Isolation of Phytotoxic Phenols and Characterization of a New 5

Polyphenols were characterized from Dothiorella vidmadera (DAR78993), which was isolated from a grapevine in Australia. In total, six polyphenols were...
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Isolation of phytotoxic phenols and characterization of a new 5-hydroxymethyl-2-isopropoxyphenol from Dothiorella vidmadera, a causal agent of grapevine trunk disease Pierluigi Reveglia, Sandra Savocchia, Regina Billones-Baaijens, Alessio Cimmino, and Antonio Evidente J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05248 • Publication Date (Web): 04 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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Journal of Agricultural and Food Chemistry

Isolation of phytotoxic phenols and characterization of a new 5-hydroxymethyl2-isopropoxyphenol from Dothiorella vidmadera, a causal agent of grapevine trunk disease

Pierluigi Reveglia,†,‡ Sandra Savocchia,‡* Regina Billones-Baaijens,‡ Alessio Cimmino,† Antonio Evidente† †

Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario

Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy ‡

National Wine and Grape Industry Centre, School of Agricultural and Wine Sciences, Charles

Sturt University, Locked Bag 588 Wagga Wagga NSW 2678, Australia

*Corresponding author. Tel.: +61 2 69334341 E-mail (address): [email protected] (S. Savocchia) ACS Paragon Plus Environment

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ABSTRACT

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Polyphenols were characterized from Dothiorella vidmadera (DAR78993) which was isolated from

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a grapevine in Australia. In total, six polyphenols were isolated including a new polyphenol

4

characterized by spectroscopic method (essentially NMR and HR ESIMS) as 5-hydroxymethyl-2-

5

isopropoxyphenol.

6

protocatechuic alcohol, the latter being the main metabolite, were also isolated. Although these are

7

already known as naturally occurring compounds in microorganisms and plants, this is the first time

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they have been isolated from a fungal organisms involved in grapevine trunk disease. When assayed

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on tomato seedlings all the compounds show similar phytotoxic effects. However, when assayed on

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grapevine leaves (Vitis vinifera cv Shiraz) resorcinol was the most toxic compound followed by

11

protocatechuic alcohol and 5-hydroxymethyl-2-isopropoxyphenol.

Tyrosol,

benzene-1,2,4-triol,

resorcinol,

3-(hydroxymethyl)phenol

and

12 13

Keywords: grapevine, Botryosphaeria dieback, Dothiorella vidmadera, phytotoxins, protocatechuic

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alcohol isopropyl ether

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INTRODUCTION

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Different phytotoxins are produced by plant pathogenic fungi involved in grapevine trunk diseases

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and these have been previously chemically characterized and tested for their toxicity on the leaves

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of various Vitis species and on non-host plants.1 These phytotoxins belong to different classes of

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organic compounds.

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Pathogenic fungi belonging to the family Botryosphaeriaceae are involved in various

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diseases that affect grapevines worldwide.2 One of the most important diseases caused by this

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family of fungi is Botryosphaeria dieback, and over the past few decades, the incidence of disease

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symptoms has increased causing economic and yield losses worldwide. Currently, no curative

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methods are available for this disease and only methods to prevent the disease are available. The

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disease symptoms include dieback of the wood, cankers and a characteristic wedge-shaped wood

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lesion of the trunk and cordons. Furthermore, foliar symptoms associated with the disease have also

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been reported.2, 3 At least 27 Botryosphaeriaceae species have been isolated from grapevines and all

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are implicated in Botryosphaeria dieback.4 To date, 10 Botryosphaeriaceae species have been

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isolated from vineyards in winegrowing regions of eastern Australia and these include Diplodia

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seriata, Diplodia mutila, Lasiodiplodia theobromae, Neofusicoccum parvum, Neofusicoccum

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australe, Botryosphaeria dothidea, Dothiorella viticola (syn. Spencermartinsia viticola),

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Dothiorella vidmadera, Neofusicoccum luteum and Neofusicoccum ribis.4-7

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Several phytotoxins have been isolated and characterized from a number of

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Botryosphaeriaceae species. Melleins, phytotoxic metabolites belonging to the isocoumarin family,

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are known to be produced by D. seriata and N. parvum, two of the most widespread and virulent

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pathogens.1,

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produced structurally different secondary metabolites in vitro, such as a new phytotoxic

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cyclohexenone oxide named cyclobotryoxide, together with 3-methylcatechol and tyrosol.9 In a

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recent study, phytotoxic metabolites produced in liquid culture by six species of Lasiodiplodia

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isolated from infected grapevine wood in Brazil and causing Botryosphaeria dieback, were

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Neofusicoccum australe haplotype H4 associated with grapevine cordon dieback,

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chemically identified.10 As ascertained by LC-MS, L. brasiliense, L. crassispora, L. jatrophicola, L.

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pseudotheobromae produced jasmonic acid, and L. brasiliense, synthesized jasmonic acid and

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(3R,4S)-4-hydroxymellein. Lasiodiplodia euphorbicola produced (-)-mellein, (3R,4R)-(-)- and

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(3R,4S)-(-)-4-hydroxymellein and tyrosol, while L. hormozganensis synthesized tyrosol and p-

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hydroxybenzoic acid.10 While the role of the phytotoxins in pathogenicity and symptomology is still

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not completely clear, an interesting hypothesis could be that they are involved in the expression of

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foliar symptoms and after production by the pathogen the phytotoxins are translocated to the leaves

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from the wood.11 So far, no foliar symptoms have been detected in Australian vineyards affected by

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Botryosphaeria dieback.4 The absence of these symptoms raises questions about the capability of

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Botryosphaeriaceae isolated from grapevines in Australia to produce phytotoxins. Dothiorella

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vidmadera is usually classified as a weak pathogen,5 however it is one of the most widespread

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Botryosphaeriaceae species in South Australian vineyards.12 To date, there is no information

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reported on the phytotoxic metabolites produced by D. vidmadera in liquid culture.

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This manuscript reports the isolation, and chemical and biological characterization of a new

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phytotoxic 5-hydroxymethyl-2-isopropoxyphenol, isolated from the culture filtrates of D.

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vidmadera (DAR78993)5 together with benzene-1,2,4-triol, resorcinol, 3-(hydroxymethy)phenol,

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protocatechuic alcohol and tyrosol.

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MATERIALS AND METHODS

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General Experimental Procedure. IR spectra were recorded as a deposit glass film on a Thermo

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Electron Corporation Nicolet 5700 FT-IR spectrometer (Madison, WI, USA) and UV spectra were

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measured in MeCN on a Jasco (Tokyo, Japan) V-530 spectrophotometer; 1H and 13C NMR spectra

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were recorded at 400 or 500 and 100 and 125 MHz, respectively, in CDCl3, unless otherwise noted,

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on Bruker (Karlsruhe, Germany) and Varian (Palo Alto, CA, USA) instruments. The same solvent

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was used as an internal standard. The multiplicity were determined by DEPT spectra.13 The same 4 ACS Paragon Plus Environment

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solvent was also used as an internal standard. DEPT, COSY-45, HSQC and HMBC were performed

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using Bruker and Varian microprograms.13 HR ESIMS and LC/MS analyses were performed using

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the LC/MS TOF system (AGILENT 6230B, HPLC 1260 Infinity, Milan, Italy) column

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Phenomenex LUNA (Torrance, CA, USA) (C18 (2) 5u 150x 4.6 mm). Analytical and preparative

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TLCs were carried out on silica gel (Kieselgel 60, F254, 0.25 and 0.5 mm respectively) and on

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reverse phase (Kieselgel 60 RP-18, F254, 0.20 mm) plates (Merck, Darmstadt, Germany). The spots

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were visualized by exposure to UV radiation, or by spraying first with 10% H2SO4 in MeOH, and

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then with 5% phosphomolybdic acid in EtOH, followed by heating at 110 °C for 10 min. Column

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chromatography was performed using silica gel (Kieselgel 60, 0.063-0.200 mm) (Merck).

76 77

Fungal Strains and Culture Conditions. The isolate of D. vidmadera (DAR78993) used in this

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study was obtained from a grapevine showing symptoms of trunk diseases in a South Australian

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vineyard and was stored at the Australian Scientific Collections Unit (Orange, NSW, Australia).5, 12

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The isolate was grown under stationary conditions in four flasks containing 2 L of modified

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Czapek–Dox medium with 0.5% yeast and 0.5% malt extract (pH 6.8). Each flask containing the

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medium was inoculated with 15 mycelial plugs of the isolate grown on potato dextrose agar (PDA)

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for 1 week. The cultures were incubated at 25°C in the dark for 14 days after which the mycelial

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mats were removed by filtration through four layers of filter paper and kept at -20°C until further

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processing.

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Extraction and Purification of Phytotoxins. The lyophilized residues of the culture filtrates of D.

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vidmadera were dissolved in 500 mL of distilled of water. The organic phase was extracted with

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EtOAc (3×60 mL) at pH 5.7. The organic extracts were combined, dried (Na2SO4), filtered, and

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evaporated under reduced pressure, yielding a brown oily residue (1.62 g). This residue was

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bioguided purified by column chromatography, eluted with CHCl3-i-PrOH (9:1, v/v), resulting in

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eight groups of homogeneous fractions. The fractions that were phytotoxic on lemon fruits as 5 ACS Paragon Plus Environment

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described in the bioassays were further purified as described below. The residue (189.1 mg) of

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fraction 4, purified by silica gel chromatographic column using CH2Cl2-i-PrOH (95:5, v/v) as the

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eluent, yielded a new compound of an amorphous solid identified as 5-hydroxymethyl-2-

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isopropoxyphenol (1, Figure 1, 2.3 mg, 0.23 mg/L), a white amorphous solid identified as tyrosol

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(6, Figure 1, 3 mg, 0.3 mg/L) and benzene-1,2,4-triol (2, Figure 1, 1.3 mg, 0.13 mg/L). The residue

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(30 mg) of fraction 3 of this latter column was further purified by preparative TLC, using CHCl3-

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MeOH-AcOH (9:0.8:0.2, v/v) as an eluent, resulting in a white homogeneous solid which was

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identified as resorcinol (3, Figure 1, 8.9 mg, 0.89 mg/L). The residue (165 mg) of fraction 5 of the

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original column, purified by column chromatography using n-hexane-EtOAc (6:4, v/v) as the

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eluent, yielded four groups of fractions (A-D). Residues of fractions A and B were combined (12.6

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mg) and further purified by preparative TLC, CHCl3-MeOH-AcOH (9:0.8:0.2, v/v) as an eluent,

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yielding a white homogeneous solid identified as 3-(hydroxymethyl)phenol (4, Figure 1 8.6 mg,

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0.86 mg/mL). Residues of fractions C and D were combined (14 mg) and further purified in the

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same conditions as above and yielded crystal prisms identified as protocatechuic alcohol (5 Figure

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1, 19 mg). The residues of fraction 6 and 7 of the original column were combined (321 mg) and

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further purified by column chromatography (n-hexane-EtOAc 8:2, v/v), yielding a further amount

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of 5 (for a total of 122 mg, 12.2 mg/L).

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Identification of compounds 1-6.

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spectroscopic data (1H NMR and ESI/MS) with those already reported in the literature and as

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reported below.

Compounds 2-6 were identified by comparing their

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5-Hydroxymethyl-2-isopropoxyphenol (1). UV λmax nm (log ε) 296 (2.13); IR νmax 3358,

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1635, 1502, 1465; 1H and 13C NMR: see Table 1; HR ESI-MS (+) spectrum m/z: 221.0019 [M+K]+

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(calcd. for C10H14KO3 221.0011).

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Benzene-1,2,4-triol (2). 1H NMR, δ: 6.58 (1H, dd, J = 8.6 and 2.8 Hz, H-3), 6.55 (1H, d, J =

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2.8 Hz, H-6), 6.45 (1H, dd, J = 8.6 and 2.8 Hz, H-5). ESI/MS (+), m/z: 149 [M + Na]+ . These data

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are in agreement with those previously reported.14

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Resorcinol (3). 1H NMR, δ: 7.28 (2H, br s, OH), 7.04 (1H, t, J = 8.0 Hz, H-5), 6.86 (1H, br

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s, H-2), 6.82 (2H, dd, J=8.0 and 2.6 Hz, H-4,6). ESI/MS (+), m/z: 133 [M + Na]+. These data are in

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agreement with those previously reported.14

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3-(Hydroxymethy)phenol (4). 1H NMR, δ: 7.23 (1H, t, J = 8.1 Hz, H-5), 6.91 (1H, d, J = 8.1

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Hz, H-4), 6.87 (1H, bs, H-2), 6.76 (1H, dd, J = 8.1 and 2.4 Hz, H-6), 4.66 (2H, s, H-7). ESI/MS

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m/z: 125 [M + H]+. These data are in agreement with the data previously reported.15

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Protocatechuic alcohol (5). 1H NMR (CD3OD), δ: 8.55 (2H, Ar-OH), 6.74 (1H, d, J = 2.8

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Hz, H-6), 6.54 (1H, d, J = 8.5 Hz, H-3), 6.41 (1H, dd, J = 8.5 and 2.8 Hz, H-4), 4.88 (1H, t, J = 5.0

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Hz, OH), 4.40 (2H, d, J = 5 Hz, H-7). ESI/MS m/z: 583 [4M + Na]+ , 723 [5M + Na]+. These data

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are in agreement with those previously reported.16

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Tyrosol (6). 1H NMR, δ: 7.20 (d, J = 8.0 Hz, H-3 and H-5), 6.80 (d, J = 8.0 Hz, H-2 and H-

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6), 4.90 (s, OH), 3.80 (t, J = 6.4 Hz, H2-8), 2.80 (t, J = 6.4 Hz, H2-7). ESI/MS (+), m/z: 295 [2 M +

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Na]+, 159 [M + Na]+. These data are in agreement with those previously reported.17

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1,2,4-O,O’,O”-Triacetyl protocatechuic alcohol (7). 7 mg (0.05 mmol) of 5 were acetylated with

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pyridine (300 µL) and acetic anhydride (100 µL). The reaction was stirred overnight at room

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temperature. After 24 h, MeOH and C6H6 were added and the azeotrope formed was evaporated

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under reduced pressure. The crude residue (10 mg) was purified by preparative TLC using n-

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hexane- Me2CO (7:3 v/v) as the eluent, yielding the corresponding triacetyl derivative of 5 (7, 6.5

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mg,). 1H NMR δ: 7.19 (1H, br s, H-6), 7.10 (2H, br s, H-3 and H-4), 5.05 (2H, s, H-7), 2.32 (3H, s,

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MeCO) 2.29 (3H, s, MeCO), 2.08 (3H, s, MeCO). ESI/MS m/z: 267 [M + H]+. These data are in

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agreement with the data previously reported.16 7 ACS Paragon Plus Environment

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Phytotoxicity Bioassays. The phytotoxic activity of the crude extract chromatographic fractions

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was initially assayed on non-host lemon fruits and fractions 4, 5, 6 and 7 were shown to be

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phytotoxic. The samples were dissolved in DMSO and diluted in sterile distilled water (SDW), up

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to a final concentration of 1 mg/mL and 4% DMSO. The lemon fruits were surface sterilized with

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NaClO (50 µg/mL) and subsequently washed with three rinses of SDW. The surface of the fruit was

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wounded three times using a sterile needle and treated with a 10 µL droplet of the test solution.

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SDW and 4% DMSO solution were used as negative controls. The treated fruit was maintained at

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room temperature (16-22°C) and visually assessed after 72 h for necrotic spots. Each experiment

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was conducted in triplicate.

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Compounds 1-5 and 7 were tested on grapevine leaves for their phytotoxicity (Vitis vinifera

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cv. Shiraz). The compounds were dissolved in 100 µL methanol and the volume adjusted to 3 ml in

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SDW (100 µg/mL, 10-3 M solution). The petioles of glasshouse-grown grapevine leaves were

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immersed in 1 mL of the phytotoxic solutions for 20 h. SDW and SDW with 3% MeOH were used

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as negative controls. The leaves were then transferred to a new vial with 2 mL SDW, placed in a

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growth chamber with 12 h light /12 h darkness period at 28 °C and maintained for an additional 28

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h period. Lesions on the leaf surface were evaluated using a 0 to 3 scoring scale: 0, no symptoms; 1,

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slight wilting of the leaf; 2, moderate wilting of the leaf; 3, severe wilting of the leaf (with

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occasional necrosis). Each treatment was conducted in triplicate.

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The toxicity of compounds 1-5 and 7 were assayed on tomato seedlings of cv. Grosse Lisse.

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The compounds were dissolved in 100 µL methanol and the volume adjusted to 3 mL SDW (10-3 M

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solution). The 2 week-old rootless tomato seedlings were immersed in the solution and kept in an

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incubator with 12 h light /12 h darkness period at 28 °C for 24 h. Seedlings were transferred to

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distilled water under the same light and temperature conditions for a further 6 hours. Symptoms

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were evaluated 30 h after immersion in SDW using the same scoring scale reported above. Each

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treatment was conducted in triplicate.

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RESULTS AND DISCUSSION

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Chromatographic column fractions of purified organic extracts obtained from the culture filtrates of

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D. vidmadera were initially tested for their phytotoxicity on lemon fruits. One new metabolite,

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named 5-hydroxymethyl-2-isopropoxyphenol (1, Figure 1), was isolated together with benzene-

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1,2,4-triol, resorcinol, 3-hydroxymethyphenol, protocatechuic alcohol and tyrosol (2-6, Figure 1).

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Compounds 2-6 have already been reported as fungal and plant metabolites and were identified by

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comparing their spectroscopic (1H-NMR and ESIMS) properties with those reported in the

174

literature.14-17

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5-Hydroxymethyl-2-isopropoxyphenol (1) had a molecular formula of C10H14O3 as deduced

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from its HRESIMS and was consistent with four hydrogen deficiencies. Its 1H and COSY spectra

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(Table 1) showed a broad singlet and a two broad doublet (J = 8.0 Hz) resonated at δ 6.52 (H-6),

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6.66 (H-3) and 6.75 (H-4), as expected for the signals system of a trisubstituted benzene ring, while

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a singlet, due to the protons of a hydroxylated methylene (H2-7) was observed at δ 4.64. Similarly, a

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quartet (J = 6.1 Hz) and a doublet (J = 6.1 Hz), typical signals of an isopropyl group, resonated at

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δ 3.75 (H-8) at 1.25 (H-9 and 10’) including two broad singlet due to two hydroxyl groups were

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recorded at δ 7.46 (OH-C-2) and 4.35 (HO-C-7).18 These signals were also in full agreement with

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the hydroxyl and aromatic bands observed in the IR spectrum19 as well as with the absorption

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maximum recorded in the UV spectrum.18 The couplings observed in the HSQC spectrum (Table 1)

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allowed the assignment of the carbons resonating in the

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115.7, 114.6, 72.0, 69.5 and 22.9 due to the protonated carbons C-3, C-4, C-6, C-8, C-7 and C-9/C-

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10. The two tertiary sp2 carbons observed at δ 145.1 and 146.1 were assigned to C-1 and C-2 for

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their couplings observed in the HMBC spectrum (Table 1)13 with HO-C-1, H-3, H-6 and HO-C-1,

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H-3, H-4 and H-6, respectively. Finally, the quaternary sp2 carbon observed at δ 137.3 was assigned

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to C-5 for the long range coupling observed in the same spectrum with H-6 and H-7.20

13

C NMR spectrum (Table 1) at δ 117.2,

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The structure of 1 was confirmed by the data of its HR ESIMS spectrum which showed a

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sodium cluster [M+H]+ at m/z 183.2239.

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5-hydroxymethyl-2-isopropoxyphenol (1) and metabolites 2-6 are polyphenols and thus

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belong to the group of polyketides that also include a diverse group of natural products. The

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function of the extracts is unknown, however it is believed that they function as pigments, virulence

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factors, info-chemicals or for defence.21 To our knowledge this is the first time that compounds 1-5

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have been isolated and characterized from a fungal organism isolated from a grapevine with

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symptoms of trunk disease.

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Benzene-1,2-4-triol (2,) is already known as a fungal and plant secondary metabolite. 2 has

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been isolated from fungi belonging to species of Aspergillus 22, from Gardenia jasminoides fruits23

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and from the leaves of Cinnamoum parthenoxylon (Jack).24 Resorcinol (3) is another well-known

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phytotoxic metabolite isolated from medicinal plants14 and it was also found as a decomposed

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product of corn and rye residues in the soil.25 Several secondary metabolites with diverse biological

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activity isolated from fungi and bacteria possess a structure directly related to a resorcinol moiety.26,

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27

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free radical scavenging activity.28 4 has been isolated from fungi29, including different species of

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Penicillium15 and from the secretions of Nicrophorus vespilloides.30 Protocatechuic alcohol (5) was

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the main phytotoxic compound isolated from D. vidmadera and has been already reported as fungal

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metabolite produced by a mangrove fungus BYY-1. 16 The same authors also assayed the antitumor

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activity of 5, showing that it significantly inhibits the proliferation of Hela cells. Tyrosol (6,) is a

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well-known phytotoxic metabolite produced by plants and by several fungi, including N. parvum

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and D. seriata. This phytotoxic metabolite was more recently isolated from different strains of

213

Lasiodiplodia involved in grapevine trunk disease in Brazil. Their phytotoxicity on grapevine

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leaves and tomato seedlings has been already reported. 10, 31-33

3-(hydroxymethy)phenol (4) is a secondary metabolite usually associated with antioxidant and

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Compounds 1-5 and 7 were assayed on grapevine leaves (V. vinifera cv Shiraz) and tomato

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seedlings at concentrations of 100 µL (10-3 M) as described above (Table 2). The most phytotoxic 10 ACS Paragon Plus Environment

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compound on grapevine leaves was resorcinol (3), causing severe shrivelling of the leaves while

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protocatechuic alcohol (5) and 5-hydroxymethy-2-isopropoxyphenol (1) caused moderate

219

shrivelling of the leaves (Figure 2). 3-(hydroxymethy)phenol (4) and 1,2,4-O,O’,O”-triacetyl

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protocatechuic alcohol (7) caused only slight wilting (Figure 2). 1,2,4-Benzene triol (2) did not

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show any phytotoxic symptoms on grapevine leaves under the test conditions. Conversely, when

222

assayed on tomato seedlings, compounds 1-5 and 7 showed essentially the same level of

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phytotoxicity. These results suggest that the grapevine leaves may be less susceptible to the

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phytotoxic metabolites assayed because they may be structurally similar to polyphenols which are

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involved in the plants’ defence against trunk disease pathogens. 34

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This study provides new knowledge on the ability of D. vidmadera to produce phytotoxins

227

in vitro. Considering the absence of foliar symptoms in Australian vineyards, further investigations

228

are required to clarify the role of phytotoxic metabolites in the pathogenicity and symptom

229

expression of Botryosphaeria dieback pathogens in grapevines.

230

231

AUTHOR INFORMATION

232

Corresponding Author

233 234 235 236 237 238

Phone: +61 2 69334341 Fax: +61 2 69334341

239

The authors declare no competing financial interest.

E-mail: [email protected]

Notes

240 241

FUNDING SOURCES

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This work was supported by academic grants from the Dipartimento di Scienze Chimiche,

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Università di Napoli Federico II and Charles Sturt University.

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REFERENCES

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1.

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fungi associated with grapevine trunk diseases. Toxins 2011, 3, 1569-1605.

250

2.

251

Mediterr. 2011, 50, 5-45.

252

3.

253

Phytopathol. Mediterr. 2001, 40, 336-342.

254

4.

255

species isolated from grapevines in Australia. Australas. Plant Pathol. 2013, 42, 573-582.

256

5.

257

grapevines in Australia and notes on Spencermartinsia. Fungal Divers. 2013, 61, 209-219.

258

6.

259

from declining grapevines in sub tropical regions of Eastern Australia. Vitis 2007, 46, 27.

260

7.

261

grapevine trunk disease fungi with the reproductive structures of Vitis vinifera. Vitis 2011, 50, 89-96.

262

8.

263

metabolites by five species of Botryosphaeriaceae causing decline on grapevines, with special interest in the

264

species Neofusicoccum luteum and N. parvum. Eur. J. Plant Pathol. 2008, 121, 451-461.

265

9.

266

Melck, D.; Evidente, A., Cyclobotryoxide, a phytotoxic metabolite produced by the plurivorous pathogen

267

Neofusicoccum australe. J. Nat. Prod. 2012, 75, 1785-1791.

268

10.

269

Surico, G.; Evidente, A., Phytotoxic lipophylic metabolites produced by grapevine strains of Lasiodiplodia

270

species in Brazil. J. Agric. Food Chem. 2017, 65, 1102-1107.

271

11.

272

vinifera L.]. Phytopathol. Mediterr. 2000, 39, 156-161.

Andolfi, A.; Mugnai, L.; Luque, J.; Surico, G.; Cimmino, A.; Evidente, A., Phytotoxins produced by

Urbez-Torres, J. R., The status of Botryosphaeriaceae species infecting grapevines. Phytopathol.

Larignon, P.; Dubos, B.; Cere, L.; Fulchic, R., Observation on black dead arm in French vineyards.

Pitt; Huang, R.; Steel, C.; Savocchia, S., Pathogenicity and epidemiology of Botryosphaeriaceae

Pitt, W. M.; Úrbez-Torres, J. R.; Trouillas, F. P., Dothiorella vidmadera, a novel species from

Savocchia, S.; Steel, C.; Stodart, B.; Somers, A., Pathogenicity of Botryosphaeria species isolated

Wunderlich, N.; Ash, G.; Steel, C.; Raman, H.; Savocchia, S., Association of Botryosphaeriaceae

Martos, S.; Andolfi, A.; Luque, J.; Mugnai, L.; Surico, G.; Evidente, A., Production of phytotoxic

Andolfi, A.; Maddau, L.; Cimmino, A.; Linaldeddu, B. T.; Franceschini, A.; Serra, S.; Basso, S.;

Cimmino, A.; Cinelli, T.; Masi, M.; Reveglia, P.; da Silva, M. A.; Mugnai, L.; Michereff, S. J.;

Tabacchi, R.; Fkyerat, A.; Poliart, C.; Dubin, G., Phytotoxins from fungi of esca grapevine [Vitis

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Journal of Agricultural and Food Chemistry

273

12.

Pitt; Huang, R.; Steel, C.; Savocchia, S., Identification, distribution and current taxonomy of

274

Botryosphaeriaceae species associated with grapevine decline in New South Wales and South Australia.

275

Aust. J. Grape Wine Res. 2010, 16, 258-271.

276

13.

277

2004.

278

14.

279

Nat. Med. 2009, 7, 37-39.

280

15.

281

Penicillium novae-zeelandiae displaying radical-scavenging activity and oxidative mutagenicity: isolation of

282

gentisyl alcohol. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 2003, 539, 187-194.

283

16.

284

antitumor activity of a phenol derivative from mangrove fungus BYY-1. Jimei Daxue Xuebao, Ziran

285

Kexueban 2011, 16, 424-428.

286

17.

287

from Gloeosporium laeticolor. Agric. Biol. Chem. 1973, 37, 2925-2925.

288

18.

289

determination of organic compounds. Springer: 2000.

290

19.

Nakanishi, K., Solomon, P.H., , Infrared Absorption Spectroscopy. second ed.; 1977.

291

20.

Breitmaier, E.; Voelter, W., Carbon-13 NMR spectroscopy. 1987.

292

21.

Hertweck, C., The biosynthetic logic of polyketide diversity. Angewandte Chemie International

293

Edition 2009, 48, 4688-4716.

294

22.

295

natural products by LC-DAD-TOFMS. J. Nat. Prod. 2011, 74, 2338-2348.

296

23.

297

jasminoides (III). Zhong yao cai= Zhongyaocai= Journal of Chinese Medicinal Materials 2014, 37, 1196-

298

1199.

Berger, S.; Braun, S., 200 and more NMR experiments: A practical course. Wiley-Vch Weinheim:

Feng, W.-S.; Gao, L.; Zheng, X.-K.; Wang, Y.-Z., Polyphenols of Euphorbia helioscopia. Chin. J.

Alfaro, C.; Urios, A.; González, M. C.; Moya, P.; Blanco, M., Screening for metabolites from

Du, X.-p.; Zhao, B.-b.; Zheng, Z.-h.; Xu, Q.-y.; Su, W.-j., Study on the isolation identification and

Kimura, Y.; Tamura, S., Isolation of L-β-phenyllactic acid and tyrosol as plant growth regulators

Pretsch, E.; Buehlmann, P.; Affolter, C.; Pretsch, E.; Bhuhlmann, P.; Affolter, C., Structure

Nielsen, K. F.; Månsson, M.; Rank, C.; Frisvad, J. C.; Larsen, T. O., Dereplication of microbial

Luo, Y.; Zuo, Y.; Zhang, Z.; Cai, M.; Luo, G., Study on chemical constituents of Gardenia

13 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 20

299

24.

Wei, X.; Li, G.-H.; Wang, X.-L.; He, J.-X.; Wang, X.-N.; Ren, D.-M.; Lou, H.-X.; Shen, T.,

300

Chemical constituents from the leaves of Cinnamomum parthenoxylon (Jack) Meisn.(Lauraceae). Biochemic.

301

Syst. Ecol. 2017, 70, 95-98.

302

25.

303

decomposition of corn and rye residues in soil. J. Chem. Ecol. 1976, 2, 369-387.

304

26.

305

3‐diol) from Basidiomycetes Albatrellus confluens. Helv. Chim. Acta 2001, 84, 259-262.

306

27.

307

Oates, J. E.; Bloemberg, G. V., Biocontrol of avocado dematophora root rot by antagonistic Pseudomonas

308

fluorescens PCL1606 correlates with the production of 2-hexyl 5-propyl resorcinol. Mol. Plant-Microbe

309

Interact. 2006, 19, 418-428.

310

28.

311

of hydroxybenzyl alcohols. Biochemical and pulse radiolysis studies. Chem.-Biol. Interact. 2009, 182, 119-

312

127.

313

29.

314

N.; Sakayaroj, J., Epoxydons and a pyrone from the marine-derived fungus Nigrospora sp. PSU-F5. J. Nat.

315

Prod. 2008, 71, 1323-1326.

316

30.

317

Nicrophorus vespilloides: chemical analyses and possible ecological functions. J. Chem. Ecol. 2011, 37, 724-

318

735.

319

31.

320

produced by Neofusicoccum parvum, a grapevine canker agent. Phytopathol. Mediterr. 2010, 49, 74-79.

321

32.

322

phytotoxic effects of polyphenols from vegetable waste waters. Phytochemistry 1992, 31, 4125-4128.

323

33.

324

1628-1630.

Chou, C.-H.; Patrick, Z., Identification and phytotoxic activity of compounds produced during

Zhi‐Hui, D.; Ze‐Jun, D.; Ji‐Kai, L., Albaconol, A Novel Prenylated Resorcinol (= Benzene‐1,

Cazorla, F. M.; Duckett, S. B.; Bergström, E. T.; Noreen, S.; Odijk, R.; Lugtenberg, B. J.; Thomas-

Dhiman, S. B.; Kamat, J. P.; Naik, D. B., Antioxidant activity and free radical scavenging reactions

Trisuwan, K.; Rukachaisirikul, V.; Sukpondma, Y.; Preedanon, S.; Phongpaichit, S.; Rungjindamai,

Degenkolb, T.; Düring, R.-A.; Vilcinskas, A., Secondary metabolites released by the burying beetle

Evidente, A.; Punzo, B.; Andolfi, A.; Cimmino, A.; Melck, D.; Luque, J., Lipophilic phytotoxins

Capasso, R.; Cristinzio, G.; Evidente, A.; Scognamiglio, F., Isolation, spectroscopy and selective

Venkatasubbaiah, P.; Chilton, W. S., Phytotoxins of Botryosphaeria obtusa. J. Nat. Prod. 1990, 53,

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Journal of Agricultural and Food Chemistry

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34.

Lambert, C.; Bisson, J.; Waffo-Téguo, P.; Papastamoulis, Y.; Richard, T.; Corio-Costet, M.-F.;

326

Mérillon, J.-M.; Cluzet, S. p., Phenolics and their antifungal role in grapevine wood decay: focus on the

327

Botryosphaeriaceae family. J. Agric. Food Chem. 2012, 60, 11859-11868.

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Figure Legend

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Figure 1. Structure of protocatechuic alcohol isopropyl ether (1), benzene-1,2,4- triol (2), resorcinol

332

(3), 3-(hydroxymethyl)phenol (4), protocatechuic alcohol (5), tyrosol (6) and triacetyl

333

protocatechuic alcohol (7).

334

Figure 2. Symptoms caused by compounds 1, 3 and 4 on leaves of Vitis vinifera cv. Shiraz, after in

335

vitro bio-assaying at 10-3 M of 1, 3 and 4: a severe necrosis and shrivelling caused by 3; b moderate

336

wilting and necrotic spots caused by 1; c slight wilting caused by 4: d symptomless leaf (negative

337

control SDW).

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Table 1. 1H and 13C NMR data of 5-Hydroxymethy-2-isopropoxyphenol (1)a,b Position HMBC δCc δH (J in Hz) 1 145.1 C HO-C-1, H-3, H-6 2 146.1 C HO-C-1, H-3, H-4, H-6 3 117.2 CH 6.66 (1H) br d (8.0) HO-C-1, H-4, H-7 4 115.7 CH 6.75 (1H) br d (8.0) 5 137.3 C H-6, H-7 6 114.6 CH 6.52 (1H) br s H-7 7 69.5 CH2 4.64 (2H) s 8 72.0 CH 3.75 (1H) q (6.1) H-6, H-9 9,10 22.9 CH3 1.24 (6H) d (6.1) HO-C-1 7.46 br s HO-C-7 4.35 br s a The chemical shifts are in δ values (ppm) from TMS. b2D 1H,1H (COSY) and 2D 13 1 C, H (HSQC) NMR experiments delineated the correlations of all protons and the corresponding carbons. cMultiplicities were assigned by DEPT spectra.

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Table 2. Level of toxicity induced 28 h after treatment on tomato seedlings cv. Grouse and Vitis vinifera cv. Shiraz leaves by metabolites (1-5 and 7) produced by Dothiorella vidmadera.

Compound 1 2 3 4 5 7

Level of toxicitya Tomato seedlings Grapevine leaves 2.5 2.0 2.5 0.0 2.5 3.0 2.5 1.0 2.5 2.5 2.0 1.0

a

Severity scale: (0) no symptoms; (1) slight wilting; (2) moderate wilting, necrotic spots; (3) severe necrosis and shrivelling.

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Figure 1. HO 7

OH 5

4

HO

6

HO

OH

1

3

OH

OH 9

2 O

OH

HO

8 10

3

2

4

1

OH HO

OAc

OH OH 5

OAc OH

OAc 7

6

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Figure 2.

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Table of Contents Graphics

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