Isolation of Phytotoxic Phenols and Characterization of a New 5

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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

8

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

9

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

10

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

14

alcohol isopropyl ether

15

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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

19

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

20

organic compounds.

21

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

25

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

28

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,

36

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

40

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

8

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).

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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.

86 87

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

188

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

189

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

190

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,



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

191 192

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

193

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

194

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

196

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

202

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

204

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

207

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

209

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

210

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

211

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

212

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

214

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

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

215

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

220

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

221

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

224

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

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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.

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Mediterr. 2011, 50, 5-45.

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

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

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

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

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

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

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

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9.

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

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

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

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

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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.

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

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

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Figure Legend

331

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).



Page 16 of 20

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.



Page 18 of 20

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


Isolation of Phytotoxic Phenols and Characterization of a New 5

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