Antifungal Nafuredin and Epithiodiketopiperazine Derivatives From the Mangrove-Derived Fungus Trichoderma harzianum D13

A new polyketide derivative, nafuredin C (1), a novel heterocyclic dipeptide, trichodermamide G (3), together with four known biogenetically related compounds nafuredin A (2), trichodermamide A (4), aspergillazin A (5), and peniisocoumarin H (6), were isolated from the mangrove-derived fungus Trichoderma harzianum D13. Their structures, including their absolute configurations, were determined by spectroscopic analysis and time-dependent density functional theory-electronic circular dichroism (ECD) calculations. Trichodermamide G was found to be a novel epithiodiketopiperazine derivative with an unprecedented cyclic system containing a sulfur bridge, and nafuredin C represented the third nafuredin derivative of these homologous compounds. The new compound nafuredin C exhibited obvious antifungal activity against Magnaporthe oryzae with a minimum inhibitory concentration (MIC) of 8.63 μM, which is on the same order of magnitude as the positive control carbendazim (MIC = 3.27 μM).

Introduction

Rice blast disease is the most serious disease affecting cultivated rice, a staple food for nearly 50% of the world’s population, and seriously threatens global food security. Each year, rice blast disease is responsible for the loss of 10–30% of the rice harvest, which is enough to feed more than 60 million people (Martin-Urdiroz et al., 2016; Zhou, 2016). Rice blast is caused by Magnaporthe oryzae, an ascomycete fungus that causes diseases in a wide range of economically important crops including barley, oats, rye grass, and millets. It was first reported in Brazil in 1985 and then rapidly spread to other South American countries, causing significant yield losses. The disease captured public attention in 2016 when it appeared in Bangladesh, resulting in thousands of rice fields being burned to prevent further spread of the disease (Ma and Xu, 2019). In China, rice blast occurs everywhere rice is cultivated, particularly in hilly areas. In epidemic years of the disease, yield loss can reach 40–50% and in severe cases can result in complete losses in major rice production areas (Zhao X. X. et al., 2019). Therefore, searching for new biological pesticides to control rice blast is necessary for sustainable development of the rice industry and food security worldwide.

Beneficial microbes can function as biocontrol organisms that help plants defend themselves from attack by pathogens. Fungi in the genus Trichoderma are excellent mycoparasites of plant pathogens and directly protect plants against them. In addition, Trichoderma spp. can enhance the plant defense system, which enables the plant to respond in a fast and strong manner to pathogen attack (Guzman-Guzman et al., 2019). These fungi are also prolific producers of numerous secondary metabolites with pharmaceutical and biotechnological importance, including polyketides, non-ribosomal peptides, siderophores, peptaibols, and volatile and non-volatile terpenes (Contreras-Cornejo et al., 2016). Several studies have focused on the application of Trichoderma spp. for controlling rice blast disease with apparent effects; however, marine-derived Trichoderma spp. have not been evaluated for use in plant protection, and few studies have reported their antifungal effects against M. oryzae.

During our ongoing search for new anti-phytopathogenic fungal secondary metabolites from marine-derived fungi in the Yellow Sea and South China Sea (Huang et al., 2018; Zhao et al., 2018; Zhao D. L. et al., 2019), we found that the extract of mangrove-derived fungus Trichoderma harzianum D13, collected from Hainan province, China, displayed a strong activity against fungal plant pathogens. Further chemical investigation of the ethyl acetate (EtOAc) extracts led to the isolation of six compounds (Figure 1), including two nafuredin derivatives, nafuredins C and A (1 and 2); three epithiodiketopiperazine derivatives, trichodermamide G (3), trichodermamide A (4), and aspergillazin A (5); and one isocoumarin, peniisocoumarin H (6). Among them, nafuredin C and trichodermamide G are new compounds. Herein, we report the isolation, structural elucidation, and antifungal activities of these compounds.

FIGURE 1

Figure 1. Chemical structures of compounds 1–6.

Materials and Methods

General Experimental Procedures

Specific rotations were measured on a JASCO P-1020 digital polarimeter (Tokyo, Japan). ECD spectra were obtained on a JASCO J-815-150S spectropolarimeter. UV spectra were recorded on a Techcomp UV2310II spectrophotometer (Shanghai, China). The 1D (¹H, ¹³C, and NOE) and 2D NMR spectra [HMQC, correlation spectroscopy (COSY), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY)] were acquired on a DD2 500 MHz NMR spectrometer (Agilent Technologies, Santa Clara, CA, United States). A Micromass Q-TOF spectrometer (Waters, Milford, MA, United States) and Thermo Scientific LTQ Orbitrap XL spectrometer (Waltham, MA, United States) was used to measure the electrospray ionization mass spectrometry (ESIMS) and HRESIMS. High-performance liquid chromatography (HPLC) was performed using a Waters e2695 separation module with a Waters 2998 detector and Waters X-Bridge C₁₈ (5 μm, 10 × 250 mm) column. Column chromatography (CC) was performed over silica gel (200–300 mesh; Qing Dao Hai Yang Chemical Group Co., Qingdao, China), octadecylsilyl silica (ODS) gel (RP18, 40–63 μm; Merck, Billerica, MA, United States), and Sephadex LH-20 (GE Healthcare, Little Chalfont, United Kingdom). Precoated silica gel plates (G60, F-254; Yan Tai Zi Fu Chemical Group Co., Yantai, China) were used for analytical thin-layer chromatography. Analytical and HPLC-grade solvents were used for isolation.

Fungal Material

The fungus was isolated from the internal tissues of the root of mangrove plant Excoecaria agallocha Linn. collected from Hainan province, China, in 2016, and was identified as T. harzianum (GenBank accession number MG827165) by sequence analysis of the internal transcribed spacer region of the rDNA. A voucher strain of this fungus was deposited in the Marine Agriculture Research Center, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao, China.

Extraction and Isolation

The fungus T. harzianum D13 was cultured on plates of potato dextrose agar medium at 28°C for 3 days. Plugs of agar supporting mycelium growth were cut and transferred aseptically into 200 × 1000-mL Erlenmeyer flasks each containing 400 mL of potato dextrose water liquid medium. The flasks were incubated at 28°C under static conditions for 30 days. The cultures (80 L) were filtered through gauze to separate the mycelial layer from the aqueous layer. The filtrate was then extracted twice with EtOAc, whereas the mycelium were mechanically broken and ultrasonically disrupted for 10 min and then extracted twice with CH₂Cl₂:MeOH (1:1, v/v). After removing CH₂Cl₂ and MeOH by evaporation under vacuum, the remaining aqueous solution was extracted three times with EtOAc. The combined EtOAc extracts were concentrated under reduced pressure to yield the total EtOAc extract (13.8 g). This extract was subjected to vacuum liquid chromatography on silica gel for elution with a gradient of EtOAc in petroleum ether (EtOAc/petroleum ether, 0–100%), and then with MeOH in EtOAc ranging from 10 to 50% to give eight fractions (Fractions 1–8). Fraction 3 was initially fractionated using an ODS gel column with a step gradient elution of MeOH–H₂O (60–90%) to afford Fr. 3-1 and Fr. 3-2. Fraction 3-1 was subsequently applied to a Sephadex LH-20 CC (CH₂Cl₂/MeOH, v/v, 1/1), and finally purified by reversed-phase-HPLC eluting with 80% MeOH–H₂O to obtain compound 2 (10.6 mg). Fraction 4 was separated on an ODS column eluting with 50–90% MeOH–H₂O to obtain Fr. 4-1 and Fr. 4-2. Fraction 4-1 was then afforded to silica gel CC (EtOAc/petroleum ether, 5% to 35%), followed by purification by HPLC (MeOH/H₂O, 80/20) to yield 1 (10.0 mg). Fraction 6 was subjected to ODS CC using a gradient elution of 30–90% MeOH–H₂O, followed by separation on Sephadex LH-20 (CH₂Cl₂–MeOH, v/v, 1/1) to afford subfractions Fr. 6-1–6-3. Fraction 6-1 was subjected to silica gel CC (MeOH/CH₂Cl₂, v/v, 1/50 to 1/10), and finally purified by semipreparative HPLC eluting with 35% MeCN–H₂O to give 6 (4.0 mg). Fractions 6-2–6-4 were purified by HPLC using 40, 40, and 35% MeOH–H₂O to give 5 (9.0 mg), 4 (68.5 mg), and 3 (32.0 mg), respectively.

Nafuredin C (1): white, amorphous powder; [α]²⁰_D +40.0 (c 0.37, MeOH); UV (MeOH) λ_max (log ε) 240 (4.38) nm; ECD (0.93 mM, MeOH) λ_max (Δε) 222 (-17.40), 244 (+21.99) nm; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 385.2349 [M +Na]⁺ (calculated for C₂₂H₃₈O₄Na, 380.2795).

TABLE 1

Table 1. ¹H NMR data (500 MHz, DMSO-d₆, δ in ppm, J in Hz) and ¹³C NMR data (125 MHz, DMSO-d₆, δ in ppm) for 1.

Trichodermamide G (3): yellow, amorphous powder; [α]²⁰_D −116.5 (c 0.50, MeOH); UV (MeOH) λ_max (log ε) 204 (4.40), 274 (3.96), 324 (4.28) nm; ECD (1.55 mM, MeOH) λ_max (Δε) 226 (−3.08), 243 (+1.09), 325 (−5.07) nm; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 449.1026 [M +H]⁺ (calculated for C₂₀H₂₁O₈N₂S, 449.1013).

TABLE 2

Table 2. ¹H NMR data (500 MHz, DMSO-d₆, δ in ppm, J in Hz) and ¹³C NMR data (125 MHz, DMSO-d₆, δ in ppm) for 3.

Antifungal Assays

Antifungal activities were evaluated using the conventional broth dilution assay (Appendino et al., 2008). Five phytopathogenic fungal strains, including Botrytis cinerea, Magnaporthe grisea, Phytophthora parasitica, Pestallozzia theae, and Valsa mali were used. Carbendazim was used as a positive control.

Results and Discussion

Structural Elucidation of the Isolated Compounds

Nafuredin C (1) was isolated as a white, amorphous powder. Its molecular formula, C₂₂H₃₄O₄, was established from high-resolution ESIMS (HRESIMS), ¹H and ¹³C nuclear magnetic resonance (NMR) data (Supplementary Figures S1, S2, S7), indicating six indexes of hydrogen deficiency. The ¹H NMR (Table 1) spectrum displayed seven olefinic protons at δ_H 6.24 (1H, dd, J = 15.0, 10.5 Hz), 6.17 (1H, dd, J = 15.0, 11.0 Hz), 6.04 (1H, dd, J = 15.0, 10.5 Hz), 5.74 (1H, d, J = 11.0 Hz), 5.64 (1H, dd, J = 15.0, 7.5 Hz), 5.59 (1H, dd, J = 15.0, 6.0 Hz), and 5.43 (1H, dd, J = 15.0, 8.0 Hz), two hydroxyl signals at δ_H 5.82 (1H, d, J = 6.5 Hz) and 5.50 (1H, d, J = 5.0 Hz), two oxygenated methines at δ_H 4.46 (1H, m) and 3.95 (1H, t, J = 6.0 Hz), and five methyls at δ_H 1.67 (3H, s), 1.24 (3H, s), 0.95 (3H, d, J = 7.0 Hz), 0.92 (3H, d, J = 6.5 Hz), and 0.82 (3H, t, J = 7.0 Hz). The ¹³C NMR and distortionless enhancement by polarization transfer (DEPT) spectra revealed 22 carbon signals, including one ester carbonyl group (δ_C 177.2), two quaternary carbons (one oxygenated, one olefinic), three methylene groups, 11 methines (seven olefinic, two oxygenated), and five methyl groups. These spectroscopic data are similar to those of nafuredin B, isolated from a mixed culture of the deep-sea-derived fungus Talaromyces aculeatus and mangrove-derived fungus Penicillium variabile (Zhang et al., 2017). The major differences were replacement of the sp² double bond at C-2 and C-3 in nafuredin B by a hydroxyl group at C-2 in 1, which were consistent with the downfield shifts of C-1/C-4 (δ_C 163.3/68.0 in nafuredin B vs 177.2/84.8 in 1) and upfield shifts of C-2/C-3 (δ_C 118.0/156.3 in nafuredin B vs 68.4/37.9 in 1), supported by the COSY correlations of H-2/H-3, and key HMBC from H-2 to C-1/C-4, from H-3 to C-1/C-5, from H-5 to C-3/C-4, from 2-OH to C-1/C-2/C-3, and from H₃-19 to C-3/C-4/C-5 (Figure 2 and Supplementary Figures S4, S5).

FIGURE 2

Figure 2. COSY, key HMBC, 1D NOE, and NOESY correlations of 1 and 3.

The relative configuration of the δ-lactone ring was established based on the NOESY spectrum (Figure 2 and Supplementary Figure S6). The correlations of 4-OH with 2-OH and H-5 and of H-6 with H₃-19 indicated a cofacial relationship among 4-OH, 2-OH, and H-5. The geometry of double bonds in the olefinic chain was E elucidated from the J values and NOESY correlations (Table 1 and Figure 2). Considering the biosynthesis and coisolation of nafuredin A (2), whose absolute configuration was determined based on asymmetric synthesis (Takano et al., 2001; Zhang et al., 2017), the chirality of C-10 and C-16 was determined to be 10R and 16S, which was supported by the identical NMR data. Comparing the computed ECD spectra with experimental results is a valid method of assigning the absolute configurations of natural products (Cao et al., 2020). To determine the absolute configuration of the δ-lactone ring in 1, ECD computations for all B3LYP/6-311+G(d)-optimized conformers (50 structures) were carried out at the B3LYP/6-311++G(2d,p) level, and six structures with relative energy less than 2.5 kcal/mol were obtained. Boltzmann statistics were performed for ECD simulations with a standard deviation of σ 0.25 eV. The experimental and calculated ECD spectra for the (2R,4S,5R,10R,16S)-1 showed good agreement (Figure 3 and Supplementary Table S1), thus suggesting the absolute configuration.

FIGURE 3

Figure 3. Experimental and theoretical ECD spectra of compound 1.

Trichodermamide C (3) was obtained as a yellow, amorphous powder, with the molecular formula C₂₀H₂₀N₂O₈S based on its HRESIMS, ¹H and ¹³C NMR data, indicating 12 degrees of unsaturation (Supplementary Figures S8, S9, S15). The ¹³C NMR and DEPT spectroscopic data (Table 2) revealed 20 carbon signals containing two carbonyl groups, two methoxyl groups, 10 methines (with five olefinic or aromatic, two thiogenated, one nitrogenated, two oxygenated), and six quaternary carbons (with five sp² and one sp³). In examining the ¹H NMR and HSQC data (Supplementary Figures S8, S10), an amide proton at δ_H 10.09, two ortho-aromatic protons attributed to a 1,2,3,4-tetrasubstituted phenyl unit at δ_H 7.04 (1H, d, J = 9.0 Hz) and 6.61 (1H, d, J = 9.0 Hz), and three olefinic protons including two derived from a cis-coupled double bond at δ_H 6.50 (1H, t, J = 8.0 Hz) and 6.04 (1H, d, J = 8.0 Hz) were observed in the deshielded region of the spectrum, whereas shielded signals for five methines at δ_H 5.18 (1H, s), 4.02 (1H, s), 3.63 (1H, s), 3.42 (1H, d, J = 9.5 Hz), and 3.29 (1H, s) were also present. Additionally, two O-methyl groups (δ_H 3.81 and 3.69) were observed in the ¹H NMR spectrum of 3. Inspection of the above NMR data indicated that the isocoumarin portion of 3 was similar to that of aspergillazine A, a modified dipeptide isolated from the marine-derived fungus Spicaria elegans (Liu et al., 2005), whereas the signals of oxazine were obviously changed, possibly because of the different location of the sulfur bridge. The characteristic NMR data of C-3 (δ_H 3.29, δ_C 44.6) and C-7 (δ_H 3.42, δ_C 37.8) indicated they were linked by a sulfur atom, which was confirmed from the COSY correlation of H5/H6/H7/H8/H9 and HMBC correlations from H-3 to C-5/C-7/C-9, from H-5 to C-7/C-9, from H-6 to C-4/C-8, and from H-7 to C-9 (Figure 2 and Supplementary Figures S11, S12). In the NOE difference spectra (Figure 2 and Supplementary Figures S13, S14), irradiation of H-2 and H-3 enhanced the resonance of H-9. This information, in addition to the similar coupling constant of H8/H-9 (brs) to trichodermamide A and aspergillazine A (Capon et al., 2005; Liu et al., 2005), indicates a syn relationship among H-2, H-3, H-9, and 8-OH. 4-OH was assigned on the opposite face of the sulfur bridge, as the cis relationship did not lead to a reasonable model according to 3D simulations.

To determine the absolute configuration of 3, ECD calculations were carried out. Monte Carlo conformational searches were applied using Spartan’s 10 software with the Merck molecular force field. Conformers with a Boltzmann-population of over 10% were selected for ECD calculations and were initially optimized at the B3LYP/6-31g (d,p) level in MeOH using the conductor-like polarizable continuum calculation model. Theoretical calculation of the ECD was conducted in MeOH using time-dependent density functional theory at the B3LYP/6-31+g (d,p) level for all conformers of compound (2R,3S,4S,7S,8S,9S)-3. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the programs SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (GraphPad, Inc., La Jolla, CA, United States) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV. The predicted ECD spectrum agreed well with the experimental result (Figure 4 and Supplementary Tables S2, S3), indicating that the absolute configuration of 3 was 2R,3S,4S,7S,8S,9S.

FIGURE 4

Figure 4. Experimental and theoretical ECD spectra of compound 3.

Epipolythiodiketopiperazine alkaloids are fungal metabolites with a highly complex molecular architecture comprising a densely functionalized core structure with many stereogenic centers. In the past decade, an increasing number of studies have discovered powerful new biological processes involving these molecules, including cytotoxic, antileukemic, antiviral, antibiotic, and antinematodal activities (Kim and Movassaghi, 2015); however, epithiodiketopiperazines have not been widely examined. Notably, trichodermamide G is a novel epithiodiketopiperazine derivative with an unprecedented cyclic system containing a sulfur bridge.

Compounds 2 and 4–6 were identified as nafuredin A (Ui et al., 2001), trichodermamide A (Liu et al., 2005), aspergillazine A (Liu et al., 2005), and peniisocoumarin H (Cai et al., 2018) by comparison of their spectroscopic data with those in the literature.

Antifungal Activity

In the present study, all isolated compounds were evaluated to determine their anti-phytopathogenic fungal activities against B. cinerea, M. oryzae, P. theae, P. parasitica, and V. mali. The nafuredin derivatives 1 and 2 exhibited obvious antifungal activities against M. oryzae, with minimum inhibitory concentrations (MICs) of 8.63 and 17.4 μM, respectively. These data indicate that the antifungal activity toward M. oryzae of new compound 1 was the same magnitude as that of the positive control carbendazim (MIC = 3.27 μM). Compounds 1 and 2 also displayed weak antifungal activity against V. mali and P. theae compared to carbendazim (Table 3).

TABLE 3

Table 3. Antifungal activity of 1 and 2.

Conclusion

In summary, we report three polyketide derivatives, nafuredin C (1), nafuredin A (2), and peniisocoumarin H (6), and three epithiodiketopiperazine derivatives, trichodermamide G (3), trichodermamide A (4), and aspergillazin A (5), isolated from the mangrove-derived fungus T. harzianum D13. Among them, nafuredin C (1) and trichodermamide G (3) are new compounds. Their structures were assigned based on extensive NMR spectroscopic data, time-dependent density functional theory ECD calculations together with comparison of their ECD spectra. Trichodermamide G contains a unique sulfur bridge compared to the homologous compounds. The polyketide derivatives nafuredin C (1) and nafuredin A (2) exhibited distinct antifungal activity against M. oryzae. T. harzianum have been widely used as biocontrol agents and commercially marketed as biopesticides. Our study expands the source of Trichoderma which used for biocontrol, and provides basic material for the discovery of new antifungal pesticides.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Author Contributions

D-LZ and C-SZ conceived and designed the experiments. X-FZ R-HH, DW, X-QW, and PZ performed the experiments. Y-QL, C-JZ, and PZ analyzed the data. D-LZ wrote the manuscript. All authors reviewed the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (41806194), the Major Agricultural Application Technology Innovation Projects of Shandong Province (SD2019ZZ002), and the Foundation of Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education (RDZH2019001).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank Dr. Fei Cao (College of Pharmaceutical Sciences, Hebei University) and Dr. Chao Liu (Sun Yat-sen University Cancer Center) for their efforts in the ECD analysis.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.01495/full#supplementary-material

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