J. srored Prod. Rrs. Vol. 25, No. 4, pp. 231-237. 1989 0022-474X:89 $3.00 + 0.

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Prmted in Great Britain. All rights reserved Copyright <: 1989 Pergamon Press plc

EVALUATION OF EXTRACTS OF NEEM (AZADIRACHTA

INDICA A. JUSS) FOR THE CONTROL OF
SOME STORED PRODUCT PESTS

W. A. MAKANJUOLA
Department of Biological Sciences. University of Lagos. Lagos. Nigeria

(Received .for publication 2 .4ugust 1989)

Abstract-Laboratory investigations on the activity of neem leaf and seed extracts in water or methylated
spirit on Callosobruchus maculatus, Sitophilus oryzae and C’ylas puncticollis were made at 27 k 2’C and
65 + 5% r.h. Field trials were conducted to determine their effectiveness as protectants of cowpeas and
maize in storage at 28 + 4’C and 82 + 7% r.h for 5 months.
The results showed that the effectiveness of neem is affected by differences in insect behaviour.
The extracts were more active as suppressants of C. macularus than Sitophilus spp; there was no effect
on C. puncticollis. All of the extracts tested resulted in a significant reduction in oviposition, % egg hatch
and % adult emergence in C. macularus and in adult emergence of Sitophilus.
Field trials showed that the extracts mostly gave good protection to cowpea against c‘. maculatus for
5 months. but only moderate protection to maize against S. xamai.s. Seed extracts were more effective
than leaf extracts.

INTRODUCTION

The widespread use of synthetic insecticides poses a serious hazard to both man and wildlife
because of adverse effects on the environment. The detrimental effects on the ecosystem are well
demonstrated when beneficial insects, predators and parasites die, but often pests, themselves,
survive treatments because the population has developed resistance to the chemicals used. One
solution to these problems might be to replace synthetic chemicals with compounds which occur
naturally in plants.
Many insects are unable to infest certain plants because of the presence of particular noxious
substances (Fraenkel, 1969). The neem tree, Azadirachta indica A. Juss, is known to be rarely
infested by insects (Bhatia and Sikka, 1957) and to have repellent or antifeedant effects (Ruscoe,
1972; Jilani and Malik, 1973; Leuschner, 1975). Active components have been isolated from
neem and these include the triterpenoids, azadirachtin (Butterworth and Morgan, 1968. 1971) and
melantriol (Lavie et al., 1967), both of which suppress feeding.
Some work has been undertaken to assess the effects of neem on storage pests (Ivbijaro,
1983a, b). but very little has been carried out under practical field conditions. The experiments
reported here were set up to determine the effectiveness of relatively crudely extracted neem leaves
and kernels as protectants of cowpea and maize.
As a result of this work, it is hoped that a method of extraction and utilization of liquid neem
extracts can be made available to farmers for their regular use.

MATERIALS AND METHODS

Preparation of crude extract

Leaves and matured kernels of the neem plant were collected and air dried for 10 days, followed
by further drying in a Gallenkamp hot box oven at 45°C for 24 and 72 hr respectively until
constant weights were obtained. The oven dried leaves and kernels were ground into powder using
an electric blender. Three hundred grams each of the leaf and kernel powders were separately
soaked in 1 litre of water or in 1 litre of laboratory methylated spirit for 24 hr. The 4 mixtures
were then filtered into separate conical flasks through Whatman No. 1 filter paper. The filtrates
were centrifuged in a Sroval super speed RC2-B automatic refrigerated centrifuge at 5000 rpm for
231
232 W. A. MAKANJSJOLA

5 min at 4’C, remove the residue and evaporated under to one-third their
volumes. few drops Teepol were to act surfactant to for even because
of oily seed The extracts stored in refrigerator until

Rearing of insects
Three species of insect, Caliosobruchus maculatus (F.), Sitophilus oryzae (L.) and Cylas
puncticollis (Boh.), used for this study were reared on cowpea seeds, maize seeds and sweet potato
tubers respectively. The cultures were placed in a Percival incubator maintained at 27 t_ 2°C and
65 F 5% r.h.. Experiments were conducted using O-7 day-old adults.

~pp~i&ation of neem extracts on cowpea, maize and sweet potato surfaces

Seventeen lots of 200 undamaged clean seeds each of cowpeas and maize were selected for surface
treatment with the extracts. Seventeen lots of 10 sweet potato tubers each of approximately the
same size were also selected. The produce was treated with solutions which contained 40, 60 and
80% concentrations of the stock solutions of the extract. These concentrations were determined
in preliminary tests.
Two hundred seeds each of cowpeas and maize were soaked for 30 set in each dilution of the
stock solutions. For the sweet potato treatment, 10 tubers were each sprayed with every dilution.
After the treatments, the seeds and tubers were air dried for 5 hr. The same numbers of seeds and
tubers were used for two sets of controls except that only water or methylated spirit, each with
a few drops of Teepol, were applied.

Feeding tests
Ten O-7 day-old adults of C. maculatus and S. oryzae were confined for 5 days in petri dishes
containing 50 treated seeds of cowpea and maize respectively for each treatment and control.
A 5 cm diameter hole covered with fine nylon mesh was made in the lid of each petri dish to provide
ventilation and the dishes secured using rubber bands. Both the treated and control experiments
were replicated 4 times.
One mated female C. puncticoZlis was placed in a 5 x 5 x 3 cm rectangular plastic cage. The insect
was exposed for 5 days, through a 2 cm2 opening in the side of the cage, to the treated surface of
one sweet potato tuber for each concentration for the extracts used. Two sets of control experiments
were set up in the same manner using tubers treated with water or methylated spirit respectively.
Both the treated and control experiments were replicated 10 times.
The experiments were carried out in a Percival incubator maintained at 27 k 2°C and
65 h 5% r.h.
The foliowing data were recorded for the three species:
Ca~losobr~~}~~ maeulatus. Number of eggs laid; percent of hatched eggs; development period and
percent of adult emergence. All emerging adults were removed from dishes daily in order to prevent
further mating and egg laying. Counts were taken for 20 days after the first emergence. No adult
emerged on the 21st day.
Sitophilus oryzae. Number of dead parent adults; number of damaged seeds by parent adult
feeding; development period from the day of parent adult exposure; and the number of adults
emerging. All emerging adults were removed from dishes daily to prevent further mating and egg
laying. Counts were taken for 26 days after the first emergence. No adult emerged on the 27th day.
Cylas punet~collis. Number of egg holes; number of adult feeding holes; development period and
percent of aduit emergence. All emerging adults were removed from dishes daily to avoid further
mating and egg laying. Counts were taken for 20 days after the first emergence. No adults emerged
on the 21st day.
Angular transformation was used for data obtained in percentages. Analysis of variance was
estimated for each parameter. Means were ranked using Duncan’s multiple range test.

Field trials of neem products

Trials of water and methylated spirit extracts of leaves and kernels were carried out under field
conditions. Because preliminary experimentation showed that the neem extracts were more sensitive
 Neem and stored product pests 23.3

under field conditions. a wider range of concentrations, namely 100, 80, 70, 60, 50 and 40% was
used in these trials.
Clean cowpea and maize seeds were treated with each concentration of leaf and kernel extracts
by shaking thoroughly for 30 sec. After treatment, the seeds were spread out in a thin layer and
air dried for 5 hr.
A total of 500 g of cowpea and maize seeds of each of the extract treatments were each placed
in 80 x 60cm jute bags. Fifty O-7 day-old adult C. maculutus and Sitophilus xamais Motsch..
respectively, were placed in the bags containing the treated cowpea and maize seeds. C. nruculatu.v
were taken from the same culture stock used in the laboratory experiment, whilst 5’. xumais were
obtained from infested maize in local cribs, The open ends of the bags were tied with twine. Bags
of untreated seeds with the same number of insects were included as controls. Each treatment was
replicated 10 times. The bags were stacked randomly in a grain store-house for 5 months at
28 + 4-C and 82 & 7% r.h.
The baseline moisture content of the grains at the start and end of the experiment was measured
using a Steinlite moisture meter to determine difference in weight due to moisture.
The population of adult insects was recorded after 5 months storage. The weight loss was
determined by taking the difference in weight between the control and treated replicates after 5
months of storage and correcting for weight change due to moisture content to obtain the actual
weight loss due to insect damage.
Angular transformation was used for data obtained in percentages. Analysis of variance was
estimated for each parameter. Means were ranked using Duncan’s multiple range test.

RESULTS
lZfect of neem extracts on C. maeu~~tus
The numbers of eggs laid, the percentage hatched eggs and percentage adult emergence were
significantly higher on the controls than on the treated cowpeas (Table 1). However, no significant
difference was found in the development period between the controls and treatments. There was
no significant difference in the results of the different parameters measured between water and
methylated spirit extracts.
Generally, the neem seed extract appears to have suppressed the development of c’. muculutus.
The results show that the number of eggs and the percentage adult emergence were lower in cowpea
seeds treated with neem seed extract than with neem leaf extract. The aqueous extract of the neem
seed appeared to be the most effective in reducing egg laying, hatching and adult emergence.

Table I Summary of the analysis of variance on the effect of necm (A. in&u) extracts on C. ~u~~~~~~~.~
Concentration Mean number of % Mean eggs Mean development
Treatment (%) eggs hatched period (days)
-
,vw?t /w/ <‘.rm,<‘/
Water 40 209 a .z?a 2x
60 200 a 48a 74
80 153 b 35a 31
Control 300 c 70 c ?I
Variance ratio 0.43** 4.83; 2.9 NS
Methylated spirit 40 107a 57 aa 28 b
60 126a 52.5 b 29 b
80 134a 42.5 b 31 b
Control 307 b 68.9 a to a
Variance ratio 10.71** 4 X9* 4.87’

,vwm .wrr/ t’lfll,( f

Water 40 45 b 29a 28
60 11 B 27a 32
80 23 a 22.9 a 32
Control 2l3c 70.9 b 24
Vanance ratio 24.1*** 5.n9* 2.7 NS
Methylated spirit 40 IOOC 5Ob 28
60 39a 31 a 29
80 53 b 29a 25
Control 197d 7oc 23
Variance ratio 16.9** 5.17* 0.9 NS

Each datum is a mean of 4 replicates. Percent adult emergence is a percentage of number of hatched eggs.
NS. not significant: *significant at 0.05%; **significant at 0.01%; ***significant at 0.001%.
Means followed by the same letter are not si~ific~n&ly different.
234 W. A. MAKANJIJOLA

The results obtained from all the concentrations of neem and kernel extracts were significantly
different from the controls (Table 1). There was inhibition of oviposition, hatching and adult
emergence of C. ~uc~~ar#s at all concentrations of each extract.

Eflect of neem extracts on S. oryzae

Except in the case of aqueous extract of neem seed, there were no significant differences between
the treatments and controls in the number of seeds damaged or the number of dead parent adults
(Table 2) indicating that there was no general inhibition of S. oryzue adult feeding when seeds were
treated with extracts of neem. However, the aqueous extract was active in inhibiting feeding on
the treated maize seeds at the 60 and 80% concentrations with a correspondingly significant
difference in the number of dead parent adults. There was a significant difference in the number
of emerging S. oryzae between the respective treatments and their controls, both extracts
significantly suppressing adult emergence at all concentrations. There was, however, no significant
difference in the developmental period between treatments and controls. The aqueous extract
appeared to be the most effective in suppressing feeding and development of S. oryzae.

Efleect of neem extract on Cyias pun~tico~iis

Cyh punctieollis was not inhibited by any of the neem extracts (Table 3). There were no
significant differences in the number of feeding holes, number of egg holes, developmental period
or percent adults emerging between the respective treatments and their controls.

l@ect of neem products in field trials

All of the neem extracts tested gave significant protection to cowpea against C. maculutus damage
for 5 months at all concentration levels (Table 4) both the final population of C. macufatus and
the weight loss being significantly lower in all treatments than in the control. Neem seed extract
appeared to be more effective than neem leaf extract.
In the case of maize, although the population of S. zeamais was high on treated maize,
particularly with the leaf extract treatment, there was a significant difference in population between
treatments and the control (Table 5). Neem seed extract was clearly more effective in suppressing
the population of S. zeamais than the leaf extract. There was no significant difference in weight
loss between the leaf extract treatments and the control (Table 5). The leaf extract treated maize
was badly damaged and mouidy. The moisture content both in the control and leaf extract treated
maize had increased from 12.3 to 21.8 and 22.7% respectively. Although the results from the
moisture content anatysis cannot be said to be conclusive at this stage, the significant increase in

Table 2. Summary of the analysis of variance on the effect of “eem (A. indica) extracts on S. orvzae
Mean number of Mean number of Mean development Mean number
Concentratmn seeds damaged by dead parent period of adults
Treatment (X) adult feeding adults (days) emerged
Neerr1 lea/- rxlrarl
Water 40 32 4 30 5a
60 36 4 31 2a
80 38 3 33 4a
Control 40 5 29 15b
Variance ratio I.1 NS 2ONS 2.3 NS 12.3’*
Methylated spirit 40 36 4 31 8b
60 32 4 32 I
80 30 2 33 2
Control 38 3 31 17c
Variance ratio 2.3 NS 2.1 NS 3.1 NS 11.4**
Neem seed e.xtrzct
Water 40 42 b 4a 35 It b
60 22a 9b 38 la
80 28a 7b 38 la
Control 40b 2a 2x 2oc
Variance ratio 4.89’ 4.9Sf l.SNS 24.5***
Methylated spirit 40 34 3 33 7a
60 40 4 33 6a
80 36 8 34 6a
Control 46 3 33 22 b
Variance ratio 3.3 NS 3.4 NS 1.ONS 10.5**
NS. not significant; *significant at 0.05%; **significant at 0.01%; ***significant at 0.001%.
Means followed by same letter are not significantly different. Each datum is a mea” of 4 replicates.
 Neem and stored product pests 235

moisture content in the leaf extract treated maize and control appears to have been caused by the
intense activities of the insects and fungi which were probably enhanced by the high ambient
relative humidity of the store-house. However, there was a significant difference in weight loss
between the control and kernel extract treated maize. The moisture content in the seed extract
treatment had only increased from 12.3 to 14.8%.

DISCUSSION

In laboratory experiments, all extracts of neem leaf and seed significantly reduced oviposition.
percent hatched eggs and percent adult emergence of C. rnaculatus on treated cowpeas. It appears

Table 3. Summary of the analysis of variance on the effect of neem (A. indiea) extracts on C. pu~cficoffi.~
Concentration Mean number of Mean number of Mean development C Mean adutt
Treatment (Oh) feeding holes egg holes period (days) emergence

40 5 6 13 8X. 1
60 4 3 15 X8.5
80 3 3 13 88.4
Control 5 5 I? XX
Variance ratio 1.79 NS I .45 NS 2.03 I.5 NS
Methylated spirit 40 4 5 14 X7
60 4 13 x4
X0 4 4 14 X2
Contr01 5 5 14 X9
Vanance ratio l.hSNS I .05 NS 2.4 2.17NS
seed exwwt
>V<w?r
Water 40 i 17 x7
60 I I7 X3
80 I I6 X3
Control 4 5 14 X5
Variance ratio 2.41 NS 3.17NS 3.1 ?,liNS
Methylated spirit 40 4 17 XX
60 4 15 Xl
80 4 16 XX
Control 4 I5 XX
Varunce ratio 1.89NS 1.79 NS 2.7 1.77 NS
NS. not signi~~~t. Each datum is a mean of IO replicates. Percent adult emergence is a percentage of number of egg holes

Table 4. Summary of the analysis of variance on the effect of neem products on C. maru/arus in field trials
Extract concentration
-.-___~~~.__---- __..
Neem formulation 100 80 70 60 SO 40 Control F-value
Popularion of’ f’. n~eulut~~.~ajim 5 monrhs srornge
Water leaf extract 19Sb 179b 203b 19% 2% 258bc 3700a X3X.X***
Methylated spirit leaf extract 189b 2Olb 256c 30ld 379de 323d 370&i 604 01***
Water seed extract 45b 68b 73k ?1bc 103d I13d 3700a 946.56***
Methylated spwit seed extrxt 87b 89b 69b 93c 213d 205d 3700a 773.45”’

Percent weight loss of rowpeas uljcr 5 monrhs storage-

water leaf extract 1. I Xb 1.2ib I .34b 1.39b 1.3lb LOlb 25.3a 6384.X***
Methylated spirtt leaf extract I.llb ?.Olc 2.17c I.852 3.12de 2.17d 25.3a 10x0.4***
Water seed extract 0.61~ 0.37b 0.30b 0.29b 0.58~ 0.74cd 25.3a 87X8.X***
Methylated spirit seed extract 0.73b 0.8% 0.66b 0.54b 0.89c 0.79c 35.3a 7334.?8+**
***Significant at 0.001. Means followed by same letter are not significantly different. Each datum is a mean of IO replicates.

Table 5. Summarv of the analysis of variance on the efkct of neem products on S. xwnais in field trials
Extract concentration
ll---_~_-~._ _...._I._
Neem formulation loo 80 70 60 50 40 Control
Popularion o/ S. xamais after 5 months srorage
Water leaf extract 129lb 94% 1258b lOl3c 13’79ab 1413a 158Sa 3 IX’
Methylated spirit leaf extract 884d 1245ab 891d ll23c 1279ab 138lab I585a 7 41**
Water seed extract 375bc 238d 313c 518b 394bc 435b 158% 113 79***
Methylated spirit seed extract 398c 374c 418b 391c 487b 473b I585a 99.X1’m*
Percenr Wright Ioss oj~mairr nfier 5 months smrage
Water leaf extract 32.98 32.79 32.47 32.58 32.72 32.83 33. t I.1 NS
Methylated spirit leaf extract 33.30 32.87 32.88 32.92 32.99 33.16 33. I 0.89 NS
Water seed extract 8.5Lc 8.7lbc 8.33c 9.47b 8.1 Ic 8.6lbc 33.t9 I I .8x***
~eth~lat~ s&it seed extract 9.43bc 7.2ld IO.43b 9.39b.z 7.49d 8.88cd 33.19 9.77*+*
NS. not significant; *Significant at 0.05: **Significant at 0.01: ***Sigmficant at 0.001,
Means followed by same letter are not significantly different. Each datum is a mean of 10 replicates
236 W. A. MAKANJUOLA

that neem acts not only as an oviposition suppressant but also as an ovicide. Fagoone (1980)
reported that egg laying was delayed and reduced in ~roudo~omia binotal~~sZell, on neem treated
cabbage leaves. Similarly, Ivbijaro (1983a) reported a significant reduction in egg laying on cowpea
mixed with neem seeds.
Similarly, field trials showed that all extracts protected cowpea from C. maculatus damage for
5 months. Pereira (1983) reported that a major part of the toxic activity of neem oil was retained
after 3 months of storage.
The neem extracts were, however, not as effective on two species of Sitophilus. With the exception
of higher concentrations of the aqueous seed extract, there was no toxic effect on S. oryzae as shown
by the level of adult feeding and the number of dead adults. SimilarIy, in field trials, only the neem
seed extracts were effective in suppressing S. zeamais population build-up on maize, suggesting that
this formulation may have some application as a control agent against Sitophiius species. In
contrast to the results obtained in the present study, Ivbijaro (1983b) reported that maize grains
mixed with dry ground neem seed were protected for 6 months in storage from damage by
S. oryzae, post-embryonic development being completely halted at all doses, suggesting that this
method of application may be more effective.
It would appear that the effect of neem depends on the part of the plant used, In the present
work, seed extracts gave better protection to both cowpea and maize than the leaf extract. This
agrees with the observation of Jilani and Malik (1973) that neem seeds exhibited maximum
repellency when compared with the leaves and flowers. In the later paper, Malik and Naqvi (1984)
reported antifeedant activity in azadirachtin isolated from fruits against stored product insects. The
differential effect may be due to the presence of a higher proportion of active components in the
seed than the leaf. There is also the possibility that the active components in the leaf may be
different or in different proportions and less effective than those in the seed. Consideration of this
question was beyond the scope of the present study.
With regard to formulation, it appeared that aqueous extracts were more effective than
methylated spirit extracts, although not all published results are in agreement. For example,
McMillan ef al. (1969) reported that a chloroform extract of neem leaves inhibited and retarded
the growth and development of Spodoptera frugiperda (J. E. Smith) larvae although a water extract
had no effect. However, Joshi and Ramaprasad (1975) used a neem seed suspension in water and
found that this reduced feeding in Spodoptera litura (F). Butterworth and Morgan (1971) reported
that azadirachtin, the repellent agent, is easily extracted with water. The method of extraction and
formulation used, therefore, plays an important role.
The importance of insect behaviour in determining the effectiveness of such treatments is
demonstrated by the lack of any inhibitory or antifeedant effect on C. puncticollis when exposed
to sweet potato surfaces treated with neem extracts. The adult of this species is not a surface feeder
and, unlike Sitophilus which inserts its eggs just below the surface of the maize grain, it embeds
its eggs within the tuber in egg holes. Any effect on eggs would, therefore, be dependent upon the
absorption of the neem extract into the tuber.
This work has demonstrated that neem is an effective protectant for some stored grains. Since
it is widely grown in many African countries, further consideration should be given to its
integration with other control measures.

Acknowledgements-I wish to express my gratitude to the International Institute of Tropical Agriculture, Nigeria for
initiating this study. Thanks are also due to Dr K. Leuschner for the encouragement and interest he took in the study.

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