Efficacy of organic extracts of Nicotiana tabacum (Solanaceae) leaf and Jatropha curcas (Euphorbiaceae) seed against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) under laboratory conditions

The maize weevil is a pest of stored maize controlled with synthetic insecticides. The widespread use of these insecticides, however, is harmful to the environment and human health. In this study, solvent extracts (SEs) and powder treatments (PTs) of Nicotiana tabacum leaves and Jatropha curcas seeds were tested for their efficacy against Sitophilus zeamais Motschulsky. The experiment was designed in a completely randomized design with three replications. For extraction, ethyl acetate and dichloromethane were used. Extract concentrations of 25, 50, 75 and 100% were evaluated at dosages of 4, 8, and 12 ml. PT of botanicals was evaluated at dosages of 4, 8, and 12 g. Mortality was calculated at 1, 3, 5, 7, and 14 days after treatment. At the end of the experiment, the percentage of grain damage, weight loss, and germination percentage was calculated. Dichloromethane SEs of N. tabacum leaves and J. curcas seeds at 50, 75, and 100% levels of extract concentrations (LEC) in all doses caused 100% mortality. The J. curcas seed PTs at 12 g and its SEs of ethyl acetate and dichloromethane in 100% LEC at 12 ml experienced the least grain weight loss of 1.36, 0.79 and 0.64%, respectively. The results suggest that these plant-based products are very promising, generally available, cost-effective, non-toxic to non-target organisms, and simple to produce. Thus, dichloromethane SEs of N. tabacum leaves and J. curcas seeds at 50% LEC and above in all doses can be recommended for the management of S.


INTRODUCTION
Maize (Zea mays L.) is a member of the grass family (Poaceae).It is one of the world's most significant annual cereal crops, serving as staple food and a source of income for many people in developing countries (Tandzi and Mutengwa, 2019).Maize grain is damaged by insects, reducing its benefits (Hiruy and Getu, 2020).Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) is one of the most damaging insects to stored grain, particularly in tropical areas (Bhusal and Khanal, 2019).
To protect their maize from insect pests, farmers and maize traders use a variety of pest control methods.Synthetic insecticides are the most commonly used grain protection option (Njoroge et al., 2014).On the other hand, insecticide exposure has been linked to several human acute and chronic diseases (Kandel et al., 2021).Hence, it is critical to develop more effective alternatives that control pest populations while minimizing the negative impact of synthetic insecticides (Goldel et al., 2020).Plants and plant-derived products have long been used to control pests in underdeveloped countries (Grzywacz et al., 2014) because they are a more environmentally friendly and cost-effective pest control method than synthetic insecticides (George et al., 2014;Tavares et al., 2021).They are non-toxic to non-target organisms and have a specific mode of action.Furthermore, farmers can easily develop these (Iqbal et al., 2021).
Tobacco, Nicotiana tabacum (Solanaceae), is an herbaceous annual or perennial plant in the Solanaceae family (Rao et al., 2016).It has been known for a long time that tobacco leaves are used as insecticides and pesticides (Weber et al., 2019;Kanmani et al., 2021;Prommaban et al., 2022).Jatropha curcas belongs to the family Euphorbiaceae (Abdelgadir and Staden, 2013).It is rich in phytochemicals and studied for toxic effects (Oseni and Akindahunsi, 2011).Its extract has a wide range of biological effects, including molluscicidal, acaricidal, and insecticidal effects (Cordova-Albores et al., 2016).Organic extracts of J. curcas seeds are shown to be toxic to a wide range of insect orders (Lepidoptera, Coleoptera, Diptera, and Hemiptera) and can be used to protect field crops and stored grains (Temitope, 2014;Eisa et al., 2020).The current study aimed to determine the efficacy of organic extracts of N. tabacum leaves and J. curcas seeds on maize weevil (S. zeamais s) mortality, adult emergence, and grain loss.

Experimental site and laboratory condition
The study was carried out between July and November, 2022 in the crop protection laboratory of the Bako National Maize Research Centre (BNMRC).Bako is located at 9°6' North and 37°9' East at an elevation of 1650 m above sea level.The experiments were carried out in a laboratory at 25 to 28°C, 65 to 70% relative humidity, and a12:12 (light:dark) photoperiod.The artificial climate chamber controlled the indoor air temperature and relative humidity with errors of ±0.3°C and ±2% throughout the experiment.The experiment was designed in a completely randomized design (CRD) with three replications.As indicated in Table 2, there were 24 treatments (2 botanicals × 4 LECs × 3 doses) for solvent extracts (SEs) and 6 treatments (2 botanicals × 3 doses) for powder treatments (PTs), as indicated in Table 7.

Plant samples collection
Tobacco, N. tabacum leaves, and J. curcas seeds were collected from gardens near Bako town and dried in shade for 3 to 4 weeks.It was ground into a fine powder using a mortar and pestle.To prevent quality loss, the powder was sealed in polythene bags and kept in the refrigerator (Khan et al., 2014).The botanicals were chosen because they were easily accessible in the area, and their powder tests showed a promising result for storage and insect pest control (Prommaban et al., 2022).

Preparation of dry extracts
To prepare the dry extracts, the solvents ethyl acetate (ETOAc) and dichloromethane (DCM) were used to extract the powdered leaves and/or seeds.For ETOAc extraction, 500 g of powdered leaves and/or seeds were put separately in conical flasks.1000 mL of ETOAc were added to each flask and corked.The mixture was left for 12 h.After 12 h, the extract filtrates were filtered into separate, labeled bottles using a funnel and Whatman filter paper No. 1. Again 500 mL of solvent was added to each flask (decanted extract), which was then kept for 24 h.After 24 h, the filtration process was repeated.Once more, 500 mL of ETOAc was added to each decanted extract and left for 48 h; the final extract filtrates were collected.Spinning was sometimes done to ensure extraction was complete.To keep solvent from escaping, cotton wool, and aluminium foil were always used to cover the flasks.Dry extracts were created after vacuum-concentrating extract filtrates with a Heidolph rotary evaporator.Dry extracts were formed by drying the concentrates further and removing any remaining solvents (Khan et al., 2014;Gitahi et al., 2021).For DCM extractions, a similar procedure was used.Both organic extracts were then stored in bottles in a refrigerator at 4°C until use.

Preparation of different levels of extract concentrations (LECs) from dry extract
Various plant extract concentrations were prepared, mostly according to Gitahi et al. (2021).The dry extracts were diluted with ETOAc and DCM at a concentration of 1 g/ml, and this was called the "stock solution" (100% w/v concentration).The plant extract concentrations used were 25, 50, 75, and 100% (w/v), which were prepared differently.For the 25% (w/v) plant extract concentration, 1 mL of the stock solution was diluted with 3 mL of solvent to produce 4 mL; for the 50% (w/v) plant extract concentration, 2 mL of the stock solution was mixed with 2 mL of solvent to make 4 mL; and for the 75% (w/v) plant extract concentration, 1 mL of solvent was added to 3 mL of the stock solution to make 4 mL (Table 1)

Mass rearing of the test insects
The maize grain used in the experiment (BH-661 hybrid maize) was obtained from the Bako National Maize Research Centre and was cleaned and disinfested by being placed in a deep freezer for two weeks at temperatures ranging from 0 to -20°C to remove internal infestations.It was kept for two more weeks in experimental settings for acclimatization by Hiruy and Getu (2020).Initial stock (weevils) was obtained from infested maize purchased from the local market.They were cultured on clean and disinfested maize grain stored in eight 1.5-kg-capacity plastic jars covered with muslin cloth.This was to allow for aeration and prevent weevil escape.
After two weeks, all weevils, both alive and dead, were removed from each jar, and the jars were kept in the same laboratory setting.Then, after 30 days, 0 to 3-day-old S. zeamais progeny were sieved out and used for the experiment.

Toxicity bioassay of the plants' solvent extracts (SEs)
Following Danga et al. (2015) methods, 100 g of disinfected maize grains were added to 250 cm 3 capacity glass jars with brass screen lids that allow ventilation.Three different rates of each extract (4, 8, and 12 ml) were added to each jar from the four levels of extract concentrations (LECs): 25, 50, 75, and 100%.The jar contents were shaken thoroughly for 5 min to ensure uniform distribution of the solution over the grain surface.The treated grains were then left for 36 h for ETOAc and 24 h for DCM to allow the solvents to completely evaporate before the bioassay.The effect of the solvents (ETOAc and DCM) without a bioassay (the untreated control) was compared to Malathion 5% dust at a recommended rate of 0.05 g/100 g of maize grains (standard check).

Toxicity bioassay of the plants' powder treatments (PTs)
Disinfested maize grains (100 g) were added to 250 cm 3 capacity glass jars with brass screen lids that allow ventilation, and three different rates of each botanical (4, 8, and 12 g) were weighed and added to the grain in each glass jar and shaken well to ensure even distribution (Danga et al., 2015).Following that for both OEs and PTs, 25 pairs of unsexed, laboratory-reared (0-3-day-old) S. zeamais were introduced into each jar at the rate of one weevil per 2 g of maize seeds (1:2 g; 50 weevils per 100 g of maize).The jars were kept in the laboratory.When the insects did not respond to a sharp pin inserted into the abdomen, it was determined that they were dead (Ileke and Bulus, 2012).

The effect of SEs and PTs on adult mortality
The mortality data of SEs or PTs-treated jars were collected at 1, 3, 5, and 7 days after treatment application, based on Kidane (2011).Dead adults (S. zeamais) were removed and counted during each assessment from each jar.During each assessment day, alive S. zeamais returned to their respective colonies.

The effects of SEs and PTs on the emergence of S. zamias
On the 14th day of treatment, all the dead and alive S. zamias were sieved, counted, and discarded.After that, the grains were placed back in their respective jars and kept under the same experimental conditions for progenies' emergence.F 1 progenies were monitored, counted, and removed every two days for 28 days.Percentage reductions in S. zamias emergence or inhibition rate (% IR) were determined using the following formula as adopted by previous researchers (Aboalola et al., 2020).
where C n is the number of newly emerged insects in the untreated check (control) jars and T n is the number of newly emerged insects in the treatments.

The effects of SEs and PTs on grain weight loss
At the end of the experiment, after 42 days, the percentages of grain damage, weight loss, and germination percentage were calculated.To estimate the percentage of weight loss, 100 maize grains were randomly taken from each jar.Based on the count and weigh method described by Hiruy and Getu (2020), the percentage of weight loss was calculated by counting and weighing the damaged and undamaged grains.

The effects of SEs and PTs on grain damage
Counting methods were used to calculate the grain damage percentage.One hundred grains were randomly selected from each maize sample.The number of damaged grains and undamaged grains was counted for holes or insect burrows using a hand lens.The percentage of grain damage was then calculated as follows (Shiferaw, 2011): No. of damaged grains % damaged grains = × 100 Total No. of grains

The effects of PTs on grain germination
The experiment was completed with a germination test after data on many factors were collected.Twenty grains were randomly chosen from both the treatment and control groups for the germination test.These grains were then placed one by one in each sterilized Petri dish containing moistened filter paper (Whatman No. 1) and kept at room temperature around 20 to 22°C (68-72°F).Each treatment was repeated three times.Untreated seeds served as the control.After seven days, the number of seedlings that emerged from each Petri dish was counted and recorded.The percentage of germination was calculated using the formula (Talská et al., 2020).
where N G = number of grains germinated and T G = total number of grains tested in each Petri dish.

Statistical analysis
Data on percentage of adult mortality, grain weight loss, and grain damage were angularly transformed (arcsine √proportion) to reduce variance heterogeneity, but data on F 1 progeny emergence and percentage of seed germination were square root transformed.The transformed data were examined using the Statistical Package for Social Sciences (SPSS, 2016).A one-way ANOVA was used to perform inferential statistics on this data, followed by Tukey's post hoc test for separation and pairwise mean comparisons.P < 0.005 was used to indicate that there was a significant difference between the treatment groups.Back transformed data are shown in the tables

Effects of N. tabacum leaf and J. curcas seed SEs on the mortality of S. zeamais
Tables 2 and 3 show the mortality of S. zeamais caused by N. tabacum leaf and J. curcas seed organic extracts at different concentration levels (LECs) and application dosages for 14 days of exposure.The treatments used in these trials resulted in S. zeamais mortality beginning on the first day of treatment.J. curcas seed organic extracts of ethyl acetate (ETOAc) in 100% LEC at a dosage of 12 ml showed 92% mean mortality on the 14th day of exposure, while the lowest mortality of 63.3% was recorded from N. tabacum leaf extract of ETOAc in 25% LEC at a dose of 4 ml (Table 2).Moreover, all dosages of J. curcas seed and N. tabacum leaf SEs of ETOAc at different LECs applied were significantly (p<0.05)different in S. zeamais mortality from the untreated control.Only the chemical insecticide Malathion (5% dust) at a rate of 2 g caused 100% weevil mortality on the first day of treatment application (Tables 2 and 3).
N. tabacum leaf and J. curcas seed of DCM SEs in 50, 75, and 100% LECs at all dosages were the most effective, causing 100% mean mortality (Table 3).All

Temesgen and Getu 277
LECs were effective insecticides.S. zeamais mortality was closely related to each extract concentration level; increases in treatment dose and exposure time were linked to a significant increase in cumulative insect mortality.
Following the 14-day powder treatment, a different mortality percentage was recorded.The least toxic dose of N. tabacum leaf powder resulted in only 38% mortality in S. zeamais (Table 7).Comparing the toxicity of leaf/seed PTs and SEs from the same plant revealed that SEs were substantially more harmful to test insects than the former (Tables 2, 3, and 5).In the untreated control, test insects were alive for the duration of the study, with only 30% mortality (Tables 2 and 3).
Tables 4 and 5 show the mean emergence of S. zeamais after 28 days of exposure to N. tabacum leaf and J. curcas seed SEs at different LECs.There was no emergence of S. zeamais in maize grains treated with Malathion 5% dust at 2 g.N. tabacum leaf SEs of ETOAc had the highest emergence (62±0.0) of S. zeamais, in 25% LEC at a dose of 4 ml, while it had the lowest emergence (18.3±0.7) of S. zeamais in 100% LEC at a dose of 12 ml.N. tabacum leaf SEs of DCM caused the highest emergence (60.3±0.3) of S. zeamais in 25% LEC at the dose of 4 ml, but it induced the lowest emergence (16±0.0) of S. zeamais in 100% LEC at the dose of 12 ml (Table 5).The results of this study showed that N. tobacco leaf SEs of ETOAc had comparatively higher emergence of S. zeamais, since about 62% of S. zeamais emergence was recorded in 25% LEC at a dose of 4 ml after 28 days of treatment (Tables 4 and 5).
On the other hand, the number of emerging S. zeamais in untreated grains was far too high (134).J. curcas seed SEs of ETOAc in 100% LEC at a dose of 12 ml inhibited 88.56% of the emergence of S. zeamais, N. tabacum leaf SEs of DCM, and J. curcas seed SEs of ETOAc in 100% LEC at a rate of 12 ml suppressed 87.3% of the emergence of S. zeamais.Malathion 5% dust at 2 g inhibited 100% of S. zeamais adult emergence (Table 5).Table 6 shows the mean percentage of weight loss and grain damage to maize grains due to S. zeamais.This is influenced by N. tabacum leaf and J. curcas seed SEs at various LECs and application dosages at the end of the 42-day experiment.The grain weight loss treated with J. curcas seed SEs of DCM was lower than that of grains treated with N. tabacum leaf SEs of ETOAc and the untreated control.N. tabacum leaf SEs of ETOAc in 25% LEC at a dose of 4 ml caused the most grain damage, followed by J. curcas seed SEs of ETOAc in 25% LEC at a dose of 4 ml, then N. tabacum leaf SEs of ETOAc in 25% LEC at a dose of 8 ml, and N. tabacum leaf SEs of ETOAc in 25% LEC at a dose of 12 ml (30.0, 29.2, 28.4, and 26.93%, respectively) (Table 6).Similarly, J. curcas seed SEs of DCM in 100% LEC gave pronounced antifeedant effects with significantly lower grain damage of 12.4% at a dose of 4 ml, 9.73% at a dose of 8 ml, and 6.8% at a dose of 12 ml when compared with untreated   controls (66.27%).In conclusion, J. curcas seed powder at 12 g and its SEs with ETOAc and DCM in 100% LEC at a dose of 12 ml experienced the least grain weight loss (1.36, 0.79 and 0.64%, respectively) (Tables 6 and 9).Untreated controls had the greatest grain weight loss (5.88%).

The efficacy of N. tabacum leaf and J. curcas seed PTs against S. zeamais
Table 7 shows the mean percent mortality of S. zeamais on maize grains treated with N. tabacum leaf and J. curcas seed PTs.The results indicated that the PTs of    concentration increased to 12 g.Only 47.3% of the S. zeamais mortality was recorded when the insects were exposed to the J. curcas seed powder at the study's lowest concentration (4 g) perished within 14 days.The 12 g powder concentration showed a mortality of 65.3% within 14 days of insects' exposure to the powder, while the 8 g powder concentration killed 56.7% of S. zeamais within the same 14 days (Table 7).The percentage mortality recorded at 4, 8, and 12 g powder concentrations was significantly different for the   emergence.The toxicity of these plant powders increased as the dosage and duration of exposure increased.J. curcas seed PTs were more toxic than N. tabacum leaf PTs to insects (Table 7).
Table 8 shows the mean number of F 1 progeny of S.
zeamais that emerged from N. tabacum leaf and J. curcas seed PTs at different concentration doses.When maize was treated with N. tabacum leaf PTs at doses of 4 to 12 g, 106 to 67 adult S. zeamais were produced.Conversely, when maize was treated with J. curcas seed PTs, 94.7 to 57.7 adult S. zeamais were produced using N. tabacum leaf PTs (Table 8).At high doses, both N. tabacum leaf and J. curcas seed PTs significantly reduced S. zeamais emergence.
Compared to grains treated with J. curcas seed powder, grains treated with N. tabacum leaf powder had the highest number of F 1 progeny on the 14th day.At a dose of 4 to 12 g, 53.3 to 35 adults of S. zeamais were recorded.At the same dose, however, 50.7 to 30.7 adults of S. zeamais were found.For the next 28 days, the outcome followed the same pattern.The untried (control) group produced the most F 1 S. zeamais (134), followed by N. tabacum leaf powder at a dose of 4 g (106) adults (Table 8).
When the efficacy of the two plants was compared after 28 days, the maize grains treated with J. curcas seed powder produced the fewest F 1 S. zeamais.In general, N. tabacum leaf powder was less effective than J. curcas seed powder.
Table 9 shows the mean percentage of grain damage, weight loss, and germination caused by S. zeamais on maize grains treated with N. tabacum leaf and J. curcas seed powders at different concentration doses.The assessment of grain damage showed that grains treated with N. tabacum leaf powder were most damaged (14.4-42.67%),whereas the least damaged grains were those treated with J. curcas seed powder (9.6-27.47%).
After the untreated control, the percentage of seed weight loss of maize grain treated with N. tabacum leaf powder (4.35% at 4 g treatment and 1.67% at 12 g treatment) was the second highest number of weight losses.On the other hand, this result significantly differed from the grain weight loss recorded on grains treated with J. curcas seed powder (3.36% at 4 g treatment and 1.36% at 12 g treatment).Both results were statistically different from untreated seeds (the control), which recorded a weight loss of 5.88%.
The percentage germination of maize grains treated with J. curcas seed powder (72.8% at 4 g, 75.9 at 8 g, and 90.8% at 12 g) was significantly higher than seeds treated with N. tabacum leaf powder at all dose rates (57.6% at 4 g, 75.87 at 8 g, and 85.87% at 12 g).N. tabacum leaf and J. curcas seed powders had a significant effect on maize seeds germination compared with the control group (Table 9).

DISCUSSION
The current study showed that both leaf and seed solvent extracts of DCM and ETOAc had the strongest protective capacity against insects.They caused mortality and a complete reduction of S. zeamais F 1 progeny.These results agree with Kavallieratos et al. (2023), who reported that in grains treated with J. curcas L. oil (Euphorbiaceae), there was a lower emergence of S. oryzae and Ephestia küehniella Zeller (Lepidoptera: Pyralidae) compared with untreated grains.The high concentration of phenol ester in the seeds may explain the superior biocidal activity of J. curcas oil extracts (Saosoong et al., 2016).Application of the extracts directly was more efficient since extract molecules are absorbed by insects' integuments, affecting their central nervous system and leading to their death (Ubulom et al., 2021).
Nicotine quickly kills insects, causing intense tremors, convulsions, and paralysis.It competes with the major neurotransmitter, acetylcholine, by binding to acetylcholine receptors at nerve synapses and causing uncontrolled nerve firing (Kanmani et al., 2021).Nicotine, as an alkaloid, disrupts biological membranes, malfunctions internal organs and metabolism, redox imbalance, disruptions in insect development and reproduction, and inhibition of food intake (El-Wakeil, 2013;Akbar et al., 2022).
Grain weight loss is an indicator of insect pest damage during storage (Ngom et al., 2020).A small weight loss of grains was observed when maize was treated with J. curcas seed and N. tobacum leaf SEs and powder formulations.The plant materials demonstrated excellent protection against maize damage caused by S. zeamais.Temitope (2014) found seed SEs of J. curcas exhibited antifeedant and grain protectant effects, producing excellent results against S. zeamais.Likewise, Viteri-Jumbo et al. (2018) and Idoko and Ileke (2020) showed that sub-lethal doses of essential oil significantly reduced grain damage since oviposition rates were reduced.
This study revealed that the number of S. zeamais present was related to maize grain weight losses.This means that because the majority of weevils died shortly after introduction, there was less adult emergence in seeds treated with SEs and powder formulations.In addition, feeding activities were reduced, and thus fewer weight losses were recorded.The present results are in agreement with Gariba et al. (2021) findings in which less progeny and grain weight loss were recorded in maize grains treated with 0.05 and 0.1 g/mL organic extracts of Lantana camara, Hyptis suaveolens, Citrus sinensis, and Moringa oleifera.In addition to their toxicity, the botanicals' chemical constituents may have inhibited weevil feeding on the treated maize grains, thereby protecting the grains from damage (Oboho et al., 2016).
Overall, the results showed that treating maize with N. tabacum leaf and J. curcas seed powders and SEs at the majority of the investigated dosages reduced the emergence of S. zeamais, decreased weight loss, and reduced seed damage.So, because these plant resources are inexpensive, readily available, simple and safe to use, farmers should be encouraged to use them to control S. zeamais.They should also protect stored maize.

Conclusion
For a long time, plant materials, crude organic plant extracts, and essential oils have been used to protect crops from insect pests.This is due to the fact that these plant-based products are very promising, generally available, economical, and simple to produce and apply to grains.They are non-toxic to non-target organisms and have a specific mode of action.Based on the findings of the present study, it can be concluded that N. tabacum leaves and J. curcas seeds SEs of ETOAc and DCM could be used for the control of S. zeamais under farmers' storage conditions.Hence, farmers are advised to utilize native plant organic extracts to control S. zeamais and safeguard stored maize.Further studies to isolate pure compounds from N. tabacum leaf and J. curcas seed organic extracts and determine the mode of action are suggested.

Table 1 .
Preparation of solvent extracts (SEs) protocols for determination of the toxicity activities of N. tabacum leaf and J. curcas seed on S. zeamais.

Table 2 .
Adult mortality of S. zeamais on exposure to N. tabacum leaf and J. curcas seed SEs of ETOAc.

Table 3 .
Percent adult mortality of S. zeamais on exposure to N. tabacum leaf and J. curcas seed SEs of DCM.

Table 4 .
Mean number of adult S. zeamais emerging and percent protection in N. tabacum leaf and J. curcas seed SEs of ETOAc.

Table 5 .
The mean number of S. zeamais emerged and the percent protection in N. tabacum leaf and J. curcas seed SEs of DCM.

Table 6 .
The mean number of grain damage and grain weight loss caused by S. zeamais on maize grains treated with N. tabacum leaf and J. curcas seed SEs of ETOAc/DCM.

Table 7 .
The mean number of mortality of S. zeamais on maize grains treated with N. tabacum leaf and J. curcas seed PTs.

Table 8 .
The mean number of F 1 progeny of S. zeamais produced and the percent protection of N. tabacum leaf and J. curcas seed PTs of maize grains.

Table 9 .
Mean numbers of percent grain damage, weight loss, and germination caused by S. zeamais on maize grains treated with N. tabacum leaf and J. curcas seed powders.
Values followed by the same superscript within the same column are not significantly different by oneway ANOVA (P ≤ 0.05) followed by Tukey`s test.GD= grain damage, WL= weight loss, UC= Untreated Control.M5%= Malathion 5% dust.Source: Authors.