Buprestids are an emerging threat to broadleaf forests across the world. Bronze birch borer (Agrilus anxius, BBB) poses a serious threat to European birch species if the insect were to be introduced. Due to their cryptic lifestyle feeding on the vascular tissue of their host plants, buprestids and other wood borers can be difficult to observe or detect. Early detection tools are vital to swiftly implement eradication measures and prevent the establishment of introduced species. In this study, we developed novel qPCR and LAMP assays for BBB and investigated the specificity and sensitivity for their use as early detection tools in European forests. Plant chemicals may limit these assays, so we conducted sensitivity testing with extracted foliage and plant vascular tissues to determine potential inhibition effects on DNA amplification. Both assays were specific to the target species when tested against the DNA of 17 other European Agrilus/buprestid species, two Scolytinae, and five Cerambycids (N = 24). Both assays varied in sensitivity with the qPCR assay amplifying at a concentration as low as 20 fg/mu L, whereas the LAMP assay amplified as low as 3.2 pg/mu L. Plant chemicals in DNA extracts from leaves did not impact the sensitivity of either assay, reaching similar detection levels. In contrast, vascular tissue reduced the sensitivity of the LAMP assay, amplifying as low as 0.04 ng/mu L compared with 0.008 ng/mu L in the control. These results demonstrate that both assays are highly specific and sensitive tools that can be used to detect frass and identify larvae as well as monitor the spread of A. anxius. qPCR resulted in more sensitive than LAMP overall. Thus, if results are needed quickly to make fast management decisions or as an initial screening of samples, the LAMP method is optimal. However, if fine detection is critical, then qPCR is preferential.

In June, 2021, laurel willow (Salix pentandra) near Slave Lake, Alberta, was found to be infected by a Melampsora sp. that produced bright yellow urediniospores in uredia that were present on catkins, leaves, and stems. All Melampsora species previously reported in Canada are recorded as infecting leaves; therefore, further investigation was undertaken to ascertain the identity of this pathogen. To assess the relationship between this specimen and other Melampsora spp. previously collected from Canada, samples of willow leaves infected by Melampsora spp. were sourced from mycological herbariums located at the Laurentian Forestry Centre (QFB) and the Northern Forestry Centre (CFB, WINF(M)). DNA sequence data from the internal transcribed spacer (ITS) ribosomal RNA region of the fresh specimen, herbarium specimens, and DNA sequence data deposited within GenBank, were used to conduct a phylogenetic analysis. Sequencing and BLAST analysis of the material from the sample resulted in a 99.3% sequence identity match to Melampsora epitea “Mel J” collected from Larix laricina in New York State. The ITS sequence from the herbarium sample WINF(M)7356 (described as M. abieti-capraearum from Manitoba) had 100.0% identity with the Alberta sample. Additionally, specimens WINF(M)11892 (Melampsora sp. from Manitoba) and CFB8931 (Melampsora sp. from the Yukon) had 99.0% sequence identity with the Alberta sample. From these results we applied the identity of M. epitea to the rust discovered in Slave Lake, AB. With the current emphasis on willows for bioenergy production in Canada, growers must remain vigilant for this pathogen and the damages it could cause to willow plantations.

Fungi play key roles in forest soils and provide benefits to trees via mycorrhizal symbioses. After severe disturbance, forest regrowth can be impeded because of changes in fungal communities. In 2013–2014, soil fungi in forest floor and mineral soil were examined by Roche 454 pyrosequencing in undisturbed, harvested, and burned jack pine stands in a forested area near Fort Chipewyan, Alberta. These fungal communities were compared with jack pine, white spruce, and larch stands in Gateway Hill, a nearby certified reclaimed area. In 2014, a more detailed sampling of forestry and reclamation jack pine sites examined fungi in soil fractions using two high-throughput sequencing platforms and a sporocarp survey. The significances of compositional and functional differences in fungal communities between the forested and reclamation sites were assessed using permutation tests of partially constrained ordinations, accounting for confounding factors by variance partitioning. Taxa associated with the forestry area were primarily ectomycorrhizal. Fungal richness and diversity were greater in soils from the reclamation sites and included significantly more pathogenic taxa and taxa with unknown functional properties. Fungal community dissimilarities may have been artefacts of historical legacies or, alternatively, may have resulted from contrasting niche differentiation between forestry and reclamation sites.

Variation in rate of infection and susceptibility of Pinus spp. to the fungus Cronartium harknessii (syn. Endocronartium harknessii), the causative agent of western gall rust, has been well documented. To test the hypothesis that there is a coevolutionary relationship between C. harknessii and its hosts, we examined genetic structure and virulence of C. harknessii associated with lodgepole pine (P. contorta var. latifolia), jack pine (P. banksiana), and their hybrids. A secondary objective was to improve assessment and diagnosis of infection in hosts. Using 18 microsatellites, we assessed genetic structure of C. harknessii from 90 sites within the ranges of lodgepole pine and jack pine. We identified two lineages (East and West, FST = 0.677) associated with host genetic structure (r = 0.81, P = 0.001), with East comprising three sublineages. In parallel, we conducted a factorial experiment in which lodgepole pine, jack pine, and hybrid seedlings were inoculated with spores from the two primary genetic lineages. With this experiment, we refined the phenotypic categories associated with infection and demonstrated that stem width can be used as a quantitative measure of host response to infection. Overall, each host responded differentially to the fungal lineages, with jack pine exhibiting more resiliency to infection than lodgepole pine and hybrids exhibiting intermediate resiliency. Taken together, the shared genetic structure between fungus and host species, and the differential interaction of the fungal species with the hosts, supports a coevolutionary relationship between host and pathogen.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Loop-Mediated Isothermal Amplification (LAMP) assay was developed for the detection of the pine pathogen Dothistroma septosporum (G. Dorog.) M. Morelet. The specificity of the LAMP assay was tested using a selection of pine needle fungi, including Dothistroma pini Hulbary, and Lecanosticta acicola (Thüm.) Syd.; only D. septosporum DNA was amplified by the test. In terms of sensitivity, the assay was able to detect as little as 1 pg of total D. septosporum DNA. This assay enables DNA extracted from diseased host needles to be rapidly tested for the presence of D. septosporum using relatively simple to operate equipment away from a fully equipped molecular biology laboratory.

The ascomycete fungi Dothistroma septosporum (Dorogin) M. Morelet and D. pini Hulbary are both responsible for Dothistroma needle blight disease (DNB) on pines. Using a DNA-barcoding approach, we confirmed that the DNB infection of lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.), jack pine (P. banksiana Lamb.) and their hybrid in four research sites in northern Alberta (Canada) was caused by D. septosporum, confirming that the range of D. septosporum extends east of the Rocky Mountains into Alberta. Analysis of mating-type (MAT) genes showed the presence of both mating types in each of the pine plantations, indicating that the pathogen has the potential to complete its lifecycle in this region. Controlled inoculation confirmed that the two native pines found in this area (P. contorta var. latifolia and P. banksiana) and their hybrid are susceptible to D. septosporum, suggesting that continuous monitoring of the disease needs to be carried out to prevent any future outbreak in natural stands.

In Canada, forest biosecurity is primarily under federal jurisdiction as the federal government is the signatory to the International Plant Protection Convention and other international trade agreements. The Canadian Food Inspection Agency (CFIA), which is Canada’s National Plant Protection Organization, has the mandate of analyzing risks, setting policy, and managing incursion responses related to forest biosecurity. Other federal government agencies play important roles; the Canada Border Services Agency (CBSA) enforces regulations at international ports of entry and the Canadian Forest Service of Natural Resources Canada conducts research and analysis in support of the development and implementation of phytosanitary regulations. The provinces and territories also manage invasive species through implementing regulations to prevent the spread of established forest pests. This paper outlines the regulatory framework for forest biosecurity within Canada, and provides case studies of species that have invaded Canadian forests or are anticipated to do so in the near future.

Seedlings of lodgepole pine ( Pinus contorta var. latifolia ), jack pine ( P . banksiana ), and their hybrid were infected by Dothistroma septosporum under natural conditions in Alberta, Canada, after planting these taxa within a heavily infected stand of lodgepole pine. This experiment has further proven that all three of these taxa are hosts for the pathogen. This information is necessary for assessing the risk of eastward movement of the pathogen from Alberta through populations of P . banksiana in the boreal forest.

Leaf spots of poplar (Populus) species and hybrids in North America commonly are caused by three Drepanopeziza species (previously referred to by their asexual morph names in genus Marssonina). A fourth species, Marssonina balsamiferae, is known only from herbarium specimens and was described from balsam poplar (Populus balsamifera) leaves collected in 1966 in Manitoba, Canada. Balsam poplar leaves collected in 2014 in Minnesota, USA bore spots with acervuli and conidia consistent with those of M. balsamiferae, and isolates were obtained from these leaves. Conidial morphology and analysis of nuclear ribosomal ITS sequences distinguished the Minnesota strains from the very common leaf spot pathogen of poplars in the northcentral region of the USA, Drepanopeziza brunnea. Inoculation of rooted balsam poplar cuttings with conidia of M. balsamiferae strains from Minnesota resulted in necrotic leaf spots. Acervuli bearing conidia consistent with those of M. balsamiferae were observed on symptomatic leaves, and the fungus was reisolated from infected tissues. Contemporary recognition of existence and pathogenicity of M. balsamiferae, including presence in the USA, should prompt further investigation of its geographic distribution and relationships with hosts and related fungal species.

A conceptual framework for climate involvement in forest tree diseases was applied to seven examples to demonstrate its suitability for different disease types: cases where climate favours pathogen biology which then leads to tree mortality or where diseases are caused primarily by climate-driven physiological injury or stress to trees. Hypotheses for climate involvement are derived from detection and monitoring data to express associations of weather or climate factors with disease development at several spatial and temporal scales. Research findings contribute to an understanding of temperature, precipitation and related climate variables that influence biotic and abiotic diseases. To demonstrate use of the framework, we accessed information from the literature which exposed data and information gaps. Among various simulated approaches to test associations of climate and disease, we found disease risk factor models that use climate inputs derived from monitoring and research provide the best understanding of climate-disease relationships. These model outputs project future disease scenarios that can be used to inform climate adaptation strategies. Conservation and management implications for current and likely future climatic conditions are provided for each disease example. The most common guidance in managed landscapes is to move the imperilled tree species to areas of lower projected climate risk and to favour non-host, climate-adapted tree species where the disease is occurring.

Gateway Hill is a 104-ha area in the oil sands region of Alberta, which was operationally reclaimed and reforested in the late 1980s and certified as reclaimed in 2007/08. In 2014, similar to 30 years after reclamation was completed, we began studies with the main objective to determine the soil fungi present at the site. Sporocarp surveys in 2014 and 2016 were used to supplement metagenomic analyses of soil fungi in jack pine (Punts banksiana), white spruce (Picea glauca), and Siberian larch (Larbc sibirica) stands. Fungal internal transcribed spacer amplicons of DNA extracted from forest floor, root, and fine soil fractions were sequenced using Roche 454 pyrosequencing in 2014. Coarse soil reclamation samples and soil samples from a nearby unmined spruce stand were sequenced with Illumina in 2016. Sequenced community assemblages were first assessed using unconstrained multivariate analyses, and then using (partially) constrained ordination techniques to directly account for the effects of tree species and other confounding factors. Based on soil DNA analyses the relative abundance of ectomycorrhizal taxa was greater in spruce stands, saprotrophs were more abundant in pine stands, and pathogens were more abundant in larch stands. Associations of fungal taxa, mostly saprotrophs and ectomycorrhizae, with tree species were especially prominent in root fractions. The sporocarp survey found sporcarp abundance was greatest in pine stands and they tended to have more ectomycorrhizal taxa and spruce and larch stands more saprotrophic taxa. The effects of tree species on fungal taxa differed between the sporocarp surveys and soil DNA analyses: many of the fungi identified by sporocarp presence were not detected by soil DNA sequencing, whereas the latter method detected many more species in all trophic groups. Both DNA sequencing methods showed that many operational taxonomic units were preferentially associated with either spruce or pine stands. Though not definitive, preliminary examination of soil fungi using the Illumina platform showed that a natural spruce stand at the base of Gateway Hill had greater species richness than did reclaimed spruce stands. The results of this study show that communities of soil fungi have become established since the initiation of reclamation. Further work is underway to compare this reclamation site to other unmined forest sites.

Many current tree improvement programs are incorporating assisted gene flow strategies to match reforestation efforts with future climates. This is the case for the lodgepole pine (Pinus contorta var. latifolia), the most extensively planted tree in western Canada. Knowledge of the structure and origin of pathogen populations associated with this tree would help improve the breeding effort. Recent outbreaks of the Dothistroma needle blight (DNB) pathogen Dothistroma septosporum on lodgepole pine in British Columbia and its discovery in Alberta plantations raised questions about the diversity and population structure of this pathogen in western Canada. Using genotyping-by-sequencing on 119 D. septosporum isolates from 16 natural pine populations and plantations from this area, we identified four genetic lineages, all distinct from the other DNB lineages from outside of North America. Modeling of the population history indicated that these lineages diverged between 31.4 and 7.2 thousand years ago, coinciding with the last glacial maximum and the postglacial recolonization of lodgepole pine in western North America. The lineage found in the Kispiox Valley from British Columbia, where an unprecedented DNB epidemic occurred in the 1990s, was close to demographic equilibrium and displayed a high level of haplotypic diversity. Two lineages found in Alberta and Prince George (British Columbia) showed departure from random mating and contemporary gene flow, likely resulting from pine breeding activities and material exchanges in these areas. The increased movement of planting material could have some major consequences by facilitating secondary contact between genetically isolated DNB lineages, possibly resulting in new epidemics.

Soil fungi are important components of boreal forest ecosystems; for example, saprotrophic fungi regulate nutrient cycling, and mycorrhizal species facilitate nutrient uptake by plants. This study aimed to assess soil fungal communities in a reclaimed area and an adjacent natural mixedwood forest and to identify the distribution of taxa available for seedling colonization. Soil fungal microbiomes were assessed along three transects (from 10 m inside the interior of the undisturbed forest to 40 m inside the reclaimed area) and in the roots of small aspen within the natural forest. Using high-throughput deoxyribonucleic acid (DNA) sequencing of internal transcribed spacer amplicons, a total of 2796 unique fungal taxa were detected across fine roots, forest floor, and mineral soils collected along the transects, whereas 166 taxa were detected in the aspen roots from the natural forest. Within the interior of the forest, ectomycorrhizal fungi were more common, whereas in the reclaimed areas, arbuscular mycorrhizae and saprophytes were more common. This survey showed that natural areas of adjacent undisturbed forest can act as a source of ectomycorrhizal fungi for dispersal into reclaimed areas. Notably, soil fungal taxa colonizing the root systems of small aspen included species that are specifically associated with soils from the undisturbed forest (primarily ectomycorrhizae) or the reclaimed clearing (saprotrophs and plant pathogens).

Earthworms that are invasive to Canada's boreal forest are mainly from the family Lumbricidae and they have the potential to change ecosystem carbon sequestration and biodiversity. Traditional methods used to survey earthworms rely on finding specimens, consume large amounts of time and labour, and are expensive. A sensitive genetic method to detect earthworm DNA in soil would provide a fast, relatively low cost method for earthworm survey. In this study, PCR primers were used to detect earthworm DNA from earthworm tissues and soil samples. Though single PCR yielded only faint signals from soil samples, a nested PCR method allowed strong detection in native and cultivated forest soils, including those archived for more than 30 years, providing a promising technique for genetic monitoring of earthworms in the boreal region of Canada.

Symbiotic relationships between insects and fungi are known to cause tree mortality either through direct damage by larval feeding that can be facilitated by symbiotic fungi, or through insects vectoring pathogens directly to healthy trees. Within their native ranges, the impacts of many insect-fungus symbioses are restricted to weakened and declining trees; however, within the last century tree mortality caused by globally invasive insect-fungus associations has had a devastating impact on trees in both urban and natural forest ecosystems. Unfortunately, Canadian forests have been seriously affected by invasive organisms and an emerging threat is the expansion of a native bark beetle into the boreal forest of Alberta. This paper reviews the symbiotic relationships between selected invasive insects and pathogens that cause tree mortality within the urban and forested landscapes of Canada; it uses these case studies to illustrate potentially damaging new evolutionary trajectories.

More than 1180 non-native species, mostly of Palaearctic origin, have been recorded from the boreal zone of Canada, with the highest diversity on the island of Newfoundland and in the southern boreal zone of Ontario and Quebec. The non-native biota of the boreal zone (and of Canada in general) is poorly known in terms of species composition and distribution. A large proportion of species are associated with disturbed anthropogenic habitats such as urban areas, agricultural landscapes, transportation and communication corridors, and industrial developments. Natural habitats in the boreal zone have a high degree of resistance to invasion compared with those of other Canadian zones, likely owing to harsh climates, low light levels, poor soil nutrient availability, low soil pH, low productivity, and dense covering of the ground by plants, especially bryophytes. Of the relatively few non-native species that have successfully colonized the boreal zone, many decline greatly in abundance after a few years, suggesting biotic resilience. To date the boreal zone has shown the least resistance and resilience to large vertebrates (moose and white-tailed deer) translocated to islands, diseases of vertebrates, and earthworms. In general, the ecological impacts of non-native species on the boreal zone have been poorly studied, and there are few examples where such impacts are evident. Likewise, there has been little attempt to quantify the economic impacts of non-native species in either the boreal zone or in Canada as a whole. In the few cases where management measures have been implemented for highly destructive non-native species, results have been somewhat successful, especially where classical biological control measures have been implemented against insects on trees. Chemical and mechanical management measures have had only limited success in localized situations. Management resources are most effectively applied to reducing the risk of introduction. The risk to the boreal zone posed by future new non-native species is increasing with the warming climate and the fast and direct transport of goods into the boreal zone from points of origin. Five recommendations are provided to address recognized gaps concerning non-native species.

Background: Nectria flute canker is an important disease of Pinus radiata in the South Island of New Zealand. The causal agent of the disease, Neonectria fuckeliana, is a known wound invader of Picea abies in Europe. To test the hypothesis that pruning wounds are necessary for infection of Pinus radiata by N. fuckeliana, the presence of the fungus was assessed in pruned and unpruned trees.Methods: The presence of the fungus was investigated in a total of 180 trees (90 pruned and 90 unpruned) using both DNA and microbial culturing techniques over three consecutive years. The data was analysed using a logistic regression analysis.Results: It was found that there was no significant difference in the presence of the fungus in pruned and unpruned trees.Conclusions: These results indicate that pruning wounds are not the primary infection court for N. fuckeliana and that the fungus is able to enter the tree through an alternative infection point.

Ectomycorrhizal (ECM) associates of the exotic plantation species Pinus radiata were investigated above and below ground over two years in the North Island of New Zealand. ECM species were identified using morphological and molecular (restriction fragment length polymorphism and DNA sequencing) analysis. Eighteen ECM species were observed fruiting above ground; 19 ECM species were identified below ground. In the above ground study, Wilcoxina mikolae, Rhizopogon pseudoroseolus and Inocybe sindonia were noted for the first time as ECM associates of P. radiata in New Zealand. Below ground, the species W. mikolae, R. pseudoroseolus, Rhizopogon luteorubescens, Pseudotomentella sp., Pseudotomentella tristis and Tomentella sp. were found as new associates of P. radiata in New Zealand. Additionally, six ECM types were found that could not be identified with molecular analysis. The putative ECM taxa Tricholoma pessundatum, Laccaria laccata and Hebeloma crustuliniforme were examined by molecular analysis, and species identifications were proposed to be changed to Tricholoma sp., L. laccata and Hebeloma sp. for specimens associated with P. radiata in New Zealand. The species identity of I. sindonia, previously unidentified to species level, was determined with direct sequencing.

Peridermium harknessii is not present in the southern hemisphere yet it poses a serious threat to Pinus radiata cultivation in exotic forest plantations there. If a suspected incursion were to occur, it would be necessary to rapidly confirm the presence of P. harknessii within non-sporulating galls. With this in mind, we have developed a DNA-based identification system, using the first intergenic spacer region (IGS-1), that is able to detect the presence of the pathogen within galled tissue. The PCR primers are highly specific and with the exception of the closest relative, Cronartium quercuum f. sp. banksianae , they did not cross react with any of the 11 species within the closely related genera Cronartium and Peridermium that were tested. Phylogenetic analysis of the IGS-1 region confirmed that C. quercuum f. sp. banksianae is the closest relative to P. harknessii . The PCR primers and protocol reported here should prove useful in the event of a suspected western gall rust disease outbreak in exotic P. radiata plantations.