Fight fire with fire: Endophytic fungi as biocontrol agents against the Plane tree canker stain (Ceratocystis platani).

Ceratocystis platani, an invasive vascular phytopathogen causing canker stain disease, poses a major threat to Platanus × acerifolia populations, particularly in urban environments across Europe. Fifty-three fungal strains were isolated from different plant tissues collected on asymptomatic Platanus x acerifolia, across two seasons. ITS-based phylogenetic analysis revealed 18 genera distributed across 16 families, mainly belonging to Pleosporales and Xylariales orders. Several taxa included known plane tree-associated pathogens as well as ubiquitous endophytes with documented biocontrol potential. Dual-culture assays against C. platani revealed diverse antagonistic outcomes, including distance inhibition, contact inhibition and overgrowth. Eight strains corresponding to eight different species were selected for in-depth metabolomic profiling using LC-HRMS/MS: Biscogniauxia mediterranea, B. nummularia, Paraconiothyrium brasiliense, Epicoccum nigrum/italicum, Diaporthe foeniculina, Sordaria fimicola, Alternaria sp. and Cytospora sp.. Chemometric analyses and molecular networking revealed strain-specific metabolic signatures and annotated metabolites significantly upregulated during fungal confrontation. Co-cultivation triggered extensive metabolic reprogramming, with several metabolites strongly correlated with pathogen growth inhibition. Notably, a putative dihydro-aryl-isoquinoline, structurally related to antifungal naphthalene-isoquinoline derivatives, was annotated. This study highlights the ecological complexity of endophyte–pathogen interactions and underscores the value of integrative metabolomic approaches for uncovering cryptic chemical crosstalk and identifying candidate biocontrol metabolites within tree-associated microbiomes.