Efficacy of Thuja orientalis L. (Cupressaceae) Essential Oil on the Two Spotted Spider Mite, Tetranychus urticae Koch (Acari: Teteranychidae)

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1 Journal of Essential Oil Bearing Plants ISSN Print: X Online: Jeobp 15 (4) 2012 pp Efficacy of Thuja orientalis L. (Cupressaceae) Essential Oil on the Two Spotted Spider Mite, Tetranychus urticae Koch (Acari: Teteranychidae) Fatemeh Mozaffari 1, Habib Abbasipour 1, Aziz Sheikhi Garjan 2, Alireza Saboori 3 and Mohammad Mahmoudvand 4 * 1 Department of Plant Protection, Faculty of Agricultural Sciences, Shahed University, Tehran, Iran 2 Institute of Plant Protection of Iran, Tehran, Iran 3 Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran 4 Department of Plant Protection, Khorramabad Branch, Islamic Azad University, Khorramabad, Iran Received 10 June 2011; accepted in revised form 27 January 2012 Abstract: Tetranychus urticae Koch is the major pest of various plants worldwide. Plant compounds are known as biopesticides recently. Essential oil of the oriental arbor-vitae, Thuja orientalis was researched on repellent and oviposition inhibition of T. urticae. The LC 50 values of fumigant toxicity of this oil on adults and eggs of the twospotted spider mite were and 8.05 μl L -1 air, respectively. Also LT 50 value of essential oil at 100 μl L -1 air was 2.06 h. On the other hand, all mites were killed after treatment with 1000 μl L -1 concentration. In addition, different concentrations of the essential oil of T. orientalis significantly affected oviposition deterrence and repellency of adults. The results of this study indicated that essential oil of T. orientalis may be considered as an biopesticide to control spider mites. Key words: Thuja orientalis, essential oil, Tetranychus urticae, oviposition detterence, repellency. Introduction The two spotted spider mite, Tetranychus urticae Koch (Acarina: Tetranychidae) is a serious pest and it infests many crops causing reduction in plant productivity or even kills the host plants 23,34. It attacks numerous greenhouse plantations causing a great problem. The ideal areas for spider mites are greenhouses that they can complete a generation in one week 13. Conventional synthetic pesticides such as chlorinated hydrocarbons, organophosphates, carbamates and pyrethroids showed a temporary success in controlling this mite pest but later posed serious problems like pesticide resistance 5, secondary pest outbreak 14, pest resurgence and toxic residues in the environment 15 as well as effect on non-target organisms 10. Spider mites have evolved resistance to more than 80 acaricides to date, and resistance has been reported from more than 60 countries 11. Plants have acquired effective defense mechanisms that ensure their survival under *Corresponding author (Mohammad Mahmoudvand) < msco_1381@yahoo.com > 2012, Har Krishan Bhalla & Sons

2 Fatemeh Mozaffari et al. / Jeobp 15 (4) adverse environmental factors. In addition to morphological mechanisms, plants have also developed chemical defense mechanisms towards organisms such as insects that affect the biochemical and physiological functions 26. An evergreen and conifer shrub, Thuja orientalis L. (Syn.: Platycladus orientalis (L.)) (in Persian: Nush, Sarv-e-Khomrehi and sarv-e-tabari) is native of Korea, Manchuria, North of China and Iran and belongs to the Cupressaceae family with a maximal height of more than 20 m. It has flat branches and twigs in one plane 4,6,21,28. One of the alternative approaches, within pest control, which may be suitable for greenhouses, is using natural fumigants, because of their safety and capability to reach every point in high-density plantation. Fumigant toxicity of several volatile oils was investigated against different pests 1,2,12,1928,29,31. Up to now there are no published reports on acaricidal activity of the essential oil of the oriental arbor vitae, T. orientalis. Being a part of systematic evaluation of the acaricidal potential of the plants, the objective of this work investigated acaricidal properties of T. orientalis leaves essential oil. They included fumigant bioassay on eggs and adults, oviposition deterrence and repellency activity against T. urticae. Materials and methods Essential oil extraction The leaves of the oriental arborvitae, Thuja orientalis are collected for extraction of the essential oil. All the collected parts were dried under indoor condition and were subjected to hydrodistillation using Clevenger type distiller 8. For extraction of essential oils, 50 g of air-dried plant material was put in water (1:12 w:v) for distillation during 4h. Extracted essential oils were dried via anhydrous sodium sulphate and stored at 4 C in darkness. For fumigant bioassay tests, pure essential oil was used. Tested mites The strain of the two spotted spider mite, Tetranychus urticae Koch was supplied from Acarology laboratory in Agriculture Faculty of Tehran University. It was reared as a test strain as the following: lima bean (Phaseolus vulgaris) seeds were planted in plastic jars (15 cm in diameter) at a rate of 4-5 seeds per jar, and incubated under muslin cage to prevent any infestation. Jars containing lima bean seedlings (15-20 cm long) were taken to the laboratory, then infested leaves of T. urticae were transferred to these plants and left to reproduce under laboratory condition at 25 ± 1 C. This colony was supplied with fresh lima bean plants from time to time according to necessity. Calculating LC 50 and LT 50 values of the oil The fumigant toxicity of essential oil on T. urticae was tested in plastic vials (140 ml) and in each of them 10 adults of mite (1-3 days old) was released on the leaves of lima bean. Filter paper discs (Whatman N 1) were cut into 2 cm diameter and were then attached to undersurface of screw caps of plastic vials. Filter papers were impregnated with series of concentrations of T. orientalis essential oil. After 24h, the dead mites were recorded. Four replications were run for each concentration and for control groups. All tests were carried out in the growth chamber at 25 ± 1 C and 70 ± 5 % RH with a 16L:8D h photoperiod. To investigate the lethal time (LT 50 ) of T. urticae, high concentrations of the essential oil was obtained and used from bioassay tests (100 and 1000 μl L -1 air). Selected concentrations similar to the mentioned method for bioassay test were tested and for each dose, four replications were performed. The mortality of mites was counted after 3, 6, 9, 12, 15, 18, 21 and 24 h and after that in 3 h intervals, mortality recording continued until 100 % mortality was reached. Toxicity of oil to the eggs For contact toxicity assay with eggs, five individuals of T. urticae females were allowed to oviposit for 24 h on the 3 cm diameter of lima bean leaf discs resting on wet cotton pads in a Petri dish (9 cm in diameter). Then they were removed and the eggs were counted. The leaf discs containing 10 eggs were put in plastic vials and for 24 h submitted to different concentrations. The

3 Fatemeh Mozaffari et al. / Jeobp 15 (4) method of this test was similar to the one used for the adulticidal assay. Oviposition deterrence To investigate the effect of essential oil on the oviposition reduction, four concentrations (500, 2500, 5000 and mg L -1 ) of essential oil of T. orientalis were prepared in mix of water and ethanol (70:30). For each treatment, 20 μl of various ethanol-water solutions was applied on the surface of leaves (3 cm in diameter). After evaporation of water and ethanol, two pairs (2 males and 2 females) of adults (3-old-days) of T. urticae for each concentration and control were selected and were placed inside a plastic vial (140 ml). For control, only ethanol and water were separately used. For each concentration, 4 replications were performed. The number of laid eggs was recorded after 24 h and oviposition deterrence was calculated with formulae as follows 25. Oviposition deterrence NE t =Number of eggs in treatment NE c =Number of eggs in control Repellency test of oil The repellency test of oil was made according to the method described by Kogan and Goeden 20. Leaf discs of lima bean of 3 cm diameter were used to evaluate the repellency of the oil. Half of the disc was immersed into an ethanolic solution of the oil in four concentrations (500, 2500, 5000 and mg L -1 ) for 5 sec. and after drying, the other half of the disk was immersed in the ethanol and water solution, which served as the control. Ten mite males and females were placed on each disc (five mites on each half of the leaf disc) and each treatment was repeated 4 times. The evaluation was made after 24 h when the numbers of mites present in each half of the leaf were counted. The repellency percentage (%R) of the oils was calculated according to the following equation: % R= C T N 100 T = mite number in the treatment C = number of mites in the control N = number of used mites in the test. Statistical analysis The lethal concentrations (LC 50 and LC 90 ) and lethal time (LT 50 ) values of mortality were assessed by Probit analysis using SAS software 30. The data from oviposition deterrence and repellency percentage were subjected to one-way ANOVA (P<0.05) after checking for normality. Means were compared by Duncan s new multiple Range Test, admitting significant differences P<0.05. The SAS software was used for all analyses 30. Results Acaricidal activities of the essential oil The LC 50 values of fumigant assay of T. orientalis essential oil on adults and eggs of the two spotted spider mite after 24 h are indicated in Table 1. Values of LC 50 bioassay on eggs and adults were and 8.05 μl L -1 air, respectively. The results show that eggs may be more susceptible than adults. Lethal time of mortality (LT 50 ) Lethal time of mortality (LT 50 ) of oil of T. orientalis on T. urticae adults was 2.06 h for 100 Table 1. Fumigant toxicity of essential oil of Thuja orientalis against eggs and adults of T. urticae after 24 h. n Df LC 50 LC 90 Slope ± SE X 2 μl L -1 air Adult ( ) ( ) 6.74 ± Eggs ( ) ( ) 0.90 ±

4 Fatemeh Mozaffari et al. / Jeobp 15 (4) mg L -1 concentration. One hundred percent mortality was found if higher concentration (1000 mg L -1 ) was used short time after treating. [Table 2] Repellency effect of essential oil Figure 1 shows the repellency effect of T. orientalis essential oil on adults of the two spotted spider mite. Essential oil had a significant repellency on adults of T. urticae. Efficacy of the high dose (10000 mg L -1 ) was more than others (df=3, 13, F=6.42, P=0.01) (Fig. 1). Oviposition deterrence of essential oil Percentage of oviposition deterrence of various water concentrations and ethalonic solutions of T. orientalis essential oil of on adults of T. urticae are shown in Table 3. There were significant differences between different tested concentrations (df=3, 13, F=21.5, P<0.0001) (Table 3). The highest reduction in oviposition rate was observed at mg L -1. Discussion and conclusion Efficacy of plant materials (extracts and Table 2. Values of lethal time of mortality (LT 50 ) of Thuja orientalis essential oil on adults of T. urticae after 24 h. Concentration (mg L -1 ) df LT 50 LT 90 Slope ± SE X ( ) ( ) 0.03 ± * * All subjects dead after a short time Table 3. Percentage (±SE) of oviposition deterrence of Thuja orientalis essential oil on adults of T. urticae Concentrations of essential oil (mg L -1 ) Mean of laid eggs ± 5.30 a ± 4.53 a ± 5.32 b ± 8.31 c Means marked with the different letters within the same row are significantly different (P<0.05; Duncan). (df=3, 13, F=21.5, P<0.0001) Repellency (%) Concentrations (mg L -1 ) Fig. 1. Repellency (%) effect of Thuja orientalis essential oil on adults of T. urticae (df=3, 13, F=6.42, P=0.01)

5 Fatemeh Mozaffari et al. / Jeobp 15 (4) essential oils) on various pests was reported in previous studies 1,22,33. Many researchers investigated efficiency of volatile oils from different families of plants against different mite species. In the current study, effect of essential oil of T. orientalis on the adults, eggs, oviposition inhibition and detergency of T. urticae were investigated. Tunc and Sahinkaya 32 proved the toxic effect of vapor of the essential oil of cumin, Cuminum cyminum L. to the carmine spider mite, Tetranychus cinnabarinus. They also reported that greenhouse pests were much more sensitive to the vapors of essential oils than stored-product pests. Oils from Artemisia absinthium and Tanacetum vulgare were toxic against T. urticae 9. Cavalcanti et al. 8 reported that materials of essential oil of Lippia sidoides Cham. had good efficiency on T. urticae. Calmasur et al. 7 investigated the acaricidal activity of Micromeria fruticosa L., Nepeta racemosa L. and Origanum vulgare L. (Lamiaceae). Efficacy tests of T. orientalis on adults and eggs of the spider mites show that this oil has a good vapour fumigant effect on this important pest. Fumigant toxicity of this essential oil on eggs was higher than on adult stage of T. urticae. The LT 50 value of 100 μl L -1 was 2.06 h and it showed the effect of this compound was fast. A few minutes after treatment by 1000 μl L -1, mites were killed. This is the first study on the toxicity of T. orientalis on a mite pest, although the oil was tested on the some insects in a few reports. Fungicidal activity of T. orientalis was approved by Guleriaa et al. 15. Similarly to our results, Papachristos and Stamopoulos 24 stated that essential oil of T. orientalis had repellent action and reduced fecundity of Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae). Jeon et al. 17 showed that T. orientalis essential oil had good larvicidal activity against the 4th-instar larvae of Aedes aegypti and Culex pipiens pallens. Kéïta et al. 18 found that another Thuja species, T. occidentalis had good fumigant toxicity on Callosobruchus maculatus (Coleoptera: Bruchidae). The tested essential oil in this study could be considered as potential alternative for synthetic miticides as their structures could lead to the development of a new type of miticidal compounds. However, further studies need to be conducted to evaluate the mode of action and costefficacy of these essential oils on wide range of pests in commercial greenhouses. Acknowledgements This work was supported by a grant from the Shahed University of Iran and carried out in Faculty of Agricultural Sciences. References 1. Abbasipour, H., Mahmoudvand, M., Rastegar, F. and Basij, M. (2010a). Insecticidal activity of Peganum harmala seed extract against the diamondback moth, Plutella xylostella. Bull. Insectol. 63: Abbasipour, H., Mahmoudvand, M., Rastegar, F. and Hosseinpour, M.H. (2011). Bioactivities of the jimsonweed extract, Datura stramonium L. (Solanaceae) on Tribolium castaneum (Coleoptera: Tenebrionidae). Turkish J. Agric. For. 35(6): Anonymous (2005). The database of arthropods resistance to pesticides [Online], Vol Available: Michigan State University, Center for Integrated Plant Systems. 4. Assadi, M. (1998). Flora of Iran. Res. Ins. For. Rangelands 21: Brattsten, L.B., Holyoke, C.W., Leeper, J.R., and Raffaet, K.F. (1986). Insecticide resistance: challenge to pest management and basic research. Science 231: British-herbal-pharmacopoeia (1983). Thuja: West Yorks, UK: British Herbal Medicine Association. 7. Calmasur, O., Aslan, I. and Sahin, F. (2006). Insecticidal and acaricidal effect of three Lamiaceae plant essential oils against Tetranychus urticae Koch and Bemisia tabaci Genn. Ind. Crops Prod. 23:

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