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Diterpenoids from Streptomyces sp. SN194 and Their Antifungal Activity against Botrytis cinerea Yuhui Bi† and Zhiguo Yu*,†,‡ †

College of Plant Protection, Shenyang Agricultural University, Shenyang 110866, People’s Republic of China Engineering & Technological Research Center of Biopesticide for Liaoning Province, Shenyang 110866, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: Botrytis cinerea is a serious phytopathogen affecting a wide range of crops around the world. Many fungicides targeting Botrytis cinerea have failed due to the pathogen’s genetic plasticity. In an effort to search for new fungicides from natural products, two new diterpenoids, named chloroxaloterpin A, 1, and B, 2, were isolated from culture broth of Streptomyces sp. SN194 along with four known diterpenoids, viguiepinol, 3, and oxaloterpins C−E, 4−6. Their structures were elucidated based on extensive MS, NMR, and X-ray crystallography analyses. Both the [(2-chlorophenyl)amino]carbonyl carbanic acyl group in 1 and the 2-[(2-chlorophenyl)amino]-2-oxo-acetyl group in 2 are discovered in natural products for the first time. All six compounds were tested against Botrytis cinerea, and chloroxaloterpin A, 1, and B, 2, demonstrated strong inhibitory activity against spore germination with EC50 of 4.40 and 4.96 μg/mL, respectively. KEYWORDS: Streptomyces, diterpenoids, single-crystal X-ray diffraction, Botrytis cinerea, antifungal activity, spore germination

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INTRODUCTION

spore germination assay and a mycelial inhibition assay, leading to discussions on their structure−activity relationships.

Botrytis cinerea is a serious phytopathogen causing gray mold disease in over 200 plant species around the world.1 Its long quiescent period and the ability to rot fruit tissues postharvest is especially detrimental to cash crops like grapes and berries. Many infections are discovered only after crops were transported to the market, which results in heavy economic losses.2 Although many fungicides have been developed to counter Botrytis cinerea, few remain potent due to genetic plasticity of the pathogen.3 The Fungicide Resistance Action Committee (FRAC) has placed Botrytis cinerea at high-risk of fungicide resistance.4 The scientific intricacy and economic damage conferred by Botrytis cinerea has earned it a place among the top 10 fungal plant pathogens.5 Thus, novel fungicides are constantly in demand to combat the damaging effects of Botrytis cinerea. Natural products, with their wide spectrum of bioactivities and environmentally friendly attributes, remain the most promising source of lead molecules for agricultural chemicals.6−9 As the largest class of natural products, terpenoids perhaps have the most structural diversity and significant biological properties.10 Terpenoids are commonly produced by secondary metabolism in actinomycetes, one of the prominent sources for novel bioactive compound discovery.11−14 Therefore, actinomycetes terpenoids are important targets for agricultural lead compounds discovery. As part of our ongoing efforts to seek new bioactive natural products,15−17 Streptomyces sp. SN194 was isolated from a soil sample collected in Fengcheng City of Liaoning Province, China, and was cultured in the laboratory subsequently. From the CH2Cl2 extract of the fermentation broth of Streptomyces sp. SN194, two new diterpenoids were isolated along with four other known diterpenoids. Their structures were determined based on MS, NMR, and X-ray crystallography analyses. All the isolated compounds were tested against Botrytis cinerea in a © 2016 American Chemical Society

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MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were measured with an AP-300 polarimeter (Atago, Tokyo, Japan). The melting points of the products were determined on an X-5 micro melting point apparatus (Beijing Tech Instrument Co. Ltd., Beijing, China). NMR spectra were recorded on an Avance-600 NMR spectrometer (Bruker, Karlsruhe, Germany) at room temperature. Carbon signals and the residual proton signals of CDCl3 (δC 77.0 and δH 7.26) were used for calibration. High-resolution electrospray ionization mass spectrometry (HRESIMS) data were recorded on a 6500 series quadrupole-time-of-flight (Q-TOF) mass spectrometer (Agilent, Santa Clara, CA). X-ray diffraction intensity data were collected on an APEX-II charge-coupled device (CCD) area detector diffractometer (Bruker, Karlsruhe, Germany) with graphite monochromated Cu Kα radiation (λ = 1.54178 Å) at 173 K and with graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 296 K. The number of spores germinated at each concentration was counted under an Eclipse 80i microscope (Nikon, Tokyo, Japan), and photos were taken by the imaging software NIS-Element F3.2. Highperformance liquid chromatography (HPLC) analysis was performed on a 1260 Infinity LC system (Agilent, Santa Clara, CA), and the column used was a 250 mm × 4.6 mm i.d., 5 μm, ZORBAX Eclipse XDB (Agilent, Santa Clara, CA). Semipreparative HPLC was performed on a 1260 series system (Agilent, Santa Clara, CA), and the column used was a 250 mm × 9.4 mm i.d., 5 μm, ZORBAX Eclipse XDB (Agilent, Santa Clara, CA). Column chromatography was performed using silica gel (100−200 mesh) (Qingdao Ocean Chemical Co. Ltd., Qingdao, China) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). All chemical reagents were purchased Received: Revised: Accepted: Published: 8525

August 15, 2016 October 26, 2016 October 29, 2016 October 29, 2016 DOI: 10.1021/acs.jafc.6b03645 J. Agric. Food Chem. 2016, 64, 8525−8529

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Journal of Agricultural and Food Chemistry

Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data of Compounds 1 and 2 in CDCl3

from chemical reagent company (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and used without further purification. Actinomycete Material. Streptomyces sp. SN194 was isolated from a soil sample collected in May 2014 from Fengcheng City of Liaoning Province, China (40° 35′ 49.2″ N, 124° 15′ 29.8″ E) at a height of 87 m. The actinomycete isolate was identified by phylogenetic analysis, comparing to 16S rRNA sequences available on the EzTaxon database. The result showed that the strain had genetic resemblances to Streptomyces pluricolorescens AB184162, Streptomyces globisporus AB184203, Streptomyces parvus AB184756, Streptomyces badius AY99978, Streptomyces sindenensis AB184759, and Streptomyces rubiginosohelvolus AB184240. The strain was subsequently named Streptomyces sp. SN194 (Genbank accession no. KX619648) and was deposited in the Laboratory of Microbial Metabolites, College of Plant Protection, at Shenyang Agricultural University, China. Fermentation and Extraction. The Streptomyces sp. SN194 was preserved as a spore solution at −80 °C. A two-stage fermentation was performed, and in both stages, F medium (i.e., sucrose, 100 g; glucose, 10 g; casamino acids, 0.1 g; yeast extract, 5 g; 3-(N-morpholino) propanesulfonic acid (MOPS), 21 g; trace elements, 1 mL; K2SO4, 0.25 g; MgCl2·6H2O, 10 g, in a final volume of 1 L of H2O, pH 7.0) was used.17 In the first stage, a 250 mL Erlenmeyer flask, containing 50 mL of the F medium, was inoculated with 10 μL of Streptomyces sp. SN194 spore solution and incubated with shaking (180 rpm) at 28 °C for 2 d to prepare the seed culture. In the second stage, 24 2-L Erlenmeyer flasks, each containing 400 mL of F medium, were inoculated with 20 mL of the seed culture and left for fermentation for 7 d under identical conditions. The production cultures were centrifuged at 5000 rpm and 4 °C for 30 min to remove mycelia, and the broth was extracted with 3% Amberlite XAD 16 resin for 4 h at room temperature with agitation. Resin was harvested by centrifugation and extracted four times with methanol. The combined methanol extracts were then concentrated under reduced pressure to afford the crude extract. Isolation and Purification. The dried extract was redissolved in 50% MeOH (600 mL). The solution was extracted four times with equal volumes of CH2Cl2. The CH2Cl2 extract was collected and concentrated on a rotary evaporator under vacuum at 28 °C to yield 3.65 g of solid brown residue. The CH2Cl2 extract was subjected to silica gel chromatography (350 mm × 25 mm i.d.) eluted stepwise with CH2Cl2−MeOH (100:0, 50:1, 20:1, 10:1, and 0:100, 2 L each) as the mobile phase to afford five fractions, F1−F5. F2 was separated by silica gel column chromatography using CH2Cl2−MeOH (100:1) as the mobile phase to yield compounds 1 (6.1 mg) and 2 (7.4 mg). Fraction F3 was subjected to gel chromatography on Sephadex LH-20 eluted with MeOH to yield compound 3 (37.8 mg) and a mixture. The mixture was then purified by reverse-phase semipreparative HPLC applying a CH3CN−H2O gradient (with 0.1% HCOOH added to both solvents) from 70% to 80% CH3CN and a flow rate of 3.6 mL/ min for 35 min, UV detection was at 210 nm. Pure compounds 5 (11.8 mg), 6 (16.4 mg), and 4 (13.4 mg) were eluted at 19.9, 25.7, and 29.8 min, respectively. X-ray Crystallography of Compounds 1 and 2. A colorless crystal of compound 1 was obtained from an n-heptane solution. Crystal data of 1 (Cu Kα radiation) was collected on an APEX-II CCD area detector diffractometer (Bruker, Karlsruhe, Germany) with graphite monochromated Cu Kα radiation (λ = 1.54178 Å). A colorless crystal of compound 2 was obtained from an acetone solution. Crystal data of 2 (Mo Kα radiation) was collected on an APEX-II CCD area detector diffractometer (Bruker, Karlsruhe, Germany) with graphite monochromated Mo Kα radiation (λ = 0.71073 Å). Chloroxaloterpin A, 1. Colorless crystal; [α]24 D -27.03 (c 0.74, CHCl3); mp, 204.4−204.8 °C; 1H NMR and 13C NMR spectroscopic data (CDCl3), see Table 1; HRESIMS m/z 507.2383 [M + Na]+ (calcd C28H37ClN2O3Na, 507.2390). X-ray crystallographic data: monoclinic, space group P21 with a = 12.157(2) Å, b = 6.0191(12) Å, c = 17.705(4) Å, β = 104.10(3)°, V = 1256.5(4) Å3, Z = 2, Dcalc = 1.282 g/cm3, R = 0.0367, wR2 = 0.0943, T = 173(2) K. Crystal size, 0.05 × 0.05 × 0.28 mm3. Absolute structure parameter 0.039(15).

1

2

δC, type

δH, mult (J in Hz)

δC, type

1

38.2, CH2

1.95, dt (13.8, 3.42) 1.45, td (13.4, 4.4)

38.2, CH2

2 3

24.5, CH2 84.1, CH

1.78, m 4.56, dd (11.5, 4.7)

24.2, CH2 85.4, CH

4 5

38.0, qC 44.9, CH

6 7 8 9 10 11 12 13 14

17.9, CH2 26.4, CH2 29.2, CH 150.2, qC 37.2, qC 116.2, CH 37.4, CH2 34.7, qC 41.3, CH2

15

150.0, CH

16

109.0, CH2

17 18 19 20 21 22 23 24 25 26 27 28 NH NH

22.2, CH3 25.1, CH3 16.1, CH3 27.5, CH3 153.9, qC 150.2, qC 134.5, qC 123.1, qC 129.0, CH 124.2, CH 127.3, CH 121.1, CH

position

1.53, dd (12.6, 6.1) 1.78, 1.60, m 1.72, 1.22, m 2.29, m

5.37, m 2.03, 1.78, m 1.40, m; 1.03, t (11.7) 5.82, dd (17.5, 10.7) 4.94, d (17.5); 4.87, d (10.7) 0.97, s 1.11, s 0.96, s 0.95, s

7.38, dd (8.0, 1.3) 7.03, td (7.9, 1.4) 7.25, m 8.31, dd (8.3, 1.3) 7.63, br s 10.49, br s

38.2, qC 44.9, CH 18.0, CH2 26.4, CH2 29.2, CH 150.2, qC 37.3, qC 116.3, CH 37.4, CH2 34.7, qC 41.3, CH2 150.0, CH 109.0, CH2 22.3, CH3 25.1, CH3 16.2, CH3 27.6, CH3 159.6, qC 153.8, qC 133.2, qC 123.0, qC 129.0, CH 125.6, CH 127.7, CH 120.8, CH

δH, mult (J in Hz) 1.98, dt (13.7, 3.3); 1.47, dd (13.7, 3.8) 1.82, m 4.72, dd (11.7, 4.4) 1.57, m 1.82, 1.63, m 1.71, 1.29, m 2.31, m

5.39, m 2.31, 1.82, m 1.42, m; 1.05, t (11.8) 5.82, dd (17.5, 10.7) 4.94, d (17.5); 4.87, d (10.7) 0.98, s 1.14, s 1.11, s 0.95, s

7.42, 7.12, 7.32, 8.48, 9.48,

d (8.0) t (7.6) t (7.7) d (8.2) br s

Chloroxaloterpin B, 2. Colorless crystal; [α]24 D -30.61 (c 0.98, CHCl3); mp, 138.6−139.4 °C; 1H NMR and 13C NMR spectroscopic data (CDCl3), see Table 1; HRESIMS m/z 492.2279 [M + Na]+ (calcd C28H36ClNO3Na, 492.2281). X-ray crystallographic data: triclinic, space group P1 with a = 6.1082(12) Å, b = 10.691(2) Å, c = 10.829(2) Å, α = 71.00(3)°, β = 73.75(3)°, γ = 75.22(3)°, V = 631.3(2) Å3, Z = 1, Dcalc = 1.236 g/cm3, R = 0.0552, wR2 = 0.1510, T = 296(2) K. Crystal size, 0.11 × 0.12 × 0.16 mm3. Absolute structure parameter 0.04(9). Effect of Compounds on the Spore Germination of Botrytis cinerea. The phytopathogenic fungus included in the assay was grown on PDA plates in darkness at 25 °C for 2 weeks. The spores were harvested from sporulating colonies and suspended in sterile distilled water containing 0.1% (v/v) Tween 80. The concentrations of spores in suspension were determined using a hemocytometer and adjusted to 1 × 106 spores per mL.18,19 The stock solutions of the tested compounds dissolved in DMSO were diluted 100-fold with sterile distilled water, which were further prepared as 2-fold dilutions for the assay, in which the final concentration of DMSO was 1% (v/v).20 Sterile distilled water with 1% DMSO was used as negative control. A 30-μL spore suspension was placed on separate glass slide containing 30 μL of prepared test solutions for a final volume of 60 μL. The inoculated glass slides were 8526

DOI: 10.1021/acs.jafc.6b03645 J. Agric. Food Chem. 2016, 64, 8525−8529

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Journal of Agricultural and Food Chemistry incubated in a moisture chamber at 25 °C for 12 h. A spore was considered to be germinated if the germ tube length was longer than the short radius of the spore,19 and the number of spores germinated at each concentration was counted under the microscope. The percentage of spore germination was calculated, and the half maximal effective concentration (EC50) values were derived from the data analysis of the concentration−inhibition rate. The experiment was independently performed three times under the same conditions. The antifungal agent pyrimethanil was used as the positive control.21 Effect of Compounds on the Mycelial Growth of Botrytis cinerea. The mycelial inhibition assay was performed using the mycelial growth inhibition method.19 PDA medium with 1% DMSO and 1% Tween 80 was used as the negative control. The antifungal agent pyrimethanil was used as the positive control.

signals from 28 carbons: 20 carbon signals were attributed to the viguiepinol skeleton, and the remaining eight carbon signals belong to a substituent. Further analyses of the 13C NMR and HSQC spectra of 1 attributed the eight carbon signals to four olefinic methine groups (δC 129.0, 124.2, 127.3, and 121.1), two olefinic quaternary carbons (δC 134.5 and 123.1), and two carbonyl groups (δC 153.9 and 150.2). The 1H NMR data also showed six hydrogens belonging to the substituent, including four olefinic protons [δH 7.38 (1H, dd, J = 8.0, 1.3 Hz, H-25), 7.03 (1H, td, J = 7.9, 1.4 Hz, H-26), 7.25 (1H, m, H-27), and 8.31 (1H, dd, J = 8.3, 1.3 Hz, H-28)] and two broad singlet protons [δH 7.63 (1H, br s) and 10.49 (1H, br s)]. These results provided evidence for the presence of a 1,2-disubstituted benzene ring in the substituent, which was also confirmed by the COSY correlations between H-25/H-26, H-26/H-27, and H-27/H-28 as well as the HMBC correlations of H-25/C-23, C-27; and H-28/C-24, C-26 (Figure 2). Long-range couplings were observed between the broad singlet proton [δH 10.49 (1H, br s)] to C-24 and C-28 in the HMBC spectrum of 1, leading to the conclusion that the secondary nitrogen coupled to the singlet proton [δH 10.49 (1H, br s)] was attached to the quaternary carbon C-23. Moreover, the long-range couplings of the skeletal oxymethine proton signal [δH 4.56 (1H, dd, J = 11.5, 4.7 Hz, H-3)] to carbonyl carbon C-21 (δC 153.9) of the substituent provided evidence that the carbonyl carbon C-21 is attached to C-3 via an oxygen atom. Given the downfield chemical shift of the broad singlet proton signal [δH 10.49 (1H, br s)], the chlorine atom was assigned to be attached to C-24. The nitrogen atom coupled to the singlet proton [δH 7.63 (1H, br s)] was suggested to be attached to carbonyl carbon C-21. The connection between the two secondary nitrogens through carbonyl carbon C-22 was thus established by elimination. These deductions were further supported by the degrees of unsaturation. To verify the proposed structure, compound 1 was subject to a single-crystal X-ray diffraction analysis (Figure 3). Computed structure from the single-crystal X-ray diffraction analysis matched flawlessly with the NMR and HRESIMS data and thus established the absolute configuration of compound 1 without doubt. This is the first report of the presence of [(2chlorophenyl)amino]carbonyl carbamic acyl substituent in a natural product, and compound 1 was thus named chloroxaloterpin A. Compound 2 was determined to have the molecular formula C 28 H36ClNO3 according to HRESIMS data. A careful comparison of the 1H and 13C NMR spectroscopic data of 1 and 2 (Table 1) revealed remarkable similarities: compound 2 also possesses a viguiepinol skeleton with a substituent containing a 1,2-disubstituted benzene ring. However, there are also some striking differences in the data for the two compounds. 1H NMR spectrum of 1 showed two active hydrogens in the low field (δH 10.49 and 7.63), whereas compound 2 only had one active hydrogen [δH 9.48 (1H, br s)]. In the 13C NMR spectra, compound 1 possessed two carbonyl signals at δC 153.9 and 150.2, whereas 2 had two signals at δC 159.6 and 153.8. It was thus reasonable to deduce that the NH group located between C-21 and C-22 in 1 is not present in 2, and the two carbonyls (C-21 and C-22) connect directly to each other in 2. Finally, a single-crystal X-ray diffraction analysis was employed to reveal the absolute configuration of 2 (Figure 3). The obtained structure was in accordance with the NMR and HRESIMS data. To the best of our knowledge, this is the first report of the presence of 2-[(2-

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RESULTS AND DISCUSSION Structure Elucidation. The CH2Cl2 extract of the fermentation broth of Streptomyces sp. SN194 was subjected to silica gel column chromatography and further purified by gel chromatography on Sephadex LH-20 or by HPLC to afford two new, 1 and 2, and four known compounds, 3−6 (Figure 1). The known compounds were identified to be viguiepinol, 3,22,23 oxaloterpin C, 4,23,24 oxaloterpin D, 5,24 and oxaloterpin E, 6,23,24 by consulting published spectroscopic data.

Figure 1. Structures of compounds 1−6.

Compound 1 was assigned the molecular formula C28H37ClN2O3 in accordance to its HRESIMS data, indicating 11 degrees of unsaturation. The existence of the chlorine element was supported by an isotope peak at m/z 509.2366 [M + Na]+ in the mass spectrum. The 1H NMR spectrum of 1 revealed four tertiary methyl groups [δH 1.11 (s, 3H, H3-18), δH 0.97 (s, 3H, H3-17), δH 0.96 (s, 3H, H3-19), and δH 0.95 (s, 3H, H3-20)]; furthermore, resonances attributed to aliphatic moieties were found between δH 1.20 and 2.30, and resonances attributed to double bonds or oxygen-linked methane groups were found between δH 4.50 and 6.00 (Table 1). These results suggested the presence of a viguiepinol skeleton in 1, which was confirmed by careful comparison of the 1H and 13C NMR spectra data of 1 and 3 and analysis of the HMBC and COSY spectra of 1 (Figure 2).23 The 13C NMR spectrum of 1 showed

Figure 2. Key 1H−1H COSY (bold) and HMBC (arrows) correlations of compound 1. 8527

DOI: 10.1021/acs.jafc.6b03645 J. Agric. Food Chem. 2016, 64, 8525−8529

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Journal of Agricultural and Food Chemistry

Figure 3. Oak Ridge thermal ellipsoid plot (ORTEP) of compounds 1 and 2.

μg/mL, respectively (Table 2). However, the four known compounds (3−6) showed little inhibitory activity (Table 2). By comparing the structures of compounds 1−6, we deduced that the composition of the side chain contributes to the inhibitory activity. The oxalyl amide derivative oxaloterpin D (5) and carbamoyl ester derivative oxaloterpin E (6) have shown much lower activities even compared to viguiepinol (3). The inhibitory activity further decreased with oxaloterpin C (4) where the oxalyl amide residue in 5 was substituted by an nhydroxyoxalyl amide residue. Both chloroxaloterpin A, 1, and B, 2, demonstrated industrially relevant inhibitory activity against Botrytis cinerea. The substituted chlorobenzene ring, which is unique to both chloroxaloterpin A, 1, and B, 2, is thought to be the key structural entity to the inhibitory activity against Botrytis cinerea spore germination. Due to the paucity of compounds 1 and 2, other aspects of bioactivity were not tested. Botrytis cinerea is a potent phytopathogen with strong tendency to develop resistance against known fungicides.3 Therefore, novel controlling agents are in constant need. Chloroxaloterpin A, 1, and B, 2, have shown promising bioactivity against Botrytis cinerea while showcasing structural diversity in natural products produced by actinomycetes. Both the [(2-chlorophenyl)amino]carbonyl carbamic acyl group in 1 and the 2-[(2-chlorophenyl)amino]-2-oxo-acetyl group in 2 are discovered in natural products for the first time. Natural products remain a diverse source of bioactive lead molecules for both agricultural and pharmaceutical uses.25,26 Moreover, natural products are usually environmentally benign and therefore are suitable alternatives to synthetic chemicals as crop protection agents.9,27 With all these benefits, natural products will continue to be explored by plant protection scientists to create environmentally safe and effective phytopathogen control agents.

chlorophenyl)amino]-2-oxo-acetyl group in a natural product, and compound 2 was subsequently named chloroxaloterpin B. Antifungal Activity Assay. Compounds 1−6 were evaluated for their antifungal activities against Botrytis cinerea in a spore germination assay and a mycelial inhibition assay, the results of which were listed in Table 2. Pyrimethanil was used as Table 2. Inhibitory Effects of Tested Compounds on Botrytis cinereaa compound chloroxaloterpin A, 1 chloroxaloterpin B, 2 viguiepinol, 3 oxaloterpin C, 4 oxaloterpin D, 5 oxaloterpin E, 6 pyrimethanilc

spore germination inhibition EC50b, μg/mL (±SD) 4.40 4.96 83.94 259.16 128.27 149.80 5.94

± ± ± ± ± ± ±

0.07 0.07 0.70 0.36 0.62 0.09 0.06

mycelial growth inhibition EC50b, μg/mL (±SD) 44.78 45.69 128.05 54.82 38.01 33.90 43.49

± ± ± ± ± ± ±

3.54 0.90 2.63 1.29 1.81 3.16 1.55

a

Data are mean values of three independent experiments, each with three replicates. bThe half maximal effective concentration values. c The positive control compound.

positive control. Compounds 1, 2, 5, and 6 showed moderate effect in the mycelial inhibition assay, with the EC50 values estimated to be 44.78, 45.69, 38.01, and 33.90 μg/mL, respectively (Table 2). In the spore germination assay, chloroxaloterpin A, 1, and B, 2, demonstrated remarkable inhibitory activities surpassing that of the positive control (Figure 4) with the EC50 values estimated to be 4.40 and 4.96

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b03645. The 1H and 13C NMR data of chloroxaloterpin A, 1, and viguiepinol, 3, in CDCl3 Physicochemical Properties and Spectroscopic Data of compounds 3-6 NMR and HRESIMS spectra for chloroxaloterpin A, 1, and B, 2 (PDF)

Figure 4. In vitro effect of chloroxaloterpin A, 1, and B, 2, against spore germination of Botrytis cinerea with pyrimethanil as positive control. 8528

DOI: 10.1021/acs.jafc.6b03645 J. Agric. Food Chem. 2016, 64, 8525−8529

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Journal of Agricultural and Food Chemistry

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 24 88342209. Fax: +86 24 88487038. E-mail: zyu@ syau.edu.cn. Funding

This work was financially supported by Liaoning Pandeng Scholar Program in 2012, China. Notes

The authors declare no competing financial interest. Crystallographic data for compounds 1 and 2 have been deposited at the Cambridge Crystallographic Data Center under supplementary publication numbers CCDC 1482300 and 1482303, respectively. Copies of the data can be obtained, free of charge, upon application to the CCDC, 12 Union Road, Cambridge CB21EZ, U.K. (Fax: + 44 (0) 1223-336033 or email: [email protected]).

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ACKNOWLEDGMENTS We are grateful to Miss Suzie Hsu for proofreading the manuscript, who is a graduate working with Dr. Michael J. Smanski in University of Minnesota - Twin Cities. We thank Shenyang Pharmaceutical University and Dalian Institute of Chemical Physics, Chinese Academy of Sciences, for technical assistance with NMR and MS spectra, respectively. We also thank Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, for technical assistance with X-ray single-crystal diffraction.

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DOI: 10.1021/acs.jafc.6b03645 J. Agric. Food Chem. 2016, 64, 8525−8529