Volatile organic compounds associated with red-stripe pathogens inhibit mycelial growth of <i>Morchella sextelata</i> by disrupting membrane integrity and inducing oxidative stress

ABSTRACT The commercial cultivation of Morchella sextelata is severely threatened by red-stripe disease, caused by the pathogenic bacteria, with Bacillus subtilis M-5 and Pseudomonas chlororaphis subsp. aureofaciens M-B being among the causative agents. This study revealed that volatile organic compounds (VOCs) emitted by these pathogens completely inhibited the mycelial growth of M. sextelata , with scanning electron microscopy revealing severe hyphal shrinkage and collapse. Headspace-gas chromatography-ion mobility spectrometry identified 2,3-butanedione and 2-methylpropanoic acid as the predominant antifungal VOCs from strains M-B and M-5, respectively. Exogenous application confirmed their potent inhibitory activity, with minimum inhibitory concentrations (MIC) of 150 µL/mL and 250 µL/mL. Mechanistic investigations demonstrated that these VOCs, particularly 2,3-butanedione and 2-methylpropanoic acid, disrupt cell membrane integrity, leading to significant leakage of intracellular nucleic acids and proteins, inducing lipid peroxidation, and triggering an antioxidant defense response characterized by elevated superoxide dismutase and peroxidase activities. Our findings elucidate that bacterial VOCs are volatile pathogenic factors responsible for growth arrest in red-stripe disease, providing a crucial theoretical foundation for developing novel management strategies for this devastating disease. IMPORTANCE The cultivation of morels, particularly Morchella sextelata , is significantly constrained by the emergent red-stripe disease, yet effective and environmentally friendly control strategies are lacking. This study reveals a previously underappreciated mechanism of pathogenesis: VOCs produced by the causative bacteria, Bacillus subtilis M-5 and Pseudomonas chlororaphis subsp. aureofaciens M-B. We identify 2,3-butanedione and 2-methylpropanoic acid as key antifungal VOCs that exerts its effect by critically damaging hyphal membrane integrity, inducing the leakage of cellular contents, and disrupting redox homeostasis. These findings shift the conventional perspective on bacterial-fungal interactions in morel disease and highlight the critical role of pathogen-derived volatiles. Consequently, our work provides a direct theoretical foundation for optimizing ventilation management in greenhouse cultivation to disperse these harmful VOCs. This insight is pivotal for developing novel, green prevention tactics and ensuring the sustainable and productive cultivation of morels.