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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. [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 . 

Abstract Plants of the same species harbor shared enemies that can limit population growth and promote plant species coexistence, a phenomenon known as conspecific negative density dependence (CNDD). Host‐specific microbial pathogens are known drivers of CNDD, yet how these plant–microbe interactions vary with environmental context is less explored. Further, microbial pathogens and mutualists may jointly contribute to the relative strength of CNDD, yet most studies focus only on pathogens. We used ITS metabarcoding to characterize soil and foliar fungal functional groups for individual trees of three dominant conifers in forests where the strength of CNDD has been shown to be greater at lower elevations relative to higher elevations. We hypothesized that trees within mesic low elevation forests accumulate more host‐specific fungal pathogens compared to trees found in the more xeric high elevation forests. Conversely, we hypothesized that trees found in the high elevation forests accumulate more host‐specific mutualists compared to those found at low elevations. We tested these hypotheses with the fungal community sequencing data by evaluating three independent metrics of plant‐associated fungal functional groups—alpha diversity, community weighted mean (CWM), and host specificity—that together address the degree to which each functional group is associated with each focal tree. We found soil pathogen alpha diversity, CWM, and host specificity to all decline in climates associated with increasing elevation. We also found foliar fungal pathogen host specificity, but not diversity or CWM, to decline in climates associated with increasing elevation. In contrast, we found no significant relationships between any of our three metrics for ectomycorrhizal fungi and climate. Our results suggest that across a gradient known to negatively covary with CNDD strength, pathogen accumulation is most prevalent in the mesic, lower elevation climates. These results are consistent with pathogenic fungi contributing to observed landscape level variation in CNDD. 

Summary The scope of plant control over its microbiome is a central question in evolutionary biology and agriculture. Leaf traits are known to shape pathogen colonization and disease development, but their impact on the broader community of largely non‐pathogenic fungi that colonize plant leaves remains an open question.We used reciprocal common gardens of the model tree,Populus trichocarpa(black cottonwood), to examine relationships between leaf traits and the leaf mycobiome in two strongly contrasting environments. We measured six leaf traits (stomatal length, stomatal density, carbon‐to‐nitrogen ratio, leaf thickness, leaf dry matter content, and specific leaf area) and used fungal marker gene sequencing to characterize leaf fungal communities for 57 tree genotypes replicated in one mesic and one xeric common garden (809 trees).Several leaf traits covaried with the leaf mycobiome, yet one relationship was paramount: plant genotypes with longer, sparser leaf stomata hosted a greater richness and diversity of more similar fungal species compared to plant genotypes with shorter, denser leaf stomata.These relationships, while modulated by the environment plants were sourced from and grown in, suggest that stomatal traits may be a general mechanism through which plants and the leaf mycobiome influence one another. 

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. 

Headwater streams are reliant on riparian tree leaf litterfall to fuel brown food webs. Terrestrial agents like herbivores and contaminants can alter plant growth, litter production, litter quality, and the timing of litterfall into streams, influencing aspects of the brown food web. At Mount St. Helens (USA), early successional streams are developing willow (Salix sitchensis) riparian zones. The willows are attacked by stem-boring herbivores, altering litter quality and the timing of litterfall. Within a established experimental plots, willows (male and female plants) were protected from herbivores using insecticides and provided with experimental additions of nitrogen. This enabled us to test the interacting influences of herbivores, nitrogen deposition, and willow sex on leaf litter quality, aquatic litter decomposition, and microbial and invertebrate detritivores. We found weak litter quality effects (higher N and lower C:N) for the herbivore treatment, but no effect of nitrogen deposition. Although litter decomposition rates were not strongly affected by litter treatments, detritivore communities were altered by all treatments. Nitrogen deposition resulted in decreased bacterial richness and decreased fungal diversity in-stream. Aquatic macroinvertebrate communities were influenced by the interacting effects of herbivory and nitrogen addition, with abundances highest in herbivore litter with the greatest N addition. Shredders showed the highest abundance in male, herbivore-attacked litter. The establishment of riparian willows along early successional streams and their interacting effects with herbivores and nitrogen deposition may be influencing detritivore community assembly at Mount St. Helens. More broadly, global changes like increased wet and dry N deposition and expanded ranges of key herbivores might influence tree litter decomposition in many ecosystems. 

Summary Elucidating the factors controlling plant microbiome assembly is a major research goal in plant biology given a growing awareness of microbial community contributions to host plant health and fitness. While stomata have long been recognized to influence pathogen colonization, less is known about whether or how stomatal traits regulate diverse communities of nonpathogenic microbes that make up the majority of the leaf microbiome. In this Viewpoint, we propose that stomata are a primary filter by which plants influence the assembly of leaf‐associated microbial communities. We discuss three nonmutually exclusive hypotheses for how stomatal traits influence leaf microbes, including preliminary support for each based on published studies of foliar fungi and bacteria. The stomatal density hypothesis argues that a greater density of pores increases the rate of microbial entry into the leaf, while the stomatal function hypothesis posits that the duration and speed of stomatal opening and closing regulate microbial access into the leaf. The stomatal covariation hypothesis recognizes that many other leaf traits covary with stomatal traits and thus could contribute to observed relationships between stomatal traits and leaf microbiome structure. Finally, we propose research priorities to improve our understanding of stomatal control over leaf microbiome assembly. 

Tree planting and natural regeneration contribute to the ongoing effort to restore Earth's forests. Our review addresses how the plant microbiome can enhance the survival of planted and naturally regenerating seedlings and serve in long-term forest carbon capture and the conservation of biodiversity. We focus on fungal leaf endophytes, ubiquitous defensive symbionts that protect against pathogens. We first show that fungal and oomycetous pathogen richness varies greatly for tree species native to the United States ( n = 0–876 known pathogens per US tree species), with nearly half of tree species either without pathogens in these major groups or with unknown pathogens. Endophytes are insurance against the poorly known and changing threat of tree pathogens. Next, we review studies of plant phyllosphere feedback, but knowledge gaps prevent us from evaluating whether adding conspecific leaf litter to planted seedlings promotes defensive symbiosis, analogous to adding soil to promote positive feedback. Finally, we discuss research priorities for integrating the plant microbiome into efforts to expand Earth's forests.