASSA AND T. G. FETCH. Morden Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 101 Route 100 Morden, MB R6M 1Y5, Canada; (M.N.R., J.N.) United States Department of Agriculture-Agricultural Research Service (USDA-ARS) Cereal Disease Laboratory and University of Minnesota Department of Plant Pathology, 1551 Lindig Street, St. Paul, MN 55108, USA; (M.T.K.) National Research Council, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada; and (T.G.F.) Brandon Research and Development Centre, AAFC, 2701 Grand Valley Road, Brandon, MB R7A 5Y3, Canada
Stem rust, caused by Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. & E. Henn. (Pgt), is a serious disease of wheat (Triticum aestivum L.). Stem rust can be controlled by utilizing effective stem rust resistance (Sr) genes in wheat cultivars. Over the past decade a number of virulent races of Pgt have evolved in Africa and pose a threat to much of the global wheat producing areas. One such example is race TRTTF which was discovered in Yemen and is virulent to approximately half of the Canadian wheat cultivars tested. ‘Harvest’ was identified as a resistant cultivar. A doubled haploid (DH) population from the cross LMPG-6S/‘Harvest’ revealed a single gene for resistance to race TRTTF which was mapped to the terminal region of chromosome arm 6DS using DNA markers. Cultivars assayed with race TRTTF were genotyped with DNA markers close to the resistance on chromosome arm 6DS. Resistant cultivars either carried the same resistance on 6DS as ‘Harvest’ or were known carriers of SrCad, a gene known to confer resistance to the Ug99 group of Pgt races. A DH population from the cross RL6071/‘Peace’ was assayed with race TRTTF. This population was previously used to map SrCad. Resistance to race TRTTF and TTKSK (Ug99) co-segregated in the population. Thus, resistance to TRTTF in Canadian wheat cultivars is conferred by a gene on chromosome arm 6DS that is likely to be an allele of Sr8 and by SrCad.
Genetic diversity and colonization patterns of Onnia tomentosa in a plantation of black spruce (Picea mariana) in north-western Ontario. Z. R. W. HOEGY, D. MORRIS, D. REID AND L. J. HUTCHISON. Faculty of Natural Resources Management, Lakehead University, Thunder Bay, ON P7B 5E1, Canada; and (D.M., D.R.) Centre for Northern Forest Ecosystem Research, 421 James Street S., Suite 103, Thunder Bay, ON P7E 2V6, Canada
Onnia tomentosa (Fr.) Karst. is prevalent throughout North America, Europe and Asia and is responsible for causing a significant root-rot disease of conifers commonly known as stand-opening disease. Although the disease infects both spruce and pine, it is more severe on the former. In the late summer of 2014, the spatial coordinates of 124 basidiomata were taken, and the basidiomata collected from a 50-year-old black spruce plantation near Limestone Lake, north of Nipigon, Ontario that had undergone thinning treatments 6 years prior. The three thinning treatments were light thinning (25% tree removal), heavy thinning (45% tree removal) and control (no thinning occurred). There was also a clear-cut treatment, however, no basidiomata of O. tomentosa were found. Using extracted DNA from each of the basidiomata, single strand conformational polymorphism polymerase chain reaction (SSCP-PCR) of two nuclear loci, and DNA sequencing of two mitochondrial loci were used to measure genetic diversity and consequently genet size in order to see if stand density had an effect on O. tomentosa’s colonization patterns. One hundred and sixteen genetically distinct individuals were found, suggesting that the majority of the basidiomata represented unique genets. Stand thinning does seem to negatively influence O. tomentosa colonization, however, it is inconclusive whether the light or heavy thinning treatments are better at countering the fungal pathogen.
Use of genotype-by-sequencing to characterize populations of Plasmodiophora brassicae. M. D. HOLTZ, S. F. HWANG, J. ZANTINGE AND S. E. STRELKOV. Field Crop Development Centre, Alberta Agriculture and Forestry, 6000C and E Trail, Lacombe, AB T4L 1W1, Canada; (S.F.H.) Alberta Agriculture and Forestry, Crop Diversification Centre North, 17507 Fort Road, Edmonton, AB T5Y 6H3, Canada; and (S.E.S.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada
Plasmodiophora brassicae Woronin is an obligate and soil-borne parasite that causes clubroot disease in canola (Brassica napus L.) and other cruciferous crops. Recently, new aggressive populations of the pathogen, virulent on clubroot resistant canola cultivars, have been found in Alberta, Canada. In order to determine the relationship of members of these aggressive populations to other P. brassicae populations present in Canada, a genotyping-by-sequencing (GBS) method was employed. Twenty-one populations or single-spore isolates were analysed. DNA was extracted from spores purified from infected roots and used for GBS. Over 10 million sequences were generated using the Ion Torrent PGM™ platform. After variant calling and filtering over 15 000 variable loci were retained for analysis. Phylogenetic tree and population structure analysis clearly identified and separated the new aggressive populations from other P. brassicae populations. Thirty-six per cent of the loci were group-specific, with no shared alleles between the aggressive populations and the other samples. Of these group-specific alleles, over a third caused non-silent mutations. The sequence information produced should allow for the development of markers and assays for the detection of resistance-defeating P. brassicae populations.
Genome analysis and pathogenicity of a new potential biothreat, Pantoea allii, to onion production in Canada. S. HSIEH, R. XU, T. J. AVIS AND J. T. TAMBONG. Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (S.H., T.J.A.) Department of Chemistry, Food Science and Nutrition Program, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
Onion is a root vegetable that is commonly consumed all over the world. It is easily grown in different types of soil. In 2004, a new species of Pantoea, Pantoea allii Brady et al., isolated from onions in South Africa and USA, was found to cause pathogenic infections to onions. There is currently no evidence of the presence of P. allii in Canada. Given that onion is an important export/import crop, the introduction of this new pathogen into Canada could have severe economic consequences. Thus, it is important to proactively study this ‘new’ pathogen, especially at the genome level, to generate new knowledge that can be used to develop diagnostic tools. Illumina MiSeq sequencing technology was used to generate paired-end reads and de novo assemblies were performed using CLC Genomics Workbench and ABySS platforms. In addition, the pathogenicity of P. allii DOAB 206 on commercially available Canadian bulb and green onions was investigated. The genome data based on the number of contigs, N50 and total sum of nucleotides, concluded that ABySS is the better platform. The size of the draft genome is about 5.2 Mb with 4880 protein-encoding sequences. In silico DNA-DNA hybridization of the draft genome sequence of P. allii with the closest phylogenetic relative, Pantoea ananatis revealed a 35.7–36.5% similarity. Pathogenicity tests showed that this pathogen can infect Canadian bulb and green onions. Precautionary measures are required to prevent infected onion imports from introducing P. allii into Canada.
Effects of dazomet on clubroot and root rot of canola. S. F. HWANG, H. U. AHMED, S. E. STRELKOV, Q. ZHOU, B. D. GOSSEN, M. R. MCDONALD, G. PENG AND G. D. TURNBULL. Crop Diversification Centre North, Alberta Agriculture and Forestry, 17507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; (S.E.S.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (B.D.G., G.P.) Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; and (M.R.M.) Department of Plant Agriculture University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada
Experiments were conducted to examine the effectiveness of dazomet (trade name Basamid) in preventing infection of canola (Brassica napus L.) by Plasmodiophora brassicae Woronin. Greenhouse studies showed that seedling emergence and plant height increased, and infection (both primary and secondary) and clubroot severity decreased with increasing rates of dazomet pre-treatment of P. brassicae-infested soil. Under field conditions, clubroot severity was reduced and seed yield increased with increasing dosage of dazomet. However, seedling emergence, root mass and seed yield were reduced, especially at 400–800 kg ha−1. Dazomet also increased emergence, plant survival and plant biomass in P. brassicae-infested soils that were inoculated with the soil-borne pathogens Fusarium avenaceum (Fr.) Sacc., Pythium ultimum Trow and Rhizoctonia solani Kühn. These results indicate that dazomet may be an effective tool for the management of both clubroot and seedling blight of canola.
Mining the microbiomes of crop wild progenitors for co-evolved beneficial microbes. G. IRIARTE, I. HALE AND K. BRODERS. (G.I.) Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH 03824, USA; (I.H.) Department of Biological Sciences, University of New Hampshire, Durham, NH 03824, USA; and (G.I., K.B.) Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523, USA
Wild progenitors of crop plants are potentially valuable to modern plant improvement efforts not only as sources of ‘lost’ genetic diversity but also, if grown in situ, as assays for beneficial co-evolved microbial associations lost during the processes of domestication and translocation from centres of origin. To explore this idea, we investigated genotypes belonging to the Mesoamerican pool of common bean (Phaseolus vulgaris) due to the fact that both wild and cultivated forms of P. vulgaris can still be found growing in central Mexico, the centre of diversity and one of the centres of domestication of the species. Replicated common gardens of six wild accessions and six cultivated varieties were grown in five locations: three in situ among wild Phaseolus populations in central Mexico and two ex situ under agricultural conditions in the north-eastern USA. Bacterial and fungal communities of the soil, rhizosphere and roots were characterized via metagenomic analyses of the 16S and ITS regions, respectively. Sample type (bulk soil vs. rhizosphere vs. root) was associated with significant variation in microbial diversity across locations. Of particular interest was the varying compositions of microbial communities between rhizosphere and root samples, as such differences indicate the root colonization of both bacteria and fungi through either active or passive means in all locations. Finally, we identified and cultured potentially novel microbial species that associated with cultivated beans grown in situ in Mexico but were not found on cultivated beans when grown in the USA. These organisms represent targets for future inoculation studies.
Genes differentially expressed during pathogenesis by two Plasmodiophora brassicae pathotypes on canola (Brassica napus). J. JIANG, R. FREDUA-AGYEMAN, S. F. HWANG AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (R.F.A., S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Forestry, 17507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada
Gene expression studies offer a means of understanding pathogenesis of clubroot disease caused by Plasmodiophora brassicae Woronin. In this study, the commercial canola cultivar ‘45H29' was inoculated with two P. brassicae pathotypes 5x and 5 and the pathogen/plant biomass was determined 7, 14 and 21 days after inoculation (dai) by quantitative real-time PCR (qPCR). To identify genes differentially expressed during the infection process, 205 genes encoding signal peptides were screened on total RNA extracted from the 14 dai samples. It was observed that the P5x/plant biomass increased across the time course while the P5/plant biomass decreased. The biomass and phenotypic data confirmed that P5x was virulent on 45H29 while P5 was avirulent. The qPCR results showed that only one of the 205 genes was up-regulated in samples inoculated with P5x than P5 while 15 genes were up-regulated in samples inoculated with P5 than P5x. At 21 dai, 12 of the above 16 genes were differentially expressed in the two pathotypes with nine being expressed higher in P5x and three being higher in P5. On the other hand, none of the 16 genes showed significant (α = 0.05) expression difference in the two pathotypes at 7 dai. Nine of the 16 genes were exclusively present in P. brassicae and may offer important information on the canola–P. brassicae interaction.
What’s on my weed? Preliminary insights into the cannabis-powdery mildew pathosystem. D. L. JOLY, N. PÉPIN, F. SORMANY, A. ROY AND N. HACHÉ. Université de Moncton, 18 avenue Antonine-Maillet, Moncton, NB E1A 3E9, Canada; and (A.R., N.H.) OrganiGram Inc, 35 English Drive, Moncton, NB E1E 3X3, Canada
Despite its longstanding history of cultivation as a source of fibre, food and medicine, knowledge on Cannabis L. diseases and pests is lacking, mainly because marijuana represents the most commonly used illicit drug. In Canada, the current number of licensed producers of medical marijuana is limited, but the number of consumers has increased intensely, raising concerns about the capability to meet demand. A better understanding of diseases and pests affecting Cannabis thus offers tremendous opportunities for increasing production and reducing crop losses, and also securing a continuous access to medical marijuana. Among diseases affecting Cannabis grown for medical purposes, powdery mildew is a recurring issue, especially with the high humidity and lack of air movement found in indoor growth rooms, which provide the perfect conditions for the germination and proliferation of fungal pathogens. In order to gain insight into the identity of powdery mildew species affecting Cannabis, sequence comparisons have been made with isolates from hops (Humulus lupulus L., the closest relative of Cannabis) and well-known powdery mildews. On the host side, a genome-wide analysis of the Mildew resistance Locus O (MLO) gene family has been conducted, and follow-up studies will investigate whether loss-of-function mutations in one or more of these candidate genes leads to powdery mildew resistance.
The dynamics of biomass accumulation by Ptr ToxA and Ptr ToxB producing isolates of Pyrenophora tritici-repentis on wheat. X. MA, R. ABOUKHADDOUR, S. F. HWANG AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Forestry, 17507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada
The fungus Pyrenophora tritici-repentis (Died.) Drechs. causes tan spot of wheat. Fungal virulence is mediated by the production of several necrotrophic effectors that selectively induce foliar necrosis or chlorosis on sensitive host genotypes. These include the small proteins Ptr ToxA and Ptr ToxB, which cause necrosis and chlorosis, respectively, and are encoded by the ToxA and ToxB genes. While sensitivity to both proteins is widespread in Canadian wheat cultivars, Ptr ToxA producing isolates of the fungus are much more common than Ptr ToxB producers. In order to assess whether or not Ptr ToxA confers a greater competitive advantage to the fungus than Ptr ToxB, the accumulation of fungal biomass was compared following the inoculation of ToxA+ and ToxB+ isolates in various combinations onto a wheat genotype sensitive to both proteins. Fungal biomass was measured over a 5-day period by quantitative PCR analysis with ToxA, ToxB and chitin synthase gene-specific primers. When the isolates were inoculated individually, fungal biomass accumulation was significantly greater for the ToxA+ vs. the ToxB+ isolate. However, when the isolates were inoculated together, the amount of biomass of the ToxA+ isolate decreased while that of the ToxB+ isolate increased, although it remained lower than the ToxA+ isolate. The results suggest that production of Ptr ToxA allows for greater accumulation of fungal biomass and/or host colonization by P. tritici-repentis than production of Ptr ToxB, and that colonization by the ToxA+ isolate facilitated growth by the ToxB+ isolate.
Nematology research at Agriculture and Agri-Food Canada: renewed emphasis on an old pest. C. KORA, Q. YU, B. MIMEE AND T. A. FORGE. Pest Management Centre, Agriculture and Agri-Food Canada (AAFC), 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; (Q.Y.) Ottawa Research and Development Centre, Canadian National Collection of Nematodes, AAFC, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; (B.M.) Saint-Jean-sur-Richelieu Research and Development Centre, AAFC, 430 Gouin Blvd, St-Jean-sur-Richelieu, QC J3B 3E6, Canada; and (T.A.F.) Summerland Research and Development Centre, AAFC, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada
Agriculture and Agri-Food Canada (AAFC) scientists, through their research and discoveries, have made remarkable contributions to the advancement of the science of plant nematology in Canada. From the early 1960s to the late 1980s numerous AAFC nematologists expanded our knowledge of the biology, management and impact of nematodes on important crops, particularly for plant parasitic nematode species of concern to Canadian agriculture. However, with the extensive use of broad spectrum fumigant nematicides through the 1990s and early 2000s, the problems associated with nematode damage seemed to decline, allowing growers to shift their attention to other pest issues of priority. With this shift, the applied nematology research within AAFC was also marked by a period of decline in activity and scope. However, changes in cropping systems, production practices and regulations over the past 15 years are having an impact. The increase in production of crops susceptible to nematodes, several nematode outbreaks with large economic impact, as well as restrictions and phase-outs to the use of fumigants which were found to have unacceptable environmental impacts, have led to renewed awareness of the importance of plant parasitic nematodes and the need for new effective non-fumigant nematode management practices. As a result, nematology research programmes within AAFC have gained new strength and are once more the focus of expertise and capacity building. Besides providing an historic perspective, this poster highlights current AAFC nematology research and development activities which aim to deliver sustainable nematode management options for the agricultural sector.
Re-classification of Clavibacter michiganensis subspecies into multiple new species. X. LI, J. TAMBONG, X. YUAN, H. XU AND S. H. DE BOER. Canadian Food Inspection Agency, Charlottetown Laboratory, 93 Mt. Edward Road, Charlottetown, PE C1A 5T1, Canada; and (J.T.) Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
The bacterial genus Clavibacter is monospecific consisting of only one species, C. michiganensis, subdivided into five subspecies, each with specificity for a single host crop. Clavibacter michiganensis subsp. michiganensis (Smith) Davies et al. (Cmm) causes bacterial canker of tomato (Solanum lycopersicum), C. m. sepedonicus (Spieck. & Kotth.) Davies et al. (Cms) causes bacterial ring rot of potato (Solanum tuberosum), C. m. insidiosus (McCulloch) Davies et al. (Cmi) causes bacterial wilt of alfalfa (Medicago sativa), C. m. nebraskensis (Vidauer & Mandel) Davies et al. (Cmn) causes the Goss’s bacterial wilt of maize (Zea mays) and C. m. tesselarius (Carlson & Vidaver) Davies et al. (Cmt) causes bacterial mosaic disease of wheat (Triticum spp.). The taxonomic position of the subspecies was evaluated using whole genome sequences of multiple strains of Cmm, Cms, Cmi, Cmn and Cmt, including type strains. Genomes were decoded using either BacBio single molecule real-time sequencing (McGill University and Genome Quebec Innovation Centre, Montreal, QC) or paired-end Illumina HiSeq sequencing with True Seq version 3 chemistry (National Research Council Canada, Saskatoon, SK, Canada). The assembled sequences were annotated and compared with sequences of Cmm, Cmi and Cms, and other Clavibacter spp. deposited in GenBank. Average nucleotide identity (ANI) values ranged from 91–95% among subspecies while pair-wise comparison among strains of the same subspecies had ANI values of 99–100%. Based on ANI values, 16S rRNA sequences, multi-locus sequence typing of house-keeping genes, and phenotypic characteristics, the taxonomic position of the phytopathogenic clavibacter subspecies should be raised to species status within the genus Clavibacter as C. michiganensis sp. nov., C. sepedonicus sp. nov., C. insidiosus sp. nov., C. nebraskensis sp. nov. and C. tessellarius sp. nov.
Identification of Albugo candida causing white blister rust of Wasabia japonica in British Columbia. J. L. MACDONALD AND Z. K. PUNJA. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada; and (Z.K.P.) Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Commercial plantings of Wasabia japonica (Miq.) Matsu. are expanding in the Pacific Northwest, where it is utilized in the culinary market and in sushi restaurants. The high moisture environment required to cultivate the crop has led to an increasing incidence of disease and occurrence of a number of destructive root and foliar pathogens. In 2015, wasabi (‘Daruma’) grown in a research polyethylene house in Agassiz, BC showed symptoms of white blister rust, including severe blistering and sori development. Sporangiospores were collected from white blister leaf samples into a water suspension and were morphologically identified as either Albugo candida (Pers. ex Lev.) Kuntze or A. wasabiae Hara. Sporangiospores were collected from white rust infected Capsella bursa-pastoris (L.) Medik in close proximity to the crop, and were shown to be morphologically indistinct. Genomic DNA was extracted and PCR conducted using ITS1 F-ITS4 fungal primers. Sequence comparison in GenBank showed both isolates were A. candida. Inoculations with sporangiospores collected from W. japonica could not confirm pathogenicity on either W. japonica or C. bursa-pastoris; however, inoculations on W. japonica with sporangiospores collected from C. bursa-pastoris resulted in small galls. Albugo white rust can be a major foliar disease of wasabi in parts of Asia, but fungicide applications can control it. In Canada and the USA there are no products currently registered for the control of white rust on wasabi, and the growing complex of diseases will continue to be a major obstacle for the industry.
Transcriptomic insight into emerging wheat leaf rust races of Ontario. K. M. MARSH, B. D. MCCALLUM, X. WANG, A. TENUTA AND B. J. SAVILLE. (K.M.M., B.J.S.) Environmental & Life Sciences Graduate Program, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, ON K9 J 7B8, Canada; (B.D.M., X.W.) Morden Research and Development Centre, Agriculture and Agri-Food Canada, 101 Route 100, Morden, MB R6M 1Y5 Canada; (A.T.) Ontario Ministry of Agriculture and Rural Affairs, Ridgetown Resource Centre, Agronomy Building, Main Street East, Ridgetown, ON N0P 2C0, Canada; and (B.J.S.) Forensic Science Program, DNA Building, Trent University, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada
Puccinia triticina Eriks., wheat leaf rust fungus (WLR), is the most economically damaging rust pathogen on a global scale. The emergence of a WLR race in Ontario with a new virulence type enabled a study of the transcriptome changes occurring in the newly emerged race relative to its putative ancestors. This investigation utilized wheat near isogenic lines (NIL) carrying different WLR resistance genes, the isolated new WLR race, and other races thought to be its potential ancestors. Illumina paired-end RNA-seq was carried out on specific infections of wheat NILs with WLR races. Fungal transcripts were aligned to 15 686 genes from the WLR reference sequence. The identification of potential effector genes whose expression level was altered in the new WLR race relative to presumed ancestors was completed using a conservative approach. The selection of altered candidate effectors included ascertaining which genes were 2-fold up- or down-regulated, identifying transcript ORFs containing secretion signals and detecting SNPs which altered protein secondary structure. This process resulted in a list of 97 candidate effector-encoding genes. Of these, seven unique genes were identified to have at least one non-synonymous SNP which altered the amino acid sequences and secondary structure of the resulting protein. If these gene alterations influence virulence, then we will have identified a basis for new WLR virulence types. Predicted orthologues to these candidate effector genes were found in Ustilago maydis (DC.) Corda, a model plant pathogen. Functional investigation of the U. maydis orthologues is currently underway.
Developing a wheat germplasm collection with diverse pathogen and pest resistance. B. D. MCCALLUM, C. W. HIEBERT, T. G. FETCH, C. A. MCCARTNEY, M. A. HENRIQUEZ, H. S. RANDHAWA AND S. J. CLOUTIER. Morden Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 101 Route 100, Morden, MB R6M 1Y5, Canada; (T.G.F.) Brandon Research and Development Centre, AAFC, 2701 Grand Valley Road, Brandon, MB R7A 5Y3, Canada; (H.S.R.) Lethbridge Research and Development Centre, AAFC, 5403 1st Avenue South, P.O. Box 3000, Lethbridge, AB T1J 4B1, Canada; and (S.J.C.) Ottawa Research and Development Centre, AAFC, K.W. Neatby Building, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
Drs Peter Dyck and Eric Kerber requested bread wheat (Triticum aestivum L.) and durum wheat (Triticum durum Desf.) germplasm from genetic banks across the world during the 1970s and 1980s. These diverse collections were screened at the time for leaf rust [Puccinia triticina Eriks.] and stem rust [Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. & E. Henn.] resistance. In 2014, we initiated a project to identify promising lines from this material for leaf rust resistance by growing 200 lines in a leaf rust nursery. Resistant lines were harvested and retested in 2015, along with a new group of 200 lines. One hundred and forty six were found to have resistance to leaf rust in the field in 2014 and/or 2015. These lines will be tested in 2016 for resistance to leaf rust, stem rust, stripe rust [Puccinia striiformis Westend.], Fusarium head blight [Fusarium graminearum Schwabe] and orange wheat blossom midge [Sitodiplosis mosellana Gehin]. A new group of 200 lines from the Kyoto wheat collection will be screened in 2016 for leaf rust resistance, and resistant lines will be added to the germplasm collection for screening with multiple pests in 2017. We plan to generate a large and diverse collection of wheat germplasm, characterized for resistance to major pests of wheat in Canada. This should be an excellent resource for the development of resistance in wheat breeding programmes. The genetic basis for resistance will be determined for many of the lines with the best levels of resistance to multiple pathogens.
Variation in boron tolerance and clubroot severity in Brassica napus and B. rapa lines in a field trial in 2015. A. MCLEAN, 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
In a previous study, boron (B) applied at 4 kg ha−1 reduced clubroot [Plasmodiophora brassicae Woronin] incidence and severity, but higher rates produced severe toxicity on canola (Brassica napus L.). Variability in B-tolerance and clubroot severity were assessed on lines of B. napus and B. rapa in a field trial on muck (~70% organic matter) soil infested with P. brassicae at the Muck Crops Research Station in Bradford, Ontario in 2015. Seedlings of 88 accessions were grown in a greenhouse for 3 weeks, then transplanted to the field. Boron (Solubor, 20.5% B) was applied at 8 kg B ha−1 in 1500 L ha−1 of water using a backpack CO2 sprayer 3 days after transplanting. Boron was reapplied 5 days after the initial application because leaching by heavy rainfall had reduced the effective rate of applied B. Toxicity was assessed 5 days after the second boron application and categorized by degree of leaf cupping and marginal burning (0–3 scale). Plants were harvested 9 weeks after seeding. Clubroot severity was rated (0–3 scale) and phytotoxicity and clubroot severity indices were calculated for each line. The above-ground growth (fresh and dry weight) of selected B-susceptible and B-tolerant lines was assessed. There were differences in boron tolerance among accessions. The only difference in clubroot severity was observed in the selected subset of B-tolerant accessions, where application of B reduced clubroot severity and increased fresh weight. Also, the water content and fresh weight of B-treated and non-treated plants from the B-tolerant and B-susceptible subsets differed.
Genomic screens to identify next-generation MAMPs and their cognate pattern recognition receptors. A. G. MOTT, S. THAKUR, E. SMAKOWSKA, P. W. WANG, Y. BELKHADIR, D. S. GUTTMAN AND D. DESVEAUX. (A.G.M., S.T., D.S.G., D.D.) Department of Cell & Systems Biology, University of Toronto, 25 Willcocks Street, Toronto, ON M5S 3B2, Canada; (E.S., Y.B.) Gregor Mendel Institute, Austrian Academy of Sciences, Vienna Biocenter, Dr Bohr Gasse 3, Vienna 1030, Austria; and (P.W.W., D.S.G., D.D.) Centre for the Analysis of Genome Evolution and Function, University of Toronto, 25 Willcocks Street, Toronto, ON M5S 3B2, Canada
The front line of plant defence against pathogens depends on the action of extracellular leucine-rich repeat, receptor-like kinases (LRR-RLKs), which serve as Pattern Recognition Receptors (PRRs) to recognize essential and therefore evolutionarily conserved features of pathogens called Microbe-Associated Molecular Patterns (MAMPs). MAMP recognition by PRRs activates PRR-triggered immunity (PTI) which suppresses the growth of nearly all ‘non-host’ microbes, as well as many potential pathogens. Next generation sequencing of Pseudomonas syringae van Hall pathovars has allowed the successful in silico prediction of MAMPs through the identification of positive selection signatures on proteins of the core genome. Although these ‘next-generation MAMPs’ induce the hallmark responses of PTI, including virulence suppression, the PRRs that recognize them remain to be identified. I will present our latest genomic approaches to identify novel MAMPs and their cognate PRRs, as well as our efforts to translate resistance conferred by these genes into agricultural crops.
Downy mildew and cone diseases of hop in Ontario in 2015. A. MUNAWAR, A. F. SHI, M. FILOTAS, C. BAKKER AND M. R. MCDONALD. Department of Plant Agriculture, Simcoe Research Station, University of Guelph, 1283 Blueline Road, P.O. Box 587, Simcoe, Ontario, N3Y 4N5, Canada; (A.F.S.) Ontario Ginseng Growers Association, Simcoe Research Station, 1283 Blueline Road, P.O. Box 587, Simcoe, ON N3Y 4N5, Canada; and (M.F.) Ontario Ministry of Agriculture, Food and Rural Affairs, 1283 Blueline Road, P.O. Box 587, Simcoe, ON N3Y 4N5, Canada
Hops (Humulus lupulus L.) is a re-emerging crop in Ontario with an increasing demand for high quality cones by both industrial and craft beer brewers. However, downy mildew (DM, caused by Pseudoperonospora humuli (Miyabe & Takah.) G.V. Wilson) and cone diseases are affecting the quality of hops. This project is focused on evaluating the spread of hop downy mildew, its effects on crop quality and effects of cone diseases at harvest. In summer 2015, three commercial hop yards and one replicated hop cultivar trial at the Simcoe Research Station (SRS) in Ontario, Canada were scouted for hop diseases. Most of the plants in all three yards showed initial DM symptoms in the form of occasional basal spikes and foliar DM lesions, however the disease did not progress further and impact on yield remained low. Alternaria cone disorder was the most prevalent disease as it was found in seven of the nine hop cultivars grown at the SRS. Other than Alternaria spp., Botrytis spp. was also isolated from cones of ‘Sterling’ and ‘Chinook’ at SRS. Alternaria was also found in most of the cone samples from the commercial hop yards. At the SRS, powdery mildew caused severe damage on ‘Bertwell’ cones, resulting in no marketable yield. Powdery mildew was not found in cones collected from commercial yards. In order to determine if P. humuli was present in hop rhizomes used for propagation, four rhizomes were collected from growers in 2015. The morphological and molecular analysis confirmed the presence of P. humuli in one of the rhizomes.
An investigation of fungal isolates associated with ginseng diseases. A. MUNAWAR, A. F. SHI, S. WESTERVELD AND M. R. MCDONALD. Department of Plant Agriculture, Simcoe Research Station, University of Guelph, 1283 Blueline Road, P.O. Box 587, Simcoe, ON N3Y 4N5, Canada; (A.F.S.) Ontario Ginseng Growers Association, Simcoe Research Station, 1283 Blueline Road, P.O. Box 587, Simcoe, ON N3Y 4N5, Canada; and (S.W.) Ontario Ministry of Agriculture, Food and Rural Affairs, 1283 Blueline Road, P.O. Box 587, Simcoe, ON N3Y 4N5, Canada
Ginseng, which is grown for its medicinal value, is a valuable horticultural crop in Ontario. Due to the 3–4 year life of a ginseng garden, roots are affected by a number of soil-borne pathogens. Although major pathogens for ginseng have been characterized, many unknown diseases were observed in the field. Therefore, there is a need to do a baseline study to investigate causal agents of ginseng diseases which may lower its marketable yield. In autumn 2015, 3-year-old ginseng roots were harvested from 10 commercial gardens. Roots with symptomatic rots were separated into individual disease categories including: Cylindrocarpon root rot [C. destructans (Zins) Scholten], Phytophthora root rot [P. cactorum (Leber & Cohn) Schröeter], root lesion nematode (Pratylenchus penetrans) and an unknown disease category. The symptoms in the unknown disease categories ranged from eroded flaccid roots, dry crater-like rot, yellow patches, brown sunken lesions, rusty roots and superficial veins or netting on the roots. Over 35 various morphotypes of fungi were isolated from this category. Some isolates were identified using morphological characteristics including Rhexocercosporidium panacis Reeleder, Alternaria spp., Fusarium spp., Chaetomium spp., Thielavia spp., Curvularia spp., Epicoccum sp. and Phoma spp. Species of Penicillium, Eupenicillium and Eurotium have also been found. Identification will be further tested using molecular techniques. In order to understand whether any of the isolated fungi are pathogenic on ginseng, further experiments are being carried out. The final outcome of this study will help build better IPM strategies for Ontario ginseng.
Development and validation of high-resolution DNA melting (HRM)-based markers derived from Rysto-STS markers YES3-3A and YES3-3B for high-throughput marker-assisted selection of potatoes carrying Rysto. X. NIE, D. SUTHERLAND, V. DICKISON, M. SINGH, A. MURPHY AND D. DE KOEYER. Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada; (M.S.) Agricultural Certification Services, 1030 Lincoln Road, Fredericton, NB E3B 8B7, Canada; and (D.D.K.) International Institute of Tropical Agriculture, PMB 5320, Oyo Road, Ibadan 200001, Oyo State, Nigeria
Sequence analysis of the chromosome region harbouring the Rysto STS markers YES3-3A and YES3-3B in tetraploid potato cultivars ‘Barbara’ (Rrrr) and ‘AC Chaleur’ (rrrr) as well as 5 progeny selections revealed 3 sequence variants in ‘Barbara’ and the resistant (R) selections, and 2 variants in ‘AC Chaleur’ and susceptible (S) selections. Further analysis of the sequence variant ratio as well as in silico PCR with YES3 primers indicates that a variant with a 21-nucleotide (nt) deletion is likely the chromosome copy harbouring the Rysto. Two primer pairs, one targeting the region containing the 21-nt deletion and the other targeting the region anchoring the YES3-3A reverse primer, were designed. As anticipated, pair one produced two visible fragments in ‘Barbara’/R pool and one visible fragment in ‘AC Chaleur’/S pool; pair two produced one visible fragment in all samples. When subjected to high-resolution DNA melting (HRM) analysis, two distinct melting profiles for R and S samples were observed. Analysis of 147 progenies of ‘Barbara’ × ‘AC Chaleur’ revealed 72 and 75 progenies with R and S melting profiles, respectively, which was 100% consistent with YES3-3A and YES3-3B assays and phenotyping analysis. The results demonstrate the potential of HRM profiles as novel molecular markers for Rysto. The efficacy of the newly developed HRM markers for marker-assisted selection (MAS) was further validated with three populations involving ‘Barbara’ as the resistant parent. HRM markers offer rapid and accurate detection of potatoes carrying Rysto and can be used for high-throughput MAS in potato breeding to identify Rysto-conferred extreme resistance to Potato virus Y.
Development of a grapevine trunk diseases macroarray. D. T. O’GORMAN, J. FRASER, J. DICK AND J. R. ÚRBEZ-TORRES. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada
Grapevine trunk diseases (GTD) are caused by a wide range of taxonomically unrelated fungal pathogens (c. 80 different species). The GTD complex includes: black-foot and Petri disease, affecting young grapevines, collectively known as young vine decline (YVD); as well as Esca, and dieback (Eutypa, Botryosphaeria and Phomopsis), primarily found in mature vines. Grapevine trunk diseases are relatively new in British Columbia (BC). The situation parallels that in other grape-growing regions of the world. In order to evaluate the magnitude of GTD in BC, field surveys were conducted. Overall, 30 GTD species were identified in BC, with 95.8% of all vineyards surveyed showing symptomatic vines, with 7.8% and 10.2% expressing YVD and dieback, respectively. Because of the large numbers of GTD pathogens found locally and globally, an accurate and rapid detection method is needed to facilitate the development of effective disease management strategies. Therefore, we have designed a DNA macroarray, with probes targeting β-tubulin gene sequences specific for the detection of 61 taxa including 34 YVD pathogens. The macroarray was shown to be accurate and sensitive, detecting as little as 10–6 ng of pathogen DNA. Work is currently being conducted to expand the DNA macroarray to include species-specific probes selected from the β-tubulin and elongation factor 1-α genes. The array will detect an additional 20 different fungal pathogens responsible for Botryosphaeria dieback and Eutypa dieback in mature vines. Once complete, the macroarray will be a rapid and comprehensive tool available for vineyard and nursery GTD management.
Slip-skin disorder on sweet cherry (Prunus avium). D. T. O`GORMAN, G. HEALY, P. M. TOIVONEN AND J. R. ÚRBEZ-TORRES. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada
Cherry slip-skin is a problem affecting the health, quality and sale-ability of (primarily) late season sweet cherry (Prunus avium L.). Reports of this condition causing major problems for producers in the 2012 harvest came from both British Columbia (BC) and Washington State (WA), with some orchards continuing to experience the problem in 2013–2015. The condition is not easily visible until just after harvest and may not show up until after shipping. Following harvest, the shoulder of the cherry becomes noticeably soft and the skin disassociates from the inner tissue, which has become macerated. With time, while the rest of the cherry remains firm, the symptoms develop radially causing breakage of the skin. During shipping, the affected areas may dehydrate forming sunken craters on the fruit’s surface. Presently, the exact cause of this condition is unknown, but preliminary investigations in both BC and WA suggest the involvement of one or more different yeast species. Our survey results indicate yeast populations are found to increase in number on the surface of developing fruit throughout the growing season, however yeast cannot always be isolated from symptomatic fruit. To help elucidate key factors involved in cherry slip-skin, several field trials were set up in 2015. The first was a spray trial, incorporating both the commercial fungicide propiconazole (0.125 ppm) and potassium metabisulfite (5000 ppm) at different spray intervals. The second trial, involved canopy management to increase the light penetration and alter the microenvironment of the fruiting zone. Preliminary results of both trials were presented.
Comprehensive assessment of grapevine virus diseases in British Columbia. S. POOJARI, T. D. LOWERY, J. BOULÉ, N. DELURY, M. ROTT, A.-M. SCHMIDT AND J. R. ÚRBEZ-TORRES. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada; and (M.R., A.-M.S.) Canadian Food Inspection Agency, Centre for Plant Health, Sidney Laboratory, Sidney, BC V8L 1H3, Canada
Virus and virus-like diseases are considered one of the major constraints to the sustainability of the wine industry worldwide. British Columbia (BC), with over 10 000 acres of wine-grapes (Vitis vinifera L.), is the second largest grape-production region in Canada. To determine the current health status of established vineyards in BC, a comprehensive survey was conducted between 2013 and 2015 and the incidence of economic important virus diseases was recorded using ELISA and PCR/RT-PCR. Over 3000 samples (including random-composite and single-targeted samples) representing the most prevalent white and red grape cultivars were tested for the presence of non-regulated grapevine viruses in BC. Diagnostic test results showed Grapevine leafroll to be the most widespread virus disease in BC. Among all Grapevine leafroll associated viruses (GLRaVs), GLRaV-3 was the most prevalent (23.9%) followed by GLRaV-2 (7.1%), GLRaV-1 (2.7%) and GLRaV-4 (2.7%). In addition, high incidence (29.2%) of Grapevine fleck virus was also detected. Results indicated a low incidence of Grapevine red blotch associated virus (1.6%), Grapevine fanleaf virus (0.5%), and Grapevine Pinot Gris virus (0.14%) and no positive samples were detected for Arabis mosaic virus. Molecular analyses also confirmed the presence of Grapevine leafroll insect vectors in BC vineyards, including the grape mealybug (Pseudococcus maritimus) and the European fruit lecanium Scale (Parthenolecanium corni). These results, along with information generated on the genetic diversity and spatial distribution patterns of major grapevine viruses in correlation with insect vector population dynamics will contribute to develop sustainable management practices for grapevine virus diseases in BC.
Using genomics approaches for rapid development of species-specific diagnostics for cucurbit downy mildew. L. QUESADA-OCAMPO. Box 7612, North Carolina State University, Raleigh, NC 27695, USA
Advances in Next Generation Sequencing (NGS) allow for rapid development of genomics resources needed to generate molecular diagnostics assays for infectious agents. NGS approaches are particularly helpful for organisms that cannot be cultured, such as the downy mildew pathogens, a group of biotrophic obligate oomycetes that infect crops of economic importance. Unlike most downy mildew pathogens that are highly host-specific, Pseudoperonospora cubensis (Berkeley & Curtis) Rostovtsev causes disease on a broad range of crops belonging to the Cucurbitaceae. In this study, we identified candidate diagnostic markers for P. cubensis by comparing NGS data from a diverse panel of P. cubensis and Pseudoperonospora humuli isolates, two very closely related oomycete species. Pseudoperonospora cubensis isolates from diverse hosts and geographic regions in the USA were selected for sequencing to ensure that candidates were conserved in P. cubensis isolates infecting different cucurbit hosts. Genomic regions unique to and conserved in P. cubensis isolates were identified through bioinformatics. These candidate regions were then validated using PCR against a larger collection of isolates from P. cubensis, P. humuli, and other oomycetes. Overall seven diagnostic markers were found to be specific to P. cubensis. These markers could be used for pathogen diagnostics on infected tissue, or adapted for monitoring airborne inoculum with real-time PCR and spore traps.
Diverse population of Septoria linicola causing pasmo disease in flax. K. Y. RASHID. Morden Research and Development Centre, Agriculture and Agri-Food Canada, 101 Route 100, Morden, MB R6M 1Y5, Canada
Pasmo caused by the fungus Septoria linicola (Speg.) Garassini (sexual state Mycosphaerella linorum Naumov.) is a major disease affecting flax (Linum usitatissimum L.), and is prevalent in most flax growing areas worldwide. The incidence and severity of this disease have been on the rise in Canada causing major reductions in yield and quality of the seed and fibre. This research aimed at studying the virulence in the pathogen population in western Canada. Disease specimens have been collected from Manitoba and Saskatchewan and hundreds of single spore isolates established. Each of seven representative isolates was tested on a set of 94 flax genotypes from diverse origin. Each isolate was inoculated onto 20-day-old seedlings of the 94 flax lines and incubated for 48 h under controlled growth cabinet conditions equipped with a misting system. The disease incidence, severity and defoliation were recorded at 10, 20 and 30 days after inoculation. The data collected showed some similarities among the interactions of the seven isolates on some of the 94 flax genotypes. The specific host–pathogen interactions were summarized into 42 virulence combinations indicating the complexity of the virulence genes in the pathogen and the matching resistance genes in the 94 flax genotypes. Gene-pyramiding of the major race-specific genes will provide improved flax genotypes with wider genetic base for resistance to the diverse pathogen population in the flax crops in western Canada.
Major shift in the virulence of sunflower rust races in Manitoba. K. Y. RASHID. Morden Research and Development Centre, Agriculture and Agri-Food Canada, 101 Route 100, Morden, MB R6M 1Y5, Canada
Puccinia helianthi Schwein is a worldwide pathogen causing rust disease in sunflower (Helianthus annuus L.) with major epidemics and great losses in seed yield and quality. Annual disease surveys of the sunflower crops in Manitoba revealed a wide range in rust incidence and severity from year to year. Local severe rust epidemics occurred in various regions in Manitoba in most years since 2002. Prior to 2009, such epidemics were caused by the predominant rust race-group 300 including the races 324, 326, 327 and 336. Clear shifts in the virulence pattern of the sunflower rust population have been observed after 2009 with the prevalence of the race-group 700 including the races 723, 736, 766, 776 and 777. The most virulent race 777 appeared in 23% of the 2009 isolates but was not detected in 2010 and 2011. However, race 777 appeared at 5% in the isolates collected in 2012–2013, and rose sharply to 74% of the collected isolates in 2015. Race 777 is virulent on all the nine sunflower rust differential genotypes used to identify sunflower rust races, and on most commercial sunflower hybrids grown in Manitoba.
Efficacy of two fumigants against clubroot [Plasmodiophora brassicae] in three field trials. J. ROBSON, B. D. GOSSEN, F. AL-DAOUD 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
Seed treatments, biocontrol agents and soil amendments do not provide effective management of Plasmodiophora brassicae Woronin (clubroot) on canola (Brassica napus L.) in Canada. Field trials were conducted in a naturally infested mineral soil and a high organic content (muck) soil site in Ontario to assess the efficacy of two soil fumigants, metam sodium (Vapam HL, Busan 1236) or chloropicrin (Pic Plus) at selected rates on clubroot. Efficacy was assessed in plant bioassays with a susceptible cultivar of Shanghai pak choi. Furthermore, propidium monoazide (PMA) assisted qPCR was used to quantify the viability of resting spores in the soil after treatment. Disease pressure at the mineral soil site in 2014 was very low; there were no clubroot symptoms in any treatment that received metam sodium or chloropicrin, and only 8% severity in the non-treated control. In trials in 2014 and 2015 at the muck soil site, chloropicrin reduced clubroot severity when applied at 128 kg a.i. ha−1 or higher. Metam sodium was only effective at 150 kg a.i. ha−1 or higher in 1 year (2015) at this site. However the lack of efficacy in 2014 may have been due to ineffective sealing of the metam sodium treatments within the soil after application. Unexpectedly, the fumigants did not have a consistent effect on resting spore viability assessed with PMA-PCR. We conclude that fumigants can reduce clubroot, but that efficacy depends on both rate of application and effectively sealing the fumigant in the soil after application.
Identification of Ustilago maydis RNA helicases and investigation of their function in the teliospore. A. M. SETO, M. E. DONALDSON AND B. J. SAVILLE. Environmental and Life Sciences Graduate Program, Trent University, 1600 West Bank Drive, Peterborough, ON K9L 0G2, Canada; and (M.E.D., B.J.S.) Forensic Science Program, Trent University, 2140 East Bank Drive, DNA Building, Peterborough, ON K9L 0G2, Canada
Ustilago maydis (DC.) Corda is dispersed as thick-walled dormant diploid teliospores, which germinate, complete meiosis and initiate new rounds of infection. We hypothesize that the stored mRNAs in teliospores code for proteins important for germination and/or the subsequent developmental events. Some of these mRNAs are stabilized through the formation of double-stranded RNAs (dsRNAs). RNA-seq and gene ontology (GO) enrichment analyses were carried out to identify the patterns of transcript level change during germination and the functional categories of genes represented by the stored RNAs. RT-qPCR on RNA isolated from teliospores at distinct stages of germination will be used to confirm the patterns indicated by RNA-seq data. The presence of RNA helicases among the teliospore expressed genes suggested that unwinding dsRNAs was an early molecular event in germination. RNA helicases are widely conserved proteins involved in RNA strand annealing, RNA duplex unwinding, protein displacement from RNAs and other aspects of RNA function. Among the 46 RNA helicase we identified in U. maydis, four had expression patterns, revealed by RNA-seq, consistent with a role in teliospore germination. RT-PCR supported the expression pattern of an orthologue to S. cerevisiae RNA helicase, ded1. The creation of ded1 deletion strains and functional analysis of this protein are underway. We will present the RNA-seq analyses of teliospore transcripts, and our initial investigation of RNA helicases in relation to a model of the molecular events of teliospore formation and germination.
A molecular method for determining the viability of Synchytrium endobioticum. D. S. SMITH AND U. SINGH. Canadian Food Inspection Agency, Charlottetown Laboratory, 93 Mount Edward Road, Charlottetown, PE C1B 1M2, Canada
Determining the viability of Synchytrium endobioticum (Schilb.) Percival in infested soil is a key component of potato wart disease management, and is necessary for achieving the long-term goal of eradicating this pathogen from Prince Edward Island. Towards this end, a molecular method for determining the viability of the potato wart pathogen, S. endobioticum, was developed. The test targeted two messenger RNA sequences in resting sporangia of S. endobioticum that were identified as highly expressed in a set of whole mRNA transcriptome data. One of the targets, Locus 852, had high homology to a ribosomal protein. The second target, Locus 166, could not be identified, but had weak homology to a nematode late embryogenesis abundant protein. Real-time reverse-transcriptase polymerase chain reaction assays were developed for these targets, and transcript-specific primers were designed to cross intron-exon borders in order to prevent amplification of DNA. Viability of S. endobioticum in sieved soil samples and in heat-treated spore preparations was determined using a sprout infection bioassay. Detection of the target mRNA transcripts was highly correlated with S. endobioticum viability for both soil and spore preparations. The molecular test was more sensitive and much more rapid than the sprout-infection bioassay for detecting viable S. endobioticum. This molecular method may be useful in screening soil samples for viable sporangia in infested fields, and aid in the evaluation of risk-mitigating strategies.
Non-race specific resistance to blackleg by Canadian canola cultivars shows delayed or reduced pathogen spread from infected cotyledons into petioles and stems. W. M. SOOMRO, H. R. KUTCHER AND G. PENG. Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; and (H.R.K.) Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
In western Canada, the majority of canola (Brassica napus L.) cultivars carry the resistance (R) gene Rlm3, Rlm1 or both to blackleg [Leptosphaeria maculans (Desmaz.) Ces. & de Not.]. Since the corresponding avirulence (Avr) genes (AvrLm3, AvrLm1) are rare in the current pathogen population, these R genes are no longer effective. However, widespread and severe damage by blackleg is still uncommon, suggesting that additional resistance mechanisms are present in commercial canola cultivars (CCCs). Three CCCs carrying Rlm1 and/or Rlm3 were assessed with ‘Westar’ (susceptible) against virulent L. maculans isolates carrying no AvrLm1 or AvrLm3. The infection of cotyledons and spread of the pathogen into the stem via the petiole were evaluated using a 09 scale and fluorescence microscopy. Droplet digital PCR (ddPCR) was used to quantify the DNA of spreading pathogen in petiole and stem tissues. At 7 days post-inoculation (dpi), a virulent isolate of L. maculans carrying a green fluorescent protein gene showed more limited hyphal spread in inoculated CCC cotyledons than in ‘Westar’. At 14 dpi, all inoculated cotyledons showed infection symptoms, but the mean severity was 5–6 for CCCs and 8–9 on ‘Westar’. ddPCR results showed that the amount of L. maculans DNA was substantially lower in petioles and stems of inoculated CCCs relative to ‘Westar’. It appears that non-race specific resistance associated with the CCCs plays a role against blackleg by delaying or reducing the spread of fungal hyphae from infected cotyledons into stems, which is where the most severe impact on plant yield occurs.
Re-emergence and rapid spread of the Goss’s wilt disease pathogen of corn: possible scenarios. J. T. TAMBONG, R. XU, A. SOLIMAN AND F. DAAYF. Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (F.D., A.S.) Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Goss’s wilt disease of corn is caused by the Gram-positive bacteria, Clavibacter michiganensis subsp. nebraskensis corrig. (Vidaver and Mandel) Davis et al. (Cmn). Yield losses as high as 50% have been reported during systemic xylem infections. First reported in 1969, in south central Nebraska, the disease was mainly confined in four Midwestern states in the USA. It became sporadic after partially resistant cultivars were identified and cultivated. Recently, the Goss’s wilt disease has re-emerged and is spreading rapidly into major corn growing regions of USA and Canada. The re-emergence of Cmn may be due to the use of corn hybrids selected solely based on high yield potential, without a robust disease-resistance/tolerance component. Secondly, it could be as a result of changes in weather systems such as wind patterns, rainfall, temperature and humidity. It could also be a phenomenon of host–pathogen coevolution. Bacterial pathogens such as Cmn are constantly changing their genetic capacity to enhance their virulence and adaptation to their hosts. In this presentation, genome sequences of Cmn strains collected in Manitoba (Canada) during the 2014 growing season will be compared with that of the type strain isolated over 40 years ago in Nebraska (USA). Pathogenicity of these strains will be compared in association with variations of their genome sequences, and the potential impact on Goss’s wilt disease trends was discussed.
Epidemiology and management of stemphylium leaf blight on onion in the Holland Marsh, Ontario. S. C. TAYVIAH, 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, caused by Stemphylium vesicarium (Wallr.) Simmons (telemorph; Pleospora allii (Rabenh) Ces. & de Not.), is a new disease of onion in Ontario. It is presently managed with multiple applications of fungicide. To understand its epidemiology, air-borne spore concentrations were monitored continuously for 120 days in 2015 using a Burkard 7-day volumetric sampler. Hourly spore counts revealed a diurnal pattern, with 48% of ascospores and 73% of conidia captured between 0500–1200 h. The highest concentrations of ascospores (54 ascospores m−3 air day−1) were captured prior to disease onset, but capture of conidia was highest (97 conidia m−3 air day−1) during disease development. Spore concentrations increased dramatically 24–72 h after precipitation. The first appearance of blight symptoms coincided with high conidia numbers, rainfall and warm days (temp ≥ 18°C for ≥ 9 h). Conidia appear to be important in the development of blight epidemics, but the role of ascospores is not yet clear. A foliar fungicide (fluopyram 12.5%, pyrimethanil 37.5%) was applied based on calendar spray timings, initiation of calendar application based on spore trapping, spray prediction models (BOTCAST threshold 1, TOMCAST DSV 15, and a model modified specifically for stemphylium leaf blight) and a non-sprayed control. TOMCAST prompted six sprays compared with 10 with the spore trapping treatment and eight applications with the other models. Early spray applications reduced foliar blight severity, but no treatment increased marketable yield. More effective fungicides and spray timings are required to provide effective management of stemphylium leaf blight.
Strawberry nursery stock as a source of virus inoculum and on-farm spread of strawberry viruses in New Brunswick, Canada. M. T. TESFAENDRIAS, C. MAUND AND R. J. A. TREMBLAY. New Brunswick Department of Agriculture, Aquaculture and Fisheries, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada
Strawberry decline disease (SDD) is caused by a combination of two or more viruses infecting the strawberry (Fragaria x ananassa Duchesne) plant. The infection results in a reduction of yield and fruit quality. Strawberry viruses and the strawberry aphid, Chaetosiphon fragaefolii (Cockerell), which transmits these viruses, were detected in New Brunswick (NB) surveys conducted in 2013 and 2014. The most common viruses identified were the strawberry mild yellow edge virus (SMYEV) and the strawberry mottle virus (SMoV). In 2015, a survey was conducted to identify the source of SDD viruses in NB strawberry fields and to follow on-farm spread of these strawberry viruses. In order to determine if the introduction of infected nursery stock into strawberry fields was the source of virus, 100–120 nursery stock plants were collected from each of six strawberry farms before transplanting. Subsequent samples were also collected twice during the growing season from strawberry fields at the end of June or early July and at the end of August. Plant tissue samples were tested for presence of the five strawberry viruses (SMYEV, SMoV, strawberry vein banding virus (SVBV), strawberry crinkle virus (SCV) and strawberry pallidosis associated virus (SPaV)) using reverse transcription polymerase chain reaction. Results showed the presence of SMYEV in nursery stock from two farms and SPaV in nursery stock from one farm. The most common viruses identified in both field sampling periods were SMYEV and SMoV. Virus incidence was lower during the first sampling period compared with the second sampling period, indicating on-farm spread of strawberry viruses. On one farm, there was an increase in SMYEV incidence from 5 to 45%. Survey results were used to develop best management practices for SDD management in New Brunswick.
Investigating the aetiology of tree fruit decline in British Columbia. J. R. ÚRBEZ-TORRES, J. BOULÉ AND D. T. O’GORMAN. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada
Tree fruits in British Columbia (BC) include 15 000 acres of apples, apricots, cherries, nectarines, peaches, pears and plums, which generate an annual farm-gate value of approximately $80 million. Fruit tree decline and variation in vigour have been recognized as an important problem for the BC industry. Much of this decline is associated with symptoms ranging from root rots, cankers, dieback or a failure to thrive. However, a general lack of knowledge exists regarding the range of organisms associated with these disease symptoms. Accordingly, a preliminary survey to assess and identify the diseases and causal agents affecting cherry and apple tree health in BC was conducted between 2011 and 2013. In total, over 200 diseased samples showing characteristic decline symptoms were collected from 66 blocks. The most prevalent symptoms observed were perennial cankers affecting the trunk and branches of declining trees but also root and crown rot was observed in some young orchards. Morphological characterization along with DNA analyses of the ITS1-5.8S-ITS2, β-tubulin and translation elongation factor 1-α partial gene regions, allowed the identification of 15 fungal pathogens belonging to 13 different genera. Pathogenicity studies showed species within the Cytopsora and Leucostoma genera in cherries and within the Diplodia and Neonectria in apples to be the most virulent causing perennial cankers in those hosts. Species within the Ilyonectria and Fusarium genera were the most prevalent fungi isolated from symptomatic roots. Results from this study will significantly contribute to the development and implementation of effective management strategies against these pathogens in BC.
Grapevine trunk diseases studies in British Columbia. J. R. ÚRBEZ TORRES AND D. T. O’GORMAN. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada
Grapevine trunk diseases (GTD), caused by a wide range of taxonomically unrelated fungi, are considered the primary biotic cause of grapevine decline and mortality worldwide and thus one of the major threats to the industry’s future economic sustainability. The British Columbia (BC) grapevine industry is internationally recognized for its award-winning wines and contributes over $2.1 billion into the Canadian economy. However, the presence of GTD in BC has long been overlooked. Accordingly, studies to determine the current status of GTD in BC started in 2010. Field surveys were conducted between 2010 and 2013 and over 200 vineyards were visited throughout all grape-growing regions of the Province. Surveys also included assessment of foliar symptomatology from over 60 000 vines and fungal isolations from over 500 symptomatic vines (young and mature). Field results revealed the presence of all GTD in BC, including black-foot and Petri disease in young vines, and Botryosphaeria dieback, esca and Eutypa dieback in mature vines. Morphological characterization along with DNA analyses and multi-locus phylogenetic studies of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rDNA, and part of the ACTIN, β-tubulin and translation elongation factor 1-α genes, allowed the identification of over 30 fungal pathogens from 15 different genera associated with GTD in BC. This study represents the first attempt to identify and characterize the GTD causal agents in BC and provides the foundation to further investigate the impact of these diseases with the aim to develop and implement effective management strategies.
Management of plant-parasitic nematodes on carrots grown in organic (muck) soils in Ontario. D. VAN DYK, K. JORDAN AND M. R. MCDONALD. Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada
Carrots (Daucus carota L., subsp. sativus (Hoffm) Arcang) can be particularly susceptible to plant-parasitic nematode damage, especially during taproot formation. In Ontario, two common nematode pests of carrot are the root-knot nematode (Meloidogyne hapla Chitwood) and carrot cyst nematode (Heterodera carotae Jones). Restrictions around fumigant use are increasing and many nematode control products are no longer available to vegetable growers, creating a need to identify effective non-fumigant nematicides. To evaluate some of these new products, field trials were conducted in the Holland Marsh, ON during 2014 and 2015 in fields with a history of nematode damage. A randomized complete block design with six replicates per treatment was used. The treatments were: PicPlus (chloropicrin 86%) at 78 kg ha−1, Vapam (metam sodium 42%) at 275 L ha−1, Nimitz (fluensulfone 15%) at 8.3 L ha−1, PicPlus at 78 kg ha−1 + Vapam at 275 L ha−1, PicPlus at 78 kg ha−1 + Nimitz at 8.3 L ha−1, MustGrow (oriental mustard seed meal 100%) at 1680 kg ha−1, Dazitol (capsaicin 0.42%, oleoresin of capsicum 3.7%) at 60 L ha−1, AgriMek (abamectin 2%) at 20 L ha−1, and an untreated check. Carrots, cv. ‘Cellobunch’ (~65 seeds m−1) were direct seeded on raised beds. Each experimental unit consisted of three rows, 66 cm apart and 13 m long. PicPlus, Vapam, Nimitz and a combination of these products increased carrot yield and per cent marketable carrots while reducing disease severity. The non-fumigant nematicide, Nimitz, reduced damage and increased yields comparable to the grower standard fumigants in 2014 but not in 2015, so further research is required.
Intra-host interactions of the pea root rot pathogens Aphanomyces euteiches and Fusarium spp. T. WILLSEY, J. THOMAS AND S. CHATTERTON. University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada; and (S.C.) Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403–1 Avenue South, Lethbridge, AB T1J 4B1, Canada
Pea root rot complex (PRRC) describes a group of closely associated soil-borne fungi that cause root rot disease in field pea (Pisum sativum L.). The oomycete Aphanomyces euteiches Drech. and several Fusarium spp. are frequently the most prevalent and damaging microorganisms within this complex, and cause severe reductions in the quality and quantity of crop yield. Fungicidal application, crop rotation and resistance breeding have been unsuccessful in managing either microorganism. Both pathogen groups have been studied extensively in isolation, but the effect of intra-host interactions on disease development remains largely unexplored. Therefore, interactions between A. euteiches and three Fusarium spp. were examined in greenhouse trials, in which P. sativum was exposed to root pathogens under controlled conditions. Results from three independent trials indicate an increase in disease symptoms in the presence of multiple pathogen species compared with inoculations with a single pathogen. Further trials have indicated that application of the fungicides fludioxonil and ethaboxam as seed treatments may inhibit proliferation of Fusarium spp. and A. euteiches, respectively, but efficacy is influenced by initial inoculum concentration. Results from controlled seed treatment trials will be confirmed in field experiments conducted in 2016. Insight into the interactions between A. euteiches and Fusarium spp. is necessary to inform timely mitigation strategies aimed at controlling both pathogens simultaneously.
Thirty years of postharvest biological control research: The journey from simplicity to complexity. M. WISNIEWSKI. USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV 25430, USA
Finding safe and effective alternatives to synthetic fungicides for reducing postharvest losses of harvested commodities has been a focus of much research over the past three decades. Identifying alternatives that are widely accepted and commercially viable has, however, been a challenge. The search to identify alternative approaches to postharvest disease management must be viewed in relation to a complex regulatory environment, the need to address disease problems in a wide array of commodities and conditions, industry and consumer acceptance, and last but not least, commercial viability. In order for companies to invest in new technologies, they must be able to clearly see both the value of a product and the financial return on their investment. This presentation will attempt to highlight how the search for alternative postharvest disease management technologies has been a journey from simplicity to complexity. Plant pathologists developing alternative technologies have been slowly moving away from the ‘silver bullet’ concept where a single intervention can be used to control a disease to viewing plant disease as a process where multiple interventions may be required at different points in the disease process. Over the past 30 years, alternatives have moved from the simple idea of applying high concentrations of biocontrol agents to a harvested commodity, to using a wide array of other alternatives, and integrating them together into a systems approach based on the multiple decrement or multiple hurdle concept. The ease of sequencing genomes and obtaining related genotypic, transcriptomic, proteomic and metabolomics information is leading to the development of new commercial technologies where problems are solved ‘biologically’.
Identification of microsatellite markers linked to quantitative trait loci associated with partial resistance to Aphanomyces root rot in field pea. L. F. WU, R. FREDUA-AGYEMAN, K. F. CHANG, R. L. CONNER, S. F. HWANG, D. FEINDEL, K. B. MCRAE AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (R.F.A., K.F.C., S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Forestry, 17507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; (R.L.C.) Morden Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 101 Route 100, Morden, MB R6 M 1Y5, Canada; and (K.B.M.) Kentville Research and Development Centre, AAFC, 32 Main Street, Kentville, NS B4N 1J5, Canada
The development of partial resistance to Aphanomyces root rot of pea, caused by Aphanomyces euteiches Drechs., is considered to be the most durable method to manage this destructive soil-borne disease. In pea, partial resistance to A. euteiches has been reported to be controlled by multiple loci. In this study, two recombinant inbred lines (RILs) populations, obtained by single-seed descent from the crosses F6: ‘00-2067ʹ × ‘Reward’ and F8: ‘Carman’ × ‘Reward’, respectively, were used to identify the QTLs associated with the partial resistance as well as to evaluate the effectiveness and stability of genetic loci controlling the disease. A greenhouse study demonstrated that the resistant cv. ‘00-2067’ remained highly resistant to A. euteiches, relative to the susceptible ‘Reward’, while the resistance in ‘Carman’ eroded as inoculum concentration increased. This indicated that ‘00-2067’ contained at least one major QTL for resistance that ‘Carman’ did not have. A total of 160 microsatellite markers were tested for polymorphism in the three parental cultivars. Forty-eight and 51 polymorphic markers were detected in the parents of the two RIL populations, respectively. Nine SSR markers from four chromosomes showed association with resistance to A. euteiches. The marker AB-64 had a significant association (P < 0.05) with the field phenotype data of the first population. As part of ongoing research, more SSR markers will be screened to better map the QTLs. In addition phenotyping will be conducted under controlled conditions in order to confirm the field data.
Development and validation of diagnostic procedures based on the next generation sequencing technology for screening potato accessions imported to Canada. H. XU, S. LI, D. L. HAMMILL, S. CODY AND J. NIE. Canadian Food Inspection Agency, Charlottetown Laboratory, 93 Mt. Edward Road, Charlottetown, PE C1A 5T1, Canada
Small quantities of Solanum spp. germplasm may be introduced into Canada, for vegetative propagation under the potato post-entry quarantine (PPEQ) programme. Potato accessions entered into the PPEQ programme are routinely propagated in vitro followed by multiplication in greenhouse followed by serial testing using biological, serological and molecular methods for detecting any possible potato pathogens. The entire process of PPEQ quarantine testing is costly and time consuming. In 2015, mini potato tubers (5 kg) illegally brought into Canada, were seized and entered in the PPEQ facility for quarantine testing. The mini tubers were grown out in the greenhouse and total RNA was extracted from leaf samples of 8 plants. The RNA was then used for cDNA library construction followed by next generation sequencing (NGS) analysis. Over 300 million reads were obtained from the paired-end sequencing in a MiSeq platform (Illumina) and used for de novo assembly using CLC Genomics Workbench. After filtration against the potato genome sequence, over 350 assembled contigs from these samples were blasted against all known sequences available in the NCBI database. A number of viruses including Potato leafroll virus, potato viruses M, S, X and Y and Potato aucuba mosaic virus were detected based on nucleotide identity (>90%). The NGS results were then validated by bioassay using 19 indicator species and RT-PCR using gene specific primers. NGS followed by molecular confirmation provides a new strategy that can significantly reduce the time (5 vs 55 weeks) and the cost for the quarantine testing. In addition, Tomato chlorosis virus and some others were also identified on the basis of nucleotide identity and genome structional analysis, and are subject to further confirmation.
Blackleg resistance by Rlm1 may be triggered by localized activation of salicylic acid and suppression of abscisic acid and auxin pathways. C. ZHAI, X. LIU, T. SONG, F. YU AND G. PENG. Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada
Host resistance plays a critical role in managing blackleg [Leptosphaeria maculans (Desmaz.) Ces. & de Not] on canola (Brassica napus L.), but the information on resistance mechanism is lacking. A transcriptome study was conducted by inoculating a double-haploid canola line carrying the resistance genes Rlm1 and Rlm3 with an avirulent (AvrLm1) or virulent (AvrLm4-7) isolate of L. maculans. RNA sequencing identified 70,709 genes in this line by mapping the result to a reference B. napus genome, among which 3015 and 6999 were differentially expressed genes (DEGs) in the local tissues of incompatible and compatible interactions, respectively. Gene ontology found many of them are related to plant-pathogen interaction or plant hormone signalling; many DEGs involved in salicylic acid (SA) pathways were significantly activated in the incompatible interaction relative to a water control, while those involved in abscisic acid (ABA) and auxin signalling were triggered exclusively in the compatible interaction. This result indicates that recognition of the avirulence gene by Rlm1 triggers a localized increase in SA, which possibly limits the infection via suppression of ABA and auxin signalling pathways. When systemic responses were assessed, RNA sequencing identified far fewer DEGs in the non-inoculated cotyledon of plants where the other cotyledon had been inoculated earlier. Additionally, few genes involved in SA were activated in the non-inoculated cotyledons and those involved in the ABA pathway were not suppressed relative to the control. When challenging the non-inoculated cotyledons with the virulent L. maculans isolate, the prior inoculation of the other cotyledon with the avirulent isolate showed no effect on infection.