Diversity, Isolation and Bioactive Secondary Metabolites of Endophytic Fungi in Duchesnea indica (Andr.) Focke.

Endophytic fungi in medicinal plants serve as important microbial resources, known to produce bioactive compounds resembling those of their host plants. Duchesnea indica, a traditional Chinese medicinal herb with centuries of application, demonstrates multiple therapeutic properties including detumescence, detoxification, antimicrobial and anticancer effects. Despite its medicinal value, the diversity and community composition of endophytic fungi in this plant species remain insufficiently characterized. This study comprehensively examined the diversity, biological activities and secondary metabolites of endophytic fungi across different tissues (roots, stems, leaves and fruits) of D. indica collected from Hangzhou during April 2023, employing both culture-dependent and culture-independent methodologies. High-throughput Illumina sequencing of the ITS1 region (culture-independent methodology) established Ascomycota and Basidiomycota as the dominant fungal phyla colonizing D. indica. Root tissues displayed the highest fungal abundance and diversity, whereas stems showed the most pronounced tissue specificity. Through culture-dependent methods, 42 fungal strains were successfully isolated, with genomic DNA extracted using commercial kits and subsequently analyzed through ITS sequencing, revealing classification into 9 genera. Antimicrobial evaluation via Oxford cup assays identified 22 fungal strains exhibiting inhibitory activity against Staphylococcus aureus and 12 strains effective against Escherichia coli. CCK-8 cytotoxicity assays demonstrated significant cytotoxic potential among most isolates, with strain PS4 (Aspergillus sp.) showing particularly strong antimicrobial and cytotoxic properties. GC-MS analysis of PS4's ethyl acetate extract detected 33 bioactive constituents. Notably, Colletotrichum and Cercospora appeared potentially involved in enhancing triterpene content in D. indica, offering novel perspectives for further investigation of endophytic fungi in medicinal plants.