Signaling in the gas phase: comparative volatile profiling of biocontrol Trichoderma spp. and aflatoxin-producing Aspergillus spp. in isolation and co-culture using HS-SPME GC-MS.

INTRODUCTION

Food contamination by aflatoxin-producing Aspergillus spp. remains a major food safety and public health concern. Trichoderma spp. and their metabolites are promising biocontrol agents, yet the volatile organic compound (VOC)-mediated interactions underlying growth inhibition of Aspergillus spp. are not fully understood.

METHODS

We applied untargeted metabolomics using Headspace Solid Phase Microextraction Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) and multivariate analyses to profile VOCs from three Trichoderma strains (T. asperellum BTU, T. virens OSK13, T. harzianum OSK34) in monoculture and in co-culture with Aspergillus flavus NRRL 3357 and A. parasiticus B62. Principal component analysis (PCA), ANOVA, PLS-DA, and Spearman correlation were used to characterize VOC profiles and identify discriminant compounds.

RESULTS

Co-cultures produced 30 VOCs across 9-12 chemical classes, dominated by sesquiterpenes, alcohols, and ketones. Among the Trichoderma strains, BTU (against A. flavus 3357) and OSK34 (against A. parasiticus B62) showed the greatest VOC abundance and diversity. PCA confirmed Trichoderma-driven changes in VOC profiles during co-cultures, with OSK13 and OSK34 sharing greater VOC profile similarity compared to BTU. In addition, Trichoderma spp. produced both species-specific and shared VOCs, with some also shared with Aspergillus spp. ANOVA further identified significant upregulation and downregulation of VOCs during co-culture. Finally, PLS-DA, ANOVA, and Spearman correlation identified four key discriminant VOCs: 6-pentyl-2H-pyran-2-one, 2-pentylfuran, 1-decanol, and 1-ethenyl-4-methoxybenzene. These VOCs were most abundant in BTU-Aspergillus co-cultures with observed antifungal activity.

DISCUSSION: These findings provide insight into potential mechanisms of Aspergillus growth inhibition by Trichoderma spp. through comparison of monocultures to co-cultures. In addition, the four identified key discriminant VOCs make promising VOC targets for selecting Trichoderma strains for biocontrol of toxigenic Aspergillus, particularly A. flavus. Together, this study establishes a metabolomic framework for VOC-mediated biocontrol and a basis for optimizing Trichoderma selection for biocontrol purposes. Future research should validate the bioactivity roles of these key VOCs and their effect on aflatoxin production, test their antifungal mechanisms, examine synergistic effects, and assess environmental influences to strengthen metabolomics-informed biocontrol strategies for reducing aflatoxin contamination in food systems.