Annual Meeting, 2010/Réunion annuelle, 2010: The Canadian Phytopathological Society

Annual Meeting, 2010/Réunion annuelle, 2010: The Canadian Phytopathological Society ^†

Effect of multiple repeated applications of low rates of fish emulsion to soils from different commercial fields on potato scab and tuber yield. P. A. ABBASI. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada

Fish emulsion (FE) is a liquid co-product of fish processing and is mainly used as a foliar fertilizer. Addition of FE to pathogen-infested soils or substrates prior to planting has been reported to provide protection against some soil-borne diseases including potato scab. The broadcast rates (20,000 L ha⁻¹) of FE that effectively reduced scab symptoms on tubers can be too costly for commercial use. Long-term field and micro-plot trials were conducted to see if multiple repeated applications of much lower rates of FE could improve scab suppression and tuber yield of potatoes. Diluted FE (100 and 200 L ha⁻¹ or 0.05% and 0.1%) was applied to the same field and micro-plots twice a year after harvesting and before planting potatoes starting in autumn of 2007 for three years. The tubers harvested in 2008 and 2009 were rated for scab severity and their yields determined. In the field trial in both years, both rates (100 and 200 L ha⁻¹) of FE consistently reduced scab severity by 18–57%, reduced the percentage of tubers with deep-pitted scab by 18–51%, and increased tuber yield by 14–20%. In the micro-plots, FE (0.1% rate) consistently reduced scab severity in both years but only in one (BL site) of the four soils. No effect on tuber yield was observed in any soil in any year in the micro-plots. The number of soil bacteria including those of potential bio-control agents belonging to the genera Pseudomonas and Bacillus was higher in the FE-treated plots. FE is an excellent organic product to enhance biological control potential of natural soils by enriching soil microbes and generating disease suppressive conditions against soil-borne diseases. These results suggest that economically feasible rates of FE applied more frequently may provide longer lasting disease suppression.

Simple sequence repeats and diversity of globally distributed Pyrenophora tritici-repentis populations. R. ABOUKADDOUR, S. CLOUTIER, L. LAMARI AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada; (S.C.) Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada; and (L.L.) Department of Plant Science, University of Manitoba, 66 Dafoe Road, Winnipeg, MB R3T 2N2, Canada

Genetic variability and relatedness in Pyrenophora tritici-repentis (Died.) Drechs., the causal agent of tan spot of wheat, was studied in 80 isolates representing the eight races of the pathogen and originating from different regions of the world, including North America, North Africa, the Caucasus, and the Fertile Crescent. Thirty-one simple sequence repeat markers were designed to represent the genome of P. tritici-repentis and amplified 31 different loci in the isolates tested. Data analysis was carried out using: (i) Nei's analysis of genetic diversity, genetic differentiation and genetic distance, (ii) analysis of molecular variance (AMOVA), and (iii) clustering by the unweighted pair group method using arithmetic averages (UPGMA). A significant genetic differentiation (Φ_PT = 0.153, P ≤ 0.001) was detected among populations, with isolates from different origins having similar levels of genetic diversity. Genetic distance and cluster analysis based on pooled allele frequency revealed that isolates of P. tritici-repentis can be grouped into four distinct populations based on region of origin. The AMOVA analysis showed that North American isolates have less than 9% genetic dissimilarity with isolates from the Caucasus region, while they have up to 32% genetic dissimilarity with isolates from the Fertile Crescent. Isolates were also compared by race, genetic distance and cluster analysis based on pooled allele frequency, which revealed that Ptr ToxA non-producing isolates clustered closely to one another and were distantly related to Ptr ToxA-producing isolates. This suggests that the host-specificity imposed by the different Ptr toxins could lead to differentiation among races of P. tritici-repentis.

Deciphering plant – pathogen interactions applying metabolomics – principles and applications. K. A. ALIFERIS AND S. JABAJI. Department of Plant Science, McGill University, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada

Metabolomics has been defined as the comprehensive qualitative and quantitative profiling of a large number of metabolites of biological systems serving as the linkage between genotypes and phenotypes. Although metabolomics approaches have been successfully applied in almost all fields of sciences, its application in the study of plant–pathogen interactions is still in its infancy. Metabolomics is a holistic bioanalytical approach that includes not only chemical analyses and data mining but also experimental design and execution, chemical analyses of samples, and data pre-processing, analyses and biological interpretation. Thus, the success of a metabolomics experiment depends on all of the above components. Here we describe step-by-step a metabolomics protocol for the study of plant–pathogen interactions in the pathosystems potato (Solanum tuberosum L.)–Rhizoctonia solani AG3 and soya (Glycine max)–R. solani AG4. Key factors that should be taken into consideration during the execution of experiments and data analyses, as well as freely available sources for metabolomics are presented.

Construction of subtractive cDNA library and identification of differentially expressed genes from wheat ‘Sumai3’ under Fusarium graminearum infection . K. AL-TAWEEL, X. GUO, W. G. D. FERNANDO AND A. BRÛLÉ-BABEL. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada

Fusarium head blight (FHB), primarily caused by Fusarium graminearum Schwabe, is a destructive disease of wheat (Triticum aestivum L.). Although several genes related to FHB resistance have been reported, global analysis of gene expression in response to FHB infection remains to be explored. To identify differentially expressed genes corresponding to FHB resistance, wheat spikes of FHB-resistant cultivar ‘Sumai3’ was inoculated with a macroconidia spore suspension of F. graminearum and collected at 6, 12, 24, 36, 48 and 72 h, and six days after inoculation. A full-length cDNA library was constructed using pooled mRNA isolated from the infected samples. Suppressive subtractive hybridization (SSH) was carried out using pooled mRNAs from the infected (tester) and the water-inoculated (driver) samples. The subtracted cDNA library was differentially screened by Dot Blot hybridization using forward and reverse subtracted cDNAs as probes. Clones that showed differential signals based on their specific hybridization with the probe of the SSH tester but not the driver were isolated and sequenced. Sequence analysis showed key genes related to the host–pathogen interaction including the deoxynivalenol detoxifying UDP-glucosyltransferase gene which is developmentally regulated and induced by deoxynivalenol as well as salicylic acid, ethylene and jasmonic acid.

Efficacy of fungicides on 3ADON and 15ADON Fusarium graminearum isolates infecting spring wheat. C. AMARASINGHE, L. TAMBURIC-ILINCIC, 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; and (L.T.-I.) Department of Plant Agriculture, University of Guelph, Ridgetown Campus, 120 Main Street East, Ridgetown, ON N0P 2C0, Canada

Fusarium graminearum Schwabe causes fusarium head blight (FHB), an important disease in wheat. Deoxynivalenol (DON) is the most frequently produced mycotoxin. There are two chemical derivatives of DON, 15ADON and 3ADON. The more virulent 3ADON chemotype is becoming more prevalent than the less virulent 15ADON chemotype in North America. This chemotypic shift may influence current FHB management strategies such as fungicide applications. The fungicides Folicur, Proline, Prosaro and Caramba are used to control FHB. The objectives of this study were (1) to investigate the efficacy of fungicides on the 3ADON and 15ADON chemotypes and their influence on DON accumulation and (2) to determine whether there is a correlation between total DON content and quantity of F. graminearum DNA in infected wheat grains. All four fungicide treatments significantly reduced the FHB index, FDK and DON and increased yield in ‘Glenn’ (moderate resistance) compared with the fungicide control. Prosaro, Proline and Caramba were more effective than Folicur in reducing the FHB index, FDK and DON, and increasing the yield. In ‘Roblin’ (highly susceptible) although all fungicides significantly reduced the FHB index, FDK and DON, and increased the yield compared with the fungicide control, fungicides were not as effective as on ‘Glenn’, confirming that the host resistance plays an important role in host–pathogen–fungicide interaction. No significant differences were found in the management of 3ADON and 15ADON chemotypes with respect to the fungicide treatments. Therefore all four fungicides can be used to control FHB regardless of the F. graminearum chemotype in that particular field or region. Strong positive correlations were found between the FHB index and DON (R = 0.85), FDK and DON (R = 0.88). Real-time PCR is being used to determine whether fungicides are controlling the amount of pathogen biomass in infected grains and if there is a correlation between the amount of pathogen DNA and DON content in infected grains.

Pest Management Centre – successes in plant pathology. K. BEDFORD AND C. KORA. Pest Management Centre, Agriculture and Agri-Food Canada. 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada

The Pest Management Centre's Minor Use and Pesticide Risk Reduction Programs work together to improve grower access to new minor uses and reduced risk pest management tools in grower-identified priority areas. Since its inception, the Minor Use Pesticides Program has completed over 65 submissions for new uses of 23 fungicides. In 2009–2010, 20 new submissions were completed and 15 registrations resulting in 23 new uses were registered. The Pesticide Risk Reduction Program has provided funding for 98 projects totalling about $ 6.1M to address priority plant disease issues in a number of crops. Highlights of a few of many successful outcomes achieved to date through the Pest Management Centre's activities in the disease management area are presented.

Identification of quantitative trait loci for tolerance against sclerotinia stem rot in three double haploid populations of Brassica napus . R. S. BEHLA, W. G. D. FERNANDO, P. B. E. MCVETTY AND G. LI. Department of Plant Science, University of Manitoba, 222 Agriculture Building, 66 Dafoe Road, Winnipeg, MB R3T 2N2, Canada

Sclerotinia stem rot (SSR) is a fungal disease of Brassica napus L. Four methods, detached leaf, cotyledon inoculation, stem inoculation and petiole inoculation, were compared to find the most suitable method. The petiole inoculation method was identified as most appropriate. We identified quantitative trait loci (QTL) against SSR through 3 DH populations (H1, H2 and H3). The genetic maps were prepared using SRAP markers. 462 SRAP markers were allocated to 19 linkage groups (LG) in H1with total genetic distance covering 1374.2 cM. In H2, 487 SRAP markers covered a total genetic distance of 1333.4 cM in 19 LG. The H3 map with 1055 SRAP markers covered a total genetic distance of 1604.8 cM in 19 LG. The phenotypic evaluation of H1 and H2 showed significant differences among the replicates (Rep). QTL analyses for these populations were done on each replicate. In H1, 5 QTL in Rep1, 4 in Rep 2 and 3 in Rep 3 were identified. In H2 population, 3 QTL in Rep 1, 4 in Rep 2 and 4 in Rep 3 were identified. The phenotypic evaluation of H3 did not show significant differences among the replicates and two QTL were identified on chromosome N12 and N16.

Structural and functional genomics of the Dutch elm disease fungus Ophiostoma novo-ulmi . L. BERNIER, V. JACOBI, J. DUFOUR, K. V. PLOURDE, G. F. BOUVET, W. E. HINTZ, R. C. LEVESQUE, V. FORGETTA AND K. DEWAR. Institut de Biologie Intégrative et des Systèmes, Université Laval, Québec, QC G1V 0A6, Canada; (W.E.H.) Department of Biology, Centre for Forest Biology, University of Victoria, Victoria, BC V8W 3N5, Canada; and (V.F., K.D.) Department of Human Genetics, McGill University, Montréal, QC H3A 1A4, Canada

The ascomycete fungus Ophiostoma novo-ulmi Brasier is the causal agent of the second pandemic of Dutch elm disease, a vascular wilt that has devastated elm populations worldwide. Genetic, molecular and genomic approaches are being used to unravel the mechanisms of parasitic fitness in this highly aggressive pathogen. The genome of O. novo-ulmi reference strain H327 was sequenced using a combination of whole genome shotgun and 8 kb paired ends using the Roche/454 system. Total length of the nuclear genome is 31.8 Mb, distributed over eight scaffolds, with over 8400 putative genes identified in silico. A genetic map consisting of 200 markers was constructed and selected markers were hybridized to chromosomes separated by pulsed-field gel electrophoresis. Several types of mobile elements were identified. Over 3000 expressed sequence tags (ESTs) were recovered from expression libraries constructed from mycelium, yeastlike cells, synnemata and perithecia. Strains altered for mycelial or yeast growth kinetics, production of protoperithecia, or virulence were recovered after insertional mutagenesis of strain H327. Mutated genes are being further characterized, as well as selected genes identified through EST analysis and annotation of the genome. Additional tools are being developed for targeted gene deletion, which remains difficult to achieve in O. novo-ulmi.

Genetic diversity of pathotypes of Plasmodiophora brassicae , causal agent of clubroot of crucifers, from Canada. T. CAO, S. XUE, S. F. HWANG, D. AN AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; and (D.A.) Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050021, China

Plasmodiophora brassicae Woronin is a soilborne pathogen causing clubroot of crucifers. Numerous pathotypes have been identified on canola in Alberta, which complicates efforts to develop clubroot-resistant cultivars. Investigations on DNA polymorphisms were carried out on 28 isolates of the pathogen, in order to identify possible cleaved amplified polymorphic sequence markers. The P. brassicae-specific primer pairs PbsF1/PbsR1 and PbsF2/PbsR2, which were based on the small subunit ribosomal RNA gene, amplified a PCR product approximately 1.6 to 2.1 kb in size from galled roots. Digestion of the amplicon with MluI and PvuII yielded two major bands from tissues infected with pathotype P₃, whereas five bands were observed from pathotypes P₂ and P₆. Sequence analysis of the P₃ amplicon revealed the presence of a 374-bp intron, which was absent in the amplicons from pathotypes P₂ and P₆. The P. brassicae-specific primer pair PbSerF/PbSerR, derived from a serine protease gene, consistently yielded no PCR products from isolates CDCN-ss1, CDCN-ss3 or ORCA-ss5, in contrast to the strong 2 kb product amplified from the remaining 25 isolates. Another set of P. brassicae-specific primers, PbHypF/PbHypR, based on a gene encoding a hypothetical protein, consistently yielded a 3 kb PCR product from most isolates except CDCN-ss1 and ORCA-ss5, from which no products were obtained. These data suggest the occurrence of differential DNA sequences in the genomes of these isolates, which could be of value in the development of pathotype-specific markers.

Inhibitory effect of aluminium salts on mycelial growth of postharvest pathogens and development of potato dry rot. C. CENATUS, 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, Pavillon de l'Envirotron, Université Laval, Québec, QC G1V 0A6, Canada

Organic and inorganic salts are widely used in the food industry as preservatives and antimicrobial agents and have shown promise as potential alternatives to synthetic fungicides. In this work, six aluminium salts were assayed for their ability to inhibit mycelial growth of postharvest pathogens and development of potato (Solanum tuberosum L.) dry rot (Fusarium sambucinum Fuckel). In liquid culture, growth (dry weight) of F. sambucinum, Aspergillus niger Tiegh. and Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. were significantly inhibited by 1 mM of aluminium chloride or aluminium sulphate. Aluminium acetate, aluminium lactate, aluminium ammonium sulphate, and aluminium potassium sulphate also significantly inhibited R. stolonifer growth at 1 mM. Aluminium sulphate was the most efficient of all the tested salts; it completely inhibited the in vitro growth of F. sambucinum, R. stolonifer and A. niger at 1 mM, 5 Mm and 100 mM, respectively. Application of the aluminium salts (5 mM) to tubers artificially inoculated with F. sambucinum showed that aluminium ammonium sulphate and aluminium potassium sulphate significantly increased dry rot symptoms. Conversely, aluminium chloride and aluminum sulphate significantly decreased dry rot symptoms when compared with the control. This study points to the possibility of exploiting specific aluminium salts as postharvest disease control agents.

Transcriptome analysis between a plant pathogen and a biocontrol agent. R. CHAMOUN AND S. JABAJI. Department of Plant Science, McGill University, Raymond Building, 21,111 Lakeshore Road, Ste. Anne de Bellevue, QC H9X 3V9, Canada

Biocontrol is typically viewed from the perspective of how antagonists affect pathogens. Just as microbial antagonists use a diverse arsenal of mechanisms to dominate interactions with pathogens, pathogens also have diverse responses when they are under attack. Understanding the pathogen's reaction mechanisms provide an innovative approach to improving the durability of biologically based disease control strategies. Differential gene expression of mycoparasitism-induced genes was monitored during interaction between the mycoparasite Stachybotrys elegans (Pidopl.) Gams and different structures (hyphae and sclerotia) of the host, Rhizoctonia solani Kühn. Using QRT–PCR assays, comparative analyses demonstrated that hyphal and sclerotial forms triggered different gene expression patterns encoding pathogenicity, replication and toxins production. In the presence of both host's structures, genes encoding oxidoreductase and Histone H2A were highly upregulated. In contrast, three toxin encoding genes were substantially expressed only in the presence of the hyphae. These results indicate that structural anatomy of fungal structures may affect gene expression and also that multiple regulatory mechanisms may be involved. From the host's point of view, all transcripts encoding genes belonging to the respiration chain, apoptosis, transport and metabolism categories were suppressed with the exception of the pyridoxal reductase gene. This finding clearly highlights the role of a possible antioxidant activity from the host against invasion of the mycoparasite.

Occurrence, impact, and management of root rot on faba bean in Alberta. K. F. CHANG, R. L. CONNER, S. F. HWANG, B. D. GOSSEN, D. L. MCLAREN, S. E. STRELKOV AND G. D. TURNBULL. Field Crop Development Centre, Alberta Agriculture and Rural Development (ARD), 6000 C & E Trail, Lacombe, AB T4L 1W8, Canada; (R.L.C.) Morden Research Centre, Agriculture and Agri-Food Canada (AAFC), Unit 100-101, Route 100, Morden, MB R6M 1Y5, Canada; (S.F.H., G.D.T.) Crop Diversification Centre North, ARD, 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; (B.D.G.) Saskatoon Research Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (D.L.M.) Brandon Research Centre, AAFC, R.R.#3, Brandon, MB R7A 5Y3, Canada; and (S.E.S.) Department of Agriculture, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada

In a survey of commercial faba bean (Vicia faba L.) fields in Alberta in 2009, the incidence of root rot ranged from 0 to 30% of plants. Diseased plants were usually shorter, had thinner foliage, and produced only 31% of the number of pods and seeds of healthy plants. The number of root nodules was also reduced. Seed from diseased plants was often shrunken, and yield and seed weight were 21% and 69%, respectively, relative to healthy plants. Fusarium spp. were the most commonly isolated pathogens from diseased roots, but Pythium spp. and Rhizoctonia solani Kühn were also isolated frequently, and the pathogens often occurred in combination. In a field trial to examine the impact of inoculation with Fusarium spp. on faba bean ‘Earlibird’, seedling emergence and nodulation declined and root rot increased with increasing inoculum concentration, but yield was not affected. In a seed-treatment fungicide trial, Apron Maxx, Vitaflo 280 and Trilex improved emergence compared with the inoculated control, while Vitaflo 280 also improved yield. Root disease caused by Fusarium spp. is an important constraint to faba bean production in Alberta, but seed-treatment fungicides may be useful in reducing the impact of these ubiquitous pathogens.

Isolation and characterization of new Pseudomonas strains antagonistic to major fungal pathogens of wheat. Q. CHEN, T. T. T. TRAN, R. XU AND J. T. TAMBONG. Environmental Health Program (Biodiversity), Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; (Q.C.) Ministry of Education Key Laboratory for Crop Genetic Resources and Improvement in Southwest China, Sichuan Agricultural University, Yaan, Sichuan 625014, China; and (T.T.T.T.) Programme de baccalauréat en technologie appliqué – Biotechnologie, Secteur Sciences de la Santé, La Cité collégiale, Ottawa, ON K1K 4R3, Canada

Wheat production is highly dependent on agricultural inputs such as pesticides. Due to adverse effects of chemical pesticides on the environment and public health, a global priority in agriculture is the development of low-risk, environmentally sustainable disease control and management practices. The aim of this project was to isolate strains of Pseudomonas that are antagonistic to three important fungal pathogens of wheat: Fusarium graminearum Schwabe, Rhizoctonia solani Kühn and Gaeumannomyces graminis (Sacc.) Arx and Olivier. A total of 148 bacterial strains were isolated from soil samples collected from Woodslee, Ontario, Canada after serial dilution plating on Pseudomonas-specific medium. Based on 16S rRNA and gacA nucleotide sequences, all strains were identified as Pseudomonas spp. with 96.5–100% homology to GenBank entries belonging to the genus. Phylogenetic analyses of partial sequences of 16S rRNA and gacA grouped the bacteria into two major clusters, which correlated with groupings based on colony morphology. In vitro antagonistic activities of 148 Pseudomonas strains against F. graminearum, R. solani and G. graminis were evaluated by dual culturing on casamino acid agar medium. Fourteen strains were antagonistic against at least two of the fungal pathogens. Four of the fourteen strains effectively suppressed all the three pathogens while seven strains were antagonistic against F. graminearum and R. solani. Antagonistic microbe–microbe interactions mediated by Pseudomonas species are driven by the gacA gene. Phylogenetic analysis of the gacA sequences of our new potential bioagents showed a close relationship to previously reported biocontrol strains (e.g. P. fluorescens strains pf-5 and BSB1-2-37). Further research will focus on the protection of wheat plants against fungal root pathogens using selected strains of Pseudomonas.

Pyrolysis of plant biomass to produce chemicals of bactericidal and fungicidal value. K. L. CONN, M. M. HOSSAIN, L. A. CACERES, C. J. BOOKER, I. M. SCOTT, B. D. MCGARVEY, K. K. -C. YEUNG, C. BRIENS AND F. BERRUTI. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada; (M.M.H., L.A.C., C.B., F.B.) Department of Chemical and Biochemical Engineering, and Institute for Chemicals and Fuels from Alternative Resources, Faculty of Engineering, The University of Western Ontario, London, ON N6A 5B9, Canada; and (C.J.B., K.K.-C.Y.) Department of Chemistry, Chemistry Building, 1151 Richmond Street, The University of Western Ontario, London, ON N6A 5B7, Canada

Pyrolysis of plant biomass is being investigated around the world for converting bulky, low-profit plant waste into a valuable source of renewable energy and fine chemicals. Pyrolysis is the process of heating biomass in the absence of oxygen at temperatures ranging from 300–600 °C generating bio-oil, char and gases. The objective of this study is to determine if bio-oils contain chemicals with antimicrobial activity and then to isolate and characterize these chemicals. Bio-oils from sunflower, tomato, lignin and cellulose heated at either 450 or 550 °C were collected from two different parts of a pyrolyser to give three bio-oils: an organic fraction from the electrostatic precipitator (ESP) and both organic and aqueous fractions from the condenser. Bio-oils and seven plant pathogenic bacteria and10 plant pathogenic fungi were mixed together in 96-well microtitre plates along with appropriate control treatments. Three replicate wells were used for each mixture. The amount of growth of the microorganisms was determined after three to seven days. Each experiment was carried out twice. The ESP fraction from all the sources was the most active, consistently inhibiting growth of the majority of bacteria and fungi tested. The organic condenser fraction had intermediate activity, inhibiting several of the bacteria and fungi, depending upon the bio-oil. The aqueous condenser fraction had minimal antimicrobial activity. There was no difference in antimicrobial activity between bio-oils produced at 450 or 550 °C from either the lignin or cellulose. Since antimicrobial activity was found in bio-oils made from lignin and cellulose, this means that pyrolysis of any plant material should yield antimicrobial compounds. Thus, pyrolysis of plant material is a potential source of new antimicrobial compounds. Isolation and identification of the active compounds is underway.

Effect of water activity on the production of antimicrobial volatile compounds by Muscodor albus . R. CORCUFF, J. MERCIER, R. J. TWEDDELL AND J. ARUL. Centre de recherche en horticulture, Pavillon de l'Envirotron, Université Laval, Québec, QC G1V 0A6, Canada; and (J.M.) AgraQuest Inc., 1540 Drew Avenue, Davis, CA 95618, USA

The fungus Muscodor albus Worapong, Strobel & Hess produces a mixture of volatile organic compounds with antimicrobial activity. These compounds were shown to inhibit the growth of Pectobacterium atrosepticum (van Hall) Gardan et al., Fusarium sambucinum Fuckel and Helminthosporium solani Dur. & Mont. and to reduce the development of soft rot [P. atrosepticum], dry rot [F. sambucinum] and silver scurf [H. solani] on potato tubers. The optimization of the production of organic volatiles by the fungus appears as an interesting approach to improve the biocontrol activity of M. albus and to favour its use as biofumigant for the control of postharvest potato diseases. In this work, we investigated how water availability or water activity (A_w) of rye grain culture of M. albus (isolate 620) affects its volatile production. Rye grain cultures of M. albus were moistened with water to obtain different A_w and placed in closed containers with a capillary pore for gas exchange. Volatiles were sampled by static headspace at intervals and analysed by GC-MS. The most abundant volatiles were isobutyric acid; bulnesene, a sesquiterpene; a non-identified terpenoid; 2 and 3-methyl butanol; and ethanol. However, the level of each of those volatiles varied with A_w. The production of alcohols increased and that of aldehydes decreased with increase in A_w. Maximum emission of acids, esters and terpenoids occurred at A_w of 0.96. The results also showed that substrate A_w affects the production dynamics of each group of volatiles by the fungus. This study suggests that biocontrol activity of M. albus could be enhanced by optimizing A_w of the culture.

Induced systemic resistance against three foliar diseases of Agrostis stolonifera by an isoparaffin mixture. A. M. CORTES-BARCO, T. HSIANG AND P. H. GOODWIN. School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Induced systemic resistance (ISR) is a defence mechanism by plants against their pathogens. ISR was found to be activated by PC1, a mixture of food-grade synthetic isoparaffins. In controlled environment tests, application of PC1 to the soil reduced the area of diseased leaves of Agrostis stolonifera by 20 to 40% for the fungal pathogens, Microdochium nivale (Fr.) Samuels & Hallett, Rhizoctonia solani Kühn, or Sclerotinia homoeocarpa Benn. compared with the water control. In A. stolonifera, expression of the jasmonate synthesis-related genes, AsAOS1, encoding an allene oxide synthase, and AsOPR4, encoding a 12-oxo-phytodienoic acid reductase, and expression of a pathogenesis-related protein gene, AsGns5, encoding an acidic β-1,3-glucanase, were primed by PC1 for activity with increased expression when M. nivale was inoculated seven days later. In addition, expression of AsAOS1 was induced directly after application of PC1 and before pathogen inoculation. These results indicate that PC1 can induce or prime ISR in A. stolonifera.

Raging hormones – the role of hormone defence signalling in the potato– Verticillium dahliae interaction. H. DERKSEN, M. BADAWI, M. A. HENRIQUEZ AND F. DAAYF. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada

Phytohormones are known to activate signalling pathways in response to abiotic and biotic stresses such as pathogen attack. The role of plant hormone signalling in pathogen defence is complex and varies among plant–pathogen systems. The mainly known hormone signalling pathways involve jasmonic acid (JA), ethylene (ET) and/or salicylic acid (SA). In many systems, there are documented examples of crosstalk between these hormone pathways, including cooperation and/or antagonism. Other hormones such as abscisic acid and auxins also seem to be involved in disease signalling. Verticillium dahliae Kleb. is a soilborne pathogen that infects a wide range of hosts including potato (Solanum tuberosum L.). Verticillium dahliae is a major contributor to the potato early dying complex which can cause yield losses up to 50%. Gene expression analyses were performed to investigate the hormone signalling pathways involved in potato defence against V. dahliae. Semi-quantitative PCR results have identified genes related to both the JA/ET pathway and the SA pathway that show increased expression in the moderately resistant reaction versus the susceptible reaction. These results indicate the possibility that both of these pathways are involved in defence in this interaction. Further analysis of these genes is required in order to determine the specific role that they play. It is proposed that information gathered from this research can be used to improve control methods of V. dahliae in potato via manipulation of hormone signalling.

Uncovering a putative function for natural antisense transcripts in the fungal pathogen Ustilago maydis . 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

Analyses of expressed sequence tag (EST) libraries from different cell types and nutritional conditions uncovered natural antisense transcripts (NATs) corresponding to a number of open reading frames in the fungal plant pathogen Ustilago maydis (Persoon) Roussel. Described roles of NATs in regulating gene expression include: (1) transcriptional interference, (2) RNA masking and (3) dsRNA-dependent mechanisms such as the broadly conserved RNA interference (RNAi) pathway. Phylogenetic and functional analyses have revealed that, while plants, animals and most fungi contain functional RNAi machinery, select yeast species and U. maydis do not. Currently, the role of NATs in U. maydis is unknown. We are testing the hypothesis that U. maydis NATs influence gene expression in an RNAi-independent manner. We have characterized over 200 NATs by fully sequencing their corresponding antisense cDNAs. Using strand-specific RT-PCR, we have determined that NATs may be differentially expressed across a range of cell types, or expressed in a cell type-specific manner. Interestingly, cell type-specific NAT expression in the dormant teliospore was detected. The relationship between sense-antisense transcript (SAT) pairs at four loci has been examined in detail using antisense constitutive over-expression mutants. Preliminary data on strand-specific quantitative-PCR will be presented from select over-expression studies. As a whole, these experiments led to the hypothesis that specific antisense transcripts in U. maydis have the ability to stabilize sense transcripts. This action may be linked to the maintenance of mRNA integrity during teliospore dormancy and the transition to actively translated mRNAs upon germination. Finally, using strand-specific RT-PCR, we detected conserved NATs in Ustilago hordei (Pers.) Lagerh. (which unlike U. maydis, contains functional RNAi-machinery). This finding suggests a functional role for those NATs conserved between U. hordei and U. maydis.

Bioinformatic analysis of full-length cDNAs to identify transcript features in Ustilago maydis . C. DOYLE 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 corn smut pathogen, Ustilago maydis (Persoon) Roussel, is an established model for the study of biotrophic plant pathogens. The genome of U. maydis has been fully sequenced and an annotation was published in 2006. This annotation was aided by comparison to sequences of approximately 30,000 cDNA clones, which were created by the Saville lab. These clones were created emphasizing the isolation of full-length cDNAs (FLcDNA). We have now compiled a sub-library of all confirmed full-length transcript sequences and used them to identify conserved sequences relevant to the control of transcription and to directing mRNA processing. For the genes represented in this dataset we have defined the 3’ and 5’ untranslated regions (UTRs), and we also isolated the sequences upstream of the initiation sites for transcription. These three sets of sequences were then used to identify conserved motifs that may act as 3’ end processing signals, potential polyadenylation sites and signals involved in transcriptional control. The sequence elements we identified around the U. maydis poly A addition site were found to resemble those of yeast and plants more closely then they resembled those of mammals. This may indicate that the events that are involved in mRNA 3’ end processing in U. maydis bear greater similarity to those in yeast and plants. This work sets the stage for further functional analysis of the control of gene expression in U. maydis.

Isolation, cloning, and sequence analysis of four pathogenicity-related genes in highly vs. weakly Verticillium dahliae aggressive isolates. A. F. EL-BEBANY, Z. YAO, A. VENTURINO AND F. DAAYF. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada

Verticillium dahliae Kleb. is a soilborne fungus that causes a vascular wilt in a wide range of plant species. Using comparative transcriptomics and proteomics analyses, we previously identified many transcripts/proteins that are related to pathogenicity in V. dahliae. Four of these genes/proteins were selected for further investigation, stress response regulator (SrrA), exopolygalacuronase, isochorismatase hydrolase, and tetrahydroxynaphthalene reductase. Genomic DNA was extracted both from one highly aggressive and one weakly aggressive V. dahliae isolate and the full length genes were isolated. Then, the genes were cloned into pGEM^®-T Easy vector system. We will present sequence analyses that showed single nucleotide mutations, which may explain the differences between the two isolates in their final protein make up and their level of pathogenicity.

Stopping the enemy in its tracks – towards understanding the role of monolignol biosynthesis in resistance of Camelina sativa to Sclerotinia sclerotiorum. C. EYNCK, W. CLARKE, G. SÉGUIN-SWARTZ AND I. PARKIN. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

The crucifer Camelina sativa (L.) Crantz (false flax) has recently gained renewed interest as an alternative oilseed crop due to its potential value for human and animal nutrition as well as industrial applications owing to the unique fatty acid composition of its seeds. Among numerous other valuable agronomic attributes, it is generally considered to be resistant to many fungal diseases. However, it is susceptible to sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary with only a few genotypes showing an enhanced level of resistance. Nothing is known to date on the molecular basis of the defence of false flax to this pathogen. Histochemical studies comparing a partially resistant and a susceptible genotype revealed a more rapid deposition of lignin-like material at the point of infection in partially resistant plants, hampering fungal entry, particularly into the vascular system of the plant host. To elucidate the mechanisms underlying this defence response, expression changes associated with S. sclerotiorum infection of 21 individual genes or members of gene families involved in monolignol biosynthesis were studied in a partially resistant and a susceptible genotype using quantitative real-time PCR. The most pronounced differences in gene regulation patterns were found for genes encoding cinnamoyl-CoA reductase-like proteins (CCRs), the first enzyme specific to monolignol biosynthesis. Thus, upon infection transcript levels of CsCCR2 were elevated to a greater extent in partially resistant than in susceptible plants. Furthermore, a constitutive 12-fold difference in the basal level of CsCCR4 transcripts was observed. These results indicate that genes encoding plant cell wall related proteins may play a role in the defence response of C. sativa to S. sclerotiorum and that both constitutive and inducible defence responses may contribute to reduced symptom development in the partially resistant genotype.

Molecular characterization of a triglyceride lipase Lip1 from Stagonospora nodorum . J. FENG, R. HWANG, S. F. HWANG, D. GAUDET AND S. E. STRELKOV. Crop Diversification Centre North, Alberta Agriculture and Rural Development (ARD), 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; (R.H., S.E.S.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (D.G.) Lethbridge Research Centre, Agriculture and Agri-Food Canada, P.O. Box 3000, Lethbridge, AB T1J 4B1, Canada

A triglyceride lipase gene (LIP1) was cloned from the wheat glume blotch pathogen, Stagonospora nodorum (Berk.) Castell & Germano. LIP1 encoded a 546 amino-acid preproprotein with a predicted N-terminal signal peptide. Its expression was upregulated during pathogenesis and in culture media supplemented with saturated fatty glycerides. Recombinant Lip1 possessed lipolytic activity against a broad range of lipid substrates. When applied to the wheat leaf surface, recombinant Lip1 increased the hydrophilicity of the leaf surface, probably by liquefaction of epicuticular wax. Pretreatment of wheat leaves with Lip1 severely compromised the attachment of conidia. Gene knockout was conducted by protoplast transformation. Relative to the wild-type, the gene replacement strain showed decreased lipase activity but similar cutinase activity. The attachment of the conidia of the gene replacement strains to the wheat leaf surface was impaired and thus, the virulence was lower than the wild-type. Collectively, our data suggest that the secreted lipase Lip1 is important for fungal development during the early stages of infection.

Influences of organic mulches on development of root-lesion nematode populations in relation to indicators of soil food web structure during apple orchard establishment. T. A. FORGE, D. NEILSEN, G. NEILSEN AND E. HOGUE. Pacific Agri-Food Research Centre (PAFRC), Agriculture and Agri-Food Canada (AAFC), 6947 Highway 7, Agassiz, BC V0M 1A0, Canada; and (D.N., G.N., E.H.) PAFRC, AAFC, P.O. Box 5000, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada

Root-lesion nematodes (RLN, Pratylenchus spp.) are pathogens of apple, contributing along with certain fungal pathogens to poor replant establishment of orchards. In the Pacific Northwest and British Columbia, orchards are often fumigated prior to replanting in order to ameliorate replant disease. Organic mulches have been observed to improve soil quality, reduce RLN populations and improve growth and yield of established apple trees. The primary objective of this study was to assess effects of organic mulches on buildup of RLN populations during early establishment of an apple orchard on fumigated soil. A secondary objective was to assess relationships between indicators of soil food web structure and RLN populations. The site was fumigated in autumn of 1999. Alfalfa hay, dairy manure solids and a combination of alfalfa hay applied over manure were surface-applied to replicate plots in spring 2000, immediately after planting. Nematode communities were assessed from composite soil samples taken from each plot in May and October of five years, 2000 through 2004. RLN populations increased through time in control and manure plots but not alfalfa and alfalfa/manure plots; population densities in control and manure plots were significantly greater (P < 0.01) than in alfalfa and alfalfa/manure plots through 2003 and 2004. Nematode indicators of soil food web structure were affected by treatments: the Enrichment Index (EI) was high for all treatments early in the study (2000, 2001) but then decreased in all treatments except alfalfa mulch. The Structure Index (SI) increased in all treatments as the complexity of the soil food web developed after fumigation. Development of the SI was not consistently influenced by mulch treatments nor related to RLN populations.

Carrot's central root rot. L. FUCIKOVSKY. Colegio de Postgraduados, Instituto de Fitosanidad, Carretera México-Texcoco, km 36.5, Montecillo, Edo. de México, C.P. 56230, México

Carrot, Daucus carota L. var. sativa DC, mostly the Nantes variety is one of the preferred vegetables cultivated each year in the Valley of Toluca, State of Mexico and other close-by areas. The altitude varies between 2000 and 2600 m above sea level and the carrot is usually sowed in June before the rainy season and harvested in December. At harvest time, two internal soft rots occur in carrot. The first soft rot appears with a frequency of 1–2% and is found internally in the core, starting at the point of growth by Sclerotinia sclerotiorum (Lib.) de Bary and later rotted rapidly by Erwinia carotovora subsp. atroseptica (van Hall) Dye. The second soft rot, with a frequency of 10% starts from the carrot aerial part, where dipterous flies place their eggs and later the larvae penetrate inside the core, introducing the bacteria during the penetration. The aim of this study was to determine the type of bacteria associated with the rot. Eight separate isolates of fluorescent bacteria were isolated. The bacterium was identified as a type of Pseudomonas fluorescens Migula where only argenine dihydrolase did not agree. This situation, as far as known, has not been described before.

Towards map-based cloning of AvrLepR1 , a novel avirulence gene in Leptosphaeria maculans corresponding to LepR1 resistance gene in Brassica napus. K. GHANBARNIA, N. LARKAN, S. R. RIMMER, G. LI, H. R. KUTCHER, P. B. E. MCVETTY, W. G. D. FERNANDO AND D. LYDIATE. Saskatoon Research Centre, Agriculture and Agri-Food Canada (AAFC), 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (G.L., P.B.E.M., W.G.D.F.) Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; and (H.R.K.) AAFC, Melfort Research Centre, Box 1240, Melfort, SK S0E 1A0, Canada

A new avirulence gene, termed AvrLepR1, which interacts with the resistance gene LepR1 was characterized genetically following an in vitro cross of virulent and avirulent isolates of the pathogen and subsequent pathology test on cotyledons of line ddm-12-6s-1. A molecular genetic map was constructed using sequence-related amplified polymorphism (SRAP) and PCR-amplified markers for Leptosphaeria maculans (Desmaz.) Ces. & De Not. The results suggest the occurrence of a novel avirulence gene, AvrLepR1, corresponding to the LepR1 resistance gene in line ddm-126s-1, which is linked to the cluster, AvrLm3-4-7-9, on linkage group 2. Sequencing of two flanking markers with 29.1 and 3 cM apart, revealed that the physical distance in this region is about 340kb. Therefore the ratio of physical to genetic distance will be about 16 kb cM⁻¹ which is similar to previous studies. Sequencing of two other markers, PT65-490 and FT158-199 with 21.3 and 3 cM are under study to confirm the order of the markers in this region. Additional markers in this region are being produced to further refine the interval and locate the gene of interest.

Impact of climate change on clubroot of canola on the Canadian prairies. B. D. GOSSEN, M. R. MCDONALD, K. C. Kalpana, S. F. HWANG, S. E. STRELKOV AND G. PENG. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (M.R.M., K.K.C.) Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada; (S.F.H.) 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

Clubroot of canola (Brassica napus L.), caused by the protist Plasmodiophora brassicae Woronin, was first reported in western Canada in 2003. It is spreading rapidly across the prairie region and options for clubroot management are limited. Our studies on canola and other Brassica species in a controlled environment have shown that infection and pathogen development occur slowly at low temperatures (≤ 17 ^ºC), and so do symptoms. Field studies in 2007–09 using short season vegetable Brassicas have demonstrated that adjusting the timing of planting to avoid the warm conditions of summer can reduce or eliminate clubroot, even for susceptible species under high pathogen inoculum pressure. In canola, field trials in 2008 showed that early seeding can reduce clubroot severity and yield loss. In 2009, severe spring drought reduced stand establishment and delayed pathogen activity, but the disease increased rapidly when rains came several weeks after seeding. Subsequent losses in seed yield were high despite the initial delay in symptom development. We conclude that the longer growing season, higher temperatures and more variable rainfall predicted for the Canadian prairies in most climate scenarios will reduce the effect of early seeding and other cultural approaches for clubroot management. These conditions will also place more pressure on the limited resources for genetic resistance to clubroot, so development of an Integrated Pest Management strategy for managing this damaging disease is an urgent priority.

Identification of type I polyketide synthases from fungal proteomes and transcriptomes. R. S. GOSWAMI, J. A. DELGADO, O. AL-AZZAM, A. M. DENTON AND S. G. MARKELL. Department of Plant Pathology, and (O.A.-A., A.M.D., S.G.M.) Department of Computer Sciences, North Dakota State University, 315 Morrill Hall, P.O. Box 6050, Fargo, ND 58108, USA

Polyketides are natural products valued for their antibacterial, antifungal, immunosuppressant and other biological activities. They are synthesized by proteins known as polyketide synthases (PKSs). Fungi appear to be one of the most promising natural producers of polyketides which are involved in the biosynthesis of secondary metabolites and pigments known to be important in their survival, competition and pathogenicity. However, fungal PKSs have been studied to a much lesser extent than bacterial PKSs. There are three types of PKSs among which type I PKSs are most common in fungi. Traditional gene cloning of type I PKSs using either bacterial or degenerative primers designed for PKS genes is not always successful due to the wide diversity of nucleotide sequence of PKS genes. The goal of this study was to design a method to identify PKSs through in silico sequence analysis. This was conducted by creating fungal databases of two major PKS protein domains, the beta-ketoacyl synthase and acyltransferase domains and using the profile hidden Markov model to identify potential PKS genes in fungal genomes. The model was initially tested on the well-annotated proteome of Fusarium graminearum Schwabe and subsequently used to identify PKSs from the predicted proteomes of the recently sequenced Alternaria brassicicola (Schwein.) Wiltshire, Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyder & Hansen, Verticillium albo-atrum Reinke & Berthold and Verticillium dahliae Kleb. genomes. We were also able to distinguish the identified protein sequences into reducing and non-reducing PKS. The predictions were validated through PCR amplification and sequencing. The models performed with high sensitivity, specificity and precision. The databases created were also used for comparative analysis that led to the identification of potential PKSs from EST sequences of libraries created during the infection of chickpea by Ascochyta rabiei (Pass.) Lab. and in culture.

Antimicrobial efficacy of cinnamon extracts against spoilage pathogens. S. E. GREEN, R. J. TWEDDELL, A. TSOPMO AND T. J. AVIS. Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6, Canada; (S.E.G.) Département de biotechnologie, La Cité Collégiale, Ottawa, ON K1K 4R3, Canada; and (R.J.T.) Centre de recherche en horticulture, Pavillon de l'Envirotron, Université Laval, Québec, QC G1V 0A6, Canada

Spices have been previously used as antimicrobial products both in traditional and herbal medicine and have shown promise as potential alternatives to synthetic fungicides. In this work, aqueous cinnamon (Cinnamomum verum J. Presl) extracts were assayed for their ability to inhibit mycelial growth of spoilage pathogens and development of carrot (Daucus carota L.) cavity spot (Pythium sulcatum Pratt & Mitch.) and potato (Solanum tuberosum L.) dry rot (Fusarium sambucinum Fuckel). In general, cinnamon extracts inhibited the tested pathogens in a dose-dependent manner. Results showed that 0.05 g mL⁻¹ cinnamon extracts completely inhibited the mycelial growth of P. sulcatum and Aspergillus niger Tiegh. The complete inhibition of the mycelial growth of F. sambucinum and Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. occurred at higher extract concentrations (0.10 and 0.15 g mL⁻¹, respectively). Application of cinnamon extracts to mature carrot roots artificially inoculated with P. sulcatum demonstrated that 0.15 g mL⁻¹ extracts significantly reduced the severity of cavity spot by 31% when compared with the control. Cinnamon extracts also decreased the development of dry rot severity by approximately 50% when 0.15 g mL⁻¹ extracts was applied to potato tubers artificially inoculated with F. sambucinum. This study points to the possibility of exploiting cinnamon extracts as a control method against specific postharvest diseases.

Evolution de novo of stable, heritable resistance to leaf rust [ Puccinia triticina ] in susceptible wheat following cycles of infection with wheat streak mosaic virus . S. HABER, D. L. SEIFERS, P. SETO-GOH AND B. MCCALLUM. Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada; and (D.L.S.) Agricultural Research Center, Kansas State University, Hays, KS 67601-9228, USA

Stable, heritable genetic resistance to infection with wheat streak mosaic virus (WSMV) can be evolved de novo in selfing bread wheat lines subjected to cycles of virus inoculation and selection of seed from the best-performing individual plants or tillers. Similar iterative protocols might also succeed in inducing heritable changes in host responses to other pathogens such as leaf rust [Puccinia triticina Eriks.]. We examined the responses to leaf rust infection of succeeding generations of the susceptible cultivars ‘Lakin’ and ‘Thatcher’ subjected to WSMV inoculation. After as few as three cycles individual plants were identified that expressed resistance to leaf rust and were subsequently confirmed as founders of sublines with uniform and heritable resistance. These sublines, into which no genetic source of resistance had been introgressed, conferred their resistance to progeny of crosses with susceptible parents. Crosses between, respectively, leaf rust-susceptible and resistant ‘Lakin’ sublines 4-3-3 and 4-12-3 produced F₁ populations that were either predominantly or uniformly resistant when inoculated at seedling stage with race TDBG. Progeny of F₂ generations descended from susceptible progenitors were uniformly susceptible while those descended from resistant F₁ plants comprised resistant and susceptible individuals in a 3:1 ratio, consistent with the action of a single, dominant gene.

New Canadian records for Gymnosporangium sabinae and Gymnosporangium yamadae . S. HAMBLETON, M. LIU, J. A. PARMELEE, M. J. CELETTI AND J. LLEWELLYN. Biodiversity (Mycology), Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (M.J.C., J.L.) Ontario Ministry of Agriculture, Food and Rural Affairs, Edmund C. Bovey Building, University of Guelph, Guelph, ON N1G 2W1, Canada

The threat to agriculture from the accidental or deliberate introduction and establishment of invasive plant pathogens is of global concern. Losses can arise from lower yields, increased pest management costs and loss of markets to trade embargos. The first official record of a non-native species can be misleading, if the species was already present at a low frequency but was overlooked, or caused little damage and escaped detection. Two Gymnosporangium spp. were studied based on ITS sequence data and examination of morphological characters, and identified as occurring in unexpected regions of Canada. The pycnial and aecial stages of Pear Trellis Rust (Gymnosporangium sabinae (Dicks.) Winther) were collected on Pyrus in the Toronto and Niagara regions and on the University of Guelph campus (ON) in 2007. In spring 2009, the telial stage was found in Guelph on the alternate host Juniperus, in close proximity to Pyrus. Already known to occur in BC, this is the first formal report of G. sabinae in ON, although herbarium records and observational reports suggest that the fungus may have been present for many years. Japanese apple rust (Gymnosporangium yamadae Miyabe ex Yamada) was first collected in 2005 in the Fletcher Garden, CEF Ottawa (ON) and was the subject of an intensive study to characterize all spore stages on Malus and Juniperus. The fungus is widely distributed on Malus based on samples collected from 25 crab-apple trees, while only one of three possible telial host trees is infected. Sequencing of herbarium specimens is underway to determine if the fungus has been previously collected but misidentified. This is the first report of G. yamadae in Canada.

A Canadian spore trapping network – monitoring Asian soybean rust and beyond. S. HAMBLETON, K. OLIVER, H. SAEED, R. TROPIANO, J. CHAPADOS, C. LEWIS, C. A. LÉVESQUE, C. VAN HERK AND A. TENUTA. Biodiversity (Mycology), Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (C.V.H., A.T.) Ontario Ministry of Agriculture, Food & Rural Affairs, Agronomy Building, 120 Main Street East, Ridgetown, ON N0P 2C0, Canada

Since 2006, a network of spore-trapping equipment, deployed in nine sites in Ontario, three in the Prairie Provinces and two in Québec, has been used to monitor a South-to-North migration of Asian soybean rust (Phakopsora pachyrhizi Syd. & Syd.) towards Canada. Three types of equipment were used: LODA or Yankee Environmental Systems active precipitation collectors, Burkard Cyclone air samplers, and passive rainfall collectors made in-house. Real-time PCR screening of total DNA extracted from all samples collected was done weekly during the growing season each year. In 2007, soybean rust spores were detected in Canadian samples for the first time, and in October of that year, increased field monitoring located infected plants in SW Ontario. This success proved that pathogen pathways can be monitored and infection potential can be detected before infected plants are found. In 2008, a major weather system deposited spores in Canada in late June/July, indicating that long-range transport of spores is possible early in the growing season. In 2009, conditions in the US were not conducive for inoculum build-up until early August, resulting in few positive Canadian spore trap samples until late season. We are now expanding our network to include sites in BC and PEI, and expanding our scope of interest. For selected sites we will apply next-generation 454 pyrosequencing technology to ribosomal DNA (rDNA) amplicons derived from archived and new air and rain samples, to develop a baseline profile of harmful and beneficial fungi and bacteria from 2007 to 2011. In a successfully completed pilot pyrosequencing trial, markers for P. pachyrhizi were detected among 47,500 sequences obtained for a 2008 air sample that tested positive for the fungus.

Reducing incidence and severity of tomato diseases using novel, silver-based compounds. M. W. HARDING, R. J. HOWARD, M. A. ROBINSON, M. J. UNRUH, D. A. SOWA AND M. E. OLSON. Innovotech, Inc. Suite 101, 2011 – 94 Street, Edmonton, AB T6N 1H1, Canada; and (R.J.H.) Crop Diversification Centre South, Alberta Agriculture and Rural Development, 301 Horticultural Station Road East, Brooks, AB T1R 1E6, Canada

Bacteria and fungi routinely cause destructive disease outbreaks in commercial tomato production systems. For example, tomato diseases such as spot, speck and canker are caused by the bacteria Pseudomonas syringae pv. tomato Van Hall, Xanthomonas campestris pv. vesicatoria (Doidge) Dye, and Clavibacter michiganensis pv. michiganensis (Smith) Dye & Kemp, respectively. Additionally, gray mould is caused by the fungus Botrytis cinerea (De Bary) Whetzel. Previous successes have been reported for the silver-based compounds oxysilver nitrate and sodium diperiodato argentite (II) as seed treatments capable of controlling seed-borne bacterial blights in dry edible bean. Based on those successes, this present study was designed to evaluate the potential of silver-based compounds to control or reduce bacterial and fungal disease symptoms on artificially inoculated tomato foliage and stems in a greenhouse. We report significant reductions in disease incidence and severity of spot, speck and canker on tomato leaves and fruit, and reductions of incidence and severity of gray mould on stems. In all cases, the silver compounds reduced disease relative to the negative control treatments and, in some cases, the reduction in symptoms was superior to registered industry standard treatments. These preliminary results validate the concept that silver-based chemistries could potentially be effective products for management of tomato diseases.

Can phylogenetically useful oligonucleotide primers be obtained from comparative genomic analyses of filamentous ascomycetes? C. H. HUANG AND T. HSIANG. School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Basic Local Alignment Search Tool (BLAST) is a commonly used program that queries databases for matching sequences. The output shows the best matches, and provides expectation values (e-values) which indicate the similarity of the match. We hypothesized that the e-values might reflect the amount of phylogenetically useful information between the query sequence and the respective genomic database sequences, and that primers based on genes of different tiers of similarity might be useful at different taxonomic levels. To test this, genome-wide TBLASTN was conducted using protein sequences of Magnaporthe oryzae Couch against 12 almost completely sequenced fungal genomes individually. The top matching results for each genome were placed into a spreadsheet, and geometric means for e-values calculated. After sorting the 11000 genes by mean e-value, 50 genes were systematically selected for phylogenetic analyses by retrieving homologues from 30 filamentous ascomycetes genomes, conducting multiple sequence alignments, and analysing with neighbour-joining tree building methods. With the taxa available, class and order level trees could be constructed, and these were compared to rDNA 18S trees for congruency as rDNA is the most widely sequenced genomic region among fungi. Among five genes chosen from the top tier of matching e-values, three resolved the filamentous ascomycetes at the class and order levels with bootstrap values greater than 80%. This implies that even among very highly conserved sequences, there may be sufficient variation for resolution at higher taxonomic levels. Additional work is being conducted on more genes to confirm these observations, and to further evaluate whether mean e-values of a single gene compared to its homologues in other species can be used to predict the phylogenetic utility at different taxonomic levels.

Plant growth promotion and disease suppression by a biocontrol strain of Bacillus cereus . C. J. HUANG, H. P. YANG, H. D. CHANG, C. F. TSAI AND C. Y. CHEN. Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 10617, Taiwan; and (H.D.C.) Department of Environmental Safety Engineering, Ming Chi University of Technology, Tai-Shan, Taipei 24301, Taiwan

The biocontrol strain, Bacillus cereus Fr. & Fr. C1L, was isolated from the rhizosphere of Formosa lily (Lilium formosanum Wall.) in Taiwan. Application of B. cereus C1L effectively decreased disease severity of Botrytis leaf blight in Formosa lily and Oriental lily cv. Star Gazer. Suppression of corn leaf blight was also observed in the fields. In addition, plant growth-promoting activities of strain C1L were demonstrated in maize, Arabidopsis and tobacco. Volatile compound of strain C1L were effective in plant growth promotion of Arabidopsis thaliana L. and tobacco. Volatile compound of strain C1L were also effective on disease suppression in tobacco. One of the volatile compounds produced by strain C1L was identified as dimethyl disulfide (DMDS) by Gas Chromatography-Mass Spectrometry. Moreover, application of DMDS could enhance growth of tobacco plants and reduce disease severity caused by B. cinerea.

Influence of cultivar resistance and pathogen inoculum concentration on root hair colonization by Plasmodiophora brassicae on canola. S. F. HWANG, H. AHMED, S. E. STRELKOV, B. D. GOSSEN, G. PENG AND G. D. TURNBULL. 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 Agriculture and Forestry, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (B.D.G., G.P.) Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Clubroot of canola (Brassica napus L.), caused by Plasmodiophora brassicae Woronin, has spread rapidly since it was first observed in western Canada in 2003. Bioassay and PCR are currently used to qualitatively confirm the presence of the pathogen, but quantitative estimation of inoculum through resting spore extraction from soil samples can be slow, tedious and often produces highly variable results. A quick, cost-effective technique is needed to determine resting spore density in infested fields. To evaluate the influence of host resistance and inoculum density on root hair infection, two canola cultivars, ‘45H26’ (susceptible) and ‘45H29’ (resistant), and two methods of inoculation were used. In one experiment, powdered clubroot galls were amended with soilless-mix to produce spore concentrations of 0, 10³, 10⁵ and 10⁷ spores mL⁻¹ of the mix. In a parallel trial, the same cultivars were seeded in clubroot-infested soil diluted with soilless mix to adjust spore densities at 0:1, 1:8, 1:1 and 1:0. The seedlings were uprooted at 4, 6, 8, 10, 12 or 14 days after seeding, and 1.5 cm root segments were examined microscopically for infection. The mean incidence of infection for individual root hairs was 37 and 31% in the susceptible cultivar, compared to 10 and 15% in the resistant cultivar, respectively, when inoculating seedlings by powdered gall or sowing them in infested soil. In the susceptible cultivar, root hair infection increased significantly with each 100-fold increase in inoculum concentration, and peaked at six to eight days after inoculation by the root dip method. Therefore, P. brassicae inoculum concentration in the soil may be estimated within two orders of magnitude by examination of root hairs six to eight days after seeding.

Effects of seed maturity, seed size and seeding depth on seedling blight of canola. S. F. HWANG, Q. ZHOU, R. HWANG, S. E. STRELKOV, B. D. GOSSEN AND G. D. TURNBULL. Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; (R.H., S.E.S.) Department of Agriculture and Forestry, University of Alberta, 410 Agriculture/Forestry Centre, Edmonton, AB T6G 2P5, Canada; and (B.D.G.) Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

Successful seedling establishment is an on-going challenge for canola (Brassica napus L.) producers in western Canada. Greenhouse trials were conducted to assess the effect of seed size on the severity of seedling blight caused by Fusarium, Rhizoctonia and Pythium spp., and the effect of seeding depth on the severity of seedling blight caused by Rhizoctonia solani Kühn. Inoculum (mycelium grown on sterilized wheat grains, dried and ground to powder) was mixed with soil prior to planting to provide consistent disease pressure. Seed size did not affect seedling emergence or root rot incidence and severity in soil inoculated with Fusarium or Pythium spp. In soil inoculated with R. solani, emergence was lower for large and small seed compared with mid-sized seed (0.7–2 mm diam.) and root rot incidence and severity was lower for mid-sized seed compared with large seed. Plant height and shoot weight generally increased with seed size, regardless of treatment. In the seeding depth study, seedling emergence was lower at 1.6 or 2.4-cm seeding depth compared with 0.8 cm. Damping-off incidence, plant height and shoot dry weight were highest at the deepest seeding depth, but seeding depth did not affect root rot incidence or severity. In another trial, canola seed was harvested at six dates and planted in non-inoculated soil to examine the effect of seed maturity on seedling vigour. Seedling emergence, and plant height and weight increased as the timing of seed harvest approached full maturity. The results demonstrate that seed maturity and size affect seedling establishment and vigour, and that seeding depth affects seedling mortality and vigour.

Chestnut fruit rot caused by an anthracnose fungus, Colletotrichum gloeosporioides , and their chemical control. L. S. HYUN, S. S. TAE, K. K. HEE AND L. J. KYU. Department of Forest Disease and Insect Pests, Korea Forest Research Institute, Seoul, Korea; and (L.J.K.) Department of Forest Environment Protection, Kangwon National University, Chuncheon, 200-701, Korea

Chestnut is one of the economically important tree species widely cultivated in Korea. Chestnut blight [Cryphonectria parasitica], chestnut ink disease [Phytophthora katsurae], and chestnut fruit rot [Colletotrichum gloeosporioides] are major diseases for chestnut tree or fruit reported in chestnut plantations in southern parts of Korea. Recently, early defoliation of chestnut burs and fruit rot caused by an anthracnose fungus were recognized in the mid parts of the Korean peninsula. Damage rate of chestnut fruit rot was investigated and compared among chestnut cultivars. The cultivar ‘Riheiguri’ was the most severely damaged cultivar with a damage rate of 14.7%. Two other cultivars, ‘Tanzawa’ and ‘Ishizuchi’, were 8% and 6%, respectively. Damage rate of the stored chestnut fruit after harvest was recorded by 10–18%. Necrotic area formed by artificial inoculation of the pathogenic fungus on stigma part of the nut was much larger than that on the front part. Damage by artifical inoculation caused softening and discoloration into brownish black of the infected tissues. For control of the disease, eight effective fungicides were selected and compared in control efficiency. Fluazinam (50%) and Tebuconazole (25%) showed relatively higher control efficiency while azoxystrobin showed lower efficiency. Dipping treatment of the nut in Tebuconazole (25%) solution for 30 s before storage at 3 °C showed relatively lower damage rate of 5% as compared with the untreated cultivars, ‘Riheiguri’ (26.1%) and ‘Ishizuchi’ (17.4%).

Genetics and pathogenicity of Microdochium nivale . L. E. JEWELL AND T. HSIANG. School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Microdochium nivale (Wollenw.) Samuels & Hallett is a common pathogen of turfgrasses and cereal crops in Europe and North America. Traditionally it has been divided into two subspecies, vars. majus and nivale, although some researchers have elevated the subspecies to different species, M. nivale and M. majus (Wollenw.) Glynn & Edwards. To investigate the genetic differences between these taxa, portions of the ITS (internal transcribed spacer) region and the EF-1α (elongation factor 1α), RPB2 (RNA polymerase II) and β-tubulin genes from North American isolates were amplified by PCR and sequenced. Preliminary sequence comparisons between putative majus and nivale isolates revealed genetic differences. The infection process was also investigated on detached leaves of Poa pratensis L., Agrostis palustris Huds., and Lolium perenne L. using four North American isolates of M. nivale var. nivale. In each case, infection proceeded via stomatal infiltration. Additionally, infection was observed by 10 h post inoculation (hpi) on P. pratensis, while A. palustris and L. perenne did not display signs of infection until 48 hpi. Future work will further investigate the pathogenic and genetic differences between the varieties of this common pathogen, as well as the genetic differences between North American and European isolates.

Early dying innate immunity involves ligand dependent endocytosis of cell surface receptors . M. KALISCHUK, I. KOVALCHUK, A. FUSARO, P. WATERHOUSE, D. PRÜFER AND L. KAWCHUK. Lethbridge Research Centre, Agriculture and Agri-Food Canada, P.O. Box 3000, Lethbridge, AB T1J 4B1, Canada; (I.K.) Department of Biological Sciences, University of Lethbridge, 4401 University Drive, AB T1K 3M4, Canada; (A.F., P.M.) School of Biological Science, University of Sydney, NSW 2006, Australia; and (D.P.) University of Münster, 48149, Germany

All eukaryotic cells exhibit endocytosis as a mechanism to communicate and respond to external stimuli. We previously described the positional cloning of two closely linked inverted Ve genes for early dying resistance from tomato and showed that expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum Reinke & Berthold. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLφ or YXXφ endocytosis signals where φ is an amino acid with a hydrophobic side chain. A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. To identify and characterize the signalling involved in early dying disease resistance, subcellular localization of the verticillium wilt Ve innate immunity disease resistance receptors was examined immunologically through a series of site-directed amino acid substitutions. We show microscopically and biochemically that the Ve receptors are located at the cell surface and respond to the pathogen by ligand dependent endocytosis.

Forecast distribution and severity of clubroot of canola in the Canadian prairies under incremental temperature and precipitation, and potential climate change scenarios. H. KLEIN-GEBBINCK, T. K. TURKINGTON, O. O. OLFERT, R. M. WEISS, D. KRITICOS, H. R. KUTCHER, K. C. FALK AND S. E. STRELKOV. Lacombe Research Centre/Beaverlodge Research Farm, Agriculture and Agri-Food Canada (AAFC), 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada; (O.O.O., R.M.W., H.R.K., K.C.F.) Saskatoon Research Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (D.K.) CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia; and (S.E.S.) Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada

Clubroot of canola, caused by Plasmodiophora brassicae Woronin, was first reported in the St. Albert region of Alberta in 2003 and has subsequently appeared over a broad area around Edmonton, Alberta, and also in individual fields elsewhere in the province. A CLIMEX^TM model was used to predict potential distribution and severity of clubroot of canola in the Canadian prairie region under: (1) incremental temperature and precipitation scenarios using long-term climate normal data (LCND); and (2) using three General Circulation Model (GCM) scenarios (Micro-H, NCAR-CCSM, CSIRO Mark 3.0), and a baseline scenario (CRU World V2). Initial predictions of clubroot disease occurrence and severity based on LCND were consistent with observations on cruciferous vegetables in the lower mainland of British Columbia and central Canada, and canola in Alberta. Based on current conditions with either the LCND or CRU World V2 scenarios, the model predicted clubroot could affect canola over a wide area of the prairie ecosystem, especially wetter regions of Alberta, Saskatchewan and Manitoba. Predicted distribution and seasonal clubroot development in the prairie region was greater, especially for the NCAR-CCSM scenario, compared to the baseline scenario and exhibited the following trend NCAR-CCSM > CSIRO Mark 3.0 > Micro-H. Incremental temperature increases of 1 to 3 °C resulted in the projected expansion of the area potentially affected by economic levels of clubroot, mainly in the cooler and wetter regions of the prairie ecosystem. Overall, a more northerly expansion and an overall greater economic impact of clubroot may be expected with combined increases in temperature and precipitation.

Hydrolytic enzymes production by the plant pathogenic bacterium Streptomyces scabiei EF-35. D. KOMEIL, A. -M. SIMAO-BEAUNOIR AND C. BEAULIEU. Université de Sherbrooke, Biologie, 2500 boulevard de l'Université, Sherbrooke, QC J1K 2R1, Canada

Streptomyces scabiei (ex Thaxter) Lambert & Loria EF-35 is the causal agent of common scab of potato. The bacterium penetrates the periderm surfaces of its host and produces a range of degrading enzymes. The ability of S. scabiei EF-35 to produce extracellular enzymes in the presence of the insoluble plant biopolymer suberin was tested in liquid culture media. Different extracellular proteins produced by the bacterium in the presence of suberin were sequenced. Streptomyces scabiei EF-35 was able to produce different glysosidases, such as xylanases and cellulases, and various hydrolases, including esterases. Furthermore, S. scabiei EF-35 was cultivated in minimal medium supplemented with different substrates (suberin, cutin, lignin, xylan, glucose and mannitol). The ability of culture supernatants to hydrolyse p-nitrophenol butyrate was determined by spectrophotometric assays. The esterase activity was highly inducible in the presence of potato suberin and was repressed by the presence of glucose and mannitol. Reverse transcription-PCR (RT-PCR) methods were used for detecting the expression of previously identified genes of the bacterium with the tested substrates. Most of the identified genes were transcribed in the presence of various plant polymers but a cutinase gene was specifically expressed in the presence of suberin.

The race structure of Leptosphaeria maculans in western Canada. H. R. KUTCHER, D. CROSS, B. MARQUER, C. L. KIRKHAM, A. -M. CHÈVRE, R. DELOURME, M. -H. BALESDENT, T. ROUXEL AND H. BRUN. Agriculture and Agri-Food Canada, Melfort Research Centre, Box 1240, Melfort, SK S0E 1A0, Canada; (B.M., A.-M.C., R.D., H.B.) Institut National de la Recherche Agronomique (INRA), Domaine de la Motte – B.P. 35327, 35653 Le Rheu CEDEX, France; and (M.-H.B., T.R.) INRA, Avenue Lucien Brétignières, B.P. 01, 78850 Thiverval-Grignon CEDEX, France

Specific resistance genes are an effective means of disease control when the pathogen population is mainly avirulent on the cultivated varieties carrying the corresponding resistance gene. Development of new races of Leptosphaeria maculans (Desmaz.) Ces. & de Not., the species responsible for the most damaging symptoms of blackleg disease of canola (Brassica napus L.), increases the risk that host genetic resistance may be rapidly overcome. This study aims to elucidate the race structure of L. maculans by determining, under controlled conditions, the frequency of avirulence alleles at eleven avirulence loci in pathogen populations collected from nine locations across western Canada. The avirulence allele at AvrLm6 was present in all isolates tested (63) from three locations. At eight locations > 88% of the isolates (423) carried the avirulence allele at AvrLm2, but only 37% at another location. For all other avirulence genes (AvrLm1, AvrLm3, AvrLm4, AvrLm7, AvrLm9, AvrLmLepR1, AvrLmLepR2 and AvrLmLepR3), avirulence allele frequency varied from 0 to 99% depending on the loci and the location (300–600 isolates). Knowledge of avirulence allele frequency and the race structure of L. maculans in canola-producing regions of western Canada will be crucial to develop strategies to maintain the efficacy of resistance genes.

Investigation on modes of action for the biofungicides Serenade and Prestop in clubroot control. R. LAHLALI, G. PENG, B. D. GOSSEN, R. H. HYNES, S. M. BOYETCHKO, S. F. HWANG AND M. R. MCDONALD. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; (M.R.M.) Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

Clubroot, caused by the protist pathogen Plasmodiophora brassicae Woronin, is an emerging threat to canola (Brassica napus L.) production on the Canadian prairies. The biofungicides Serenade^® (Bacillus subtilis QST 713) and Prestop^® (Gliocladium catenulatum Gilman & E. Abbott, strain J1446) were investigated for clubroot control in growth cabinet and field conditions. When applied as an in-furrow treatment at10 L and 1.4 kg ha⁻¹ rates, respectively (in 500 L ha⁻¹ water volume), Serenade and Prestop reduced clubroot severity by 80–90% on canola when compared with untreated controls in growth-cabinet trials but failed to control the disease substantially in a field trial that experienced four weeks of drought after seeding. Suspensions of these biofungicides made with pure bacterial or fungal cultures were as efficacious as the formulated products, while cell-free product filtrates were slightly less effective. Trials were conducted in growth cabinets to determine the effect of different biofungicide components on the infection process of P. brassicae on canola. The impact of soil-dryness duration after biofungicide treatments was also investigated. Results indicated that the biofungicides, whether of the formulated product or cell-free filtrate, have limited impact on the germination of P. brassicae resting spores. It is proposed that the biocontrol reduces clubroot by suppressing root and/or root hair infection by zoospores. Extended dryness may reduce the survival of both biofungicide agents in the soil, hence reduce the efficacy of clubroot control when conditions conducive to infection occur following the dry period.

Polymerase chain reaction protocol for detection of Pyrenophora teres in natural field soils. R. T. LARTEY, T. C. CAESAR-TONTHAT, A. J. CAESAR, A. W. LENSSEN, S. HANSON AND R. G. EVANS. USDA-ARS, Northern Plains Agricultural Research Lab., Sidney, MT 59270, USA

Net blotch of barley, a widely distributed foliar disease, occurs extensively in the Northern Great Plains of the USA. The causal agent, Pyrenophora teres Drechs., survives in infested straw in field soils which produces conidia and ascospores, probably the most important sources of primary inoculum. We developed and present a PCR technique for direct detection in field soils. Total DNA was purified from P. teres amended and barley planted field soils using PowerSoil DNA Kit (MO BIO Lab. Carlsbad, CA) as per manufacturer's instructions. The purified DNA was subjected to PCR reaction in Extract-N-Amp PCR mix (Sigma Aldrich, St Louis MO) with PTACTIN, a P. teres actin primer. The amplicons were resolved by electrophoresis in 1% agarose gels. The amplified fragment sizes of P. teres from amended and the natural field soils correlated with the expected size from an amplified control P. teres pure culture. All amplicons were excised from the gel, purified with QIAquick Gel Extraction Kit (QIAGEN, Valencia CA), and sequenced. Sequence alignment confirmed the detection of P. teres from soils. This will enable rapid pre- and postplanting screening of soil for inocula of P. teres and determination of the effect of soil-applied chemicals, biological control agents or other management techniques.

Detection of DMI fungicide resistance in grape powdery mildew in the Okanagan valley. C. LEUNG, P. HAAG, P. L. SHOLBERG AND D. T. O'GORMAN. Pacific Agriculture Research Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada

Resistance of the grape powdery mildew pathogen Uncinula necator (Schw.) Burr. to demethylation-inhibiting (DMI) fungicides is common in most grape growing regions of the world. To confirm the presence of DMI resistance in British Columbia, grape leaves showing symptoms of powdery mildew were collected from six Okanagan vineyards. Uncinula necator conidia were harvested from the infected leaves and used in a bioassay to determine fungicide sensitivity/resistance status. DNA was also extracted from the conidia for subsequent molecular characterization. The resistance to DMI fungicides is due in part to a single DNA point mutation; this mutation is found in the gene encoding the enzyme cytochrome P-450 demethylase, which is targeted by this class of fungicides. The cytochrome P-450 gene was therefore sequenced for both resistant and sensitive isolates and the findings were in agreement with literature and the bioassay results. In resistant isolates a point mutation was identified as an A to T transition giving TTT (phenylalanine) in place of TAT (tyrosine) at codon 136. In conjunction with the sequence data, a quantitative PCR (QPCR) assay was developed with a dual hydrolysis probe specific for the point mutation found in DMI-resistant U. necator isolates. The QPCR assay was evaluated using DNA extracted from known sensitive and resistant isolates and subsequently used to analyse spore trap samples collected from local Okanagan vineyards.

The genome of Pythium ultimum provides insight into pathogenicity mechanisms and evolution of oomycetes. C. A. LÉVESQUE, G. P. ROBIDEAU, A. W. A. M. DE COCK, K. BALA, Q. A. EGGERTSON, T. L. RINTOUL, J. HAMILTON, N. TISSERAT AND C. R. BUELL. Biodiversity (Mycology), Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; (G.P.R., Q.A.E.) Department of Biology, Carleton University, Ottawa, ON K1S 5B6, Canada; (A.W.A.M.C.) CBS KNAW Fungal Biodiversity Centre, 3508 AD Utrecht, the Netherlands; (J.H., C.R.B.) Department of Plant Biology, Michigan State University, East Lansing MI 48824-1312, USA; and (N.T.) Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, CO 80523-1173, USA

Genome sequencing and annotation of Pythium ultimum Trow var. ultimum was recently completed and is available at http://pythium.plantbiology.msu.edu. Comparison to Phytophthora genomes showed that several of the core genes involved in pathogenicity are present but some unique features compatible with a necrotrophic lifestyle were also revealed. A comparative genome analysis of two strains of P. ultimum var. ultimum was done and used for identification of genes with high rates of single nucleotide polymorphisms. These identified genes were used to assess the P. ultimum species complex that contains another variety, P. ultimum var. sporangiiferum Drechsler. Multiple gene genealogies were created using some of the highly variable orthologous genes to test the phylogenetic species concept in this group. The species status of this second variety is being further clarified by comparison of the genome of P. ultimum var. sporangiiferum to P. ultimum var. ultimum. Genomes of other representative species from the different clades of Pythium are also being sequenced. P. vexans from Pythium clade K is of high interest as it is from a clade that is phylogenetically closer to Phytophthora than Pythium. The genus name Phytopythium has been proposed for this unique clade. This work in comparative genomics should provide insight into the evolution and pathogenicity mechanisms in oomycetes.

Exploring bacterial communities that influence plant health under different cropping systems using terminal-restriction fragment length polymorphism and pyrosequencing. R. LI, E. KHAFIPOUR, D. O. KRAUSE, M. H. ENTZ, T. R. DE KIEVIT AND W. G. D. FERNANDO. Department of Plant Science, 222 Agriculture Building, 66 Dafoe Road, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; (T.R.D.) Department of Microbiology, 418 Buller Building, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; and (E.K., D.O.K.) Department of Animal Science, Faculty of Agriculture & Food Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada

Plant health can be influenced by soil, and the function of soil is determined by many microorganisms including bacterial communities that play an important role in maintaining the health of plants. Many antibiotic-producing biocontrol strains and plant growth-promoting rhizobacteria (PGPR) belong to the Phylla Actinobacteria, Firmicutes and Proteobacteria. The objective of this study was to investigate how bacterial communities shift under different cropping systems (rotation vs. monoculture; zero vs. conventional tillage; organic vs. conventional) in Manitoba by using terminal-restriction fragment length polymorphism (T-RFLP) and pyrosequencing. The T-RFLP profile of V4 region of 16S rDNA gene demonstrated that Actinobacteria, Firmicutes, Proteobacteria, Bacteroidetes and Acidobacteria were the major populations in Manitoba's arable land. However, there are bacterial shifts among different cropping systems. Compared with wheat monoculture, wheat-oat-canola-pea rotation increased populations of Proteobacteria, Firmicutes and Acidobacteria by 150%, 340%, 279% respectively. Interestingly, when compared with the canola monoculture, wheat-oat-canola-pea rotation showed a similar microbial community, except for group Bacteroidetes, with an increase of 789% in the rotation. Canola monoculture supported much higher populations of Proteobacteria, Firmicutes and Acidobacteria than did wheat monoculture. Zero tillage was associated with an increase of 5-fold for Proteobacteria, and a 2.18-fold increase for Firmicutes. It was found that the group of Actinobacteria was quite stable and did not show significant shifts under the above cropping systems. Higher richness and diversity were observed in organic systems compared with the conventional systems. Pyrosequencing is a new, high-throughput technique to characterize microbial communities to the genus level using 16S rRNA sequences. The pyrosequencing data are being analysed at the present time. The study showed that different cropping systems influence the antibiotic-producing and PGPR communities, which influence plant health.

Genetic analyses of crown rust resistance in Avena sterilis accessions. Y. LIN, C. A. MCCARTNEY AND J. CHONG. Crop Development Centre/Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada; and (J.C.) Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada

Crown rust, caused by Puccinia coronata Corda f.sp. avenae Eriks., is an economically important disease of cultivated oat (Avena sativa L.) in western Canada. The development of resistant varieties requires the identification of new resistance genes because crown rust resistance genes are typically not durable. The objective was to discover new effective crown rust resistance genes in Avena sterilis L. accessions. Six genetic populations were generated by crossing ‘AC Morgan’ with six A. sterilis accessions. F₂ populations were inoculated with isolate CR259 (highly virulent on Pc 38/39/40/91) at the two leaf stage and rated for disease reaction 11 days post-inoculation. Chi-square analysis of F₂ generation data indicated single gene control of resistance in AC Morgan/PI334672 (3R: 1S), AC Morgan/PI375506 (3R: 1S) and AC Morgan/PI375547 (3R: 1S), and two-gene control of resistance in AC Morgan/PI311623 (15R:1S), AC Morgan/PI333561 (15R: 1S) and AC Morgan/PI333562 (15R: 1S). Evaluation of F₃ families in field test and seedling test will help to confirm F₂ generation data. These preliminary data suggested that putative new effective crown rust resistance genes have been identified.

Genetic diversity of pea downy mildew [ Peronospora viciae f.sp. pisi ] in Alberta, Canada. J. F. LIU, K. F. CHANG, S. F. HWANG AND S. E. STRELKOV. Department of Agriculture, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (K.F.C.) Field Crop Development Centre, Alberta Agriculture and Rural Development (ARD), 6000 C & E Trail, Lacombe, AB T4L 1W8, Canada; and (S.F.H.) Crop Diversification Centre North, ARD, 17 507 Fort Road N.W., Edmonton, AB T6G 2P5, Canada

Downy mildew, caused by the obligate parasite Peronospora viciae f.sp. pisi Boerema & Verh., is a common disease of pea. In 2009, 37 commercial pea fields located in the Mannville, Fort Saskatchewan and Vermillion areas of central Alberta were surveyed for downy mildew, with disease severity found to range from 0% to 26%. As the genetic structure of P. viciae populations from Alberta is not known, RAPD markers were used to investigate genetic diversity among 23 isolates of the pathogen collected from 19 infested fields. A total of 47 polymorphic bands were produced using nine arbitrary primers. Based on analysis with the POPGENE program, the 23 isolates were clustered into six groups, which were not associated with pea cultivar or location of origin. Five of the P. viciae isolates collected from the most severely infested field clustered in three groups. Studies of the population structure of P. viciae in Alberta may be useful in helping to manage downy mildew of field pea, by providing an understanding of the diversity of pathogen populations.

Genetic association study in the white pine blister rust pathosystem. J. -J. LIU, R. A. SNIEZKO AND A. K. M. EKRAMODDOULLAH. Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada; and (R.A.S.) USDA Forest Service, Dorena Genetic Resource Center 34963 Shoreview Road, Cottage Grove, OR 97424, USA

To dissect molecular mechanisms underlying western white pine (Pinus monticola Douglas ex Don) genetic resistance against the white pine blister rust (WPBR) pathogen (Cronartium ribicola Fisch.), a group of pathogenesis-related (PR) genes were selected as candidates for DNA re-sequencing in white pine populations. Along with other plant PR families, PR3 chitinase genes are important components with the potential to contribute to white pine partial resistance against C. ribicola. The P. monticola gene family encoding for class IV endochitinases (PmCh4) consists of at least nine genes (PmCh4A to PmCh4I) with two pseudogenes (PmCh4G and PmCh4I). Single nucleotide polymorphisms (SNPs) of the PmCh4 genes were characterized in seven open-pollinated seed families of diverse geographical distribution and variable levels of quantitative resistance to C. ribicola infection. PmCh4 loci were revealed with average pairwise nucleotide diversity (π = 0.00193–0.00965), similar to those of other conifer genes related to environmental stresses. Based on the PmCh4 gene variations, we detected moderate to high levels of population structure (average Fst = 0.163, P < 0.001) among these seven seed families currently used in breeding programs. The PmCh4 intragenic linkage disequilibrium (LD) levels were low, but most of them were statistically significant within ∼800 bp PmCh4 gene sequences. Partial resistance phenotypes in tree stems were quantitatively assessed using a series of parameters, including presence of aecia, canker damage and severity, number of stem symptoms by type (normal canker, partial bark reaction, complete bark reaction), or ‘clean’/stem-symptom free. An association analysis found a significant genetic association between PmCh4B nucleotide variations and partial resistance phenotypes. Our results suggest that LD-based association analysis is a feasible strategy to dissect partial resistance in the WPBR pathosystem.

Study of silicon transport to improve plant disease resistance. J. MONTPETIT, J. VIVANCOS, W. REMUS-BOREL, C. GREGOIRE, J. G. MENZIES, F. BELZILE AND R. R. BELANGER. Phytologie, 2425, rue de l'Agriculture, Université Laval, Québec, QC G1V 0A6, Canada; and (J.G.M.) Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada

Silicon (Si) is not considered as an essential element for plant growth yet its uptake is known to be beneficial in alleviating abiotic and biotic stresses, namely fungal diseases. However, positive effects are variable since accumulation differs among plant species and a direct correlation between benefits and absorption has been shown. Some Gramineae can accumulate up to 10% on a dry weight basis while most dicots accumulate less than 0.1%. Recently, specific genes involved in Si uptake have been discovered in rice with homologues reported in maize and barley. The objective of this project was to investigate, identify and characterize the presence of Si-transport genes in wheat, a species known to accumulate Si, and to determine their functionality and localization. Our results have allowed the identification and the cloning of a putative Si-transport gene presenting high homology (> 80%) with the Si-influx protein in rice known as Lsi1. Transient expressions of the wheat Lsi1 Si transporter (TaLsi1) coupled with GFP in Nicotiana benthamiana indicated that this protein was localized across the plasma membrane, a feature typical of other members of the Lsi1 family. The Si transport activity of TaLsi1 was confirmed in a heterologous system, Xenopus laevis oocytes, and its efficiency at transporting Si was comparable to that of the rice Lsi1. These results are of particular interest since breeding wheat plants for Si transport activity could result in cultivars more naturally tolerant or resistant to environmental stresses.

Antimicrobial efficacy of ginger extracts against spoilage pathogens. H. N. MVUEMBA, R. J. TWEDDELL, A. TSOPMO AND T. J. AVIS. Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6, Canada; and (R.J.T.) Centre de recherche en horticulture, Pavillon de l'Envirotron, Université Laval, Québec, QC G1V 0A6, Canada

Spices have been previously used as antimicrobial products both in traditional and herbal medicine and have shown promise as potential alternatives to synthetic fungicides. In this work, aqueous ginger (Zingiber officinale Roscoe) extracts were assayed for their ability to inhibit mycelial growth of spoilage pathogens and development of potato (Solanum tuberosum L.) dry rot (Fusarium sambucinum Fuckel) and carrot (Daucus carota L.) cavity spot (Pythium sulcatum Pratt & Mitch.). In general, ginger extracts inhibited the tested pathogens in a dose-dependent manner. Results showed that 0.05 g mL⁻¹ ginger extracts completely inhibited the mycelial growth of P. sulcatum. At the highest tested extract concentration (0.15 g mL⁻¹), F. sambucinum growth was significantly decreased whereas Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. growth was completely inhibited. Aspergillus niger Tiegh. was not inhibited at any of the tested ginger extract concentrations. Application of ginger extracts (0.05 to 0.15 g mL⁻¹) to potato tubers artificially inoculated with F. sambucinum demonstrated that none of the tested concentrations reduced dry rot severity. Conversely, 0.15 g mL⁻¹ extracts significantly reduced cavity spot severity on mature carrot roots artificially inoculated with P. sulcatum by 35% when compared to the control. This study points to the possibility of exploiting ginger extracts as a control method against specific postharvest diseases.

Wax and pigment for turfgrass disease control. B. NASH, T. HSIANG AND P. H. GOODWIN. School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

A novel synthetic isoparaffin-based mixture (wax) produced by Petro-Canada has been tested for turfgrass disease control in the field since 2004. Previous research has demonstrated that the isoparaffin mixture can induce resistance in plants against diseases. In 2007, a copper-containing green pigment dispersion was added to improve the effectiveness of this product against turfgrass diseases. Current lab trials are aimed at determining the potential mode of action of the pigment dispersion. Initial trials have shown direct inhibition on fungal growth of turfgrass pathogens such as Sclerotinia homoeocarpa Benn. (EC₅₀ = 9.1%), as well as reduced disease symptoms on creeping bentgrass inoculated with S. homoeocarpa. We hypothesize that part of the disease suppression caused by the pigment may be due to acquired resistance since low concentrations of metal-containing compounds can enhance stress responses in plants through activation of stress responsive genes. In addition, a significant reduction in disease was observed on creeping bentgrass inoculated seven days after treatment with 2% pigment, relative to creeping bentgrass inoculated immediately after treatment. Current work aims to identify genes which may be responsive to application of the pigment dispersion, as well as the effect of combining the pigment dispersion with the isoparaffin-based mixture on disease resistance and defence gene expression.

Response of potato cultivars to Potato virus Y strain groups. X. NIE, B. NIE, M. SINGH, R. P. SINGH, A. SULLIVAN AND C. XIE. Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada; (M.S.) Agricultural Certification Services, 1030 Lincoln Road, Fredericton, NB E3B 8B7, Canada; (A.S.) Plant Propagation Centre, New Brunswick Department of Agriculture and Aquaculture, 598 Lincoln Road, Fredericton, NB E3B 5H1, Canada; and (B.N., C.X.) College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, Hubei 430070, China

Potato virus Y (PVY, genus Potyvirus, family Potyviridae) is one of the most economically important viruses of the potato crop worldwide, causing significant yield loss and quality degradations. Multiple strains/substrains have been recognized and characterized. The ordinary (common) strain (PVY^O) and the tobacco veinal necrosis strain (PVY^N) were the basic strain groups, and from which, various recombinant strain groups including PVY^N:O (with one recombinant event) and PVY^NTN (with three recombinant events) emerged. To better understand the interactions between potato cultivars and PVY strain groups, 14 common potato varieties were individually mechanically inoculated with PVY^O-FL (a severe PVY^O isolate), PVY^O-RB (a mild PVY^O isolate), PVY^N:O-Mb58, PVY^N-Jg or PVY^NTN-1 in the greenhouse. The infection was confirmed by enzyme-linked immunosorbent assay (ELISA) for PVY at three weeks post-inoculation. Foliar symptoms were monitored daily after the inoculation until harvest; and tuber symptoms, mainly potato tuber necrotic ringspot disease (PTNRD), were checked at the harvest and at two months post-harvest. The symptoms in plants varied significantly, depending on potato cultivar and virus strain. Cultivars ‘Cal White’ and ‘Red La Soda’ did not develop obvious symptoms regardless of PVY strains/isolates; and ‘Russet Burbank’ and ‘Russet Norkotah’ developed mild mosaic by PVY^O, PVY^N:O or PVY^NTN. On the other hand, ‘Jemseg’, ‘Ranger Russet’, ‘AC Chaleur’ and ‘Yukon Gold’ developed local lesions and systemic necrosis in leaves by PVY^O, mild mosaic/mottle by PVY^N, severe mosaic by PVY^N:O, and milder but visible systemic necrosis by PVY^NTN. PTNRD was only observed in PVY^NTN-infected ‘AC Chaleur’, ‘Cherokee’ and ‘Yukon Gold’.

Screening for potato genes expressed differentially in the compatible and incompatible virus–potato interactions. B. NIE, C. XIE AND X. NIE. Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada; and (B.N., C.X.) College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, Hubei 430070, China

Plant–virus interaction is a complicated process. When virus and host are compatible, systemic infection takes place and the plants exhibit a susceptible phenotype. When virus and host are incompatible, systemic infection is disrupted and the plants demonstrate a resistant phenotype. Potato cultivar ‘Shepody’ is resistant to potato virus A (PVA) but susceptible to potato virus Y (PVY). Graft-inoculation of ‘Shepody’ with PVA led to hypersensitive resistant responses in stems and tubers in the plants; whereas inoculation with PVY, either by mechanical rubbing or grafting, led to systemic infection and mosaic symptoms in leaves in the plants. To reveal the molecular aspects of the interactions, a gene screening technology termed ACP™ (Annealing Control Primer) was employed to identify differentially expressed genes (DEGs). A total of 53 DEGs were selected, cloned and sequenced. Most of the DEGs exhibited significant similarities with known genes or expression sequences tags (ESTs) from potato and other plant species. Some of them showed high identities with genes involved in senescence, wounding, and drought as well as pathogen invasions. Further expression analysis of the DEGs is underway.

Recognition and molecular discrimination of severe and mild PVY^O variants of potato virus Y in potatoes. B. NIE, M. SINGH, A. SULLIVAN, R. P. SINGH, C. XIE AND X. NIE. Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada; (M.S.) Agricultural Certification Services, 1030 Lincoln Road, Fredericton, NB E3B 8B7, Canada; (A.S.) Plant Propagation Centre, New Brunswick Department of Agriculture and Aquaculture, 598 Lincoln Road, Fredericton, NB E3B 5H1, Canada; and (B.N., C.X.) College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, Hubei 430070, China

A field isolate of potato virus Y (PVY) was collected in New Brunswick due to its unusual pathogenicity in different varieties including FL 1533, FL 1579, Gold Rush and Atlantic in 2007. Enzyme-linked immunosorbent assay (ELISA) analysis using various virus-specific antibodies revealed that PVY was the only virus present in the samples. To unveil the strain identity, tobacco/potato-based bioassay, PVY^O and PVY^N-specific antibody-based ELISA and reverse transcription-polymerase chain reaction (RT-PCR)-based genotyping were carried out. All the assays demonstrate that the isolate, now designated as PVY^O-FL, belongs to the PVY^O strain group. The complete genome sequences of PVY^O-FL and PVY^O-RB, a mild PVY^O isolate, were determined. BLAST analysis revealed that the two shared 97% and 98% identities at the nucleotide and polyprotein levels, respectively. Further BLAST analysis unveiled that PVY^O-FL shared 99.7% nucleotide sequence identity with PVY^O-Oz, an isolate reported in Maine, USA; whereas the PVY^O-RB shared 99.1% with PVY^O-139, a PVY^O isolate reported in New Brunswick, Canada. The phylogentic tree of available full-length sequences of PVY demonstrated that two subgroups were formed under the PVY^O branch, possibly representing the mild (PVY^O-139, -RB, -SASA-110) and severe (PVY^O-Oz, -FL, -SCRI-O) isolate subgroups, respectively. Group-specific sense primers for differentiation of the two groups were developed and evaluated. A limited survey using the newly developed PCR primers indicated that the majority of PVY^O from a field at the research centre belonged to the severe isolate group.

Characterization of resistance against white pine blister rust using in vitro techniques. D. NOSHAD, J. N. KING AND A. EKRAMODDOULLAH. BC Forest Service, P.O. Box 9519, Stn Prov Govt, Victoria, BC V8W 9C2, Canada; and (A.E.) Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada

White pine blister rust (WPBR) caused by the rust fungus Coronartium ribicola J.C. Fischer is one of the most devastating diseases of western white pine (Pinus monticola Dougl.) trees. Different types of resistance with a very low frequency have been observed in a four-year field screen programme. The resistant plants have been categorized in four major groups: difficult-to-infect (DI), bark reaction (BR), slow canker growth (SCG) and needle shed (NS). A disease assessment index (DAI), based on both in vitro and in vivo techniques, was used to evaluate specific reactions to the pathogen. The in vitro method provides a new approach to study inoculation in an axenic environment under controlled conditions. The results from electron microscopic study of different tissues and organs from several samples of the DI resistant family collections indicated a significant difference (P <0.05) in their needle structure from those of control/susceptible families. The statistical analysis of the amount of epicuticular wax indicated that there is a significant difference (P < 0.05) between DI resistant families more than the control families. Evaluation of epicuticular wax using chloroform method confirmed the electron microscopic results in that the majority of the stomata in DI resistant families were occluded.

Induced fruiting of Phellinus sulphurascens . K. W. PELLOW AND R. N. STURROCK. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada

Phellinus sulphurascens Pilát (syn. P. weirii (Murrill) Gilb.) is an important root pathogen in the western forests of Canada and the USA. In nature, sporophore formation by P. sulphurascens is rare and inconsistent. Recently, molecular methods have been used to study mating and phylogenetic relationships in this and other Phellinus species. To facilitate our understanding of the genetics of P. sulphurascens we developed a method to initiate fruit body formation as a means of obtaining single spore cultures. Briefly, sterilized stem sections of red alder (Alnus rubra Bong.), inoculated with liquid cultures of P. sulphurascens and sufficiently colonized inside mushroom spawn bags, were placed over plastic bins containing moistened peat moss and exposed to ambient light and temperature conditions in a shade house; bags of blocks were covered with a plastic sheet to maintain a high relative humidity. It took an average of two weeks for sporophore tissue to form and for sporulation to occur. We also tested and had success using this fruit body induction method with other Phellinus species; including P. weirii (formerly known as the cedar form of P. weirii sensu lato), an isolate of P. nigrolimitatus (Romell) Bourdot and Galzin, and with a Japanese isolate of P. sulphurascens. We are continuing with a variety of sporophore induction trials.

Managing late blight and pink rot of potatoes with foliar and post-harvest applications of phosphorous acid. R. D. PETERS, R. H. COFFIN, H. W. PLATT, K. I. AL-MUGHRABI, G. WANG-PRUSKI, D. PINTO, S. LIM, I. K. MACDONALD, K. A. DRAKE, R. POIRIER, S. VEENHUIS-MACNEILL, W. HARDY AND T. ASTATKIE. Crops and Livestock Research Centre, Agriculture and Agri-Food Canada, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; (R.H.C., S.V.-M., W.H.) Cavendish Farms, Summerside, PE C1N 5J5, Canada; (K.I.A.-M., R.P.) Potato Development Centre, New Brunswick Department of Agriculture and Aquaculture, 39 Barker Lane, Wicklow, NB E7L 3S4, Canada; (G.W.-P., S.L., T.A.) Nova Scotia Agricultural College, P.O. Box 550, Truro, NS B2N 5E3, Canada; and (D.P.) National Research Council-IMB, Halifax, NS B3H 3Z1, Canada

Late blight [Phytophthora infestans (Mont.) de Bary] and pink rot [Phytophthora erythroseptica Pethyb.] are devastating diseases of potatoes that occur worldwide and cause significant crop losses annually. Over the past six years, we have conducted a number of trials that have examined the efficacy of phosphorous acid-based products (PA) for control of these diseases. During harvest operations, pathogen spores can be splashed from diseased to healthy tubers resulting in potential rot during storage. When PA is applied as a post-harvest spray to potatoes entering storage, complete suppression of late blight tuber rot and pink rot caused by these splashed spores can be achieved, if application occurs prior to tuber infection. When applied to the foliage, PA has significantly delayed late blight epidemic development in our studies. The combination of PA with Bravo® was a particularly effective approach likely due to the synergies captured by combining a truly systemic (PA) with a protectant (Bravo®) product. Foliar application of PA also provided excellent suppression of both late blight and pink rot development in tubers in the field. As well, tubers from plants receiving PA in the field were less susceptible to disease than control tubers when inoculated with the oomycete pathogens after harvest. Since PA-based products are recognized as being environmentally friendly, their incorporation into disease management programs may significantly reduce the usage of other fungicides. In fact, we have conducted additional studies which indicate that the rate of a protectant fungicide (Bravo®) can be reduced by 50% and still achieve excellent disease control if PA is included in the disease management program. In this way, environmental risk of pesticide application, particularly to aquatic organisms, can be lessened.

Interaction between flax genotypes and Septoria linicola isolates from Western Canada. K. Y. RASHID. Agriculture and Agri-Food Canada, Morden Research Station, Unit 100 101 Route 100 Morden, MB R6M 1Y5, Canada

Septoria linicola (Speg.) Garassini (sexual state Mycosphaerella linorum Naumov), is the causal agent of the Pasmo disease in flax (Linum usitatissimum L.) worldwide. This is a stubble-borne pathogen that infects the leaves and stems causing severe defoliation, stem break and lodging. Pasmo is prevalent in all flax growing areas in western Canada causing 10–30% loses in yield, and degrading the quality of the harvested seed. None of the registered commercial flax cultivars in Canada have resistance to pasmo. Research studies to identify sources of resistance among hundreds of flax accessions using four single spore isolates of the fungus under controlled growth room conditions resulted in identifying several flax genotypes with reduced levels of infections on leaves and stems or both indicating partial resistance to this pathogen. The results also showed a strong interaction between specific S. linicola isolates and flax genotypes. Several flax genotypes have been identified with resistance to one, two, three and four isolates of S. linicola, confirming the specific genetic interaction. Crosses among the various flax genotypes are underway to study the inheritance of resistance and pyramid resistance genes in future cultivars for a durable field resistance.

Comparative histological examination of morphologically distinct clubroot galls in crucifers . D. C. RENNIE, V. P. MANOLII, M. PLISHKA, S. F. HWANG AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Sciences, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; (S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada; and (M.P.) Department of Biology, University of Alberta, Edmonton, AB T6G 2P5, Canada

Clubroot [Plasmodiophora brassicae Woronin] is a cosmopolitan disease of crucifers. Disease development is typified by the proliferation of the pathogen in the roots of host plants. Subsequent division and enlargement of infected cells is responsible for gall formation. While typical galls are often diffuse and spindle-shaped, discrete spheroidal galls have also been observed in particular host–pathogen combinations. These spheroidal galls have historically been regarded as resistance structures, although they have been found to contain P. brassicae plasmodia and infective resting spores. We hypothesize that the morphological differences observed between spheroid and typical spindle-shaped galls result from the restriction or limitation of the plasmodia during the early development of the spheroid galls. This restriction may underpin the discrete, spheroidal gall morphology that is regularly observed. Studies on the developmental anatomy of spheroid galls relative to typical spindle-shaped galls, using Safrinin/Fast Green-stained thin sections of infected roots, are underway to test this hypothesis.

Biofumigation against endogenous and quarantine plant parasitic nematodes of vegetable crops in the Pacific Northwest. E. RIGA. IAREC, Washington State University, Prosser, WA 99350, USA

The endophytic fungus, Muscodor albus Worapong, Strobel & Hess, brassica green manures and mustard seed meals were tested against plant parasitic nematode species including the quarantined potato cyst nematode, Globodera pallida (Stone) Behrens, from economically important vegetable crops in the Pacific Northwest. The Columbia root knot nematode, Meloidogyne chitwoodi Golden et al., the northern root knot nematode, Meloidogyne hapla Chitwood, the stubby root nematode Paratrichodorus allius (Jensen) Siddiqi and the lesion nematode Pratylenchus penetrans (Cobb) were exposed in the laboratory, greenhouse and/or field conditions to the above treatments, while G. pallida was tested only in the greenhouse. The mean per cent mortality of nematodes exposed to M. albus in the greenhouse was 82.9% for P. allius, 82.1% for P. penetrans and 95% for M. chitwoodi; mortality in the controls was 9%, 7% and 3.9% respectively. Only 21.6% of M. hapla juveniles died due to M. albus in comparison to 8.9% in controls, however, 69.5% of the treated M. hapla juveniles displayed reduced motility in comparison to the controls. Green manures from brassica crops reduced all of the above plant parasitic nematodes between 80 and 100% in the greenhouse and field with exception of M. chitwoodi. The Columbia root knot nematode has five to seven life cycles per growing season, therefore, green manure on their own were not able to reduce this species. However, green manures in combination with reduced rates of synthetic nematicides reduced it to similar levels as synthetic fumigants. Mustard seed meals significantly reduced all of the above plant parasitic nematode species in the greenhouse and in the field in comparison to the controls. In addition, mustard seed meals caused 100% mortality to G. pallida in all treated pots in comparison to the untreated controls.

Foliage and stem susceptibility of six eastern Canadian forest species to Phytophthora ramorum . D. RIOUX, M. SIMARD, A. JINEK, S. C. BRIÈRE, A. K. WATSON AND R. J. TWEDDELL. Natural Resources Canada, Laurentian Forestry Centre, Québec, QC G1V 4C7, Canada; (S.C.B.) Canadian Food Inspection Agency, Ottawa, ON K2H 8P9, Canada; (A.K.W.) Department of Plant Science, McGill University, Ste-Anne-de-Bellevue, QC H9X 3V9, Canada; and (R.J.T.) Centre de recherche en horticulture, Université Laval, Québec, QC G1V 0A6, Canada

Although Phytophthora ramorum Werres et al. (Pr) is absent in the wild in eastern North America, there is concern regarding its possible introduction and spread into this area. In order to assess this risk, foliage and stems of six eastern Canadian forest species were inoculated with Pr. Abies balsamea (L.) Mill. (Ab), Acer saccharum Marshall, Betula alleghaniensis Britt. (Ba), Fraxinus americana L. (Fa), Larix laricina (Du Roi) Koch (Ll) and Quercus rubra L. (Qr) were the species tested. Wounded detached leaves/needles and plant-dipping of wounded and unwounded leaves/needles were used to study the effect of Pr on foliage. The foliage of Ba, Fa and Ab were the most susceptible among the species tested and allowed the highest level of sporulation following plant-dipping. After stem inoculation, nearly 25% of Ll and Ab seedlings died. Necrotic areas were obvious on Qr stems. Preliminary microscopic examinations revealed for the first time compartmentalization reactions in the foliage and stem of some species that apparently limit the colonization of Pr in host tissues. The significance of these data and their interpretation relative to the natural potential for Pr to establish and spread on these species in eastern Canada will be discussed.

Physical separation of germinating Ustilago maydis teliospores for gene expression analysis. A. M. SETO AND B. J. SAVILLE. Environmental and Life Sciences Graduate Program, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada; and (B.J.S.) Forensic Science Program, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada

Ustilago maydis (Persoon) Roussel is a pathogenic basidiomycete that infects maize and its ancestor teosinte causing tumour formation in the developing stems, leaves, tassels and ears of the plant. This pathogen can have a significant economic impact on corn production and it serves as a model for less tractable fungal pathogens. Ustilago maydis differentiates inside the tumour and forms diploid teliospores. Teliospores are the dormant dispersal agents of U. maydis and are the only cell type capable of meiotic cell division. Germination can be induced in the teliospore and meiosis can be completed on plates or in liquid culture. A goal of the laboratory is to isolate genes that are expressed at specific stages of germination. However, germination is asynchronous and therefore a physical means of separating teliospores at different stages of germination are required. We are investigating two primary means of physical separation to enable us to pool viable teliospores at the same stage of germination. Centrifugal elutriation has been successful in separating populations of budding Saccharomyces cerevisiae Meyen ex Hansen spores suitable for molecular analysis. Size-selective sieving has been used to separate Tilletia indica (Mitra) Mund. teliospores for PCR assays. Obtaining synchronized spore populations will enable isolation of stage specific transcripts and proteins. This, in turn, will form the basis for investigating mechanisms U. maydis uses to control germination and complete meiosis. We reported on our progress in isolating stage specific teliospores as well as our gene expression analysis from asynchronous populations.

From laboratory bench to marketplace – the ‘Chontrol®’ story – a legacy to government–academia–industry partnerships. S. F. SHAMOUN. Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada

Environmental concerns have called for the reduction of chemical herbicide use in managed forests and given support to the concepts of integrated forest vegetation management, sustainable forestry and biodiversity. There is an urgent need to develop cost-effective, efficacious and environmentally acceptable techniques for management of forest weeds. One viable option is the utilization of naturally occurring plant pathogenic fungi as an inundative biocontrol approach or mycoherbicides. Chondrostereum purpureum (Pers.) Pouzar is a wood-inhabiting fungus, which has been tested as a potential biocontrol agent for weedy hardwood species in conifer plantations and utility rights-of-way. The Canadian Forest Service's Pacific Forestry Centre and it's partners MycoLogic Inc./UVic. and BC Hydro have collaborated since 1995 in order to generate scientific data relevant to the registration guidelines of C. purpureum as a biocontrol product. The regulatory agencies in Canada and the USA require the following data: (1) genetic characterization of the active ingredient; (2) determination of the environmental fate and risk; (3) development of solid- and liquid-based formulations; and (4) experimental field trials to determine the efficacy on target species in several forest ecosystem zones in Canada and the USA. This scientific database has been the key to the joint registration of the Chontrol® Paste of C. purpureum isolate PFC2139 in Canada and the USA. Development and registration of Chontrol® as the first commercial biocontrol agent for management of weedy hardwood species in North American forests represents a major breakthrough in forest vegetation management practices. In this presentation, I will focus on history of C. purpureum use worldwide, the scientific and technological challenges encountered during the development of Chontrol® and research collaboration among government, academia and industry partners.

Identification, tissue susceptibility and chemical control of volutella blight of boxwood in Ontario. F. SHI AND T. HSIANG. School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

In collections since 2008, a fungus tentatively identified as Volutella buxi (Corda) Berk. was most commonly recovered out of 180 symptomatic samples of boxwood from several Ontario nurseries. Because the highly divergent sequences annotated as V. buxi in the GenBank database are likely from multiple species, the type species, V. ciliata (Alb. & Schwein.) Fr., was obtained to compare the ribosomal DNA sequences and morphological features with our isolates. The comparisons confirmed the tentative identification of our isolates as a species of Volutella. To test the resistance of different tissues of boxwood to Volutella blight, inoculation tests were done in the laboratory, which showed that wounded boxwood tissues were more susceptible than non-wounded boxwood tissues, and that one-month-old leaves were more susceptible to infection than one-year-old leaves. For fungicidal control, three treatments (benomyl, propiconazole and iprodione) were applied separately to boxwood plants at different periods, preventively or curatively. The preventive activity of the fungicides was more effective than curative activity, and all three fungicides were able to reduce disease on average by 60%.

Impacts of ring nematodes ( Mesocriconema xenoplax ) on self-rooted Merlot and selected rootstocks under Okanagan Valley growing conditions. R. SMIT, T. A. FORGE, C. KOCH, G. H. NEILSEN AND D. NEILSEN. Pacific Agri-Food Research Centre (PAFRC), Agriculture and Agri-Food Canada (AAFC), 6947 Highway 7, Agassiz, BC V0M 1AO, Canada; and (G.N., D.N.) PAFRC, AAFC, P.O. Box 5000, 4200 Highway 97, Summerland, BC VOH IZO, Canada

Several species of plant parasitic nematodes, including Mesocriconema xenoplax (Raski) Loof & De Grisse have been found in Okanagan Valley vineyards that appear to be declining in productivity and have sparse, often necrotic root systems. Research conducted on relatively fine-textured soils in coastal Oregon and California indicated that M. xenoplax can cause significant reductions in grapevine root growth and vigour, but the impact of M. xenoplax on grapevines under Okanagan Valley growing conditions is unknown. Our research objectives were to: (1) determine if M. xenoplax has detrimental effects on growth of self-rooted vines and three rootstocks growing in a sandy soil typical of many Okanagan Valley vineyards, and (2) determine if application of compost to the root zone of grape affects M. xenoplax population densities. In spring 2007, field microplots were fumigated, inoculated with the nematode (control microplots were not inoculated), and planted with self-rooted Merlot or plants of Merlot grafted onto rootstocks of Riparia Gloire, 44-53M or 3309C. In spring 2009, M. xenoplax trunk diameters and pruning weights of self-rooted vines were lower in M. xenoplax-inoculated microplots than in non-inoculated microplots. In contrast, rootstocks were not affected by the nematode. Composted poultry manure was applied as the primary source of nitrogen to experimental plots in two different mature vineyards; the application rate was intended to result in similar available N as fertilizer-treated control plots. The compost was applied for three years and then nematode populations were assessed at the end of the third year and at three dates in the fourth year. Population densities of M. xenoplax were greater in compost-amended plots than in fertilizer-treated plots.

Epidemiology and management of potato late blight in Punjab, India. S. S. SOKHI AND P. S. SEKHON. Markfed, Punjab, Chandigarh and Punjab Agricultural University, Ludhiana, India

Late blight of potato caused by Phytophthora infestans (Mont.) de Bary occurs in severe form in the October-planted crop in northern Punjab. The favourable weather conditions for the development of the disease are: temperature, 10–22 °C; per cent relative humidity, more than 80; and frequent rains in November–December coupled with cloudy/foggy weather. Incubation period of the disease varies from 4–6 days at mean temperature of 16 ± 2 °C, and 12–14 days at 8.8 ± 2 °C. Leaf wetness period of 18–24 h results in shorter incubation periods, increased sporulation and more generations of the fungus. Seed potato having 5–10% infection is the major cause of primary infection. Removal of the infected seed tubers before storing in cold store and subsequently before planting minimizes the chances of primary infection. During favourable weather conditions, three sprays of preventive fungicides like mancozeb or chlorothalonil or copper oxychloride at weekly intervals, followed by two sprays of fungicide like Ridomil MZ (metalaxyl + mancozeb) or Curzate M (cymoxanil + mancozeb) and subsequently two more sprays of preventive fungicides control the disease effectively. This schedule works well if there is a 45-day favourable period for the disease development. Under less favourable weather conditions, the disease could be arrested with two to three sprays of preventive fungicides. No spray schedule is recommended in the southern Punjab where the weather conditions are not conducive for the disease. Site-specific forecasting of late blight incidence during the crop season is necessary to ensure judicious execution of the spray schedule. Cultivation of late blight resistant varieties like Kufri Jyoti, Kufri Badshah, Kufri Pushkar and Kufri Pukhraj delays the initiation of the disease.

Re-emergence of spotted net blotch in Manitoba. A. TEKAUZ AND M. DESJARDINS. Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg MB R3T 2M9, Canada; and (M.D.) Crop Diagnostic Centre, Manitoba Agriculture, Food and Rural Initiatives, 545 University Crescent, Winnipeg, MB R3T 5S6, Canada

Two forms of net blotch, netted net blotch (NNB) and spotted net blotch (SNB), caused by Pyrenophora teres f. teres Drech. (Ptt) and P. teres f. maculata Smed.-Pet. (Ptm), respectively, have been known to affect Manitoba barley crops since the early 1970s. Systematic, annual monitoring for barley leaf spot diseases was initiated in 1994 to document disease levels and the pathogens involved. Net blotch and spot blotch (Cochliobolus sativus (Ito & Kurib.) Drechsler ex Dastur were common each year. Based on symptoms on collected leaves, most ‘net blotch’ lesions appeared to be those of NNB. This assumption was tested in 2009 by isolating the pathogen(s) from randomly selected field collections to obtain 14 single-spore isolates of P. teres. The isolates were inoculated onto a maximum of 24 barley differential lines. Surprisingly, 9 of 14 isolates produced SNB symptoms on barley plants, indicating they were isolates of Ptm. The three Ptt isolates tested on the entire differential set had distinct virulence phenotypes, which also differed from those of standard Ptt isolates WRS102 and WRS858. The four Ptm isolates tested likewise were unique. These results suggest a comprehensive re-evaluation of the P. teres population in Manitoba, and western Canada, is warranted to assess the current virulence in the pathogen and review the effectiveness of genetic sources used in breeding for NNB and SNB resistance.

Comparison of methods to assess the sensitivity of Ascochyta rabiei to strobilurin fungicides. N. H. THAHER, 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

Strobilurin fungicides provided effective management of ascochyta blight caused by Ascochyta rabiei (Pass.) Labrousse on chickpea (Cicer arietinum L.) in western Canada in the mid 2000s. However, repeated application on this crop resulted in a shift to insensitivity to these fungicides in the A. rabiei population across the region, noted first in 2006. A study was conducted to assess factors that affect the assessment of sensitivity of A. rabiei to strobilurins. One component of the study compared conidial germination and mycelial growth assays of sensitivity. Ten isolates of A. rabiei (three sensitive, four intermediate, and three insensitive) were selected based on reduction in radial growth at 5 μg mL⁻¹ pyraclostrobin compared with a non-treated control. The isolates were grown on potato dextrose agar (PDA) medium amended with Headline (0.1–1000 μg pyraclostrobin mL⁻¹ for the radial growth assay, 0.0001–10 μg a.i. mL⁻¹ for the conidial germination assay), with and without salicylhydroxamic acid (SHAM) at 100 μg mL⁻¹. EC₅₀ values were calculated for each isolate in each of three replicates. There was a strong positive correlation of response in isolates assessed with and without SHAM (r = 0.97, P = 0.0001 for radial growth; r = 0.88, P = 0.0001 for conidial germination), and a strong correlation between techniques (r = 0.67, P = 0.0001). A second component of the study compared the sensitivity of single-spore and mass-transfer isolates using radial growth assessments at 5 μg mL⁻¹ pyraclostrobin. Four single-spore subcultures were developed from each of the original 10 mass-transfer isolates. The pattern of response at this discriminatory dose did not differ substantially between single-spore and mass-transfer isolates. We conclude that assessment of mass-transfer isolates in a radial growth assay without SHAM provides a quick and accurate assessment of sensitivity to pyraclostrobin in A. rabiei.

Forecasting the potential distribution and severity of fusarium head blight of wheat in the Canadian prairies under potential climate change scenarios. T. K. TURKINGTON, O. O. OLFERT, R. M. WEISS, H. KLEIN-GEBBINCK, K. XI AND D. KRITICOS. Lacombe Research Centre/Beaverlodge Research Farm, Agriculture and Agri-Food Canada (AAFC), 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada; (O.O.O., R.W.) Saskatoon Research Centre, AAFC, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (K.X.) Alberta Agriculture and Food, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada; and (D.K.), CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia

Fusarium graminearum Schwabe is the most important causal agent of fusarium head blight (FHB) of wheat resulting in yield and quality losses, and until recently was mainly a production issue of cereals in the eastern prairies. A bioclimatic model using CLIMEX^TM (Hearne Software, Inc.) was constructed and used to predict potential distribution and severity of FHB in the prairie region under: (1) incremental temperature and moisture scenarios using long-term climate normal data (LCND); and (2) using three General Circulation Model (GCM) scenarios (Micro-H, NCAR-CCSM, CSIRO Mark 3.0) and a baseline scenario (CRU World V2). Based on current conditions with either the LCND or the baseline scenario, the model predicted that FHB caused by F. graminearum would readily develop in the cereal-growing areas of western Canada and present a significant risk to cereal production, especially in moister regions. Predicted FHB development in the prairie region tended to be greater, especially for the NCAR-CCSM compared to the baseline scenario. A similar trend, but with a reduced increase in FHB severity, was observed with the CSIRO Mark 3.0 and Micro-H scenarios, especially for the western areas of the central and northern prairies. Incremental temperature increases of 1 to 3 °C with a 20% increase in seasonal rainfall resulted in the greatest projected increase in the potential range and severity of F. graminearum in the prairie cereal growing ecozone. In contrast, a 20% decrease in precipitation resulted in a substantial reduction in the area potentially affected by economic levels of FHB. Moreover, the area was further reduced when the decrease in precipitation was combined with a 1 to 3 °C increase in temperature.

Semi-quantitative-PCR and fluorescence microscopy analysis of the progression Puccinia triticina in the compatible and incompatible interaction using Thatcher near-isogenic wheat lines with leaf rust resistance genes Lr2a , Lr3 and LrB . X. WANG, B. D. MCCALLUM, T. FETCH, G. BAKKEREN, G. F. MARAIS AND B. SAVILLE. Cereal Research Centre, Agriculture and Agri-Food Canada (AAFC), 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada; (G.B.) Pacific Agri-Food Research Centre, AAFC, P.O. Box 5000, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada; and (B.S.) Forensic Science Program, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, ON K9J 7B8, Canada

The progression of wheat leaf rust fungus, Puccinia triticina Eriks, in the compatible and incompatible interaction was investigated using a set of ‘Thatcher’ near-isogenic wheat lines with leaf rust resistance genes Lr2a, Lr3 and LrB. ‘Thatcher’ near-isogenic lines were inoculated separately with P. triticina virulence phenotypes BBBD, MBDS and FBDJ. Phenotype BBBD was avirulent to all three genes, MBDS was avirulent only to Lr2a and FBDJ was avirulent to Lr2a and LrB. Inoculation were performed at three different concentrations (1.0, 2.5 or 5.0 × 10⁶ urediniospores mL⁻¹) for each interaction and plants were sampled at 0, 7, 14, 21 and 28 days after inoculation. Infection processes were observed under the fluorescence microscopy after staining with Uvix2B. For the semi-quantitative PCR analysis, total genomic DNA was extracted from infected leaves and fungal biomass accumulation as a determinant of virulence in each interaction was measured as the ratio of fungal to plant DNA by performing multiplex PCR using two sets of wheat and P. triticina specific primers. Our results showed that the highest relatively proportion of fungal DNA was found using the highest inoculum density on susceptible host lines. In susceptible interactions 21 d after inoculation, 40–50% of the total genomic DNA extracted from infected leaves was fungal, whereas in resistant or incompatible interactions only 10–20% of total genomic DNA extracted from infected leaves was fungal in origin. Rust development in the leaves, determined microscopically, corresponded to the molecular results.

Effect of root exudates on the germination of resting spores of Plasmodiophora brassicae under laboratory conditions. Q. XIAO, S. F. HWANG, H. AHMED AND S. E. STRELKOV. Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (H.A., S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17 507 Fort Road N.W., Edmonton, AB T5Y 6H3, Canada

Clubroot of canola, caused by Plasmodiophora brassicae Woronin, has emerged as an important disease of canola (Brassica napus L.) in Alberta, Canada. Clubroot management is difficult, largely as a consequence of the long survival of P. brassicae resting spores in the soil. Root exudates of host and non-host plants have been reported to induce resting spore germination in the soil. The cropping of bait plants, which can induce germination of resting spores and thereby decrease the soil inoculum load, has been proposed as a tool for the management of clubroot. The germination of P. brassicae resting spores was investigated in a nutrient solution supplemented with root exudates from two host species [Chinese cabbage (B. rapa chinensis L.) and canola] and a non-host species [perennial rye grass (Lolium perenne L.)]. Relative to nutrient solution alone or water controls, there was a significantly greater percentage of resting spore germination in treatments in which root exudates from these plant species were included. However, root exudates from canola and Chinese cabbage were more effective than those from perennial rye grass in stimulating spore germination. The results indicate that the root exudates can stimulate P. brassicae resting spore germination and the stimulation effect varies depending on the specific plant species.

Biocontrol of fusarium head blight in wheat using ACM941-CL01, a formulated product of Clonostachys rosea strain ACM941. A. G. XUE, Y. H. CHEN, H. D. VOLDENG, G. FEDAK, M. E. SAVARD AND S. Z. ZHANG. Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (S.Z.Z.) Soybean Research Institute, Key Laboratory of Soybean Biology of Chinese Education Ministry, Northeast Agricultural University, Harbin, Heilongjiang 150030, China

Fusarium head blight (FHB), caused by Gibberella zeae (Schwein.) Petch (anamorph Fusarium graminearum Schwabe), is a destructive disease of wheat in Canada. Previous studies demonstrated that Clonostachys rosea (Link:Fr.) Schroers et al. strain ACM941 was an antagonist against G. zeae. The objective of this research was to evaluate the efficacy of ACM941-CL01, a formulated product of ACM941, on the reduction of perithecial production and control of FHB in comparison to registered fungicide Folicur^® (tebuconazole). When applied to corn, soybean and wheat residues in two separate field trails in 2009, ACM941-CL01 reduced the daily perithecial reproduction over a period of 42 days by an average of 90% on corn, 90% on soybean and 71% on wheat. These effects were better but not significantly different from tebuconazole in the same trials. Five concentrations of ACM941-CL01, ranging from 10⁴ to 10⁸ CFU mL⁻¹, were tested for the control of FHB in two greenhouse trials and in a FHB nursery in 2009. All the concentrations significantly reduced the FHB severity. ACM941-CL01 at 10⁸ CFU mL⁻¹ was the most effective treatment, reducing the area under the disease progress curve (AUDPC) by 79%, infected spikelets (IS) by 30%, and Fusarium damaged kernels (FDK) by 91% in the greenhouse experiments. Under the field conditions, ACM941-CU at 10⁸ CFU mL⁻¹ significantly reduced the AUDPC by 43%, FHB index by 52%, IS by 45% and FDK by 43%. These effects were less but not significantly different from those achieved with tebuconazole, which reduced AUDPC by 44%, FHB index by 70%, IS by 59% and FDK by 50%. Results of this study suggest that ACM941-CL01 is a promising biocontrol product and may be used for managing FHB in wheat.

Morphological and molecular characterization of Ditylenchus dipsaci infesting garlic in Ontario, Canada. Q. YU, W. YE, A. BADISS AND F. SUN. Environmental Health Program/Invertebrate Biodiversity, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, Ontario K1A 0C6, Canada; (W.Y.) Nematode Assay Section, Agronomic Division, North Carolina Department of Agriculture & Consumer Services, Raleigh, NC 27607, USA; and (F.S.) Canadian Food Inspection Agency, Ottawa, ON K2H 8P9, Canada

Ditylenchus dipsaci (Kühn) Filipjev (Nematoda: Anguinidae), the stem nematode or stem and bulb nematode, is an internationally quarantined nematode pest for many countries in the world. Recent years, there are indications that it has become a more serious pest on garlic in Ontario. Morphological studies reveal that the population have significant morphological variations from the classical description of the nomenclature types: the cuticle has longitudinal ridges; the lateral fields are flanked by areas with half the annules, and without longitudinal ridge; hemizonid is 1–3 annules anterior to the excretory pore; the anterior end of the bursa is 20 μ ahead from the base of specula; and some old males and females have needle-like crystals in their respective reproductive organs. Other morphological characters match the descriptions of the type. The ITS sequence had 100% match to sequences of many isolates from Europe. Phylogenetic study of the sequence of the ITS places the population in the well-supported monophylic clad of the populations associated with various crops in Europe.

Notes

^†This meeting was held jointly with the Pacific Division of the American Phytopathological Society in Vancouver, British Columbia on 20–23 June 2010.