Chemical Composition Andcomparative Study of Insecticidal Activity

Advances in Environmental Biology, 10(11) November 2016, Pages: 60-69 AENSI Journals

Advances in Environmental Biology

ISSN-1995-0756 EISSN-1998-1066

Journal home page: http://www.aensiweb.com/AEB/

Chemical Composition andComparative study of insecticidal activity by Contact and vapeurof four essential oils against Sitophilus oryzae (L.) (Coleoptera: Curculionidae).

Dalila Saheb and Fazia Mouhouche

Laboratory of Phytopharmacology, Zoology agricultural and forest department, High School of Agronomy, Hassan Badi, 16200 El Harrach, Algiers, Algeria

Address For Correspondence: Dalila Saheb, Laboratory of Phytopharmacology, Zoology agricultural and forest department, High School of Agronomy, Hassan Badi, 16200 El Harrach, Algiers, Algeria.

This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Received 23 August 2016; Accepted 1 November 2016; Published 20 November 2016

ABSTRACT To find a new and safe alternative to conventional insecticides, we evaluated the contact and fumigant toxicityactivity of essential oil (EO) of ginger (Zingiberofficinale), clove (Syzygium aromaticum), thyme (Thymus vulgaris) and rosemary (Rosmarinus officinalis)against the adult rice weevil ofSitophilus oryzaeandinvestigates their chemical composition.The respective methodology use to assess oils was impregnated filter paper disk bioassay to contact bioassay (dose range: (2.5μl.ml-1,5 μl.ml-1,10 μl.ml-1, 20 μl.ml-1) and closed chamber exposure to fumigant bioassay (with a single high dose:0.24ml.L-1) and seven times exposure. Thechemical constituentsof these essential oils were identified using GC-MS technology. Qualitative and quantitative analysis of the four EOs revealed that the major chemical compound of ginger EO was αzingiberene (23.8%).Clove EOs contained 89% eugenol; thyme EO contained 43.2% thymol and 34.4% carvacrol ; rosemary EO contained 15.3% α-pinene and 14.2%camphor. In contact toxicity test on S.oryzae, at the highest concentration (0.31μl.cm-2), clove EO induced 100% mortality, thyme and rosemary produced respectively 82.9%, 90.8% and 88.2%mortality after 6days exposure. In Fumigant test, thyme EO produced 50% mortality after 120 min exposure time, whereas clove and ginger EO produced 62,8% and 53,9% mortality after 4 h exposure, respectively. With Rosemary EO, 52,6% mortality was observed only after 6 h exposure. Clove and thyme Eos in Fumigant test showed the highest efficiency causing 100%mortality in Sitophilus oryzaeet can be useful as promising agent in insect pest management programme.

KEYWORDS: essential oil, insecticidal activity, contact activity, inhalation test, Sitophilusoryzae,

INTRODUCTION

The insect living in the stored food products can cause importantlossesbyreducingthequalityandthequantityofthestoredproducts. Losses due to harmful insects correspond to 35% of the world agricultural production [6]. Because of the efficiency, easy and practical application chemical insecticides are the most used to fight against the harmful insects [5], however their use for a long time(since their creation) appeared dangerous toward ecosystems andhumain healt. Problems caused by intense use of pesticides to control store d grain insects have been the matter of concern for both scientists and public in the past twenty years, for two main reasons: a high toxicity and non biodegradable properties of chemical pesticides and the persistence of their residues that may affect public health [7]. In addition, it always appears insect resistant strains for each new pesticide molecule in use which forced the phytosanitary industries to change regularly the active ingredient in use. These problems also led to

Copyright © 2016 by authors and Copyright, American-Eurasian Network for Scientific Information (AENSI Publication). 61 Dalila Saheb and Fazia Mouhouche, 2016 Advances in Environmental Biology, 10(11) November 2016, Pages: 60-69 the pollution of the stored grain ecosystem because most of the registered pesticides used in stored grain protection are leaving residues that may persist in the cereal food chain up to cereal food consumption. The limitation of the use of persistent pesticides is a priority objective of governmental policies in order to reinforce the preservation of consumer health and to promote sustainabledevelopment [34]. The development of non harmful pesticide for the consumer and the environment became consequently, the essential concern of the researchers. In nature, raw matériel sallowing to lead to biodegradable and non toxic pesticide are sought. Recent studies have examined the potential for botanical substances withinsecticidal properties. Among these substances, essential oils (EOs) were extensively studied for plant protection in order to include these substances as a tool for integrated pest management in stored products whether in developed or developing countries [20]. Additionally, plant essential oils are a mixture of numerous chemical compounds with different levels of volatility. Among these major compounds some of them are sufficiently volatile to exhibit a significant fumigant activity on stored products insect pests [38,33,10,30,27]. The use of EOs for insect control has been the subject of a large number of studies and has attracted much scientific interest. This has been reflected in numerous publications dealing with the effectiveness of the EOs in protecting stored grain and foodstuffs; [11,19,15,40,32,9,28,36]. The aim of this study was to evaluate the insecticidal activity of the EOs offour Mediterranean plants against the riceweevil Sitophilusoryzae(L.) and to relate this efficacy to qualitative compositionof each EO in major compounds.A comparison between contact andfumigant toxicity of EOs against this pest insect is included to tentativelyrelate the insecticidal activity to the specific properties of each EO.

MATERIAL AND METHODS

2.1 Insects: The mass rearing S. oryzae was conducted in a dark oven at a temperature of30 ± 0.5°C and a relative humidity of 70 ± 5%. Eighty young adults wereplaced in a glass jar containing 250g of wheat. The strainoriginated fromwheat flour stored in a flour-bag warehouse at the Technical Institute of Large Crops of OuedSmar (ITGC), El-Harrach (Algeria).The variety of wheat used in S. oryzaelivestock was Hd 1220 Khroubcultivated in Constantine (Algeria) geographical area, and was provided by the same Institute.

2.2 Plant material and essential oil extraction method: The mass rearing: Sitophilus oryzae was conducted in a dark oven at a temperature of30 ± 0.5°C and a relative humidity of 70 ± 5%. Eighty young adults were placed in a glass jar containing 250g of wheat. The strain originated from wheat flour stored in a flour-bag warehouse at the Technical Institute of Large Crops of Oued Smar (ITGC), El- Harrach (Algeria).The variety of wheat used in S. oryzae livestock was Hd 1220 Khroub cultivated in Constantine (Algeria) geographical area, and was provided by the same Institute.

Plant material: four plants were selected: Tuber of Ginger Zingiberofficinale (Zingiberales;Zingiberaceae),glove Syzygium aromaticum (Myrtales; Myrtaceae) ; cloves are the flower buds of the clove that are shaped nail. They are hand- picked before flowering and dried in the sun were imported from Morocco; The aerial parts of rosemary, Rosmarinus officinalis (Lamiales; Lamiaceae) and thyme Thymus vulgaris L. (Lamiales; Lamiaceae) were harvested just before inflorescence stage on May 2013 in Wilaya (District) of Blida distant 50 km from Algiers (Algeria) (36° 29′ 00″ Nord 2° 50′ 00″ Est- elevation: 229m)

Essential oil extraction from dried plants: Essential oils were extracted from dried plant batches. The extraction of essential oils was performed at the factory, Extral-Bio (Chiffa, Wilaya of Blida, Algeria) by hydrodistillation in a Clevenger-type apparatus according to the European Pharmacopoeia method. The distillation time was 3 hours. The essential oils were dried over anhydrous sodium sulfate (Na2SO4). After drying, the EO was conditioned in taint glassvials, tightly sealed to prevent tampering. Vials filled with essential oils were kept in a refrigerator at 4°C until further use.

Chemical analysis: The constituents of essential oils were analysed at the Moubydal Development Center andresearch Laboratory (Algiers, Algeria) by gas chromatography, mass spectrometry (GC-MS)Agilent Technologies / HP 5973 GC/MSD system. The GC-MS conditions were as follows:GC injector with split-splitless injection mode; MS in capacities up to 650 Dalton and electronimpact mode work; detector type, mass selective detector (MSD); chromatographic separationcapillary column pH 5 (30m x 0.25mm; inner coating film thickness: 0.25μm); helium ascarrier gas at flow rate 1.5ml/min (constant flow); injector temperature: 250°C; MS detector

62 Dalila Saheb and Fazia Mouhouche, 2016 Advances in Environmental Biology, 10(11) November 2016, Pages: 60-69 temperature: 230° C for the source and 50° C for the quadrupole; column initial temperature:50° C(maintained for 2 min) and final temperature 250 °C with a ramp of 6°C min-1. The injected volume was 0.2μl in split-splitless mode. Essential oils were injected in their crude form (without purification) in order to identify all components without loss of volatile molecules on one hand, and on the other hand, to allow accurate determination of relative retention time (for the confirmation of MS identification). Quantitative data on the relative amount of constituents of crude EOs were derived from the GC- MS analytical results. Confirmation of such identification was done by comparing their mass spectral fragmentation patterns with those stored in the MS database (NIST 2005 and Wiley 7N libraries) (Adams,2007).

2.3.1 Bioassays protocol: Contact toxicity: Four doses in contact toxicity tests were adjusted from preliminary studies: 2, 5, 10 and 2010μL/cm2. Every concentration of EO was diluted in 1ml acetone and was uniformly distributed by a micro-vaporizer on filter paper disk (9 cm diameter) placed in open Petri dish (9cm diameter and 2cm high) to evaporate acetone thus creating an essential oil film. The Petri dish controls are treated with pure acetone. The mortality was recorded each 24 h for 6 days. After evaporation of solvent, 20 adults of Sitophilus oryzae aged from 15 days are deposited in the box treated by essential oil; the Petri dish thus prepared is protected by a very fine mesh fabric allowing its ventilation (four replicates for essential oils and control).mortality criterion used is total immobility of the insect after delicate tickling legs with an entomological pin. LC50 and LC90 which represent the concentrations causing respectively the death of 50% and 90% ofindividuals of the same dish six days after treatment

Fumigant toxicity: The insecticidal activity by vapor phase (fumigant activity) was tested with a single dose of pure essential oil: 0.24μl/cm3 deposit on filter paper disk (3cm diameter) placed into a screw-cap vial to saturate.Thevolume of glass vial is 82 cm3. 10 min after treatment 20 adults of Sitophilus oryzae are deposit in glass vial,for fumigant test, 7 exposure time are study (60, 120, 180, 240, 300, 360and 1440 minutes). Fourrepetitions were realized for every exposure time and control.LT50 which represent the time causing the death of 50% of individuals of the same glass vial was determined for every essential oil study contact and fumigant tests, control series with pure acetone were included in the experimental design as EO “free”treatment allowing the correction of observed mortality rate in EO treated series if needed byAbbott formula [1] (if mortality rate in control series was less than15%).LC50, LC90, LT50 and LT 90 were determined by probit analysis tested using the method of Finney [12].

2.3.2 EO insecticidal activity assessment: The insect mortality rate from 24 to 144h exposure time by contacttoxicity was recorded and mean mortality rate at each exposure time was calculated.Differencebetween insecticidal activity of each EO as well as the pattern of mortality increase withexposure time were compared. In fumigant test, mortalities were recorded 60, 120, 180, 240,300 and 360, 1440 minutes after treatment, evaluation of lethality wasspread up to 24hours to estimate the rapid action of Essential oils on the target insect.

2.4 Statistical: Statistical Analysis: Statistical analysis was performed with the SPSS 18.0 software(SPSS Inc). Statistical comparisons were made with one-way ANOVA followed by Student Newman-Keuls (SNK) test at P < 0.05.test. The level of significance was set at p <0.05.

RESULTS AND DISCUSSION

3.1Essential oil major compounds (chemotypes): The GC-MS analysis of the four essential oils showed a very variable composition in major compounds on a qualitative and quantitative basis (Table 1). Rosemary was mainly constituted fromalpha-pinene (15.3%), camphor(14.2%), neo-3-thujanol (11.9%), and verbenone (10.1%). Thymol (43.2%) and carvacrol(34.5%) were the major constituents of thyme, followed by carvone (8.4%). the thymolhas shown analgesic effect through its action on the α 2adrenergic receptors of the nerve Shabnum and Wagay [35]. carvacrol are the most responsible of this insecticidal activity.

63 Dalila Saheb and Fazia Mouhouche, 2016 Advances in Environmental Biology, 10(11) November 2016, Pages: 60-69

Eugenol is the main bioactive compound of clove, which is found in concentration of 89% followed by Eugenol acetate (13%)and β-Caryophyllene (9%).Of all the compounds found in the ginger, the percentage of the α-zingiberene was the highest(23.8%). The main other compounds were Geranial (10%) andgeraniol (9.3%). The other constituentsof the four EOs are givenin Table 1.  In comparing the chemotypes of our plant EOs with the ones found by other authors, we found large differences. Thus, in a study of rosemary obtained from plant grown in Morocco, Imelouane et al. [14] found 35 compounds, with eucalyptol among the major compounds. The same author suggested that there could be two main chemotypes for essential oil composition in rosemary: one with high concentrations of 1,8-cineol and the other characterized by low levels of this compound associated to a high level of alpha-pinene (29%) followed by camphor (12.7%). In our study, the rosemary was composed by four terpenoids in the range from 10% to more than 15% relative concentration (alpha-pinene (15.3%), camphor (14.2%), neo-3-thujanol (11.9%), and verbenone (10.1%)). Khia et al., [17] identified in rosemaryα et β- pinènes, camphène, 1,8-cinéole et camphre. Boutekdjiret et al. (1998), haveworked on qualitative and quantitative composition of EO of Algerian Rosemary from Monts of babors, 35 constituents were identified , in this case, Eucalyptol were the major compound.for the thyme EO, Hudaib et al., [13]; Zubiri, and Baaliouamer, [42] identified by GCMS , thymol and carvacrol as major compounds in thyme EO extracted from Thymus vulgaris. Alaoui et al., [3] have studied the insecticidal activities ofThymus oils and agree to say that the components thymol and carvacrol are the most responsible of this bioactivity. In chemotype of clove’s EO, we found eugenol as major compound at a rate of 89% according to Armati et al. [4], Thymus zygis is characterized by the presence of thymol (33.02%), o-cymene (32.02%) and β-E-ocimene (11.90%)accompanied by other compounds in relatively low contents: linalool (3.99%), carvacrol (2.69%), β-pinene (2.08%), borneol (1.56%), E-caryophyllene (1.51%), 1,4-cineole (1.49%) and tricyclene (1.34%). showed the presence of Zingibrene and geranial as the main constituents with 37,58% and 38,96% respectively, followed by b-myrcene ( 5,9%), 2,3 dehydro-1,8 -cineol (0,08%), 1,8- cineol, limonene (0,56%), p-cimene (0,08%), linaloo (1,50%) citonellel (0,57%), 6-methyl-5-hepten-2-one (3,16%), dehydro-p- cymene (0,36%), nerol/geraniol (0,05%), geraic acid (0,02%), 2,2- di-me-7- methoxy-benzo-pyon (0,22%), 2- tridecanone (0,45%). The focus over the last few years has been on the determination of the insecticidal activity of isolated chemical compounds from plant extracts in order to find out the most biologically active chemical components, Positive results for contact and fumigant activity ofmonoterpenes were obtained against S. oryzae. Amarti et al. [4 examined the fumigant toxicity of different essential oils towards the rice weevil, S. oryzae. The essential oil from eucalyptus contained 1,8-cineole (81.1%), limonene (7.6%) and α-pinene (4.0%). The oil generated LD50= -1 -1 28.9 μl L air. 1, 8- cineole was more active (LD50=23.5 μl L air) than limenone andα-pinene. Benzaldehyde -1 (LD50=8.65 μl L air) occurring in peach and almond kernels had also a potent fumigant toxicity towards the rice weevils. Lee et al. [21] evaluated the fumigant toxicity oftwenty naturally occurring monoterpenoidsagainstS. oryzae, T. castaneum, O. surinamensis, the house fly, Musca domestica L., and the German cockroach, Blattellagermanica L. Cineole, l-fenchone, and pulegone at 50 μg ml-1air caused 100% mortality in all five species tested. Wang, [41] investigated the toxicity of 1,8-cineole, camphor, eugenol, linalool, carvacrol, thymol, borneol, bornyl acetate and linalyl acetate against adults of S. oryzae, R. dominica and T. castaneum. The most sensitive species was S. oryzae, followed by R. dominica. T. castaneum was highly tolerant of the tested compounds. 1,8-Cineole, borneol and thymol were highly effective against S. oryzae when applied for 24 h at the lowest dose (0.14 μl L-1). For R. dominica camphor and linalool were highly effective and produced 100% mortality in the same conditions. Against T. castaneum no oil compounds achieved more than 20% mortality after exposure for 24 h, even with the highest dose (139 μl L-1). However, after 7 days exposure, 1,8-cineole produced 92.5% mortality, followed by camphor (77.5%) and linalool (70.0%)Camphor is believed to be toxic to insects and is thus sometimes used as a repellent.In studies conducted by Stamopoulos and al. [38], vapor form of monoterpenoids terpinen-4-ol, 1,8-cineole, linalool, R-(+)-limonene and geraniol were tested against different -1 stages of T. confusum. The LC50 values ranging between 1.1 and 109.4 μl L air for terpinen-4-0l, from 4 and -1 -1 278 μl L air for (R)-(+)-limonene (with LC50 and from 1,8-cineole 3.5 and 466 μl L air were the most toxic to -1 all stages tested, followed by linalool (with LC50 values ranging between 8.6 and 183.5 μl L air) while the least -1 toxic monoterpenoid tested was geraniol with LC50 values ranging between 607 and 1627 μl L air

3.2 Essential oils activity against target insect: 3.2.1Contact toxicity: The tests carried out to evaluate the toxicity of essential oils by contact released the insecticidal power of these products on S.oryzae, at all the tested concentrations, mortalities slowly increased in time from a low level to a high one which were recorded at the highest concentration (0,31/cm2or20µl/ml) after 144 h exposure time, results are given in table 2 and represented in figure1;figure 2; figure 3 and figure 4. The essential oil of Rosemary caused a percentage of mortality higher than 50% only at the highest dose ( 51.28%) after 48 h of exposure to the treatment, mortalities then spread out in the time from 57,14%,65,79%

64 Dalila Saheb and Fazia Mouhouche, 2016 Advances in Environmental Biology, 10(11) November 2016, Pages: 60-69

78.95% to 88.16%. respectively at 72h, 96h, 120h. and 144h. thyme EO has shown insecticidal activity on the target insect at the dose of 10 µl/ml where we recorded 52,63%, this EO became toxic at the highest dose and gave 58.44% of mortalities after 72 h of treatment, this rate continued to increase to reach 72.37% after 96h, 85.53% after 120h and 90.79% after144h.The essential oil of the ginger had a toxic effect on S.oryzae at the highest dose were mortality has reached 50% at 96h, this rate was 72.37% at 120 h and 82.89% at 144h of treatment. the application of 10 µl/mL, 56.58% was the mortality rate noted after 144 h exposure time , that is not the case for times 120h, 96h, 72h, 48h and 24h where obtained results were lower than 50%. Apparently, clove EO is the most efficient since it showed toxicity against the target insect at 5µl/ml where mortality values were estimated at 51.32% after 120h exposure time and Reached 57.89% after 144h. they continued to evaluate in time as the doses increased, indeed, at 10 µl/ml, we recorded 51,32%,63,16% and 81.58% at 96h, 120h, and 144h respectively, the highest dose became toxic after 96h and given 59.21% of mortality, this percentage Increased in time and reached 80.26% after 120h, application of this dose led to a rate of 100% after 144h of treatment. For all the Eos tested mortalities recorded were very low at the dose of (2,5µl/ml)corresponding to 0,039 μl/cm2, also, for the control treatment with acetone , results wereweak.Analysis data revealed a significant difference for the two factors EO-doses (p<0.005). the four Eos have the same effect since their Effectiveness depends tightly on the doses applied so that mortality values increased in time, consequently, in the test of newman and keuls, five homogenous groups appeared and in each one are gathered four Eos. Several laboratory studies were conducted to evaluate the insecticidal activity by contact of Eos and their compounds on stored products insects, Khani and Rahdari,[16] studies and showthat the essential oil extracted from coriander, Coriandrum sativum L. (Apiaceae), seeds againstadults of Tribolium confusum: The mortality of 1–7-day-old adults of the insect pests increased withconcentration from 43 to 357 μL/L air and with exposure time from 3 to 24 h.. a concentration in air. The menthol extracted from 30 plants species generated 90% to 100% of mortality after two to four days of treatment according to the plant species [37]. Kim et al. [18], carried out a study on 30 aromatic and medicinalplants at several forms (powders and Eos), results showed that these plants were toxic to Callosobuchuschinensis and Sitophilus oryzae, among them, essential oils of Brassica juncea L. and Cinnamomumcassicawere more effective with percentages of mortality of 84.2% and 98% respectively. El-Guedoui, [11] testing the essential oil of Thymus fontanesiiBoiss. &Reut. on Rhyzoperthadominica(F.) finds this oil more toxic by contact (100% kill at 0.69mg/cm2) than by inhalation (40.9% kill at 1.44mg/cm3), Owabali et al, [25], carried out tests on the effectiveness by contact of Eos of Ginger on S. zeamais, they shown 2 weak toxicity toward this target insect, DL 50 values was 0.7 µl.cm .

3.2.2 Fumigant toxicity: Fumigant Toxicity of tested essential oils is shown in figure. 5. with percentage of mortality included, the rice weevil was very sensitive to the applied dose (0,24 ul/cm3) with all Eos within 1440 min (24h) exposure time, thyme and clove had done complete lethality, while rosemary and ginger induced 97,33% and 98,67% respectively. Under this exposure time, obtained results recorded from 60 to 360 min after treatment proved the rapid action of Eos vapors, indeed, Thyme was the first which has done 50% of mortality after 120 min of exposure time, whereas, Clove and Ginger gave at 240min 62,82% and 53,85% respectively. With Rosemary Eos, it is until 360min that the rate of mortality reached 52, 63%. For a long time, tests on fumigant toxicity of plant essential oils and their constituents against stored- product insects have been realized and conducted with a large scale of plants (mainlybelonging to Apiaceae, Lamiaceae, Lauraceae and Myrtaceae) and their components (cyanohydrins, monoterpenoids, sulphucompounds, thiocyanates andothers) andhave largely focused on beetle pests such as Triboliumcastaneum, Rhyzoperthadominica, Sitophilusoryzae and Sitophilus zeamais. In the last years, several researches in this context knew a revolutionary development, as example; Mondal and Khalequzzaman 2009,tested the ovicidal activity of essential oil vapours ofcardamom (Elletaria cardamomum L.), cinnamon (Cinnamomum zeylanicumBlume), clove (Sygium aromaticum L. Merrill. et. Perry), Eucalyptus spp. and neem (Azadirectica indica A. juss) against the eggs of red flour beetle, Triboliumcastaneum (Herbst). The exposure to vapours of essential oilscardamom andcloveresulted in 100% mortality of the eggsat a concentration of 5.769 mg/l air and exposureperiod of 24h. Sung-Woong(2016) assessed the toxicity of vapours of the essential oils fromhyssop (Hyssopus offcinalis), majoram (Origanum majorana), and Thymus zygis essential oils showed strong fumigant toxicity against S. oryzae adults at 25 mg/L airconcentration.The essential oil vapours were toxic to all immature stages tested with LC50 values ranging between 0.6 and 76 μl L-1air, depending on oil and development stages. Lee and al. [22] studied the potent fumigant toxicity of 42 essential oils and found out that six of them extracted form Eucalyptus nicholi (Maiden and Blakely), E. codonocarpa(Blakely andMcKie), E. blakely(Maiden), Callistemon sieberi(F.Muell.), Melaleuca fulgens (R.Br.) and M. armillary (R.Br.) were toxic to S. oryzae, R. dominica and T. castaneum. The -1 LD 50 and LD 95 against the adults of S. oryzae were between 19.0 to 30.6 and 43.6 to 56.0 μg ml air,

65 Dalila Saheb and Fazia Mouhouche, 2016 Advances in Environmental Biology, 10(11) November 2016, Pages: 60-69 respectively. The LD95 of 1,8-cineole was for S. oryzae 47.9, for R. dominica 30.4 and for T. castaneum 21.0 μg ml-1 air. The fumigant toxicity of five oils in the space 50% filled up with wheat was 3 to 5 times lower in 50% filled up the space than in an empty space and in a case of EO extracted from E. codonocarpa in 50% filled up the space with wheat, even 9 times less toxic.Tapondjou et al. (2005) investigated the toxicity of cymol and essential oils of Cupressussempervirens (L.) and Eucalyptus saligna (Sm.) against S. zeamais and T. confusum. -2 Eucalyptus oil was more toxic than Cupressus oil to both insect species (LD50=0.36 μl cm for S. zeamais and -2 0.48 μl cm for T. confusum) on filter paper discs, and was more toxic to S. zeamais on maize (LD50=38.05 μl per 40 g grain).In other fumigant tests on C. maculatus, the DL50 values of Eos of Eucalyptus sp, Cymbopogonnardus, Rosmarin us officinalis,Elettariacardamomum(L.) andGeranium sp, were evaluated at 11.66, 16.25, 21.35, 22.07 and 25.11 μl l-1air respectively [36].

Table 1: Qualitative and quantitative analysis of essential oils extracted from four Mediterranean aromatic plants .

Clove Ginger Rosemary Thyme Compounds Rate Compounds Rate Compounds Rate Compounds Rate (%) (%) (%) (%) -pinène 15,25 Thymol 43,22 eugénol 89% α-zingibérène 2 3.8 camphore 14,15 Carvacrol 34,49 eugénol acetate 13% Géranial 10 Néo-3-thujanol 11,89 Carvone 8,35 β-Caryophyllène 9% Géraniol 9.3 Verbenone 10,13 Bornéol 2,41 α-humulène 2,56 β-bisabolène 7.9 Cis-thujone 7,02 Para –cymène-8-ol 1,61 β-pinène 0, 31 Nérol 7.1 Bornéol 5,68 spathulénol 1,13 limonène 0,29 α-terpinéol 5.6

Camphène 3,52 Thymoquinone 0,97 Bornéol 5.4 limonène 3,08 Sabinène 0,48 1.8-cinéole 6.2 Iso-3-thujanol 2,15 Eucalyptol 0,73 β-phellandrène 3.1 Carvone 1,41 -elemène 0,33 Linalol 1.7 2-Undécanone 1.6

Table 2: Mean mortality of S. oryzae in filter paper contact test with different EOs. Essential Oils Exposure Control Doses of Essential Oils Time 2,5μl/mL 5μl/mL 10μl/mL 20 μl/mL 24 0 3.75 7.5 6.25 26.25 48 2.5 3.85 15.38 17.95 51.28 72 3.75 3.89 15.26 24.67 57.14 Rosemary 96 5 5.26 15.79 31.58 65.79 120 5 6.58 21.05 40.79 78.95 144 5 7.89 25.00 47.37 88.16 24 0 1.25 2.5 5 20 48 2.5 1.28 5.13 11.54 33.33 72 3.75 2.59 10.38 18.18 58.44 Thyme 96 5 2.63 15.79 30,26 72.37 120 5 5.26 18.42 42,11 85.53 144 5 7.89 23.68 52,63 90.79 24 0 10 11.25 13.75 11.25 48 2.5 10.26 20.51 21.79 23.08 72 3.75 14.28 27.27 28.75 35.06 Ginger 96 5 14.47 34.21 40,79 50.00 120 5 19.74 46.05 47,37 72.37 144 5 28.95 48.68 56,58 82,89 24 0 13.75 1750 13.75 11.25 48 2.5 15.38 26.92 30.77 28.21 Glove 72 3.75 19.48 33.76 41.55 41.55 96 5 25.00 36.84 51.32 59.21 120 5 36.84 51.32 63.16 80.26 144 5 47.37 57.89 81.58 100.00

66 Dalila Saheb and Fazia Mouhouche, 2016 Advances in Environmental Biology, 10(11) November 2016, Pages: 60-69

100

80 2,5 60 5 40 10

20 20 Correctedmortality % 0 24 48 72Time 96 (h) 120 144

Fig. 1: Evolution in time of mortality of S. oryzae treated with different dose of romarin Oil expresed as μl/mL

120 100 80 2,5 60 5 40 20 10 0 20

-20 24 48 72 96 120 144 Corrected mortality (%) mortality Corrected Time (h)

Fig. 2: Evolution in time of mortality of S. oryzae treated with different dose of thyme Oil expresed asμl/mL 100 80

60 2,5 40 5 20 10 0 20 -20 24 48 72 96 120 144

Corrected mortality (%) mortality Corrected Time (h )

Fig. 3: Evolution in time of mortality of S. oryzae treated with different dose of Ginger Oil expresed as μl/mL

67 Dalila Saheb and Fazia Mouhouche, 2016 Advances in Environmental Biology, 10(11) November 2016, Pages: 60-69

120

100

60 2,5 5 40 10 20 20 Corrected mortality (%) mortality Corrected 0 24 48 72 96 120 144 -20 Time (h)

Fig. 4: Evolution in time of mortality of S. oryzae treated with different dose of Clove Oil expresed as μl/mL

120

100

60 Rosmary Thyme Ginger

40 correctedmortlity(%) Clouve

0 60 min 120 min 180 min 240 min 300 min 360 min 1440min Time (min)

Fig. 5: Fumigant toxicity of Esential EOs againstSitophilus oryzae

Conclusion: The tests carried out on the insecticidal activity of EOs against S. oryzaeby the two mode of action contact and inhalation, had shown that all Eos tested are efficient at the highest dose were rate of mortality has reached 100% for the Clove Eoafter 6daysexposure, followed by theginger Eos with 82.89%, for thyme and rosemary, we recorded respectively,90.79% and 88.16% mortalities after 6d exposure. Whereas, in the fumigant effect, the application of the same dose (the highest) has done great level of mortality within 24h, complete lethality was observed for clove and thyme, while rosemary and ginger induced 97, 33% and 98, 67% respectively, that lets to conclude that because of their volatile compounds, Eos have a high and rapid actionon target stored insects by fumigation Thus, they could be a safer fumigant to control stored-graininsect pests than those currently used.

68 Dalila Saheb and Fazia Mouhouche, 2016 Advances in Environmental Biology, 10(11) November 2016, Pages: 60-69

ACKNOWLEDGEMENTS

We are grateful to Francis Fleurat-Lessard for profitable advice for the writing of the manuscript.

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