Phytochemical Constituents and Insecticidal Efficacy of the Root and Leaf Powders of Mimosa diplotricha and Aspilia africana against Callosobruchus maculatus (Fab.) (Coleoptera: Chrysomelidae)

This study investigated the efficacy of the root and leaf powders of Aspilia africana and Mimosa diplotricha against the cowpea beetle, Callosobruchus maculatus. Newly emerged adults of C. maculatus were exposed to grains treated with one of four treatments (powders: 0.5, 1.0, 1.5 and 2.0 g) of the two plants at different exposure period of 24, 48, 72 and 96 hours. Both A. africana and M. diplotricha exhibited a high level of mortality which was a function of treatment concentration and of exposure time. At 96 hours of exposure, 2 g of the root and leaf powders of A. africana caused 70% and 54% mortality respectively, in C. maculatus. When C. maculatus was exposed to 2 g for a 96-hour exposure, the root and leaf powders of M. diplotricha, however, resulted in 52% and 50% mortality respectively. Although powders from all four treatment types exhibited insecticidal activities by causing varying levels of mortality in C. maculatus, the highest death rate was caused by the root powder of A. africana. Qualitative analysis of the plants revealed that alkaloids, flavonoids, saponins, steroids, tannins, terpenoids and glycosides were present in the leaf extracts of A. africana and M. diplotricha. The moderately high insecticidal activity demonstrated by the root powders suggest that they hold more potential in the control of C. maculatus compared to the leaf powders. DOI: https://dx.doi.org/10.4314/jasem.v24i4.16 Copyright: Copyright © 2020 Uti et al. This is an open access article distributed under the Creative Commons Attribution License (CCL), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dates: Received: 11 February 2020; Revised: 14 March 2020; Accepted: 22 March 2020

The increased and persistent usage of synthesized insecticides in the control of pests have created a lot of ecological as well as environmental challenges. The use of pesticides pose a big threat to food security and human existence (Ghosh et al., 2007), thus, there is need to develop an environmental friendly alternative that would help combat this challenge while maintaining a balance in the flora and fauna of the ecosystem (Akinneye et al., 2006). Vigna unguiculata (L) (Fabaceae), commonly known as cowpea, is a highly profitable crop of economic importance in Nigeria (Abba, 2013) where it is usually and commonly referred to as 'beans'. It is a very nutritious crop that serves as a good and cheap source of protein.
The usefulness of cowpea to Nigerians cannot be over emphasized as all parts of the plant are fully utilized to serve many purposes. Like other grain crops, V. unguiculata is not free from pest attacks. The cowpea bruchid, Callosobruchus maculatus (Fab.) (Coleoptera, Chrysomelidae) is one of the most notorious pests of cowpea (Deshpande et al., 2011). It is reported to be the most destructive pest in storage (Deshpande et al., 2011) due to the massive destruction caused to seeds and losses in yield (NARO, 2012). Several attempts have been made to control the effects of C. maculatus, most of which have been with the use of synthetic pesticides (Agaba et al., 2015). The ineffectiveness and the several negative effects of the chemical control measures employed thus far has prompted the search for a sustainable method of controlling these pests and a divergence into the virtually surrealistic world of botanicals and their uses in pest control (Mishra, 2013;Onunkun, 2013) since it leaves no harmful trace in the environment. Botanicals involve the utilization of plant extracts such as, essential oils or powders in insect pest control. Although this appears to be a better prospect in pest control, the efficacy of its use only has little empirical evidence (Udebuani et al., 2015;Uyi and Igbinoba, 2016), even though there exists studies on the insecticidal properties of plants (Uyi and Obi, 2017;Opuba et al., 2018;Uyi and Samugana, 2018).
The giant sensitive plant, Mimosa diplotricha C. Wright ex Sauvalle (Mimosaceae) and Aspilia Africana (Pers.) C. D. Adams (Astaraceae) were both used for this study. Mimosa diplotricha is a leguminous shrub native to the Neotropics (Holm et UYI, OO;UDEOGWU CC;ROTIMI J al., 1977;Waterhouse and Norris, 1987) and invasive to Africa. Its presence in Nigeria dates back to the early or late 1980s (Uyi, 2020). It is an annual and fast growing scrambling but erect shrub that can form dense and problematic thickets within a short period of time (Uyi, 2020) while seriously competing with indigenous vegetation (Lockett and Albin, 1990). It has the potential of acclimatizing quickly and adapting to any weather condition as well as pH range (Ekhator et al., 2013;Uyi, 2020). However, A. africana is indigenous to Africa and grows extensively across the tropics (Dalziel, 1973). It is used extensively in traditional medicine to treat a wide variety of ailments and health related issues due to its high medicinal value (Elufioye and Agbedahunsi, 2004;Moronkola et al., 2007). In Nigeria, the leaves of A. africana has been used in treating malaria , Diabetes mellitus, sore throat, liver and menstrual pains (Elufioye and Agbedahunsi, 2004;Moronkola et al., 2007), tuberculosis, corneal opacities, stomach disorders, haemostatic and skin rashes (Olowokudejo et al., 2008) and cleaning wounds (Okoli et al., 2007).
Although studies have shown that certain plants such as Chromolaena odorata (L.) King and Robinson (Asteraceae) and Jatropha curcas (L.) (Euphorbiaceae) possess repellent and insecticidal properties (Uyi and Igbinoba, 2016;Uyi and Obi, 2017;Opuba et al., 2018), however, recent studies (Uyi and Samugana, 2018;Uyi et al., 2018) have reported the repellent and insecticidal properties of M. diplotricha extracts and powders but studies on the insecticidal activities of A. africana are scarce or nonexistent. This study, therefore, investigated the phytochemical constituents and insecticidal activities of the root and leaf powders of M. diplotricha and A. africana, against C. maculatus.

MATERIALS AND METHODS
Insect culture: Infested and un-infested cowpea seeds were purchased from New Benin market, Benin City, Edo State, Nigeria. While the un-infested cowpea seeds were freeze dried, pending further use, infested seeds were transferred to a 4-litre plastic container and kept in the laboratory. From the above, a culture of C. maculatus was grown in the laboratory at an ambient temperature of 25 ± 4 ˚C and the adults were separated from the cowpea seeds, after the occurrence of mating and oviposition had been confirmed. The cowpea seeds were placed in clean transparent but aerated containers and left undisturbed in the laboratory pending the hatching of oviposited eggs and their use for the trials.
Collection and preparation of plant powders: Mimosa diplotricha and A. africana plants were collected from an overgrown farmland at Ekosodin Community, Ovia North East Local Government Area, Edo State, and from a sparse vegetation at the Faculty of Life Sciences, University of Benin, Benin City, (6˚23'N, 5˚61'E), respectively. They were identified by Taxonomists from the Department of Plant Biology and Biotechnology, University of Benin, Benin City, Nigeria. After identification, the leaves and roots were chopped off and rinsed in water before being air dried for 15 days at room temperatures ranging between 25˚C to 30˚C. The dried plant parts were blended into fine particles using an electric blender and then stored in an airtight and water-proofed container pending further use.

Mortality bioassay:
In performing the mortality bioassay, four different treatment types; roots and leaves of A. africana and roots and leaves of M. diplotricha were used and the experiment was performed at an ambient temperature of 23 ± 4˚C and a relative humidity of 80 ± 5 % at the laboratory of the Department of Animal and Environmental Biology, University of Benin, Benin City, Nigeria. Different quantities of these plants powders (0.5 g, 1.0 g, 1.5 g and 2.0 g) were weighed using a AWS SC-2KGA sensitive balance and added to 20 g of un-infested cowpea seeds placed in properly labeled 100 ml plastic containers (commonly known as disposable cups) and then sealed up with aluminum foil held tightly in place with rubber band. This was thoroughly shaken to ensure that the seeds mix up with the treatment. No treatment was administered to containers meant for control. Using a blade, a small rectangular incision was made at the top of the aluminum foil through which, ten pairs of 1-3 days old sexed but unmated adults of C. maculatus were introduced into the containers. The openings were carefully sealed with masking tape and the treatments, including the control, were replicated five times. The containers, including controls, were arranged in a completely randomized pattern. The insect mortality was counted and noted in a properly prepared data sheet at 24, 48, 72 and 96 hours, after insects were confirmed dead when they do not respond to probing with a soft brush. After each counting, dead insects were removed from each container and discarded.
Phytochemical analysis of plant material: 5 g of plants samples were subjected through qualitative phytochemical analysis using methods reported in Keay et al. (1964) and Ejikeme et al. (2014).

UYI, OO; UDEOGWU CC; ROTIMI J Test for alkaloids (dragendroff test):
To 1 ml of methanolic extract in two different test tubes, 2-3 drops of Dragendroff's and Meyer's reagents were added into the different test tubes. The presence of orange red precipitate/turbidity with dragendroff's reagent or white precipitate with Meyer's reagent inferred the presence of alkaloids.
Test for flavonoids (magnesium ribbon test): To 4 ml of the plant extract, a piece of magnesium ribbon was added followed by few drops of concentrated HCl. The presence of color ranging from crimson to magenta indicated that flavonoids are present.

Test for glycosides (Keller Killianoi test): Keller
Killianoi method was used to test for the presence of glycosides. To 2ml of plant extract, 1ml of glacial acetic acid with Iron (III) chloride and conc. H2SO4 was added. The appearance of blue color was observed indicating the presence of glycosides.
Test for saponins (frothing test): 1 ml of plant extract was measured in a test tube and 5ml of distilled water was added and vigorously shaken. A persistent froth that lasted for at least 15 minutes was observed and this formed an emulsion when few drops of olive oil was added, confirming the presence of saponins.
Test for tannins (ferric chloride test): 2 ml of the extract diluted with distilled water was measured into separate test tubes and to these was added 2-3 drops of 5% Iron (III) chloride (FeCl3) solution. A green-black or blue-black coloration indicated the presence of tannins.
Test for terpenoids: A mixture of 2ml chloroform and 3ml concentrated H2SO4 acid was added to 5ml of each extract to form a layer. The presence of a reddishbrown coloration at the interface shows positive results for the presence of terpenoids.
Test for steroids: To 2ml of the plant extract, 1ml of concentrated H2SO4 was added carefully along the sides of the test tube. A red color produced in the chloroform layer infers the presence of steroids.
Test for phenols: Phenols were determined spectrophotometrically using 2.0ml of the plant extract.
Statistical analysis: The percentage mortality effects of control treatment and four concentrations (0.5 g, 1.0 g, 1.5 g and 2.0 g) of different plant parts (leaf and stem) powders of M. diplotricha and A. africana on C. maculatus were analyzed with General Linear Model Analysis of Variance (GLM ANOVA). When the overall results were significant in the GLM analysis, the difference among the treatment means were compared using the Tukey's Honest Significant Difference (HSD) test. All data were analyzed using SPSS Statistical software, version 16.0 (SPSS, Chicago, USA).

RESULTS AND DISCUSSION
Mortality bioassay: The leaf and root powders of M. diplotricha exhibited insecticidal activities by causing varying levels of mortality to C. maculatus (Figures  1a-d). When C. maculatus was exposed to different concentrations (0.5 g, 1.0 g, 1.5 g and 2.0 g) of the leaf and root powders of M. diplotricha for 24 hours, percentage mortality differed as a function of concentration (F4,40=7.83; P=0.001) but not as a function of plant part (F4,40=1.75; P=0.193) ( Figure  1a). The 0.5 g treatment caused the least mortality (8 and 12% for the leaf and root powders, respectively) while the 1.0 g, 1.5 g and 2.0 g caused high mortality (14-24%) but the difference was not significant between the three treatments ( Figure 1a). Similarly, beetle mortality differed as a function of concentration (F4,40=8.64; P=0.001) but not as a function of plant part (F4,40=0.74; P=0.394) when the insects were exposed to different concentrations of the leaf and root powders of M. diplotricha for 48 hours (Figure 1b). Apart from the control treatment that caused the least mortality (8-10 %), the 0.5 g treatment caused low mortality (18 and 24% for leaf and root, respectively) while the 1.0 g, 1.5 g and 2.0 g caused high mortality (26-32%) (Figure 1b). When C. maculatus was exposed to different concentrations of the leaf and root powders of M. diplotricha for 72 hours, percentage mortality differed as a function of concentration (F4,40=13.65; P=0.001) but not as a function of plant part (F4, 40=0.55; P=0.463) (Figure 1c). Apart from the control treatment that caused the least mortality (12%), the 0.5 g treatment caused low mortality (24 and 28% for leaf and root, respectively) while the 1.0 g, 1.5 g and 2.0 g caused high mortality (36-46%) but the difference was not significant between the three treatments ( Figure 1c). Similarly, beetle mortality differed as a function of concentration (F4, 40=22.03; P=0.001) but did not differ as a function of plant part (F4, 40=2.5; P=0.121) when the insects were exposed to different concentrations of the leaf and root powders of M. diplotricha for 96 hours (Figure 1d). Apart from the control treatment that caused the least mortality (12%), the 0.5 g treatment caused low mortality (26 and 32% for leaf and root, respectively) while the 1.0 g, 1.5 g and 2.0 g caused high mortality (40-52%) (Figure 1d). The leaf and root powders of A. africana UYI, OO; UDEOGWU CC; ROTIMI J exhibited insecticidal activities by causing varying levels of mortality to C. maculatus (Figure 2a-d).
When C. maculatus was exposed to different concentrations of the leaf and root powders of A. africana for 24 hours, percentage mortality differed as a function of concentration (F4,40=7.71; P=0.001) but not as a function of plant part (F4,40=0.04; P=0.834) (Figure 2a).. Beetle mortality differed as a function of concentration (F4, 40=41.22; P=0.001) and as a function of plant part (F4, 40=24.00; P=0.001) when the insects were exposed to different concentrations of the leaf and root powders of A. africana for 48 hours with the root powder causing higher mortality compared to the leaf powder (Figure 2b).

UYI, OO; UDEOGWU CC; ROTIMI J
The 2.0 g treatment recorded the highest mortality (28 and 34% for leaf and root powders, respectively) while the control treatment accounted for the least mortality (4-6% mortality) (Figure 2b). On 72-hours exposure of C. maculatus to different concentrations of the leaf and root powders of A. africana, mortality differed significantly as a function of concentration (F4, 40=31.09; P=0.001) and as a function of plant part (F4, 40=10.53; P=0.002). While control treatment caused the lowest mortality (6% each of leaf and root), 0.5 g, 1.0 g, 1.5 g and 2.0 g resulted in high mortality even though the difference was not significant between the three treatments (30-50%), with the root powder causing the highest mortality ( Figure 2c).  (Fig 3).
This study was conducted to investigate the insecticidal activities of the root and leaf powders of M. diplotricha and A. africana, against C. maculatus and also to determine the phytochemical constituents of the plants. The study revealed that powders from roots and leaves of M. diplotricha and A. africana exhibited some levels of insecticidal activities, although these were a function of both the treatment concentrations and plant parts used. Earlier studies  have documented the insecticidal activities of extracts from M. diplotricha and C. odorata on the worker caste of Macrotermes species using root extracts. Also, Uyi and Samugana (2018) demonstrated the potentials of using leaf powder of M. diplotricha in controlling Sitophilus zeamais. Furthermore, the control of C. maculatus has been attempted by several researchers (Olufumilayo, 2015;Ofuya et al., 2015;Iloba et al., 2016;Uyi and Obi, 2017;Opuba et al., 2018) using different concentrations of different plant powders and extracts. For example, Uyi and Igbinoba (2016) demonstrated the potency of C. odorata in the control of the beetle. The authors reported more than 80% mortality in a 48 hour exposure trial to the root powder of C. odorata. Likewise, Opuba et al. (2018) demonstrated the toxicological effects of Jatropha curcas on C. maculatus and recorded more than 90% mortality and a significant alteration in insect ovipositional tendency. As shown in other studies (e.g. Ojo and Ogunleye, 2014;Iloba et al., 2016;Opuba et al., 2018), this current research demonstrates the relationship between percentage mortality of insect pest, concentration and exposure period to plant materials. Similarly, leaf and root powder of A. africana resulted in percentage mortality that was dependent on the plant parts and treatment concentrations used. Although the use of A. africana in insect pest control has not been documented, the plant has been employed, however, in medicinal purposes (Okoli et al., 2007;Eweka, 2008;Olowokudejo et al., 2008). For instance, Agonihotri et al. (2010) reported its use in the treatment of gonorrhea, tuberculosis, cough, rheumatic pains, stomach trouble, corneal opacity, wounds and insect bites. Also, its toxicological effect has been investigated in mice (Oko et al., 2011) where it was reported to cause nervous and respiratory disorders. However, studies relating the use of extracts and or powders from A. africana in insect pest control are non-existent therefore, this study is the first to report the insecticidal activities of A. africana.  (Denloye, 2010). Secondly, plant powders have been known to negatively affect insects by eroding their cuticle and causing dehydration (Kedia et al., 2013). Furthermore, Ofuya and Dawodu (2002) reported the impairment of physiological processes by penetration into the insect body via the respiratory or alimentary system. Thirdly, plants are usually characterized by the presence of bioactive phytochemicals which can alter the biochemistry, physiology and metabolism of insects (Udebuani et al., 2015). Consequently, the phytochemical screening of these plants reveals the presence of secondary metabolites including alkaloids, tannins, saponins, flavonoids, glycosides, steroids, and phenols. These are elemental phytochemicals that gives evidence to the toxicological tendencies of plants. Saponins possess detergent and haemolytic properties that make them highly toxic and enzymatically reactive when injected into the blood stream. Tannins are known to possess astringent and detergent properties as well as inhibitory effects on many enzymes due to protein precipitation (Trease and Evans, 2002). Flavonoids, a class of phenolic compounds, are toxic to insects, fungi, bacteria, nematodes and weeds (Carlsen and Fomsgaard, 2008) because of their antifeeding and deterrent properties.
The alkaloid content of the plants indicate their nematostatic and nematicidal effects on plant-parasitic nematodes (Thoden et al., 2009) as alkaloids are wellknown as feeding deterrent against herbivores and are toxic for a wide range of non-adapted animals (Thoden et al., 2009) while also acting as protease inhibitors (Wen et al., 2013). Despite all these plausible reasons, the presence of secondary metabolites holds a better explanation for the observed mortality. However, all of these reasons could be summed up to explain why A. africana exhibited a higher level of mortality than M. diplotricha. Meanwhile, the potency shown by the root powders of both plants could be the result of a larger concentration of secondary chemicals in them (Biller et al., 1994). This is to be expected as roots are the life wire of the plants.