Ovipositional deterrent in the sweet pepper, Capsicum annuum, at the mature stage against Liriomyza trifolii (Burgess)

Article abstract of DOI:10.1271/bbb.69.1831

Liriomyza trifolii (Burgess), the American serpentine leafminer fly, is well known as a serious pest throughout the world. This insect attack over 21 different plant families including solanaceae plants. The mature sweet pepper, Capsicum annuum (Solanaceae), however, shows resistance to this leafminer fly. This resistance is based on the ovipositional deterrent in the sweet pepper leaf against the fly species. Based on bioassay-guided fractionation, luteolin 7-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside was isolated and identified as the ovipositional deterrent against this insect species. This compound completely deterred L. trifolii females from laying their eggs on a host plant leaf treated at 4.90 μg/cm².

Full text of DOI:10.1271/bbb.69.1831

Biosci. Biotechnol. Biochem., 69 (10), 1831–1835, 2005
Ovipositional Deterrent in the Sweet Pepper, Capsicum annuum,
at the Mature Stage against Liriomyza trifolii (Burgess)
*
Takehiro KASHIWAGI, Yoh HORIBATA, Daniel Bisrat MEKURIA,
y
Shin-ich TEBAYASHI, and Chul-Sa KIM
Department of Bioresources Science, Faculty of Agriculture, Kochi University,
B200 Monobe, Nankoku 783-8502, Japan
Received March 14, 2005; Accepted July 11, 2005
Liriomyza trifolii (Burgess), the American serpentine
leafminer fly, is well known as a serious pest throughout
the world. This insect attack over 21 different plant
families including solanaceae plants. The mature sweet
pepper, Capsicum annuum (Solanaceae), however,
shows resistance to this leafminer fly. This resistance
is based on the ovipositional deterrent in the sweet
pepper leaf against the fly species. Based on bioassay-
guided fractionation, luteolin 7-O-ꢀ-D-apiofuranosyl-
(1!2)-ꢀ-D-glucopyranoside was isolated and identified
as the ovipositional deterrent against this insect species.
This compound completely deterred L. trifolii females
from laying their eggs on a host plant leaf treated at
4.90 ꢀg/cm2.
as one of the hosts.8) In fact, this insect species fiercely
attacks young C. annuum. However, we have observed
that L. trifolii seldom attacked and laid their eggs on
mature sweet pepper in our greenhouse.
We report in this paper the factors that contributed to
the resistance of mature C. annuum against L. trifolii.
A fresh kidney bean leaf was soaked in a methanol
solution of C. annuum at the mature stage (1 g of fresh
leaf equivalent/ml) and then submitted to a bioassay.
Only a few ovipositional marks (1:85 marks/cm2 ꢀ
0:72; mean ꢀ S. E.) were observed on the treated leaf.
On the other hand, a significant number of marks
(64:83 marks/cm2 ꢀ 2:85) were observed on the control
leaf. There were no larvae hatching from the treated
leaves after keeping them for a few days in the
incubator. These results are similar to our previous
observations in the greenhouse and clearly show that the
leaf of C. annuum at the mature stage exerted an
ovipositional deterrent against this insect species. Dur-
ing the bioassay, the female flies landed briefly on the
kidney bean leaf that had been treated with the methanol
extract. Upon landing, the females walked over the leaf
and drummed the surface of the leaf, before attempting
to insert their ovipositors into the leaf.
The active methanol extract was separated into the
hexane, diethyl ether, ethyl acetate, water-saturated
butanol and water layers by liquid-liquid partition. Of
these, the hexane (9:03 marks/cm2 ꢀ 1:88) and the
water layers (7:31 marks/cm2 ꢀ 0:13) showed intense
activity. The other fractions (diethyl ether: 49.35
marks/cm2 ꢀ 4:27, ethyl acetate: 45:71 marks/cm2 ꢀ
4:04, and water-saturated butanol: 37:53 marks/cm2 ꢀ
6:38) did not show any activity against this insect
species (Fig. 1). The active compound in the hexane
layer has recently been determined to be the terpene
alcohol, (ꢁ)-phytol. This compound showed oviposi-
tional deterrent activity against the insect species at
35.2 mg/cm2 (5:91 marks/cm2 ꢀ 2:04). Since (ꢁ)-phytol
is a common compound in various plants and sweet
pepper contained as much of the phytol as that in kidney
Key words: Liriomyza trifolii; ovipositional deterrent;
Capsicum annuum; luteolin 7-O-ꢀ-D-apio-
furanosyl-(1!2)-ꢀ-D-glucopyranoside
Liriomyza trifolii (Burgess), the American serpentine
leafminer fly, is well known as a serious pest to many
vegetables and crops throughout the world. The fly has a
broad host range and can attack over 120 plant species in
more than 21 families.1) Females of L. trifolii puncture
the leaf surface with their ovipositor for feeding and
oviposition. The larvae, after hatching from the eggs,
feed on the mesophyll tissue in the leaf and form a
serpentine mine which can significantly reduce the
photosynthetic capacity of the plant.2)
The fly, which originated in North America, has
quickly spread its distribution area throughout the world
and was first found in Japan in 1990 in Shizuoka
prefecture.3,4) In addition to the broad host range, the
leafminer shows resistance to most insecticides and can
develop all year round in a greenhouse,3,5–7) making
control of the leafminer pest more difficult. The
development of an integrated pest control method
against this species is therefore necessary.
Sweet pepper, Capsicum annuum (Solanaceae) has
been reported, along with many other solanaceae plants,
y
To whom correspondence should be addressed. Tel: +81-88-864-5185; Fax: +81-88-864-5186; E-mail: cs-kim@cc.kochi-u.ac.jp
Present address: Department of Biomechanical Systems, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan
*

1832
T. KASHIWAGI et al.
Fig. 1. Scheme for Fractionation of the Methanol Extract of C. annuum.
bean, this compound may have been a by-product
generated during the extraction procedure with meth-
anol.9) When the water layer (0.70 g: 20 g of fresh leaf
equivalent) was dissolved in 1 ml of water, as educt was
generated. After separating this educt by centrifugation,
the precipitate and supernatant were submitted to a
bioassay. The precipitate (11:93 marks/cm2 ꢀ 2:16) and
supernatant (21:25 marks/cm2 ꢀ 2:33) both showed
strong activity against this insect species. The precipitate
was separated into three fractions (A, B and C) by
reverse-phase HPLC. Of these three fractions, only B
showed high activity (A, 52:93 marks/cm2 ꢀ 5:80; B,
3:87 marks/cm2 ꢀ 0:95; C, 41:69 marks/cm2 ꢀ 5:74).
Fr. B was a yellow powder and gave a single peak
(compound 1) in the HPLC analysis. However, this
compound was hardly detectable in the supernatant,
indicating that several active compounds, which deter-
red this insect from laying its eggs on the leaf, were
present in the leaf of mature C. annuum.
Table 1. Chemical Shifts Values for Compound 1 and Analogous
Flavonoids in Their 13C-NMR Spectra
luteolin
7-O-glucoside11)
C-Position Compound 1 Luteolin10)
Apiin12)
Aglycon
C-2
C-3
Luteolin
164.5
103.2
181.9
161.2
99.4
Luteolin
164.5
103.3
182.2
162.1
99.2
Luteolin
164.9
103.6
182.4
161.6
100.0
163.4
95.2
Apigenin
164.3
103.1
181.9
161.3
99.4
C-4
C-5
C-6
C-7
162.7
94.7
164.7
94.2
162.7
94.8
C-8
C-9
157.0
105.4
157.9
104.2
157.4
105.8
156.9
105.4
C-10
C-10
C-20
C-30
C-40
C-50
C-60
121.4
113.5
145.8
150.0
116.0
119.2
119.3
113.8
146.2
150.1
116.4
122.1
121.8
114.0
146.3
150.4
116.5
119.7
121.0
128.5
116.0
161.1
116.0
128.5
The LC–MS data (positive m=z 581 ½M þ Hꢂþ;
negative m=z 579 ½M ꢁ Hꢂꢁ), indicated the molecular
weight of compound 1 to be 580. Since a set of ABX
systems at ꢁ 6.96 (d, J ¼ 8:8, H-50), ꢁ 7.48 (d, J ¼ 1:2,
H-20) and ꢁ 7.50 (dd, J ¼ 8:8, and 1.2, H-60), two
aromatic proton signals (ꢁ 6.48, J ¼ 2:0, H-6; and 6.81,
J ¼ 2:0, H-8) and one aromatic proton signal (ꢁ 6.80, s,
H-3) were observed in the 1H-NMR spectrum of
compound 1, this compound was considered to be a
luteolin derivative. As shown in Table 1, 11 carbon
signals, including two anomeric carbons (98.1 ppm and
108.8 ppm), were observed, in addition to those signals
due to the luteolin moiety, in the 13C-NMR spectrum.
This compound seemed to be a luteolin diglycoside. A
comparison of the 13C-NMR spectrum of 1 with that of
luteolin10) showed significant downfield shifts at C-6
(þ0:2 ppm), C-8 (þ0:5 ppm) and C-10 (þ1:2 ppm), and
upfield shifts at C-5 (ꢁ0:9 ppm), C-7 (ꢁ0:2 ppm) and C-
Glucose
C-100
98.1
75.9
76.8
69.8
77.0
60.6
100.3
73.8
76.8
70.0
77.6
61.1
98.2
75.9
76.1
69.8
77.0
60.6
C-200
C-300
C-400
C-500
C-600
Apiose
C-1000
C-2000
C-3000
C-4000
C-5000
108.8
76.0
79.3
74.0
64.2
108.7
76.7
79.1
73.9
64.2
9 (ꢁ0:9 ppm). Such shifts are typical of O-glycosylation
at the C-7 position. It is therefore reasonable that this
compound had a luteolin aglycon with a di-O-glycoside
at position 7. Comparing the 13C-NMR spectrum with

Ovipositional Deterrent against Liriomyza trifolii
1833
Fig. 2. Structure of Compound 1.
NOEs and HMBC relationships observed are shown by solid lines and arrows, respectively.
that of apiin (apigenin 7-O-ꢀ-D-apiofuranosyl-(1!2)-ꢀ-
D-glucopyranoside),12) the chemical shift values of the
sugar moieties were very similar. Hence, this compound
was determined to be luteolin 7-O-ꢀ-apiofuranosyl-
(1!2)-ꢀ-glucopyranoside. As shown in Fig. 2, the H–H
and C–H COSY, HMQC and HMBC data indicated the
same structure. Methanolysis of compound 1 was con-
ducted to confirm the absolute configuration of both
sugars. The obtained mixture of 1-O-methyl-ꢂ- and ꢀ-
glucopyranose and 1-O-methyl-ꢀ-apiofuranose showed
specific rotation values of þ86:0ꢃ and ꢁ88:8ꢃ, respec-
tively. These values are similar to those of an authentic
sample (½ꢂꢂD þ87:1ꢃ) and literature data (ꢁ95ꢃ).13) Each
absolute configuration was determined as being D-form
from these results. This compound was therefore
determined to be luteolin 7-O-ꢀ-D-apiofuranosyl-
(1!2)-ꢀ-D-glucopyranoside, which completely deterred
the fly from laying its eggs on a leaf treated at 4.90 mg/
cm2 (Fig. 3). On the other hand, luteolin 7-O-ꢀ-D-
glucopyranoside isolated from the same plant leaves did
not show any activity against this fly insect species at all.
These results indicate that the apiose moiety was very
important to the activity.
Fig. 3. Ovipositional Deterrent Activities of Luteolin.
7-O-ꢀ-D-Apiosyl-(1!2)-ꢀ-D-glucoside and Luteolin 7-O-ꢀ-D-
glucoside against L. trifolii.
levels were not strong enough to have any ovipositional
deterrent effect towards the adult females of L. trifolii.
In contrast, the distribution of compound 1 in the leaves
at the mature stages (twenty-leaf stage and fruiting
stage) was 130–3000 times as much as that at the young
stages. This dose range was enough to exert an oviposi-
As shown in Table 2, the concentration and calculated
distribution of compound 1 in the leaves of C. annuum
increased with plant growth. The calculated distribution
of compound 1 in the cotyledon and leaves at the four-
leaf stage was 0.1 and 0.7 mg/cm2, respectively. These
Table 2. Concentration and Calculated Distribution of Compound 1 in the Leaves at Various Stages and in the Fruit of the Sweet Pepper
Cotyledons at
four-leaf stage
Four-leaf
stage
Six-leaf
stage
Ten-leaf
stage
Twenty-leaf
stage
Fruiting
stage
Fruit
417.9
—
Concentration of compound 1
(mg/g)
Calculated distribution of
4.1
0.1
61.2
0.7
2116.3
26.3
4147.7
123.4
8250.3
222.3
12,135.6
329.5
compound 1
(mg/cm2)

1834
T. KASHIWAGI et al.
tional deterrent effect against this insect spices.
Compound 1 was initially isolated and identified from
parsley (Petroselinum crispum) as graveobioside A by
under vacuum, the residue (33.9 g) was dissolved at a
concentration of 1 g of fresh leaf equivalent/ml in
methanol.
Nordstrom.14) The biological activity and spectroscopic
¨
data for this compound are reported here for the first
time. This flavonoid glycoside is present in a large
amount (more than 12,000 ppm) in mature C. annuum
leaves. The fruit of sweet pepper also contains 417.9 mg
of compound 1 per 1 g of fresh weight. This compound
can be very easily isolated and is consumed in daily
food. These results might contribute to the development
of an integrated pest control method against this insect
that is safe and of suitable concentration for the natural
environment. Active compounds in the supernatant from
the water-soluble fraction now need to be investigated,
and the mechanism for changing the resistance of
C. annuum with growth stage also needs to be eluci-
dated.
Bioassay of the plant extract. A fresh kidney bean leaf
was dipped into the test solution (1 g of fresh leaf
equivalent/ml) for 30 s. After removing the solvent, the
leaf was put onto a moistened filter paper at the bottom
of a Petri dish (10 mm in high, 90 mm in i.d) to maintain
the humidity. Five adult female flies at least 24 h old
were put into a screw vial (28 mmꢃ) and it was placed
on treated or control leaves so that the mouth of the vial
touched the leaf and allowed to oviposit for 24 h at 27 ꢃC
under 16:8(L:D) illumination. The number of oviposi-
tional marks was counted on each leaf under a micro-
scope. Each test was replicated five times. The control
leaf was treated in the same manner with only a
methanol solution.
Experimental
Isolation of compound 1. The methanol extract
(17.8 g, 320 g of fresh leaf equivalent) was dissolved
in water (430 ml), and then successively extracted with
hexane (300 ml ꢄ 4, 3.80 g), diethyl ether (300 ml ꢄ 4,
0.45 g), ethyl acetate (300 ml ꢄ 4, 0.53 g) and water-
saturated butanol (300 ml ꢄ 4, 3.1 g). The water-soluble
fraction (0.70 g: 20 g of fresh leaf equivalent) was
dissolved in 1 ml of water and then divided into the
supernatant and precipitate by centrifuging (10,000 rpm
at 4 ꢃC for 10 min). The precipitate was separated
into three fractions, A (Rt ¼ 0{11:1 min), B (Rt ¼
11:1{12:8 min) and C (Rt ¼ 12:8{15:0 min), by re-
versed-phase HPLC (SHISEIDO CAPCELLPAK C18
Instruments. LC–MS data were recorded with a
Shimadzu LCMS-2010 mass spectrometer when using
a reversed-phase column (SHISEIDO CAPCELLPAK
ꢀ
C18 UG120 A, 250 mm ꢄ 4:6 mm i.d.) eluted with 10–
90% acetonitrile in water containing 1% acetic acid in
the APCI-positive and APCI-negative modes. Optical
rotation was measured with a HORIBA SEPA-200
spectropolarimeter. 1H-NMR and 13C-NMR spectra,
including two-dimensional correlation spectra, were
measured with a JEOL JNM-AL 400 (400 MHz) instru-
ment. TMS was used as an internal standard. Letters
(br.)s, d, t, q, and m represent (broad)singlet, doublet,
triplet, quartet, and multiplet, respectively, and coupling
constants are given in Hz.
ꢀ
UG120 A column, 250 mm ꢄ 10 mm i.d.), eluting with
20% acetonitrile in water at a flow rate of 2 ml/min and
detecting at UV 254 nm. Compound 1 was isolated at
Rt ¼ 11:8 min from fraction B. The leaf of sweet pepper
at the twenty-leaf stage contained compound 1 at
8,250 mg/g of fresh leaf equivalent.
Insect and plant. Stock colonies of L. trifolii were
collected from Kochi Pref. Agricultural Center and
successively reared on 10- to 14-d-old seedlings of the
kidney bean, Phaseolus vulgaris L. (Daikintoki) at
27 ꢀ 2 ꢃC and a relative humidity of 60–70% with
16:8(L:D) illumination. After emerging, 1-d-old females
were used for the bioassay.
Forty seeds of C. annuum var. angulosum (Sakigake
2-go) were planted in a plastic tray (20 cm ꢄ 27 cm in
length, 1 cm in depth) containing nursery soil in a plant-
growth incubator at 27 ꢀ 2 ꢃC. After four weeks, the
seedlings were transplanted into individual pots (6.6 cm
in height, 5.0 cm in i.d.) containing nursery soil and
grown in a greenhouse without any application of
insecticides. Plants at the twenty-leaf stage (approx-
imately 17 weeks old) were used for the extract. Ten- to
14-d-old seedlings of P. vulgaris were used for the
bioassay.
Methanolysis of compound 1. Compound 1 (120 mg)
was mixed with 1 ml of 5% hydrochloric acid in
methanol, and heated at 80 ꢃC for 1 h. After vacuum
concentration, the reaction mixture was dissolved in
10 ml of water and extracted three times with 10 ml of
ethyl acetate. The water layer diluted with water (20 ml)
was passed through a Sep-pak C18 cartridge (Waters)
and successively eluted with 10 ml of water and 10 ml of
40% aqueous methanol. The 1-O-methylsugars were
recovered from the water eluate, and luteolin (45.6 mg)
was obtained from the 40% aqueous methanol eluate
and the ethyl acetate layer. 1-O-Methylapiofranose
(2.7 mg) and 1-O-methylglucopyranose (16.8 mg) were
isolated by HPLC (Nakarai tesque waters Cosmosil
5NH2-MS column; 10 mmꢃ ꢄ 250 mm, 70% acetoni-
trile in water at 3 ml/min; refractive index detector).
ꢂ- and ꢀ-D-1-O-Methylglucopyranose were also pre-
pared from an authentic D-glucose sample by the same
procedure.
Preparation of the plant extract. Fresh sweet pepper
leaves (420 g) were twice extracted with 80% methanol
in water (1 liter) for 3 d in darkness. After concentrating

Ovipositional Deterrent against Liriomyza trifolii
1835
Compound 1 (luteolin 7-O-ꢀ-D-apiofranosyl-(1!2)-
References
22
ꢀ-D-glucopyranoside). ½ꢂꢂD ꢁ82:0ꢃ (c 1.0, DMSO).
LC–MS (APCI-positive) m=z (%): 581 (87.5, M þ Hþ),
449 (8.6, M þ Hþ-apiose), 319 (17.3), 287 (74.1,
luteolin þ Hþ), 157 (100.0). LC–MS (APCI-negative)
m=z (%): 579 (72.2, Mꢁ ꢁ H), 447 (9.6, Mꢁ ꢁ H-
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ꢁ
1
apiose), 285 (100.0, luteolin ꢁ H). H-NMR (DMSO-
d6) ꢁ: 7.50 (1H, dd, J ¼ 8:8, and 1.2, H-60), 7.48 (1H, d,
J ¼ 1:2, H-20), 6.96 (1H, d, J ¼ 8:4, H-50), 6.81 (1H, d,
J ¼ 2:0, H-8), 6.80 (1H, s, H-3), 6.48 (1H, d, J ¼ 2:0,
H-6), 5.41 (1H, s, H-1000), 5.23 (1H, d, J ¼ 6:8, H-100),
3.97 (1H, d, J ¼ 9:2, H-4000-a), 3.79 (1H, s, H-2000), 3.77
(1H, d, J ¼ 9:6, H-600-a), 3.72 (1H, d, J ¼ 9:2, H-4000-b),
3.58 (1H, t, J ¼ 9:2, H-300), 3.56 (1H, dd, J ¼ 9:6 and
9.2, H-600-b), 3.55 (1H, t, J ¼ 9:2, H-500), 3.52 (1H, dd,
J ¼ 9:0 and 6.8, H-200), 3.34 (2H, s, H-5000), 3.25 (1H, t,
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1
Luteolin from compound 1. H-NMR (DMSO-d6) ꢁ:
7.50 (1H, dd, J ¼ 8:8, and 1.2, H-60), 7.48 (1H, d,
J ¼ 1:2, H-20), 6.96 (1H, d, J ¼ 8:4, H-50), 6.81 (1H, d,
J ¼ 2:0, H-8), 6.80 (1H, s, H-3), 6.48 (1H, d, J ¼ 2:0,
H-6). 13C-NMR (DMSO-d6) ꢁ: 181.9 (s, C-4), 164.5 (s,
C-2), 162.7 (s, C-7), 161.2 (s, C-5), 157.0 (s, C-9), 150.0
(s, C-40), 145.8 (s, C-30), 121.4 (s, C-10), 119.2 (d, C-60),
116.0 (d, C-50), 113.5 (d, C-20), 105.4 (s, C-10), 103.2
(d, C-3), 99.4 (d, C-6), 94.7 (d, C-8).
ꢂ- and ꢀ-D-1-O-Methylglucopyranose from com-
22
pound 1. Rt ¼ 7:42 min, ½ꢂꢂD þ86:0ꢃ (c 1.0, H2O).
9) Kashiwagi, T., Mikagi, E., Daniel, B. M., Aman, D. B.,
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(d, ꢂ-3), 76.8 (d, ꢂ-5), 74.2 (d, ꢀ-3), 74.2 (d, ꢂ-2), 72.7
(d, ꢀ-5), 72.3 (d, ꢀ-2), 70.7 (d, ꢂ-4), 70.6 (d, ꢀ-4), 61.8
(t, ꢂ-6), 61.6 (t, ꢀ-6), 58.3 (q, ꢂ-O-Me), 56.1 (q, ꢀ-O-
Me).
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ꢂ- and ꢀ-D-1-O-Methylglucopyranose from an au-
22
thentic sample. Rt ¼ 7:42 min, ½ꢂꢂD þ87:1ꢃ (c 1.0,
H2O). 13C-NMR (D2O) ꢁ: 104.3 (d, ꢂ-1), 100.3 (d, ꢀ-1),
77.0 (d, ꢂ-3), 76.9 (d, ꢂ-5), 74.2 (d, ꢀ-3), 74.2 (d, ꢂ-2),
72.7 (d, ꢀ-5), 72.3 (d, ꢀ-2), 70.8 (d, ꢂ-4), 70.7 (d, ꢀ-4),
61.9 (t, ꢂ-6), 61.7 (t, ꢀ-6), 58.4 (q, ꢂ-O-Me), 56.2 (q, ꢀ-
O-Me).
22
ꢀ-D-1-O-Apiofuranose. Rt ¼ 6:16 min, ½ꢂꢂD ꢁ88:8ꢃ
(c 0.18, H2O). 13C-NMR (D2O) ꢁ: 109.8 (d, A-1), 79.8
(s, A-3), 77.0 (d, A-2), 74.0 (t, A-4), 63.9 (t, A-5), 56.6
(q, -O-Me).
Acknowledgments
We gratefully thank the Insect Department of Kochi
Pref. Agricultural Center for providing the insects.
14) Nordstrom, C. G., and Swain, T., The flavonoid glyco-
¨
sides of Dahlia variabiles. Part I, General introduction.
Cyanidin, apigenin, and luteolin glycosides from the
variety ‘‘dandy’’. J. Chem. Soc., 2764–2773 (1953).