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Integrated Protection of Stored Products IOBC-WPRS Bulletin Vol. 81, 2012 pp.

111-121

Relation between the composition of four essential oils from Algerian plants and their insecticidal activity against Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae)
Dalila Saheb1, Fazia Mouhouche1*, Sidali Benazzedinei1, Francis Fleurat-Lessard2 1 Ecole Nationale Suprieure Agronomique, Laboratoire de Phytopharmacie, INA ElHarrach, 16200 Algiers, Algeria; 2UR INRA 1264, Mycology and Food Safety (MycSA), Ple Qualis - 71, Avenue Edouard Bourlaux, 33883 Villenave dOrnon Cedex, France * Corresponding author e-mail: fmouhouc@gmail.com

Abstract: Several essential oils (EOs) from Mediterranean plants are known to present a significant biological activity on insect pests. The insecticidal activity of essential oils extracted by hydrodistillation from four aromatic plants from Algeria: spearmint (Mentha viridis), thyme (Thymus vulgaris), rosemary (Rosmarinus officinalis), and eucalyptus (Eucalyptus globulus), was tested either 2 from contact test exposure (dose range: 0.05-0.4l/cm ) or vapour phase exposure (single high dose: 3 0.582l/cm ) against Sitophilus oryzae and Tribolium confusum. The respective methodology of the two bioassays was the impregnated filter paper disk bioassay in a Petri dish and the closed chamber exposure. The chemical composition of the essential oils was established by GC/MS analysis. Qualitative and quantitative analysis of the four EOs revealed that their major chemical compounds were mainly monoterpenes. eucalyptus EO contained 83.3% of three substances simultaneously eluted: limonene, 1,8-cineole and p-cymene; rosemary EO contained 15.3% -pinene and 14.2% camphor; thyme EO contained 43.2% thymol and 34.4% carvacrol; spearmint contained 24.3% carvone, 8.3% 1,8-cineole and 8.1% limonene. In contact toxicity test, the four EOs exhibited more or less a similar level of toxicity on both insect species. At the highest concentration (0.4l/cm), all the four EOs had similar average activity against S. oryzae with more than 88% mortality observed after 6 d exposure. For T. confusum, thyme, spearmint and rosemary EOs induced more than 95% mortality meanwhile eucalyptus EO caused significantly less mortality observed at 72.6%. Among the four tested EOs, rosemary and mint EO showed the highest inhalation efficiency. The mortality rate reached 100% after 24h exposure time, either on S. oryzae or T. confusum. The analysis of the relationship between insecticidal activity level on the two target insects and qualitative composition of the different essential oils showed that the presence of oxygenated monoterpenes was linked to higher insecticidal activity. To expect a future development of botanicals as a tool for integrated management of pest in stored products new studies will be focused on identified pure bioactive compounds. Key words: whole essential oil, insecticidal activity, contact activity inhalation test, Sitophilus oryzae, Tribolium confusum

Introduction
The widespread use of pesticides has led to pollution of various ecosystems including the stored grain ecosystem. This use is the main cause of the emergence of resistant strains in insect populations which became insensitive to the pesticides currently used (Leonard and Ngamo, 2004). 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

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order to reinforce the preservation of consumer health and to promote sustainable development. Consequently, the current research of alternatives to chemical pesticides for insect pest control attracted much scientific interest on insecticidal properties of phytochemicals or plant essential oils (generally recognized as safe, i.e. with toxicity per os for rats generally higher than 2000mg/kg body weight, associated to a short delay of persistence). Several recent studies have examined the potential for botanical substances with insecticidal properties respectful for human and environment health. 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 (Lahlou, 2004). 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 (Regnault-Roger and Hamraoui, 1995; Papachristos and Stamopoulos, 2002; Lee et al., 2004; Negahban et al., 2007; Stamopoulos et al., 2007; Sahaf et al., 2008; Ebadollahi et al., 2010). The use of numerous EOs for insect control has been the subject of a large number of studies during the past two decades 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, and has often applied to situations in African countries (Shaaya et al., 1997; Tun et al., 2000; Isman, 2000; Hummelbrunner and Isman, 2001, Huang et al., 2002; Kim et al., 2003; Tapondjou et al., 2003, Tripathi et al., 2003; Koona and Njoya, 2004; Kellouche and Soltani, 2004; Tapondjou et al., 2005; Tiaiba, 2006; Owabali et al., 2009; Camara, 2009). The main objective of this work was to evaluate the insecticidal activity of the EOs of four Mediterranean plants against two major stored product insects Sitophilus oryzae (L.) and Tribolium confusum (Jacquelin du Val) and to relate this efficacy to qualitative composition of each EO in major compounds. This study included a comparison between contact and fumigant toxicity of EOs against these two grain primary pest insect species to tentatively relate the insecticidal activity to the specific properties of each EO.

Material and methods


Insects The mass rearing S. oryzae and T. confusum was conducted in a dark oven at a temperature of 30 0.5C and a relative humidity of 70 5%. For S. oryzae, eighty young adults were placed in a glass jar containing 250g of wheat. The strain of T. confusum originated from wheat flour stored in a flour-bag warehouse at the Technical Institute of Large Crops of Oued Smar (ITGC), El-Harrach (Algeria). Forty adults were placed in a glass jar on 250g white flour from the market. 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 and essential oil extraction method For the purpose of this study, four plants were selected: eucalyptus, Eucalyptus globulus Labillardiere (Myrtales; Myrtaceae); rosemary, Rosmarinus officinalis (Lamiales; Lamiaceae); spearmint, Mentha viridis L. (Lamiales; Lamiaceae) and thyme, Thymus vulgaris L. (Lamiales; Lamiaceae). All were harvested in Wilaya (District) of Blida (Algeria). Essential oils were extracted from dried plant batches by hydrodistillation.

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Essential oil extraction from dried plants The extraction of essential oils was performed at the factory, Extral-Bio (Chiffa, Wilaya of Blida, Algeria) by hydrodistillation in a Clevenger-type apparatus. The extraction method may be summarised as follows: after drying and grinding plant samples, 100g of powder introduced into a 2l capacity glass balloon. The plant material was then immersed in distilled water up to two-third of the balloon (i.e. from 1200 to 1500ml of distilled water). Then, the balloon was placed on a balloon-flask-heater with heating-regulation electronic system. The distillation time was 2 to 3 hours. The light essential oil (supernatant) was separated from water (recovering the supernatant phase) and then water in the EO was removed by anhydrous sodium sulfate (Na2SO4) in a desiccator. After drying, the EO was conditioned in taint glass vials, tightly sealed to prevent tampering. Vials filled with essential oils were kept in a refrigerator at a temperature ranging from 0 to 8C until further use. Chemical analysis The constituents of essential oils were analysed at the Moubydal Development Center and Research 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 electron impact mode work; detector type, mass selective detector (MSD); chromatographic separation capillary column pH 5 (30m x 0.25mm; inner coating film thickness: 0.25m); helium as carrier gas at flow rate 1.5ml/min (constant flow); injector temperature: 250C; MS detector 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 6C min-1. The injected volume was 0.2l in split 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. Bioassays protocol Essential oils insecticidal activity was tested either from contact toxicity or inhalation activity (fumigant effect). The dose in contact toxicity tests was adjusted in preliminary studies in the range 0.05-0.40l/cm2. The EO total amount was diluted in 1.3ml acetone and was uniformly distributed by a micro-vaporizer on a 90mm diameter filter paper disk placed in the bottom of an open Petri dish. Only the results obtained with the highest dose (0.4l/cm2, i.e. 19.7l EO on 64cm2 disk) are reported. The insecticidal activity by vapour phase (fumigant activity) was tested with the same amount of EO as in the contact test, but introduced on a 3cm diameter filter paper into a screw-cap vial with a 44ml capacity. This high amount of EO (corresponding to theoretical concentration 0.582l/cm3) was designed to saturate the air in the exposure vessel. Vapour / liquid equilibrium was established as EO was released during the exposure time (24h up to 144h). Thus, each EO was tested at a single dose with four replicates. The mortality rate was observed each day during six consecutive days after insect containment on either the treated filter paper disk or in inhalation mini-chamber. Parallel 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 by Abbott formula (1925) (if mortality rate in control series was higher than 15%).

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EO insecticidal activity assessment The insect mortality rate from 24 to 144h exposure time by both contact toxicity and inhalation was recorded and mean mortality rate at each exposure time was calculated. Differences in insecticidal activity of each EO as well as the pattern of mortality increase with exposure time were compared. The mortality rate in control treatments after 144h exposure time was rarely more than 15%. The comparison of insecticidal activity at each EO dose was performed by XLStat software (Addinsoft, France). The variance analysis (ANOVA) was completed by Bonferronis means comparison test. In order to respect the conditions of variance classical analysis (Gaussian distribution), the mortality data were transformed by angular conversion (Asin(square root of the raw mortality percentage).

Results and discussion


Essential 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). Eucalyptus was mainly constituted from three co-eluted monoterpenes: para-cymene, limonene and 1,8-cineol (83.3%), followed by minor compounds such as alpha-pinene (3.9%) and gamma-terpinene (6.7%). Rosemary contained four major compounds: alpha-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%). This latter monoterpene was the major component of spearmint (24.3%) followed by 1,8-cineol and limonene (both around 8%). The major constituents in the four EOs were a large set of monoterpenes. Nevertheless, very few common major compounds were found common in these efour EOs. Alpha-pinene was found in eucalyptus and rosemary in different concentrations: 4% and more than 15%, respectively. 1,8-cineol and limonene were found in eucalyptus and spearmint. Carvone was found in thyme and spearmint at a very different relative amount of 8.4 and 24% respectively. Oxygenated monoterpenes such as 1,8-cineol, camphor, thymol and carvone were observed in rosemary and thyme (Table 1). In comparing the chemotypes of our plant EO with the ones found by other authors in the same plant species harvested in North African countries, we found large differences. Thus, in a study of rosemary obtained from plant grown in Morocco, Imelouane et al. (2010) 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%)). Makhlouf (2002) identified 36 constituents in rosemary representing 90.6% of the oil, including verbenone (9.2%) and limonene (5.4%). In the chemotype of thyme, the GC-MS analysis revealed that the major compound was thymol (43.2%) followed by carvacrol (34.5%). Other constituents were in a minor but significant concentration such as carvone (8.4%) and borneol (2.4%). In the fight against the various pests and parasites, thyme has shown promising results. Thymol is a recognised bioactive molecule with remarkable bactericidal, fungicidal, insecticidal, and acaricidal properties. With regard to the insecticidal activity on stored product insects, Regnault-Roger and Hamraoui (1995) in testing carvacrol and thymol activity against Acanthoscelides obtectus (Say) show that these phenolic compounds expressed high toxicity by inhalation, inhibit larval development, and decrease fertility and oviposition of females. El-Guedoui, (2003)

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testing the essential oil of Thymus fontanesii Boiss. & Reut. on Rhyzopertha dominica (F.) finds this oil more toxic by contact (100% kill at 0.69mg/cm2) than by inhalation (40.9% kill at 1.44mg/cm3). In our case, the major compound in spearmint essential oil, carvone, was found at a rate of 24.3%; so this corresponded to the chemotype: mint carvone. It also contained a low percentage of 1,8-cineol ( 8.3%), limonene (8.1%), caryophyllene (4.1%), trans-dihydrocarvone (3.6%), and iso-menthol (3.4%). The chemotype of essential oils may vary according to geographical origin, botanical sample, season and year of harvest. Table 1. Qualitative and quantitative analysis of essential oils extracted from four Mediterranean aromatic plants (major components representing more than 1% total components)
Eucalyptus Rate (%) 3.94 Rosemary Alphapinene Camphor Neo-3thujanol Rate (%) 15.25 Thyme Rate (%) 43.22 Spearmint Rate (%) 24.28

Alphapinene Myrcene Alphaphellandrene Paracymene Limonene

Thymol

Carvone

1.27 1.45

14.15 11.89

Carvacrol Carvone

34.49 8.35

1,8-cineole Limonene

8.30 8.08

Verbenone 83.28 Cisthujone

10.13

Borneol

2.41

Caryophyllene Transdihydrocarvone Iso-menthol Bicyclogermacrene

4.05

7.02

Spathulenol

1.13

3.61

1,8-cineole Gammaterpinene 6.68

Borneol

5.68 3.52

3.38 2.42

Camphene

Essential oils activity against target insects At the highest dose studied (0.40l/cm2) the contact activity of the four EOs slowly increased from a low mortality level up to almost complete kill after 6 d exposure time (Table 2). The two species exhibited a similar level of sensitivity to the four EOs without significant differences between explanatory sources of variation (Table 3).

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Table 2. Mean mortality of S. oryzae and T. confusum in filter paper contact test with different EOs (SD = Standard deviation).
Dose (l/ cm2) 24h Eucalyptus Rosemary Thyme Mint T. confusum 24h Eucalyptus Rosemary Thyme Mint 0.40 0.40 0.40 0.40 10.25 70.00 67.5 58.75 SD 4.08 12.91 13.23 8.54 48h 27.5 70.0 73.75 77.5 SD 6.45 14.72 13.15 11.9 72h 50.0 87.5 82.5 90.0 SD 96h SD 120h 68.75 98.75 100 100 SD 144h SD 0.40 0.40 0.40 0.40 16.25 26.25 20 33.75 SD 8.54 6.29 10.80 16.52 48h 41.25 52.5 35 52.5 SD 14.93 6.45 12.91 21.02

S. oryzae

Exposure time (h) 72h 67.5 58.75 60 65 SD 96h SD 120h 93.75 80.0 86.25 85.0 SD 12.5 144h 97.5 SD 5

21.02 83.75 20.16 6.29 67.5 6.45

9.13 88.75 8.54 9.46 91.25 8.54 14.72 96.25 4.79

10.0 73.75 8.54 26.77 78.75 19.31

10.80 61.25 8.54 6.45 93.75 6.29 12.58 91.25 11.81 9.13 98.75 2.5

8.54 73.75 6.29 2.5 0 0 98.75 100 100 2.5 0 0

Table 3. Variance global analysis of insecticidal activity of the four EOs (contact toxicity) Asin conversion applied to mortality rate Square Sum (SS) 13.663 1.443 2.028 3.298 20.433 4.912 25.345

Source Expotime Species Plant EO Species*Plant EO Model Error Corrected Total

DF 1 1 3 3 8 183 191

SS Mean 13.663 1.443 0.676 1.099 2.554 0.027

F 509.027 53.764 25.183 40.957 95.151

Pr > F < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Calculated against the model Y=Mean(Y)

Bonferronis test comparing plant EO insecticidal activity by contact effect Plant EO Mint Thyme Rosemary Eucalyptus Estimated Mean 1.178 1.103 1.098 0.900 Groups A A A A

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Bonferronis test comparing insect species susceptibility to the four EOs by contact effect Species T. confusum S. oryzae Estimated Mean 1.157 0.983 Group A A

Fumigant type activity (through vapor phase) The vapour toxicity of rosemary was high for the two species which were killed after 24h exposure only (Table 4). T. confusum was also very sensitive to mint, which was also lethal after 24h exposure time, when S. oryzae was completely killed after 96h exposure time to this EO. Nevertheless, mint and rosemary were significantly more active on T. confusum and S. oryzae than the two others EOs (Table 4). The fumigant effect of essential oils on S. oryzae and T. confusum at a dose of 0.582l/cm3 showed that all oils caused 100% mortality after 6 days. For S. oryzae, rosemary induced 100% mortality after 48h exposure time. For the three other EOs, the complete kill was observed after 96h for spearmint, 120h for eucalyptus and 144h for thyme (Table 4). For T. confusum, rosemary and spearmint induced 100% mortality after 24 h of exposure by inhalation, and the same result was observed with thyme and eucalyptus after 120h (Table 4). Table 4. Mean mortality of S. oryzae and T. confusum in inhalation test (fumigant toxicity) with different EOs (SD = Standard deviation).
Dose (l/cm3) 24h Eucalyptus Rosemary Thyme Mint T. confusum 24h Eucalyptus Rosemary Thyme Mint 0.582 0.582 0.582 0.582 SD 48h SD 72h SD 9.46 0 96h 98.75 100 SD 2.5 0 9.46 0 120h 100 100 100 100 SD 0 0 0 0 144h 100 100 100 100 SD 0 0 0 0 0.582 0.582 0.582 0.582 SD 48h 67.5 100 SD 11.90 0 17.5 9.46 72h 80 100 80 98.75

S. oryzae

Exposure time (h) SD 9.13 0 16.83 2.5 96h 91.25 100 85 100 SD 6.29 0 13.69 0 120h 100 100 90 100 SD 0 0 144h 100 100 SD 0 0

42.5 11.90 42.5 10.41 45 70

16.83 66.25 10.8 93.75

10.80 93.75 9.46 0 100 0

56.3 14.93 81.25 11.09 93.75 100 0 100 70 100 0 9.13 0 100

52.5 10.41 100 0

83.75 13.77 93.75 100 0 100

SD = Standard deviation

The interaction between plant EO and insect species mortality was not significant, indicating that the two target insect species have a similar susceptibility to the four tested EOs (Table 5). The statistical analysis for classification of EO insecticidal activity by inhalation by Bonferroni test showed a first group of high fumigant activity with mint and rosemary, and two other groups with less fumigant activity with eucalyptus and thyme (Table 5b). It has

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been proved by many authors, notably Regnault-Roger (1997), that several plant species belonging to the Labiatae family (mint family) exhibit inhalation toxicity for stored product insects such as bruchids or flour beetles. The plant belonging to this botanical family with the highest activity of their EO are Thymus vulgaris L. and Rosmarinus officinalis L. Plant species belonging to the Myrtaceae family (e.g. Eucalyptus spp.) are known to have EO with a good insecticidal activity on the bean weevil, A. obtectus. Tun et al. (2000) studies show that the essential oil of oregano at a concentration in air of about 200l/l causes 89% mortality in T. confusum after 96h exposure, to be compared with rosemary essential oil with which the level of mortality falls to 65% in the same conditions. Furthermore, El-Guedoui (2003) evaluating the inhalation toxicity of rosemary and thyme on R. dominica asserts that rosemary is more toxic than thyme by inhalation (fumigant effect) at a high dose of 1.44l/cm3. Chaumont et al. (2001) and Koba et al. (2004) also show after bioassay and chemical analysis that citronella oil (citronellal, geraniol and citronellol major compounds) is active against microbes rather than against insects, even though citronella has good repellent properties against flying insects and is largely used in domestic environments. Shakarami et al (2004) studied the toxicity and repellency of essential oil of Artemisia aucheri Boiss. on C. maculatus, T. castaneum, S. oryzae and S. granarius at concentrations of 0.03, 0.18, 0.37, 0.55, 0.74, and 0.921/cm3. They deduced that the most sensitive species to this oil was C. maculatus from the LD50 values recorded for these four species, respectively at: 0.10, 0.12, 0.13, and 0.14l/cm3. Recently, Camara (2009) examined the effectiveness of essential oils of Ocimum basilicum L., O. gratissimum L., and Cymbopogon citratus (D.C.) Stapf by fumigation and contact to grain insect pests S. oryzae and Tribolium castaneum (Herbst) but failed to observe satisfactory efficiency at an economic dosage to expect a practical development of plant EO as an alternative of grain chemical protectants. Table 5a. Variance global analysis (all qualitative and quantitative variables) of fumigant toxicity of four EOs against S. oryzae and T. confusum.
Square Sum Source DF (SS) Exposure time 1 6.1 Insect species 1 0.866 3 3.554 Plant EO Species*Plant EO 3 0.0017 Model 8 10.521 Error 183 6.754 Corrected Total 191 17.2755 calculated against the model Y=Mean(Y)

SS Mean 6.1 0.866 1.185 0.0006 1.315 0.037

F 165.282 23.462 32.096 0.015 35.635

Pr > F < 0.0001 < 0.0001 < 0.0001 0.997 < 0.0001

Table 5b. Bonferronis test for sorting homogeneous groups of EO insecticidal activity Sorting of plant EO insecticidal activity by fumigant effect (inhalation test)
Essential oil Mint Rosemary Eucalyptus Thyme Estimated mean 1.504 1.499 1.274 1.196 Groups A A B C

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Conclusion
We observed that among the five essential oils we studied, rosemary and spearmint showed the highest inhalation efficiency on both test-insects: S. oryzae or T. confusum. Test results about EOs contact activity were disappointing since only exposure time of 6d was needed to expect almost complete kill of the two insects at the highest tested dosage. In rosemary and spearmint it was found bioactive compounds such as 1,8-cineol, camphor and carvone which are known for their high antimicrobial activity (Imelouane et al., 2010). These oxygenated monoterpenes are more or less volatile compounds and they likely confer to these EOs their insecticidal properties by fumigant effect rather than by contact. This is for this reason that we started some months ago a new study of the individual monoterpenes substances existing in various plant essential oils exhibiting demonstrated antibacterial, antifungal or insecticidal activities. Our long-term objective will be to include the bioactive compounds in a controlledrelease formula specifically designed according to the physical-chemical properties of the bioactive molecules in order to optimize the ratio release rate / fumigant toxicity.

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