Ran X, et al. J Vet Sci. 2023 May;24:e48. https://doi.org/10.4142/jvs.22307

White-nose syndrome is caused by Pseudogymnoascus destructans, leading to the near extinction of multiple bat populations in North America. This fungal pathogen has also been detected in China, but the prevalence and loads are relatively low in the hosts and environment. Previous studies have screened bat skin microbiomes in China to identify microbes that inhibit the growth of P. destructans . However, there is limited information on bacterial genera that possess properties that inhibit P. destructans in bat cave environments in China, particularly regarding antifungal metabolic pathways. We isolated 29 bacterial strains that have the ability to inhibit growth of P. destructans from the skin of bats and soil samples in China. These strains primarily belonged to several genera, including Acinetobacter , Pseudomonas , and Serratia . Gas chromatography-mass spectrometry analysis identified volatile organic compounds from strains that inhibit P. destructans , showing that 100 µL of α-Pinene, 2-Undecanone, 2-Nonanone, 2,5-Dimethylpyrazine, as well as 10 µL Benzaldehyde and Thujone, completely inhibited the growth of P. destructans and caused morphological damage to the mycelium. The soluble secondary metabolites from the antagonistic strains indicated that the bioactive compounds were predominantly small-molecule organic substances. Whole-genome sequencing of these antagonistic strains revealed that the most enriched potential antifungal gene clusters were associated with bacteriocins, siderophores, and β-lactones. β-Lactones were the primary gene cluster against P. destructans , and chitinases may play a crucial role in the antifungal process. IMPORTANCE: Bat skin and environmental microbiota may influence the colonization and persistence of Pseudogymnoascus destructans , thereby potentially affecting the occurrence of white-nose syndrome. Examining differences in these gene clusters contributes to understanding variation in the capacity of bacterial groups to have characteristics that inhibit P. destructans . This study lays the foundation for further exploration and elucidation of the mechanisms by which bacteria from bat skin and roosting environments suppress P. destructans growth in vitro .

White-nose syndrome, caused by the psychrophilic fungus Pseudogymnoascus destructans, 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 P. destructans , 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 in vitro inhibition of P. destructans . 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 P. destructans and offers a scientific basis for combating white-nose syndrome. IMPORTANCE: White-nose syndrome, driven by the cold‐adapted fungus Pseudogymnoascus destructans , 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 P. destructans 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.

White-nose syndrome (WNS) is an infectious disease of bats caused by the psychrophilic fungus Pseudogymnoascus destructans. Phenazine-1-carboxylic acid (PCA) is a microbial secondary metabolite with broad-spectrum antifungal activity. Previous studies show that PCA suppresses the growth of P. destructans at low concentrations, yet its mechanism remains unclear. Here, we evaluated the in vitro antifungal activity of PCA. We then investigated its potential mechanism using physiological and biochemical assays, as well as integrated transcriptomic and metabolomic analyses. PCA showed effective antifungal activity against P. destructans (EC₅₀ = 32.9 μg/mL). Physiological and biochemical assays indicated that PCA perturbed cell wall organization and increased membrane permeability, leading to leakage of intracellular contents. It also induced oxidative stress, DNA damage, and apoptosis. Multi-omics integration revealed that PCA markedly perturbed cell wall and membrane metabolism, virulence factor expression, and energy metabolism. It provoked oxidative stress while downregulating genes involved in the cell cycle, DNA replication, and repair. Together, these findings delineate the inhibitory effects of PCA on P. destructans in vitro, provide initial mechanistic insights into its antifungal action, and suggest that PCA merits further evaluation as a possible component of environmentally compatible strategies for WNS management.

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 Pseudogymnoascus destructans , 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 P. destructans in vitro 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 P. destructans growth. Taken together, our study elucidates the antifungal potential of environmental VOCs and offers new insights and application prospects for preventing and controlling WNS. IMPORTANCE: White-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 Pseudogymnoascus destructans in vitro . 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.

Pseudogymnoascus destructans (Pd) invades the skin tissue of bats, leading to severe population declines. The skin microbiome plays a crucial role in protecting hosts from fungal infection and exhibits pronounced spatiotemporal dynamics in its structure and function. Meanwhile, metabolites derived from microbial communities reflect the host physiological state and participate in microbe–pathogen interactions. In this study, we investigated the spatiotemporal dynamics of skin bacterial communities and metabolites during hibernation in Rhinolophus ferrumequinum by integrating 16S rRNA sequencing with untargeted metabolomics and experimentally verified the antifungal effects of microbially derived potential metabolites against Pd. Our results revealed that the structure of the skin bacterial community varied significantly across sampling contexts, with its assembly primarily governed by stochastic processes. Bacterial diversity reached its lowest level during middle hibernation, accompanied by a simplified co-occurrence network dominated by cooperative or mutualistic interactions. Additionally, metabolomic analyses demonstrated systematic metabolic remodeling of bat skin across hibernation stages, marked by significant enrichment of multiple pathways closely involved in host antimicrobial defense. Furthermore, metabolite profiles differed across locations, and the abundance patterns of several metabolites were strongly correlated with Pd infection levels. Integrated analyses identified multiple metabolites that showed significant correlations with bacterial genera capable of synthesizing the corresponding compounds. In vitro validation confirmed that nine metabolites effectively inhibited the growth of Pd, among which melatonin exhibited the strongest antifungal activity. Collectively, this study reveals the dynamics of the skin microbiome and metabolites of R. ferrumequinum during hibernation, providing novel insights into the defensive role of skin-associated microbes and metabolites in maintaining population health and resilience against fungal pathogens.