Fungicidal Activity of Secondary Metabolites from Trichoderma hamatum B-3

1. Introduction

The continuous growth of the global population exerts tremendous pressure on agricultural production systems. Concurrently, the food and economic losses caused by plant pathogens in agriculture have raised serious concerns [1,2]. Chemical control can mitigate plant diseases and enhance grain yield, making it one of the most effective methods for prevention and control. However, the frequent use of chemical pesticides can result in pesticide residues in crops; contaminate soil and water sources; as well as pose risks to human health [3,4]. In contrast, biological control involves managing plant diseases through microorganisms and their metabolites, offering a safer, more effective, and environmentally friendly alternative to chemical control [5]. Trichoderma strains are widely utilized in the biological control of plant diseases within agriculture, employing diverse mechanisms such as competition, antibiosis, induced systemic resistance and mycoparasitism [6,7,8,9].

Antibiosis is a primary biological control mechanism employed by Trichoderma, which involves the production of secondary metabolites that inhibit the growth of pathogenic fungi [10,11]. Trichoderma strains are known to produce a diverse array of natural products, including epipolythiodioxopiperazines [12], peptaibols [13], pyrones [14], butenolides [15], koninginins [16], steroids [17], lactones [18], and trichothecenes [19,20]. Our research team has been dedicated to the study of fungal secondary metabolites with bioactive properties over an extend period, resulting in the isolation of several previously undescribed compounds from fungal strains such as Chaetomium elatum [21], and Nigrospora sphaerica [22]. In this study, we investigated the secondary metabolites of Trichoderma hamatum, leading to the isolation of two new harziane diterpenes (1-2) and five cyclonerane sesquiterpenes (3-7). The structures of these compounds were determined by HRESIMS, NMR, ECD, Mosher's method [23] combined with chemical calculation. Compounds 1-10 were tested for their in vitro fungicidal activity against six agriculture pathogenic fungi. Compound 10 was then tested for in vivo fungicidal activities against some pathogenic fungi. Herein, we report the details of isolation, elucidation and fungicidal activity of above-mentioned secondary metabolites.

2. Materials and Methods

2.1. General Experimental Procedures

Optical rotation was measured on a Perkin-Elmer 241 Polarimeter (Perkin Elmer, Inc., Waltham, MA, USA). UV, CD and ICD spectra were measured on a Chirascan circular dichroism (Applied Photophysics Ltd., Leatherhead, UK). IR spectrum was acquired from an Agilent Cary 600 FT-IR infrared spectrometer (Agilent Technologies, Santa Clara, USA). Mass spectrum was obtained on an Ultra-performance liquid chromatography coupled with Q Exactive Quadrupole-Electrostatic field Orbital trap high resolution mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). NMR spectra were measured with a Bruker Ascend 600 MHz (Burker, Karlsruhe, Germany) with TMS as internal standard. Fractionation was conducted on a Column chromatography silica gel (200-300 mesh). Sample fractionated by dynamic axial compression column (Hanbon Sci.& Tech, Huaian, China). Purification was performed with a NP7000 preparative high performance liquid chromatograph (Hanbon Sci.& Tech, Huaian, China) with a kromasil C18 5 μm semi-preparative column (10×250 mm). Methanol, acetone, ethyl acetate are analytical pure and dimethyl sulfoxide (chromatographic pure) were purchased from Chengdu Kelong Chemical Co., Ltd. Dichloromethane (analytically pure), methanol (chromatography preparative pure) and acetonitrile (chromatography preparative pure) were purchased from Chengdu Jinshan Chemical Reagent Co., Ltd. Deuterated chloroform was purchased from Adamas Reagent Co., Ltd. N, N-dimethylformamide (analytically pure) was purchased from Tianjin Fuyu Fine Chemical Co. Ltd. Pyrimethanil and dimethachlon (analytically pure) were purchased from Alta Scientific Co., Ltd.

2.2. Fungal Material and Fermentation

The strain (GenBank accession no. OR553890, sequence no. NR134371.1) was isolated from the rhizomes of Bergenia purpurascens collected from Mount Emei and identified to be T. hamatum by Shanghai Sangong Bioengineering Co., Ltd. through morphological analysis and BLAST comparison.

Fungus 2 medium (maltose 20 g; monosodium glutamate 10 g; dextrose 10 g; yeast paste 3 g; corn syrup 1 g; mannitol 20 g; KH₂PO₄ 0.5 g; MgSO₄.7H₂O 0.3 g; CaCO₃ 20 g; 5-azacytidine 10 mg; distilled water 1 L) was used for the cultivation of T. hamatum b-3[24]. The experimental strains were inoculated onto potato dextrose agar (PDA) medium for duration of 5 days. Subsequently, five pieces (0.5×0.5 cm²) of mycelial agar plugs were transferred into sterilized and cooled Fungi No. 2 liquid medium contained in 500 mL conical flasks, with 200 mL medium per flask. These flasks were then placed in a constant temperature shaker set at 28 ℃ and 120 rpm/min for 7 days to obtain the T. hamatum seed cultures. Following this, the seed cultures were inoculated into the sterilized Fungus 2 medium for expansion cultures, with 2.5 mL of seed cultures added into each 100 mL of medium. The inoculated medium was cultured in a constant temperature shaker at 28 ℃ and 120 rpm/min for 14 days to produce the fermentation broth of T. hamatum b-3 resulting in a total culture volume of 564.4 L.

2.3. Extraction and Isolation

The cultured fermentation broth was centrifuged at 4000 r/min in a high-speed centrifuge to separate the fermentation broth. The broth was then treated with an equal volumn of ethyl acetate for 24 h. Subsequently, the extraction process was repeated three times using an equal volume of ethyl acetate. The ethyl acetate layer was collected and concentrated under reduced pressure, yielding 126 g of ethyl acetate extract.

The ethyl acetate extract was separated by silica gel CC, eluted with petroleum ether and acetone (30: 1~1: 1, V/V), yield seven fractions (Fr.1~Fr.7). Fr.1 was separated through column chromatography on dynamic axial compression column (MeOH/H₂O, 90: 10) and semi-pHPLC (MeOH/H₂O, 68: 32) to obtain 9 (11.5, t_R 20.3 min). Fr.2 was separated via RP-18 CC (MeOH/H₂O, 95:5) to afford three fractions (Fr.2.1~ Fr.2.3). Fr.2.1 was then separated through semi-pHPLC (MeOH/H₂O, 50: 50 to 100:0) to obtain eleven fractions (Fr.2.1.1~ Fr.2.1.11), Fr.2.1.5 was then separated through semi-pHPLC (MeOH/H₂O, 40: 60) to yield 10 (87 mg, t_R 25.8 min). Fr.2.1.6 was subjected to silica gel CC and semi-pHPLC (MeOH/H₂O, 70: 30) to yield 1 (1.8 mg, t_R 28.0 min). Fr.2.1.8 was separated via Sephadex LH-20 CC (CH₂Cl₂/MeOH, 70: 30) and purified by semi-pHPLC (MeCN/H₂O, 20: 80) to produce 8 (13.5 mg，t_R 30.3 min) and semi-pHPLC (MeCN/H₂O, 24: 76) to produce 4 (1.8 mg, t_R 19.7 min) and 5 (9.3 mg, t_R 26.6 min). Fr.3 was separated through dynamic axial compression column chromatography (MeOH/H₂O, 50:50 to 50:100) to give six fractions (Fr.3.1~ Fr.3.6), Fr.3.4 was further purified by CC on Sephadex LH-20 (MeOH) as well as semi-pHPLC (MeCN/H₂O, 57:43) to afford 3 (1.3 mg, t_R 8.3 min). Fr.3.4.1.10 was further purified by semi-pHPLC (MeCN/H₂O, 35: 65) to obtained 6 (1.4 mg, t_R 35.4 min). Fr.5 was separated via dynamic axial compression column chromatography (MeOH/H₂O, 60: 40 to 100: 0) to give five fractions (Fr.5.1~ Fr.5.5), Fr.5.2 was then purified by CC on Sephadex LH-20 (CH₂Cl₂/MeOH, 70:30) and semi-pHPLC (MeCN/H₂O, 40:60) to yield 7 (5.7 mg, t_R 19.4 min).

Compound 1: Colorless oil; ${[\alpha ]}_D^{20}$ = +81.8 (c = 0.011, MeOH); UV (MeOH) λ_max 248 (0.58); IR (KBr): 3449, 2920, 2851, 1740, 1384, 1234, 1026 cm^-1; CD (c 2.23 mM, MeOH) λ_max (Δε) 238 (-1.21), 295 (0.53), 342 (0.67) nm; HRESIMS m/z 359.2212 [M+H]⁺ (calcd. For C₂₂H₃₁O₄⁺, 359.2217); For ¹H-NMR, ¹³C-NMR data see Table 1 and Table 2.

Compound 2: Colorless oil; ${[\alpha ]}_D^{20}$ = +7.1 (c = 0.028, MeOH); UV (MeOH) λ_max 255 (0.48); IR (KBr): 3439, 2936, 1730, 1643, 1386, 1199 cm^-1; CD (c 2.51 mM, MeOH) λ_max (Δε) 222 (-0.31), 251 (-0.73), 299 (0.11), 343 (0.53) nm; HRESIMS m/z 319.2268 [M+H]⁺ (calcd. For C₂₀H₃₁O₃⁺, 319.2268); For ¹H-NMR, ¹³C-NMR data see Table 1 and Table 2.

Compound 3: Colorless oil; ${[\alpha ]}_D^{20}$ = -8.0 (c = 0.025, MeOH); UV (MeOH) λ_max 209 (4.32); IR (KBr): 3436, 2958, 2926, 2850, 1666, 1622, 1386 cm^-1; CD (c 3.05 mM, MeOH) λ_max (Δε) 224 (0.33), 321 (-0.12) nm; HRESIMS m/z 263.1624 [M+Na]⁺ (calcd. For C₁₄H₂₄NaO₃⁺, 263.1618); For ¹H-NMR, ¹³C-NMR data see Table 3 and Table 4.

Compound 4: Colorless oil; ${[\alpha ]}_D^{20}$ = -24.0 (c = 0.05, MeOH); IR (KBr): 3424, 2962, 2875, 1716, 1647, 1455, 1377, 918 cm^-1; HRESIMS m/z 279.1940 [M+Na]⁺ (calcd. For C₁₅H₂₈NaO₃⁺, 279.1931); For ¹H-NMR, ¹³C-NMR data see Table 3 and Table 4.

Compound 5: Colorless oil; [α]20 D -19.7 (c 0.20, MeOH); UV (MeOH) λ_max 213 (0.49); IR (KBr): 3402, 2966, 2931, 1458, 1377, 1156, 920 cm^-1; CD (c 2.94 mM, MeOH) λ_max (Δε) 216 (-0.05) nm; HRESIMS m/z 271.1910[M-H]^- (calcd. For C₁₅H₂₇O₄^-, 271.1914); For ¹H-NMR, ¹³C-NMR data see Table 3 and Table 4.

Compound 6: colorless oil; ${[\alpha ]}_D^{20}$ = -3.2 (c = 0.06, MeOH); IR (KBr): 3453, 2964, 2937, 1737, 1459, 1383, 1211, 1169 cm^-1; CD (c 1.62 mM, MeOH) λ_max (Δε) 205 (-0.21), 243 (-0.04), 307 (0.3) nm; HRESIMS m/z 393.2249 [M+Na]⁺ (calcd. For C₂₀H₃₄NaO₆⁺, 393.2248); For ¹H-NMR, ¹³C-NMR data see Table 1 and Table 2.

Compound 7: Colorless oil; ${[\alpha ]}_D^{20}$ = -4.9 (c = 0.14, MeOH); IR(KBr): 3456, 2968, 2936, 1738, 1438, 1379, 1217, 1163 cm^-1; CD (c 2.16 mM, MeOH) λ_max (Δε) 221 (0.05) nm; HRESIMS m/z 393.2252 [M+Na]⁺ (calcd. For C₂₀H₃₄NaO₆⁺, 393.2248); For ¹H-NMR, ¹³C-NMR data see Table 1 and Table 2.

Compound 8: Colorless oil; ${[\alpha ]}_D^{20}$ = -26.8 (c = 0.28, MeOH); IR (KBr): 3408, 2960, 2873, 1719, 1647, 1455, 1372, 921 cm^-1; HRESIMS m/z 279.1941 [M+Na]⁺ (calcd. For C₁₅H₂₈NaO₃⁺, 279.1931); For ¹H-NMR, ¹³C-NMR data see Table 3 and Table 4.

2.4. NMR and ECD Calculation Methods

The chemical calculations of compounds were conducted using Gaussian 16 ¹. Initially, a conformational analysis was performed with Conflex 8 to generate conformations via Boltzmann Jump [25]. All geometric configurations with relative energies between 0 and 5.0 kcal/mol were optimized at the B3LYP/6-31G level in the gas phase, as well as at ωB97XD/DGDZVP level in methanol. Room-temperature equilibrium populations were determined based on the Boltzmann distribution law [26]. Shielding tensor calculations were conducted at the PCM/mPW1PW91/6-31+G (d, p) level (with Boltzmann distribution ≥1%) employing the GIAO method [27]. The isotropic values of TMS were calculated at the same level and used as a reference. The DP4+ parameters were computed using the Excel file provided by Sarotti [28]. ECD calculations were performed using TD-DFT at the CAM-B3LYP/DGDZVP level in methanol. The ECD spectra were generated by considering the Boltzmann distribution of each geometric conformation. Subsequently, SpecDis 1.71 was utilized to combine the individual CD spectra with a Boltzmann statistical weighting, resulting in a Gaussian curve (σ = 0.16-0.4 eV), which was then compared with experimental data.

2.5. Fungicidal Activity Assay of Compounds 1-10 In Vitro

Pathogenic fungi, including Alternaria solani, Fusarium graminearum, Phytophthora capsici, Sclerotinia sclerotiorum, Botrytis cinerea, and Rhizoctonia solani, were inoculated onto petri dishes containing a compound solution at a concentration of 50 μg/mL. The dishes were then incubated in a biochemical incubator at 25 °C in the dark. The assessment of bactericidal activity was conducted following a three days incubation period, with each experimental group replicated three times. The control group was treated with sterile water. The results of the activity assessment were quantified on a percentage scale ranging from 0 to 100, where 0 indicates no activity and 100 signifies complete eradication [29].

$$\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}(\%)={\displaystyle \frac{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k} \mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y} \mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}-\mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y} \mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{l}\mathrm{i}\mathrm{q}\mathrm{u}\mathrm{i}\mathrm{d} \mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k} \mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y} \mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}-4}}\times 100$$

2.6. Fungicidal Activity Assay of Compound 10 In Vivo

The commercial fungicides pyrimethanil and dimethachlon were utilized as positive controls in this study. Both compounds, along with the controls, were dissolved in N, N-dimethylformamide and subsequently diluted to a concentration of 200 µg/mL. Cucumber plants were then sprayed with these solutions and allowed to air dry for approximately 2 h. Following this drying period, the undersurfaces of the treated cucumber leaves were sprayed with a pathogen spore suspension containing approximately 1×10⁴ spores/mL. The plants were then placed in an incubator maintained at 20 °C with humidity levels exceeding 90% for a 5 day infection period. After this initial incubation, the plants were transferred to a greenhouse for an additional 5 days before being assessed for disease control scores [29].

3. Results

3.1. Structural Identification of Compounds

Compound 1 was isolated as a colorless oil, with its molecular formula determined to be C₂₂H₃₀O₄ by HRESIMS, revealing eight degrees of unsaturation. The FT-IR absorption bands indicate the presence of hydroxyl (3449 cm⁻¹), methyl (2920 and 2851 cm⁻¹), carbonyl (1740 cm⁻¹) groups. The ¹H-NMR, ¹³C-NMR (Table 1 and Table 2) and HSQC spectra reveal the presence of three carbonyl groups [δ_C: 214.2 (C-3), 196.5 (C-11), 170.9 (C-21)], a set of conjugated double bonds [δ_C: 153.0 (C-10), 141.1 (C-9)], six aliphatic methylene groups [δ_H: 5.12 (H-20a), 4.76 (H-20b), 2.90 (H-4a), 2.66 (H-12a), 2.51 (H-12b), 2.29 (H-8), 2.09 (H-4b), 2.05 (H-15a), 1.97 (H-7a), 1.54 (H-15b), 1.40 (H-7b); δ_C: 63.3 (C-20), 60.2 (C-12), 42.7 (C-4), 30.3 (C-7), 26.7(C-15), 23.8 (C-8)], three hypomethyl groups [δ_H: 2.90 (H-5), 2.52 (H-14), 2.29 (H-2); δ_C: 59.4 (C-2), 52.9 (C-14), 30.1 (C-5)], five methyl groups [δ_H: 2.10 (H-22), 1.54 (H-19), 1.13 (H-18), 1.01 (H-17), 1.00 (H-16); δ_C: 25.2 (C-16), 23.4 (C-17), 21.1 (C-18), 21.0 (C-22)], 20.6 (C-19)] and three quaternary carbons [δ_C: 51.8 (C-6), 49.7 (C-1), 40.4 (C-13)]. The ¹H-¹H COSY correlations of H-2/H-15/H-14; H-4/H-5/H-18; and H-7/H-8 along with HMBC correlations of H-2, H-4, H-15/C-3; H-18/C-4, C-5, C-6; H-16, H-17/C-1, C-2, C-6; H-7/C-5; H-19/C-10, C-12, C-13, C-14; H-20/C-8, C-9, C-10, C-21 and of H-22/C-21, elucidated the plannar structure of 1 as shown in Figure 1.

The relative configuration of compound 1 was determined to be 2S*, 5R*, 6R*, 13S*, 14S* based on the NOESY correlations of H-16/H-14, H-2 and H-5/H-19. The absolute configuration of 1 was confirmed to be 2S, 5R, 6R, 13S, 14S by subsequent ECD calculations.

Figure 2. Key HMBC (bold lines) and ¹H–¹H COSY (arrows) correlations of compound 1–8.

Figure 2. Key HMBC (bold lines) and ¹H–¹H COSY (arrows) correlations of compound 1–8.

Compound 2 was purified as colorless oil, and its molecular formula was deduced to be C₂₀H₃₀O₃ by (+)-HRESIMS m/z 319.2268 [M+H]⁺ (calcd. For C₂₀H₃₁O₃⁺, 319.2268), implying six degrees of unsaturation. The IR spectrum of 2 showed absorption bands for hydroxyl (3439 cm⁻¹), methyl (2936 cm⁻¹) and carbonyl (1730 cm⁻¹). The ¹H-NMR, ¹³C-NMR (Table 1 and Table 2) and HSQC spectra indicate that compound 2 is a harziane diterpenes type compound like compound 1, with the difference that the substituents of compound 2 at C-3 and C-20 are both hydroxyl groups. The entire structure was confirmed by the ¹H-¹H COSY correlations of H-14/H-15/H-2/H-3/H-4/H-5/H-18; H-7/H-8 and the HMBC correlations of H-7/C-5; H-12/C-11; H-16, H-17/C-1, C-2, C-6; H-18/C-4, C-5, C-6; H-19/C-10, C-12, C-13, C-14 as well as H-20/C-8, C-9, C-10. The relative configuration of compound 2 was assigned to be 2S*, 3S*, 5R*, 6R*, 13S*, 14S* by the NOESY correlation of H-15b/H-19, H-3; H-19/H-5. The experimental and calculated ECD spectra did match well, suggesting 2S, 3S, 5R, 6R, 13S, 14S was the correct absolute configuration (Figure 4).

Compound 3 was isolated as colorless oil, and its molecular formula was deduced to be C14H24O3 by (+)-HRESIMS m/z 263.1624 [M+Na]⁺ (calcd. For C₁₄H₂₄NaO₃⁺, 263.1618), implying three degrees of unsaturation. The FT-IR absorption band suggested the presence of hydroxy group (3436 cm⁻¹), methyl group (2958 cm⁻¹, 2926 cm⁻¹ and 2850 cm⁻¹), olefinic group (1622 cm⁻¹) and carbonyl group (1666 cm⁻¹). The ¹H-NMR, ¹³C-NMR (Table 3 and Table 4) and HSQC spectra indicate the presence of one carbonyl signal [δ_C: 198.5 (C-11)], a group of conjugated olefin signals [δ_H: 6.88 (H-9), 6.13 (H-10); δ_C: 144.1 (C-9), 134.2 (C-10)], three aliphatic methylene signals [δ_H: 2.45 (H-8a), 2.36 (H-8b), 1.91 (H-5a), 1.72 (H-4a), 1.58 (H-4b, 5b); δ_C: 43.8 (C-8), 40.4 (C-4), 24.6 (C-5)], two hypomethyl signals [δ_H: 1.86 (H-6), 1.62 (H-2); δ_C: 54.9 (C-6), 44.6 (C-2)],two oxidized quaternary carbon signals [δ_C: 81.4 (C-3), 75.0 (C-7)] and four methyl signals [δ_H: 2.27 (H-12), 1.27 (H-13), 1.19 (H-14), 1.06 (H-1); δC: 27.2 (C-12), 26.2 (C-13), 25.9 (C-14) , 14.6 (C-1)]. The ¹H-¹H COSY correlations of H-1/H-2/H-6/H-5/H-4; H-8/H-9/H-10 as well as HMBC correlations of H-1/C-3, H-13/C-2, C-3, C-4; H-14/C-6, C-7, C-8; H-10/C-11 and of H-12/C-10, C-11 generated the planar structure of 3. The configuration of the double bond at C-9 was assigned as trans by the NOESY correlations signal of H-8/H-10, and the large coupling constant between H-9 and H-10. The NOESY correlations of H-2/H-13 and H-1/H-6 indicates the relative configuration of C-2, C-3, C-6.

The absolute configuration of C-7 could not be determined by ECD. To solve this difficult stereoscopic problem, the NMR data of 3a (2S^*, 3R^*, 6R*, 7R*) and 3b (2S*, 3R^*, 6R*, 7S*) were further calculated at the PCM/mPW1PW91/6–31+G (d, p) level using the GIAO method [26]. The calculated ¹³C NMR chemical shifts of 3a showed a better agreement with the experimental values of compound 3 with a higher correlation coefficient [R² for 3a: 0.9958; R² for 3b: 0.9956, Figure S 79]. In addition, DP4⁺ probability analysis [27] based on both ¹H and ¹³C NMR data predicated 3a as the correct relative structure with 96.49 % probability (Tables S 13). The experimental and calculated ECD spectra for 2S, 3R, 6R, 7R in MeOH did match well, indicating that 2S, 3R, 6R, 7R was the correct absolute configuration (Figure 4).

Compound 4 was isolated as colorless oil, its molecular formula was determined as C₁₅H₂₈O₃ by (+)-HRESIMS m/z 279.1940 [M+Na]⁺ (calcd. For C₁₅H₂₈NaO₃⁺, 279.1931), implying two degrees of unsaturation. The FT-IR absorption band indicate that compound 4 contains hydroxyl group (3424 cm⁻¹), methyl group (2962 and 2875 cm⁻¹), olefinic groups (1647 cm⁻¹). The planar structure of 4 was deduced to be the same as that of 11-cycloneren-3,7,10- triol [30] by comparison of its ¹H- and ¹³C-NMR data (Table 3 and Table 4) with those for 4, which was supported by HMBC correlations of and ¹H-¹H COSY correlations (Figure 2).

The relative configurations of the cyclopentane in compound 4 were determined to be 2S*, 3R*, 6R* by the NOESY-related signals of H-1/H-6 as well as H-2/H-13. Since the amount of compound 4 was not sufficient for the Mosher's reaction, we determined its absolute configuration by calculating NMR and ECD. The relative configuration of the compound 4 was further determined to be 2S*, 3R*, 6R*, 7R*, 10R* based on DP4+ probability (100% for 2S*, 3R*, 6R*, 7R*, 10R*, Table S 26) analysis of ¹H-NMR and ¹³C-NMR data. The absolute configuration of compound 4 was determined to be 2S, 3R, 6R, 7R, 10R based on the comparison of calculated and experimental ECD spectra (Figure 4).

Compound 5 was separated as colorless oil, with the molecular formula C₁₅H₂₈O₄ established by the (-)-HRESIMS data at m/z 271.1910 [M-H]^- (calcd. For C₁₅H₂₇O₄^- 271.1914), indicating two degrees of unsaturation. The FT-IR absorption band indicate that compound 5 contains hydroxyl group (3402 cm⁻¹) and methyl group (2966 and 2931 cm⁻¹). The NMR data of 5 (Table 3 and Table 4) closely resembled those of 9-cycloneren-3, 7, 11-triol [30], with the main difference is that -OH at the C-10 is substituted with -OOH. This was demonstrated by the HRESIMS data and the HMBC correlations of H-10/H-11; H-12/H-10, H-11, H-15. Other HMBC and ¹H-¹H COSY correlations confirm the entire structure of the compound 5. The trans relative configuration of C-9 and C-10 was established according to the large coupling constant of H-9 and H-10 and the NOESY correlations of H-10/H-8 (Figure 3). The relative configuration of 5 was determined to be 2S*, 3R*, 6R* by NOESY cross-peaks of H-2/H-13 and of H-1/H-6.

Additionally, the relative configuration of C-7 was deduced as 7R* by NMR calculations with the DP4+ probability of 100% (Table S 35). The absolute configuration of compound 5 was determined to be 2S, 3R, 6R, 7R by comparing the calculated ECD and experimental ECD spectra (Figure 4).

Compound 6 was isolated as colorless oil, its molecular formula was deduced to be C₂₀H₃₄O₆ by (+)-HRESIMS m/z 393.2249 [M+Na]⁺ (calcd. For C₂₀H₃₄NaO₆⁺, 393.2248), with four degrees of unsaturation. The FT-IR absorption band suggested the presence of hydroxy group (3453 cm⁻¹), methyl group (2964 and 2937 cm⁻¹) and carbonyl group (1737 cm⁻¹). The ¹H-NMR, ¹³C-NMR（Table 1 and Table 2）and HSQC spectra indicate the presence of two carbonyl signal [δ_C: 172.9 (C-16), 172.4 (C-13)], a group of conjugated olefin signals [δ_H: 5.41 (H-10); δ_C: 131.2 (C-10), 129.9 (C-11)], six aliphatic methylene signals [δ_H: 2.64 (H-14, H-15), 2.19 (H-9a), 2.12 (H-9b), 1.85 (H-5a), 1.68 (H-4a), 1.56 (H-4b), 1.55 (H-5b), 1.49 (H-8)；δ_C: 40.5 (C-4, C-8), 29.3 (C-15), 29.1 (C-14), 24.5 (C-5), 22.5 (C-9)], an oxidized methylene signal [δ_H: 4.66 (H-12a), 4.60 (H-12b); δ_C: 63.6 (C-12)], two hypomethyl signals [δ_H: 1.84 (H-6), 1.60 (H-2); δ_C: 54.6 (C-6), 44.4 (C-2)], two oxidized quaternary carbon signals [δ_C: 81.4 (C-3), 74.8 (C-7)] and four methyl signals [δ_H: 1.74 (H-20), 1.26 (H-18), 1.15 (H-19), 1.04 (H-1); δ_C: 26.2 (C-18), 25.0 (C-19), 21.6 (C-20), 14.7 (C-1)] and a methoxy signal [δ_H: 3.69 (H-17); 52.0 (C-17)].

The ¹H-¹H COSY correlations of H-1/H-2/H-6/H-5/H-4; H-8/H-9/H-10; H-14/H-15 as well as HMBC correlations of H-1/C-3; H-18/C-2, C-3, C-4; H-19/C-6, C-7, C-8; H-10/C-8; H-20/C-10, C-11, C-12; H-12/C-13; H-14/C-13; H-15/C-13, C-16 and of H-17/C-16 generated the primary structure of 6. The configuration of the double bond at C-10 was assigned as cis by the NOESY correlations signal between H-9/H-12 as well as H-10/H-20. The correlations between H-2 and H-18, H-1 and H-6 suggests that the relative configurations of C-2, C-3 and C-6 are 2S, 3R, 6R.

The relative configuration of the C-7 is 7R*, as shown by the theoretical NMR calculation using the GIAO's method combined with a DP4⁺ probability analysis (Table S 44). The absolute configuration of 6 was confirmed by the similarity between the calculated ECD curve of 2S, 3R, 6R, 7R-6 and its experimental ECD spectrum (Figure 4). Thus, the structure of 6 is as shown in Figure 1.

Compound 7 was obtained as colorless oil, The molecular formula C₂₀H₃₄O₆ was given by analysis of (+)-HRESIMS m/z 393.2252 [M+Na]⁺ (calcd. For C₂₀H₃₄NaO₆⁺, 393.2248), suggesting four degrees of unsaturation. The FT-IR absorption band shows the presence of hydroxyl (3456 cm^-1), methyl (2968 and 2936 cm^-1) and carbonyl (1738 cm^-1). Its ¹H- and ¹³C-NMR (Table 1 and Table 2) as well as HREIMS data closely resembled those of 7, and ¹H-¹H COSY and HMBC correlations (Figure2) also suggested 8 has a similar planar structure to 7. The difference between compound 8 and 7 is that the geometry of double bond at C-10 is trans which was inferred from the NOESY correlations of H-9/H-20 as well as H-10/H-12. The relative configuration of C-2, C-3, C-6 was determined to be 2S, 3R, 6R based on the correlation signals of H-2/H-18 as well as H-1/H-6 in the NOESY spectra (Figure 3).

The relative configuration of C-7 was determined by NMR calculations combined with DP4⁺ probability analysis (100% for 2S*, 3R*, 6R*, 7R*, Table S 43). By contrast of the experimental and calculated ECD data, the absolute configuration was validated.

Figure 3. Key NOESY correlations of compound 1-8.

Figure 3. Key NOESY correlations of compound 1-8.

Figure 4. Calculated ECD spectra and experimental ECD curves of compound 1–8 in MeOH.

Figure 4. Calculated ECD spectra and experimental ECD curves of compound 1–8 in MeOH.

Compound 8 was obtained as colorless oil, its molecular formula was determined as C₁₅H₂₈O₃ by (+)-HRESIMS m/z 279.1941 [M+Na]⁺ (calcd. For C₁₅H₂₈NaO₃ ⁺, 279.1931), implying two degrees of unsaturation. The FT-IR absorption band indicate that compound 8 contains hydroxyl group (3408 cm⁻¹), methyl group (2960 and 2873cm⁻¹), olefinic groups (1647 cm⁻¹). The ¹H-NMR, ¹³C-NMR (Table 3 and Table 4) and HSQC spectra exhibited the presence of a group of olefinic signals [δ_H: 4.97 (H-12a), 4.86 (H-12b); δ_C: 147.7 (C-11), 111.0 (C-12)], four methylene groups [δ_H: 1.87 (H-5a), 1.71 (H-9a), 1.69 (H-4a), 1.61 (H-9b), 1.57 (H-8a, H-4b), 1.56 (H-5b), 1.50 (H-8b); δ_C: 40.5 (C-4), 35.8 (C-8), 29.1 (C-9), 24.5 (C-5)], two hypomethyl groups [δ_H: 1.87 (H-6), 1.59 (H-2); δ_C: 54.8 (C-6), 44.5 (C-2)], one oxidized hypomethyl group [δ_H: 4.09 (H-10); δ_C: 76.0 (C-10)], two oxidized quaternary carbon signals [δ_C: 81.4 (C-3), 74.8 (C-7)] and four methyl signals [δ_H: 1.72 (H-15), 1.26 (H-13) 1.16 (H-14), 1.04 (H-1); δ_C: 26.2 (C-13), 25.2 (C-14) , 18.2 (C-15), 14.6 (C-1)].

The ¹H-¹H COSY correlations of H-1/H-2/H-6/H-5/H-4; H-8/H-9/H-10 along with the HMBC correlations signals of H-1/C-3; H-13/C-2, C-3, C-4; H-14/C-6, C-7, C-8; H-10/C-11, C-12 and of H-15/C-10, C-11, C-12 demonstrated that compound 8 is 11-cycloneren-3, 7, 10-triol [24].According to previous research[33], the chirality of 11-cycloneren-3,7,10-triol at C-10 was undetermined, so the Mosher's method was used to determine its conformation. Compound 8 was esterified with (S)-MTPA-Cl and (R)-MTPA-Cl, respectively, to give Mosher ester derivatives. The Δδ_S-R of the ¹H-NMR data of each proton adjacent to C-10 in the (S)-MTPA ester and (R)-MTPA ester products were compared (Fig. 50), and the final absolute configuration of C-10 was determined to be 10S. The ECD of compound 8 was calculated, and the calculated ECD curves of 2S, 3R, 6R, 7R, 10S-8 were found to be in good agreement with the experimental ECD curves, thus the absolute configuration of compound 8 was determined.

Figure 5. Δδ_S-R value (ppm) of MTPA ester of 8.

Figure 5. Δδ_S-R value (ppm) of MTPA ester of 8.

3.2. Fungicidal Activities of Compounds 1-10 In Vitro

We tested the fungicidal activity of compounds 1-10 against six crop pathogens at a concentration of 50 µg/mL. Fungicidal tests showed that compounds 1-10 all showed good inhibition of S. sclerotiorum at a concentration of 50 µg/mL. In addition, compound 3 and 10 showed significant inhibition against B. cinerea with inhibition rates of 81.6% and 100%, respectively.

3.3. Fungicidal Activities of Compound 10 In Vivo

In the in vivo activity test, compound 10 was effective against two pathogens, and it showed significant inhibitory activity against B. cinerea, with a control effect of 65.8%.

Table 6. Activities of compound 10 against Botrytis cinerea and Sclerotinia sclerotiorum in vivo.

Preventative efficiency (%)
pathogenic fungi	Botrytis cinerea	Sclerotinia sclerotiorum
10	65.8 %	49.1 %
PC	88.6 %	100 %

Table 6. Activities of compound 10 against Botrytis cinerea and Sclerotinia sclerotiorum in vivo.

Preventative efficiency (%)
pathogenic fungi	Botrytis cinerea	Sclerotinia sclerotiorum
10	65.8 %	49.1 %
PC	88.6 %	100 %

Figure 6. Activities of compound 10 against B. cinerea and S. sclerotiorum in vivo.

Figure 6. Activities of compound 10 against B. cinerea and S. sclerotiorum in vivo.

4. Discussion

A variety of Trichoderma strains have been utilized worldwide as effective biocontrol agents, including T. harzianum, T. hamatum, T. asperellum, T. atroviride, T. koningii, and T. viride [31,32,33,34,35]. One of the mechanisms by which Trichoderma exerts biological control is through the production of secondary metabolites [36,37]. In the present study, we isolated and purified secondary metabolites extracted using ethyl acetate from T. hamatum and identified the structures of the previously undescribed compounds 1-7, as well as the known compound 8, utilizing HRESIMS, NMR, UV, IR, circular dichroism, and Mosher’s method in conjunction with computational chemistry. Compounds 1, 2, and 9 are harziane diterpenes characterized by a unique 6-5-4-7 tetracyclic carbon skeleton; compounds 3-8 are cyclonerane sesquiterpenes, among which compound 5 represents the first -OOH-substituted cyclonerane sesquiterpene to be discovered, while compound 10 was identified as dehydroacetic acid.

Dehydroacetic acid (10) was first isolated from Solandra nitida in 1866 and has been utilized as a food preservative due to its efficacy in inhibiting the growth of molds, yeasts, and bacteria. Its derivatives have been investigated for the development of effective antimicrobial agents against various bacteria and fungi[38]; however, research on its antagonistic effects against agricultural pathogens remains relatively limited. In this study, the fungicidal activities of compounds 1-9 and dehydroacetic acid (10) against six agricultural pathogens were assessed. The results indicated that some of these compounds exhibited promising fungicidal activities against pathogenic fungi. Notably, dehydroacetic acid (10) significantly inhibited the growth of both B. cinerea and R. solani. Furthermore, in vivo examinations of the effects of dehydroacetic acid (10) on the control of B. cinerea and S. sclerotiorum suggested its potential for development as an agricultural antibiotic. Additionally, the substantial isolation of dehydroacetic acid may partially account for the antibiosis activity observed in T. hamatum.

This work not only enriched the active material base of T. hamatum but also advanced the application of its secondary metabolites in controlling agricultural pathogenic fungi, thereby providing a valuable reference for the development of fungal biocontrol strategies.

5. Patents

Chengdu University of Traditional Chinese Medicine has filed patents ZL202311414683.0 and ZL202311251991.6 concerning the in vitro antifungal activity of compounds 2 and 3.