Role of effectors in inducing and suppressing disease resistance in barley and potato by pathogenic fungi and nematodes. S. ALI, P. A. ABBASI, J. D. LAURIE, R. LINNING, J. A. CERVANTES-CHÁVEZ, D. GAUDET, L. JAMSHAID, G. BÉLAIR, P. MOFFETT AND G. BAKKEREN. Kentville Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 32 Main Street, Kentville, NS B4N 1J5, Canada; (J.D.L., D.G.) Lethbridge Research and Development Centre, AAFC, 5401-1 Avenue South, Lethbridge, AB T1J 4B1, Canada; (R.L, J.A.C., G.B.) Summerland Research and Development Centre, AAFC, 4200 Highway 97 South, Summerland, BC V0H 1Z0, Canada; (G.B.) Saint-Jean-sur-Richelieu Research and Development Centre, AAFC, 430 Gouin Boulevard, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada; and (P.M.) Département de Biologie, Université de Sherbrooke, 2500, boulevard de l’université, Sherbrooke, QC J1K 2R1, Canada
During infection, plant pathogens secrete effector proteins to reprogram the host plant for its own benefit. In special cases, recognition of certain effectors by resistance genes, essential components of the host surveillance system, induces resistance to infection. No effectors with such avirulence function have been described for basidiomycete fungi infecting cereals. Ustilago hordei (Pers.) Lagerh. is a biotrophic basidiomycete fungus that infects barley. UhAVR1 from U. hordei functions as an avirulence protein, rendering it avirulent on barley cultivar Hannchen, having corresponding resistance gene Ruh1. We have located UhAvr1 within the genome using a deletion approach and confirmed its resistance-triggering function. We also provide evidence that transposable element (TE) activity in the UhAvr1 promoter region and translocation of the coding region are likely responsible for enabling virulence on Hannchen. This region of the genome harbours a cluster of predicted secreted proteins and is syntenic to a cluster in closely related corn pathogens, U. maydis (DC.) Corda and Sporisorium reilianum (Kühn) McAlpine. In U. maydis, deletion of this region results in dramatic reduction in virulence on corn. Potato cyst nematodes, including Globodera rostochiensis (Woll.), are major obstacles to potato production in many areas of the world. Like many other pathogens, G. rostochiensis secretes effectors, which facilitate colonization of the host plants. These proteins are delivered to the plant intercellular space as well as to the host cell cytoplasm through a specialized structure, known as a stylet. We have characterized 30 such effector proteins from G. rostochiensis by expressing in planta. The results of some of these effectors was presented.
Estimation of Verticillium and Fusarium abundance by real-time quantitative PCR using DNA extracted from plant and soil samples – pitfalls and limitations. T. BORZA, H. LIU, X. GAO, A. GOVINDARAJAN AND G. WANG-PRUSKI. Department of Plant, Food and Environmental Sciences, Faculty of Agriculture, Dalhousie University, 50 Pictou Road, Truro, NS B2N 5E3, Canada
Verticillium and Fusarium spp. infect a wide range of plants. These pathogens affect several crops grown in Atlantic Canada, including potatoes, carrots and strawberries. The main Verticillium species pathogenic to potatoes and strawberries are V. dahliae Kleb. and V. albo-atrum Reinke & Berthold while the main Fusarium species pathogenic to carrots are considered Fusarium avenaceum (Fr.) Sacc. and Fusarium oxysporum Schlecht. emend. Snyder & Hansen. Several assays using real-time quantitative PCR (qPCR) have been designed in our laboratory to assess the incidence and abundance of these pathogens. Subsequently, these approaches have been used to process a large number of plant and soil samples. The identification of pathogens using the qPCR method was much faster and reliable than the traditional methods involving plating on specific media. On the other hand, the estimation of pathogen abundance using the absolute quantification (standard curve) qPCR method, was found to be affected by several pitfalls and limitations. While generating and processing qPCR data from the above-mentioned pathogens, it has been found that several factors such as the amount of sample used for DNA isolation, gene copy number, genetic polymorphism, the amount of DNA used as template for amplification, the volume of the qPCR reaction, qPCR amplification efficiency and detection limit, and the statistical processing of pathogen distribution in soil and in plant samples, all greatly affect the outcome of this molecular approach. Therefore, the significance of the results has to be clearly spelled out and the limitations of this molecular approach have to be acknowledged.
Diseases diagnosed in commercial crops submitted to the Prince Edward Island plant disease diagnostic laboratory for the 2017 season. M. M. CLARK AND A. MACLEOD. P.E.I. Department of Agriculture and Fisheries, Plant Disease Diagnostic Laboratory, P.O. Box 2000, 23 Innovation Way, Charlottetown, PE C1E 0B7, Canada
A total of 259 disease diagnoses were completed during the period 1 June to 14 November 2017. Categories of samples received were potatoes (62.55%), cereal and oilseed crops (10.81%), vegetable and fruit crops (25.48%) and other (2.67%). The 2017 potato growing season started with overall good emergence and vigorous plant stands. However, some uneven emergence and potato seed piece decay became noticeable in potato varieties Gemstar, Russet Burbank, Piccolo, Prospect and Goldrush. The prevalent Fusaria strains involved with the seed piece decay samples were Fusarium oxysporum Schlecht. emend. Snyder & Hansen and Fusarium coeruleum Libert ex Saccardo. Both Fusaria spp. were found to be resistant to fludioxonil and in most cases sensitive to thiabendazole, difenoconazole and prothioconazole. This fungicide resistance work was completed by Dr Rick Peters and his staff at Agriculture and Agri-Food Canada (AAFC/ACC). For the first time in history, there were no confirmed cases of potato foliar late blight. Dr Sean Lee and Dr Jingbai Nie from the Canadian Food Inspection Agency (CFIA/ACIA) confirmed an identification of a phytoplasma in a commercial garlic sample and Pectobacterium atrosepticum (van Hall) Gardan, Gouy, Christen & Samson in one potato bacterial blackleg sample. Isolations from potato stem tissue confirmed early dying fungi involved included Rhizoctonia sp., Colletotrichum coccodes (Wallr.) S. Hughes, Verticillium spp. and a high level of F. oxysporum (Dr Tharcisse Barasubiye, AAFC/NFIS). Foliar potato leaf spot symptoms started to invade the wilted potato plants of potato varieties FL1879, Atlantic, Innovator, Ranger Russet and Russet Burbank. The causal agents involved included Alternaria alternata (Fr.) Keissl and some Alternaria solani Sorauer. Isolations from ‘highbush’ blueberry plants confirmed the presence of the fungal organism, Phomopsis sp. The apple acreage on Prince Edward Island is increasing and fire blight symptoms appeared in mid-July in two varieties (confirmation pending).
Review: Marker-assisted selection of disease traits integration into AAFC’s potato breeding programme. V. DICKISON, B. BIZIMUNGU, D. DE KOEYER, K. DOUGLASS AND X. NIE. Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada
The development of new potato cultivars using conventional breeding takes on average 10–12 years prior to industry integration. Breeding for disease resistance is complicated by the lack of effective screening tools in order to speed up and improve the selection process. Currently, disease traits are evaluated using phenotypic data collected after exposure to the disease and subsequent rating of the effects of the disease on each line. This process is labour intensive and time-consuming, and can take months or years to ensure accurate assessment of a single trait. Multiple high throughput molecular markers are being developed and validated for use within AAFC’s potato breeding programme. Currently, high resolution DNA melting (HRM) markers/assays for the resistances against potato cyst nematodes (PCN) (Globodera rostochiensis (Woll.) pathotype Ros1), Potato virus Y (PVY) and Potato virus X (PVX) are being integrated within the programme. The HRM assay for PCN resistance has been adopted and part of routine screening for 7 years within AAFC’s breeding programme; developed, validated and performed by AAFC colleagues, Dr D. De Koeyer and K. Douglass. The breeding team has recently increased its capacity to do their own evaluation of lines using marker-assisted selection (MAS) and will commence PCN marker evaluation. In addition to the HRM marker for the PCN resistance, respective HRM markers for extreme resistance (ER) against PVY and PVX, developed by X. Nie and his team, are being incorporated into the programme’s selection process. These gel electrophoresis-free markers/assays were developed either by converting the existing gel electrophoresis-dependent markers such as STS markers or based on ER-linked single nucleotide polymorphisms (SNPs) that were identified by next-generation sequencing (NGS). The utilization of the high throughput HRM-based marker-assisted selection within the breeding programme will assist AAFCs breeding programme to incorporate traits of interest sought by industry needs more efficiently and effectively.
Characterization of Fusarium graminearum putative virulence factors by CRISPR/Cas9 gene editing. A. J. FOSTER. Charlottetown Research and Development Centre, Agriculture and Agri-Food Canada, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada
Pathogenic fungi, such as Fusarium graminearum Schwabe (a causal agent of fusarium head blight in wheat, barley and oats), utilize small secreted cysteine-rich proteins (SSCRPs) to modulate host immunity. These proteins are commonly referred to as effectors. Currently, the function and role in virulence of only a few SSCRPs is known compared with the vast number that have been identified. One widely used approach to characterize fungal effectors is gene disruption through homologous recombination. This method works by replacing all or part of a target gene with a selectable marker and assessing the phenotype of the new ‘knock-out’ isolates. This approach is greatly limited, as only one gene can be targeted per transformation whereas many effector proteins are suspected of having functional redundancy and can exist in large gene families. The type II clustered regularly interspaced short palindromic repeat system (CRISPR) together with the CRISPR-associated, protein Cas9 recently emerged as a powerful new tool for gene editing in molecular biology. Among the different uses of the CRISPR/Cas9 system is the ability to target multiple genes for editing through insertion and/or deletion (indels). Methods have been developed and adapted to use CRISPR/Cas9 to assess SSCRP function through the selection of gene targets, the design of sgRNA, the construction of cassettes, vector assembly and fungal transformation. These approaches for CRISPR/Cas9 gene editing of SSCRPs were discussed.
Bio-protectant development to control potato pathogens. C. W. KIRBY, M. H. NABUURS, J. N. D. VACON AND S. M. BOYETCHKO. Charlottetown Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; and (S.M.B.) Saskatoon Research and Development Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada
Bioautography methods are being used to screen for bio-protectant candidates produced from an established soil bacterial collection to control potato pathogens. Current pathogens of interest are Phytophthora erythroseptica Pethybr. (pink rot), Colletotrichum coccodes (Wallr.) S. Hughes (black dot), Helminthosporium solani Durieu & Mont. (silver scurf), Streptomyces scabies Lambert and Loria (common scab), Fusarium sambucinum Fuckel (dry rot), Alternaria alternata (Fr.) Keissl. (brown spot) and Phytophthora infestans (Mont.) de Bary (late blight). Direct bioautography methods require large amounts of spores to conduct the assay in a reasonable time-frame; however, determining growth conditions to induce sporulation of our target pathogens has proved to be taxing. To overcome this problem, we are using a modified method where the culture can be taken directly from its standard growth conditions on a Petri plate to create the microbial suspension for the assay to allow rapid screening for antimicrobial compounds. These ongoing efforts to improve potato viability (both in the field and storage) through the screening and isolation of potential bio-protectant candidates for environmentally friendly treatment methods was presented.
Suberization of skin cells from potato tubers infected or not infected with common scab pathogens. Y. LIN, Z. ZHANG, R. COFFIN, J. COFFIN, T. BORZA AND G. WANG-PRUSKI. Department of Plant, Food and Environmental Sciences, Faculty of Agriculture, Dalhousie University, 50 Pictou Road, Truro, NS B2N 5E3, Canada; (R.C., G.C.) Privar Farm Inc., 909 Eliot River Road, North Wiltshire, PE C0A 1Y0, Canada; and (Z.Z.) Joint FAFU-Dalhousie Lab, College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
The suberization of potato tuber skin cells is important for water and mineral metabolisms, and disease resistance during growth and development. Observation of structural changes of suberized cell layers during tuber development can be assessed by the number of suberized cells, and the thickness of suberized layers. The outcome can provide a better understanding of the efficacy of this barrier against pathogens such as common scab (Streptomyces scabiei Lambert and Loria) in different potato varieties. A quick and effective method to observe the suberized cell layers in potato tuber skin was developed in our laboratory. The simplified method allowed the evaluation of as many as eight tubers a day; as a result, suberization could be compared in real-time throughout tuber development. Two trials were established in the 2017 season, one in the growth chamber and another in the field. Potato tuber suberization was compared in different growth stages in non-infected tubers in the growth chamber trial that comprised of the varieties Goldrush (resistant to common scab) and Prospect (susceptible to common scab). In addition, tubers from these two varieties plus Hindenburg (resistant), Jubel (resistant), and Riverdale Russet (susceptible) were collected periodically from the common scab infested field. Data confirmed that the number of suberized cell layers and the thickness of suberized cell layers were increased during tuber growth and development. Also, common scab resistant varieties were found to have a significantly higher number of suberized cell layers and thicker suberized cell layers as compared with the susceptible varieties.
Foliar selenium application induces defence response in potato plants infected with late blight. A. SOMALRAJU, J. MCCALLUM, D. MAIN, R. D. PETERS AND B. FOFANA. Charlottetown Research and Development Centre, Agriculture and Agri-Food Canada, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; and (A.S.) University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A 4P3, Canada
Late blight caused by Phytophthora infestans (Mont.) de Bary is a devastating disease, leading to complete crop loss in some cases. Although chemical control of potato late blight using fungicides is effective under conventional production systems, organic systems require different and viable disease control practices. Selenium (Se) is a mineral micronutrient, often used in crop and livestock bio-fortification because of its antioxidant and stress tolerance properties. However, the role of selenium in plant growth and defence is not fully understood. This study was designed to evaluate the defence responses induced by foliar selenium application in potato plants infected with late blight under a greenhouse setting. Our recent data on disease incidence, secondary metabolite production and phenylpropanoid pathway gene expression following selenium application was presented and discussed.
Assessment of the use of biopesticides and phosphite to reduce common scab in potatoes. G. WANG-PRUSKI, R. COFFIN, J. COFFIN, B. BEATON, C. GOYER, M. Z. ALAM, F. DESAI, T. BORZA, Y. LIU AND Y. XI. Department of Plant and Animal Sciences, Faculty of Agriculture, Dalhousie University, 50 Pictou Road, Truro, NS B2N 5E3, Canada; (R.C., G.C.) Privar Farm Inc., 909 Eliot River Road, North Wiltshire, PE C0A 1Y0, Canada; (B.B.) PEI Department of Agriculture and Fisheries, P.O. Box 2000, Charlottetown, PE C1A 7N8, Canada; and (C.G.) Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada
Potato common scab (Streptomyces scabiei Lambert and Loria) is increasingly becoming a tuber quality issue in PEI. Biopesticides are promising formulations to suppress common scab. Two commercial biopesticides, Microflora ProTM and Double NickelTM, were examined in 2016 and 2017 on a field infested with common scab at Privar Farm Inc., PEI. Four potato varieties including Goldrush (resistant), Prospect (susceptible), Green Mountain (very susceptible) and Red Pontiac (very susceptible) were used. Four replicated plots were established for each of the four varieties and treatments. Since phosphite products such as Confine, Phostrol and Rampart are widely used by growers in the region, the effect on common scab development using one of these products was also investigated. The same treatments and field design were used as described above and the plots were sprayed four times with Phostrol during the growing season. Common scab rating was recorded for each tuber by determining the percentage of common scab area, number of shallow pits, number of deep pits and necrotic patches. The number of suberized skin cell layers and the thickness were measured using a protocol developed in our laboratory. Scab rating, shallow pits, deep pits and necrotic area showed significant differences among cultivars, and their response to the treatments also showed significant variations. Histological assessments of suberized tuber skin showed significant variations in the four varieties. Although there was visible reduction of common scab in some of the varieties following the Microflora Pro and Double Nickel treatments, scab severity on tubers of the susceptible varieties was not decreased enough, therefore, these tubers are unlikely to pass for premium grade table stock potatoes.