Antimicrobial activity of fungal extracts
From a total of 46 fungal extracts assayed, 32 extracts (69.6%) showed antimicrobial activity against at least one of the indicator pathogenic microbes tested (Tables 1 and 2). The antimicrobial activity of the same isolated fungal strain was significantly different (P < 0.05) when cultured on different media, but all of the antimicrobial activities were weaker than that of the positive control.
Of the endophytic fungi isolated from R. stylosa (25 isolates, Table 1), RM was determined to be more suitable for antibiotic production in fungal isolates than the other three media (P < 0.05). Of these, 13 strains cultured on RM (52%) exhibited antimicrobial activity using 1 mg/mL extracts, and among them, 7 strains had stronger inhibitory effects on MRSA with MIC values less than 0.5 mg/mL. HHL55 showed the broadest antimicrobial spectrum against four indicator test microorganisms, and CZA culture of HHL55 was found to show the most potent antimicrobial activity against PA with an MIC value of 0.031 mg/mL. Only 2 strains fermented on the GM displayed low inhibitory activity with an MIC value of 1 mg/mL. In addition, three culture media derived from HHL94, HHL64 and HHL82 showed inhibitory effects on three indicator microorganisms.
Of the endophytic fungi isolated from R. mucronata (21 isolates, Table 2), most of the fungal strains (15 isolates, 71.4%) exhibited antimicrobial activity. A high growth inhibition rate was detected from the fungal extracts cultured on CZA (10 isolates, 47.6%) and RM (10 isolates, 47.6%) in comparison with PDA (7 isolates, 33.3%) and GM (2 isolates, 9.5%) at the selected concentration of 1 mg/mL (P < 0.05). The RM of 9 isolates and CZA of 3 isolates showed strong inhibitory effects on MRSA with MIC values less than 0.5 mg/mL. Only the extract of HQD20 cultivated on GM inhibited the growth of MRSA and MA. Moreover, HQD6 and HQD5 displayed antimicrobial activity against the growth of four indicator microorganisms tested with MIC values ranging from 0.015 to 1 mg/mL.
Cytotoxicity of fungal extracts
The cytotoxic effects of R. stylosa and R. mucronata endophytic fungal extracts were tested against HeLa, A549 and HepG2 cells using an MTT colorimetric assay. The half inhibitory concentration (IC50) values are shown in Tables 3 and 4. The results are presented as the means ± standard deviation of experiments performed in triplicate. Doxorubicin was used as the positive control, and the IC50 values were 0.17 ± 0.01 nM, 8.6 ± 0.1 nM and 137 ± 0.9 nM for A549, HeLa and HepG2 cells, respectively, which were stronger than the cytotoxicity of the fungal extracts. Cell viability using different concentrations of these extracts after 24 h of treatment was determined (Figs. 1 and 2). The cytotoxicity of fungal extracts showed a dose-dependent relationship.
Antitumour activity of endophytic fungi from R. stylosa. (A–D) Shows that the fermentation products of endophytic fungi of R. stylosa have an inhibition rate for A549 tumour cells in different media, where (A) is obtained under dextrose agar (PDA) culture conditions, (B) is obtained under Czapek’s agar (CZA) culture conditions, (C) is obtained under rice medium (RM) culture conditions and (D) is obtained under grain medium (GM) culture conditions. (E–H) Shows that the fermentation products of endophytic fungi of R. stylosa have an inhibition rate for HeLa tumour cells in different media, where (E) is obtained under dextrose agar (PDA) culture conditions, (F) is obtained under Czapek’s agar (CZA) culture conditions, (G) is obtained under rice medium (RM) culture conditions and (H) is obtained under grain medium (GM) culture conditions. (I–K) shows that the fermentation products of endophytic fungi of R. stylosa have an inhibition rate for HepG2 tumour cells in different media, where (I) is results under dextrose agar (PDA) culture conditions, (J) is results under Czapek’s agar (CZA) culture conditions, and (K) is results under rice medium (RM) culture conditions.
Antitumour activity of endophytic fungi from R. mucronata. (A–D) Shows that the fermentation products of endophytic fungi of R. mucronata have an inhibition rate for A549 tumor cells in different media, where (A) is obtained under dextrose agar (PDA) culture conditions, (B) is obtained under Czapek’s agar (CZA) culture conditions, (C) is obtained under rice medium (RM) culture conditions and (D) is obtained under grain medium (GM) culture conditions. (E–H) Shows that the fermentation products of endophytic fungi of R. mucronata have an inhibition rate for HeLa tumor cells in different media, where (E) is obtained under dextrose agar (PDA) culture conditions, (F) is obtained under Czapek’s agar (CZA) culture conditions, (G) is obtained under rice medium (RM) culture conditions and (H) is obtained under grain medium (GM) culture conditions. (I–L) Shows that the fermentation products of endophytic fungi of R. mucronata have an inhibition rate for HepG2 tumor cells in different media, where (I) is results under Dextrose Agar (PDA) cultural condition, (J) is results under Czapek’s Agar (CZA) cultural condition, (K) is results under Rice Medium (RM) cultural condition, and (L) is results under Grain Medium (GM) cultural condition.
As shown in Table 3, of the endophytic fungi isolated from R. stylosa, extracts of 10 isolates (40%) tested were cytotoxic and exhibited a viability percentage of A549 cells ≤ 50%, with all of the Phomopsis sp. and 3 isolates (50% of the Pestalotiopsis sp.) showing antitumour activity, and the IC50 values of four extracts (HHL75, HHL46, HHL10 and HHL50) were lower than 100 μg/mL (IC50 values of 64.38 ± 3.40, 11.65 ± 0.34, 97.21 ± 1.36 and 16.31 ± 0.36 μg/mL, respectively), suggesting the cytotoxic potential of these four fungal isolates; 75% of them belong to Phomopsis sp. and Pestalotiopsis sp. (Fig. 1A–D). Extracts of 9 isolates (36%) showed cytotoxicity and displayed a HeLa cell viability percentage ≤ 50%. In particular, the HHL46, HHL82, HHL52 and HHL50 extracts showed the most significant cytotoxic effect, with IC50 values of 31.03 ± 1.21, 14.38 ± 1.84, 70.55 ± 1.37 and 73.18 ± 1.64, respectively, which were lower than 100 μg/mL. The four fermentation products that inhibited HeLa cells were all from Phomopsis sp. and Pestalotiopsis sp., which coincided with the inhibition of A549 cells (Fig. 1E–H). Extracts of 13 isolates (52%) showed cytotoxicity against HepG2 cells, among which 7 extracts (HHL61, HHL75, HHL46, HHL10, HHL52 and HHL50) could significantly suppress the proliferation of HepG2 cells with IC50 values below 100 μg/mL. Eight of the 13 isolates (61.54%) belong to Phomopsis sp. and Pestalotiopsis sp. (Fig. 1I–K). Phomopsis sp. and Pestalotiopsis sp. were the dominant fungi (41.67%) in R. stylosa, with 9 strains inhibiting at least one tumour cell, suggesting that these two fungal genera could have high potential as producers of antitumour compounds.
As shown in Table 4, of the endophytic fungi isolated from R. mucronata, extracts of 13 isolates (61.9%) displayed cytotoxic activity on A549 cells, 10 isolates (47.6%) on HeLa cells, and 10 isolates (47.6%) on HepG2 cells. Of these, 9 extracts (HQD83, HQD33, HQD28, HQD48, HQD41, HQD5, HQD1, HQD6 and HQD8) exhibited significant cytotoxicity against A549 cells with IC50 values less than 100 μg/mL (Fig. 2A–D). Four extracts (HQD83, HQD48, HQD5 and HQD20) showed strong cytotoxic effects against the HeLa cell line with IC50 values below 100 μg/mL (Fig. 2E–H). Seven extracts (HQD83, HQD28, HQD48, HQD41, HQD5, HQD6 and HQD57) were potent (IC50 < 100 μg/mL) against HepG2 cells (Fig. 2I–L). Diaporthe sp. and Pestalotiopsis sp. were dominant fungi (38.09%) in R. mucronata, with all of them displaying at least one tumour cell. However, the three isolates with the strongest ability to inhibit A549, HeLa and HepG2 cells were Fusarium verticillioides, Pestalotiopsis microspore and Eutypella scoparia, respectively, and none of them were Diaporthe sp.
In an attempt to promote antitumour substance production, four different media were adopted for fungal isolate cultivation to activate biosynthetic silencing of gene expression. We found that extracts of 1 isolate (HQD28) cultured on PDA, 1 isolate (HQD48) cultured on CZA, 3 isolates (HHL82, HQD52 and HQD6) cultured on RM and 4 isolates (HHL46, HQD48, HQD5 and HQD8) cultured on GM exhibited significant antiproliferative activity against at least one of the tested carcinoma cells with an IC50 < 20 μg/mL. GM culture of R. stylosa endophytic HHL46 and RM culture of HHL61 and HHL82 were most effective against A549, HeLa and HepG2 cells, with IC50 values of 14.38 ± 1.84 μg/mL, 23.17 ± 4.26 μg/mL and 14.38 ± 1.84 μg/mL, respectively. The significant difference analysis showed that the inhibition of HeLa cells by the products of endophytic fungi of R. stylosa cultured on RM was stronger than those cultured under the other three conditions (P < 0.05). No significant difference (P > 0.05) was observed between the cytotoxicity for A549 of samples from four cultural media having equal degrees at end of 24 h (Table 3). The extracts from CZA culture of R. mucronata endophytic HQD48 and RM culture of HQD20 and HQD6 exhibited cytotoxicity towards A549, HeLa and HepG2 cells, with IC50 values of 4.83 ± 1.61 μg/mL, 14.38 ± 1.84 μg/mL and 9.58 ± 0.01 μg/mL, respectively (Table 4). The genera Pestalotiopsis and Phomopsis were demonstrated to be rich sources of antitumour secondary metabolites. Notably, RM culture of HQD6 exhibited cytotoxic and antiproliferative effects against three tested cancer cell lines with IC50 values ranging from 9.58 to 14.99 μg/mL.
Profiling of bioactive metabolites by HPLC
According to the screening for antimicrobial activity and cytotoxicity, we found that 6 endophytic fungi showed strong bioactive abilities: HHL46, HHL50, HQD5, HQD6, HQD83 and HQD48. The difference significance analysis showed that RM was to be the best to produce active metabolites and the product diversity of endophytic fungi on RM was analyzed by HPLC in further. Figure 3 shows the chromatograms of the fermented products of these endophytic fungi at 254 nm. The HPLC analysis results provide a wealth of information, and the hydrophilic compounds were found in the first 10 min. Within 10 to 15 min, the moderately polar components and hydrophobic components were washed out. RM cultures of HQD5 and HQD6 showed more diverse secondary metabolites than other cultures.
HPLC chromatogram fermented endophytic fungi produced in rice medium (RM) fermentation. (A) Shows the chromatograms of fermented product grown on the RM at 254 nm for 2 endophytic fungi of R. stylosa (HHL46 and HHL50) with strong bioactive abilities and the extraction of RM as control. (B) Shows the chromatograms of fermented product grown on the RM at 254 nm for 4 endophytic fungi of R. mucronata (HQD5, HQD6, HQD48 and HQD83) with strong bioactive abilities and the extraction of RM as control. Among them CK means, control check, the result of HPLC from the extraction of rice medium.
