Richard Hamelin

Members of the Phytophthora genus are among the most serious invasive pathogens of forests and cause devastating ecological and economic impacts. Epidemics caused by the sudden oak death pathogen, Phytophthora ramorum, and root rot of Christmas trees caused by multiple Phytophthora species are among the many causing loss of millions of dollars to the nursery industry. In addition, a large volume of manufactured products is traded in packages made of wood and wood products globally raising phytosanitary concerns for global trade. Rigorous inspections and biosurveillance involving the monitoring of pathogens before and after applying phytosanitary measures are essential to keep these organisms under control. Despite technological advances in diagnostic assays based on genomics, assessment of treatment efficacy remains challenging for these pathogens. DNA remains stable during heat treatments and can remain as a relic in environments long after the pathogen is dead. Culture methods used for viability testing are time consuming and often report false negatives. We propose a solution to the problem using a transcriptomics approach applying the heat lability of RNA for the prediction of viability and sequence variability for taxonomic identity. We measure levels of RNA degradation by simulating phytosanitary treatment and show that RNA degradation by heat can be assessed from the length of the RNA transcript transcribed and amplified by RT-PCR. Phytophthora transcript sequences identified from an RNAseq study and reference genomes of eight clades of Phytophthora are used to create a panel of oligonucleotides that can target multiple regions along the length of several transcripts commonly expressed during host infection. Significant reduction (60-99%) in mean coverage within transcripts is observed after heat treatments for the 18 genes targeted by the panel. We present the design and testing of a rapid and high-throughput transcriptomics assay for viability assessment of Phytophthoras using the power of targeted next generation sequencing. The assay can be applied for implementation of trade regulations and management of Phytophthora infections in nurseries by regulatory agencies.

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The negative impact of agricultural pesticides on the environment are well documented. This in vitro study evaluates the biological activity of seven British Columbia native plant extracts and plant extracts fermented with Saccharomyces cerevisiae, against three plant pathogens of economic importance: Phytophthora infestans, Fusarium sambucinum and Pectobacterium carotovorum. Crude plant extracts and yeast-fermented extracts using simple fermentable media and Saccharomyces cerevisiae were evaluated, while the pathogen growth development was challenged against in vitro treatments. Dry plant extracts were prepared by dissolving 5 mg of crude extract in 2.5 ml of 70% ethanol; then different concentrations were prepared in 96 wells microliter plates and tested against the selected organisms. The 4- way interaction model (yeast, extract, solvent, treatment dilution) was used for the analysis, and the significance of the interaction in the models was tested by a likelihood ratio test. Moreover, 262 combinations out of 480 of either crude extracts, or fermented extracts applied at different dilutions with different solvents (methanol or water) showed growth inhibition in comparison to controls. Arbutus menziesii treatments, including fractions of the crude extract, demonstrated the most diverse levels of growth suppression of all the pathogens. Fermentation has shown great potential for increasing the extract efficacy. In some cases, a combination of the fermentation period and the extract concentration influences the biological activity of specific treatments. Even though treatments that included water as a solvent mixed with the pathogen culture indicated pathogen suppression, when applied as a solvent, methanol showed preferable scales of inhibition. Moreover, results showed that several fermented treatments inhibited pathogen growth, whereas in many cases, the same treatments did not show the same inhibition when they were tested as crude extracts. This was observed with a 24-hour fermentation of water and methylene dichloride fractions of Arbutus menziesii, Maianthemum stellatum extract, and Rubus spectabilis extract applied against F. sambucinum and P. infestans. Further research is encouraged to identify potentially active compounds, but also to evaluate the fermentation treatments and factors influencing the combinations. Plant fermentation may offer a new opportunity in the development of more ecological and safer biopesticides.

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The Mountain pine beetle (MPB) has symbiotic relationships with Ophiostomatoid fungi (Ophiostomatales, Ascomycota), also known as blue stain fungi. The fungal symbionts associated with MPB contribute to the success of beetle survival, reproduction and outbreaks by providing a number of benefits to beetles. The multi-partite mutualism between fungi and MPB has remained remarkably stable over a long period of evolutionary time without exhibiting strong direct competition. We hypothesize that niche partitioning is a stabilizing mechanism which is important for the maintenance of coexistence and diversity of fungal species. The interspecific and intraspecific phenotypic characteristics of three fungal symbionts from a wide geographic and environmental range were investigated under different temperatures in vitro. The study found that three fungal species are differentially adapted to temperatures. The interspecific comparison indicates that Grosmannia clavigera and Leptographium longiclavatum were able to grow better at lower temperatures than Ophistoma montium, while Ophistoma montium displayed greater tolerance to extreme high temperatures. The intraspecific variation within species showed that isolates of the same species from different genetic groups have different temperature tolerances. Leptographium longiclavatum exhibited greater variability in response to temperature than Grosmannia clavigera and Ophistoma montium. The phenotypic data agrees with the genetic population structures of three fungi species. My research indicated that differential temperature tolerances of three fungal species may mediate competition among symbionts, thus influencing dynamics and compositions of the symbiosis in naturally attacked trees. Different growth responses to variable environmental conditions allow the competing species to capture resources at different times without expending energy on direct competition. By maintaining multipartite relationships with these fungi species, bark beetles are more likely to obtain benefits provided by different species of fungi under fluctuating environmental conditions. The complementarity of these nutritional symbionts could facilitate the colonization of new habitats and a broad ecological amplitude for MPB.

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Endangered whitebark pine (Pinus albicaulis Engelm.), native to high elevation forests of western North America, is declining mainly due to the introduced pathogen Cronartium ribicola J.C. Fisch, causing the disease white pine blister rust. This decline is compounded by the impacts of climate change, mountain pine beetle, and fire suppression. Low levels of genetic resistance to blister rust are present in whitebark pine. Traditional methods of inoculating seedlings to determine family-level resistance to the rust are costly, labour and time intensive. Due to the need for resistant material for planting, this presents a bottleneck in the process of restoring whitebark pine stands that have been heavily infected by the rust. In this study I tested an alternative approach to controlled inoculations that would be an effective way to screen large numbers of families without as many costs and limitations. A large genetic sample comprising 214 open-pollinated families from 44 provenances were screened at Skimikin Nursery, British Columbia, to determine: (1) the effectiveness of natural rust inoculation from Ribes nigrum L. in a common garden; (2) family and provenance level resistance to blister rust; and (3) climate variables related to height and rust resistance. Eighty-one families previously screened in Dorena, Oregon using artificial inoculation methods were also planted at Skimikin to compare with the natural inoculation. The natural inoculation was effective, with 73% of seedlings displaying stem symptoms of the disease, and 95% showing rust infection. A clear relationship was found between distance from the Ribes and severity of blister rust. Linear mixed models with spatial correlations were fitted to height and rust data using ASReml-R to estimate breeding values, heritability, and among-population differentiation (QST). Resistance was highest in the Cascade Mountains of Washington, while the most susceptible families were located mainly in the BC Coast Mountains. QST values revealed low genetic differentiation for height (0.07) and moderate differentiation for rust (0.28) while heritability was higher for height (0.42) than for rust resistance (0.23). This method of screening should be used more widely to determine families resistant to white pine blister rust and increase the availability of resistant seedlings for restoration.

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The recent discovery of a hybrid forest fungal pathogen, Cronartium x flexili, suggested to have arisen from sexual hybridization between the introduced C. ribicola and the native C. comandrae, was surprising because the parental species do not share hosts. Although the pathosystems of both parental species are well described, the impact of their hybridization is yet unknown. The purpose of this study is to determine the occurrence, level of hybridization and introgression of the hybrid pine stem rust Cronartium x flexili. A total of 831 samples from dikaryotic aecia of C. ribicola and C. comandrae were collected from 13 sites across British Columbia and Alberta and analysed both genetically and morphologically over two sampling seasons. Microscopic and genetic methods, including PCR, qPCR, and genotyping by sequencing (GBS), were used to identify hybrid samples. The results of these analyses indicate that C. x flexili is either no longer prevalent in areas where it was previously found, or else prevalent at such low frequencies that it has evaded detection in the sampling effort of this study. Two previously collected and extracted C. x flexili DNA samples were examined using a fixed loci analysis and did not demonstrate evidence of introgression, indicating that this hybrid does not facilitate gene flow between the introduced C. ribicola and the native C. comandrae. This suggests that when a hybridization event occurs between the parental Cronartium spp., first generation (F1) hybrids result but are not fertile and do not occur perennially. Some evidence suggests that this could be due to a number of factors including: low relative fitness of C. x flexili compared to the parental species if ‘hybrid breakdown’ occurs as explained by the Bateson-Dobzhansky-Muller incompatibility (BDMI) model; variations in local climate factors influencing life cycle parameters; or sexual incompatibility with the parental species. This work adds to the limited literature on the genetics of hybrid forest fungal pathogens and improves our understanding of the evolutionary mechanisms occurring when allopatrically evolved forest fungal species hybridize.

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In order to determine if living fungi of phytosanitary concern are present in wood or to evaluate the efficacy of treatments, the method of choice is to grow microbes in petri dishes for subsequent identification. However, some fungi are difficult or impossible to grow in cultures, and thus, to validate the effectiveness of existing and emerging wood treatments, a molecular methodology that can detect living fungi and fungus-like organisms is required. RNA-based molecular diagnostic assays were developed to detect the presence of living fungi and fungi-like organisms of phytosanitary concern. Since RNA represents the transcription of genes and can therefore only be produced by living organisms, it provides a marker to determine if an organism is alive. The assays were designed to target genes that are essential to vital processes, then used to assess their presence and abundance through real-time reverse transcription polymerase chain reaction (PCR). A stability analysis was conducted by comparing the RNA to DNA ratio over treatment time. The results illustrated that for treated samples, DNA remained stable over a period of 10 days post treatment, whereas RNA could not be detected after 24 hours for Phytophthora ramorum or 96 hours for Grosmannia clavigera. Therefore, this method provides a reliable way to evaluate viability of organisms following treatments and can have profound impacts on assessing both timber and non-timber forest products of commercial value.

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Rust fungi cause some of the most severe pine diseases. Cronartium ribicola (J. C. Fisch.), the causal agent of white pine blister rust, was introduced accidentally to North America from Europe in the late 1800s. Since then, it has devastated a large number of native, commercially valuable white pines, and is threatening alpine ecosystem stability by endangering high elevation white pines. In order to better understand the global epidemiology of this pathogen, we conducted a genome scan of a global collection of C. ribicola using Genotyping-by-Sequencing (GBS) to: 1) ascertain the origin and the routes of introduction of C. ribicola, and 2) uncover cryptic population structure of C. ribicola in western North America, in relation to different pine hosts, climates and landscapes. More than eight thousand single nucleotide polymorphism markers were genotyped on 192 samples of C. ribicola from three continents. The highest genetic and nucleotidic diversity were observed in Siberian samples, supporting the hypothesis that central Russia is the center of origin of C. ribicola. Diversity was reduced in all other populations and was lowest in western North America. Genetic and nucleotidic diversity were two to five times lower in western than in eastern North America. This result supports the observation of multiple introductions of the pathogen in eastern North America and contrasts with the single reported introduction in western North America. However, western populations had a larger number of rare alleles. This could represent the signature of population expansion following a bottleneck or a selective sweep. A cryptic Coast/Interior split was detected within the western cluster, most likely maintained by the scarcity of white pines in central British Columbia acting like a barrier to gene flow. Finally, western individuals with a high level of eastern admixture were discovered in two populations east of the Continental Divide. This could indicate that the eastern-western barrier to gene flow is leaky. Such information is of significance to white pine resistance breeding programs and to the monitoring of this disease.

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The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, has destroyed over 18 million ha of pine forest in Canada since 1999, the largest insect epidemic in recorded history. Fungal symbionts in the Ophiostomales (Ascomycetes) play an important role in the outbreak by reducing the tree defense response following beetle colonization, making its environment more conducive to the insect development. A better understanding of the population genetics attributes of the fungal associates could be important to elucidate their role and to explain epidemic patterns. We investigated the genetic structure of one of those fungal associates, Leptographium longiclavatum, sampled from 28 locations in western North America using 11 microsatellite markers developed from the genome of its closest relative, Grosmannia clavigera, another fungal symbiont of the MPB. We found that L. longiclavatum has a distinctive genetic population structure, and by using Bayesian clustering inference, we discovered the presence of three clusters that are concordant with geographic origin of the samples. In addition, we observed an apparent North-South pattern of genetic diversity consistent with the chronology of the epidemic. Structure output showed one cluster comprised of individuals from Northern Alberta where the beetle-fungus complex has been recently established, and a second cluster composed of individuals originating along the Rocky Mountains, and a third cluster was from populations in BC. High haplotypic diversity was found throughout the range sampled, a surprising result given that sexual fruiting structures have never been observed for this fungus. Consistent with recent introduction history, the least differentiated populations were located in northern BC and Alberta. We observed a strong correlation in the genetic diversity pattern observed in L. longiclavatum, G. clavigera and D. ponderosae, as shown by a significant correlation in the genetic distance matrices amongst L. longiclavatum, G. clavigera, and D. ponderosae. This correlation and the similar north-south population structure of these interacting organisms suggest a joint population history consistent with the dependence of fungal dispersal on their bark beetle vector. These results can help clarify the roles and close relationships between the beetles and their fungal associates.

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White pine blister rust, Cronartium ribicola (Basidiomycota, Pucciniales), is a macrocyclic (5 different spore types) heteroecious (requires two hosts) rust that alternates on Ribes spp. It is an exotic pathogen in North America and cause high levels of mortality of pine in the subsection Strobus (white pines). To better understand the epidemiology of the pathogen, the population structure of white pine blister rust in North America was investigated. Thirty one single nucleotide polymorphism (SNP) markers have been developed to genotype 1341 individuals from 76 populations from across North America including samples from diverse landscapes. In western Canada, sampling was structured to contrast different landscapes and pine hosts. Distance-based and Bayesian likelihood methods indicated the presence of two major genetic clusters: ‘eastern’ and ‘western’ in North America, separated by the Great Plains that act as a barrier to gene flow. The eastern cluster had greater genetic diversity than the western cluster, which confirms that multiple introductions occurred in eastern North America in contrast to a single introduction in the west. Two populations, New Mexico and Minnesota were each found to form a separate cluster in some assignment analyses and the distance-based analyses clearly placed them outside of the main clusters. Both of these populations displayed the hallmarks of a founder effect, i.e. low genetic diversity and/or inbreeding. The pathogen was discovered in New Mexico in the 1970’s, almost a century later than the populations in the two major clusters. Although white pine blister rust has been present for longer in Minnesota, the population parameters strongly suggest a founder effect and a barrier to gene flow between Minnesota and the populations within the eastern cluster. However, no landscape, host, or other patterns could be correlated with these clusters. A rare SNP was detected in Smithers, a population with high levels of inbreeding located at the northern most extent of the rust. Understanding the population structure will provide great knowledge of the rust for breeding programs and deployment of rust resistant pines.

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