Detection and quantification methods for Ilyonectria mors-panacis in ginseng (Panax quinquefolius L.) gardens/fields of Ontario. K. ADUSEI-FOSU, A. F. SHI, S. WESTERVELD AND M. R. MCDONALD. Department of Plant Agriculture, University of Guelph, Guelph, ON N1G 2W1, Canada; and (A.F.S., S.W.) Ontario Ministry of Agriculture, Food and Rural Affairs, Simcoe Resource Centre 1283 Blueline Road & Hwy. #3, Box 587, Simcoe, ON N3Y 4N5, Canada
Ilyonectria mors-panacis (IMP) is a soilborne pathogen that causes root rot in ginseng (Panax quinquefolius L.). The pathogen is also responsible for ginseng replant disease and subsequent crop losses. Root rot has been identified in ginseng gardens in Ontario in both replant and non-replant sites. There is a need for a rapid, robust, sensitive molecular method for detection and quantification of this pathogen in soil. A real-time quantitative polymerase reaction (qPCR) method for timely detection and quantification of I. mors-panacis was developed. To test the methodology, ginseng garden soils collected in 2016 and 2019 were tested. New primers were developed based on the internal transcribed spacer region of the rRNA operon from known IMP. The EF-1α and effector proteins genes failed to detect IMP at the primer screening and design phase. In 2016, IMP was quantified in ginseng garden soils that were untreated, or treated with solarization, anaerobic soil disinfestation (ASD) or biofumigation. Later, half of each plot was fumigated with metam sodium (Busan). More IMP DNA was detected in the untreated and solarization treatments as compared to the ASD and biofumigation treatments. These results indicate that treatments combining ASD or biofumigation with metam sodium can reduce but not eliminate IPM in ginseng soils. Analysis of 2019 soils in ongoing. This qPCR detection method will make an important contribution to research on the management of IMP to support the ginseng industry in Ontario.
Weekly treatment with pipecolic acid induces systemic acquired resistance in cabinet-grown cucumber. N. BELU, A. B. FUFENG, G. M. NUNN, N. W. XIAO, M. B. MARTIN AND R. K. CAMERON. Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4L8, Canada; and (M.B.M.) Children’s Health Research Institute, 800 Commissioners Road East, London, ON N6C 2V5, Canada
The Canadian greenhouse vegetable industry represents the largest sector of Canadian horticulture, and it has been rapidly expanding in recent years. A challenge faced in this sector is that crop plants in the humid greenhouse environment can quickly become infected with disease-causing microbes, potentially leading to large-scale outbreaks and significant losses. In response to some localized infections, plants activate the production and mobilization of long-distance signals to systemic leaves, where their perception results in the establishment of a systemic acquired resistance (SAR) of systemic tissue to a broad range of normally virulent pathogens. Exogenous application of small defence-related compounds has also been observed to activate plant-wide resistance in numerous crop species. Cucumber, the second most-harvested greenhouse crop in Canada, was selected to study the resistance induced by treatment with pipecolic acid, a putative long-distance SAR signalling compound. Plants were given one to three weekly soil-drench treatments with pipecolic acid or water, inoculated 24 h afterwards with virulent Pseudomonas syringae pv lachrymans, and bacterial growth in planta was determined 3 days later. Plants treated with pipecolic acid multiple times supported lower bacterial levels in leaves compared to plants treated once and to water-treated controls. Expression of the cucumber SAR marker genes peroxidase (PRX) and chitinase (PR8) was found to be enhanced in pipecolic acid-treated plants compared to water-treated controls, indicating that exogenous pipecolic acid activated the SAR pathway. These findings may inform the development of alternative approaches to protect greenhouse cucumber from diseases while reducing the use of more hazardous pesticides.
Identifying effective nematode extraction methods for the stem and bulb nematode (Ditylenchus dipsaci) from garlic field soil. T. BLAUEL, M. J. CELETTI (ret’d), K. S. JORDAN AND M. R. MCDONALD. University of Guelph, Department of Plant Agriculture, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (M.J.C.) Ontario Ministry of Agriculture, Food and Rural Affairs, 1 Stone Road West, Guelph, ON N1G 4Y2, Canada
Stem and bulb nematodes, Ditylenchus dipsaci (Kühn) Filip’ev, are important plant parasites affecting the rapidly growing garlic (Allium sativum L.) industry in Ontario. Stem and bulb nematodes can survive in field soil and garlic seed cloves. Infections in seed cloves can easily go unseen, but can result in substantial damage to the garlic crop. It is also important to avoid infested soil. An effective method to quantify the nematode in soil is needed. There are two common methods for nematode extraction and quantification, the sugar centrifugal flotation method and the Baermann pan method, and there are variations in both methods. Two different sugar centrifugal flotation methods and four Baermann pan methods were compared using soil inoculated with 200 stem and bulb nematodes per 100 cc of soil. One sugar centrifugal flotation method collected the first supernatant, with other alterations, while the other did not. The Baermann pan methods used either two paper towels or six tissues and an extracting time of two or seven days. Both sugar centrifugal flotation methods recovered significantly more stem and bulb nematodes (≥ 49.1% of nematodes applied) than the other methods. The Baermann pan method which used paper towel and extracted for seven days recovered more nematodes (31.2%) than the other Baermann pan methods (≤ 12.7%). These results indicate that the sugar centrifugal flotation extraction method should be used by laboratories and diagnostic clinics to quantify stem and bulb nematodes in soil. Fields should be tested before planting to prevent unforeseen yield losses at harvest.
Mn-reducing bacteria: A powerful tool for biocontrol of important fungal phytopathogens. E. CARVALHAIS, M. MAZHAR, K. LY, S. ALI, S. SALDIAS, S. KANDASAMY AND G. LAZAROVITS. A&L Biologicals, Agroecological Research Services Centre, 2140 Jetstream Road, London, ON N5V 3P5, Canada; and (G.L.) Department of Biology, University of Western Ontario, 1151 Richmond Street, London, ON N6A 3K7, Canada
Bacterial biocontrol involves harnessing disease-suppressive bacteria to improve plant health. Mn-reducing bacteria can provide available Mn to plants that can be used to improve photosynthesis, production of root exudates that enhance biocontrol, production of phenols and lignin, and consequently inhibit fungal exoenzymes. The severity of many fungal diseases thus can be managed by providing a broad-spectrum antifungal formulation of Mn-reducing bacteria. We isolated eight bacterial strains on manganese reduction media from the rhizosphere of corn plants growing in southwestern Ontario soil. A dual plate inhibition assay was carried out to screen the biocontrol ability of these isolates for eight important and prevalent plant pathogens. Results indicated that 7/8 of these strains significantly inhibited the growth of Alternaria tenuissima, 8/8 inhibited Colletotrichum coccodes, 2/8 inhibited Fusarium graminearum, 3/8 inhibited Fusarium oxysporum, 2/8 inhibited Fusarium solani, 5/8 inhibited Pythium aphinadermatum, 8/8 inhibited Rhizoctonia solani, and 3/8 inhibited Sclerotinia sclerotiorum growth in dual plate assay. Two strains Bacillus subtilis Mn-6 and Paenibacillus polymyxa Mn-13 showed significant fungal growth inhibition on all fungal pathogens. Growth room trials with mock infested soils with these fungal pathogens are underway. Bioformulations based on these Mn-reducing bacteria can provide an environmentally friendly method to attain sustainable agriculture.
Impact of alternative spray programmes for management of Cercospora beticola on sugarbeet (Beta vulgaris L. spp. vulgaris). C. DERVARIC, M. R. MCDONALD AND C. TRUEMAN. Department of Plant Agriculture, University of Guelph, Ridgetown Campus, 120 Main Street East, Ridgetown, ON N0P 2C0, Canada; and (M.R.M.) Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada
Cercospora beticola Saccardo, the causal agent of Cercospora leaf spot (CLS), is one of the most serious pathogens of sugarbeets. Management of CLS in Ontario is challenged by the presence of fungicide resistance in C. beticola to Group 1 and 11 fungicides, increasing insensitivity to Group 3 fungicides such as prothioconazole (PC), and an anticipated change in use pattern of the contact fungicide mancozeb in Canada. Thus, spray schedules incorporating alternative fungicides including a phosphites-based product and copper hydroxide + canola oil (CHCO) were evaluated in a field experiment at Ridgetown, Ontario in 2020. Spray schedules using combinations and alternations of PC, mancozeb, phosphites, and CHCO were applied using intervals based on the BEETcast™ moderate and susceptible models, and a 12 to 14-day Calendar interval. Area under the disease progress curve (AUDPC) was greater in the nontreated control than all other spray schedules, and greater in the phosphites-only Calendar schedule than all other spray schedules. When phosphites were used to replace all mancozeb applications using the BEETcast™ susceptible model, AUDPC was greater than the standard PC-mancozeb programme applied using the same model. For the remainder of the modified treatments all spray schedules applied using the Calendar interval and BEETcast™ moderate model were equivalent to the mancozeb and PC-mancozeb standards, respectively. No differences were found among treatments for total beet yield. Analysis of sugar quality and yield is underway and will help determine the value of these programs for CLS management in the future.
The plasmodesmal protein AtOSM34 promotes turnip mosaic virus infection. R. HE, M. A. BERNARDS AND A. WANG. London Research and Development Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada; and (M.A.B.) Department of Biology, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 3K7, Canada
In plants, plasmodesmata (PD) are plasmamembrane-lined pores that traverse the cell wall to establish cytoplasmic and endomembrane continutiy between neighbouring cells. Plant viruses take advantage of PD to spread from infected cells to non-infected cells. This intercellular movement is required for an invading virus to develop systemic infection, and relies on the coordinated action of virus-encoded proteins and host factors, especially PD-localized ones. To better understand the involvement of PD in viral infection, we conducted a quantitative proteomic analysis of the PD-enriched fraction from Nicotiana benthamiana leaves in response to turnip mosaic virus (TuMV) infection. A pathogenesis-related protein named osmotin was identified to be significantly accumulated in the PD fraction isolated from TuMV-infected leaves, when compared with the protein level in the corresponding fraction from the healthy control. To characterize the possible role of osmotin in TuMV infection, we chose osmotin34 (OSM34), an ortholog from Arabidopsis thaliana (AtOSM34) for detailed analyses. Subcellular localization assay revealed that AtOSM34 is indeed localized to PD. In Arabidopsis, AtOSM34 expression is upregulated in response to TuMV infection, consistent with the previous quantitative proteomic data derived from N. benthamiana. Overexpression of AtOSM34 promotes viral replication and facilitates TuMV intercellular movement. Confocal microscopy results show that AtOSM34 is recruited to the viral replication complex (VRC) of TuMV. Protein-protein interaction assays indicated that AtOSM34 interacts with TuMV-encoded proteins VPg and 6K2, both of which are essential for TuMV replication and cell-to-cell movement. Taken together these data suggest that AtOSM34 may be recruited by TuMV via the interactions with VPg and/or 6K2 to promote TuMV infection in plants.
Corn root mycobiome diversity and its link to plant health and productivity. K. LY, S. KANDASAMY, N. WEERASURIYA, R. G. THORN AND G. LAZAROVITS. (K.L., S.K., N.W., G.L.) A & L Canada Laboratories Inc., 2140 Jetstream Road, London, ON N5V 3P5, Canada; and (K.L., S.K., N.W., R.G.T., G.L.) Department of Biology, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 3K7, Canada
Soil biome are integral parts of healthy ecosystem functioning in production agriculture. The mycobiome abundance and diversity on crop productivity is poorly explored in comparison with bacterial biomes. We sampled 42 corn sites at V10 growth stage across southwestern Ontario, using aerial infrared imaging, identified zones of healthy/stressed plants. Root samples from low and high-yield zones, soil physical and chemical properties were measured in conjunction with sequencing of the mycobiome communities using Illumina MiSeq amplification of four rRNA amplicons, two targeting general fungi, and one each for Ascomycetes and arbuscular mycorrhizal fungi. Sequence analyses identified indicator species associated with high and low productive sites within a farm, but communities varied across locations, demonstrating the impact of soil texture and chemistry in shaping the mycobiome. Across all four primer sets, roots from high-yielding sites shared 61 OTUs including Trichoderma, Chalara, Penicillium, Exophiala, Claroideoglomus, Neomassariosphaeria and Gibellulopsis. Low-yielding sites shared 52 OTUs including Fusarium, Pythium, Setophoma, Myrmecridium and Neonectria. Modelling for yield differences across farms with varying soil physicochemical parameters indicated that soil that most predicted the yield and its links to mycobiome diversity across farms are %clay, %sand, %P saturation, CEC, pH, OM and C:N ratio. Balance in soil physicochemical properties not only affects the productivity directly but also indirectly influence it by changing the abundance and diversity of both beneficial and disease-causing fungal biomes. Different combinations of microbial and soil heterogeneity lead to differences in plant health and productivity, a common conclusion for many other studies in this field.
Two plants, one pathogen: a novel system gives insight on prune dwarf virus, an understudied virus. A. J. SIMKOVICH, J. B. RENAUD, S. E. KOHALMI AND A. WANG. (A.J.S., S.E.K., A.W.) Department of Biology, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 5B7, Canada; and (J.B.R., A.W.) London Research and Development Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada
Prune dwarf virus (PDV) is an important viral pathogen of stone fruits such as sweet cherry (Prunus avium L.). To better understand PDV pathogenesis and molecular PDV-host interactions, we used label-free quantitative (LFQ) proteomics to study protein level changes in P. avium following PDV infection. A total of 791 proteins were identified from infected and uninfected leaves of trees located in an Ontario orchard. Many proteins showing significant accumulation changes (P < 0.05) were linked with responses to other pathogens or environmental stressors suggesting that they are part of a general stress response rather than a specific response to PDV. To better associate P. avium proteins with PDV infection, a model using cucumber (Cucumis sativus L.) as the host was employed. A total of 1597 proteins were identified, with 87 having significant accumulation changes upon PDV infection. 83% of the differentially accumulated proteins (n = 111) in C. sativus were orthologous to those identified in P. avium. Interestingly, tetraspanin 8 (TSPAN8), implicated as an antiviral protein has not been studied in PDV infection. TSPAN8 showed significant accumulation in C. sativus but was not detected in P. avium. The P. avium ortholog was cloned and co-localized with the PDV coat protein. This demonstrates the power of using a model host to circumvent challenges of studying crop trees under uncontrollable field conditions. This ‘two plant, one pathogen’ approach provides a better understanding of the plant-pathogen interactome which is essential for the development of novel antiviral strategies.
Heat shock protein 70 family proteins are involved in Turnip mosaic virus infection potentially through interaction with Eukaryotic translation initiation factor 4E isoform. Z. TANG, S. LYV, M. A. BERNARDS AND A. WANG. Department of Biology, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 3K7, Canada; and (S.L., A.W.) London Research and Development Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada
Potyviruses including Turnip mosaic virus (TuMV) represent the largest group of known plant-infecting RNA viruses and cause damage to a wide range of crops. To complete their infection process, viruses must interact with and subvert various host factors. Understanding the functional roles of host factors facilitates antiviral strategy development. Eukaryotic translation initiation factor 4E or its isoform (eIF4E/eIF(iso)4E) are the only known host factors absolutely indispensable for the infection of many potyvirus and even some non-potyvirus. The essential and multiple functional roles of eIF4E/eIF(iso)4E in potyvirus infection render the possibility to identify new host factors involved in potyvirus infection through their interaction with eIF4E/eIF(iso)4E. Previously, we performed immunoaffinity chromatography to isolate the eIF(iso)4E protein complex from TuMV infected Arabidopsis thaliana plants, and further determined the protein components in the isolated complex using liquid chromatography-tandem mass spectrometry. Several Heat shock protein 70 proteins (HSP70s) were identified as potential ingredients of the eIF(iso)4E protein complex. Arabidopsis T-DNA insertion mutants corresponding to these identified HSPs were used to evaluate the requirement of HSPs for TuMV infection. Plant infection and protoplast transfection assays revealed that TuMV viral genome replication was inhibited in HSP70-8 or HSP70-1/2 knockout and HSP70-11/12 knockdown plants. These results indicate that several members of the HSP70 proteins are host factors involved in the TuMV infection process, potentially through a direct interaction with eIF(iso)4E.
Effect of a deposition aid, nozzle type, and carrier volume on spray coverage and penetration within the sugarbeet canopy. K. THORNTON, J. DEVEAU, A. SCHAAFSMA AND C. TRUEMAN. Department of Plant Agriculture, University of Guelph, 120 Main Street East, Ridgetown, ON N0P 2C0 Canada; and (J.D.) Ontario Ministry of Agriculture, Food and Rural Affairs, 1283 Blueline Road Simcoe, ON N3Y4N5 Canada
Cercospora leaf spot (CLS; Cercospora beticola Sacc.) reduces sugarbeet (Beta vulgaris L.) yield and sugar quality. Efficacy of the contact fungicide mancozeb (Manzate Pro-Stick) relies on good canopy coverage. Interlock, a modified vegetable oil-based spray adjuvant (MVO), may improve spray deposition by optimizing droplet size. To investigate the potential of MVO to improve mancozeb efficacy, three sets of field trials, repeated two or three times, were completed in 2019 and 2020 at the Ridgetown Campus, University of Guelph. Canopy coverage was assessed by analysing the concentration of Rhodamine WT dye recovered from leaf samples collected from six locations within the canopy: the tip and base of the leaves in the inner, middle, and outer canopy. In the first trial, mancozeb and MVO were applied independently and together. MVO alone provided greater coverage than water and mancozeb alone, but for MVO + mancozeb, coverage was not improved compared to mancozeb alone for the canopy locations sampled. In the second and third trials, three nozzle types (ISO injet, XR110, and AI3070) and three carrier volumes (115, 235, and 350 L ha−1) were evaluated with and without MVO + mancozeb, respectively. Including MVO with mancozeb did not improve canopy coverage for any canopy locations compared to mancozeb alone, regardless of the nozzle or carrier volume tested. Thus, across all sets of trials, canopy penetration was not improved using MVO + mancozeb compared to mancozeb alone. Additional trials evaluating MVO, mancozeb, and carrier volume are being conducted to evaluated effects CLS severity.