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Biological Performance of Eucalyptus Camaldulensis Leaf Oils from Thailand Against the Subterranean Termite Coptotermes Formosanus Shiraki

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Biological Performance of Eucalyptus Camaldulensis Leaf Oils from Thailand Against the Subterranean Termite Coptotermes Formosanus Shiraki J Wood Sci (2009) 55:41–46 © The Japan Wood Research Society 2008 DOI 10.1007/s10086-008-0990-4 ORIGINAL ARTICLE Pornpun Siramon · Yoshito Ohtani · Hideaki Ichiura Biological performance of Eucalyptus camaldulensis leaf oils from Thailand against the subterranean termite Coptotermes formosanus Shiraki Received: April 16, 2008 / Accepted: August 20, 2008 / Published online: October 10, 2008 Abstract The antitermitic activities of leaf oils and their countries such as Japan, Taiwan, and southern parts of the constituents, taken from three clones of Eucalyptus camal- United States. Thus, it has been used for bioassays in many dulensis Dehnh. in Thailand, against Coptotermes formosa- research studies.2–4 nus Shiraki were investigated in contact and noncontact Synthetic chemicals are currently used for controlling tests. The termiticidal mechanism was also examined. termites. However, the conventional termiticides, such as Antitermitic tests demonstrated that E. camaldulensis leaf aldrin, chlordane, dieldrin, endrin, heptachlor, and mirex, oils were both contact toxicants and fumigants to C. formo- which are the chemicals identifi ed as persistent organic pol- sanus with LC50 values ranging between 12.68 and 17.50 mg/ lutants (POPs), have been known to cause negative impacts g by the contact method, and between 12.65 and 17.50 mg/ on global environments, including health hazards to humans petri dish (100 cm3) by the noncontact method. p-Cymene and other nontarget species. The application of these chem- and g-terpinene were primarily responsible for the contact icals now tends to be restricted.5 To avoid those impacts, toxicity and 1,8-cineole was responsible for fumigation. there is a trend toward identifying naturally occurring pes- From the investigation of termiticidal mechanism, E. camal- ticides. Many phytochemicals are known to possess anti- dulensis leaf oils exhibited the inhibition of acetylcholines- termitic or repellent activities. Among them, plant essential terase activity and showed the common symptoms of a oils may provide potential alternatives to termite control- neurotoxic mode of action against C. formosanus. ling agents currently in use because they constitute a rich source of bioactive chemicals. Recently, essential oils have Key words Antitermitic activity · Coptotermes formosanus been the subject of increasing attention as sources of effec- Shiraki · Essential oil · Eucalyptus camaldulensis tive and safe insecticides in which the main components are monoterpenes and sesquiterpenes, and some of them have been found to be termiticidal.6,7 Introduction Eucalyptus camaldulensis Dehnh. is the most well- known Eucalyptus species for cultivation in Thailand. It is mainly planted for use as pulpwood and utilized as the Termites seriously damage a variety of materials, not only main raw material even at ages of 3–5 years because of wooden products but also noncellulosic materials. Damage the high growth rate. During the process of papermaking, to wooden structures and other cellulosic materials caused large amounts of waste such as leaves are generated and by termites has been estimated to exceed $3 billion annually 1 disposed of. The utilization of these waste materials is in the United States. Among many species of termites, crucial from an ecological viewpoint. Siramon and Ohtani8 Coptotermes formosanus Shiraki is the subterranean termite found that the leaf essential oils of E. camaldulensis from responsible for most destruction of wooden materials in Thailand have antioxidant properties, and suggested the potential of these materials as alternatives to synthetic antioxidants. In this study, we investigated the bioactivity of the P. Siramon · Y. Ohtani (*) · H. Ichiura essential oils from E. camaldulensis leaves against C. for- Department of Forest Science, Faculty of Agriculture, Kochi mosanus. The antitermitic activities of the essential oils University, B-200 Monobe, Nankoku, Kochi 783-8502, Japan Tel. +81-88-864-5143; Fax +81-88-864-5143 and each component against C. formosanus were examined e-mail: [email protected] using two different testing methods, namely, contact and noncontact methods. The termiticidal mechanism of these Part of this report was presented at the 58th Annual Meeting of the essential oils and their main constituents were also partly Japan Wood Research Society, Tsukuba, March 2008 investigated. 42 Materials and methods dissolved in 1 ml of methanol and then applied to 1 g of fi lter paper (Advantec No.2, 8.7 cm in diameter). A piece of fi lter paper treated with methanol only was used as a Chemicals control. After the solvent was removed from the treated fi lter papers by air drying at ambient temperature, 30 active Acetylthiocholine iodide, dithiobisnitrobenzoic acid, a- termites (28 workers and 2 soldiers) above the third instar pinene, a-terpineol, terpinen-4-ol, 1-decanol, methanol, were placed on each piece of fi lter paper in a petri dish and phosphate buffer solution were purchased from Nacalai (8.7 cm in diameter × 1.7 cm in height). Distilled water was Tesque (Kyoto, Japan). 1,8-Cineole was purchased from added occasionally onto each fi lter paper and the covered Wako (Osaka, Japan). p-Cymene and g-terpinene were pur- petri dishes were incubated in darkness at 25°C. Three chased from Tokyo Kasei Kogyo (Tokyo, Japan). replicates were made for each test sample. The mortality of the termites was counted daily for 14 days and the 50% Preparation of essential oil lethal concentrations (LC50) were estimated from linear cor- relation between the sample concentrations and termite Three clones S1, S2, and S3 of Eucalyptus camaldulensis mortality. were selected through several generations based on their The noncontact method developed by Ohtani et al.10 was pulp yields and adaptability to circumstance, etc. by Siam employed to distinguish the respiratory poisoning functions Forestry, Kanchanaburi Province, Thailand. The leaves of from termiticidal activity of essential oils. A small glass dish each clone were collected in mid-April (summer season) (2.8 cm in diameter × 1.5 cm in height) in which a treated 2007. paper disk with either 10 mg or 20 mg of essential oils was About 1 kg of E. camaldulensis fresh leaves was extracted held, was placed on the center of a fi lter paper in a larger by water and steam distillation for 5 h (until no more essen- petri dish (100 cm3 in volume). Twenty-eight workers tial oil was obtained). The essential oils were collected, and two soldiers were introduced into the outer part from dried over anhydrous sodium sulfate, and stored in sealed the inner small dish and these samples were incubated in vials at low temperature before analysis. The moisture darkness at 25°C for 7 days. Distilled water was supplied content of the leaves was determined by the weights before occasionally. and after drying at 105°C for 12 h in an oven. Termiticidal activities of each component in E. camal- dulensis leaf oils, namely, p-cymene, g-terpinene, 1,8- cineole, terpinen-4-ol, a-pinene, and a-terpineol were Gas chromatography-mass spectrometry analysis tested by the aforementioned two methods with the authen- tic chemicals. The chemical compositions of the essential oils were ana- lyzed by gas chromatography-mass spectrometry (GC-MS). The analysis used a GC-17A gas chromatograph (GC) Determination of concentration of volatile components in coupled to a QP5050A mass spectrometer (Shimadzu, the headspace air Kyoto, Japan), and the GC used a fused-silica capillary column TC-1 (0.25 mm i.d. × 15 m, 0.25 μm fi lm thickness; A paper disk treated with 0.1 g of oil sample was placed in GL Sciences, Tokyo, Japan) as described by Siramon and a 320-ml closed bottle. The bottle was left to stand for 1 h Ohtani.8 Identifi cation of the compounds was based on at room temperature. The air inside the bottle was evacu- comparison of GC-MS data with the NIST database library, ated through charcoal tubes (organic gas sampler, Shibata and most compounds such as p-cymene, g-terpinene, a- Scientifi c) with a minipump (MP-S30, Shibata Scientifi c) at pinene, 1,8-cineole, terpinen-4-ol, and a-terpineol obtained a fl ow rate of 0.2 l/min for 5 min. Thereafter, the charcoal as reagents were directly compared with authentic com- tubes were taken out and soaked in 5 ml acetone for 12 h. pounds. The calculated chemical compositions of the oils The liberated volatile substances were then analyzed and were based on the peak areas of GC chromatogram using quantifi ed by GC-MS. 1-decanol as an internal standard. Analysis of the termiticidal mechanism Antitermitic activities The termiticidal mechanism of the essential oils from E. Coptotermes formosanus was collected in May 2007 camaldulensis leaves against C. formosanus was investi- and June 2008 from fi elds in Kochi Prefecture, Japan. The gated by the three procedures described below. termites collected were reared in an incubator at 25°C. Three worker termites were placed in small glass dishes, Distilled water and fi lter papers were used as foodstuffs. and the dishes were placed on ice for 2 min. The oil samples The antitermitic activities of the essential oils and their or their main constituents (0.4 μl) were applied topically to constituents against the termites were examined using the termite abdomens using a Hamilton 701-N micro-syringe contact and noncontact methods. The contact method (Hamilton, Reno, NV, USA) for estimation of the contact developed by Ohtani et al.9 with some modifi cation was toxicity against termites. The mortality of the termites employed
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