Culture conditions and microbial interactions shape VOC profiles and reveal 6-pentyl-α-pyrone and 2-oxindole as modulators of Colletotrichum gloeosporioides growth and pathogenicity.

Colletotrichum gloeosporioides, the causal agent of anthracnose, significantly reduces the productivity of high-value crops, and volatile organic compounds (VOCs) are gaining attention as a disease-management approach. This study assessed how nutrient sources and microbial interaction systems shape VOC-mediated antagonism against C. gloeosporioides. Culture medium strongly affected antifungal performance, with Luria-Bertani (LB) promoting up to a 4.28-fold increase in inhibition compared with Potato Dextrose Agar (PDA). Interaction systems also shaped antagonism: bipartite systems generated up to 1.31-fold stronger inhibition than tripartite systems. Volatilome profiling of bipartite interactions involving Trichoderma strains (T1, T2, T3) and Bacillus subtilis (Bs) revealed distinct VOC signatures. Isopropyl alcohol, 6-pentyl-α-pyrone (6PP), 2-oxindole (2OI), dimethyl disulfide, 2-pentylfuran, and α-phellandrene were assessed individually; 6PP and 2OI showed the strongest inhibition, reaching 43%. While 6PP is a well-established antifungal VOC, 2OI has not been previously reported as a natural antifungal volatile metabolite, making this study the first to provide functional evidence of its inhibitory role. Combined application of 6PP and 2OI at 125 µM each maintained inhibition, suggesting a concentration-sparing effect, whereas half-concentrations produced additive inhibition, comparable to that of the individual treatments. In avocado leaf assays, low VOC concentrations increased disease severity despite reducing in vitro growth, whereas higher concentrations reduced lesion development and colonization by 30-50%. At 1000 µM, 2OI significantly inhibited hyphal growth and sporulation. These findings demonstrate that media composition and microbial interactions modulate VOC production and that specific VOC can suppress fungal growth and pathogenicity, highlighting their potential for sustainable anthracnose management.