Novel Endophytic Fungi from Euchresta tubulosa Dunn: Characterization of Their Bioactive Secondary Metabolites and Extracellular Enzymes

The endangered ethnomedicinal plant <i>Euchresta tubulosa</i> harbors a valuable community of endophytic fungi, demonstrating significant potential for biotechnological applications. Endophytic fungi were isolated from <i>E. tubulosa</i> and identified to characterize their secondary metabolites and extracellular enzyme activities. Endophytic fungi were isolated from <i>E. tubulosa</i> using tissue explant culture and identified by morphological and molecular (ITS) analysis. The chemical profiles of strain fermentation products were analyzed by LC-MS/MS, while extracellular enzyme production (cellulase, protease, amylase) was assessed through chromogenic plate assays and liquid fermentation. The results indicated that a total of 55 endophytic fungi were isolated from <i>E. tubulosa</i>, assigned to 17 genera. Among these, three genera (<i>Colletotrichum</i>, <i>Fusarium</i>, and <i>Penicillium</i>) constituted the dominant groups, while four strains (including three novel species) represented potential new taxa. LC-MS/MS analysis revealed that fermentation products of the three novel endophytic fungal species contained bioactive compounds such as flavonoids and alkaloids; furthermore, bioactivity assays indicate that they exhibited significant degrees of antibacterial and antioxidant activity. Extracellular enzyme assays demonstrated that three <i>E. tubulosa</i>-derived endophytic strains exhibited multi-hydrolytic enzyme production capabilities. Notably, strain ETG-1-2-1 showed the highest amylase and cellulase activities (10.95 U/mL and 9.68 U/mL, respectively), while strain ETXG-1-1-1 displayed the highest protease activity (2.34 U/mL). This study provides the first systematic report on the diversity of endophytic fungi in <i>E. tubulosa</i>, their secondary metabolite profiles, and extracellular enzyme activities, establishing a theoretical foundation for discovering novel bioactive compounds and developing microbial resources, while also highlighting their ecological roles and biotechnological potential.