Evaluation of resistance and tolerance of interior Douglas-fir ( Pseudotsuga menziesii var. glauca ) seedlings to fungal pathogens and drought. E. M. BECKER, M. G. CRUICKSHANK, K. BLEIKER, K. PELLOW 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 a commercially important tree species in the Pacific Northwest, Europe and New Zealand. In cooperation with researchers in the BC Forest Service, we have initiated a first-of-its-kind multiple stressor trial of interior Douglas-fir (var. glauca = Fdi). Seedlings from four breeding zones, representing distinct physical and biological environments in B.C., have previously been screened for their resistance to armillaria root disease. We are evaluating 99 half-sibling Fdi families, as well as wild-type breeding stock from the four breeding zones, for their responses to drought stress, infection by Phellinus sulphurascens Pilát (the fungus causing laminated root rot), and infection by an Ophiostomatoid fungus vectored by the Douglas-fir bark beetle (Dendroctonus pseudotsugae). “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. We are interested in measuring both resistance and tolerance of Fdi to these disease agents. Over much of its range, Fdi receives little or no summer precipitation, and this major geographic race has thus adapted to grow under drought stress conditions. Seedlings in PFC greenhouses have been subjected to four levels of drought stress over the 2012 growing season. Our experimental methods and preliminary results of drought screening were presented and discussed. Results of this trial will increase our knowledge of conifer responses to multiple disease agents, and inform and guide breeding strategies for Douglas-fir trees.
Comparative transcriptome analysis between compatible and incompatible interactions of poplar and Melampsora larici-populina . Z. J. CHEN, N. FEAU, J. LAMANTIA, P. E. TANGUAY, J. F. MAO AND R. C. HAMELIN. (Z.J.C., N.F., J.L., J.F.M., R.C.H.) Forest Sciences Centre, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada; (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; and (Z.J.C.) College of Forestry, Northwest A&F University, Yangling, Shaanxi 712100, China
Poplar is a valuable natural resource for the lumber and paper industry and may have a future role in bioenergy and carbon sequestration. Poplar leaf rust disease is caused by the fungal species, Melampsora larici-populina (Mlp) Kleb. In European poplar plantations, Mlp epidemics greatly reduce the photosynthetic capacity, biomass production, and overall economic value of poplar trees. Understanding the molecular mechanisms associated with fungal biotrophy and host resistance is critical to the effective control and management of this disease. In order to identify genes involved in host defense and pathogen virulence, we used the RNA-seq technique to generate simultaneous transcriptome profiles of a P. deltoides and Mlp incompatible interaction. These profiles were then compared to compatible interactions with Mlp and two other poplar species and their hybrids. Over one million of cDNA reads were assembled into more than 10,000 transcripts that were mapped onto the poplar and rust genomes. Comparative transcriptome analyses over time (at 48, 96, and 168 hpi) revealed 147 P. deltoides and 109 Mlp genes were differentially expressed. Comparative analysis of poplar transcripts between compatible and incompatible interaction revealed defense response genes including the canonical leucine-rich repeat proteins, kinase-related, disease resistance-responsive genes, several transcriptional factors, kinase-related genes and oxidase proteins. On the other hand, rust transcripts involved in plant colonization and biotrophic growth encode secreted proteins, transporters, proteases and lipases, showed higher expression during the compatible interaction. Our approach makes it possible to simultaneously identify host and pathogen candidate genes and gene networks involved during the interaction.
Molecular characterization of a novel endornavirus in the plant pathogenic fungus Chalara elegans ( Thielaviopsis basicola ). X. CHEN AND Z. K. PUNJA. Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Double-stranded (ds) RNA elements are found in strains of the plant pathogenic fungus Chalara elegans Nag Raj & Kend. (Thielaviopsis basicola (Berk. & Broome) Ferraris). A 11,602 bp cDNA sequence was obtained from overlapping cDNA clones produced by RT-PCR of approx. 12 kb ds RNA in strain NC1527. Northern blot analysis confirmed that 15 individual cDNA clones that covered the entire length of the cDNA sequence all hybridized to the 12 kb dsRNA. Open reading frame (ORF) search revealed one large ORF of 11,575 bp nucleotides, which potentially encodes a large putative polyprotein of 3858 aa. Specialized BLAST searches of conserved domains indicated that the putative polyprotein contained six domains, including viral RNA helicase1 (Hel), RecD producing ATP-dependent exoDNAse (exonuclease V), GT1_Gtf_like and glycosyl transferase (GT), and two RNA-dependent RNA polymerases (RdRp) domains. BLASTP searches in the protein database showed that the cloned dsRNA had homology to endornaviruses, which are present in some plant species and in a few fungi. There was 28–34% amino acid identity in the RdRp domain region, 23–32% in the helicase region, and 29–30% in GT region. The identified typical motifs from RdRp, Hel and UGT confirmed that CeEV1 belongs to the genus Endornavirus. We designate this dsRNA element as a new endornavirus, Chalara elegans endornavirus 1 (CeEV1). Phylogenetic analysis of the endornavirus sequences of RdRP, helicase and GT indicated that CeEV1 is relatively distant from other described endornaviruses and may be an ancestral form.
Phytophthora species associated with urban and agricultural waterways in Vancouver and Victoria, BC. A. DALE, N. FEAU, G. J. BILODEAU AND R. C. HAMELIN. Forest Sciences Centre, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada; and (G.J.B.) Canadian Food Inspection Agency, P.O. Box 11300, 3851 Fallowfield Road, Ottawa, ON K2H 8P9, Canada
Phytophthora de Bary, a genus of Oomycetes, has historically been known as a plant pathogenic genus. The most well-known species infect a wide range of hosts including economically valuable tree species, both angiosperm and gymnosperm, and many important agricultural species. Many Phytophthora are invasive and have been introduced through nursery and agricultural trade. Recent studies on Phytophthora suggest diverse communities in natural ecosystems. In the past decade the genus has doubled to over 100 described species. Many newly recognized species rarely cause noticeable disease and appear to occupy a more saprophytic niche in their natural habitats. Little is known about the natural diversity of Phytophthora in British Columbia, and how that diversity is affected by agriculture or urban activities. As part of a larger study comparing Phytophthora diversity between natural and urban environments in southwest British Columbia, waterways in five sites around agricultural or residential areas were baited with mesh bags containing Rhododendron leaves. Phytophthora species were cultured from symptomatic leaves and identified based on internal transcribed spacer (ITS) DNA sequences. To date, 17 different Phytophthora species have been found, representing six major clades (out of 10 known) of the genus. There are minor differences in species composition between sites and early trends show slightly higher diversity in residential compared to agricultural areas. This data will be compared to the diversity in natural forests and will give an understanding of how urban activities influence Phytophthora diversity and if urban activities result in the introduction of invasive species into the landscape.
Comparative genomics of poplar pathogens from natural pathosystems. B. DHILLON, N. FEAU, P. E. TANGUAY, M. SAKALIDIS, S. BEAUSIEGLE, R. OHM, A. AERTS, I. GRIGORIEV, G. H. J. KEMA, S. B. GOODWIN AND R. C. HAMELIN. Forest Sciences Department, Faculty of Forestry, University of British Columbia, 2424 Main Hall, Vancouver, BC V6T 1Z4, Canada; (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; (R.O., A.A., I.G.) Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA; (G.H.J.K.) Plant Research International B.V., Wageningen, The Netherlands; and (S.B.G.) United States Department of Agriculture–Agricultural Research Service, Purdue University, West Lafayette, Indiana, USA
Mycospharella populorum Thomps. and M. populicola Thomps. are closely related fungal pathogens of poplars that have co-evolved with two different hosts. Mycospharella populorum causes leaf-spots and cankers on poplars with Aegiros parentage and is also responsible for severe damage in hybrid poplar plantations. Mycospharella populicola, on the other hand, only causes leaf spots on poplars in the Tacamahaca section. To identify and understand the evolution of pathogenicity and host-specificity factors in these pathogens, their genomes were sequenced to a coverage of 18–38X, using next-gen sequencing technology. Comparative sequence analysis estimated their divergence time to be ∼6.4 Mya, which agrees with the divergence time estimates for the poplar botanical sections (6.8- 7.8 Mya). This recent divergence is also reflected in whole genome comparisons that show a remarkably high macro-synteny, lacking in other Mycosphaerella genomes. However, disruption of synteny at the micro-level aided in the identification of genomic regions specific to each genome, which may potentially explain differences in their etiology and host-specificity. A consistent expansion of several pathogenicity-related gene families was observed in M. populorum, suggesting a role for gene-dosage in determining its ability to cause cankers. Preliminary enzyme assays show significant differences in beta-glucosidase and xylanase activities between these two fungi.
Colonization behaviour and toxin production by Pseudomonas syringae pv. syringae isolated from highbush blueberry. A. DICARLO AND Z. K. PUNJA. Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Pseudomonas syringae pv. syringae (Pss) van Hall, is a highly adapted epiphyte readily isolated from blueberry plants in the Fraser Valley of British Columbia. Although it is the causal agent of bacterial blight, large populations have been found on apparently healthy blueberry plants. One isolate (Pss9) collected from a blighted stem sample was tested for its ability to colonize tissue-cultured lilac plantlets and blueberry leaf discs under laboratory conditions and to produce necrosis-inducing toxin in vitro and in planta. Colonization of leaf discs depended on cultivar and phenological status of the host. Pss9 was able to establish 6 × 105 CFU/mm2 on ‘Duke’ leaf tissue collected from the field in May, 3.8 × 104 CFU/mm2 in June and 5.8 × 101 CFU/mm2 in July. Necrosis of leaf disks was significantly greater in treated vs. controls in May and June but not in July. Pss9 colonized leaf tissue from ‘Elliott’, a putatively resistant cultivar, to a lesser extent on all dates. Inoculum dose was found to affect both severity and onset of disease symptoms in lilac, with doses of 105-104 CFU producing symptoms of significantly greater severity more rapidly than doses of 103-102 CFU. Extracts from Pss9 cultures grown under toxin-inducing conditions showed fungistatic effects on Botrytis, Sclerotinia and Phomopsis in vitro. Expression of a toxin production gene (syrB) was detected in Pss9 cultures and in inoculated blueberry leaf tissue and lilac plantlets. Epiphytic survival of Pss may be enhanced by toxin production and competence in colonizing susceptible host tissues.
Susceptibility of Populus trichocarpa provenances in the Pacific Northwest to Septoria musiva and Septoria populicola. J. C. Y. FUNG, B. LAI, S. BEAUSEIGLE, P. HERATH, B. DHILLON, B. NELSON AND R. C. HAMELIN. Faculty of Forestry, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada; and (B.N.) British Columbia Ministry of Forests, Land and Natural Resource Operations, P.O. Box 9513 Stn Prov Govt, Victoria, BC V8W 9C2, Canada
A Populus trichocarpa Torr. & A. Gray provenance trial in Harrison Mills, BC, had tested positive in 2009 and 2010 for Septoria musiva Peck (telomorph: Mycosphaerella populorum Thomps.), a pathogen that causes necrotic spots on the leaf surface and cankers on branches and stem; cankers can potentially girdle and kill the host. A related pathogen, Septoria populicola Peck (telomorph: Mycosphaerella populicola Thomps.), is also present in the provenance trial; it is native to British Columbia and causes only non-lethal leaf lesions, but is difficult to distinguish from S. musiva without DNA-based analyses. This study was aimed to assess the frequency of S. musiva and S. populicola infections on P. trichocarpa provenances and determine if there was difference in Septoria susceptibility. The provenances ranged from Alaska, through British Columbia and into Oregon. The proportions of S. musiva and S. populicola infections were compared to determine if there is a difference in infection proportions along the latitudinal gradient of the provenance. The number of infections was higher in north than in south provenance; however, no significant differences in infection proportions were found between the north and south provenance for either S. musiva or S. populicola in 2010 or 2011. Comparison between 2010 vs. 2011 S. musiva and S. populicola infections yielded a significant difference only in the 2010-north and 2011-north for S. populicola.
Detection and molecular characterization of dsRNA viruses in North American populations of Heterobasidion irregulare and Heterobasidion occidentale . C. HAMMETT AND S. F. SHAMOUN. Forest Sciences Department, Faculty of Forestry, University of British Columbia, 2424 Main Hall, Vancouver, BC V6T 1Z4, Canada; and (S.F.S.) Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
Heterobasidion spp. are root and butt rot fungi that infect several tree species across the Northern Hemisphere. There are two species in North America H. occidentale Otrosina & Barbel. (S-type) and H. irregulare Otrosina & Barbel. (P-type). H. occidentale is found on the Pacific coast of North America from Alaska to Southern Mexico west of the Rocky Mountains infecting many tree genera, preferably Tsuga, Abies, and Pseudotsuga. Heterobasidion irregulare is found in pine forests across North America from Ontario and Quebec, through the USA and Mexico and in the Northern Caribbean. Heterobasidion populations around the world have been isolated and surveyed for the presence of dsRNA mycoviruses. However, North American mycoviruses have not been well studied with only three previously surveyed and one confirmed to be from the partitivirus genus. We screened 140 isolates, 62 H. irregulare and 78 H. occidentale from around North America for dsRNA viruses. Analysis showed 19 H. irregulare isolates contain dsRNA bands with sizes around 2–5 kbp, including five isolates with consensus sequences of the partitivirus HetRV1 species. Preliminary analysis of H. occidentale isolates indicates the presence of dsRNA bands in 13% of the population. Future sequencing of these viruses will reveal the family of virus and possibly identify new viruses present in North American Heterobasidion. Potential testing of the effects of viral presence in Heterobasidion may allow for use as a biological control agent or a vector for hypovirulence factors to help control and decrease the threat of Heterobasidion to North American forests.
Incidence of Septoria musiva and Septoria populicola on Populus trichocarpa provenances. P. HERATH, C. TSUI, B. DHILLON, G. BILODEAU, S. BEAUSEIGLE, B. LAI, J. FUNG, C. LIU, N. FEAU, H. KOPE, S. ZEGLEN AND R. C. HAMELIN. Forest Sciences Department, Faculty of Forestry, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada;(G.B.) Canadian Food Inspection Agency, P.O. Box 11300, 3851 Fallowfield Road, Ottawa, ON K2H 8P9, Canada; and (H.K., S.Z.) British Columbia Ministry of Forests, Land and Natural Resource Operations, P.O. Box 9513 Stn Prov Govt, Victoria, BC V8W 9C2, Canada
Septoria musiva Peck is an important pathogen of poplar causing both leaf spots and canker. Canker can be especially devastating leading to tree death and reduced biomass in commercial poplar plantations. Septoria populicola Peck, on the other hand, causes leaf spots and does not cause extensive damage. While S. musiva was originally not considered a serious threat to BC poplars, recent studies have detected this pathogen in the Fraser Valley hybrid poplar plantations. In order to identify resistant poplar provenances and investigate the distribution of S. musiva in BC, we developed a high throughput, real-time PCR assay to detect both Septoria spp. and mapped the presence of infected trees throughout major poplar provenance trials within the province. The assays are conducted directly on the leaf spots, without the need for culturing the fungus. In 2011, 1467 trees belonging to 477 provenances were tested and 42 trees tested positive for S. musiva. The majority of the evaluated provenances were infected with S. populicola only. But some provenances were notably more susceptible, e.g. provenances 7.5, 25.3, and 121.4 which were infected by S. musiva repeated times. However, the natural incidence level of S. musiva was low and the provenances not infected by S. musiva could be escapes. Septoria musiva appears to be confined, for now, to the Fraser Valley region of BC. More extensive, recurrent surveys for pathogen incidence and the planting of resistant provenances are recommended as necessary steps for keeping the BC poplars protected from this devastating pathogen.
New and emerging plant health issues in British Columbia in 2012. V. JOSHI, M. JEFFRIES AND C. WIENS. Plant Health Laboratory, Plant and Animal Health Branch, B.C. Ministry of Agriculture, Abbotsford Agriculture Centre, 1767 Angus Campbell Road, Abbotsford, BC V3G 2M3, Canada
The Plant Health Laboratory at the Abbotsford Agriculture Centre provides plant health diagnostics services for all commercial crops grown in British Columbia (B.C.). Plant samples are submitted to the laboratory by growers, consultants, extension staff and industry personnel. Ornamentals (outdoor and greenhouse) and berry crop samples dominate the total number of samples submitted to the laboratory. A very wet spring in 2012 resulted in high incidence of bacterial blight [Pseudomonas syringae van Hall] problems on ornamentals as well as small fruit crops. Later in the summer, powdery mildew was observed on many ornamentals. New disease detections for B.C. included stem rot [Colletotrichum dracaenophilum Farr & Palm] in Dracaena sanderiana, corm rot [Ceratocystis fimbriata Ellis & Halst] in the indoor tropical plant Ficus microcarpa, stem rot [Pythium splendens Braun] and stem canker [Lasiodiplodia theobromae Ellis & Everh] in Pachira. Boxwood blight caused by Cylindrocladium buxicola Henricot (Synonym C. pseudonaviculatum) was the predominant disease issue in the ornamental industry causing significant losses to the industry. All infected boxwoods (Buxus spp.) have been destroyed and monitoring continues. Other new detections in some minor specialty crops included downy mildew [Peronospora belbahrii Thines] in greenhouse and field grown Basil and foliar blight [Itersonilia perplexans Derx] on Dill. All new detections were confirmed by molecular tests. Canker diseases such as phomopsis and godronia cankers are continuing to cause significant damage to blueberry crop especially in young plantings and new varieties. Research projects are underway to understand the epidemiology of canker and dieback issues in berry crops.
Septoria musiva on Populus trichocarpa in British Columbia. H. H. KOPE AND S. ZEGLEN. British Columbia Ministry of Forests, Lands and Natural Resource Operations (BCMFLNRO), 727 Fisgard Road, Victoria, BC V8W 1R8, Canada; and (S.Z.) BCMFLNRO, 2100 Labieux Road, Nanaimo, BC V9T 6E9, Canada
Septoria musiva Peck (teleomorph Mycosphaerella populorum Thomps.) is native to eastern North America and causes leaf spot and, more importantly, stem cankers that often lead to breakage in Populus spp. Since 2007 in British Columbia (B.C.), S. musiva has been repeatedly detected in leaf spots and cankers on hybrid Populus in a nursery and in clonal plantations along the lower Fraser River. Septoria musiva has also been detected and isolated from leaf spots and cankers of native black cottonwood (Populus trichocarpa Torr. & A. Gray). Surveys of over 400 different, young and mature P. trichocarpa trees, along the Fraser River were carried out in 2008, 2009 and 2012. Over the 3 sample years S. musiva was detected in leaf samples of 14 different trees. The distribution of trees with S. musiva leaf spot was wide spread along the Fraser River, between Chilliwack and Hope. This could indicate multiple introductions, rapid spread from a single introduction, or undetected slow spread over a long period. In 2012, leaves were sampled from a provenance trial with over 180 families of P. trichocarpa collected throughout its native range in B.C. and from limited collections in Washington, Oregon and California. Over 40 families tested positive for S. musiva. These results indicate that S. musiva under favourable conditions can infect multiple families of P. trichocarpa from western North America. Populus trichocarpa is an important riparian species, and the impact that S. musiva could have on its growth and ecology is currently unknown.
Association analysis identifies Melampsora x columbiana poplar leaf rust resistance SNPs. J. LA MANTIA, J. KLAPŠTE, Y. A. EL-KASSABY, S. AZAM, R. D. GUY, C. J. DOUGLAS, S. D. MANSFIELD AND R. C. HAMELIN. Forest Sciences Department, Faculty of Forestry, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada;(C.J.D.) Department of Botany, 3529–6270 University Boulevard, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; and (S.D.M.) Department of Wood Science, University of British Columbia, 2424 Main Hall, Vancouver, BC V6T 1Z4, Canada
Populus spp. are currently being domesticated through intensive time- and resource-dependent programs to maximize biomass for biofuels. Severe infections of polar leaf rust disease caused by Melampsora spp. leads to decreased photosynthetic capacity, premature defoliation, reduced biomass and fiber volume, and increased susceptibility to additional pathogens. Genetic resistance is effective in reducing economic losses but major resistance loci have been race-specific and can be readily defeated by the pathogen. Developing durable disease resistance requires the identification of non-race-specific loci. We performed association analysis for area under the disease progress curve (AUDPC) on 412 unrelated P. trichocarpa Torr & A. Gray genotypes in a common garden experiment representing the species’ natural range. AUDPC was measured on natural infection of M. xcolumbiana G. Newc. over 11 weeks in 2009, 2010, and 2011. Association analysis was performed using two stages with 29,355 SNPs. First SNPs were pre-selected in a simple SNP-trait regression to reduce the constraints of multiple testing corrections and then principal components (PC) where used in place of Q matrix in a PC regression with pre-selected SNPs. A total of 45 SNPs achieved experiment-wide significance at P < 1.45 × 10−5 across the 3 years. Each of these loci explains between 2.2 – 4.5% of the phenotypic variance. In Arabidopsis, orthologs of the significant loci indicate functionality in pathogen induced transcriptome reprogramming, Ca2+/ calmodulin signaling, salicylic acid signaling, and tolerance to reactive oxygen species. In this long-lived organism, these associated loci further our understanding of poplar: rust interactions and lay the foundation for more efficient breeding of durable disease resistance.
Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect living pinewood nematode, Bursaphelenchus xylophilus , in wood. I. LEAL, E. ALLEN, C. REISLE, J. ANEMA, A. UZUNOVIC, A. VARGA AND D. JAMES. Natural Resources Canada, Canadian Forest Service, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada; (A.V., D.J.) Sidney Laboratory, Centre for Plant Health, Canadian Food and Inspection Agency, 8801 East Saanich Road, North Saanich, BC V8L 1H3, Canada; and (A.U.), FPInnovations, Forintek Division 2665 East Mall, Vancouver, BC V6T 1W5, Canada
Pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, is an inhabitant of native pine species of North America, where its presence in trees is non-pathogenic. In contrast, the introduction of this nematode to forests in Asia and Europe has devastated some pine stands and is recognized as a pest of phytosanitary concern by the National Plant Protection Organization of several countries. The ability to detect PWN in internationally traded wood products is crucial to reduce the spread of this organism. Current molecular techniques for the detection of PWN rely on the presence of genomic DNA and thus fail to differentiate between living and dead PWN. The detection of dead nematodes could lead to unnecessary trade disruption. Therefore, accurate techniques for the detection of and differentiation between living and dead PWN are critical. We have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, which specifically identifies living PWN in wood by detecting the presence of expansin mRNA as a viability marker. This diagnostic method was found to be more sensitive, amplifies in a fraction of the time, requires less laboratory equipment, and allows for simple visual detection compared to PCR. This method will help resolve disputes over the detection of PWN by clarifying whether it originates from live or dead organisms, avoiding unnecessary trade disruption and thus protecting market access of Canadian wood products. It can also be used to evaluate the efficiency of wood treatment procedures.
Analysis of Pinus monticola transcriptome in interaction with Cronartium ribicola via RNA-seq and de novo assembly. J.-J. LIU AND R. STURROCK. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
White pine blister rust (WPBR), caused by the exotic fungus Cronartium ribicola Fischer, has resulted in extremely high mortality of western white pine (Pinus monticola) and other eight native five-needle pines in North America. Incorporation of multiple resistance mechanisms including major gene (Cr2) resistance and quantitative disease resistance in elite seed orchards is one of the main objectives in soft pine breeding and is considered as the most effective approach for long-term disease resistance. Use of next generation sequencing (NGS), such as RNA-seq, is very effective for the collection of comprehensive transcriptomic data for discovery of tree genes and alleles involved in genetic resistance. We sequenced transcriptomes from cDNA libraries constructed from pooled needles of P. monticola seedlings with Cr2-resistant and cr2-susceptible genotypes using paired-end reads on an Illumina GAII system. A total of 120M-150M reads were generated from RNA-seq samples. De novo assembly produced 39K-45K contigs with average length ranging from 855-bp to 899-bp. After comparison of samples, we created a consensus transcriptome that contained 39,439 contigs with average length of 1300-bp and total length of 51 MB. This consensus transcriptome will be used as a reference to reveal resistance-related genes differentially expressed in compatible and incompatible WPBR interactions as well as to discover SNPs linked to Cr2-genotype. Our results demonstrate that NGS is a rapid and affordable strategy for gene discovery and expression profiling in conifers. We believe that similar strategies are feasible to investigate resistance to WPBR in whitebark pine (P. albicaulis Engelm.) and other white pines.
Molecular breeding of white pine resistance against white pine blister rust. J.-J. LIU, A. ZAMANY AND M. GIRARD-MARTEL. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
Major gene (Cr2) resistance (MGR) and quantitative disease resistance (QDR) are present, but very rare in natural stands of western white pine (Pinus monticola). How to identify and select genetic resources with different resistance mechanisms is a long term challenge in traditional breeding programs. Molecular breeding via biochemical diagnosis tools and biomarkers is able to evaluate high numbers of young seedlings to predict the field performance of resistance traits. Our lab used genomics, proteomics, and bioinformatics technologies to identify tree genes that contribute to genetic resistance. Hundreds of genes were annotated in functional categories of defence-response, signal transduction, and transcription by random Sanger sequencing and RNA-seq analysis of cDNA libraries. Comparative proteomics identified 100 proteins regulated differentially between Cr2-resistant and cr2-susceptible genotypes post rust infection. Resistance candidates included R gene analogs (RGAs), pathogenesis-related proteins, anti-microbial peptides, and R gene-triggered signalling factors. Candidate genes/proteins provide genomic targets for us to develop DNA markers for marker-assisted selection (MAS). A genetic linkage map was constructed for the Cr2 gene using SCAR, RGA-related sequences, and DNA markers of other types. DNA markers tightly linked to Cr2 provide molecular tools for MAS in breeding or as reference map for comparative MGR mapping among five-needle pines. To develop molecular tools for QDR, genetic variations of candidate genes were investigated in pine populations. Association genetics revealed that single nucleotide polymorphisms (SNPs) and indels of PmAMP1, PmCh4B, and PmPR10 genes significantly contributed to phenotypic variations of the QDR levels, providing potential markers for breeding selection of QDR.
Neofusicoccum arbuti associated with the decline of Arbutus menziesii in BC. R. R. MCGREGOR, M. L. SAKALIDIS AND R. C. HAMELIN. TAIGA, Forest Sciences Centre, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada
The iconic tree species, Arbutus menziesii Pursh has been in decline for the last 30 years. The decline is likely the result of multiple factors including climate change, fire suppression, and pathogens. One of the primary pathogens attributed to the decline in the northern range of A. menziesii is Neofusicoccum arbuti (Farr & Elliot) Crous, Slippers & Phillips; a latent pathogen in the Botryosphaeriaceae family. Little is known about the additional hosts of N. arbuti in the native range of A. menziesii. Although some research has been done to determine whether N. arbuti is an indigenous species or introduced, the information available is inconclusive. The goals of this project are: 1) establish the extent of decline of A. menziesii in Lighthouse Park. 2) identify the primary pathogen causing arbutus cankers and other endophytic fungi, 3) determine if N. arbuti is present in non-arbutus hosts in the native ecosystem of A. menziesii, and 4) confirm whether N. arbuti is an indigenous or introduced species. To accomplish these goals, a survey of A. menziesii at Lighthouse Park in West Vancouver, BC was conducted. Samples from A. menziesii and other hosts were taken from ten sites distributed throughout the park. Once pure cultures were obtained samples were sorted based on colony morphology. The dominant morphology associated with cankers was similar to N. arbuti. Future work is focused on identifying other fungi and characterizing the genetic diversity of N. arbuti.
Post-harvest fungal pathogens of greenhouse tomatoes – what effect does Rhapsody ( Bacillus subtilis ) have on disease? Z. K. PUNJA, S. FORMBY, A. TIRAJOH AND G. RODRIGUEZ. Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Isolations were made from diseased greenhouse-grown fresh market tomato fruits sampled from greenhouses and retail outlets in the Fraser Valley of BC during 2011 and 2012. The most prevalent fungi recovered were species of Penicillium and Botrytis cinerea Pers.:Fr., followed by Rhizopus stolonifer (Ehrenb.:Fr.) Vuill., Alternaria alternata (Fr.) Keissler and Geotrichum candidum Link ex Pers. In pathogenicity tests, P. olsonii Bainier & Sartory and B. cinerea caused the most extensive lesions on wounded and nonwounded fruits. Swabs taken of the surface of ripening tomato fruits followed by streaking onto potato dextrose agar revealed an abundance of fungi, including species of Penicillium, Aspergillus, Cladosporium and Rhizopus. These genera were also recovered from decomposing plant material and leaf litter placed on the greenhouse floor. Applications of Bacillus subtilis (Rhapsody, 0.5% v/v) were made at monthly intervals to tomato plants in two commercial greenhouses during July-October, 2012. Populations of fungi and bacteria on the fruit surface were assessed from fruit swabs taken at weekly intervals. Subsequent post-harvest disease levels were determined from harvested fruit that were incubated in a growth chamber maintained at 21 °C and 70% relative humidity. Populations of B. subtilis reached 1 × 144 CFU/cm2 of fruit surface and were maintained at high levels over the ensuing 4 weeks in the greenhouse. Populations of fungi on the fruit surface were reduced to zero. Application of the biocontrol agent significantly (P < 0.05) reduced post-harvest disease development on fruit after 2 weeks of storage at 21 °C. The most commonly recovered fungi were Penicillium species and B. cinerea. Application of Rhapsody has the potential to reduce fruit surface contamination by potential fungal pathogens and also reduce post-harvest decay of treated fruits.
West of Eden - the origin of Mycosphaerella populorum in British Columbia. 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 Thomps. is a leafspot and canker causing pathogen of poplar. This pathogen occurs in natural stands of Populus deltoides but it can also infect a further 26 poplar taxa and several of 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 programs. 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. We wanted to determine the source of M. populorum in BC and examine the migration pathways of this pathogen across the US and Canada. For this task eight genes were sequenced on a geographically diverse set of 65 strains of M. populorum, generating a panel of 294 Single Nucleotide Polymorphisms (SNPs). Genetic diversity estimates and population structure analyses do not support the scenario of a recent introduction followed by a population bottleneck. Instead the level of genetic diversity is higher in BC than the rest of the continent. The results also illustrate a split between eastern and western Canada. One explanation of these results suggest a combination of a) endemic M. populorum in western North America recently expanding onto planted poplar and b) exchange events between eastern and western populations. Resolving these issues is important with regard to potential eradication of the disease.
Roles of the fungal and water mold species in the decline of birch in South-central Kootaney area in British Columbia, Canada. C. SARMIENTO, A. DALE, S. BEAUSEIGLE, R. C. HAMELIN AND M. MURRAY. Forest Sciences Department, Faculty of Forestry, University of British Columbia, 2424 Main Hall, Vancouver, BC V6T 1Z4, Canada; and (M.M.) BC Forest Service, Ministry of Forests, Lands and Natural Resource Operations, #401 - 333 Victoria Street, Nelson, BC V1L 4K3, Canada
Increasing reports of large-scale birch dieback in British Columbia could have huge implications towards the health of future forest stands. Birch also has valuable economic value and can be used for the production of veneers and sawlogs. Many studies suggest that birch decline is caused by stress from changing climatic conditions. However, recent studies of birch decline in Europe have determined fungal and water mold species can play a key role in the birch decline. In this study, a survey of logs, soils and roots associated with birch species was conducted around the South-central part of the Kootenay Area of British Columbia, Canada, to determine the role of fungal and water mold species in the current decline of birch. Pure fungal cultures were isolated from roots and small sections of wood was sampled from the logs. Phytophthora spp. were isolated from the soil using a baiting technique. DNA barcoding was used for species identification. From the 32 isolates extracted from the logs collected, eight species were identified and were found to be mostly decay fungi. Fomes fomentarius (L.) Fr. was the most common species found (15 of 32 isolates). Cryptosporella tomentella (Peck) Mejia, which has been determined to cause necrotic lesion and broken tops in silver birch (Betula pendula) in Estonia, was found at one site. Analyses of soils for Phytophthora spp. as well as root pathogens, including Armilaria spp. are still in progress.
Global threat from Phytophthora forest diseases with a Canadian perspective on sudden oak death disease caused by Phytophthora ramorum . S. F. SHAMOUN AND D. RIOUX. Natural Resources Canada (NRC), Canadian Forest Service (CFS), Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada; and (D.R.) NRC, CFS, Laurentian Forestry Centre, 1055 du P.E.P.S., Québec, QC G1V 4C7, Canada
Phytophthora is a genus of more than 100 described species, and is probably the most destructive group of plant pathogens throughout forests and landscape areas worldwide. Diseases such as jarrah dieback [P. cinnamomi Rands] in western Australia, needle blight [P. pinifolia Durán, Gryzenh. & Wingf.] of radiata pine planted in Chile, root rot [P. lateralis Tucker & Milbrath] of Port Orford cedar in western North America, chestnut ink disease [P. cambivora (Petri) Buisman and P. cinnamomi] and root and collar rot of alder [P. alni Braisier & Kirk] in Europe cause severe damage. Diseases caused by P. ramorum (Pr) Werres et al. and P. kernoviae Braisier et al. have also drawn the attention of several regulating international agencies. For instance, in 2009, Pr, which usually affects broadleaf species, was found on Japanese larch in the U.K. and so far 3 million trees have been killed or felled in an attempt to control the disease. In Canada, Pr was introduced a few times in nurseries of British Columbia but the pathogen is not considered established yet. At the Canadian Forest Service, research is being carried out in order to understand the biology, molecular differentiation among clonal lineages, and development of mitigation measures to help assess the risk this pathogen represents to Canada. Our presentation summarized the results about: 1) PCR-RFLP molecular markers to identify the three Pr lineages; 2) efficacy of biocontrol products and fungicides; 3) assessment of the aggressiveness among isolates and lineages; 4) evaluation of susceptibility of some forest tree species common in eastern Canada; and 5) research of putative resistance mechanisms in trees to this pathogen.
Infection of western redcedar roots by the fungal pathogen Phellinus weirii – preliminary results. R. N. STURROCK AND K. W. PELLOW. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
Western redcedar (WRC; Thuja plicata Donn) is a species of significant ecological and economic value occurring in both coastal and interior wet belt forests of western North America. Despite their reputation for resistance to decay, standing WRC trees often have extensive heartwood decay, which negatively impacts the value of harvested trees. The fungal root pathogen, Phellinus weirii (Murrill) Gilb., is commonly associated with root and butt decay in WRC. To gain a better understanding of the infection biology of P. weirii we infected roots of 29-year-old WRC trees using an inoculation technique that we developed. Briefly, bags of sterilized stem sections of red alder (Alnus rubra Bong.), colonized using liquid cultures of P. weirii, were placed atop individual, carefully exposed, WRC roots. Excavation and evaluation of roots after seven years showed that P. weirii had invaded bark tissues at several points, resulting in lesions, callus tissue, and other host responses in sapwood of infected roots. Furthermore, advanced, pitted decay was evident in a significant portion one of the largest primary roots and the fungus had grown internally in wood at least 40 cm up the stem. More inoculation trials are needed to fully document the WRC-P. weirii interaction.
Molecular identification of three new strains of Cherry mottle leaf virus from infected sweet cherry trees. L. SU, B. BHAGWAT, M. BERNARDY, P. WIERSMA, Z. H. CHENG AND Y. XIANG. Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC VOH IZO, Canada; and (Z.H.C.) College of Horticulture, Northwest A&F University, Yangling, Shaanxi, 712100, China
Cherry mottle leaf virus (CMLV, genus Trichovirus, family Flexiviridae) is a severe pathogen of sweet cherry in certain regions of North America. Three new CMLV strains were identified by 454 pyrosequencing and molecular cloning from the accessions in the Canadian Plant Virus Collection's Virus Orchard (CPVC-VO) located at PARC-AAFC, Summerland, BC, Canada. Nucleotide sequence identities among the three strains and a previously reported CMLV isolate Sa1162-21 (Arch Virol 145:995–1007) ranged from 78 to 84%. All three strains have the same genomic structure as the CMLV Sa1162-21 and contain the same four putative open reading frames that code for a putative viral replicase, a movement protein, a coat protein (CP) and a nucleic acid-binding protein, P4 of unknown function. The deduced amino acid sequence identities among the strains ranged from 87.7 to 93.1% for the replicase, 81.5 to 88.9% for the movement protein, 94.8 to 98.4% for the coat protein, and 80.0 to 90.4% for the P4. The coat proteins of the three strains, with an estimated size of 21.6 kDa were detected from the infected sweet cherry trees by western blot using a polyclonal antibody raised against a peptide corresponding to a conserved region in the deduced CP amino acid sequences.
Genomics for the detection of fungal pathogens. C. K. M. TSUI, R. C. HAMELIN AND THE TAIGA TEAM. TAIGA, Forest Sciences Centre, University of British Columbia 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada
Many species of fungi can cause disease in plants, animals and humans. Accurate and robust detection and quantification of fungal pathogens are essential for diagnosis, modeling and management. Direct detection of fungi also enables a deeper understanding of natural microbial communities, particularly as many fungi are difficult or impossible to cultivate. We are currently utilizing next generation sequencing technology to generate genomic profiles of tree pathogens to develop novel assays/molecular markers for accurate identification of fungal pathogens. The genome and transcriptome of important forest pathogens have been re-sequenced and sequenced de novo, to identify the ‘core gene set’ as well as “unique genes sets” by genome comparison to related pathogens and non-pathogenic relatives. Multiple assays/targets will be integrated onto a single chip to perform up to thousands of individual reactions simultaneously. This high level of redundancy will result in an increase in sample processing and a reduction in false negatives and false positives.