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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 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 ofPopulus trichocarpaandP. balsamiferarepresents 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 moreP. trichocarpaancestry 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. 

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.