3.1 NaBr supplementation enhances antibacterial activity and metabolite diversity in T. virens

Since the isolated strain originated from a marine environment, it is likely to produce diverse secondary metabolites upon culture under halogen salt stress. The growth of the marine-derived T. virens strain B211 was investigated in response to the addition of three different halogen salts on solid (agar) culture conditions. Supplementation with 3% halogen salts altered the pigmentation of B211 colonies (Fig. 1A). When NaBr was added, B211 colonies secreted a darker brown pigment (Fig. 1A). Fungal growth was not significantly affected by the addition of NaCl and NaBr; however, KI supplementation resulted in a slower growth rate without complete inhibition (data not shown).

Fig. 1

Effects of halogen salt supplementation on Trichoderma virens B211. A Colony morphology on potato dextrose agar supplemented with 3% halogen salts (NaCl, NaBr, and KI) versus control (CK). B Antibacterial activity of ethyl acetate extracts from agar (AE) and liquid (LE) cultures against Erwinia amylovora, with or without 3% halogen salts. Data are shown as mean ± SD. Different letters above the bars indicate statistically significant differences (Student t-test; *P < 0.05; **, P < 0.01; and ***, P < 0.001). C HPLC chromatograms of secondary metabolites under different culture conditions, showing that NaBr supplementation markedly increased metabolite diversity, especially in liquid culture

Ethyl acetate extracts obtained from agar and liquid cultures (AEs and LEs) were evaluated for their antibacterial activity against E. amylovora. As shown in Fig. 1B, when cultured with the addition of NaBr, the activity of AE and LE was significantly increased. In contrast, addition of NaCl decreased the activity of AE and LE compared to CK (without halogen salt). The addition of KI severely impaired antibacterial activity (Fig. 1B). Overall, LEs completely inhibited the growth of E. amylovora at lower concentrations (MICs of 25–50 μg/mL) than AEs (MICs of 100 μg/mL). In particular, the LE from NaBr-containing PDB medium showed the most potent activity against E. amylovora (MIC = 12.5 μg/mL). HPLC analysis revealed differences in the production of secondary metabolites between the solid and liquid cultures (Fig. 1C). When cultured with 3% NaBr supplementation, B211 produced a variety of secondary metabolites, which was particularly maximized under liquid culture conditions (Fig. 1C). When the effects of NaBr concentration on activity were investigated, LEs of PDB supplemented with 1%, 3%, and 6% NaBr showed MIC values of 15.6, 12.5, and 32 µg/mL, respectively (Table S2). HPLC analysis further revealed that increasing NaBr supplementation in the liquid medium led to greater diversification of metabolites in the LEs (Fig. S2).

The enhanced antibacterial activity observed in NaBr-supplemented cultures correlated well with pronounced changes in metabolite profiles, particularly under liquid culture conditions. Liquid extracts consistently exhibited stronger activity against E. amylovora than agar extracts, indicating that submerged fermentation favors the biosynthesis or accumulation of bioactive metabolites in B211. Notably, while increasing NaBr concentrations promoted greater chemical diversification, maximal antibacterial activity was achieved at an intermediate supplementation level (3%), suggesting that optimal bioactivity depends not only on metabolite diversity but also on the relative abundance and composition of active constituents.

3.2 Isolation and identification of secondary metabolites from T. virens

From large-scale cultures grown on PDA (6 kg) and PDB (8 L) supplemented with 3% NaBr, fourteen compounds (1–14) were purified (Figs. S1 and S3). Spectroscopic analyses and comparison with literature data identified two new metabolites (1 and 5) and twelve known ones, including nodulisporiviridin G (2), viridin (3), β-viridin (4), 1β-hydroxy-2α-hydroasterogynin A (6), asterogynin A (7), gliotoxin (8), bisdethiobis(methylthio)gliotoxin (9), (3S)−2,3-dihydro-6-hydroxy-3-(hydroxymethyl)−2-methylpyrazino[1,2-α]indole-1,4-dione (10), (±)−2,3-dihydro-3-(hydroxymethyl)−2-methylpyrazino[1,2-α]indole-1,4-dione (11), hydroheptelidic acid (12), xylaric acid B (13), and 5-hydroxy-3-hydroxymethyl-2-methyl-7-methoxychromone (14) [21,22,23,24,25,26,27,28,29,30,31]. The chemical structures of 1–14 are shown in Fig. 2.

Fig. 2

Chemical structures of compounds 1–14 isolated and identified from Trichoderma virens B211 under culture conditions supplemented with 3% NaBr. Compounds 1–8 were obtained from liquid culture, whereas compounds 8–14 were derived from agar culture. Notably, compound 8 was isolated from both liquid and agar cultures

Compound 1 was obtained as white wax. The HRESIMS analysis (Fig. S4) revealed quasi-molecular ions at m/z 341.1391[M + H]+ and 363.1207 [M + Na]+ (calculated m/z 341.1389 and 363.1208 for C20H21O5 and C20H20O5Na, respectively), indicating the molecular formula of C20H20O5 with eleven degrees of unsaturation. The 1H and 13C NMR spectra, along with COSY and HSQC spectra of 1 (Figs. S5–S8), revealed the presence of two methyls, five aliphatic methylenes, an aliphatic methine, an olefinic methine, an oxygenated methine, and ten quaternary carbons, including two aliphatic quaternary carbons at δC 37.9 and 51.0 ppm; five sp2 at δC 122.1, 132.9, 145.5, 146.7, and 160.9 ppm; and three ketone carbons at δC 174.0, 191.5, and 221.3 ppm (Table 1).

Table 1 1H and 13C NMR data of compounds 1 and 5 measured at 400 MHz

The structure of compound 1 was established using 1H–13C HMBC and 1H–1H COSY spectra (Figs. 3A, S7, and S9). COSY correlations of H-1/H-2, H-11/H-12, and H-14/H-15/H-16 defined partial fragments. The structures of rings A and E were confirmed by HMBC correlations from H-1 to C-2, C-3, and C-19; from H-2 to C-1, C-3, and C-4; from H-19 to C-1, C-5, and C-10; and from H-20 to C-3, C-4, C-5, and C-6 (Fig. 3A). The structures of the rings C and D were supported by correlations from H-11 to C-12; from H-12 to C-11, C-13, C-14, C-17, and C-18; from H-14 to C-8, C-15, and C-18; from H-15 to C-8, C-16, and C-17; from H-16 to C-17; and from H-18 to C-12, C-13, C-14, and C-17 (Fig. 3A). Correlations from H-1 and H-19 to C-9 and from H-11 to C-10 indicated that rings A and C are connected through the C-9/C-10 bond (Fig. 3A). Weak correlations from H-11 and H-20 to C-7 suggested that rings E and C ring are linked through the ketone carbon C-7, forming the B ring and completing the planar structure of compound 1 in agreement with the degree of unsaturation (Fig. 3A).

Fig. 3

Structure determination of compound 1 by NMR and ECD analyses. A Key COSY (bold lines), HMBC (blue solid arrows), and NOESY (black dashed arrows) NMR correlations. B ECD spectra of compound 1 and nodulisporiviridin G (2)

The relative stereochemistry of 1 was determined from NOESY experiments. Because of overlapping signals in the 1H NMR spectrum, 1D-NOESY was employed to observe correlations between irradiated and enhanced signals. Irradiation of the C-18 methyl protons enhanced the H-11β, H-15β, H-16β, and H-19, while irradiation of the oxygenated proton H-12 enhanced the H-11α and H-14 (Figs. 3A and S10). These data indicated that H-11α, H-12, and H-14 are oriented in the same direction, whereas protons H-11β, H-15β, H-16β, H-18, and H-19 are oriented oppositely, similar to the pattern observed in compound 2 (Fig. S10). Thus, the relative stereochemistry of compound 1 was assigned as 10R*,12R*,13R*,14S* (or the enantiomeric configuration) (Fig. 3A).

Given the similarity of the planar structure and relative configuration of compound 1 to nodulisporiviridins G (2) and H, the absolute configuration was further determined by ECD spectroscopy (Fig. 3B). The ECD spectrum of compound 1 exhibited negative Cotton effects (CEs) at 220 and 260 nm and positive CE at 310 nm, closely matching those of nodulisporiviridins G (2) and H [21]. Accordingly, the absolute configuration of the new compound 1 was established as 10R,12R,13R,14S, and it was named neoviridin.

Compound 5 was obtained as yellow wax. The HRESIMS analysis (Fig. S11) showed a quasi-molecular ion at m/z 329.1020 [M + H]+ and 351.0841 [M + Na]+ (calculated m/z 329.1026 and 351.0845 for C18H17O6 and C18H16O6Na, respectively), indicating the molecular formula of C18H16O6 with eleven degrees of unsaturation. The 1H and 13C NMR spectra, along with COSY and HSQC spectra of compound 5 (Figs. S12–S15), revealed the presence of a methyl, a methoxy, two aliphatic methylenes, three olefinic methines, an oxygenated methine, and ten quaternary carbons, including two oxygenated quaternary carbons at δC 79.7 and 71.4 ppm; five sp2 at δC 127.2, 137.8, 155.6, 157.5, and 179.2 ppm; and three ketone carbons at δC 195.4, 202.7, and 205.9 ppm (Table 1).

The 1H–1H COSY correlations of H-10/H-11 and H-14/H-15 (Fig. 4A) defined partial fragments of compound 5. The connectivity of ring A was confirmed by HMBC correlations from H-1 to C-2, C-3, and C-9 and from H-3 to C-2, C-4, and C-9 (Fig. 4A). A methoxy group was assigned to C-3 based on the correlations from H-3 to C-18 and from H-18 to C-3. The structures of rings C and D were supported by HMBC correlations from H-10 to C-11, C-12, and C-6; from H-11 to C-7, C-13, and C-16; from H-14 to C-6, C-12, C-13, C-15, and C-16; and from H-15 to C-13, C-14, and C-16 (Fig. 4A). In addition, strong HMBC correlations were observed from H-1 to C-8; and H-10 to C-8; from H-3 to C-5; and from H-17 to C-7, C-8, and C-9, together with weaker correlations from H-10 to C-5 (Fig. S16). Integration of these data with the degree of unsaturation indicated that C-4 and C-6 are connected through the ketone carbon C-5, while C-7 and C-9 are linked via a quaternary carbon C-8, which carries a methyl group (C-17) and a hydroxy group. These features completed the B ring and established the planar structure of compound 5, as shown in Fig. 4A.

Fig. 4

Structure determination of compound 5 by NMR and ECD analyses. A Key COSY (bold lines) and HMBC (blue solid arrows) NMR correlations. B Experimental vs. calculated ECD spectra of 3R,4S,8S- and 3S,4R,8R- stereoisomers, confirming stereochemical assignment

The absolute configuration of compound 5 was determined through a combined analysis of ECD and DP4 + NMR chemical shift calculations. Considering the presence of three stereogenic centers, eight possible stereoisomers were generated: (3R,4R,8R)-, (3S,4S,8S)-, (3R,4R,8S)-, (3S,4S,8R)-, (3R,4S,8R)-, (3S,4R,8S)-, (3S,4R,8R)- and (3S,4R,8S)-5. were generated. Comparison of the calculated and experimental ECD spectra revealed that the (3S,4R,8R)-5 stereoisomer exhibited the closest resemblance to the observed curve, followed by (3S,4R,8S)-5 (Figs. 4B and S17). To further distinguish between these two possibilities, 1H and 13C NMR chemical shift calculations were performed. The calculated chemical shifts of (3S,4R,8R)-5 showed superior agreement with the experimental data compared to those of (3S,4R,8S)-5, as confirmed by DP4 + probability analysis (Table S4). Based on these combined computational and spectroscopic results, the absolute configuration of compound 5 was unambiguously established as 3S,4R,8R. The newly identified compound was subsequently named norviridin.

Compound 11 was obtained as white wax. The low-resolution ESIMS spectrum (Fig. S18) displayed a quasi-molecular ion at m/z 244.9 [M + H]+. The 1H NMR spectrum in DMSO-d6 (Fig. S19) showed five olefinic protons at δH 8.35 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.52 (ddd, J = 8.3, 7.2, 1.3 Hz, 1H), 7.41 (td, J = 7.7, 1.3 Hz, 1H), and 7.35 (s, 1H) ppm; three oxygenated protons at δH 4.53 (t, J = 2.3 Hz, 1H), 4.00 (dd, J = 11.6, 2.8 Hz, 1H), and 3.87 (dd, J = 11.6, 2.0 Hz, 1H) ppm; and a N-methyl protons at δH 3.04 (s, 3H) ppm. The 13C NMR and DEPT spectra of compound 11 (Fig. S19) revealed thirteen carbons, including one N-methyl carbon, one oxygenated methylene carbon, one aliphatic methine carbon, five olefinic methine carbons, and five quaternary carbons. The chemical shift of the olefinic protons and carbons supported the presence of a 1,2-disubstituted benzene ring. Two carbonyl carbons at δC 165.2 and 156.3 ppm indicated a diketopiperazine moiety. Comparison of the 13C NMR data of compound 11 with literature values (Table S5) led to its identification as (±)−2,3-dihydro-3-(hydroxymethyl)−2-methylpyrazino[1,2-α]indole-1,4-dione [29]. The specific rotation [α]D20 (c 0.1, methanol) was measured to be zero, indicating that compound 11 is a racemic mixture. To the best of our knowledge, this is the first report of compound 11 being isolated from a natural source.

3.3 Antimicrobial activity of compounds 1–14 against plant pathogens

The isolated compounds 1–14 were evaluated for their activity to inhibit the growth of plant pathogenic bacteria and fungi using the microdilution assay (Tables 2 and 3). Among the furanosteroids (1–4), viridin and β-viridin (3 and 4) exhibited broad-spectrum antibacterial activity with MICs of 0.4–50 μg/mL and antifungal activity with MICs of 25–100 μg/mL. Neoviridin (1) exhibited antifungal activity against A. brassicicola, C. coccodes, P. infestans (MICs = 25–50 μg/mL), whereas its structural isomer, nodulisporiviridin G (2), displayed antibacterial activity against A. citrulli and R. solanacearum (MICs = 50 μg/mL). Interestingly, most of steroid-like compounds 1–7, except for norviridin (5), were effective in inhibiting the growth of P. infestans (MICs = 0.4–200 μg/mL). Gliotoxin (8), an epipolythiodioxopiperazine featuring a 2,5-diketopiperazine ring system bridged by a transannular disulfide bond, displayed the most potent activity against all bacterial and fungal pathogens, with MIC values ranging from 0.8 to 25 μg/mL. In contrast, bisdethiobis(methylthio)gliotoxin (9), lacking the disulfide bridge, showed markedly reduced activity, being active only against E. amylovora and C. coccodes (MICs = 200 μg/mL). Other diketopiperazine derivatives (10 and 11) exhibited weak or no antifungal activity, with MICs of 200–400 μg/mL. Compounds 12–13 showed no activity against all tested plant pathogens at concentrations up to 400 μg/mL.

Table 2 Minimum inhibitory concentrations (MICs) of the isolated compounds 1–14 against plant pathogenic bacteriaTable 3 Minimum inhibitory concentrations (MICs) of the isolated compounds 1–14 against plant pathogenic fungi3.4 Disease control efficacy of extracts and gliotoxin derivatives against fire blight

To investigate the fire blight control efficacy, Chinese pearleaf crab apple plants were treated with two crude extracts (LE and AE) and their respective major active compounds gliotoxin (8) and bisdethiobis(methylthio)gliotoxin (9) prior to inoculation with E. amylovora (Fig. 5). At 8 days after inoculation, untreated plants (CK) showed severe fire blight symptoms, including necrosis of terminal buds, petioles, and leaves, while no symptoms were observed in the positive control treated with streptomycin sulfate (100 μg/mL). As shown in Fig. 5, plants treated with LE and AE extracts (500 μg/mL) displayed high levels of disease suppression, with control values of 93% and 88%, respectively. Gliotoxin (8) and bisdethiobis(methylthio)gliotoxin (9) achieved comparable disease suppression, with control values of 85% and 89% at lower concentration of 100 and 500 μg/mL, respectively. No phytotoxic effects were observed in any treated plants (Fig. 5).

Fig. 5

A Fire blight control efficacy of crude extracts (LE and AE, 500 μg/mL) and their major active compounds, gliotoxin (8) (20 and 100 μg/mL) and bisdethiobis(methylthio)gliotoxin (9) (100 and 500 μg/mL), against E. amylovora infection in Chinese pearleaf crab apple. Plants were inoculated with a cell suspension of E. amylovora (4 × 108 CFU/mL) 1–2 h after treatment. Treatment with the 0.025% Tween 20 solution served as the negative control (CK), while streptomycin sulfate (100 μg/mL) was used as the positive control (PC). Disease development was assessed at 8 days after inoculation. Bars represent the mean ± standard deviation of two runs with three replicates. The different letters above the bar graphs indicate significant differences (Tukey's HSD test; P < 0.05). B–D Representative images of apple plants treated with crude extracts and compounds