From Contact to Stalemate: MAPK-Associated Chemical and Enzymatic Defenses Shape a Stable Barrage in the Co-Culture of Trametes sp. D and Aspergillus niger L14

The co-culture between Trametes sp. D and Aspergillus niger L14 resulted in a distinct orange-brown antagonistic band at their interface. Direct hyphal contact was associated with markedly enhanced production of numerous secondary metabolites (SMs), some of which were absent or decreased in monocultures. T. sp. D induced indolic compounds and cyclic dipeptides, such as Indole-3-acetamide and Cyclo-(Pro-Phe), whereas A. niger L14 overproduced polyketide-derived pigments and organic acids, such as Fonsecin and Kojic acid. These SMs did not inhibit their producer but suppressed the opponent’s growth, indicating reciprocal chemical antagonism. Transcriptomic analysis revealed upregulation of stress-related and metabolic genes, consistent with each fungus activating defense pathways. Biochemical assays showed that the confrontation zone had the highest oxidative stress markers, cell wall-degrading enzyme activity, and acidification (notably by A. niger L14), reflecting intense interfungal antagonism. The stress-response mitogen-activated protein kinase (MAPK) pathway was also activated in both fungi. Our findings supported a mechanistic model of fungal competition involving direct contact, chemical exchange, enzymatic attack, and stress signaling, highlighting that physical interactions likely contributed to triggering cryptic secondary metabolism and robust defense responses.