Research Article Received: 21 June 2009 Revised: 28 October 2009 Accepted: 6 January 2010 Published online in Wiley Interscience: (www.interscience.wiley.com) DOI 10.1002/ps.1927 Bioactivity against Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) of Cymbopogon citratus and Eucalyptus citriodora essential oils grown in Colombia Jesús Olivero-Verbel,a,b∗ Luz S Nerioa,b and Elena E Stashenkob Abstract BACKGROUND: Essential oils isolated from Cymbopogon citratus (DC) Stapf. and Eucalyptus citriodora Hook grown in Colombia were analysed by gas chromatography–mass spectrometry (GC-MS) and tested for repellent activity and contact toxicity against Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae). RESULTS: The main components of C. citratus oil were geranial (34.4%), neral (28.4%) and geraniol (11.5%), whereas those of E. citriodora were citronellal (40%), isopulegol (14.6%) and citronellol (13%). The mean repellent doses after 4 h exposure were 0.021 and 0.084 mL L−1 for C. citratus and E. citriodora oils respectively – values lower than that observed for the commercial product IR3535 (0.686 mL L−1 ). CONCLUSION: These studies showed the composition and repellent activity of essential oils of C. citratus and E. citriodora, suggesting that these are potential candidates as insect repellents. c 2010 Society of Chemical Industry
Keywords: Cymbopogon citratus; Eucalyptus citriodora; Tribolium castaneum; repellency; essential oil; Colombia 1 INTRODUCTION 2 Stored-product insects are a recurrent problem in retail stores, where they damage and contaminate food products. Tribolium castaneum (Herbst), one of the most economically important stored-product pests,1 is distributed worldwide and attacks many flour mills, warehouses and grocery stores in the tropical area. Control of this kind of insect relies heavily on the use of gaseous synthetic insecticides and fumigants, which has led to problems such as ozone depletion, environmental pollution, increasing costs of application, pest resurgence and resistance and hazard effects on non-target organisms in addition to direct toxicity to users.2,3 In many storage systems, fumigants such as methyl bromide (MeBr) and phosphine (PH3 ) are the most economical and convenient chemicals for managing stored-product insect pests, not only because of their ability to kill a broad spectrum of pests but also because of their easy penetration into the product while leaving minimal residues.4 The wide use of MeBr and PH3 is being restricted owing to potential ozone-depleting properties and to increasing resistance in stored-product insects respectively.5,6 In order to control this kind of species without disturbing the environment, natural products have been screened for potential sources of repellents and insecticides;7 in fact, plant essential oils have traditionally been used to kill or repel insects,8 being considered as an alternative to stored-grain conventional pesticides because of their low toxicity to warm-blooded mammals and their high volatility.9,10 Pest Manag Sci (2010) www.soci.org MATERIALS AND METHODS 2.1 Plant materials Plant material was obtained from the Industrial University of Santander (UIS) in Bucaramanga, Colombia, at the experimental pilot agroindustrial station, from species cultivated from certified seeds under controlled conditions. Identification of the plants was carried out by botanist Prof. Humberto García, and voucher specimens 00 446 and 00 447, corresponding to Eucalyptus citriodora Hook and Cymbopogon citratus (DC) Stapf. respectively, were deposited in the Herbarium of the CENIVAM centre at the UIS. Only leaves were used to obtain essential oil from E. citriodora (Myrtaceae), whereas in the case of C. citratus (Poaceae) the whole plant was used for the extraction. 2.2 Essential oil isolation The essential oils were obtained from plant material (ca 150 g in 1 L of water) by microwave-assisted hydrodistillation carried out in a ∗ Correspondence to: Jesús Olivero-Verbel, Environmental and Computational Chemistry Group, Department of Chemistry, Campus of Zaragocilla, University of Cartagena, Cartagena, Colombia. E-mail: joliverov@unicartagena.edu.co; jesusolivero@yahoo.com a EnvironmentalandComputationalChemistryGroup,DepartmentofChemistry, Campus of Zaragocilla, University of Cartagena, Cartagena, Colombia b Chromatography Laboratory, Research Centre of Excellence, CENIVAM, Industrial University of Santander, Bucaramanga, Colombia c 2010 Society of Chemical Industry
www.soci.org Clevenger-type apparatus, the round flask of which was contained in a domestic microwave oven (LG Intelowave, 2450 MHz, 720 W) as described elsewhere.11 Four consecutive 10 min heating periods were employed. Each extraction was repeated in triplicate. 2.3 Essential oil characterisation Essential oils were characterised as previously reported.11 In short, a gas chromatography–mass spectrometry (GC-MS) system was employed – an 6890 Plus gas chromatograph (Agilent Technologies, Palo Alto, CA) equipped with a 5973N mass selective detector (Agilent Technologies). A 7863 automatic injector was used in split/splitless mode (split ratio 1 : 50), and an MS-ChemStation G1701-DA data system that included the spectral libraries WILEY 138K, NIST 2002 and QUADLIB 2004. A DB-5 MS fused-silica capillary column (J&W Scientific, Folsom, CA) of 60 m × 0.25 mm ID × 0.25 µm df was employed. The oven temperature was programmed from 45 ◦ C (5 min) to 150 ◦ C (2 min) at 4 ◦ C min−1 , then to 250 ◦ C (5 min) at 5 ◦ C min−1 and finally to 275 ◦ C (15 min) at 10 ◦ C min−1 . The ionisation chamber and transfer line temperatures were kept at 230 and 285 ◦ C respectively. Compound identification was based on chromatographic criteria (retention times, retention indices, standard compounds) and spectroscopic criteria (spectral interpretation, comparison with databases and standards). 2.4 Test procedures 2.4.1 Insects Tribolium castaneum were obtained from a 10 kg package of oat flakes from a grain store located in the main market of Cartagena, Colombia. They were reared on insecticide-free ground oats. For the experiments, adults belonging to the second generation were selected. 2.4.2 Repellent activity The repellent effects of the different essential oils and a commercial repellent against T. castaneum were tested by the area preference method.12 Solutions were prepared at concentrations of 0.001, 0.01, 0.1, 1.0 and 10 mL L−1 using acetone as solvent. Test areas consisted of 9 cm diameter Whatman No. 1 filter papers cut in half. As positive control, a 150 mL L−1 formulation of IR3535 [ethyl 3-(N-acetyl-N-butylamino)propionate], a well-known arthropod repellent, was used under the same conditions as the oils. A volume of 0.5 mL of each solution was applied to one half of the paper discs as uniformly as possible. The other half was treated with an equal volume of acetone alone as a control. The treated and control half-discs were air dried for 10 min in order to evaporate the solvent completely. Treated and untreated halves were attached to their opposites using adhesive tape and placed in 90 mm glass petri dishes. Twenty adult T. castaneum of mixed J Olivero-Verbel, LS Nerio, EE Stashenko sex were released at the centre of each filter paper disc. The dishes were then covered and placed in darkness at 26 ± 2 ◦ C and 75 ± 10% RH. Five replications were made for each concentration. The number of T. castaneum present on the treated and untreated portions of the experimental paper halves was recorded after 2 and 4 h of exposure. The percentage repellency (PR) values were computed as follows: PR = [(Nc − Nt )/(Nc + Nt )]100 where Nc and Nt are the number of insects after the exposure period on the untreated and treated areas respectively. 2.4.3 Contact toxicity on filter papers The contact toxicity of both oils against T. castaneum was tested through filter paper discs (Whatman No. 1, 9 cm diameter) placed in glass petri dishes.12 Oils were dissolved in acetone to concentrations of 10, 20, 30 and 40 ml L−1 , and 1 mL of each solution was applied on the paper’s surface. After 10 min, once the solvent had been evaporated, 20 unsexed adults were released into each disc and kept in darkness at 26 ± 2 ◦ C and 75 ± 10% RH. Three replications were made for each treatment, recording insect mortality daily up to 5 days. 2.5 Data analysis For repellent activity, the mean number of insects on the treated portion of the filter paper was compared with the number on the untreated portion using the paired t-test at P < 0.05 using GraphPad InStat v.3.00.13 In the case of contact toxicity, ANOVA was performed to compare means of dead insects for the different treatments, after normal distribution and equality between variances had been evaluated by the Kolmogorov–Smirnov and Barlett tests respectively. Probit analysis14 was used to calculate the mean repellent dose (RD50 ), the dose that repelled 50% of the exposed insects, along with 95% confidence intervals. Results are presented as the mean of percentage repellency ± standard error (X ± SE). 3 RESULTS AND DISCUSSION The chemical compositions of the examined essential oils are presented in Table 1. Both essential oils are rich in oxygenated compounds (monoterpenoids and phenolic compounds). Cymbopogon citratus oil is mainly made up of geranial (34.4%), neral (28.4%) and geraniol (11.5%), while Eucalyptus citriodora has citronellal (40.0%), isopulegol (14.6%) and citronellol (13%) as major components. Percentage repellence and RD50 values are shown in Tables 2 and 3 respectively. Both essential oils exhibited repellent activity Table 1. Chemical composition (%) of tested essential oils No. 1 2 3 4 5 6 Kovat’s index DB-5 (Ik ) Compound Eucalyptus citriodora 932 933 969 974-977 988 1029 α-Pinene 6-Methyl-5-hepten-2-one Sabinene β-Pinene Myrcene p-Cymene 0.8 www.interscience.wiley.com/journal/ps c 2010 Society of Chemical Industry
Cymbopogon citratus 0.6 0.1 2.2 0.4 1.0 8.1 Pest Manag Sci (2010) Repellent activity of essential oils of C. citratus and E. citriodora www.soci.org Table 1. (Continued) No. Kovat’s index DB-5 (Ik ) Compound Eucalyptus citriodora 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 28 29 30 31 32 33 34 35 36 1034 1036 1039 1049-1047 1063 1075 1089 1091 1100 1113 1154 1157 1177 1191 1202 1220 1231 1244 1252 1253 1261 1273 1275 1293 1313 1351 1377 1399 1435 1471 1599 Limonene cis-β-Ocimene 1,8-Cineole trans-β-Ocimene γ -Terpinene p-Mentha-3,8-diene Terpinolene 6,7-epoxi Myrcene Linalool cis-Rose oxide Photocitral A Citronellal Isopulegol 4-Terpineol α-Terpineol Verbenone Citronellol Neral Nerol Geraniol Methyl citronellate Geranial Citronellyl formate 2-Undecanone 3,7-Dimethyl-6-octenoic acid 5-Methyl-2-(2-hydroxy-2-propyl)-cyclohexanol Geranyl acetate cis-Jasmone trans-β-Caryophyllene α-Humulene Caryophyllene oxide 1.1 Table 2. Repellency of Eucalyptus citriodora and Cymbopogon citratus essential oils against Tribolium castaneum after two exposure times 2h 4h Eucalyptus citriodora 0.001 0.01 0.1 1.0 10 18 (±13) 28 (±14) 76 (±4)∗∗ 80 (±9)∗∗ 90 (±3)∗∗ 8 (±16) 18 (±27) 72 (±7)∗∗ 76 (±12)∗∗ 84 (±9)∗∗ Cymbopogon citratus 0.001 0.01 0.1 1.0 10 14 (±12) 38 (±7)∗∗ 84 (±10)∗∗ 100 (±0)∗∗ 100 (±0)∗∗ 8 (±18) 36 (±13)∗ 80 (±11)∗∗ 98 (±2)∗∗ 100 (±0)∗∗ Sample IR3535 Pest Manag Sci (2010) 0.7 0.8 3.4 2.2 0.2 1.2 40.0 14.6 1.0 1.0 0.3 13.0 28.4 2.2 11.5 0.1 2.0 34.4 0.2 0.7 1.9 4.7 1.1 0.4 1.8 0.1 0.3 Repellencya,b (%) after exposure time Sample against T. castaneum, with higher RD50 values than the positive control; nevertheless, this activity had a small tendency to decrease with increase in exposure time. Cymbopogon citratus essential oil was the most active, with 100% repellency at 2 h exposure of the 1.1 3.4 0.2 1.5 0.6 1.6 Table 2. (Continued) Repellencya,b (%) after exposure time Concentration (mL L−1 ) Cymbopogon citratus Concentration (mL L−1 ) 2h 4h 0.001 0.01 0.1 1.0 10 2 (±8) 16 (±9)∗∗ 54 (±12)∗∗ 60 (±13)∗∗ 78 (±5)∗∗ −6(±18) 4 (±22) 40 (±11)∗∗ 50 (±5)∗∗ 75 (±8)∗∗ a Values are mean ± SE of five replicates. Differs significantly from control (P < 0.05) according to paired t-test. ∗∗ Differs significantly from control (P < 0.01) according to paired t-test. b∗ oil diluted to 1.0 mL−1 as against 80% repellency by Eucalyptus citriodora. Nevertheless, comparison of the RC50 values shows no significant differences. The results for the contact lethality on filter paper for the examined essential oils are shown in Figs 1 and 2. The oil of C. citratus presented more potency and efficacy than that of c 2010 Society of Chemical Industry
www.interscience.wiley.com/journal/ps www.soci.org J Olivero-Verbel, LS Nerio, EE Stashenko Table 3. RD50 values of the essential oils tested against Tribolium castaneum Regression parameters Exposure time (h) RD50 (95% CL) (mL L−1 ) R P Cymbopogon citratus 2 4 0.016 (0.008–0.033) 0.0211 (0.011–0.0421) 0.9840 0.9887 0.0160 0.0113 Eucalyptus citriodora 2 4 0.0373 (0.012–0.113) 0.084 (0.030–0.235) 0.9418 0.9307 0.0167 0.0217 IR3535 2 4 0.256 (0.085–0.774) 0.686 (0.242–1.947) 0.9751 0.9846 0.0047 0.0023 Sample Figure 1. Mortality of Tribolium castaneum exposed to the essential oil from Cymbopogon citratus. lal, α-pinene, β-pinene and 1,8-cineole, have been previously described as active constituents of botanical insecticides.8 Moreover, α-pinene and β-pinene presented higher toxicity against T. castaneum.1 Essential oils and ethanolic extracts of C. citratus have been successfully tested as a mosquito repellent, alone or mixed with other materials that could increase the protection time,19 – 22 although it was not a good larvicide against Ae. aegypti.18 Insect bioactivity of an essential oil depends on insect species and chemical composition, which varies with the part and age of the plant, geographical area and extraction method. The main compounds of C. citratus essential oil are geranial, neral and geraniol (Table 1). Geranial and neral were described as the major mosquito-repellent metabolites of an essential oil isolated from C. citratus leaves;23 probably, these compounds could be responsible for higher bioactivity of C.citratus essential oil against T. castaneum. In short, results presented here suggest that the essential oils of Eucalyptuscitriodora and Cymbopogoncitratus are good candidates for use as repellents against Tribolium castaneum. ACKNOWLEDGEMENTS This work was possible owing to the support of Colciencias, Bogotá, Colombia (Grant RC 432-2004), and the University of Cartagena, Cartagena, Colombia. REFERENCES Figure 2. Mortality of Tribolium castaneum exposed to the essential oil from Eucalyptus citiodora. E. citriodora, and in both cases the toxicity was dose and time dependent. Eucalyptus citriodora has been traditionally used as a flying insect repellent. In Africa, burning of its leaves provides a cost-effective method of household protection against mosquitoes.15 The pest control properties of essential oils isolated from this species have been previously studied against other insect species. Eucalyptus citriodora has been found to be toxic against Sitophilus zeamais Motsch.16 However, when it was tested for fumigant and repellent properties against Pediculus humanus capitis Deg. among other eucalyptus species, this species was the least effective.17 Moreover, it did not have larvicidal activity against Aedes aegypti L.18 According to the chemical composition of E. citriodora essential oil (Table 1), the compounds present in this mixture, citronel- www.interscience.wiley.com/journal/ps 1 Garcia M, Donadel OJ, Ardanaz CE, Tonn CE and Sossa ME, Toxic and repellent effects of Baccharis salicifolia essential oil on Tribolium castaneum. Pest Manag Sci 61:612–618 (2005). 2 Jembere B, Obeng-Ofori D, Hassanali A and Nyamasyo GNN, Products derived from the leaves of Ocimum kilimanndscharicum (Labiatae) as post-harvest grain protectants against the infestation of three major stored product insect pests. Bull Entomol Res 85:361–367 (1995). 3 Okonkwo EU and Okoye WI, The efficacy of four seed powders and the essential oils as protectants of cow-pea and maize grain against infestation by Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae) and Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) in Nigeria. Internat J Pest Manag 42:143–146 (1996). 4 Mueller DK, Fumigation, in Handbook of Pest Control, ed. by Mallis A. Franzak & Foster Co., Cleveland, OH, pp. 901–939 (1990). 5 Bell CH and Wilson SM, Phosphine tolerance and resistance in Trogoderma granarium Everts (Coleoptera: Dermestidae). J Stored Prod Res 31:199–205 (1995). 6 Lee BH, Annis PC, Tumaalii F and Choi WS, Fumigant toxicity of essential oils from the Myrtaceae family and 1,8-cineole against 3 major stored-grain insects. J Stored Prod Res 40:553–564 (2004). 7 Sukumar K, Perich MJ and Boobar LR, Botanical derivatives in mosquito control; a review. J Am Mosq Control Assoc 7:210–237 (1991). c 2010 Society of Chemical Industry
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