Postharvest Control of the Date Moth Ectomyelois ceratoniae Using Eucalyptus Essential Oil Fumigation

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1 Postharvest Control of the Date Moth Ectomyelois ceratoniae Using Eucalyptus Essential Oil Fumigation Soumaya Haouel and Jouda Mediouni-Ben Jemâa, Laboratoire de Protection des Végétaux, INRAT, 2080 Ariana, Université de 7 Novembre à Carthage, Tunisia, and Mohamed Larbi Khouja, Laboratoire d Ecologie et Amélioration Sylvo- Pastorale, INRGREF, 2080 Ariana, Université de 7 Novembre à Carthage, Tunisia ABSTRACT Haouel, S., Mediouni-Ben Jemâa, J., and Khouja, M.L Postharvest control of the date moth Ectomyelois ceratoniae using eucalyptus essential oil fumigation. Tunisian Journal of Plant Protection 5: This work aims to investigate the chemical composition and to assess for the first time the fumigant toxicity of two eucalyptus (Eucalyptus camaldulensis and ) essential oils against the date moth Ectomyelois ceratoniae in storage as alternative to methyl bromide. GC-MS analyses showed that E. rudis essential oil presented α-pinene (14.49%), 1,8-cineole (19.87%), β-pinene (3.91%), γ-terpinene (6.04%), terpinene-4-ol (4.46%) and α-terpineol (4.32%) as major compounds whereas, E. camaldulensis essential oil had α-pinene (16.49%), 1,8-cineole (20.62%), γ-terpinene (4.08%), terpinene-4-ol (4.45%), aromadendrene (3.93%) and isothymol (7.3%) as major compounds. Results showed that fumigant toxicity depends on oil species, concentrations and exposure time. The fumigant toxicity potential of (LC 50 = µl/l air) was greater than E. camaldulensis (LC 50 = µl/l air). Moreover, the median lethal time values (LT 50 ) were and h, respectively. Results suggested that and E. camaldulensis essential oils could be used as an alternative to the synthetic fumigant in postharvest treatment program for the control of E. ceratoniae. Keywords: Essential oil, Eucalyptus camaldulensis,, Ectomyelois ceratoniae, GC-MS, lethal concentration LC 50, lethal time LT 50 In South of Tunisia, dates present the main resource of oasis and have a major role in shorting the agriculture and developing national economy. Tunisian dates occupy the third position in agricultural exports. Date production has increased by 17% compared to last season. The date s industry represents 5% of the total value of agricultural production and 16% of the total value of agricultural exports. Tunisian dates are ranked the 4 th in terms of exported quantities and the first in terms of foreign exchange earnings (17). Nevertheless, dates are subjected to many diseases and pests that decrease their yield and deteriorate their quality. In Tunisia, the date moth Ectomyelois ceratoniae Corresponding author: Jouda Mediouni- Ben Jemâa (Lepidoptera, Pyralidae) is the major joudamediouni@lycos.com insect pest of dates both in field and in Accepted for publication 20 October 2010 storage (13, 20). Date moth infests 20% of the harvestable crop annually (17). This devastating insect degrades the Tunisian Journal of Plant Protection 201 Vol. 5, No. 2, 2010

2 stored dates and causes weight loss and downgrading of the commercial value of the fruit (2, 14). Dates intended for export undergo the process of conditioning to preserve and improve their quality. Chemical control using fumigation is the most economical tool for managing this stored pest in postharvest treatment (3). Methyl bromide is still the primary insecticide of postharvest insect control for dates in Tunisia and in several other countries (19, 53). Nevertheless, the use of this fumigant is being restricted because of its health and environmental damages (6). Moreover, the Montreal Protocol of the United Nations Environment Program (49) recommends the phasing out of methyl bromide by 2005 in developed countries and by 2015 in developing countries (33). Consequently, research of effective alternative methods is needed. In recent years, essential oils have received much attention as pest control agents because of their insecticidal properties (42). Essential oils are volatile and can act like fumigants offering the prospect for use in storedproduct protection (7, 15, 41, 44, 46). Eucalyptus (Myrtaceae) is one of the most cultivated genera in the world including more than 700 species. Various biological properties have already been attributed to the genus Eucalyptus, among them insecticidal activity against beetles (8), repellent action against Phlebotomus papatasi (51) and larvicidal activity on culicids (10). Besides, eucalyptus essential oils are used for medicinal and pharmaceutical purposes (11, 12, 18, 26, 37, 43). In Tunisia, 177 Eucalyptus species were introduced and acclimatized into more than 30 arboretums since 1957 (23). The objectives of this study were to determine the chemical composition of the essential oils of Eucalyptus camaldulensis and and to evaluate their fumigant toxicity against new emerged adults of E. ceratoniae. MATERIALS AND METHODS Insect rearing. A laboratory rearing colony was established in the Laboratory of Plant Protection at the Institut National de la Recherche Agronomique de Tunisie (INRAT) from infested field-collected dates. The moth was reared on an artificial diet based on wheat bran (60%), sucrose (12%), salt mixture (2%), yeast (1.3%), lysine (1.23%), methyl paraben (0.13%), vitamin C (0.67%), aureomycine (0.67%), glycerine (150 ml) and distilled water (150 ml) (for details see 34). Rearing was conduced in plastic boxes ( cm) placed in a rearing room. The rearing conditions were: temperature of 25 ± 1 C, photoperiod of 15: 9 (L: D) and 65 ± 5% relative humidity. Plant material. E. camaldulensis and leaves were collected from natural populations at the flowering stage on August 25, 2009 from the arboretum of Korbus (North Tunisia) of the Institut National des Recherches en Génie Rurale, Eaux et Forêts (INRGREF). The harvested material was air-dried at room temperature (20-25 C) for one week and then stored in cloth bags. Essential oil extraction and analysis. The essential oils were extracted by hydrodistillation of dried plant material (100 g of each sample in 500 ml of distilled water) using a Clevenger-type apparatus for 4 h. The oils were dried over anhydrous sodium Tunisian Journal of Plant Protection 202 Vol. 5, No. 2, 2010

3 sulphate and stored in sealed glass vials at 4-5 C prior to analysis. Yield based on dry weight of the sample was calculated. GC-MS analysis. Chemical analyses were performed at the Laboratory of Bioprocesses, Centre de Biotechnologie de Sfax (CBS), Tunisie. The essential oils were analyzed using an Agilent-Technologies 6890 N Network GC system equipped with a flame ionization detector and HP-5MS capillary column (30 m 0.25 mm, film thickness 0.25 µm; Agilent-Technologies, Little Falls, CA, USA). The injector and detector temperatures were set at 220 and 290 C, respectively. The column temperature was programmed from 80 to 220 C at a rate of 4 C/min, with the lower and upper temperatures being held for 3 and 10 min, respectively. The flow rate of the carrier gaz (Helium) was 1.0 ml/min. A sample of 1.0 µl was injected, using split mode (split ratio, 1:100). All quantifications were carried out using a built-in data-handling program provided by the manufacturer of the gas chromatograph. The composition was reported as a relative percentage of the total peak area. The identification of the essential oils constituents was based on a comparison of their retention times to n- alkanes, compared to published data and spectra of authentic compounds. Compounds were further identified and authenticated using their mass spectra compared to the Wiley version 7.0 library. The volatile compounds were ranged into groups (Monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene oxygenated, sesquiterpene hydrocarbons, other compounds). Major compounds in each group were marked in bold form. Fumigant toxicity bioassays. To assess fumigant toxicity of E. camaldulensis and essential oils, 2 cm diameter filter papers (Whatman No.1) were impregnated with the different oil doses. Doses were converted to give equivalent fumigant concentrations of to µl/l air. The impregnated filter paper was then attached to the screw caps of a 38 ml Plexiglas bottle. Caps were screwed tightly on the vials, each of which contained 10 unsexed adults (1-2 days old). Each treatment and control was replicated five times. Mortality was recorded each hour. When no leg or antennal movements were observed, insects were considered dead. Mortality data were corrected for control mortality by using Abbott s formula (1). Lethal dose bioassays. Trials were designed to assess 50% lethal doses (LC 50 values). Ten unsexed adult insects were put into 38 ml Plexiglas bottles with screw lids. Oil amounts tested were 0.5, 1, 2, 3, 4, and 5 µl corresponding to concentrations of 13.16, 26.31, 52.63, 78.95, , and µl/l air. Control insects were kept under the same conditions without any essential oil. Each dose was replicated five times. The number of dead and alive insects in each bottle was counted 24 h after initial exposure. The mortality was evaluated by direct observation of the insects every hour until total mortality. Probit analysis (16) was used to estimate LC 50 values. Lethal time bioassays. To be effective, fumigation trials should be based on the use of lower fumigant concentrations leading to high mortality in short time. Therefore, a bioassay was designed to determine median effective Tunisian Journal of Plant Protection 203 Vol. 5, No. 2, 2010

4 times (LT 50 ) to cause mortality of 50% of tested insects at the lowest doses (13.16 and µl/l air). The mortality was assessed by direct observation of insects every hour until all the insects were dead. Data were analyzed using Finney s method (16). RESULTS Essential oils composition. GC and GC-MS analysis of E. camaldulensis and essential oils were respectively reported in Tables 1 and 2. The oil yields based on dry matter weight were respectively 1.32% for E. camaldulensis and 2.09% for. Table 1. Chemical fractions, others constituents and total identified compounds of the essential oil obtained from leaves of E. camaldulensis collected at flowering stage (%) N Compound Percent. (%) RT KI Monoterpene hydrocarbons α-pinène γ-terpinene Dehydro-p-cymene Oxygenated monoterpenes ,8-cinéole Terpinéne-4-ol Isothymol Piperitone Sesquiterpene hydrocarbons Aromadendrene Oxygenated sesquiterpenes Spathulenol Viridiflorol t-muurolol Other compounds Total RI, KI were respectively Retention Index and Kováts Index calculated on a HP-5MS capillary column (30 m 0.25 mm 0.25 µm). A total of 94.02% from the constituents of E. camaldulensis leaf essential oil were identified (Table 1). Among them, 22.52% were monoterpene hydrocarbons, 33.82% oxygenated monoterpenes, 6.65% oxygenated sesquiterpenes and 3.93% sesquiterpene hydrocarbons. The major compounds were α-pinene (16.49%), 1,8-cineole (20.62%), p-cymene (21.69%), isothymol (7.3%), γ-terpinene (4.8%), and terpinene-4-ol (4.45%). Tunisian Journal of Plant Protection 204 Vol. 5, No. 2, 2010

5 Table 2. Chemical fractions, others constituents and total identified compounds of the essential oil obtained from leaves of collected at flowering stage (%) N Compound Percent. (%) RT KI Monoterpene hydrocarbons α-pinene β-pinene γ-terpinene Oxygenated monoterpenes ,8-cineole Terpinene-4-ol α-terpineol Sesquiterpene hydrocarbons Aromadendrene Bicyclogermacrene Oxygenated sesquiterpenes Isospathulenol t-muurolol Others compounds Total RI, KI were respectively Retention Index and Kováts Index calculated on a HP-5MS capillary column (30 m 0.25 mm 0.25 µm). For leaf essential oil, a total of 78.11% of the constituents was identified (Table 2). The oxygenated monoterpenes represented 28.85% followed by the monoterpene hydrocarbons 24.44%, the sesquiterpene hydrocarbons 9.22% and the oxygenated sesquiterpenes 5.47%. The main constituents were α-pinene (14.49%), β- pinene (3.91%), 1,8-cineole (19.87%), γ- terpinene (6.04%), terpinene-4-ol (4.46%), α-terpineol (4.32%) and aromadendrene (6.37%). ceratoniae adults exposed for various periods of time to essential oils from E. rudis and E. camaldulensis. Results showed that E. camaldulensis and essential oils were toxic to E. ceratoniae adults (Fig. 1). E. camaldulensis was more toxic than for the concentrations exceeding µl/l air. At the lowest concentration (13.16 µl/l air), achieved 25% of mortality after 24 h of exposure compared with 10% of mortality for E. camaldulensis. However, at the highest concentration ( µl/l air), 100% mortality was recorded for E. camaldulensis and essential oils Fumigant toxicity. Mortality was corrected for control mortality by using Abbott s formula (1). Results were presented as percentage of mortality of E. after only 12 h of exposure (Fig. 1). Tunisian Journal of Plant Protection 205 Vol. 5, No. 2, 2010

6 Dose µl/ l air E. camaldulensis Control Dose µl/l air E. camaldulensis Control Mortality (%) Mortality (%) Exposure time (h) Exposure time (h) Dose µl/l air E. camadulensis Control Dose µl/l air E. camaldulensis Control Mortality (%) Mortality (%) Exposure time (h) Exposure time (h) Mortality (%) Dose µl/l air E. camaldulensis Control Mortality (%) Dose µl/l air E. camaldulensis Control Exposure time (h) Fig. 1. Percentage of mortality of E. ceratoniae adults exposed for various periods of time to different doses of essential oils from and E. camaldulensis Exposure time (h) Probit analysis showed that E. ceratoniae was more sensitive to essential oil than E. camaldulensis one. The corresponding LC 50 values were and µl/l air, respectively (Table 3). The LT 50 values ranged from h for the concentration µl/l air to h for the concentration µl/l air for essential oil. Nevertheless, for E. camaldulensis essential oil, the LT 50 values were and h for the concentrations and µl/l air, respectively (Table 4). Tunisian Journal of Plant Protection 206 Vol. 5, No. 2, 2010

7 Table 3. LC 50 values calculated for mortality within 24 h of exposure of E. ceratoniae adults to and E. camaldulensis essential oils Plant species E. camaldulensis LC a,b 50 (LD 50) ( ) ( ) Slope ± SEM 2.17 ± ± 0.88 ( ) ( ) Degree of freedom χ a Units LC 50 = µl/ air, applied for 24 h at 25 C. b 95% lower and upper confidence limits are shown in parenthesis. Table 4. LT 50 values calculated for E. ceratoniae adults exposed to and E. camaldulensis essential oils Essential oil Concentration LT (µl/l air) 50 (h) a Degree of Slope ± SE freedom χ ± 0.47 ( ) ( ) ± 0.31 ( ) ( ) ± E. ( ) ( ) camaldulensis ± ( ) ( ) a 95% lower and upper confidence limits are shown in parenthesis. DISCUSSION cineole content in eucalyptus essential This study showed that the essential oils and their toxicity to insects. oils of E. camaldulensis and Furthermore, this compound when collected from Korbus were rich in present in eucalyptus essential oils monoterpenoids. Moreover, the major showed promising fumigant toxicity (36, common compounds of these two oils 38, 40). Additionally, Yang et al. (52) were 1,8-cineole ( %), α- attributed the toxicity of E. globulus pinene ( %), γ-terpinene (4.8- essential oil against the eggs and adults of 6.04%) and terpinene-4-ol ( %). Pediculus humanus capitis (Anoplura: Among various compounds of Pediculidae) to the action of 1,8 cineole. eucalyptus essential oils, 1,8-cineole is Besides, Lee et al. (24) reported that the most important. This compound is essential oils rich in 1,8-cineole presented characteristic of the Eucalyptus genus and a strong toxicity against Sitophilus is mainly responsible of its insecticidal oryzae, Tribolium castaneum and proprieties (5). Indeed, 1,8 cineole which Rhyzopertha dominica. is the major compound of E. Moreover, the pesticidal activity of camaldulensis and essential oils eucalyptus oils has been due to the was recognized to be toxic on several components such as α-pinene and γ- insect species. Lucia et al. (31) indicated terpinene (4, 27, 28, 29, 39, 47). In this the presence of correlation between 1,8- Tunisian Journal of Plant Protection 207 Vol. 5, No. 2, 2010

8 context, several works reported that α- and Pistacia lentiscus essential oils pinene is listed among major constituents against E. ceratoniae. The author associated with pesticidal activities (9, 30, indicated that 100% of mortality was 45). Besides, Su et al. (47) indicated that obtained for these two oils at γ-terpinene is involved in biological concentrations and µl/l air activities of eucalyptus essential oils. after 18 and 36 h of exposure, On the other hand, terpinen-4-ol which also represents a major compound of these two essential oils was reported to respectively. On the other hand, E. camaldulensis essential oil was reported to be toxic be toxic on several insect species. Indeed, against stored-product beetles (35). Kordali et al. (21) indicated that terpinen- 4-ol was the most toxic against Sitophilus granaries adults after 12 h of exposure. Furthermore, this compound showed promising fumigant toxicity on the rice Moreover, Tunç et al. (48) indicated that E. camaldulensis essential oil induced high mortality to E. kuehniella and Tribolium confusum. Besides, Winters et al. (50) demonstrated that and E. weevil Sitophilus oryzae (25). camaldulensis essential oils were Additionally, terpinen-4-ol showed characterized by similar emission of relatively strong toxicity against the monoterpenes with respective values of larvae and adults of Leptinotarsa and µg C m -2 h -1. decemlineata (22). The present work reported first This work demonstrated that investigations on the fumigant activity of essential oils of and E. E. camaldulensis and essential camaldulensis displayed strong fumigant oils against E. ceratoniae in storage. toxicity against new emerged adults of Results clearly demonstrate the the date moth E. ceratoniae. The insecticidal activity of these essential oils insecticidal activity depends on oil against this major pest of stored dates. species, concentration and exposure time. Few works have been reported on the fumigant activity of essential oils against the date moth E. ceratoniae. Mansouri (32) first investigated the fumigant toxicity of Lavandula officinalis Thus, they could be used as an alternative to the synthetic fumigant in postharvest treatment programs. Accordingly, there is an interest to further investigate the essential oil toxicity on other stored date s pests. RESUME Haouel S., Mediouni-Ben Jemâa J. et Khouja M.L Lutte en post-récolte contre la pyrale des dates Ectomyelois ceratoniae moyennant la fumigation aux huiles essentielles d'eucalyptus. Tunisian Journal of Plant Protection 5: Ce travail a pour objectifs d étudier la composition chimique et d évaluer pour la première fois l effet fumigène des huiles essentielles de deux espèces d eucalyptus (Eucalyptus camaldulensus et ) contre la pyrale des dattes Ectomyelois ceratoniae en entrepôts comme alternative au bromure de méthyle. Les analyses CPG-SM ont montré que l huile essentielle d présente α-pinène (14.49%), 1,8-cinéole (19.87%), β-pinène (3.91%), γ-terpinène (6.04%), terpinéne-4-ol (4.46%) et α- terpinéol (4.32%) comme constituants majeurs alors que celle d E. camaldulensis présente α-pinène (16.49%), 1,8-cineole (20.62%), γ-terpinène (4.08%), terpinène-4-ol (4.45%), aromadendrene (3.93%) Tunisian Journal of Plant Protection 208 Vol. 5, No. 2, 2010

9 et isothymol (7.3%) comme composés majeurs. Les résultats ont montré que l activité fumigène dépend de l espèce d huile, de la concentration et du temps d exposition. Le potentiel fumigène de l huile essentielle d (CL 50 = µl/l air) était supérieur à celui de l huile essentielle d E. camaldulensis (CL 50 = µl/l air). En outre, les valeurs respectives des temps létaux médians (TL 50 ) étaient de et h. Les résultats suggèrent que les huiles essentielles d et E. camaldulensis peuvent être utilisées comme alternatives aux fumigants de synthèse en traitement postrécolte pour la lutte contre E. ceratoniae. Mots clés: Concentration létale CL 50, CPG-SM, Ectomyelois ceratoniae, Eucalyptus camaldulensis, Eucalyptus rudis, huile essentielle, temps létal LT 50 ملخص حوال سمية وجودة مديوني-بن جماعة ومحمد العربي خوجة المكافحة في مرحلة ما بعد الجني لعثة التمر باستعمال التبخير بالزيوت العطرية للا وآالبتوس. Tunisian Journal of Plant Protection 5: Ectomyelois ceratoniae يهدف هذا العمل إلى دراسة الترآيب الكيمياي ي وتقيم الفعالية السمية بالاختناق للزيوت العطرية/الا ساسية لنوعين من الا وآليبتوس Eucalyptus camaldulensis و.E rudis لمكافحة عثة التمر Ectomyelois ceratoniae لا ول مرة في تونس في المخازن آطريقة بديلة لبروميد الميثيل. بينت التحاليل الكروماتوغرافية (GC-MS) أن الزيت الا ساسي ل.E β-pinene (3.91%) (19.87%) و 1,8-cineole و (14.49%) α-pinene يحتوي علي المكونات الا ساسية التالية: rudis و (6.04%) γ-terpinene و (4.46%) terpinéne-4-ol و (4.32%) α-terpineol بينما احتوى الزيت الا ساسي ل.E γ-terpinene (20.62%) و 1,8-cineole و (16.49%) α-pinene على العناصر الا ساسية التالية: camaldulensis (4.08%) و (4.45%) terpinene-4-ol و (3.93%) aromadendrene و (7.3%). isothymol بينت النتاي ج أن الفعالية السمية بالاختناق للزيوت الا ساسية تختلف حسب نوع الا وآليبتوس وترآيز الزيت ومدة التعرض. آانت الفعالية السمية للزيت الا ساسي ل (LC 50 = µl/l air).e rudis أفضل من الفعالية السمية للزيت الروحي ل.E camaldulensis air).(lc 50 = µl/l إضافة إلي ذلك بلغ الوقت القاتل ) 50 (TL على التوالي و ساعة بالنسبة لهذين الزيتين وأظهرت النتاي ج أنه يمكن استعمال الزيوت الا ساسية ل.E rudis و ل.E camaldulensis آطريقة بديلة للمبخرات الصناعية في برنامج معاملة ما بعد القطاف لمكافحة عثة التمر.E. ceratoniae آلمات مفتاحية: التحاليل الكرومتغرافية الترآيز القاتل الزيوت الروحية عثة التمر الوقت القاتل Eucalyptus camaldulensis, LITERATURE CITED 1. Abbott, W.S A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: Al-Azawi, A.F., El-Haidari, H.S., Aziz, F.M., Murad, A.K., and Al-Saud, H.M The effect of high temperatures on the dried fruit beetle Carpophilus hemipterus L., a pest of stored dates in Iraq. Date Palm Journal 3: Azelmat, K., El Garrouj, D., Mouhib M., and Sayah F Irradiation of Bouffegous dates: Effects on chemical composition during storage. Postharvest Biology and Technology 39: Batish, D.R., Singh, H.P., Setia, N., Kaur, S., and Kohli, R.K Chemical composition and phytotoxicity of volatile essential oils from intact and fallen leaves of Eucalyptus citriodora. Z. Naturforsch. 61: Batish, D.R., Sing, P.H., Kohli, K.R., and Kaur, S Eucalyptus essential oil as a natural pesticide. Forest Ecology Management 256: Bell, C.H Fumigation in the 21 st century. Crop Prot. 19: Bignell, C.M., Dunlop, P.J., and Brophy, J.J Volatile leaf oils of some Queensland and northern Australian species of the genus Eucalyptus (series II) part II. Subgenera (a) Tunisian Journal of Plant Protection 209 Vol. 5, No. 2, 2010

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