Dereplication-Guided Isolation of 3- <i>O</i> -Methylfunicone from Endophytic Fungal Co-culture and Its Inhibitory Effect on <i>Phytophthora palmivora</i>

<i>Phytophthora palmivora</i> is a destructive pathogen infecting more than 200 plant species, including cocoa, coconut, and papaya. Endophytic fungal co-cultures provide a promising strategy to activate silent biosynthetic pathways and generate bioactive metabolites for crop protection, as endophytes naturally compete for limited resources through the production of specialized metabolites. In this study, we examined the co-culture of <i>Aspergillus pseudonomiae</i> J1 and <i>Talaromyces pinophilus</i> J6, isolated as endophytes from <i>Euphorbia umbellata</i> leaves. Ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) data analysis, processed with MS-DIAL and supported by SIRIUS 5 and manual fragmentation proposition, enabled the annotation of quinone, phenylpropanoid, iridoid, and funicone-type natural products. Guided by this analysis, 3-<i>O</i>-methylfunicone was isolated, and its chemical structure was confirmed by Nuclear Magnetic Resonance (NMR) and High-Resolution Mass Spectrometry (HRMS) data analysis. Bioassays demonstrated its antifungal activity against <i>P. palmivora</i>, inhibiting mycelial growth by 54.9 ± 6.1, 30.9 ± 5.1, and 15.6 ± 6.2% at concentrations of 1.0, 0.5, and 0.25 mM, respectively. Our findings show that metabolomics combined with dereplication offers a powerful strategy to optimize the discovery of bioactive metabolites from endophyte co-cultures.