Subscriber access provided by UNIV OF DURHAM
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 20
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
Journal of Agricultural and Food Chemistry
Page 2 of 20
1
ABSTRACT
2
Polyphenols were characterized from Dothiorella vidmadera (DAR78993) which was isolated from
3
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
2 ACS Paragon Plus Environment
Page 3 of 20
Journal of Agricultural and Food Chemistry
16
INTRODUCTION
17
Different phytotoxins are produced by plant pathogenic fungi involved in grapevine trunk diseases
18
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
22
diseases that affect grapevines worldwide.2 One of the most important diseases caused by this
23
family of fungi is Botryosphaeria dieback, and over the past few decades, the incidence of disease
24
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
26
disease symptoms include dieback of the wood, cankers and a characteristic wedge-shaped wood
27
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
29
are implicated in Botryosphaeria dieback.4 To date, 10 Botryosphaeriaceae species have been
30
isolated from vineyards in winegrowing regions of eastern Australia and these include Diplodia
31
seriata, Diplodia mutila, Lasiodiplodia theobromae, Neofusicoccum parvum, Neofusicoccum
32
australe, Botryosphaeria dothidea, Dothiorella viticola (syn. Spencermartinsia viticola),
33
Dothiorella vidmadera, Neofusicoccum luteum and Neofusicoccum ribis.4-7
34
Several phytotoxins have been isolated and characterized from a number of
35
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
37
pathogens.1,
38
produced structurally different secondary metabolites in vitro, such as a new phytotoxic
39
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
41
isolated from infected grapevine wood in Brazil and causing Botryosphaeria dieback, were
8
Neofusicoccum australe haplotype H4 associated with grapevine cordon dieback,
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 20
42
chemically identified.10 As ascertained by LC-MS, L. brasiliense, L. crassispora, L. jatrophicola, L.
43
pseudotheobromae produced jasmonic acid, and L. brasiliense, synthesized jasmonic acid and
44
(3R,4S)-4-hydroxymellein. Lasiodiplodia euphorbicola produced (-)-mellein, (3R,4R)-(-)- and
45
(3R,4S)-(-)-4-hydroxymellein and tyrosol, while L. hormozganensis synthesized tyrosol and p-
46
hydroxybenzoic acid.10 While the role of the phytotoxins in pathogenicity and symptomology is still
47
not completely clear, an interesting hypothesis could be that they are involved in the expression of
48
foliar symptoms and after production by the pathogen the phytotoxins are translocated to the leaves
49
from the wood.11 So far, no foliar symptoms have been detected in Australian vineyards affected by
50
Botryosphaeria dieback.4 The absence of these symptoms raises questions about the capability of
51
Botryosphaeriaceae isolated from grapevines in Australia to produce phytotoxins. Dothiorella
52
vidmadera is usually classified as a weak pathogen,5 however it is one of the most widespread
53
Botryosphaeriaceae species in South Australian vineyards.12 To date, there is no information
54
reported on the phytotoxic metabolites produced by D. vidmadera in liquid culture.
55
This manuscript reports the isolation, and chemical and biological characterization of a new
56
phytotoxic 5-hydroxymethyl-2-isopropoxyphenol, isolated from the culture filtrates of D.
57
vidmadera (DAR78993)5 together with benzene-1,2,4-triol, resorcinol, 3-(hydroxymethy)phenol,
58
protocatechuic alcohol and tyrosol.
59 60
MATERIALS AND METHODS
61
General Experimental Procedure. IR spectra were recorded as a deposit glass film on a Thermo
62
Electron Corporation Nicolet 5700 FT-IR spectrometer (Madison, WI, USA) and UV spectra were
63
measured in MeCN on a Jasco (Tokyo, Japan) V-530 spectrophotometer; 1H and 13C NMR spectra
64
were recorded at 400 or 500 and 100 and 125 MHz, respectively, in CDCl3, unless otherwise noted,
65
on Bruker (Karlsruhe, Germany) and Varian (Palo Alto, CA, USA) instruments. The same solvent
66
was used as an internal standard. The multiplicity were determined by DEPT spectra.13 The same 4 ACS Paragon Plus Environment
Page 5 of 20
Journal of Agricultural and Food Chemistry
67
solvent was also used as an internal standard. DEPT, COSY-45, HSQC and HMBC were performed
68
using Bruker and Varian microprograms.13 HR ESIMS and LC/MS analyses were performed using
69
the LC/MS TOF system (AGILENT 6230B, HPLC 1260 Infinity, Milan, Italy) column
70
Phenomenex LUNA (Torrance, CA, USA) (C18 (2) 5u 150x 4.6 mm). Analytical and preparative
71
TLCs were carried out on silica gel (Kieselgel 60, F254, 0.25 and 0.5 mm respectively) and on
72
reverse phase (Kieselgel 60 RP-18, F254, 0.20 mm) plates (Merck, Darmstadt, Germany). The spots
73
were visualized by exposure to UV radiation, or by spraying first with 10% H2SO4 in MeOH, and
74
then with 5% phosphomolybdic acid in EtOH, followed by heating at 110 °C for 10 min. Column
75
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
78
study was obtained from a grapevine showing symptoms of trunk diseases in a South Australian
79
vineyard and was stored at the Australian Scientific Collections Unit (Orange, NSW, Australia).5, 12
80
The isolate was grown under stationary conditions in four flasks containing 2 L of modified
81
Czapek–Dox medium with 0.5% yeast and 0.5% malt extract (pH 6.8). Each flask containing the
82
medium was inoculated with 15 mycelial plugs of the isolate grown on potato dextrose agar (PDA)
83
for 1 week. The cultures were incubated at 25°C in the dark for 14 days after which the mycelial
84
mats were removed by filtration through four layers of filter paper and kept at -20°C until further
85
processing.
86 87
Extraction and Purification of Phytotoxins. The lyophilized residues of the culture filtrates of D.
88
vidmadera were dissolved in 500 mL of distilled of water. The organic phase was extracted with
89
EtOAc (3×60 mL) at pH 5.7. The organic extracts were combined, dried (Na2SO4), filtered, and
90
evaporated under reduced pressure, yielding a brown oily residue (1.62 g). This residue was
91
bioguided purified by column chromatography, eluted with CHCl3-i-PrOH (9:1, v/v), resulting in
92
eight groups of homogeneous fractions. The fractions that were phytotoxic on lemon fruits as 5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 20
93
described in the bioassays were further purified as described below. The residue (189.1 mg) of
94
fraction 4, purified by silica gel chromatographic column using CH2Cl2-i-PrOH (95:5, v/v) as the
95
eluent, yielded a new compound of an amorphous solid identified as 5-hydroxymethyl-2-
96
isopropoxyphenol (1, Figure 1, 2.3 mg, 0.23 mg/L), a white amorphous solid identified as tyrosol
97
(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
98
(30 mg) of fraction 3 of this latter column was further purified by preparative TLC, using CHCl3-
99
MeOH-AcOH (9:0.8:0.2, v/v) as an eluent, resulting in a white homogeneous solid which was
100
identified as resorcinol (3, Figure 1, 8.9 mg, 0.89 mg/L). The residue (165 mg) of fraction 5 of the
101
original column, purified by column chromatography using n-hexane-EtOAc (6:4, v/v) as the
102
eluent, yielded four groups of fractions (A-D). Residues of fractions A and B were combined (12.6
103
mg) and further purified by preparative TLC, CHCl3-MeOH-AcOH (9:0.8:0.2, v/v) as an eluent,
104
yielding a white homogeneous solid identified as 3-(hydroxymethyl)phenol (4, Figure 1 8.6 mg,
105
0.86 mg/mL). Residues of fractions C and D were combined (14 mg) and further purified in the
106
same conditions as above and yielded crystal prisms identified as protocatechuic alcohol (5 Figure
107
1, 19 mg). The residues of fraction 6 and 7 of the original column were combined (321 mg) and
108
further purified by column chromatography (n-hexane-EtOAc 8:2, v/v), yielding a further amount
109
of 5 (for a total of 122 mg, 12.2 mg/L).
110
Identification of compounds 1-6.
111
spectroscopic data (1H NMR and ESI/MS) with those already reported in the literature and as
112
reported below.
Compounds 2-6 were identified by comparing their
113
5-Hydroxymethyl-2-isopropoxyphenol (1). UV λmax nm (log ε) 296 (2.13); IR νmax 3358,
114
1635, 1502, 1465; 1H and 13C NMR: see Table 1; HR ESI-MS (+) spectrum m/z: 221.0019 [M+K]+
115
(calcd. for C10H14KO3 221.0011).
6 ACS Paragon Plus Environment
Page 7 of 20
Journal of Agricultural and Food Chemistry
116
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 =
117
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
118
are in agreement with those previously reported.14
119
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
120
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
121
agreement with those previously reported.14
122
3-(Hydroxymethy)phenol (4). 1H NMR, δ: 7.23 (1H, t, J = 8.1 Hz, H-5), 6.91 (1H, d, J = 8.1
123
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
124
m/z: 125 [M + H]+. These data are in agreement with the data previously reported.15
125
Protocatechuic alcohol (5). 1H NMR (CD3OD), δ: 8.55 (2H, Ar-OH), 6.74 (1H, d, J = 2.8
126
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
127
Hz, OH), 4.40 (2H, d, J = 5 Hz, H-7). ESI/MS m/z: 583 [4M + Na]+ , 723 [5M + Na]+. These data
128
are in agreement with those previously reported.16
129
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-
130
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 +
131
Na]+, 159 [M + Na]+. These data are in agreement with those previously reported.17
132
133
1,2,4-O,O’,O”-Triacetyl protocatechuic alcohol (7). 7 mg (0.05 mmol) of 5 were acetylated with
134
pyridine (300 µL) and acetic anhydride (100 µL). The reaction was stirred overnight at room
135
temperature. After 24 h, MeOH and C6H6 were added and the azeotrope formed was evaporated
136
under reduced pressure. The crude residue (10 mg) was purified by preparative TLC using n-
137
hexane- Me2CO (7:3 v/v) as the eluent, yielding the corresponding triacetyl derivative of 5 (7, 6.5
138
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,
139
MeCO) 2.29 (3H, s, MeCO), 2.08 (3H, s, MeCO). ESI/MS m/z: 267 [M + H]+. These data are in
140
agreement with the data previously reported.16 7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 20
141
Phytotoxicity Bioassays. The phytotoxic activity of the crude extract chromatographic fractions
142
was initially assayed on non-host lemon fruits and fractions 4, 5, 6 and 7 were shown to be
143
phytotoxic. The samples were dissolved in DMSO and diluted in sterile distilled water (SDW), up
144
to a final concentration of 1 mg/mL and 4% DMSO. The lemon fruits were surface sterilized with
145
NaClO (50 µg/mL) and subsequently washed with three rinses of SDW. The surface of the fruit was
146
wounded three times using a sterile needle and treated with a 10 µL droplet of the test solution.
147
SDW and 4% DMSO solution were used as negative controls. The treated fruit was maintained at
148
room temperature (16-22°C) and visually assessed after 72 h for necrotic spots. Each experiment
149
was conducted in triplicate.
150
Compounds 1-5 and 7 were tested on grapevine leaves for their phytotoxicity (Vitis vinifera
151
cv. Shiraz). The compounds were dissolved in 100 µL methanol and the volume adjusted to 3 ml in
152
SDW (100 µg/mL, 10-3 M solution). The petioles of glasshouse-grown grapevine leaves were
153
immersed in 1 mL of the phytotoxic solutions for 20 h. SDW and SDW with 3% MeOH were used
154
as negative controls. The leaves were then transferred to a new vial with 2 mL SDW, placed in a
155
growth chamber with 12 h light /12 h darkness period at 28 °C and maintained for an additional 28
156
h period. Lesions on the leaf surface were evaluated using a 0 to 3 scoring scale: 0, no symptoms; 1,
157
slight wilting of the leaf; 2, moderate wilting of the leaf; 3, severe wilting of the leaf (with
158
occasional necrosis). Each treatment was conducted in triplicate.
159
The toxicity of compounds 1-5 and 7 were assayed on tomato seedlings of cv. Grosse Lisse.
160
The compounds were dissolved in 100 µL methanol and the volume adjusted to 3 mL SDW (10-3 M
161
solution). The 2 week-old rootless tomato seedlings were immersed in the solution and kept in an
162
incubator with 12 h light /12 h darkness period at 28 °C for 24 h. Seedlings were transferred to
163
distilled water under the same light and temperature conditions for a further 6 hours. Symptoms
164
were evaluated 30 h after immersion in SDW using the same scoring scale reported above. Each
165
treatment was conducted in triplicate.
166 8 ACS Paragon Plus Environment
Page 9 of 20
Journal of Agricultural and Food Chemistry
167
RESULTS AND DISCUSSION
168
Chromatographic column fractions of purified organic extracts obtained from the culture filtrates of
169
D. vidmadera were initially tested for their phytotoxicity on lemon fruits. One new metabolite,
170
named 5-hydroxymethyl-2-isopropoxyphenol (1, Figure 1), was isolated together with benzene-
171
1,2,4-triol, resorcinol, 3-hydroxymethyphenol, protocatechuic alcohol and tyrosol (2-6, Figure 1).
172
Compounds 2-6 have already been reported as fungal and plant metabolites and were identified by
173
comparing their spectroscopic (1H-NMR and ESIMS) properties with those reported in the
174
literature.14-17
175
5-Hydroxymethyl-2-isopropoxyphenol (1) had a molecular formula of C10H14O3 as deduced
176
from its HRESIMS and was consistent with four hydrogen deficiencies. Its 1H and COSY spectra
177
(Table 1) showed a broad singlet and a two broad doublet (J = 8.0 Hz) resonated at δ 6.52 (H-6),
178
6.66 (H-3) and 6.75 (H-4), as expected for the signals system of a trisubstituted benzene ring, while
179
a singlet, due to the protons of a hydroxylated methylene (H2-7) was observed at δ 4.64. Similarly, a
180
quartet (J = 6.1 Hz) and a doublet (J = 6.1 Hz), typical signals of an isopropyl group, resonated at
181
δ 3.75 (H-8) at 1.25 (H-9 and 10’) including two broad singlet due to two hydroxyl groups were
182
recorded at δ 7.46 (OH-C-2) and 4.35 (HO-C-7).18 These signals were also in full agreement with
183
the hydroxyl and aromatic bands observed in the IR spectrum19 as well as with the absorption
184
maximum recorded in the UV spectrum.18 The couplings observed in the HSQC spectrum (Table 1)
185
allowed the assignment of the carbons resonating in the
186
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-
187
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,
9 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
The structure of 1 was confirmed by the data of its HR ESIMS spectrum which showed a
191 192
Page 10 of 20
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
195
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
197
have been isolated and characterized from a fungal organism isolated from a grapevine with
198
symptoms of trunk disease.
199
Benzene-1,2-4-triol (2,) is already known as a fungal and plant secondary metabolite. 2 has
200
been isolated from fungi belonging to species of Aspergillus 22, from Gardenia jasminoides fruits23
201
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
203
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,
205
27
206
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
208
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
216
seedlings at concentrations of 100 µL (10-3 M) as described above (Table 2). The most phytotoxic 10 ACS Paragon Plus Environment
Page 11 of 20
Journal of Agricultural and Food Chemistry
217
compound on grapevine leaves was resorcinol (3), causing severe shrivelling of the leaves while
218
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
223
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
225
involved in the plants’ defence against trunk disease pathogens. 34
226
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
242
This work was supported by academic grants from the Dipartimento di Scienze Chimiche,
243
Università di Napoli Federico II and Charles Sturt University.
244 11 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 12 of 20
245 246 247
REFERENCES
248
1.
249
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
12 ACS Paragon Plus Environment
Page 13 of 20
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,
14 ACS Paragon Plus Environment
Page 15 of 20
Journal of Agricultural and Food Chemistry
325
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.
328
329
330
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).
15 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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.
16 ACS Paragon Plus Environment
Page 17 of 20
Journal of Agricultural and Food Chemistry
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.
17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
18 ACS Paragon Plus Environment
Page 19 of 20
Journal of Agricultural and Food Chemistry
Figure 2.
19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 20 of 20
Table of Contents Graphics
20 ACS Paragon Plus Environment