Stilbene Derivatives from Photorhabdus temperata SN259 and Their

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Stilbene Derivatives from Photorhabdus temperata SN259 and Their Antifungal Activities against Phytopathogenic Fungi Danshu Shi, ran an, wenbo zhang, Guilong Zhang, and Zhiguo Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04303 • Publication Date (Web): 14 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

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Journal of Agricultural and Food Chemistry

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Stilbene Derivatives from Photorhabdus temperata SN259 and Their Antifungal Activities against Phytopathogenic Fungi

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Danshu Shi,† Ran An,† Wenbo Zhang,† Guilong Zhang, ‡ and Zhiguo Yu*,†,§

5

†

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People’s Republic of China

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‡

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People’s Republic of China

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§

1 2

College of Plant Protection, Shenyang Agricultural University, Shenyang 110866,

Agro-Environmental Protection Institute, Ministry of Agriculture, Tianjin 300191,

Engineering & Technological Research Center of Biopesticide for Liaoning

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Province, Shenyang 110866, People’s Republic of China

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Affiliation and address: College of Plant Protection, Shenyang Agricultural

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University, 120 Dongling Road, Shenyang Liaoning 110866, China.

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*

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[email protected].

Corresponding Author: Tel: +86 24 88342209. Fax: +86 24 88487038. E-mail:

1

ACS Paragon Plus Environment


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ABSTRACT

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Chemical investigation of an insect pathogenic enterobacterium Photorhabdus

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temperata SN259 led to the isolation and identification of seven metabolites, which

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include three new compounds, 3-hydroxy-2-isopropyl-5-phenethylphenyl carbamate,

19

1,

20

2-(1-hydroxypropan-2-yl)-5-[(E)-2-phenylethenyl]benzene-1,3-diol, 3, and four

21

known metabolites (4-7). Their structures were elucidated on the basis of MS and

22

NMR data and by comparison with those reported previously. The activities of

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compounds 1-7 were evaluated against four phytopathogenic fungi (Pythium

24

aphanidermatum, Rhizoctonia solani Kuhn, Exserohilum turcicum, and Fusarium

25

oxysporum). In an agar medium assay, compounds 1 and 7 showed strong inhibition

26

against P. aphanidermatum with EC50 values of 2.8 and 2.7 µg/mL, respectively. By

27

comparing the structure of compounds 1-7, we deduced that the acylamino group in

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compound 1 and the isopropyl group in compound 7 contribute to the inhibitory

29

activity.

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KEYWORDS: Photorhabdus temperata SN259, stilbene derivatives, antifungal

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activity, Pythium aphanidermatum, Rhizoctonia solani Kuhn, Exserohilum turcicum,

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Fusarium oxysporum

2-(1-hydroxypropan-2-yl)-5-[2-phenylethyl]benzene-1,3-diol,

2


2,

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INTRODUCTION

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Plant diseases caused by fungi can lead to heavy losses in agriculture and

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therefore constitute a serious threat to the global food security.1 Considerable

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postharvest losses of fruits and vegetables have been attributed to fungal pathogens,

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which often cause crops to rot and sometimes produce mycotoxins that are harmful

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to human and animals. 2,3 P. aphanidermatum and F. oxysporum are two major fungi

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that can cause postharvest decay in crops.3 Exserohilum turcicum is a common plant

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pathogen that causes leaf blight and is responsible for serious reduction in

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agriculture production in the Northeast China. Rhizoctonia solani Kuhn is another

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notorious phytopathogenic fungal that causes rice sheath blight, a destructive disease

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that causes significant economic damage to rice crops.

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Currently, a great variety of chemical fungicides are produced to control these

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diseases. However, repeated and exclusive application of chemical-based fungicides

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often results in increased chemical resistance in pathogens, undesirable effects on

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nontarget organisms, and the potential risks to human health and environmental

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pollution.4,5 Therefore, it is particularly desirable to search for biologically active

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natural products and develop them into new antifungal agents to effectively control

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these agricultural diseases. In previous studies, Photorhabdus, an insect pathogenic

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enterobacterium that maintains a mutualistic interaction with Heterorhabditis

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nematodes was identified as a rich source of secondary metabolites.6 These

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metabolites have diverse chemical structures which could be divided into four

3



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classes, including carbapenems,7 anthraquinones,8 siderophore photobactin9 and

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stilbenes.10 Stilbenes exhibit a wide spectrum of biological activities, such as

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antibacterial,11-16 antifungal,13,17,18 insecticidal,19-21 nematicide,22 antioxidant23 and

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anticancer, 23 and have become a major source of novel antibiotics.

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In this study, seven stilbene derivatives were isolated from the crude extract of

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Photorhabdus temperata SN259. These compounds were tested for their antifungal

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activities against four phytopathogenic fungi. Although these stilbene derivatives

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have similar structure, their bioassays revealed different activities which provided

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information on their structure-activity relationships.

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MATERIALS AND METHODS

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General Experimental Procedures. NMR spectra were recorded on a Bruker

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Avance-600 NMR spectrometer (Bruker, Karlsruhe, Germany) at room temperature.

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Carbon signals and the residual proton signals of CDCl3 (δC 77.0 and δH 7.26) and

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DMSO-d6 (δC 39.5 and δH 2.50) were used for calibration. High-resolution

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electrospray ionization mass spectrometry (HRESIMS) spectra data were recorded

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on a 6500 series quadrupole-time-of-flight (Q-TOF) mass spectrometer (Agilent,

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Santa Clara, CA). High-performance liquid chromatography (HPLC) analysis was

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performed on a 1260 Infinity LC system (Agilent) coupled with a C18 column

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(Agilent ZORBAX Eclipse XDB, 4.60×250mm, 5µm). Semi-preparative HPLC was

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performed on an Agilent 1260 series system coupled with a C18 column (Agilent

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ZORBAX Eclipse XDB, 9.4×250 mm, 5µm). Column chromatography was

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performed using silica gel (100-200 mesh) (Qingdao Ocean Chemical Co. Ltd.,

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Qingdao, China) or Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). All

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chemical reagents were purchased from Sinopharm Chemical Reagent Company

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(Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and used without further

79

purification.

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Bacteria Material. P. temperata SN259 was isolated from a soil sample collected

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in June, 2015, from Fengcheng City of Liaoning Province, China (40°30′46″N,

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124°51′49″E) at a height of 200 m. The bacterium was identified by phylogenetic

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analysis, through comparison to 16S rRNA sequences available on the EzTaxon

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database. The sequence was most similar (100) to the sequence of P. temperata

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(GenBank accession number EU136626) and was named P. temperata SN259

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(GenBank accession number KU240002). The strain was deposited in the Laboratory

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of Microbial Metabolites, College of Plant Protection, at Shenyang Agricultural

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University, China.

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Fermentation and Extraction. The P. temperata SN259 was preserved in

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glycerol suspensions (10%, v/v) at -80 °C. A two-stage fermentation procedure was

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employed to grow the P. temperata SN259. In the first stage, a 250-mL Erlenmeyer

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flask, containing 50 mL of LB medium (i.e., tryptone 10 g, yeast extract 5 g, NaCl

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10 g, in a final volume of 1.0 L H2O, pH 7.0), was inoculated with 200 µL of P.

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temperata SN259 bacterial suspension and incubated with shaking (180 rpm) at

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28 °C for 18 h to prepare the seed culture. In the second stage, twenty-four 2-L

5



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Erlenmeyer flasks, each containing 400 mL of M medium (i.e., glucose 6.13 g,

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peptone 21.29 g, MgSO4·7H2O 1.50 g, (NH4)2SO4 2.46 g, KH2PO4 0.86 g, K2HPO4

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1.11 g, NaSO4 1.72 g, in a final volume of 1.0 L H2O, pH 7.2), were inoculated with

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40 mL of the seed culture and left for fermentation for 5 d under identical conditions.

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The fermentation cultures were centrifuged at 6,500 rpm and 4 °C for 30 min to

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remove bacteria, and the broth was extracted with 3% Amberlite XAD 16 resin for 4

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hours at room temperature with agitation. Resin was harvested by centrifugation and

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eluted four times with methanol. The combined methanol elution was then

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concentrated under reduced pressure to afford the crude extract.

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Isolation and Purification. The dried extract was redissolved in 50% MeOH (600

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mL). The solution was extracted four times with equal volume of CH2Cl2. The

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CH2Cl2 extract was collected and concentrated on a rotary evaporator under vacuum

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at 28 °C to yield 5.8 g of solid brown residue.

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The CH2Cl2 extract was subjected to silica gel chromatography (350 mm × 25 mm

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i.d.) eluted stepwise with petroleum ether : ethyl acetate (100:5, 100:10, 100:20,

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100:30, and 0:100, v/v, 2 L of each) as the mobile phase to afford five fractions, S1

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to S5. Fraction S4 was subjected to gel chromatography on Sephadex LH-20 eluted

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with MeOH, then purified by reverse-phase semi-preparative HPLC applying a 51%

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MeOH-H2O (with 0.1% HCOOH added to both solvents) and a flow rate of 3.0

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mL/min for 60 min, UV detection was at 254 nm. Pure compounds 1 (16 mg), 2 (200

116

mg), and 3 (15 mg) were eluted at 52.7 min, 34.2 min, and 29.7 min, respectively.

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Fraction S3 was repeatedly chromatographed on a Sephadex LH-20 gel column

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(1000 mm × 20 mm i.d.) with MeOH (600ml) as eluent to yield compound 7 (600

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mg) and a mixture. The mixture was then isolated by reverse-phase semi-preparative

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HPLC applying a 72% MeOH-H2O (with 0.1% HCOOH added to both solvents) to

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give compound 6 (50 mg, tR=37.1 min). Fraction S2 was separated by reverse-phase

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semi-preparative HPLC eluted with 50% CH3CN-H2O (with 0.1% HCOOH added to

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both solvents) to give compound 4 (15 mg, tR=12.8 min). Fraction S1 was subjected

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to a silica gel column (350 mm × 20 mm i.d.), eluted with petroleum ether：ethyl

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acetate (100:2, v/v, 600ml) to give compound 5 (118 mg) .

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3-hydroxy-2-isopropyl-5-phenethylphenyl carbamate, 1. colorless oil; 1H NMR

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and 13C NMR spectroscopic data (DMSO-d6), see Table 1; HRESIMS m/z 322.1418

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[M + Na]+ (calcd for C18H21NO3Na, 322.1419).

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2-(1-hydroxypropan-2-yl)-5-[2-phenylethyl]benzene-1,3-diol, 2. colorless oil;

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1

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295.1308 [M + Na]+ (calcd for C17H20O3Na, 295.1310).

H NMR and

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C NMR spectroscopic data (CDCl3), see Table 1; HRESIMS m/z

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2-(1-hydroxypropan-2-yl)-5-[(E)-2-phenylethenyl]benzene-1,3-diol, 3. colorless

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oil; 1H NMR and 13C NMR spectroscopic data (DMSO-d6), see Table 1; HRESIMS

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m/z 293.1150 [M + Na]+ (calcd for C17H18O3Na, 293.1153).

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In Vitro Effect on Mycelial Growth of Phytopathogenic Fungi. Strains of P.

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aphanidermatum, R. solani Kuhn, E. turcicum, and F. oxysporum were provided by

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the Laboratory of Microbial Metabolites, College of Plant Protection, at Shenyang

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Agricultural University. The effects of compounds 1-7 on the mycelial growth of

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phytopathogenic fungi were performed by the agar medium assay as described

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previously.24,25 Test compounds were dissolved in acetone, then mixed with sterile

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molten potato dextrose agar to obtain final concentrations of 3.125, 6.25, 12.5, 25,

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and 50 µg/mL. The PDA was poured into 9 cm plates (15 mL) then the 5 mm plugs

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of plant pathogens were placed in the center of each plate. Three replicate plates

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were used per treatment, and PDA containing a corresponding concentration of

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acetone was used as control dishes. Chlorothalonil was used as the positive

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comparison. Experiments were performed three times. When the fungal mycelium

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reached the edges of the control dishes, the antifungal activities were calculated. The

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formula for calculating the percentage of growth inhibition was as follows:

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inhibition rate of mycelial growth (%) = (1-Da/Db) ×100

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where Da is the diameter of the pathogen colony in the plate containing a test

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compound and Db is the diameter of the colony in the control plate.

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Microdilution

Broth

Assay.

The

antifungal

activities

against

four

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phytopathogenic fungi (P. aphanidermatum, R. solani Kuhn, E. turcicum, and F.

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oxysporum) were evaluated in 96-well microtiter plates using a modification of the

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broth microdilution method.26,27 Arrayed stock solutions of the tested compounds

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dissolved in DMSO were diluted 100-fold with RPMI-1640 medium and tested at

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final concentrations between 1 and 128 µg/mL. Under the sterile environment, fungal

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suspensions (100 µL) of each pathogenic fungi were poured into the wells containing

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100 µL of 2-fold serially diluted single compounds in the RPMI-1640 medium for a

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final volume of 200 µL. The wells containing DMSO (≤1%) were run as negative

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controls. Chlorothalonil was used as positive control. Under the same concentrations,

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the blank wells were prepared with RPMI-1640 medium containing the tested

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compounds. The inoculated plates were incubated at 28 °C. After an incubation of

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48 h, the optical density (OD) of each well was measured using a microplate reader

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(MULTISKAN GO, Thermo Fisher Scientific, Vantaa, Finland) at 620 nm. The

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growth inhibition of each dilution was calculated using the following formula:

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inhibition (%) = (1- OD of treated well / OD of negative control well) ×100

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where OD values of the negative control well and the OD of the treated well were

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corrected with the OD of the blank wells corresponding to each concentration. The

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IC50 values were derived from Probit analysis of the concentration-response data,

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with serially diluted concentrations of the pure compounds.

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RESULTS AND DISCUSSION

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Structure Elucidation. The CH2Cl2 extract of the fermentation broth of P.

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temperata SN259 was subjected to silica gel column chromatography and further

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purified by gel chromatography on Sephadex LH-20 or by HPLC to afford three new

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compounds (1-3), one new natural product (4) and three known compounds (5-7)

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(Figure 1). The known compounds were identified by comparison of spectroscopic

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data with those reported in the literature as 2-isopropyl-5-[2-phenylethyl]benzene-1,

9



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3-diol, 5,28 2-ethyl-5-[(E)-2-phenylethenyl]benzene-1,3-diol, 6,10 and 2-isopropyl-5-

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[(E)-2-phenylethenyl]benzene-1,3-diol, 7.10

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Compound 1 had the molecular formula determined to be C18H21NO3 according to

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HRESIMS data, indicating nine degrees of unsaturation. The 1H NMR spectrum of 1

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(Table 1) displayed two methyl protons at δH 1.21 (6H, d, J = 7.1 Hz), two

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methylene protons at δH 2.71 (2H, br dd, J = 10.2, 6.2 Hz), 2.81 (2H, br dd, J = 10.1,

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6.2 Hz), one aliphatic methine at δH 3.18 (1H, m), one monosubstituted benzene ring

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[δH 7.27 (4H, m), 7.18 (1H, t, J = 7.0 Hz)], and one 1,2,3,5-tetrasubstituted benzene

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ring [δH 6.54 (1H, brs), 6.33 (1H, brs)].

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carbon resonances ascribed to two methyls, two methylenes, eight methines (seven

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aromatic including two overlapped and one aliphatic), six quaternary carbons (one

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amide carbonyl, five aromatic including two oxygenated). The structural

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construction of 1 was performed by 2D NMR experiments. The presence of

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1,2-diphenylethane was supported by the 1H-1H COSY correlations of H2-7/H2-8 and

193

the HMBC correlations of H2-7/C-4, C-6, and C-9；H2-8/C-10, C-14, and C-5 (Figure

194

2). Furthermore, the presence of a 1,2,3,5-tetrasubstituted benzene ring including

195

3,5-dihydroxy substituent was established by a combination of the HMBC

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correlations of H-4/C-2, C-6; H-6/C-4, C-2, together with the downfield chemical

197

shift of C-1 (δC 156.2) and C-3 (δC 149.6). This inference was further supported by

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the HMBC correlations of H3-16, H3-17/C-2; H-15/ C-1, C-3, while the HMBC

199

correlations information indicated that the isopropyl was linked to C-2 by C-15. In

The

13

C NMR spectrum of 1 showed 18

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addition, the presence of the amide group were determined by the chemical shifts of

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C-18 (δC 155.2), CONH2 (δH 7.08, brs; 6.73, brs) indicated the amide group located

202

at C-1 by an oxygen atom based on the molecular formula C18H21NO3. We proposed

203

the structure of 1 as depicted in Figure 1, and it was thus named

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3-hydroxy-2-isopropyl-5-phenethylphenyl carbamate.

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Compound 2 was assigned the molecular formula C17H20O3 in accordance to its

206

HRESIMS data, indicating that the compound 2 possessed one more oxygen atom

207

than the known compound 5. A careful comparison of the 1H and

208

spectroscopic data of 2 (Table 1) and 5 revealed that 2 was very similar to 5, the only

209

difference between them was that 2 had one more hydroxyl group than 5. The methyl

210

group (δC 20.8, δH 1.40) in 5 was replaced by an oxygen-bearing methylene group

211

(δC 67.6, δH 3.88 and 3.95) in 2. The proposal was supported by the HMBC

212

correlations of H2-17/C-2, C-16. All the above data suggested that compound 2 was

213

C-17 hydroxylated analogue of compound 5, and named

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2-(1-hydroxypropan-2-yl)-5-[2-phenylethyl]benzene-1,3-diol.

13

C NMR

215

Compound 3 had a molecular formula C17H18O3 on the basis of its positive

216

HRESIMS data, suggesting that 3 possessed one more oxygen atom than 7. By

217

comparison of the 1H NMR spectrum of 3 with that of 7, compound 3 showed one

218

additional methylene group at δH 3.59(1H, dd) and 3.72(1H, dd). In the

219

spectrum, compound 7 processed two methyl signals at δC 20.6, whereas 3 had

220

signals at δC 15.4 and 64.6. It was thus reasonable to deduce that the methyl group

11


13

C NMR


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[δC 20.6, δH 1.26(3H, d)] in 7 was replaced by an oxygen-bearing methylene group

222

[δC 64.6, δH 3.59(1H, dd) and 3.72(1H, dd)] in 3. The chemical value of the C-15 at

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δC 23.7 in 7 was downfield shifted to δC 32.2 in 3, further demonstrating that the

224

extra hydroxyl group was attached to C-17 in 3. The proposal was also supported by

225

the HMBC correlations of H2-17/C-2, C-16. Taken together, the structure of 3 was

226

determined as shown (Figure 1) and named

227

2-(1-hydroxypropan-2-yl)-5-[(E)-2-phenylethenyl]benzene-1,3-diol.

228

The structure of 4 was determined by comparison of the NMR spectra with

229

compound 6. The 1H NMR spectrum of 6 revealed that the C-7 and C-8 showed

230

olefinic proton signals at δH 6.89 and 7.02, while that of 4 revealed the C-7 and C-8

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possessed methylene signals at δH 2.77 and 2.88, indicating that the double bond

232

between C-7 and C-8 in 6 was reduced in 4. The structure of compound 4 was

233

therefore

234

2-ethyl-5-(2-phenylethyl)benzene-1,3-diol. Compound 4 was synthesized previously,

235

29

236

Antifungal Activity Assay.

237

activities against four phytopathogenic fungi by using the agar medium assay and the

238

96-well microdilution broth assay, the results of which were listed in Table 2 and

239

Table 3. Compounds 1 and 7 demonstrated strong inhibition against mycelial

240

growth of P. aphanidermatum with the EC50 values estimated to be 2.8 and 2.7

241

µg/mL, respectively, which was comparable to that of the positive control (Figure 3).

established

a

shown

in

Figure

1,

and

named

as

but this is the first time for the compound to be identified as a natural product. Compounds 1-7 were evaluated for their antifungal

12


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In the 96-well microdilution broth assay, compounds 1 and 7 exhibited significant

243

antifungal properties against the P. aphanidermatum, with IC50 values of 2.6 and 2.0

244

µg/mL, respectively. Additionally, compounds 1 and 7 also possessed high levels of

245

antifungal activity against R. solani Kuhn. Compounds 4, 5 and 6, exhibited

246

moderate antifungal activities, and compounds 2 and 3 showed even weaker

247

inhibitory activities.

248

An overall evaluation of the relationship between the structures and antifungal

249

activity of the compounds suggested that the fungicidal activities of these analogues

250

were strongly influenced by the substituents attached to the benzene ring, as well as

251

the double bond between the benzene rings. Comparing the structures of 2 and 5,

252

introduction of one hydroxyl group at the C-17 position greatly reduced the

253

antifungal activities against all of the tested fungi, and the same trend is observed

254

between compounds 3 and 7. Compounds 5 and 7 were more active than compounds

255

4 and 6, suggesting that alkylated derivatives of C-15 slightly increased the

256

antifungal activity. By comparing structures 7 and 5, the reduction of the olefin

257

between C-7 and C-8 to alkane decreased antifungal activity, as well as that

258

observed between compounds 6 and 4. Comparison of compounds 1 and 5 revealed

259

that the replacement of the hydrogen of the hydroxyl group at C-1 with an acylamino

260

group increased the antifungal activity, so that this group plays an important role in

261

its activity. The structure-antifungal activity relationships of these compounds

262

provide a new template for further discovery of novel antifungal agrochemicals.

13



263

Currently, only a few stilbene analogs have been isolated from Photorhabdus.6

264

The three newly discovered stilbene derivatives has increased the structural diversity

265

of stilbene compounds. Compounds 1 and 7 showed strong bioactivity against P.

266

aphanidermatum and R. solani Kuhn. This is the first report that stilbenes exhibit

267

inhibitory activity against P. aphanidermatum, which provides further proof that

268

stilbene compounds are good candidate agents for antifungal agrochemicals

269

discovery.

270

ASSOCIATED CONTENT

271

Supporting Information

272

The HRESIMS, 1H NMR,

273

new compounds 1-3, the 1H NMR of compounds 4-7, the 13C NMR of compounds 4,

274

5 and 7. This material is available free of charge via the Internet at

275

http://pubs.acs.org.

276

AUTHOR INFORMATION

277

Funding

278

This work was supported in part by Liaoning Pandeng Scholar Program in 2012, and

279

the Natural Science Foundation of Tianjin Municipal (Grant No. 13JCYBJC254 00),

280

China.

281

Notes

282

The authors declare that there are no conflicts of interest.

283

ACKNOWLEDGMENTS

13

C NMR, HMQC, HMBC and 1H-1H COSY spectra of

14


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The authors thank Shenyang Pharmaceutical University for technical assistance with

285

NMR and MS spectra.

286

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FIGURE CAPTIONS Figure 1. Structures of compounds 1-7 Figure 2. Key 1H-1H COSY (bold) and HMBC (arrows) correlations of compound 1 Figure 3. In vitro effect of compounds 1 and 7 against mycelial growth of P. aphanidermatum with chlorothalonil as positive control

21



Table 1.1H (600 MHz) and

position

δC 156.2 123.8 149.6 113.9 139.4 112.5 36.5 36.6 141.6 128.2 128.3 125.8 128.3 128.2 24.6 20.7 20.6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 -CONH2 -CONH2 1-OH 3-OH 17-OH a

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C (150 MHz) NMR Data for Compounds 1-3

1a δH (J in Hz)

δC 154.9 115.3 154.9 6.33 s 108.8 141.8 6.54 s 108.8 2.71 br dd (6.2,10.2) 37.2 2.81 br dd (6.2,10.1) 37.2 141.8 7.27 m 128.3 7.27 m 128.3 7.18 t (7.0) 125.9 7.27 m 128.3 7.27 m 128.3 3.18 m 30.9 1.21 d (7.1) 15.0 1.21 d (7.1) 67.6

2b δH (J in Hz)

6.23 s 6.23 s 2.70 br dd (6.4,10.1) 2.81 br dd (6.4,10.2) 7.16 m 7.25 t (7.50) 7.16 m 7.25 t (7.50) 7.16 m 3.62 m 1.32 t (7.8) 3.95 dd (4.6,10.2) 3.88 dd (2.4,10.2)

δC 156.6 117.5 156.6 105.2 137.0 105.2 126.9 128.9 135.2 126.4 128.6 127.4 128.6 126.4 32.2 15.4 64.6

3a δH (J in Hz)

6.48 s 6.48 s 6.91 d (16.3) 7.01 d (16.3) 7.56 d (7.6) 7.35 t (7.6) 7.24 t (7.3) 7.35 t (7.6) 7.56 d (7.6) 3.42 m 1.20 d (7.1) 3.72 dd (7.3,10.1) 3.59 dd (4.9,10.1)

155.2 7.08 s 6.73 s 9.32 s 9.32 s

9.43 s

Recorded in DMSO-d6.

b

Recorded in CDCl3.

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Table 2. EC50 Values of Compounds 1-7 against the Test Phytopathogens measured by agar medium assay (µg/mL)a

Compound 1 2 3 4 5 6 7 Chlorothalonilb a

P. aphanidermatumis 2.8±0.3 47.8±0.4 41.2±0.7 16.7±1.0 6.0±0.2 8.6±0.5 2.7±0.1 2.1±0.2

R. solani Kuhn 6.1±0.5 49.2±0.4 44.1±0.2 15.6±0.6 7.3±0.4 9.4±0.7 5.8±0.0 3.3±0.6

E. turcicum 13.8±0.4 >50 48.1±0.6 32.3±1.5 21.1±1.9 24.0±0.6 10.5±0.4 2.0±0.3

F. oxysporum 14.5±1.7 >50 >50 26.9±1.3 17.7±0.2 19.3±0.6 11.8±0.6 2.8±0.3

Experiments were performed three times, and data were presented as mean values ±

standard deviation. Differences among different treatments were analyzed using SPSS version 22.0 at the 5%. b

Chlorothalonil was co-assayed as a positive control.

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Table 3. IC50 Values of Compounds 1-7 against the Test Phytopathogens measured by microdilution broth assay (µg/mL) IC50 (95% CI)a Compound 1 2 3 4 5 6 7 Chlorothalonilb a

P. aphanidermatumis 2.6(1.6-4.1) 41.0(29.8-56.4) 32.6(23.4-45.5) 14.5(7.3-28.4) 4.4(2.6-7.4) 6.4(3.8-10.9) 2.0(1.3-2.7) 1.0(0.6-1.5)

R. solani Kuhn 4.8(4.1-6.2) 43.1(36.3-50.1) 37.3(35.2-39.8) 15.4(14.6-18.8) 6.7(5.2-8.9) 7.7(7.2-9.3) 6.3(5.8-8.4) 0.7(0.5-1.0)

E. turcicum 8.8(7.7-10.4) >50 38.7(35.9-41.7) 28.3(24.5-32.1) 9.1(7.7-12.3) 16.6(14.7-19.1) 8.1(7.35-9.1) 1.1(0.8-1.5)

F. oxysporum 5.4(4.9-6.2) >50 34.5(30.1-39.1) 27.0(23.0-31.17) 7.8(6.5-9.5) 21.3(18.1-24.8) 5.3(4.8-5.9) 2.2(2.0-2.5)

The antifungal activity is expressed as the IC50 (concentration causing 50% growth

inhibition) as determined by the broth microdilution method. 95% CI, 95% confidence interval. b

Chlorothalonil was co-assayed as a positive control.

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Figure 1

25



NH2

O O

OH

Figure 2

26


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Figure 3

27



Graphic for table of contents

28


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Stilbene Derivatives from Photorhabdus temperata SN259 and Their

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