Cucumber necrosis virus coat protein interacts with heat shock proteins both in vitro and in vivo. S. B. ALAM AND D. ROCHON. University of British Columbia, Faculty of Land and Food Systems, MCML 248–2357 Main Mall, Vancouver, BC V6T 1Z4, Canada; and (S.B.A, D.R.) Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada
Viruses are obligate intracellular parasites and must recruit a variety of host components including HSP70/HSc70 homologues for performing various functions during multiplication. Identification of such host components could assist in the development of therapeutic targets to mitigate virus disease. HSP70 protein levels increase significantly during Cucumber necrosis virus (CNV) infection in Nicotiana benthamiana. We have also found that HSP70/HSc70 coimmunoprecipitates with CNV coat protein expressed in leaves and copurifies with CNV particles. CNV/HSc70 interaction was observed in virus overlay assays. The down-regulation of HSP70 in N. benthamiana leads to an aggravated and rapid induction of necrotic lesions which is consistent with the involvement of HSP70 in the inhibition of apoptosis. To examine whether HSP70 could potentially participate in CNV disassembly, HSP70 was overexpressed via heat shock (HS) treatment in the local lesion host Chenopodium quinoa and leaves were then inoculated with virus particles. We observed a statistically significant increase in the number of local lesions in HS versus non-HS plants. However, a significant increase was not observed when virion RNA was used as an inoculum suggesting that HSP70 may play a role in CNV disassembly. A CNV CP mutant lacking the β-region in the arm domain did not show a statistically significant difference in the number of local lesions on HS versus non-HS plants suggesting that the β-region may interact with HSP70. We have also found that incubating HSP70/HSc70 with virus in vitro renders the particles more sensitive to trypsin digestion suggesting a role in conformational change of virions and possibly virus disassembly.
Leaf anatomy and pathogen resistance: the case of Thuja plicata and Didymascella thujina. J. A. ALDANA, J. H. RUSSELL AND B. J. HAWKINS. Centre for Forest Biology, University of Victoria, P.O. Box 3020 STN CSC, Victoria, BC V8W 3N5, Canada; and (J.H.R.) British Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, 7060 Forestry Road, Mesachie Lake, BC V0R 2N0, Canada
Western redcedar (Thuja plicata Donn ex D. Don, WRC), an important tree of British Columbia, is susceptible to cedar leaf blight (Didymascella thujina (Durand) Marie, CLB). No major resistance gene appears to exist since all trees are prone to infection. There is quantitative variation among WRC populations in resistance to CLB; however, little information exists on the underlying mechanisms of resistance. This study examined the relationship between the anatomy of uninfected leaves of WRC families and susceptibility to infection by CLB. Twenty seedlings, one and a half years old, from four resistant (399, 685, 687 and 689), and four susceptible families (525, 528, 582 and 583) were exposed to CLB in the field during natural sporulation in summer 2012, and scored for disease severity after symptoms appeared. Five uninfected plants per family were used to measure stomatal density, cuticle thickness, epidermal thickness, leaf thickness, leaf area and frequency of resin glands in leaf cross-sections using light microscopy. Mesophyll to leaf thickness, and mesophyll to leaf area ratios were also calculated. When analysed by class (resistant vs. susceptible), only disease severity rendered significant differences in non-parametric ANOVAs. However, when compared by family, there were significant differences in leaf thickness, cuticle thickness and leaf area. No significant differences were found among families in other traits. These results suggest that leaf anatomy plays only a minor role in WRC family resistance to CLB, and other mechanisms are likely involved due to the quantitative nature of CLB resistance.
Methods for screening survival, resistance and tolerance traits 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
Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) is an ecologically and commercially important tree species native to the Pacific Northwest. Over much of its range, interior Douglas-fir (var. glauca = Fdi) receives little or no summer precipitation, and this major geographic race has thus adapted to grow under drought stress conditions. In cooperation with researchers in the BC Forest Service, we have initiated greenhouse trials to study the responses of Fdi to drought and biotic stressors. Seedlings from four breeding zones, representing distinct environments in BC, have been previously screened for their resistance to Armillaria root disease. In separate experimental trials, we are evaluating 99 half-sibling Fdi families for their responses to drought stress, infection by Phellinus sulphurascens (the fungus causing laminated root rot), and infection by an Ophiostoma fungus vectored by the Douglas-fir bark beetle (Dendroctonus pseudotsugae). We are interested in measuring both resistance and tolerance of Fdi to these disease agents. ‘Resistance’ is defined as a plant's ability to limit damage by a pathogen, while ‘tolerance’ is the ability to sustain damage without limiting fitness or growth. In one experiment, seedlings were subjected to four levels of drought stress over the growing season. Another experiment involved withholding all water and analysing the factors that contributed to survival over time. Our experimental methods and preliminary results of drought screening were presented and discussed. Results of these trials will increase our knowledge of conifer responses to multiple disease agents, and inform and guide specific breeding strategies for Douglas-fir trees.
Biological control of tanoak and bay laurel resprouts using Chondrostereum purpureum. M. ELLIOTT, G. CHASTAGNER, S. F. SHAMOUN, G. SUMAMPONG, E. GOHEEN, A. KANASKIE, E. ORRE AND A. BERNHEISEL. Washington State University, Puyallup Research and Extension Center, 2606 W. Pioneer, Puyallup, WA 9837 USA; (S.S., G.S.) Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada; (E.G.) USDA Forest Service, Forest Health Protection, Central Point, OR 97502, USA; (A.K.) Oregon Department of Forestry, Salem, OR 97310, USA; and (E.O., A.B.) CALFIRE, Soquel Demonstration State Forest, Soquel, CA 95073, USA
The invasive Oomycete pathogen Phytophthora ramorum (Werres et al.) has killed millions of susceptible oak and tanoak trees in California and southern Oregon forests and is responsible for losses in revenue to the nursery industry through mitigation activities. In addition, infestation of forests in the UK by this organism has resulted in the destruction of many hectares of larch plantations. Resprouting stumps can be a reservoir for inoculum of P. ramorum persisting on a site. In areas where the application of herbicides is not permitted, a biocontrol treatment would be an indispensable alternative. Treatment of stumps with the sap-rotting fungus Chondrostereum purpureum (Pers.) Pouzar has been shown to be an effective tool for suppression of resprouting on several species, most notably red alder. In this project, the ability of C. purpureum to suppress resprouting is being evaluated on stumps of two host species, tanoak and California bay laurel. In autumn 2009 our research team established a field trial near Brookings, Oregon on tanoak, and in spring 2013 a trial on bay laurel in the Soquel Demonstration State Forest near Soquel, California. Laboratory testing of three California isolates of C. purpureum indicates that the fungus can colonize bay laurel stems. Early results of field testing showed that C. purpureum was able to colonize the stumps of tanoak following treatment, and was found occurring naturally on tanoak logs and stumps. Formulations of C. purpureum appear to have some effect on reducing resprouting in tanoak, but the most effective and rapid treatment is the hack and squirt method of applying the herbicide imazapyr. Over time, applications of C. purpureum may be a more permanent solution as the stumps begin to decay.
Systemic acquired resistance and induced systemic resistance and the discovery of the mode of a novel plant defence activator. P. H. GOODWIN, T. HSIANG AND A. C. BARCO. School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada
Systemic induced resistance is a way to pre-trigger innate immunity in plants resulting in greater resistance against later attack by pathogens. While there are many forms of systemic induced resistance, the two major types are systemic acquired resistance (SAR) and induced systemic resistance (ISR). This seminar will review these two types in terms of their biological inducing agents (pathogens versus non-pathogens), signalling molecules (salicylate (SA) versus jasmonate (JA) and ethylene (ET)), effect on gene expression (gene induction versus priming), effectiveness against types of pathogens (biotrophs versus necrotrophs). In addition to biological agents, systemic induced resistance can be activated by pathogen-associated molecular patterns (PAMPs), effectors and synthetic analogues. While there are several synthetic analogues reported for activating SAR, previous reports had only described rhizobacteria, rhizofungi and their PAMPs for activating ISR. An example will be given as to how a mixture of isoparaffins was found to induce resistance in plants and then show how it was linked to ISR by demonstrating that it affected JA/ET-dependent defence gene expression similar to the volatile ISR PAMP activator, (2R,3R)-butanediol. In contrast, the isoparaffin mixture did not affect SA-dependent defence gene expression like the SAR synthetic analogue activator, S-methyl 1,2,3-benzothiadiazole-7-carbothioate (sold commercially as Actigard by Syngenta).
Can blister rust resistant Idaho western white pine be used below 1000 m in coastal British Columbia? R. S. HUNT. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
White pine blister rust [Cronartium ribicola Fisch.] alternates between currants (Ribes spp.) and white pines, and in British Columbia (BC) it is often devastating to western white pine (Pinus monticola Donn) unless silvicultural treatments and/or resistant stocks are used. Resistance from a seed orchard in Idaho (ID) improves with increasing age and a decreasing growing season. The recommendation for the BC coast is to plant above 1000 m where the growing seasons are short. Perhaps the recommendation could be relaxed if there were good data from lower sites. Four tree row plots of ID stock and one tree of unselected BC stock were replicated 25 times at four elevation classes (300 m, 450 m, 650 m and 775 m) across three sites. Four to 5 years post-planting the BC and ID stock had a mean of 75% and 73% canker-free (CF) trees respectively. One site, Chilliwack, was covered with currants (R. sanguineum Pursh) and by 11 years post-planting only 11 CF trees were observed. In contrast, the plots at Ladysmith lacked or had few Ribes spp. and the incidence of CF trees was 96% in ID stock and 93% in BC stock. The ID stock appeared superior to BC stock above 628 m at Woss/Vernon (75% and 63% CF trees respectively) and above 775 m at Ladysmith (96% and 87% CF respectively) at 10 and 11 years post-planting, respectively. This contrast is expected to increase with time. Apparently, the gain in resistance by planting at higher elevations can be trumped by increasing Ribes spp. on site, therefore the Ribes spp. incidence must be considered when choosing this ID stock.
The effects of dsRNA in North American Heterobasidion spp. on growth rate, sporulation and germination. I. KASSATENKO, S. F. SHAMOUN, G. SUMAMPONG AND C. HAMMETT. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
Heterobasidion annosum sensu lato is a basidiomycete fungus considered to be the most economically important forest pathogen in the northern hemisphere, causing losses of over $1 billion annually. Heterobasidion annosum sensu lato is a complex consisting of five closely related species. Three are European: H. parviporum (S), H. annosum sensu stricto (P) and H. abietinum (F), and two species are North American: H. irregulare (P) and H. occidentale (S). Heterobasidion populations around the world have been isolated and surveyed for the presence of dsRNA viruses. dsRNA viruses are widespread in fungi and their influence ranges from a three-way mutualistic symbiosis with a fungal host and a plant host, for example, the virus CThTV in Curvularia protuberata helping a panic grass to survive in hot temperatures, to causing hypovirulence, for example, the virus in Cryphonectria parasitica. It is very important to understand the relationship between dsRNA viruses and their plant pathogenic hosts, since there is a potential to use hypovirulent viruses as biological control agents in the future. While the main goal of our research is screening North American populations of H. occidentale and H. irregulare for dsRNA viruses and characterization of them, this presentation will focus on the influence of dsRNA in North American populations of H. occidentale and H. irregulare on host fitness. A total of 140 isolates throughout North America were screened for dsRNA viruses and 26 dsRNA positive isolates were detected, 23 from H. irregulare (37%) and 3 from H. occidentale (4%). Four isolates were cured from dsRNA (2 of H. irregulare and 2 of H. occidentale) using the hyphal tip method. Initial results show significant difference in mycelia growth, sporulation and germination rate between wild (dsRNA positive) and cured (dsRNA negative) strains of H. occidentale isolate PFC5190.
Discovery and application of single nucleotide polymorphisms of disease resistance-related genes for selection of Pinus monticola with genetic resistance against Cronartium ribicola. J.-J. LIU, H. WILLIAMS, A. ZAMANY, R. N. STURROCK, R. A. SNIEZKO AND A. KEGLEY. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada; and (R.A.S., A.K.) USDA Forest Service, Dorena Genetic Resource Center, 34963 Shoreview Road, Cottage Grove, Oregon, 97424, USA
The invasive fungus Cronartium ribicola Fisch. causes white pine blister rust (WPBR), the most destructive disease affecting native five-needle pines in North America. Phenotype-based selection of genetic resistance currently used in conifer breeding programmes is a long-term, high cost process. Genotype-based or genomic selection would provide a more effective breeding strategy. Using RNA-seq technology we compared transcriptomes between major gene (Cr2) resistant and susceptible genotypes (Cr2/- vs. cr2/cr2) of western white pine (WWP, Pinus monticola). In-silico analysis of raw RNA-seq data revealed ~100 000 single nucleotide polymorphism (SNP) sites from orthologous conifer genes and disease resistance-related genes among WWP populations. To validate putative SNPs and select those genetically associated with Cr2, Sequenom iPLEX high-throughput SNP genotyping analysis was used to individually genotype each of 434 SNPs in 282 seedlings with known needle and stem phenotypes. About 70% of SNPs (301/434) were successfully genotyped. Of these 301 SNPs, 227 (75%) were polymorphic in the tested populations. Association analysis of the polymorphic SNPs with disease resistance phenotypes identified four disease resistance-related genes with tight linkage to Cr2. TaqMan and HRM qPCR genotyping assays were then investigated for selection of genetic resistance to WPBR in WWP. Our project demonstrates that a combined approach using RNA-Seq and high-throughput SNP genotyping can be used to discover functional SNP markers and genotype-based selection can be applied to breeding selection of a desirable trait without phenotyping in non-model plants.
Single nucleotide polymorphism discovery in Cronartium ribicola using Genotyping-by-Sequencing approach. T. PU, B. DHILLON AND R. C. HAMELIN. Department of Forest Sciences, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada
Cronartium ribicola Fisch. is the causal agent of white pine blister rust, a branch and stem canker disease of five-needle pines. Since its introduction from Europe in the early 1900s, it has been associated with severe epidemics. All North American white pines are susceptible with mortality rates as high as 95% in some stands, drastically reducing the volume of these trees in Canada. To understand the evolutionary history of C. ribicola populations in North America, we generated genetic variation at the genome level by using the Genotyping-by-Sequencing technique (GBS). GBS offers an efficient and cost-effective approach to studying genetic diversity in large-genome organisms such as barley and maize. It is based on Illumina sequencing of restriction enzyme (RE) digested genomic DNA segments. This sequence information can then be used to discover single nucleotide polymorphisms (SNPs). Our objectives were to: (1) assess the feasibility of GBS for SNP discovery in C. ribicola, (2) determine the right enzyme combination and DNA concentration. For this, a pilot project of seven samples from western Canada and one sample from eastern Canada were genotyped at five different DNA concentrations. We obtained 158 million single-end Illumina reads resulting in 1125 shared SNPs. A principal component analysis showed that the number of SNPs did not vary significantly across concentrations as the clustering of isolates was concentration-independent. These results indicate that GBS is a feasible approach for SNPs discovery in C. ribicola and any DNA concentrations tested could be used for future experiments.
Genetic patterns reveal introduction pathways of the invasive poplar pathogen Mycosphaerella populorum. M. L. SAKALIDIS, N. FEAU, B. DHILLION, P. E. TANGUAY AND R. C. HAMELIN. TAIGA, Forest Sciences Centre, University of British Columbia 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada; and (P.E.T.) Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 rue du P.E.P.S., P.O. Box 10380, Sainte-Foy Quebec, QC G1V 4C7, Canada
Mycosphaerella populorum Thompson (anamorph Septoria musiva Peck) is a leafspot and canker causing pathogen of poplar. This pathogen occurs in natural stands of Populus deltoides but it can also infect P. trichocarpa, P. balsamerifera and their hybrids. Reports of M. populorum have mainly come from central North America and eastern Canada, the assumed endemic range. In 2006, M. populorum was reported for the first time in British Columbia (BC). It is important to determine the source of this introduction to prevent further incursions and to enforce eradication programmes. The recent sequencing of the M. populorum genome has allowed us to target new genes of interest beyond the traditional ‘housekeeping’ genes commonly associated with fungal genetic studies. Our objective was to determine the source of M. populorum in BC and examine the migration pathways of this pathogen across the USA and Canada. For this task eight genes were sequenced on a geographically diverse set of 73 strains of M. populorum, generating a panel of 342 Single Nucleotide Polymorphisms (SNPs). Genetic diversity estimates, population structure and Approximate Bayesian Computation analyses do not support an introduction from eastern Canada. Instead the results reveal distinct east and west Canadian populations. One explanation of these results suggests multiple introduction events from west Canadian and US populations of M. populorum into British Columbia. Resolving these issues is important with regard to potential eradication of the disease.
Design and development of a DNA array for detection and identification of fungal pathogens associated with young vine decline. J. R. ÚRBEZ-TORRES, P. HAAG, J. DICK AND D. T. O’GORMAN. Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada
Large investments in vineyard establishment and maintenance are needed before a full crop is obtained. Therefore, a long lifespan for vines is essential for economic success in vineyards. Hence, the declining of young vines can cause a substantial untenable economic loss to the grape industry. Young vine decline (YVD) is associated with petri disease, caused by Phaeomoniella chlamydospora Crous and Gams and several Phaeoacremonium spp., and black-foot disease, caused by species in the genera Campylocarpon, Cylindrocarpon, Cylindrocladiella and Ilyonectria. Additionally, several species in the genus Cadophora have also been recently shown to be involved in YVD. Short DNA (16–25 bases long) probes from part of the β-tubulin gene region were developed to specifically identify fungal species associated with YVD. The probes were attached to a nylon membrane by an amine modified linker arm and arranged in a precise pattern to form an array for detecting all possible fungal pathogens associated with YVD. Probe’s specificity was determined by hybridizing pure cultures of the pathogens from different geographical grape-growing regions to the probes. Additionally, known ex-type specimens from the different species were also included as positive controls. The DNA array correctly identified over 60 different fungal species in the genera Phaeomoniella, Phaeoacremonium, Campylocarpon, Cylindrocarpon, Ilyonectria and Cadophora including those shown to cause YVD symptoms. The array was validated by testing artificially inoculated grapevine cuttings, soil and field samples from commercial vineyards. The high specificity of this DNA array showed a promising detection system for accurate and rapid identification of YVD pathogens in a single test.
Grapevine trunk diseases in British Columbia: Incidence and characterization of the fungal pathogens associated with the decline and consequent death of young and mature vines. J. R. ÚRBEZ-TORRES, P. HAAG, P. BOWEN, J. DICK AND D. T. O’GORMAN. Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada
Grapevine trunk diseases (GTD), caused by a wide range of taxonomically unrelated fungi, are currently recognized as one of the major disease issues the grapevine industry is facing worldwide, threatening, in many grape-growing regions, the future sector’s economic sustainability. To date, no information was available regarding the status of GTD in British Columbia (BC), the second largest grape-growing province in Canada. Therefore, field surveys were conducted in 191 vineyards throughout all grape-growing regions in BC that included assessment of foliar symptomatology from over 60 000 vines and isolations from 463 symptomatic vines. These studies revealed the presence of all GTD in BC, including black-foot, botryosphaeria dieback, esca, eutypa dieback and petri disease. Colony and conidia phenotypical characterization, along with DNA analyses 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 us to identify: (i) five different black foot fungi in the genera Cylindrocarpon and Ilyonectria; (ii) seven species associated with esca and petri disease, including the novel species Phaeoacremonium canadensis Úrbez-Torres, Haag & O’Gorman and Phaeoacremonium roseum Úrbez-Torres, Haag & O’Gorman; and (iii) six Botryosphaeriaceae and ten Diatrypaceae taxa associated with botryosphaeria dieback and eutypa dieback, respectively. This study represents the first attempt to identify and characterize the causal agents of GTD in BC, the current incidence and geographical distribution of GTD, and the role that the different fungi play on grapevine health based on pathogenicity studies.