1H NMR-based profiling and candidate prioritization of interaction-associated metabolites from fungal co-cultures

Abstract

Fungal communities mediate antagonism and emergent, interaction-specific inhibition through a dense chemical dialogue, yet linking ecological phenotypes to specific metabolites remains a central challenge in natural product discovery. Conventional bioassay-guided fractionation is iterative and often biased toward abundant constituents. We addressed this challenge using a consortium of protease-rich, fruit-derived fungi ( Neofusicoccum ribis , Fusarium sacchari and F. verticillioides ). A hypothesis-generating metabolomics workflow was applied, coupling controlled axenic, pairwise and three-way co-culture phenotyping with 1 H NMR-guided orthogonal projections to latent structures-discriminant analysis (OPLS-DA). An exploratory OPLS-DA model compared the 1 H NMR spectra of bioactive crude extracts with those of 31 purified metabolites to rank compounds by spectral similarity as a hypothesis-generating prioritization strategy. The five highest-ranked compounds were then screened in a preliminary antifungal assay at a single concentration of 50 μg/mL. Potent allelopathy of axenic F. verticillioides (V18) (∼70% inhibition against V6) was significantly attenuated in the V6/V18 duo co-culture (adjusted p < 0.0001, Cohen’s d = 3.1), whereas a unique three-way (trio) emergent inhibition arose against an otherwise resistant competitor. The trio culture produced the highest biomass, and the endophyte (V6) grew substantially faster than either pathogen. The OPLS-DA model (Q 2 = 0.748) prioritized several candidates, including a constitutively produced isocoumarin (Compound 5) and Compound 27 [7-hydroxy-3-(hydroxymethyl)-5-methyl-1H-isochromen-1-one], which was isolated exclusively from the trio co-culture—the only condition that inhibited F. sacchari . Preliminary testing confirmed statistically significant activity of the five highest-ranked compounds against at least one target fungus (Table S5). Compound 27 showed moderate activity against F. sacchari (20.9% inhibition), while Compounds 11, 5 and 6 exhibited comparable or higher activity against F. verticillioides . These findings generate chemically explicit hypotheses and provide initial experimental support for the prioritization workflow, but they do not establish causality or confirmed potency of individual compounds. Emergent bioactivities in this system are best interpreted as the combined outcome of ecological competition and interaction-triggered secondary metabolism. Dose-response assays are required before functional relevance can be claimed.