Repellency and Fumigant Toxicity of Aloe Vera, Astragalus Sarcocolla

Repellency and Fumigant Toxicity of Aloe vera, Astragalus sarcocolla , Commiphora myrrha and Ferula assa-foetida L Gum Resin Powders and Methanol Extracts against Trogoderma granarium Everts

Al-Fuhaid, Nawalabdulaziz

Department of Biology, Sattam Bin Abdul-Aziz University College of Science and Humanities, Kharj, Saudi Arabia, Phone: +966506437231 For Correspondence:[email protected]

ABSTRACT

The biological activity of Ferula assa-foetida, Aloe vera, Astragalus sarcocolla and Commiphora myrrha gum resin powders and their methanol extracts were tested on 5th, fifth instar larvae of the Trogoderma granarium beetle. This evaluation was conducted using bioassays of the repellency effect of the gum resin powders at concentrations of 0.5,0.9,2 and 4%and the fumigants toxicity of the methanol extracts at concentrations of0.2,0.4,0.6 and 0.8%at exposure periods of 24, 48 and 72 hours. The descending order of effective powders starts with the repellency effect at applied concentrations: F.assa-foetida >A.vera >A.sarcocolla >C.myrrha. The F.assa-foetida gum resin powder lead in repellency by providing 100% repellency at all concentrations after 24 hours of exposure. A.vera, A.sarcocolla and C.myrrha

(µ1/L air) of the methanol extracts had a descending rating for effectiveness: 0.99, 0.99, 0.98 and 8.33 µ1/L air for F.assa-foetida >A. vera >C. myrrha >A. sarcocolla, respectively, after 72 hours of exposure to the fumigants. This study discussed the potential biological activity of some chemical compounds that possess plant gum resin.

Keywords: monoterpenoids, neurotoxicity, polysulfides, sarcocolla, hing

INTRODUCTION products, oilseeds, dried fruits and dry animal products including milk powder and meat and fat The pests that afflict stored grains are considered an powder and dry fish. Chemical control for this pest is economic hindrance for both developed and complicated because the larvae can starve for a long developing countries. The most dangerous of these time, and they infect food for a long duration(Banks, pests is the beetle Trogoderma granarium Everts, 1977; Benz, 1987). which belongs to Coleoptera class and the Dermestidae family. The presence of this beetle has been recorded in Southern Europe, Western Africa, The use of chemical pesticides as well as fumigants Asia and South America. for the control of the discovered species of this insect are methyl bromide and phosphine (OEPP/ EPPO, Control measures for Trogoderma granarium Everts 1986), but there is a danger of direct, delayed or began in 1966 in America because the damage remaining impacts of the pesticides on humans and caused by the beetle led to an economic loss of $15 animals. Additionally, the repeated use of these m for the United States of America (Kerr, 1988). chemicals causes biological resistance in controlling insects (Champ and Dyte,1977;Lindgren,1988; There is a high probability that the larvae stage of this Rahman et al., 2009; Boyer et al.,2012). beetle is the destructive phase for this insect because of its ability to live and survive without food for up to Using the natural alternative of temperature by 23 months(Kerr, 1988), as well as its ability to feed exposing the seeds to 60 °C for 30 seconds caused a on different types of stored food, such as flour 100% mortality rate in T. granarium insects(Morner et

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al., 1987). Additionally, botanical alternatives with Preparation of methanol extract: For the crude insecticidal properties, despite their use in cooking or extraction, 100% methanol was used as a solvent, medicine, especially in agricultural or rural areas, and 500 g of each gum powder sample was soaked draws attention to the possibility of use in repelling in 750 ml of methanol and kept in the lab for 5 days some insects including arthropods or vermin due to with continuous manual stirring of the extract. The the daily observations or odors of the plants. The extract was then filtered by using a rotary evaporator. most prominent of these plants include Chrysanthemum cinerariifolium (Trevir.) Pyrethrum Preparations of solutions: According to (Asrar et (Bruce, 1940; Foil and Hogsette, 1994), Azadirachta al., 2016),solutions of crude methanol extract at 2%, indica neem(Chagas et al., 2010; Khater, 2012), 4%, 6% and 8% concentrations were prepared by Mentha piperita( Ehrh.)(Lachance and Grange, dissolving 0.2,0.4,0.6 and 0.8ml of the crude 2014),and Ociumum suaveWilld(Obeng-Ofori et al., methanol extract In 0.8, 0.6, 0.4 and 0.2 ml of 1998)because they succeed in toxicity, antifeedant methanol. and bioefficacy to many of the pests in stored grains. Repellency bioassay: The (Naworth,1973) method Therefore, the objective of this study was to evaluate was followed with some modifications in assessing the repellency effect and the toxic fumigants of Aloe the repellency effect of the gum resin plant powders vera, L. Burm.f., Astragalus sarcocolla Dymock, against the 5 th instar larvae of the studied insect. Commiphora myrrha L and Ferula assa-foetida L. We used a wide plastic dish with a diameter of 15 cm These plants have world-wide therapeutic uses in folk and a height of 2.5 cm and a small dish with a and local medicine (Eigner and Scholz, 1999; Abd El- diameter of 8.5 cm and a height of 1.3 cm. The small Razek et al., 2001; Jaradat et al., 2017),as well as in dish was placed and fixed in the middle of the large the Kingdom of Saudi Arabia(Alqasoumi, 2012; dish with adhesive material after separately placing Alshammari, 2016), against 5th, fifth instar larvae of 15 g of wheat grain in each small dish. Then, the Trogoderma granarium Everts. plant powder concentrations were mixed with a

weight / weight ratio for each dish with three MATERIALS AND METHODS replicates, and 10 fifth-age newly molted larvae were then inserted into the small dish. The nozzle of the Breeding the insect: The insects were collected large dish was covered with a muslin cloth and tied from different types of stored grains including wheat, with a rubber band. After that, the number of larvae grit, and oats and were cultured for three months in that escaped from the small dish to the large plate the incubator at a temperature of 28 ± 2 °C with a after a period of 24,48 and 72 hours was recorded. relative humidity of 70-60%.The insects were placed The percentage for repulsion (PR)was calculated per in 200 g of sterile wheat in 500-ml containers. The (Talukder and Howse, 1994)where cultures were covered with a muslin cloth, and the PR =Percent repellency cap was fixed with a rubber band. The containers NT=The number of larvae trapped in the dietary were incubated in the incubator and renewed after medium each generation. The newly molted 5 th instar larvae NE=The number of larvae escaped from the treated were used in all experiments with three replicates, dietary medium with 10 larvae in each replicate. According to Musa et PR% = NT-NE x100 al.(2009),5 th instar larvae are considered the most NT+NE gluttonous age. NT:

Preparation of plant powders: The finest gum types The repellency was assessed and classified in were purchased for the study, as shown in Table I, according to McGovern et al.(1977): from the medicinal plants markets in Alkharj city. The Class I= 0.1-20 weak repellence plants were washed and dried in the shade for one Class II= 20.1-40 repellent to some extent month, and the fresh Ferul aassa-foetida gum took Class III= 40.1 -60 medium repellent six complete months to dry. Then, the plants were Class IV = 60.1 – 80 very good repellent ground by a high-voltage electric mill to a very fine Class V= 80.1-100 high repellent powder. The resulting powder was passed through a 20 mess sieve to obtain a fine powder and was then kept refrigerated in a glass container until use.

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Table I. Names the Gums resins tested of the Repellency and Fumigant Toxicity.

Scientific name Family Plant Common Active compound References name part name Guenther 1972; Sesquiterpene Karr et al.,1990; oxygenated compounds: Nerio et al.,2010 lactones , limonene, Celestino et al 2013; Aloe vera ,L. Burm.f. Liliaceae leaves aloe gum B-caryophyllen, Khan et al.,2014; Glycosides (alion, Boudreau et al .,2006 barbalion)

Triterpene, monoterpenes Guenther 1972; Nerio et sarcocolla glycosides and al.,2010; Khan et al.,2014 Astragalus sarcocolla Leguminosa roots oxygenated compounds: Alshammari 2016; Jaradat Dymock e flavonoids et al.,2017

saponins

Monoterpenes and Guenther 1972; Karr et Commiphora myrrha L Burseraceae trunks myrrha oxygenated compounds: al.,1990; Nerio et al.,2010; Ugenol, Limonene Wisniak 2013

Guenther 1972; Monoterpenes, Shankaranarayana et al Ferula assa-foetida L Umbelliferae roots Hing sesquiterpenes 1982; Khajeh et al.,2005; and oxygenated Abd El-Razek et al.,2001; compounds: Karr et al.,1990; Nerio et phenols, polysulfides, al.,2010; Iranshahy et limonene, al.,2011; Alshammari flavonoids, limonene, 2016 polyphenolic, myrcene

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Table II. Repellent activity of A.sarcocolla A.vera, C.myrrha and F.assa-foetida gum resins powders on Larvae (5st instar) T.granarium after 72h.

Treatment Con. Repellent% Repellen F L.S.D. L.S.D.0. Slope ± Intercep 95%confidence µg/cm ± S.E cy class 0.05 01 SE t ²

0 .00±.000 Lower upper

Ferula 4 30.00±.000 ------4.590± -1. 267 assa-foetida 0.483 -1.459- -1.076- 2 30.00 ± .000 V

0.9 30.00 ± .000 0.5 30.00 ± .000

0 .00±.000 Aloe vera 4 24.67±1.667 2 24.66 0.506 ± 0.05 -0.856 18.33±4.667 IV 5.907* 14.33 -.952- -0.760- 0.9 14.33±4.667 0.5 11.00±5.000

0 .00±.000 Commiphor 4 15.00±5.000 a myrrha 1.618 15.00 15.00 0.276±0.044 -0.949 2 12.67±1.667 III -1.047- -0.852- 0.9 7.33±7.333 0.5 11.00±4.933

0 .00±.000 Astragalus 4 sarcocolla 21.67±.333 2 22.00 10.00 0.497±0.048 -1.000 22.00±.577 IV 12.89** -1.099- -0.901- 0.9 10.00±3.512 0.5 7.67±4.702 The means followed by the same letter (in the same column) are not significantly different at the 0.05 level of probability. **Differences between means are highly significant at 0.01 level of probability.

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Table III. Fumigant toxicity of A.sarcocolla ,A.vera, C.myrrha and F.assa-foetida gum resins Methanol Extracts on Larvae (5st instar) of T.granarium after 24 and 48 h.

95% Confidence Interval

Treatment Interval Mean± S.E F Lower Bound Upper Bound

Ferula assa- 24 a 9.600 ±1.794 4.528 3.889 15.311 foetida 48 7.200 a±1.131 9 3.599 10.801 24 .000a±.00 -- .000 .000 Aloe vera 48 3.400a±.392 37.696 2.154 4.646

Commiphora 24 5.600a± 0.535 50.860 3.896 7.304 myrrha 48 7.000a±0.748 24.143 4.618 9.382 Astragalus 24 2.200a± 0.216 34.714 1.513 2.887 sarcocolla 48 2.400a±1.068 7.737 -.998- 5.798 The means followed by the same letter (in the same column) are not significantly different at the 0.05 level of probability

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Table.IV. Fumigant toxicity of A.sarcocolla ,A.vera, C.myrrha and F.assa-foetida gum resins Methanol Extracts on Larvae of T.granarium after 72h.

LC50 LC95 Treatment Con.µ1/ Mortality % ± F L.S.D.0.0 (µ1/ L (µ1/ L Slope ± 95%confidence L SE 5 air) air) SE

0 .00±.000 Lower Upper

F.assa- .80 foetida 10.00±2.309 10** 10 0.997 2.497 1.097± 0.240 -1.219- -0.969- .60 10.00 ±0.577 .40 10.00 ±0.577 .20 10.00 ±2.000

A.vera 0 .00±.000

.80 10.00±7.211 0.984 2.212 1.340 ± .60 -1.454- -1.183- 9.33±6.566 0.452 - 0.253 .40 8.67±6.766 .20 7.00±6.506

C.myrrha 0 .00±.000

.80 10.00±0.577 .60 2.256 0.997 2.497 1.097± 10.00± 0.577 0.240 10 -1.219- -0.969- .40 10.00 ± 2.517 .20 10.00±6.110

A.sarcocolla 0 .00±.000

.80 8.33± 2.333 .60 22 8.333 1.843 2.102± - - 5.00± 2.000 3.762* 0.353 2.443- 2.016- .40 3.00± 2.082 .20 1.33± 0.667

The means followed by the same letter (in the same column) are not significantly different at the 0.05 level of probability. **Differences between means are highly significant at 0.01 level of probability.

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Fumigants bioassay: In accordance with (Ling et 72 hours when treated with A. sarcocolla al., 2009), with some modifications, the activity of the concentrations of 2% and 4%, respectively. fumigants for the methanol extracts was tested on 30 There is gradual increase for the activity of repellency newly molted 5 th instar larvae. The methanol for all treatments after 72 hours. One of the extracts were distributed evenly over three 500-ml behavioral changes observed on the larvae after 24 glass jars with a 20 g cotton ball with a diameter of 1 hours of treatment with all concentrations of F. assa- cm treated with 1 µof each of the concentrations of foetida and high concentrations 2 and 4% of C. the methanol extracts for all tested resins. The myrrha, A. sarcocolla and A. vera was the escape of control cotton ball was treated with methanol only. the larvae to the cloth cover with a clinging action, The oil was injected in the middle of each cotton ball and with an increased treatment time, there was a to allow volatilization to avoid direct contact with the disturbance in their motion and curling. However, the remaining residues. Before being placed inside the larvae exposed to F. assa-foetida were clinging to jars, both the oil and methanol were left to enter the the cover of the treatment dishes and were calmer., treated inserts after being left to dry for five minutes Table II depicts the rate of repellency from the 6 in the laboratory conditions in the fume hood. Food (a levels of repellency for F. assa-foetida C. myrrha, A. few wheat grains) was placed in the treatment and vera and A .sarcocolla at levels V, IV and III. All control containers, and three replicates were used treatments proved significant effects at levels of per concentration and the control in a completely probability 0.05 and 0.10 for A. vera, A. sarcocolla, randomized design. The mortality number was and. F. assa-foetida recorded after 24, 48 and 72 hours of the exposure time by average and accumulative percentages, The toxic effects of methanol extract fumigants: respectively. Tables III and IV compare the effectiveness of gum resin methanol extracts on 5 th instar T. granarium Statistical analysis: All data were statistically larvae. The LC50 (µ1/L air) of the methanol extracts analyzed using SPSS statistical analysis one way are listed in a descending rating for effectiveness: analysis of variance (ANOVA, version 11) (Bluman, 0.99, 0.99, 0.98 and 8.33 for the F. assa-foetida>A. 2007). Means were compared using the least vera>C. myrrha>A. sarcocolla after 72 hours of significant difference (L.S.D) at the 0.10 level. exposure to their respective fumigants. The treatments proved significant effects at levels of 0.05 RESULTS and 0.10 for A. sarcocolla and F. assa-foetida .

The repellency impact of the gum resin plants The F. assa-foetida methanol extract exhibited a powders on the 5 th instar larvae are displayed in high toxicity effectiveness that killed100 percent of 5 Table II ,depicting that all treatments have the mean th instar larvae after 24 hours at all concentrations of significant difference at the 0.10 level. The F. assa- F. assa-foetida , and this also occurred the highest foetida powder is superior in the repellency activity to concentration of 0.8% percent for A. vera after the the larval exposed for all exposure periods and at all same duration and at all concentrations after 72 tested concentrations, while treatment with A.vera, hours of exposure to C. myrrha. The fumigants of the A.sarcocolla and C.myrrha methanol extracts from F. assa-foetida and C. myrrha demonstrated a considerable effective repellency after 72 hours.In contrast, the highest toxicity after 24 hours of larval exposure at the tested concentration gave a 4% repellency ratio: concentration of 8%, with a mortality rate of 14% and 83.3%, 76.7%, and 73.3% when treated with A. 10% for the two plants. In contrast to the toxic effect sarcocolla, A. vera and C. myrrh after 72 hours of of the methanol extract of A. vera, which exposure to the powders of these plants. It is demonstrated a 70% mortality rate after 72 hours, the interesting to note that the direct repellent impact on methanol extract of A. sarcocolla had a lethal effect, the larvae after 24 hours of exposure to 4% gradually increasing with increased concentration concentration of F. assa-foetida, A. vera, A. and exposure time, with a mortality rate of13.3% and sarcocolla and C. myrrh had repellent ratios of 83.3% for concentrations of 2% and 8%, respectively, 100%,76.7%,70% and 36.7%, respectively. after the 72 hours of exposure. Changes in the larvae Additionally, another important observation is the low behavior during their exposure to the methanol rate of repellency from 76.7 to 73.3 and the extract fumigants for the experimental plants included corresponding stability at a ratio of 73.3 after 48 and impaired mobility and appearing semi-narcotic with

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the treatment of F. assa-foetida , with black coloring 1989). Banchio et al.(2003), Tunón et al.(2006), of the body and a movement disorder with curling Pulpati et al.(2008) and IIeke and Oni(2011) reported during the treatments with A. sarcocolla, A. vera and toxicity, growth inhibition, feeding deterrent, molting C. myrrha. disorders and repellent properties as a result of some medicinal plants because they possess a variety of DISCUSSION biological compounds; moreover, plants induce antimicrobial, antioxidant, cytotoxic, phytotoxic, The powders and extracts of plant methanol tested in insecticidal and anticancer growth properties. this study proved effective in the repulsion and Accordingly, those biological impacts could be a main induction of toxicity of vapors in the 5th instar larvae reason for the effectiveness of the tested risen plants of T. granarium. Based on previous research on the in this study. efficacy of certain plant products for the safe control of human food pests in the store and the field without Additionally, the studied plants possess the biological residues harmful to humans, animals, plants and compounds. Table I summarizes some of the environmental health, this activity can be attributed to biological compounds that have potent insecticide the biochemical content of tested plants and their effects, as demonstrated in previous studies(Shaaya toxic effects on insects(Ansari et al.,2000;Chaubey, et al., 1997; Erler, 2005; Bakkali et al., 2008; Asrar et 2007;Sultana et al.,2016; Showler, 2017). al., 2016). The monoterpenoids are the main toxic component of plant extracts used against insects The effectiveness of the repellent was evident from (Coats et al., 1991; Ahn et al., 1998; Khan et al., the beginning of the experiment, especially for the F. 2014).The monoterpenoids have a high volatility and assa-foetida, A. vera and C. myrrha powders, which are lipophilic and these characteristics could have included persistence with the progression of the effects on the physiological functions of the treated exposure time. Related to F. assa-foetida, Rafi larvae by penetrating the insect's cuticle(Richards, (2002),evaluated the high repellent and toxicity 1978; Lee et al., 2002). activity of the F. assa-foetida resin extract to Moreover, the monoterpenoids may be responsible Tribolium castaneum and Ephestia kuebneilla . for the treated larvae mortality and cause the color Additionally, Peyrovi et al.(2011), indicated that there changes in the body of the larvae prior to death via was a strong repellent and toxicity activity for the F. the methanol extracts. The methanol extract of the A. assa-foetida resin essential oil against the adult sarcocolla caused to the appearance the white spots carob moth. Bhalerao (2014) reported a toxicity with some scratches in the larval cuticle, although the efficiency of Aloe vera gum extract when mixed with larvae were not fed the food that was served in the 4 g/4 g of Tribolium castaneum food after 72 hours fumigant experiment jars. The scratches may be due of treatment. The methanol extracts have a rapid to the loss of water content from the larval bodies, efficiency during the first a hours of the exposure for which then distributed the physiological all tested plants. functions(Sousa et al., 2005; Asrar et al., 2016).All

the tested plants are rich with oxygenated According to Wei et al.(2007), some plants use their compounds (see Table I). Tunón et biochemical defenses for protection against attacking al.(2006),indicated high repellent activities of the insects, while Schardl and Chen(2010), and oxygenated compounds on the tick Ixodes ricinus. Subramaniam and Kaushik(2014),considered the biochemical defense compounds to be secondary The expected shocking effect or impairment of the metabolites since they include phenols, resins, and respiratory activity for treated larvae for the F. assa- saponins. Adeyemi and Mohammed(2014)reported foetida and A. vera treatments is likely caused by that the secondary metabolites contain various the strong odor due to a high content of as concentrations of pathogens and cellular structures. disulphide(Kapoor, 1990)and by the bitter taste of While Nawrot et al.(1986),Jacobson(1966)and glycoside alion, Celestino et al.(2013),from F. assa- Chapman(2003),indicated the mixture of secondary foetida and A. vera, which may also be responsible plant metabolites increases diversification, which for the strong odor. provides plants with an ability to attract or deter most natural enemies, including arthropods or insects, and Bisseleua et al.(2008), Kumar et al.(2008) and Muto- this treatment has led to a decrease in the Fujita et al.(2017)considered the important role of the development of resistance to insecticides (Chiu, host plant odors; the air pollution by the odors of the non-host plant impacts the food searching behavior

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of insects. This suggests that the strong odors cause benefits for generations of humanity and agricultural impairment on respiratory activity by overlapping with wealth. the air around the treated larvae, and this led to the ACKNOWLEDGMENTS compulsory closing the respiratory spiracle and thus a high mortality of larvae when exposed to methanol I would like to thank M. Al-roees, A. Al-Dosari, extracts fumigants F. assa-foetida, A. vera, and C. S.Qahtaniand, M. Al-Dosari for the technical myrrha. This conclusion agrees with an opinion from assistance from the Prince Sattam Bin Abdul-Aziz Ivbijaro(1984),that plant products that distinguish University, the College of Science and Humanities, larvicidal activity may have a chemical and physical and the Department of Biology for their invaluable toxicity. Clearly, the resin plants tested in this study help to complete this study. have a larvicidal activity. REFERENCES The continued reduction of the repellency activity of Abd El-Razek, M., Ohta, S., Ahmed, A. and Hirata, T. the A. sarcocolla powder on the larvae with time (2001).Sesquiterpene coumarins from the roots of progression in comparison with the beginning of Ferula assafoetida. Phytochemistry 58(8): 1289–1295. treatment indicates that the plant powder loses ’repellency activity or become acceptable to the Adeyemi, M.M. and Mohammed, M. (2014). Prospect of larvae since a number of larvae that previously antifeedant secondary metabolites as post harvest escaped at the beginning of the treatment returned. material. International Journal of Innovative Research This result is consistent with previous studies, which in Science, Engineering and Technology 3(1): 8701– revealed a high mortality versus a lower repellency 8708. with increased exposure times after treatment(Haq et Ahn, Y.-J., Lee, S.-B., Lee, H.-S. and Kim, G.-H. (1998). al., 2005; Nana et al., 2014; Asrar et al., 2016). Insecticidal and acaricidal activity of carvacrol and β- thujaplicine derived from Thujopsis dolabrata var. Neurotoxicity is apparent for the treated larvae with hondai sawdust. J. Chem. Ecol. 24(1): 81–90. the methanol extracts fumigants for all the tested plants. The most important and prominent evidence Alqasoumi, S. (2012). Anxiolytic effect of Ferula is the disturbance in movement, coiling and lying on assafoetida L. in rodents. J. Pharmacogn. Phytother. the back, where the larvae were less active and weak 4(6): 86–90. at the first hours of their exposure when treated with the methanol extract from F. assa-foetida. Alshammari, S. (2016). Cytotoxic activity of Ferula assafoetida and Astragalus sarcocolla against cervical epithelial and colon carcinoma cell lines. Dissertations A number of previous researchers have explained & theses. Tennessee State University. that the occurrence of neurotoxicity is the result of the effect of the monoterpenoide, which inhibits the Ansari, M.A., Vasudevan, P., Tandon, M. and Razdan, acetyle-cholinesterase enzyme activity(Enan, 2001) R.K. (2000). Larvicidal and mosquito repellent action of and cytochrome P450 (De-Oliveira et al., 1997). peppermint (Mentha piperita) oil. Bioresour. Technol. However, (Omolo et al., 2004; Odalo et al., 2005; 71(3): 267–271. Wang et al., 2006, 2014) suggest that the terpenoids, sesquiterpenoid and some compounds from Asrar, M., Ashraf, N., Hussain, S.M., Zia, K. and Rasool, B. (2016). Toxicity and repellence of plant oils against monoterpenoide are repellants against Anopheles Tribolium castaneum (Herbst), Rhyzopertha dominica gambia(Diptera) and coleopterans(Papachristos and (F.) and Trogoderma granarium (E.). Pak. Entomol. Stamopoulos, 2002; Khan et al., 2014). 38(1): 55–63. CONCLUSION Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. This study highlights the effectiveness and high (2008). Biological effects of essential oils – A review. capacity of methanol extract powders from the gum Food Chem. Toxicol. 46(2): 446–475. resins of A. sarcocolla, C. myrrha, A. vera and F. assa-foetida for the protection of stored wheat grains Banchio, E., Valladares, G., Defagó, M., Palacios, S. and Carpinella, C. (2003). Effects of Melia azedarach, against the T. granarium beetle larvae. The success (Meliaceae) fruit extracts on the leaf miner Liriomyza in our discovery and development of the plants for huidobrensis, (Diptera, Agromyzidae): assessment in use as natural insecticides have great and direct laboratory and field experiments. Ann. Appl. Biol. 143(2): 187–193.

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