Targeting of Cucumber necrosis virus coat protein to chloroplasts is associated with attenuation of necrotic symptoms during infection of Nicotiana benthamiana. S. B. ALAM, R. READE, A. MAGHODIA, B. GHOSHAL, J. THEILMANN AND D. ROCHON. (S.B.A., D.R.) Faculty of Land and Food Systems, University of British Columbia, 248-2357 Main Mall, Vancouver, BC V6T 1Z4, Canada; and (R.R., A.M., B.G., J.T., D.R.) Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada
Cucumber necrosis virus (CNV) is a (+)ssRNA virus, belonging to the Tombusvirus genus, that elicits a spreading, systemic necrosis phenotype in Nicotiana benthamiana Domin. Previous work showed that 1–5% of CNV coat protein (CP) enters chloroplasts during infection and the CP arm region functions as a chloroplast transit peptide, targeting the CP shell (S) and protruding (P) domains to the stroma. Chloroplasts play a prominent role in host defence responses, hence we hypothesized that entry of CNV CP into the stroma may interfere with chloroplast-mediated defence. We show that several CNV CP arm mutants that do not efficiently enter chloroplasts, along with a CP mutant lacking the S and P domains [SP(-)], show a more rapid induction of necrosis, a more localized lesion phenotype and higher, more sustained levels of the hypersensitive response (HR) marker gene PR1a. Also, unlike WT CNV, SP(-) failed to infect plants systemically by 7 days post inoculation suggesting that the SP region of the CP that enters the chloroplast stroma partially interferes with systemic necrosis. We also found that CNV CP attenuates the HR induced by Tomato bushy stunt virus p19 in agroinfiltrated Nicotiana tabacum L. and also reduces PR1a mRNA levels induced by Agrobacterium tumefaciens (Smith & Townsend) Conn in N. benthamiana. Additionally, we provide evidence that several mRNAs related to the HR response and plant defence are upregulated in infection induced by a CNV CP transit peptide mutant, as compared with WT CNV. Together, our data strongly suggest that the presence of CNV CP in the chloroplast stroma suppresses host defence responses, leading to a spreading necrosis phenotype.
New and recurring pathogens of wasabi in British Columbia. E. C. BETZ, J. L. MACDONALD AND Z. K. PUNJA. Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada; and (J.M.) Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada
Wasabi (Wasabia japonica (Miq.) Matsumura) is grown for its highly valuable rhizome in greenhouses with high humidity, providing ideal conditions for disease development. Symptoms of leaf spot, root rot, rhizome rot and petiole blight are commonly observed. We surveyed five different wasabi greenhouses during 2015–2016 to determine the pathogens that may be causing these various diseases. Symptomatic tissues were surface-sterilized and plated onto PDA and V8 juice agar each containing 100 mg L−1 streptomycin sulphate. Recovered fungi were identified based on morphology and ITS1–ITS4 rDNA sequences. Fungi identified included Colletotrichum higginsianum Sacc. (causing leaf spots and blight), Leptosphaeria biglobosa Shoemaker & H. Brun (causing leaf spots, petiole infection and rhizome discolouration), Botryotinia fuckeliana (de Bary) Whetzel (causing leaf blight), Erysiphe cruciferarum Opiz ex L. Junell (causing powdery mildew), Fusarium solani (Mart.) Sacc. and Fusarium avenaceum (Fr.) Sacc. (causing root rot), as well as Verticillium isaacii Inderb., Bostock, R.M. Davis & K.V. Subbarao (causing wilt and rhizome blackening). Three additional pathogens were identified: Albugo candida (Pers. ex Lev) Kuntze (causing white rust), Phytophthora sansomeana E.M. Hansen (causing root rot), and a Pythium sp. N. Pringsheim (causing root rot). Pathogenicities of C. higginsianum, L. biglobosa, A. candida and B. fuckeliana were confirmed by spraying spore suspensions of 1–4 × 105 spores mL−1 onto healthy plants, which resulted in leaf spot and blight development after 7–14 days. The appearance and spread of previously unreported pathogens on wasabi implies inoculum introduction and persistence is occurring within current production areas.
Influences of nitrogen inputs on development of plant-parasitic nematode populations in the root zone of blueberry. T. A. FORGE, D. EHRET, B. FREY, M. DORAIS AND P. RANDALL. Summerland Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada; and (D.E., B.F., M.D.) Agassiz Research and Development Centre, AAFC, 6947 Highway 7, P.O. Box 1000, Agassiz, BC V0M 1A0, Canada
Several genera of plant-parasitic nematodes are known to parasitize highbush blueberry and have been found in poorly growing blueberry plantations in British Columbia. Little is known, however, of the dynamics of population development in relation to root growth in new plantings, or how they may be affected by cultural practices. An experimental block of highbush blueberry cv ‘Duke’ was planted in autumn of 2008, with six replicate plots of each of four nitrogen (N) fertilizer treatments: (1) untreated control and broadcast applications of (2) 100, (3) 150 and (4) 200% of recommended annual N application rates. Commencing in 2009, composite soil samples were taken from root zone soil in each plot in autumn of each year. Nematodes were extracted from the soil samples and plant-parasitic nematodes were identified and enumerated. Three species of interest were recovered at low population densities (< 100 nematodes per L soil) from a small proportion of the plots through 2014: Rotylenchus robustus de Man, Paratrichodorus renifer Siddiqi and Pratylenchus crenatus Loof. In 2015 there was substantial increase in frequency of occurrence and population densities of R. robustus, to levels in excess of 1500 nematodes per L soil. Population densities of P. renifer and P. crenatus also increased during 2015 but not to the same extent as R. robustus. Through 2016 there has been no significant effect of N application rate on population densities of any of the species being monitored, while root biomass has increased with time and N application rate.
Explosive seed discharge in the lodgepole pine dwarf mistletoe Arceuthobium americanum is correlated with a peak in daytime temperature and a drop in relative humidity. D. L. FRASER, M. J. PAETKAU AND C. ROSS FRIEDMAN. Department of Biological Sciences, Thompson Rivers University (TRU), 900 McGill Road, Kamloops, BC V2C 0C8, Canada; and (M.P.) Department of Physical Sciences (Physics), TRU, 900 McGill Road, Kamloops, BC V2C 0C8, Canada
Arceuthobium americanum Nutt. ex Engelm., the lodgepole pine dwarf mistletoe, parasitizes lodgepole pines (Pinus contorta subsp. latifolia Engelm.) in western Canada, causing considerable damage to the host trees, which leads to premature mortality. Dwarf mistletoes are renowned for their ability to explosively discharge their seeds, a process that occurs around the end of August in the south-central interior of British Columbia. Previous work has suggested that seasonality, developmental stage, and high relative humidity are all triggers for discharge. A recent study has also implicated fruit thermogenesis as having a role in dispersal. Here, we examined the environmental variables potentially associated with explosive discharge. Using a weather station and time-lapse cameras that we installed in a stand of lodgepole pines heavily infected with dwarf mistletoe near Kamloops, we measured standard environmental variables (temperature, relative humidity, wind speed, etc.) as well as irradiance. We found that discharge from late August through early September is correlated with a peak in daytime temperature, which usually occurs between 1400 and 1800 h. We also noted that seed discharge could be predicted by a drop in relative humidity in the five or so minutes preceding discharge. Additionally, explosive discharge typically occurred in sunlight with an irradiance value of around 5000 lux. Future work will involve correlating the internal temperature of fruit with the ambient temperature in order to best understand how the internal processes are linked to environmental cues.
Fungal endophytes and surface pathogens in the lodgepole pine dwarf mistletoe (Arceuthobium americanum) and its lodgepole pine (Pinus contorta subsp. latifolia) host. E. M. GALARY, C. M. HORST, N. CHEEPTHAM, L. D. HAMPEL, D. J. ZIEGLER AND C. ROSS FRIEDMAN. Department of Biological Sciences, Thompson Rivers University, 900 McGill Road, Kamloops, BC V2C 0C8, Canada
The lodgepole pine dwarf mistletoe (Arceuthobium americanum Nutt. ex Engelm.) is a parasite that infects coniferous forests in North America. This parasite obtains water, nutrients and photosynthate from its host lodgepole pine (Pinus contorta subsp. latifolia Engelm.) via a system of root-like tissues that penetrate the host’s cortex and vasculature. Mistletoe infection reduces conifer growth, reproductive fitness and wood quality, which affects the timber industry. Endophytic fungi and fungal surface pathogens have previously been described as associated with A. americanum. There is no evidence that fungal endophytes are pathogenic, however, previous research has indicated that endophytes may protect the mistletoe from other pathogenic fungi. Additionally, the degree to which fungal endophytes and fungal pathogens are shared between the mistletoe and the pine has not been explicitly determined. This study aims to examine the relationship of endophytic and pathogenic fungi possibly found associated with both the dwarf mistletoe and the lodgepole pine. Arceuthobium americanum shoots and lodgepole pine needles were collected in Stake Lake, BC and cultured onto potato dextrose agar. Fungal colonies were subcultured and incubated at 25.0°C for 2 weeks. Fungal pathogens and endophytes were identified by colony characterization as well as by spore analysis via brightfield and scanning electron microscopy. Preliminary results indicate that Cladosporium cladosporioides (Fresen.) G.A. de Vries and Ulocladium sp. Preuss are associated with both A. americanum and its pine host. Further work aims to determine if there is a common vascular conduit between any internally shared fungi.
A novel approach to detect fungal and wheat genes involved in leaf rust disease by expression associations during various race-specific interactions. H. B. KHALIL, X. WANG, R. LINNING, D. L. JOLY, D. CRAM, N. THIESSEN, G. TAYLOR, B. MCCALLUM, B. SAVILLE AND G. BAKKEREN. Summerland Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada; (X.W., B.M.) Morden Research and Development Centre, AAFC, 101 Route 100, Morden, MB R6M 1Y5, Canada; (D.J.) Department of Biology, Université de Moncton, 18 Antonine-Maillet Avenue, Moncton, NB E1A 3E9, Canada; (D.C.) Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada; (N.T., G.T.) Genome Sciences Centre, 570W 7th Avenue #100, Vancouver, BC V5Z 4S6, Canada; and (B.S.) Trent University, 1600 West Bank Drive, Peterborough, ON K9J 0G2, Canada
We designed a high-resolution RNA-seq experiment to compare global gene expression reprogramming of wheat leaf rust fungus Puccinia triticina Erikss. (Pt) and its wheat host Triticum aestivum L (Ta) upon infection. Four Pt isolates, race 1 (virulence phenotype BBBD), 12–3 (MBDS), 9 (SBDG) or 161 (FBDJ), were inoculated on susceptible cv. Thatcher and two near-isogenic lines carrying the leaf rust resistance genes Lr2a and Lr3. Different combinations represent various infection types (ITs): highly susceptible (IT3 or 4), intermediate resistant (IT2), and highly resistant triggering necrosis (IT1). Sampling of all interactions using Illumina-based RNA sequencing allowed association of ITs with gene expression profiles. Relative expression levels of both Pt and Ta genes were calculated using the race 1 reference genome sequence and the draft Ta genome (International Wheat Genome Sequencing Consortium). In-depth analysis of candidate Pt effector protein genes revealed sequential waves of their expression across time (24 hours to 14 days). Focusing on highly expressed effectors, we identified Pt genes whose expression varied significantly among various ITs. By correlating these expression patterns with those of host genes, we detected wheat genes likely affected by and therefore possibly interacting with those effectors. Fluorescent chimers of five Pt effectors, whose transcript levels were highly associated with wheat transcripts across various interactions, were targeted to diverse plant organelles in a heterologous N. benthamiana plant system. Using a bimolecular fluorescent complementation assay, two Pt effectors, PTTG_08468 and PTTG_10273, were found to interact with Ta phosphoethanolamine methyltransferase and glycogenin-2 like protein, respectively. Verification of these interactions is in progress.
Comprehensive transcriptome analysis of Pinus flexilis stems for understanding host susceptibility to Cronartium ribicola. J.-J. LIU, A. W. SCHOETTLE AND R. A. SNIEZKO. Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada; (A.W.S.) USDA Forest Service, Rocky Mountain Research Station, 240 West Prospect Road, Fort Collins, CO 80526, USA; and (R.A.S.) USDA Forest Service, Dorena Genetic Resource Center, 34963 Shoreview Road, Cottage Grove, Oregon, 97424, USA
Cronartium ribicola J.C. Fisch. causes white pine blister rust (WPBR), a deadly forest disease on limber pine (Pinus flexilis E. James) and other native five-needle pines in North America. The fungal basidiospores, released from leaves of Ribes L spp., infect pine needles through the stomata. The rust mycelium grows into the branches and stem and can kill trees of all ages as cankers girdle the main stem. The molecular interaction of white pine-blister rust in the host stem tissues remains largely unknown. Therefore, RNA-seq analysis was performed on limber stem tissues to identify the genes and transcriptional regulators involved in host susceptibility during stem canker development. The transcriptome was sequenced using Illumina Hi-seq 2000 platform which generated about 650 and 900 million 100-bp paired-end reads from healthy non-infected and cankered seedlings, respectively. Transcripts were de novo assembled using Trinity program, resulting in an N50 at 1.5-Kb for the assembled transcriptomes. A total of 309 529 and 394 430 transcripts were detected in healthy non-infected and cankered seedlings, respectively. Dual RNA-seq of cankered seedlings revealed gene expression changes simultaneously in both host tissues and pathogenic cells. A comprehensive gene ontology (GO) analysis was further carried out on the transcriptomes for functional annotation of expressed genes. Differential expression analysis indicated that a series of biological processes and metabolic pathways are enriched in the cankered tissues, thereby providing novel insight into molecular plant-microbe interactions for understanding host susceptibility in the WPBR pathosystem.
Characterizing the genomic organization of Canadian isolates of strawberry mottle virus: identification of cleavage sites and mapping of functional protein domains within the polyproteins. K. S. MANN, M. WALKER AND H. SANFACON. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada
Strawberry mottle virus (SMoV, family Secoviridae) is one of the viruses found in association with the strawberry decline disease in Eastern Canada. The two genomic RNAs each encode a single large polyprotein, which are presumably cleaved by the RNA1-encoded 3 C-like protease (PRO). Using in vitro processing assays, we confirmed the position of three cleavage sites in the RNA1 polyprotein, which are processed at various efficiencies between the putative helicase and viral genome-linked protein domains (VPg) (+++), VPg and PRO domains (+), and PRO and RNA-dependent RNA polymerase domains (±). Mutation of the predicted cysteine of the PRO catalytic triad abolished these cis-cleavages. A consensus cleavage site sequence was determined to be AxEQ/G. This consensus sequence was found in two additional locations in the N-terminal region of the RNA1 polyprotein and cleavage at these sites is under investigation. The consensus sequence is not present in the RNA2 polyprotein, although a related sequence (AxEE/G) between the predicted domains for the movement and coat proteins (CP) was found to be cleaved by intermediate RNA1-encoded polyproteins that contained the PRO domain (e.g. VPg-PRO). Finally, a new cleavage event was detected close to the predicted C-terminus of the CP domain. Interestingly, this cleavage was not dependent on the RNA1-encoded protease. Rather, deletion of the C-terminal region of the RNA2 polyprotein eliminated this cleavage, suggesting the presence of a second protease downstream of the coat protein. Proteolytic processing of the RNA2 polyprotein by a second protease is the first to be reported within the family Secoviridae.
Unravelling cherry slip-skin maceration disorder. D. T. O'GORMAN, M. WALKER, J. FRASER, J. BOULE, P. M. TOIVONEN AND J. R. ÚRBEZ-TORRES. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada
Cherry slip-skin is a problem affecting the health and quality of primarily late season sweet cherry (Prunus avium L.). Reports of this condition causing major problems in the 2012 harvest came from both British Columbia and Washington State producers, with significant but variable incidence in following years. Previously in the literature slip-skin maceration disorder has been described as a post-harvest disease caused primarily by Rhizopus arrhizus Fischer and R. stolonifer (Ehrenb. ex Fr.) Lind in French prunes and Mucor piriformis A. Fisch., in cherries. Preliminary investigations reported here differ somewhat and suggest both physiological and pathological components, involving several different yeast species within the genera Hanseniaspora Zikes, Aureobasidium Viala & G. Boyer, Cryptococcus Vuill, Candida Berkhout and Rhodotorula F.C. Harrison. The condition is not easily visible until just after harvest and may not show up until after shipping. Following harvest, the shoulder of the cherry becomes noticeably soft and the skin disassociates from the inner tissue which has become macerated. With time, while the rest of the cherry remains firm, the affected tissue develops radially causing breakage of the skin and during shipping the affected areas may form sunken craters on the fruit’s surface. In this current study, trials involving canopy management and early season calcium sprays have both produced improvements in fruit health: 20% and 60% respectively. Fungicide trials conducted in a research cherry block (cv. Staccato) identified several products, Oxidate® 2.0 and Tilt® 250E, that reduced cherry slip-skin development by 70% and 51% respectively, while other products such as potassium metabisulphite only reduced symptoms by 18% and Quintec® which showed a slight increase when compared with controls.
Differential regulation of the accumulation of an Argonaute protein (NbAGO2) is correlated with symptom recovery outcome in plants infected with two isolates of Tomato ringspot virus. D. B. PAUDEL, B. GHOSHAL, S. JOSSEY AND H. SANFACON. (D.B.P., B.G., H.S.) Department of Botany, University of British Columbia, Vancouver, BC Canada; and (S.J., H.S.) Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada
Symptom recovery is a characteristic outcome of nepovirus infection in herbaceous plants. In Nicotiana benthamiana Domin, a severe isolate of Tomato ringspot virus (ToRSV-Rasp1, family Secoviridae) causes lethal systemic necrosis at 21°C. In contrast, plants infected with a mild isolate (ToRSV-GYV) recover from infection after an initial systemic necrotic response. Early in infection, both isolates accumulate to similar levels. Viral coat protein (CP) and RNA levels subsided in ToRSV-GYV infected plants at the onset of recovery but continued to increase to high levels in ToRSV-Rasp1 infected plants, suggesting the induction of a defence mechanism that functions against ToRSV-GYV but not ToRSV-Rasp1. We examined the expression of two N. benthamiana Argonaute genes known to play a role in plant antiviral RNA silencing. While NbAGO1 mRNA levels were not significantly affected by ToRSV infection, NbAGO2 expression was transiently induced early in infection (~40-fold increase in mRNA levels with either isolate). ToRSV infection also enhanced accumulation of microRNAs miR403 and miR168 which are known to downregulate NbAGO2 and NbAGO1 mRNA accumulation, respectively. This probably prevents sustained overexpression of NbAGO2 and NbAGO1. Interestingly, a spike in NbAGO2 protein accumulation was observed early in infection in ToRSV-GYV infected plants but not in ToRSV-Rasp1 infected plants, suggesting differential regulation of the translation of NbAGO2 mRNAs and/or of the stability of NbAGO2 protein. Plants silenced for NbAGO2 accumulated higher levels of ToRSV-GYV CP and displayed more severe symptoms. Together, these results suggest a role for NbAGO2 in regulating symptom expression in plants infected with ToRSV-GYV at 21°C.
Prevalence of Grapevine red blotch associated virus in British Columbia. S. POOJARI, J. BOULE, N. DELURY, D. T. LOWERY, M. ROTT, A.-M. SCHMIDT AND J. R. ÚRBEZ-TORRES. Agriculture and Agri-Food Canada, Summerland Research and Development Centre, Summerland, British Columbia V0H 1Z0, Canada; and (M.R., A.-M.S.) Sidney Laboratory, Canadian Food Inspection Agency, 8801 East Saanich Road, North Saanich, BC V8L 1H3, Canada
Grapevine red blotch-associated virus (GRBaV) is a recently identified virus of both table and wine grapes. GRBaV has been primarily found in grape-growing regions of North America and it is known to significantly affect fruit sugar levels in certain cultivars and rootstocks. To understand GRBaV prevalence in British Columbia, a total of 2200 composite leaf samples randomly collected from 128 vineyard blocks spread across different grape-growing regions were tested by end-point PCR using GRBaV specific primers. Overall, there was a low (1.6%) incidence of GRBaV in BC vineyards. Interestingly, most of the GRBaV positive samples were detected in vineyard blocks planted between 2011 and 2014, which indicates a recent introduction and primary spread of GRBaV through infected planting material. Phylogenetic analysis based on the full-length genome of eight representative GRBaV isolates indicated the presence of genetic variants belonging to two different clades. Putative recombination events were observed at multiple locations spanning the entire genome with divergent isolates as major and minor parents indicating the natural recombination between isolates influencing the evolution of GRBaV. This study represents the first comprehensive quantitative data on the current status of GRBaV in BC and underscores the need for the development of a domestic clean plant programme and understanding the epidemiological significance of GRBaV in cool climatic conditions of BC.
Induction of retrotransposons during Cucumber necrosis virus (CNV) infection in Nicotiana benthamiana. R. READE, S. B. ALAM, J. THEILMANN AND D. ROCHON. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada; and (S.B.A., D.R.) Faculty of Land and Food Systems, University of British Columbia, 248-2357 Main Mall, Vancouver, BC V6T 1Z4, Canada
Retrotransposons are mobile genetic elements that transpose via an RNA intermediate. Most retrotransposons are dormant but stress induces their transposition. Virus infection has not yet been identified as a major stressor for retrotransposition in either plants or animals. RNA-seq analysis of Nicotiana benthamiana Domin infected with the Tombusvirus CNV revealed that transcript levels of specific long terminal repeat (LTR)-type retrotransposons are dramatically elevated. A similar phenomenon was observed for three other plant viruses. Phylogenetic analyses indicated that several CNV-induced retrotransposons are closely related to the retrotransposon ONSEN, previously described to be induced by heat stress. ONSEN possesses heat shock elements (HSEs) in the LTR promoter that are activated by heat shock factors (HSFs). Analysis of CNV-induced retrotransposons indicated that most contain canonical HSE motifs. CNV infection induces HSE-containing HSP70 mRNA as well as HSF mRNA leading to the hypothesis that CNV infection induces transcription of retrotransposons via activation of their HSEs by HSFs. Nicotiana benthamiana plants were heat-shocked and tested for induction of those retrotransposons induced by CNV using ddPCR. Each of the five tested retrotransposons was found to be heat-induced and their relative induction levels paralleled that observed during CNV infection. To ascertain the involvement of HSEs, the LTRs of two retrotransposons were fused to green fluorescent protein (GFP) coding sequence and used to agro-infiltrate N. benthamiana. Both heat shock and CNV infection resulted in significant induction of GFP suggesting a plausible mechanism for induction of retrotransposons by CNV. Since retrotransposition can induce mutations in nuclear genes, virus infection may alter evolution of plant genomes.
Development of root-lesion nematode populations in the rootzone of Gisela series semi-dwarfing cherry rootstocks. C. REITH, T. A. FORGE, D. NEILSEN, G. NEILSEN AND P. RANDALL. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada
Historically, sweet cherry production in British Columbia has utilized ‘Mazzard’ rootstock which is susceptible to the root-lesion nematode, Pratylenchus penetrans (Cobb) Filipjev & Schuurmans-Stekhoven (Pp). Interest in the use of ‘Gisela’ (G) series semi-dwarfing rootstocks is growing; however, little is known of the susceptibility of these rootstocks to Pp infestation. An experimental block of ‘Skeena’ sweet cherry on G3, G5 and G6 rootstocks was planted in spring of 2010. There were 18 four-tree plots of each rootstock, six of which were subjected to each of three different training systems. Twice per year from 2013 through 2016, root zone soil was sampled from each plot. Biomass of root fragments was quantified and nematodes were extracted from fine roots and soil. There was a significant date × rootstock interaction for Pp populations in roots; G3 harboured greater numbers of Pp than both G5 and G6 at three of the first four sample dates. Soil populations did not differ among rootstocks at any date. There was also a significant date × rootstock interaction effect on fine root biomass, with G3 having lower root biomass density than G6 at the first sample date, and greater biomass at the seventh sample date. Overall, Pp populations in both roots and soil declined steadily between spring 2013 and summer 2016. We speculate that either ‘Gisela’ rootstocks become more resistant to Pp with age or that soil at the site has become suppressive with time.
Pathogenomics analyses of the Heterobasidion species complex. S. F. SHAMOUN, J.-J. LIU, X. LI, M. ROTT, G. SUMAMPONG AND C. HAMMETT. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada; (X.L.) Canadian Food Inspection Agency, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada; and (M.R.) Canadian Food Inspection Agency, Centre for Plant Health, 8801 East Saanich Road, Sidney, BC V8L 1H3, Canada
The basidiomycete species complex Heterobasidion annosum (Fr.) Bref., sensu lato, are regarded as some of the most destructive pathogens of conifers worldwide. Recent work has classified the complex into five species based on their genetic diversity, inter-sterility between mating groups, geographic distribution and host preference. In North America, there are two species, H. irregulare Garbel. & Otrosina and H. occidentale Otrosina & Garbel., while H. annosum s.s., H. parviporum Niemelä & Korhonen and H. abietinum Niemelä & Korhonen occur in Eurasia. The Canadian Food Inspection Agency has identified all Heterobasidion spp. as forest pest species of significant regulatory concern because of the invasive risks they pose. We have developed PCR primers specific for conserved alleles of glyceraldehyde 3-phosphate dehydrogenase and elongation factor 1α genes, for identification and differentiation of H. irregulare and H. occidentale. The method is sensitive enough to detect either species from infected wood. In addition, we have screened 140 isolates of H. irregulare and H. occidentale from North America for presence of dsRNA viruses. Analyses showed 26 putative viruses including 23 from H. irregulare (37%) and three from H. occidentale (4%). Ongoing research work is focusing on designing and validating Loop-Mediated Isothermal Amplification (LAMP) assays for detection and identification of Heterobasidion spp. Furthermore, genome sequencing of H. occidentale is underway to search for candidate virulence genes and marker development for detection and identification of individual species of the Heterobasidion complex. Research and development of dsRNA viruses detection and discovery pipeline using next generation sequencing (NGS) for Heterobasidion spp. is being proposed.
Gene expression in dwarf mistletoe during the explosive seed dispersal with special attention to aquaporins. J. URBAN, R. KALDENHOFF, C. ROSS FRIEDMAN, B. OTTO AND A. BOUDICHEVSKAIA. Department of Biological Sciences, Thompson Rivers University, 900 McGill Road, Kamloops, BC V2C 0C8, Canada
Arceuthobium americanum (lodgepole pine dwarf mistletoe) is found as a pathogen of coniferous trees across North America, ultimately killing the host tree by redistributing water and nutrients from healthy areas of the tree to those infected with the parasite. Similarly, Arceuthobium oxycedri (DC.) M.Bieb. (juniper dwarf mistletoe) is detrimental to Juniperus L. spp. (junipers) in Eurasia. Arceuthobium spp. employ a unique method of seed dispersal whereby the seed is explosively discharged from the fruit. The molecular mechanism of explosive seed discharge and, more specifically, how aquaporins and other proteins could be involved in establishing the hydrostatic pressure responsible for propelling the seed was investigated. Total RNA was extracted from fresh A. americanum as well as from A. oxycedri plants using a MasterPure™ Plant RNA Purification Kit and an RNA Easy Plant Extraction Kit with the addition of PEG, respectively, from samples collected in spring and just before the dispersal of seed (early autumn). Extraction was followed by cDNA synthesis and library construction. The cDNA library was screened for aquaporins using MicroHybridization Kit; a Southern Blot was performed with a mixture of Digoxigenin-11-dUTP-labelled probes. Positive results obtained in the hybridization were sent for sequencing. Using NCBI BLAST, sequence similarity to an aquaporin PIP2:1 gene as well as a few other genes were found. As Arceuthobium’s genome project has not yet been started and its genome is not sequenced, a reverse approach grounded on a heterologous array was used in which RNA from fruits of A. americanum was subjected to commercial Arabidopsis Gene 1.0 ST Arrays (Affymetrix, USA) gene chip analysis to probe for differences in genes expression before and during explosive seed dispersal.
Quantification of Agrobacterium vitis in nursery planting material. T. M. VOEGEL AND L. M. NELSON. Department of Biology, University of British Columbia, Okanagan, 1177 Research Road, Kelowna, BC V1V 1V7, Canada
Agrobacterium vitis Ophel and Kerr is the causal agent of grapevine crown gall, an economically important disease in cold climate viticulture regions. The disease occurs frequently in Okanagan vineyards and limits grape production and lifespan of vineyards. The pathogen spreads through cuttings from grapevine nurseries or infects grapevines planted in soil harbouring A. vitis. Wounds in the grapevine trunk or canes due to winter freezing or mechanical injuries lead to gall formation. Current detection techniques are time intensive and no methods are available to quantify A. vitis populations in plant tissues. In this study, a rapid protocol (1–2 days) was developed to quantify A. vitis virA gene copy numbers in roots of grapevine planting material using digital droplet PCR. The method was tested on dormant grapevine samples received from Canadian and international nurseries. Detectable virA gene concentrations ranged from 1 to 3311 copies μL−1. Currently, the tested plants are being grown in a greenhouse to assess the development of crown gall symptoms. Our method can be applied to screen new planting material from nurseries or be implemented as part of a clean plant network.
Soil amendments and irrigation type influence Pratylenchus penetrans populations and growth of newly planted sweet cherry. T. T. WATSON, L. M. NELSON, D. NEILSEN, G. H. NEILSEN AND T. A. FORGE. The University of British Columbia – Okanagan Campus, 1177 Research Road, Kelowna, BC V1V 1V7, Canada; and (T.T.W., D.N., G.H.N., T.A.F.) Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC V0H 1Z0, Canada
Pratylenchus penetrans (Cobb) Filipjev & Schuurmans-Stekhoven is an economically important plant parasite capable of causing significant yield loss of stone fruits. Restrictions on soil fumigants have generated interest in alternative management strategies, particularly those associated with promotion of a suppressive rhizosphere. This study evaluated the impacts of organic soil amendments (compost and bark mulch) and irrigation type (drip emitter and micro-sprinkler) on: (1) early growth of sweet cherry, (2) P. penetrans populations in roots and (3) the abundance of rhizobacteria with antibiotic biosynthesis genes for 2,4-diacetylphloroglucinol and pyrrolnitrin. ‘Skeena’ sweet cherry on Gi.6 rootstock was planted into an old apple orchard site. Entire tree rows (whole plots) were irrigated with drip emitters or micro-sprinklers. Soil treatments were applied as split-plots and included: (1) compost, (2) bark mulch, (3) compost and bark mulch, (4) fumigation and (5) untreated control. Fumigation and pre-plant incorporation of compost combined with surface application of bark mulch increased trunk cross-sectional area compared with untreated control plots. Drip irrigation increased trunk cross-sectional area compared with micro-sprinkler irrigation. Compost and bark mulch suppressed P. penetrans populations in roots relative to fumigation or the control. Drip irrigation suppressed P. penetrans populations relative to micro-sprinkler irrigation. Compost increased the abundance of 2,4-diacetylphloroglucinol and pyrrolnitrin-producing bacteria in the rhizosphere relative to fumigation and the control. Overall, organic soil amendments in combination with drip irrigation show potential as a non-fumigant alternative for suppression of P. penetrans on sweet cherry, with promotion of antibiotic-producing rhizobacteria a possible contributing mechanism in compost-induced soil suppressiveness.
A thirst for more: morphological changes in Arceuthobium americanum indicate substantial water theft from its host, Pinus contorta subsp. latifolia. D. J. ZIEGLER AND C. M. ROSS FRIEDMAN. Department of Biological Sciences, Thompson Rivers University, 900 McGill Road, Kamloops, BC V2C 0C8, Canada
In western North America, Pinus contorta subsp. latifolia Engelm. (lodgepole pine), an important lumber species, is parasitized by the dwarf mistletoe Arceuthobium americanum Nutt. ex Engelm. This infection reduces the host’s reproductive viability, growth rate and wood quality, reducing timber availability. Water relations are extremely important in this parasite’s life cycle in many ways. Dwarf mistletoe taps into the vasculature of the host and consequently draws water and vital nutrients from the tree through perpetually open stomata. Furthermore, A. americanum explosively discharges its seeds by accumulating hydrostatic pressure within the fruit. Due to the importance of stomata in this parasitism, the arrangement, organization and distribution of these stomata are of great importance to understanding the plant’s pathological implications on the host. Using environmental scanning electron microscopy, we studied the anatomy and distribution of the stomata as well as the stomatal density on the flowers/fruit, while also noting developmental changes of the fruit. We found that the fruit increases significantly in both length and diameter, while the stomatal density declines significantly. The initial stomatal density was quite high (55 stomata mm−2), which is unusual for floral components. The significant increase in fruit diameter and length, compounded with a declining stomatal density indicated that a large amount of water is being drawn into, and eventually retained, in the mistletoe fruits. Due to the absence of developed leaves, the transpiration role for A. americanum is largely filled by the flowers. Therefore, we suggest the flowers are analogous in transpirational function to the leaves of other angiosperms.