J.Natn. Sci.

Foundation S r i Lanka 2003 31(3 & 4): 445-458

EVALUATION OF VOLATILE CONSTITUENTS OF NEEM (AZADIRACHTA .

INDICA A. JUSS.) LEAF EXTRACTS AGAINST CALLOSOBRUCHUS
....
MACULATUS (I?. )

P.A. PARANAGAMA1*,A. A. C. K. ADHIKARI1, K.P. ABEYWICKRAMA2and

K. A. N. P. BANDARA3
Department of Chemistry, University of Kelaniya, Kelaniya.
Department of Botany, University of Kelaniya, Kelaniya.
Entomology Division, Horticultural Crop Research and Development Institute, Gannoruwa.

(Received: 17 September 2002 ;accepted: 20 May 2003)

A b s t r a c t : The effect of neem (Azadirachta indica A. Juss.) leaf volatiles was

investigated against the cowpea bruchid, Callosobrc~chusmaculatus (F.) to test
fumigant toxicity, contact toxicity and repellency using olfactometer and choice
chamber bioassays. Callosobruchw maculatus is more susceptible to contact toxicity
t h a n to fumigant toxicity. During fumigant and contact toxicity bioassays, the
percentage mortality increased, while oviposition and the number of F, adults that
emerged were significantly inhibited by neem leaf volatiles a t concentrations higher
than 23.0 g/l and 3.8 g/l respectively a t 3 days after treatment. The LC,, values for
contact and fumigant toxicity were 1.14 gll and 8.45 g/l respectively. The results for
t h e toxicity bioassays revealed that the contact effect of leaf volatiles on bruchids is
higher t h a n t h e fumigant effect. Choice chamber and olfactometer bioassays
demonstrated the repellent effect of neem leaf volatiles against cowpea bruchids. At
a dose of 160 mg, the mean number of bruchids that responded in the olfactometer
and choice bioassays were 3.83 and 0.20 respectively. These findings indicate the
suitability of neem leaf volatiles as a botanical insecticide and a repellent in the
control of cowpea bruchids. Further studies on the active compounds in the volatiles
and their effect on bruchids need to be investigated before recommending neem leaf
volatiles as a suitable commercial product.

Key words:Azadirachta indica, Bruchid, Callosobruchus maculatus, cowpea, insect

repellent, neem leaf.

INTRODUCTION

Cowpea [Vignaunguiculata (L.) Walpl is one of the major sources of protein in many
developing countries, including Sri Lanka. Cowpea is a seasonal crop and farmers
store the harvest for considerable periods of time.

The major pests encountered in stored cowpea in Sri Lanka are the bruchids,
CalLosobruchus maculatus (F.) (Southern cowpea weevil) and Callosobruchus
chinensis (L.) (Adzuki bean weevil). Pulses stored for prolonged periods under high
moisture and relative humidity become infested with the above two bruchid species.
Infestation by bruchids is evident from the characteristic emergence holes in
seeds.l Damage to seed by bruchids cause quantitative and quality losses,
* Corresponding author
446 PA. Paranaganza et al.

reducing the economic value and the viability of seeds. Caswell reported a 50 %
loss in cowpea stored for 3-4 months due to infestation by C. m a c u l a t ~ s . ~

At present, in Sri Lanka and in many other countries, pulses such as cowpea
and mungbean stored in warehouses are fumigated using chemicals such as methyl
bromide and phosphine. Methyl bromide, now recognized as a potent ozone depletor,
is most likely to be banned or at least be highly restricted by the year 2005. Phosphine
(PH,) is the most widely used gas fumigant for disinfecting stored grains; however,
there are disadvantages such as aluminium hydroxide residues in grain and the
need for sophisticated equipment to circulate the phosphine gas.3

Therefore, it is essential to develop safe, effective and simple alternative

methods to control insect pests in stored grains. Plant extracts such as Azadirachta
indica, Zingiber perfurium (Roscoe),Eucalyptus camaldulensis, Didax procumbens,
Lantana camara and Cinnamomum camphora have been used in the recent past to
develop natural, biodegradable, environmentally friendly pesticide^.^^^,^ Although
the efficacy of plant extracts are sometimes inferior to those of toxic, broad
spectrum, synthetic insecticides, the degree of the reduction of the insect
population is quite sufficienL7

It is a traditional practice of rural farmers in developing countries to mix

dried leaves of neem with stored cowpea for protection against insects. Neem
oil, neem seed kernel, neem fruit dust, neem seed powder and neem leaves are
known to control several stored grain pests including Callosobruchus sppS8Neem
seed oil a t 10 % w/w protects cowpea seeds from bruchid infestation for a t
least 4 months.9*lo Effects of neem seed extracts against insects, include
antifeedancy, reduced oviposition, egg hatch, emergence and direct lethality.".'2
The use of powdered neem leaves in controlling Rhyzopertlza dominica (F.) has
been demonstrated."

Even though insecticidal properties of the powder or the volatile constituents

of neem seeds, and the powder of neem leaves have been tested against different
stored grain pests, the insecticidal1 repellent nature of neem leaf volatiles have
not been investigated against stored grain pests. The present study was
conducted with the aim of identifying toxicity and insect repellant properties of
volatile constituents of neem leaves against Callosobruchus maculatus in stored
cowpea. Once the efficacy of neem leaf volatiles is identified, they could
subsequently be formulated and marketed to manage weevils and other insect
pests in cowpea, mungbean and other stored grains.

METHODS AND MATERIALS . .

Source of cowpea: Newly harvested, non-fumigated cowpea (Vigna unguiculata) seeds

were collected from the Anuradhapura district in Sri Lanka. Cowpea thus obtained
446 PA. Paranagama et al.

reducing the economic value and the viability of seeds. Caswell reported a 50 %
loss in cowpea stored for 3-4 months due to infestation by C. m a c ~ l a t u s . ~

At present, in Sri Lanka and in many other countries, pulses such as cowpea
and mungbean stored in warehouses are fumigated using chemicals such as methyl
bromide and phosphine. Methyl bromide, now recognized as a potent ozone depletor,
is most likely to be b a ~ e or
d at least be highly restricted by the year 2005. Phosphine
(PH,) is the most widely used gas fumigant for disinfecting stored grains; however,
there are disadvantages such as aluminium hydroxide residues in grain and the
need for sophisticated equipment to circulate the phosphine gas.3

Therefore, it is essential to develop safe, effective and simple alternative

methods to control insect pests in stored grains. Plant extracts such as Azadirachta
indica, Zingiberperfurium (Roscoe),Eucalyptus camaldulensis, Didaxprocumbens,
Lantana camara and Cinnamomum carnphora have been used in the recent past to
develop natural, biodegradable, environmentally friendly pesticide~.~.~,%Although
the efficacy of plant extracts are sometimes inferior to those of toxic, broad
spectrum, synthetic insecticides, the degree of the reduction of the insect
population is quite sufficient."

It is a traditional practice of rural farmersin developing countries to mix

dried leaves of neem with stored cowpea for protection against insects. Neem
oil, neem seed kernel, neem fruit dust, neem seed powder and neem leaves are
known to control several stored grain pests including Callosobruchus ~ p pNeem.~
seed oil a t 10 % w/w protects cowpea seeds froin bruchid infestation for a t
least 4 lo Effects of neem seed extracts against insects, include
antifeedancy, reduced oviposition, egg hatch, emergence and direct lethality.'l~12
The use of powdered neem leaves in controlling Rhyzopertha dominica (I?.) has
been demonstrated.ll

Even though insecticidal properties of the powder or the volatile constituents

of neem seeds, and the powder of neem leaves have been tested against different
stored grain pests, the insecticiday repellent nature of neem leaf volatiles have
not been investigated against stored grain pests. The present study was
conducted with the aim of identifying toxicity and insect repellant properties of
volatile constituents of neem leaves against Callosobruchus maculatus in stored
cowpea. Once the efficacy of neem leaf volatiles is identified, they could
subsequently be formulated and marketed to manage weevils and other insect
pests in cowpea, mungbean and other stored grains.

METHODS AND MATERIALS

Source of cowpea: Newly harvested, non-fimigated cowpea (Vigna unguiculata) seeds

were collected from the Anuradhapura district in Sri Lanka. Cowpea thus obtained
Bioactivity of neem leaf volatiles against cowpea bruchids 447

was used in the experiments and also for rearing bruchids.

Laboratory rearing of bruchids: Adults of Callosobruchus maculatus were obtained

from the Entomology Division, Horticultural Crop Research and Development
Institute, Gannoruwa, Sri Lanka, and the rearing of bruchids was carried out at
the Department of Chemistry, University of Kelaniya, under laboratory conditions
of 28 + 3 "C and 75 f 5 % r. h. using the method described by Bandara and
Saxena.13

Clear, transparent 1 L plastic bottles (with 8-cm diameter screw lid) were
used to breed bruchids. A hole (3 cm diameter) was made in the lid of each bottle
and a fine gauze (25 mesh/ cm2) was pasted over the hole from the inside of
the lid to provide sufficient ventilation for -the bruchids. Twenty-five pairs of
adult bruchids were placed in each container, with 250 g of seeds. Under the
above laboratory conditions, a new generation of bruchids emerged after 3-4
weeks from eggs laid by the introduced females. Five to ten hour old adults were
obtained from the laboratory cultures.

Plant materials: Fresh leaves ofAzadirachta indica A Juss. (Neem) were collected
from plantations in the Anuradhapura and Gampaha.districts. Seven hundred grams
offresh neem leaves were steam distilled for 3 hours and the aqueous distillate (500
ml) was extracted with CH,Cl, (200 ml) to obtain the volatile constituents. The
extract was concentrated to 20 ml using a rotavapour (BUCHI, Rotavopor, R-114,
Waterbath, B-480, Beckman Instruments Inc, Westbury, N.Y. 11590) a t 35 OC
and dried with anhydrous Na2S0,. The solvent was evaporated by passing a N,
steam. The Clavenger arm was not psed to collect volatiles from neem leaf, as
the percentage yield was very low (0.15 %) and there was no separable amount
of volatiles collected after the distillation of 700 g of neem leaves. The modified
Lickens and Nickerson apparatus was used with a cold-water condenser to check
whether highly volatile constituents i n neem leaf are lost during the steam
distillation process. The GC analysis of the two extracts indicates that the chemical
constituents of neem leaf were not significantly different from each other.

Toxicity of neem leaf volatiles on Callosobruchus maculatus

..

Bioassay for contact toxicity: Bioassays were performed using a modified method
Huang et al.14 Varying concentrations of neem leaf extracts (0.35 - 3.80 g/l) were
prepared using ethanol as the solvent (100 yl). Extracts of known concentration
were evenly applied to the inner surfaces of a glass vial (6.5 ml). The solvent was
evaporated using N2 gas. Five pairs of 5-10 hour old adult weevils were introduced
into each vial and the caps were tightly screwed. After 24 hours, the weevils were
transferred into clean vials with 50 untreated, fresh cowpea seeds and kept in the
insect room under prevailing environmental conditions of 28 + 3 "C and 75 5 % +
r. h. The same procedure was carried out with vials coated with equal amount of
448 PA. Paranagarna et al.

ethnol and vials without any treatment were considered as controls. Percentage
mortality of bruchids and the number of eggs laid were recorded daily during the
incubation period of 10 days. When the first generation adults (F,) emerged, the
numbers of F, adults were also recorded. Experimental design was a completely
randomised design (CRD) with six-replicates.

Bioassay for fumigant toxicity: Fumigant toxicity was tested using a modified method
described by Bandara and Seneviratne? Filter paper discs (1.5 cnl diameter) were
impregnated with each concentration of neem volatiles dissolved in ethanol (100 pl)
and the ethanol was allowed to evaporate for 10 minutes. The filter paper was then
attached to the inner surface of the screw cap of a-glass vial. Five pairs of 5-
10 hour old bruchids were introduced, and the neck of the vial was blocked with
a piece of metal mesh to prevent bruchids coming in contact with the treated
filter paper. The vials were tightly screwed and, kept for 24 hours at 28 k 3 "C
and 75 f 5 % r. h. Thereafter, the bruchids were transferred into clean vials with
50 untreated, fresh .cowpea seeds. Six different concentrations (0.75 - 23.00
g/l) of extracts were used to coat the vials. The same procedure was carried
out with vials coated with equal amount of ethnol and vials without any treatment
were considered as controls. The experimental design was a CRD with six replicates
per treatment.

Percentage mortality of bruchids and the number of eggs laid were recorded
daily during the experimental period of 10 days. When the first generation of adults
(F,) emerged, the numbers.of F, adults were recorded.

Repellent action of volatiles

~ i o a s s a with
j olfactometer: The repellent action of volatiles was tested using a
modified Y-shaped olfactometer. The olfactometer consisted of 3 glass arms each
10 cm long having a diameter of 1cm. At the point where the three arms meet, a
fourth tube was connected (0.5 diameter- and 3 cm long), which was directed to the
vacuum pump through a 1m long rubber tube (Figure 1).

Two perforated plastic containers (300 ml) were fured to the ends of two arms
of the Y-tube. A filter paper strip (2.5 cm x 5.0 cm) held by copper wire was
placed in the center of each container. A known volume (10 - 160 p1) of the test
sample was placed on the strip; the solvent was allowed to evaporate and the
strip was then placed in the container (baited arm). A filter paper strip treated
with an equal volume of ethanol was placed in the non-baited arm. The airflow
through the olfactometer was regulated using a vacuum pump (Model DYNAX,
Charles Austen Pump Limited, England). Subsequently, a round-bottomed flask
(100 ml) with 50 unsexed adult bruchids was connected to the third arm of the
olfactometer.
Bioactivity of neem leaf uolatiles against cowpea bruchids 449

The experimental apparatus was kept in an upright position with the baited
and non-baited arms directed towards a 40 W fluorescent light source. The arm
connected to the round-bottomed flask was kept in the dark by placing it inside
a cardboard box. The sequence of placing the test samples and ethanol in the
two arms was interchanged randomly after replication. The bioassays were
conducted between 7 a.m. and 11 a.m. After a duration of half an hour, the
number of bruchids that had moved into each container was counted.

Figure 1: Set- up f o r t h e olfactometer bioassay

Five different doses (10 - 160 mg) of volatiles were tested separately with 6
replicates. The olfactometer was thoroughly cleaned using CH,Cl, and detergent,
and dried in subsequent replicates.

Bioassay with clzoice chamber: The choice chamber consisted of eight transparent
plastic bottles (300 ml), placed equidistant to each other. The 8 plastic bottles were
connected to a large transparent bottle (1L) placed in the center of the chamber
using glass tubing (diameter 1cm and length of 8 cm). The experimental set-up was
placed in a plastic basin (diameter of 42 cm and depth of 18 cm) and the sides
covered with black paper.

In each experiment, five different doses (10 mg - 160 mg) ofneem leafvolatiles
and the solvent were used. Each volatile sample was placed on a filter paper
strip (2.5 cm x 5.0 cm) and the solvent allowed to evaporate. Strips having
different concentrations were placed in different bottles, each containing 50
cowpea seeds. Two of the bottles each containing 50 cowpea seeds withbut
any treatment were considered a s the controls. Two hundred and fifty adult
bruchids (unsexed, 1-3 days old) were introduced into the central bottle, and
the chamber was placed in a dark room. After 24 hours, the number of bruchids that
 450 PA. Paranagama et al.

moved through the arms into the baited bottles and the number of eggs deposited in
each bottle was recorded. Each experiment with 5 doses of the extract, ethanol and
,
the control were replicated 5 times. The choice chamber apparatus was thoroughly
cleaned using CH,Cl, and detergent and dried in subsequent replicates.

Statistical analysis: Data obtained following bioassays for toxicity and repellency
were analysed statistically using One-way ANOVA and the means compared using
Tukey's Multiple Range Comparison tests. The results of the olfactometer and choice
chamber experiments were analysed using Chi Squared tests. The LC,, values of
neem leaf volatiles were calculated using Probit Analysis.

RESULTS

Contact and fumigant toxicity of neem leaf volatiles against Callosobruclcus

maculatus

Contact toxicity

The mortality of bruchids increased in the different treatments with the increasing
concentrations of volatiles. Figures 1 , 2 and 3 illustrate the effect of contact toxicity
of neem leaf volatiles on C. maculatus. Percentage mortality in the solvent treated
and control groups was 1.70 %. When the extract concentration was increased to
3.80 g/l, the mortality reached 100 %. The LC,, value for contact toxicity was
1.14 g/l. As shown in Figures 2 and 3, the mean number of eggs laid and the F,
adults were significantly reduced with the increasing volatile concentrations
(p<0.05).Egg laying and F, generation were completely inhibited at a concentration
of 3.80 g/l. The results revealed that neem leaf volatiles significantly inhibited the
number of F, adults ( ~ ~ 0 . 0produced,
5) than the number of eggs laid. The highest
number of eggs and I?, adults were observed in solvent treatments and controls and

0.35 0.75 I I .5 2.3 3.8 Control Solvcnl

Trcatnicna (!$I)

Figure 2: Percentage mortality of Callosobruchus maculatus o n 3rdd a y

after t r e a t m e n t following contact toxicity bioassay. E a c h bar represents
+
t h e m e a n S.E. of 6 replicates (F= 118.93)
Bioactivity of neem leaf volatiles against cowpea bruchids 451

there was no significant difference between these two treatments (p>0.05),indicating

that the use of ethanol as a solvent has no significant effect on bruchid behaviour.

0 35 0.75 I 1.5 2.3 3.8 Control Solvent

Treatments (61)

Figure 3: Number of eggs by Callosobruchus maculatus on 3 * day after

'
treatment following contact toxicity bioassay. Each bar represents the mean
f S. E. of 6 replicates (F = 59.34)

0.35 0.75 I 1.5 2.3 3.8 Control Solvent

Trcatmcnts (611)

Figure 4: Number of F, adults of Callosobruchus maculatus following

contact toxicity bioassay. Each bar represents the mean fS.E.of 6 replicates
(F=27.98)

80
70
$ GO
i 50
40
' 30
20

& rjh
15 3X 77 154 23 Cobrtml Solvcnt
Trcnlmcnlr (ell)

Figure 5: Percentage mortality of Callosobruchus maculatus on 3rdday

after treatment following fumigant toxicity bioassay. Each bar represents
t h e mean f S. E. of 6 replicates (F=46.04)
452 PA. Paranagama et al.

Fumigant toxicity

The mortality of bruchids increased with increasing volatile concentrations. Bruchid

mortality reached 85 % a t a concentration of 23.00 gA (Figure 4). Figures 5,6 and 7
show percentage mortality, the mean number of eggs laid and the mean number of
F, adults. At concentrations higher than 1.50 g/l, the number of eggs laid and the
number of F, adults were significantly low compared to the control and solvent
) . LC,, for fumigant toxicity test was 8.45 gA. Significantly
treatment ( ~ ~ 0 . 0 5The
low mortality of bruchid (1.70 %) and a significantly higher number of eggs (73.0)
were recorded in both the control and solvent treated samples.

0.75 1.5 3.8 7.7 15.4 23 Control Solvent

Treat~ncnts(gll)

Figure 6: Number of eggs laid b y Callosobruchus maculatus on 3 r d d a y

after t r e a t m e n t following fumigant toxicity bioassay. E a c h b a r represents
t h e m e a n k S.E. of 6 replicates (F=20.70).

0.75 1.5 3.8 7.7 15.4 23 Control Solvcnt

Trcatmcnts (gtl)

F i g u r e 7: Num-ber of F, a d u l t s of Callosobruchus maculatus following

+
f u m i g a n t toxicity bioassay. E a c h bar r e p r e s e n t s t h e m e a n S.E. of 6
replicates (F= 21.94).
Bioactivity of neem leaf volatiles against eowpea bl-uchids 453

Table 1: Response of Callosobruchus maculatus to leaf volatiles of neem

following olfactometer bioassay

Extract dose Responding (mean f S. E.)

(mg) N Baited arm Non-baited arm C

10 6 8.50k1.92 7.33f 1.49 0.08

20 6 9.33k1.58 10.83f 1.91 0.11
40 6 8.66k1.20 18.16k1.46 3.40'
80 G 6.33f1.08* 17.00+3.03* 4.88

Fifty insects were used in each experiment. Insects in each arm were counted 30 min after introduction.
Except a t 10, 20 and 40 mg doses, the mean numbers of insects i n baited and non-baited arms were
significantly different (p<0.05,3.84* Significant a t 5 8,Chi square test).

Table 2: Response of Callosobruchus maculatus to leaf volatiles of neem

following choice chamber bioassay

Extract dose Responding (mean f S. E.) Eggs laid in baited arm

(mg) N Baited container C (mean k S.E.)

Solvent 5 30.4k8.4 0.42 135.6f 11.3'

Control 5 34.8k8.2 0.81 141.6f8.0~
Two hundred and fifty insects were used i n each experiment. Insects in each container were counted
24 hours after introduction. Mean response of the test insects was analyzed using Chi square tests (p<
0.05,14.07, Significant a t 5%)
The mean number of eggs laid was analysed usingANOVA and Tukey's Multiple Range Tests. The number
of eggs laid in each treatment (denoted by similar letters) was not significantly different (p<0.05).
454 PA. Paranagana et al.

Repellent effect of neem leaf volatiles against bruchids

Olfactometer

The movement of bruchids towards the baited arm was considered a positive
response. Results presented in Table 1show that the number of bruchids that moved
to the treated arm was significantly low in the neem leaf volatiles treated arm
compared to the non-baited arm. A higher number of bruchids had moved into the
solvent treated arm (non-baited arm) indicating that use of ethanol has no repellent
effect on bruchid behaviour. The number of bruchids that responded decreased from
8.50 to 3.83 when the concentrations of the neem leafvolatiles were increased from
10 to 160 mg. At doses higher than 40 mg, the repellent effect of neem volatiles was
significantly higher compared to the control (p<0.05).

Choice chamber bioassay

Table 2 gives the results of the choice chamber bioassay where different doses of
neem leaf volatiles, solvent and a control were used. Some degree of repellency was
shown in all the treatments. The number of bruchids in the neem treatment was
significantly different from the control and the solvent treatments (pe0.05). The
response to the neem volatile treatment (40-160 mg) revealed that the number of
bruchids that responded was 3.4-0.20, whereas in the solvent and the control
treatments it was 30.4 - 34.8 % (not significantly different, p>0.05).When the volatile
concentration was increased from 10 mg to 40 mg, the number of eggs laid decreased
from 21.5 to 2.2 (at doses higher than 80 mg the number of eggs laid was zero). The
number of eggs laid in the solvent treatment and control was not significantly
different (135.6 and 141.6 respectively) (p>0.05)and these results were significantly
different ( ~ ~ 0 . 0from
5 ) neem treated samples.

DISCUSSION

Semiochemicals such as plant volatiles have been used in the past as fumigants to
control stored grain pest^.^.^*" Several authors have reported the use of volatile
constituents containing plant materials and vegetable oils to control Callosobruclzus
spp. in stored ~ o w p e a . ~ , ~

Traditionally, for the control of stored grain pests, Indo-Pakistan fanners mixed
2 - 5 kg of dried neem leaves1100 kg grainl%r soaked empty sacks overnight in
water containing 2 - 10 kg of neem leaves I 100 L and dried these sacks before filling
them with grain.16Avariety of biologically active constituents, including triterpenoids
azadirachtin, salanin and meliantriol are found in'neem leaf, fruit, bark and seed."
These compounds reportedly control more than 100 species of insects, mites and
nematodes.18 Modes of control include antifeedant, growth regulatory, repellent or
pesticidal action on larva and I or adult stages of these pests.lgNeem leaves,I6water
Bioactivity of neem leaf volatiles against cowpea bruchids 455

extract of crushed neem seeds,20neem and neem "cake,"22appear particularly

promising for pest-control use in developing countries. Powdered neem seed kernels
have been tested against adult Rhyzopertha dominica, adult Sitophilus oryzae and
larva of Dogodermagranariu~nin wheat seeds. The results revealed that powdered
neem seeds at the rate of 1to 2 parts per 100 parts of seeds protect wheat seeds,
from these insects for a t least 10 - 12 months.20

During the present survey, toxic effects of neem leaf volatiles were investigated
by monitoring the mortality, oviposition and F, adults of C. maculatus. The results
of the bioassay clearly indicate that the contact toxicity of neem leaf volatiles is
significantly high when compared to the untreated control. During the present study,
the egg laying capacity and F, adults in the neem leafvolatile treatments decreased
significantly. Over 85 % mortality was observed a t concentrations higher than
2.30 g/l and 23.00 g/l during contact and fumigant toxicity bioassays. The present
study also revealed that the neem leaf volatiles bring about contact and fumigant
toxicity in bruchids a t low LC,, values such as 1.14 gtl and 8.45 g/l.

The study of repellent effects of leaf volatiles using a Y shaped olfactometer

and the choice chamber bioassay indicated that the repellent effect of neem leaf
volatiles varies with the dose. The number of insects in all treatments was
significantly low when compared to the control and the solvent treatments. At a
dose of 160 mg, the number of bruchids that responded to the olfactometer bioassay
and the choice chamber bioassays was 3.83 and 0.20 % respectively, indicating that
the effect of the volatile is more pronounced after 24 hours than after 30 minutes.
Jilani and Su and Jilani et al., studied extracts of neem leaves and seeds against
Dibolium castaneum (Herbst.),Sitophilusgranaries (L.) and Rhyzopertlza donzinicn
(l?.).239"
Stored rice and maize insects were used in their study and the findings are
in agreement with the present study, although different insect species had been
used.

Neem extracts have generally been found to be safe for mammals and the
environment. For example, the incorporation of 20 % neem cake in their diet resulted
in a higher growth rate in sheep.25Hence, contact, fumigant and repellent properties
of neem leaf volatiles could be used to develop environmentally friendly pesticides
from neem leaf volatiles.

Neem is used for medicinal purposes in Sri Lanka and in many other Asian
countries. Botanicals with insecticidal or repellent properties are, therefore, an
alternative to the expensive synthetic pesticides and fumigants. Volatile neem based
repellent products, if developed and introduced to the local market, would no doubt
be adapted by consumers owing to the environmentally friendly, medicinal properties
of the products. It is evident by the present study, that the volatile constituents of
neem leaf make it a safe control method against insect pests in stored cowpea and
similar grains. Experiments are being conducted to identify the bioactive components
456 PA. Paranagama et al.

in neem leaf volatiles using gas chromatography. Thereafter, GC-EAG studies will
be carried out to assess the response of bruchids to selective bioactive components
of neem volatiles.

Acknowledgement

The authors thank the Council for Agricultural Research Policy (CARP) (1214401
329) and the National Research Council (NRC) for financial support. The authors
acknowledge Dr. Upali Chandrasekara for assistance provided during statistical
analysis, and Mr. A. Gunawardane of Puwakpitiya, Henegama for technical
assistance.

References

1 Giga D.P. & Smith R.H. (1983). Comparative life history studies of four
Callosobruchus species infesting cowpea with special reference to
Callosobruchus rhodesianus (PIC) (Coleoptera; Bruchidae). Journal of Stored
Products Research 19: 189-198.

2 Caswell G.H. (1981). Damage to stored cowpea in Northern Nigeria. Samaru

Journal of Agricultural Research 1:11-19.

3 Waterford C.J., Whittle C.P. &Winks R.G. (1994). New aluminium phosphide
formulation for controlled generation of phosphine. Proceedings of the GL"
International Working Conference on Stored Product Protection 1:226-229.

4 Bandara K.A.N.P. & Seneviratne D.K.H.W.P.W.R. (1993).Effect of wild ginger

Zingiberperfurium Roscoe on cowpea.seed bruchid, Callosobruchus maculatus
(I?.) in stored cowpea. Dopica1 Agriculturist 149: 89-100.

5 Loganathan B. & Ahangama D. (1996). Alternative materials for the control

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