Biology subjects

Hamelin, R. C.

Publications and source records attributed to Hamelin, R. C..

6 recordsLinked to original sources

bioRxiv2024

Heterogeneity in establishment of polyethylene glycol-mediated plasmid transformations for five forest pathogenic Phytophthora species

Plasmid-mediated DNA transformation is a foundational molecular technique and the basis for most CRISPR-Cas9 gene editing systems. While plasmid transformations are well established for many agricultural Phytophthora pathogens, development of this technique in forest Phytophthoras is lacking. Given our long-term research objective to develop CRISPR-Cas9 gene editing in a forest pathogenic Phytophthora species, we sought to establish the functionality of polyethylene glycol (PEG)-mediated plasmid transformation in five species: P. cactorum, P. cinnamomi, P. cryptogea, P. ramorum, and P. syringae. We used the agricultural pathogen P. sojae, a species for which PEG-mediated transformations are well-established, as a transformation control. Using a protocol previously optimized for P. sojae, we tested transformations in the five forest Phytophthoras with three different plasmids: two developed for CRISPR-Cas9 gene editing and one developed for fluorescent protein tagging. Out of the five species tested, successful transformation, as indicated by stable growth of transformants on a high concentration of antibiotic selective growth medium and diagnostic PCR, was achieved only with P. cactorum and P. ramorum. However, while transformations in P. cactorum were consistent and stable, transformations in P. ramorum were highly variable and yielded transformants with very weak mycelial growth and abnormal morphology. Our results indicate that P. cactorum is the best candidate to move forward with CRISPR-Cas9 protocol development and provide insight for future optimization of plasmid transformations in forest Phytophthoras.

microbiology↗

bioRxiv2024

Genetic architecture underlying response to the fungal pathogen Dothistroma septosporum in lodgepole pine, jack pine, and their hybrids

In recent decades, Dothistroma needle blight (DNB), a pine tree disease caused by the fungal pathogen Dothistroma septosporum, has severely damaged lodgepole pine (Pinus contorta Dougl. ex. Loud.) in British Columbia, Canada, and raised health concerns for jack pine (Pinus banksiana Lamb.). The pathogen has already shown signs of host shift eastward to the hybrid populations between lodgepole pine and jack pine (Pinus contorta x P. banksiana), and possibly into pure jack pine. However, we have little knowledge about mechanisms of resistance to D. septosporum, especially the underlying genetic basis of variation in pines. In this study, we conducted controlled inoculations to induce infection by D. septosporum and performed a genome-wide case-control association study with pooled sequencing (pool-seq) data to dissect the genetic architecture underlying response in lodgepole pine, jack pine, and their hybrids. We identified candidate genes associated with D. septosporum response in lodgepole pine and in hybrid samples. We also assessed genetic structure in hybrid populations and inferred how introgression may affect the distribution of genetic variation involved in D. septosporum response in the studied samples. These results can be used to develop genomic tools to evaluate DNB risk, guide forest management strategies, and potentially select for resistant genotypes.

plant biology↗

bioRxiv2022

Lifestyles shape genome size and gene content in fungal pathogens

Fungi display a wide range of lifestyles and hosts. We still know little about the impact of lifestyles, including pathogenicity, on their genome architecture. Here, we combined and annotated 552 fungal genomes from the class Sordariomycetes and examined the association between 13 genomic and two lifestyle traits: pathogenicity and insect association. We found that pathogens on average tend to have a larger number of protein-coding genes, including effectors, and tRNA genes. In addition, the non-repetitive size of their genomes is larger than that of non-pathogenic species. However, this pattern is not consistent across all groups. Insect endoparasites and symbionts have smaller genome sizes and genes with longer exons; moreover, insect-vectored pathogens possess fewer genes compared to those not transmitted by insects. Our study shows that genes are the main contributors to genome size variation in Sordariomycetes and that pathogens can exhibit distinct genome architectures, depending on their host and vector interactions.

evolutionary biology↗

bioRxiv2021

Secondary metabolism drives ecological breadth in the Xylariaceae

Global, large-scale surveys of phylogenetically diverse plant and lichen hosts have revealed an extremely high richness of endophytes in the Xylariales, one of the largest clades of filamentous fungi and a significant source of novel secondary metabolites (SMs). Endophytes may produce host protective antimicrobial or insecticidal SMs, as well as compounds that facilitate symbiotic establishment through suppression or degradation of host immune response, but the ecological roles of most SMs are unknown. Here we characterized metabolic gene clusters in 96 genomes of endophytes and closely related saprotrophs and pathogens in two clades of Xylariales (Xylariaceae s.l. and Hypoxylaceae). Hundreds of genes appear horizontally transferred to xylarialean fungi from distantly related fungi and bacteria, including numerous genes in secondary metabolite gene clusters (SMGCs). Although all xylarialean genomes contain hyperabundant SMGCs, we show that increased gene duplications, horizontal gene transfers (HGTs), and SMGC content in Xylariaceae s.l. taxa are linked to greater phylogenetic host breadth, larger biogeographic distributions, and increased capacity for lignocellulose decomposition compared to Hypoxylaceae taxa. Overall, our results suggest that xylarialean endophytes capable of dual ecological modes (symbiotic and saprotrophic) experience greater selection to diversify SMGCs to both increase competitiveness within microbial communities and facilitate diverse symbiotic interactions.

genomics↗

bioRxiv2019

In Situ Processing and Efficient Environmental Detection (iSPEED) of pests and pathogens of trees using point-of-use real-time pcr

The increase in global trade is responsible for a surge in foreign invasive species introductions across the world. Early detection and surveillance activities are essential to prevent future invasions. Molecular diagnostics by DNA testing has become an integral part of this process. However, for environmental applications, there is a need for cost-effective and efficient point-of-use DNA testing that would allow for the collection of results in real-time away from laboratory facilities. To achieve this requires the development of simple and fast sample processing and DNA extraction, room-temperature stable reagents and a portable instrument. We conducted a series of tests using a crude buffer-based DNA extraction protocol and lyophilized, pre-made, reactions to address the first two requirements. We chose to demonstrate the use of this approach with organisms that cover a broad spectrum of known undesirable insects and pathogens: the ascomycete Sphaerulina musiva, the oomycete Phytophthora ramorum, the basidiomycetes Cronartium ribicola and Cronartium comandrae and the insect Lymantria dispar. Tests performed from either infected leaf material or spores (pathogens), or legs and antenna (insects). We were able to obtain positive amplification for the targeted species in all the samples tested. The shelf-life of the lyophilized reactions was assessed, confirming the stability of over a year at room temperature. Finally, successful tests conducted with portable thermocyclers and disposable plastics, demonstrating the suitability of the method, named in Situ Processing and Efficient Environment Detection (iSPEED), for field testing. This kit is ideally adapted to field testing as it fits in a backpack and can be carried to remote locations.

plant biology↗

bioRxiv2019

Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera

Abstract/KeywordsTo determine if living microorganisms of phytosanitary concern are present in wood after eradication treatment and to evaluate the efficacy of such treatments, the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can therefore only be produced by living organisms, providing a proxy for viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of wood infected with the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated wood samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and test the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade.

microbiology↗