Strawberry decline syndrome – an emerging threat to strawberry production in Atlantic Canada. P. A. ABBASI. Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, 32 Main Street, Kentville, NS B4N 1J5, Canada
Strawberry nurseries in the Annapolis valley, Nova Scotia, are major suppliers of bare-root transplants or plug plants for the commercial growers in Canada and the USA. However in 2012 and 2013, acute decline symptoms appeared in strawberry plants in some of these nurseries, gravely threatening strawberry production in both countries. Strawberry growers noticed uneven growth patterns, stunted foliage, reddening of older leaves, and yellowing of leaf margins in their fields. The strawberry decline syndrome has also been reported from several US states and the source of infection was traced back to infected plants from nurseries in Nova Scotia. Analysis of infected plants revealed the presence of two main viruses, strawberry mild yellow edge virus (SMYEV) and strawberry mottle virus (SMoV). Both these viruses are vectored by aphids in a persistent or semi-persistent manner. It was also confirmed that the samples showing severe decline symptoms were doubly infected by both SMYEV and SMoV. When the viruses are present by themselves in a plant, there is little to no disease development, but when they are both present, there is a sudden shift in severity of symptoms for unexplained reasons. Several strawberry plantings have already been discarded to prevent further spread and both strawberry fruit and nursery production sectors are at great risk to this disease. Characterization of interactions between strawberry viruses, host and aphid vectors and their roles in acute decline disease would lead to development of a disease management strategy to mitigate the economic losses to the national strawberry industry.
Gene expression analysis of changes induced by phosphites in Phytophthora infestans. T. BORZA, G. SAKTHIVEL AND G. WANG-PRUSKI. Department of Plant and Animal Sciences, Faculty of Agriculture, Dalhousie University, 50 Pictou Road, Truro, NS B2N 5E3, Canada
Phosphite-containing fungicides are increasingly used to control the development of the fungal-like plant pathogens from the Oomycetes group. The oomycete Phytophthora infestans (Mont.) de Bary is responsible for the occurrence of late blight disease in potatoes, which causes enormous economic damage worldwide. Phosphites (Phi) inhibit oomycetes by two different modes of action; directly, by inhibiting the pathogen’s development, and indirectly, by inducing plant defence mechanisms. The way Phi suppresses the development of P. infestans in plants is still not well documented. With the aim to determine the effects of Phi on P. infestans we analysed, using quantitative RT-PCR (qPCR), the expression pattern of 14 genes involved in pathogen-plant interaction, calcium-mediated signal transduction, energy production, oxidative stress, translational regulation and protein synthesis. Phytophthora infestans mating type A2, genotype US-8 was grown in vitro, on a pea medium. After 5 days of growth, two different concentrations of Phi, i.e. 100 µg mL−1 and 150 µg mL−1, were added to the medium. To ascertain the fungitoxic effects, samples were taken 24 h and 48 h after the addition of Phi. Both concentrations triggered a similar overall response pattern. After 24 h the expression of most genes was found to be down-regulated. A completely opposite trend was documented after 48 h, when the expression of nearly all genes was found to be up-regulated. A possible explanation for this pattern is that after a short period of stress, P. infestans aims to compensate the toxic effects by turning on the expression of a wide array of genes.
The development of genetic markers as a screening tool for resistance to late blight [Phytophthora infestans] in potatoes (Solanum tuberosum). J. COFFIN, R. H. COFFIN, G. WANG-PRUSKI AND R. D. PETERS. Privar Farm Inc., 909 Eliot River Road, North Wiltshire, PE C0A 1Y0, Canada; (G.W.-P.). Department of Plant and Animal Sciences, Faculty of Agriculture, Dalhousie University, Truro, NS B2N 5E3, Canada; and (R.D.P.) Crops and Livestock Research Centre, Agriculture and Agri-Food Canada, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada
Late blight [Phytophthora infestans (Mont.) de Bary] is a serious disease. The objectives were to develop genetic markers related to the late blight resistance genes in parental materials and to determine if these genetic markers could be used to distinguish breeding progeny for resistance. The late blight resistant material was derived from Solanum bulbocastanum. Two resistant lines, F02005 and F02006, were accessed from Agriculture and Agri-Food Canada, Fredericton, NB. Seven DNA markers were successfully developed within the two R genes. The two R genes, Rpi-BLB1 and Rpi-BT1, were tested using four pairs of primers for Rpi-BLB1 and three pairs of primers for Rpi-BT1. The seven DNA markers successfully detected two of the functional R genes present in the two resistant parents, but not in the two susceptible ones. The next step was to see if the progeny produces the proper PCR products from the DNA markers, which confirms the progeny inherited the resistant genes. Progeny were selected from survivors of offspring of the resistant × susceptible cross by inoculation with a suspension of spores of P. infestans, A2-US8. Leaves were collected from the field-grown progeny and immediately preserved in liquid nitrogen. Six of the seven markers, three for the Rpi-BLB1 gene and three for the Rpi-BT1 gene, were examined using the four parents and nine offspring. Of the nine progeny tested, seven contain two functional R genes and two do not. This outcome indicates that these DNA markers can distinguish between progeny with the R genes and those without.
Resistance to metalaxyl-m in populations of Phytophthora erythroseptica causing pink rot of potato in Canada. B. CRANE, R. D. PETERS, L. M. KAWCHUK, A. MACPHAIL, K. A. DRAKE, D. GREGORY AND K. MACDONALD. Crops and Livestock Research Centre, Agriculture and Agri-Food Canada (AAFC), 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; and (L.M.K.) Lethbridge Research Centre, AAFC, 5403 1st Avenue South, Lethbridge, AB T1J 4B1, Canada
Pink rot, caused by Phytophthora erythroseptica Pethybr., is a common disease of potatoes in Canada. It is particularly prevalent when high levels of moisture in autumn contribute to pathogen spore release and tuber infection. Management of pink rot has relied heavily upon application of metalaxyl-m (Ridomil Gold ®), either at planting or as a foliar spray during the growing season. In recent years, isolates of P. erythroseptica with resistance to metalaxyl-m have been recovered in New Brunswick and in 2012, resistant strains of the pathogen were found in Prince Edward Island. A national survey to assess the distribution of resistant strains of P. erythroseptica was initiated in 2013. Samples of infected tubers from across Canada were used to obtain isolates of the pathogen for subsequent testing for metalaxyl-m sensitivity using an in vitro agar assay. To date, isolates of P. erythroseptica with resistance to metalaxyl-m have been recovered from Prince Edward Island, Nova Scotia, New Brunswick, Ontario and Manitoba. Therefore, an expansion of the range and distribution of metalaxyl-m resistant isolates of the pink rot pathogen is occurring in Canada. The widespread occurrence of metalaxyl-m resistance raises concerns about the efficacy of applications of Ridomil Gold ® for pink rot control and may add importance to the role played by phosphites in the management of this disease.
Detection and quantification of Verticillium dahliae and V. albo-atrum in soils and potato plants by qPCR. X. GAO, T. BORZA, Z. GANGA AND G. WANG-PRUSKI. Department of Plant and Animal Sciences, Faculty of Agriculture, 50 Pictou Road, Truro, NS B2N 5E3, Canada; and (Z.G.) Cavendish Farms, New Annan, PE C1N 5J5, Canada
Verticillium wilt, mainly caused by the soil-borne pathogens Verticillium dahliae Kleb. and V. albo-atrum Reinke & Berth., is a vascular wilt disease causing declining yields in worldwide potato production areas. Pre-planting risk assessment of Verticillium pathogens in soils or early diagnosis of infection in potato plants are necessary to determine the best crop production management strategies. This study seeks to develop a rapid PCR-based Verticillium test system for detecting and quantifying both V. dahliae and V. albo-atrum in soils and potato plant tissues. Based on the ribosomal DNA (rDNA) intergenic spacer (IGS) and β-tubulin gene sequences, species-specific SYBR Green real-time quantitative PCR (qPCR) assays were modified or developed for the detection and quantification of V. dahliae and V. albo-atrum, respectively. Our results showed that the primer pairs can be used to distinguish the V. dahliae and V. albo-atrum pathogens grown in in vitro culture, or from soil and plant tissues samples. The lowest detection limits of Verticillium DNA in plant and soil samples are approximately 10 and 100 fg of DNA, respectively. These amounts are equivalent to less than one Verticillium cell in plant samples and to one–three cells in soil samples, depending on the genes (IGS or tubulin) used for identification. Repeated experiments confirmed that the methodology is sensitive, species-specific, and reliable. It can be widely used in field assessment, crop production and evaluation of treatment options.
Molecular detection, identification and phylogenetic study of Heterobasidion species complex. X. LI, K. YUAN, S. F. SHAMOUN, J. NIE, H. ARSENAULT, G. SUMAMPONG, C. HAMMETT AND S. H. DE BOER. Canadian Food Inspection Agency, Charlottetown Laboratory, Mount Edward Road, Charlottetown, PE C1A 5T1, Canada; and (S.F.S., G.S., C.H.) Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
Heterobasidion annosum (Fr.) Bref. sensu lato causing root and butt rot of coniferous trees is a species complex comprising of five species with various geographic distributions. In North America, H. irregulare Otrosina & Garbel. causes disease on pine trees and was known as the traditional P-intersterility group (P-ISG), and H. occidentale Otrosina & Garbel. infects spruce and was named S-ISG. The other three species, H. annosum (Fr.) Bref. sensu stricto, H. parviporum (Fr.) Niemelä & Korhonen and H. abietinum Niemelä & Korhonen, are widely distributed in European and Eurasian countries and known as P-ISG, S-ISG and F-ISG (pathogenic on fir), respectively. Morphological differentiation among these species remains a long-standing challenge for classification, and risk is, therefore, unknown for accidental introduction of a new, potentially more aggressive root and butt rot pathogen of coniferous trees via trading of forest products and commodities. In this study, 22 Canadian isolates from British Columbia, Quebec and Ontario, and 55 European and Eurasian isolates were collected for developing species-specific PCR, real-time PCR and loop-mediated isothermal amplification (LAMP) assays. A real-time PCR assay was modified and evaluated for detection and identification of all five species of Heterobasidion species complex while four LAMP assays were designed for identification of H. irregulare, H. occidentale, H. annosum sensu stricto and H. abietinum with various specificities. Phylogenetic data based on eight housekeeping gene markers indicated that 10 isolates from British Columbia and 15 from Quebec and Ontario formed two North American lineages, resembling H. occidentale and H. irregulare, and three clades formed by all European isolates belonged to species of H. annosum sensu stricto, H. parviporum and H. abietinum, respectively. The phylogenetic data also showed that the European and North American species formed two monophyletic sister clades: one consists of H. abietinum, H. parviporum and H. occidentale, and the other includes H. annosum and H. irregulare. Both geographic separation and host specificity are the main factors contributing to species formation of Heterobasidion species complex.
Fusarium head blight in the Atlantic Region – a 30-year retrospective. R. A. MARTIN, H. VOLDENG, T. M. CHOO AND W. YAN. Crops and Livestock Research Centre, Agriculture and Agri-Food Canada (AAFC), 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; and (H.V., T.M.C., W.Y.) Eastern Cereal and Oilseed Research Centre, AAFC, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
Fusarium head blight (FHB) [Fusarium graminearum Schwabe] has been an on-going important disease problem in cereals in the Atlantic Region of Canada since the early 1980s. Severity has ranged from trace to highly severe between years and or between areas within the region; with mycotoxin (deoxynivalenol, DON) levels in excess of 10 ppm. Spring wheat and barley have been the major affected cereals in the region, while infected oats is impacted to only a minor extent. Winter wheat cultivars are highly susceptible but the crop appears to miss critical infection periods and field issues are fewer than with spring cereals. The challenge has been providing adequate risk-management strategies which address regional epidemic development patterns while being economically viable and address field symptoms and mycotoxin contamination. Primary emphasis in research and development has been directed towards the development of superior germplasm with resistance to FHB and resultant mycotoxin contamination. While a slow process, success with resistant germplasm has started to occur, with release of cultivars with good resistance maintaining high agronomic and other disease resistant characteristics; for example, AAC Scotia, breed in Charlottetown, with some of the best resistance available in a good high-yielding and quality spring wheat line. In barley, research has consistently demonstrated that two row cultivars, Island particularly, are far superior to six row cultivars, and this has resulted in a heavier use of two row barley, in regional production. While fungicide studies have identified superior materials for foliar/head application for increasing yield in wheat and barley, the impact on FHB control has been disappointing, with DON reductions ranging from 0–50%. Some basic epidemiology studies have demonstrated that infection periods are not limited to anthesis but continue throughout the season, resulting in challenges to development of superior germplasm and other disease reduction strategies. This has meant that resistance strategies or germplasm developed in other regions are not always effective in the Atlantic Region.
Breeding for resistance to Potato virus Y – an update from the Potato Research Centre. A. M. MURPHY, D. H. WILSON AND S. ALLABY. Potato Research Centre, Agriculture and Agri-Food Canada, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada
Several strategies have been used to increase the frequency of extreme resistance to Potato virus Y (PVY) conferred by Ry genes in potato selections. Progeny from resistant parents were screened by mechanical inoculation followed by graft inoculation to verify extreme resistance. Extreme resistance is effective against all strains of PVY. However, several parents used to confer resistance to PVY were found to be highly susceptible to common scab. For this reason, a project was undertaken to develop improved parents which have both extreme resistance to PVY and common scab. Approximately 1000 clones from 12 families were subjected to common scab and PVY challenges over several cycles; survivors were retained on the basis of their disease reactions. These survivors were also assessed for the presence of a marker associated with resistance to golden nematode (Globodera rostochiensis (Woll.) Behrens). As of October 2013, 23 selections have been retained, 20 of which have extreme resistance to PVY, 15 have good scab scores and five carry a marker for golden nematode resistance. Their agronomic performance has been assessed and they will be evaluated for parental value. Several potato selections recently released through the Accelerated Release process have extreme resistance to PVY and are currently undergoing evaluation by industry participants.
PVX and PVYNTN co-infections incite stronger synergistic reactions than mixed infections with PVX and other strains of PVY in solanaceous plants. X. NIE, M. SINGH, Z. LIANG AND X. XIONG. (X.N., Z.L.) Potato Research Centre, Agriculture and Agri-Food Canada, 850 Lincoln Road, Fredericton, NB E3B 8Z7, Canada; (M.S.) Agricultural Certification Services, 1030 Lincoln Road, Fredericton, NB E3B 8B7, Canada; and (Z.L., X. X.) College of Horticulture and Landscape, Hunan Agricultural University, Changsha, China
Post-harvest test of potato cv. ‘Kennebec’ revealed a Potato virus Y (PVY) incidence at 15.8%, a rate that is unusually high for a cultivar possessing a high level of field resistance to the virus. Randomly selected tubers were planted in a field plot and the resulting plants were monitored. Approximate 16% of plants developed symptoms ranging from mild mosaic to severe necrosis/rugosity/stunting. ELISA and RT–PCR analysis revealed that infections with Potato virus S (PVS), Potato virus X (PVX) and PVY, mostly in mixed-infections, occurred commonly in the 14 sampled plants. Two strains, namely PVYO and PVYNTN, were identified in the PVY-positive plants. In general, mild mosaic was associated with infections with PVX and PVS; intermediated mosaic was associated with PVS and PVYNTN infections; whereas severe leaf deformation/necrosis/drop were associated with PVYNTN and PVX co-infections, or with PVYO and either PVS or PVX co-infections. Virus-free plantlets were mechanically inoculated with PVX, PVYO and PVYNTN alone or with PVX + PVYO or PVX + PVYNTN combination in the greenhouse. Infections with PVYO, either alone or with PVX, or with PVX + PVYNTN incited severe mosaic and systemic necrosis soon after the inoculation. The most severe symptoms occurred in the mixed-inoculation with PVX + PVYNTN, demonstrating dramatic synergism between PVX and PVYNTN. Profound PVX and PVYNTN synergism was also found in tobacco, tomato and Physalis floridana plants, suggesting that the genetic makeup of PVY plays an important role in the level of synergistic reactions between PVX and PVY on solanaceous plants.
De novo assembly and analysis of the mRNA transcriptome in resting spores of the potato wart pathogen Synchytrium endobioticum. D. S. SMITH AND U. SINGH. Canadian Food Inspection Agency, Charlottetown Laboratory, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada
Potato wart, caused by the soilborne fungal pathogen Synchytrium endobioticum (Schilbersky) Percival, is a serious potato disease with the potential to cause significant economic damage. The overwintering stage of S. endobioticum is a resting spore, which can remain viable in soil for decades. Synchytrium endobioticum is a regulated, quarantine pest, and infested fields must be declared free from viable sporangia before they can be released from quarantine. The current viability test is a bioassay, which is labour-intensive and takes 3 months to perform. It has been proposed that a molecular test based on the detection of mRNA transcripts may provide a more efficient supplementary or replacement viability test for S. endobioticum in soil. A cDNA library from resting spore RNA was constructed and sequenced, generating 16 million 150 base paired-end reads. The reads were quality-checked and assembled de novo into 7285 transcripts representing 6663 loci. Four previously sequenced genes were found within the assembly, thus validating the sequence and assembly data. The vast majority of transcripts were expressed at a low level, and only 283 (0.04%) were expressed at 500 transcripts per million or higher. Of these, 166 sequences demonstrated significant homologies and could be annotated using blast2GO. Predominant biological processes and molecular functions associated with the more highly expressed transcripts included response to stress, signal transduction, intracellular transport, oxidoreductase activity, ribosome structure, and translation.
Modernization of Canadian potato post-entry quarantine programme – validation and implementation of molecular diagnostic methods. H. XU AND S. CODY. Canadian Food Inspection Agency, Charlottetown Laboratory, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada
The importation of tuber-producing species of Solanum spp., either as true seed or plant parts for vegetative propagation, is prohibited from all countries and some regions of the USA. However, small quantities of Solanum spp. germplasm may be introduced into Canada, for vegetative propagation while minimizing the risk of introducing quarantine pathogens 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 before serial testing is conducted for detecting any possible potato pathogens. For a long time, the detection has relied on bioassay, ELISA and electron microscopy. R-PAGE was implemented in the 1990s for the detection of viroids. The entire process of the PPEQ testing is costly and time consuming. Efforts have been made in recent years to modernize the PPEQ programme by introducing molecular diagnostic procedures. Since 2010, standard protocols based on RT-PCR and real-time PCR were validated and implemented for detecting Potato mop-top virus and Clavibacter michiganensis subsp. sepedonicus (Spieck. & Kotth.) Davis et al. in potato germplasm. Projects have also been initiated for the evaluation and validation of multiplex RT-PCR procedures for detecting various viruses in microplantlets and grown-out plants. Methods for the efficient extraction of nucleic acids from various potato tissues and for the verification of primary quarantine testing results have also been evaluated and incorporated into standard diagnostic protocols. These molecular methods are superior to the bioassay and ELISA in sensitivity, specificity and rapidity and their implementation will greatly improve the quality and efficiency of potato quarantine testing.
Comparative genomics of pectobacteria and development of species-specific assays against Pectobacterium wasabiae. K. YUAN, Z. ADAM, J. TAMBONG, W. CHEN, C. LEWIS, S. H. DE BOER AND X. LI. Canadian Food Inspection Agency, Charlottetown Laboratory, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada; and (W.C., C.L., J.T.) Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
Pectobacterium wasabiae (Goto & Matsumoto) Gardan et al. (previously classified as Erwinia carotovora subsp. wasabiae) is one of the soft rot bacteria belonging to the Gram negative Enterobacteriacae family and responsible for significant economic losses of potato and ornamental plants. Pectobacterium wasabiae has unique features compared with other soft rot bacterial species and genera and is well suited for studying the ecology, speciation and pathogenicity. A draft genome sequence of a Canadian potato isolate Pw1002 was generated using paired-end Illumina HiSeq sequencing technology. De novo assembly resulted in 42 scaffolds of a total size of 5.0 Mb. Further analysis on the RAST server identified a number of predicted virulence factors. Comparative genomic analysis of P. wasabiae Pw1002 with two other potato strains, SCC3193 (Europe) and WPP163 (USA), and one horseradish strain, CFBP3304 (Japan), was executed using MAUVE (v2.3.1). Eighteen hypervariable regions bearing pathogenicity related factors were grouped to ten INDELs and eight highly diversified regions. Among these regions only six loci have unique sequences for developing specific assays using AlleleID7 (v7.8). Pectobacterium wasabiae specificity test was performed in preliminary PCR amplificaton of all six loci on closely related species and subspecies of pectobacteria (P. atrosepticum, P. carotovorum subsp. brasiliense, P. carotovorum subsp. carotovorum, Dickeya spp. (E. chrysanthemi) and P. carotovorum subsp. odorifera). Further evaluation indicated that two loci could be used as targets for P. wasabiae specific real-time PCR assays. Analyses of genome sequences of strains isolated from different hosts and geographic regions have provided a blueprint for phylogenetic and functional studies of this widely distributed pathogen.