Insecticidal Activity of Pistacia lentiscus Essential Oil on Tribolium castaneum as Alternative to Chemical Control in Storage Olfa Bachrouch, Unité de Recherche de Physico-Chimie Moléculaire, IPEST, BP 51, 2070 La Marsa, Tunisia, Jouda Mediouni-Ben Jemâa and Ikbal Chaieb, Laboratoire de Protection des Végétaux, INRAT, 2080 Ariana, Tunisia, Thierry Talou, Laboratoire de Chimie Agro-industrielle, 118 route de Narbonne, 31077 Toulouse Cedex, France, Brahim Marzouk, Unité des Plantes Aromatiques et Médicinales, Centre de Biotechnologie du Technopole de Borj Cedria, BP901, 2050 Hammam Lif, Tunisia, and Manef Abderraba, Unité de Recherche de PhysicoChimie Moléculaire, IPEST, BP 51, 2070 La Marsa, Tunisia _______________________________________________________________________ ABSTRACT Bachrouch, O., Mediouni-Ben Jemâa, J., Chaieb, I., Talou, T., Marzouk, B., and Abderraba, M. 2010. Insecticidal activity of Pistacia lentiscus essential oil on Tribolium castaneum as alternative to chemical control in storage. Tunisian Journal of Plant Protection 5: 63-70. This study reported first investigations on fumigant toxicity of Pistacia lentiscus essential oil against third instar larvae and adults of the red flour beetle Tribolium castaneum. The essential oil tested was found to be toxic to larvae and adults. The oil showed 51 and 100% mortality of larvae and adults, respectively, at the concentration of 1023 µl/l air after 24 h of exposure. Great differences in insect mortality were observed depending on developmental stage, oil concentrations and exposure time. The fumigant toxicity potential of P. lentiscus on adults was higher (LC50 = 28.03 µl/l, LC95 = 63.46 µl/l) than on the third instar larvae (LC50 = 112.12 µl/l, LC95 = 253.53 µl/l). Results suggested that P. lentiscus essential oil may have potential as a control agent against this major stored product insect. Keywords: Anacardiacae, fumigant toxicity, lethal dose, Pistacia lentiscus, stored-product insects, Tribolium castaneum
__________________________________________________________________________ Insects are the main problem in stored grains because they affect their quantity and their quality (18). The susceptibility of stored grains to insect infestation depends on some factors such as harvest, environmental conditions,
bulk purity, storage facilities and pest control methods (17). Insect damage may account for 10-40% of loss in stored grains, worldwide (19). It is obvious that the red flour beetle Tribolium castaneum is a cosmopolitan and polyphagous stored product pest. It is among the major pests of stored grains and stored products throughout the world (32, 33). In Tunisia and North Africa, Jarraya (14) reported that this insect is amid the most important and destructive pests in mills.
Corresponding author: Olfa Bachrouch Email:
[email protected]
Accepted for publication 15 January 2010 Tunisian Journal of Plant Protection
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Fumigation is the most economical tool for managing these stored pests (3). Currently, methyl bromide is the common fumigant worldwide used for stored product protection. However, the ozone depleting effect of methyl bromide has led to restrictions on its use. The Montreal Protocol of the United Nations Environment Program (36) recommends the phasing out of methyl bromide by 2005 in developed countries and by 2015 in developing countries (20). There is, therefore, a great need, for the development of alternative control methods that would be both effective and friendly to the environment. Several alternatives have been tested as replacement for methyl bromide in the control of stored products. These include fumigants such as phosphine, sulfuryl fluoride and carbonyl sulphide (9), as well as ethyl formate (7) and newer compounds like alkylphosphines (5), cyanogens (24) and isothiocyanates (30). Moreover, carbon dioxide (CO2) has been used for disinfesting storage commodities. Annis (2) reported the toxic effects of CO2-rich atmospheres on several insect pests of stored products. Furthermore, Gamma radiation using Cobalt 60 could be employed to disinfest stored product insects (26). In addition, insect growth regulators (IGR) may be used to manage stored insect pests (21). Furthermore, potential use of semiochemicals was reported to protect stored products from insect infestation (6). Although, the effectiveness of these methods seems good, there are of global concerns about their negative effects (15, 22, 25). Therefore, among Integrated Pest Management tactics, plants played a significant role because they constitute an important source of insecticides (11). In recent years, essential oils received a great deal of attention as pest control Tunisian Journal of Plant Protection
agents. They are characterized by a low toxicity to human and animals, high volatility, and toxicity to stored grain insect pests (4, 27, 28, 31). Thus, this study was undertaken to investigate the bioactivity of Pistacia lentiscus (Anacardiacae) essential oil collected from Jebel Mansour (North Tunisia) against third instar larvae and adult insects of T. castaneum (Coleoptera: Tenebrionidae).
MATERIALS AND METHODS Insect rearing. The red floor beetle T. castaneum was reared on wheat floor (Triticum aestivum). The rearing conditions were darkness in 25 ± 1°C and 65 ± 5% (Relative Humidity). Adult insects, 1 day old and third instar larvae were used for fumigant toxicity tests. Plant material. P. lentiscus leaves were collected from natural populations at the flowering stage during May 2008 in the region of Jebel Mansour in Tunisia. A voucher specimen (P.l.08003) was deposed in the Aromatic and Medicinal Plants Laboratory (Borj Cedria Biotechnology Center). The harvested material was air-dried at room temperature (20-25°C) for one week and then stored in cloth bags. Extraction of essential oil. Essential oil was extracted from leaves (100 g of dry matter) subjected to hydrodistillation during 90 min using a modified Clevenger-type apparatus. This time was fixed after a kinetic survey. Anhydrous sodium sulphate was used to remove water after extraction. The extracted oil was stored at 4°C. Fumigant toxicity. To determine the fumigant toxicity of P. lentiscus essential oil, 2 cm diameter filter papers 64
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(Whatman No.1) were impregnated with the tested oil doses, calculated to give equivalent fumigant concentrations of 227.3-1023 µl/l air for larvae and adults of T. castaneum, respectively. The impregnated filter paper was then attached to the screw caps of a 44 ml Plexiglas bottle. Caps were screwed tightly on the vials, each of which contained separately 10 adults (1-7 days old) of each species. Each treatment and check was replicated five times. Mortality was recorded after 3, 6, 9, 12 and 24 h of exposure. When no leg or antennal movements were observed, insects were considered dead. The mortality was calculated using the Abbott correction formula (1). A second experiment was designed to assess 50% and 95% lethal doses. A series of dilutions was prepared to evaluate mortality of insects after an initial dose-setting experiment. Ten adult insects were put into 44 ml Plexiglas bottles with screw lids. Oil amounts tested on T. castaneum were 10, 15, 20, 25, 30 and 45 µl corresponding to concentrations of 227, 341, 454, 568 and 1023 µl/l air, respectively. Control insects were kept under the same conditions without any essential oil and 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 till total mortality. Probit analysis (11) was used to estimate LC50 and LC95 values. Moreover, a bioassay was designed to determine median effective time to cause mortality of 50% of test insects (LT50 values) at 227, 341 and 454 µl/l air. The mortality was assessed by direct observation of insects every hour up to total mortality. Data showing time Tunisian Journal of Plant Protection
mortality for each experiment were analyzed by Finney’s method (10).
RESULTS Fumigant toxicity. P. lentiscus essential oil was toxic for both larvae and adults of T. castaneum (Fig. 1). The fumigant impact was clearly more significant to adults than to larvae. The results showed that fumigant toxicity varied depending on developmental stage, oil concentration and exposure time. The highest concentration of the oil (1023 µl/l air) caused 100% mortality of adults and 51% mortality of larvae after 24 h of exposure. Meanwhile, at the concentration of 568 µl/l air, P. lentiscus essential oil led to 32.5 and 16% mortality for adults and larvae, respectively, after 24 h exposure. At the lowest concentration of the oil (227 µl/l air), no mortality was recorded until 24 h of exposure where 5 and 3% mortality of adults and larvae, respectively, were accomplished (Fig. 1). Lethal concentrations. Probit analysis showed that T. castaneum adults were more susceptible to P. lentiscus essential oil than larvae. The corresponding LC50 and LC95 were respectively 28.03 and 63.46 µl/l air against 112.12 and 253.84 µl/l air (Table 1). Lethal time. According to LT50 bioassay results, the dose response of the red floor beetle adults was better than the larvae. The LT50 values of adults ranged from 14.79 h for the lowest dose (227 µl/l air) to 41.05 h for the highest dose (454 µl/l air). Although for larvae, the LT50 values ranged from 31.09 h to 12.7 h for the lowest and the highest doses, respectively (Table 2). 65
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100
100
Dose : 227 µl/l air
Adults Larvae
Dose : 341 µl/l air
60 40 20 0
60 40 20 0
0
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Dose : 454 µl/l air
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Mortaliy (%)
Mortality (%)
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80 60 40 20
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Dose : 682 µl/l air
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Dose : 1023 µl/l air
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60 40 20 0
0 0
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6 9 Exposure time (h)
Fig. 1. Percentage of mortality of larvae and adults of T. castaneum exposed to various periods of time to essential oil from P. lentiscus
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Table 1. LC50 and LC95 values of P. lentiscus essential oil against larvae and adults of T. castaneum Developmental Slope ± Degree of Chi square LC50a,b LC95a,b stage SEM freedom ( χ 2) 112.12 253.84 Larvae 2.3 ± 0.59 10 12.13 (99.09-125.15) (220.6-287.08) 28.03 63.46 Adults 4.64 ± 0.69 10 22.52 (20.14- 35.93) (48.56-105.97) a Units LC50 and LC95 =µl/ air, applied for 24 h at 25°C. b 95% lower and upper fiducial limits are shown in parenthesis
Table 2. LT50 values of P. lentiscus essential oil against larvae and adults of T. castaneum Developmental stage
Concentration (µl/l air)
LT50 (h)1
568 31.09 (27.50-35.35) 682 22.03 (19.23-27.73) 1023 12.7 (10.5-16.02) 227 41.05 (37.51-46.21) Adults 341 21.44 (18.23-26.87) 454 14.79 (12.27-18.65) 1 95% lower and upper fiducial limits are shown in parenthesis Larvae
Degree of freedom
Chi square ( χ 2)
3.90 ± 0.98 2.03 ± 0.25 1.69 ± 0.14 4.70 ± 0.88 2.06 ±0.35 1.78 ± 0.29
5 5 5 5 5 5
4.32 3.67 2.57 2.35 5.98 1.87
castaneum and two other stored-product insects (23). Essential oil from Ellatoria cardamomum showed significant fumigant toxicity against different development stages of T. castaneum (12, 13). Vitex pseudo-negundo essential oil was also active against T. castaneum (29). According to our results, larvae of T. castaneum were tolerant to fumigant toxicity of P. lentiscus essential oil compared to adults. These findings are in agreement with those obtained in other studies (13, 37). On the other hand, P. lentiscus essential oil has potent insecticidal activity. In this context, it was indicated that significant effects were detected with the combination of P. lentiscus + Mentha microphylla and P. lentiscus + Myrtus communis essential oils using the topical
DISCUSSION In this work, the essential oil of P. lentiscus demonstrated fumigant toxicity against the red flour beetle T. castaneum. The insecticidal activity depends on developmental stage, oil concentration and exposure time. The results showed that adults were more susceptible than the third instar larvae. Many studies have been reported on the insecticidal activity of several essential oil species against this pest. It was indicated that Artemisia tridentata and A. vulgaris essential oils have an important fumigant effect against adults, larvae and eggs of T. castaneum (8, 37). Essential oil of A. annua showed fumigant activity against T. castaneum (35). Fumigant toxicity of A. sieberi essential oil was investigated on T. Tunisian Journal of Plant Protection
Slope ± SE
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application technique, so they achieved activity against the red flour beetle T. total mortality against the mosquito Culex castaneum. Thus, it could be used as an pipiens (34). In addition, it was alternative to the synthetic fumigant in mentioned that P. lentiscus essential oil current usage in Integrated Pest Management Program. Consequently, was active against eggs and adults of the Hessian fly Mayetiola destructor (16). there is an interest to further investigate This work demonstrated that P. the essential oil bioactivity on other lentiscus essential oil has a fumigant stored product pests. __________________________________________________________________________ RESUME Bachrouch O., Mediouni-Ben Jemâa J., Chaieb I., Talou T., Marzouk B. et Abderraba M. 2010. Activité insecticide de l’huile essentielle du pistachier lentisque Pistacia lentiscus contre Tribolium castaneum comme alternative au traitement chimique en stockage. Tunisian Journal of Plant Protection 5: 63-70. Cette étude concerne une évaluation de l’effet fumigène de l’huile essentielle du pistachier lentisque Pistacia lentiscus sur le troisième stade larvaire et les adultes du charançon roux de la semoule Tribolium castaneum. L’huile essentielle testée a montré une toxicité vis-à-vis des larves et des adultes. Le taux de mortalité est de l’ordre de 51% pour les larves et de 100% pour les adultes à une concentration de 1023 µl/l air après 24 h d’exposition. Les résultats ont démontré une différence significative en terme de mortalité et qui dépend du stade de développement de l’insecte, de la concentration de l’huile et du temps d’exposition. L’effet fumigène de l’huile essentielle de P. lentiscus est plus important sur les adultes (LC50 = 28.03 µl/l, LC95 = 63.46 µl/l) que sur les larves (LC50 = 112.12 µl/l, LC95 = 253.53 µl/l). Les résultats indiquent que l’huile essentielle de P. lentiscus présente un potentiel insecticide qui pourrait être utilisé dans le cadre de la lutte contre cet important insecte des denrées stockées. Mots clés: Anacardiacae, dose létale, effet fumigène, insecte des denrées stockées, Pistacia lentiscus, Tribolium castaneum
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