Inhibitory and synergistic effects of volatile organic compounds from bat caves against <i>Pseudogymnoascus destructans in vitro</i>

Fungi are ubiquitous in natural ecosystems, and environmental reservoirs such as bat hibernacula can harbor fungal pathogens and shape disease dynamics. Beyond serving as pathogen reservoirs, these environments may also contain volatile organic compounds (VOCs) with antifungal properties that help a host resist infection. Studies have shown that various VOCs from bat caves significantly inhibit the growth of <i>Pseudogymnoascus destructans</i>, the pathogen responsible for white-nose syndrome (WNS), although the underlying mechanisms remain unclear. This study investigates two VOCs isolated from bat cave environments-isovaleric acid (IVA) and ethyl methyl carbonate (EMC)-to evaluate their single-agent and combination activities against <i>P. destructans in vitro</i> and to explore the underlying mechanisms. The results show that both IVA and EMC significantly inhibit mycelial growth in a dose-dependent manner and exhibit synergistic antifungal effects. Physiological and biochemical analyses revealed that VOC treatment disrupts cell wall and membrane integrity, induces apoptosis, elevates reactive oxygen species levels, and causes DNA damage. Concentrations of adenosine triphosphate, malondialdehyde, ergosterol, and NADPH also increased significantly. Transcriptomic and metabolomic analyses showed disruption of the mycelial structure, modulation of virulence-associated pathways, induction of oxidative stress and apoptosis, and interference with purine metabolism, cAMP signaling, and energy metabolism. Notably, combined IVA-EMC treatment enhanced DNA damage and suppressed heat shock protein expression, effectively inhibiting <i>P. destructans</i> growth. Taken together, our study elucidates the antifungal potential of environmental VOCs and offers new insights and application prospects for preventing and controlling WNS.IMPORTANCEWhite-nose syndrome has devastated bat populations across North America, yet effective control measures remain limited. This study highlights the potential of naturally occurring volatile organic compounds from bat cave environments as antifungal agents against <i>Pseudogymnoascus destructans in vitro</i>. By uncovering the physiological and molecular mechanisms of the action of isovaleric acid and ethyl methyl carbonate, individually and in combination, this work paves the way for novel, environmentally derived strategies for managing white-nose syndrome and fungal pathogens more broadly.