Background

Recent studies have shown that increasing insecticide resistance, among all other things, continues to threaten the efficacy of the most commonly used insecticides employed in the management of insect pests. In most cases, the over-reliance on a single class of insecticide, to suppress insect pest populations below economic injury level, has been singled out as cause for the development of resistance in insects (Ffrench-constant, 2004). Insect response to this monotherapy has been a prelude to major infestations of stored agricultural crops such as cowpea (Boeke et al., 2004; Umeozor, 2005; Jeyansakar et al., 2016). Specifically, in the tropics, this has allowed pests such as Callosobruchus chinensis (Linnaeus), a member of the family Chrysomelidae; subfamily Bruchinae, and a worldwide storage pest of cowpea, Vigna unguiculata (L.) Walp, and other legumes, to continually thrive (Taylor, 1981; Kang et al., 2013).

The attendant proliferation of Callosobruchus species in field and in storage, where they wreak havoc on cowpea seeds (Deshpande et al., 2011; Soundararajan et al., 2012), has informed the need for control measures. In times past, synthetic insecticides have been largely used due to their ability to achieve result within a short term, however, the significant adaptive responses of these species to these chemical pressures have altered the efficacies of these insecticides (Kareiva, 1999; Despres et al., 2007). Simply put, more of the insecticides are required to achieve less success.

As alternatives, natural pesticides (which are based on plant essential oils) with little or no cost (Magaji et al., 2005; Ukeh, 2009), have achieved striking results as crop protectants (Keita et al., 2001; Isman, 2008; Parugrug and Roxas, 2008). But there has been no compelling evidence, yet, to prove that the continual usage and over-reliance, as with the case with synthetic insecticides, will not produce a similar phenomenon. Feng and Isman (1995) argued that the whole of a botanical insecticide is able to deter resistance development unlike a single isolated component of such plant material. Also, phytochemicals identified thus far are commercialized as single, concentrated compounds, despite the bulk of available evidence pointing that compound mixtures reduce pest resistance than do single compounds. Koul et al. (2008) posited that the potency of the secondary metabolites in mixture of botanicals is much higher than those in single combination. These have been informative models to investigate the diversity of plant chemicals present in a single and mixture combinations. The mix of several components in botanicals, could play a role in overcoming resistance and increase susceptibility in insects. It has been demonstrated that resistance development is rapid in a single isolated component of a plant material compared to the whole of the botanical (Feng and Isman, 1995).

In that regard, as a contribution towards suppressing the effect of insect pests of agricultural importance using biopesticides, this research work screened single, and mixtures of ethanolic extracts of Moringa oleifera and Zingiber officinale for protection of cowpea seeds. Also, attempts were made to obtain the profiles of compounds present in these extracts using Gas Chromatography coupled with Mass Spectrometry (GC-MS). Information about any previous work investigating the efficacy of single and mixture of ethanolic extract of Moringa oleifera and Zingiber officinale for protection of cowpea seeds against Callosobruchus chinensis in storage is scanty in literature.

1 Results

1.1 Comparative inhibitory potential of Z. officinale and M. oleifera oil on egg count and adult emergence of C. chinensis

Irrespective of the oil used, the number of eggs laid by C. chinensis decreased with increasing concentration of Z. officinale and M. oleifera extract used (Figure 1). Bruchids exposed to Z. officinale, however, showed to be the least affected by the toxicity of the oil at concentrations 10 µl, 20 µl and 30 µl; while those exposed to M. oleifera, at those concentrations, showed the highest inhibitory effect on egg count. On the other hand, lower number of eggs were laid by C. chinensis exposed to M. oleifera oil than Z. officinale at higher concentrations; 40 µl (40.00 to 66.33) and 50 µl (30.67 to 53.67).

Likewise, the number of adults emerged decreased with increasing concentrations of the plant oil used (Figure 2). Only at the highest concentration (50 µl) did lower number of C. chinensis adults emerged in treatment exposed to Z. officinale than M. oleifera. While greater percentage of adults initially emerged in bruchids exposed to Z. officinale, the results show that increasing the extract concentration achieve different results (Figure 3). Thus, at higher concentrations (40 µl and 50 µl), M. oleifera extract exhibited greater inhibitory effect on percentage adult emergence compared to Z. officinale (Figure 4).

1.2 Comparative toxicity of Z. officinale and M. oleifera extracts on C. chinensis

Irrespective of the plant extract used, the activity of C. chinensis, as measured by the mortality data, decreased with increasing exposure time. Also, the toxicity of the plant extract to the bruchids was observed to be concentration dependent (Figure 5). M. oleifera, however, showed the least toxicity to the bruchids over the period of exposure; while Z. officinale showed the highest toxicity effect.

Likewise, there was a significant effect of concentration (C) (p < 0.000,1) on the susceptibility of C. chinensis to the botanicals assayed. At 24 hours, there was no mortality recorded in the population of the bruchids subjected to 10 µl of both extracts. Only M. oleifera caused mortality (6.67) at 20 µl of the extract. Upon increasing the concentration of the extracts, higher mortality values, however, were observed in bruchids exposed to Z. officinale than those subjected to M. oleifera. At 48 hours, again, M. oleifera extract was observed to be more toxic to the bruchids than Z. officinale at lower concentrations (10 µl and 20 µl). However, at concentrations 40 µl and 50 µl, Z. officinale was observed to be more toxic to C. chinensis than M. oleifera. The same trend in response of the bruchids to the extracts investigated at 48 hours was observed in those exposed for 72 hours. At 96 hours, however, Z. officinale extract was seen to be more toxic to C. chinensis than M. oleifera at every level of concentration used. These responses (response of the bruchids to extracts at every period of exposure and concentration) although higher in one instance and lower in another, were however not significantly different (p > 0.05), one from another.

1.3 Relationship between concentrations and percentage mortality at different exposure periods of extracts of Z. officinale and M. oleifera

Regressing percentage mortality (Y-axis) against increasing concentration levels (X-axis), showed significant (p < 0.05) positive correlation at the different exposure periods with prediction equations shown in (Table 1). The relationship between the toxicity of the extracts on C. chinensis showed that bruchids mortality positively correlated with increasing concentration at 24 h (r = 0.920), 48 h (r = 0.931), 72 h (r = 0.994) and 96 h (r = 0.980) (Table 1).

1.4 Median lethal concentration (LC₅₀ in µl/20 g of cowpea) of extracts of Z. officinale and M. oleifera

Table 2 shows the lethal concentration (LC₅₀ in µl/20 g of cowpea) of ethanolic extracts of Z. officinale and M. oleifera and the corresponding confidence limits. The highest LC₅₀ value (38.00) was observed in bruchids exposed to M. oleifera. However, bruchids exposed to Z. officinale had the steepest slope (2.01) indicating high homogeneity of the response of the C. chinensis to Z. officinale extract.

1.5 Variable factors and comparative toxicity of extracts to C. chinensis

There was highly significant effect (P ≤ 0.05) of time (T) (F_{3, 192} = 191.35), concentration (C) (F_{5, 192} = 198.89) and extracts (E) (F_{1, 192} = 208.73) on the mortality of C. chinensis exposed to Z. officinale and M. oleifera extracts. Likewise, there were significant impact (P ≤ 0.05) of the interactions of time X concentration (T X C) (F_{15, 192} = 5.95), time X extracts (T X E) (F_{3, 192} = 7.77), concentration X extracts (C X E) (F_{5, 192} = 9.54). Also, the interactions of time X concentration X extracts (T X C X E) (F_{15, 192} = 2.13), were highly significant (P ≤ 0.05).

1.6 Synergistic toxicity of extracts of Z. officinale and M. oleifera to C. chinensis

The mixture of the extracts initiated 78.15% mortality in the bruchids at 96 h post-treatment (Table 3) compared with 75.19 and 60.74% recorded for the toxicity bioassay with extracts of Z. officinale and M. oleifera at 50 µl 96 h post-treatment respectively (Figure 5). ANOVA showed no significant difference between mortalities obtained at 48 and 72 h post-treatment (P = 0.051). The lowest mortality (16.7%), by the extract mixture, was obtained at 24 h post-treatment.

By contrast, the median lethal time (LT₅₀) required to kill 50% of C. chinenesis by extract mixture (Z. offcinale and M. oleifera) (36.36) was significantly lower (p < 0.05) than those gotten at singular exposure of Z. officinale (64.61) and M. oleifera (76.44) (Table 4). In that order, the results demonstrate that more time is required for M. oleifera extract to kill 50% of the bruchids.

1.7 Chemical components of ethanolic extracts of Z. officinale and M. oleifera

Chromatograms of ethanolic extracts of Z. officinale and M. oleifera are shown in Figure 6 and Figure 7. GC-MS revealed varied chemical components present in the extracts tested. Forty-one (41) chemical components were identified in ethanolic extract of Z. officinale rhizome (Table 5). 1-(4-Hydroxy-3-methoxyphenyl)-dec-en-3-one (12.40%) and 1-(4-Hydroxy-3-methoxyphenyl) tetradec-4-en-3-one (9.05%) were the most abundant components. Thirteen (13) chemical components were identified in ethanolic extract of M. oleifera seed (Table 6). Hexadecanoic acid, ethyl ester (22.33%) and 11-Octadecenoic acid, methyl ester (18.09%) were the most abundant components.

2 Discussion

As plant-based pesticides, the individual biopotential of ethanolic extracts of M. oleifera and Z. officinale oil, as well as their combined toxicity on some aspects of the developmental biology of C. chinensis was investigated. Also, in order to identify responsible chemical structures, the chemical composition of the oils was characterized. Both essential oils, at examined concentrations, were, to a moderate extent, effective at suppressing the number of eggs, number of adults emerged and percentage emergence of the bruchids, giving suggestive clues on their likely usage as insecticides to suppress bruchids population below economic injury level should the need arise.

The peculiarity of this study showed that each plant extract assayed exhibited considerable degree of toxicity to adult C. chinensis; however, the degree of toxicity varied. The analysis on the oviposition results, were similar to that of adult emergence. In both instances, at the highest concentration investigated, extract of Z. officinale was superior to that of M. oleifera. This was not surprising as an egg would always hatch into an adult on completion of life cycle of an insect. For the individual toxicity, as revealed by the LC₅₀ values, ethanolic extract of Z. officinale were more toxic to C. chinensis than M. oleifera extract. This could be due to a number of reasons. One of which may include the presence of higher number of phenolic components in the plant samples. Shelly and McInnis (2001) reported the presence of phenolic compounds in ginger root oil. Also, a number of phenolic compounds were identified in moringa seeds (Kundu et al., 2009). In this study, forty-one (41) components were identified in Z. offficinale extract and twenty-one (21) components were identified in M. oleifera extract as revealed by GC-MS analyses. Grzanna et al. (2005) evinced the richness in bioactive compounds found in ginger rhizome over other tested plant materials to be flaunted for reasons over their heightened superiority. Likewise, in a study conducted on green peach aphids, Feng and Isman (1995) stressed the importance of bioactive richness by demonstrating the superiority in the effectiveness of more bioactive components over lesser bioactive components. Another reason for the superior toxicity of Z. offficinale extract could be due to the experimental condition. Kim et al. (2003) investigated insecticidal bioactivity of extracts of a similar polar solvent, methanol, from 30 plants including Z. officinale and M. oleifera against C. chinensis. He reported that methanolic extract of Z. officinale showed remarkable effectiveness as regards increase in mortality and reduction in percentage adult emergence than M. oleifera in closed container than in open ones. This condition, could possibly be a reason for the difference in toxicity, especially given the similarity of the experimental conditions vis-à-vis closed container and solvent polarity.

Furthermore, the presence of endo-Borneol, curcumene (1-(1, 5-dimethyl-4-hexenyl)-4-methylbenzene) and 1-(4-Hydroxy-3-methoxyphenyl)-dec-en-3-one, a derivative of shogaol, in Z. officinale may account for the superiority in toxicity. Borneol, a bicyclic organic compound and a terpene derivative, is a natural insect repellent and chemical component of many essential oils (White et al., 2002; Wong et al., 2006). The function of curcumene as insecticide, repellent, and insect-feeding deterrent has been previously reported. Mashael et al. (2017) reported that ar-curcumene and epi-beta-bisabolol of Hedychium larsenii (Zingiberaceae) essential oil showed high toxicity (larvicidal) against early third instars of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Low doses of the compounds were effective as oviposition deterrents against the three tested mosquito species. Since only the larvae of C. chinensis fed, the significant reduction in percentage emergence of adult C. chinensis, as observed with ethanolic extract of Z. officinale could be linked to the feeding of the insects’ larvae on the pre-treated cowpea seeds containing the harmful chemical compounds, particularly endo-Borneol (Chen et al., 2005). These secondary plant metabolites may act both as insecticides and antifeedants, thus influencing insect’s locomotion, oviposition, feeding behaviour, developmental and physiological processes, as well as behavioural patterns (Govindarajan, 1982; Shelly and McInnis, 2001; Kundu et al., 2009).

In addition, GC-MS analyses revealed the presence of gingerol and 1-(4-Hydroxy-3-methoxyphenyl) tetradec-4-en-3-one (another derivative of shogaol), may have elicited a boosting synergistic toxicity effect. Previous studies have shown that this compound elicits strong bioactivity towards storage insects (Sahayaraj, 1998; Shelly and McInnis, 2001; Shelly et al., 2003). More so, Agarwal et al. (2001) reported that gingerol and dehydroshogaol exhibited maximum Insect Growth Regulator (IGR) and antifeedant activity against the insect pest, Spilosoma obliqua. Ajayi and Lale (2001) reported that pre and post-oviposition application of essential oils of Z. officinale significantly suppressed oviposition and adult emergence of adult C. maculatus. The superior toxicity elicited by ethanolic extract of Z. officinale over M. oleifera may also be attributed to the higher number of phenolic compounds found in Z. officinale.

A significant positive correlation between the adulticidal activities of Z. officinale and M. oleifera on C. chinensis populations was observed in this study. This implies that bruchids susceptible to Z. officinale might also be susceptible to M. oleifera. There is the need to increase the number of botanical types to ascertain the universality of this indication as this study is on botanicals with cocktail of (probable) synergistic chemicals. The essential oils used in this study are representative members of plant materials routinely used in West Africans for culinary, aesthetic or other purposes. This should make them more attractive especially to small-scale farmers as protectants of stored pulses against bruchid depredation (Lale, 1992). Their adoption is, however, only feasible if the cost-benefit of extracting and using essential oils for this purpose is favourable, and this aspect has not been fully evaluated.

One of the reasons while synthetic insecticide usage is desired over other insect management approaches is due to their ability to achieve quicker result in less time. The data on the median lethal time of the mixture of the extracts compared to individual extract showed similar remarkability. Also, the synergistic potential of the extracts observed in this study toxicity gives suggestive clues on the possibility of combining the extract mixtures. This could be a line of defense in conditions where resistance and or tolerance needs to be delayed/suppressed as soon as possible. The mixtures of ethanolic extracts of the plant materials are synergistic in action because it achieved a significant mortality of the tested insects. The result demonstrates that these mixtures were more toxic than would have been in individual assays. While the mechanism behind such synergisms are not clearly understood, it is assumed that it may likely involve the ability of one component of a mixture to inhibit the detoxification of others or to enhance the absorption of others from or the perfect miscibility of the chemical components present in the extracts of the plant materials (Oladipupo, 2017). Mixtures of plant components reduce the evolution of resistance to natural insecticides, compared to a single component. Feng and Isman (1995) argued that the whole of a botanical insecticide is able to deter resistance development unlike a single isolated component of such plant material.

In conclusion, we investigated the comparative individual and possible synergistic toxicity of ethanolic extracts of Z. officinale over M. oleifera. On the whole, it is apparent that both oils have the propensity to inhibit C. chinensis; giving cues on their applicability as biopesticide should the need arise. While both oils inhibited aspects of the developmental biology of C. chinensis, Z. offcinale was however more toxic to the bruchid than M. oleifera. When combined, the results confirm the synergistic potentials of the oils. Given this, additional studies are needed to determine the role of all components in the overall synergistic influence. This knowledge may facilitate the discovery of components that are essential in the design of an effective cum sustainable biopesticide with multiple mode of actions.

3 Materials and Methods

3.1 Preparation of cowpea seeds

Cowpea seeds (Ife-brown cultivar) used for this study were sourced from the Institute of Agricultural Research and Training (IAR&T), Moor Plantation, Ibadan, Oyo State, Nigeria. The cowpea seeds were sorted and disinfested in freezer at -18°C for two weeks as in Koehler (2003). The disinfested seeds were then air-dried in the laboratory to prevent mouldiness before the introduction of insects. The seeds were kept in sealed polythene bags and placed in a safe, water-free container until when required.

3.2 Insect rearing

One-to-two days old Callosobruchus chinensis adult used for culturing in this study were obtained from already infested stock in the Postgraduate Research Laboratory (PRL) of the Department of Biology. They were introduced into 200 g each of disinfested cowpea seeds in insect rearing jars. The containers were covered with perforated lids and muslin cloth to prevent the escape of insects and to allow air into the container. It was setup in the laboratory at temperature of 28 ± 2°C and 75 ± 5% Relative humidity starting from December, 2015. After 24 h, adult insects were removed from the culture.

3.3 Plant materials and extraction of essential oil

Mature seeds of M. oleifera and rhizomes of Z. officinale were sourced from a market in Akure metropolis, Nigeria. The essential oils were obtained by cold extraction with ethanol using standard methodology. 300 g of M. oleifera and Z. officinale powders were soaked separately for 72 h in round-bottomed glass jar containing ethanol. The oils obtained were filtered using muslin cloth. Substantial amount of solvents was removed using a rotary evaporator according to Udo (2011). The resulting extracts were air-dried and stored in the refrigerator till further use.

3.4 Comparative influence of plant extracts on C. chinensis activity

The oils were tested at 10, 20, 30, 40 and 50 µl v/w. One ml of each oil (M. oleifera and Z. officinale) was applied to 20 g of cowpea seeds in 250 ml round-bottomed transparent plastic containers. Uniform coating of the cowpea seeds with oils was achieved by shaking the plates containing treated seeds for 5-10 min. Five pairs of 0-24 h old C. chinensis adults were introduced into each replicate and immediately covered with transparent lid. Untreated and solvent controls were similarly set up. All experiments were replicated three times in Completely Randomized Design (CRD). Adult mortality was recorded daily for 96 h. The beetles were confirmed dead when there was no response to prodding of the abdomen with sharp pin. At the end of 96 h, all insects (dead or alive) were removed from each container and the number of eggs laid on the seeds in each container was recorded. Experimental set up was left for additional 26 days to allow for emergence of first filial (F₁) generation. Number of adults that emerged from each replicate was recorded. Percent reduction in adult emergence of F₁ progeny was calculated according to the method described by Tapondju et al. (2002) stated below:

Where C_n is the number of emerged insects in the control and T_n is the number of emerged insects in the treated containers.

3.5 Synergistic influence of plant extracts on C. chinensis activity

To investigate the possible synergistic influence of the botanicals, toxicity bioassays using mixtures of ethanolic extracts of Z. officinale and M. oleifera was conducted. Oils were combined using LC₅₀ recorded in toxicity test with individual botanical in ratio 1:1. LC₅₀ value for oil of Z. officinale and M. oleifera. Adult mortality was recorded on 24 h basis (daily) for a period of 96 h (four days).

3.6 GC-MS analyses of extracts

Gas Chromatography coupled with Mass Spectrometry (GC-MS) analysis was used to reveal profiles of components in the oils. The extracts were further purified through liquid-liquid chromatography with sodium sulphate on separating funnel packed with silica gel. Gas chromatographic analysis of 1 µl of Z. officinale and M. oleifera oils were analyzed using Agilent 7890A Gas Chromatograph (GC) system (Agilent Technologies, USA) with a Mass spectrometer (5975C VLMSD) and Injector (7683B series). The carrier gas was Helium. The capillary column used was HP-5MS and the dimensions were: 30 cm in length, 0.320 mm internal diameter, and film thickness was 0.25 µm. The GC oven temperature was set at 80°C for two min. The temperature increased steadily at 6°C per min to 240°C and was held for 6 min. The run time of each sample was 36 min. The peak of each chemical component was expressed based on its retention time and abundance. The identification of components was achieved by searching the mass spectra database and checking for direct similarities with identified components in the system (Adams, 2001).

3.7 Analysis of data

Adult mortality data was corrected with mortality obtained in control (untreated) using Abbott formula (Abbott, 1925). Data generated in count were square-root transformed while those generated in percentage were arc-sined transformed (Tukey, 1977) for normality. The transformed data were analyzed using One-way Analysis of Variance (ANOVA). Tukey’s posthoc test was used to separate the means at α = 0.05. Also, factorial analysis (Payton et al., 2006) was conducted on mortality data to compare all variable factors for possible interactions using Minitab version 17. Probit analysis (Finney, 1971) was used to determine LC₅₀ of each plant extracts. The relationship between concentrations and percentage mortalities, at different exposure periods (24-96 h post-treatment) for both oils was determined using regression analysis, by plotting percentage mortalities against increasing concentration levels. The corresponding correlation coefficients (r) for prediction equations were also established. The linear regression equation is as stated below:

Where Y is the dependent variable, a is the intercept, b is the slope, and X is the independent variable. All analysis was done in IBM SPSS software version 20 (IBM SPSS Inc., 2011).

Authors’ contributions

AOE and JOJ conceived, designed and conducted the research. AOE and OSO analyzed data. All authors drafted, revised, read and approved the final manuscript.

Acknowledgements

The authors are grateful to one anonymous professor who assisted in the analysis of the data.

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