Phytotoxic α-pyrones produced by Pestalotiopsis guepinii, the causal agent of hazelnut twig blight

Full text of DOI:10.1038/ja.2011.134

The Journal of Antibiotics (2012) 65, 203–206
2012 Japan Antibiotics Research Association All rights reserved 0021-8820/12
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&
NOTE
Phytotoxic a-pyrones produced by Pestalotiopsis
guepinii, the causal agent of hazelnut twig blight
Antonio Evidente1, Maria Chiara Zonno2, Anna Andolfi1, Ciro Troise1, Alessio Cimmino1 and
Maurizio Vurro2
The Journal of Antibiotics (2012) 65, 203–206; doi:10.1038/ja.2011.134; published online 1 February 2012
Keywords: 6-(1-hydroxypentyl)-4-methoxy-pyran-2-one; 6-pentyl-4-methoxy-pyran-2-one; Pestalotiopsis guepinii; phytotoxins;
a-pyrones; twig blight of hazelnut
Pestalotiopsis guepinii (Desm.) is the fungal causal agent of the mechanism typical of a-pyrones.9 The structure assigned to 1 was also
so-called ‘twig blight’, one of the most serious diseases of hazelnut supported by the data of its NOESY spectrum in which a significant
(Corylus avellana L.) in Turkey, and one of the main causes of yield coupling was observed between the two a-pyrone protons H-5 and
losses. P. guepinii has been also isolated from walnut (Juglans spp.) and H-3 and the methoxy group; furthermore, H-5 also coupled with the
gum mastic tree (Pistacia lentiscus var. Chia). Recently, the main greater part (H2-1¢, H2-2¢ and H2-3¢) of the 1-hydroxypentyl side
lipophilic phytotoxic metabolite produced by P. guepinii in vitro chain protons and these latter themselves coupled.
culture was isolated and identified by spectroscopic methods as
pestalopyrone (7),1 a pentaketide already known as a minor toxin corresponding monoacetyl and oxidized derivatives (2 and 3, respec-
produced by Pestalotiopsis oenotherae.2
tively, Figure 1), whose spectroscopic data were fully consistent with
Some key derivatives were prepared by converting 1 into the
When the fungus was grown on a different culture medium,3 some those previously reported in literature. Indeed, 2 showed optical
fractions obtained by the purification of the culture filtrate, not rotation IR, UV and 1H NMR spectra very similar to those previously
containing pestalopyrone, proved to be phytotoxic. Their further reported when PC2 was acetylated by a similar procedure.4 The ESIMS
purification yielded other two amorphous phytotoxic metabolites spectrum of 2 showed, beside the sodium cluster and the pseudomo-
(1 and 6, Figure 1) that by preliminary spectroscopic experiments lecular ion at m/z 277 [M+Na]+, 255 [M+H]+, the significant
(including 1H and 13C NMR, IR and UV) appeared to be closely fragmentation peak at m/z 195 [M+H-AcOH]+, which was generated
related to a-pyrones.
by the pseudomolecular ion through the typical loss of the acetic acid
Indeed 1 was identified as 6-(1-hydroxypentyl)-4-methoxy-pyran- residue.9 The oxidized derivative of 3 showed IR, UV and H NMR
2-one and showed optical rotation, IR, UV, 1H and 13C NMR spectra data very similar to those previously reported in the literature for the
very similar to those previously reported in literature for the meta- product obtained by the reaction of N-bromosuccinimide with the
bolite named PC-2 isolated from a Penicillium sp.4,5 The same 5-hydroxy-3-methoxy-6-oxo-2-decenoic acid d-lactone, a metabolite
a-pyrone (1) was successively isolated also from Galiella rufa together isolated from an unidentified fungus10 and close related to the
with (-)-gallielactone and its biogenetic precursor.6 Penicillium monosubstituted 5,6-dihydro-a-pyrone pestalotin. This latter com-
nordicum, P. olsonii and P. verrucosum7 and recently a marine-derived pound is a fungal metabolite with giberellin synergistic activity
fungus Xylaria sp. PSU-F1008 also showed to produce 1. The structure isolated originally from the phytopathogenic fungus Pestalotia crypto-
assigned to 1 was supported by the data observed in its COSY, HSQC meriaecola11,12 and successively from a Penicillum sp. and designed
and HMBC spectra (Table 1) as well as also by the data of the ESIMS. LL-P880a.4,13 Some closely related a-pyrones and 5,6-a-dihydropyr-
Beside both sodium clusters of the dimmer and the toxin itself, and one were isolated from the same two fungi4,5 and also from Penicillium
the pseudomolecular ion observed at m/z 447 [2XM+Na]+, 235 citro-viride.7
1
[M+Na]+, 213 [M+H]+, the ESIMS spectrum also showed the
The absolute stereochemistry of the secondary hydroxylated carbon
significant fragmentation peak [M+Na-CO2]+ at m/z 191 generated C-1¢ of 1-hydroxypentyl side chain at C-6 of 1 was determined
from the sodium cluster by loss of CO2, which is a fragmentation by applying the modified Mosher’s method.14 By reaction with the
1
Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Universita di Napoli Federico II, Portici, Italy and 2Istituto di Scienze delle Produzioni
Alimentari, CNR, Bari, Italy
`
`
Correspondence: Professor A Evidente, Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Universita di Napoli Federico II, via Universita
`
100, 80055 Portici, Italy.
E-mail: evidente@unina.it
Received 14 September 2011; revised 21 November 2011; accepted 8 December 2011; published online 1 February 2012

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R-(-)-a-methoxy-a-trifluoromethyl-a-phenylacetate (MTPA) and
The a-pyrone 6 was identified as 6-pentyl-4-methoxy-pyran-2-one
S-(+)MTPA chlorides, 1 A was converted in the corresponding on the basis of its spectroscopic data below described in detail.
diastereomeric S-MTPA and R-MTPA esters (4 and 5, respectively), Although 6 was originally reported as a synthetic compound15 in a
whose spectroscopic data were consistent with the structure assigned work aimed at preparing natural polyketides, and as a metabolite of
1
to 1. The comparison between the H NMR data (Table 1) of the Galiella rufa, no spectroscopic data had been reported for this
1
S-MTPA ester (4) and those of the R-MTPA ester (5) of 1 allowed to compound. Its H and 13C NMR data (Table 2) were very similar to
assign to C-1¢ a R-configuration. This unambiguously and directly those of 1, as they differed only for the absence of any secondary
assigned absolute configuration, which allowed to formulate 1 as hydroxylated carbons in the side chain at C-6. The assignment of the
6-(1R-hydroxypentyl)-4-methoxy-pyran-2-one, was the same of that chemical shift to all carbons and corresponding protons was also
of metabolite PC-2, which was previously indirectly determined.6
based on the coupling observed in the COSY, HSQC and HMBC
spectra. In the NOESY spectrum a significant coupling was observed
between the methoxy group with both two a-pyrone protons H-5 and
H-3 with, as well as H-5 also coupled with the greater part (H2-1¢,
H2-2¢ and H2-3¢) of the n-pentyl side chain protons and these latter
themselves coupled. This structure of 6 was also supported by ESIMS
data, which showed the sodium cluster and the pseudomolecular ion
at m/z 219[M+Na]+and 197 [M+H]+, respectively.
OCH3
4
5
4'
2'
2
5'
1'
3'
6
O
O
R1 R2
No biological properties were reported for a-pyrones 1 and 6
except some bioassays carried out on the metabolite 6 for a possible
nematocidal activity,16,17 but with negative evidences. For this reason,
1
2
3
R1=H, R2=OH
R1=H, R2=OAc
R1+R2=O
OCH3
OCH3
Table 2 1H and 13C NMR data of 6-pentyl-4-methoxy-pyran-2-one
(6)a,b
1'
1'
O
O
O
O
O
O
C
dC mc
dH
HMBC
O
H
H
O
2
164.3 s
87.3 d
170.9 s
100.0 d
165.1 s
32.7 t
H-3
3
5.43 d (J¼2.2 Hz)
5.79 d (J¼2.2 Hz)
OCH3
CF3
OCH3
Ph
Ph
4
H-5, H-3, OMe
H2-1¢
H-5, H2-1¢, H2-2¢
H2-2¢, H2-3¢, Me-4¢
H2-1¢
CF3
5
4
5
6
1¢
2¢
3¢
4¢
5¢
OMe
2.46 t (J¼7.5Hz)
1.67 (2H) m
OCH3
OCH3
25.9 t
22.0 t
1.34 (2H) m
H2-2¢, Me-4¢
6
30.0 t
1.34 (2H) m
1'
O
O
O
O
14.2 q
57.1 q
0.92 (3H) t (J¼6.8Hz)
3.82 s
6
7
aThe chemical shifts are in d values (p.p.m.) from TMS.
Figure 1 Structures of 6-(1-hydroxypentyl)-4-methoxy-pyran-2-one and 6-
pentyl-4-methoxy-pyran-2-one (1 and 6), four derivatives (2-5) of 1, and
pestalopyrone (7).
b2D 1H, 1H (COSY) 13C, 1H (HSQC) NMR experiments delineated the correlations of all the
protons and the corresponding carbons.
cMultiplicities were assigned by DEPT spectra.
Table 1 1H NMR data of 6-(1-hydroxypentyl)-4-methoxy-pyran-2-one (1)a,b its (S)- and (R)-MTPA esters (4 and 5)a
1
4
5
Position
dH
HMBC
H-3
dH
dH
2
3
5.43 d (J¼2.1Hz)
6.07 d (J¼2.1Hz)
H-5, OMe
5.450 d (J¼2.0Hz)
5.805 d (J¼2.0Hz)
5.480 d (J¼2.0 Hz)
5.981 t (J¼6.5Hz)
4
H-5, H-3, OMe
5
H-3, H-1¢
H-5, H-1¢
6
1¢
2¢
4.38 dd (J¼7.8 and 4.7 Hz)
1.86 m
H-5, H2-2¢, H2-3¢
H-1¢, H2-3¢, Me-5¢
5.610 t (J¼6.3 Hz)
1.960 (2H) m
5.630 t (J¼6.5Hz)
1.929 (2H) m
1.71 m
3¢
4¢
5¢
MeO
MeO
Ph
1.35 (2H) m
H-1¢, H2-2¢, Me-5¢
H-1¢, H2-2¢, H2-3¢, Me-5¢
H2-3¢
1.333 (2H) m
1.333 (2H) m
0.901 (3H) t (J¼6.9 Hz)
3.811 s
1.210 (2H) m
1.210 (2H) m
0.853 (3H) t (J¼7.0Hz)
3.828 s
1.35 (2H) m
0.90 (3H) d (J¼7.0Hz)
3.82 s
3.560 s
3.570 s
7.530–7.420 m
7.550–7.250 m
aThe chemical shifts are in d values (p.p.m.) from TMS.
b2D 1H, 1H (COSY) 13C, 1H (HSQC) NMR experiments delineated the correlations of all the protons and the corresponding carbons.
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Phytotoxic a-pyrones from Pestalotiopsis guepinii
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a preliminary assessment of their phytotoxic and antimicrobial activ- Solvent systems: (A) EtOAc-n-hexane (3 : 2); (B) EtOAc-n-hexane (1 : 1); (C)
EtOAc-n-hexane (2 : 3).
ities was performed and compared with those of the two derivatives (2
and 3) of 1. When tested by puncture on leaves of a number of plant
species (that is: Convolvulus arvensis, Mercurialis annua, Chenopodium
album and Ailanthus altissima) at 20mg per droplet, 1 proved to be
highly phytotoxic, causing the fast appearance of large necrosis on the
leaves of all the species tested. 6 proved to be almost as toxic as 1,
although it was unable to cause necrosis on leaves of C. arvensis,
probably owing to a lower sensitivity of this plant. The acetyl
derivative 2 was less active compared with 1, whereas the oxidized
The strain of P. guepinii used in this study was isolated from naturally
diseased hazelnut leaves as previously reported3 and deposited in the collection
of the Istituto di Scienze delle Produzioni Alimentari, CNR, Bari, Italy, with the
number ITEM 13203. The fungus was grown on a mineral defined liquid media
named M1-D.5 The culture filtrate having high phytotoxic activity on leaves
(3.250l, pH 4.80) was lyophilized, resuspended in distilled water (1/10 of its
original volume) and then extracted by EtOAc (four times with 330 ml each).
The organic extracts were combined, dried with Na2SO4 and the solvent
evaporated under reduced pressure, yielding a brown oil (141.9 mg). This oil
one 3 proved to be completely ineffective to all the tested leaves. was purified by column chromatography (solvent system A), yielding seven
groups of homogeneous fractions. The first three fractions were combined
(11.0 mg) and further purified by TLC (solvent system B). Three metabolites
were obtained as amorphous solids; that is: the already described pestalopyrone
(7) (4.3mg, Rf 0.28); 6-(1-hydroxypentyl)-4-methoxy-pyran-2-one (1, 1.9 mg,
Probably some plants, as C. arvensis and M. annua, have the capability
to hydrolyse 3 into 1. Assayed by comparison at the same concentra-
tion, the main metabolite pestalopyrone proved to be on average less
toxic then the two a-pyrones 1 and 6. These results make possible to
suppose that the n-pentyl side chain is important for the activity. If a
substituent is present in this side chain it could be a nucleophilic
group as are the hydroxy or the double bond present, respectively, in
1 and 7. Conversely, the presence of an electrophilic group, as is the
carbonyl present in 3, determines the loss of activity. However, 7
0.6 mg lꢀ1
, Rf 0.57); and 6-pentyl-4-methoxy-pyran-2-one (6, 1.7 mg,
0.5 mg lꢀ1, Rf 0.49). The fourth fraction (8.0mg) was also purified by TLC
(solvent system A) giving a further amount of 1 A (6.4mg, total 10.7mg,
3.3 mg lꢀ1).
6-(1-hydroxypentyl)-4-methoxy-pyran-2-one (1): Amorphous solid; [a]2D5
+59.8 (c 0.29); IR nmax 3396, 1689, 1645, 1562, 1455, 1409 cmꢀ1; UV lmax
showed a reduced phytotoxicity as a 2-butenyl group instead of a 281 nm (log e 4.25), 224 nm (sh); (lit. 4: [a]2D3 +78.5 (c 1.0 MeOH). IR
nFilmmax cmꢀ1: 3460, 1720, 1700, 1650, 1570, 1410, 1255. UVEtOH
lmax nm (e)
n-pentyl side chain was bonded at C-6. In the bioassay on Lemna
minor L., carried out at the concentration of 100 mg per well the most
toxic compounds proved to be 6 and the acetyl derivative of 1, which
caused the complete desiccation of the plantlets already 24h after their
immersion in the test solution. This effect was similar to that observed
in the case of fumonisin B1, a powerful phytotoxin18 used for
comparison at the same concentration. a-Pyrone 1 and its oxidized
derivative 3 proved to be less toxic and slower acting, as they caused a
clear chlorosis of the plantlets 48h after immersion. In this bioassay
227 (sh. 3650) 289 (9850), 224 nm); 1H NMR see Table 1; 13C NMR was very
similar to that previosly reported in literature;20 ESIMS: m/z 447 [2XM+Na]+,
235 [M+Na]+, 213 [M+H]+, 191 [M+Na-CO2]+.
6-pentyl-4-methoxy-pyran-2-one (6): Amorphous solid; IR nmax 1725, 1649,
1569, 1456, 1411cmꢀ1; UV lmax 282 nm (log e 3.37), 224 nm (sh); 1H and 13
NMR spectra: see Table 2; ESIMS (+) m/z 219 [M+Na]+, 197 [M+H]+.
C
1¢-O-Acetyl derivative of 1 (2): 6-(1-hydroxypentyl)-4-methoxy-pyran-2-
one (1, 3.7mg) was converted into the corresponding 1-O¢-acetyl derivative by
usual reaction with pyridine and acetic anhydride. Derivative 2 had: [a]D25 +61.3
pestalopyrone proved to be ineffective. None of these compounds, (c 0.24); IR nmax 1731, 1655, 1572, 1455, 1412, 1225cmꢀ1; UV lmax 282 nm
(log e 3.49), 224 nm (sh) (lit. 4: [a]23 +85.3 (c 0.19, MeOH); IR nFilmmax cmꢀ1
:
when tested up to 100 mg per diskette, showed any antibiotic activities
when assayed on Bacillus subtilis (gram +) and Escherichia coli
(gram-), and neither a fungitoxic activity when assayed against
Geotrichum candidum.
In conclusion, the two a-pyrone, 6-(1-hydroxypentyl)-4-methoxy-
pyran-2-one; 6-pentyl-4-methoxy-pyran-2-one (1 and 6) were
isolated for the first time as metabolites of P. guepinii together
pestalopyrone, another a-pyrone recently isolated from the same
fungus.1 Pestalopyrone, named demehtyl nectriapyrone A, was pre-
1730, 1650, 1565, 1405, 1245, 1225, 1035, 1020); 1H NMR, d 5.97 (1H, d,
J¼2.1 Hz, H-5), 5.47 (1H, d, J¼2.1Hz, H-3), 5.44 (1H, dd, J¼7.3 and 6.0 Hz,
H-1¢), 3.82 (3H, s, OMe), 2.14 (3H, s, MeCO), 1.89 (m, H2-2¢), 1.34 (4H, m,
H2-3¢ and H2-4¢), 0.93 (3H, t, J¼6.9 Me-5¢). EIMS (rel. int) m/z: 277 [M+Na]+,
255 [M+H]+, 195 [M+H-AcOH]+.
1-Oxo-derivative of 1 (3): 6-(1-hydroxypentyl)-4-methoxy-pyran-2-one (1,
2.0 mg) was oxidized at room temperature in anhydrous CH2Cl2 with MnO2
(24.6 mg). Derivative 3, obtained as homogeneous solid (1.1mg) had: IR nmax
1721, 1699, 1637, 1567, 1465, 1412 cmꢀ1; UV lmax 310 nm (log e 3.29), 220 nm
viously isolated with nectriapyrone B from an unidentified fungus (log e 3.86) (lit. 10: i.r. (KBr) 1721, 1702, 1639, 1569, 1273 and 1257cmꢀ1
;
(CCl4) 1746, 1711, 1638 and 1247cmꢀ1; uv lmax (EtOH) 223 (e 18100) and
309 nm (e 5350)). 1H NMR, d 6.78 (1H, d, J¼2.3 Hz, H-5), 5.73 (1H, d,
J¼2.3 Hz, H-3), 3.88 (3H, s, OMe) 2.92 (2H, t, J¼7.4 Hz, H2-2¢), 1.64-130.
(4H, m, H2-3 and H2-4¢), 0.96 (3H, t, J¼7.4 Me-5¢). ESIMS (+) m/z: 233
[M+Na]+, 211 [M+H]+.
isolated from the indo-pacific sponge Stylotella sp.19,20 Furthermore, a
suitable substituted n-pentyl side chain at C-6 of pyrone ring appeared
to be a structural feature important for the phytotoxicity.
EXPERIMENTAL PROCEDURE
(S)-a-Methoxy-a-trifluoromethyl-a-phenylacetate (MTPA) ester of a-pyr-
Optical rotations were measured in CHCl3 solution on a Jasco P-1010 one 1 (4). 6-(1-hydroxypentyl)-4-methoxy-pyran-2-one (1, 2.6 mg) was con-
(Tokyo, Japan) digital polarimeter; IR spectra were recorded as glassy film on verted into the corresponding a-Methoxy-a-trifluoromethyl-a-phenylacetate
a Perkin-Elmer Spectrum (Norwak, CT, USA). One FT-IR Spectrometer and (MTPA) ester of a-pyrone 1 (4) by reaction with (R)-(-)-MPTA-Cl dry
UV spectra were taken in MeCN solution on a Perkin-Elmer Lambda 25 UV/ pyridine. The usual work-up of the reaction yielded 4 as a homogeneous solid
Vis spectrophotometer. 1H and 13C NMR spectra were recorded at 600 and (2.9mg). It had: [a]2D5 +32.0 (c 0.26); IR nmax 1732, 1658, 1573, 1455,
400, and 100 and 150MHz, respectively, in CDCl3 on Bruker spectrometers 1413 cmꢀ1; UV lmax 282 nm (log e 3.79) 227 nm (sh); for 1H NMR, see
(Kalsruhe, Germany), unless otherwise noted. The same solvent was used as Table 1; ESIMS (+) m/z 879 [2xM+Na]+, 451 [M+Na]+, 429 [M+H]+.
internal standard. Carbon multiplicities were determined by DEPT spectra.
DEPT, COSY-45, HSQC, HMBC and NOESY experiments were performed one
(R)-a-Methoxy-a-trifluoromethyl-a-phenylacetate (MTPA) ester of a -pyr-
(5). 6-(1-hydroxypentyl)-4-methoxy-pyran-2-one (1, 2.6 mg) was
1
using Bruker microprograms. ESI MS spectra were recorded on Waters converted into the (R)-a-methoxy-a-trifluoromethyl-a-phenylacetate (MTPA)
Micromass Q-TOF Micro -(Milford, MS, USA) instruments. Analytical ester of a-pyrone 1 (5) by reaction (S)-(+)-MPTA-Cl. The reaction was carried
and preparative TLC were performed on Si gel (Kieselgel 60 F254, 0.25 and out under the same conditions used for preparing 4 from 1. 5, obtained as a
0.50 mm, respectively, Merck, Darmstadt, Germany) plates; the spots were homogeneous solid (2.6mg), had: [a]2D5 +60.8 (c 0.23); IR 1732, 1658, 1573,
visualized by exposure to UV light and/or by spraying first with 10% H2SO4 1455, 1413, UV lmax 281 nm (log e 3.77) 227 nm (sh) and ESIMS very similar
in MeOH and then with 5% phosphomolybdic acid in EtOH, followed by to those of 4. For 1H NMR, see Table 1; ESIMS (+) m/z 879 [2xM+Na]+, 451
heating at 110 1C for 10min. CC: Si gel (Kieselgel 60, 0.063-0.200mm, Merck). [M+Na]+, 429 [M+H]+.
The Journal of Antibiotics

Phytotoxic a-pyrones from Pestalotiopsis guepinii
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The bioassay of culture filtrates, organic extracts, chromatographic fractions
and pure compounds were assayed by using a leaf puncture assay on different
non-host plants; that is, Convolvulus arvensis, Mercurialis annua, Chenopodium
album and Ailanthus altissima. The test solutions were dissolved in a small
volume of MeOH (final concentration of MeOH ¼2%) and then diluted with
distilled water. The pure compounds were tested at a final concentration of
2 mgmlꢀ1, by applying 10ml of solution to detached leaves previously punc-
tured with a needle. Leaves were kept in a moistened chamber under
continuous fluorescent lights. Symptoms were estimated visually 3 days after
droplet application, using a score from 0 (no symptoms) to 4 (very wide
necrosis - 1 cm diameter).
3
4
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7
Pure compounds were tested at a concentration of 2mg mlꢀ1 on Lemna
minor by adapting a protocol already described.18 Briefly, the wells of sterile,
plastic 96-well microtitre plates were filled with 50 ml aliquot of solutions
containing the metabolites to be tested, at the concentration of above reported.
One frond of actively growing axenic L. minor was placed into each well.
Control wells were included in each plate. At least three replications were
prepared for each compound. The plates were incubated in a growth chamber
with 12/24 h fluorescent lights and observed daily up to 4 days. One day after
the application of the test solution, 100ml of distilled water was added to each
well. Appearance of necrotic or chlorotic symptoms was assessed visually by
comparison of the treated plants with the control appearance.
8
9
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The pure compounds were also tested for the antifungal activity on
Geotrichum candidum, for the antibiosis against Bacillus subtilis (gram +)
and Escherichia coli (gram-) according to the protocols already described, up to
100 mg per diskette.21
ACKNOWLEDGEMENTS
The authors thank Dr Pierluigi Mazzei, Universita di Napoli Federico II, for
¨
16 Kopcke, B., Johansson, M., Sterner, O. & Anke, H. Biologically active secondary
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