www.thaiagj.org Thai Journal of Agricultural Science 2009, 42(1): 27-33

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

Ko Ko1, W. Juntarajumnong2 and A. Chandrapatya2,*

1 Protection Division, Myanma Agriculture Service Bayint Naung Road, West Gyogon Insein P.O. 11011, Yangon, Myanma 1Department of Entomology, Faculty of Agriculture, Kasetsart University Chatuchak, Bangkok 10900,

*Corresponding author. Email: [email protected]

Abstract Melaleuca cajuputi (Cajuput tree) which can be found in (Burma) and Thailand was examined for insecticidal properties against Sitophilus zeamais and Tribolium castaneum. The results showed that M. cajuputi 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 -1 fumigant assays, LC50 value for S. zeamais was 178.23 µL L and for T. castaneum was 213.17 µL -1 L . The LD50 values for S. zeamais and T. castaneum in contact toxicities were 0.062 and 0.143 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 internal feeder causing considerable loss to cereals affecting the quantity as well as quality of the Insect pest damage to stored grains is known to grains (Gupta et al., 1999). Tribolium castaneum is cause major economic losses to warehouse keepers, a major pest in storage of grain-based products, but the milling industry and small scale farmers milled grain products such as flour appears to be throughout the world. This problem is greatest in preferred food as well (Campbell and Runnion, developing countries. The global post-harvest grain 2003). losses caused by insect damage and other bio- At present, the natural compounds such as agents vary from 10% to 40% (Raja et al., 2001; essential oils are alternatives to synthetic pesticides Papachristos and Stamopoulos, 2002). In order to to control post-harvest insects since they are more reduce the losses in postharvest systems from insect acceptable both environmentally and to the infestation, synthetic insecticides that are used as consumer (Markham, 1999). The toxicity of a large fumigants, are routinely employed despite their number of essential oils and their constituents has undesirable side effects such as ozone depletion and been evaluated against a number of stored-product environmental pollution (World Meteorological insects (Paranagama et al., 2003). Organization (WMO), 1995), toxicity to non-target Melaleuca cajuputi Powell () naturally organisms, pest resistance (Mohan and Fields, occurs in Myanmar (Burma) and Thailand through 2002) and pesticide residues (Kostyukovsky et al., Southeast East Asia to northern (Weiss, 2002; Ogendo et al., 2003). 1997). The of M. cajuputi possess antibacterial, Sitophilus zeamais Motschulsky and Tribolium anti-inflammatory and anodyne properties and are castaneum (Herbst) are two of the most important reputed to have insect-repellent properties. It is pests in stored grains. Sitophilus zeamais is an also used as flavor in cooking and as a fragrance 28 Ko Ko et al. Thai Journal of Agricultural Science and freshening agent in the soaps, cosmetics, Repellent Activity detergents and perfumes (Doran, 1999). So, leaf The repellent activity was performed using petri essential oil of M. cajuputi is considered to be used dishes (9 cm in diameter) to confine the insects as safe alternative botanical insecticide for during the experiment. Essential oils of M. cajuputi controlling stored product insects. were diluted in ethanol to different concentrations At present, there is no report on insecticidal (0.5%, 1%, 1.5% and 2% or 0.16, 0.31, 0.47 and activities of M. cajuputi against stored product 0.63 µg cm-2) and absolute ethanol was used as the pests. Therefore, this experiment was carried out to control. Each filter paper of 9 cm diameter was cut determine the possible insecticidal activities of the in half. One ml of the tested material was then leaf essential oil of M. cajuputi on S. zeamais and applied separately onto one half of the filter paper T. castaneum. as uniformly as possible. The other half (control) was treated with 1 ml of absolute ethanol. Both the Materials and Methods treated half and the control half were air-dried to evaporate the solvent completely. A full disc was Insects carefully remade by attaching the tested to the Sitophilus zeamais and T. castaneum from control halves with tape. Precaution should be Department of Agriculture, Ministry of Agriculture taken so that the distance between the filter-paper and Co-operatives, Thailand were used throughout halves was sufficient to prevent seepage of test this study. Sitophilus zeamais was reared on rice samples from one half to another. Each remade 12–13% moisture content while T. castaneum was filter paper was placed in a petri dish with the seam reared on rice bran. The cultures were maintained oriented in one of four randomly selected different in the laboratory at 29-32˚C and 70–80% RH. directions to avoid any insecticidal stimuli affecting the distribution of insects. Extraction of the Essential Oils Ten insects were released in the center of each Fresh leaves of M. cajuputi were collected at filter-paper disc and a cover was placed on petri Kasetsart University, Bangkhen campus, (13º98’N, dish. Five replicates were used and the experiment 48º18’E) in June 2007 and the voucher specimens was repeated once. The number of insects (#CHKU 00028) was deposited at the Bangkok presented on each strip was counted at hourly Herbarium, Botanical Research Unit, Department interval up to the fifth hour. The percent repellency of Agriculture, Bangkok, Thailand. of the essential oil was calculated using the formula

The essential oil was extracted by water- PR (%) = [(Nc – Nt)/(Nc + Nt)] x 100 where Nc was distillation using a Clevenger-type apparatus for 6 the number of insects on the control half and Nt h. The superior phase was collected from the was the number of insects on the treated half. condenser, dried over anhydrous sodium sulphate and stored in amber-colored vials at 10-12ºC for Fumigant Toxicity further experiments. Essential oil was analyzed by Filter paper discs of 2-cm diameter were GC/MS (Shimadzu capillary GC-quadrupole MS impregnated with oil at doses calculated to give system QP 5050A) equipped with a DB-5 capillary equivalent fumigant concentrations of 37, 56, 94, column (60 m, 0.25 mm, 0.25 µm film thickness) 130, 185, 296, 370, 444 and 556 µL L-1 in air. The ZJ and W Scientific. The column temperature was impregnated filter paper was then attached to the programmed at 60ºC for 5 min then increased at under-surface of the screw cap of a glass vial (27 1ºC min-1 to 80ºC, finally 4ºC min-1 to 200ºC and mL). Ten 1-7 days old adults of either S. zeamais held for 10 min. The injector and detector or T. castaneum were placed in each vial before the temperatures were 250ºC. Helium was the carrier caps were screwed tightly. Each treatment was gas, at a flow rate of 1.2 mL min-1. The injection replicated five times. Number of dead insects was volume was 1 µL with split ratio 1:7. Essential oil determined at 3, 6, 9, 12 and 24 h after exposure. components were identified by comparing their GC Insects were considered dead when no leg or retention times and their mass spectra with those antennal movements were observed. Mortality rate presented in the MS library. was calculated using the Abbott’s formula for Vol. 42, No.1, 2009 Melaleuca cajuputi against S. zeamais and T. castaneum 29 natural mortality in untreated controls (Abbott, Results 1925). Probit analysis was used to estimate the

LC50 and LC95 values. The experiment was Repellent activity arranged by randomized complete block design and Melaleuca cajuputi moderately repelled S. ANOVA was computed using SPSS 16.0 software zeamais and T. castaneum except the highest package. concentration where 100% repellency could be detected for T. castaneum. The repellency for S. Contact Toxicity zeamais and T. castaneum was not statistically Aliquots of 0.5 mL of each dilution (10%, 20%, different in all concentrations (Table 1). Still, the 30% and 40%) were applied topically to the thorax repellency for T. castaneum was obvious when of S. zeamais and T. castaneum adults using a compared to the lowest concentration and the Burkard Arnold microapplicator (Burkard highest one (36 and 96% respectively). Generally, Manufacturing Company Ltd., England). Control repellency increased with concentration. The 100% insects were treated with absolute ethanol. Both repellency could occur in T. castaneum at 2 h and 5 treated and control insects were transferred to glass h. However, 100% repellency did not occur in S. vials (10 insects vial-1) (2 cm dia. and 5.5 cm zeamais during 5 h duration. height) and kept in incubators set at 27-28˚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.

Table 1 Percent repellency (PR) of the leaf essential oil of Melaleuca cajuputi to Sitopilus zeamais and Tribolium castaneum using treated filter paper test.

PR (Mean% ± SD) hours after insect release1/ Insect species Oil PR2/ 1 2 3 4 5 (µg cm-2) (Mean %) S. zeamais 0.16 52 ± 36a 60 ± 42a 68 ± 18a 68 ± 39a 72 ± 39a 64.0 0.31 52 ± 22a 48 ± 22a 68 ± 23a 68 ± 33a 56 ± 33a 58.4 0.47 56± 43a 80 ± 28a 90 ± 33a 72 ± 32a 60 ± 32a 71.6 0.63 56 ± 45a 72 ± 22a 72 ± 23a 72 ± 33a 76 ± 33a 69.6

F(3, 16) 0.018 1.077 0.120 0.053 0.387 P 0.996 0.387 0.947 0.983 0.764 T. castaneum 0.16 40 ± 57a 64 ± 41a 44 ± 77a 48 ± 69a 44 ± 77a 36.0 0.31 44 ± 43a 64 ± 38a 56 ± 54a 56 ± 52a 48 ± 64a 53.6 0.47 52 ± 18a 68 ± 11a 68 ± 18a 68 ± 18a 56 ± 33a 62.4 0.63 88 ± 18a 100± 0a 96 ± 9a 96 ± 9a 100 ± 0a 96.0

F(3, 16) 1.678 1.854 1.083 1.132 1.203 P 0.212 0.178 0.385 0.366 0.34 1/ 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).

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.

Degree of Insect species LC 1/ LC 1/ Slope ± SE Chi-square (x2) 50 95 freedom S. zeamais 178.23 408.54 0.007 ± 0.00 8 95.41 (119.23-243.04) (321.52-604.84) T. castaneum 213.17 376.1 0.010 ± 0.001 8 82.04 (168.95-266.33) (311.67-503.37) 1/ -1 Units LC50 and LC95 = µL L air, applied for 24 h at 27˚C; 95% lower and upper fiducial limits are shown in parenthesis.

Fumigant Toxicity constituents were presented in our tested plant, we Melaleuca cajuputi leaf essential oil showed could conclude that these constituents might also fumigant toxicity against S. zeamais and T. exert fumigant effect on S. zeamais and T. castaneum. However, S. zeamais (LC50 = 178.23 µL confusum (Table 4). Moreover, α-pinene (4.26%), L-1) was considerably more susceptible than T. limonene (2.91%), α-terpinene (4.44%) and α- -1 castaneum (LC50 = 213.17 µL L ) (Table 2). terpineol (1.09%) found in M. cajuputi leaf Essential oil of Evodia rutaecarpa Hook f. et essential oil also known to exhibit insecticidal Thomas had fumigant toxicity to S. zeamais and T. activity. For example, ρ-cymene had fumigant castaneum with the LC50 values of 41 and 11.74 µL toxicity on Acanthosceloides obtectus (Say) L-1 air, respectively (Liu and Ho, 1999). Negahban (Regnault-Roger and Hamraoui, 1995) and α- et al. (2006) also demonstrated that Artemisia pinene was reported to be toxic to T. confusum sieberi Besser essential oil had fumigant toxicity to (Ojimelukwe and Alder, 1999). Prates et al. (1998) -1 T. castaneum with the LC50 value of 16.76 µL L also showed fumigant activity of limonene against air. In addition, Don-Pedro (1996) showed that T. confusum. In addition, the most repellent limepeel oil exhibited fumigant toxicity against S. compound in Baccharis salicifolia (Ruiz and Pav.) -1 zeamais (LC50 = 11.75 µL L ). We could conclude Pers. Essential oil against T. confusum was α- that fumigant toxicity of our studies was not so terpineol (Garcia et al., 2005). Lee et al. (2001a) good as their findings. demonstrated that ρ-cymene, α-terpinene, α– terpeneol and terpine-4-ol had the possible Contact Toxicity fumigant toxicity to S. oryzae. Hence, these early Contact toxicity of M. cajuputi oil was very findings supported the present experiment. obvious when applied topically to the dorsal surface Recently, Rajendran and Sriranjini (2008) of S. zeamais and T. castaneum. On the basis of the mentioned that essential oils of (mainly

LD50 and LD95 values, S. zeamais adults were more belonging to Apiaceae, Lamiaceae, Lauraceae and susceptible to the essential oil of M. cajuputi than Myrtaceae) and their components (monoterpenoids T. castaneum (Table 3). and others) were tested for fumigant toxicity where many of them indicated positive results against Discussion stored insect pests including S. oryzae and T. castaneum. M. cajuputi is in the family Myrtaceae. The result on chemical constituents revealed that Many researchers have demonstrated the terpiniolene, γ–terpinene and ρ-cymene were major insecticidal activities of numerous plant species components of M. cajuputi leaf essential oil. Other from the Myrtaceae family on several stored minor constituents were terpine-4-ol, α-pinene, product insects. For example, Lee et al. (2004) limonene, α-terpinene and α-terpineol. Erler (2005) studied 42 essential oils extracted from several showed that γ-terpinene and terpine-4-ol were the species of the Myrtaceae family found in Australia promising fumigants against T. confusum and and reported that 2 Melaleuca species namely Ephestia kuehniella Zeller. As these two chemical 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.

LD50 LD95 Insect species (95% fiducial limit) (95% fiducial limit) Slope ± S.E Y-intercept ± S.E (µL insect-1) (µL insect-1) S. zeamais 0.062 0.111 33.858 ± 2.929 - 2.099 ± 2.929 (0.101 - 0.114) (0.159 – 0.183) T. castaneum 0.143 0.296 10.771 ± 1.010 -1.543 ± 1.010 (0.082 – 0.282) (0.209 – 0.982)

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 5.92 - α-Pinene 4.26 9.38 β - Myrcene 1.38 - α -Phellandrene 3.76 3.92 α -Terpinene 4.44 4.52 p-Cymene 8.39 8.41 Limonene 2.91 1.10 γ – Terpinene 25.25 22.84 Terpineolene 29.77 24.74 Terpinol-4-ol 4.06 2.57 α - Terpineol 1.09 0.99 β– elemene 1.88 1.51 (Z) β – Famesene 5.00 - α - Caryopphyllene 1.63 - Germacrene B 0.25 - Linalool - 0.27 β-elemene - 1.51

Melaleuca armillaris (So. Ex. Gaetn.) Sm. and M. ericifolia Sm., Melaleuca lanceolata Otto, M. fulgens K. Rule showed the fumigant toxicity to T. linariifolia Sm. and Melaleuca thymifolia Sm. to S. castaneum with the LD50 equalling 30.6 and 28.6 oryzae had a LD50 ranging between 30.6 to > 50 µL µL L-1, respectively. Moreover, they showed that L-1 at 24 h after exposure. The results of our the fumigant effects of M. armillaris, M. fulgens, experiments were less toxic as compared to their Melaleuca decussate (Totem Poles), Melaleuca experiments. 32 Ko Ko et al. Thai Journal of Agricultural Science

Don-Pedro (1996) and Lee et al. (2001b) Doran, J.C. 1999. Melaleuca cajuputi Powell, pp. 126- suggested that the toxicity of essential oils to 131. In P.C.M. Janson, E. Westphal and N. Wulijarni-Soetipto, eds., Plant Resources of South stored-product insects was influenced by the East Asia: Essential-Oil Plants. Procea Foundation, chemical composition of the oil, which in turn Bogor, . depended on the source, season and ecological Erler, F. 2005. Fumigant activity of six monoterpenoids conditions, method of extraction, time of extraction from aromatic plants in Turkey against two stored- and plant part used. Brophy et al. (2002) studied product pests confused flour beetle, Tribolium confusum, and Mediterranean flour moth, Ephestia the chemical compounds of volatile leaf essential kuehniella. J. Plant Dis. Prot. 112: 602-611. oil of M. cajuputi in Thailand and their chemical Garcia, M., O.J. Donadel, C.E. Ardanaz, C.E. Tonn and constituents were found not to be different from our M.E. Sosa. 2005. Toxic and repellent effects of results. However, some constituents were not Baccharis salicifolia essential oil on Tribolium castaneum. Pest Manage. Sci. 61: 612-618. present in their findings as our findings and vice Gupta, A.K., S.R. Behal, B.K. Awasthi and R.A. Verma. versa. For example, α-Thujene and β-Myrcene 1999. Screening of some maize genotypes against could be found in our tested plant species, but these Sitophilus oryzae. Indian J. Entomol. 61: 265-268. constituents were not present in their findings. Kostyukovsky, M., U. Ravid and E. Shaaya. 2002. The Similarly, although their results showed that there potential use of plant volatiles for the control of stored product insects and quarantine pests in cut flowers. In were Linalool and β-elemene in M. cajuputi, these J. Bernath, E. ZamborineNemeth, L. Crakerm and O. constituents were not found in our studies. Kock, eds. Proceedings of International Conference on In summary, M. cajuputi leaf essential oil had Medicinal and Aromatic Plants Possibilities and repellency, fumigant and contact toxicities against Limitations of Medicinal and Aromatic Plant Production in the 21st Century, July 8–11, 2001, S. zeamais and T. castaneum. These findings Budapest, Hungary . demonstrated the potential of M. cajuputi leaf Lee, B.H., W.S. Choi, S.E. Lee and B.S. Park. 2001a. essential oil for further development into a Fumigant toxicity of essential oil and their biopesticide in the control of stored-product insects. constituent compounds towards the rice weevil, Sitophilus oryzae (L.). Crop Prot. 20: 317-320. Lee, S.E., B.H. Lee, W.S. Choi, B.S. Park, J.G. Kim and Acknowledgments B.C. Campbell. 2001b. Fumigant toxicity of volatile natural products from Korean spices and medicinal We would like to thank the Oil Crops plants towards the rice weevil, Sitophilus oryzae (L). Development Project (UTF/MYA/006/Mya) in Pest Manage. Sci.57: 548-553. Myanmar laid down by Ministry of Agriculture and Lee, B.H., P.C. Annis, F. Tumaalii and W.S. Choi. 2004. Fumigant toxicity of essential oils from the Irrigation, Myanmar and FAO for granting Myrtaceae family and 1,8-cineole against 3 major fellowship for the first author. This work was also stored-grain insects. J. Stored Prod. Res. 40: 553- supported by the Thailand Research Fund under 564. Grant #RTA 4880006. Liu, Z.L. and S.H. Ho. 1999. Bioactivity of the essential oil extracted from Evodia rutaecarpa Hook f. et Thomas against the grain storage insects, Sitophilus References zeamais Motsch. and Tribolium castaneum (Herbst). J.Stored Prod. Res. 35: 317-328. Abbott, W.S. 1925. A method for computing the Markham, J.L. 1999. Biological activity of tea tree oil, effectiveness of an insecticide. J. Econ. Entomol.18: pp. 169-190. In I. Southwell and R. Lowe, eds., Tea 265–267. Tree: The Genus Melaleuca. Hardwood Academic Brophy, J.J., S. Thubthimthed, T. Kitirattrakarn and C. publishers, Australia. Anantachoke. 2002. Volatile leaf oil of Melaleuca Mohan, S. and P.G. Fields. 2002. A simple technique to cajuputi, pp. 304-313. Proceedings of Forest assess compounds that are repellents or attractive to Conference. September 16-17, 2002, Bangkok, stored product insects. J. Stored Prod. Res. 33: 289- Thailand (In Thai). 298. Campbell, J.F. and C. Runnion. 2003. Patch exploitation Negahban, M., S. Moharramipour and F. Sefidkon. 2006. by female red flour beetles, Tribolium castaneum. J. Insecticidal activity and chemical composition of Insect Sci. 3 (20): 8 Artemisia sieberi Besser essential oil from Karaj, Don-Pedro, K.N. 1996. Fumigant toxicity of citrus peel Iran. J.Asia- Pacific Entomol. 9: 61-66. oils against adult and immature stages of storage Ogendo, J.O., S.R. Belmain, A.L. Deng and D.J Walker. insect pests. Pestic. Sci. 47: 213–223. 2003. Comparison of toxic and repellent effects of Lantana camara L. with Tephrosia vogelii Hook and Vol. 42, No.1, 2009 Melaleuca cajuputi against S. zeamais and T. castaneum 33

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