Effect of Neem Powder (Azadirachta indica A. Juss) on the Control of Cowpea Weevils [Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)] in Cowpea Beans

Abstract

The cowpea weevils [Callosobruchus maculatus (F.)] are the primary pest affecting grain and seeds of stored cowpea beans. The control of this insect comprises expensive methods such as fumigation or spraying of chemicals, which are unfeasible for small farmers. The use of insecticidal plants, such as the neem tree (Azadirachta indica), may stand out as a cheaper alternative. This study evaluates the bioactivity of neem powder on the control of weevils in cowpea seeds. We tested four types of powders according to the part of the plant from which it originated: leaves, fruits, bark, and the mixture of these three parts in the same proportion. The bioassay of the action spectrum and the insecticidal effects were assessed using four doses of each type of powder: 0.25%, 0.50%, 0.75%, and 1.00% per 20g of beans. The fruit powder repelled weevils at the lower doses used, while leaf powder, bark, and the mixture were neutral. Although neem powder reduced the survival of insects, the reduction was slow, showing mild toxicity. Neem powder may be an alternative for the control of cowpea weevils in storage units. However, the efficiency of the control depends on the part of the plant and dosage used.

Full text

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*Corresponding author: E-mail: bdeandrade3@gmail.com, b-deandrade@hotmail.com;
Journal of Experimental Agriculture International
30(2): 1-7, 2019; Article no.JEAI.46051
ISSN: 2457-0591
(Past name:
American Journal of Experimental Agriculture,
Past ISSN: 2231-0606)
Effect of Neem Powder (Azadirachta indica A. Juss)
on the Control of Cowpea Weevils [Callosobruchus
maculatus (F.) (Coleoptera: Bruchidae)] in
Cowpea Beans
Eduardo Pereira de Sousa Neto
1
, Anderson Bruno Anacleto de Andrade
2*
,
Ewerton Marinho Costa
3
, Patrício Borges Maracajá
3
, Alex Béu Santos
3
,
José Lucas Guilherme Santos
3
and Thiago Alves Pimenta
2
1
Universidade Federal do Ceará, Fortaleza, Brazil.
2
Universidade Federal de Alagoas, Maceio, Brazil.
3
Universidade Federal de Campina Grande, Pombal, Brazil.
Authors’ contributions
This work was carried out in collaboration between all authors. Author EPSN carried out the research
in the laboratory and wrote the text with the authors ABS and JLGS. Authors ABAA, EMC, PBM and
TAP contributed with the statistical analysis and as advisors for the writing and bibliographic revision
of the manuscript. All authors read and approved the final manuscript.
Article Information
DOI: 10.9734/JEAI/2019/46051
Editor(s):
(1)
Dr. Slawomir Borek, Professor, Department of Plant Physiology, Adam Mickiewicz University, Poland.
Reviewers:
(1)
Sunita Bhatnagar, Regional Plant Resource Centre, India.
(2)
Aba-Toumnou Lucie, University of Bangui, Central African Republic.
Complete Peer review History:
http://www.sdiarticle3.com/review-history/46051
Received 17 October 2018
Accepted 11 January 2019
Published 29 January 2019
ABSTRACT
The cowpea weevils [Callosobruchus maculatus (F.)] are the primary pest affecting grain and seeds
of stored cowpea beans. The control of this insect comprises expensive methods such as fumigation
or spraying of chemicals, which are unfeasible for small farmers. The use of insecticidal plants, such
as the neem tree (Azadirachta indica), may stand out as a cheaper alternative. This study evaluates
the bioactivity of neem powder on the control of weevils in cowpea seeds. We tested four types of
powders according to the part of the plant from which it originated: leaves, fruits, bark, and the
mixture of these three parts in the same proportion. The bioassay of the action spectrum and the
Original Research Article

Sousa Neto et al.; JEAI, 30(2): 1-7, 2019; Article no.JEAI.46051
2
insecticidal effects were assessed using four doses of each type of powder: 0.25%, 0.50%, 0.75%,
and 1.00% per 20g of beans. The fruit powder repelled weevils at the lower doses used, while leaf
powder, bark, and the mixture were neutral. Although neem powder reduced the survival of insects,
the reduction was slow, showing mild toxicity. Neem powder may be an alternative for the control of
cowpea weevils in storage units. However, the efficiency of the control depends on the part of the
plant and dosage used.
Keywords: Vigna unguiculata; alternative control; insecticidal plants; bioactivity.
1. INTRODUCTION
Cowpea [Vigna unguiculata (L.) Walp.]
comprises an essential food source in the tropics
and subtropics, mainly in Africa, Central America,
and South America [1,2]. The northern and
northeastern region of Brazil leads the national
cowpea production, where family farmers
cultivate these beans in subsistence agricultural
systems. Cowpea is a low-cost food supply, rich
in proteins and essential amino acids [3].
Among the phytosanitary problems affecting
cowpea, the pest insects both attack the crop in
the field and damage stored grains and seeds.
The cowpea weevil [Callosobruchus maculatus
(F.) (Coleoptera: Bruchidae)] is the primary
storage pest of cowpea, with widespread
worldwide occurrence [4,5].
The infestations of weevil in cowpea compromise
seeds viability, grains physiology, and its
nutritional quality, as well as contaminate the
product with excrement. Such problems cause
qualitative and quantitative losses through, which
reduces beans commercial value. Cowpea
weevil causes annual losses between 30 and
50% and sometimes above 90% [6,7,8,9,10].
The control of cowpea weevil has been carried
out by fumigation or spraying with chemicals
of different toxicological classes. Synthetic
insecticides are expensive for small farmers and
require equipment and training for their use [11].
The massive use of these products in recent
years has driven to many problems, such as the
emergence of resistant populations and high
amount of insecticides residues in foodstuffs,
which harm consumers’ health and the
environment [7,12].
In addition to the problems mentioned above,
many producers, especially in family farms,
neglect control the weevil due to lack of financial
resources. In this scenario, the use of insecticidal
plants stands out as a promising alternative for
weevil control since these plants usually have
low cost, easy application, biodegradability, and
may be available on the producer's property
[13,14,15].
Among the promising vegetable species for the
control of cowpea weevil, products derived from
neem (Azadirachta indica A. Juss) stand out
because they contain substances, especially
Azadirachtin, that act as an insecticide [16].
Neem leaf powder caused increased adult
mortality of weevil in cowpea seeds [17], without
causing changes in the viability characteristics of
the seeds [18,19]. However, there are still few
studies evaluating the effect of powders made
from different parts of the neem tree on the
mortality of cowpea weevil.
Given the above, this work aimed to evaluate the
bioactivity of the powder of different parts of the
neem plant in the control of adult cowpea weevils
in stored seeds of cowpea.
2. MATERIALS AND METHODS
The study was carried out at the Laboratory of
Entomology of the Agriculture Sciences
Academic Unit (UAGRA) of the Center of
Agrifood Science and Technology of the Federal
University of Campina Grande (CCTA-UFCG),
Campus of Pombal, Paraíba. The experiment
occurred under controlled conditions of
temperature (32 ± 2°C) and relative humidity (70
± 5%).
Cowpea weevils used in the bioassays were
reared in the Laboratory of Entomology following
the methodology of Freire et al. [20]. The insects
were kept in glass cages with a capacity of 1.5
liters (21.0 x 10.5 x 10.5 cm), top coated with anti
aphid screen, containing cowpea 'Canapu'
seeds.
Leaves, fruits, and bark were collected from
neem plants in the CCTA-UFCG (6°48'16"S;
37°49'15"W; 144 m of altitude). The material was
packed in kraft paper bags and dried in a forced
air circulation oven at 40°C for 48 h. After that,

Sousa Neto et al.; JEAI, 30(2): 1-7, 2019; Article no.JEAI.46051
3
the different parts of the plant were crushed
separately in a food processor and sieved (0.5
mm mesh) until the production of powder with
uniform granulometry.
The action spectrum bioassay was carried out to
verify the behavior index of cowpea weevils
relative to the presence of neem powder. We
used multiple-choice experimental arenas
consisting of six plastic containers with 10 cm in
diameter and 4 cm high (Fig. 1) [21]. The set
comprised a central container symmetrically
interconnected by 0.5 cm diameter plastic tubes
to another five diagonally arranged containers.
We released 50 non-sexed adult insects in the
central container. The peripheral containers
contained 20 g of cowpea 'Canapu' with the
powders at 0.0% (control), 0.25%, 0.50%, 0.75%,
and 1.00% dosages. Each assay tested one
type of powder, leaves, fruits, bark, and
leaf+fruit+bark (proportion 1:1:1), in three
replicates.
Fig. 1. Experimental arenas used in the action
spectrum bioassay
We counted the live and dead insects in each
container after 24, 48, and 72 hours from the
begging of the experiment. At the end of each
count, the dead insects were removed from the
container and discarded. The Behavior Index (BI)
was used to compare treatments according to
the following equation: BI = (% of insects in the
test-plant - % of insects in control) / (% of insects
in the test-plant + % of insects in control). When
BI lies between -1.00 and -0.10 the plant is a
repellent, a BI between -0.10 and +0.10 indicates
a neutral effect and a BI between +0.10 and
+1.00 an attractive effect [21].
Evaluations of the insecticidal effect of the
powders followed the same experimental design
described above (four types of powder at the four
concentrations, and one control). Each treatment
was performed in 4 replicates. The insects were
exposed to the treatments in round plastic
arenas of 500 mL (120 mm diameter and 78 mm
height) containing 20 g of cowpea inside. The
upper part of the containers was perforated for
air circulation. Twenty adult insects were
released in each container, evaluating mortality
and behavior every 24 hours until all insects
died.
For the analysis of the insecticidal action of the
powder, we elaborated curves showing the
mortality of the insects over time by the Kaplan-
Meier method with application of the non-
parametric Log-Rank Test to compare the curves
and the mean lethal time for the death of 50% of
the insects was estimated using non-linear
regression models in the GraphPad Prism
®
6
software [22].
3. RESULTS AND DISCUSSION
The neem fruit powder repelled weevils in
cowpea seeds under all doses (Fig. 2B). The use
of products with repellent effects to control
cowpea weevils comprises a primary technique
in the management of this pest. Considering that
the attractive odor of an alcohol (2-Ethylhexanol)
present in cowpea mediates the preference of
cowpea weevils [23], the neem fruit powder may
act confusing the insect perception or emitting an
unattractive odor.
Several studies report repellent effects against
cowpea weevil cause by some plant species of
the Caatinga Brazilian ecoregion such as
Amburana cearensis A. C. Smith, Croton
sonderianus Müll. Arg., Cleome spinosa Jacq.,
Mimosa tenuiflora Benth., Anadenanthera
macrocarpa (Benth.) Brenan, Aspidosperma
pyrifolium Mart., Senna occidentalis (L.) H.S.
Irwin & R.C. Barneby, Hyptis suaveolens (L.)
Poit., and Ziziphus joazeiro Mart. [15], showing
the potential of these plant products as an
alternative control of this pest.
The powders made from leaves, bark and the
mixture had a neutral effect in most doses, with
no potential for insect repellency or attraction,
especially at lower doses (Fig. 2).
Boeke et al. [24], treating cowpea with leaf
powder of neem in the proportion 5g/kg, found an
attractive effect on the weevil, which opposes our
neutral result.

Sousa Neto et al.; JEAI, 30(2): 1-7, 2019; Article no.JEAI.46051
4
Schumacher et al. [25] state that botanical
bioactivity on insects can have attractive and
insecticide effects at the same time, while others
can be repellent and do not cause an insecticidal
effect. However, an ideal product should repel
and kill the insects, because the repellent effect
decreases the oviposition and consequently the
number of insects that will hatch, and still cause
a substantial decrease in the pest population
through the insecticidal action.
The evaluation of insecticidal action of neem
powder resulted in significant differences (P <
0.01) in the comparison between the mortality
curves of all doses with the control treatment,
even though in some doses the observed
difference was unexpressive (Table 1; Fig. 3).
The leaves powder at 1.0% (10 g/kg) provided
the total death of insects in 144 hours (6 days),
the shortest time recorded but not showing
immediate action of toxicity (Fig. 3). In the control
treatments, the longest survival time was 288
hours (12 days). In the insecticidal activity of
neem powder on cowpea weevils, the mortality
time is dose-dependent, the highest being 10
g/kg. The neem powder efficiency on the
mortality of this pest was observed by Silva et al.
[26] with the use of 150 g/kg. Tofel et al. [27],
using a dose of 83.27 g/kg, recorded mortality of
50% of the weevil population in 3 days.
The powder of the leaves from Solanum
melongena and Capsicum annuum promoted the
death of all weevils in 120 hours (5 days). The
researches look for products that cause insect
mortality as soon as possible so that population
decrease occurs and hinders oviposition [20].
For the mortality of 50% of the insect population
(TL50), the use of different parts of neem in the
powder caused similar results, with the highlight
only for the treatment with the mixture (Leaves +
Fruits + Bark) that had a faster action at the
concentration of 0.75% (7.5 g/kg) with the time of
60 hours (2.5 days), while the control had TL50
of 114 hours (5 days) (Fig. 3). Thus, showing that
the use of powder from other parts of neem,
besides the leaves, may contribute to the
management of cowpea weevil, as the protection
by powders of seeds and roots [28].
The neem tree, through the use powders
from leaves, fruits, and bark comprises an
alternative for the management of cowpea
weevil in storages, but the plant part and the
doses used are decisive for efficiency in the
control.
Fig. 2. Behavior of cowpea weevil (Callosobruchus maculatus) on cowpea seeds with
increasing doses of the powder made from the following parts of the neem tree (Azadirachta
indica). (A) Leaves; (B) Fruits; (C) Bark; (D) Mixture (Leaves + Fruit + Bark)
-1 -0.75 -0.5 -0.25 0 0 .25 0.5 0.75 1
0.25%
0.50%
0.75%
1.00%
Behavior Index
repellent neutral attractive
(A)
-1 -0.75 -0.5 -0.25 0 0. 25 0.5 0.75 1
0.25%
0.50%
0.75%
1.00%
Behavior Index
repellent neutral attractive
(B)
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
0.25%
0.50%
0.75%
1.00%
Behavior Index
repellent neutral attractive
(C)
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
0.25%
0.50%
0.75%
1.00%
Behavior Index
repellent neutral attractive
(D)

Sousa Neto et al.; JEAI, 30(2): 1-7, 2019; Article no.JEAI.46051
5
Table 1. Significant variation between mortality curves of cowpea weevil between control
treatment and use of neem parts powder
Parts
Comparison with control
mortality curve
DF
Chi-square
P value
Leaves 0.25% 1 16.03 <0.0001
**
0.50% 1 19.89 <0.0001
**
0.75% 1 14.31 0.0002
**
1.0% 1 36.21 <0.0001
**
Fruits 0.25% 1 22.85 < 0.0001
**
0.50% 1 9.119 0.0025
**
0.75% 1 13.54 0.0002
**
1.0% 1 10.82 0.0010
**
Bark 0.25% 1 11.10 0.0009
**
0.50% 1 7.472 0.0063
**
0.75% 1 8.645 0.0033
**
1.0% 1 14.46 0.0001
**
Mixture (leaves +
fruits + bark)
0.25% 1 17.86 < 0.0001
**
0.50% 1 21.60 < 0.0001
**
0.75% 1 32.55 < 0.0001
**
1.0% 1 18.01 < 0.0001
**
DF: Degree of freedom. **significant value at the 1% probability level by the non-parametric Log-Rank Test
Fig. 3. Mortality curves of cowpea weevil (Callosobruchus maculatus) on cowpea beans
treated with neem powder tree (Azadirachta indica) in increasing doses. (A) Leaves; (B) Fruits;
(C) Bark; (D) Mixture (Leaves + Fruits + Bark)
4. CONCLUSION
The powder from neem fruit repels cowpea
weevils, and the powder from fruit, leaves, and
bark has an insecticide action.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.

Sousa Neto et al.; JEAI, 30(2): 1-7, 2019; Article no.JEAI.46051
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REFERENCES
1. Amusa OD, Ogunkanmi AL, Bolarinwa K,
Ojobo O. Evaluation of four cowpea lines
for bruchid (Callosobruchus maculatus)
Tolerance. Journal of Natural Sciences
Research. 2013;3(13).
2. Oliveira GB, Kunza D, Peres TV, Leal RB,
Uchôa AF, Samuels RI, et al.
Variantvicilins from a resistant Vigna
unguiculata lineage (IT81D-1053)
accumulate inside Callosobruchus
maculatus larval midgut epithelium.
Comparative Biochemistry and Physiology,
Part B. 2014;168:45–52.
DOI: 10.1016/j.cbpb.2013.11.001
3. Freire Filho FR, Ribeiro VQ, Rocha MM,
Silva KJD, Nogueira MSR, Rodrigues EV.
Production, breeding and potential of
cowpea crop in Brazil. Teresina: Embrapa
Mid-North; 2012.
[Accessed 20 June 2018]
Available:https://ainfo.cnptia.embrapa.br/di
gital/bitstream/item/84470/1/feijao-
caupi.pdf
4. Sousa AH, Maracajá PB, Silva RMA,
Moura AMN, Andrade WG. Bioactivity of
vegetal powders against Callosobruchus
maculatus (Coleoptera: Bruchidae) in
caupi bean and seed physiological
analysis. Revista de Biologia e Ciências da
Terra. 2005;5(2):1519-5228.
5. Ekeh FN, Oleru KI, Ivoke N, Nwani CD,
Eyo JE. Effects of Citrus sinensis Peel Oil
on the Oviposition and Development of
Cowpea Beetle Callosobruchus maculatus
(Coleoptera: Chrysomelidae) in Some
Legume Grains. Pakistan Journal of
Zoology. 2013;45(4):967–974.
6. Udo IO, Harry GI. Effect of groundnut oil in
protecting stored cowpea (Vigna
unguiculata) from attack by cowpea weevil
(Callosobruchus maculatus). Journal of
Biology, Agriculture and Healthcare. 2013;
3(1).
7. Radha R, Susheela P. Efficacy of plant
extracts on the toxicity, ovipositional
deterrence and damage assessment of the
cowpea weevil, Callosobruchus maculatus
(Coleoptera: Bruchidae). Journal of
Entomology and Zoology Studies. 2014;2:
16–20.
8. Mogbo TC, Okeke TE, Akunne CE.
Studies on the Resistance of Cowpea
Seeds (Vigna unguiculata) to Weevil
(Callosobruchus maculatus) Infestations.
American Journal of Zoological Research.
2014;2(2):37–40.
9. Ahmad T, Haile A, Ermias A, Etbarek R,
Habteab S, Teklai S. Eco-friendly
approaches for management of bruchid
beetle Callosobruchus chinensis
(Coleoptera: Bruchidae) infesting faba
bean and cowpea under laboratory
conditions. Journal of Stored Products and
Postharvest Research. 2015;6(3):25–29.
10. Ekeh FN, Odo GE, Nkiru E, Agwu EJ,
Ikegbunam C, Haruna AS. Effects of
biopesticides on developmental stages and
longevity of Callosobruchus maculatus in
some leguminous grains. Journal of
Parasitology and Vector Biology. 2015;
7(1):9–21.
DOI: 10.5897/JPVB2014.0161
11. Ilesanmi JO, Gungula DT. Preservation of
cowpea (Vigna unguiculata (L.) Walp)
grains against cowpea bruchids
(Callosobruchus maculatus) using neem
and moringa seed oils. International
Journal of Agronomy, 2010;8.
DOI: 10.1155/2010/235280
12. Owolabi MS, Padilla-Camberos E,
Ogundajo AL, Ogunwande IA, Flamini G,
Yusuff OK, et al. Insecticidal activity and
chemical composition of the Morinda lucida
essential oil against pulse beetle
Callosobruchus maculatus. The Scientific
World Journal. 2014;14:1-7.
DOI: 10.1155/2014/784613
13. Ekeh FN, Onah IE, Atama CI, Ivoke N, Eyo
JE. Effectiveness of botanical powders
against Callosobruchus maculatus
(Coleoptera: Bruchidae) in some stored
leguminous grains under laboratory
conditions. African Journal of Biotechno-
logy. 2013;12(12):1384–1391.
14. Campos ACT, Radunz LL, Radünz AL,
MossiI AJ, Dionello RG, Ecker SL.
Insecticide and repellent activity of the
essential oil from carqueja doce on bean
weevil. Revista Brasileira de Engenharia
Agrícola e Ambiental. 2014;18(8):861–865.
DOI:10.1590/1807-1929/agriambi
15. Melo BA, Molina-Rugama AJ, Haddi K,
Leite DT, Oliveira EE. Repellency and
bioactivity of caatinga biome plant powders
against Callosobruchus maculatus
(Coleoptera: Chrysomelidae: Bruchinae).
florida entomologist. 2015;98(2):417–423.
DOI: 10.1653/024.098.0204
16. Schmutterer H. Properties and potential of
natural pesticides from theneem tree,

Sousa Neto et al.; JEAI, 30(2): 1-7, 2019; Article no.JEAI.46051
7
Azadirachta indica. Annu. Rev. Entomol.
1990;35:271–297.
DOI:
10.1146/annurev.en.35.010190.001415
17. Medeiros DC, Andrade Neto RC, Figueira
LK, Nery DKP, Maracajá PB, Nunes GHS.
Powder of dried and fresh leaves of to
control in cowpea seeds. Revista Científica
Eletrônica de Engenharia Ambiental. 2007;
6(10).
18. Araujo EC, Medeiros Filho S, Vieira FVA,
Bezerra AME. Physiological quality oi
cowpea bean seeds treted with nim
powdel. Ciência Agronômica. 2001;32(1):
60–68.
19. Medeiros DC, Andrade Neto RC, Figueira
LK, Nery DKP, Maracajá PB. Quality seeds
of cowpea submitted powder of neem
dryness and green leaves. Revista
Caatinga. 2007;20(2):94–99.
20. Freire GF, Leite DT, Pereira RA, Melo BA,
Silva JF, Maracajá PB. Bioactivity of
Solanum melongena L. and Capsicum
annuum L. on Callosobruchus maculatus
(Coleoptera: Bruchidae) ACTA Biológica
Colombiana. 2016;21(1):123-130.
DOI: 10.15446/abc.v21n1.45775
21. Procópio SO, Vendramim JD, Ribeiro
Júnior JI, Santos JB. Bioactivity of powders
from some plants on Sitophilus zeamais
Mots. (Coleoptera: Curculionidae) Ciência
e Agrotecnologia. 2003;27(6):1231–1236.
DOI: 10.1590/S1413-70542003000600004
22. Motulsky MDH. Intuitive biostatistics, New
York: Oxford University Press, 1995;386.
23. Ajayi OE, Balusu R, Morawo TO, Zebelo S,
Fadamiro H. Semiochemical modulation of
host preference of Callosobruchus
maculatus on legume seeds. Journal of
Stored Products Research. 2015;63:31–
37.
DOI: 10.1016/j.jspr.2015.05.003
24. Boeke SJ, Baumgart IR, van Loon JJA,
van Huis A, Dickea M, Kossou DK. Toxicity
and repellence of African plants
traditionally used for the protection of
stored cowpea against Callosobruchus
maculatus. Journal of Stored Products
Research. 2004;40:423–438.
DOI: 10.1016/S0022-474X(03)00046-8
25. Schumacher M, Cerella C, Reuter S,
Dicato M, Diederich M. Antiinflammatory,
pro-apoptotic, and anti-proliferative effects
of a methanolic neem (Azadirachta indica)
leaf extract are mediated via modulation of
the nuclear factor-kappa B pathway.
Genes Nutrition. 2011;6:149-160.
DOI: 10.1007/s12263-010-0194-6
26. Silva WA, Oliveira JV, Barbosa DRS,
Breda MO, Esteves Filho AB. Bioactivity of
vegetable powders on biological
parameters of Callosobruchus maculatus
(Fabr.) (Coleoptera: Chrysomelidae,
Bruchinae) in Vigna unguiculata. Indian
Journal of Agricultural Sciences. 2016;
86(1):128–132.
27. Tofel KH, Nukenine EN, Stähler M, Adler
C. Insecticidal efficacy of Azadirachta
indica powders from sun- and shade-dried
seeds against Sitophilus zeamais and
Callosobruchus maculatus. Journal of
Entomology and Zoology Studies. 2015;
3(1):100–108.
28. Ahmed IA, Kutama AS, Hassan KY, Abdul
I. Comparative efficacy of neem
(Azadirachta indica A. Juss) powder
against cowpea beetle (Callosobruchus
maculatus Fab.) on stored cowpea seeds.
Standard Research Journal of Agricultural
Sciences. 2014;2(4):49–53.
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