Potential of selected plant essential oils in management of Sitophilus oryzae (L.) and Rhiyzopertha dominica (F.) on wheat grains

Two plant essential oils, basil (Ocimum basilicum L.) and petitgrain mandarin (Citrus reticulate L.), and one pyrethroid chemical insecticide, lambda-cyhalothrin, were evaluated against two of the most important stored product insect pests, Sitophilus oryzae and Rhyzopertha dominica, on wheat grains under laboratory conditions using mixing with feeding method. The effect of the tested materials was studied on F1 progeny of the tested insects. Chemical components of the oils and side effects on protein content percentage, total carbohydrates of wheat grain and germination were investigated. Results obtained showed that the two oils had a good insecticidal effect on the tested insects. Petitgrain mandarin oil was the premier. The two tested essential oils reduced the F1 progeny. In addition, the progressive of periods after storage (0–180 days) significantly reduced protein content (%) and total carbohydrates compared to control. Nearly, there were no significant differences between treatments (LC50 and LC95) of basil and (petitgrain) mandarin on the studied parameters. Germination percentages from 0 to 180 days post-storage had slight differences between control and treatments for oils. Lambda-cyhalothrin significantly reduced germination, especially with the LC95 from 60 to 180 days. Chemical analysis demonstrated that the main components of basil oil were linalool, eugenol, methyl chavicol and cani, while the main components of petitgrain mandarin oil were dimethyl panthenalate, α-terpinene, β-cinem and citral accounting for 71.1 and 95% of the two oils, respectively. The results obtained proved that the two tested plant essential oils had an obvious role in suppressing the activity of the two tested insects and can be used in an integrated pest management program. The two tested essential oils can be used as green pesticides and can be effective alternatives for chemical pesticides.

pests, and they can destroy crops and food products or contaminate them, making food unfit for human consumption. Insecticides are often non-specific and also affect non-target organisms (Spochacz et al. 2018). In this context, the use of insecticides based on botanical extracts is attracting considerable interest both among researchers and consumers. Among botanical extracts used as insecticides, essential oils (Eos) are a promising alternative because of worldwide availability and relative cost-effectiveness (Campolo et al. 2018). Essential oils are secondary metabolites synthesized by plants, and they play a very important role in plant defense (both against biotic and abiotic stresses) (Smith et al. 2006;Walters 2010;Zuzarte and Salgueiro 2015). Plant species that produce essential oils are called aromatic plants and are distributed worldwide; these plants (over 17,000 species) belong to a limited number of families: Asteraceae, Cupressaceae, Lamiaceae; Lauraceae, Rutaceae, Myrtaceae, Piperaceae and Poaceae (Svoboda and Greenway 2003;Bruneton 1999). Essential oils are mainly constituted by monoterpenes and sesquiterpenes synthesized in the cytoplasm and plastids (Zebec et al. 2016). Stored product sector seems to be a perfect candidate for the development of new essential oils-based alternative pest control strategies. Therefore, the objectives of this study aimed to evaluate the insecticidal activity of two plant oils (basil and petitgrain mandarin) and one pyrethroid insecticide, lambda-cyhalothrin, against S. oryzae and R. dominica using mixing with medium methods. In addition, determine the effect on F 1 offspring and reduction of progeny. The side effects of the tested oils and the chemical insecticide were evaluated on the protein content, total carbohydrate and germination of wheat grain.

Rice weevil Sitophilus oryzae (L.)
The adults of rice weevil, S. oryzae (L.) 1-2 weeks old, used in this study were obtained from laboratory colony, established and reared on wheat grain (Triticum aestivum) under laboratory constant conditions of 26 ± 1 °C and (65 ± 5 R.H.). New adults 2-3 weeks old were selected for toxicity evaluation test (Feng et al. 2009).

Lesser grain borer Rhyzopertha dominica (F.)
The adults of lesser grain borer, R. dominica (F.), were collected from Kafr El-Sheikh Rice and Wheat Mills Companies and reared on wheat grain (T. aestivum) under laboratory constant conditions of 26 ± 1 °C and 65 ± 5 R.H. in dark. New adults of 2-3 weeks old were used for the next experiments (Teodoro et al. 2012).

Wheat samples
Wheat grain samples (Triticum vulgare L.), Sakha 69, were taken from the untreated area, Field Crops Research Institute, Agricultural Research Center.

Plant oils
Basil oil, Ocimum basilicum L.
Petitgrain mandarin oil, Citrus reticulate L. Both oils were purchased from Hashem Brothers Company for Essential Oils and Aromatic Products (69 Abdel Moneim Riad St., Giza, Egypt).

Analysis of essential oils
Chemical analyses were performed on a gas chromatography (Hewlett Packard 5890)/mass spectrometry (Hewlett Packard 5989B) (GC-MS) apparatus. Essential oils were diluted in diethyl ether, and a sample amount of 0.5 µl was injected. The GC column was a 30 m (0.25 mm i.d., film thickness 0.25 um) HP-5MS (5% diphenyl) dimethylpolysiloxane capillary column. The GC conditions were as follows: injector temperature, 240 °C; column temperature, isothermal at 70 °C and held for 2 min, then programmed to 280 °C at 6 °C/min and held at this temperature for 2 min; ion source temperature, 200 °C; detector temperature, 300 °C. Helium was used as the carrier gas at the rate of 1 ml/min. The effluent of the GC column was introduced directly into the ion source of the MS. Spectra were obtained in the El mode with 70 eV ionization energy. The sector mass analyzer was set to scan from 40 to 400 amu for 5 s. The oil components were identified by comparison of their retention indices and mass spectra with the NIST Mass Spectral Library.

Bioassay procedures Insecticidal bioassay (mixing with feeding medium)
Plant oils and lambda-cyhalothrin insecticide Twenty grams of wheat grains was placed in a glass jar 250 ml and was replicated three times. The two plant oils (O. basilicum and C. reticulata) were diluted in acetone and added to the wheat grains at rates of 50,75,100,125,150,175,200 mg/kg for oils, and seven levels of 1, 10, 50, 100, 200, 400, 800 mg/kg of lambda-cyhalothrin were weighed and admixed with grains at required doses. Jars were mechanically shaken for adequate and fixed time (15 min.) to ensure complete mixing. The treated grains were allowed to dry at room temperature. Twenty unsexed adults of both tested insects (2-3 weeks old) for S. oryzae and R. dominica were separately transferred to each jar. The same numbers of insects were transferred to glass jar containing acetone-treated wheat grains (untreated control) (Ragaa et al. 2012). Mortality was recorded after 7, 14 and 21 days for all tested compounds. Results were recorded and corrected by Abbott's formula (Abbott 1925). LC 50 and LC 95 values were calculated by the method of Finney (Finney 1952). This method was described by Harein and Soderstorm (Harein et al. 1966) and Davis and Bry (Davis et al. 1985).

Effect on progeny
A laboratory experiment was established to evaluate the affection of the tested toxicants on the progeny of S. oryzae and R. dominica. Batches (50 g) of wheat grain were placed in small jars and treated with the considerable concentrations, for oils (1000, 2000, 4000, 6000, 8000, 10,000 and 20,000 ppm) and (1, 10, 50, 100, 200, 400 and 800 ppm), and then were infested with 20 adults for each jar. After two weeks, all insects were removed. Jars were held in an incubator at 26 + 1 °C and 65 + 5% R.H. The untreated grains were used as control. The treatments (treated and control) were repeated three times. The newly emerged adults were recorded for two weeks, and adult reduction % was calculated according to the following equation: MNEC-Mean number of those which emerged in the control.
MNET-Mean number of those which emerged in the treatment.

Determination of carbohydrates
Total carbohydrate content in wheat grains was determined. The procedure was carried out according to Smith et al. (1964). Carbohydrates were determined colorimetrically using a Hunter colorimeter (Hunter Lab.D 25L) by the phenol-sulfuric acid. 0.5 g of fine dry powder was placed in a tube, and 10 mL of 1 N from H 2 SO 4 was added. The tube was boiled and condensed in a boiling water bath for 60 min and traced of barium carbonate (0.1 g). The solution was filtered into a measuring flask (100 mL) by Whatman No. 1 filter paper. The residue was washed several times with distilled water. The total sugar was determined calorimetrically. A known volume (1 mL) of sugar solution was quantitatively transferred in a test tube and (1 mL) of 5% aqueous phenol solution was added followed by 9 mL of concentrated sulfuric acid, and the tube was then left to cool at room temperature. Measurement of the intensity of the yellow-orange color was carried out by spectrophotometer (Shimadzu UV-Vis spectrophotometer Model UV-240) at 490 nm. A standard curve was prepared by the known concentration of glucose. The blank was distilled water (1 mL) instead of the sugar solution. Total carbohydrates were expressed as g/100 g dry weight.

Determination of nitrogen and crude protein
Kjeldahl method as described by A.O.A.C. (1980) was used. Briefly, 0.5 gm of sample was weighed and placed on free filter paper, then folded and dropped into a Kjeldahl digestion tube, and 3.0 gm of digesting mixed catalyst (CuSO 4 ± Na 2 SO 4 ) and 25 mL of conc. Na 2 SO 4 were added to sample in the digestion tube. The mixture was transferred to the Kjeldahl digestion apparatus. The heater was regulated at a temperature below the boiling point of the acid. The mixture was boiled vigorously as the temperature was increased, until the green color was obtained. The digests were allowed to be cooled and then 10 cm 3 transferred into volumetric flasks and diluted with distilled water to make up 100 cm 3 , and 10 ml of digests was introduced into the distillation jacket of the microsteam distillation apparatus. 20 mL of 40% NaOH was added to each digest in the distillation jacket, 50 mL of 40% boric acid was measured into two 250 mL, and four drops of methyl red indicator was added. The conical flasks containing the mixture were placed into the distillation apparatus, and NH 3 was collected through the condenser. The distillation continued till 25 mL of the distillate was trapped into the boric acid solution, and the color changes from red to yellow. The distillates were then titrated with 0.02 m HCl, and the titer value was recorded. The percentage of nitrogen and crude protein was calculated by multiplying nitrogen percent by a factor 6.25.

Seed germination
Untreated sterilized wheat grains were mixed with the tested, plant oils, basil oil and petitgrain mandarin oil and pesticide, lambda. All tested materials were at concentration of LC 50 and LC 95 . After ensuring mixing 20 seeds from each treatment was allowed to germinate on medical cotton on Petri dishes (9 cm diameter), according to the technique stated by International Seed Testing Association (1976). All treatments are stored at 20 °C. Germination was made after treatment at zero time up to 180 days. Samples were taken after 0, 15, 30, 60, 90, 120, 150 and 180 days. Successful germination was noticed after the appearance of more than 3 mm of the radical.

Statistical analysis
All data were analyzed using the procedure (SAS, 2008) program of the version 9.2 to determine single or interaction effects of factors, according to Duncan's multiple range test (Duncan 1955).

Results
Laboratory experiments were conducted to evaluate the tested plant's oils (basil oil and petitgrain mandarin oil) against S. oryzae and R. dominica.

Toxicity of the tested plant oils
LC 50 data obtained in Table 1 showed that petitgrain mandarin was stronger than basil oil with the two tested insects at all times post-treatment except that of 7 days for S. oryzae. In addition, R. dominica was more susceptible than S. oryzae with the two tested oils at all exposure times from 7 to 21 days. Results represented that the toxicity increased with the increase of exposure time, and LC 50 reduced from 132.75 to 108.08 and from 135.30 to 104.45 with basil oil and petitgrain mandarin, respectively, for S. oryzae. For R. dominica, LC 50 decreased from 129.10 to 91.44 and from 125.17 to 89.36 mg/kg from 7 to 21 days post-treatment for both the two tested insects.

Toxicity of the tested insecticides
Unlike the results presented in Table 1, data in Table 2 cleared that S. oryzae adults are more susceptible than R. dominica in response to lambda-cyhalothrin. The toxicity in Table 2 had the same trend as that in Table 1, where the effectuation increased with the increase of exposure period. The different impacts between oils and chemical insecticides may be due to the composition of each, the physiology and the behavior of the two tested insects. Results in Table 2 demonstrated that lambda-cyhalothrin recorded LC 50 of 48.56 mg/kg and 45.28 mg/kg after 24 and 48 h, respectively. Table 3 cleared that basil oil had a greater effect than petitgrain mandarin oil on emergence of S. oryzae where the concentration of 20,000 mg/kg reduced the emerged progeny from 144 in control to 14 and 27 adults with basil and petitgrain mandarin oil, respectively.

Data in
In addition, basil oil significantly reduced the reduction of progeny to 90.72% as compared to petitgrain mandarin oil which achieved 81.25%. In general, the two tested oils significantly reduced the number of emerged adults and increased the reduction of progeny with all of the tested concentrations of the two tested oils.

Effect of lambda insecticide
S. oryzae Data in Table 4 summarized the influence of lambda insecticide on emerged progeny which significantly inhibited the newly emerged adults of S. oryzae at all tested concentrations; for example, concentration 400 mg/kg scored 68 insects compared to control 187 by 63.3% reduction. Generally, the tested insecticide achieved a significant reduction in emerged adults number and increasing in % reduction of progeny with all tested concentrations. Data summarized in Table 4 indicated that increasing the concentration of lambda-cyhalothrin significantly decreased the number of progeny and increased the reduction percentage, where the mean number of progeny ranged from 206 to 76 with 8.03% to 66.07%, of reduction at the concentrations from 1 to 800 mg/kg as compared to control which produced 224 individual of R. dominica adults.

Effect of basil and petitgrain mandarin oils
Data in Table 5 cleared that basil oil has more influence than petitgrain mandarin oil at all concentrations on the two tested parameters.
For example, the highest concentration 200 mg/kg produced 38 and 46 adults and increased reduction of progeny to 82.15% and 78.40% with basil and petitgrain mandarin oils, respectively. All tested concentrations of both basil and petitgrain mandarin oils significantly reduced the number of progeny which ranged from 173 to 38 and from 191 to 46 as compared to control which produced 213 adults.

Side effects On protein and carbohydrates
An experiment was conducted to study the effect of the tested, basil, petitgrain mandarin oils and lambdacyhalothrin on the total crude protein, total carbohydrates and germination of wheat grain at LC 50 and LC 95 concentrations when stored for 180 days. Samples of treated wheat grain were withdrawn at different intervals (zero, 15, 30 till 180 days).

Protein content Effect of basil and petitgrain mandarin oils
Results obtained in Table 6 cleared that there were no significant differences between the treatments of LC 50 and LC 95 compared to control at all periods of storage except that of LC 95 at 150 and 180 days. Meanwhile, periods of storage had an obvious effect on the protein content percentage throughout the duration of the experiment, whether treatment or control samples. For  example, the protein content decreased from 12.33 at zero time to 11.19 and 11.18 and 11.09% at 180 days poststorage for basil oil with the control and treatments. For petitgrain mandarin, the protein content inhibited from 12.15 to 11.26, 11.23 and 11.19% at the same times and treatments.

Effect of lambda-cyhalothrin
Likewise, data obtained in Table 7 had the same trend of oils, where the period of storage dramatically affects the percent of protein content. There is no significant difference between control and LC 50 levels till 120 days, while a significant effect is shown at 120 and 150 days. For LC 95 , the protein content (%) significantly differed from 60 to 180 days.

Effect of oils and insecticides
Similarly, data obtained in Tables 8 and 9  In addition, the findings obtained clarified that no significant changes were found between control and (LC 50 and LC 95 ) of oils and insecticide concerning the effect on carbohydrates content except for LC 90 of lambda at 90-180 days.
In the same column, means followed by the same letter are not significantly different at 0.05 level according to Duncan multiple range test.

On germination percentages
A laboratory experiment was conducted to study the induction of the used materials on germination percentages of wheat grains using two levels of concentration, LC 50 and LC 95 , at intervals of zero to 180 days post-treatment.

Effect of basil and petitgrain mandarin oils
Results given in Table 10 illustrated fluctuation in the germination percentage with the control and plants oils at LC 50 and LC 95 through the intervals of the experiment from 0 to 180 days. There were slight significant differences like LC 50 and LC 95 of basil at 60 and 150 days, but the remainder values were nearly equal. For petitgrain mandarin, the germination percentage significantly differed from 60 to 180 days with the two levels of concentration LC 50 and LC 95 . Concerning the mode of action that may be due to monoterpenes, it is well known to have phytotoxic effects or cause their effect by inhibition of respiratory.

Effect of lambda-cyhalothrin
There were no significant differences in control from the beginning to the end of experiment (Table 11). In the same column, means followed by the same letter are not significantly different at 0.05 level according to Duncan multiple range test. Except the percent of germination of LC 50 from 0 to 30 days, there were significant differences between the effect of LC 50 and LC 95 on the germination percentage. Likewise, the control of lambda had the same trend of plant oils where the time did not affect the percent of germination (0-180 days). This decrement of germination percentages may be attributed to the effect of the tested pesticide metabolites which penetrated inside wheat grains.

Discussion
In agreement with the current study, basil oil showed insect repelling for insects (Dube et al. 1989). Cosimi et al. 2009 reported that extracts from tested oil are very toxic to mosquitoes; also, it was repellent to adults of S. zeamais. Repellency was dependent on insect species and oil origin. Petitgrain mandarin oil has insecticidal activity against insect pests, where C. reticulate showed more toxicity at 48 h exposure against adults of S. oryzae (Bhuwan and Tripathi 2011;Stefanazzi et al. 2011;Mumtaz et al. 2013). In the light of previous studies, plant materials extracts, oil and powders have deterrent effects against stored product pests. Kampouzia et al. 2009 reported that toxicity of 42 essential oil extracts from Moraceae was tested against S. oryzae. Also, essential oils toxicities are used as fumigants from Iranian Moraceae species (Lee et al. 2004). Worthily, the essential oil of plants is less harmful to human and environment (Yegen et al. 1998). Many essential oils and their constituents may have potential as alternative compounds to currently used compounds as fumigants (Huang et al. 2000;Tunc et al. 2000;Lee et al. 2001;Chayengia et al. 2010). All of those authors reported the toxicity effect of some plant oils. Also, Rutaceae family has important fruits and many essential oils products, and it has been widely used in many fields such as flavoring, perfume and pharmaceutical applications.
In the current study, the results cleared that petitgrain mandarin oil and basil oil had good toxicity against the tested stored grain insect pests by contact toxicity. Essential oils from C. reticulate may be recommended as easily available for former level, low mammalian toxicity and alternative to synthetic insecticides. It could reduce the application of insecticides (Bhuwan and Tripathi 2011). Dhram et al. 1998;Golob and Hanks 1990 reported that organophosphorus compounds are insufficiently effective against S. oryzae, whereas pyrethroids are very good efficacy. So, pyrethroids compounds are recommended to S. oryzae and other pests such as lambda-cyhalothrin. Wohlgemuth et al. 1993;Samson et al. 1989;El-Lakwah et al. 2000 observed the efficacy of lambda against S. oryzae and R. dominica.
Our results are agreed with Umoetok and Gerard 2003;Khalequzzaman and Nazeenm 2010. In this investigation, all treatments with insecticide gave good protection for stored grain under testing. Lambda-cyhalothrin was effective against S. oryzae and R. dominica. Current results agreed with Dhram et al. 1998). Golob and Hanks 1990 showed that lambda-cyhalothrin applied at 7 ppm was completely successful against S. oryzae for 3 months. After this time, a rapid decrease in efficiency occurred. Many plant volatile essential oils and their constituents have been studied to possess potential as alternative compounds and gaining tremendous importance for the management of stored products, and these are ecologically safe and biodegradable (Cosimi et al. 2009;Rajendran and Sriranjini 2008;Bareth 1989    Citrus reticulate (Peel) extract had significant insecticidal activity against T. castaneum. Also, they claimed that due to cheap and easy availability of this plant, its extract or powder can be the promising substitutes of chemical insect ides in pest management programs. Abouelatta et al. 2020 studied the chemical composition of geranium essential oils and studied the fumigant and repellent and contact toxicity of geranium essential oils against R. dominica and found that all tested essential oils had fumigant and repellent effect against R. dominica. Mode of action for the essential oils against insects may be due to inhibition of acetyl-cholinesterase (AChE) (Ryan and Byrene 1988) by determined five monoterpenes which inhibited AChE activity. The obtained results are in agreement with Bhuwan and Tripathi 2011and Ragaa et al. 2012. Ahmad et al. 2006Lopez et al. 2008 studied the effect of basil oil and other oils on emerged and reduction percentages of adults of S. oryzae and R. dominica and stated high effects of basil on reduction percentage for R. dominica. The two commercialized basil and orange oils showed stored fumigant and contact activities against S. zeamais and T. castaneum (Kim and Lee 2013). These results demonstrated that lambda-cyhalothrin had strong effect on the tested insects (Abo-Arab et al. 2014;Salem et al. 2015). These results of lambda are in agreement with (Afridi et al. 2009;Sayed and Shaker 2010). These results are in agreement with Nagi 1979 who found that crude protein content in wheat grains was gradually decreased when pirimiphos methyl was used as protectant. Furthermore, Magdoline 1985 observed that insecticide concentration as well as storage period significantly affected the total nitrogen in wheat grains. The major components of O. basilicum oil were reported by Lopez et al. 2008;Ogendo et al. 2008. Similar to those previously described by Ahmad et al. 2006;Tandorost and Karimpour 2012. The constituents of the basil oil were linalool (21.83%), estragole (74.29%) and α-humulene (2.17%) (Kim and Lee 2013). These results are in disagreement with our study. These differences may be due to the date of harvest, analysis method and location of collected plant.

Conclusions
The results obtained proved that the two tested plant essential oils had an obvious role in suppressing the activity of the two tested insects and can be used in integrated pest management program. The two tested essential oils can be used as green pesticides and can be effective alternatives for chemical pesticides.

Abbreviations
LT 50 : Is the time that will kill 50 percent of the sample population under scrutiny,; LC 50 : Is the concentration of a chemical that will kill 50 percent of the sample population under scrutiny.; C.L.: Confidence limits; S.V.: Slope value; S.E.: Standard error; Mg: Milligram; Kg: Kilogram.