Annual Meeting, 2012/Réunion annuelle, 2012 The Canadian Phytopathological Society¹

Managing potato scab and enhancing tuber yield with low rates of fish emulsion applied as a pre-plant soil amendment. P. A. ABBASI. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada

Soil amendment of fish emulsion (FE) can provide suppression of seedling damping-off, potato scab, and verticillium wilt diseases. However, the application rates (20,000 L/ha) of FE suppressive to potato scab in the field may not be feasible. The aim of this 4-year study was to see if much lower rates of FE could suppress potato scab and increase tuber yield. Diluted FE (1000 and 2000 L/ha or 0.05 and 0.1%) was applied to the field plots or micro-plots twice a year before planting and after harvesting potatoes starting in fall of 2007 and ending in spring of 2010. In the micro-plots, amendment of high rate (0.1%) of FE to an infested soil from a potato field (site BL) consistently reduced scab severity by 44.9% in 2008, 44.8% in 2009, and 30.9% in 2010 compared to the control. Scab severity on tubers harvested from such plots was also low in 2011 when no further fish emulsion was applied. In the field trials, the high rate of FE (2000 L/ha) consistently reduced scab severity by 45% in 2008, 53% in 2009, 44% in 2010, and 38% in 2011; reduced the percentage of tubers with deep-pitted scab by 48% in 2008, 51% in 2009, 66% in 2010, and 77% in 2011; and increased the percentage of marketable tubers by 37% in 2008, 83% in 2009, 20% in 2010, and 8% in 2011. FE treatments increased total tuber yield by 7-20% in first 3 years, but there was no effect in the fourth year. Marketable tubers were consistently higher in the plots treated with high rate of FE. FE soil treatments increased soil bacteria but the numbers dropped to the control level after FE application was stopped. There was no change in total fungal numbers. These results suggest that economically feasible rate of FE can provide disease suppression and enhance tuber yield and this effect may last longer for continuous potatoes without any further FE application.

Bactericidal activity of some natural and synthetic compounds against phytopathogenic bacteria, Erwinia carotovora sub sp. carotovora . M. A. ABBASSY, S. A. M. MASOUD AND A. M. K. NASSAR. Department of Plant Protection, Faculty of Agriculture, Damanhour University, Egypt

Current study aimed to search for safe and effective natural products to control the potato soft rot disease Erwinia carotovora subsp. carotovora (Jones) Bergey et al. Antibacterial efficiency of edible mushroom extracts and garlic essential oil were compared with two bactericides (Streptrol and Oxolinic acid) and two fungicides (Mancopper and Copper Oxychloride) against soft rot bacteria. The paper disc diffusion and potato slices methods were used. All of the natural and synthetic compounds exhibited marked in vitro antibacterial activity particularly Streptrol and the chloroform extract of mushroom which showed 3.4 folds inhibitory effect compared with garlic essential oil. Also, the chloroform extract of mushroom showed similar activity to Mancopper and Copper Oxychloride. In vivo studies revealed that concentrations of 2, 3, 3 and 6.5 mg/mL from the chloroform, petroleum ether, ether extractives of mushroom and garlic oil, respectively, prevent the development of soft rot symptoms on potato slices as compared with the synthetic bactericides and fungicides. Mixing of garlic oil (1 mg/mL) with Streptrol or Oxolinic acid (0.05 mg/mL) synergized the inhibitory effect of both synthetic bactericides. The overall results suggest the potential use of the edible mushroom extracts and garlic essential oil for the control of soft rot disease on potatoes.

Pathogenic diversity of Manitoba isolates of Phytophthora infestans on potato and tomato. H. ALKHER, L. M. KAWCHUK, L. R. ADAM, M. R. ISLAM, K. F. DOBINSON, K. L. CONN, R. D. PETERS, K. I. AL-MUGHRABI AND F. DAAYF. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; (L.M.K.) Lethbridge Research Centre, Agriculture and Agri-Food Canada (AAFC), 5403 1^st Avenue South, Lethbridge, AB T1J 4B1, Canada; (K.F.D., K.L.C.) Southern Crop Protection and Food Research Centre, AAFC, 1391 Sandford Street, London, ON N5V 4T3, Canada; (R.D.P.) Crops and Livestock Research Centre, AAFC, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; and (K.I.A.-M.) Potato Development Centre, New Brunswick Department of Agriculture, Aquaculture and Fisheries, 39 Barker Lane, Wicklow, NB E7L 3S4, Canada

Late blight caused by Phytophthora infestans (Mont.) de Bary can affect various solanaceous plant species. However, it is on potato and tomato crops that it is considered one of the most destructive diseases. Shifts in the population structure of P. infestans in North America and elsewhere have been recorded in the last two decades along with an increase in the frequency and severity of late blight epidemics. The 1990s saw the old US-1 displaced by the US-8 genotype, which rapidly became the most dominant genotype in Canada. In 2009-2011, new outbreaks of late blight were caused by new strains (US-22, US-23, and US-24) detected in potato and tomato growing areas. Phytophthora infestans strains collected from potato and tomato in Manitoba were characterized based on their mating type, GPI allozyme genotype, RG57 finger printing, and sensitivity to two formulations of metalaxyl fungicide. Thirteen isolates from different genotypes and hosts (potato, tomato) were selected to infect potato cultivar ‘Kennebec’ and Sunrise tomato hybrid using whole plants in a growth chamber as well as tubers in vitro. Most of the potato isolates, that are US-24 genotype, showed higher pathogenicity on Sunrise tomato even at 3-day post inoculation, compared to the ‘Kennebec’ potatoes. The level of infection was also compared on ‘Kennebec’ tuber slices results. In general, there was a high level of diversity in pathogenicity of the tested isolates. However, it was clear that US-24 induced more symptoms compared to the other genotypes (US-8, US-11, US-22, and US-23).

Comparative gene expression analysis of susceptible and resistant wheat infected by Fusarium graminearum. K. ALTAWEEL, A. BRÛLÉ-BABEL AND W. G. D. FERNANDO. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada

Wheat fusarium head blight (FHB), caused by the fungus Fusarium graminearum Schwabe, is a destructive disease of wheat (Triticum aestivum L.). Identification of host genes involved in defense responses is one of most critical steps leading to the elucidation of disease resistance mechanisms in plants. In order to understand the mechanisms of wheat resistance and have a clear insight to the expression of host genes involved in defense response against FHB disease, a study on genome-wide gene expression was conducted comparing the responses of resistant ‘Sumai3’ and susceptible ‘Caledonia’ wheat cultivars to FHB infection. To identify differentially expressed genes corresponding to FHB resistance, wheat spikes of ‘Sumai3’ were inoculated with a macroconidia suspension of F. graminearum. A full-length cDNA library was then constructed and a subtracted cDNA library was synthesized using suppressive subtractive hybridization method. The gene expression patterns of the two cultivars were investigated 6, 12, 24, 36, 48, 72, and 144 h after inoculation using quantitative real-time PCR (qRT-PCR). From this, we have identified and analyzed some differentially expressed genes between ‘Sumai3’ and ‘Caledonia’.

Phylogenetic relationships of Fusarium species complex inferred from TRI101 gene and ISSR markers. C. C. AMARASINGHE AND W. G. D. FERNANDO. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada

The Fusarium graminearum species complex consists of phylogenetically distinct species that cannot be classified based on their morphological characteristics. Their geographic distributions and chemotypes are dramatically different, and they pose new challenges in fusarium head blight disease management, breeding and quarantine strategies. Therefore the objective of this study is to determine the molecular genetic diversity among the F. graminearum isolates from Canada, USA, UK, Germany, Switzerland, France, Netherlands and China based on TRI101 gene sequences and ISSR markers. A collection of 25 ISSR markers were tested to detect the genetic diversity among 312 Fusarium isolates. Among these markers only ISSR1 and ISSR4 showed genetic differences between the isolates collected from different countries. The strains were analyzed with multiplex PCR in the TRI12 sequence of the trichothecene gene cluster and a higher percentage of 3-ADON and NIV isolates were found from the countries representing the European continent. The 15-ADON chemotypes were more prominent in North America. The SCAR grouping of the isolates showed that 81% of the isolates belong to SCAR group I (lineage 7) and 19% belong to SCAR group V (lineage 6). To further this study, the genetic diversity among the isolates is being analysed based on the sequence differences in TRI101 gene.

Field research permits - the first step in the regulatory process. K. ANDERSON. Development and Licensing, Bayer CropScience Inc., 295 Henderson Drive, Regina, SK S4N 6C2, Canada

Biological control agents are regulated in Canada under the Pest Control Products Act (PCPA) and are subject to all PCPA regulatory processes. Potential biological control candidates are often initially identified by researchers with little experience in the regulatory system. These individuals may be unaware that the regulatory process begins prior to the initiation of field trials with the application for field research permits. This presentation provided insight to the regulations, processes and timelines that affect field based research of biological control agents.

How temperature profiles may alter the efficacy of the bioherbicide Phoma macrostoma. K. L. BAILEY AND J. DERBY. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Phoma macrostoma Montag. was registered as a microbial bioherbicide for broadleaved weed control in turfgrass in Canada in 2011. A granular formulation is used to broadcast this fungus to soil whereby it grows towards the plants, infecting the roots and releasing phytotoxic macrocidins which are lethal to several broadleaved weed species. It was hypothesized that temporal soil temperature could affect growth of P. macrostoma, which in turn would alter the efficacy of weed control. Phoma macrostoma was grown on PDA plates at a range of temperature regimes between 5/5 and 38/38 °C (day/night) to measure the radial growth for 14 days. These plates were then used in an inoculum-mat bioassay to determine efficacy (% chlorosis and % weed reduction) against dandelion seedlings. The experiment examined the effects on four fungal strains at 11 temperature combinations with four replicates, and was conducted twice. Radial growth, chlorosis and weed reduction was much lower at temperature extremes (5/5, 15/5, 15/10 and 30/30, 38/38 °C) than at moderate temperatures (15/15, 25/5, 25/10, 30/5, 30/15. 25/25). The data suggest that the temperature optima to maximize the efficacy of P. macrostoma are in the moderate, temperate range. Applications outside of this range may reduce the level of weed control.

Initial molecular investigation of key stages in the establishment of Pythium aphanidermatum infection in divergent crop hosts. K. BALA, C. A. LEVESQUE AND B. J. SAVILLE. Forensic Science Program and Environmental and Life Sciences Graduate Program, Trent University, Peterborough, ON K9J 7B8, Canada; and (C.A.L.) Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada

Pythium aphanidermatum (Edson) Fitzp. causes root rot disease, a major threat to greenhouse vegetables and cereal crop production worldwide. Unlike many plant pathogens, P. aphanidermatum is capable of infecting divergent host species. To determine whether there is a host influence on gene expression and pathogenic development by P. aphanidermatum, we selected three hosts that vary in susceptibility to infection. Using specific protocols that we developed, P. aphanidermatum zoospores were applied to the roots of cucumber, wheat and corn under conditions highly conducive to infection. RNA was isolated from infected host tissue at time points ranging from 0 - 72 h. Transcript levels of pathogenicity, housekeeping and effector-like genes, identified through bioinformatics analysis, were determined by RT-qPCR. Housekeeping gene transcript levels were used as a measure of relative host and pathogen representation in the total RNA. The expression data analyses showed that the key transitions in gene expression occurred between 24 and 36 h in all infections, however, the relative amount of pathogen to host RNA increased only in the most susceptible cucumber host. These analyses also indicated that P. aphanidermatum effector genes transcript levels were higher at early time points in all infections, suggesting their possible roles in the initiation of infection. This preliminary study sets the stage for full transcriptome analyses of the infection process.

Fungicide effects on field pea foliar and root diseases and yield in Saskatchewan. S. BANNIZA, H. R. KUTCHER AND T. WARKENTIN. Crop Development Centre, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada

Diseases of field pea were identified as impediments to sustainable production in the Saskatchewan Parkland. This project evaluated seed- and foliar-applied fungicides from 2009 to 2011 at three locations each year. Two field experiments were conducted, one to assess the efficacy of foliar fungicides against ascochyta blight [Mycosphaerella pinodes (Berk. & Blox.) Vestergr.; Phoma medicaginis Malbr. & Roun. in Roun. var. pinodella (L.K. Jones) Boerema], and the other to assess the efficacy of seed treatments for control of root rot. Foliar application of Bacillus subtilis (Serenade ASO) did not reduce ascochyta blight severity or increase seed yield in any site-year; pyraclostrobin (HEADLINE) significantly reduced disease severity in four of eight site-years. Yield was increased by pyraclostrobin in three site-years, but in only one of these site-years was disease severity reduced. This indicates that pyraclostrobin has activity other than disease reduction. Seed treatment had an effect on seedling emergence in only one of nine site-years and yield was not improved in any site-year. We conclude that root rot pathogens were not a critical constraint to field pea production in our study and that foliar fungicides had inconsistent yield benefit not always associated with disease control.

The National Fungal Identification Service at ECORC - challenges and opportunities. T. BARASUBIYE, K. A. SEIFERT AND C. A. LÉVESQUE. Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada

The National Fungal Identification Service at ECORC-Ottawa provides accurate identifications for mycology and bacteriology supported by reliably identified reference material from the National Mycological Herbarium and Canadian Collection of Fungus Cultures (CCFC). The staff includes a manager and associated research scientists with taxonomic expertise. Clients submit the samples directly to the manager and must provide data on substrates, geographical origins, and dates of collection or isolation for entry into our in-house databases. All material submitted for identification is sequenced if possible. In-house databases for specific genera, including Cladosporium spp., Fusarium spp., Penicillium spp., Phytophthora spp., Pythium spp., and Trichoderma spp., including unpublished data, supplement GenBank BLAST searches allowing for more reliable identifications. Identifications are confirmed by micro-morphological examination if possible. Interesting cultures are deposited in CCFC, a world-class repository for vouchers used to validate research and a source of cultures for research. As the collections grow and better represent the Canadian microbiota, the associated DNA sequence databases also grow. Increased regulations for import permits and the release of microbes into the environment emphasize the importance of maintaining and developing a national collection with a broad representation of plant pathogens isolated in Canada.

The influence of sclerotial origin and three biological control agents on the survival of sclerotia of Botrytis squamosa , Sclerotinia sclerotiorum , and Sclerotium cepivorum . L. C. BARBISON, M. R. MCDONALD AND G. J. BOLAND. Department of Environmental Biology University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (M.R.M.) Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Sclerotia of Botrytis squamosa Walker, Sclerotinia sclerotiorum (Lib.) de Bary, and Sclerotium cepivorum Berk. were tested for natural long term survival and the efficacy of three biological control agents (BCAs): Coniothyrium minitans Campbell, Microsphaeropsis ochracea (Carisse & Bernier), and Trichoderma atroviride (Karsten). Laboratory- and field-produced sclerotia of S. sclerotiorum and S. cepivorum were untreated and used to evaluate long term survival. Laboratory-produced sclerotia of B. squamosa, S. sclerotiorum, and S. cepivorum were treated with spore suspensions of the individual BCAs. All sclerotia were buried 5-10 cm deep in pots containing muck soil and placed under field conditions on December 12, 2011. Percent survival was assessed at one and three months post burial. Once recovered, sclerotia were plated onto agar and observed for germination and formation of daughter sclerotia. After 3 months, laboratory-produced sclerotia of S. sclerotiorum had greater survival than field-produced sclerotia, which were heavily contaminated with Fusarium spp. In contrast, field-produced sclerotia of S. cepivorum had greater survival than laboratory-produced sclerotia. Activity of BCAs varied among pathogens. Trichoderma atroviride demonstrated the greatest and most consistent reduction in percent survival of sclerotia, with reductions of 7, 8, and 64% for B. squamosa, S. cepivorum and S. sclerotiorum, respectively, after 3 months. Results representing 5 months post burial are currently being analysed.

rpo B and rpo D are reliable genetic markers for phylogenetic differentiation of Pseudomonas syringae pv. syringae from Pseudomonas syringae pv. tomato . M.-A. BLAIS, R. XU AND J. T. TAMBONG. La Cité Collègiale, 801 Aviation Parkway, Ottawa, ON K1K 4R3, Canada; and (R.X., J.T.T.) Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada

The gram-negative bacterium Pseudomonas syringae van Hall is pathogenic to more than 50 different economically important crops and trees. The species is divided into pathovars primarily on the basis of host specificity. Host specificity assays for routine differentiation of P. syringae pathovars require growing and infecting different plants. This method requires several weeks and can be unreliable, leading to inaccurate taxonomic characterisation. As a pilot study, partial 16S rRNA, rpoB and rpoD genes of 20 strains of pv. syringae and pv. tomato were sequenced and analysed to determine their suitability as taxonomic markers. Alignments of rpoB and rpoD sequences showed significant variations that could be exploited for accurate and reliable differentiation of pv. syringae from pv. tomato. Phylogenetic analyses of rpoB and rpoD clustered the pathovars in two distinct clusters, corroborating the alignment differences, except for strain PSS 91-1A, which formed a separate cluster. This supports our previous report, based on cyoA and gyrase B analyses, that strain PSS 91-1A is not pv. syringae. The 16S rRNA gene provided some degree of differentiation, but could not differentiate strain PSS 91-1A from pv. tomato. All known pathovars are now being sequenced to determine whether rpoB and rpoD could become universal genetic markers for rapid differentiation of pathovars of P. syringae.

Characterizing Fusarium isolates from asparagus fields and managing fusarium crown and root rot with soil organic amendments. A. BORREGO-BENJUMEA, J. M. MELERO-VARA, M. J. BASALLOTE-UREBA AND P. A. ABBASI. (A.B., P.A.A.) Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada; (A.B., J.M.M.) Department of Crop Protection, Institute of Sustainable Agriculture, Spanish National Research Council (CSIC), P.O. Box 4084, 14080 Córdoba, Spain; and (M.J.B.) Andalusian Institute of Agricultural Research and Training, Fisheries, Food and Organic Production (IFAPA) Las Torres-Tomejil, Apdo. Oficial, 41200 Alcalá del Río, Sevilla, Spain

Fusarium crown and root rot (FCRR) of asparagus has a complex etiology with several soilborne Fusarium spp. as causal agents. Twenty three Fusarium isolates, obtained from plant and soil samples collected from Ontario commercial asparagus fields with a history of FCRR, were identified as F. oxysporum Schlecht. emend Snyder & Hans. and F. solani (Mart.) Sacc. based on morphological or cultural characteristics and PCR analysis with species-specific primers. The isolates were further characterized by inter-simple-sequence repeat (ISSR) PCR analysis. In the in vitro pathogenicity screening tests, three isolates of F. oxysporum and one isolate of F. solani caused severe symptoms on asparagus. The management of FCRR with soil organic amendments [pelleted poultry manure (PPM), olive residue compost (ORC), and fish emulsion (FE)] was evaluated in the greenhouse trials using a susceptible cultivar of asparagus in soils infested with one of the three pathogenic isolates of F. oxysporum (F-5). Lower root rot severity and higher plant weights were observed for all treatments (28, 35, and 37% root rot severity; and 2.6, 2.1, and 1.4 g plant weights with the treatments 1% PPM, 6% ORC, and 0.5% FE, respectively) compared to the Fusarium control treatment (66% root rot severity and 0.8 g plant weight). Populations of Fusarium and total bacteria were enumerated after 1, 3, 7 and 14 days of soil amendment. In amended soils, the population of Fusarium gradually decreased while the population of total culturable bacteria increased, especially in soils amended with 1% PPM. These results indicate that soil organic amendments, especially 1% PPM, can increase disease suppressiveness and promote plant growth possibly by decreasing pathogen population and enhancing bacterial activity in the soil.

Assessment of bacterial strains for biological control of pathogens causing post-harvest and storage decay of fruits and vegetables. S. M. BOYETCHKO, T. ZHOU, P. AUDY AND A. M. SVIRCEV. Saskatoon Research Centre, Agriculture and Agri-Food Canada (AAFC), 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (T.Z.) Guelph Food Research Centre, AAFC, 93 Stone Road West, Guelph, ON N1G 5C9, Canada; (P.A.) Soils and Crops Research and Development Centre, AAFC, 2560, boul. Hochelaga, Québec, QC G1V 2J3, Canada; and (A.M.S.) Southern Crop Protection and Food Research Centre, AAFC, Vineland Research Farm, 4902 Victoria Avenue North, P.O. Box 6000, Vineland, ON L0R 2E0, Canada

The value of post-harvest losses caused by diseases and pests in fruits and vegetables is over $300 billion each year worldwide. Important storage pathogens include Monilinia fructicola, (Winter) Honey, Botrytis cinerea Pers.:Fr., Sclerotinia sclerotiorum (Lib.) de Bary and Phytophthora infestans (Mont.) de Bary. The principal management strategy for these diseases is application of chemical fungicides, but issues related to health and safety, pesticide residues in food and fungicide resistance are a major concern. Several bacterial isolates belonging to Bacillus spp., Pseudomonas spp., and Pantoea spp. were selected for evaluation as possible biological control candidates against these pathogens. These bacteria were tested for their ability to inhibit spore germination and mycelial growth, and to reduce infection on fruits and tubers of peach, tomato, and potato. Nine of the bacterial isolates inhibited conidial germination of M. fructicola and 10 of the isolates inhibited mycelial growth. Pre- and post-harvest application of Bacillus amyloliquefaciens BM01 reduced the incidence of brown rot by over 80%. More than 50 bacterial isolates belonging to Bacillus and Pseudomonas significantly reduced mycelial growth of P. infestans, while 8 to 10 bacterial isolates reduced spore germination, mycelial growth, and sclerotial formation of S. sclerotiorum and B. cinerea. In some cases, inhibition of fungal growth was 100%. The mode of action for the biocontrol activity by the bacteria, including the role of extracellular antimicrobial substance(s) and induced resistance is being explored.

Survey of viruses and Clavibacter michiganense subsp. tessellarius on winter wheat in Illinois. C. A. BRADLEY AND V. CHAPARA. Department of Crop Sciences, University of Illinois, 1102 South Goodwin Avenue, Urbana, IL 61801, USA

Winter wheat is an important crop grown in Illinois, with an average of 320,000 ha being harvested annually in 2007-2011. Symptoms of virus infection generally are observed every year in Illinois wheat fields, and include discolored leaves, mosaic patterns, and stunting. To better understand which viruses are most prevalent in Illinois, wheat leaves were collected from fields in 2009-2011. In total, leaves from 241 fields were collected. Leaves were sent to Agdia Inc. (Elkhart, IN), where they were tested with ELISA assays that screened for several viruses that included different strains of Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV), High plains virus (HPV), Wheat spindle streak mosaic virus (WSSMV), and Wheat streak mosaic virus (WSMV). In addition, an ELISA assay tested for the presence of Clavibacter michiganense subsp. tessellarius (Carlson & Vidaver) Davis et al. (Cmt), the causal agent of bacterial mosaic. Only BYDV-PAV, CYDV-RPV, HPV, WSSMV, WSMV, and Cmt were detected. Of these, Cmt was detected most often (79.3% of leaf samples), followed by BYDV-PAV (22%), WSSMV (19.1%), CYDV-RPV (6.6%), WSMV (2.9%), and HPV (0.8%). The high level of Cmt detection was somewhat surprising, and more research is needed to better understand the impact of Cmt on wheat in Illinois.

Extreme resistance to Cowpea mosaic virus - virus-encoded elicitor and cognate cowpea resistance gene. G. BRUENING, R. V. PENMETSA, J. GAO, N. CARRASQUILLA-GARCIA, P. A. FELDSTEIN AND D. R. COOK. Department of Plant Pathology, University of California, One Shields Avenue, Davis, CA 95616-8751, USA

Cowpea (Vigna unguiculata) is an important crop in West Africa, valued for its drought tolerance and quality protein. One μg/mL Cowpea mosaic virus (CPMV) uniformly infects cowpea seedlings of susceptible lines, but introgressing the dominant Cpa locus gave lines producing neither symptoms nor virions at 10,000 μg/mL CPMV inoculum. Fan et al. (Virology 417, 71-78) transiently expressed segments of CPMV genomic RNA1 in Cpa cowpea leaves. Only segments encoding active CPMV protease (24KPro) induced necrosis. Conserved orthologous set (COS) markers, based on priming sites that flank introns, were developed from expressed sequence tags of cowpea that matched both Medicago truncatula and soybean or lotus. Synteny-aided fine mapping (average marker separation 0.65 cM), based on inheritance of CPMV resistance and COS markers, placed Cpa near one end of linkage group 3. Corresponding 70 kbp regions of Cpa-introgressed and susceptible cowpea lines were sequenced, revealing one incomplete and two complete resistance gene homologs (RGHs 0, 1 and 2, respectively) for Cpa and four for the susceptible line. Opposite leaves of Cpa seedlings were infiltrated with Agrobacterium tumefaciens bearing plasmids designed to direct double-stranded RNA production for sequences unique to Cpa RGH1 or RGH2. Subsequent agroinfiltration introduced 24KPro. RGH1, but not RGH2, sequences interfered with 24KPro-induced necrosis, implicating RGH1 as the Cpa gene.

Analysis of incidence – severity relationships for strawberry powdery mildew caused by Podosphaerea aphanis as influenced by cultivar, cultivar type and production systems. O. CARISSE, A. LEFEBVRE, H. VAN DER HEYDEN, L. ROBERGE AND L. BRODEUR. Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, St-Jean-sur-Richelieu, QC J3B 3E6, Canada; and (H.V.D.H., L.R., L.B.) Compagnie de recherche Phytodata Inc., 111 Rg. Saint-Patrice, Sherrington, QC J0L 2N0, Canada

The relationship between strawberry powdery mildew incidence (I) and severity (S) were investigated for various cultivars, cultivar types and production systems. Data were collected from 2006 to 2011 at 11 sites (2326 observations). For the cultivars grown in open fields, higher severity was observed for ‘Seascape’, followed by ‘Chambly’, ‘Cavendish’, ‘Darselect’ and ‘Jewel’ with 29.9, 13.3, 11.5, 11.1, and 10.5% leaf area diseased, respectively. Linear complementary log-log models provided a good fit of the data (R² _adj from 0.82 to 0.96). A covariance analysis indicated that the sampling year, site and cultivar within the June-bearing ones did not significantly influence the estimated slope of the I-S relationship. However, the production system and the cultivar type significantly influenced the estimated slope. From these analyses, it was possible to develop three specific models; one for open field grown June-bearing cultivars, one for the open field grown day-neutral cultivars and one for June-bearing cultivars grown in plastic tunnel. It was concluded that strawberry powdery mildew leaf severity can be accurately estimated from incidence of diseased leaves. These models may be used in making practical management decisions, especially for management programs that include information on the level of disease in the field to initiate fungicide spray programs or time interval between sprays.

Analysis of the relationships between weather, airborne inoculum of Podosphaera aphanis , powdery mildew foliar severity and strawberry yield losses. O. CARISSE, V. MORISSETTE-THOMAS, H. VAN DER HEYDEN, L. ROBERGE AND L. BRODEUR. Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, St-Jean-sur-Richelieu, QC J3B 3E6, Canada; (V.M.T.) Department of Mathematics, Sherbrooke University, Sherbrooke, QC J1K 2R1, Canada; and (H.V.D.H, L.R., L.B.) Compagnie de recherche Phytodata Inc., 111 Rg. Saint-Patrice, Sherrington, QC J0L 2N0, Canada

Several fruit diseases, including strawberry powdery mildew (SPM), develop as two concomitant lagged and/or concurrent epidemics on foliage and on fruits. However, for most of these diseases, epidemiological information is mostly derived from studies on disease development on foliage. The purpose of this study was to investigate the relationship between weather, airborne inoculum concentration, foliar disease severity and subsequent yield losses for day-neutral strawberries. The experiment was conducted at two, five, and four sites in 2006, 2007, and 2008, respectively, for a total of 12 epidemics. At each site, data were collected on 25 plants at 2-day intervals from the end of May to early October for a total of 60-62 samplings annually. First, the relationships between weather, disease and yield losses were statistically described and secondly a lagged regression model to predict yield losses from the parameters that significantly influenced losses was developed. The variables that most influenced crop losses were, the concentration of airborne inoculums (> 50 conidia/m³), alternation of cool and humid nights (10-15 °C and RH >90%) with hot and dry days (20-28 °C RH <70%), and foliar severity of powdery mildew (>10% leaf area diseased). These results suggest that information on foliar powdery mildew must be considered while making disease management decisions.

Transgenic over-expression of the verticillium wilt resistance gene Ve1 confers partial resistance to susceptible tomato plants. C. D. M. CASTROVERDE, R. N. NAZAR AND E. J. ROBB. Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON N1G 2W1, Canada

The Ve resistance locus from tomato (Solanum lycopersicum) confers resistance against virulent strains of Verticillium dahliae Kleb. and V. albo-atrum Reinke & Berthold, the causative agents of verticillium wilt. This locus consists of two genes, Ve1 and Ve2, encoding putative receptor-like proteins. Previous studies in the laboratory show that endogenous Ve1 expression is dramatically induced in infected ‘Craigella’ resistant (CR) tomato plants compared to that in ‘Craigella’ susceptible (CS). In order to investigate the functional significance of this Ve1 induction, the Ve1 CR allele was over-expressed in CS plants. The generated 35S:Ve1 transgenic plants were evaluated in terms of plant architecture, Ve1 expression levels, transgene copy number and resistance to V. dahliae race 1 (Vd1). We found that 35S:Ve1 plants had comparable root and shoot sizes to non-transformed plants. The Ve1 transcript levels were six to twelve times higher than in non-transformed plants. The transgene copy number ranged from two to five, consistent with the low copy numbers associated with Agrobacterium-mediated transformation. However, Ve1 over-expressing tomatoes only possessed partial Verticillium resistance. Fungal levels and symptom expression were lower compared to non-transformed CS plants, but were slightly higher compared to non-transformed CR plants. These preliminary results suggest that Ve1 over-expression alone is not enough to bring about complete resistance in the plant.

Effect of soaking stem and bulb nematode ( Ditylenchus dipsaci ) - infested garlic seed in abamectin on bulb damage, yield and nematode population at harvest. M. J. CELETTI AND M. PAIBOMESAI. Ontario Ministry of Agriculture, Food and Rural Affairs, Room 3110, Edmund Bovey Building, University of Guelph, Guelph, ON N1G 2W1, Canada

Abamectin applied to stem and bulb nematode (Ditylenchus dipsaci (Kühn) Filipjev)-infested garlic cloves prior to planting was evaluated for reduction of bulb damage, yield losses and nematode populations in garlic bulbs at harvest. Two lots of garlic cloves cv. ‘Music’ infested with stem and bulb nematodes were used in the trial. One lot had 617 nematodes/g dry cloves (high infestation level) and the other lot had 17 nematodes/g dry cloves (low infestation level). Cloves were soaked in 1) a solution containing 0.072 g abamectin/L water, 2) water alone for 4 h just prior to planting, or 3) left untreated. Cloves with high nematode infestation that were soaked in the abamectin solution produced bulbs with significantly lower bulb damage and nematode populations at harvest than bulbs produced from cloves that were untreated or soaked in water prior to planting. Garlic yields from plots planted with highly infested cloves soaked in an abamectin solution prior to planting were higher than the yields from plots planted with cloves soaked in either water or left untreated. No differences in bulb damage, yield, or nematode populations were detected at harvest among treatments applied to cloves with a low level of nematodes prior to planting.

Using benefit information and use history to address value data requirements - implications for biopesticide registration submissions. L. DE LUNA. Pest Management Regulatory Agency, Health Canada, 2720 Riverside Drive, Ottawa, ON K1A 0K9, Canada

In late 2010, the Pest Management Regulatory Agency (PMRA) of Health Canada issued a regulatory proposal with the objective to reduce the regulatory burden on stakeholders by providing a more flexible approach by which applicants could meet the value requirements for registration of pest control products. While PMRA assessments must take into account the value of a product (the Pest Control Product Act states that only pest control products which are determined to be of acceptable value can be approved for use in Canada), the new proposal recognizes that there is more than one way to address this requirement. In particular, the new proposal provides guidance on how use history and benefit information for a product can be used to address regulatory data requirements. The application of this approach in the context of biopesticide submissions was discussed.

Expression of resistance to Plasmodiophora brassicae in four resistant canola cultivars. A. DEORA, B. D. GOSSEN AND M. R. MCDONALD. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; and (M.R.M.) Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

The timing and expression of resistance in four canola (Brassica napus L.) cultivars with resistance to Plasmodiophora brassicae Woronin, the causal agent of clubroot, was assessed against four pathotypes. Each of the resistant cultivars was highly resistant to all of the pathotypes, and their response to infection was the same. Root hair infection was high, but pathogen development was slower compared to the susceptible cultivar (control). Secondary infection and development in cortical cells was severely inhibited in each of the resistant cultivars; only a few bi-nucleated plasmodia were observed at 12 days after inoculation (DAI), but were rarely observed at 18 and 24 DAI. In contrast, pathogen development in the susceptible cultivar had reached the resting spores stage at 24 DAI. A dense ring of reactive oxygen species (ROS) accumulation was observed in the endodermis, pericycle and vascular cambium of non-inoculated controls and inoculated plants of the resistant cultivars, but rapidly broke down in infected plants of the susceptible cultivar. The absence of any specific points for ROS accumulation or lignification in response to resistance indicates that a hypersensitive response is not the main mechanism driving clubroot resistance in these cultivars. The uniform response of these four new clubroot-resistant cultivars to all four P. brassicae pathotypes provides an indication that the resistance in these cultivars is conditioned by a gene that confers broad resistance from a single source, because most sources of resistance to P. brassicae are pathotype specific.

Changes in canola root anatomy following infection by Plasmodiophora brassicae . A. DEORA, B. D. GOSSEN AND M. R. MCDONALD. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; and (M.R.M.) Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Infection by Plasmodiophora brassicae Woronin stimulates abnormal cell division and enlargement in the cortex and stele region of roots, which results in the development of a characteristic root symptom (clubbing) in susceptible host species. A study was conducted to track the progression of P. brassicae over time in roots of the susceptible canola (Brassica napus L.) cultivar ‘46A76’. Root hair infection occurred rapidly after seedling inoculation and the primary (root hair) phase of pathogen development was essentially completed by 12 days after inoculation (DAI). By 18 DAI, secondary plasmodia had extensively colonized the root cortex. Cells in and around the infested areas were enlarged and undifferentiated. A pre-existing reactive oxygen species barrier in the endodermis, pericycle and vascular cambium of healthy plants disappeared, likely as a result of detoxification by the pathogen. Plasmodia penetrated into the stele 28 DAI. Within the stele, the plasmodia preferentially colonized xylem parenchyma cells, stimulating cell division and enlargement, and synthesis of vascular strands. Clusters of rapidly dividing xylem parenchyma cells pushed into and between areas of the xylem cells with lignified secondary walls, leaving the lignified cells as isolated islands within a mass of infected cells at 35 DAI. At an advanced stage, cell wall expansion occurred in these lignified cells and they become hypertrophied and infected.

Characterization of Phytophthora infestans from tomato in southwestern Ontario. K. F. DOBINSON, R. D. PETERS, K. L. CONN, K. I. AL-MUGHRABI, F. DAAYF AND L. M. KAWCHUK. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada (AAFC), 1391 Sandford Street, London, ON N5V 4T3, Canada; (R.P.) Crops and Livestock Research Centre, AAFC, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; (K.I.A.-M.) Potato Development Centre, New Brunswick Department of Agriculture, Aquaculture and Fisheries, 39 Barker Lane, Wicklow, NB E7L 3S4, Canada; (F.D.) Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; and (L.M.K.) Lethbridge Research Centre, AAFC, 5403 1^st Avenue South, Lethbridge, AB T1J 4B1, Canada

Late blight, caused by the oomycete Phytophthora infestans (Mont.) de Bary, is infamous as the cause of the Irish potato famine in the mid-1800s. In recent years the disease has re-emerged in various parts of the world including North America, and in 2009/2010 late blight caused substantial crop loss across Canada for both potato and tomato industries. In the course of a national survey of the emerging late blight population in Canada, several P. infestans isolates were collected in 2010 from plants in Ontario tomato fields, and two genotypes were identified, US-22, and a potentially new US-22a, possibly derived from US-22 by sexual reproduction. Both genotypes were metalaxyl sensitive and of the A2 mating type. The presence of both potato and tomato hosts in the same growing region raises questions about the introduction and evolution of P. infestans strains on each crop, host specificity/preference of different genotypes, and potential movement between the two crops. In 2011 the annual survey was therefore expanded to include the broader collection and characterization of isolates from tomato. Isolates collected that year from southwestern Ontario, a major tomato-growing region, indicated the presence of US-11 A1 (A1 mating type), and prevalence of US-22 (A2 mating type) and a new US-25 (A1) genotype. The occurrence of both mating types in the same region is of concern as it provides opportunities for sexual recombination and development of new strains of the pathogen that may complicate disease prevention.

Genetic description of the haloparasitic plant; Cistanche phelypaea . H. E. ELWAKIL, N. R. ABDELSALAM, R. M. ABD EL-AZEEM, A. A. HEMADA, N. Y. ABBAS AND A. M. K. NASSAR. Department of Agricultural Botany, Faculty of Agriculture-Saba Basha, Alexandria University, Egypt; (R.M.A.) Department of Molecular Biology, Institute of Genetic Engineering and Biotechnology, University of Minufiya; (A.A.H.) Department of Bioinformatics, Institute of Genetic Engineering and Biotechnology, University of Minufiya; and (A.M.K.N.) Department of Plant Protection, Faculty of Agriculture, Damanhour University, Egypt

Broomrape; Cistanche phelypaea (L.) Coutinho (Family: Orbanchaceae) is an obligate haloparasitic plant attaching itself to the roots of its host. It depends on its host for water, minerals and organic nutrients. The main natural hosts are the perennial dicots woody species for example Arthrocnemum macrostachyum and Atriplex leucoclada (Family: Chenopodiaceae) and Zygophyllum album (Family: Zygophyllacea). Cistanche phelypaea L. grows in Egypt in low density and scattered on a wide land area. It was identified in the Nile region (Faiyum), the Oases of the Western desert including Siwa, the Red Sea coastal strip, and the entire Sinai Peninsula including the Mediterranean coast. It grows in sandy and alluvial soil on the edges of cultivated fields as a parasite on plant roots. In this study the Cistanche species were surveyed and photographed at Siwa Oasis and different other obliterated Oases around Siwa Depression. Morphological studies and molecular and biochemical genetic analyses were carried out in order to identify the genetic and morphological description of the C. phelypaea and to study the genetic variation among studied population.

Phenotyping and genotyping of fungicide resistant Botrytis cinerea from table grapevine berries. D. ERRAMPALLI AND T. BARASUBIYE. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada (AAFC), Vineland Research Farm, 4902 Victoria Avenue North, P.O. Box 6000, ON L0R 2E0, Canada; and (T.B.) Eastern Cereal and Oilseed Research Centre, AAFC, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada

Botrytis cinerea Pers.:Fr. the causal agent of gray mold, is an economically important pathogen. Twenty two single-spore isolates from grapevine (Vitis vinifera L.) berries collected from Ontario and two reference isolates of B. cinerea B05.10 and BC-34R were identified based on the sequences from benomyl-A (Ben-A) β-tubulin gene. The isolates were also tested for sensitivity to the benzimidazole thiabendazole, the anilinopyrmidine cyprodinil and penbotec, the phenylpyrrole fludioxonil and the dicarboximide iprodione, on PDA media amended with a discriminatory concentration of 5 ug/mL of each of the fungicides. Sensitivity determinations to the fungicides revealed a total of five phenotypes: each of the isolate was resistant to at least one fungicide; all were resistant to anilinopyramidines; and three isolates were resistant to all four chemical groups. As expected, cross resistance was observed between two of the anilinopyramidine fungicides, cyprodinil and penbotec. All isolates were pathogenic on grapevine berries. The genotyping of the isolates with BenA gene and BcOS1 gene showed that all benzimidazole resistant isolates carried the 198 Glu to Ala (E198A) mutation in BenA gene in β -tubulin and all dicarboximide resistant isolates carried 365 Ile to Ser (I365S) mutation in BcOS1 gene that encodes histidine kinase. We report multiple fungicide resistance in B. cinerea collected from grapevine berries.

Effect of methyl jasmonate on PAL defense gene expression in Botrytis cinerea -infected grapevine berries. D. ERRAMPALLI, P. H. GOODWIN AND A. SHARON. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada (AAFC), Vineland Research Farm, 4902 Victoria Avenue North, P.O. Box 6000, ON L0R 2E0, Canada; (P.H.G.) School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (A.S.) Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv 69978, Israel

Botrytis cinerea Pers.:Fr., a necrotrophic pathogen, causes gray mould on grapevine berries (Vitis vinifera L). Methyl jasmonate (MeJA) occurs naturally in host plant tissues and has a signalling role in eliciting induced systemic resistance (ISR) against disease. Here we report the effect of exogenous MeJA, on phenylalanine ammonia lyase (PAL) defense gene expression in B. cinerea-infected grapevine berries. The ‘Thompson Seedless’ grape berries were spray-treated with 5 mM of MeJA and 3 days after the MeJA treatment, the grapevine berries in the bunch was wounded and inoculated with 1 x 10⁴ spores of B. cinerea B05.10 and incubated in the dark at 20 ºC and 85% RH. Control treatment did not receive MeJA. The lesion diameter was recorded at 7 days after inoculation. The elicitor, MeJA induced defense response by significantly suppressing the Botrytis gray mould disease. Maximum levels of induction of PAL gene was observed at 48 h post inoculation. A significantly lower level of PAL defense gene was expressed in MeJA treated and B. cinerea-infected grapevine berries, as compared to B. cinerea only infected berries. Postharvest treatment with MeJA may be incorporated as a potential tool in the grape postharvest disease management strategies.

Spatial variation of airborne sporangia of Phytophthora infestans of potato production of New Brunswick, Prince Edward Island and Quebec - characterization and implications for monitoring networks implementation. M. L. FALL, H. VAN DER HEYDEN, L. BRODEUR, Y. LECLERC AND O. CARISSE. Department of Biology, Sherbrooke University, Sherbrooke, QC J1K 2R1, Canada; (H.V.D.H., L.B.) Compagnie de recherche Phytodata Inc., 111 Rg. Saint-Patrice, Sherrington, QC J0L 2N0, Canada; (Y.L.) McCain Foods Canada, 107 Main Street, Florenceville, NB E7L 1B2, Canada; and (M.L.F., O.C.) Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, St-Jean-sur-Richelieu, QC J3B 3E6, Canada

An effective control of potato late blight involves considering and understanding the spatial distribution of Phytophthora infestans (Mont.) de Bary airborne sporangia concentration (ASC). This study evaluated the ASC at the regional scale of potato production of New-Brunswick (NB), Prince Edward Island (PEI) and Quebec (QC) by using a regional spore-trap network. This experiment was undertaken in 2008, 2009, 2010 and 2011. ASC of P. infestans was measured with rotating-arms spore samplers placed at the canopy level (QC) and at 3 m height (NB and PEI) to measure the field-born and incoming ASC respectively. In all locations, the negative binomial distribution fit the data better than the Poisson distribution, indicating heterogeneity. Based on the Taylor's power law, the results suggest that heterogeneity was influenced by increasing ASC and according to analysis of covariance; the slopes of Taylor's regressions were not significantly different between years in NB (P = 0.1494, R² _adj = 0.92) while they were in QC (P< 0.0001, R² _adj = 0.98). Thus, it is important to orient the scouting activities and build an effective network of airborne spore trapping for detecting and monitoring P. infestans sporangia. Therefore, the next step of this study will be establishing the link between ASC and infection efficiency.

Analysis of expressed sequence tags derived from a compatible pea- Peronospora viciae interaction. J. FENG, K. F. CHANG, S. F. HWANG, S. E. STRELKOV, R. L. CONNER, B. D. GOSSEN AND D. L. MCLAREN. Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; (S.E.S.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (R.L.C.) Morden Research Station, Agriculture and Agri-Food Canada (AAFC), Unit 100-101, Route 100, Morden, MB R6M 1Y5, Canada; (B.D.G.) Saskatoon Research Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; and (D.L.M.) Brandon Research Centre, AAFC, 18^th Street North and Grand Valley Road, P.O. Box 1000A, R.R. #3, Brandon, MB R7A 5Y3, Canada

A cDNA library was constructed from field pea leaves infected by the downy mildew pathogen, Peronospora viciae f. sp. pisi Boerema & Verh., using a suppression subtractive hybridization approach. The library consists of 399 expressed sequence tags, from which 207 unisequences were obtained after sequence assembly. Of the unisequences, six were shown to be of P. viciae f. sp. pisi origin . The remaining unisequences were subjected to gene ontology analysis and their functions were predicted in silico. Eleven of these unisequences (representing 24 clones) shared significant sequence similarities with Arabidopsis genes known to be involved in downy mildew resistance, including the well characterized genes RPP5, RPP6 and RPP27, and therefore appear to be of host origin.

Investigation of the Plasmodiophora brassicae infection process on canola. J. FENG, S. F. HWANG AND S. E. STRELKOV. Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; and (S.E.S.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada

The early stages of infection of canola roots by the clubroot pathogen Plasmodiophora brassicae Woronin were investigated. Primary infections began to be observed 12 h after inoculation (hai) with a suspension of 1 × 10⁵ resting spores/mL. Secondary infection began to be observed at 72 hai. Inoculation with secondary zoospores produced primary infections similar to those produced by inoculation with resting spores, both with respect to morphology and stage development. Secondary zoospores caused secondary infections earlier than resting spores, but concurrent with their own primary infection. When the plants were inoculated with 1 × 10⁷ resting spores/mL 2 days after being challenged with 1 × 10⁴ or 1 × 10⁵ resting spores/mL, secondary infections were observed on the very next day, which was earlier than the secondary infections observed after inoculation with the 1 × 10⁷ resting spores/mL suspension alone, and later than secondary infections produced by the 1 × 10⁴ or 1 × 10⁵ resting spores/mL suspensions alone. Compared with the 1 × 10⁷ resting spores/mL alone and the 1×10⁴ or 1 × 10⁵ resting spores /mL alone, secondary infection on plants that had received both inoculations remained at higher levels throughout a 7-day time course. These data indicate that primary zoospores can cause secondary infection directly when the host is under primary infection. Information from this study will facilitate our understanding of the relationship and the relative importance of the two infection stages of P. brassicae, which will provide new clues for resistance breeding and fungicide development.

Inheritance of resistance to Ug99 stem rust in ‘Triumph 64’ winter wheat. T. FETCH JR., C. HIEBERT AND T. ZEGEYE. Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada

Stem rust of wheat (Triticum aestivum), caused by Puccinia graminis Pers. f. sp. tritici Eriks. & Henn., has inflicted significant crop loss worldwide. Recently, strain Ug99 (race TTKSK) arose in east Africa with virulence to most Sr genes, and is spreading. However, the differential line ‘Triumph 64’ has been resistant to all variants of Ug99 in numerous virulence tests and was postulated to be due to gene SrTmp. Thus, a study was conducted to determine the gene(s) in ‘Triumph 64’ that condition resistance to stem rust. A doubled haploid (DH) population was developed from F₁ plants of the cross LMPG-6S x ‘Triumph 64’. Since ‘Triumph 64’ is a winter wheat, DH progeny with a winter habit were discarded and those with a spring-type growth habit were retained. Progeny were inoculated with races RKQS and TTKSK in separate experiments. The DH population fitted a 1:1 (resistant:susceptible) ratio to race RKQS (χ² = 0.78, p = 0.78, n = 196) and to race TTKSK (χ² = 1.00, p = 0.32, n = 144), indicating single gene inheritance. Seedling reaction to RKQS and TTKSK co-segregated for most DH progeny, with nine exceptions which were attributed to segregating lines that were a result of out-crossing. Thus, it is likely that the same gene conferred resistance to both races. SrTmp has been reported to be located on chromosome 4B, but molecular marker analysis of our DH population found no linkage between seedling resistance and DNA markers previously mapped to 4B. The DH population will be tested with additional P. graminis f. sp. tritici races for further genetic analysis and DNA markers will be used to identify the chromosomal locations of these gene(s). Since few effective stem rust genes to Ug99 are derived from common wheat, resistance from ‘Triumph 64’ will be very useful to wheat breeders worldwide.

Ring nematodes ( Mesocriconema xenoplax ) affect early establishment of self-rooted ‘Merlot’ grapevines under Okanagan Valley growing conditions. T. A. FORGE, R. SMIT, C. KOCH, G. H. NEILSEN, D. NEILSEN AND K. HANNAM. Pacific Agri-Food Research Centre (PARC), Agriculture and Agri-Food Canada (AAFC), 6947 Highway 7, Agassiz, BC V0M 1AO, Canada; and (G.N., D.N., K.H.) PARC, AAFC, 4200 Highway 97, Summerland, BC VOH IZO, Canada

Mesocriconema xenoplax (Raski) Loof & De Grisse is an ectoparasitic nematode that has been implicated in reduced grapevine vigour in coastal Oregon and California. The species has recently been found to be relatively widespread in Okanagan Valley, British Columbia vineyards that appear to be declining in productivity. Our research objective was to experimentally assess the impacts of a BC population of M. xenoplax on growth of self-rooted ‘Merlot’ grapevines and three rootstocks under field conditions. In spring of 2007, 100 L field microplots were fumigated and then inoculated with the nematode or not (controls), and planted with self-rooted ‘Merlot'vines or ‘Merlot’ vines grafted onto rootstocks of Riparia Gloire, 44-53M or 3309C. Nematode populations increased on all genotypes. In fall of 2009 and 2010, trunk diameters and pruning weights of self-rooted vines were smaller in M. xenoplax-inoculated microplots than in non-inoculated microplots, but the nematode did not appear to affect any of the rootstocks. In 2011, trunk diameters of self-rooted and 3309C vines were smaller in inoculated microplots than in non-inoculated microplots. Root biomass, which was assessed in 2011, was reduced by the nematode on self-rooted vines but not any of the rootstocks. This research indicates that some rootstocks are more tolerant of M. xenoplax than self-rooted grapevines and would be preferred when establishing a vineyard on ring nematode-infested soil.

Fine mapping of the Leptosphaeria maculans avirulence gene, AvrLepR1 , corresponding to LepR1 resistance gene in Brassica napus . K. GHANBARNIA, N. LARKAN, D. LYDIATE AND H. BORHAN. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Leptosphaeria maculans (Desmaz.) Ces. & de Not. isolate 99-56 is avirulent on Brassica lines possessing the LepR1 resistance gene due to the presence a single dominant gene named AvrLepR1. Using a mapping population of 94 F1 progenies from a cross between 99-56 and the virulent isolate 87-41, the position of the AvrLepR1 locus was determined to be on linkage group 4, close to the AvrLm4-7 gene. To define the physical location and clone the AvrLepR1gene, the mapping population was increased to 198 individuals. Four markers (FT161-223, P1341-300, PT65-499 and PPM63-440) closely linked to the AvrLepR1locus were sequenced and their positions on the genome of Leptosphaeria maculans isolate JN3 were identified. The order of the markers and their locations on the genome showed that AvrLepR1 is located at the end of SuperContig-12-v2. To clone the gene, BAC libraries of both parental isolates were constructed and BACs spanning the AvrLepR1 intervals were identified. The next step toward cloning AvrLepR1 is sequencing of the positive BACs, work which is currently in progress.

Ratio of 3-ADON and 15-ADON isolates of Fusarium graminearum recovered from wheat kernels in fusarium head blight field nurseries at Glenlea, Manitoba from 2008 to 2011. J. GILBERT, R. CLEAR, S. PATRICK, K. SLUSARENKO AND C. WOLFE. Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada; and (R.C., S.P.) Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, MB R3C 3G8, Canada

Fusarium head blight (FHB) of wheat, caused principally by Fusarium graminearum Schwabe in Canada, causes accumulation of mycotoxins in the grain. The fungus produces deoxynivalenol (DON) and its acetylated forms 3-ADON or 15-ADON. More isolates of F. graminearum collected in Manitoba between 1998 and 2004 were of the 3-ADON chemotype, whereas prior to 1998 the 15-ADON chemotype was considered the only significant cause of FHB in North America. Strains of F. graminearum were isolated from the Glenlea FHB nursery from 2008 to 2011 to monitor the current ratio of 3-ADON to 15-ADON chemotypes. Up to 40 isolates, for which monospore cultures were established, were taken from infected kernels of each of six check wheat lines (240 isolates in each year). DNA was extracted and isolates identified to chemotype using PCR. In 2008, the ratio of 3-ADON to 15-ADON was 79:21%. However, the ratio changed dramatically to 55:45%, 51:49% and 43:57% in 2009, 2010, and 2011, respectively. Under controlled conditions, the mean percentage of 3-ADON isolates recovered from the same six checks rose with increasing temperatures from 32% at 20 °C to 70% at 28 °C. However, an examination of temperatures and precipitation for the 4 years of the field study showed no correlation between recovery of chemotype and maximum, minimum and mean temperatures, or precipitation.

Evidence for tolerance to Plasmodiophora brassicae in a cabbage cultivar. T. V. GLUDOVACZ, B. D. GOSSEN AND M. R. MCDONALD. Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (B.D.G.) Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Several cultivars of cabbage (Brassica oleracea L. var. capitata) and other crucifers with resistance to clubroot, caused by Plasmodiophora brassicae Woronin, have recently been released. The resistance mechanism(s) in these cultivars is not well understood. The reaction of the cabbage cvs. ‘Bronco’, ‘Kilaherb’, and ‘B-2819’ to P. brassicae was assessed under controlled and field conditions. In field trials at a site where pathotype 6 was predominant, ‘Bronco’ was susceptible to clubroot (100% incidence), ‘Kilaherb’ was highly resistant (0%), and ‘B-2819’ was intermediate (63%). Under controlled conditions, seedlings were inoculated with resting spores of P. brassicae pathotypes 3 and 6, and harvested at 4, 12 and 28 days after inoculation (DAI). Pathogen development was assessed in root hairs stained with aniline blue at 4 and 12 DAI, and the percent area colonized by the pathogen was assessed using image analysis of root cross-sections stained with methylene blue at 28 DAI. At 4 DAI, there were no differences in incidence of root-hair infection among the lines; however at 12 DAI, root-hair infection was higher in ‘B-2819’ (82% incidence) than ‘Kilaherb’ (73%), and ‘Bronco’ was not different than the other two cultivars (75%). At 28 DAI, ‘Bronco’ had a higher area of colonization (21%) than ‘Kilaherb’ (6%) or ‘B-2819’ (8%). ‘Kilaherb’ (resistant) did not develop clubroot symptoms, even though the root cortex was colonized at the same level as ‘B-2819’ (intermediate).

Effect of fluctuating temperature on the development of Plasmodiophora brassicae . T. V. GLUDOVACZ, B. D. GOSSEN AND M. R. MCDONALD. Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East Guelph, ON N1G 2W1, Canada; and (B.D.G.) Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Previous controlled environment studies on the effect of temperature on development of clubroot caused by Plasmodiophora brassicae Woronin have used only constant temperatures, and the effect of diurnal temperature fluctuation has not been assessed. Canola (Brassica napus L.) seedlings inoculated with P. brassicae were grown on a unique apparatus (temperature gradient plate) that permits independent temperature control of each experimental unit. In one trial, constant temperatures from 12.5 to 30 °C at 2.5 °C increments, and fluctuating temperatures around the same means (10 °C range from diurnal min to max) were assessed. Assessment of infection incidence and pathogen growth stage at 10 days after infection (DAI) in root hairs stained with aniline blue was used to quantify pathogen development. There were no differences in the incidence of root-hair infection between constant and fluctuating temperatures. The optimum temperature for root-hair infection was 23 °C for constant temperature and 22 °C for fluctuating temperature, based on quadratic regression. In another trial, the effect of temperature fluctuation (0, 5, 15 °C) around selected means (15, 17.5, 20 °C) on pathogen development at 14 DAI was assessed using quantitative PCR to quantify pathogen genomic DNA in planta. In this trial, temperature fluctuation had a very substantial effect on development. The amount of gDNA recovered was consistently higher where the temperature range included 25 °C (optimal for pathogen development, based on previous studies) relative to a constant temperature regime.

Development and application of KASPar markers for marker-assisted selection of the Pc91 oat crown rust resistance gene. B. N. GNANESH, C. A. McCARTNEY, J. M. FETCH, J. MENZIES, A. D. BEATTIE AND P. E. ECKSTEIN. Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada; (A.D.B.) Crop Development Centre, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada; and (P.E.E.) Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada

Crown rust is an important disease of oat caused by Puccinia coronata Corda f. sp. avenae Eriks. Race-specific seedling resistance genes are the primary means of controlling crown rust of oat. Pc91 is a seedling crown rust resistance gene that is highly effective against the current crown rust population in North America. Many race-specific resistance genes have been mapped and markers that are closely linked to them have been identified. However, the use of these markers in oat breeding practice has been limited due to the economics of marker-assisted selection (MAS) deployment. The primary objective of this study was to develop and deploy markers linked to Pc91 for purposes of high throughput marker-assisted selection in oat breeding programs. The KBiosciences Competitive Allele Specific PCR (KASPar) assay is simple and cost-effective compared to other genotyping assays. A KASPar assay was developed from a previously reported DArT marker that co-segregated with Pc91. The KASPar assay accurately postulated the Pc91 status of 16 North American oat breeding lines. Three populations segregating for Pc91 were validated using the KASPar assay which was subsequently implemented in an oat marker-assisted breeding program.

Risk potential of clubroot on the Canadian prairies, based on soil type and pH. B. D. GOSSEN, H. KASINATHAN, S. E. STRELKOV, V. MANOLII, S. F. HWANG, G. PENG, T. CAO AND M. R. MCDONALD. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (H.K., M.R.M.) Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; (S.E.S., V.M., T.C.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (S.F.H.) Alberta Agriculture and Food, Crop Diversification Centre North, 17 507 Fort Road N.W., Edmonton, AB T5B 4K3, Canada

Studies were conducted to examine the effect(s) of soil pH and soil type on clubroot of canola (Brassica napus L.) caused by Plasmodiophora brassicae Woronin. In controlled environment studies across a range of temperatures, clubroot levels were highest at pH ≤ 6.5 and declined as pH increased. At pH 8.0, clubroot levels at 25 °C (optimum temperature) were about 50% of the maximum at low pH. This indicates that clubroot levels are likely to be lower in fields with pH > 7.0 compared to those at lower pH, but that high soil pH alone does not eliminate the risk of clubroot. In other studies, clubroot levels in soil-less mix were lower than in the other treatments, but there was little difference among sand, mineral soil and muck soil under saturated conditions. Also, soil compaction may increase clubroot levels. This indicates that soil type likely has a small impact on clubroot levels in saturated soil, e.g., after heavy precipitation. The relatively small effect of pH on clubroot was supported by assessments of pH and clubroot in 267 commercial canola fields in Alberta, where the correlation between soil pH and clubroot incidence was significant, but weak (r = 0.30, P < 0.05). These results indicate that many regions of the Canadian prairies could be at risk of clubroot epidemics on canola if the long-lived resting spores are distributed by human activities, wind or water.

Epidemiology of Plum pox virus ‘D’ strain in Canada and US. T. R. GOTTWALD AND E. J. WIERENGA. U.S. Department of Agriculture, Agricultural Research Service, 2001 S. Rock Road, Fort Pierce, FL 34949, USA; and (E.J.W.) Canadian Food Inspection Agency, 174 Stone Road West, Guelph, ON N1G 4S9, Canada

The successful 10-year Pennsylvania Plum pox virus (PPV) eradication program was based on vigilant survey followed by removal of all Prunus within 500 m of confirmed PPV + trees, resulting in declaration of eradication in October 2009. Whereas, in Ontario the low incidence but widely dispersed PPV epidemic extended throughout the stone fruit industry when first discovered in 2000. The Canadian program was predicated on vigilant survey plus PPV + trees and block removal to reduce PPV incidence initially, followed by increasingly stringent eradication protocols over time. However, eradication was not achieved prior to program termination. Retrospective analyses of the Canadian epidemic indicated that the estimated PPV distribution from known sources followed a pulse-peak-decay function. The function indicated that viruliferous aphids transmitted infections most commonly at 43 m (peak) from prior infections, but PPV distribution also has a long (decaying) tail. A Weibull model was fitted to of the proportion of new PPV + trees detected demonstrated that 95% of new infections occurred within 629 m, 465 m, and 317 m, for 1, 2, and 3-year moving averages, respectively. Results suggest that eradication might be achievable by employing a 317-629 m cull radius. A risk-based survey methodology has been developed and deployed to sample Prunus orchards throughout New York that emphasizes proximity to prior PPV discoveries, and proximity to the Canadian border.

What ‘fusarium head blight-resistance QTLs’ might really be telling us - the curious tale of shifts in SSR -marker alleles in resistant wheat lines descended from susceptible progenitors. S. HABER, A. MALIHIPOUR, J. GILBERT AND S. ROBINSON. Cereal Research Centre, Agriculture and Agri-Food Canada (AAFC), 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada; (A.M.) Seed and Plant Improvement Institute, Karaj, Iran; and (S.R.) Saskatoon Research Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Heritable traits can be evolved de novo in germplasm subjected to systemic stresses. As demonstrated in repeated rounds of indoor and field testing, this allows fusarium head blight (FHB)-resistant sublines to be selected from descendants of a susceptible progenitor such as the cultivar ‘Roblin’. Since analyses of AFLP patterns confirmed that exotic genes had not been accidentally introduced into these sublines, SSR alleles should be identical in the susceptible progenitor and in individual plants of descendant sublines expressing strong resistance. We compared SSR alleles linked to FHB QTLs in individual plants of FHB-resistant 6^th-cycle sublines with those of their susceptible ‘Roblin’ progenitor and observed discrete allele shifts. Moreover, for 7 of 23 examined SSR markers, different tillers sampled from the same individual plants also exhibited such discrete shifts. We suggest the formation of these SSR alleles might be sensitive to epigenetic effects and that these effects might in some instances vary among tillers of a plant. The mechanism mediating the epigenetic effect on SSRs remains to be determined. Perhaps FHB-resistance QTLs should be seen not as identifying genomic regions that contain specific resistance genes but rather as regions responsive to epigenetic variation. From such variation associated with altered gene expression, selection then identifies those individuals that become founders of more resistant sublines.

Biology and management of box blight caused by Cylindrocladium buxicola . S. E. HEALY AND T. HSIANG. School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Box blight causes a serious foliar disease on Buxus spp. and the causal pathogen Cylindrocladium buxicola Henricot, has recently been found to occur in Ontario creating concerns for nursery growers. In April 2012, we received two potted plants of ‘Green Mountain’ from a nursery in Strathroy, Ontario which showed symptoms of defoliation. Diseased leaves showed dark brown lesions and cankers visible on the stems. Diseased leaves were incubated on PDA and were allowed to grow for 7 days; the colonies were then subcultured and allowed to grow for up to 2 weeks to confirm identity morphologically. The infection process of C. buxicola was investigated on detached leaves of ‘Green Gem’ using hyphal plugs as inoculum. Four days after inoculation lesions began to appear, and all inoculated leaves showed blight symptoms within 2 weeks. The pathogen was re-isolated from these lesions completing Koch's postulates. The ITS region of the fungal DNA was amplified and the PCR products were sequenced. Comparison of the sequence with those in GenBank showed 98% identity to sequences labeled as C. buxicola. Further work will focus on understanding the infection process and determining the susceptibilities of boxwood cultivars in Ontario. Focus will also be placed on establishing chemical and cultural control methods for this disease.

Survey for head blight of cereals and stalk rot of corn caused by Fusarium graminearum in Alberta in 2010-11. R. J. HOWARD, G. C. DANIELS, M. W. HARDING, T. K. TURKINGTON AND P. LAFLAMME. Crop Diversification Centre South, Alberta Agriculture and Rural Development, 301 Horticultural Station Road East, Brooks, AB T1R 1E6, Canada; (M.W.H.) Innovotech, Inc., Crop Diversification Centre South, 301 Horticultural Station Road East, Brooks, AB T1R 1E6, Canada; (T.K.T.) Lacombe/Beaverlodge Research Centre, Agriculture and Agri-Food Canada, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada; and (P.L.) Pest Surveillance Branch, Alberta Agriculture and Rural Development, 307 J. G. O'Donoghue Building, 7000-113 Street, Edmonton, AB T6H 5T6, Canada

Fusarium head blight (FHB), caused by Fusarium graminearum (Fg) Schwabe and a number of other pathogenic Fusarium species, is a serious disease of small grain cereals in Western Canada. Fg can also cause stalk and ear rots in field corn. It has become established on wheat and silage corn in the irrigated districts of southern Alberta over the past decade. A coordinated provincial survey was carried out in 2010-11 to update the records of Fg occurrence obtained during comparable surveys conducted in 2001-03 and 2006. In 2010-11, more than 1300 fields were surveyed between July and October for Fg infections in cereal heads, cereal stubble or corn stalks. In 2010, the largest survey year (907 fields), the average percentage of fields infected with Fg was 6.0% for cereal stubble samples, 13.6% for cereal grain samples, and 42.2% for corn stalk samples. The majority of infected fields were in seven municipalities in southern Alberta. Four cereal fields in central Alberta were also found to have low levels of Fg infection. Of 289 Fg isolates obtained from the cereal and corn samples collected during the 2010 survey, 90% were the 15ADON chemotype and 9.7% were the 3ADON type. The 3ADON chemotype appears to be gradually displacing the 15-ADON type in southern Alberta.

How many genes should be used in multigene phylogenetic trees? V. C. H. HUANG, T. HSIANG AND G. MORENO-HAGELSIEB. School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (G.M.H.) Faculty of Science (Biology), Wilfrid Laurier University, Waterloo, ON N2L 3C5, Canada

Sequencing technologies have improved significantly while becoming much more affordable. This has led to an inundation of data allowing phylogenetic studies to transition from single-gene analyses to multigene analyses. Although it has been demonstrated that trees based on multiple genes may provide better resolution than single-gene trees, the optimal number of genes that should be used is unclear. Given the abundance of sequenced genomes, it may be possible to pinpoint an optimal range for the number of genes required for informative multigene trees. Using BLAST, the ∼10,000 genes in the genome of Neurospora crassa Shear & Dodge were compared against 48 other filamentous ascomycete genomes to identify genes present in all these genomes. From the ∼8,000 genes identified, ten independent sets of 5, 10, 15, 20, and 25 genes were randomly sampled. The selected genes were individually aligned, and then concatenated into superalignments to make multigene trees using maximum likelihood (ML). Out of the 50 ML trees, 45 shared the same topology (branching patterns). Four of the five incongruent topologies were found among the ten 5-gene superalignment trees. These results suggest that the number of genes that should be used for multigene trees may lie between five and ten genes as five appear insufficient, but ten may be sufficient.

Stem and bulb nematode ( Ditylenchus dipsaci) in Ontario-grown garlic. B. HUGHES, M. CELETTI, M. PAIBOMESAI AND Q. YU. New Liskeard Agricultural Research Station, University of Guelph, New Liskeard, ON P0J 1P0, Canada; (M.C., M.P.) Ontario Ministry of Agriculture, Food and Rural Affairs, Room 3110, Edmund Bovey Building, University of Guelph, Guelph, ON N1G 2W1, Canada; and (Q.Y.) Eastern Cereal and Oilseed Research Center, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada

Stem and bulb nematode, Ditylenchus dipsaci (Kühn) Filipjev, is a serious pest of garlic, onions and leeks which has become established in Ontario garlic in recent years. A survey to determine the extent of stem and bulb nematode infestation in Ontario garlic was conducted in 2011. Garlic samples, each consisting of 10 bulbs, were contributed by growers or collected by the researchers from across Ontario. A total of 123 samples, from 79 growers in 33 counties/districts, were included in the survey. These were analyzed at the University of Guelph Pest Diagnostic Clinic for stem and bulb nematode using the Baermann-funnel and mist chamber method. Nematode counts were expressed per gram of dry bulb. Positive samples were retained for random amplified polymorphic DNA (RAPD) analyses. Stem and bulb nematodes were found in garlic from across the province with 73% of the samples testing positive. Eighty two percent of the samples of the variety ‘Music’ had stem and bulb nematode, while 54% of the other varieties were infested. Nematode counts ranged from < 1 to > 3,000 per gram. Stem and bulb nematodes were not found in 5 of the 33 counties/districts. The infestation seemed to be clustered in two parts of the province, southwestern Ontario and eastern Ontario.

Genetic diversity of Plum pox virus - strains, disease, and related challenges for control. D. JAMES. Centre for Plant Health, Canadian Food Inspection Agency, 8801 East Saanich Road, Sidney, BC V8L 1H, Canada

The genome of Plum pox virus (PPV) consists of positive sense, single stranded RNA that is approximately 10,000 nucleotides long, encapsidated by a protein coat to form virions. The virus is classified as a member of the genus Potyvirus, family Potyviridae. The disease associated with the virus can cause crop losses as high as 95-100% in some susceptible stone fruit cultivars. The genome of the virus was considered to act as a single gene, encoding one large open reading frame (ORF). However, recently a short essential gene (PIPO, Pretty Interesting Potyviridae ORF) was discovered embedded in the P3 cistron of potyviruses. The main ORF is translated into a single polyprotein that is cleaved post-translation into 10 functional proteins including the coat protein and a RNA-dependent RNA polymerase (RdRp). The RdRp is responsible for genome replication, but it lacks proof reading capability which results in the introduction of errors or mutations in the genome. Genome variation or genetic diversity is caused also by deletions, insertions, and recombination events. As a consequence seven strains of PPV (D, M, EA, C, Rec, W, and T) have been described to date, with distinct genetic and serological identities which allow unique identification using nucleic acid-based or protein-based assays, respectively. The strains differ in their disease severity, natural host range, transmissibility, and geographic distribution. There seems to be some host preference among certain strains. This means therefore that strain identification is very important for effective control/management of the virus and associated disease. There is, also, genetic and biological diversity within strains. All together this information is essential for understanding the epidemiology of this important virus and disease.

Pear rootstock effects on seasonal colonization pattern of pear decline phytoplasma. M. KAVIANI, D. M. HUNTER, P. H. GOODWIN AND A. NASSUTH. Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; (D.M.H.) Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, Vineland Research Farm, 4902 Victoria Avenue North, P.O. Box 6000, Vineland, ON L0R 2E0, Canada; (P.H.G.) School of Environmental Science, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (A.N.) Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Pear decline (PD) is a serious disease of the cultivated European and pear (Pyrus communis). The disease is widespread in Europe and North America and is caused by the phytoplasma Candidatus. P. pyri which is phylogenetically closely related to Ca. P. mali (apple proliferation). The seasonal variation of PD phytoplasma colonization was studied in pear trees cv. ‘HW620’ grown on three pear rootstocks: ‘Bartlett’, PD-resistant ‘OHF87’ and PD-susceptible ‘OHF69’. TaqMan Real-time PCR assays were performed to quantify the Ca. P. pyri titre over 1 year in leaves, branches and roots of asyptomatic but PD-positive pear trees planted in a commercial orchard in Niagara-on-the-Lake, Ontario, Canada. Colonization showed a seasonal pattern with the phytoplasma titre decreasing in the leaves (until leaf fall) and shoots during winter, surviving in the roots over winter, and then reappearing in the spring followed by a systematic increase throughout summer and fall. Trees with different rootstocks had different phytoplasma titres in leaves and shoots as well as in roots. The pattern of colonization and amount of phytoplasma varied greatly in different samples and different rootstocks. The complex pattern of colonization and seasonality of the PD phytoplasma should be considered when sampling for diagnostic purposes.

Biological control potential of soil bacteria isolated from a commercial potato field for the management of seedling damping-off and root rot diseases. S. E. KHABBAZ AND P. A. ABBASI. (S.E.K., P.A.A.) Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada; and (S.E.K.) General Commission for Scientific Agriculture Research, Hama, Syria

Fish emulsion (FE) and other soil organic amendments reduce the impact of soilborne diseases possibly through biological control. Field plots treated with FE had a higher level of bacterial activity in the soil samples. More than 150 of these bacteria were screened for biological control potential against soilborne pathogens in laboratory and growth room tests. The selected bacteria were identified and characterized based on phenotypic characteristics, sequence analysis of 16S-23S rDNA gene, and biochemical tests. Two strains (Pf 9A-14 and Pf 8D-45) identified as Pseudomonas fluorescens and one (Bs 8B-1) as Bacillus subtilis showed very good bio-control and plant growth promotion activity in the growth room assays. All three strains produced hydrogen cyanide and siderophores (Pf strains only) in growth media, and their culture filtrates showed positive activity for indole acetic acid, salicylic acid, and β-1,3-glucanase. When applied as pre- or post-planting treatments to the infested peat-based mix, all three strains of bacteria protected cucumber seedlings from damping-off by improving the percentage of healthy cucumber seedlings (44-81% healthy vs 20-22% healthy in the control) and reducing disease severity (1.2-1.84 vs 2.2-2.6 in the control). The average fresh weights of cucumber plants produced in pots receiving pre- or post-planting application of the three bacteria were greater than those of plants produced in the pathogen-infested (113-184% increase) and non-infested (10-38% increase) control pots. Peat and talc-based formulations of these bacteria applied as pre-planting or seed treatments to the infested substrate also provided effective control of damping-off and root rot of cucumber and enhanced plant growth.

Comparison of RNA purification methods from challenging plant samples and the effect on viroid detection. W.-S. KIM AND Y. HAJ-AHMAD. (W.-S.K., Y.H.-A.) Norgen Biotek Corp., 3430 Schmon Parkway, Thorold, ON L2V 4Y6, Canada; and (Y.H.-A.) Brock University, 500 Glenridge Avenue, St. Catharines, ON L2S 3A1, Canada

A viroid consists of a small, single-stranded, circular RNA (240 bp - 410 bp). Viroids are known to be highly contagious, thus their rapid and reliable detection is the only way to control their spread, by eradicating all infested material at a very early stage of infection. Unlike viruses, viroids are not encapsulated in a protein coat, thus molecular hybridization techniques and PCR are most frequently used detection methods in practice. High quality plant RNA purification is a prerequisite for many downstream applications, such as reverse transcription polymerase chain reaction (RT-PCR), cDNA library construction, RNA sequencing and gene expression profiling using microarrays. Purification of high quality RNA from plant tissues is often a challenging process, due to the high concentration of polysaccharides, polyphenols and other secondary metabolites which can become complexed with RNA. In this study, a phenol/chloroform-based RNA purification method, which is known as the gold standard protocol, was compared to two market-leading spin column methods. RNA quality and quantity, RIN (RNA Integrity Number) values, gDNA contamination levels and small RNA recovery were all evaluated, using difficult and moderately challenging plant samples. Large differences in RNA yield and quality among protocols were found, especially with the more challenging plant samples. The effect of RNA purification method on viroid detection will also be discussed.

Exploitation of the antifungal activity of aluminum-containing salts for the control of carrot cavity spot. E. A. KOLAEI, R. J. TWEDDELL AND T. J. AVIS. Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6, Canada; and (R.J.T.) Centre de recherche en horticulture, Université Laval, 2480, boul. Hochelaga, Québec, QC G1V 0A6, Canada

Cavity spot is caused by different species of Pythium (including P. sulcatum Pratt & Mitch.). Symptoms of the disease generally appear in the field as the carrot root matures and lesions continue to develop during storage. Few efficient chemical options are available to control cavity spot. In this context, aluminum-containing salts (aluminum acetate, aluminum ammonium sulfate, aluminum chloride, aluminum lactate, aluminum potassium sulfate, aluminum sulfate) were assessed for their effectiveness to inhibit the mycelial growth of P. sulcatum and for their ability to control carrot cavity spot (P. sulcatum). Salts were incorporated into agar culture medium at 0 (control), 0.1, 1, 5, and 10 mM to determine inhibition of mycelial growth. In order to evaluate the effect of the salts on cavity spot development, 5 mM salt solutions or water (control) were sprayed on carrot roots artificially-inoculated with P. sulcatum. Results showed that only aluminum chloride and aluminum sulfate significantly inhibited P. sulcatum growth when tested at 1 mM. Minimal inhibitory concentrations for all tested aluminum salts were 5 mM. Aluminum sulfate completely inhibited the development of cavity spot. Aluminum lactate also caused a significant reduction in cavity spot lesions. These results indicate that specific aluminum salts could be effective at controlling carrot cavity spot and could find application as postharvest treatments.

Sulfur–containing salts for the control of carrot cavity spot and potato dry rot. E. A. KOLAEI, R. J. TWEDDELL AND T. J. AVIS. Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6, Canada; and (R.J.T.) Centre de recherche en horticulture, Université Laval, 2480, boul. Hochelaga, Québec, QC G1V 0A6, Canada

Postharvest diseases are responsible for significant economic losses worldwide. The application of synthetic fungicides to control potato dry rot (Fusarium spp.) and carrot cavity spot (Pythium spp.) is becoming less effective as many strains of the pathogens are becoming resistant to these fungicides. As alternatives to synthetic fungicides, sulfur-containing salts (ammonium sulfate, calcium sulfate, magnesium sulfate, potassium metabisulfite, potassium sulfate, sodium metabisulfite, and sodium sulfate) were evaluated for their inhibition of mycelial growth of Pythium sulcatum Pratt & Mitch. and Fusarium sambucinum Fuckel and for their suppression effects on carrot cavity spot (P. sulcatum) and potato dry rot (F. sambucinum). Our results showed that metabisulfite-containing salts provided strong inhibition of both pathogens. Among the sulfate-containing salts, calcium sulfate, magnesium sulfate and sodium sulfate were effective in inhibiting the growth of P. sulcatum and magnesium sulfate and sodium sulfate decreased the growth of F. sambucinum. Both metabisulfite salts provided 100% inhibition of cavity spot (at 50 mM) and dry rot (at 200 mM). At a concentration of 50 mM, calcium sulfate and sodium sulfate also significantly reduced carrot cavity spot development whereas ammonium sulfate, magnesium sulfate, potassium sulfate, and sodium sulfate significantly reduced potato dry rot development at 200 mM. The study indicated that sulfate and metabisulfite salts could be effective at controlling these pathogens.

Getting rich or going broke? Economic considerations to guide research decisions on microbial pest control products. T. LAENGLE. Pest Management Centre, Agriculture and Agri-Food Canada, 308 Brookfield Road, Building 5, St. John's, NL A1E 0B2, Canada

Promising micro-organisms with antagonistic properties against key pests are frequently discovered through targeted research or accidental discovery. Yet relatively few microbial pest control products have successfully made an impact in the market place. Among the key factors determining the success of a biopesticide product is cost efficiency and profitability. This presentation explored economic factors to consider when gauging a potential products viability, and will provide researchers with guidance to make go/no-go decisions at early stages of product development.

Induced host defense responses are involved in suppressing clubroot on canola with Bacillus subtilis , Clonostachys rosea and Heteroconium chaetospira . R. LAHLALI, G. PENG, L. MCGREGOR, M. R. MCDONALD, F. YU, B. D. GOSSEN, S. F. HWANG, R. K. HYNES, S. M. BOYETCHKO AND J. GEISSLER. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (M.R.M.) Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road, Edmonton, AB T5Y 6H3, Canada

The commercial biofungicides Serenade^® (Bacillus subtilis), Prestop^® (Clonostachys rosea f. catenulata (Gilman & Abbott) Schroers, Samuels, Seifert & Gams), and the endophytic fungus Heteroconium chaetospira (Grove) M.B. Ellis reduced the severity of clubroot [Plasmodiophora brassicae Woronin] on canola (Brassica napus L.) by >85% when applied to the soil under controlled conditions. The treated plants were examined for gene expression related to jasmonic acid (JA), ethylene (Et), auxin (IAA), PR protein, and phenylpropanoid (Pp) pathways using qRT-PCR to determine whether induced host-defence responses were involved in biocontrol. In plants treated with Serenade^® or Prestop^®, genes encoding JA (BnOPR2), Et (BnACO) and Pp (BnOPCL, BnCCR) were up-regulated while genes encoding IAA and PR proteins were down-regulated in roots relative to non-treated controls. The same pattern was also observed in leaves, but only the increase in Pp gene expression was significant. Plants treated with H. chaetospira showed higher transcript levels for Et, JA, IAA, PR-2 and Pp in roots, but the expression of Pp genes in leaves was insignificant based on a microarray study. These results indicate that induced host resistance is possibly involved in clubroot suppression by these biocontrol agents via the modulation of JA, Et and Pp pathways in root tissues. The up-regulation of Pp pathways in leaves by Serenade^® or Prestop^® implies that the acquired resistant response is systemic. The Pp pathways influence the production of several host-defense secondary metabolites including phenolics, salicylates and flavonoid phytoalexins.

Microsatellite DNA markers indicate quantitative trait loci controlling resistance to pea root rot caused by Fusarium avenaceum . W. J. LI, J. FENG, K. F. CHANG, R. L. CONNER, S. F. HWANG, S. E. STRELKOV, B. D. GOSSEN AND D. L. MCLAREN. Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; (R.L.C.) Morden Research Station, Agriculture and Agri-Food Canada (AAFC), Unit 100-101, Route 100, Morden, MB R6M 1Y5, Canada; (S.E.S.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (B.D.G.) Saskatoon Research Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; and (D.L.M.) Brandon Research Centre, AAFC, 18^th Street North and Grand Valley Road, P.O. Box 1000A. R.R. #3, Brandon, MB R7A 5Y3, Canada

To identify quantitative trait loci (QTL) controlling root rot resistance of field pea, 213 microsatellite markers were screened against a population of recombinant inbred lines (RIL) derived from crosses between a moderately resistant cultivar ‘Carman’ and a susceptible cultivar ‘Reward’. Phenotypic data were obtained following artificial infection of pea plants by Fusarium avenaceum (Fr.) Sacc. in field experiments conducted in 2009 and 2010. Linkage analysis based on a single factor ANOVA indicated that four markers were associated with root rot resistance. QTL analysis based on these four markers identified a QTL on Chromosome VII that explained 21.7% of the variance in resistance. The microsatellite markers that are closely linked to this QTL may be useful for stacking QTLs from ‘Carman’ and other sources to develop cultivars with superior fusarium root rot resistance.

Genetic variation of the Leptosphaeria maculans/Leptosphaeria biglobosa species complex in Alberta, Canada. Y. LIANG, S. F. HWANG AND S. E. STRELKOV. (Y.L., S.E.S.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (Y.L.) Institute of Agriculture and Life Science, Chongqing University, Chongqing 400044, China; and (S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada

Canola is an economically valuable oilseed crop in Canada and diseases can have devastating effects on its yield and quality. Blackleg or phoma stem canker, caused by the Leptosphaeria maculans (Desmaz.) Ces. & de Not./L. biglobosa species complex, is one of the most important diseases of canola and cruciferous vegetables worldwide. The population biology of L. maculans/L. biglobosa in western Canada, with an emphasis on Alberta, is being studied. A representative population composed of over 100 single-conidial isolates was obtained from stubble of canola plants showing symptoms of infection, which were collected from canola crops across Alberta. The isolates are being characterized according to morphological diversity, as well as proportion of L. maculans to L. biglobosa, pathogenicity grouping, mating type, frequencies of avirulence genes, and variation in phytotoxin production. Although a considerable amount of time and effort has gone into investigating the L. maculans/L. biglobosa complex and phoma stem canker, this study may contribute to the development of enhanced crop protection strategies for canola production in Alberta and Canada.

Characterization of Leptosphaeria maculans race structure in Western Canada. S. H. LIBAN, D. J. CROSS, G. PENG, H. R. KUTCHER AND W. G. D. FERNANDO. Department of Plant Science, University of Manitoba, 66 Dafoe Road, Winnipeg, MB R3T 2N2, Canada; (D.J.C.) Melfort Research Centre, Agriculture and Agri-Food Canada (AAFC), P.O. Box 1240, Melfort, SK S0E 1A0, Canada; (G.P.) Saskatoon Research Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; and (H.R.K.) Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada

The phoma stem canker causal agent Leptosphaeria maculans (Desmaz.) Ces. & de Not. is a ubiquitous fungal pathogen and the most destructive disease affecting oilseed rape and canola (Brassica napus L.). Genetic resistance has proven to be an effective means of disease control in western Canada and a cultivar carrying a specific resistance gene will be resistant to the pathogen carrying the corresponding avirulence gene. However, host genetic resistance can be overcome with population shifts and the emergence of new races of the pathogen. Thus information on the genetic variability in the pathogen population is essential for the development of an effective control strategy. This study sampled isolates of L. maculans, the aggressive pathogen species, in 2010 and 2011 across the three Prairie Provinces Alberta, Saskatchewan, and Manitoba. The race structure was assessed by differentials and/or PCR on avirulence alleles AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm6, AvrLm7, AvrLm9, LepR1, LepR2, and LepR3. The frequency of certain alleles was relatively consistent with AvrLm6 present in 62%-83% of isolates and AvrLm9 near 0% across all sites. However, some loci differed greatly across geographic locations. AvrLm2 ranged from 90% in Vegreville (AB) to 38% in Plum Coulee (MB). Selection pressure from different race-specific resistance genes in canola cultivars is possibly the cause of the variation in virulence observed.

Characterization of the fungi associated with ascochyta blight of field pea in Alberta, Canada. J. LIU, T. CAO, J. FENG, K. F. CHANG, S. F. HWANG AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (J.F., K.F.C., S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road, Edmonton, AB T5Y 6H3, Canada

Ascochyta blight, caused by a complex of Mycosphaerella pinodes (Berk. & Blox.) Vestergr., Phoma pinodella (Jones) Morgan-Jones & Burch, Ascochyta pisi Lib., and/or Phoma koolunga Davidson et al., is a devastating disease of field pea. A survey in central Alberta in 2011 revealed the occurrence of ascochyta blight in all 54 pea crops visited. A total of 162 single-spore fungal isolates were obtained from diseased pea samples collected from various counties or municipalities in the region, including Strathcona, Westlock, Parkland, Smoky River, High Prairie, Sturgeon, Minburn, and Big Lakes. The virulence of 159 of the isolates was tested on the pea cv. ‘Midas’, on which they were found to range from strongly to weakly virulent. Random amplified polymorphic DNA analysis revealed six groups of isolates, with Group II, the largest, consisting of 85 isolates. Most of the isolates from Parkland, Strathcona, Smoky River, and Big Lakes were clustered into Group II, while the majority of the isolates from Sturgeon, Minburn, and Westlock were clustered into Group I. Analysis of the internal transcribed spacer region from 144 of the isolates revealed that it was identical among those isolates and to a sequence from M. pinodes. Morphological characterization of these isolates is underway to better understand the composition of the ascochyta blight fungal complex in Alberta.

Baseline sensitivity of Leptosphaeria maculans to strobilurin fungicides. C. LIU, W. G. D. FERNANDO, Y. T. GAN, H. R. KUTCHER AND G. PENG. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; (Y.T.G.) Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada (AAFC), P.O. Box 1030, Swift Current, SK S9H 3X2, Canada; (H.R.K.) Department of Plant Sciences, 3C02 Agriculture Building, 51 Campus Drive, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada; and (G.P.) Saskatoon Research Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Blackleg disease (phoma stem canker), caused by Leptosphaeria maculans (Desmaz.) Ces. & de Not., is the most widespread fungal disease of canola (Brassica napus L.) in western Canada. Strobilurin fungicides, which belong to the quinone outside inhibitors (QoI), are registered to control blackleg in Canada. They have a single-site mode of action and act as a respiration inhibitors against the pathogen. Single-site fungicides are at high risk to become ineffective due to genetic changes in the pathogen, and selection pressure on the pathogen population. In 2011, 27 L. maculans isolates were collected from infected canola residue from three fields where azoxystrobin and pyraclostrobin (QoI fungicides) were applied between the 2-4 leaf stage and bolting. The isolates were tested in vitro on fungicide-amended media to assess the effective fungicide concentration at which the growth of pathogen mycelia would be reduced by 50% (EC₅₀), relative to that on non-amended media. The baseline EC₅₀ values for azoxystrobin ranged from 0.097 to 0.449 ug/mL (mean – 0.214, median - 0.195) and for pyraclostrobin from 0.126 to 0.477 ug/mL (mean – 0.312, median - 0.356). Sensitivity to these fungicides will be monitored for the next 2 years from treated field sites to detect any shift in sensitivity in L. maculans.

The first infectious cDNA clone of a widespread grape virus and its potential utilization in biotechnology. B. MENG, S. VENKATARAMAN, C. LI, W. WANG, C. DAYAN-GLICK AND M. MAWASSI. Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON N1G2W1, Canada; and (C.D.G., M.M.) The Plant Pathology Department - The Virology Unit, Plant Protection Institute, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50250, Israel

As a member of the genus Foveavirus within the brand new family Betaflexiviridae, Grapevine rupestris stem pitting-associated virus (GRSPaV) has a positive-sense, single-stranded RNA genome. GRSPaV is among the most prevalent virus, has a worldwide distribution in grapevines, and is associated with ‘Rupestris stem pitting’, ‘vein necrosis’ and ‘Syrah decline’. In this study, we have engineered a full-length cDNA clone of GRSPaV and a GFP-tagged variant, both of which are under the transcriptional control of the CaMV 35S promoter. We demonstrated that both clones were infectious in grapevine and Nicotiana benthamiana. Kinetics of viral replication was studied through fluorescence microscopy, Western blotting, RT-PCR and Northern blotting. Furthermore, GRSPaV virions were observed in plants inoculated with both viral clones. Interestingly, GRSPaV did not cause systemic infection in four of the most commonly used herbaceous experimental hosts, even in the presence of movement proteins from two other viruses that have demonstrated the ability to complement the movement function of movement-defective mutant viruses of diverse taxonomic groups. These infectious clones are the first among members of the Foveavirus. Investigation on GRSPaV may facilitate revelations on the molecular mechanisms governing various stages of virus replication for members of the Foveavirus and perhaps those of the Betaflexiviridae family. Work in underway to test the possibility of rendering GRSPaV into vectors to express foreign proteins and to elucidate plant gene functions through virus-induced gene silencing.

Verticillium dahliae hydrophobins and their roles in development and pathogenicity. N. P MORALES-LIZCANO AND K. F. DOBINSON. (N.P.M.-L., K.F.D.) Department of Biology, University of Western Ontario, London, ON N6A 5C1, Canada; and (K.F.D.) Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada

The broad host range, soil-borne fungus Verticillium dahliae Kleb. is the causal agent of an economically significant vascular wilt disease of dicotyledonous plants. Verticillium dahliae produces persistent resting structures, known as microsclerotia (MCS), which are the primary source of disease inoculum in the field. Five hydrophobin-like proteins (VDH1 to 5) were identified in the genome of V. dahliae. The gene encoding VDH1 was previously studied by the characterization of a vdh1 (gene disruptant) strain. An amicrosclerotial phenotype was shown by the vdh1 strain, and it also produced disease faster in inoculated tomato plants than did the wild type (WT) strain. Bioinformatic analyses suggest secretion of the other four proteins, and indicate that they are, like VDH1, class II hydrophobins. Differential expression of the genes encoding these proteins suggests that each one may have different roles rather than redundant functions. Agrobacterium tumefaciens-mediated transformation was used to generate a gene deletion mutant (knock out; KO) for Vdh5. The vdh5 (mutant) strains showed a delayed production of MCS, as well as a less aggressive pathogenic phenotype when compared to the WT. The results obtained with the mutants studied support the hypothesis that the hydrophobin genes in V. dahliae have different functions in development and pathogenicity.

Uncovering novel pathogenesis genes during analysis of expressed sequence tags from an Ustilago maydis filamentous dikaryon cDNA library. E. N. MORRISON, M. E. DONALDSON AND B. J. SAVILLE. Environmental & Life Sciences Graduate Program, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada; and (B.J.S.) Forensic Science Program, DNA Building, Trent University, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada

The work presented here aids in the annotation of the genome sequence of the model fungal pathogen Ustilago maydis (DC.) Corda. Analyses of 4425 expressed sequence tags derived from a filamentous dikaryon cDNA library allowed confirmation and correction of gene models and the documentation of transcript structural features. Normalization of the cDNA library provided a greater depth of coverage than previous U. maydis cDNA libraries and enabled the identification of antisense and noncoding RNAs. Further, candidate pathogenesis genes were identified based on their representation in the U. maydis dikaryon cDNA library only or in the U. maydis dikaryon and filamentous diploid cDNA libraries, followed by comparative analysis with the genome sequences of other plant pathogens. Of these genes, six were conserved only among pathogenic fungi and six were unique to U. maydis. The expression of these genes during in planta growth was confirmed using reverse transcriptase PCR. The transcript level of three of these genes varied during fungal development within the plant suggesting previously unrecognized changes in fungal gene expression. Some newly identified U. maydis noncoding RNAs showed patterns of cDNA library representation suggestive of a role in pathogenesis. The deletion of one such ncRNA in a solopathogenic strain reduced virulence, revealing a new class of fungal pathogenesis genes.

Identification of the avirulence gene Avr1d from the soybean root rot pathogen Phytophthora sojae. R. NA, W. YIN, S. DONG, D. QUTOB, D. YU, J. ZHAO, Y. WANG AND M. GIJZEN. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON Canada; (W.Y., S.D., D.Y., Y.W.) Nanjing Agricultural University, Nanjing, China; and (J.Z.) Inner Mongolia Agricultural University, Huhhot, China

Phytophthora sojae Kaufmann and Gerdemann is an oomycete and a pathogen of soybean that causes root rot. During infection, P. sojae delivers effector proteins into host cells to suppress defense responses. However, effector-triggered immunity (ETI) results when effectors are recognized by host resistance (R) proteins. Thus effectors are the key factors that determine cultivar specificity. Now we have identified the P. sojae Avr1d gene, encoding a predicted effector protein with a host-targeting amino acid motif Arg-X-Leu-Arg (RXLR). Genetic mapping of 16 different P. sojae isolates, and of a segregating F₂ population of 40 individuals shows that the predicted RXLR effector gene Avh6 precisely co-segregates with the Avr1d phenotype. The Avh6 gene is present in cultures that are avirulent on Rps1d cultivars, whereas the gene is absent from the genome of virulent cultures. Analysis by reverse transcriptase polymerase chain reaction (RT-PCR) detected Avh6 transcripts in avirulent but not in virulent P. sojae isolates. Transient expression and co-bombardment with a reporter gene confirms that Avh6 triggers cell death in Rps1d soybean plants. Thus, all results indicate that Avh6 corresponds to Avr1d. Two sequence variants of the Avr1d gene encoding different protein products are present in different strains of P. sojae, but both are recognized by Rps1d and cause ETI.

Genetic diversity of Entomosporium mespili and its interaction with Saskatoon berry. A. NAOUI, A. MINTENKO, L. R. ADAM AND F. DAAYF. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; and (A.M.) Manitoba Agriculture, Food and Rural Initiatives, Agri-Industry Development and Innovation, Crops, Carman, MB R0G 0J0, Canada

The saskatoon berry (Amelanchier alnifolia Nutt.) is a perennial woody shrub, from the Pomoidae subfamily of Rosaceae. It is mainly cultivated in the Canadian and Northern US prairies due to its resistance to low temperatures and drought. The Saskatoon berry industry has great potential for growth due to the health benefits that the fruit provides. The cultivated area of saskatoons has increased by 9.7% between 2001 and 2006. However, the production of berries is seriously affected by entomosporium leaf and berry spot (ELBS), caused by the biotrophic ascomycete Entomosporium mespili (DC.) Sacc. The disease causes a loss of $5-8 Million every year to Saskatoon fruit industry. The genetic diversity of 46 isolates of E. mespili, isolated from leaves of Saskatoon Berry across the Canadian prairies, was carried out using Random Amplified Polymorphic DNA (RAPD). Analysis of data has revealed a high level of intraspecific diversity, based on the diverse banding profiles among isolates. The spectrum of amplicons ranged between 400 and 2000-bp. The genetic distance between isolate pairs was ranged from 0.06 to 0.88. In order to confirm such genetic diversity, we are currently carrying out Amplified Fragments Length Polymorphism (AFLP) tests using these isolates. These results will be compared with the pathogenicity status of the tested isolates.

Evaluation of air sampling and detection methods to quantify airborne ascospores of Sclerotinia sclerotiorum . M. PARKER, M. R. MCDONALD AND G. J. BOLAND. Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (G.J.B.) School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Detection and quantification of airborne ascospores as a component of the sclerotinia rot of carrot (SRC) forecast model is currently done using the blue plate test (BPT), which uses a semi-selective medium. A quantitative polymerase chain reaction (qPCR) assay was developed to reduce the time to quantify ascospores of Sclerotinia sclerotiorum (Lib.) de Bary from air samples collected using a Burkard Sampler. The qPCR assay was highly sensitive, detecting DNA from 0.5 to 5 x 10⁴ ascospores within a linear range (R ² = 0.99). The test was used to quantify ascospores of S. sclerotiorum in air samples collected over three growing seasons. SRC was observed from 10 to 35 days following detection of two to four ascospores m⁻³ of air. Results from air samples collected using an Andersen Sampler and the qPCR assay were compared to the BPT. Ascospore counts from the Burkard Sampler and the BPT followed similar trends, and fewer ascospores were consistently detected from the Anderson Sampler. Three days are required to confirm the number of ascospores using the BPT, while results from a Burkard Sampler coupled with a qPCR assay can provide results within five hours of air sampling. The choice of method will depend on the available resources and the need for a quick result.

Flooding reduces survival of Leptosphaeria spp. on canola stubble. C. PELUOLA, W. G. D. FERNANDO, C. HUVENAARS, H. R. KUTCHER AND G. PENG. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; (C.H., H.R.K.) Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada; and (G.P.) Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Blackleg, caused by Leptosphaeria maculans (Desmaz.) Ces. & de Not., is an important disease of canola and rapeseed (Brassica napus L.) in many countries, including Canada. However, L. maculans has not been reported in China, where blackleg is caused by a less aggressive pathogen, L. biglobosa R.A. Shoemaker & H. Brun. Strategies that mitigate the risk of blackleg epidemics may help alleviate China's concern about accidental introductions of L. maculans. In China, winter rapeseed is the main oilseed crop followed often by a paddy rice or cotton. Paddy rice fields normally are flooded for weeks during late spring and summer. This study was conducted to assess the effect of flooding temperature (12–40 °C) and duration (2–12 weeks) on survival of Leptosphaeria spp. in canola stubble. Pieces of infested stubble were submerged in water in small glass jars containing 20 cc soil on a thermogradient plate capable of maintaining up to 96 independent temperature regimes simultaneously. Flooded stubble pieces were sampled every 2 weeks, surface sterilized, and incubated on V8-juice agar for 10 days to recover the pathogen. Flooding for 2 weeks reduced the pathogen recovery substantially relative to non-flooded controls, irrespective of temperature and no pathogen was recovered after 6 weeks of flooding. The pathogen was eliminated more rapidly at flooding temperatures >20 °C compared to 12–16 °C. There was no difference between L. maculans and L. biglobosa in ability to survive flooding. Stem tissues degraded rapidly during the first 2 weeks of flooding, corresponding to a rapid decline in pathogen survival in the same interval. These results indicate that a paddy rice crop following winter rapeseed should minimize the impact of blackleg by eradicating the inoculum of Leptosphaeria spp. in stubble.

Assessment of crop rotation and host resistance in combination with Bacillus subtilis for management of clubroot on canola. G. PENG, R. LAHLALI, D. PAGEAU, S. F. HWANG, R. K. HYNES, K. ANDERSON, M. R. MCDONALD, B. D. GOSSEN, S. E. STRELKOV, T. K. TURKINGTON, F. Q. YU, K. C. FALK, S. M. BOYETCHKO, L. MCGREGOR, D. HUPKA AND J. GEISSLER. Saskatoon Research Centre, Agriculture and Agri-Food Canada (AAFC), 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (D.P.) AAFC Research Farm, 1468, Saint-Cyrille St., Normandin, QC G8M 4K3, Canada; (S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road, Edmonton, AB T5Y 6H3, Canada; (K.A.) Bayer CropScience, 295 Henderson Drive, Regina, SK S4N 6C2, Canada; (M.R.M.) Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; (S.E.S.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (T.K.T.) Lacombe Research Centre, AAFC, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada

Two field studies were conducted to assess the effect of crop rotation and cultivar resistance for management of clubroot on canola, together with potentially complementary biofungicide (Bacillus subtilis). In the first study, two granular formulations of B. subtilis were applied in-furrow at 50 kg/ha on resistant and susceptible commercial cultivars in three field trials near Leduc and Edmonton, Alberta and Normandin, Quebec. In a second study, B. subtilis was applied as seed dressing at 1 × 10⁵ to 5 × 10⁶ cfu/seed and evaluated on a susceptible cultivar in plots with a 1, 3, or 11-year break from the previous canola crop in Normandin. Clubroot disease pressure was high in each of the trials, with disease severity indices (DSI) ranging from 69% to 98% on the susceptible cultivar. None of the B. subtilis formulations reduced clubroot substantially in either study. In the first study, the resistant cultivar reduced DSI to below 15% and doubled the yield over that of the susceptible cultivar. In the second study, there was a higher level of pathogen inoculum in plots with a 1-year break from canola than in the other two rotations, based on bioassay and qPCR assessment. Although DSI for all of the rotation treatments were high, approaching 100% in 1-year-break plots, longer crop rotations (3 and 11 years) reduced gall size slightly, showed less above-ground disease impact on canola plants, and yielded significantly more than the 1-year rotation. Even the 3-year break from canola alleviated the impact of clubroot substantially, with seed yield doubled when compared to the 1-year break.

DNA polymorphism in the stem and bulb nematode Ditylenchus dipsaci in garlic in Ontario. Y. QIAO, M. ZAIDI, Q. YU, A. BADISS, B. HUGHES AND M. J. CELETTI. Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; (B.H.) New Liskeard Agricultural Research Station, University of Guelph, New Liskeard ON P0J 1P0, Canada; and (M.J.C.) Ontario Ministry of Agriculture, Food and Rural Affairs, Room 3110, Edmund Bovey Building, Guelph, ON N1G 2W1, Canada

Stem and bulb nematode, Ditylenchus dipsaci (Kühn) Filipjev, is a serious pest in many crops including garlic. It is internationally regulated in many crops but not in garlic in North America. A survey for the presence of stem and bulb nematode in garlic was conducted throughout Ontario during 2011. The results indicate it is widespread in the garlic producing areas in the province. Over 90 nematode isolates were collected from garlic samples and the identification of Ditylenchus species was confirmed based on morphological characteristics and the sequencing of the ITS and 18S regions. The genetic diversity and phylogeny of the 90 garlic isolates as well as 1 isolate from onion and 1 isolate of D. destructor as the out-group species were investigated based on random amplified polymorphic DNA (RAPD). One primer screened from 21 RAPD primers was used in the PCR amplification. Hierarchical cluster analysis was performed to demonstrate the intraspecies and interspecies relationships. The average coefficient (SM) among isolates of D. dipsaci was 71.6%, which was much higher than the average similarity among interspecies (57.0%). All the isolates were separated into 2 clusters: a small cluster consisting of 19 isolates from counties of the southern part of the province, and a large cluster with a high degree of genetic similarity (>0.81) was mostly from the eastern region. The DNA sequencing of all garlic isolates were different from the onion isolate tested. More studies are required to determine if the stem and bulb nematode isolates from Ontario garlic will also infest onion. No correlation was found between the DNA sequence of nematode isolates and the garlic cultivars.

Prevalent races of Puccinia helianthi on sunflower in Manitoba. K.Y. RASHID. Morden Research Station, Cereal Research Centre, Agriculture and Agri-Food Canada, Unit 100 – 101 Route 100, Morden, MB R6M 1Y5, Canada

Sunflower rust [Puccinia helianthi Schwein] is a major disease affecting sunflower (Helianthus annuus L.) worldwide. This rust is macrocyclic, autoecious rust, on sunflower, and readily produces new races. Rust infections drain the plant of its energy, cause leaf burning and defoliation and the disease reduces the yield and seed quality. Infected leaves were collected from 20-60 sunflower crops from 2003-2011 in Manitoba. Urediospores were suspended in petroleum oil and inoculated onto the seedlings of the international set of nine rust differential genotypes under controlled growth room conditions. The resistant/ susceptible reactions to each isolate determined the identity of the races based on the international nomenclature system for sunflower rust. Severe rust epidemics occurred in 2002, 2003, 2006, 2008, and 2009. A shift occurred in the prevalent races from the low virulent race groups, 100 and 500, to the highly virulent race groups, 300 and 700. Races 324, 326, 327 and 336 were prevalent up to the year 2008, after which races 726, 736, 776, and 777 became the most prevalent. The most virulent race 777 appeared in 2009 as 23% of the isolates collected, but was not detected in 2010 and 2011 due perhaps to the lack of fitness in this race and a sharp decline in the sunflower acreage.

Fine scale clubroot mapping in Alberta. D. C. RENNIE, J. M. LEBOLDUS, T. K. TURKINGTON AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (T.K.T.) Lacombe Research Centre, Agriculture and Agri-Food Canada, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada; and (J.L.) North Dakota State University, 1340 Administration Avenue, Fargo, ND 58102, USA

Clubroot [Plasmodiophora brassicae Woronin] is a significant pathogen of crucifers production in Alberta. Management of the disease by wide-scale application of pesticides is both financially and environmentally costly. Clubroot mapping would enable precise application of pesticides, alleviating these significant costs. Directly assessing plant symptoms (plant pulling) in the field can guide pesticide application, however this direct assessment of plant symptoms is not proactive. By using polymerase chain reaction techniques combined with geoprocessing methods, we were able to develop a method of finely resolved clubroot mapping. The resolution of these maps is dependent on the number of sample points; we utilized 50 and 100 sampling points to gauge the distribution of the pathogen in variously sized farm and research plots. Our mapping suggests that patches with elevated resting spore numbers exist in many of the farm fields, but resting spores can be detected at low levels at other points in the fields.

Dust monitoring for clubroot [ Plasmodiophora brassicae ] in Alberta. D. C. RENNIE, T. K. TURKINGTON, J. M. LEBOLDUS, R. J. HOWARD, H. R. KUTCHER AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (T.K.T.) Lacombe Research Centre, Agriculture and Agri-Food Canada, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada; (J.L.) North Dakota State University, 1340 Administration Avenue, Fargo, ND 58102, USA; (R.J.H.) Crop Diversification Centre South, Alberta Agriculture and Rural Development, 301 Horticultural Station Road East, Brooks, AB T1R 1E6, Canada; and (H.R.K.) Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada

Clubroot [Plasmodiophora brassicae Woronin] is a biotrophic, soil-borne disease of many Brassicaceae species. The extent of spread in Alberta is an indication that long range dispersal mechanisms exist, yet most hypotheses have not been supported by data. Wind borne dust is likely a factor in the spread of P. brassicae resting spores. However, the significance of this mechanism in commercial production is unknown. Big springs number eight (BSNE) samplers were deployed at five fields in central Alberta and one field in southern Alberta. Three fields were in commercial crop production, while three were clubroot research plots. BSNE samplers collected dust at five different heights at four to five different locations at each site. Accumulated dust was collected at biweekly intervals and analyzed for both dust loads and resting spore presence/quantity. Data from 2011 indicated that clubroot resting spores can be moved in wind borne dust and dust loads varied according to sampler height. Clear relationships between dust quantity, BSNE height or position, and resting spores could not be determined. This project is continuing into 2012 with an optimized sampler arrangement and positioning. Soil saltation samplers and PM ₁₀ air monitoring stations are being employed for 2012 to improve data collection.

First report and management of basil downy mildew in Ontario. C. SAUDE, S. WESTERVELD, M. FILOTAS AND M. R. MCDONALD. Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph ON N1G 2W1, Canada, and (S.W., M.F.) Ontario Ministry of Agriculture, Food and Rural Affairs, Simcoe Resource Centre, 1283 Blueline Road, Simcoe ON N3Y 4N5, Canada

Basil (Ocimum spp.) is one of the most commercially significant fresh culinary herb crops worldwide. Basil infection by Peronospora belbahrii, the causal agent of basil downy mildew has been reported since 2001, and it was first reported in the field in Ontario in 2010. Disease symptoms include clear to black sporulation on the lower leaf surface, yellow discoloration on the upper surface, and leaf senescence. In 2011, infected leaves were collected from basil grown at Simcoe, ON. Sporangia (n = 50) dislodged from leaf lesions were round, olive to brown color, 25-35 μm long, 20-30 μm diameter. Sporangiophores (n = 15) were hyaline, 150-360 μm long and ended in branchlets 5-27 μm long. Pathogenicity of P. belbahrii was tested on leaves of 5-week-old Basil ‘Genovese’ plants inoculated with a 1 x 10⁵ conidial suspension/mL and kept in a growth chamber maintained at 23/18 °C, 60-80% relative humidity and 12/12 h light/dark. Non-inoculated plants served as controls. Typical basil downy mildew symptoms developed after 8 days on the inoculated plants and the non-inoculated controls remained healthy. The DNA of 10 isolates was extracted and the ITS region sequenced using ITS4 and ITS5 primers. The obtained sequences showed 98-100% similarity with sequences of P. belbahrii isolated from Florida, Hungary and Germany.

Comparison of basil varieties and fungicides for management of basil downy mildew in Ontario. C. SAUDE, S. WESTERVELD, M. FILOTAS AND M. R. MCDONALD. Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph ON N1G 2W1, Canada, and (S.W., M.F.) Ontario Ministry of Agriculture, Food and Rural Affairs, Simcoe Resource Centre, 1283 Blueline Road, Simcoe ON N3Y 4N5, Canada

Basil (Ocimum spp.) is a herb crop widely cultivated and used as a flavoring, spice, health product, fragrance, and medicine. Basil downy mildew, caused by the Oomycete Peronospora belbahrii, is a destructive disease of basil. Once symptoms develop, plants are no longer marketable. Basil downy mildew was reported in mainland United States in 2007 and in Ontario in 2010. In field trials established in Simcoe, ON, the virulence of P. belbahrii on 18 basil cultivars and the efficacy of six fungicides were evaluated. Products were applied weekly and downy mildew was assessed weekly by counting the number of lesions per leaf and using the lesion numbers in a rating scale where 0 = no lesions, 5 = >5 lesions/plant. The rating values were used to calculate the disease severity index (DSI). All cultivars were susceptible to downy mildew, but cultivars ‘Medinette’ and ‘Mrs. Burns’ had slightly less disease. The fungicides Zampro (ametoctradin+ dimethomorph), QGU42 (experimental) and Presidio (fluopicolide) reduced downy mildew and increased yields. Treatments with the biofungicides Regalia (Reynoutria sachalinensis extract 20%), Serenade Max (Bacillus subtilis 7.3 x 10⁹ CFU/g) and Organocide (Sesame oil 5%) slightly reduced downy mildew on cultivar ‘Genovese’, but on cultivar ‘Sweet Basil’, these treatments were not significantly different from the untreated control.

Effect of clubroot [ Plasmodiophora brassica e] on shoot and root weight in Brassica crops. K. SHARMA, B. D. GOSSEN, T. GLUDOVACZ, A. DEORA AND M. R. MCDONALD. Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; and (B.D.G.) Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Information on the impact of clubroot [Plasmodiophora brassicae Woronin] on crop growth and yield is limited and often contradictory. There is a consistently-held assumption that larger plants produce larger clubs, so gall size is flawed indicator of susceptibility or impact on yield. The effect of P. brassicae on root and shoot weight was evaluated on canola (Brassica napus L.) and selected vegetable Brassica crops (all B. oleracea L. except Shanghai pak choy, B. rapa L.) under controlled and in field conditions. At 6 weeks after seeding/inoculation or at crop maturity, plants were uprooted, separated by clubroot severity class (0–3), and assessed for shoot and root weight relative to resistant cultivars or non-inoculated controls. In resistant cultivars of canola, broccoli, and Brussels sprouts there was a positive correlation between shoot and root weight, but there was no correlation in resistant cultivars of cabbage. In field trials, clubroot reduced shoot weight and increased root weight in susceptible cultivars relative to resistant cultivars in each crop species. In infected plants of susceptible cultivars, root weight was not correlated with shoot weight in canola, or broccoli, but there was a positive correlation in Brussels sprouts, Shanghai pak choy, kale and one of two cabbages. Clubroot increased the root: shoot ratio in canola but did not have a consistent effect in vegetable Brassica crops.

Mating type genes in Pseudonectria buxi . F. SHI AND T. HSIANG. School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Volutella blight of boxwood caused by the fungus Pseudonectria buxi (DC.) Seifert, Gräfenhan & Schroers has seen in Ontario since over 10 years. Although the sexual stage of P. buxi has been observed in Europe and the U.S., no sexual structures have been found on samples collected in southern Ontario. In Ascomycetes, fungal sexual reproduction is controlled by two mating-type idiomorphs called MAT1-1 and MAT1-2. The complete genome of an Ontario isolate of P. buxi was sequenced in this lab, and was found to have a MAT1-2 idiomorph. Primers were designed from this sequence, and DNA of 16 out of 28 isolates was amplified. To determine the content of the MAT locus from the remaining 12 isolates, the neighboring genes were used. The MAT locus in P. buxi as with other Sordariomycetes lies between conserved homologs of APN2 and SLA2. Primers were designed to amplify a 12 kb fragment between APN2 and SLA2, and a conserved MAT1 primer was used in semi-nested PCR to amplify a 597 bp fragment that was shown to match MAT1-1 from other fungi. Sexual reproduction of P. buxi thus may be possible in nature, and may occur more frequent than observed because of the 16:12 ratio of mating type genes.

Role of bacterial exopolysaccharides and monosaccharides in Erwinia amylovora resistance to bacteriophages. D. R. SJAARDA, D. R. ROACH, A. I. YAGUBI, A. J. CASTLE AND A. M. SVIRCEV. Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St. Catharines, ON L2S 3A1, Canada; and (A.M.S.) Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, Vineland Research Farm, 4902 Victoria Avenue North, P.O. Box 6000, Vineland, ON L0R 2E0, Canada

Fire blight is a disease caused by the phytopathogenic bacterium Erwinia amylovora (Burrill) Winslow et al., an economically important pathogen in the commercial production of apples and pears. Bacteriophages have been proposed as a commercial biopesticide to relieve the pressures on apple and pear production and provide alternatives to existing biological control options. Concerns of phage resistant bacteria resulting from use of phages-based biopesticides require further research investigate mechanisms of phage resistance. Erwinia amylovora produces a loose capsule of two exopolysaccharides (EPS), amylovoran and levan, which are involved in the bacterium's virulence. EPS deficient bacterial mutants were generated through recombineering to investigate the role of EPS in E. amylovora bacteriophage adsorption and infection. Erwinia amylovora mutants that were deficient in amylovoran production were avirulent and resistant to infection by Podoviridae phages. Levan deficient bacterial mutants resulted in reduced phage titers in some phages from the Myoviridae family. Exopolysaccharide mimetic monosaccharides were used to further demonstrate that levan and amylovoran play an important role in phage attack of E. amylovora. Bacteriophages of E. amylovora may bind to the bacterial EPS as a first step of phage infection.

Distribution of dogwood anthracnose in Canada. M. STANESCU AND T. HSIANG. School of Environmental Sciences, University of Guelph, 50 Stone Road, Guelph, ON N1G 2W1, Canada

Dogwoods (Cornus spp.) play important roles in the forest ecosystems of North America and are also valued as landscape ornamentals. The most devastating disease of dogwood is anthracnose caused by the fungus Discula destructiva Redlin, which belongs to the Ascomycete order Diaporthales. In North America, the disease has been reported on two major dogwood species: Cornus florida (flowering dogwood) in the east and C. nuttallii (Pacific dogwood) in the west. Infected trees have reduced esthetic value and reduced fruit production, and severe forms of the disease can kill entire trees. Previous reports of the disease in Canada have relied on symptoms and cultural morphology of the fungus. This study aimed to confirm the distribution of dogwood anthracnose in Southern Ontario with molecular methods, and examine disease development. A total of 18 sites were sampled for dogwood anthracnose across SW Ontario in 2010 and 2011, covering five counties. Only two counties were found to be positive for D. destructiva: Middlesex and Norfolk. The pathogen was isolated from symptomatic samples collected in Ontario in early July to early September, and most commonly from early July until early August. From British Columbia, the fungus has been isolated from samples of C. nutallii collected throughout the growing season, from flowers, leaves and twigs.

Factors affecting the transmission, detection, and management of Plum pox virus . L. W. STOBBS, D. T. LOWERY, N. GREIG, P. M. VICKERS AND L. A. BITTNER. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada (AAFC), Vineland Research Farm, 4902 Victoria Avenue North, P.O. Box 6000, Vineland, ON L0R 2E0, Canada; and (D.T.L.) Pacific Agri-Food Research Centre, AAFC, 4200 Highway 97, South, Summerland, BC V0H 1Z0, Canada

Unlike most members of the family Potyviridae, Plum pox virus (PPV) is a disease primarily of Prunus trees. We have infected herbaceous plants in the laboratory, but extensive testing of plants in and around commercial peach orchards did not identify any naturally infected herbaceous hosts. Woody perennials are likely to invest more energy in disease defense mechanisms and there is less urgency for the virus to quickly acquire new hosts. As a consequence, PPV infection results in delayed symptom expression and a relatively slow decline in tree vigour. Delayed and mild expression of symptoms makes early detection and removal of infected trees difficult and has likely contributed to the spread of PPV throughout the world in infected nursery material. We have demonstrated that the susceptibility of Peach (Prunus persica) trees varies throughout the season. Detached peach leaves were sampled biweekly over the growing seasons from 2008-2012, each inoculated with 25 viruliferous aphids and maintained in culture plates for 4 weeks. When assayed, leaves sampled from mid to late summer demonstrated reduced susceptibility to infection with PPV. Biweekly testing of new season growth in peach orchards showed that virus was predominately found in basal leaves on the lower one third of the shoot, and it is suggested that these leaves should be targeted when sampling for infected trees. As shoots elongated over the summer, PPV was generally restricted to the lower part of the shoots. In plum and nectarine, PPV was detected further up the shoot. Studies on dormant wood demonstrated that cambial tissues provided better virus detection than bud tissues in peach varieties. Seasonal changes in susceptibility of peach limits the spread of PPV by aphids mostly to a brief period in spring, which explains why the relatively high rate of transmission achieved in the laboratory is not reflected in a rapid yearly increase in disease incidence in the field. Numbers of aphid vectors occurring in spring when trees are most susceptible to infection has also been reduced by the recent introduction of the Multicolored Asian Ladybeetle (Harmonia axyridis). Control measures involving the use of foliar oil sprays are most effective when applied during the short period of time before trees become resistant and after aphid vectors have begun flying. Future research is required to better explain the mechanisms of host resistance to PPV and to assist in the development of treatments that might induce resistance earlier in the season or contribute to other means of control.

Screening house-keeping genes for phylogenetic differentiation of subspecies of Pantoea stewartii . J. T. TAMBONG, N. LOAIZA, R. XU, E. SANCHEZ, L. GOMEZ-ALPIZAR, C.-A. KANEZA AND L. BEAUPRÉ. Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; (N.L., E.S., L.G.-A.) Centro de Investigaciónes Agronomicas, Universidad de Costa Rica, Apdo. 2060 San Pedro, Montes de Oca, San José, Costa Rica; (E.S.) Centro de Investigación en Estructuras Microscópicas, Universidad de Costa Rica; and (C.-A.K., L.B.) La Cité Collégiale, 801 Aviation Parkway, Ottawa, ON K1K 4R3, Canada

Pantoea stewartii subsp. stewartii (Smith) Mergaert et al. is a quarantine-regulated pathogen of corn, while Pantoea stewartii subsp. indologenes Mergaert et al. is thought to cause leaf spot of foxtail millet and pearl millet and rot of pineapple. In 2011 using a polyphasic taxonomic approach, we proposed a third subspecies for isolates from Costa Rican peach palm. Traditional taxonomic differentiation of taxa using biochemical and plant pathogenicity tests requires specialized data interpretation and is prone to subjectivity. To circumvent these shortcomings, a multi-locus sequence screening and analysis approach was adopted and seven housekeeping genes (fusA, gyrB, leuS, pyrG, infB, rpoB, and rplB) were screened for their potential to discriminate the three proposed subspecies of P. stewartii. Analyses of a concatenated data matrix comprising all seven genes supported recognition of the subspecies previously differentiated using non-molecular data. Analyses of individual genes indicated that only rpoB and gyrB were highly discriminatory, leuS showed moderate discriminatory power, while the remaining genes were unsuitable as phylogenetic markers. Nucleotide sequences of rpoB and gyrB will be exploited for development of molecular tools for specific, rapid and reliable detection and identification of subspecies and species of Pantoea.

Fusarium head blight of barley – is there one best time to assess it? A. TEKAUZ, M. SMITH, M. STULZER AND M. BEYENE. Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada

Fusarium head blight (FHB) occurs annually on barley in Manitoba, Canada, and since 1994, its presence and severity has been documented from field surveys. Ultimate damage from FHB, including aberrant kernels, fungal seed infestation, and mycotoxin accumulation, is likely best measured at maturity; in mid-season, the Fusarium pathogens can be identified and disease severity (FHB-I) estimated. In 2010, ten barley crops were sampled mid-season and again at maturity to evaluate and compare FHB. Fusarium graminearum Schwabe dominated (70% of total Fusarium) in most mid-season crops, and FHB-I was estimated at 2.8%. The maximum severity (13.4%) was later found to have resulted from high seed-borne levels of Bipolaris sorokiniana (Sacc.) Shoem. At maturity, F. graminearum remained dominant (55%), but F. poae (Peck) Wollenw. rose from 12% to 27%. Discoloured fusarium damaged kernels (FDK) averaged 11.5% and reflected infection by both F. graminearum and B. sorokiniana. Deoxynivalenol (DON) levels were <1.0 ppm in most crops but reached 7.8 ppm in one; the mean was 1.5 ppm. DON accumulation was highest in two crops with the most F. graminearum. DON and F. graminearum levels at maturity were highly correlated (0.91; P < 0.001), and DON was also correlated with total Fusarium (0.70; P = 0.026). Neither FHB-I or FDK were correlated significantly (P <= 0.05) with any other FHB components. In 2010, the presence of B. sorokiniana in Manitoba confounded estimation of FHB in crops of barley. This pathogen should be considered when evaluating FHB in environments where both it and Fusarium can flourish.

Biocontrol products decrease germination and survival of sclerotia of Sclerotinia sclerotiorum and Sclerotium cepivorum . M. T. TESFAENDRIAS AND M. R. MCDONALD. Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Trials were conducted in organic soil in the Holland Marsh, Ontario to evaluate the efficacy of biocontrol products on germination, recovery and survival of sclerotia of Sclerotinia sclerotiorum (Lib.) de Bary and Sclerotium cepivorum Berk. Treatments were: Contans (Coniothyrium minitans 1 x 10⁶ spores/mL), Trichoderma atroviride A (1 x 10⁸ spores/mL), Trichoderma atroviride B (1 x 10⁸ spores/mL), Microsphaeropsis ochracea (1 x 10⁸) and untreated control. The biocontrol solutions were applied to soil in sacs containing 50 sclerotia of S. cepivorum, which were buried in an onion plot and recovered 2, 4 and 6 months later. Sclerotia of S. sclerotiorum were soaked in biocontrol solutions for 24 h and placed in grids on soil in a carrot crop. Germination was monitored weekly. Contans, T. atroviride A and T. atroviride B reduced germination of S. sclerotiorum sclerotia. No apothecia were formed in the Contans treated sclerotia and most of the sclerotia disintegrated over the season (88.7%) compared to all the other treatments (53 – 59%). There was no mycelial growth of recovered sclerotia of S. sclerotiorum treated with Contans, T. atroviride A and T. atroviride B. All the biofungicides reduced germination and survival of S. cepivorum sclerotia compared to the untreated control. Contans and both strains of T. atroviride reduced the survival of S. sclerotiorum and S. cepivorum.

Fusarium root rot and crown rot of carrots in Ontario and Prince Edward Island. M. T. TESFAENDRIAS, M. R. MCDONALD, M. M. MACDONALD, R. D. PETERS, T. BARASUBIYE AND K. A. SEIFERT. Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; (M.M.M., R.D.P) Crops and Livestock Research Centre, Agriculture and Agri-Food Canada (AAFC), 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; and (T.B., K.A.S.) Eastern Cereal and Oilseed Research Centre, AAFC, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada

Root rot and crown rot caused by Fusarium species was first identified on carrots in the field in Ontario in 2008 and in Prince Edward Island (PEI) in 2011. In PEI, the disease mainly affected the crown and in Ontario it affected both the crown and the root. Isolates from Ontario were identified as F. coeruleum Lib. ex Sacc. and those from PEI were identified as F. avenaceum (Fr.) Sacc. Fusarium species have not been previously recognized as causing disease of carrots in the field. In PEI during 2011, in some fields up to 70% of carrots were rejected due to crown decay. In Ontario, Koch's postulates were performed to confirm pathogenicity on carrots. Mycelial plugs of F. coeruleum grown on potato dextrose agar were placed on wounded and non-wounded carrot roots. Non-inoculated carrot roots served as controls. Lesions ranging from 9-41 mm and 3-12 mm developed on wounded and non-wounded carrot roots, respectively. Non-inoculated carrot roots remained healthy. Field trials were conducted at commercial carrot fields in Ontario to evaluate the efficacy of fungicides for control of fusarium root rot. Treatments Maxim XL (fludioxonil 21%) and Scholar (fludioxonil 230 g/L) were applied at, or shortly after seeding. An untreated control was also included. There were no differences in disease incidence and disease severity among the treatments.

Streptomyces biocontrol of thielaviopsis and colletotrichum root rot in tomato transplants and fusarium fruit rot in sweet bell pepper in greenhouses. J. A. TRAQUAIR, B. SINGH AND S. SABARATNAM. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada; and (S.S.) Abbotsford Agriculture Centre, British Columbia Ministry of Agriculture, 1767 Angus Campbell Road, Abbotsford, BC V3G 2M3, Canada

Thielaviopsis basicola (Berk. & Broom) Ferraris and Colletotrichum coccodes (Wallr.) Hughes can cause root rot and compromise the quality of greenhouse-grown tomato plug transplants. Fusarium solani (Mart.) Sacc. (teleomorph Nectria haematococca Berk. & Broome) infects wounds on the stems and fruit of greenhouse-grown bell peppers. Streptomyces griseocarneus Benedict et al. (isolate Di944) inhibits mycelial growth of Rhizoctonia solani Kühn and other fungal pathogens in dual culture and suppresses rhizoctonia damping-off in tomato plug transplants. Our objective was to extend the target efficacy of Di944 to thielaviopsis and colletotrichum root rot of tomato and to fusarium fruit rot of bell pepper. Rootballs of 4-week-old seedlings (cv. ‘Bonnie Best’) grown in Pro-Mix® previously amended with chitin and cells of Di944, were artificially infested with conidia of T. basicola or C. coccodes. Mechanical wounds on pepper fruit were treated with cells of Di944 for 24 h prior to infestation with conidia of F. solani. After 21 days in the greenhouse, the incidence of four levels of tomato root rot severity between zero and 75% was assessed. Rot by T. basicola ≥ 25% was reduced by 100% in tomato plugs, while incidence of root rot severity ≤ 25% was reduced by 13%. Incidence of C. coccodes rot severity ≥ 25% was reduced by 67%, while incidence of root rot ≥ 50% was reduced by 80%. Streptomyces applications to pepper wounds decreased the incidence of fusarium fruit rot by 100% after 21 days at 22 °C. These preliminary results indicate a potential for using S. griseocarneus (isolate Di944) as a supplement to commercial, peat-based potting media and for developing suitable formulations as protective sprays in organic pepper greenhouses.

Copper alternatives for the management of bacterial spot [ Xanthomonas gardneri ] and bacterial speck [ Pseudomonas syringae pv. tomato ] in processing tomatoes. C. L. TRUEMAN. University of Guelph, Ridgetown Campus, 120 Main Street East, Ridgetown, ON N0P 2C0, Canada

Bacterial spot [Xanthomonas gardneri syn. Xanthomonas campestris pv. vesicatoria Group D (Doidge) Dye] and bacterial speck [Pseudomonas syringae pv. tomato (Okabe) Young et al.] cause significant economic damage to tomatoes. Field trials were completed to evaluate the efficacy of copper alternatives for disease management in processing tomato cv. ‘H9909’ in 2010 and 2011. Nine and eight applications of standards Kocide 2000 (copper hydroxide) and Kocide 2000 + Dithane (mancozeb) were compared to alternatives applied alone, or tank-mixed or alternated with Kocide 2000. Products included Kasumin (kasugamycin) in 2010, Actigard (acibenzolar-S-methyl) in 2011, and Serenade Max (Bacillus subtilis) and Regalia Maxx (extract of Reynoutria sachalinensis) in both years. Applications of Kocide 2000 + Dithane, Kocide 2000, and Regalia Maxx + Kocide 2000 resulted in lower area under the disease progress curve (AUDPC) than the nontreated control in both years, the Kasumin + Kocide 2000 and Kasumin alternating with Kocide 2000 treatments in 2010, and the Serenade Max + Kocide 2000 and Actigard + Kocide 2000 treatments in 2011. Alternatives applied without Kocide 2000 did not suppress disease below levels observed in the nontreated control, and none of the Kocide 2000 tank mixes provided better control than a Kocide 2000-only program. Additional research on application timing and spray programs is ongoing.

The prevalence of different strains of Rhizoctonia solani associated with rhizoctonia crown and root rot symptoms in Ontario sugarbeet fields. C. L. TRUEMAN, L. E. HANSON AND J. E. LEBOEUF. University of Guelph, Ridgetown Campus, 120 Main Street East, Ridgetown, ON N0P 2C0, Canada; (J.E.L.) Ontario Ministry of Agriculture, Food, and Rural Affairs, 120 Main Street East, Ridgetown, ON N0P 2C0, Canada; and (L.E.H.) United States Department of Agriculture, 1066 Bogue Street, #494 Michigan State University, East Lansing, MI 48824, USA

Rhizoctonia crown and root rot (RCRR) [Rhizoctonia solani Kühn] is an important disease of sugarbeets in southwestern Ontario, Canada. A survey of commercial sugarbeet fields was completed in 2010 and 2011 to determine the range of R. solani anastomosis groups (AGs) and inter-specific groups (ISGs) associated with RCRR in this region. Soil immediately surrounding plants with RCRR symptoms was collected and stored at 4 °C. Sugarbeet cv. ‘Crystal 824’ was seeded in pots containing the collected soil, grown in a greenhouse, and monitored for damping-off. Rhizoctonia solani cultures from 55 and 45 soil samples were obtained in 2010 and 2011 respectively by isolating the fungus from symptomatic seedlings. Standard hyphal fusion assays showed that 69 and 80% of cultures isolated in 2010 and 2011 belonged to AG-2-2. ISG identification using differential temperature assays for 2011 samples is ongoing; however 35 and 24% of all isolates collected in 2010 were AG-2-2 IIIB and AG-2-2 IV. AG-2-2 IIIB is considered more aggressive than AG-2-2 IV, and has a wider host range that includes corn and soybeans. In fields where AG-2-2 IIIB is present, there may be a higher risk of losses from RCRR when host crops precede sugarbeet plantings in the crop rotation.

Evidence for tomato disease suppressive soils in southwestern Ontario. A. L. TURNBULL, K. DELANEY, A. MARSHALL, A. WOJCIK AND G. LAZAROVITS. A&L Biologicals, 2136 Jetstream Road, London ON N5V 3P5, Canada

Over two field seasons 19 commercial tomato field sites were visited; ten showed symptoms of tomato vine decline (TVD; a new field tomato disease complex) while nine were healthy fields for comparison. Bacteria were isolated from the rhizoplane of healthy and diseased plants and subsequently tested for antibiotic production against the fungal pathogens implicated in TVD. In 2010 diseased fields, 10% (19 of 192 isolates) of isolates had any level antibiotic activity, while 24% (31 of 128 isolates) had antibiotic activity in healthy fields. In 2011, there was a smaller difference in the proportion of antibiotic producing bacteria between healthy and diseased field (38% compared to 31%, respectively), however there were fewer colonies and less bacterial diversity observable in the diseased samples. Thus, 99 antibiotic producing bacteria were isolated from five healthy fields (n = 263 total isolates) while 55 were isolated from four diseased fields (n = 175 total isolates). Of the pathogens tested in both years (Rhizopycnis vagum Farr, Pyrenochaeta lycopersici Schneider and Gerlach, Collectotrichum coccodes (Wallr.) Hughes), the most frequent activity was observed against C. coccodes. Two isolates from 2010 have shown activity in diseased field soil by increasing shoot mass 22-38%. Additionally, pyrosequencing was done on the rhizoplane of 25 plants from five fields in 2010 (three diseased fields and two healthy fields).

Exploring spatial relationships between mutations related to fungicide resistance among Botrytis cinerea populations. H. VAN DER HEYDEN, P. DUTILLEUL, L. BRODEUR AND O. CARISSE. (H.V.D.H., O.C) Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, St-Jean-sur-Richelieu, QC J3B 3E6, Canada; (H.V.D.H., P.D.) Department of Plant Science, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, QC H9X 3V9, Canada; and (H.V.D.H., L.B.) Compagnie de recherche Phytodata Inc., 111 Rg. Saint-Patrice, Sherrington, QC J0l 2N0, Canada

Knowledge and monitoring of single nucleotide polymorphisms (SNPs) related to fungicide resistance are the foundation of modern anti-resistance strategies. The objectives of this project were to study the occurrence and distribution of different SNPs and to explore their spatial interactions within Botrytis cinerea Pers.:Fr. populations isolated from grapes. B. cinerea isolates were collected following a quadrat- based design (100 10 x 10 m quadrats) in two commercial vineyards. The presence of nine SNPs related to resistance to iprodione, boscalid, azoxystrobin and fenhexamid was detected using PCR-RFLP, AS-PCR and RT-qPCR assays. The data were spatially referenced and considered as bivariate point patterns. They were analysed by pairs, using an extension of Diggle's procedure for the analysis of nearest-neighbour distances. Our results show that 94.8%, 66.1%, 55.8% and 3.1% of the B. cinerea isolates were resistant to azoxystrobin, iprodione, boscalid, and fenhexamid, respectively. For boscalid, one of the five monitored SNPs was prevailing and reported to have a low resistance factor. The results also suggest that among the SNP pairs, some were exclusive while others were inclusive. The next steps will be to formally study the spatial pattern of SNPs related to fungicide resistance and to develop a sampling scheme based on the minimum sampling number required to estimate the mean incidence of these SNPs.

Development of genetic resistance to Plum pox virus . A. WANG AND D. C. W. BROWN. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, N5V 4T3, Canada; and (D.C.W.B.) Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, 351 Taché Avenue, Winnipeg, MB R2H 2A6, Canada

In 2000, shortly after Plum pox virus (PPV) was first detected in the Niagara fruit growing region of Canada, a national research program was initiated in support of the Canadian Food Inspection Agency-led program to eradicate the virus from the region. A biotechnology tool box for the development of genetic resistance to PPV has been developed. Progress on in vitro production of and gene transfer into Prunus species was advanced to the point where genetic sequences introduced into plum targeting PPV genomic sequences such as P1, HCpro and CP regions have been shown to be expressed and confer resistance to the virus after three years of testing. New molecular approaches in the design of genetic constructs have resulted in the increased ability to commercialise new plants coming out of these programs as well as introduce mechanisms for the control of pollen release from the plants. Along with the implementation of the new Plum Pox Monitoring and Management Program in 2011, our research program has been concentrated on developing durable virus resistance strategies. Significant progress has been made in generating Prunus mutants resistant to PPV, developing an attenuated recombinant virus against PPV through virus-induced gene silencing, and inducing PPV resistance in non-transgenic scion through transmissible RNA silencing signals produced in transgenic rootstock.

Production of 3-hydroxypropionaldehyde by Bacillus subtilis s train CU12. C. WISE, L. NOVITSKY, A. TSOPMO AND T. J. AVIS. Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada

Bacillus subtilis strains are known to produce a vast array of antimicrobial compounds. However, structures of all bioactive molecules remain to be elucidated. The objectives of this study were therefore to characterize the antifungal activity of B. subtilis strain CU12 and to isolate, purify, and characterize the compound(s) responsible for its activity. The antagonistic activity of B. subtilis strain CU12 tested in vitro showed a significant reduction in mycelial growth of Alternaria solani Sorauer, Botrytis cinerea Pers.:Fr., Fusarium sambucinum Fuckel, and Pythium sulcatum Pratt & Mitch. Crude B. subtilis culture filtrates were subsequently extracted with ethyl acetate and butanol. Thin layer chromatography combined with a direct bioassay revealed the presence of one major antifungal compound in the butanol extract. Isolation and purification of the compound were performed using reverse-phase C18 column chromatography. ¹H and ¹³C NMR data showed that the main antimicrobial compound was the cyclic dimer form of 3-hydroxypropionaldehyde (3-HPA). Other isomeric forms of 3-HPA were also present. This study indicates that 3-HPA could be at least in part responsible for the antimicrobial activity of B. subtilis strain CU12 against phytopathogenic microorganisms. This indicates that B. subtilis strain CU12 could be effective at controlling pre- and post-harvest pathogens through antibiosis.

Sequence analysis of CRISPR spacers in selected Erwinia amylovora isolates. A. I. YAGUBI, A. J. CASTLE AND A. M. SVIRCEV. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, Vineland Research Farm, 4902 Victoria Avenue North, P.O. Box 6000, Vineland, ON L0R 2E0, Canada; and (A.J.C.) Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St. Catharines, ON L2S 3A1, Canada

Phage-based biological control of the fire blight pathogen, Erwinia amylovora (Burrill) Winslow et al., shows promise in field based trials. Further research is required to understand the biological relationship between phages and their hosts. The emergence of phage-resistant bacteria due to the use of phage-based biopesticide is a valid concern. The various mechanisms by which E. amylovora may gain resistance against phages may include: adsorption blocks, the presence of restriction modification, abortive infections, lysogeny and CRISPR immunity. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are novel aspects of the bacterial defense system. Bacterial CRISPRs are sites containing conserved short direct repeats found in the genomes of some bacteria. The repeats are separated by sequences “spacers”. The spacers are found to match sequences in phage genomes. Spacers are utilized by the CRISPR system to recognize and silence exogenous genetic elements via a mechanism possibly similar to RNA-interference in eukaryotes. Bacterial hosts may acquire resistance or immunity against invading phages following previous exposure to the same phage. The role of the CRISPR system in protecting E. amylovora against phages has not been investigated. In the present study, the composition of CRISPR loci of nine different E. amylovora isolates was examined and the incidence of phage sequence integrated in CRIPR loci was explored.

Identification and mapping of clubroot resistance genes in Brassica species. F. YU, M. CHU, K. FALK AND G. PENG. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Clubroot, caused by Plasmodiophora brassicae Woronin, is one of the most economically important diseases in Brassica spp. worldwide, and an emerging threat to canola production in western Canada. Cultivar resistance is the cornerstone for effective management of clubroot on canola. A total of more than 1,000 accessions of Brassica spp. were evaluated and 34 were identified highly resistant to the pathotype 3 race of P. brassicae in B. rapa, B. oleracea, B. nigra and B. napus. Selected resistant lines from different Brassica spp. were crossed with respective susceptible doubled haploid (DH) lines and segregating populations (test-cross, F₂ and BC₁) were produced for identification and mapping of clubroot resistance (CR) genes. Parents, F₁, F₂ and BC₁ were evaluated for resistance to P. brassicae pathotype 3. DNA samples were analyzed with microsatellite markers. Single dominant resistance genes controlling clubroot disease were found in four of the lines, two in B. rapa (pak choy and Chinese cabbage) and two in B. nigra. Two resistance genes from the pak choy lines FN and Chinese cabbage line JNC, herein designated Rpb1 and Rpb2, respectively, were mapped to different genomic regions on B. rapa linkage group A3. Molecular markers closely linked to these CR genes were developed to facilitate marker-assisted breeding. Introgression of CR genes from the FN and JNC lines into canola (B. napus, B. juncea and B. rapa) and from B. nigra into B. carinata and B. juncea is in progress.

Characterization of major resistance genes against blackleg in Canadian canola germplasm. X. ZHANG, W. G. D. FERNANDO, H. R. KUTCHER AND G. PENG. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; (H.R.K.) Department of Plant Sciences, 3C02 Agriculture Building, 51 Campus Drive, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada; and (G.P.) Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Blackleg, caused by Leptosphaeria maculans (Desmaz.) Ces. & de Not., is the most economically important disease of canola in Canada. The disease is mainly controlled by cultivation of resistant cultivars and adequate crop rotation. In order to manage the disease efficiently, it is important to understand the occurrence of major resistance genes (R-genes) in canola germplasm. In this study, 14 L. maculans isolates harbouring different known avirulence genes were used as differentials to characterize the major resistance genes in Canadian canola cultivars/lines. Forty-nine canola cultivars/lines provided by five research institutions/companies were analyzed using a cotyledon-inoculation assay. The differential isolates detected the presence of R-genes Rlm1 to Rlm10 and RlmS. The results indicated that 16% of tested germplasm were fully susceptible to all isolates and had no R-genes, whereas 84% of the genotypes were resistant to at least one of the isolates indicating they had at least one R-gene. One canola cultivar from University of Manitoba was resistant to six of the L. maculans isolates, whereas the others showed resistance against 1 to 4 of the isolates. The most common R-genes in tested germplasm were Rlm1 (10%) and Rlm3 (69%), whereas other R-genes were not frequent. The range of resistance genes appears to be narrow in the canola genotypes evaluated.

Population structure of a Rhizoctonia solani population in a single field using ISSR markers. L. ZHENG, F. SHI AND T. HSIANG. Key Lab of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan, 430070, China; and (F.S., T.H.) School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Brown patch, caused by Rhizoctonia solani Kühn, is an important disease of cool-season grasses, including bentgrasses, bluegrasses, fescues, and ryegrasses. The objective of this work was to examine the genetic diversity and population structure of R. solani in a single bentgrass (Agrostis stolonifera) field. In August 2011, 23 isolates of R. solani were obtained with a toothpick method from 17 patches of infected creeping bentgrass at the Guelph Turfgrass Institute, Guelph, Ontario. Genetic variation was analyzed by using five inter-simple sequence repeat (ISSR) primers. The five primers generated 34 polymorphic loci out of 50 band positions (68.0% polymorphism). The size of amplified bands ranged from 400 to 2000 bp. Cluster analysis of the resulting data was calculated with the unweighted pair group method using arithmetic averages (UPGMA). The resulting dendrogram grouped the 23 isolates into four main clusters, with genetic distance (Dice coefficient) ranging from 0 to 12%. Almost all isolates from different parts of the same patch were clustered more closely and showed less genetic distance in the UPGMA tree. In the southeast corner of the plot area, a group of patches (#7-#12) in a 2.8 m by 1.2 m area contained ISSR-haplotypes that were also found in other areas of the field (54 m by 17 m). One explanation is that isolates of R. solani spread from this area to the other areas, which is consistent with the mowing pattern that usually starts in this corner. In conclusion, abundant genetic variation among R. solani isolates is present in a small field of creeping bentgrass, but the relationship of isolates as determined by ISSR fingerprinting revealed a population structure that showed evidence of transport between the patches.

Development of a quantitative PCR detection technique for Sclerotinia sclerotiorum on canola. B. R. ZIESMAN, T. K. TURKINGTON, U. BASU AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (T.K.T.) Lacombe Research Centre, Agriculture and Agri-Food Canada, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada

Sclerotinia stem rot is a sporadic disease of canola caused by the necrotrophic fungus Sclerotinia sclerotiorum de Bary. This disease can cause yield losses greater than 50% and the current primary control method used by growers is the application of fungicides, typically without any indication of potential disease risk. A reliable, timely forecasting method is needed as a tool to assist canola producers in western Canada in making spray decisions and to minimize the unnecessary application of fungicides. A quantitative PCR (qPCR) detection system based on the known correlation between petal infestation and final disease levels would fit the qualifications of a valuable decision making tool. A hypothetical novel protein that may serve as a virulence factor for S. sclerotiorum has been found to be orthologous to a secreted protein in Botrytis cinerea Pers.:Fr. and unique to these two fungi. A set of primers was designed that was specific to the form of the encoding gene found in S. sclerotiorum, and which was found to amplify S. sclerotiorum DNA with high specificity and sensitivity in conventional PCR assays. This primer set shows promise for the development of a qPCR detection technique that could be used for sclerotinia forecasting in Western Canada.

Notes

¹ This meeting was held jointly with the International Plum Pox Virus Meeting in Niagara Falls, Ontario on 24–27 June, 2012.