Metabolomics and image interpolation reveal secondary metabolite regulation in co-cultures of endophytic Penicillium cataractarum and Sclerotinia sclerotiorum.

Competition among microorganisms can activate cryptic genes, leading to the production of secondary metabolites that play roles in microbial defense and communication. Co-culture is an efficient strategy to induce metabolite biosynthesis, modulate biosynthetic gene clusters, and change metabolite abundance, thereby providing insights into microbial interactions with phytopathogens and potential approaches for agricultural pest control research. In this study, we investigated the co-culture interaction between the endophytic fungus Penicillium cataractarum 5IP2F4 (PC) and the phytopathogen Sclerotinia sclerotiorum (SS) to evaluate co-cultivation-induced metabolic changes. Fungal strains were cultivated in triplicate under monoculture and co-culture conditions. Extracts were obtained from control monocultures of S. sclerotiorum (CTRL-SS, CTRL-SSSS) and P. cataractarum (CTRL-PC, CTRL-PCPC), and from co-cultures (CNF-SC, CNF-PC, CNF-SS, and CNF-SSPC). These extracts were analyzed by LC-DAD-MS/MS. The acquired data were processed (67 metabolomic features), and statistical analyses were performed. The data were also submitted to the GNPS platform for molecular networking and metabolite annotation, which was supported by comparison with spectral data reported in the literature. Images of S. sclerotiorum (SS) and P. cataractarum (PC) were processed, and the ion intensities of annotated compounds were interpolated onto the images, predicting the spatial distribution across the culture dishes. Principal component analysis (PCA) revealed clear separation between control and co-culture samples, indicating pronounced chemical changes induced by fungal-fungal interaction. Specifically, the co-culture samples showed increased production of dipeptides and fumisoquin A derivatives, including oxo-prolyl-proline (2), N,N-dimethyl[N'-(1-methylethyl)glycyl]-prolinamide (3), asparaginyl-fumisoquin A (4), fumisoquin A (6), prolyl-hydroxyproline (7), oxo-prolyl-leucine (8), and acetyl asparaginyl-fumisoquin A (9), whereas the cyclopeptide fungisporin A (10) exhibited a reduction in intensity compared to the control samples. These metabolites are associated with interaction-driven chemical responses and likely contribute to microbial defense and interspecies communication. In conclusion, our results demonstrate that the interaction between P. cataractarum and S. sclerotiorum selectively upregulates the biosynthesis of specific dipeptides and fumisoquin A derivatives, while suppressing fungisporin A. These concrete metabolic shifts, successfully mapped via spatial analysis, reveal the primary chemical countermeasures deployed during fungal warfare, establishing these specific metabolites as key targets for future functional characterization.