Agricultural and Food Science in Finland, Vol. 10 (2001): 243–259 243 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 243–259. © Agricultural and Food Science in Finland Manuscript received April 2001 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 243–259. Review 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 The interest in the use of monoterpenes for insect pest and pathogen control originates from the need for pesticide products with less negative environmental and health impacts than highly effective synthetic pesticides. The expanding literature on the possibility of the use of these monoterpenes is reviewed and focused on the effects of limonene on various bioorganisms. Limonene is used as in- secticide to control ectoparasites of pet animals, but it has activity against many insects, mites, and microorganisms. 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, in few cases limonene-treated plants have become attractive to plant damaging insects and phytotoxic effects on cultivated plants have been observed. As a plant-based natural product limonene and other monoterpenes might have use in pest and weed control in organic agriculture after phyto- toxicity on crop plants and, effects on non-target soil animals and natural enemies of pest have been investigated. Key words: monoterpenes, limonene, essential oil, natural pesticides, plant protection, deterrent, in- sect control mailto:ibrahim@uku.fi 244 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Ibrahim, M.A. et al. Limonene in pest control 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 dis- tillation of plant parts, lent to them the term es- sential oil. The monoterpenes with to a lesser extent the sesquiterpenes, comprise the major components of essential oils. Monoterpenes op- erate as a chemical defence against herbivores and diseases, fragrances attractive to pollinators and allelopathic inhibition of seed germination and plant growth (Gershenzon and Croteau 1991, Langenheim 1994). Well-documented records show that before 1850, 20 plant species belong- ing to 16 different families were used for con- trol of agricultural and horticultural pests in Western Europe and China (Needham 1986, Smith and Secoy 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, lina- lool, or a crude citrus oil extract have worked their way into the market place. Two of these oils, (+)-limonene and linalool are long-stand- ing 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 re- garded 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 and Croteau 1999). Hydrocarbon variations that differ only in the arrangement of atoms are called isomers. De- scription of isomers and their chemical and bi- ological functions are summarized in Table 1 according to Fessenden et al. (1998). Enantiom- ers are chiral molecules that have the same mo- lecular 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 enan- tiomers (R)–(+)-limonene and (S)–(–)-limonene, but the enantiomers of carvone are (R)–(–)-car- vone 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 S- limonene is close to that of pine turpentine (Las- ka and Teubner 1999). Natural occurrence and functions of monoterpenes More than 1000 naturally occurring monoterpe- nes have been isolated from higher plants (Ger- shenzon and Croteau 1991). Monoterpenes are volatile and responsible for the characteristic 245 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 243–259. odours of many plants. Most monoterpenes oc- cur 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, represent- ing one of the largest and most diverse families of natural compounds (Croteau 1987). They ex- ist as hydrocarbons or as oxygenated moieties with aldehyde, alcohol, ketone, ester, and ether functionalities. Overall monoterpenes are insol- uble in water, however, monoterpenes contain- ing oxygen have greater solubility than hydro- carbons with comparable skeletons (Weidenham- er 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). Monoterpe- nes are most widely recognized constituents of conifers, mints (Lamiaceae), composites (Aster- aceae), and citrus (Rutaceae). α-pinene and β- pinene 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 essen- tial oil composition is determined both by ge- netic 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 in- sects (Brattesten 1983). Some exhibit acute tox- icity 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 Saxe- na 1974) and, interfere with physiological and biochemical processes (Gershenzon and Croteau 1991). Since monoterpene composition of plant spe- cies is very distinctive, specialist herbivore in- sects that have only one plant species or one plant genus as host plant, use highly volatile monote- rpenes as a cue to locate their specific host plant. Table 1. Description of isomers summarized from Fessenden et al. (1998) and chemical designation of some monoterpenes. Type of the isomers Nomenclature Description Old New A. Structural isomers Different compounds that have the same molecular formula, but differ in order of attachment of atoms B. Stereoisomers 1. Enantiomers Chiral molecules are mirror images of each other. (optical isomers) Chemical properties are similar, but physiological properties differ. The prefixes R (clockwise) and S (counter clockwise) D R indicate absolute configuration of group around chiral L S carbon. The prefixes (+)- and (–)- are used to designate the D + sign of optical rotation of plane-polarized light by the L – enantiomers 2. Diastereomers Cis Z Molecules are not mirror images of each other. including achiral Trans E Geometric isomers result from groups being Z (same) geometric isomers and E (opposite) side. 246 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Ibrahim, M.A. et al. Limonene in pest control For many oligophagous and polyphagous insect herbivores monoterpenes have been demonstrat- ed to act as toxins, feeding and oviposition de- terrents. Thus, monoterpenes appear to play an important role in protecting plants from insect attack (Gershenzon and Croteau 1991, Langen- heim 1994, Phillips and Croteau 1999). The best- known insect neurotoxins among monoterpenes are the pyrethroids, a group of monoterpene es- ters 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 re- viewed in this paper with a focus on the monot- erpenes 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 patho- gens, 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 cyto- plasmic membranes of Gram-negative bacteri- um Escherichia coli AG 100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325. These essential oils also cause potassium leak- age (Cox et al. 2000). The essential oil of Cym- bopogon densiflorus showed a wide spectrum of activity against Gram positive and Gram nega- tive 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-piperi- tenone, p-mentha-1 (7), 8-dien-ol, and p-men- tha-2, 8-dien-1-ol. (Takaisi-Kikuni et al. 2000). The main constituents of the oil of Cala- mintha nepeta (limonene, menthone, pulegone, menthol) were tested against some bacteria spe- cies, and only pulegone showed antimicrobial activity, particularly against all Salmonella spe- cies (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%, includ- ing 17% limonene) against several microorgan- isms showed antibacterial activities towards Gram-positive and Gram-negative bacteria. Streptococcus pyogenes and Micrococcus sp. were the more sensitive in the case of Gram-pos- itive bacteria and Shigella sonnei in Gram-neg- ative 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 vul- gare (dulce or sweet, vulgare or bitter, and azo- ricum or Florence) against 25 microorganisms was evaluated. The essential oil from sweet fen- nel (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 orega- no were active against Bacillus subtilis, E. coli, Hafnia alvei, Micrococcus luteus, Proteus vul- garis, 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 in- hibitory activity of some essential oils, includ- ing (+)-carvone against E. coli 0157:H7 and Sal- monella typhymurium, and determined that, (+)- 247 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 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. The essential oil of Tagetes minuta inhibited the multiplication of Gram-positive and Gram- negative bacteria showing 95–100% of inhibi- tion (Hethelyi et al. 1987). Similarly, Hyptis sua- veolens’ essential oil including limonene inhib- its the growth of both Gram-positive and Gram- negative 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 activi- ty 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, ter- pinolene and E-β-ocimene) were active against Gram-positive bacteria, yeast and fungi (Jans- sen et al. 1984). Fungi Cox et al. (2000) reported that fungal toxicity of M. alternifolia essential oils to the yeast Can- dida albicans is based on increased permeabili- ty of the plasma membranes. Essential oil of H. suaveolens including limonene has mild an- tifungal activity against C. albicans (Iwu et al. 1990). Monoterpenes (1R, 2S, 5R)-Isopulegol, (R)-carvone and Isolimonene showed good fun- gistatic 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 ac- tivity against Aspergillus niger (Marotti et al. 1994). Lippia alba essential oil was the most effec- tive 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 Colletotri- chum 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, Cer- atocystis paradoxa, Rhizoctonia solani, Curvu- laria lunata, Periconia atropurpuria and Epic- occum nigrum (Singh et al. 1998). A positive correlation between the monoter- pene content of the oils (other than limonene and sesquiterpenes) and fungal inhibition was ob- served in an experiment testing the effect of vol- atile components of citrus fruit essential oils on Penicillium digitatum and P. italicum. P. digi- tatum was found to be more sensitive to the in- hibitory action of the oils than P. italicum (Cac- cioni et al. 1998). Essential oils extracted from leaves of Oci- mum canum (O. americanum) and seeds of Anethum graveolens and Pimpenella anisum completely inhibited the growth of fungi at 3000 ppm. P. anisum oil showed fungicidal activity at 3000 ppm against C. falcatum, C. paradoxa and P. solani (Singh et al. 1998). (+)-limonene, cin- eole, β-myrcene, α -pinene, β-pinene and cam- phor showed high antifungal activity against Botrytis cinerea (Wilson et al. 1997). Volatiles from crushed tomato leaves inhibited hyphal growth of Alternaria alternata isolated from le- sions of tobacco leaves and Botrytis cinerea iso- lated from infected strawberry fruit. Aldehydes, including C 6 and C 9 compounds, formed by lipoxygenase enzyme pathway upon wounding leaves, inhibited growth of both species. Terpene hydrocarbons, 2-carene, and limonene had no significant effect on hyphal growth (Hamilton- Kemp et al. 1992). Cardamom oil inhibited the growth of A. flavus, A. parasiticus A. ochraceus, Penicillium sp., P. patulum, P. roquefortii and P. citrinum, and of its components α-terpinyl ac- etate had the greatest antifungal spectrum, fol- lowed by linalool, limonene, and cineole (Badei 1992). In agar diffusion experiments the essen- tial oil of Tagetes minuta inhibited the multipli- cation of fungi and showed 100% of inhibition (Hethelyi et al. 1986, 1987). 248 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Ibrahim, M.A. et al. Limonene in pest control Nematodes (+)-limonene at 100 ppm reduced the Heterod- era 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% mortal- ity in 30 mins. The nematicidal activity is there- fore 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 oviposi- tion 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 oc- curring monoterpenes were evaluated against T. urticae and most of the monoterpenes were lethal to the mite at high concentrations; carvo- menthenol and terpinen-4-ol were especially ef- fective (Lee et al. 1997). Insects Secondary plant metabolites play an important role in plant resistance to insects. Monoterpe- nes 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 suc- cessfully applied for the control of insect para- sitoids 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 kid- ney function (Tonelli 1987). (+)-limonene was toxic to all life stages of the cat flea, Cteno- cephalides felis (Hink and Fee 1986). (+)- limonene has shown to have efficacy against malathion-resistant fleas (Collart and Hink 1986), but dogs developed toxicity effects in- cluding extensive erythema, and therefore it should be used cautiously (Rosenbaum and Ker- lin 1995). Limonene has shown insecticidal prop- erties against human blood-sucking insects when tested against early 4th instar larvae of the mos- quito 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 Culi- coides 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, euca- lyptol, 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 249 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 243–259. germanica) decreased significantly the probabil- ity of young emerging from them, but did not affect female mortality (Karr and Coats 1992). Untreated diet was significantly preferred com- pared with diet treated with high levels of (+)- limonene, linalool, and α -terpineol. However, (+)-limonene among other tested compounds reduced significantly the time required by cock- roaches’ nymphs to reach the adult stage (Karr and Coats 1992). Limonene has been shown to be toxic to sev- eral 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. pondero- sae (Raffa and Berryman 1983). Laboratory bi- oassay indicated that myrcene, limonene and β- phellandrene applied topically at 20 ppm were toxic to 60% of adult spruce beetles, D. rufipen- nis (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 ter- raenovae in a net cage study. Other compounds like eucalyptol, limonene, p-cymene, gamma- terpinene, dihydrocarvyl-acetate, β-pinene, β- myrcene, 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 essen- tial 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 phyto- toxicity, and are manifested in several ways. Leaf tips, margins, or the entire leaf surface can ap- pear burned, growing tips and buds can be killed and roots can also be burned. Chlorosis or yel- lowing of leaves (in spots, along margins) or a general chlorosis of the entire leaf or leaf dis- tortion may appear as curling, crinkling, or cup- ping 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 abnor- mal 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 de- ficiencies, 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 tu- bers during storage with low persistence. Car- vone 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 250 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Ibrahim, M.A. et al. Limonene in pest control Table 2. Summary of reports indicating negative or positive effects of internal limonene concentrations in plants or in artificial food on plant damaging insects. Insect species Host plant Response Reference Reduced activity of pest insect Pissodes strobi Paired agar disc Limonene inhibited feeding at high Alfaro et al. 1980 (Coleoptera: bioassay concentration. Curcullionidae) – Pine shoot feeder Diaphania nitidalis Treated on artificial A concentration of 20 µl of (–)- Peterson et al. 1994 (Lepidoptera, Pyralidae) sites limonene 96% caused slight but – Pickleworm moth significant reduction in oviposition. Trioza apicalis Various Apiacea Large amounts of either (–) or (+)- Valterova et al. 1997 (Homoptera: Psyllidae) species limonene were released by the – carrot sucker Apiacea species of low preference of T. apicalis. Dioryctria zimmermanni, Scots pine, Pinus Resistant proveniences emitted Sadof & Grant 1997 (Lepidoptera: Pyralidae) sylvestris relatively high levels of limonene. – pine trunk borer Increased activity of pest insect Pissodes strobi Paired agar disc Limonene stimulated feeding at low Alfaro et al. 1980 (Coleoptera: bioassay concentration. Curcullionidae) – Pine shoot feeder Pissodes strobi Eastern white pine, Limonene concentration was highest Wilkinson 1980 – pine shoot feeder Pinus strobus in trees attacked most frequently. Dioryctria abietivorella Eastern white pine, Ten microliters of a test solution Shu et al. 1997 (Lepidoptera: Pyralidae) Pinus strobus containing 10 µg (–)-limonene 95% – twig borer elicited a significant oviposition response, but was the least stimulating monoterpene in EAG tests. Cydia strobilella Norway spruce, Limonene elicited the highest Åhman et al. 1988 (Lepidoptera: Tortricidae) Picea abies electroantennogram response, – spruce seed moth probably cue compound for ovipositing females. Dioryctria amatella Loblolly pine, Combination of (+)-limonene, Hanula et al .1985 (Lepidoptera: Pyralidae) Pinus taeda α-pinene, and myrcene was attractive – Southern pine coneworm to females. Dioryctria sylvestrella Maritime pine, Attacked trees contained a significantly Jactel et al. 1996 (Lepidoptera: Pyralidae) Pinus pinaster higher percentage of limonene together – maritime stem borer with longipinene and copaene. Thecodiplosis japonensis Japanese pine, Higher limonene and β-pinene contents Kim et al. 1976 (Diptera: Cecidomyiidae) Pinus thunbergii were associated to resistance. – Pine gall midge (Bouwmeester et al. 1995). Limonene oxide and linalool both inhibited sprouting and fungal growth but tubers were soft after exposure. Limonene and α -pinene did not inhibit sprout- ing, and fungal growth was present on every tu- ber treated (Vaughn and Spencer 1991). Because 251 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 243–259. of the phytotoxicity, monoterpenes may be a potential source of products used for haulm kill- ing and weed control. (+)-limonene is highly phytotoxic to sugar- beet seedlings (CV. SSY 1) at high concentra- tions (Viglierchio and Wu 1989). Carvone, (+)- limonene, and (–)-limonene were either less ef- fective 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 ob- servations suggest that cabbage and carrot seed- lings are sensitive to limonene at the concentra- tion of 9% (Ibrahim et al., unpublished results). In general, there is not much information avail- able on the phytotoxicity threshold values of monoterpenes to different cultivated plants. Suitability of limonene in control of plant-damaging insect pests Insecticidal use Experiments dealing with the use of monoterpe- nes extracted from plants like insecticides in plant protection are scarce. For insect pests that associated with plant roots, drenching with tox- ic monoterpene solution might increase larval mortality and reduce damage, but may also af- fect other soil animals (Karr et al. 1990). A wide range of monoterpenes has larvicidal effects on the western corn rootworm in the soil and effec- tively protects corn roots from attack by this lar- va 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 concentra- tion of limonene (Kainulainen et al. 2001). This observation suggests that, the selection of car- rot varieties with high limonene contents can be used to reduce carrot psyllid damages in the ar- eas, where the risk of damage is high. The se- lection 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 spe- cies 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 (Ho- neoye 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 cylin- der 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. sag- ittariae was immediately released into the mid- dle 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 Hone- oye 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 mix- ture significantly reduced feeding when leaf disks were offered to larvae 24 hrs after spray- ing (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 252 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Ibrahim, M.A. et al. Limonene in pest control T ab le 3 . S um m ar y of r ep or ts o f ex og en ou s ap pl ic at io n of l im on en e ag ai ns t pl an t da m ag in g in se ct s. In se ct s pe ci es H os t pl an t L im on en e ap pl ic at io n R es po ns e R ef er en ce R ed u ce d a ct iv it y o f p es t in se ct T h a u m et o p o ea p it yo ca m p a P in e (P in u s n ig ra ) E m ul si fi ed w it h w at er a nd (+ )- li m on en e re du ce d th e nu m be r T ib er i et a l. 1 99 9 (L ep id op te ra : N ot od on ti da e) sp ra ye d on t he f ol ia ge o f of e gg c lu st er o n pl an ts s pr ay ed pi ne s ee dl in gs w it h it H yl o b iu s a b ie ti s P in e (P in u s sy lv es tr is ), E xp os ur e to l im on en e H ig h li m on en e co nc en tr at io ns L in dg re n et a l. 1 99 6 (C ol ., C ur cu li on id ae ) S pr uc e (P ic ea a b ie s) va po ur s ex hi bi te d si gn s of p oi so ni ng w it hi n a fe w h ou rs D el ia a n ti q u a , O ni on , S ha ll ot , L ee k V ol at il e m ix tu re 3 -c ar en e, M on ot er pe ne m ix tu re s de te rr ed N ti am oa h et a l. 1 99 6 (D ip te ra , A nt ho m yi id ae ) li m on en e, a nd p -c ym en e ov ip os it io n of a du lt f li es re le as er f ro m c ap il la ry tu be s ne ar o ni on p la nt s T ri oz a ap ic al is C ar ro t S pr ay ed o n th e ca rr ot i n R ed uc ed d am ag e an d in cr ea se d A al to ne n et a l. 2 00 0 (H om op te ra , p sy ll id ae ) th e fi el d in 1 –6 % s ol ut io ns yi el d (s m al l am ou nt s of c ar vo ne as c on ta m in an t) M eg a st ig m u s p in u s an d W hi te f ir ( A b ie s co n co lo r) O lf ac to ry r es po ns es t o pu re L im on en e si gn if ic an tl y ac te d as L iu k et a l. 1 99 9 M . ra fi n i (H ym en op te ra : al ph a- pi ne ne a nd l im on en e re pe ll en t T or ym id ae ) – S ee d w as ps D en d ro ct o n u s ru fi p en n is , S pr uc e sp p. ( e. g. S it ka , B io as sa ye d fo r th ei r to xi ci ty (+ )- li m on en e at 6 0 pp m k il le d W er ne r 19 95 D . si m p le x (C ol ., S co ly ti da e) w hi te a nd E ng el m an n 10 0% o f th e pe st s af te r 24 h rs o f – S pr uc e be et le , E as te rn l ar ch sp ru ce ) ex po su re be et le r es pe ct iv el y D el ia r a d ic u m B ra ss ic ac ea e (e .g . R ad is h, O lf ac to ry s ti m ul i fo r L im on en e fr om t he s ur fa ce p ar t of K os ta l 19 92 (D ip te ra , A nt ho m yi id ae ) tu rn ip , c ab ba ge , c au li fl ow er , or ie nt at io n be ha vi ou r pl an t ho st w as r ep el le nt – C ab ba ge m ag go t ra pe ) A cr o le p io p si s a ss ec te ll a L ee k & o ni on , b ut a bl e O lf ac to m et er w it h tw o L im on en e w as r ep el le nt A l- R ou z et a l. 1 98 8 (L ep id op te ra , p lu te ll id ae ) de ve lo p on a ll A ll iu m c ro p pa ra ll el a ir c ur re nt s – L ee k m ot h sp ec ie s co nt ai ni ng a Y -s ha pe d ny lo n fi br e co n ti n u ed o n t h e n ex t p a g e 253 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 243–259. co n ti n u ed f ro m t h e p re ce d in g p a g e In se ct s pe ci es H os t pl an t L im on en e ap pl ic at io n R es po ns e R ef er en ce C o st el yt ra z ea la n d ic a P as tu re g ra ss es H os t se le ct io n te st i n gl as s L im on en e ha d so m e re pe ll en t ef fe ct O sb or ne & B oy d (C ol . S ca ra ba ei da e) ch am be rs 19 74 – G ra ss g ru b In cr ea se d a ct iv it y o f p es t in se ct P ra ys c it ri ( L ep id op te ra : C it ru s li m o n u m , C it ru s E le ct ro an te nn og ra m L im on en e ac ti va te d ov ip os it io n E l- S ay ed e t al . 1 99 4 H yp on om eu ti da e) d ec u m a n a & C it ru s re sp on se t o pu re l im on en e a u ra n ti u m H el ic o ve rp a a rm ig er a P ol yp ha go us m ot h (e .g . E le ct ro an te nn og ra ph y A tt ra ct iv e to 1 –2 d ay s ol d m ot hs D in g et a l. 1 99 7 (L ep id op te ra , N oc tu id ae ) co tt on , m ai ze c uc ur pi ta ce ae , us ed t o in ve st ig at e – C ot to n bo ll w or m to m at oe s, l eg um in ou s cr op s, el ec tr op hy si ol og ic al co ni fe rs ) re sp on se s Ip s ty p o g ra p h u s S pr uc e (e .g . N or w ay s pr uc e) F ie ld b io as sa y us in g (+ )- li m on en e in m ix tu re w it h R ed de m an n & (C ol ., S co ly ti da e) P he ro m on e ba it ed t ra ps α -p in en e at tr ac te d ad ul ts S ch op f 19 96 S pr uc e ba rk b ee tl e w it h a m ix tu re o f li m on en e an d α -p in en e P a p il io d em o le u s F ab ac ea e (e .g . C u ll en l en a x, O ri en ta ti on r es po ns es t o (– )- li m on en e sh ow ed m ax im um S ax en a et a l. 1 97 5 (L ep id op te ra , P ap il io ni da e) P sr a le a s p p .) a nd R ut ac ea e di ff er en t od ou rs i n O lf ac to ry at ta ra ct io n to t he l ar va e of t hi s pe st – C it ru s S w al lo w ta il (e .g . M u rr a ya k o en ig ii , c it ru s) N o e ff ec t H yl o b iu s a b ie ti s P in e (P in u s sy lv es tr is ), E xp os ur e to l im on en e L ow l im on en e le ve ls d id n ot a ff ec t L in dg re n et a l. 1 99 6 (C ol ., C ur cu li on id ae ) S pr uc e (P ic ea a b ie s) va po ur s fe ed in g ac ti vi ty H yl o b iu s a b ie ti s P in e (P in u s sy lv es tr is ), S co ts p in e (P in u s sy lv es tr is ) N o an ti fe ed in g ef fe ct s in 4 8h K le pz ig & S ch ly te r (C ol ., C ur cu li on id ae ) S pr uc e (P ic ea a b ie s) tw ig s w er e tr ea te d w it h fe ed in g tr ia l w it h ad ul t be et le s 19 99 li m on en e di ss ol ve d in e th yl ac et at e so lv en t 254 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Ibrahim, M.A. et al. Limonene in pest control confirmation of results, field tests are needed, since high concentration of volatile compounds inside closed Petri dishes may reduce larval feed- 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 oleores- in extracts of P. cembra cones significantly re- duced 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 macula- tus (Coleoptera: Bruchidae) (Don-Pedro 1996). Limonene, mixed with the combination of α- pinene and ethanol on old clear cuttings, inhib- ited completely the catch of Hylobius pinastri (Coleoptera: Curculionidae) and that of H. abi- etis 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 con- tain over 70% limonene, were highly toxic to 2nd 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 de- fensive compounds against the western spruce budworm (Zou and Cates 1997). Ntiamoah and Borden (1996) found that, a ternary mixture of carene, limonene and p- cymene in the choice bioassay significantly de- terred the oviposition of cabbage maggots, but the deterrence was slight in the non-choice bio- assay. Increasing the complexity of the blend to six monoterpenes increased the deterrent effect markedly. These results indicate that, as for on- ion maggots (Ntiamoah et al. 1996), monoter- penes are oviposition deterrents for cabbage maggots. The results also suggest that monoter- penes may be oviposition deterrents for other anthomyiids. Combining various deterrents used in different areas may develop a way of pest con- trol more valuable than by using single deter- rent. 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 spray- ing (grey bars). Asterisk above bar indicate significant (P < 0.05) difference from the control (water) treatment accord- ing to oneway anova. 255 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 10 (2001): 243–259. Effects on non-target organisms and manipulation of natural enemies Although the acute toxicity of monoterpenoids is low relative to that of conventional insecti- cides (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 clari- fied more extensively, since potential pesticide use of limonene in organic farming may be harm- ful for whole agroecosystem if earthworm pop- ulations are reduced. A l s o s e l e c t i v i t y a n d h a r m l e s s n e s s o f limonene toward natural enemies and other bio- logical agents needs testing. Interaction with entomopathogenes such as Bacillus thuringien- sis 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 pred- ator ant Myrmicaria eumenoides uses an efficient recruitment communication system based on the poison gland secretion that includes mainly (+)- limonene (Kaib and Dittebrand 1990). This ob- servation 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 arthro- pod 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 agri- culture. Possible attractive effects of limonene to natural enemies of pests may offer novel ap- plications to use natural compounds for manip- ulation of beneficial animals in organic agricul- ture. However, since monoterpenes are phyto- toxic to several cultivated plants, critical thresh- olds of limonene for plant physiology and limonene sensitivity should be determined. 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Holopainen Kuopion yliopisto ja MTT (Maa- ja elintarviketalouden tutkimuskeskus) Kiinnostus luonnonmukaisiin, kasveista peräisin ole- viin ja vähemmän terveys- ja ympäristöhaittoja ai- heuttaviin torjunta-aineisiin on lisääntynyt luomuvil- jelyn yleistyessä. Tässä katsauksessa selvitetään mo- noterpeenien käyttömahdollisuuksia kasvinsuojelus- sa ja arvioidaan erityisesti limoneenin vaikutuksia eri eliöryhmiin. Limoneenilla on torjuttu lemmikkieläin- ten ulkoloisia, mutta sen on todettu tehoavan myös moniin muihin hyönteisiin, punkkeihin ja mikrobei- hin. Sekä karkottavaa että myrkkyvaikutusta on ha- vaittu. Limoneenin houkuttavuus tuhohyönteisten luontaisille vihollisille voi tarjota mahdollisuuden käyttää sitä luomuviljelyyn sopivana biologisena tor- juntamenetelmänä, jossa tuholaisten luontaisia vihol- lisia houkutellaan kasvustoon ennen tuholaisia. Jois- sain tapauksissa limoneenilla käsitellyt kasvit voivat kuitenkin altistua tuholaisille limoneenikäsittelyn seurauksena, ja korkeilla pitoisuuksilla limoneeni on kasveille myrkyllinen. Kasviperäisinä luonnontuottei- na limoneenista ja muista monoterpeeneistä voi tul- la luomuviljelyyn sopivia tuhoeläinten ja rikkakas- vien torjunta-aineita. Tämä kuitenkin edellyttää, että aineiden mahdolliset haittavaikutukset viljelykasvei- hin, maaperäeliöstöön ja tuholaisten luontaisiin vihol- lisiin ensin selvitetään. Title Introduction Effects of monoterpenes on bio-organisms Suitability of limonene in control of plant-damaging insect pests Summary and future perspectives References SELOSTUS