Unraveling the antagonistic mechanisms of Hanseniaspora volatile organic compounds against Botrytis cinerea, a causal agent of postharvest decay

• Hanseniaspora spp. VOCs show highly strain-specific antagonistic activity. • Hva9, Hoc16, and Hsi23 are outstanding biocontrol candidates against B. cinerea . • Potent antagonism is linked to specific organic acids and alcohols. • Top strains trigger distinct molecular stress responses in B. cinerea . Yeast of the genus Hanseniaspora , through their production of volatile organic compounds (VOCs) are promising biocontrol agents against Botrytis cinerea , although strain-specific mechanisms remain poorly understood. In this study, 25 Hanseniaspora strains were screened for antagonistic activity using a multi-parametric approach that integrated physiological impacts (growth, sporulation) with cellular (glycerol, ergosterol) and molecular stress responses (HOG and Cell Wall Integrity pathways via Hog1 and Rho1 genes) in B. cinerea . The volatilome was characterised by GC–MS, and multivariate and correlation analyses were used to link VOC profiles to antagonistic efficacy. Results confirmed that antagonistic activity is highly strain-specific. Three strains, Hanseniaspora valbyensis 9, H. occidentalis 16, and H. singularis 23, emerged as the most potent antagonists. Crucially, these strains exhibited distinct mechanisms of action, inducing diverse and sometimes paradoxical stress responses in B. cinerea . These included a classic osmotic and cell wall stress response (upregulation of Hog1 and Rho1 ), a ’signalling paralysis’ characterized by the downregulation of stress-response pathways, and an uncoupling of Hog1 transcription from its metabolic output. The volatilome was dominated by esters and alcohols, but principal component analysis revealed that antagonistic efficacy was not driven by the quantity of VOCs but by the quality of the blend, with organic acids and branched-chain alcohols being characteristic of the most effective strains. This study provides a novel insight into the complex chemical and molecular interactions in yeast-mould antagonism. By linking VOC profiles to physiological and genetic responses, our findings lay the groundwork for rational selection of biocontrol agents, balancing high antagonistic efficacy with the sensory compatibility for target food applications.