Abstract
Freshwater lignicolous fungi represent an underexplored reservoir of bioactive secondary metabolites, yet their chemical diversity and antimicrobial potential remain insufficiently characterized. In this study, we investigated the antibacterial and antibiofilm properties of two recently described species, Longipedicellata megafusiformis and Wicklowia fusiformispora, by integrating in vitro biological assays with high-resolution LC-QQQ-MS-based metabolomic profiling. Crude mycelial ethyl acetate (EtOAc) extracts of both fungi exhibited inhibitory activity against pathogenic bacteria, while L. megafusiformis additionally displayed measurable biofilm-preventive effects. Untargeted LC-MS annotation revealed 27 metabolites in L. megafusiformis and 33 in W. fusiformispora, spanning alkaloids, peptides, polyketides, terpenoid derivatives, saponins, and carotenoid-related pigments. Several annotated metabolites in L. megafusiformis have well-documented antibacterial activities, including glucosinalbin, distomadine B, agelasidine A, sugikurojin B, 6,8-diprenylorobol, septacidin, ambiguine isonitrile, bachitrocin C, epipachysamine D, spirilloxanthin, nodulisporic acid E, and anhydrorhodovibrin, providing a coherent chemical rationale for the observed antibacterial phenotype. Notably, compounds with validated antibiofilm activity were restricted to spiroleucidine-type scaffolds and carotenoid derivatives such as spirilloxanthin. Importantly, the present study does not assign antibiofilm activity to individual compounds, and the observed effects should be interpreted at the crude-extract level. The metabolome of W. fusiformispora was dominated by structurally distinct antibacterial metabolites, including betaine, bengamide E, aspidocarpine, cyanocycline B, jantithrem G, chrysosporide, ajudazol A, plakinamine F, and several saponin-type compounds. However, no metabolites with experimentally confirmed antibiofilm function were detected in this species. The combined datasets reveal two complementary antimicrobial chemotypes: one enriched in multi-scaffold antibacterial and antibiofilm constituents (L. megafusiformis), and another characterized by a broad yet exclusively antibacterial metabolome (W. fusiformispora). These findings highlight freshwater fungi as promising sources of chemically diverse antimicrobial agents and demonstrate the value of integrating untargeted metabolomics with classical bioassays as a foundation for future isolation-guided and mechanistic studies.