Fumigant Toxicity of Volatile Natural Products from Korean Spices And
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Pest Management Science Pest Manag Sci 57:548±553 2001) DOI: 10.1002/ps.322 Fumigant toxicity of volatile natural products from Korean spices and medicinal plants towards the rice weevil, Sitophilus oryzae (L) Sung-Eun Lee,1* Byoung-Ho Lee,2 Won-Sik Choi,2 Byeoung-Soo Park,2 Jeong-Gyu Kim3 and Bruce C Campbell1 1Plant Protection Research Unit, WRRC, USDA-ARS, 800 Buchanan St, Albany, CA 94710, USA 2Department of Life Science, Soonchunhyang University, Asan-si, Chungnam 337–745, Korea 3Department of Agricultural Chemistry, Korea University, Seoul 136-701, Korea Abstract: The fumigant toxicity of various volatile constituents of essential oils extracted from sixteen Korean spices and medicinal plants towards the rice weevil, Sitophilus oryzae L Coleoptera: Curculionidae), was determined.The most potent toxicity was found in the essential oil from Mentha À1 arvensis L var piperascens LC50 =45.5ml litre air).GC±MS analysis of essential oil from M arvensis showed it to be rich in menthol 63.2%), menthone 13.1%) and limonene 1.5%), followed in abundance by b-pinene 0.7%), a-pinene 0.6%) and linalool 0.2%). Treatment of S oryzae with each À1 of these terpenes showed menthone to be most active LC50 =12.7ml litre air) followed by linalool À1 À1 LC50 =39.2ml litre air) and a-pinene LC50 =54.9ml litre air).Studies on inhibition of acetylcho- linesterase activity of S oryzae showed menthone to have a nine-fold lower inhibitory effect than menthol, despite menthone being 8.1-fold more toxic than menthol to the rice weevil. Different modes of toxicity of these monoterpenes towards S oryzae are discussed. # 2001 Society of Chemical Industry Keywords: Sitophilus oryzae; rice weevil; fumigant toxicity; essential oils; Mentha arvensis; menthone; menthol; acetylcholinesterase inhibition 1 INTRODUCTION possibly rendering grain pests completely resistant to Infestation of grain by various stored-product pests the pesticides. may occur at various stages from time of harvest to In many storage systems the use of fumigants is the procurement by consumers. The susceptibility of most economical tool for managing stored-grain insect stored grain to insect infestation is dependent on a pests.5 Ideal fumigants should leave no residues number of factors, including condition of the grain at hazardous to humans and should not adversely affect harvest, environment ie temperature, humidity, etc), the nutritional quality, ¯avour, or processing charac- cleanliness of on-farm and bulk storage facilities, and teristics of stored grains.1 Fumigants should be bio- treatment of the grain by protectants, usually fumi- logically active, suf®ciently volatile to be removed by gants. The grain industries in developing countries use aeration, not absorbed by grain, not ¯ammable and a range of methods to minimize insect infestation on not corrosive. Currently, few chemicals are available stored grains, but generally rely on fumigants.1 for use as fumigants that meet these constraints. Protectants or insecticides are important tools in the Methyl bromide, currently the most effective fumi- integrated management of insect pests of stored grains. gant, will soon be restricted due to its potential ozone- These chemicals are used extensively by all sectors of depleting properties.6 Moreover, it is highly toxic to the grain industry. Currently, a mixture of organo- warm-blooded animals.7 The fumigation of stored phosphate OP) and pyrethroid insecticides, with grains with phosphine is likely to become more widely piperonyl butoxide added as a synergist, is also used used in the future because of its ef®cacy and rapid to control stored-grain insect pests. However, ®eld desorption. However, phosphine fumigation may populations of stored-grain insect pests have markedly become increasingly limited in use because resistance increased in their resistance to OP insecticides over the of stored grain insects to phosphine has now been past decade.2±4 Thus, if current strategies of using discovered in more than 45 countries.8,9 In addition, these pesticides to control insect pests of grain con- phosphine has been argued to be genotoxic to tinue, increasing amounts of pesticide will be required, occupationally exposed fumigators.10 Because of the * Correspondence to: Sung-Eun Lee, Plant Protection Research Unit, WRRC, USDA-ARS, 800 Buchanan Street, Albany, CA 94710, USA E-mail: [email protected] Contract/grant sponsor: Soonchunhyang University, Korea (Received 24 July 2000; accepted 19 February 2001) # 2001 Society of Chemical Industry. Pest Manag Sci 1526±498X/2001/$30.00 548 Toxicity of Mentha arvensis to rice weevil increasing drawbacks in continued use of today's Aldrich Co Milwaukee, WI). Acetylthiocholine io- conventional fumigants an effort is needed to develop dide ATChI) and 5,5'-dithiobis2-nitrobenzoic acid) new compounds to replace those currently used. DTNB) were purchased from Sigma Co St Louis, Essential oils are potential sources of alternative MO). All chemicals were of highest grade commer- compounds to currently used fumigants. Essential oils cially available. have low toxicity to warm-blooded animals, high 11±13 volatility and toxicity to stored-grain insect pests. 2.4 General procedure Essential oils are easily distilled from suitable plant Identi®cation of constituents in essential oils was material. Their major constituents, monoterpenes, are determined using electron impact mass spectra also of interest to industrial markets because of other EI-MS) obtained by gas chromatography±mass spec- potent biological activities in addition to their toxicity trometry GC±MS). The GC±MS identi®cation of to insects.14±19 terpenes was con®rmed by co-injection with authentic Herein, we report toxicities of essential oils as samples. GC±MS was performed on a Shimadzu fumigants from sixteen Korean medicinal and spice CLASS 5000 system interfaced with a Shimazdu plants, and of monoterpenes identi®ed from the most QP-5000 GC±MS ®tted with a CBP-5 25m active essential oil of Mentha arvensis L var piperascens, 0.25mm ID) capillary column. Chromatography against the rice weevil, Sitophilus oryzae L). Rice conditions were as follows: column temperature, weevils are one of the chief stored-grain pests, world- pro®le 70°Cto80°Cat1°C minÀ1, to 190°Cat wide.20±22 We also report the inhibition by mono- 10°C minÀ1, and then to 250°Cat30°C minÀ1; in- terpenes of acetylcholinesterase activity in S oryzae to jector temperature, 250°C; detector temperature, establish a primary mode of action for their toxicity. 280°C; carrier gas, helium at 30cmsÀ1. 2.5 Fumigation bioassays 2 METHODS AND MATERIALS Fumigation chambers were 3.4-liter glass round- 2.1 Insects bottom ¯asks with glass stoppers ®tted with a hook. Rice weevils were from a colony maintained by the Test fumigant materials deposited on a piece of ®lter Department of Agricultural Biology, Korea Univer- paper Whatman No 1) were placed, together with a sity, Korea. Weevils were reared to the adult stage small cage containing insects, into the fumigation using techniques described by Ryoo and Cho.23 chamber. The aqueous solution of terpenes were made freshly before bioassay. To obtain even distribution of 2.2 Essential oils from Korean medicinal plants test fumigants during assays, a magnetic stirrer was Sixteen Korean medicinal plants were purchased from used at the bottom of each ¯ask. Twenty adult insects, Kyeng-Dong traditional market in Seoul, Korea, and aged 10±15 days, were placed in each of three cages identi®ed by Professor KH Yoon, Department of 4cm in length and 1.5cm ID) perforated with small Biology, Soonchunhyang University, Asan, Korea. holes to enable penetration of any volatiles emanating After drying at room temperature, leaves of the from the test source. Small amounts of polished rice medicinal plants were subjected to steam distillation were placed in each cage as a food source for the according to protocols outlined by Marcus and weevils. Each treatment was duplicated. Mortality was 24 Lichtenstein. Species and yields %) of essential oils determined after 24h of treatment. The LC50 and LC95 of each dried plant are: Adenophora remoti¯ora M values were calculated by Probit analysis.25 Control Campanulaceae), <0.01%; Adenophora triphylla var mortality was accounted by Abbott's formula.26 japonica Hara Campanulaceae), <0.01%; Artemisia princeps var orientalis Hara Asteraceae), 0.54%; Aster 2.6 Inhibition study scaber T Compositae), <0.01%; Caranga sinica R Inhibition of acetylcholinesterase AChE) was as- Annonaceae), <0.01%; Chrysanthemum coronarium L sessed by the method of Ellman et al. 27 Test mono- var spatiosum Benth Compositae), <0.01%; Chry- terpenes were dissolved in absolute ethanol 5ml) and santhemum zawdskii var latilobumK Compositae), the total volume made to 50ml with 0.1 M phosphate 0.24%; Crisium japonicum var ussurienseK Com- buffer, pH 8.0, to give a 0.09 M stock solution of each positae), <0.01%; Leonurus sibiricus L Lamiaceae), test compound. Assay concentrations of monoter- 0.04%; Liriope muscari Benth Liliaceae), <0.01%; penes were diluted serially so that the concentration of Lonicera japonica Thunb var japonica H Caprifolia- ethanol never exceeded 10ml litreÀ1, a concentration ceae), <0.01%; Mentha arvensis L var piperascens M we assessed to inhibit AChE by less than 5%. Each Labiatae), 1.05%; Perilla frutescens L) Britt var assay was repeated three times. The reaction mixture orientalis Labiatae), 0.21%; Pimpinella bursa-pastoria contained a crude AChE enzyme preparation from M Araliaceae), <0.01%; Pueraria thunberginana adults of S oryzae 100ml), ATChI 0.5mM), DTNB Benth Leguminosae), <0.01%; Taraxacum platycar- 0.33mM) and phosphate buffer 92.7mM) containing pum Darlst Asteraceae), 0.02%. the test monoterpene, to a total volume of 3ml. The reaction was initiated by addition of crude enzyme, 2.3 Chemicals followed by incubation at 25°C for 5min. Levels of Monoterpenes used in bioassays were purchased from AChE activity were estimated by determining levels of Pest Manag Sci 57:548±553 2001) 549 S-E Lee et al thiocholine through measuring absorbance at 412nm.