Ovipositional Responses of Chilo partellus
J. Agric. Food Chem., Vol. 51, No. 14, 2003 4009
next morning, females that had laid eggs were selected for oviposition
bioassays to be used in the evening. Each test was replicated five times,
and each replicate consisted of the average number of eggs laid by
three females. Only those bioassays in which the eggs attained the
“blackhead stage” were considered as valid replicates and were selected
for statistical analysis.
Seeds of maize cultivars, Basilocal (susceptible) and Kisan (resistant),
were obtained from the Indian Agricultural Research Institute (IARI),
New Delhi. These were sown in experimental plots of the Zoology
Department, University of Delhi. The plants were raised in pesticide-
free conditions, under standard farming practice.
Extraction of Plant Foliage Chemicals. Fully expanded, freshly
excised young leaves, located on the lower half of 3-5 week old maize
plants, which is the preferred site for oviposition by C. partellus moths
(7), were used for extraction. From each test cultivar (Basilocal and
Kisan), 500 g of leaves were soaked overnight in 2.5 L of hexane. The
extracts were then decanted. The leaves and jars were rinsed three times
with 500 mL of hexane each time. The pooled extracts were
concentrated to near dryness in vacuo and the residue was redissolved
in hexane to prepare a solution containing 1gfle (1 g of fresh leaf
extract) per mL of the extract. The extracts were stored at 4 °C until
workup.
R-methylene protons and a multiplet at δ 1.52 was assigned for
â-methylene protons. Because the remaining methylene groups are
similar, the chemical shift appeared as a broad singlet at δ 1.28. A
triplet, observed at δ 0.88 for three protons, was assigned for the
terminal methyl group. On the basis of the above-mentioned spectral
data and comparison of observed melting point with that reported in
the literature, compound 2 was identified as heptadecanol. On the basis
of similar spectral studies, compound 3 was characterized as nonade-
canol.
Heptadecanol (2). White solid, mp 55-56 °C. IR νmax (KBr): 3445,
2924, 1378, 1060, 665 cm-1. 1H NMR (δ, CDCl3, 250 MHz): 0.88 (t,
3H, -CH3), 1.28 (brs, 28H), 1.52 (m, 2H, -CH2CH2O-), 3.58 (t, 2H,
-CH2OH). EIMS m/z (%): 256 (M+, 8), 238 (M+ -H2O, 5), 210 (8),
199 (16), 185 (6), 171 (9), 156 (6), 143 (6), 129 (15), 111 (36), 97
(60), 83 (85), 69 (82), 55 (92), 43 (100).
Nonadecanol (3). White solid, mp 58-59 °C. IR νmax (KBr): 3440,
2925, 1380, 1062, 649 cm-1. 1H NMR (δ, CDCl3, 250 MHz): 0.88 (t,
3H, -CH3), 1.28 (brs, 32H), 1.52 (m, 2H, -CH2CH2OH), 3.61 (t, 2H,
-CH2OH). EIMS m/z (%): 284 (M+, 9), 266 (M+ -H2O, 6), 241 (16),
227 (18), 213 (6), 199 (16), 185 (26), 171 (16), 157 (14), 143 (3), 129
(42), 95 (42), 83 (63), 73 (96), 71 (100), 69 (42), 61 (88), 57 (66), 55
(76).
Isolation and Characterization of Active Components from
Hexane Extract of Kisan Leaves (HEKL). The solvent free extract
from Kisan leaves was found to be a mixture of several components
of varying polarity on TLC and was, therefore, further fractionated by
column chromatography. The column was prepared in petrol using silica
gel (60-80 mesh) as an adsorbent and eluted with petrol/chloroform
mixtures of increasing polarity. Thirteen fractions were collected, and
the following compounds were isolated.
Compound 1 was obtained from the fractions eluted with petrol/
chloroform (70:30). On treatment with acetic anhydride and dry
pyridine, it yielded an acetate (compound 1a). Compounds 2 and 3
were isolated from the crude extract by column chromatography using
petrol/chloroform (10:90) as the eluent.
The natural compounds 2 and 3 were also synthesized to confirm
their proposed structures and to obtain them in sufficient amounts to
test the effect of individual compounds on the ovipositional responses
of C. partellus. Synthetic heptadecanol and nonadecanol were obtained
by reducing their corresponding methyl esters with lithium aluminum
hydride. Also, heptadecinonitrile (4), a model compound, was synthe-
sized by the reaction of 1-bromo hexadecane with potassium cyanide
in absolute methanol and fully characterized by its spectral data as given
below.
Heptadecinonitrile (4). White solid, mp 34 °C. IR νmax (KBr): 2919,
1
2852, 2245, 1472, 1421, 1376, 718 cm-1. H NMR (δ, CDCl3, 250
MHz): 0.88 (t, 3H, -CH3), 1.28 (brs, 26H), 1.65 (m, 2H, -CH2CH2-
CN), 2.40 (t, 2H, -CH2CN). EIMS m/z (%): 251 (M+, 14), 222 (25),
208 (35), 194 (31), 180 (26), 166 (23), 152 (28), 138 (40), 124 (67),
110 (100), 97 (92), 83 (74), 71(86), 69 (81), 57 (96).
Characterization of Compounds 1 and 1a. Preliminary examina-
tion of compound 1 indicated it to be aliphatic in nature. The absence
of a molecular ion peak in the mass spectrum and the presence of a
peak at m/z 448 for (M+ - H2O) indicated that it contained an -OH
group and was therefore analyzed for C32H66O. The presence of a
hydroxyl group was further supported by 13C NMR, which showed a
signal at δ 63.04 for the carbon carrying the hydroxyl group, and this
was confirmed by its IR spectrum, which showed a broad absorption
Other compounds isolated from HEKL but not yet identified are
coded as MR-1, MR-7, MR-22a, MR-22b, and MR-27. Fractions
obtained from silica gel chromatography, in the case of hexane extract
of Basilocal leaves (HEBL), were analyzed along with those from
HEKL by TLC. It was observed that, while the spots of compounds
from HEKL were very prominent, the corresponding spots could not
be seen in HEBL on TLC, using solvent systems CHCl3/petrol (1:1,
v/v) and benzene/EtOAc (9:1, v/v) and spraying with 5% alcoholic
solution of polymolybdic acid and heating the plate as well as exposing
the plate to iodine vapors. Therefore, further investigation of HEBL
was not carried out.
Bioassay Equipment. The oviposition chamber was made of a
vertical emery paper cylinder (6 cm ht × 6.8 cm diam) closed at both
ends by glass Petri dishes (1 cm ht × 7 cm diam). Rough surface of
emery paper provided a contrast to the smooth surface of glass Petri
dishes, which is preferred by Chilo females for egg laying (14).
Moreover, test stimuli were presented on the bottom Petri dish, because
preliminary studies showed that the stem borer females, on the second
night of emergence, laid a higher percentage of eggs on the bottom
surface, compared to the top surface.
1
band at 3446 cm-1. Its H NMR spectrum exhibited signals for the
presence of a terminal methyl group at δ 0.90 and a chain of methylene
protons at δ 1.26. A triplet at δ 3.63 showed the presence of a methylene
linked to oxygen atom. All these spectral data, when coupled together,
indicated compound 1 to be a saturated primary alcohol containing
C32 carbon atoms. On the basis of the above spectral studies, compound
1 was characterized as dotriacontanol (1). The proposed structure was
confirmed by analyzing its acetate (1a).
Dotriacontanol (1). White solid, mp 87-88 °C. IR νmax (KBr):
1
3446, 2920, 1465, 1360, 1061, 670 cm-1. H NMR (δ, CDCl3, 250
MHz): 0.90 (t, 3H,-CH3), 1.26 (brs, 58H), 1.54 (m, 2H, H - 2), 3.63
(t, 2H, -CH2OH). 13C NMR (δ, CDCl3, 62.89 MHz): 14.01 (-CH3),
25.67-32.76 (-CH2-), 63.04 (-CH2OH). EIMS m/z (%): 448 (M+
-18, 36), 421 (8), 365 (5), 153(18), 71(80), 57(100).
Dotriacontanyl Acetate (1a). White solid, mp 76 °C. 1H NMR (δ,
CDCl3, 250 MHz): 0.90 (t, 3H, -CH3), 1.27 (brs, 58H), 1.60 (m, 2H,
H-2), 2.25 (s, 3H, -COCH3), 4.10 (t, 2H, -CH2O-). EIMS m/z (%):
508 (M+, 75), 507 (48), 479 (56), 449 (58), 446 (82), 421(47), 390
(42), 364 (46), 335 (45), 307 (51), 293 (59), 279 (55), 265 (42), 252
(58), 238 (55), 211 (45), 197 (55), 183 (35), 151 (26), 123 (92), 99
(100), 83 (92), 69 (91), 57 (92).
Characterization of Compounds 2 and 3. Compound 2 was
purified as a white solid having melting point 55-56 °C. Its IR spectrum
showed a characteristic absorption at 3445 cm-1, indicating the presence
of a hydroxyl group. It gave a molecular ion peak at m/z 256 along
with a peak at 238 (M+ - H2O), supporting the presence of a hydroxyl
group and was assigned the molecular formula C17H36O. In its 1H NMR
spectrum, a triplet for two protons at δ 3.58 was assigned for the
Bioassay Protocol. The ovipositional responses to hexane extracts
of foliage of test cultivars and to chemicals extracted from HEKL were
studied in two-choice bioassays. The test solution was smeared in one-
half of the bottom Petri dish, so as to deposit 100 µg of residue per
cm2 of the substrate, and the other half (control) was smeared with an
equal volume of hexane.
In bioassays where hexane extracts of Basilocal as well as those of
Kisan foliage were tested simultaneously, one-half of the bottom Petri
dish was smeared with HEBL and with HEKL in the other. The solvent
was allowed to evaporate for nearly 30 min before releasing adults
into the oviposition chamber. The test chambers were arranged
randomly, so as to eliminate positional bias.
A pair of mated male and female moths was released in the
oviposition chamber at 6 p.m. Moths were removed the next morning,