We present NEEDL-Bench, a microscopy detection benchmark for Swiss Needle Cast (SNC), a fungal disease of Douglas-fir trees. Douglas-fir is a keystone species of major ecological and economic importance as a softwood timber resource, and SNC affects productivity by forming sexual reproductive structures (pseudothecia) that emerge through the gas exchange pores (stomata) of the needles, thereby blocking gas exchange and compromising needle function. To date, there is no dataset for automatic computer vision detection of these structures, despite computer vision being well poised to standardize and viably scale severity measurements. To address this, we present NEEDL-Bench, a dataset of 3250 annotated images from 1082 Douglas-fir needles, annotated for both keypoints and bounding-box detectors. This dataset exhibits a challenging collection of features, including blur, poor object contrast, small objects of interest, and occlusions. To better capture both the nominal distribution of the data and the full breadth of rare structures, we present two distinct evaluation splits: either random sampling from the collected images or sequential sampling to maximize structural diversity. We evaluate multiple popular keypoint and bounding box methods for detection on this dataset as a baseline and observe a maximum F1 score of 0.8479, suggesting significant potential for gains from future development on this problem. Further, we find that larger models generally do not show commensurate gains in performance on this dataset, indicating that improvements on this problem will not come from scaling laws but rather from domain-specific inductive biases.

Biscogniauxia species are typically recognized by carbonaceous stromata on woody substrates, but some also occur as asymptomatic endophytes and may shift ecological roles across hosts and substrates. Here, Biscogniauxia barriae sp. nov . is described, first isolated as a foliar endophyte from asymptomatic Douglas-fir ( Pseudotsuga menziesii ) needles in coastal British Columbia and later found producing stromata on dead hardwoods, including bigleaf maple ( Acer macrophyllum ), red alder ( Alnus rubra ), and hawthorn ( Crataegus sp.). Phylogenetic analyses of ITS, BenA , and RPB2 resolved B. barriae as a distinct, well-supported lineage within Biscogniauxia s.s., sister to B. capnodes and allied with a clade including B. anceps and B. nummularia . The species is characterized by applanate to pulvinate, carbonaceous stromata with coarsely papillate ostioles and dark brown, broadly ellipsoid ascospores with a conspicuous straight germ slit; a nodulisporium-like asexual morph is produced in culture. The highly complete 42.7 Mb draft genome contained 10,986 predicted protein-coding genes and was broadly comparable to available Biscogniauxia genomes. Orthogroup analyses indicated extensive conservation of protein-coding gene content, while identification of a MAT1-2 idiomorph suggests heterothallism. The discovery of B. barriae through both Douglas-fir endophyte surveys and hardwood stromatal collections highlights a dual life history linking conifer foliar endophytism with hardwood-associated saprotrophy or possible latent pathogenicity. Strong inhibition of the in vitro growth of Nothophaeocryptopus gaeumannii , the causal agent of Swiss needle cast, by culture-filtrate extracts of B. barriae suggests potential ecological relevance within the Douglas-fir needle mycobiome.

Mycoviruses, viruses infecting fungi, have been identified in several ascomycetes as single infections or mixed infections. These interactions with plant pathogenic fungi have been associated with hyper- or hypovirulence. This report focuses on single and mixed mycovirus infection of the plant pathogenic fungus Cryptostroma corticale, the causal agent of sooty bark disease (SBD) affecting maple trees in Europe and North America. In humans, exposure of C. corticale spores causes maple bark disease (MBD), causing hypersensitive pneumonitis and allergic asthma with viral-like symptoms. In this study, we identified four species of single-stranded, positive-sense (ss+) RNA mycoviruses that are phylogenetically diverse, including one Mitovirus: Cryptostroma corticale mitovirus 1 (CcMV1) and three Narnavirus species: Cryptostroma corticale narnavirus 1, 2, and 3 (CcNV1, 2, and 3). Notably, CcMV1 shows predicted secondary structures in both 5' and 3' UTRs, while CcNV1 and CcNV3 exhibited strong secondary structures at the 3' UTR. These four mycoviruses were prevalent in 45% (n = 35) of the isolates tested, and growth assays of single infection and the most complex mixed infection showed faster radial expansion compared to a virus-free isolate. These findings reveal single and mixed mycoviruses infections in C. corticale differentially affecting fungal growth, with potential implications for SBD pathogenesis and human MBD risk.

Phytophthora abietivora is a recently described root rot pathogen of true firs (Abies spp.) and a major contributor to fir plantation decline in eastern North America. We present a 62.17-Mbp draft genome assembly of P. abietivora generated using Oxford Nanopore and Illumina sequencing technologies. The assembly comprises 2,917 scaffolds with an N50 of 43.8 kb, 99.7% completeness, and 15,906 predicted protein-coding genes, including 1,210 secreted proteins. Single-nucleotide polymorphism analysis revealed extended runs of homozygosity and allele frequency shifts, suggesting dynamic extreme aneuploidy. This reference genome will serve as a critical resource for exploring the evolution and pathogenicity of P. abietivora and developing control strategies.Copyright (c) 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Climate change is fundamentally reshaping forest disease dynamics through direct effects on pathogen biology and indirect impacts on host physiology. Rising temperatures, altered precipitation patterns, and extreme weather events are driving disease emergence by disrupting ecological relationships between trees and their microbial associates. This review examines how climate change compounds biotic and abiotic risks to forest health, distinguishing between climate-pathogen diseases, where climatic shifts directly favor pathogen activity, and climate-stress diseases, where physiological stress predisposes trees to decline. We explore the continuum from native pathogens gaining new opportunities to exotic pathogens establishing in previously unsuitable environments while considering distinctions among endophytes and latent and nonlatent pathogens. The review emphasizes critical knowledge gaps and highlights emerging research directions, including integration of genomics, remote sensing, and predictive modeling for disease surveillance, adaptive forest management strategies balancing disease mitigation with climate adaptation and new solutions for enhancing forest resilience under accelerating environmental change.

Red alder (Alnus rubra Bong.) is the most abundant broadleaf tree species along the Pacific Northwest (PNW), including coastal British Columbia (BC), Canada, where it plays an important role as a pioneer species providing a wide range of benefits to riparian ecosystems. Its symbiotic relationship with nitrogen-fixing actinomycetes (Frankia sp.) contributes to soil fertility and ecosystem regeneration. Alder dieback has been attributed to multiple interacting biotic and abiotic agents, including oomycetes of the genus Phytophthora, which have been acting as predisposing or contributing factors in declining stands in the PNW (Sims et al. 2015; Feau et al. 2022) and Europe (Husson et al. 2015). In June 2024, a survey was conducted in a 0.7 ha stand of declining red alder trees in Daajing Giids (53.2569°N, 132.0919°W) on the Haida Gwaii archipelago in costal BC. Three Phytophthora-like cultures were obtained by plating root lesions on PARP(H)-CMA selective media (Ivors 2015) and by baiting soil samples collected from beneath declining red alder trees with pears. On V8 agar, colonies displayed a characteristic chrysanthemum-like growth pattern. Sporangia were predominantly ovoid, occasionally reniform, averaging 30.9 ±19.4 × 26.2 ±16.7 µm (n = 198), with a size range of 1.0-68.2 × 1.6-65.5 µm. Cultures were identified by DNA barcoding after growth in clarified V8-PARP(H) liquid medium. Genomic DNA was extracted from freeze-dried mycelia using DNeasy® Plant Mini Kit (Qiagen, Toronto, Ontario). The internal transcribed spacer (ITS) of the rDNA, elongation factor 1 alpha (EF1α) and beta tubulin (Btub) were amplified in three separate PCR reactions. PCR amplicons were sequenced in both directions using Sanger sequencing at the CHUL sequencing platform in Quebec City. BLASTn analysis of all three loci showed 99.82-100% identity with the P. siskiyouensis Reeser & E.M. Hansen ex-type isolates CPHST BL 56 (ITS) and 41B7 (EF1α and Btub). Sequences were deposited in GenBank under accession numbers PX875328-PX875330 (ITS), PX903677-PX903679 (EF1α), and PX883747-PX883749 (Btub). To verify Koch’s postulates, six one-year old A. rubra seedlings were inoculated with a single P. siskiyouensis isolate by inserting a mycelial plug into a small stem wound 10 cm above the soil line Seedlings were maintained at 20 °C in a growth chamber. Black, sunken lesions became visible externally three weeks after inoculation, and internal examination revealed phloem necrosis extending up to 4 cm above and below the inoculation point. The pathogen was successfully re-isolated from all inoculated A. rubra seedlings. Cultures and infected stems were deposited at the Pacific Forestry Centre’s Herbarium under accession numbers DAVP29865 to DAVP29867. No lesions developed on the three control seedlings inoculated with sterile V8-PARP(H) plugs. To our best knowledge, this is the first report of P. siskiyouensis associated with declining red alder in BC. This pathogen was first described from myrtlewood and tanoak in Oregon (Reeser et al. 2007) and has since been reported causing disease on Alnus spp. in Oregon and California (Rooney-Latham et al. 2009; Sims et al. 2015) and on grey alder in the UK (Perez-Sierra et al. 2015). This finding extends the known geographic distribution and host range of P. siskiyouensis in BC and highlights its potential as an emerging threat to red alder ecosystems, with possible implications for riparian stability and alder-associated ecological functions.

Seedborne fungal pathogens, either native or exotic, spread through seed trade and pose serious risks to reforestation efforts. Traditional pathogen detection methods, such as seed plating assays, are limited in scope and sensitivity. We assessed the potential of DNA-metabarcoding with Oxford Nanopore Technologies (ONT) to detect and identify fungal pathogens in conifer seed lots. Using ONT-based sequencing of the internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1) loci, we analyzed 20 seed lots from Douglas fir (Pseudotsuga menziesii) and interior spruce (Picea engelmannii x glauca). Our results were benchmarked against culture-based and real-time PCR assays. The ITS ONT assay detected a broad range of fungal taxa, including conifer pathogens such as Fusarium spp., Sirococcus conigenus, and Caloscypha fulgens. Although the TEF1 ONT assay showed lower detection sensitivity, it increased the robustness of species complex resolution within the Fusarium genus. We observed a strong correlation between ITS ONT read counts and real-time PCR quantification cycle (Cq) values, indicating the potential for quantitative pathogen load assessment. Differences between detection methods highlighted the importance of optimizing seed sampling strategies to improve pathogen detection consistency. The portability, affordability, and ongoing improvements in ONT technology suggest a promising future for its application in forest seed diagnostics and biosecurity monitoring.Copyright (c) 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

BACKGROUND:Ash dieback, caused by Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya, is a serious fungal disease affecting Common ash (Fraxinus excelsior) populations across Europe and posing a global risk to ash trees worldwide. Traditional polymerase chain reaction (PCR)-based detection methods often target conserved gene regions, limiting the development of species-specific probes. However, the increasing availability of whole-genome sequences for H. fraxineus and its close relatives enables the identification of unique genes or genomic regions. RESULTS:This study utilized such genomic data to develop a sensitive, species-specific loop-mediated isothermal amplification (LAMP) assay for detecting H. fraxineus in Fraxinus mandshurica samples. The LAMP assay demonstrated a sensitivity of 2.53 fg/μL and proved effective in early detection of the pathogen. CONCLUSION:Altogether the developed LAMP detection method could be quite useful for further studies on the characteristics of the life cycle and colonization process of H. fraxineus in Asian ash host species. © 2025 Society of Chemical Industry.

Climate significantly influences the distribution, composition, and diversity of fungal communities, impacting the growth, spread, and virulence of fungal forest pathogens. This study employs advanced landscape genomics methods to explore the genomic adaptations of three major fungal pathogens: Those responsible for Dutch elm disease, dothistroma needle blight, and Swiss needle cast. Our findings reveal that precipitation and humidity are primary drivers of adaptation in these species. We use these insights to forecast potential adaptations under future climate scenarios (genomic offset) and identify specific genes and pathways associated with climate responses in each pathogen. Notably, we detect a convergence in moisture adaptation across these distantly related species, particularly in genes related to the cytoskeleton and transporters. This study enhances our understanding of fungal pathogen evolution in response to climate change, offering crucial insights for forest disease management.

Douglas-fir (Pseudotsuga menziesii) is one of the most economically important softwood trees grown worldwide. However, the past several decades has seen a decline in its productivity due to increased infection by the fungus Nothophaeocryptopus gaeumannii, the causal agent of Swiss needle cast disease, for which there are currently no feasible control methods. We studied Douglas-fir endophyte extracts to direct future investigations profiling their biologically active natural products. Endophytes were isolated from asymptomatic Douglas-fir needles, and 63 strains representing 32 unique species across 20 families in 14 orders were selected for investigation. A bioassay was developed to assess the antifungal properties of culture filtrate extracts from the endophyte collection against the three distinct N. gaeumannii lineages. Depending on the P value stringency, 26 (P <= 0.05) or 8 (P <= 0.01) strains significantly inhibited the growth of N. gaeumannii, most notably Lachnum virgineum and a putatively novel species of Biscogniauxia. In addition, eight metabolites from the Douglas-fir endophytes Xylaria hypoxylon and Coleophoma sp. were purified and characterized, and an additional 10 metabolites were identified by high-resolution mass spectrometry. Endophytes that inhibited the N. gaeumannii lineages will be prioritized for future studies to determine their potential to modify foliar microbiomes and assist forest disease management.Copyright (c) 2025 His Majesty the King in Right of Canada, as represented by the Minister of Natural Resources Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Sooty bark disease (SBD), caused by the fungus Cryptostroma corticale, is causing a widespread outbreak on sycamore maple in Europe and is presently emerging as a threat to urban and native maple trees in the Pacific Northwest. Rising temperatures and prolonged drought conditions probably exacerbate SBD disease severity. Current detection methods for C. corticale rely on laboratory-based PCR and morphological identification, which require specialised equipment and are not suited for rapid field deployment. We developed a portable, species-specific PCR-nucleic acid lateral flow immunoassay (PCR-NALFIA) for point-of-care detection of C. corticale. The assay can be performed in 90 min by incorporating a 5-min crude DNA extraction using cellulose dipsticks, a species-specific ITS1-targeted PCR with internal probe labelling on a portable PCR machine, and a lateral flow dipstick for visual detection. The method yielded accurate detection from infected wood, bark and conidia, with detection thresholds as low as 10 conidia or 0.1 mg of stroma in 0.5 mL buffer. The PCR-NALFIA achieved 100% specificity when tested on 87 fungal samples, outperforming other available PCR assays. Furthermore, lyophilised reaction mixes were stable for 60 days at room temperature, making the assay viable for field applications. This tool offers a rapid and reliable diagnostic solution for managing SBD in forestry and urban environments.

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 × 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.

CRISPR-Cas9 gene editing has become an important tool for the study of plant pathogens, allowing researchers to functionally characterize specific genes involved in phytopathogenicity, virulence, and fungicide resistance. Protocols for CRISPR-Cas9 gene editing have already been developed for Phytophthoras, an important group of oomycete plant pathogens; however, these efforts have exclusively focused on agricultural pathosystems, with research lacking for forest pathosystems. We sought to develop CRISPR-Cas9 gene editing in two forest pathogenic Phytophthoras, Phytophthora cactorum and P. ramorum, using a plasmid-ribonucleoprotein (RNP) co-transformation approach. Our gene target in both species was the ortholog of PcORP1, which encodes an oxysterol-binding protein that is the target of the fungicide oxathiapiprolin in the agricultural pathogen P. capsici. We delivered liposome complexes, each containing plasmid DNA and CRISPR-Cas9 RNPs, to Phytophthora protoplasts using a polyethylene glycol-mediated transformation protocol. We obtained two ORP1 mutants in P. cactorum but were unable to obtain any mutants in P. ramorum. The two P. cactorum mutants exhibited decreased resistance to oxathiapiprolin, as measured by their radial growth relative to wild-type cultures on oxathiapiprolin-supplemented medium. Our results demonstrate the potential for RNP-mediated CRISPR-Cas9 gene editing in P. cactorum and provide a foundation for future optimization of our protocol in other forest pathogenic Phytophthora species.IMPORTANCECRISPR-Cas9 gene editing has become a valuable tool for characterizing the genetics driving virulence and pathogenicity in plant pathogens. CRISPR-Cas9 protocols are now well-established in several Phytophthora species, an oomycete genus with significant economic and ecological impact globally. These protocols, however, have been developed for agricultural Phytophthora pathogens only; CRISPR-Cas9 systems have not yet been developed for any forest pathogenic Phytophthoras. In this study, we sought to establish CRISPR-Cas9 gene editing in two forest Phytophthora pathogens that cause widespread tree mortality: P. cactorum and P. ramorum. We successfully obtained gene mutations in P. cactorum and demonstrated a decrease in fungicide resistance, a trait that could impact the pathogen’s ability to cause disease. However, the same protocol did not yield any mutants in P. ramorum. The results of our study will serve as a baseline for the development of CRISPR-Cas9 gene editing in forest Phytophthoras and other oomycetes.

Sooty bark disease is an invasive disease causing significant mortality of sycamore maple (Acer pseudoplatanus L.) in Europe, where it is emerging due to increasing drought and heat events. The causal agent, Cryptostroma corticale (Ellis & Everh.) P. H. Greg. & S. Waller, is a fungus endemic to the Great Lake region in eastern Canada, where it does not appear to cause disease within its natural host range (e.g. sugar maple, Acer saccharum Marshall). Sooty bark disease was reported causing mortality on sycamore maple and additional species within Washington State beginning in 2017. In summer 2022, sooty bark disease was found on a sycamore maple near Vancouver, representing the first report of the disease and causal agent within the province of British Columbia (BC). In this study, we identify the causal agent of sooty bark disease by morphological and molecular methods and confirm its pathogenicity in a controlled growth chamber experiment fulfilling Koch's postulates on sycamore maple. Cryptostroma corticale has so far been found in BC on sycamore maple, Norway maple (A. platanoides L.) and bigleaf maple (A. macrophyllum Pursh). Sooty bark disease has been previously shown to increase in severity and occurrence under drought and warm conditions; we anticipate rising sooty bark disease cases as BC experiences increasingly frequent and extreme summer droughts and heat events.

We report an annotated draft genome of Heterobasidion occidentale, a fungus (Basidiomycota, Agaricomycetes) that has pathogenic and saprophytic lifestyles. This fungus belongs to the H. annosum (Fr.) Bref. sensu lato species complex that comprises several root rot pathogens. Heterobasidion occidentale causes annosus root and butt rot primarily in true fir (Abies spp.) and spruce (Picea spp.) species throughout western North America.

Ophiostoma haidanensis is described as a new species of the Ophiostoma piceae complex isolated from yellow-cedar (Callitropsis nootkatensis (D. Don) Oerst. ex D.P. Little) sapwood in the Haida Gwaii island archipelago and the North Coast of British Columbia, Canada. The fungus is characterized by the production of a typical sporothrix-like asexual morph but is distinguished morphologically from other members of the O. piceae species complex by its large, multiseptate primary conidia. Phylogenetic analysis of DNA sequences from the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and the beta-tubulin (BTUB) and translation elongation factor 1-alpha (TEF1) genes supports the inclusion of O. haidensis as a distinct member within the O. piceae complex. To our knowledge, this is the first report of a blue stain fungus infecting yellow-cedar, an ecologically, culturally, and economically important conifer naturally distributed along the coastal forests of the Pacific Northwest in North America.

Western white pine (Pinus monticola) with genetic tolerance to white pine blister rust disease caused by Cronartium ribicola constitutes an invaluable resource for the mitigation of this disease as well as the restoration of the species and its utilization in managed forests. In the last several years, typical root-rot symptoms and sudden tree death have been observed in some western white pine plantations located in British Columbia (BC) in Canada, and a few Phytophthora species were found to be associated with symptomatic trees. Our objectives were to survey one of the plantations located in south-west BC on Vancouver Island to confirm the presence of these Phytophthora species and other oomycetes and test their pathogenicity on western white pine by completing Koch's postulates. We used direct plating of root samples on PARP(H)-CMA media and by baiting for oomycetes in soil and root samples. Ten oomycetes, including four Phytophthora species (Phytophthora cinnamomi, P. cactorum-like, P. cryptogea and P. pseudocryptogea) were isolated from roots and soil samples of diseased Pi. monticola trees. The Phytophthora species were identified using ITS, beta-tubulin and TEF1 gene regions. Koch's postulates were validated with controlled inoculations of 1-year old seedlings; mortality occurred as early as 15 days after inoculation with P. cinnamomi when the seedlings were maintained in warm temperature conditions (25 degrees C). Molecular typing of two P. cinnamomi cultures using Oxford Nanopore MinION (TM) sequencing indicated that they belong to one of the two main clonal clusters of mating type A2 that have been recognized as the main drivers of the global P. cinnamomi epidemic.

International trade in wood products is an important component of the global economy. However, wood and wood products may have pests associated with them that could be introduced into importing countries, posing phytosanitary risks and leading to the implementation of regulatory restrictions that affect wood trade. The application of heat to kill wood-associated pests has been a successful phytosanitary method to reduce their spread. To evaluate the efficacy of wood heat treatment to kill fungal and fungus-like pathogens, the method of choice has been to grow organisms in cultures for subsequent identification. However, some plant pathogens can be difficult or impossible to grow in axenic cultures, and a molecular method can still be useful for assessing pathogen viability after heat treatment. RNA is a single-stranded molecule that is responsible for the transcription of genes. Since it becomes rapidly unstable after cell death, it provides a measure of viability. We therefore designed and tested RNA-based molecular diagnostic assays targeting essential genes and assessed their presence after heat treatment in wood colonized by four Phytophthora species of phytosanitary concern (P. xmultiformis, P. cinnamomi, P. lateralis, and P. ramorum) through reverse transcription and real-time polymerase chain reaction (RT-qPCR). Our assays differentiate between genomic and mRNA as the TaqMan probes span exon-intron junctions. We validated these RT-qPCR assays to assess heat treatment efficacy of Phytophthora-inoculated wood. These assays can be very useful tools to assess the effectiveness of current and emerging phytosanitary wood treatments.