Antiviral Activities of Trichothecenes Isolated from Trichoderma

Article pubs.acs.org/JAFC

Antiviral Activities of Trichothecenes Isolated from Trichoderma albolutescens against Pepper Mottle Virus Seung Mok Ryu,† Hae Min Lee,‡ Eun Gyeong Song,‡ Young Hye Seo,†,§,∥ Jun Lee,§,∥ Yuanqiang Guo,⊥ Beom Seok Kim,† Jae-Jin Kim,# Jin Sung Hong,¶ Ki Hyun Ryu,*,‡ and Dongho Lee*,† †

Department of Biosystems and Biotechnology and #Division of Environmental Science and Ecological Engineering, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea ‡ Plant Virus GenBank, Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women’s University, Seoul 01797, Republic of Korea § Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Republic of Korea ∥ Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea ⊥ State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300350, People’s Republic of China ¶ Department of Applied Biology, College of Agriculture and Life Sciences, Kangwon National University, Chunchon 24341, Republic of Korea S Supporting Information *

ABSTRACT: A bioassay-guided isolation using a green fluorescence protein (GFP)-tagged pepper mottle virus (PepMoV-GFP) based leaf-disk method to obtain new antiviral agents led to the isolation of trichodermin, 1, and a new compound trichoderminol, 2, from EtOAc extract of Trichoderma albolutescens culture medium. The structures of compounds 1 and 2 were determined by MS and NMR experiments, and the absolute configurations of the compounds were established by experimental and calculated vibrational circular dichroism spectra. Compounds 1 and 2 were evaluated for their anti-PepMoV potential in systemic host plants, such as tobacco and pepper, by PepMoV-GFP based systemic host method. All compounds exhibited inactivation effects against PepMoV. Furthermore, compound 1 showed protective effects against PepMoV. KEYWORDS: Trichoderma albolutescens, trichothecene, vibrational circular dichroism, Nicotiana benthamiana, Capsicum annuum, pepper mottle virus, antiviral agent

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INTRODUCTION

In our continuous search for natural product derived antiviral agents against PepMoV, an EtOAc extract of Trichoderma albolutescens13 culture medium exhibited anti-PepMoV activities by a green fluorescence protein (GFP)-tagged PepMoV, pSP6PepMoV-Vb1/GFP (PepMoV-GFP), based leaf-disk method.14,15 A bioassay-guided isolation of EtOAc extract of T. albolutescens culture medium led to the isolation of two trichothecenes, and the structures of these compounds were determined by applying spectroscopic techniques including vibrational circular dichroism (VCD). The two compounds were evaluated for their anti-PepMoV potential in systemic host plants, such as tobacco and pepper, by PepMoV-GFP based systemic host method. The isolation, structural identification, and biological evaluation of these compounds are described herein.

Plant virus diseases are reported worldwide, and they cause severe damage to agricultural crops. The pepper mottle virus (PepMoV), of the genus Potyvirus belonging to the family Potyviridae, is one of the common plant viruses affecting Solanaceae species, such as pepper (Capsicum spp.) and tobacco (Nicotiana spp.).1−3 The symptoms of PepMoV infection include mottling, vein clearing, leaf distortion, and fruit deformation in various host plants.4,5 PepMoV causes tremendous economic losses. However, to date, no antiviral agents that can protect host plants from PepMoV infection have been developed. Therefore, the development of effective antiviral agents against PepMoV is critically needed. Trichoderma species are filamentous fungi that are commonly found in various habitats, including soil, marine sediments, and woods,6 and they have been used as biocontrol agents against many crop pathogens.7 They produce a wide range of bioactive secondary metabolites; among these, trichothecenes are a wellknown class of sesquiterpene-based metabolites and reported to have various biological activities, such as antifungal, anticancer, antibiotic, antileukemic, antimalarial, and antiviral activities against animal viruses.8−12 © 2017 American Chemical Society

Received: Revised: Accepted: Published: 4273

March 6, 2017 May 10, 2017 May 11, 2017 May 11, 2017 DOI: 10.1021/acs.jafc.7b01028 J. Agric. Food Chem. 2017, 65, 4273−4279

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Journal of Agricultural and Food Chemistry

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(ATR): 3444, 2928, 1725, 1433, 1375, 1245, 1074, 1030, 963 cm−1; ESIMS (negative) m/z 353.1 [M + HCOO]−; ESIMS (positive) m/z 309.1 [M + H]+, 617.3 [2 M + H]+, 925.5 [3 M + H]+; HRESIMS m/z 309.1694 [M + H]+ (calcd for C17H25O5, 309.1702). Computational Methods. NOESY spectra and Chem3D modeling were used to build the 3D models of compounds 1 and 2. Conformational analysis was performed by the Merck Molecular Force Field (MMFF), as implemented in Spartan’14 software (Wavefunction, Inc., Irvine, CA). The selected conformers were optimized at the density functional theory (DFT) [B3LYP functional/6-31+G(d,p) basis set] level of theory using the Gaussian 09 software (Gaussian, Inc., Wallingford, CT). IR and VCD spectra were simulated using the same method chosen for the optimized geometries. The calculated frequencies were scaled using a factor of 0.98, and the calculated intensities were converted to Lorentzian bands with half-width of 6 cm−1 for comparisons to experimental data. The IR and VCD spectra of the conformers were summed based on a Boltzmann statistical weighting, and calculated spectra were compared with experimental IR and VCD. Preparation of Screening Materials. Tobacco (Nicotiana benthamiana L.) and hot pepper (Capsicum annuum L., P915 inbred line, Nong-woo Bio Co., Ltd., Suwon, Korea) were cultivated in a greenhouse of Seoul Women’s University, Seoul, Korea. GFP-tagged PepMoV (pSP6PepMoV-Vb1/GFP)14 was obtained from Plant Virus GenBank, Seoul Women’s University, Seoul, Korea. Anti-PepMoV Biological Assays. The anti-PepMoV activity of the extract and purified compounds was tested using our previously reported method (leaf-disk method and systemic host method).15 To prepare the inoculum, PepMoV-GFP-infected pepper leaves (0.1 g) were ground with 1 mL of phosphate buffered saline (0.01 M PBS, pH 7.2) in a mortar. The plants treated with solvent alone [0.1% dimethyl sulfoxide (DMSO)] were taken as the positive control, and healthy plants were used as the negative control. All samples were tested in triplicate to ensure the consistency of results. PepMoV-GFP Based Leaf-Disk Method. PepMoV-GFP infected pepper leaves were used as the source of leaf disks. After attaining sufficient growth, the leaves were cut into disks of 0.5 cm diameter, and the leaf disks were floated on Murashige−Skoog liquid culture medium containing solutions of crude extracts in 24-well plates. All leaf disks were kept at 25 °C for 7 days. PepMoV-GFP Based Systemic Host Method (Inactivation Effects). Tobacco and pepper plants at the 5−6 leaf stage were used for this experiment. Different concentrations of each of the purified compounds 1 (50, 10, 5, and 1 μM) and 2 (2, 1, 0.5, and 0.1 mM) were premixed with PepMoV-GFP inoculum. After 30 min, the plants were inoculated with the inoculum mixture. All plants were kept at 25 °C for 1 month. PepMoV-GFP Based Systemic Host Method (Protective Effects). Pepper plants at the 5−6 leaf stage of pepper were used in this method. Pepper leaves were pretreated with solutions of compound 1 (2 mM), and after 1 h, the treated leaves were inoculated with PepMoV-GFP inoculum. All plants were kept at 25 °C for 1 month. RT-PCR and Western Blot Analysis. RT-PCR and Western blot analysis were performed as described previously.14,15

MATERIALS AND METHODS

General Experimental Procedures. Column chromatography was performed using C18 reversed phase (RP) silica gel (12 μm) (YMC, Kyoto, Japan) and silica gel (230−400 mesh) (Merck, Darmstadt, Germany). Thin-layer chromatography was performed using precoated silica gel 60 F254 plates (0.25 mm) (Merck). All organic solvents used for the extraction and isolation of samples were of extra-pure grade (Dae-jung, Siheung, Korea). ESIMS was carried out using a Q-TOF micromass spectrometer (Waters, Milford, MA). NMR spectra were obtained using a 500 MHz NMR spectrometer (Varian, Palo Alto, CA) with tetramethylsilane as an internal standard. IR spectra were measured with a 640 FT-IR spectrometer (Varian), and optical rotations were obtained using a P-2000 (JASCO, Tokyo, Japan). VCD spectra were recorded on a ChiralIR-2X TM FT-VCD spectrometer (BioTools, Jupiter, FL). Fungal Material. T. albolutescens was collected from Odaesan National Park, Gangwon-do, Korea, in April 2013, and authenticated by Professor Jae-Jin Kim (Division of Environmental Science and Ecological Engineering, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea). A voucher specimen (KACC 83007BP) was deposited at the Korean Agricultural Culture Collection, National Academy of Agricultural Science, Wanju, Korea. T. albolutescens was cultivated on potato dextrose agar medium (Difco, Detroit, MI) in Petri dishes (150 mm × 2 cm × 45 plates) at 28 °C for 10 d in the dark. Extraction and Isolation. The fungal media were extracted with MeOH (3 × 2.0 L) at room temperature (25−28 °C) and evaporated in vacuo. The residue was suspended in H2O (2.0 L) and partitioned with EtOAc (3 × 2.0 L) to yield an EtOAc-soluble extract (806.1 mg). The EtOAc extract was fractionated on a glass column (58 × 3 cm) packed with C18 RP silica gel, using stepwise gradient elution with MeOH−H2O (5:5, 6:4, 7:3, 8:2, 9:1, and 10:0) to afford six fractions (Fr 1 to Fr 6). Fr 2 (102.0 mg) was further separated into three subfractions, Fr 2.1 to Fr 2.3, by silica gel column chromatography (28 × 1 cm) with CHCl3−MeOH gradient (9.8:0.2, 9.5:0.5, 9:1, and 7:3). Fr 2.1 (30.6 mg) was purified by silica gel column chromatography (12 × 1 cm) with an n-hexane−EtOAc gradient (8:2, 7:3, and 5:5) to afford trichoderminol, 2 (15.5 mg). Fr 3 (312.0 mg) was purified by silica gel column chromatography (38 × 1 cm) with an n-hexane− EtOAc gradient (8:2, 6:4, and 5:5) to give trichodermin, 1 (243.9 mg). Trichodermin 1 (Figure 1): colorless oil; [α]26 D −10.8 (c 0.8, CHCl3); VCD (c 1.0 M, CDCl3) (Figure 2).

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Figure 1. Structures of compounds 1 and 2.

RESULTS AND DISCUSSION Anti-PepMoV Activities of Crude Extracts. The fungal and plant extracts were evaluated for anti-PepMoV activity using the PepMoV-GFP based leaf-disk method. The EtOAc extract of T. albolutescens culture medium exhibited antiviral activities against PepMoV. The intensity of green fluorescence declined in PepMoV-GFP-infected leaf disks treated with EtOAc extract of T. albolutescens culture medium (Figure 3). These results indicate that the EtOAc extract of T. albolutescens culture medium has anti-PepMoV potential. Therefore, this extract was selected for further studies to identify its active compounds.

Trichoderminol 2 (Figure 1): colorless oil; [α]26 D −5.7 (c 0.8, CHCl3); VCD (c 0.2 M, CDCl3) (Figure 2); 1H NMR (500 MHz, CDCl3) δ 5.69 (1H, m, H-10), 5.58 (1H, dd, J = 8.0, 3.5 Hz, H-4), 4.05 (2H, br d, J = 3.5 Hz, H-16), 3.82 (1H, d, J = 5.5 Hz, H-2), 3.67 (1H, br d, J = 5.5 Hz, H-11), 3.12 (1H, d, J = 4.0 Hz, H-13a), 2.84 (1H, d, J = 4.0 Hz, H-13b), 2.54 (1H, dd, J = 15.5, 8.0 Hz, H-3a), 2.09 (2H, m, overlap, H-8), 2.09 (3H, s, overlap, H-OAc), 1.99 (1H, m, H3b), 1.93 (1H, m, H-7a), 1.48 (1H, m, H-7b), 0.95 (3H, s, H-15), 0.73 (3H, s, H-14); 13C NMR (125 MHz, CDCl3) δ 171.0 (C-OAc), 143.0 (C-9), 118.6 (C-10), 79.3 (C-2), 75.0 (C-4), 70.0 (C-11), 66.2 (C-16), 65.5 (C-12), 49.0 (C-5), 47.8 (C-13), 40.9 (C-6), 36.7 (C-3), 24.1 (C7), 23.4 (C-8), 21.1 (C-OAc), 16.0 (C-15), 5.8 (C-14); IR ν max 4274

DOI: 10.1021/acs.jafc.7b01028 J. Agric. Food Chem. 2017, 65, 4273−4279

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Figure 2. Comparison of experimental and calculated IR and VCD spectra of compounds (A) 1 and (B) 2.

Figure 3. Photograph of leaf disks treated with the EtOAc extract of T. albolutescens culture medium at different concentrations (20, 10, 5, and 2.5 mg/mL). The photographs were taken at day 7 under UV and visible lights. C, control (PepMoV-GFP infected leaf disks with DMSO); H, healthy.

Identification of Compounds 1 and 2. Two trichothecene compounds, 1 and 2, were obtained from the EtOAc extract of T. albolutescens culture medium by a bioassay-guided isolation using the PepMoV-GFP based leaf-disk method. Compound 1 was identified as trichodermin by comparing spectroscopic data with the published values.16−18 Compound 2 was obtained as colorless oil, and its elemental formula was determined to be C17H24O5 by HRESIMS analysis, suggesting six degrees of unsaturation. 1H and 13C NMR spectroscopic data of compound 2 were similar to those of trichodermin, 1, which has a typical trichothecene skeleton, except for the absence of methyl group signals of compound 1 [δH 1.71 (3H, s, H-16); δC 23.0 (C-16)] and the presence of oxygenated methylene signals of compound 2 [δH 4.05 (2H, br d, J = 3.5 Hz, H-16); δC 66.2 (C-16)]. The HMBC cross-peaks of H-16/C-8, C-9, and C-10 indicated that the oxygenated methylene group in compound 2 was located at the C-9 position. Furthermore, the IR spectrum exhibited an absorption band at 3444 cm−1. Collectively, the results obtained from NMR, MS, and IR spectra indicated that compound 2 contains an alcohol group at the C-9 position. The relative configurations of all stereogenic centers of compound 2 were similar to those of trichodermin, 1, as indicated by the close similarity of 1D NMR chemical shifts and NOESY data. The

NOESY correlations of H-4 with H-15 and H-11 allowed them to be located on the same face of the ring system. The correlations of H-2 with H-13a and those of H-13b with H-14 indicated that they are on the opposite face of the ring system. Consequently, the structure of compound 2 was determined as shown, and has been given the trivial name trichoderminol (Figure 1). The absolute configurations of compounds 1 and 2 were established by comparison of their experimental VCD spectra with those calculated using the DFT method. A conformational search was performed using MMFF in Spartan’14 software. Geometry optimization and VCD calculation for selected conformers were performed at the DFT level [B3LYP functional/6-31+G(d,p) basis set] in Gaussian 09 software. The calculated IR and VCD spectra of compounds 1 and 2 matched well with the respective experimental results (Figure 2). The absolute configurations of compounds 1 and 2 were 2R, 4R, 5S, 6R, 11R, and 12S (Figure 1). Inactivation Effects of Compounds 1 and 2 on PepMoV. Compounds 1 and 2 were evaluated for their inactivation effects against PepMoV in systemic host plants. The PepMoV-GFP inoculum premixed with different concentrations of each isolated compound (1 and 2) were used to infect tobacco (N. benthamiana L.), which is an indicator host 4275

DOI: 10.1021/acs.jafc.7b01028 J. Agric. Food Chem. 2017, 65, 4273−4279

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Figure 4. (A) Inactivation effects of compounds 1 and 2 at different concentrations (1, 50, 10, 5, and 1 μM; 2, 2, 1, 0.5, and 0.1 mM) in tobacco (Nicotiana benthamiana L.). The plants were photographed under UV and visible lights at 6 and 9 dpi. (B) RT-PCR and Western blot analysis of upper leaves in tested plants. M, marker; C, control (PepMoV-GFP with 0.1% DMSO); H, healthy.

concentrations (MIC) of compounds 1 and 2 were 10 μM and 1 mM, respectively. However, at 9 dpi, green fluorescence was observed from the inoculated leaves toward the upper leaves.

of PepMoV. The results are shown in Figure 4. The two compounds exhibited inactivation effects against PepMoV-GFP at 6 days post inoculation (dpi), and the minimum inhibitory 4276

DOI: 10.1021/acs.jafc.7b01028 J. Agric. Food Chem. 2017, 65, 4273−4279

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Figure 5. (A) Inactivation effects of compound 1 at different concentrations (2, 1, 0.5, and 0.1 mM) in hot pepper cultivar (Capsicum annuum L.). The plants were photographed under UV and visible lights at 9 and 12 dpi. (B) RT-PCR and Western blot analysis of upper leaves in tested plants. M, marker; C, control (PepMoV-GFP with 0.1% DMSO); H, healthy.

fluorescence was first observed at 7 dpi in plants treated with compound 1. Furthermore, viral RNA and coat protein bands were detected at 7 dpi using RT-PCR and Western blot analysis. These results indicate that compound 1 delayed PepMoV spreading in host plants, and it can protect host plants from PepMoV infection. In summary, bioassay-guided isolation using a PepMoV-GFP based leaf-disk method led to the isolation of trichodermin, 1, and a new compound, trichoderminol, 2, from an EtOAc extract of T. albolutescens culture medium. The structures of compounds 1 and 2 were determined by spectroscopic techniques, and the absolute configurations of the compounds were established by VCD. The two compounds were found to exhibit effective anti-PepMoV activities in systemic hosts (tobacco and pepper) by a PepMoV-GFP based systemic host method. This is the first study to report the inhibition of plant viruses by trichothecenes. The trichothecenes are a large family of fungal secondary metabolites produced by various species of Fusarium and Trichoderma.19 Among the trichothecenes, T-2 toxin and deoxynivalenol are reported to have diverse toxicities in humans and animals, including neurotoxicity, immunotoxicity, and hematotoxicity.20−23 However, trichodermin, 1, has been reported to possess various bioactivities, such as antifungal, antibacterial, anticancer, and nematicidal, without significant toxicities in humans and animals.24−27 Thus, further study is needed to confirm the mechanisms underlying the antiviral action of these compounds, as well as their antiviral activity in field trial.

Furthermore, to confirm the existence of PepMoV-GFP in all tested plants at 6 and 9 dpi, RT-PCR and Western blot analysis were carried out. Viral RNA (822 bp) and coat protein (33 kDa) bands were not detected in plants treated with compounds 1 (50 and 10 μM) and 2 (2 and 1 mM) at 6 dpi, whereas all bands were detected in the upper leaves at 9 dpi (Figure 4). These results showed that both compounds significantly delayed PepMoV-GFP spreading in whole host plants, and compound 1 showed stronger anti-PepMoV activity than compound 2. Compound 1 possessing stronger anti-PepMoV activity was selected for further evaluation of its potential inactivation effects in hot pepper cultivar (C. annuum L.) inbred line. The results revealed that compound 1 delayed the spread of PepMoV-GFP with an MIC of 1 mM in pepper. In addition, RT-PCR and Western blot results were consistent with the visual results (Figure 5). Collectively, these results indicate that the two trichothecene compounds from the EtOAc extract of T. albolutescens culture medium inhibited PepMoV spreading in systemic host plants, including tobacco and pepper. Protective Effects of Compound 1 on PepMoV. The protective effects of compound 1 on PepMoV were evaluated by pretreating pepper leaves with a solution of compound 1 (2 mM) or solvent alone (0.1% DMSO with 0.01 M PBS buffer) for 1 h before inoculation with PepMoV-GFP inoculum. The results are shown in Figure 6. In the solvent pretreated plants, green fluorescence was first observed at 4 dpi, and it was shown to gradually spread to the whole plant. However, green 4277

DOI: 10.1021/acs.jafc.7b01028 J. Agric. Food Chem. 2017, 65, 4273−4279

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Figure 6. (A) Protective effects of compound 1 (2 mM) in hot pepper cultivar. The photographs were taken under UV light at 3 to 7 dpi. (B) RTPCR and Western blot analysis of upper leaves in tested plants. M, marker; C, control (PepMoV-GFP with 0.1% DMSO); +, PepMoV-GFP; H, healthy.

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(2) Vance, V. B.; Moore, D.; Turpen, T. H.; Bracker, A.; Hollowell, V. C. The complete nucleotide sequence of pepper mottle virus genomic RNA: comparison of the encoded polyprotein with those of other sequenced potyviruses. Virology 1992, 191, 19−30. (3) Purcifull, D.; Zitter, T.; Hiebert, E. Morphology, host range, and serological relationships of pepper mottle virus. Phytopathology 1975, 65, 559−562. (4) Pernezny, K.; Roberts, P. D.; Murphy, J. F.; Goldberg, N. P. Compendium of pepper diseases; APS Press: St. Paul, MN, 2003; pp 33− 34. (5) Kim, Y.-J.; Jonson, M. G.; Choi, H. S.; Ko, S.-J.; Kim, K.-H. Molecular characterization of Korean pepper mottle virus isolates and its relationship to symptom variations. Virus Res. 2009, 144, 83−88. (6) Harman, G. E.; Howell, C. R.; Viterbo, A.; Chet, I.; Lorito, M. Trichoderma speciesopportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2004, 2, 43−56. (7) Reino, J. L.; Guerrero, R. F.; Hernandez-Galan, R.; Collado, I. G. Secondary metabolites from species of the biocontrol agent Trichoderma. Phytochem. Rev. 2008, 7, 89−123. (8) Peres de Carvalho, M.; Weich, H.; Abraham, W.-R. Macrocyclic trichothecenes as antifungal and anticancer compounds. Curr. Med. Chem. 2016, 23, 23−35. (9) Kupchan, S. M.; Jarvis, B. B.; Dailey, R. G., Jr; Bright, W.; Bryan, R. F.; Shizuri, Y. Tumor inhibitors. 119. Baccharin, a novel potent antileukemic trichothecene triepoxide from Baccharis megapotamica. J. Am. Chem. Soc. 1976, 98, 7092−7093. (10) Godtfredsen, W.; Vangedal, S. Trichodermin, a new sesquiterpene antibiotic. Acta Chem. Scand. 1965, 19, 1088−1102. (11) Isaka, M.; Punya, J.; Lertwerawat, Y.; Tanticharoen, M.; Thebtaranonth, Y. Antimalarial activity of macrocyclic trichothecenes isolated from the Fungus myrothecium v errucaria. J. Nat. Prod. 1999, 62, 329−331. (12) Garcia, C.; Rosso, M.; Bertoni, M.; Maier, M.; Damonte, E. Evaluation of the antiviral activity against junin virus of macrocyclic

ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge at The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01028. NMR data, NMR and MS spectra, computational conformation analysis, and calculated VCD spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(K.H.R.) Phone: +82 2 970 5618. Fax: +82 2 970 5610. Email: [email protected] *(D.L.) Phone: +82 2 3290 3017. Fax: +82 2 953 0737. E-mail: [email protected]. ORCID

Yuanqiang Guo: 0000-0002-5297-0223 Dongho Lee: 0000-0003-4379-814X Funding

This research was supported by a grant from the iPET (Korea Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries, 115090021SB010) and a grant from National Research Foundation of Korea (NRF2015R1D1A1A01060321). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.7b01028 J. Agric. Food Chem. 2017, 65, 4273−4279