Sesquiterpenes with Phytopathogenic Fungi Inhibitory Activities from a

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Bioactive Constituents, Metabolites, and Functions

Sesquiterpenes with Phytopathogenic Fungi Inhibitory Activities from a Fungus Trichoderma virens from Litchi chinensis Sonn. Zhibo Hu, Yiwen Tao, Xingyu Tao, Qinhua Su, Jiachun Cai, Can Qin, Weijia Ding, and Chunyuan Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04053 • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 4, 2019

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Journal of Agricultural and Food Chemistry

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Sesquiterpenes with Phytopathogenic Fungi Inhibitory Activities

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from a Fungus Trichoderma virens from Litchi chinensis Sonn.

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Zhibo Hu,†, Yiwen Tao,‡, Xingyu Tao, † Qinhua Su, † Jiachun Cai, † Can Qin, † Weijia

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Ding,*, † and Chunyuan Li*, †

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†

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510642, China

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‡

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Pharmaceutical Sciences and the Fifth Affiliated Hospital, Guangzhou Medical

College of Materials and Energy, South China Agricultural University, Guangzhou

Key Laboratory of Molecular Target & Clinical Pharmacology, School of

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University. Guangzhou 511436, China

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*Corresponding Author (Tel: +86-20-85280319; Fax: +86-20-85282366; E-mail:

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[email protected])

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ACS Paragon Plus Environment


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ABSTRACT: A new mono-sesquiterpene diacetylgliocladic acid (1), a new dimeric

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sesquiterpenes divirensol H (9), and two exceptionally novel trimeric sesquiterpenes

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trivirensols A and B (11 and 12), together with another eight known congeners were

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purified from an endophytic fungus Trichoderma virens FY06, derived from Litchi

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chinensis Sonn. whose fruit is a delicious and popular food. All of them were identified

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by comprehensive spectroscopic analysis, combined with biosynthetic considerations.

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Trivirensols A and B are the unprecedented trimers of which three subunits were

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connected by two ester bonds of the sesquiterpene class. Relative to the positive control

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triadimefon, all the tested metabolites showed strong inhibitory activities against at

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least one phytopathogenic fungus among Penicillium italicum, Fusarium oxysporum,

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Fusarium graminearum, Colletotrichum musae, and Colletotrictum gloeosporioides.

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Notably, as metabolites of the endophytic fungus from L. chinensis, they all presented

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strong antifungal activities against C. gloeosporioides which causes anthracnose in L.

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chinensis.

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KEYWORDS:

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Antifungal activity; Litchi chinensis Sonn.

Plant-derived fungi; Trichoderma virens; Trimeric sesquiterpene;


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INTRODUCTION

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Nowadays, plant endophytic fungi have been realized as sustainable sources to

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produce novel metabolites with agricultural or medical applications.1-6 Litchi chinensis

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Sonn. is an evergreen plant originated and widely planted in the south of China.7 Its

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fruit called lychee, is delicious and popular with lots of people.8 However, litchi

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anthracnose caused by Colletotrictum gloeosporioides Penz., is an important disease

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that affects lychee production.9 It not only causes leaves withering, spikes browning

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and rotting, early flowers and fruits dropping, but also results in fruits browning and

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rotting during storage and transportation after harvest.10 Trichoderma is a filamentous

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ascomycetes that had been found almost ubiquitously, producing various bioactive

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metabolites.11-13 Moreover, some species have been utilized as biocontrol agents to

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manage soil-borne diseases and plant pathogens.14 During our search for novel

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fungicidal leads with agricultural value from endophytes,15-18 a fungus Trichoderma

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virens (collection No. FY06) obtained from the root of L. chinensis provoked our

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attention because of its inhibitory activity against C. gloeosporioides in vitro. Therefore,

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we investigated the metabolites and separated twelve sesquiterpenes (1‒12), four of

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which were new, including one mono-, one dimeric, and two trimeric sesquiterpenes.

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The following is a description of the isolation, structure elucidation, and antifungal

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bioassay of 1‒12.

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EXPERIMENTAL

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General Experimental Procedures

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Optical rotations were measured on a MCP 300 (Anton Paar, Shanghai, China) digital



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polarimeter. UV and CD were determined via a Chirascan instrument (Applied

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Photophysics, London, UK). HRESIMS were measured on a LCMS-IT-TOF

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(Shimadzu, Japan) mass spectrometer. NMR data were detected on a Bruker AVIII

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(600MHz) NMR spectrometer (Bruker BioSpin GmbH company, Rheinstetten,

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Germany), with tetramethylsilane (TMS) as an internal standard. HPLC separation was

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using a P230II system (Elite Analytical Instrument Co., Ltd., Dalian, China) with a C18

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column (250 × 10 mm, 5 μm, H&E Co., Ltd). The types of silica gel for separation and

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TLC were 200-300 mesh and GF254, respectively (Qingdao Haiyang Co., Ltd.,

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Qingdao, China), Sephadex LH-20 (GE Healthcare, Sweden) and RP18 (AAG12S50,

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YMC company, Kyoto, Japan). All reagents were of analytical grade except the

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chromatographic pure methanol.

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Fungal Material

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Trichoderma virens FY06 was purified from the root of L. chinensis and stored in

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the College of Materials and Energy, South China Agricultural University. This strain

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was identified on the basis of its ITS sequence (No. MN102106 in GenBank) analysis.

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After the initial culture on potato dextrose agar plates, the fermentations were scaled

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up at 25 °C for 30 days in 75×1 L Erlenmeyer flasks statically, each containing the

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autoclaved rice medium (120 mL water, 65 g rice).

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Extraction and Isolation

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The resulting cultivation was extracted repeatedly using 95% ethanol. Then the

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ethanol concentrate was extracted four time with ethyl acetate (EtOAC) to afford a

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crude extract (20.7 g). It was divided into five fractions (Fr. 1-Fr. 5) on a silica gel


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column chromatograph (CC) (45×6 cm) by eluting with gradient solvent composed of

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petroleum ether (PE)/ EtOAc (v/v, 10:1, 5:1, 1:1, 1:5, 1:10). Fr. 2 was eluted (PE/EtOAc,

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v/v, 2:1) by CC (45×2.5 cm) and then purified by Sephadex LH-20 (MeOH) to yield

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compounds 1 (9.8 mg), 4 (8.4 mg), and 5 (6.3 mg). Fr.3 was subject to Sephadex LH-

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20 (MeOH) CC (105× 4.1 cm), to give 6 subfractions (Fr.3.1-Fr.3.6) on the basis of

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TLC properties (PE/EtOAc, v/v, 1:1). Fr.3.2 and Fr.3.3 was purified by semi-

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preparative HPLC with MeOH/H2O (v/v, 65:35 to 100:0, 2.5 mL/min, 20 min) to

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provide compounds 2 (8.6 mg, tR=13.2 min), 3 (7.5 mg, tR=16.8 min), 6 (13.2 mg,

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tR=9.5 min), and 7 (1.8 mg, tR=11.6 min). Fr. 4 was chromatographed on RP18 CC

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(MeOH/H2O, v/v, 60:40 to 80:20), to obtain compounds 8 (8.8 mg), 9 (8.5 mg) and 10

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(6.4 mg) and other 6 subfractions (Fr.3.1-Fr.3.6) according to TLC properties

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(PE/EtOAc, v/v, 1:3). Fr.4.4 and Fr.4.5 was purified through semi-preparative HPLC

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with MeOH/H2O (v/v, 77:23, 2.4 mL/min) to give compounds 11 (8.4 mg, tR=8.4 min)

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and 12 (6.6 mg, tR=10.2 min).

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Antifungal Assay

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The phytopathogenic fungi: Penicillium italicum (P. italicm), Fusarium oxysporum

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(F. oxysporum), Fusarium graminearum (F. graminearum), Colletotrichum musae (C.

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musae), C. gloeosporioides were obtained from the College of Agriculture, South China

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Agricultural University. The antifungal activities were determined by the broth dilution

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method, identical to previously described16, 18 using triadimefon (Aladdin Bio-Chem

103

Technology company Shanghai, China) and the solvent (v:v, 5% DMSO/H2O:potato

104

dextrose broth=1:1) as positive and negative controls, respectively.



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ECD Calculations

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Conformational analysis was undertaken by Spartan'10 (Wavefunction Inc., Irvine,

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CA) with the MMFF94 force field. Conformers within a 10 kcal/mol were optimized

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using DFT method at the 6-31 G (d, p) level. TD-DFT calculation were run for the

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resulting stable conformers using Gaussian 09 (Gaussian Inc., Wallingford. CT) with

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B3LYP/6-311+G (d, p) functionals. 30 excited states were calculated with IEFPCM

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solvent model in MeOH. ECD curves were simulated via SpecDis 1.64 (university of

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Wurzburg. Wurzburg, Germany) with a half-bandwidth of 0.3 eV.

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Diacetylgliocladic acid (1). Colorless gum, [ɑ] 25D = 42.0 (c=0.10, MeOH), UV

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(MeOH) λmax (log ε)=218 (0.39). HRESIMS m/z 339.1807 [M + H]+ (calcd. for

115

C18H27O6, 339.1808), 1H and 13C NMR (Table 1).

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Divirensol H (9). Colorless gum, [ɑ]25D = 31.00 (c=0.15, MeOH), UV (MeOH) λmax

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(log ε)=214 (1.03). HRESIMS m/z 673.2992 [M − H]− (calcd. for C33H50ClO12,

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673.2991), 1H and 13C NMR (Table 2).

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Trivirensol A (11). Colorless gum, [ɑ]25D = 5.23 (c=0.13, MeOH), UV (MeOH) λmax

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(log ε)=218 (1.10). HRESIMS m/z 857.3957 [M − H]− (calcd. for C45H61O16, 857.3960),

121

1

H and 13C NMR (Table 3).

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Trivirensol B (12). White solid, m.p.165.4–167.6 °C, [ɑ]25D = 4.39 (c=0.12, MeOH),

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UV (MeOH) λmax (log ε)=200 (0.50). HRESIMS m/z 813.4058 [M − H]−- (calcd. for

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C44H61O14, 813.4061), 1H and 13C NMR (Table 3).

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RESULTS AND DISCUSSION

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The molecular formula C18H26O6 of diacetylgliocladic acid (1) (Figure 1), was


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elucidated by HRESIMS spectrum (m/z 339.1807 [M + H]+, calcd. 339.1808), identical

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with a diacetylated derivate of gliocladic acid (2).12 Comparing the 1D NMR spectra of

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1 (Table 1) and 2, it was shown that 1 has two more acetyl groups (δH 2.03, δC 19.9,

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169.8; δH 2.00, δC 19.8, 169.8) than 2. The HMBC correlations (Figure 2) from H-16

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and H-3 to C-15, H-14 and H-18 to C-17, supported this deduction and indicated the

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structure of 1 to be 4, 14-diacetylgliocladic acid. NOE correlations (Figure 3) between

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H-3 and H-5, and between H-5 and H-11 revealed the E geometry of the ∆2, 4 double

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bond and the trans orientation of H-5 and H-10, respectively. The absolute

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configuration of 1 was ascribed to be 5R, 10R, consistent with the co-metabolite 2

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whose absolute configuration was resolved in 2011 by CD analysis,12, 19 based on the

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same relative configurations, similar optical rotations and CD spectra (∆ε: 1, 226 (+),

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263 (-); 2, 228 (+), 264 (-)) (Figures S49 and S50). This was also supported by the

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biosynthetic pathway relationships between them (Figure S58).

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Compounds 4 and 8 (Figure 1) were known sesquiterpenes which happened to be

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identical with two very recently reported metabolites rhinomilisins B and A from a

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mangrove fungus Rhinocladiella similis, respectively.20 Their structures were

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elucidated by comparison the NMR, HRESIMS and optical rotation data with those of

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rhinomilisins B and A, and further confirmed by detailed inspections of their 2D NMR

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spectra (Figures S11-S14, and Figures S19-S22). The similar curves of the measured

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and calculating ECD spectra of 4 (Figure 4) also supported the absolute configuration

147

of 4 was 5S, 6S, 7S, 10R, consistent with that of rhinomilisin B. The NMR data of 4 and

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8 were also included in Tables 1 and 2, respectively, because they were firstly detected



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in the solvent of acetone-d6 herein.

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Divirensol H (9) possesses the molecular formula C33H51ClO12 (10 double-bond

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equivalents) determined by HRESIMS at m/z 673.2992 ([M − H]−, calcd 673.2991). It

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could be speculated that 9 was a chlorinated sesquiterpene ester formed by dimerization

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of 4 to satisfy its molecular weight and similar NMR spectral data of each subunit to 4.

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However, there are three more oxygenated methyl groups (δH 3.80/δC 52.1, 1-OCH3; δH

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3.68/δC 52.3, 15-OCH3; δH 3.65/δC 52.4, 15'-OCH3) in 9, whose positions were assigned

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by HMBC correlations from H-3 and H-1-OCH3 to C-1, H-6 and H-15-OCH3 to C-15,

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H-6' and H-15'-OCH3 to C-15', respectively. Additionally, the chlorine atom at C-14'

158

was revealed by H-14' to C-6', C-7' and C-8'. HMBC correlation from H-14 to C-1′

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indicated that C-1′ and C-14 were connected through an oxygen bridge to form the ester

160

bond (Figure 2). NOE correlations between H-3 and H-5, and between H-3' and H-5',

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disclosed E configurations of ∆2, 4/∆2', 4'. NOE correlations between H-11/H-14 and H-

162

5, between H-11'/H-14' and H-5', between H-6 and H-10, and between H-6' and H-10',

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along with J5,6 12.0 Hz and J5',6' 12.6 Hz, required cis H-5/H-14, cis H-5'/H-14', cis H-

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6/H-10, cis H-6'/H-10', trans H-5/H-10, and trans H-5'/H-10' geometries, suggesting

165

the chair configuration of each six-membered ring. Thus, each partial structure of 9

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(Figure 2) had the same relative configuration as 4. The similar CD spectra (∆ε: 4, 214

167

(+), 248 (-); 9, 211 (+), 241 (-) ) (Figures 4 and S55) and biosynthetic relationships

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between 9 and 4 (Figure S58) further allowed determination of the identical absolute

169

configurations at their corresponding chiral carbons.

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Trivirensol A (11) had the molecular formula C45H62O16 (15 double-bond equivalents)


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deduced from its HRESIMS at m/z 857.3957 ([M − H]−, calcd 857.3960), consistent

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with a trimeric sesquiterpene ester formed by condensation of compound 8 and

173

hydroheptelidic acid (6)21 to remove a hydrogen chloride. Comparison of the 13C NMR

174

data for 11 (Table 3) with 8 (Table 2) and 6 showed great similarity, except the chemical

175

shift of C-14' changing from 47.7 in 8 to 65.2 in 11. This result supported the

176

assumption deduced from the molecular formula mentioned above. Detailed HMBC

177

and 1H–1H COSY spectral inspection (Figure 2) confirmed the presence of three

178

sesquiterpene subunits, corresponding to 8 and hydroheptelidic acid (6). Two ester

179

bonds connecting the three sesquiterpenes were ambiguously assigned by HMBC

180

correlations from H-14 to C-1', and from H-14' to C-1'', respectively. The relative

181

configurations of the subunits in 11 (Figure 2) were in common with 6 and 7,22 deduced

182

from their similar coupling constants of J5,6, J5',6' and J5'',6'' and NOE correlations (Figure

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3) between H-3/H-11/H-14α and H-5, between H-3'/H-11'/H-14'α and H-5', between H-

184

3''/H-11''/H-14'' and H-5'', between H-6 and H-10, between H-6' and H-10', and between

185

H-6'' and H-10''. Biosynthetic relationship considerations among the co-metabolites 6,

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7 and 11 (Figure S58) and their similar CD spectra (∆ε: 6, 205 (+), 234 (-); 7, 214 (+),

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248 (-); 11, 212 (+), 250(-)) (Figures S52, S53 and S56) further confirmed the absolute

188

configuration for 11, which was also consistent with 6 and 7.

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Trivirensol B (12) had the molecular formula C44H62O14 (14 double-bond equivalents)

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inferred from the HRESIMS at m/z 813.4058 ([M − H]−, calcd 813.4061). A

191

comparable analysis of the 13C NMR spectra of 11 and 12, revealed that 12 was also a

192

trisesquiterpene ester with a subunit corresponding to 8. The most obvious differences



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of NMR data in 12 compared to 11 in the remaining subunit (C-1''‒C-15'') were the

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lacks of a carbonyl (δC 177.2), an oxygenated quaternary carbon (δC 75.0) and an

195

alkylmethine (δC 52.4, δH 2.20), and the appearance of two olefinic carbons (δC 121.8;

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δC 140.2.0). Moreover, the double-bond equivalent of 12 was one less than that of 11.

197

These assigned the remaining partial structure of 12 to be gliocladic acid (2), supported

198

by their similar

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NOESY spectral inspection (Figures 2 and 3) confirmed the planar structure and

200

relative configuration as shown. Similar to 11, the positions of two ester linkages were

201

assigned by HMBC correlation from H-14 to C-1', and from H-14' to C-1'', respectively.

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NOE correlations of the subunit corresponding to 8 in 12 were identical to those of 7,

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8 and the same moiety in 11. NOE correlations of the remaining structure in 12 were

204

consistent with those of 2. These and the coupling constants of J5,6 12.6 Hz, and J5',6'

205

12.6 Hz in 12, revealed the relative configurations of the subunits in 12 as shown

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(Figure 2). Biosynthetic considerations among 2, 7, 8 and 12 (Figure S58) and their

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similar CD spectra (∆ε: 2, 228 (+), 264 (-); 7, 214 (+), 248 (-); 8, 223 (+), 252 (-); 12,

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226 (+), 262 (-)) (Figures S50, S53, S54 and S57) allowed determination of the absolute

209

configuration for 12, consistent with 2, 7 and 8.

13

C NMR spectral data. Comprehensive HMBC, 1H–1H COSY and

210

The rest of sesquiterpenes were identified as gliocladic acid (2),12 3-acetylgliocladic

211

acid (3),12 xylaric acid B (5),19 hydroheptelidic acid (6),21 chlorine heptelidic acid (7),22

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and divirensol D (10)12 by comparison with the spectroscopic data previously reported.

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All compounds were evaluated using the broth dilution assay as previous

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descriptions16,

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for their inhibitory activities against phytopathogenic fungi F.


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oxysporum, C. gloeosporioides, C. musae, P. italicm, and F. graminearum except 7 due

216

to the small amounts. As shown in Table 4, they all exhibited moderate to high activities

217

on these fungi except 6, presenting no inhibition towards F. oxysporum (MIC > 400

218

μg/mL). Notably, compounds 9 and 11 belonging to dimeric and trimeric

219

sesquiterpenes respectively, both showed potent inhibitory activities against all the

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pathogens, and their activities were superior to those of the positive control triadimefon.

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In addition, 9 was more active, whose activities towards F. oxysporum, C.

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gloeosporioides, C. musae, P. italicm, and F. graminearum were four, four, one, eight,

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sixteen times as high as those of 11, and eight, eight, three point two, eight, twenty-four

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times as high as those of triadimefon.

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Compounds 1‒6 are mono-sesquiterpenes. Especially, the only difference between 4

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and 5 is that the substituents at C-15 are -Cl and –OCH3, respectively. They both

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exhibited strong activities towards F. oxysporum with the same MIC values as

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triadimefon and moderate activities towards P. italicm with MIC values equivalent to

229

each other, suggesting that –Cl and –OCH3 substituted at C-15 had the same impacts

230

on the antifungal effects toward F. oxysporum and P. italicm. Meanwhile, compound 4

231

showed higher activities against other three fungi than triadimefon. However,

232

compound 5 only showed equivalent activity towards C. gloeosporioides and lower

233

activity towards C. musae and F. graminearum, relative to triadimefon. It suggested

234

that -Cl substituted at C-15 exerted better enhancing effects on these antifungal

235

activities than –OCH3. Compounds 1 and 3 are 4, 14-diacetylated and 3-acetylated

236

derivatives of 2, respectively. But no obvious structure-activity relationship was



237

observed for the three analogues. The inhibitory activities of all of them towards F.

238

graminearum, of 1 towards C. gloeosporioides and C. musae, and of 3 towards F.

239

oxysporum were stronger than those of triadimefon. Meanwhile, the activities of 1 and

240

2 towards F. oxysporum, and of 2 and 3 towards C. gloeosporioides, were equal to those

241

of triadimefon. The remaining compound 6, showed selective strong activity against C.

242

gloeosporioides with the same MIC value as triadimefon.

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Compounds 8 and 10 belong to the dimeric sesquiterpenes. They both showed strong

244

activity against F. oxysporum, with MIC values equal to triadimefon. Compound 10

245

also exhibited equivalent activity towards C. gloeosporioides as triadimefon, whereas

246

compound 8 exhibited higher activities against both C. gloeosporioides (MIC, 25

247

μg/mL) and F. graminearum (MIC, 100 μg/mL) than triadimefon (MICs, 50 and 150

248

μg/mL). Compound 12 was another trimeric sesquiterpene ester besides 11. It showed

249

strong activities against C. gloeosporioides (MIC=50 μg/mL) and F. oxysporum

250

(MIC=100 μg/mL), consistent with triadimefon.

251

The results revealed the potentials of the sesquiterpenes to be phytopathogenic

252

fungicides. Interestingly, all the sesquiterpenes showed strong antifungal activities

253

against C. gloeosporioides, suggesting that the sesquiterpenes produced by this

254

endophytic fungus from its host L. chinensis might act as a defensive role by inhibiting

255

some invasive phytopathogenic fungi, especially C. gloeosporioides.

256

Trivirensols A and B (11 and 12) are the unprecedented sesquiterpene trimers of

257

which three subunits were connected by two ester bonds. Additionally, this is the first

258

report for rhinomilisins B (4) and A (8) as the metabolites from Trichoderma sp. To the


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best of our knowledge, divirensol H (9) is a new member of such a rare kind of dimeric

260

sesquiterpene ester. Till now, only approximately ten examples including trichoderonic

261

acid B,23 divirensols A−G,12 rhinomilisin A,20 and (4'E, 1S, 6S, 7R,10S, 6'S, 7'R)-

262

aspergilloid A24 had been previously found in this class. All the isolated co-metabolites

263

can be regarded as the derivatives of heptelidic acid25 which had been previously found

264

to be generated from a precursor farnesyl pyrophosphate through 1,10- and 1,6-

265

cyclization.26 Heptelidic acid was transformed into the intermediate A via hydrolysis

266

and further changed into 7 via chlorination. 4 was derived from 7 by hydrolysis. The

267

intermediate B was formed by hydrolysis of heptelidic acid twice, and transformed into

268

5 and 6 via methylation and intramolecular esterification, respectively. 2 was produced

269

from B by dehydration and decarboxylation, and converted into 3 and 1 by acetylation

270

and diacetylation, respectively. The dimeric sesquiterpenes 8 and 10 were produced via

271

esterification of A and 7, and A and 2, respectively. 9 was generated by esterification

272

of B and 4, followed by methyl esterification. The trimeric sesquiterpenes 11 and 12

273

were yielded by trimerization through esterification of A, A and 6, and of A, A and 2,

274

respectively. The plausible biogenetic relationship discussed above agreed with

275

previous reports related to such a kind of sesquiterpene class,20 and was summarized in

276

Figure S58. Since the esterification reaction did not change the configuration of chiral

277

carbons of each involved mono-sesquiterpene, the above-mentioned biogenetic

278

pathway indicated that the absolute configurations of the isolated dimeric and trimeric

279

sesquiterpenes were consistent with those of the corresponding co-metabolic mono-

280

sesquiterpenes. Previously, such kind of molecules had been found to possess



281

glyceraldehyde-3-phosphate dehydrogenase inhibitory,27 X DNA polymerase

282

inhibitory,23 antitumor,22,

283

antibacterial and cytotoxic 20 activities.

284

FUNDING SOURCES

28

antimalarial,29 insecticidal30, hepatic protective24,

285

This work was supported by Guangdong Natural Science Foundation

286

(2018A030313582, 2018A0303130201, 2016A030313588), Science and Technology

287

Project of Guangzhou (201707010342), and Special Fiscal Fund of Guangdong

288

Provincial Oceanic and Fishery Administration in 2017 (A201701607).

289

SUPPORTING INFORMATION

290

This material is available free of charge via the Internet at http://pubs.acs.org.

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1D，2D NMR, HRESIMS, and UV spectra of compounds 1, 4, 8, 9, 11, and 12, CD

292

spectra of compounds 1–3, 6–9, 11–12, and plausible biosynthetic pathway for

293

compounds 1–12 (Figures S1‒S58).

294

Corresponding Authors:

295

*Telephone: +86-20-85280319. E-mail: [email protected].

296

* Telephone: +86-20-85280319. E-mail: [email protected]

297

Author Contributions:

298

Zhibo Hu and Yiwen Tao contributed equally to this work.


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299

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FIGURE CAPTIONS Figure 1. Structures of compounds 1‒12. Figure 2. Key 1H-1H COSY and HMBC of compounds 1, 9, 11 and 12. Figure 3. Key NOESY of compounds 1, 9, 11 and 12. Figure 4. Calculated and experimental ECD curves of compound 4.



Position

a

1 2 3 4 5 6 7 8

δC 166.8 128.1 57.9 151.6 39.2 125.1 135.7 25.7

9

20.9

10 11 12 13 14 15

44.8 28.5 20.7 16.2 67.1 169.8

16 17 18

19.9 169.8 19.8

Table 1. NMR Data of Compounds 1 and 4 1 4 δH/ppm, multi (J/Hz) δC δH/ppm, multi (J/Hz) 167.9 133.6 4.86, s 56.5 4.28, s 6.78, d (10.8) 144.8 6.50, d (10.8) 3.27, t (10.8) 40.4 2.90, dd (10.8, 10.8) 5.37, s 58.6 2.75, d (11.6) 71.9 α2.12, m 34.9 α2.40, dt (12.0, 3.6) β2.09, m β1.44, dt (12.0, 3.6) α1.81, m 20.2 α1.66, m β1.35, m β1.21, m 1.41, m 46.3 1.42, m 1.69, m 28.2 1.68, m 0.97, d (6.6) 21.0 0.90, d (6.6) 0.83, d (6.6) 15.2 0.79, d (6.6) 4.45, s 174.1 48.6 α4.07, d (12.0) β3.91, d (12.0) 2.03, s 2.00, s

Measured in acetone-d6


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Page 21 of 30


Position 1 1-OCH3 2 3 4 5 6 7 8 9 10 11 12 13 14 15 15-OCH3 1' 2' 3' 4' 5' 6' 7' 8' 9' 10' 11' 12' 13' 14' 15'

Table 2. NMR Data of Compounds 8 and 9 8a δH/ppm, multi δC δC (J/Hz) 166.0 167.4 52.1 129.5 133.5 61.0 α5.02, d (14.4) 57.5 β5.26, d (14.4) 146.1 7.31, d (5.4) 144.5 39.7 2.75, m 40.0 53.0 3.68, d (13.2) 57.4 72.4 72.3 35.0 α1.49, m 35.7 β2.25, m 20.7 α1.32, m 20.2 β1.75, m 48.4 1.66, m 46.3 27.4 2.14, m 28.3 20.8 0.99, d (7.2) 15.5 14.7 0.95, d (7.2) 14.7 66.0 α4.48, d (12.6) 65.6 β5.28, d (12, 6) 171.8 173.8 52.3 165.4 166.7 129.4 133.5 61.2 α5.02, d (14.4) 57.2 β5.35, d (14.4) 146.0 7.31, d (5.4) 145.2 39.9 2.69, m 40.2 52.8 3.82, d (12.6) 57.5 72.4 72.3 171.8 α1.43, m 35.4 β2.42, m 20.9 α1.39, m 20.4 β1.75, m 48.1 1.66, m 46.1 27.3 2.14, m 28.2 21.0 0.99, d (7.2) 15.5 14.7 0.97, d (7.2) 14.7 47.7 α3.99, d (12.0) 48.4 β4.83, d (12.0) 171.6 173.5


9b δH (J in Hz) 3.80, s α4.29, d (12.6) β4.35, d (12.6) 6.45, d (10.8) 2.88, m 2.66, d (12.0) α1.40, m β2.04, m α1.26, m β1.67, m 1.32, m 1.64, m 0.75, d (6.6) 0.91, d (6.6) α4.39, d (12.0) β4.60, d (12, 0) 3.68, s

4.37, t (10.8) 6.51, d (10.8) 2.80, m 2.79, d (12.6) α1.44, m β2.41, m α1.15, m β1.72(m) 1.37, m 1.64, m 0.77, d (6.6) 0.92, d (6.6) α3.91, d (12.0) β3.89, d (12.0)


15'-OCH3 a

52.4

Measured in acetone-d6; b Measured in CDCl3.


Page 22 of 30

3.65, s

Page 23 of 30


Table 3. NMR Data of Compounds 11 and 12 11a Position

δC

1 2 3

166.3 129.7 61.2

4 5 6 7 8

145.8 39.7 52.9 72.6 35.1

9

20.8

10 11 12 13 14

48.1 27.4 20.7 14.7 66.0

15 1' 2' 3'

171.9 165.4 129.7 61.2

4' 5' 6' 7' 8'

146.8 39.6 52.7 72.5 35.0

9'

21.1

10' 11' 12' 13' 14'

48.1 27.4 20.8 14.8 65.2

15' 1''

171.9 166.9

δH/ppm, multi (J/Hz)

α5.01, d (14.4) β5.26, d (14.4) 7.27, d (4.2) 2.73, m 3.68, d (12.6) α1.49, m β2.28, m α1.37, m b1.72, m 1.66, m 2.15, m 0.98, d (7.2) 0.94, d (7.2) α4.48, d (12.6) β5.30, d (12,6)

α5.05, d (14.4) β5.33, d (14.4) 7.35, d (4.8) 2.76, m 3.64, d (12.6) α1.47, m β2.32, m α1.38, m β1.74, m 1.66, m 2.12, m 0.98, d (7.2) 0.94, d (7.2) α4.50, d (12.0) β5.20, d (12.0)

δC 166.5 129.7 61.3 145.6 39.7 52.9 72.6 34.9 20.7 48.1 27.3 20.7 14.8 66.0 172.0 165.5 129.5 61.2 146.5 39.7 52.6 72.5 34.9 21.0 48.1 27.3 20.7 14.7 65.4 171.9 167.4


12a δH/ppm, multi (J/Hz)

α5.01, d (14.4) β5.26, d (14.4) 7.27, d (4.2) 2.73, m 3.69, d (12.6) α1.45, m β2.30, m α1.40, m b1.71, m 1.64, m 2.14, m 0.98, d (7.2) 0.94, d (7.2) α4.50, d (12.6) β5.28, d (12.6)

α5.03, d (14.4) β5.33, d (14.4) 7.34, d (4.8) 2.75, m 3.67, d (12.6) α1.48, m β2.26, m α1.42, m β1.73, m 1.64, m 2.13, m 0.98, d (7.2) 0.94, d (7.2) α4.51, d (12.0) β5.17, d (12.0)


a

2'' 3''

133.7 56.2

4'' 5'' 6'' 7'' 8''

144.4 40.2 52.4 75.0 32.0

9'' 10'' 11'' 12'' 13'' 14''

20.9 45.1 28.2 15.0 20.8 74.7

15''

177.2

α4.17, d (12.0) β4.26, d (12.0) 6.67, d (11.2) 2.71, m 2.20, d (10.8) 1.76, m 1.33, m 1.41, m 1.77, m 0.75, d (7.2) 0.95, d (7.2) α4.52, d (9.6) β4.08, d (9.6)

132.1 56.0 149.5 38.7 121.8 140.2 25.5 21.0 45.4 28.6 16.4 21.2 65.0

Measured in acetone-d6.


Page 24 of 30

α4.39, d (12.0) β4.43, d (12.0) 6.64, d (10.2) 3.30, m 5.28, d (6.6) α1.98, m β1.96, m 1.35, m 1.34, m 1.70, m 0.84, d (7.2) 0.95, d (7.2) α4.54, d (9.6) β3.93, d (9.6)

Page 25 of 30


Table 4. The Antifungal Activity Compounds 1‒6，and 8‒12

Compounds

F. oxysporum

C. gloeosporioides

C. musae

P. italicm

F. graminearum

MIC, μg/mL 1

100

25

50

100

100

2

100

50

100

200

100

3

50

50

100

200

100

4

100

25

12.5

100

25

5

100

50

100

100

200

6

＞400

6.25

100

100

200

8

100

25

100

100

100

9

12.5

6.25

25

6.25

6.25

10

100

50

100

200

200

11

50

25

25

50

100

12

100

50

100

100

200

Triadimefona

100

50

80

50

150

a

positive control towards fungi.



Figure 1


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Figure 2



Figure 3


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Page 29 of 30


Figure 4



TABLE OF CONTENTS GRAPHICS


Page 30 of 30

Sesquiterpenes with Phytopathogenic Fungi Inhibitory Activities from a

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