Effects of aqueous and oil leaf extracts of Pterocarpus santalinoides on the maize weevil , Sitophilus zeamais pest of stored maize grains

The effects of aqueous and oil leaf extracts of Pterocarpus santalinoides at concentrations of 5, 10, 15 and 20%v/v, each as proctectant in the control of the maize weevil, Sitophilus zeamais, were compared with a conventional insecticide, pirimiphos-methyl at a rate of 0.5 ml / 50 g maize grains of Oba Super II variety in a Completely Randomized Design with four replicates. Parameters assessed, include adult mortality, rate of oviposition and adult emergence, grain damage and weight loss, and seed viability. The data were subjected to analysis of variance and means separated by using Duncan’s Multiple Range Test. Mortality of adult S. zeamais increased with increased concentration of the extracts as well as with days of exposure. Emergence rate was significantly (p<0.05) reduced from 41.82% pt in the control values to 19.35 to 2.14% in P. santalinoides aqueous and 9.30 to 0.90% in P. santalinoides oil with pirimiphos methyl having the least number of emerged adults (0.60%). Damaged maize seeds varied from 12.32 to 1.98% in P. santalinoides aqueous extract and 4.86 to 0.61% in P. santalinoides oil extract while 0.59% was recorded in Pirimiphos methyl-treated seed grains. Grain weight loss significantly varied between treatments after two and a half months (F11, 24= 1499.2, p<0.0001). Highest seed germination percentage (78.60%) was observed in seeds treated with 5%v/v P. santalinoides aqueous extract and the control, followed by 72.40% in P. santalinoides oil concentrations at 5, 10 and 15%v/v. The findings showed that almost all treatments except un-treated control decreased (weevil reproduction) grain weight loss and grain damage (holes on grains) in maize.

Apart from food, maize is also useful as medicines and as raw materials for industries.According to Oladejo and Adetunji (2012), levulinic acid, a chemical derived from maize, is used as ingredient in antifreeze and is capable of replacing the toxic petroleum-based ingredients normally used.Plastic and fabrics are made from corn stocks, ethanol obtained from maize can be used as a biomass fuel and stigmas from female corn-flowers, known as corn silk, can be used as herbal supplements (Oladejo and Adetunji, 2012).Also, the leaves of the maize plant serve as forage for livestock.Maize is used extensively as the main source of calories in animal feeding and feed formulation, and also in making silage after fermentation of corn stocks (Oladejo and Adetunji, 2012).
Despite the vast uses of maize grains and its products, there are problems to its sustainable production.One of the most important limitations to maize production is the effect of pests (both field and storage pests).Some of them include: the bacterium Pantoea stewartii, which causes Stewart's Bacterial Wilt,; the pathogenic fungus Ustilago maydis that causes smut on maize (Hoopen and Maïga, 2012).
Nevertheless, by far the most important pest of maize is the maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae).The insect is a major pest of stored maize grains in the tropics and temperate regions of the world (Adedire, 2001;Yeshaneh, 2015).Its infestation causes severe post-harvest losses of staple food crops in Nigeria leading to major economic losses (Oni and Ileke, 2008).Various strategies aimed at checking the menace of pest infestation, especially with the maize weevil, S. zeamais in stored grains have been employed.These strategies include physical, biological and chemical control among others.
However, because of serious health and environmental concerns as well as genetic resistance by insect species, pest resurgence and residual toxicity related to the use of these chemicals, most societies have questioned their use and are in search of more favourable/acceptable pest control methods (Adedire, 2002;Adedire et al., 2011;Ekeh et al., 2013).The main advantage of botanical pesticides centers majorly on their eco-friendliness, easily bio-degradable and plant-derived natural products that are toxic to pests and they could be produced from locally available organic materials.
Currently, attention is being given to the use of edible plant materials as grain protectants (Ivbijaro and Agbaje, 1986;Adedire and Lajide, 2003;Akinkurolere et al., 2006;2009Adedire et al., 2011)).Farmers, especially the peasant ones in developing countries had over the years been using selected indigenous plants materials believed to possess insecticidal properties as crop protectants by mixing them with the stored grains (Edelduok et al., 2015).And one of the plants employed is Pterocarpus santalinoides, (French: Ouokisse; Hausa: Gyadar kurmi, Gunduru; Igbo: Nturukpa; Yoruba: Gbengbe) a tree in the family fabaceae.The tender leaves of the plant are used as vegetables in soup making while the stem bark is used in making "pepper soup" (Okwu and Ekeke, 2003).
The aqueous extract of the stem bark of P. santalinoides, has been established to have effects on Pseudomonas aeruginosa which is responsible for such infections as pneumonia, urinary tract infections (UTIs) and bacteraemia (Eze et al., 2012).According to Anowi et al. (2012b), the methanolic extract of leaves of P. santalinoides possesses analgesic activity and Okpo et al. ( 2011) reported the anti-diarrhea property of aqueous extract of the leaves of the plant while Otitoju et al. (2014b) observed that the leaves of P. santalinoides are used in preparing soups like ogbono and egwusi (melon soup).Also, Adeleke et al. (2009) reported the larvicidal properties of the seed oil of P. santalinoides.But there has been paucity of information on the effects of aqueous and oil leaf extracts of P. santalinoides on maize weevil, S. zeamais.
Hence, the need to study this plant`s oil and aqueous leaf extracts on maize weevil, S. zeamais was borne due to its ready availability and non-toxicity to humans, local farmers use of the seed extracts, leaf extracts and stembark extracts as grain protectant.However, whether or not this has been effective is subject to investigation.Therefore, the present study investigated the P. santalinoides effectiveness by using its aqueous and oil leaf extracts on the maize weevil.S. zeamais access the rate of mortality, oviposition, rate of adult emergence, grain damage, weight loss and seed viability of S. zeamais with application of varied concentrations of aqueous, and oil leaf extracts of P. santalinoides, also compare its effectiveness with that of a synthetic pesticide (Pirimiphos methyl).
Laboratory, Department of Z o o l o g y and Environmental Biology, University of Nigeria, Nsukka under ambient temperature of 27±2°C and 65±5% relative humidity.

Stock culture of weevils
Weevils were p u r c h a s e d from traders" granary stores in town (Ogige market) as initial stock.They were first mass reared in a separate container (tins, cans covered with muslin cloth at the top).500 unsexed adult S. zeamais weevils were introduced into the maize seeds and the container kept in ideal conditions for reproduction to take place.Jars were placed on a table whose stands were dipped in plastic bowls containing oil to prevent ants from contaminating the culture.Newly emerged weevils were used for the rest of the study (Ojo and Ogunleye, 2013).

Procurement of maize grains
The Oba Super II variety of maize grains was used for this research.One kilogram of the uninfested grains was bought from Ogige Market, Nsukka L.G.A Enugu state and identified at the department of Crop Science, University of Nigeria, Nsukka.The grains were still handpicked to make sure only clean unhampered grains were picked, thereafter the clean seeds were left under the sun in order to eliminate any resident insect pest.The grains were then sieved with a 2 mm-mesh size sieve to remove any dead insect and frass.The maize seeds were then packaged in an airtight container and kept in a refrigerator, pending usage.

Procurement of Pterocarpus santalinoides leaves
The plant material used were leaves of P. santalinoides which were collected from a farm in Ugboezeji Abakpa Nike, Enugu, Enugu State, Nigeria and identified to species level at the Department of Plant Science and Biotechnology.The plant materials were dried under room atmosphere for three weeks until a constant weight was maintained. 2 kg of the leaves were then ground into very fine powder using an electric blender and kept in plastic containers with tight lids and stored in a refrigerator at 4±2°C till the time for Soxhlet extraction (Mulungu et al., 2010;Ileke and Oni, 2011).P. methyl was bought from Lavans Group Limited, Enugu, Enugu State.

Extraction of the plant material
Aqueous 800 g of the ground plant material was soaked in 5500 ml of water and left for 24 h.The solution was filtered with a muslin cloth of 1.5 mm mesh size.The filtrate was then poured into an evaporation dish and dried under fan at room temperature.

Oil
The soxhlet extraction method was used for the extraction of oil from the plant material.The soxhlet extractor was set up with preweighed soxhlet flask and the extraction procedure followed.400 g of powdered plant material was put into the thimble and the material was continuously shaked for 6 h using 800 ml distilled N-hexane (40 to 60°C) and thereafter left to cool and concentrate at room temperature for 24 h using cold pressing through decanting method at 130 rpm (Ekeh et al., 2013b).At the end of the extraction, the thimble was removed and the solvent distilled off and filtered using whatman filter paper.Crude extract of P. santalinoides were later diluted with N-hexane to obtain five different concentrations of 10, 20, 30,40, and 50% v/v with a control (0 %v/v) containing only Nhexane.The oil was transferred to glass jar with cover and kept in a refrigerator until needed.

Phytochemical test of the plant material
The qualitative phytochemical composition of the leaves of P. santalinoides was studied following the method of Ndukwe and Ikpeama ( 2013) and Otitoju et al. (2014b).
Experimental design 50 g of clean maize grains contained in 200 ml plastic plate of about 0.075 m diameter were added five pairs of a day old male and female adult Sitophilus zeamais which were obtained from a stock culture.Each of the two treatments of aqueous and oil extracts of the plant material was added to the different plates (12 plates for the aqueous treatment and 12 plates for the oil treatment) at the rates of 10, 20, 30, 40 for the aqueous and oil extracts, and each concentration was replicated three times.A total of 26 plates were used.The experimental control for each of the treatments was set up with maize grains and S. zeamais but no plant or oil extract.Each of the plate was covered with muslin cloth of about 0.2mm mesh size to permit air passage and prevent escape of the insects.The set up was allowed for six weeks at temperature of about 30±3°C with daily monitoring.Dead insects in each plate were removed and counted.Oviposition of eggs in the maize grains by S. zeamais was also monitored daily and natality was recorded as adult emergence, also number of holes and seed damage was recorded.

Evaluation of extracts for contact toxicity for determination of LC50
Twelve day-old S. zeamais were placed per petri-dish and were individually picked and treated by applying 1-2µ l drops of each concentration of both extracts on their ventral sides from a micro syringe.The contents of the petri-dishes were provided with maize grains and insects were observed at 24 h.Insects that do not respond to probing with a seeker were considered dead.The concentrations were converted into logarithmic values while mortality values were converted to probits.Probit values were plotted against logarithmic values and a regression line (of best fit) was drawn.The logarithmic dose at the median point when changed to antilogarithm was taken as the effective LC 50 for each extract (Finney, 1971).

Effects of plant extracts and P. methyl on the mortality of S. zeamais
50 g of maize grains were weighed into jars.Using a microsyringe, 0.5 ml of the concentrations (10, 20, 30 and 40% v/v) of each of the extracts and P.methyl was applied to the grains and shaken to allow for coverage.Grains in the control jar were treated with ethanol only.The grains were infested with ten (1 male: 1 female) adult s. zeamais per jar and jars covered with a lid of fine mesh to allow for aeration.Mortality was recorded at 24, 48, 72 and 96 h after infestation, with insects considered dead if they did not move when probed with a camel hairbrush.Dead adults were removed at each assessment, counted and recorded.Data on percentage mortality were corrected using Abbott"s (1925)

Effects of plant extracts and P. methyl on emergence of S. zeamais, grain damage, weight loss and seed viability
After 10 days, all adult weevils were removed and the jars left undisturbed and monitored (two and half months) until the emergence of F 1 progeny.Data on F 1 adult emergence were assessed from the commencement of adult emergence with emerged adults removed, counted and recorded.The grains were later sieved to remove the dust produced from adult feeding and re-weighed by using a Mettler Weighing balance and the percentage loss in weight determined as follows: initialwt-finalwt Per (%) weight loss= × 100 final weight The number of grains perforated in each of the treated and control jars were counted and percentage seed damage was determined as: Number of perforated grains % seed damage = × 100 Total number of grains counted In order to assess the viability of seeds, germination test was conducted using twenty seeds from each jar.The seeds were placed on moist filter paper in plastic Petri dishes kept in an incubator at 25°C and the number of germinated seed was counted and recorded, and percentage seed viability was calculated as:

Statistical analysis
All percentage data were angular transformed prior to statistical analysis, in order to equalize variances.All data were analysed and significant differences were compared at 0.05 significant level using Duncan"s Multiple Range Test (DMRT) (Zar, 1984).

Contact toxicity of plant extracts on S. zeamais
Contact effect of aqueous extracts of P. santalinoides and its oil extract on S. zeamais at various concentrations showed an increase in mortality in both extracts from 39.4 to 78.4% in P. santalinoides aqueous and to 100% in P. santalinoides oil.The lethal concentration (LC 50 ) of P. santalinoides aqueous extract was 19.95% while that of P. santalinoides oil was 13.18% (Figures 1 and 2).

Effects of plant extracts and P. methyl on the mortality of S. zeamais
Mortality of adult S. zeamais exposed to different rates of extracts of P. santalinoides aqueous extract, P. santalinoides oil and the conventional insecticide (P.methyl) was compared in Table 1.Adult mortality increased with increase in concentration and with days of exposure to both extracts and in P. methyl.There was no significant difference (p < 0.05) between P. santalinoides oil at 20%v/v and P. methyl at 72 and 96 h post-treatment.P. santalinoides oil at 20%v/v also caused 100% mortality to adult S. zeamais even though the synthetic insecticide caused 100% mortality 24 h earlier.All rates of application of both extracts and P. methyl were significantly (p <0.05) different from the control in all the days of the trials (Tables 2 and 3).

Effects of plant extracts and P. methyl on emergence of S. zeamais, grain damage, weight loss and seed viability
The effect of P. methyl and different concentrations of extracts of P. santalinoides (aqueous and P. santalinoides oil) is shown in Table 4. Mean number of emerged F 1 adults decreased with increasing concentrations of both extracts.Emergence was significantly (p<0.05)reduced from 41.82 in the control values to 19.35 to 2.14 in P. santalinoides aqueous and 9.30 to 0.90 in P. santalinoides oil with P. methyl having the least number of emerged adults (0.60).However, treatment with P. methyl did not significantly (p > 0.05) reduce emergence than those with P. santalinoides oil at 15, 20 and 20%v/v at P. santalinoides aqueous extract.All treatments were significantly better (p < 0.05) than the control in reducing adult emergence (Table 4).Damaged maize seeds in treatments of various concentrations of the extracts varied from 12.32 to 1.98% in P. santalinoides aqueous and 4.86 to 0.61% in P. santalinoides oil while 0.59% was recorded in treatment with P. methyl.All treatments proved superior to control, (28.25%) with P. methyl and 20 %v/v with P. santalinoides oil outstanding (Table 5).The percentage loss in weight of grains due to damage varied among treatments from 0.04% in pirimiphos -methyl to 4.57% in the control.20 % v/v of P. santalinoides oil and pirimiphos -methyl were significantly (p<0.05)better than the other treatments in reducing weight loss (Table 3).Highest germination percentage (78.60)was observed in  seeds treated with 5%v/v P. santalinoides aqueous extract and the control, followed by 72.40% in P. santalinoides oil concentrations at 5, 10 and 15 %v/v.Other treatments gave the least germination of 70.25%.There were no significant differences (p > 0.05) in seed germination among the treatments and the control.

Number of holes
The number of holes on maize seeds was significantly different between treatment levels (F11, 24= 631.1, p<0.0001).Number of seeds with holes were significantly different between treated and untreated maize grains (p<0.0001)(Figure 3).

Percent grain weight loss
Grain weight loss significantly varied b e t w e e n treatm ent levels after two months and a half (F11, 24= 1499.2, p<0.0001).After two and half months, percent weight loss of untreated seeds was about 35%.Treated ones did not lose weight (Figure 3).

Percent germination of seeds
Similarly, rate of seed germination significantly varied between treated and untreated seeds (F11, 24= 93.53, p<0.0001).Germination rates did not vary significantly between doses of the P. santalionis oil and P. santalionis aqueous leaf extracts (Figure 4).

DISCUSSION
The results of this study justified the role of P.s antalinoides aqueous leaf extract and P. santalinoides oil extract in the storage of maize grain against degradation by storage insects.The treatments have been observed to significantly reduce the ability of maize weevils to lay eggs on the protected seeds and thus led to a reduction in the level of damage.Weevils did not reproduce on treated maize seeds because they did not find these seeds suitable for reproduction to takes place.From the result, it could be concluded that treatments controlled maize weevil reproduction almost 100% for two months and a half.The mechanism of action may be their antifeedant or repellent nature.Antifeedants, or feeding deterrents are chemicals that inhibit feeding or those that disrupt insect feeding by rendering the treated materials unattractive or unpalatable (Saxena et al., 1998).Therefore, according to the result of the analysis of variance, damage, t h a t i s , grain weight loss and number of holes of both extracts were significantly reduced or completely prevented by using these botanical products.
Oils are used in insect control because they are relatively efficacious against virtually all life stages of insects (Adedire, 2003;Rajashekar et al., 2014).Topical application caused high mortality to S. zeamais suggesting that oils have contact toxicity on the insects.P. methyl was very effective in controlling adult S. zeamais which agreed with Asawalam and Emosairue (2006) who reported 100% mortality to S. zeamais when treated with P. methyl in stored maize.
P. santalinoides aqueous and P. santalinoides oil extracts may have been potent because of the strong odours emitted thereby disrupting normal respiratory activities of the weevils; resulting in asphyxiation and subsequent death (Adedire and Ajayi, 1996).However, their effectiveness was dependent on dosages and exposure period.Highly significant difference on the emergence of adults S. zeamais reared on treated and untreated maize indicated that insecticidal materials tested had significant effects on the developmental stages which in turn affected emergence.Arannilewa et al. (2003) reported that the oil extract on application covered the outer layer (testa) of the seeds serving as food poison to the adult insects; while some of them penetrated the endosperm and germ layer thereby suppressing oviposition and larval development.Significantly lower number of emerged F 1 progeny relative to control suggests the presence of some active principles in the plants that had contact toxicity and fumigants action on the weevils (Adedire, 2003).Tahir et al. (2015) did repellency work using four indigenous plant extracts and found that M. longifolia was the most effective repellent while C. longa was least effective repellent against R. dominica.Significant difference in repellency was observed with increasing exposure time and dose rate.The repellency action is contributed to the presence of active metabolites in extract.These metabolites are composed of essential oils which are responsible for the repellent action (Gunarathna and Karunaratne, 2009;Saljoqi et al., 2006;Al-Jabr et al., 2001;Geetha and Roy, 2014) also reported the same trend.
Application of P. santalinoides aqueous and P. santalinoides oil extracts with pirimiphosmethyl to grains resulted in significant reduction of percentage weight loss.After two and half months, about 70% of untreated maize grains had holes and treated ones did not have holes.Grain weight declined with increasing number of holes and, therefore, weight loss and number of holes were directly related (Pearson"s correlation coefficient r=0.99,P<0.0001, N=36).Infested maize seeds exhibit holes through which the adults emerge (Sahaf et al., 2008).Many indigenous plants, in powder form, effectively control cowpea seed beetles (Ofuya, 2003).Similar results have been reported earlier.For example, weight loss of wheat was prevented by applying the powder of A. indica and A. boone (Ileke and Oni, 2011).When maize weevils perforate maize grains, the weight of the grains declines.
In the present experiment, the P. santalinoides aqueous leaf and P. santalinoides oil extracts have prevented the formation of holes on seeds.This result is also supported by other researches on cowpea bruchids (Swella and Mushobozv, 2007) and common beans (Busungu and Mushobozv, 1991).Malathion treated common beans did not lose weight whereas the untreated ones did.Beans which were treated by Actellic super dust (as in the present study) and coconut oil to prevent Z. subfasciatus had the lowest number of holed seeds and the highest weight of seeds as compared to the untreated ones (Busungu and Mushobozv, 1991).
Increase in percent damaged bean seeds and weight loss is because of increasing bruchid number and the degradation of oils with time (Swella and Mushobozv, 2007).Just 2% turmeric powder provided good protection to rice or wheat and reduced grain weight loss (Saxena et al., 1998).Botanical insecticides such as pyrethrum, derris, nicotine, oil of citronella, and other plant extracts have been used for centuries (Singh and Upadhyay, 1993).In the plant powder, 99.1% mortality was recorded in V. nugundo, 94% in N. speciosum, and 96% in A. officinuarium.Adult emergence was registered in A. indica and A. officinarum (both 18%) followed by G. superpa (20%).The lowest grain weight loss was reported in A. indica (18.55%) and A. officinarum (18.56%) (Akinnusi, 1986).
The result of an earlier study by Okonkwo and Okoye (1996) showed that percentage weight loss was related to the population of adult S. zeamais.Seed viability pretreated with the extracts showed that the treatments did not negatively affect seed germination.When maize weevils perforate maize grains, seed germination rate declines.Insect pests inflict their damage on stored products mainly by direct feeding (Malek and Parveen, 1989).
Some species feed on the endosperm causing loss of weight and quality, while other species feed on the germ, resulting in poor seed germination and low viability.P. santalinoides aqueous and oil extracts prevented the formation of holes on seeds due to their insecticidal properties.In this study, the germination rate of untreated maize grain by P. santalinoides aqueous leaf and oil extracts is lower than that of the treated one almost by 80%.
Presence of S. zeamais in maize grains led to a reduction in germination with increasing developmental stage of the insects, from 13% at the egg stage to 93% at the adult stage (Santos et al., 1990).This result is also supported by other researches on cowpea bruchids (Swella and Mushobozv, 2007) and on common beans (Busungu and Mushobozv, 1991).This agrees with the report of Adedire et al., (2011) which gave no significant differences in viability of seeds pre-treated with 0.5 and 2.0% of four plant extract concentrations and the control.Results obtained from this study demonstrates active potentials of this plant products as plant-derived insecticides against maize weevil and provide a scientific rationale for the use of these botanicals as alternative to synthetic insecticides in post harvest protection.
The problems posed by broad spectrum synthetic pesticides have led to the need for effective biodegradable pesticides with greater selectivity (Dayan et al., 2009).The efficacy of the products tested in the present study indicates their potential for replacing synthetic pesticides.
The two plant extracts regardless of dose prevented reproduction.That was a great leap forward.Synthetic insecticides not only do they pollute the environment but they also speed up weevil resistance to synthetic pesticides (Ileleji et al., 2007).On the other hand, pest insects have little chance of developing resistance to botanical products.
Botanical products are receiving more and more attention for pest control.What is needed is refining those using conventional scientific procedures.They have been with grain producers and traders for centuries.For example, Egyptian and Indian farmers used ash and leaves and seeds of neem for the control of stored grain pests (Varma and Dubey, 1999;Ahmed and Koppel, 1985).In eastern Africa, leaves of the wild Ekeh et al. 625 shrub O. suave and the cloves of Eugenia aromatic are traditionally used as stored grain protectants (Powell, 1989).In Rwanda, farmers store edible beans in a traditional closed structure and whole leaves of O. canum are usually added to the stored foodstuff to prevent insect damage within these structures.
Essential oil constituents such as thymol, citronellal and αterpineol are effective as feeding deterrents against tobacco cutworm, Spodoptera litura (Hummel and Isman, 2001).Synergism or additive effects of a combination of monoterpenoids from essential oils have been good against S. litura larvae.The H. spicigera essential oils showed fumigant toxicity against S. zeamais.The mortality rate of S. zeamais increased with the concentration and duration of exposure to the essential oils (Wekesa et al., 2011).

Conclusions
So, protecting our food from storage insects is a priority to ensure food security.The treatments decreased weevil reproduction; grain weight loss and grain damage (holes on grains) and increased mortality.No loss of weight and perforation of holes were observed on treated maize grains.However, untreated grains sustained huge weight loss, the greatest number of offspring and holes.As the amount of P. santalinoides oil applied increased, the rate of germination was affected unlike that of P. santalinoides aqueous extracts, which does not have a negative impact on the rate of germination of maize grains.Generally, P. santalinoides extracts treatments were found to be effective against the attack of S. zeamais.This provides good arguments for carrying out this study on natural pesticides.Thus, the tested products could serve as potential tools for the management of storage insect pests.Future efforts should focus on product optimization, packaging and marketing.

Figure 3 .
Figure 3. Number of holes and percentage weight loss.

Table 1 .
Percentage proximate composition of stored maize grain.

Table 3 .
Effects of plant extracts and P. methyl on mortality of S. zeamais.Means followed by common letters in the same column are not significantly different at 5% level.

Table 4 .
Effects of plant extracts and Pirimiphos methyl on emergence of S. zeamais and grain damage.
a Means followed by common letters in the same column are not significantly different at 5% level.

Table 5 .
Effects of plant extracts and P. methyl on percentage weight loss and germination of maize seeds.
LSD (0.05); 0.12; 0.2.Means followed by common letters in the same column are not significantly different at 5% level.