Introduction
The common bean (Phaseolus vulgaris L.) is a food crop originating from Central and South America (Chacón, Pickersgill, & Debouck, 2005) . It is rich in starch, protein and plays an essential role in the human diet, especially in some tropical regions. Its high protein content makes it one of the most important food crops for people in the South (Blair, Muñoz, Garza, & Cardona, 2006; Broughton et al., 2003) . Globally, the common bean is ranked as the most essential consumed food crop, with an estimated annual production of 26,902,672 tons on an area of 33,066,183 hectares in 2019 (Anonymous, 2019).
Cameroon is the second most cultivated leguminous after groundnuts, with a national estimated production of 413,723 tons on 307,020 hectares (Anonymous, 2019). The Western highland is the central production zone with a production of 284,676 tons on an area of 183,592 hectares in 2016 (Anonymous, 2017). Beans belong to the group of crops capable of fixing and using atmospheric nitrogen thanks to the rhizobium located in the nodules (Doucet, 1992) .
However, due to lthe oss of weight and nutritional value, the control of storage pest is gaining importance (Baoua et al., 2015; Czembor, Stepien, & Waskiewicz, 2015). Insect pests are therefore the major constraint to seed storage, and among these storage pests of common beans, Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae: Bruchinae), is a common pest on legumes especially common bean (Phaseolus vulgaris L.). Several papers have been reported a important yield loss with the pest infection (Hagstrum & W, 2014; Savkovic´, Stojkovic´, & Stojković, 2019).
In order to solve these problems and ensure food security, many countries are using synthetic pesticides. Though effective and easy to use, their intensive and uncontrollable use has many drawbacks (Salim, 2011). These include; the appearance of resistant insect strains, consumer poisoning and environmental pollution (Belkebir, 2018; Guèye, Seck, Wathelet, & Lognay, 2011). Faced with these nuisances, the need for friendly alternatives to human health and the environment is necessary. Therefore, plant extracts could present a solution in regulating insect pests of grains stocks (Oliveira et al., 2020; Shah, Razaq, Ali, Han, & J, 2017; Sherin, 2018; Sujatha, Prabhudas, & P, 2012) . The antiparasitic activity of Moringa oleifera seeds has already been the subject of numerous studies, at the end of which it has been identified with fungicidal (Ayirezang, Azumah, & Achio, 2020) , bactericidal properties (Valarmathy, Gokulakrishnan, Kausar, & Kusum, 2010), insecticidal properties (Ezeaku, Ndubuaku, Ndubuaku, Ike, & Ikemefuna, 2015; Oliveira et al., 2020; Shah et al., 2017; Sujatha et al., 2012). Given the biopesticide character of this plant, this study aimed to evaluate the chemical characterization and insecticidal effect of Moringa oleifera seed extracts on the developmental stages of Acanthoscelides obtectus in stored beans at different stages of its development.
MATERIAL AND METHODS
Material
Plant material
The plant materials used were seeds of Moringa oleifera obtained in the mokolo market situated in Yaoundé. The common “MEX 142” bean variety (small white bean) was obtained from the Institute of Agricultural Research for Development (IRAD).
Methods
Preparation of organic extract
Mature Moringa oleifera seeds were previously dried at room temperature for seven days in the laboratory. These seeds were then weighed and ground to obtain the powder. 500 g of seed powder were weighed and macerated in 2 litres of organic solvent and incubated for 72 hours (Stoll, 1994). The whole solvent + solute was filtered, and the filtrate obtained was concentrated with a rotary evaporator.
Phytochemical analysis
The chemical profiles of the studied extracts was determined by a Shimadzu brand LC-MS-8040 model tandem mass spectrometer. All analytical details were given in earlier paper (Yilmaz, 2020). The detailed analytical parameters of the applied validated method were given in Appendix A (Table S1).
Insecticidal activity
Collection and rearing of insects
Mass rearing was carried out on healthy common bean seeds (large red bean with white spots, black and white). After twenty days (time required for perfect oviposition), the rearing medium was sieved to remove all live and dead insects. The rest is then kept under the same conditions to allow a new generation of adult insects.
In vivo evaluation of the insecticidal activity of the extracts
In vivo evaluation of the insecticidal effect of extracts was done at doses of 12.5, 25 and 50 µl/ml each. Extracts were obtained by diluting the volumes of crude extracts in 1 ml of ethanol. In each jar, 15 A. obtectus adults were introduced in transparent polystyrene jars containing pre-weighed 50 common bean seeds of the MEX 142 variety. To these, different doses of M. oleifera seed extracts were added. The negative control jars, prepared under the same conditions, were treated only with ethanol (1 ml). A synthetic insecticide, SINOGRAIN 2% DP (20 g pyrimiphos methyl as the active ingredient), commonly used to control insect pests of stored grains, was used as a positive control. The jars were arranged and stored randomly at a photoperiod of 12/12 h.
The mortality rate was calculated by the following formula (Singh & Jakhmola, 2011):
Mortality rate = (Number of dead insects / Total number of insects) x100
Assessment of extract activity on egg-laying and emergence
The total number of laid eggs and the percentage of hatched eggs were counted. After the death of the insects introduced in different jars, the bean seeds were observed with a magnifying glass to count the eggs laid by the insects before their death. These seeds were then reintroduced into the same jars and kept until the new generation (F1) appeared. Once they had appeared, they were also counted. This allowed us to calculate the emergence rate following the formula used bySingh et al. (2011):
ER = ((AC-AT) / AC) x 100
Where ER = Emergence rate; AC = Number of adults emerged in control jars; AT = Number of adults emerged in the treated jars
Results
Phytochemical analysis
The data collected of the three extracts' chemical composition and the concentration of specific molecules were presented in Table 1. It was noted that extraction solvent affected the chemical composition of extracts. For instance, 12 and 13 molecules were found in the methanol and ethanol extract, respectively. Remarkably, of the 53 molecules, none was found in the acetone extract. Fumaric acid and 4-OH benzoic acid levels were highest in the methanol extract (1.263 and 0.570, respectively), suggesting that methanol might be more suitable for obtaining fumaric acid, quinic acid and 4-OH benzoic acid.
Table 1
Chemical composition of the tested extracts (mg/g extract)
Chemical compounds | Methanol | Acetone | Ethanol | Chemical class | Biological activity | Reference |
Hesperidin | 0.006 | nd | 0.009 | Flavonoid | Fungicide and insecticide | (Ilboudo, Bonzi, Tapsoba, Somda, & Bonzi-Coulibaly, 2016; Kopustinskiene, Jakstas, Savickas, & Bernatoniene, 2020) |
Quinic acid | 0.933 | nd | 0.297 | Phénolic acid | Insecticide | |
Fumaric acid | 1.263 | nd | 0.153 | Organic acid | Nd | Nd |
Gallic acid | 0.014 | nd | 0.013 | Phénolic acid | Fungicide | |
Protocatechuic acid | 0.051 | nd | 0.020 | Phénolic acid | Fungicide | |
4-OH Benzoic acid | 0.570 | nd | 0.207 | Phénolic acid | Nd | Nd |
Cynaroside | nd | nd | 0.012 | Flavone | Fungicide | |
isoquercitrin | 0.037 | nd | 0.017 | Flavonol | Fungicide | |
Cosmosiin | nd | nd | 0.003 | Flavonoid | Nd | Nd |
Quercitrin | 0.089 | nd | 0.077 | Flavonol | Fungicide | |
Luteolin | 0.003 | nd | 0.002 | Flavone | Fungicide | |
Naringenin | 0.002 | nd | nd | Flavonoid | Fungicide | |
Apigenin | 0.001 | nd | 0.001 | Flavonoid | Fungicide | |
Salicylic acid | 0.030 | nd | 0.010 | Phénolic acid | Fungicide, insecticide, growth hormoneand stimulator of the natural defenses of the plant. |
Effect of M. oleifera seed extracts on the mortality rate of A. obtectus adults
The survival of A. obtectus adults on the application of M. oleifera seed extracts is presented in Table 2. In general, adults of A. obtectus can survive for at least four days. However, the mortality rate was more pronounced in batches treated with M. oleifera extracts. The analysis of the mortality rate shows significant differences (P < 0.05) between the treatments at the same concentrations. The increase in the mortality rate of treatment varies proportionally with the increase in concentration.
Methanol extract showed a higher mortality rate of 88.78%, 93.8% and 96.55% for the concentrations 12.5, 25 and 50 µl/ml respectively on day 1. Contrarily, a low mortality rate of 7.80%, 13.94% and 60.6% was recorded in control on days 1, 2 and 3, respectively. Also, treatment with chemical insecticide had a mortality rate of 100% from the first day of treatment.
Table 2
Effect of Moringa oleifera seed extracts on the mortality rate of Acanthoscelides obtectus adults
[i] The same letter markings indicate insignificantdifferences, and markings with different letters have significant differencesat the level of p ≤0.05 (Duncan test). ME: methanol extract; EE: ethanolextract; AE: acetone extract. 1: concentration 1 (12.5 µl/ml); 2: concentration2 (25 µl/ml) and 3: concentration 3 (50 µl/ml)
Effect of M. oleifera seed extracts on the egg-laying and emergence rate of A. obtectus adults
Data in Table 3 shows the number of eggs laid by Acanthoscelides obtectus adults and the emergence rate of the latter. In general, the different Moringa seed extracts significantly reduced egg-laying in A. obtectus adults and the emergence rate of A. obtectus lava (P < 0.05) compared to the negative control. However, the number of eggs laid decreased with an increased concentration of extract used. These rates vary from 236.67 % in control to 0.00 % in the acetone extract treatment at a dose of C3 (EA3). On the other hand, there was no significant difference between the results obtained with insecticidal treatment and the treatment with AE3, where no eggs were laid.
As concerns emergence rate, it decreased with an increase in the concentration of extracts used. Generally, no significant difference was observed in the control treatment (87.76 %), methanol extracts at a dose of C1 (ME1: 84.66 %) and ethanol extract at a dose of C1 (EE1: 84.39 %). Also, no significant difference was observed between treatments with insecticide and acetone at a dose of C3 (AE3), where no emerged insects were recorded
Table 3
Variation in the number of eggs laid by Acanthoscelides obtectus adults and their viability rate according to the different treatments
[i] The same letter markings indicate insignificantdifferences, and markings with different letters have significant differencesat the level of p ≤0.05 (Duncan test). ME: methanol extract; EE:ethanol extract; AE: acetone extract. 1: concentration 1 (12.5 µl/ml); 2:concentration 2 (25 µl/ml) and 3: concentration 3 (50 µl/ml)
Discussion
Three hundred gram of Moringa oleifera seeds extracted with methanol yielded 15.5 %, 19.1 % with acetone and 16.6 % with ethanol. The yield difference between the organic solvents is due to the solubility of the compounds, which depend on the properties of the solvent (Muhammad et al., 2013). Furthermore, the high polarity of organic solvents (methanol, ethanol, and acetone) would bind to many compounds in M. oleifera seeds, thereby increasing the extraction yield (Muhammad et al., 2013).
The toxicity results of M. oleifera seed extracts on Acanthoscelides obtectus show that its extracts influenced adult survival. This could be explained by the presence of secondary compounds in the extracts, such as Salicylic acid, Quinic acid, Hesperidin, Fumaric acid etc. According to (Boulogne & Sciences du Vivant [q-bio], 2011), almost 116 molecules are identified to have insecticidal activity in plant extracts and the molecules most often responsible for this are terpenoids, alkaloids and phenolic compounds. However, the insecticide activity of organic extracts of M. oleifera is due to the biological activity of the compounds present in these extracts, which have an anti-nutritional effect and cause respiratory disorders. They inhibit nutrition and cause death and malformations in future generations of phytophagous insects (Carpinella, Defago, Valladares, & Palacios, 2003). The results of this work are similar to those of (Oliveira et al., 2020). They showed that phytochemical analysis of water extract (WE) from M. oleifera seeds and water-soluble lectin (WSMoL) revealed the presence of biological active components in WE. With the application of wheat flour with WE or WSMoL, the extract exhibited toxix effect to S. zeamais. In another study conducted by (Ibrahim & Aliyu, 2014), the application with African nutmeg oil and moringa seed oil exhibited the lowest hole number in the cowpea seeds. Moreover, (Ouedraogo, Sawadogo, & &dakouo, 2016) showed that Ocimum gratissimum oil at the dose of 75 µl causes 80% mortality of Sitophilus zeamais adults within 24h compared to 99.5% when 100 µl of Cimbopogon nardus essential oil is used for 72h. After the application of 50 µl of O. gratissimum oil for 48 h on the adults of Rhyzopertha dominica, the mortality rate was 100%.
The insects were able to lay eggs during the experimental period. This was observed from the presence of eggs in control and some treated jars. However, the significant differences (P < 5%) observed between the treated and control jars showed that the extracts influenced the laying of eggs. This result can be explained because the seed extracts' toxicity at these doses would have prevented the adults from laying eggs before dying. This phenomenon is confirmed by the emergence rate where a low appearance of individuals was observed in the treated batches compared to the untreated batches. This result could be explained by the fact that the extracts caused the insects' early death, reducing egg-laying by the females or the non-viability of the eggs laid. The results are similar to those of (Sherin, 2018), who obtained 84.64% of oviposition in the control batches with little or no oviposition treated with M. oleifera, Simmondsia chinensis and Prunus dulcis oil at 25, 30 and 35% concentrations.
In the presence of Moringa extract, the emergence of F1 individuals of A. obtectus showed a decrease in the number of emerged individuals with an increase in the doses of Moringa oil applied. The maximum reduction was observed at a dose of 50μl/ml. This decrease in emerged individuals could be due to a reduction in gas exchange between the A. obtectus larva and the medium, increasing with the concentration. These results agree with those of (Wahedi, David, Edward, Mshelmbula, & Bullus, 2013) who showed that neem seed extract significantly prevented the emergence of F1 adults of C. maculatus, and no subsequent weight loss was made due to pests. Moreover, (Sherin, 2018) obtained a maximum reduction (100%) in the emergence of Callosobruchus maculatus F1 for concentrations of 25, 30 and 35% of Jojoba, Moringa, Fenugreek and sweet almond oils. (Woguem, 2017) obtained complete inhibition of F1 emergence of A. obtectus with a dose of 1.6μl/g of Mondia whitei essential oil and 0.64μl/g of Echinops giganteus essential oil.
Conclusion
This work has demonstrated the biopesticidal potential of M. oleifera extracts against Acanthocelides obtectus in stored .vulgaris. It also presented the substances present in the extracts of M. oleifera seeds that could be responsible for its biopesticidal activity and their effect on the viability and multiplication of A. obtectus in natural conditions. However, the results obtained show that the substances contained in M. oleifera seed extracts are likely to reduce the infestation of common bean seeds in storage by the pest A. obtectus. Therefore, it is clear that using M. oleifera seeds from 25 µl/ml in the control of A. obtectus is an alternative to chemical control.
Conflicts of interest
Given his role as Associate Editor, Gokhan Zengin has not been involved and has no access to information regarding the peer review of this article. Full responsibility for the editorial process for this article was delegated to Co-Editor Carlos L. Cespedes Acuña. There is no conflict of interest among the authors or any other person.
