Host-induced gene silencing (HIGS) is a common method for engineering plant protection against pathogens, although success requires double-stranded RNA (dsRNA) uptake mechanisms that may not be present in all fungi. We explored HIGS in transgenic poplar to study and control Sphaerulina musiva, the cause of Septoria stem canker disease. HIGS transgenic poplars expressing dsRNA that targeted either or both S. musiva CYP51 and DCL were developed and screened for resistance to stem canker disease in two greenhouse inoculation trials. While differences in resistance between transgenic lines and wild-type controls were not detected, there was a correlation between greenhouse-expressed disease resistance and transgene expression among HIGS lines targeting S. musiva DCL. To evaluate the likelihood that HIGS or spray-induced gene silencing might be effective under some conditions, concurrent with greenhouse screening, we studied: (i) S. musiva's capacity for uptake of environmental dsRNA; (ii) effects of in vitro silencing of CYP51 and DCL on fungal growth and target transcript abundance; and (iii) persistence of dsRNA in culture. The uptake of fluorescently tagged dsRNA was not detected with confocal imaging. In dsRNA-treated cultures, fungal growth inhibition was not detected, and RNA was rapidly degraded. Of the five target transcripts tested after dsRNA treatment, only DCL1 had reduced expression. Knockdown of DCL1 along with the enhanced resistance among high-expressing HIGS events targeting DCL suggests some HIGS may have been observed. Further determination of the factors limiting dsRNA uptake by S. musiva are needed to determine whether HIGS can be an effective technology for limiting stem canker.Copyright (c) 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Background The model woody plant Populus trichocarpa displays an atypical alkene-diverse wax cuticle likely driven by copy number variation (CNV) of 3-ketoacyl-CoA synthases ( KCS ), which has been difficult to confirm based on short-read assemblies. New long-read sequencing provides opportunities to develop telomere-to-telomere resources to detect cryptic variation, including CNVs, which are currently missed in traditional analyses. Integrating this information can improve genomic prediction for breeding and provide insights into the evolutionary basis of important traits. Results Our analysis of 78 telomere-to-telomere long-read haplotypes identified more than twice as many KCS genes as previously reported, along with numerous intragenic non-synonymous substitutions. Random forest predictive models highlighted the importance of Potri . 010G079500 in producing very long chain alkenes; however, its absence did not predict previously reported alkene-deficient phenotypes. Instead, alkene levels are best predicted by the combinations of KCS copies. Amino acid substitutions clustered around ligand and donor binding pockets, suggesting they contribute to differing wax cuticle composition. Finally, each KCS gene and copy was linked to a helitron transposon. A phylogenetic analysis indicates they are the evolutionary mechanism for generating KCS tandem arrays. Conclusions Long-read sequencing and telomere-to-telomere assembles revealed large-effect loci critical to genetic studies that are unattainable from short-reads. These approaches also have the potential to reveal novel insights into genome structure and function, such as the helitrons identified here. Our results highlight that, given current challenges in annotation and assembly, detailed and focused long-read sequences are key to interpreting complex genomic regions that contain tandem copy number variants. ### Competing Interest Statement The authors have declared no competing interest. United States Department of Energy, https://ror.org/01bj3aw27 Australian Research Council Centre of Excellence for Plant Success in Nature and Agriculture, CE200100015

In a rapidly changing environment, predicting changes in the growth and survival of local populations can inform conservation and management. Plastic responses vary as a result of genetic differentiation within and among species, so accurate rangewide predictions require characterization of genotype-specific reaction norms across the continuum of historic and future climate conditions comprising a species' range. Natural hybrid zones can give rise to novel recombinant genotypes associated with high phenotypic variability, further increasing the variance of plastic responses within the ranges of the hybridizing species. Experiments that plant replicated genotypes across a range of environments can characterize genotype-specific reaction norms; identify genetic, geographic, and climatic factors affecting variation in climate responses; and make predictions of climate responses across complex genetic and geographic landscapes. The North American hybrid zone of Populus trichocarpa and P. balsamifera represents a natural system in which reaction norms are likely to vary with underlying genetic variation that has been shaped by climate, geography, and introgression. Here, we leverage a dataset containing 45 clonal genotypes of varying ancestry from this natural hybrid zone, planted across 17 replicated common garden experiments spanning a broad climatic range, including sites warmer than the natural species ranges. Growth and mortality were measured over two years, enabling us to model reaction norms for each genotype across these tested environments. Genomic variation associated with species ancestry and northern/southern regions significantly influenced growth across environments, with genotypic variation in reaction norms reflecting a trade-off between cold tolerance and growth. Using modeled reaction norms for each genotype, we predicted that genotypes with more P. trichocarpa ancestry may gain an advantage under warmer climates. Spatial shifts of the hybrid zone could facilitate the spread of beneficial alleles into novel climates. These results highlight that genotypic variation in responses to temperature will have landscape-level effects.

Phytophthora is a long-established, well-known, and globally important genus of plant pathogens. Phylogenetic evidence has shown that the biologically distinct, obligate biotrophic downy mildews evolved from Phytophthora at least twice. Because, cladistically, this renders Phytophthora "paraphyletic," it has been proposed that Phytophthora evolutionary clades be split into multiple genera (Crous et al. 2021; Runge et al. 2011; Thines 2023, 2024). In this letter, we review arguments for the retention of the generic name Phytophthora with a broad circumscription made by Brasier et al. (2022) and by many delegates at an open workshop organized by The American Phytopathological Society. We present our well-considered responses to the genus splitting proposals, both in general terms and in terms of the specific proposals for new genera, alongside new information regarding the biological properties and mode of origin of the Phytophthora clades. We consider that the proposals are mostly non-rigorous and not supported by the scientific evidence. Further, given (i) the apparent lack of any distinguishing biological characteristics (synapomorphies) between the Phytophthora clades; (ii) the fundamental monophyly of Phytophthora in the original Haeckelian sense (Haeckel 1877); (iii) the fact that paraphyly is not a justification for taxonomic splitting; and (iv) the considerable likely damage to effective scientific communication and disease management from an unnecessary breakup of the genus, we report that workshop delegates voted unanimously in favor of preserving the current generic concept and for seeking endorsement of this view by a working group of the International Commission on the Taxonomy of Fungi. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Upon introduction, clonal pathogen populations are expected to go through a genetic bottleneck followed by gradual clonal divergence. Two distinct and purely clonal lineages of the sudden oak death pathogen Phytophthora ramorum recently emerged in forests in the Western United States, providing the unique opportunity to study a naturally replicated invasion into the same ecosystem. We characterized population genomic patterns during early invasion using whole-genome sequencing of two P. ramorum clonal lineages sampled in the first 5 years following their detection. We re-sequenced genomes from populations of two dominant clonal lineages, NA1 (n = 134; 2001 to 2005) and EU1 (n = 160; 2015 to 2019), and obtained 106,070 high-quality single-nucleotide polymorphisms in genic regions. Our results are consistent with the hypothesis of one introduction for each lineage. The NA1 population had a wider distribution of pairwise genetic distances than EU1 and higher genetic diversity, though neither NA1 nor EU1 populations clustered clearly by year. There was significant correlation between genetic distance and geographic distance for NA1 (P = 0.042), but not for EU1 (P = 0.402). The genetic diversity in NA1 is strongly driven by loss of heterozygous positions, which impacted more than one-third of the sampled NA1 population. However, loss of heterozygosity was rare in EU1. This work provides novel insights into the invasion biology and dynamics of clonal plant pathogens in natural ecosystems.

Increasingly frequent drought events can favour forest disease emergence. Sooty bark disease (SBD) of Acer pseudoplatanus (sycamore maple) is a good example of this phenomenon. Records of its causal agent, Cryptostroma corticale, invasive in Europe, have increased since the 2000s in central and southern Europe. The pathogen is found asymptomatically in host tissues and switches to a pathogenic lifestyle following abiotic stress. This latent phase hinders the pathogen's early detection. In this study, we assessed the prevalence of C. corticale in asymptomatic trees in France. Our study covered six regions, each including a city and its nearby peri-urban and forested ecosystems, representing a human gradient of influence. We assessed the pathogen's presence in 540 wood samples from asymptomatic maple trees by using real-time PCR. The prevalence was 13.6%, across all plots. The pathogen appears to be widespread in the natural sycamore maple stands of France, increasing the risk of disease development following future drought events. Host density and 3-year accumulated water deficit best explained detection of C. corticale in asymptomatic sycamore maples. However, the detection of the fungus was not related to the level of human influence, as no significant differences were observed along the urban-to-forest gradient. Furthermore, C. corticale was detected in asymptomatic hosts even in regions with limited reports of SBD, indicating that the local reporting frequency of the disease does not reflect the latent presence of the pathogen.

Natural products derived from Allium spp., such as garlic oil, garlic powder, and diallyl disulfide (DADS), are strong elicitors of sclerotia germination in the fungus Sclerotium cepivorum (syn. Stromatinia cepivora), the causal agent of Allium white rot. However, these compounds can also have broad antimicrobial activity against a wide range of bacteria, oomycetes, and other fungi when they are applied to soil. The objective of this study was to determine the potential impacts that DADS application has on soil microbial communities. DADS was applied to two soil types and incubated under aerobic and anaerobic conditions. Metabarcodes for bacterial, fungal, and oomycete communities were analyzed to identify changes. A significant effect of DADS treatment on the overall compositions of bacterial, fungal, and oomycete communities was observed compared with the mock-treated control. Soil type and incubation conditions did not have a significant effect on soil microbial communities, and significant interactions were not observed with DADS treatment in this study. Potential changes in soil microbial communities should be considered when applying DADS to field soils.

Phytophthora pseudosyringae is a self-fertile pathogen of woody plants, particularly associated with tree species from the genera Fagus, Notholithocarpus, Nothofagus and Quercus, which is found across Europe and in parts of North America and Chile. It can behave as a soil pathogen infecting roots and the stem collar region, as well as an aerial pathogen infecting leaves, twigs and stem barks, causing particular damage in the United Kingdom and western North America. The population structure, migration and potential outcrossing of a worldwide collection of isolates were investigated using genotyping-by-sequencing. Coalescent-based migration analysis revealed that the North American population originated from Europe. Historical gene flow has occurred between the continents in both directions to some extent, yet contemporary migration is overwhelmingly from Europe to North America. Two broad population clusters dominate the global population of the pathogen, with a subgroup derived from one of the main clusters found only in western North America. Index of association and network analyses indicate an influential level of outcrossing has occurred in this preferentially inbreeding, homothallic oomycete. Outcrossing between the two main population clusters has created distinct subgroups of admixed individuals that are, however, less common than the main population clusters. Differences in life history traits between the two main population clusters should be further investigated together with virulence and host range tests to evaluate the risk each population poses to natural environments worldwide.

Outbreaks of insects and diseases are part of the natural disturbance regime of all forests. However, introduced pathogens have had outsized impacts on many dominant forest tree species over the past century. Mitigating these impacts and restoring these species are dilemmas of the modern era. Here, we review the ecological and economic impact of introduced pathogens, focusing on examples in North America. We then synthesize the successes and challenges of past biotechnological approaches and discuss the integration of genomics and biotechnology to help mitigate the effects of past and future pathogen invasions. These questions are considered in the context of the transgenic American chestnut, which is the most comprehensive example to date of how biotechnological tools have been used to address the impacts of introduced pathogens on naïve forest ecosystems.

Plant domestication and movement are large contributors to the success of new diseases. The introduction of new host species can result in accelerated evolutionary changes in pathogens, affecting long-established coevolutionary dynamics. This has been observed in poplars where severe epidemics of pathogens that were innocuous in their natural pathosystems occurred following host domestication. The North American fungus Sphaerulina musiva is responsible for endemic leaf spots on Populus deltoides. We show that the expansion of poplar cultivation resulted in the emergence of a new lineage of this pathogen that causes stem infections on a new host, P. balsamifera. This suggests a host shift since this is not a known host. Genome analysis of this emerging lineage reveals a mosaic pattern with islands of diversity separated by fixed genome regions, which is consistent with a homoploid hybridization event between two individuals that produced a hybrid swarm. Genome regions of extreme divergence and low diversity are enriched in genes involved in host–pathogen interactions. The specialization of this emerging lineage to a new host and its clonal propagation represents a serious threat to poplars and could affect both natural and planted forests. This work provides a clear example of the changes created by the intensification of tree cultivation that facilitate the emergence of specialized pathogens, jeopardizing the natural equilibrium between hosts and pathogens. This article is part of the theme issue ‘Infectious disease ecology and evolution in a changing world’.

Sudden oak death (SOD) is caused by Phytophthora ramorum, an invasive oomycete pathogen. This pathogen is of major regulatory concern for nurseries, horticulture, and forestry in the United States and around the world. Three of the 12 identified lineages of P. ramorum currently occur in the United States (NA1, NA2, and EU1) affecting wildland forests and nurseries. Rapid identification and lineage determination is essential to accelerate management decisions, detect introductions of new lineages, and control the spread of SOD. The objective of this study was to develop and validate diagnostic tools to rapidly identify P. ramorum and distinguish among the four common lineages of the pathogen and to accelarate management decision making. The loop-mediated isothermal amplification (LAMP) assays developed here are species specific with no cross reaction to common Phytophthora species found in Oregon, California, and Washington. The lineage-specific assays unambiguously distinguish among the four common clonal lineages. These assays are sensitive and able to detect P. ramorum DNA ranging in concentration from 30 to 0.03 ng/μl depending on the assay. These assays work effectively on a variety of sample types including plant tissue, cultures, and DNA. They have been integrated into the SOD diagnostic process in the forest pathology lab at Oregon State University. To date, 190 samples have been correctly identified from over 200 field samples tested for lineage determination. The development of these assays will help managers in forestry and horticulture identify and rapidly respond to new outbreaks of P. ramorum.

In North America, Coniferiporia weirii causes root and butt rot of western redcedar (Thuja plicata) and yellow-cedar (Callitropsis nootkatensis). There is currently no draft genome for C. weirii. As a result, C. weirii isolate 30910 originally isolated from a Thuja plicata in Idaho, U.S.A., was sequenced using an Illumina HiSeq 3000 sequencing system. The genome was assembled into 24,918 scaffolds with a scaffold N50 length of 53,821 bp. The total size of the genome was estimated to be 42.2 Mb. This included 96% and 95% recovery of basidiomycete complete and single-copy BUSCO genes, respectively. A total of 3.2% of the assessed BUSCO genes were missing and were not recovered. The assembly contained 10,351 predicted protein-coding genes. The estimated mean gene length of the predicted genes was 1,911 bp. While much is known about the biology of this fungus, little is known about its genome. This draft genome provides a baseline resource that will help further understand the population structure, reproductive mode, and evolutionary history of this important forest pathogen.

Phytophthora species are plant pathogens responsible for many notable biological invasions in agricultural, forests, and natural ecosystems. Detection and monitoring for invasive introductions of Phytophthora spp. is time and resource intensive. Development of citizen science detection and monitoring programs can aid in these efforts focused on reducing Phythophthora impacts. There are multiple methods for monitoring and detecting Phytophthora invasions suitable for citizen science approaches such as, leaf sampling, stream baiting or soil collections. Here we summarize five active projects in western North America where citizen scientists are aiding the monitoring and research efforts surrounding Phytophthora species and their impacts. Projects varied in scope, scale, methods, and capacity, but each project increased citizen scientists’ abilities for surveillance and advanced detection or knowledge of Phytophthora species. Some projects were integrated with school programs, others involved hands-on training with small groups, and another approach invited mass participation from interested citizens. Overall, all projects had positive outcomes multiplied across education, monitoring, and research. Together these case studies demonstrate how citizen scientists can amplify surveillance efforts, advance baseline knowledge, and reduce the impacts of biological invasions.

Fungal effectors play critical roles in manipulating plant immune responses and promoting colonization. Sphaerulina musiva is a heterothallic ascomycete fungus that causes Septoria leaf spot and stem canker disease in poplar (Populus spp.) plantations. This disease can result in premature defoliation, branch and stem breakage, increased mortality, and plantation failure. However, little is known about the interaction between S. musiva and poplar. Previous work predicted 142 candidate secreted effector proteins in S. musiva (SmCSEPs), 19 of which were selected for further functional characterization in this study. SmCSEP3 induced plant cell death in Nicotiana benthamiana, while 8 out of 19 tested SmCSEPs suppressed cell death. The signal peptides of these eight SmCSEPs exhibited secretory activity in a yeast signal sequence trap assay. Confocal microscopy revealed that four of these eight SmCSEPs target both the cytoplasm and the nucleus, whereas four predominantly localize to discrete punctate structures. Pathogen challenge assays in N. benthamiana demonstrated that the transient expression of six SmCSEPs promoted Fusarium proliferatum infection. The expression of these six SmCSEP genes were induced during infection. SmCSEP2, SmCSEP13, and SmCSEP25 suppressed chitin-triggered reactive oxygen species burst and callose deposition in N. benthamiana. The candidate secreted effector proteins of S. musiva target multiple compartments in the plant cell and modulate different pattern-triggered immunity pathways. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

In North America, Coniferiporia weirii causes root and butt rot of western redcedar (Thuja plicata) and yellow-cedar (Callitropsis nootkatensis). There is currently no draft genome for C. weirii. As a result, C. weirii isolate 30910 originally isolated from a Thuja plicata in Idaho, U.S.A., was sequenced using an Illumina HiSeq 3000 sequencing system. The genome was assembled into 24,918 scaffolds with a scaffold N50 length of 53,821 bp. The total size of the genome was estimated to be 42.2 Mb. This included 96% and 95% recovery of basidiomycete complete and single-copy BUSCO genes, respectively. A total of 3.2% of the assessed BUSCO genes were missing and were not recovered. The assembly contained 10,351 predicted protein-coding genes. The estimated mean gene length of the predicted genes was 1,911 bp. While much is known about the biology of this fungus, little is known about its genome. This draft genome provides a baseline resource that will help further understand the population structure, reproductive mode, and evolutionary history of this important forest pathogen.

To identify, validate, and functionally characterize alleles that confer resistance to Septoria canker and leaf spot in Populus. The proposed research will elucidate a major mechanism of resistance to Sphaerulina musiva, the major limiting factor to plantations in eastern North America. Genome-wide association mapping, CRISPR/Cas9, and protein-protein assays will be used, enabling marker-aided breeding, reducing costs, and accelerating development of resistant varieties.

Radiata pine is an important plantation tree; more than 4 million ha are planted globally, and 90% of planted forests in Aotearoa New Zealand are radiata pine. It is susceptible to several root and foliar diseases caused by Phytophthora species, and is potentially susceptible to Phytophthora ramorum, the pathogen responsible for sudden oak death in the United States and Europe. A series of experiments were conducted to determine the potential risk of P. ramorum infection to radiata pine logs and seedlings in the context of forest harvest and replanting. In a natural inoculation experiment, bolts of radiata pine, Douglas-fir and tanoak were exposed to P. ramorum inoculum produced from sporulating tanoak canopies infected with either the NA1 or EU1 lineage of P. ramorum. The experiment occurred at four sites, two NA1 and two EU1, across 2 years. Four per cent of radiata pine bolts, 8% of tanoak bolts and 0% Douglas-fir bolts tested positive for P. ramorum. Artificial inoculations of bolts of the same species revealed a significant effect of lineage (p = .0024), species (p < .0001) and their interaction (p = .0027) on lesion length. Species was the only parameter that had a significant impact on average lesion length (p < .0001) and sporulation (p = .00144) from seedlings. No sporangia were observed on radiata pine seedlings (n = 60); few were observed on tanoak and Douglas-fir. Although radiata pine can be colonized by P. ramorum, without a sporulating host in proximity it does not appear to be at high risk from this pathogen.