Evaluation and characterization of the Leptographium procerum Canadian population from red turpentine beetle (Dendroctonus valens) G. J. BILODEAU, M.-C. GAGNON, A. POTVIN, D. SHEARLAW, T. KIMOTO AND P. TANGUAY Ottawa Plant Laboratory, Canadian Food Inspection Agency (CFIA), P.O. Box 11300, 3851 Fallowfield, Ottawa, ON K2H 8P9, Canada; (T.K.) Plant Health Science Services Division, CFIA, 4321 prom Still Creek, Burnaby, BC V5C 6S7, Canada; and (A.P., P.T.) Natural Resources Canada, Laurentian Forestry Centre, 1055 du P.E.P.S., P.O. Box 10380, STN Sainte-Foy, Québec City, QC G1V 4C, Canada
The occurrence of declining trees associated with the presence of red turpentine beetle (RTB), Dendroctonus valens, has increased over the last years around the city of Abbotsford, British Columbia (BC). This bark beetle is usually a secondary insect pest attacking weakened conifers or those that have been recently fire-scorched. We sought to verify if increased attacks by the RTB were related to environmental stresses or if they were due to the introduction of a more aggressive strain of Leptographium procerum, a blue stain fungus vectored by the RTB. The spring of 2015 was the warmest and driest ever recorded for the Vancouver area. The Abbotsford’s pine trees likely suffered drought at this time, which may have facilitated a RTB attack. To rule out the hypothesis of a newly introduced race of L. procerum, we compared the genotypes of Canadian L. procerum strains to the genotypes of strains from Europe and Asia. Fifteen microsatellite markers, showing high variability, were used to characterize a panel of DNA samples extracted from strains isolated in Ontario, Quebec, Poland and China. High levels of variation and moderate levels of heterozygosity were observed for 14 of the 15 L. procerum markers used. We determined the number of multilocus genotypes (MLGs), and then assessed the genetic distance, the relationships between MLGs and the heterozygosity. Although we were not able to isolate L. procerum specimens collected in Western Canada (BC and Manitoba) during our survey, our analyses revealed the presence of distinct American, European and Asian populations of L. procerum.
Differential production of antimicrobial lipopeptides by Bacillus subtilis B9-5 in the presence of plant pathogens S. DEFILIPPI AND T. J. AVIS Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
Bacteria from the Bacillus Cohn genus have shown success as biocontrol agents of various plant pathogens. Antagonistic strains of Bacillus subtilis (Ehrenb.) Cohn produce antimicrobial compounds, including cyclic lipopeptides. Variability in the production of these metabolites when confronted with plant pathogens has not been well studied. The objectives of this study were to determine the effect of B. subtilis strain B9-5 on growth of plant pathogens and to assess production of antimicrobial cyclic lipopeptides over time. In vitro assays were performed to determine B. subtilis B9-5 inhibition of mycelial growth of Alternaria solani Sorauer, Botrytis cinerea Pers., Fusarium oxysporum f. sp. radicis-lycopersici Jarvis & Shoemaker, Fusarium sambucinum Fuckel, Pythium sulcatum Pratt & Mitch., Rhizopus stolonifer (Ehrenb.) Vuill. and Verticillium dahliae Kleb. To assess lipopeptide production, the bacterium was grown alone and in the presence of V. dahliae, F. sambucinum and R. stolonifer in liquid media. After 24, 48 and 72 h of co-culture, lipopeptides were quantified using LC-MS. Results showed that B. subtilis B9-5 inhibited mycelial growth of all tested pathogens to varying degrees, except R. stolonifer. It was determined that both V. dahliae and R. stolonifer caused an increase in the production of the lipopeptides fengycin and surfactin up to 48 h, followed by a decrease at 72 h. Conversely, F. sambucinum caused increasing concentrations of both lipopeptides over all time periods. Overall, lipopeptide production varied substantially over time depending on the pathogen, which provides valuable information concerning the usefulness of B. subtilis as a biocontrol agent.
Effects of long-term crop rotation and tillage on Oomycete species diversity and abundance in agricultural soils A. C. GAHAGAN, C. A. LEVESQUE, M. MORRISON AND W. CHEN Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
Many oomycetes species are important crop pathogens with host range varying from specific (e.g. Phytophthora infestans) to broad (e.g. Pythium ultimum). Studies on oomycete communities and their response to agronomic treatments, such as crop rotation and soil tillage in pathogen management, have largely been limited to a subset of species. The current study used metabarcoding and physiological approaches to evaluate the effects of tillage and rotation on oomycete populations and in vivo seedling emergence. Soil was sampled from a 15-year long-term trial at the Central Experimental Farm, Ottawa, Agriculture and Agri-Food Canada, with three rotations (monoculture corn (CCC), monoculture soybean (SSS), and wheat-corn-soybean rotation (WCS)) and two tillage (conventional tillage (CT) and no tillage (NT)) treatments. The nuclear ribosomal internal transcribed spacer 1 (ITS1) was amplified from the soil DNA and sequenced using oomycete specific primers on the Illumina MiSeq platform. Species richness was higher under NT and under WCS and SSS rotation systems. At the species level, generalists, such as Pythium intermedium, were more abundant under CT; while host-specific species, e.g. Pythium hypogynum (a pathogen of soybean), were more abundant when their preferred host was included in the rotation system (SSS and WCS). Soil samples were seeded with soybean and monitored for symptoms of seedling disease in the growth cabinet. The development and severity of disease were influenced by rotation and tillage, especially the latter. Continued study of the impact of agronomic treatments on oomycete community structure in soil could facilitate the development of more effective disease management strategies.
Whole genome sequencing for identification of molecular markers to develop diagnostic detection tools for the regulated plant pathogen Lachnellula willkommii E. GIROUX AND G. J. BILODEAU Canadian Food Inspection Agency, 3851 Fallowfield Road, Ottawa, ON K2H 8P9, Canada
The filamentous ascomycete fungus Lachnellula willkommii is the causal agent of European Larch Canker (ELC), which is considered to be one of the most destructive diseases of larch in Europe. Lachnellula willkommii was first detected in North America in 1927 on a plantation of European larch (L. decidua) planted years earlier from infected nursery stock imported from Great Britain. After decades of sanitation practices eliminating the pathogen from the area, the fungus has reappeared in coastal areas of the USA and Canada. Lachnellula willkommii is under official control in Canada and is considered a quarantine pest in Canada and the USA. There is concern that ELC could spread throughout the range of eastern larch, which is a transcontinental species typical of the Boreal forest which spans the landscape from the Atlantic Provinces of Canada in the east to Alaska in the north-west of North America. Current methods to detect Lachnellula species on plant samples rely primarily on identification based on biological and morphological characteristics, however, misdeterminations between the pathogenic L. willkommii species from non-pathogenic saprophytic sister species are frequent as the fungus does not always produce the structures which would allow positive identification. Whole genome sequencing technologies were used to obtain the draft genome sequences of L. willkommii and six other closely related sister species for the purpose of generating molecular markers which may be used as a diagnostic tool to detect and distinguish this regulated pest from non-pathogenic sister species that are currently indistinguishable using existing diagnostic methods.
Whole genome phylogeny and analysis of cyclic lipopeptide biosynthesis genes in plant pathogen antagonistic strains of Bacillus E. GROULX, T. BUJAKI, A. WONG, N. RODRIGUE, M. L. SMITH AND T. J. AVIS Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada; and (A.W., N.R., M.L.S.) Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
Disease suppressive compost and soil were found to contain bacteria belonging to the genus Bacillus Cohn that were antagonistic to plant pathogens. Bacillus spp. are known to produce numerous antimicrobial compounds. More specifically, these bacteria produce three main families of antimicrobial cyclic lipopeptides named surfactins, fengycins and iturins. However, not all Bacillus isolates produce all cyclic lipopeptide families. The genetics behind the production of these lipopeptides are not well understood. The objective of this study was to confirm the identity of the bacterial isolates using whole genome phylogeny and to identify Bacillus cyclic lipopeptide biosynthesis genes in these isolates. Fifteen isolates of Bacillus subtilis (Ehrenb.) Cohn, two isolates of Bacillus megaterium de Bary and one isolate of Bacillus badius Batchelor were sequenced using an Illumina MiSeq system and phylogenetic analysis was performed using protein-coding alignments. Nucleotide and protein BLAST were used to help identify members of the main Bacillus cyclic lipopeptide gene families. Results showed that the identity of the bacterial isolates resembled the identity assigned previously using 16S rRNA sequences. Although each of the B. subtilis isolates possessed the gene operons for two or all three of the cyclic lipopeptides, they did not necessarily produce the antimicrobial compounds under previous test conditions. Results from this study give insight into the genetics behind cyclic lipopeptide production by Bacillus spp.
PO-7-Antimicrobial effects of fengycin in model membranes composed of plant pathogen lipid extracts E. MANTIL, T. CRIPPIN AND T. J. AVIS Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
Plant pathogenic moulds containing low or no sterols in their cellular membranes are typically more sensitive to fengycin, an antimicrobial cyclic lipopeptide produced by Bacillus subtilis (Ehrenb.) Cohn. Previous work using artificial ternary supported lipid bilayers exposed to fengycin showed additional distribution of microdomains and disappearance of large distinct ordered domains, creating a smoothing effect, in bilayers containing 0% and 3% ergosterol. Similar effects were seen initially in bilayers containing 6% and 12% ergosterol, but these high sterol content bilayers returned to distinct domains over time. This suggested that fengycin preferentially acts on ordered phase lipids, the region in which ergosterol resides. In the present study, bilayers were prepared with lipids extracted from Alternaria solani Sorauer, Fusarium sambucinum Fuckel and Pythium sulcatum Pratt & Mitch. The relative ergosterol content in these lipid extracts was high in A. solani and low in F. sambucinum whereas P. sulcatum did not contain ergosterol. Total Internal Reflection Fluorescence microscopy using Texas Red1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine was used to visualize domain formations and to distinguish disordered from ordered lipid domains in these model membranes following exposure to low (50 µg) and high (500 µg) doses of fengycin. At the lower fengycin dose, A. solani bilayers were the most stable, exhibiting little change. High fengycin doses led to a reduction of distinct domain distribution in all models. Results suggested that bilayers from A. solani were less affected by the presence of fengycin because of higher ergosterol content.
Fusarium poae and emerging mycotoxins associated with fusarium head blight in Canada F. VACHON, D. P. OVERY, A. HERMANS, A. JOHNSTON, A. SPROULE, A. XUE AND L. J. HARRIS Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada
While Fusarium graminearum Schwabe is considered the main causal agent of fusarium head blight (FHB) in cereals, Fusarium avenaceum (Fr.) Sacc., Fusarium equiseti (Corda) Sacc., Fusarium sporotrichioides Sherb. and Fusarium poae (Peck) Wollenw. are also commonly isolated from FHB-contaminated grain. These species are each capable of producing their own diverse array of mycotoxins. Globally, F. poae has been reported to produce type A and type B trichothecenes as well as beauvericin, cyclonerodiol and enniatins, but little is known about the mycotoxigenic potential of Canadian isolates. Recent Ontario surveys suggest F. poae and F. graminearum are the main Fusaria that can be isolated from barley while F. poae is most often found contaminating oats. We initiated a study of the genetic diversity and mycotoxin production of the Canadian F. poae population. Sampling from Ontario, Quebec and Saskatchewan oat, barley and wheat fields from 2006 to 2016 resulted in the collection of 160 monosporic F. poae isolates. Species identification was validated by sequencing the TEF1-ɑ gene. These isolates were cultured on liquid and solid growth media for metabolite profiling by UPLC-HRMS-CAD. We are surveying this collection for two unlinked mycotoxin biosynthetic gene sequences, TRI1 and TRI8, which may contribute to variations in chemotype. Phylogenetic analysis based on the TRI8 gene revealed 27 SNPs and four major groups in the Canadian population. Our goal is to define Canadian F. poae mycotoxigenic potential and explore whether genetic diversity impacts the success of F. poae on different cereal hosts.
Monitoring airborne bacterial phytopathogens using MinION Nanopore technology R. XU, J. CHAPADOS, A. SOLIMAN, F. DAAYF AND J. T. TAMBONG Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (A.S., L.A., F.D.) Department of Plant Science, University of Manitoba, 66 Dafoe Road, Winnipeg, MB R3T 2N2, Canada
Bacterial phytopathogens, such as Pantoea stewartii, Pseudomonas syringae and Clavibacter michiganensis can cause severe diseases to important agricultural crops with significant yield losses. These pathogens are airborne as well as insect vectored. Monitoring air samples can be an essential step to developing effective management strategies. We initiated work based on Nanopore technology to monitor these plant pathogens in air samples. Air samples were collected at the Ottawa Central Experimental Farm agricultural plots by direct culturing on four media (Luria-Bertani medium, King’s B, Corynebacterium Nebraskense Selective and Tryptic Soy Agar) using the Neutec air samplers. Total DNA was extracted from the enriched communities and metagenomics analysis performed using the Oxford MinION Nanopore technology targeting the 16S rRNA gene amplicons (~1500 bp). In addition, a mock bacterial community (MBC) composed of 24 known bacterial strains (eight genera) and diseased corn leaf discs were also processed. The generated sequence reads (1.3 million) were classified using the cloud-based EPI2ME software under 16S Classification Workflow with 85% alignment accuracy threshold. Seven of the eight MBC genera (Pseudomonas, Pantoea, Clavibacter, Xanthomonas, Enterobacter, Pectobacterium and Dickeya) were identified except Streptomyces, demonstrating high reliability of the method. Airborne bacterial communities obtained were dependent on the culture medium used. The diseased corn leaf discs showed 11 genera including potential phytopathogens Pseudomonas, Clavibacter, Pantoea, Microbacterium and Bacillus. The presence of Clavibacter michiganensis subsp. nebraskensis in corn leaf discs was confirmed using other methods. The nanopore technology could be a valuable tool for monitoring bacterial phytopathogens.