Brazilian Journal of Biological Sciences, 2018, v. 5, No. 11, p. 673-681. ISSN 2358-2731 https://doi.org/10.21472/bjbs.051106

Bioefficacy of boonei extract against cowpea beetle Callosobrochus maculatus infesting stored cowpea seeds in storage

Kayode David Ileke¹ and Arotolu Temitope Emmanuel¹,²

¹Department of Biology. Federal University of Technology Akure. Nigeria. Email: [email protected]. ²Centre of Conservation Medicine and Ecological safety, Northeast Forestry University, Harbin P.R. China. Email: [email protected].

Abstract. This study was conducted to investigate the efficacy of the oils of Alstonia boonei leaf extracted with n-hexane, Received petroleum ether, methane and acetone as contact insecticides October 5, 2018 against the activities of Callosobrochus maculatus in stored cowpea seed. The oils were incorporated at rates 2.0 mL per Accepted December 23, 2018 20 g of cowpea seeds. The parameters assessed include, mortality of adult insects, oviposition and adult emergence to Released ascertain the control of the beetle. All concentration of the December 31, 2018 extracts used evoked 100% mortality of C. maculatus after 72 h of post treatment. The development of Callosobrochus maculatus Full Text Article was inversely proportional to the concentration of the oil. As the ratio of Alstonia boonei leaf oil extract increased, the mortality of the beetle increased. Therefore, complete protection of seeds and complete inhibition of adult emergence in the oils extracts of Alstonia boonei leaf were effective in controlling cowpea bruchid in stored cowpea seed.

Keywords: Alstonia boonei; Leaf oil extracts; Cowpea seed; Callosobrochus maculates; Seed protection.

0000-0002-3106-4328 Kayode David Ileke 0000-0003-1330-0160 Arotolu Temitope Emmanuel

Introduction most important producing areas in Nigeria are located in the savanna Cowpea Vigna unguiculata L. (Agboola, 1979). According to Blade et al. (Walp), is an important crop in the (1997), Nigeria accounts for 70% of the tropics especially in West Africa where it world’s production. Cowpea is an is a cheap source of dietary protein extremely valuable crop both as a source (Boeke et al., 2004). Cowpea is widely of revenue and an important source of cultivated and consumed in Nigeria; the cheap dietary protein for the third world

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where meat is expensive (Ileke and on the use of alternative eco-friendly Olotuah, 2012). High protein and lysine insect pest control methods among contents of cowpea make it a natural which are the use of products supplement to staple diets of cereals, (Ileke and Oni, 2011). Lale (1995) roots, tuber and fruits (Bressani, 1985). reported that materials and local In Nigeria, the production of cowpea is traditional methods are safer than carried out largely by peasant farmers synthetic insecticides and suggested that and cowpea production is limited by their use needed exploitation. Small scale insect pests which cause serious post- farmers in the tropics have reported the harvest losses to the cowpea grain. The need to protect cowpea against pest value of cowpea lies in its high protein damage during storage (Lale, 1995; content and ability to tolerate drought Golob et al., 1999). The potential of using and also its compatibility as an intercrop plant products such as leaf, bark, root with maize, millet, sorghum, sugarcane powder from certain plants such as and cotton (Caswell, 1984). This makes Capsicum spp, Piper spp, and citrus peels the grain legumes an important have been investigated by Rajapakse and component of traditional intercropping Van Emden (1997) and Ileke and Bolus systems especially in the complex and (2012). elegant subsistence farming systems of Alstonia boonei De wild is a the dry savanna in sub-Saharan Africa. medicinal plant of West and Central Cowpea bruchid, Callosobruchus African origin which is extensively used maculatus depreciates stored cowpea for the treatment of malaria fever, (Jackai and Adalla 1997). The huge post- intestinal helminthes, snake bite, arrow, harvest losses and quality deterioration impotence, toothache and oedema caused by this insect pest are threat to (Terashima, 2003; Akinmoladun et al., food security in a developing nation like 2007). The various ethnomedical, Nigeria (Boeke et al., 2004). chemical, pharmacological and The infestation of cowpea seeds toxicological properties of Alstonia by Callosobrochus maculates (F.) boonei were recently reviewed and the accounted for the major loss of cowpea profile further revealed that it is useful in both in store and at fields (Adedire, the treatment and management of 2001; Udo, 2005). The females of the several illnesses (Adotey et al., 2012). In bruchid lay their eggs on the seed surface spite of its vast acceptability as a which hatch into larvae within 5 days. medicinal plant, the insecticidal potential The larvae bore through the seed coat of A. boonei has not been fully explored into the underlying cotyledons where compared to other botanicals used in development continues until adult storage. The objectives of this study is to emergence; the adults are already investigate the efficacy of A. boonei leaf sexually mature on emergence (Credland extract against the biological activities of and Wright, 1989; Ofuya and Credland, Callosobruchus maculatus in storage. 1995). Effective and efficient controls of Materials and method storage insect pests are centered mainly on synthetic insecticides. The use of Insect cultures these synthetic chemicals is hampered by The starter culture of many attendant problems like Callosobruchus maculatus was obtained development of resistance insect strain, from an infested cowpea seeds at toxic residues in foods and humans; Environmental Biology and Fisheries workers safety and high cost of laboratory, Adekunle Ajasin University, procurements (Adedire and Lajide 2001, Akungba Akoko, Nigeria. The cowpea Ileke and Oni, 2011, Ileke and Olotuah, were first sorted to eliminate stones, 2012). These have necessitated research chaff and other unwanted material, and

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Bioefficacy of Alstonia boonei leaf extract against cowpea beetle 675

were later disinfected by keeping them in Toxicity of A. boonei leaf oil extract on a freezer at -5 °C for 7 days. The mortality, oviposition, adult disinfected seeds were then air dried in emergence of Callosobruchus the laboratory to prevent mouldiness maculatus, weight loss, damage and before the introduction of insects from weevil perforation index of cowpea infected ones (Adedire et al., 2011). They seed were placed in kilner jars and covered The toxic effect of plants oil on with muslin cloth. The jars were placed adult C. maculatus was accomplished in insect rearing cages at ambient using 250 mL plastic containers temperature of 30±3 °C and 70%±5% containing 20 g of cowpea seeds with relative humidity. concentration of 2 mL of solvent extract of Alstonia boonei leaf. The oil and seeds Plant collection were thoroughly mixed with the aid of a Fresh of Alstonia boonei glass rod and agitated for 5-10 min to were collected once from Ijiro, Ilasa Ekiti, ensure uniform coating. The containers Ekiti State. The plant leaves were air were left open for 30 min so as to allow dried in an open laboratory and ground trace of solvent to evaporate off. Ten into very fine powder using an electric copulating pairs of C. maculatus were blender (Supermaster, Model SMB 2977, introduced into the container and Japan). The powders were further sieved mortality was observed daily for 4 days. to pass through 1 mm² perforations prior Cowpea seeds that were solvent treated to storage in a plastic with tight lids in a served as the control experiment refrigerator at 4 °C before use (Ileke and (Arannilewa et al., 2006). Similar Oni, 2011). preparation was set up for Alstonia boonei leaf extracted with N-hexane, Oil extract petroleum ether and methanol. Three Extracts of Alstonia boonei was replicates per treatment were prepared. carried out using cold extraction method. The adult mortality was assessed every About 250 g of A. boonei powders were 24 h for 96 h. Adult insects were soaked separately in an extraction bottle considered dead where no response was containing absolute acetone, petroleum observed after probing them with ether, methanol and N-hexane. The forcepts. After 96 h, all insects both dead mixture was stirred occasionally with a and alive were then removed. Number of glass rod and extraction was terminated eggs laid in each container was counted after 72 h. Filteration was carried out and recorded. using a double layer of Whatman No. 1 Progeny emergence (F1) was filter paper and solvent evaporated using then recorded at 6 weeks. The containers a rotary evaporator at 30 °C to 40 °C with were sieved out and newly emerged rotary speed of 3 to 6 rpm for 8 h (Ileke adult bruchid were counted with an and Olotuah, 2012). The resulting extract aspirator. The percentage adult was air dried in order to remove traces emergence was calculated according to of solvents. the method described by Odeyemi and Daramola (2000):

% Adult emergence = No of adults emerged x 100 No of eggs laid

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The cowpea were re-weighed by determined as described by Odeyemi and using Metler weighing balance and Daramola (2000): percentege loss in weight was

% Weight loss = Difference in weight x 100 Initial weight

After re-weighing, the numbers holes. Percentage seed damaged was of damaged cowpea seeds were calculated according to the method evaluated by counting wholesome and described by Ileke and Olotuah (2012) as bored or seed with bruchid emergent follows:

% seed damage = Number of seeds damaged x 100 Total number of seeds

Weevil Perforation Index (WPI) Ajayi (1996). The weevil perforation is were adopted from the analysis of defined as follows: damage using the method of Adedire and

WPI = % treated cowpea seeds perforated x 100 % control cowpea seeds perforated

mortality were 60%, 33%, 53%, 33%, Statistical analysis 50%, 30%, and 0%, with N-hexane pet Data were subjected to analysis ether, acetone, methanol, and control, of variance (ANOVA) in SPSS 21.0 respectively. At 48 h the percentage software and treatment means were mortality rate increases to 80%, 76.67%, separated using the New Duncan’s 70%, 53.33%, 0%, in N-hexane, pet- Multiple Range Test. ether, acetone and methanol, respectively, without any mortality in the Results control experiment while at 72 h post treatment, there was an increase in the Effects of Alstonia boonei leaf percentage mortality. The percentage extracts on adult mortality of mortality rate at 96 h of post treatment Callosobruchus maculatus (F.) was 100% in all treatment. The mortality Table 1 shows the mortality rate rate of adult C. maculatus in the control of C. maculatus in 20 g of cowpea seeds was significantly higher than those in treated with 2 mL of Alstonia boonei oil cowpea seeds treated Alstonia boonei oil. extracts. The effect of N-hexane was N-hexane extract of A. boonei leaf was significantly (P > 0.05) higher at 24 h on observed to have the highest mortality mortality rate of C. maculatus and 72 h in effect on C. maculatus. The control was all the cowpea seeds treated with 2 mL of significantly higher than those in cowpea leaf oils. At 24 h post treatment with seeds treated with other oils. 2 mL of extracts, the percentage

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Table 1. Mortality of adult C. maculatus in cowpea seeds treated with different solvents of Alstonia boonei.

Alstonia boonei leaf extract % mortality at hours post treatment 24 48 72 96 N-hexane 60.33±001c 80.00±0.00d 100.00±0.00c 100.00±0.00b Petroleum-ether 53.33±0.01bc 76.67±0.04cd 100.00±0.00c 100.00±0.00b Acetone 50.00±0.00bc 70.00±0.00c 100.00±0.00c 100.00±0.00b Methanol 30.00±0.00b 53.33±0.01b 80.00±0.00b 100.00±0.00b Control 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a Each value is a mean ±standard error of three replicates. Means within the same column followed by the same letter(s) are not significantly different at p > 0.05 using New Duncan Multiple Range Test.

Effect of Alstonia boonei leaf leaf extracted with n-hexane, pet ether, extracts on oviposition and adult acetone, methanol and control emergence of Callosobruchus respectively. The number of eggs laid in maculatus (F.) cowpea seeds treated with methanol leaf The number of eggs laid by adult extracts while control having the highest C. maculatus varied with A. boonei leaf number of eggs. There was a reduction in extract used and the concentration of the the numbers of eggs laid in cowpea seeds extracts added to the cowpea seeds, treated with n-hexane leaf extracts which (Table 2). The mean number of egg proves to be the most effective among oviposition by adult C. maculatus where other treatments. 10.00, 10.33, 10.00, 23.33 and 87.67 in

Table 2. Effects of Alstonia boonei leaf extracts on oviposition and adult emergence of Callosobruchus maculatus.

Alstonia boonei extracts No. of eggs laid (oviposition) % Adult emergence n-hexane 10.00±0.00a 0.00±0.00a Petroleum-ether 10.33±0.01a 0.00±0.00a Acetone 10.00±0.01a 0.00±0.00a Methanol 23.33±0.01b 0.00±0.00a Control 87.67±0.04c 85.28±0.11b Each value is a mean ±standard error of three replicates. Means within the same column followed by the same letter(s) are not significantly different at p>0.05 using New Duncan Multiple Range Test.

There were no adult emergences of C. maculatus in all the treated cowpea Effect of Alstonia boonei leaf oil seeds. The numbers of adult emerged in on short term storage of cowpea seeds cowpea seeds treated with various The mean percentage numbers of Alstonia boonei leaf extracts was lower cowpea seeds by different oil extracts are (p 0.05) when compared with number significantly different (Table 3). The of adult emerged in cowpea seeds in mean percentage damage by control˂ that recorded 85.28%. C. maculatus on cowpea seeds were not

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significantly different with the oil weevil perforation index (WPI) of extracts of Alstonia boonei leaf except the C. maculatus on cowpea seeds with leaf control which have high number of seed oil extracts of A. boonei with different damaged. The four oil extracts with n- solvent were good protectant (less than hexane, pet ether, acetone and methanol 50) while control is not a good from Alstonia boonei leaf are more protectant (50.00±0.00). effective in reducing the weight loss. The

Table 3. Effect of Alstonia boonei leaf oil on short term storage of cowpea seeds. Mean Mean total Mean % Weevil 0.5% v/w A. number of Mean number of seed perforation boonei extracts damaged weight loss seeds damage index seeds n-hexane 93.67 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a Petroleum-ether 95.00 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a Acetone 91.33 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a Methanol 95.33 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a Control 93.33 86.67±0.01b 92.86±0.14b 11.02±0.01b 50.00±0.00b Each value is a mean ±standard error of three replicates. Means within the same column followed by the same letter(s) are not significantly different at p>0.05 using New Duncan Multiple Range Test.

Discussion acetone oil extracts treatment on cowpea seeds show some degree effects against The resultant high mortalities of C. maculatus on oviposition and adult adults C. maculatus observed on cowpea emergence but not as high as that of treated with A. boonei leaf oil extracts n-hexane leaf oil treatment which has could be due to high toxic effect of the highest degree of effect. This confirm the product on adult C. maculatus. The report of Benner (1993) who reported A. boonei leaf oil extracts also prevented that extractions of insecticidal plants the emergence of C. maculatus adults, an powders with appropriate solvent, often effect that is in agreement with the concentrates the active minerals and observation of Osawe et al. (2007) who make their potency readily detectable. reported that the aqueous extracts of the In this study, the lethal effect of stem bark and leaves of this plant these oil extracts on cowpea beetle could adversely affected the survival and be as a result of contact toxicity. Most growth of Sesamia calamists. The results insects breathe by means of trachea show that the leaf oil extracted with n- which usually opens at a surface of the hexane, pet ether, acetone and methanol body through spiracles (Adedire et al., had varied degrees of effects on 2011; Ileke and Olotuah, 2012). This C. maculatus at 24 h, 48 h, 72 h and 96 h spiracle must have been blocked by the of post treatment. This study oil extracts thereby leading to suffocation demonstrated that A. boonei oil is toxic to of the insects. The oils on application adults and eggs of C. maculatus. This is in disrupt mating and sexual agreement with the report of Daniel communication as well as deterring (1991) who reported that plant oils are female from laying eggs (NRC, 1992). toxic to young larval, adults and eggs of Also, on application to cowpea seeds, the C. maculatus. Petroleum-ether and oil covered the testa of the grains,

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serving as food poison to the adult’s Conflicts of interest insects; while some of them penetrated into the endosperm and germ layer Authors declare that they have no thereby suppressing oviposition and conflict of interests. larval development. The non-protectant control had no effect on the mortality; References oviposition and adult emergence of C. maculatus may be attributed to Adedire, C. O. Use of nutmeg Myristica interference with normal respiration fragrans (Houtt.) powder and oil for the resulting in suffocation (Hall and Harman control of cowpea storage bruchid, C. 1991). Out of the four solvents used, maculatus (p.). Journal of Plant Diseases , v. 109, p. 193-199, 2002. acetone has been found to give highest and Protection mean value, with no or low number of Adedire, C. O.; Ajayi, T. S. Assessment of the adult emergence in n-hexane. The lower insecticidal properties of some plant extracts as grain protection against the maize weevil, the mean value observed, the more Sitophilus zeamais. Nigerian Journal of effective the solvent of leaf oil, n-hexane Entomology, v. 13, p. 93-101, 1996. oil extract from the leaf at different Adedire, C. O.; Ajayi, O. E. Insecticidal activity concentration is the most effective on of under-utilized tropical plant seed oil, Hura adult emergence of C. maculatus. crepitans L. on cowpea seed beetle, Callosobruchus maculatus (F.) (Coleoptera: Conclusion Bruchidae). Nigeria Journal of Entomology, v. 20, p. 74-81, 2003. This study revealed the Adedire, C. O.; Obembe, O. O.; Akinkurolere, bioefficacy of Alstonia boonei lesf extract R. O.; Oduleye, O. Response of Callosobruchus against cowpea beetle Callosobruchus maculatus (Coleoptera: Chysomelidae: maculatus infesting stored cowpea seeds Bruchidae) to extract of cashew kernels. in storage. Effective and efficient controls Journal of Plant Diseases and Protection, of storage insect pests are centered v. 118, no. 2, p. 75-79, 2011. mainly on synthetic insecticides. The use https://doi.org/10.1007/BF03356385 of these synthetic chemicals is hampered Adedire, C. O.; Lajide, L. Efficacy of powders by many attendant problems. These have of some plants in the control of the pulse necessitated research on the use of beetle, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). alternative eco-friendly insect pest Journal of Applied Tropical Agriculture, v. 6, p. 11-15, 2001. control methods among which are the Adotey, J. P. K.; Adukpo, G. E.; Opoku Boahen, use of plant products. The plant (Alstonia Y; Armah, F. A. A review of the ethnobotany ) is readily available, and has not boonei and pharmacological importance of Alstonia been reported to be toxic to man. boonei De Wild (). ISRN Therefore, this study suggest that Pharmacology, v. 2012, Article ID 587160, Alstonia boonei leaf oil can be used as 2012. https://doi.org/10.5402/2012/ biopesticides against cowpea beetles in 587160 storage. Agboola, S. A. An agricultural atlas of Nigeria. London: Oxford University Press, Acknowledgements 1997. Akinmoladun, A. C.; Ibukun, E. O.; Afor, E.; We are thankful for the dedicated Akinrinlola, B. L.; Onibon, T. R.; Akinboboye, support by the technologists in the A. O.; Obuotor, E. M.; Farombi, E. O. Chemical Federal University of Technology Akure constituents and antioxidant activity of Research laboratory for our research, Alstonia boonei. African Journal of most especially Mrs T. Ojo. We also thank Biotechnology, v. 6, no. 10, p. 1197-1201, the people who accompanied us to 2007. procure the plant leaves.

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