Insecticidal , repellent , antimicrobial activity and phytotoxicity of essential oils : With special reference to limonene and its suitability for control of insect pests

Mohamed A. Ibrahim Department of Ecology and Environmental Science, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland, e-mail: ibrahim@uku.fi Pirjo Kainulainen MTT Agrifood Research Finland, Plant Production Research, Plant Protection, FIN-31600 Jokioinen, Finland. Current address: Department of Ecology and Environmental Science, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland Abbas Aflatuni MTT Agrifood Research Finland, Regional Research, Tutkimusasemantie 15, FIN-92400 Ruukki, Finland Kari Tiilikkala MTT Agrifood Research Finland, Plant Production Research, Plant Protection, FIN-31600 Jokioinen, Finland Jarmo K. Holopainen MTT Agrifood Research Finland, Plant Production Research, Plant Protection, FIN-31600 Jokioinen, Finland. Current address: Department of Ecology and Environmental Science, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland


Introduction Historical perspective
Essential oil-bearing plants have been collected from the forest and cultivated areas since pre-Christian time for their flavour and fragrance properties.Their volatility, which made them easy to discover in fragrant plant material and at the same time readily obtainable by simple distillation of plant parts, lent to them the term essential oil.The monoterpenes with to a lesser extent the sesquiterpenes, comprise the major components of essential oils.Monoterpenes operate as a chemical defence against herbivores and diseases, fragrances attractive to pollinators and allelopathic inhibition of seed germination and plant growth (Gershenzon andCroteau 1991, Langenheim 1994).Well-documented records show that before 1850, 20 plant species belonging to 16 different families were used for control of agricultural and horticultural pests in Western Europe and China (Needham 1986, Smith andSecoy 1981).The rich knowledge of plants with pesticide properties was not lost in China as evidenced by a recent report stating that in China different parts or extracts of 276 plant species are used as pesticides (Yang and Tang 1988).
Insecticidal properties have been recognized in the oil of many citrus fruits and in recent years, several products containing (+)-limonene, linalool, or a crude citrus oil extract have worked their way into the market place.Two of these oils, (+)-limonene and linalool are long-standing and widely used for food additives (Hooser 1990).

Chemical properties of monoterpenes
Terpenes are hydrocarbons classified by the number of five-carbon (isoprene) units that they contain.Monoterpenes contain a basic skeleton of 10-carbon atoms derived from of fusion of two C 5 isoprene units.The other classes of ter-penoids are sesquiterpenes (C 15 ), diterpenes (C 20 ), triterpenes (C 30 ), tetraterpenes (C 40 ) and polyterpenes (C n ).The plastids of plants are regarded to be the site of monoterpene synthesis.All terpenoid compounds are biosynthesised from isopentenyl diphosphate (IPP), which may be derived by acetate/mevalonate or pyruvate/ glyceraldehydes-3-phosphate pathway.The head-to-tail condensation of one molecule of IPP with one molecule of dimethylallyl diphosphate (DMAPP), itself derived from the reversible isomerization of IPP by IPP isomerase yields the C 10 compound geranyl diphosphate (GPP) which is the immediate precursor of the monoterpenes (Lichtenthaler et al. 1997, Little andCroteau 1999).
Hydrocarbon variations that differ only in the arrangement of atoms are called isomers.Description of isomers and their chemical and biological functions are summarized in Table 1 according to Fessenden et al. (1998).Enantiomers are chiral molecules that have the same molecular formula but are mirror images of each other.Absolute configuration of chiral carbon (R or S) is not dependent on the direction of optical rotation (+ or -), but in each specific compound are always related, e.g.limonene has two enantiomers (R)-(+)-limonene and (S)-(-)-limonene, but the enantiomers of carvone are (R)-(-)-carvone and (S)-(+)-carvone.In this review we use (+ and -)-nomenclature, since optical isomers are related closely to the biological activity of monoterpenes.Even human sense of smell is able to discriminate the odours of the enantiomers of α-pinene, carvone and limonene.R-limonene is the smell of lemon or orange, while smell of Slimonene is close to that of pine turpentine (Laska and Teubner 1999).

Natural occurrence and functions of monoterpenes
More than 1000 naturally occurring monoterpenes have been isolated from higher plants (Gershenzon and Croteau 1991).Monoterpenes are volatile and responsible for the characteristic odours of many plants.Most monoterpenes occur free in plant tissue, but some of them are found as glycosides.The monoterpenes occur in a variety of acyclic, monocyclic, bicyclic, and tricyclic structural types and derivates, representing one of the largest and most diverse families of natural compounds (Croteau 1987).They exist as hydrocarbons or as oxygenated moieties with aldehyde, alcohol, ketone, ester, and ether functionalities.Overall monoterpenes are insoluble in water, however, monoterpenes containing oxygen have greater solubility than hydrocarbons with comparable skeletons (Weidenhamer et al. 1993).
Because of lipophilic properties most of monoterpenes are stored in special structures as resin ducts, secretory cavities and epidermal glands (Dell and McComb 1981).Monoterpenes are most widely recognized constituents of conifers, mints (Lamiaceae), composites (Asteraceae), and citrus (Rutaceae).α-pinene and βpinene are among the most widely distributed monoterpenes in the plant kingdom and are the major constituents of the various volatile oils (Schütte 1984).Overall, the variability in essential oil composition is determined both by genetic and epigenetic factors.
Generally, plants can produce a diverse range of secondary metabolites such as terpenoids, phenolic compounds and alkaloids (Benner 1993).Terpenoids are among the vast reservoir of secondry compounds produced by higher plants evolved in defence against herbivores and pathogens (Duke et al. 1991).Monoterpenes may interfere with basic behavioural functions of insects (Brattesten 1983).Some exhibit acute toxicity whereas others are repellents (Watanabe et al. 1993), antifeedants (Hough-Goldstein 1990), or disrupt on growth and development (Karr and Coats 1992) or reproduction (Sharma and Saxena 1974) and, interfere with physiological and biochemical processes (Gershenzon and Croteau 1991).
Since monoterpene composition of plant species is very distinctive, specialist herbivore insects that have only one plant species or one plant genus as host plant, use highly volatile monoterpenes as a cue to locate their specific host plant.

Ibrahim, M.A. et al. Limonene in pest control
For many oligophagous and polyphagous insect herbivores monoterpenes have been demonstrated to act as toxins, feeding and oviposition deterrents.Thus, monoterpenes appear to play an important role in protecting plants from insect attack (Gershenzon and Croteau 1991, Langenheim 1994, Phillips and Croteau 1999).The bestknown insect neurotoxins among monoterpenes are the pyrethroids, a group of monoterpene esters found in the leaves and flowers of certain Chrysanthemum species (Harborne 1993).Some compounds in essential oils have shown promise as natural insect pest control agents because they naturally provide plants with chemical defences against phytophagous insects and plant pathogens.These advances are reviewed in this paper with a focus on the monoterpenes and especially limonene, a compound with low toxicity to humans and having even some antitumor activity (Crowell 1999).

Effects of monoterpenes on bio-organisms
Bacteria Essential oil of plants has been shown to have activity against human, animal and plant pathogens, as well as food poisoning bacteria.The essential oils have effects on bacteria cells or their activity.The essential oils from Melaleuca alternifolia (tea tree oil) inhibit the respiration and increase the permeability of bacterial cytoplasmic membranes of Gram-negative bacterium Escherichia coli AG 100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325.These essential oils also cause potassium leakage (Cox et al. 2000).The essential oil of Cymbopogon densiflorus showed a wide spectrum of activity against Gram positive and Gram negative bacteria in the range of 250-500 and 500-1000 ppm, respectively.The main essential oil components were limonene, cymenene, p-cymene, Z-and E-carveol, carvone, iso-piperitenone, p-mentha-1 (7), 8-dien-ol, and p-mentha-2, 8-dien-1-ol.(Takaisi-Kikuni et al. 2000).
The main constituents of the oil of Calamintha nepeta (limonene, menthone, pulegone, menthol) were tested against some bacteria species, and only pulegone showed antimicrobial activity, particularly against all Salmonella species (Flamini et al. 1999).The determination of the minimal bactericidal concentration of the essential oil from the leaves of Peumus boldus (main constituents: monoterpenes 90.5%, including 17% limonene) against several microorganisms showed antibacterial activities towards Gram-positive and Gram-negative bacteria.Streptococcus pyogenes and Micrococcus sp. were the more sensitive in the case of Gram-positive bacteria and Shigella sonnei in Gram-negative bacteria (Vila et al. 1999).
The antibacterial activity of the various oils (main constituents were (E)-anethole, limonene, fenchone, and methyl chavicol) hydrodistilled from the seeds of 3 varieties of Foeniculum vulgare (dulce or sweet, vulgare or bitter, and azoricum or Florence) against 25 microorganisms was evaluated.The essential oil from sweet fennel (at the early waxy seed stage) was the most effective antibacterial agent.Essential oils of Rosmarinus officinalis (from Giza, Egypt) showed a high antimicrobial activity against Cryptococcus neoformans and Mycobacterium intracellularae (Soliman et al. 1994).
In antibacterial assays, the essential oil of Origanum onites, Thymus capitatus and oregano were active against Bacillus subtilis, E. coli, Hafnia alvei, Micrococcus luteus, Proteus vulgaris, S. aureus and Streptococcus faecalis but not against Pseudomonas aeruginosa.O. onites, T. capitatus and oregano inhibited the growth of the 5 test fungi.It is suggested that the observed antimicrobial activities may be associated with the phenolic constituents in the essential oil of O. onites, T. capitatus and oregano (Biondi et al. 1993).Helander et al. (1998) tested the inhibitory activity of some essential oils, including (+)-carvone against E. coli 0157:H7 and Salmonella typhymurium, and determined that, (+)- . 10 (2001): 243-259.carvone was among less inhibitory compounds.Chanegriha et al. (1994) reported that, limonene and terpenyl acetate both inhibited the activity of B. subtilis and E. coli.

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The essential oil of Tagetes minuta inhibited the multiplication of Gram-positive and Gramnegative bacteria showing 95-100% of inhibition (Hethelyi et al. 1987).Similarly, Hyptis suaveolens' essential oil including limonene inhibits the growth of both Gram-positive and Gramnegative bacteria (Iwu et al. 1990).
The bark essential oil of Xylopia longifolia exhibited antimicrobial properties against some microorganisms including S. aureus and E. coli (MIC values of 0.5 and 2 mg/l respectively) (Fuornier et al. 1993).The antimicrobial activity of the Cotinus coggygria oil was manifested by its strong inhibition of the multiplication of both Gram-positive and Gram-negative bacteria (Hethelyi et al. 1986).The main components of the essential oil of leaves and stems of Ducrosia anethifolia (α-pinene, myrcene, limonene, terpinolene and E-β-ocimene) were active against Gram-positive bacteria, yeast and fungi (Janssen et al. 1984).
Fungi Cox et al. (2000) reported that fungal toxicity of M. alternifolia essential oils to the yeast Candida albicans is based on increased permeability of the plasma membranes.Essential oil of H. suaveolens including limonene has mild antifungal activity against C. albicans (Iwu et al. 1990).Monoterpenes (1R, 2S, 5R)-Isopulegol, (R)-carvone and Isolimonene showed good fungistatic activities against C. albicans (Naigre et al. 1996).All essential oils hydrodistilled from the seeds of 3 varieties of Foeniculum vulgare (dulce or sweet, vulgare or bitter, and azoricum or Florence) exhibited a marked antifungal activity against Aspergillus niger (Marotti et al. 1994).
Lippia alba essential oil was the most effective of essential oils extracted from various parts of 11 higher plants for their fungitoxicity against a range of fungal sugarcane pathogens.L. alba essential oil was fungistatic against Colletotrichum falcatum (Glomerella Tucumanensis) and C. pallescens at 700 ppm or less, and fungicidal at higher concentrations against all the other test pathogens such as: Fusarium moniliforme, Ceratocystis paradoxa, Rhizoctonia solani, Curvularia lunata, Periconia atropurpuria and Epicoccum nigrum (Singh et al. 1998).
A positive correlation between the monoterpene content of the oils (other than limonene and sesquiterpenes) and fungal inhibition was observed in an experiment testing the effect of volatile components of citrus fruit essential oils on Penicillium digitatum and P. italicum.P. digitatum was found to be more sensitive to the inhibitory action of the oils than P. italicum (Caccioni et al. 1998).

Ibrahim, M.A. et al. Limonene in pest control
Nematodes (+)-limonene at 100 ppm reduced the Heterodera schachtii population to less than 3% of the control after 3 months, when sugarbeet seedlings cv.SSY1 were inoculated with 2300 freshly hatched H. schachtii J2.However, root growth was also reduced by phytotoxic effects of (+)limonene (Viglierchio and Wu 1989).Aqueous extracts of H. suaveolens leaves gave 100% mortality of Meloidogyne incognita larvae in 80 mins, whereas the whole oil gave 100% mortality in 30 mins.The nematicidal activity is therefore linked to the essential oil of H. suaveolens, of which (+)-limonene and menthol are two main constituents (Babu and Sukul 1990).

Mites
(-)-limonene, β-pinene, α-pinene and ∆ 3 -carene were toxic to adult females of the spruce spider mite Oligonychus ununguis when exposed for 24 hrs at concentrations below the calculated LC50s.All four compounds decreased oviposition in the mites while three of these compounds (limonene, β-pinene, and α-pinene) influenced movement (Cook 1992).Monoterpene vapours from peppermint have similar toxic effects on the spider mite Tetranychus urticae (Larson and Berry 1984).Acute toxicity of 34 naturally occurring monoterpenes were evaluated against T. urticae and most of the monoterpenes were lethal to the mite at high concentrations; carvomenthenol and terpinen-4-ol were especially effective (Lee et al. 1997).

Insects
Secondary plant metabolites play an important role in plant resistance to insects.Monoterpenes including limonene can be bioassayed to determine their possible fumigant, contact, and ingestion activity against insect pests and other pathogens.These substances can be toxic via penetration of the insect cuticle (contact effect), via the respiratory system (fumigant effect) and via the digestive apparatus (ingestion effect) (Prates et al. 1998).
Insecticidal use of limonene has been successfully applied for the control of insect parasitoids of pet animals.Weekly application of (+)limonene reduced flea and tick infestations by 80% in 24 dogs and one cat, with no adverse effects on blood composition or liver and kidney function (Tonelli 1987).(+)-limonene was toxic to all life stages of the cat flea, Ctenocephalides felis (Hink and Fee 1986).(+)limonene has shown to have efficacy against malathion-resistant fleas (Collart and Hink 1986), but dogs developed toxicity effects including extensive erythema, and therefore it should be used cautiously (Rosenbaum and Kerlin 1995).Limonene has shown insecticidal properties against human blood-sucking insects when tested against early 4 th instar larvae of the mosquito Culex quinquefasciatus.The LC50 was 53.80 ppm after 24 h and 32.52 ppm after 48 h.Limonene-treated water was less favourable than untreated water for oviposition by females of the mosquito (Kassir et al. 1989).The oil of Myrica gale acts as a deterrent to biting midge Culicoides impunctatus, but limonene together with camphene and terpinene-4-ol were at the bottom of the scale of the activity (Stuart and Stuart 1998).
Some monoterpene showed efficacy against food and wood pests.Components (cymol and limonene) of the essential oils of Eucalyptus camaldulensis, E. cameroni, and of the peal of Citrus aurantium have significant insecticidal action, being lethal to the stored product pests Rhyzopertha dominica and Tribolium castaneum.(+)-limonene showed more effective control of T. castaneum than of R. dominica (Santos et al. 1997).A study carried out by Sharma and Raina (1998) indicated that linalool, citronellal, and carvone showed promising toxicity against the termite Odontotermes brunneus, while, eucalyptol, terpinene, and limonene did not show much activity.Application of high doses of (+)limonene or linalool to oothecal (egg save) of gravid female of German cockroach (Blatella Vol. 10 (2001): 243-259.germanica) decreased significantly the probability of young emerging from them, but did not affect female mortality (Karr and Coats 1992).Untreated diet was significantly preferred compared with diet treated with high levels of (+)limonene, linalool, and α-terpineol.However, (+)-limonene among other tested compounds reduced significantly the time required by cockroaches' nymphs to reach the adult stage (Karr and Coats 1992).
Limonene has been shown to be toxic to several bark beetles e.g. the Southern pine beetle, Dendroctonus frontalis (Coyne and Lott 1976), the Western pine beetle D. brevicomis (Smith 1975) and the mountain pine beetle D. ponderosae (Raffa and Berryman 1983).Laboratory bioassay indicated that myrcene, limonene and βphellandrene applied topically at 20 ppm were toxic to 60% of adult spruce beetles, D. rufipennis (Werner and Illmann 1994).The vapours of the monoterpenes present in grand fir (Abies grandis) phloem caused a significant mortality of the fir engraver beetle (Scolytus ventralis).Toxicity was observed at doses normally found in the host tree, either in the attacked phloem or in the reaction tissue induced by the associated fungi (Raffa et al. 1985).
The substances dihydrocarvone and carvone were repelled the blow fly, Protophormia terraenovae in a net cage study.Other compounds like eucalyptol, limonene, p-cymene, gammaterpinene, dihydrocarvyl-acetate, β-pinene, βmyrcene, eugenol, and α-humulene seemed to have a deterrent effect mainly by contact of the fly with the treated bait at concentrations of 17-25 µmolcm -2 (Thorsell et al. 1989).The essential oils of 0.5g citrus including (+)-limonene, α-pinene, and myrcene caused rapid knockdown (KT50) on Musca domestica in 10-20s, and also inhibition of the emergence rate of the pupae increased with the increased exposure time.The same dose of oils killed all treated flies within 24h (Liao and Liao 1999).
The internal concentration of limonene in plants or in artificial food of herbivorous insects has significant effects on the behaviour and food consumption of plant feeding insects (Table 2).
Most of the studies indicate the attractive role of limonene for herbivorous insects of conifers, which have a high content of monoterpenes in their oleoresin.For specialised herbivores limonene can be a signal compound to detect the right host plant species of certain plant family as shown with Trioza apicalis by Valterova et al. (1997).The same carrot pest (T.apicalis) can even avoid carrot varieties with high limonene content (Kainulainen et al. 2001).

Phytotoxicity
Plant injuries from chemicals are called phytotoxicity, and are manifested in several ways.Leaf tips, margins, or the entire leaf surface can appear burned, growing tips and buds can be killed and roots can also be burned.Chlorosis or yellowing of leaves (in spots, along margins) or a general chlorosis of the entire leaf or leaf distortion may appear as curling, crinkling, or cupping of the leaf.Stunting of growth on all or parts of the plant is also one of the phytotoxic impacts.Phytotoxic chemicals can also stimulate abnormal either excessive growth (as aerial roots and suckering), or elimination and distortion of fruit or flowers.Symptoms of phytotoxicity can be confused with insect or mite damage, diseases, and other abiotic problems such as nutrient deficiencies, or environmental conditions (Fink 1999).
Phytotoxicity of some naturally occurring monoterpenes were tested on maize plant, and some of them have shown phytotoxicity to maize roots and leaves.On the other hand, D-Carvone was the most phytotoxic, whereas pulegone was the safest (Lee et al. 1997).But on the other hand, carvone inhibited sprouting of treated potato tubers during storage with low persistence.Carvone has to be reapplied about every 3 months during storage.The low persistence means that tubers could be consumed as soon as 15 days after treatment.Carvone also inhibited early sprouting of seed tubers (Reust 2000).In the Netherlands, carvone has been introduced as a commercial sprouting inhibitor for potatoes
Limonene and α-pinene did not inhibit sprouting, and fungal growth was present on every tuber treated (Vaughn andSpencer 1991). Because Vol. 10 (2001): 243-259. of the phytotoxicity, monoterpenes may be a potential source of products used for haulm killing and weed control.
(+)-limonene is highly phytotoxic to sugarbeet seedlings (CV.SSY 1) at high concentrations (Viglierchio and Wu 1989).Carvone, (+)limonene, and (-)-limonene were either less effective and/or more phytotoxic to wheat, barley and perennial ryegrass when seed dressing to control slugs were tested (Nijenstein and Ester 1998).Preliminary tests showed that limonene has phytotoxic activity at concentrations more than 3% to strawberry (cv.Jonsok and Honeoye) seedlings (Ibrahim 2000).Our preliminary observations suggest that cabbage and carrot seedlings are sensitive to limonene at the concentration of 9% (Ibrahim et al., unpublished results).In general, there is not much information available on the phytotoxicity threshold values of monoterpenes to different cultivated plants.

Insecticidal use
Experiments dealing with the use of monoterpenes extracted from plants like insecticides in plant protection are scarce.For insect pests that associated with plant roots, drenching with toxic monoterpene solution might increase larval mortality and reduce damage, but may also affect other soil animals (Karr et al. 1990).A wide range of monoterpenes has larvicidal effects on the western corn rootworm in the soil and effectively protects corn roots from attack by this larva under greenhouse conditions (Lee et al. 1997).

Deterrent
Internal limonene concentrations in plants have shown deterrent effects on only few insect pests (Table 2).Among these the carrot psyllid (T.apicalis) has shown a reduced oviposition rate on carrot varieties having a high concentration of limonene (Kainulainen et al. 2001).This observation suggests that, the selection of carrot varieties with high limonene contents can be used to reduce carrot psyllid damages in the areas, where the risk of damage is high.The selection of resistant varieties may be a suitable method for pest control in organic farming, but the factors determining pest deterrence should be known.
Exogenous treatment of cultivated plants with limonene extracted from other plant species have more often reduced insect attack than increased and attracted pest insects (Table 3).In leaf disk tests to estimate deterrent effects of (+)limonene on larvae of Galerucella sagittariae (Coleoptera: Chrysomelidae) (Holopainen et al. 2000), plants from two strawberry varieties (Honeoye and Jonsok) were treated with 1% (+)limonene, 1% mixture (75:25%) of (+)-limonene and (+)-carvone, and water in fumehood.The leaf discs (diam.15 mm) were cut with a cylinder tube from strawberry leaves.For choice tests three leaf discs (one from each treatment) were put into Petri dish on filter paper, 2 hours and 24 hours after the treatment, one larva of G. sagittariae was immediately released into the middle of the Petri dish.After monitoring 24 hours the eaten area from the leaf discs by the larva was estimated visually.
Spraying of leaves with limonene or mixture of limonene and carvone did not significantly reduced the feeding ability of the larvae of G. sagittariae on leaf discs of the variety Honeoye if offered to the larvae 2 or 24 hours after spraying of leaves (Fig. 1a).In the test with strawberry variety Jonsok, larvae did not prefer feeding on leaf disk offered 2h after spraying, but limonene and, limonene and carvone in mixture significantly reduced feeding when leaf disks were offered to larvae 24 hrs after spraying (Fig. 1b).The result suggests that carvone in mixture with limonene can be as effective as pure limonene to reduce G. sagittariae larval feeding on certain strawberry varieties.For the ing also on control treatment as observed with the variety Jonsok when offered leaf discs sprayed only 2 h before feeding trial (Fig. 1b).Field sprays of Pinus uncinata with oleoresin extracts of P. cembra cones significantly reduced the overall damage of specialized cone insects.None of the cones sprayed with oleoresin were attacked, whereas insects damaged 11% and 31% of the unsprayed control cones (Dormont et al. 1997).Several compounds including the major component of all citrus peel oils, (+)limonene, were found to be bioactive, having a strong vapour insecticidal activity, against the cowpea weevil beetle Callosobruchus maculatus (Coleoptera: Bruchidae) (Don-Pedro 1996).

Ibrahim, M.A. et al. Limonene in pest control
Limonene, mixed with the combination of αpinene and ethanol on old clear cuttings, inhibited completely the catch of Hylobius pinastri (Coleoptera: Curculionidae) and that of H. abietis was reduced by two thirds.On fresh clear cuttings the inhibitory effect of limonene was small or absent (Nordlander 1990).Volatile oils of baladi orange and mandarin peels, which contain over 70% limonene, were highly toxic to 2 nd larval instar stage of Spodoptera littoralis.Their toxicity consistently increased with increasing concentration of the volatile oils.The results from this experiment suggest that these volatile oils could be used as larval growth disruptors and also as repellent materials against moths for controlling programme of cotton leaf worm S. littoralis (Omer et al. 1997).Sesquiterpenes, in combination with tricyclene, camphene, myrcene, limonene, terpinolene, and the acetate fraction appear to be an effective mixture of defensive compounds against the western spruce budworm (Zou and Cates 1997).Ntiamoah and Borden (1996) found that, a ternary mixture of carene, limonene and pcymene in the choice bioassay significantly deterred the oviposition of cabbage maggots, but the deterrence was slight in the non-choice bioassay.Increasing the complexity of the blend to six monoterpenes increased the deterrent effect markedly.These results indicate that, as for onion maggots (Ntiamoah et al. 1996), monoterpenes are oviposition deterrents for cabbage maggots.The results also suggest that monoterpenes may be oviposition deterrents for other anthomyiids.Combining various deterrents used in different areas may develop a way of pest control more valuable than by using single deterrent.

Effects on non-target organisms and manipulation of natural enemies
Although the acute toxicity of monoterpenoids is low relative to that of conventional insecticides (Lee et al. 1997), the effects of field scale use on soil and water animals should be tested further.Potential drifts to aquatic ecosystem may have effects on food chains, since mortality of aquatic dipterans have increased after limonene treatment (Kassir et al. 1989).Toxic effects on earthworms (Karr et al. 1990) should be clarified more extensively, since potential pesticide use of limonene in organic farming may be harmful for whole agroecosystem if earthworm populations are reduced.Also selectivity and harmlessness of limonene toward natural enemies and other biological agents needs testing.Interaction with entomopathogenes such as Bacillus thuringiensis should be understood.The potential use of limonene and other monoterpenes in integrated pest management can be achieved with the knowledge of all these interactions.For rapid attraction of large numbers of nestmates to newly discovered food sources, the polyphagous predator ant Myrmicaria eumenoides uses an efficient recruitment communication system based on the poison gland secretion that includes mainly (+)limonene (Kaib and Dittebrand 1990).This observation suggests that a better understanding of the communication systems of predators and parasitoids may offer novel ways to increase the efficiency of natural enemies of pest insects with limonene and other monoterpenes.

Summary and future perspectives
Monoterpenes distilled from plants have effects on many bacterial, fungal, nematode and arthropod species and some compounds are effective sprouting inhibitors.The monoterpene limonene has shown deterrent and insecticide properties suggesting that as a plant-based natural product it might have use in pest control in organic agriculture.Possible attractive effects of limonene to natural enemies of pests may offer novel applications to use natural compounds for manipulation of beneficial animals in organic agriculture.However, since monoterpenes are phytotoxic to several cultivated plants, critical thresholds of limonene for plant physiology and limonene sensitivity should be determined.Also the potential harmful effects on natural enemies of pests and non-target soil animals should be investigated.After these evaluations, suitable recommendations for the use of limonene on specific crop plant species and against specific pest species can be given.

Fig. 1 .
Fig.1.The effect of 1% limonene treatment on the feeding of the larvae Galerucella sagittariae on cv.Honeoye (A) and cv.Jonsok (B) leaf discs when larvae were put into the Petri dishes for feeding 2 h (black bars) and 24 h after spraying (grey bars).Asterisk above bar indicate significant (P < 0.05) difference from the control (water) treatment according to oneway anova.

Table 1 .
Description of isomers summarized from Fessenden et al. (1998) and chemical designation of some monoterpenes.

Table 2 .
Summary of reports indicating negative or positive effects of internal limonene concentrations in plants or in artificial food on plant damaging insects.

Table 3 .
Summary of reports of exogenous application of limonene against plant damaging insects.Insect species Host plant Limonene application Response Reference Megastigmus pinus and White fir (Abies concolor) Olfactory responses to pure Limonene significantly acted as Liuk et al. 1999 Papilio demoleus Fabaceae (e.g.Cullen lenax, Orientation responses to (-)-limonene showed maximum Saxena et al. 1975 (Lepidoptera, Papilionidae) Psralea spp.) and Rutaceae different odours in Olfactory attaraction to the larvae of this pest Exposure to limonene Low limonene levels did not affect Lindgren et al. 1996 (Col., Curculionidae) Spruce (Picea abies) vapours feeding activity Hylobius abietis Pine (Pinus sylvestris), Scots pine (Pinus sylvestris) No antifeeding effects in 48h Klepzig & Schlyter (Col., Curculionidae) Spruce (Picea abies) twigs were treated with feeding trial with adult beetles 1999 limonene dissolved in ethyl acetate solvent confirmation of results, field tests are needed, since high concentration of volatile compounds inside closed Petri dishes may reduce larval feed- -Citrus Swallowtail (e.g.Murraya koenigii, citrus)No effectHylobius abietis Pine (Pinus sylvestris),