Purpose An endangered Tsuga chinensis (Franch.) Pritz. is widely used as a natural medicinal herb in many countries, but little has been reported on its culturable endophytic fungi capable of producing secondary metabolites applied in modern medicine and pharmacy. The present study aimed to evaluate the distribution of fungal endophytes and their cytotoxic and antioxidant properties. Methods This study used the surface sterilization method to isolate endophytic fungi which were then identified using morphological characteristics and ITS sequence analysis. The antimicrobial and cytotoxic potentials of fungal ethyl acetate extracts were evaluated by the minimum inhibitory concentration (MIC) and sulforhodamine B (SRB) assays, respectively. Paclitaxel-producing fungi were primarily screened using PCR-based molecular markers. Additionally, biochemical assays were used to reveal the antioxidant potencies of selected strains. Results A total of sixteen endophytic fungi that belonged to 7 known and 1 unknown genera were isolated from T. chinensis. The greatest number of endophytes was found in leaves (50%), followed by stems (31.3%) and roots (18.7%). Out of 16 fungal strains, 33.3% of fungal extracts showed significant antimicrobial activities against at least 4 pathogens with inhibition zones ranging from 11.0 ± 0.4 to 25.8 ± 0.6 mm. The most prominent cytotoxicity against A549 and MCF7 cell lines (IC50 value < 92.4 μg/mL) was observed in Penicillium sp. SDF4, Penicillium sp. SDF5, Aspergillus sp. SDF8, and Aspergillus sp. SDF17. Out of three key genes (dbat, bapt, ts) involved in paclitaxel biosynthesis, strains SDF4, SDF8, and SDF17 gave one or two positive hits, holding the potential for producing the billion-dollar anticancer drug paclitaxel. Furthermore, four bioactive strains also displayed remarkable and wide-range antioxidant activity against DPPH, hydroxyl radical, and superoxide anion, which was in relation to the high content of flavonoids and polyphenols detected. Conclusion The present study exploited for the first time fungal endophytes from T. chinensis as a promising source for the discovery of new bioactive compounds or leads for the new drug candidates.

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