Long-term effects of cedar leaf blight infection in western redcedar seedlings. J. A. ALDANA, J. H. RUSSELL, B. MOA AND B. J. HAWKINS. Centre for Forest Biology, University of Victoria, P.O. Box 3020 STN CSC, Victoria, BC V8W 3N5, Canada; (B.M.) University Systems, University of Victoria, 3800 Finnerty Road, Clearihue Loading Ramp Rm. C061a, Victoria, BC V8W 2Y2, Canada; and (J.H.R.) British Columbia Ministry of Forests, Lands and Natural Resource Operations, P.O. BOX 335, Mesachie Lake, BC V0R 2N0, Canada
Western redcedar (WRC, Thuja plicata), a preferred softwood for outdoor uses, is susceptible to the cedar leaf blight (CLB) [Didymascella thujina (E.J. Durand) Maire]. Quantitative resistance to CLB in WRC has been documented previously (Russell et al. 2007, Can. J. Forest Res. 37: 1978–1986), but the genetic basis and mechanisms of resistance are still unknown. The objective of this investigation was to determine gene expression and biochemical effects of CLB infection in WRC from infection to symptoms. One-year-old seedlings from one resistant and one susceptible full-sib family were exposed to natural CLB inoculum in 2013 and then maintained until symptoms developed; never-infected plants from the same families were used as check. Foliage from three seedlings per family and treatment was collected for chemical and gene expression analyses. Principal components (PC) analysis was used to identify compounds significantly associated with the presence or absence of symptoms, and RNA-Seq was used to study differential gene expression associated with disease development. PC 1 and 2 explained 48.9% of the variance in the chemical data; only sabinene (P ≤ 0.0001) and α-thujene (P = 0.0061) concentrations were significantly higher in resistant plants. There were 173,924 transcripts in the assembled transcriptome. Four main groups of transcripts were identified: 1. Sequences at high numbers only in resistant seedlings; 2. Those with high levels exclusively in the susceptible plants; 3. Transcripts at high levels in the susceptible infected seedlings; and 4. Sequences with no specific pattern. Currently, genes of interest in the above groups are being identified.
POLYOXIN D Zinc Salt 5SC for greenhouse cucumber and tomato disease management. J. F. ELMHIRST AND J. R. HAYES. Elmhirst Diagnostics and Research, 5727 Riverside Street, Abbotsford, BC V4X 1T6, Canada
In 2015, POLYOXIN D Zinc Salt 5SC was evaluated for control of powdery mildew of greenhouse tomato cv. ‘Torero’ and gummy stem blight of greenhouse mini-cucumber cv. ‘Jawell’, in British Columbia, Canada. POLYOXIN D was applied weekly at 0.3–1.0 L/ha and every 14 days at 1.0 and 1.5 L ha−1 and compared to an alternating programme of PRISTINE WG and NOVA every 14 days, and a water-sprayed check. At all rates, POLYOXIN D controlled powdery mildew of tomato as well as, or better than PRISTINE/NOVA, resulting in 86.1–95.4% fewer lesions and 92.5–95.5% less leaf area diseased than the water-check, at 28 days after the last application. There was no statistical difference among rates of POLYOXIN D, or application intervals. POLYOXIN D at 1.0 L ha−1 controlled gummy stem blight of cucumber as well as PRISTINE/NOVA, reducing necrotic leaf area by 85% for up to 14 days after the last application and increasing yield (weight of marketable fruit) statistically, compared with the check. There was no statistical difference between weekly or bi-weekly applications of POLYOXIN D at 1.0 and 1.5 L ha−1. Weekly applications of POLYOXIN D at lower rates, 0.3 and 0.5 L ha−1, were less effective against gummy stem blight and, at 0.3 L ha−1, marketable yield was not statistically different from the check. No phytotoxicity was observed.
Water-conserving mulch and micro-irrigation practices affect assemblages of nematodes parasitizing winegrape in the Okanagan Valley. T. A. FORGE, D. NEILSEN, G. NEILSEN, K. HANNAM, P. RANDALL AND L. WAMBOLD. Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, P.O. Box 5000, 4200 Highway 97, Summerland, BC VOH IZO, Canada; and (L.W.) Mission Hill Estate Winery,1730 Mission Hill Road, West Kelowna, BC V4T 2E4, Canada
In semi-arid regions such as the Okanagan Valley of British Columbia, growers are increasingly adopting micro-irrigation practices and considering the use of organic mulches and amendments to reduce water consumption and improve resilience to water shortages. Several species of plant-parasitic nematodes are frequently found in Okanagan vineyards, including: Mesocriconema xenoplax (Raski) Loof & DeGrisse, Meloidogyne hapla Chitwood, Pratylenchus spp. Filipjev and Xiphinema americanum-group Cobb populations. The effects of shifting irrigation and mulching practices on populations of these nematodes are poorly understood but they could have implications for root health and drought tolerance. In 2013, a field experiment was established in a commercial organic vineyard to study the interactive effects of micro-sprinkler (MS) or drip irrigation and compost mulch on populations of nematodes under ‘Merlot’ grapevines on SO4 rootstock. Soil samples were collected four times between autumn 2013 and summer 2015. Compost increased population densities of M. xenoplax across sample dates, and Pratylenchus on one sample date. Xiphinema population densities were greater under MS than under drip. There was a significant irrigation × compost interaction for M. hapla: with compost, M. hapla was more abundant under MS than under drip, but without compost there was no irrigation effect. Irrigation had no effect on M. xenoplax or Pratylenchus; these results contrast with a concurrent study at another location, in which Pratylenchus abundance was greater and M. xenoplax abundance was lower under drip than under MS. Our results show that changes in irrigation practices can affect nematode populations in a relatively short time-frame.
Identification of Colletotrichum destructivum causing anthracnose of Wasabia japonica in British Columbia. J. L. MACDONALD AND Z. K. PUNJA. Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, 6947 Highway 7, Agassiz, BC V0M 1A0, Canada; and (Z.K.P.) Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
Wasabia japonica (Miq) Matsumura is grown in the Pacific Northwest for the culinary market and use in sushi restaurants. The high moisture environments required to successfully cultivate the crop and a 15–18 month crop cycle are conducive for infection by pathogens. In 2014, a commercial greenhouse crop in Maple Ridge, British Columbia showed symptoms of leaf spot and blight. Small leaf tissue pieces were surface-sterilized and placed on potato dextrose agar. Emerging fungal colonies were grey-black and formed concentric rings. Microscopic examination revealed the acervuli with setae and abundant conidia that were characteristic of a Colletotrichum sp. Genomic DNA was extracted and amplified using fungal-specific primers, ITS1 F and ITS2. Sequence comparison in GenBank identified the species as Colletotrichum destructivum O’Gara. Wasabi leaves under humid conditions (25 ± 2°C) inoculated with a conidial solution (8.6 × 104 conidia mL−1) of C. destructivum developed brownish-black lesions within 3 days; after 8 days inoculated leaves turned yellow and lesions coalesced. The effects of temperature (5–35°C) and photoperiod (8000 ± 500 lux; 0, 12 and 24-hour light) on mycelial growth and conidia production were assessed. Maximum mycelial growth was at 25 and 30°C (mean 7.4 ± 0.4 and 7.6 ± 0.2 mm day−1, respectively); it declined at higher and lower temperatures. Maximum conidial production was under darkness (0-hour light: 100%; 12-hour: 10%; and 24-hour: 3%). Photoperiod had no effect on mycelial growth, but colony colour was white under darkness, and salmon under light conditions. Cultivar response to C. destructivum infection is currently being assessed.
Characterization of resistance against cedar leaf blight caused by Didymascella thujina. D. NOSHAD, S. L’HIRONDELLE, B. HAWKINS AND J. RUSSELL. (D.N., B.H.) Department of Biology, P.O. Box 3020 STN CSC, Victoria, BC V8W 3N5, Canada; (S.L’H., J.R.) BC Ministry of Forests, Lands and Natural Resource Operations, P.O. Box 9519, Stn Prov Govt, Victoria, BC V8W 9C2 Canada; and (D.N.)Canadian Bio-Act Ltd, 331 E. Burnside Road, Victoria, BC V9A 1A6, Canada
Cedar leaf blight (CLB), caused by the biotrophic pathogen Didymascella thujina (E.J. Durand) Maire, is an important disease of western redcedar (Thuja plicata Donn ex D. Don) in British Columbia. Our field screening programme found different kinds of presumptive resistance against CLB. In this study we collected samples from BC western redcedar populations and pathogen isolates to develop an efficient in vivo and in vitro screening method for phenotype expression of CLB infection. To better understand the disease, we established a controlled inoculation method followed by studying foliage from each family in detail using scanning (SEM) and transmission electron microscopy (TEM). Preliminary results indicate (1) large morphological differences among pathogen isolates in size and germ tube development; (2) considerable presence of a secondary pathogen, Truncatella angustata (Pers.) S. Hughes; and (3) an apparent difference in foliar reactions to the pathogen after controlled inoculation. Ongoing studies with in vitro screening will be used to further examine redcedar population differences in resistance to CLB.
Identification and characterization of the fungal pathogens inhabiting the root zone of sweet cherry (Prunus avium L.) in the Okanagan Valley of British Columbia. D. T. O’GORMAN, P. HAAG, T. A. FORGE AND J. R. ÚRBEZ-TORRES. Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, P.O. Box 5000, 4200 Highway 97, Summerland, BC VOH IZO, Canada
Differences in tree vigour within and among sweet cherry (Prunus avium L.) orchards, is a continuing problem for the BC tree fruit industry and is believed to be associated with maladies such as cankers, dieback, root rots and replant disease. However, a general lack of knowledge exists regarding the pathogens associated with these diseases of cherry. Therefore the objective of this study was to identify fungal species in the rhizosphere that are associated with root rots and replant disease of cherry. A survey of both commercial and research cherry orchards was conducted to identify and assess pathogenicity of the predominant fungi colonizing roots or inhabiting the rhizosphere. Fungi were isolated from necrotic and discoloured cherry root tissues, orchard soils and/or wooden toothpicks inserted into soils as bait. Fungal species recovered from the sampling were identified using morphological characteristics and DNA sequencing of the ITS, β-tubulin and EF1 genes. Pathogenicity testing for some of the most prominent fungal species identified (Fusarium, Ilyonectria and Rhizoctonia spp.) were conducted in the greenhouse by inoculating Mazzard rootstock. All isolates tested, produced significant regions of vascular discolouration extending from the inoculation point. Ilyonectria macrodidima (Halleen, Schroers & Crous) P. Chaverri & C. Salgado and Fusarium oxysporum Schlecht. emend. Snyder & Hansen produced the largest areas of necrosis and were re-isolated at rates of 57% and 66%, respectively. Additionally, oligonucleotide probes for the species found in our survey were designed or modified using available sequences and assembled in a DNA-macroarray as part of the development of a diagnostic assay.
Genetic diversity and molecular diagnostics of grapevine viruses in British Columbia. S. Poojari, T. Lowery, J. Boulé, N. DeLury, M. Rott, A. M. Schmidt and J. R. Úrbez-Torres. Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, P.O. Box 5000, Summerland, BC VOH IZO, Canada; and (M.R., A.M.S.) Canadian Food Inspection Agency, Centre for Plant Health, Sidney Laboratory, Sidney, BC V8L 1H3, Canada
Rapid expansion of wine grape production in British Columbia (BC) in the past two decades raised concern for virus and virus-like diseases that spread through propagating planting material and insect vectors. To understand the prevalence and diversity of grapevine viruses in BC vineyards, large-scale field surveys were carried out during 2013 and 2014 growing seasons and a total of 1,957 random-composite and 293 target-individual grapevine samples from 10 red and 12 white cultivars from 113 vineyard blocks were collected and tested for the presence of Grapevine leafroll-associated viruses (GLRaV-1, −2, −3 and 4–9), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV), Arabic mosaic virus (ArMV) and Grapevine red blotch-associated virus (GRBaV). Among the GLRaVs, the most widespread was GLRaV-3 (17.2%), followed by GLRaV-2 (5.5%), GLRaV 4–9 (4.2%) and GLRaV-1 (1.4%). High incidence of GFkV (29.2%) was detected, whereas GFLV and GRBaV were limited with 0.5% and 0.4% incidence respectively. Nucleotide sequence analysis of partial heat-shock protein (Hsp-70 h) gene of 11 GLRaV-2 isolates showed high level of identity (90.0–99.3%) among BC isolates. Phylogenetic analysis with global GLRaV-2 variants indicated the presence of a single variant of GLRaV-2 in BC vineyards belonging to the Pacific Northwest lineage. Sequence analysis of partial Hsp-70 h gene of 15 GLRaV-3 isolates showed nucleotide sequence identities in the range of 88.7 to 99.8% belonging to two distinct clades. The established sensitive and large-scale diagnostic methods would support virus-free planting material programmes for the long-term economic sustainability of the wine grape industry in BC.
Development and assessment of different molecular tools for the detection, identification and quantification of grapevine trunk diseases fungal pathogens and their potential use to develop and implement disease management strategies. J. R. ÚRBEZ TORRES, P. HAAG 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 IZ0, Canada
Grapevine trunk diseases (GTDs) are the primary biotic cause of grapevine decline and mortality around the world. With an overall economic impact of US$1.5 billion worldwide, GTDs are recognized as one of the major threats to the industry’s future economic sustainability. To date, over 80 fungal species belonging to 22 genera are associated with GTDs. This broad spectrum of taxonomically unrelated fungi makes species identification extremely challenging and time consuming. Because precise and reliable means of detecting and monitoring plant pathogens are critical to develop management strategies, research in our laboratory has been focused during the last few years on the development and assessment of molecular tools for an accurate, rapid and high throughput detection of GTDs fungi. Recently, a DNA-macroarray based on reverse dot-blot hybridization and containing over 100 species-specific oligonucleotide probes complementary to portions of the β-tubulin gene was developed. In total, 61 different fungi, including 34 GTDs pathogens can be detected and identified by the array in a single test, which has the potential to be used by the grapevine nursery industry to determine the health status of the planting material. Additionally, detection and quantification of GTDs inoculum in BC vineyards using the latest available technology (droplet-digital PCR) is currently being evaluated in our laboratory with the objective to characterize high-risk infection periods throughout the growing season and to determine the environmental conditions that could favour spore release and infection in the Okanagan Valley; all key information to implement effective control strategies for GTDs in BC.
Control of Pratylenchus penetrans on sweet cherry using organic soil amendments. T. T. WATSON, L. M. NELSON, D. NEILSEN, G. H. NEILSEN, D. O’GORMAN AND T. A. FORGE. The University of British Columbia – Okanagan Campus, 3333 University Way, Kelowna, BC V1V 1V7, Canada; and (T.T.W., D.N., G.H.N., D.O., T.A.F.) Pacific Agri-Food Research 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 beneficial microorganisms in the rhizosphere. This study aimed to investigate the impacts of amending P. penetrans-infested orchard soil with compost or bark mulch on plant growth and the abundances of P. penetrans, Pseudomonas spp. and phosphate-solubilizing bacteria (PSB). ‘Skeena’ sweet cherry on Gi.6 rootstock was planted into an old apple orchard site. Soil treatments included: (1) compost, (2) bark mulch, (3) compost and bark mulch, (4) fumigation, or (5) nothing (untreated control). The abundance of P. penetrans in fine roots and soil was monitored over the first 2 years of growth (2014–2015). Rhizosphere Pseudomonas spp. and PSB were quantified in spring and autumn of 2015. Compost improved shoot length to levels equivalent to fumigation in autumn 2014. Organic soil amendments resulted in a lower abundance of P. penetrans in roots relative to the untreated control in autumn 2014 and 2015. By autumn 2014, organic amendments had decreased the abundance of P. penetrans in soil relative to the control, whereas fumigated soil had been recolonized by P. penetrans and did not differ from the control. Pseudomonas spp. and PSB were most abundant in the rhizosphere of plots receiving organic amendments. Overall, organic soil amendments show potential as a non-fumigant alternative for control of P. penetrans and promotion of early growth of sweet cherry.