Repellency, Fumigant and Contact Toxicities of Melaleuca cajuputi Powell against Sitophilus zeamais Motschulsky and Tribolium castaneum Herbst

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1 Thai Journal of Agricultural Science 2009, 42(1): Repellency, Fumigant and Contact Toxicities of Melaleuca cajuputi Powell against Sitophilus zeamais Motschulsky and Tribolium castaneum Herbst Ko Ko 1, W. Juntarajumnong 2 and A. Chandrapatya 2, * 1 Plant Protection Division, Myanma Agriculture Service Bayint Naung Road, West Gyogon Insein P.O , Yangon, Myanma 1 Department of Entomology, Faculty of Agriculture, Kasetsart University Chatuchak, Bangkok 10900, Thailand *Corresponding author. agramc@ku.ac.th Abstract Melaleuca cajuputi (Cajuput tree) which can be found in Myanmar (Burma) and Thailand was examined for insecticidal properties against Sitophilus zeamais and Tribolium castaneum. The results showed that M. cajuputi leaf essential oil had repellency, fumigant and contact toxicities against these two insects. The 100% repellency was only occurred in T. castaneum at 2 h and 5 h. Probit analysis showed that S. zeamais adults were more susceptible than T. castaneum. In fumigant assays, LC 50 value for S. zeamais was µl L -1 and for T. castaneum was µl L -1. The LD 50 values for S. zeamais and T. castaneum in contact toxicities were and µl insect -1. Melaleuca cajuputi leaf essential oil could be used as an alternative grain protectant for stored-product insects and further investigation should be done for other stored-product insects. Keywords: contact, fumigant, grain protectant, Melaleuca cajuputi, repellency Introduction Insect pest damage to stored grains is known to cause major economic losses to warehouse keepers, the milling industry and small scale farmers throughout the world. This problem is greatest in developing countries. The global post-harvest grain losses caused by insect damage and other bioagents vary from 10% to 40% (Raja et al., 2001; Papachristos and Stamopoulos, 2002). In order to reduce the losses in postharvest systems from insect infestation, synthetic insecticides that are used as fumigants, are routinely employed despite their undesirable side effects such as ozone depletion and environmental pollution (World Meteorological Organization (WMO), 1995), toxicity to non-target organisms, pest resistance (Mohan and Fields, 2002) and pesticide residues (Kostyukovsky et al., 2002; Ogendo et al., 2003). Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst) are two of the most important pests in stored grains. Sitophilus zeamais is an internal feeder causing considerable loss to cereals affecting the quantity as well as quality of the grains (Gupta et al., 1999). Tribolium castaneum is a major pest in storage of grain-based products, but milled grain products such as flour appears to be preferred food as well (Campbell and Runnion, 2003). At present, the natural compounds such as essential oils are alternatives to synthetic pesticides to control post-harvest insects since they are more acceptable both environmentally and to the consumer (Markham, 1999). The toxicity of a large number of essential oils and their constituents has been evaluated against a number of stored-product insects (Paranagama et al., 2003). Melaleuca cajuputi Powell (Myrtaceae) naturally occurs in Myanmar (Burma) and Thailand through Southeast East Asia to northern Australia (Weiss, 1997). The leaves of M. cajuputi possess antibacterial, anti-inflammatory and anodyne properties and are reputed to have insect-repellent properties. It is also used as flavor in cooking and as a fragrance

2 28 Ko Ko et al. Thai Journal of Agricultural Science and freshening agent in the soaps, cosmetics, detergents and perfumes (Doran, 1999). So, leaf essential oil of M. cajuputi is considered to be used as safe alternative botanical insecticide for controlling stored product insects. At present, there is no report on insecticidal activities of M. cajuputi against stored product pests. Therefore, this experiment was carried out to determine the possible insecticidal activities of the leaf essential oil of M. cajuputi on S. zeamais and T. castaneum. Materials and Methods Insects Sitophilus zeamais and T. castaneum from Department of Agriculture, Ministry of Agriculture and Co-operatives, Thailand were used throughout this study. Sitophilus zeamais was reared on rice 12 13% moisture content while T. castaneum was reared on rice bran. The cultures were maintained in the laboratory at C and 70 80% RH. Extraction of the Essential Oils Fresh leaves of M. cajuputi were collected at Kasetsart University, Bangkhen campus, (13º98 N, 48º18 E) in June 2007 and the voucher specimens (#CHKU 00028) was deposited at the Bangkok Herbarium, Botanical Research Unit, Department of Agriculture, Bangkok, Thailand. The essential oil was extracted by waterdistillation using a Clevenger-type apparatus for 6 h. The superior phase was collected from the condenser, dried over anhydrous sodium sulphate and stored in amber-colored vials at 10-12ºC for further experiments. Essential oil was analyzed by GC/MS (Shimadzu capillary GC-quadrupole MS system QP 5050A) equipped with a DB-5 capillary column (60 m, 0.25 mm, 0.25 µm film thickness) ZJ and W Scientific. The column temperature was programmed at 60ºC for 5 min then increased at 1ºC min -1 to 80ºC, finally 4ºC min -1 to 200ºC and held for 10 min. The injector and detector temperatures were 250ºC. Helium was the carrier gas, at a flow rate of 1.2 ml min -1. The injection volume was 1 µl with split ratio 1:7. Essential oil components were identified by comparing their GC retention times and their mass spectra with those presented in the MS library. Repellent Activity The repellent activity was performed using petri dishes (9 cm in diameter) to confine the insects during the experiment. Essential oils of M. cajuputi were diluted in ethanol to different concentrations (0.5%, 1%, 1.5% and 2% or 0.16, 0.31, 0.47 and 0.63 µg cm -2 ) and absolute ethanol was used as the control. Each filter paper of 9 cm diameter was cut in half. One ml of the tested material was then applied separately onto one half of the filter paper as uniformly as possible. The other half (control) was treated with 1 ml of absolute ethanol. Both the treated half and the control half were air-dried to evaporate the solvent completely. A full disc was carefully remade by attaching the tested to the control halves with tape. Precaution should be taken so that the distance between the filter-paper halves was sufficient to prevent seepage of test samples from one half to another. Each remade filter paper was placed in a petri dish with the seam oriented in one of four randomly selected different directions to avoid any insecticidal stimuli affecting the distribution of insects. Ten insects were released in the center of each filter-paper disc and a cover was placed on petri dish. Five replicates were used and the experiment was repeated once. The number of insects presented on each strip was counted at hourly interval up to the fifth hour. The percent repellency of the essential oil was calculated using the formula PR (%) = [(N c N t )/(N c + N t )] x 100 where N c was the number of insects on the control half and N t was the number of insects on the treated half. Fumigant Toxicity Filter paper discs of 2-cm diameter were impregnated with oil at doses calculated to give equivalent fumigant concentrations of 37, 56, 94, 130, 185, 296, 370, 444 and 556 µl L -1 in air. The impregnated filter paper was then attached to the under-surface of the screw cap of a glass vial (27 ml). Ten 1-7 days old adults of either S. zeamais or T. castaneum were placed in each vial before the caps were screwed tightly. Each treatment was replicated five times. Number of dead insects was determined at 3, 6, 9, 12 and 24 h after exposure. Insects were considered dead when no leg or antennal movements were observed. Mortality rate was calculated using the Abbott s formula for

3 Vol. 42, No.1, 2009 Melaleuca cajuputi against S. zeamais and T. castaneum 29 natural mortality in untreated controls (Abbott, 1925). Probit analysis was used to estimate the LC 50 and LC 95 values. The experiment was arranged by randomized complete block design and ANOVA was computed using SPSS 16.0 software package. Contact Toxicity Aliquots of 0.5 ml of each dilution (10%, 20%, 30% and 40%) were applied topically to the thorax of S. zeamais and T. castaneum adults using a Burkard Arnold microapplicator (Burkard Manufacturing Company Ltd., England). Control insects were treated with absolute ethanol. Both treated and control insects were transferred to glass vials (10 insects vial -1 ) (2 cm dia. and 5.5 cm height) and kept in incubators set at C and 58-62% RH. Culture media were added to each treatment after 24 h. The number of dead insects was recorded daily until end-point mortality was reached 1 week after treatment. Results Repellent activity Melaleuca cajuputi moderately repelled S. zeamais and T. castaneum except the highest concentration where 100% repellency could be detected for T. castaneum. The repellency for S. zeamais and T. castaneum was not statistically different in all concentrations (Table 1). Still, the repellency for T. castaneum was obvious when compared to the lowest concentration and the highest one (36 and 96% respectively). Generally, repellency increased with concentration. The 100% repellency could occur in T. castaneum at 2 h and 5 h. However, 100% repellency did not occur in S. zeamais during 5 h duration. Table 1 Percent repellency (PR) of the leaf essential oil of Melaleuca cajuputi to Sitopilus zeamais and Tribolium castaneum using treated filter paper test. Insect species Oil PR (Mean% ± SD) hours after insect release (µg cm -2 ) (Mean %) S. zeamais ± 36a 60 ± 42a 68 ± 18a 68 ± 39a 72 ± 39a ± 22a 48 ± 22a 68 ± 23a 68 ± 33a 56 ± 33a ± 43a 80 ± 28a 90 ± 33a 72 ± 32a 60 ± 32a ± 45a 72 ± 22a 72 ± 23a 72 ± 33a 76 ± 33a 69.6 F (3, 16) P T. castaneum ± 57a 64 ± 41a 44 ± 77a 48 ± 69a 44 ± 77a ± 43a 64 ± 38a 56 ± 54a 56 ± 52a 48 ± 64a ± 18a 68 ± 11a 68 ± 18a 68 ± 18a 56 ± 33a ± 18a 100± 0a 96 ± 9a 96 ± 9a 100 ± 0a 96.0 F (3, 16) P Values were based on 4 levels of content (0.16, 0.31, 0.47 and 0.63 µg cm -2 ), five replicates of 10 insects in each replication. For each insect species, means in the same column followed by the same letters do not differ significantly (P > 0.05) as determined by Lsd test. 2/ Values were means of 4 levels of content (0.16, 0.31, 0.47 and 0.63 µg cm -2 ) over the 5 h duration (at 1, 2, 3, 4, 5 hours after insects were released). PR 2/

4 30 Ko Ko et al. Thai Journal of Agricultural Science Table 2 Fumigant toxicity of Melaleuca cajuputi leaf essential oil against Sitophilus zeamais and Tribolium castaneum. Insect species LC 50 S. zeamais ( ) T. castaneum ( ) LC ( ) ( ) Slope ± SE Degree of freedom Chi-square (x 2 ) ± ± Units LC 50 and LC 95 = µl L -1 air, applied for 24 h at 27 C; 95% lower and upper fiducial limits are shown in parenthesis. Fumigant Toxicity Melaleuca cajuputi leaf essential oil showed fumigant toxicity against S. zeamais and T. castaneum. However, S. zeamais (LC 50 = µl L -1 ) was considerably more susceptible than T. castaneum (LC 50 = µl L -1 ) (Table 2). Essential oil of Evodia rutaecarpa Hook f. et Thomas had fumigant toxicity to S. zeamais and T. castaneum with the LC 50 values of 41 and µl L -1 air, respectively (Liu and Ho, 1999). Negahban et al. (2006) also demonstrated that Artemisia sieberi Besser essential oil had fumigant toxicity to T. castaneum with the LC 50 value of µl L -1 air. In addition, Don-Pedro (1996) showed that limepeel oil exhibited fumigant toxicity against S. zeamais (LC 50 = µl L -1 ). We could conclude that fumigant toxicity of our studies was not so good as their findings. Contact Toxicity Contact toxicity of M. cajuputi oil was very obvious when applied topically to the dorsal surface of S. zeamais and T. castaneum. On the basis of the LD 50 and LD 95 values, S. zeamais adults were more susceptible to the essential oil of M. cajuputi than T. castaneum (Table 3). Discussion The result on chemical constituents revealed that terpiniolene, γ terpinene and ρ-cymene were major components of M. cajuputi leaf essential oil. Other minor constituents were terpine-4-ol, α-pinene, limonene, α-terpinene and α-terpineol. Erler (2005) showed that γ-terpinene and terpine-4-ol were the promising fumigants against T. confusum and Ephestia kuehniella Zeller. As these two chemical constituents were presented in our tested plant, we could conclude that these constituents might also exert fumigant effect on S. zeamais and T. confusum (Table 4). Moreover, α-pinene (4.26%), limonene (2.91%), α-terpinene (4.44%) and α- terpineol (1.09%) found in M. cajuputi leaf essential oil also known to exhibit insecticidal activity. For example, ρ-cymene had fumigant toxicity on Acanthosceloides obtectus (Say) (Regnault-Roger and Hamraoui, 1995) and α- pinene was reported to be toxic to T. confusum (Ojimelukwe and Alder, 1999). Prates et al. (1998) also showed fumigant activity of limonene against T. confusum. In addition, the most repellent compound in Baccharis salicifolia (Ruiz and Pav.) Pers. Essential oil against T. confusum was α- terpineol (Garcia et al., 2005). Lee et al. (2001a) demonstrated that ρ-cymene, α-terpinene, α terpeneol and terpine-4-ol had the possible fumigant toxicity to S. oryzae. Hence, these early findings supported the present experiment. Recently, Rajendran and Sriranjini (2008) mentioned that essential oils of plants (mainly belonging to Apiaceae, Lamiaceae, Lauraceae and Myrtaceae) and their components (monoterpenoids and others) were tested for fumigant toxicity where many of them indicated positive results against stored insect pests including S. oryzae and T. castaneum. M. cajuputi is in the family Myrtaceae. Many researchers have demonstrated the insecticidal activities of numerous plant species from the Myrtaceae family on several stored product insects. For example, Lee et al. (2004) studied 42 essential oils extracted from several species of the Myrtaceae family found in Australia and reported that 2 Melaleuca species namely

5 Vol. 42, No.1, 2009 Melaleuca cajuputi against S. zeamais and T. castaneum 31 Table 3 Contact toxicity of Melaleuca cajuputi leaf essential oil applied topically to Sitophilus zeamais and Tribolium castaneum. LD 50 LD 95 Insect species (95% fiducial limit) (95% fiducial limit) (µl insect -1 ) (µl insect -1 ) S. zeamais ( ) ( ) T. castaneum ( ) ( ) Slope ± S.E Y-intercept ± S.E ± ± ± ± Table 4 Comparison of some chemical constituents of the essential oils (%) from Melaleuca cajuputi leaves collected from Kasetsart University, Bangkhen campus and Narathiwat Province, Thailand. Compound Chemical constituent (%) Kasetsart University Narathiwat Province α - Thujene α-pinene β - Myrcene α -Phellandrene α -Terpinene p-cymene Limonene γ Terpinene Terpineolene Terpinol-4-ol α - Terpineol β elemene (Z) β Famesene α - Caryopphyllene Germacrene B Linalool β-elemene Melaleuca armillaris (So. Ex. Gaetn.) Sm. and M. fulgens K. Rule showed the fumigant toxicity to T. castaneum with the LD 50 equalling 30.6 and 28.6 µl L -1, respectively. Moreover, they showed that the fumigant effects of M. armillaris, M. fulgens, Melaleuca decussate (Totem Poles), Melaleuca ericifolia Sm., Melaleuca lanceolata Otto, M. linariifolia Sm. and Melaleuca thymifolia Sm. to S. oryzae had a LD 50 ranging between 30.6 to > 50 µl L -1 at 24 h after exposure. The results of our experiments were less toxic as compared to their experiments.

6 32 Ko Ko et al. Thai Journal of Agricultural Science Don-Pedro (1996) and Lee et al. (2001b) suggested that the toxicity of essential oils to stored-product insects was influenced by the chemical composition of the oil, which in turn depended on the source, season and ecological conditions, method of extraction, time of extraction and plant part used. Brophy et al. (2002) studied the chemical compounds of volatile leaf essential oil of M. cajuputi in Thailand and their chemical constituents were found not to be different from our results. However, some constituents were not present in their findings as our findings and vice versa. For example, α-thujene and β-myrcene could be found in our tested plant species, but these constituents were not present in their findings. Similarly, although their results showed that there were Linalool and β-elemene in M. cajuputi, these constituents were not found in our studies. In summary, M. cajuputi leaf essential oil had repellency, fumigant and contact toxicities against S. zeamais and T. castaneum. These findings demonstrated the potential of M. cajuputi leaf essential oil for further development into a biopesticide in the control of stored-product insects. Acknowledgments We would like to thank the Oil Crops Development Project (UTF/MYA/006/Mya) in Myanmar laid down by Ministry of Agriculture and Irrigation, Myanmar and FAO for granting fellowship for the first author. This work was also supported by the Thailand Research Fund under Grant #RTA References Abbott, W.S A method for computing the effectiveness of an insecticide. J. Econ. Entomol.18: Brophy, J.J., S. Thubthimthed, T. Kitirattrakarn and C. Anantachoke Volatile leaf oil of Melaleuca cajuputi, pp Proceedings of Forest Conference. September 16-17, 2002, Bangkok, Thailand (In Thai). Campbell, J.F. and C. Runnion Patch exploitation by female red flour beetles, Tribolium castaneum. J. Insect Sci. 3 (20): 8 Don-Pedro, K.N Fumigant toxicity of citrus peel oils against adult and immature stages of storage insect pests. Pestic. Sci. 47: Doran, J.C Melaleuca cajuputi Powell, pp In P.C.M. Janson, E. Westphal and N. Wulijarni-Soetipto, eds., Plant Resources of South East Asia: Essential-Oil Plants. Procea Foundation, Bogor, Indonesia. Erler, F Fumigant activity of six monoterpenoids from aromatic plants in Turkey against two storedproduct pests confused flour beetle, Tribolium confusum, and Mediterranean flour moth, Ephestia kuehniella. J. Plant Dis. Prot. 112: Garcia, M., O.J. Donadel, C.E. Ardanaz, C.E. Tonn and M.E. Sosa Toxic and repellent effects of Baccharis salicifolia essential oil on Tribolium castaneum. Pest Manage. Sci. 61: Gupta, A.K., S.R. Behal, B.K. Awasthi and R.A. Verma Screening of some maize genotypes against Sitophilus oryzae. Indian J. Entomol. 61: Kostyukovsky, M., U. Ravid and E. Shaaya The potential use of plant volatiles for the control of stored product insects and quarantine pests in cut flowers. In J. Bernath, E. ZamborineNemeth, L. Crakerm and O. Kock, eds. Proceedings of International Conference on Medicinal and Aromatic Plants Possibilities and Limitations of Medicinal and Aromatic Plant Production in the 21 st Century, July 8 11, 2001, Budapest, Hungary. Lee, B.H., W.S. Choi, S.E. Lee and B.S. Park. 2001a. Fumigant toxicity of essential oil and their constituent compounds towards the rice weevil, Sitophilus oryzae (L.). Crop Prot. 20: Lee, S.E., B.H. Lee, W.S. Choi, B.S. Park, J.G. Kim and B.C. Campbell. 2001b. Fumigant toxicity of volatile natural products from Korean spices and medicinal plants towards the rice weevil, Sitophilus oryzae (L). Pest Manage. Sci.57: Lee, B.H., P.C. Annis, F. Tumaalii and W.S. Choi Fumigant toxicity of essential oils from the Myrtaceae family and 1,8-cineole against 3 major stored-grain insects. J. Stored Prod. Res. 40: Liu, Z.L. and S.H. Ho Bioactivity of the essential oil extracted from Evodia rutaecarpa Hook f. et Thomas against the grain storage insects, Sitophilus zeamais Motsch. and Tribolium castaneum (Herbst). J.Stored Prod. Res. 35: Markham, J.L Biological activity of tea tree oil, pp In I. Southwell and R. Lowe, eds., Tea Tree: The Genus Melaleuca. Hardwood Academic publishers, Australia. Mohan, S. and P.G. Fields A simple technique to assess compounds that are repellents or attractive to stored product insects. J. Stored Prod. Res. 33: Negahban, M., S. Moharramipour and F. Sefidkon Insecticidal activity and chemical composition of Artemisia sieberi Besser essential oil from Karaj, Iran. J.Asia- Pacific Entomol. 9: Ogendo, J.O., S.R. Belmain, A.L. Deng and D.J Walker Comparison of toxic and repellent effects of Lantana camara L. with Tephrosia vogelii Hook and

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