Mechanisms of volatile organic compounds from bat cave environments against <i>Pseudogymnoascus destructans in vitro</i>

White-nose syndrome, caused by the psychrophilic fungus <i>Pseudogymnoascus destructans,</i> is a wildlife disease that infects hibernating bats, resulting in the deaths of millions of bats in North America. Previous studies have confirmed that volatile organic compounds (VOCs) effectively inhibit the growth of <i>P. destructans</i>, but the antifungal mechanisms of these compounds have not been comprehensively characterized. This study screened two VOCs, 2,5-dimethylcyclohexanol (DMCH) and nonanal, identified from bat cave environments for their potent <i>in vitro</i> inhibition of <i>P. destructans</i>. Scanning and transmission electron microscopy revealed mycelial deformations and disruptions in cellular structures following treatment with these compounds. Physiological and biochemical assays showed higher Annexin V-fluorescein isothiocyanate/propidium (Annexin V-FITC/PI) signals consistent with mycelial apoptosis, increased reactive oxygen species (ROS) levels, higher adenosine triphosphate (ATP), superoxide anion, and glutathione (GSH) contents, and lower catalase (CAT) and superoxide dismutase (SOD) activities. Integrated transcriptomic and metabolomic analyses of mycelia exposed to DMCH or nonanal indicated disruption of cell wall and membrane integrity, altered expression of virulence-associated genes, and perturbation of primary metabolism and energy homeostasis. We also observed signatures of heightened oxidative stress, overexpression of ribosomal genes, and modulation of the MAPK signaling pathway. This study provides novel insights into the antifungal effects of VOCs targeting <i>P. destructans</i> and offers a scientific basis for combating white-nose syndrome.IMPORTANCEWhite-nose syndrome, driven by the cold-adapted fungus <i>Pseudogymnoascus destructans</i>, has decimated hibernating bat populations across North America, with profound ecological and economic consequences. Although volatile organic compounds (VOCs) have emerged as promising antifungal agents, their modes of action against <i>P. destructans</i> remain poorly defined. In this study, we demonstrate that two cave-derived VOCs, 2,5-dimethylcyclohexanol (DMCH) and nonanal, not only deform fungal ultrastructure and trigger apoptosis, but also induce severe oxidative stress, disrupt energy metabolism, and dysregulate critical signaling pathways. By integrating transcriptomic and metabolomic profiling, we elucidate how DMCH and nonanal exposure compromises cell wall and membrane integrity, alters virulence gene expression, and perturbs the MAPK cascade, culminating in fungal cell death. These findings advance our mechanistic understanding of VOCs antifungal activity and highlight a novel, environmentally inspired strategy for mitigating white-nose syndrome. Moreover, our work lays the groundwork for the development of VOC-based interventions to protect vulnerable bat populations and preserve ecosystem health.