Lasiojasmonates A-C, three jasmonic acid esters produced by Lasiodiplodia sp., a grapevine pathogen

Article abstract of DOI:10.1016/j.phytochem.2014.03.016

In this study, a strain (BL 101) of a species of Lasiodiplodia, not yet formally described, which was isolated from declining grapevine plants showing wedge-shaped cankers, was investigated for its ability to produce in vitro bioactive secondary metabolites. From culture filtrates of this strain three jasmonic acid esters, named lasiojasmonates A-C and 16-O- acetylbotryosphaerilactones A and C were isolated together with (1R,2R)-jasmonic acid, its methyl ester, botryosphaerilactone A, (3S,4R,5R)-4-hydroxymethyl-3,5- dimethyldihydro-2-furanone and (3R,4S)-botryodiplodin. The structures of lasiojasmonates A-C were established by spectroscopic methods as (1R ^*,2R^*,3′S^*, 4′R^*,5′R^*)-4-hydroxymethyl-3,5- dimethyldihydro-2-furanone, (1R^*,2R^*, 3′S^*,4′R^*,5′R ^*,10′R^*,12′R^*, 13′R^*,14′S^*) and (1R ^*,2R^*,3′S^*, 4′R^*,5′R^*,10′S ^*,12′R^*,13′R^*, 14′S^*)-4-(4-hydroxymethyl-3,5-dimethyltetrahydro-furan-2- yloxymethyl)-3,5-dimethyldihydro-2-furanones jasmonates (1, 4 and 5). The structures of 16-O-acetylbotryosphaerilactones A and C were determined by comparison of their spectral data with those of the corresponding acetyl derivatives obtained by acetylation of botryosphaerilactone A. The metabolites isolated, except 4 and 5, were tested at 1 mg/mL on leaves of grapevine cv. Cannonau and cork oak using the leaf puncture assay. They were also tested on detached grapevine leaves at 0.5 mg/mL and tomato cuttings at 0.1 mg/mL. In all phytotoxic assays only jasmonic acid was found to be active. All metabolites were inactive in the zootoxic assay at 50 μg/mL.

Full text of DOI:10.1016/j.phytochem.2014.03.016

Phytochemistry 103 (2014) 145–153
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Lasiojasmonates A–C, three jasmonic acid esters produced
by Lasiodiplodia sp., a grapevine pathogen
Anna Andolfi a, Lucia Maddau b, Alessio Cimmino a, Benedetto T. Linaldeddu b, Sara Basso a,
Antonio Deidda b, Salvatorica Serra b, Antonio Evidente a,
⇑
a Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia, 4, 80126 Napoli, Italy
b Dipartimento di Agraria, Sezione di Patologia vegetale ed Entomologia, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a strain (BL 101) of a species of Lasiodiplodia, not yet formally described, which was isolated
from declining grapevine plants showing wedge-shaped cankers, was investigated for its ability to produce
in vitro bioactive secondary metabolites. From culture filtrates of this strain three jasmonic acid esters,
named lasiojasmonates A–C and 16-O-acetylbotryosphaerilactones A and C were isolated together with
(1R,2R)-jasmonic acid, its methyl ester, botryosphaerilactone A, (3S,4R,5R)-4-hydroxymethyl-3,5-
dimethyldihydro-2-furanone and (3R,4S)-botryo⁄diplodin. The structures of lasiojasmonates A–C were
established by sp⁄ectr⁄oscopic methods as (1R⁄⁄,2R ,30S⁄,40R⁄,50R⁄)-4-hydrox⁄yme⁄thyl-3,5-⁄dimethyldi⁄hydro-
2-fu⁄ranone, (1R ,2R ,30S⁄,40R⁄,50R⁄,100R⁄,120R ,130R⁄,140S⁄) and (1R⁄,2R ,30S ,40R⁄,50R ,100S⁄,120R ,130R⁄,
140S )-4-(4-hydroxymethyl-3,5-dimethyltetrahydro-furan-2-yloxymethyl)-3,5-dimethyldihydro-2-furanones
jasmonates (1, 4 and 5). The structures of 16-O-acetylbotryosphaerilactones A and C were determined by
comparison of their spectral data with those of the corresponding acetyl derivatives obtained by acetylation
ofbotryosphaerilactoneA.Themetabolites isolated, except4 and 5, weretested at1 mg/mLon leavesofgrape-
vine cv. Cannonau and cork oak using the leaf puncture assay. They were also tested on detached grapevine
leaves at 0.5 mg/mL and tomato cuttings at 0.1 mg/mL. In all phytotoxic assays only jasmonic acid was found
Received 20 December 2013
Received in revised form 9 March 2014
Available online 23 April 2014
Keywords:
Botryosphaeriaceae
Lasiodiplodia sp.
Grapevine trunk disease
Phytotoxins
Jasmonic acid
Lasiojasmonates A–C
to be active. All metabolites were inactive in the zootoxic assay at 50 lg/mL.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
disease symptoms caused by Lasiodiplodia spp. on grapevine
consist of sunken cankers associated with wedge-shaped lesions
of the vascular tissues.
Results of a recent study conducted in several Sardinian vine-
yards and aimed at clarifying the aetiology of grapevine canker
and dieback, have led to the identification and characterization of
9 different species of Botryosphaeriaceae from symptomatic grape-
vine tissues (Linaldeddu et al., unpublished data). Among these, a
Lasiodiplodia species morphologically (shape and size of conidia)
The Botryosphaeriaceae represent a well-known family of plant
pathogenic fungi associated with fruit rot, leaf spots, dieback,
cankers and root rot of Angiosperms and Gymnosperms worldwide
(Phillips et al., 2013). Over the past decades, several species of the
Botryosphaeriaceae have been recognized as important pathogens
of grapevine in the many growing areas (Larignon et al., 2001;
Phillips, 2002; van Niekerk et al., 2006; Pitt et al., 2010; Úrbez-
Torres, 2011). In particular, to date, 21 different taxa of Botryosph-
aeriaceae have been reported as weak or aggressive pathogens on
grapevine. Among these, four species belonging to the Lasiodiplodia
genus namely Lasiodiplodia crassispora T.I. Burgess & Barber,
Lasiodiplodia theobromae (Pat.) Griffon & Maubl., Lasiodiplodia
missouriana Úrbez-Torres, Peduto & Gubler and Lasiodiplodia vitico-
la Úrbez-Torres, Peduto & Gubler have been recognized to be
highly virulent on grapevine (Úrbez-Torres, 2011). The main
and phylogenetically (ITS and EF1-a sequence data) distinct from
all known species was isolated. Its formal description will be
addressed in a future publication currently in preparation.
At present, 18 species are recognized in Lasiodiplodia (Phillips
et al., 2013) and recent studies, based on ITS and EF-1
a sequence
data, have led to the clarification of systematics of this genus and
to the identification of cryptic species within the L. theobromae
species complex (Alves et al., 2008; Abdollahzadeh et al., 2010;
Begoude et al., 2010; Úrbez-Torres et al., 2012).
The nature of symptoms (vascular necrosis) caused by this new
species of Lasiodiplodia on grapevine suggests that phytotoxic
⇑
Corresponding author. Tel.: +39 081 2539178; fax: +39 081674330.
E-mail address: evidente@unina.it (A. Evidente).
http://dx.doi.org/10.1016/j.phytochem.2014.03.016
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

146
A. Andolfi et al. / Phytochemistry 103 (2014) 145–153
metabolites may be involved in the host-pathogen interaction. On
the other hand, it is well-known that L. theobromae, which is con-
sidered the type species of the Lasiodiplodia genus, biosynthesizes a
variety of lipophilic and hydrophilic metabolites that exhibit inter-
esting biological activities (Aldridge et al., 1971; Husain et al.,
1993; Nakamori et al., 1994; Matsuura et al., 1998; Yang et al.,
2000; He et al., 2004; Miranda et al., 2008; Kitaoka et al., 2009;
Abdou et al., 2010; Pandi et al., 2010; da Cunha et al., 2012).
Therefore, the main aims of this work were: (1) to isolate and
identify the main secondary metabolites produced in vitro by a
selected strain (BL 101) of this new Lasiodiplodia species; (2) to
evaluate their biological activities such as phytotoxicity and
zootoxicity.
B Rf 0.13, respectively) and 1H NMR with those of commercial sam-
ples of ( )-jasmonic acid and its methyl ester. Some other new spec-
troscopic data as ESI and/or APCIMS spectra and in particular the
13C NMR data for the trisubstituted dihydrofuranone 9 were
detailed reported in the corresponding paragraphs of the experi-
mental section. In addition, the identification of 8 was also sup-
ported by data from its COSY, HSQC and HMBC spectra. Finally, as
(3R,4S)-botryodiplodin (10) was obtained as an inseparable ano-
meric mixture (a/b, 65:35), its structure was confirmed by acetyla-
tion carried out in the usual conditions. In fact, this reaction yielded
only the 2,3-trans-botryodiplodin acetate, whose physic and spec-
troscopic data were very similar to those previously reported
(Arsenault and Althaus, 1969).
Furthermore, the preliminary 1H and 13C NMR investigation of 1
showed its close correlation with both jasmonic acid and 3,4,5-tri-
substituted dihydrofuranone 9 and, being a new compound as
described below, it was named lasiojasmonate A. These suggestions
were also confirmed by the band typical of ketone ester and olefinic
groups (Nakanishi and Solomon, 1977) and the end absorption max-
imum (Scott, 1964) observed in the IR and UV spectra, respectively.
It showed a molecular weight of 336 associated with the molecular
formula of C19H28O5 with six hydrogen deficiens as deduced from
the HRESIMS spectrum. The 1H NMR spectrum, also compared to
that of jasmonic acid (Husain et al., 1993) and of standard commer-
cial sample (Table 1), showed the signal patterns of the jasmonyl
residue. For the 2-pentenyl moiety the multiplets of the protons
(H-10 and H-9) of the cis double bond, the two allylic methylene
groups (H2-8 and H2-11) and the triplet (J = 7.4 Hz) of the terminal
methyl group (Me-12) were observed at d 5.45, 5.26, 2.37, 2.04
and 0.95, respectively. For the 2,3-disubstituted cyclopentanone
residue the multiplets of H-1 and H-2, the double doublet (J = 20.0
and 8.2 Hz) and the multiplet, and the multiplets of H2-4 and H2-5
were observed at d 2.10, 1.90, 2.74 and 2.37, and 2.33 and 1.50
2. Result and discussion
The fungal culture filtrates were exhaustively extracted with
EtOAc at pH 2, and the corresponding organic extract was purified
by combined column and TLC chromatography as detailed reported
in the experimental section, yielding 10 metabolites (1–10, Fig. 1).
The preliminary 1H NMR investigation showed that the metabolites
belong to different classes of natural compounds. The structures of
the known compounds were confirmed by physical and spectro-
scopic methods (OR, IR, UV, 1H and 13C NMR, ESI and/or APCI MS)
and by comparison of the obtained data with those reported in
the literature for (1R,2R)-jasmonic acid and its methyl ester (6
and 7) (Husain et al., 1993; Yukimune et al., 2000), botryosphaeri-
lactone A (8) (Rukachaisirikul et al., 2009), (3S,4R,5R)-4-hydroxy-
methyl-3,5-dimethyldihydro-2-furanone (9) (Ravi et al., 1979),
and (3R,4S)-botryodiplodin (10) (Ramezani et al., 2007). The identi-
fication of jasmonic acid and its methyl ester was confirmed by
comparison by their behavior TLC (silica gel eluent A Rf 0.43, and
on reversed phase eluent C Rf 0.50; silica gel eluents A Rf 0.8 and
6
O
5
11
O
1
O
8
9
O
7
8
3
15
3
5
O
1
10
7
14
R1
8'
OR2
7'
O
O
12
3'
16
5'
6'
17
1'
O
O
2 R1=β-H, R2=Ac
3 R1=α-H, R2=Ac
8 R1=β-H, R2=H
1
O
O
O
16'
8'
7
O
O
13'
COOR
10'
R
O
O
O
6 R=H
7 R=CH3
4 R=β-H
5 R=α-H
O
HO
O
O
OH
O
9
10
Fig. 1. Structures of lasiojasmonate, 16-O-acetylbotryosphaerilactone A and C, (ꢀ)-(1R,2R)-jasmonic acid and its methyl ester, botryosphaerilactone A, (3R,4R,5R)-4-
hydroxymethyl-3,5-dimethyldihydro-2-furanone and (ꢀ)-(3R,4S)-botryodiplodin (1, 4, 5, 2, 3, 6–10) produced by Lasiodiplodia sp.

A. Andolfi et al. / Phytochemistry 103 (2014) 145–153
147
Table 1
dimethyldihydro-2-furanone. This stereochemistry was also con-
firmed by the correlations observed in the NOESY spectrum
(Berger and Braun, 2004). In fact, this spectrum showed the corre-
lations between the protons of the cis double bond H-10 and H-9
and that this latter with the protons of allylic methylene H2-8,
and that of H-30 with H-50. Thus, lasiojasmonate A can be formu-
lated as (1R⁄,2R⁄,30S⁄,40R⁄,50R⁄)-4-hydroxymethyl-3,5-dimethyldi-
hydro-2-furanone jasmonate.
2 and 3, being two epimeric acetals, both showed a molecular
weight of 314, as deduced from their ESIMS. They were obtained
as an inseparable mixture (a/b, 45:55). All of the attempts carried
out for their separation, using different chromatographic methods
on direct and reverse phase and different solvent mixtures, failed.
Their 1H NMR spectrum (Table 2) showed the presence of two dou-
blets (J = 2.3 and 4.6 Hz) typical of anomeric protons (H-10) for the
NMR data of lasiojasmonate A (1).a,b
Position
1
dCc
dH (J in Hz)
HMBC
1
2
3
4
37.6 d
53.9 d
218.3 s
38.7 t
2.10 (1H) m
1.90 (1H) m
–
2.74 (1H) dd, (20.0, 8.2)
2.37(1H) m
H-2, H2-5, H2-6
H2-4, H2-8, H-9
H-1, H2-5, H-8
H2-5
5
27.2 t
2.33 (1H) m
1.50 (1H) m
2.32(2H) m
H-1, H2-4
6
7
8
9
37.9 t
171.7 s
25.5 t
124.9 d
134.2 d
20.6 t
14.1 q
177.6 s
38.6 d
50.5 d
77.0 d
19.9 q
14.1 q
62.5 t
–
H2-5, H2-6, H2-80
H-9, H-10
H2-8, H-11
H2-8, H2-11, Me-12
Me-12
2.37 (2H) m
5.26 (1H) m
5.45 (1H) m
2.04 (2H) m
0.95 (3H) t (7.4)
–
2.49 (1H) dq (11.0, 7.1)
2.04 (1H) m
4.26 (2H) m
1.47 (3H) d (6.1)
1.28 (3H) d (7.1)
4.26 (2H) m
4.21 (1H) dd (11.0, 5.7)
10
11
12
20
30
40
50
60
70
80
b and
a isomers at d 4.67 and 4.79, respectively. The comparison of
H2-11
H-30, Me-70, H2-80B
Me-70
this spectrum with that of botryosphaerilactone 8 (Rukachaisirikul
et al., 2009) indicates that 2 and 3 were the acetyl derivatives of
botryosphaeriolactones A and C, or of their respective enantiomers.
In fact, their 1H NMR spectra (Table 2) differed from that of 8 and
botryosphaerilactone C (Rukachaisirikul et al., 2009) essentially for
H-30, Me-60, Me-70
Me-60, H2-80
H-30
H-30
H-30, H-50
the downfield shift of H2-16 (
for b and isomers respectively) resonating as two double dou-
blets (J = 11.0, 5.6 and 11.0, 7.9 Hz) for b anomer (2) at d 4.15
and 4.04, while for the anomer (3) at d 4.21 and 4.06 (J = 11.3,
Dd 0.46 and 0.35; and 0.41 and 0.40,
a
a
The chemical shifts are in d values (ppm) from TMS.
b
2D 1H, 1H (COSY) 13C, 1H (HSQC) NMR experiments delineated the correlations
of all the protons and the corresponding carbons.
a
4.5 and 11.3, 7.6 Hz). Furthermore, two singlets of the acetyl
groups resonated at d 2.06 and 2.07 for 2 and 3 respectively. The
chemical shifts of all protons of two anomers were assigned
(Table 2) by comparison of their 1H NMR spectrum with that of
8. The epimeric nature of 2 and 3 was also confirmed by the usual
acetylation of botryosphaerilactone A with pyridine and acetic
c
Multiplicities were assigned by DEPT spectrum.
respectively (Pretsch et al., 2000). As regard the signal system of the
4-hydroxy-3,5-dimethyl-3,4-dihydrofurane residue, which were
also compared to those reported in literature (Ravi et al., 1979)
(Table 1) the protons of hydroxymethylene H2C-80 showed, in par-
ticular, the typical AB system appearing as a mutiplet and double
doublet (J = 11.0 and 5.7 Hz) at d 4.26 and 4.21 significantly down-
anhydride. As expected this reaction yielded both the b and
mers the latter (3) being the main product of the reaction.
a ano-
The structures assigned to 2 and 3 were also confirmed by data
of both ESI and APCIMS spectra. The ESIMS spectrum showed
dimeric sodiated form [2M+Na]+and the sodium cluster [M+Na]+
at m/z 651 and 314, respectively. The APCIMS spectrum showed
ions originating from the pseudomolecular ion [M+H]+ recorded
at m/z 315 by fragmentation mechanisms typical of asymmetrical
ethers (Pretsch et al., 2000) as reported in Fig. 2. In fact, the
pseudomolecular ion by cleavage of the acetalic bond C(10)-O
field shifted (Dd 0.43 and 0.46), as a consequence of the esterifica-
tion with the jasmonic acid. The other signals of the same moiety
appeared as a double quartet (J = 11.0 and 7.1 Hz) for H-30, two mul-
tiplets for H-40 and H-50 (the latter being overlapped with the signal
of H-80A) and two doublets (J = 6.1 and 7.1 Hz) for the two secondary
methyl groups (Me-60 and Me-70) at d 2.49, 2.04, 4.26, and 1.47 and
1.28, respectively (Pretsch et al., 2000). All these attributions were
confirmed by the couplings observed in the COSY and HSQC spectra
(Berger and Braun, 2004), with the latter also contributing to assign-
ing the chemical shifts of protonated carbons in the 13C NMR spec-
trum as reported in Table 1. The signals of the remaining quaternary
carbons observed at d 218.3, 177.6 and 171.7 typical of a ketone and
two ester carbonyl groups (Breitmaier and Voelter, 1987) were
assigned to C-3, C-20 and C-7 with the last two attributed on the
basis of the couplings observed in the HMBC spectrum (Table 1)
(Berger and Braun, 2004) between C-20 with H-30, Me-70 and H-80B
and C-7 with H2-5, H2-6 and H2-80. The coupling observed in the
HMBC spectrum between C-7 with H2C-80 was very significant in
supporting the jasmonate esterification of the 4-hydroxytmethyl-
3,5-dimethyl-3,4-dihydrofuranone.
On the basis of these results lasiojasmonate A was formulated
as 4-hydroxymethyl-3,5-dimethyldihydro-2-furanone jasmonate
(1).
The structure of 1 was confirmed by all of the other couplings
observed in the HMBC spectrum and by data from the HRESI and
APCI MS spectra which showed, respectively, the sodium cluster
[M+Na]+ and the pseudomolecular ion [M+H]+ at m/z 359.1913
and 337.
(mechanism a) originated the c-lactonyl and the trisubstituted tet-
rahydrofurane ions recorded at m/z 145 and 172, respectively. This
Table 2
1H NMR data of 16-O-acetylbotryosphaerilactones A and C (2 and 3).a
Position
2
3
dH (J in Hz)
dH (J in Hz)
2
3
4
5
6
7
8
2.53 (1H) dq (9.2, 6.7)
1.90 (1H) m
2.57 (1H) dq (11.3, 7.1)
1.91 (1H) m
4.29 (1H) dq (8.4, 6.0)
1.43 (3H) d (6.2)
1.26 (3H) d (7.1)
3.83 (1H) dd (10.3, 4.3)
3.41 (1H) dd (10.3, 4.8)
4.67 (1H) d (2.3)
3.91 (1H) dq (12.3, 6.2)
1.51 (1H) m
4.32 (1H) dq (9.2, 6.2)
1.45 (3H) d (6.2)
1.26 (3H) d (7.1)
3.84 (1H) dd (10.2, 5.4)
3.41 (1H) dd (10.2, 4.8)
4.79 (1H) d (4.6)
3.97 (1H) dq (12.3, 6.2)
1.51 (1H) m
10
12
13
14
15
16
2.03 (1H) m
2.05 (1H) m
1.13 (3H) d (7.0)
4.15 (1H) dd (11.0, 5.6)
4.04 (1H) dd (11.0, 7.9)
1.31 (3H) d (6.2)
2.06 (3H) s
1.04 (3H) d (7.0)
4.21 (1H) dd (11.3, 4.5)
4.06 (1H) dd (11.3, 7.6)
1.30 (3H) d (6.2)
2.07 (3H) s
The relative stereochemistry of 1 as depicted in Fig. 1 was
deduced by the comparison of the couplings constant measured
in its 1H NMR spectrum with those reported for (1R,2R)-jasmonic
acid (Husain et al., 1993) and (3S,4R,5R)-4-hydroxymethyl-3,5-
17
COCH3
a
The chemical shifts are in d values (ppm) from TMS.

148
A. Andolfi et al. / Phytochemistry 103 (2014) 145–153
latter, by loss of AcOH, generated a fragment ion at m/z 113 (Fig. 2).
Furthermore, the pseudomolecular ion by the alternative cleavage
of the acetalic bond C(8)-O (fragmentation mechanism b) gener-
appeared to be closely related to both botryosphaerilactones A and
C and jasmonic acid, suggesting for 4 and 5 a structure of jasmonate
esters and therefore were named lasiojasmonates B and C. Their 1H
NMR spectrum (Table 3) showed the presence of two doublets
(J = 2.0 and 4.5 Hz) typical of anomeric protons (H-100) at d 4.68
ated the c-lactonyl fragment ion observed at m/z 127 (Fig. 2).
Compounds 4 and 5 showed a molecular weight of 464, as
deduced from their HRESIMS spectrum recorded in positive mode
associated with the molecular formula of C26H40O7. They were
and 4.80 for the
differences from the 1H NMR spectra of botryosphaerilactones A
and were observed for the protons of the ABX systems
HC-(130)-H2C-(160). As expected the H2-160 protons were downfield
b and a isomers, respectively. Significant
obtained as an inseparable epimeric mixture (
a
/b, 30:70). As above
C
reported for 2 and 3, all attempts made using different chromato-
graphic methods failed. By preliminary 1H NMR investigation, they
shifted (
D
d 0.48, 0.41, and 0.43, 0.41 for 4 and 5) with respect to the
(a)
+
O
O
OCOCH3
O
+
OH2
+
m/z 145
O
O
b
a
+
OH
O
H
+
O
O
-CH3COOH
OCOCH3
OCOCH3
+
O
O
CH2
OH
m/z 172
m/z 113
2, 3
m/z 315
(b)
+
HO
O
O
+
OCOCH3
O
CH2
m/z 127
O
(e)
(c)
+
O
O
O
O
OH
O
e
O
+
+
CH2
O
+
+
O
m/z 127
OH
O
O
H2O
O
O
d
c
CH2
O
+
m/z 113
O
H
O
O
O
(d)
+
4, 5
m/z 465
O
O
+
O
OH2
O
m/z 145
+
O
O
O
O
+
O
OH
H
O
m/z 322
Fig. 2. Mechanisms of fragmentation observed for 2–5 in the APCI MS (+).

A. Andolfi et al. / Phytochemistry 103 (2014) 145–153
149
Table 3
Fifty days after inoculation, the lignified canes inoculated with
the pathogen displayed dark-brown to black discoloration on bark
and vascular tissues, measuring 7.4 2.7 cm (mean S.D.). In cross
section all canes showed a wedge-shaped necrotic sector. The path-
ogen was successfully re-isolated from the margin of symptomatic
tissues, thus fulfilling Koch’s postulates. Artificially obtained symp-
toms were congruent with field observations. Control canes inocu-
lated with sterile PDA plugs remained symptomless.
1H NMR data of lasiojasmonates B and C (4 and 5).a
Position
4
5
dH (J in Hz)
dH (J in Hz)
1
2
4
2.13 dq (17.5, 9.8)
1.91 m
2.70 dd (17.5, 6.1)
2.25 m
2.13 dq (15.5, 9.8)
1.91 m
2.70 dd (17.5, 6.1)
2.25 m
5
2.37 m
2.37 m
The phytoxicity data obtained in the leaf-puncture assay on
cork oak and grapevine leaves showed that (1R,2R)-jasmonic acid
(6) was the only active metabolite (Table 4). It had remarkable tox-
icity at 1.0 mg/mL causing necrotic lesions to leaves of both species
tested (necrosis area: 7.04 and 11.96 mm2 on grapevine and cork
oak, respectively). All of the other metabolites tested did not show
any effect in this bioassay.
All compounds, except 4 and 5, were also tested on grapevine
detached leaves at 0.25 and 0.5 mg/mL. In this bioassay only jas-
monic acid caused vein necrosis, slowly spreading to large areas
of the leaf lamina at 0.5 mg/mL. When assayed on tomato cuttings
at 0.1 mg/mL jasmonic acid caused withering within 7 days from
treatment. None of the compounds showed activity in the assay
1.63 ddd (14.3, 6.9, 4.6)
2.37 m
2.37 m
5.25 m
5.46 m
2.04 dq (7.5, 1.0)
0.96 t (7.5)
2.54 m
2.00 m
4.30 (m)
1.44 d, (6.2)
1.26 d (7.1)
3.86 dd (10.0, 4.3)
3.41 dd (10.0, 5.6)
4.68 d (2.0)
3.90 dq (12.3, 6.1, 2.8)
1.50 m
1.63 ddd (14.3, 6.9, 4.6)
2.37 m
2.37 m
5.25 m
5.46 m
2.04 dq (7.5, 1.0)
0.96 t (7.5)
2.54 m
2.00 m
4.30 m
1.45 d (6.2)
1.26 d (7.1)
3.87 dd (10.0, 4.3)
3.41 dd (10.0, 5.3)
4.80 d (4.5)
3.96 dq (12.6, 6.2, 1.6)
1.50 m
6
8
9
10
11
12
30
40
50
60
70
80
100
120
130
140
150
160
performed for zootoxicity (Artemia salina) at 50 lg/mL.
In conclusion, from the culture filtrates of Lasiodiplodia sp. were
isolated three new jasmonic acid esters, namely lasiojasmonates
A–C (1, 4 and 5), and for the first time 16-O-acetyl derivatives of
botryosphaerilactones A and C (2 and 3), together with (1R,2R)-jas-
monic and its methyl ester (6 and 7), botryosphaerilactone A (8),
2.03 m
2.03 m
1.13 d (7.2)
4.17 dd (11.0, 6.9)
4.10 dd (11.0, 7.2)
1.32 d (6.1)
1.05 d (7.0)
4.23 dd (11.3, 4.5)
4.07 dd (11.3, 5.8)
1.30 d (6.2)
170
a
The chemical shifts are in d values (ppm) from TMS.
and
(3S,4R,5R)-4-hydroxymethyl-3,5-dimethyl-dihydro-2-fura-
none (9) and (3R,4S)-botryodiplodin (10).
Jasmonic acid is the basic structure of a naturally occurring
family of compounds named jasmonates which regulate many
aspects of growth, development and defence responses in plants
(Miersch et al., 1999a). In particular, jasmonic acid is a signaling
molecule in plant defence, activating responses to wounding, her-
bivores and necrotrophic pathogens.
6 and relate compounds were also produced by fungi (Miersch
et al., 1999b and literature cited). In particular, Lasiodiplodia spp.
are among the few fungal species which synthesize enantiomeric
pure form of the different enantiomers as (1R,2S) diasteromer,
called also (+)-7-iso-jasmonic acid, isolated from L. theobromae
(Miersch et al., 1987). Jasmonic acid is biosynthesised by the octa-
decanoid pathway in plants (Schaller and Stinzi, 2009) and fungi
(Tsukada et al., 2010). Recently jasmonate biosynthesis by a linol-
enate 9R-dioxygenase and an allene oxide synthase has been
described in L. theobromae (Jernerèn et al., 2012).
values reported for the same protons in botryosphaerilactones A
and C (Rukachaisirikul et al., 2009). These protons appeared as dou-
blet of doublets (J = 11.0, 7.2, and 11.0, 6.9) and (J = 11.0, 5.8, and
11.3, 4.5) at d 4.10 and 4.17, and d 4.07 and 4.23 for 4 and 5. Thus,
all chemical shifts of all protons were assigned to 4 and 5 in compar-
ison with those reported for 2 and 3 and jasmonic acid⁄as repor⁄ted i⁄n
Table 3. Therefore, 4 and⁄5 can be for⁄mul⁄ated as (1R ,2R⁄,30⁄S ,40R⁄,
50R⁄,⁄100R⁄,⁄120R⁄,130R⁄,140S )- and (1R ,2R ,30S⁄,40R⁄,50R⁄,100S ,120R ,
130R ,140S )-4-(4-hydroxymethyl-3,5-dimethyltetrahydro-furan-2-
yloxymethyl)-3,5-dimethyldihydro-2 furanone jasmonates.
These structures were confirmed through fragmentation ions
observed in APCIMS spectrum recorded in positive mode. Also in
this case the spectrum exhibited ions originating by pseudomolec-
ular ion by two different fragmentation mechanisms generated by
the cleavage of the acetalic bond as reported in Fig. 2. The cleavage
of the bond C(80)-O (mechanism c) generated the fragment ion m/z
127 and the 4-hydroxy-3,5-dimethyl-4-hydroxy jasmonate esters.
This latter generated jasmonic acid and fragment ion m/z 113 and
H2O (mechanism e). Alternatively, the pseudomolecular ion by
cleavage of the acetalic bond C(100)-O (mechanism d) generated
two fragment ions recorded at m/z 145 and 322.
The presence in lasiojasmonates A–C of (1R,2R)-jasmonic acid
was confirmed by their mild alkaline hydrolysis carried out accord-
ing to Farmer et al. (1992). In fact jasmonic acid obtained from 1, 4
and 5, reported in detail in the experimental section, and 6 showed
the same Rf by TLC analysis (silica gel eluent A Rf 0.43, and on
reversed phase eluent C Rf 0.50) also by co-injection and OR and
1H NMR recorded in the same conditions.
In addition, the keto-enolization process of C-2 of jasmonic acid,
as reported in literature by Miersch et al. (1987), was ruled out for
the compounds 1,4–7 isolated from Lasiodiplodia sp. as a result of
their optical (OR) and/or spectroscopic (essential 1H NMR) proper-
ties. So that compounds 1, 4–7 are not artefact.
Botryosphaerilactone A and (3S,4R,5R)-4-hydroxymethyl-3,5-
dimethyl-dihydro-2-furanone (8 and 9) were recently reported as
secondary metabolites of the endophytic fungus L. theobromae
Table 4
Phytotoxicity of compounds 1–10 evaluated at 1 mg/mL by leaf punctured assay on
two plant species. Area lesions (mm2) error standard.
Compound
Plant species
Cork oak
Grapevine
1
2
3
4
5
6
7
8
9
10
n.a.
n.a.
n.a.
n.t.
n.t.
11.96 1.82
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.t.
n.t.
7.04 0.91
n.a.
n.a.
n.a.
n.a.
n.a.
Pathogenicity of the new species of Lasiodiplodia was verified by
wound inoculation of excised canes under controlled laboratory
conditions.
n.a. = not active.
n.t. = not tested.

150
A. Andolfi et al. / Phytochemistry 103 (2014) 145–153
isolated from leaves of Garcinia mangostana (Rukachaisirikul et al.,
2009). When they were assayed against Staphylococcus aureus, they
did not show antibacterial activity (Rukachaisirikul et al., 2009).
(3R,4S)-Botryodiplodin was first isolated from cultures of
L. theobromae Pat. the causal agent of fruit rot in tropical fruits
trees (Gupta et al., 1966). Subsequently, it was found in other cul-
tures of phytopathogenic fungal species such as Penicillium roque-
fortii strains (Nielsen et al., 2006), Penicillium stipitatum (Fuska
et al., 1988) and Macrophomina phaseolina (Ramezani et al., 2007).
(3R,4S)-Botryodiplodin exhibits anticancer, antibacterial, anti-
fungal, phytotoxic, mitogenic, and antifertility activities, and may
play a role in plant diseases. Unusual structural properties allow
oligomerization of (ꢀ)-botryodiplodin to pigments and cross-link-
ing agents, which may be responsible for some of its biological
activities (Shier et al., 2007).
n-hexane-Me2CO (95:5), (C) EtOH-H2O (6:4); (D) CHCl3-i-PrOH
(93:7); (E) n-hexane-Me2CO (6:4). ( )-Jasmonic acid and its methyl
ester were purchased from Sigma–Aldrich.
3.2. Fungal strains
The strain BL 101 of Lasiodiplodia sp. used in this study was orig-
inally isolated from wood tissues of a wedge-shaped canker col-
lected in a vineyard in northern Sardinia (Italy). The strain was
identified on the basis of morphological characters and analysis
of internal transcribed spacer (ITS) of rDNA and part of the transla-
tion elongation factor 1-
a gene (EF1-alpha). Fungal DNA extrac-
tion, PCR amplification reactions, and DNA sequencing were
carried out as reported by Linaldeddu et al. (2013). Representative
sequences of both loci were deposited in GenBank: (ITS: accession
This study represents the first report on the expression of second-
ary metabolites by a new species of Lasiodiplodia which is closely
related phylogenetically to L. pseudotheobromae. The results here
reported have permitted the increase of the knowledge on second-
ary metabolites produced by members of Botryosphaeriaceae, a fam-
ily of ascomycetes capable of producing a plethora of bioactive
compounds (Evidente et al., 2010, 2012; Andolfi et al., 2011, 2012).
However, they do not permit the elucidation of the true biological
function of the only phytotoxin isolated, the jasmonic acid, in the
pathogenesis process. Moreover, the data available in the literature
concerning the role of this metabolite in host–pathogen interaction
is uncertain. In a previous study it was suggested that in the Brassica
nigra – L. theobromae pathosystem, the development of systemic
acquired resistance does not occur due to the deficiency of salycilic
acid whose biosynthesis is inhibited by jasmonic acid produced by
fungus (Thakkar et al., 2004). In addition, it is possible to hypothe-
size that the lasiojasmonates and methyl esters (1, 4, 5 and 7) repre-
sent a reserve that could generate jasmonic acid by hydrolysis at
physiological pH as frequently observed in other natural metabo-
lites and this conversion is known as lethal metabolism (Hassal,
1990).
number KJ170150), (EF1-a: accession number KJ170151).
Pure cultures were maintained on potato-dextrose-agar (PDA,
Fluka, Sigma–Aldrich Chemic GmbH, Buchs, Switzerland) and
stored at 4 °C in the collection of the ‘‘Dipartimento di Agraria –
Sezione di Patologia vegetale ed Entomologia’’, University of Sas-
sari, Italy, as BL 101.
3.3. Pathogenicity test
Pathogenicity of the new species of Lasiodiplodia from grapevine
was verified by inoculating a selected strain (BL101) on nine
excised grapevine canes (30 cm in length) of cv. Cannonau. A
mycelial plug (3 to 4 mm2) taken from the margin of an actively
growing colony on PDA was placed in a shallow wound (ꢁ3 mm)
made by a scalpel on the middle of each cane. The inoculation
point was covered with cotton wool soaked in sterile water and
wrapped with ParafilmÒ. A sterile PDA plug was placed on the
wound in control canes. The inoculated canes were enclosed in a
transparent plastic bag at 25 °C. Fifty days after inoculation, the
length of the dark-brown to black necrotic lesions were measured.
Re-isolation was attempted by transferring to PDA 10 pieces of
inner bark and xylem tissue taken from the margin of each lesion.
Finally, further investigation must be addressed at acquiring a
body of data in order to speculate on the potential use of these
metabolites in the resolution of the taxonomic status of Botryosph-
aeriaceae species, in particular the cryptic species closely related
phylogenetically to L. theobromae complex.
3.4. Extraction and purification of fungal metabolites (1–10)
The fungus was grown in 2 L Erlenmeyer flasks containing
400 mL of Czapek medium amended with corn meal (pH 5.7). Each
flask was seeded with 5 mL of a mycelial suspension and then incu-
bated at 25 °C for 4 weeks in darkness. The culture filtrates (3 L)
were acidified to pH 2 with 2 N HCl and extracted exhaustively
with EtOAc. The organic extracts were combined, dried with Na2-
SO4, and evaporated under reduced pressure to give a brown-red
oil residue (2.3 g), having a high phytotoxic activity on grapevine
leaves by the puncture leaf bioassay at 4 mg/mL. The residue was
submitted to a bioassay-guided fractionation through CC on silica
gel, eluted with the solvent system A. Height homogenous fraction
groups were collected and screened for their phytotoxic activity.
The residue (18.7 mg) of the first fraction was purified by TLC on
silica gel eluted with solvent system B yielding a homogeneous
oil identified as (1R,2R)-methyl ester of jasmonic acid (7, 3.4 mg,
1.1 mg/L, Rf 0.8 and 0.13, eluent A and B respectively). The residue
(27.7 mg) of the second fraction was purified by TLC on reversed
phase eluted with the solvent system C to give three main bands.
The first band corresponded to an uncoloured oil (1.6 mg, 0.6 mg/
L, Rf 0.30, eluent C), which was characterized as lasiojasmonates
B and C (4 and 5), obtained as an inseparable mixture. The second
band corresponded to another uncoloured oil (5.2 mg, 1.7 mg/L, Rf
0.40, eluent C) which was characterized as lasiojasmonate A (1).
The third band corresponded to an amorphous solid (5.2 mg,
1.7 mg/L, Rf 0.50, eluent C), which was characterized as 16-O-bot-
ryosphaerilactone A and C acetylates (2 and 3), obtained as an
3. Experimental
3.1. General
Optical rotations were measured in CHCl3, unless otherwise
noted, on a Jasco polarimeter; IR spectra were recorded as glassy
films on a Perkin–Elmer Spectrum One FT-IR spectrometer and
UV spectra were taken in MeOH solution on a Perkin-Elmer
Lambda 25 UV/Vis spectrophotometer; 1H and 13C NMR spectra
were recorded at 600 and at 125 MHz, respectively, in CDCl3,
unless otherwise noted, on Bruker spectrometers. The same sol-
vent was used as internal standard. DEPT, COSY-45, HSQC, HMBC
and NOESY experiments (Berger and Braun, 2004) were performed
using standard Bruker microprograms. HRESI and ESI, and APICMS
spectra were recorded on Waters Micromass Q-TOF and Agilent
Technologies 6120 Quadrupole LC/MS instrument. Analytical and
preparative TLC were performed on silica gel (Merck, Kieselgel
60, F254, 0.25 and 0.5 mm respectively) or reverse phase (What-
man, KC18 F254, 0.20 mm) plates. The spots were visualized by
exposure to UV radiation (253), or by spraying first with 10%
H2SO4 in MeOH and then with 5% phosphomolybdic acid in EtOH,
followed by heating at 110 °C for 10 min. Column chromatography
(CC) was performed on silica gel column (Merck, Kieselgel 60,
0.063–0.200 mm). Solvent systems: (A) CHCl3-i-PrOH (95:5); (B)

A. Andolfi et al. / Phytochemistry 103 (2014) 145–153
151
inseparable mixture. The residue (587.4 mg) of the third fraction of
3.10. Botryosphaerilactone A (8)
the original column appeared to be the main metabolite, obtained
as a pure yellow oil (Rf 0.43, TLC of silica gel eluent A and Rf 0.50,
TLC on reversed phase eluent C), which was identified as (1R,2R)-
jasmonic acid (6). The residue (294.4 mg) of the fourth fraction
was dissolved in EtOAc and then washed with a saturated solution
of NaHCO3 to remove jasmonic acid (110.3 mg, 236.6 mg/L). The
organic phase was dried with Na2SO4, and evaporated under
reduced pressure affording a brown oil residue (175.8 mg). This
residue was further purified by CC on silica gel, eluted with solvent
system D, yielding seven homogeneous fraction groups. The resi-
due (48.3 mg) of the second fraction was purified by TLC on
reversed phase eluted with solvent system C, yielding an amor-
phous white solid (6.6 mg, 2.2 mg/L, Rf 0.68, eluent C), which
was characterized as (3R,4S)-botryodiplodin (10), and an unco-
loured oil (27.6 mg, 9.2 mg/L, Rf 0.64, eluent C), which was charac-
terized as botryosphaerilactone A (8). The residue (18.3 mg) of the
fifth fraction of the same column was purified by TLC on reversed
phase eluted with eluent system C yielding a homogeneous amor-
phous solid (4.4 mg, 1.7 mg/L, Rf 0.76, eluent C), which was charac-
terized as the (3S,4R,5R)-4-hydroxymethyl-3,5-dimethyldihydro-
2-furanone (9).
25
Compound 8: [
a
]
ꢀ4.0 (c = 0.3, MeOH); UV kmax nm (log
e)
D
209 (3.81); IR mmax cmꢀ1 3458, 1764, 1678, 1613, 1453, 1187 [lit:
27
[a
]
ꢀ1.2 (c 0.77, MeOH); UV (MeOH) kmax (log
e) 286 (1.30)
D
nm; IR (neat) mmax 3438, 1770 cmꢀ1 (Rukachaisirikul et al.,
2009)]; 1H and 13C NMR were very similar to those reported previ-
ously (Rukachaisirikul et al., 2009); ESIMS (+) spectrum m/z: 567
[2M+Na]+, 295 [M+Na]+; ESI (ꢀ) m/z 271 [M - H]-; APCIMS (+) m/
z: 145 [C7H13O3]+, 129 [C7H13O2]+, 127 [C7H11O2]+, 111 [C7H11O]+.
3.11. (3S,4R,5R)-4-Hydroxymethyl-3,5-dimethyldihydro-2-furanone (9)
25
Compound 9: [
a
]
ꢀ18 (c = 0 0.3); UV kmax final absorption; IR
D
mmax cmꢀ1 3427, 1747, 1635; 1456, 1385, 1183; lit, Ravi et al.
(1979): IR
max cmꢀ1 3400, 1770 (Ravi et al., 1979); 1H NMR spec-
trum was similar to data previously reported (Ravi et al., 1979), 13
m
C
NMR d: (179.0, s, C-2), (76.1, d, C-5), (60.7, t, C-8), (52.7, d, C-4),
(37.6, d, C-3), (20.1, q, C-6), (14.2, q, C-7); ESIMS (+) m/z: 167
[M+Na]+; ESIMS (ꢀ) m/z: 143 [MꢀH]ꢀ.
3.12. (3R,4S)-Botryodiplodin (10)
Compound 10: UV kmax nm (loge
) 205 (3.92); IR mmax cmꢀ1
3.5. Lasiojasmonate A (1)
3457, 1707, 1467, 1343; 1H NMR was similar to data previously
reported (Ramezani et al., 2007); ESIMS (+) m/z: 167 [M+Na]+, APC-
IMS (+) m/z: 145 [M+H]+, 127 [MꢀOH]+.
25
Compound 1: [
a
]
D
ꢀ15 (c = 0.4); UV kmax nm (log
e) 204
(3.98); IR mmax cmꢀ1 1747, 1695, 1634, 1240, 1207; 1H and 13C
NMR spectra: see Table 1; HRESIMS (+) m/z: 359.1913 [M+Na]+
(calcd. for C19H28NaO5 359.1834); APCIMS (+) m/z: 337 [M+H]+.
3.13. Acetylation of botryosphaerilactone A
Botryosphaerilactone
(30 L), was converted into the corresponding 16-O-acetyl deriva-
tives ( and b anomers) by acetylation with Ac2O (30 L) carried
out at room temperature for 10 min. The reaction was stopped
by addition of MeOH and the azeotrope formed by addition of ben-
zene was evaporated with a N2 steam. The oily residue (6.0 mg)
was purified by TLC on silica gel, solvent system E, yielding a mix-
A (8, 5.0 mg) dissolved in pyridine
3.6. 16-O-Acetylbotryosphaerilactone A and C (2 and 3)
l
a
l
Compounds 2 and 3: UV kmax nm (loge) 203 (4.01); IR m
max cmꢀ1
1764, 1731, 1681, 1438, 1379; 1H NMR, see Table 2; ESIMS (+) m/z:
651 [2 M+Na]+, 314 [M+Na]+; APCIMS (+) m/z: 315 [M+H]+, 172
[C9H16O3]+, 145 [C7H13O3]+, 127 [C7H11O2]+, 113 [C7H13O]+.
ture of two anomeric acetyl derivatives 3 and 2 (4.1 mg, Rf 0.39,
b, 90:10).
a/
3.7. Lasiojasmonates B and C (4 and 5)
Compounds 4 and 5: UV kmax nm (loge) 205 (4.00); IR m
max cmꢀ1
3.14. Acetylation of (3R,4S)-botryodiplodin
1765, 1701, 1624, 1238, 1211; 1H NMR (CDCl3), see Table 3; ESIMS
(+) m/z: 487.1682 [M+Na]+ (calcd. for C26H40NaO7 487.2672); APC-
IMS (+) m/z: 482 [M+H2O]+, 465 [M+H]+, 322 [C19H30O4]+, 145
[C7H13O3]+, 127 [C7H11O2]+, 113 [C7H13O]+.
(3R,4S)-Botryodiplodin (2.0 mg) was acetylated with pyridine
(35 L) and Ac2O (35 L) in the same conditions above reported
l
l
to converted 8 in the corresponding 2-O-acetyl derivative. Also
the reaction work-up is the same and the residue (2.4 mg) was
purified by TLC on silica gel, eluent system A, to give 2,3-trans-bot-
ryodiplodin acetate (0.8 mg, Rf 0.88, eluent A), whose physic and
spectroscopic data were very similar to those previously reported
(Arsenault and Althaus, 1969).
3.8. (1R,2R)-Jasmonic acid (6)
25
Compound 6: [
a]
ꢀ75 (c = 0.3, MeOH); UV kmax nm (log
e)
D
206 (3.72); IR mmax cmꢀ1 3571, 1749, 1707, 1636, 1259; [lit,
25
Aldridge et al., 1971: [
a
]
D
ꢀ73 (c = 0.1, MeOH); lit, Nielsen and
3.15. Alkaline hydrolysis of lasiojasmonates A–C
Smedsgaard, 2003: UV absorption (nm) in% of UV-max (MeOH)
end; lit, Husain et al., 1993: mmax 1740, 1700 cmꢀ1]; 1H and 13C
NMR were similar to data previously reported (Husain et al.,
1993); ESIMS (+) m/z: 233 [M+Na]+; APCIMS (+) m/z 211 [M+H]+.
Lasiojasmonate A (1, 3,0 mg) was dissolved in MeOH (200
ll)
and H2O (10 l) and hydrolyzed with K2CO3 (6.0 mg). The mixture
l
was stirred at 60 °C for 2 h following the procedure previously
reported (Farmer et al., 1992). Then the mixture was diluted with
distilled water (2 mL) and acidified to pH 4.0 with 2 N HCl and
extracted with EtOAc (2 mL ꢂ 3). The organic extracts were com-
bined, dried (Na2SO4) and evaporated under reduced pressure.
The residue (2.8 mg) was purified by TLC, eluted with solvent sys-
tem A, and the jasmonic acid (0.8 mg) obtained as an oil was com-
pared (TLC, OR and 1H NMR) with the authentic sample of (1R,1R)-
jasmonic acid. Lasiojasmonates B and C (4 and 5) were hydrolyzed
in the same conditions used to treat 1, yielding (1R,2R)-jasmonic
acid ascertained as above reported.
3.9. (1R,2R)-Methyl jasmonate (7)
25
Compound 7: [
a]
ꢀ58 (c = 0.4); UV kmax nm (log
e) 205 (3.67),
D
IR mmax cmꢀ1 1754, 1678, 1426, 1165 [lit. (Nishida et al., 1985)
25
[
a]
ꢀ58 (c = 0.2, MeOH); lit (Nielsen and Smedsgaard, 2003)
D
UV absorption (nm) in% of UV-max (MeOH) end; lit. (Takeda
et al., 2006) IR (neat)
m
max: 1738, 1438, 1198, 1160 cmꢀ1]; 1H
NMR spectrum was similar to data reported (Takeda et al., 2006);
ESIMS (+) m/z: 247 [M+Na]+; APCIMS (+) m/z 225 [M+H]+.

152
A. Andolfi et al. / Phytochemistry 103 (2014) 145–153
Andolfi, A., Mugnai, L., Luque, J., Surico, G., Cimmino, A., Evidente, A., 2011.
3.16. Biological activities
Phytotoxins produced by fungi associated with grapevine trunk diseases. Toxins
3, 1569–1605.
Grapevine bioassay: All compounds, except 4 and 5, were
assayed on detached leaves of grapevine cv Cannonau (red vine).
The leaves with their petioles were immersed in 2 mL toxic solu-
tion until complete absorption. After this they were transferred
to distilled water. Toxicity symptoms were recorded 48 h later.
Each compound was tested at 0.25 and 0.5 mg/mL.
Leaf Puncture Assay: Young cork oak and grapevine leaves were
utilized for this assay. Each compound was assayed at 1.0 mg/mL.
Compounds were first dissolved in MeOH, and then a stock solu-
Andolfi, A., Maddau, L., Cimmino, A., Linaldeddu, B.T., Franceschini, A., Serra, S.,
Basso, S., Melck, D., Evidente, A., 2012. Cyclobotryoxide, a phytotoxic metabolite
produced by the plurivorous pathogen Neofusicoccum australe. J. Nat. Prod. 75,
1785–1791.
Arsenault, G.P., Althaus, J.R., 1969. Structure of antibiotic botryodiplodin. Use of
chemical ionization mass spectrometry in organic structure determination. J.
Chem. Soc. (D) Chem. Commun., 1414–1415.
Begoude, B.A.D., Slippers, B., Wingfield, M.J., Roux, J., 2010. Botryosphaeriaceae
associated with Terminalia catappa in Cameroon, South Africa and Madagascar.
Mycol. Prog. 9, 101–123.
Berger, S., Braun, S., 2004. 200 and More Basic NMR Experiments: A Practical
Course, first ed. Wiley-VCH, Weinheim.
tion with sterile distilled water was made. A droplet (20
solution was applied on the adaxial sides of leaves that had previ-
ously been needle punctured. Droplets (20 l) of MeOH in distilled
ll) of test
Breitmaier, E., Voelter, W., 1987. Carbon-13 NMR Spectroscopy. VCH, Weinheim, pp.
183–280.
da Cunha, M.A.A., Turmina, J.A., Ivanov, R.C., Barroso, R.R., Marques, P.T., Fonseca,
E.A.I., Fortes, Z.B., Dekker, R.F.H., Khaper, N., Barbosa, A.M., 2012. Lasiodiplodan,
l
water (4%) were applied on leaves as control. Each treatment was
repeated three times. The leaves were then kept in a moist cham-
ber to prevent the droplets from drying. Leaves were observed
daily and scored for symptoms after 7 days. The effects of the tox-
ins on the leaves were observed for up to 7 days. Lesions were esti-
mated using APS Assess 2.0 software (Lamari, 2002) following the
tutorials in the user0s manual. The lesion size was expressed in
mm2.
Tomato cutting assay: Tomato cuttings were taken from 21-day-
old seedling and each compound was assayed at 0.1 mg/mL. Cut-
tings were placed in the test solutions (2 mL) for 72 h and then
transferred to distilled water. Symptoms were visually evaluated
for up to 7 days.
Artemia salina bioassay: All compounds, except 4 and 5, were
assayed on brine shrimp larvae (Artemia salina L.). The assay was
performed in cell culture plates with 24 cells (Corning) as already
described (Favilla et al., 2006). The metabolites were dissolved in
methanol and tested at 50 lg/mL. Tests were performed in quadru-
plicate. The percentage of larval mortality was determined after
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The NMR spectra were recorded by Mrs Dominique Melck in the
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gratefully acknowledges Sardinia Regional Government for the
462 financial support of his PhD scholarship (P.O.R. Sardegna
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
03.016. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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