OA10731A - Repellent compositions containing aromatic aldehydes - Google Patents

Abstract

Repellent compositions which contain falvonoid aldehydes such as cinnamic aldehyde, .alpha.-hexyl cinnamic aldehyde and/or coniferyl aldehyde are provided, together with methods for their use as repellents for pests including flies, cockroaches, aphids, silverleaf white flies, mosquitos, ticks, fleas, leafhoppers, thrips, two-spotted spider mites, snails, slugs, biting midges, earwigs, and moths.

Description

010731

REPELLENT COMPOSITIONS CONTAININGAROMATIC ALDEHYDES

INTRODUCTION

Technical Field

This invention relates to aromatic aldéhydes as pest repellents. The invention isexemplified by the use of cinnamic aldéhyde or alpha hexyl cinnamic aldéhyde as a mosquitorepellent and for repelling agricultural pests such as aphids and thrips.

Background

In many countries today, diseases such as malaria, vector-borne hémorrhagie fevers,cockroaches allergies, filth fleas, bubonic plague, ticks viruses, rickettsiae, spirochate bacteria,snails-schistosomiasis, and sand fly fever are still responsible for serious illnesses andnumerous deaths among inhabitants. The ever-growing concern for the protection ofendangered species and the downward trend in availability of the broad spectrum pesticides forpublic health are forcing scientists to look for other economical means of providing protectionfrom vectors of disease. Moreover, the cost of pesticides often is too high for many of the lessdeveloped nations and the increasing résistance to these compounds by vector populations is agrowing problem.

The use of repellents is an excellent alternate means of providing relief when otherconventional vector control methods are not feasible. For mammalian vector targets,repellents properly applied to the skin and/or clothing are an inexpensive and practical meansof reducing the biting activity of hematophagous arthropods and for the prévention of vectordisease transmission. Repellents are effective against a wide range of disease vectors, whereasa separate vaccine must be developed for each disease. Moreover, only a few vaccines areeffective against vector-borne diseases. Diseases spread by vectors also affect plants. Forexample, Dutch Elm Disease has destroyed millions of elm trees across the United States. Thedisease is caused by a fungus which is spread from tree to tree by a particularly species of 010731 insects attracted to the elms. Current methods of prophylaxis hâve had only limited success. A need exists for an effective prophylaxis for this destructive plant disease.

Retractions of the use of repellents in vector control and disease prévention usuallycenter on questions of safety and cost. For example, one of the more extensively usedrepellents was the 6-2-2 repellent which contained dimethyl phthalate, ethyl hexanediol, andIndalone in the proportion 6:2:2. Dimethyl phthalate and Indalone are still in limited use, butin 1991, the U.S. Environmental Protection Agency canceled ail registrations of ethylhexanediol at the request of the manufacturers concerned. This action was taken because ofnew information on possible adverse effects on fêtai development.

As another example, one of the most effective mosquito repellents is DEET (N.N-diethyl-1.3-methylbenzamide). This material virtually eclipsed other repellents for topical use,and it remains the principal repellent in use today, nearly 40 years after its discovery.

However, in recent years permethrin, a synthetic dérivative of pyrethrum, has largely replacedDEET for use on clothing and other fabric items. As a repellent, DEET is highly effective,but it may also cause allergie and toxic effects, especially when used on the skin repeatedly inhigh concentrations. Repellent formulations containing 90-99% DEET are considered highconcentrations, whereas repellent formulations containing 50% or less DEET are considered aseffective as a concentration of 100%. A concentration of 33% DEET is effective in providing10-14 hour protection. However, for products containing even low concentrations of DEET, itis recommended that the skin be cleaned with mild soap and rinsed with water as soon as therepellent is no longer needed, in order to minimize possible adverse reactions. Theserecommendations frequently are impractical in third world countries and for military use.

Repellent compositions exist for topical application to a mammal, as well as to repelinsects from entering a dwelling or other area. However, the safety of many of the topicalcompositions has been questioned. Moreover, many of the topical compositions are of limitedeffectiveness, especially in areas of severe infestation with insects. Treatment for externalinsect infestations of a mammal, such as lice or crabs, often involves topical application ofharsh toxic insecticidal compositions to skin or scalp. Irritation often develops, and adversehealth effects from long-term use are also known. A need exists for a safe, effective topicalrepellent composition for a mammal. Repellent compositions for the prévention of entry ofinsects are similarly ineffective. Many of the known such repellent compositions also are not 010731 safe for use in enclosed spaces due to their high toxicity, especially where children and petsmay corne into contact with them. A need exists for a non-irritating, non-toxic, effectiverepellent composition.

Insect infestations of trees and other woody plants destroy millions of ornamental andagricultural trees every year. Current treatments are only partially successful, and may rendera crop of agricultural products worthless due to their persistent toxicity. Thus, a need alsoexists for a relatively non-persistent, effective means for repelling insect infestations of treesand woody shrubs.

Relevant Literature USPN 5,093,326 discloses repellent compositions which include an ozonizedunsaturated hydrocarbon, including terpenes. USPN 5,365,017 discloses préparation of atransgenic plant having increased levels of cycloarterol. Publications relating to repellantformulations include Reifenrath et al. (1989) J. Am. Mosquito Control Association 5: 45-61and Reifenrath (1995) Cosmetics & Toiletries Magazine 110: 85-93.

SUMMARY OF THE INVENTION

The présent invention provides repellent compositions which contain aromaticaldéhydes and methods of using these compositions. The repellent compositions contain acompound which has a formula shown in (1) below:

wherein R represents -CH2OH or -CHO; n is an integer from 0 to 3; and each R’independently represents OH or an organic substituent containing from 1 to 10 carbon atomsand from 0 to 5 heteroatoms, wherein the total number of carbon and heteroatoms in ail R’substituents of said compound is no more than 15; and R4 represents hydrogen or an organicconstituent containing from 1 to 10 carbon atoms. These compounds include natural 010731 compounds, such as cinnamic aldéhyde. Also of interest are alpha substituted aldéhydes, suchas alpha hexyl cinnamic aldéhyde (HCA). In use, the compounds are applied to a surface,such as skin, clothing, bark, habitat components and the like, from which it is désirable torepel insects and other pests. The invention finds use, for example, in the prévention ofdisease and infection which can resuit from contact with a disease-carrying insect vector orother pest vector.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Methods and compositions are provided for obtaining and/or maintaining an areasubstantially free of insects and other pests. Mammals, birds, fish and their habitats, as wellas seeds, seedlings, plants, and plant parts such as fruit substantially free of pathogenicorganisms such as fungi, insects and other pests, as well as viruses, bacteria, spirochètes, andother disease-causing organisms, and sap-sucking insects are provided together with a methodto repel pests and disease-causing organisms. A surface of interest is contacted with aromaticaldéhyde in an amount sufficient to repel an insect or other pest. The amount of repellent thatis applied will dépend in part upon the nature of the surface, and to some extent upon theformulation and the spécifie compounding used and, therefore, must be empirically determinedfor best results with a particular insect or other pest.

The compositions and methods of the subject invention offer several advantages overexisting compositions and methods. A major advantage is that the formula components aregenerally regarded as safe (GRAS) and approved for food use. For example, a number of thearomatic aldéhydes which may find use in the subject invention, such as a-hexyl cinnamicaldéhyde (HCA), cinnamaldéhyde, and vanillin are GRAS synthetic flavoring agents (21 CFR§172.515). HCA was in public use before the 195O's and today is widely used in consumerpréparations (soaps, détergents, creams, lotions, perfumes) (Monographs on fragrances rawmaterials. Food Cosmet, Toxicol. 12: suppl., 915, 1974). HCA was granted GRAS status byFEMA (Flavoring Extract Manufacturers' Association. Survey of flavoring ingrédient usagelevels. No. 2569. Fd. Technol., Champaign, 19: (part 2) 155, 1965) in 1965 and is approvedby the US FDA for use in food (21CFR121.1164). The Council of Europe (1970) (Council ofEurope. Natural and Artificial Flavouring Substances. Partial Agreement in the Social andPublic Health Field. Strasbourg, List A(l), Sériés 1, no. 129, p. 55, 1970) included HCA in 010731 the list of admissible artificial flavoring substances at a level of 1 ppm.

Surfactants which can be used as emulsifiers in the subject formulations, such as theTweens (polysorbates) also already are used as food additives, as is saponin (which also hasGRAS status). In addition, formulation residuality can be managed. This is of great benefitfor integrated pest management programs with bénéficiai insects because short term residualscan be obtained. The long term control of pathogenic organisms results in a healthier plantand an improved yield of produce by the host plant as compared to nontransgenic plants. Thearomatic aldéhydes in particular hâve positive organoleptic and olefactory properties which insome cases can improve the flavor and/or smell of treated products and eliminate theunpleasant odor associated with many pest repellants. The odor of a-hexyl cinnamic aldéhyde(HCA), for example, is described as floral or jasmine-like with some herbaceous character(Technical Data Sheet).

For plants, the active ingrédients can be made by the plant following introduction of thegene(s) responsible for synthesis of the various aldéhydes into the plant cell genome. The useof transgenic plants rather than topical application of repellent to the plant decreases thelikelihood of any adverse side effects to field workers, or to animais, fish or fowl which ingestthe tissues or parts of the plants, since many of the compounds of formula (1), in particularthose of formulas (3) and (4), are GRAS food additives. In addition, the subject inventionovercomes the failure of current pesticides to translocate, for example, to roots for treatment ofphylloxéra. Also, prévention of infestation by repelling the vector which carries diseases, ordamages the target for the pest, significantly decreases the number of target animais or plantswhich will succumb to disease carried by the pest or be damaged by the activities of the pest,such as, for example, the damage done by female medflies as they oviposit on fruit.

When applied to animais, including humans, the subject formulations are non-toxic andnon-irritating to the skin at the concentrations used. For example, a-hexyl cinnemaldehyde(HCA) has an oral LD50 of 3.1 g/kg in rats and a dermal LD50 of greater than 3 g/kg (Moreno,O.M. Report to RIFM, March 24, 1971). HCA was found to be moderately irritating whenthe neat compound was applied to intact or abraded rabbit skin for 24 hours under occlusion(Moreno). When tested at 12% in petrolatum, HCA produced no irritation after a 48 hourclosed-patch test on human subjects and produced no sensitization in a maximization testcarried out on 25 human subjects (Kligman (1966) J. Invest. Dermatol. 47: 393). HCA at 010731 20% in diethylphthalate produced no positive reactions in a repeated insult patch test conductedon 100 human subjects. Jimbo tabulated allergenicity data found in the literature for 18fragrance compounds. While cinnamic aldéhyde had a positive reaction from the humanmaximization test, HCA was négative in the test. Patch test results of 2% HCA on 100eczema and dermatitis patients were négative (0 out of 100). Of 4 patients sensitive to 2%cinnamaldéhyde, none were found to cross react with 2% HCA. The skin sensitizationfrequently reported for cinnamaldéhyde is probably initiated by reaction of amino groups onproteins with the aldéhyde functional group. Substitution of bulky alkyl groups in the alphaposition (e.g. the hexyl group of HCA) relative to the aldéhyde group can reduce this reactivityby creating steric hindrance as well as reducing the electrophilicity of the aldehydic carbon.Alpha-substituted cinnamaldéhydes, to which skin sensitization is uncommon, react veryslowly or not at ail with amines in comparison with cinnamaldéhyde. In studies using themaximization test in guinea pigs, Senma and coworkers report a tendency that as the numberof hydrocarbons of alkyl groups replacing the alpha-hydrogen in cinnamaldéhyde increased,the rate of sensitization reaction declined.

The subject formulations also provide for effective control of multiple organisms, suchas both fungi and insects. The compounds also hâve been reported to hâve inhibitory activityagainst C. botulinum spore germination (Bowles and Miller, G. Food Protection (1993) 56:788-794). This multi-target efficacy reduces the need for application of multiple agents to aplant or animal to be protected, and substantially éliminâtes the need for application ofpesticides. In particular situations, such as where an insect damages an animal or a plant partor tissue and a secondary fungal or bacterial disease develops, this aspect of the invention isparticularly advantageous.

The general formulation is as shown in formula (1) above. A preferred formulation isshown in formula (2) below:

wherein Rj represents-CHO, R3 represents -H, -OH or an organic substituent containing from 1 to 10 carbon atoms, R2 represents -H, a methoxy group or organic substituent containing from 1 to 10 carbon atoms, and R4 represents a hydrogen or an organic substituent containing from 1 to 10 carbon atoms. Of particular interest are aromatic aldéhydes. Examples of aromatic aldéhydes of use in the présent invention are cinnamic aldéhyde ((3) below): 010731

CHO

and coniferyl aldéhyde ((4) below):

CHjO

CHO

Other compounds of interest include analogs of the compound of formula (1) such ascompounds substituted at the alpha position with an alkyl, such as a hexyl group, or abranched alkyl group such as an amyl group. Generally the group at the alpha position is fromC-5 to C-10. Such compounds include a-hexyl cinnamaldéhyde and a-amyl cinnamaldéhyde.The Chemical structure of a-hexyl cinnamic aldéhyde (HCA) is shown in (5) below. CH3 (ch2)5

CHO (5) 010731

The Chemical Abstracts Service (CAS) name of HCA is 2-(phenylmethylene) octanal and theCAS Registry Number is [101-86-0]. The compound is also described by the Chemical nameof 2-hexyl-3-phenyl-2-propenal. The compounds's formula is C15H20O and molecular weightis 216.3. HCA can be obtained from Firmenich; their product is composed principally of the (E)-cis isomer (93.8% maximum), and the (Z)-trans isomer (6% maximum). Among minorcomponents is the self aldol condensation product of octanal (1-1.5% (PersonalCommunication, June Burkhardt, Firmenich, Plainsboro, New Jersey).

The compounds can be used either alone or in combination with other active or inactivesubstances and can be applied by spraying, pouring, dipping, injecting, in the form ofconcentrated liquids, solutions, suspensions, powders and the like, containing suchconcentration of the active compound as is most suited for a particular purpose at hand. Theycan be applied, for example, in the form of dilute solution, in a suitable solvent directly to therhizosphere either as part of an irrigation schedule or as a separate application.

For use as a foliar spray, although the aldéhyde can be formulated alone, it can berendered substantive by including an emulsifier such as Tween 80. Other détergents which canbe used include anionic détergents such as those described in U.S. Patent Application No.4,978,686. Other compounds which can be used alone or in conjunction with détergentsinclude saponins from any of a variety of sources, particularly saponin from Yucca schidigeraor Yucca valida. Generally, détergents and other agents used in the formulation do not detractfrom the repellent properties of the aromatic aldéhydes but do increase the substantiveproperties of the formulation (see for example, U.S. Patent No. 4,477,361) and may improvethe pesticide properties, including fiingicide properties (see below). Additional componentssuch as an aqueous préparation of a sait of a polyprotic acid such as sodium bicarbonate,sodium sulfate, sodium phosphate or sodium biphosphate can be included in the formulation, toincrease the antifungal properties of the formulation. The resulting émulsion is diluted to anappropriate concentration for use.

In a preferred embodiment, the formulation includes a-hexyl cinnamic aldéhyde,cinnamic aldéhyde and/or coniferyl aldéhyde in a formulation containing Tween 80 or saponinas an emulsifier and may include sodium bicarbonate. The preferred formulation for repellingflies, mosquitoes, fleas, ticks, lice, cockroaches, two-spotted spider mites, silverleaf whiteflies, aphids, leafhoppers, thrips and ants is 10-5000 ppm; for ticks, 100-2500 ppm. 010731

Generally, the total amount of aldehyde(s) présent in the formulation is 2% or less. Theformulations are effective without the use of antioxidants; particular aldéhydes may hâveinhérent antioxidant properties, for example, coniferyl aldéhyde. Alcohols, such as glycols,including propylene glycol, are likewise not required for efficacy of the formulations and infact may be harmful to the plant.

Stability of the formulation can be evaluated by a variety of methods, includingaccelerated tests in which a formulation of interest is exposed to elevated températures over aset time. Samples of the formulations are taken at regular intervals and analyzed chemically bymethods known to those skilled in the art to détermine the rate and nature of dégradation. Forexample, HCA can be analyzed by Gas Liquid Chromatography (GLC), using a 30 meter nompolar polydimethylsiloxane capillary column (e.g. HP-1, Hewlett-Packard, or SPB-1, Supelco)and a flame-ionization detector (Personal Communication). Using hélium as a carrier gas (8ml/min.) and a column température of approximately 240°C, the (E)-cis isomer (majorcomponent) has a rétention time of approximately 6.0 minutes and the (Z)-trans isomer (minorcomponent) has a rétention time of approximately 6.3 minutes.

The most effective amount for compositions including compounds of formula (3) and/orformula (4) and/or formula (5) as well as the amount of other compounds of formula (1) whichfind use can be determined using protocols known to those of skill in the art for evaluatingrepellent efficacy of compounds. Examples of such protocols follow. These protocols alsocan be used to optimize each formulation for spécifie pathogens using any of the compoundsencompassed by formula (1) or formula (5) as well as for spécifie applications to minimizeplant phytotoxicity or skin sensitivity and other side effects for animais while maximizing theantipathogenic effect of the formulation.

In some instances, the efficacy of the formulation can be increased by adding one ormore other components, i.e., a compound other than a compound of formula (1) or (5), to theformulation where it is désirable to alter particular aspects of the formulation. As an example,it may be désirable for certain plant applications if there is an undesirable amount ofphytotoxicity to decrease the phytotoxicity effect (phytotoxicity rating of 2 or less, with 1 orless preferred, see below) or to increase the repellent effect of the formulation, or both. It ispréférable that the additional component(s) minimize any side effects to plants or animais whileincreasing the repellent effect of the formulation. Of particular interest is the use of a 10 01073î component(s) which is a synergist to increase repellency while minimizing any side effects asrelated to a particular formulation. By "synergist" is intended a component which, by virtue ofits presence, increases the desired effect by more than an additive amount. The concentrationof one or more of the other formulation ingrédients can be modified while preserving orenhancing the desired repellent effect of the formulation. Of particular interest is the additionof components to a formulation to allow for a réduction in the concentration of one or moreother ingrédients in a given formulation while substantially maintaining efficacy of theformulation. Combination of such a component with other ingrédients of the formulation canbe accomplished in one or more steps at any suitable stage of mixing and/or application of thecomposition.

Preferred additional components include saponins, as they can be substituted forsurfactants as emulsifying agents, and additionally on at least same plants hâve a growthpromotant effect at the concentrations used. Generally, the use of saponin does not interfèrewith the repellant properties of the formulation. Saponins are a class of compounds, eachconsisting of a sapogenin portion and a sugar moiety. The sapogenin may be a steroid or atriterpene and the sugar moiety may be glucose, galactose, a pentose, or a methylpentose. S.Budavari, ed., The Merck Index, llth ed., Merck & Co., Inc., Rahway, N.J., 1990, p. 1328.The saponins for use in the présent invention can be produced and/or isolated from variousplant parts including fruit, leaf, seed and/or root, using means known in the art, from a varietyof sources including the various plants known to produce them, ranging from yucca, quillaja,agave, tobacco, licorice, soybean, ginseng and asparagus to aloe woods. Saponins for use withthe présent invention are preferably non-toxic to humans and higher animais. Most preferablythe saponin for use in the présent invention is non-toxic food grade, the source being fromyucca plants. Even more preferred are the saponins from Yucca schidigera or Y. valida andtheir équivalents. The saponins are generally prepared by a cold press extraction process andthe resulting liquid extract used. The yucca fiber also can be used; it is typically sundried,mulled and sized by screening. Generally an effective amount of saponin is of the range 0.01to 0.1% and most preferably about 0.01% v/v aqueous solution of 10° brix saponin extract. A variety of structurally related saponins are known, the most variable structural feature being the glycosylation pattern. Saponins also may contain additional modifications, such as sarasaponins which are saponins with a steroid attached, and saponin structure can be il 010731 modified by any number of enzymatic, Chemical and/or mechanical means known in the art.Saponins from Yucca schidigera contain stéroïdal saponins with the major sapogenins beingsarasapogenin and tigogenin. The sarasaponin yields on hydrolysis, sarasaspogenin(sarasaspogenin 5-beta, 20-betaF, 22-deltaF, 25-betaF; also known as spirostan-3-beta-01 andparigenin), glucose and galactose. The saraspogenin has a molecular formula ofNobel, Park S., Oxford Univ. Press, New York, 1994. A variety of structurally related saponins are known, the most variable structural feature being the glycosylationpattern. S. Budavari, ed., The Merck Index, llth ed., Merck & Co., Inc., Rahway, N.J., 1990, p. 1328. Saponins also may contain additional modifications, such as the sarasaponinswhich are saponins with a steroid attached, and saponin structure can be modified by anynumber of enzymatic, Chemical and/or mechanical means known in the art. Generally, aneffective amount of saponin is of the range of about 0.01 to 3% and most preferably about0.25% v/v aqueous solution of 10° brix saponin extract. 10° brix is a terms of art in sugarchemistry. The brix degrees equals the percent by weight of sugar in the solution. Hawley,ed., The Condensed Chemical Dictionary, lOth ed., Van Nostrand Reinhold, New York, 1981,p. 149.

For applications where the formulation is to be used to préparé the ground or othergrowth substrate for planting of host plants susceptible to particular pathogens, particularlywhere the growth substrate is already infested, the formulations of the subject invention can beadded directly to the rhizosphere or the substrate or they can be bound to a solid support orencapsulated in a time release material to repel undesirable insects and other pests. Where asolid carrier is used, materials which can lead to oxidation of the active aldéhydes should beavoided. Examples of delivery Systems which can be used include starch-dextran, and the like.

See Yuan et al., Fundamental and Applied Toxicology (1993) 20: 83-87 for other examples ofappropriate materials.

In addition to the spécifie compounds of the formulas (1), (2), (3), (4) and (5) above,precursors of any of these compounds that produce a compound of the formulas identifiedabove upon action of a biological System on the precursors are considered to be équivalent tocompounds of the invention. Thus application of precursor compounds to plant parts or tissueswould be équivalent to the practice of the présent invention. Biological conversion ofprecursor compounds into aromatic aldéhydes is described in, for example, U.S. Patent 12 010731

Application No. 5,149,715 and référencés cited therein. See also Casey and Dobb EnzymeMicrob. Technol. (1992) 14: 739-747.

The method of the présent invention is carried out by introducing onto a surface ofinterest a sufficient amount of an repellent agent to repel the insect or other pest.

Alternatively, where the surface of interest is skin, fur, hair, clothing and the like, theapplication can be by way of contacting the surface of interest with a formulation that has beenrendered substantive for the surface of interest so that a repellant amount of the formulationremains on the surface so treated and is released at a rate sufficient to repel susceptible insector other pest. A formulation containing the repellent agent generally is introduced by topicalapplication to a surface. For example, the formulation is sprayed on, as a wet or dryformulation, the surface and/or underside as applicable to the surface of interest. Among theformulations suitable for application are sprays, sticks, and repellent oils or ointments. Insome instances, the surface of interest can be impregnated with the repellent formulation byabsorption into the surface. Alternately, the formulation can be applied wet or dry to therhizosphere where it can vaporize in the vicinity of the roots and associated pathogenicorganisms which colonize the roots at a rate sufficient to repel a susceptible insect or pest. Insome instances, air can be introduced into the rhizosphere to increase the vaporization process.

To prevent an ingress of insects into an area, the compositions of the invention can be appliedto surfaces within and/or surrounding the area, for example, the compositions can be appliedto doors, Windows and other openings of a building and/or to surfaces that surround theseopenings.

Where the surface of interest is a plant or plant part, the presence of the repellent agentcan be a resuit of topical application; for example, the compositions can be aerially applied tocrops, or it can be by élaboration from the host plant as a resuit of genetic modification of thehost plant,

The aromatic and aliphatic aldéhydes of the subject invention can be prepared byvarious synthetic methods known to those skilled in the art. For example, see, J. March, ed.,Appendix B, Advanced Organic Chemistry: Réactions, Mechanisms, and Structure, 2nd Ed.,McGraw-Hill, New York, 1977. Cinnamaldéhyde canbe prepared synthetically, for example,by oxidation of cinnamyl alcohol (Traynelis et al., J. Am. Chem. Soc. (1964) 86:298) or bycondensation of styrene with formylmethylanîline (Brit. patent 504,125). The subject 13 010731 aldéhydes also can be obtained by isolation from natural sources. For example, cinnamaldéhyde can be isolated from woodrotting fungus, Stereum subpileatum. Birkinshaw et al., Biochem. J. (1957) 66:188, and a-hexyl cinnamic aldéhyde (HCA) can be obtained from rice, or synthesized as described in USPN 5,055,621. A preferred method for producing a desired component of the présent formulations in aplant host is through recombinant DNA means, particularly by modifying the level of at leastone compound of interest of the formula (1), (2), (3), (4), or (5) in plant tissues of interestthrough construction of transgenic plants using recombinant techniques known in the art. Themethods involve transforming a plant cell of interest with an expression cassette functional in aplant cell comprising as operably linked components in the 5' to 3' direction of transcription, atranscriptional and translational initiation regulatory région, joined in reading frame 59 to aDNA sequence encoding and capable of modulating the production and/or required to producethe compound of interest, and translational and transcriptional termination régions. Expressionof an enzyme required to produce the compound of interest provides for an increase inproduction of the compound as a resuit of altered concentrations of the enzymes involved inthe compounds' biosynthesis. Of particular interest is the sélective control of cinnamic and/orconiferyl aldéhyde and/or HCA production in plant tissues such as leaves, roots, fruits andseeds. One or more compounds of the présent formulations can be produced by modulatingthe expression of one or more genes or a gene encoding or more enzymes or an enzymepathway or cluster required to control the level of the compound of interest in a plant, plantpart, plant cell, spécifie plant tissue and/or associated with a particular stage of plant growth.

The enzyme or enzymes can be in a biosynthetic pathway or a dégradation pathway andthe régulation will be up or down respectively; i.e., to modulate expression of an indigenousor an endogenous plant gene an indigenous plant gene is one which is native to the genome ofthe host plant. An endogenous plant gene is one that is présent in the genome of the plant hostof interest, and may be an indigenous gene or a gene that is présent as a resuit of infection ofthe plant le. g., a viral gene), or otherwise naturally incorporated into the plant genome. Thehost plant also can be modified by recombinant means or by traditional plant breeding methodsto introduce one or more genes exogenous to the host plant which encode enzymes whichcontrol the level of the compound of interest and/or are in the synthetic pathway for one ormore compounds of formula (1), (2), (3), (4) or (5). By "modulation of gene expression" is 14 010731 intended control of production of a gene product of interest at the level of transcription,translation and/or post translation. The level of the compound of interest is controlled bymodulating the expression of one or more endogenous genes or transgenes encoding one ormore enzymes required to synthesize the compound of interest.

Methods for modulating gene expression in plants are known in the art. Variation ingrowth conditions or exogenous application of compounds to a plant can affect geneexpression. At the molecular level, gene expression dépends substantially on the transcription,translation and termination control régions which regulate expression of a structural genecoding région. By exploiting the plant signais which regulate these control régions or by thedirect recombinant manipulation of the control régions, expression of a gene encoding anenzyme required to control the level of cinnamic aldéhyde, for example, can be modulated.

For use in a transgene supplied exogenously to a plant host, the transgene will include controlrégions that are selected and designed to achieve the desired tissue and/or level and timing ofgene expression. As appropriate, the control régions may be homologous (native) or non-homologous (non-native) to the gene of interest. By "homologous" it is meant that the controlregion(s) is from or substantially similar to a control région normally associated with the geneof interest. By "non-homologous" it is meant that the control region(s) originates from adifferent nucléotide source or sequence or is substantially different from the control region(s)normally associated with the gene of interest. For example, if the enzyme coding sequence isnon-homologous in source as compared to the control régions, in order to hâve expression ofthe gene in a plant cell of interest, transcriptional and translational initiation regulatory régionsor promoters functional in these plant cells must be provided operably linked to the codingsequence. Transcription and translation initiation signais functional in plant cells include thosefrom genes which are présent in the plant host or other plant species, and direct constitutive orsélective expression in a plant host.

Of particular interest are the gene control régions that selectively regulate structuralgene expression in a plant, plant part, plant cell, spécifie plant tissue and/or are associated witha particular stage of plant growth. Preferred are those control régions, that are known in theart, and in particular, transcriptional control régions or promoters, that can be used tomodulate the expression of a gene encoding an enzyme required to control the level of acompound of formula (1), (2), (3), (4) and/or (5) in a plant, plant part, plant cell, or spécifie 15 010731 plant tissue and/or are associated with a particular stage of plant growth. For example,promoters showing differential expression patterns in fruit are described in USPN 4,943,674and USPN 5,175,095; seed in USPN 5,315,001; and in rapidly developing tissues and tendershoots in USPN 5,177,011.

For sélective control of biosynthesis of cinnamic and/or coniferyl aldéhyde and/or HCAin a plant tissue of interest, plant cells are transformed with an expression cassette comprisingDNA encoding a structural gene for one or more enzymes required to synthesize cinnamicand/or coniferyl aldéhyde and/or HCA and capable of increasing the amount of these aldéhydesin the tissue of interest. Of particular interest are those genes encoding one or more enzymescapable of metabolizing a precursor compound required for the biosynthesis of cinnamic and/orconiferyl aldéhyde and/or HCA from substrates normally found in a plant cell, moreparticularly the transgenic expression of at least one compound of the formula (1), (2), (3), (4),or (5). DNA constructs for expressing a gene of interest can be prepared which provide forintégration of the expression cassette into the genome of a plant host. Intégration can beaccomplished using transformation Systems known in the art such as Agrobacterium,electroporation or high-velocity microparticle-mediated transformation. Depending upon theapplication, saponin or one of the other compounds of interest can be preferentially expressedin a tissue of interest and/or a particular organelle. Tissue specificity is accomplished by theuse of transcriptional regulatory régions having the desired expression profile. Translocationof the enzyme to a particular organelle is accomplished by the use of an appropriatetranslocation peptide. Methods for tissue and organelle spécifie expression of DNA constructshâve been described are known in the art.

To verify régulation and expression of the gene of interest, various techniques exist fordetermining whether the desired DNA sequences présent in the plant cell are integrated into thegenome and are being transcribed. Techniques such as the Northern blot can be employed fordetecting messenger RNA which codes for the desired enzyme. Expression can further bedetected by assaying for enzyme activity or immunoassay for the protein product. Mostpreferably the level of the compound of interest présent in a plant host is measured usingmethods known in the art. A desired phenotype, for example, is increased HCA content in aplant tissue of interest as measured by expression of the gene of interest and/or the level of 16 010731 HCA présent in the plant host as compared to a control plant.

For introduction of one or more compounds of the présent formulations to the targetorganism, a plant host expressing a gene encoding an enzyme required to control the level ofthe compound of interest results in the exposure of a target organism to at least one componentof the repellent formulation. At least one component of the repellent formulation can beexpressed by the plant host and optionally other components of the repellent formulation areexogenously applied to the plant host so that the combination elicits the desired repellent effect.

Transgénie plants having an increased ability to accumulate aromatic aldéhydes such ascinnamaldéhyde and coniferyl aldéhyde and HCA to provide self-protection against plant pestsor be used as a natural source of aromatic aldéhydes for extraction and subséquent use as arepellant can be prepared.

Accumulation of aromatic aldéhydes can be achieved by downregulating the expressionof spécifie plant genes that encode enzymes which either cause further metabolism of thedesired aldéhydes or divert metabolic intermediates away from the desired aldéhydes. In thecase of cinnamaldéhyde, for example, this involves downregulating the expression ofcinnamate 4-hydroxylase (CA4H) and cinnamic alcohol dehydrogenase (CAD). CA4Hordinarily diverts some cinnamic acid away from cinnamaldéhyde to produce p-coumaric acid,itself a metabolic intermediate. Reducing CA4H activity alone is not sufficient to causeaccumulation of cinnamaldéhyde because CAD can rapidly convert cinnamaldéhyde tocinnamyl alcohol, which then becomes incorporated into lignin or accumulâtes as glycosides.Simultaneously reducing both CA4H and CAD activities results in increased metabolic fluxfrom cinnamic acid into cinnamaldéhyde and decreased conversion of cinnamaldéhyde intocinnamyl alcohol. Some cinnamaldéhyde becomes incorporated into lignin but cinnamaldéhyde(either free or as glycosides) also accumulâtes to above-normal levels, particularly at timeswhen the biosynthesis of cinnamic acid is elevated. This occurs when the level ofphenylalanine ammonia lyase (PAL; the first and rate-limiting step in general phenylpropanoidmetabolism, Hahlbrock and Scheel (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:347-369) activity is high, a situation that naturally occurs in plants in response to a wide range ofstimuli including invasion by fungal pathogens and mechanical damage associated withwounding and insect feeding.

Inhibiting CAD activity in transgenic plants has been proposed as a method of reducing l7 010731

lignin synthesis in plants and thereby improving the digestibility of fodder crops (WO 93/05159). These experiments suggested that lignin biosynthesis had been altered qualitatively, but not necessarily quantitatively, but did not demonstrate or appreciate the desirability of accumulating cinnamaldéhyde as a method of increasing insect and other pest repellancy. A number of plant CA4H and CAD genes hâve been cloned and their sequences areavailable from GenBank. Portions of these genes that include nucléotide sequences that areconserved between different plant species can be used directly in a plant expression vector(antisense or sense orientation) to suppress the expression of the corresponding endogenousgenes (e.g., Pear, et al., The Plant Cell Antisense Res. and Develop. (1993) 3:181-190,

Napoli, et al., The Plant Cell (1990) 2:279-289. More preferably, these conserved genesequences are used to isolate CA4H and CAD cDNA clones from a cDNA library of the plantspecies that is to be modified. The resulting cDNA clones, or portions thereof, are thenintroduced into a plant expression vector (antisense or sense) and used to transform the plant(s)of interest. DNA constructs according to the invention preferably comprise a sequence of atleast 50 bases which is homologous to the endogenous CA4H or CAD genes. A recombinant DNA molécule can be produced by operatively linking a vector to auseful DNA segment to form a plasmid that can be used for plant transformation. A vectorcapable of directing the expression of RNA from a cloned portion of a gene is referred toherein as an "expression vector." Such expression vectors contain expression control élémentsincluding a promoter. Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the Ti plasmid of Agrobacteriumtumefaciens described by Rogers et al., Methods in Enzymology (1987) 153:253-277. Acommon promoter that is used to provide strong constitutive expression of an introduced geneis the cauliflower mosaic virus (CaMV) 35 S promoter (available from Pharmacia, Piscataway,NJ). Either constitutive promoters (such as CaMV 35S) or inducible or developmentallyregulated promoters (such as the promoter from a PAL gene or the endogenous CA4H or CADgenes) can be used. Use of a constitutive promoter will tend to affect fonctions in ail parts ofthe plant, while use of an inducible or developmentally regulated promoter has the advantagethat the antisense or sense RNA is only produced in the tissue and under the conditions it isrequired. The use of developmentally regulated promoters is preferred in the use of this 010731 invention because the down-regulation of phenylpropanoid biosynthesis is known to be capableof producing undesirable side-effects on the development of transgenic plants containing aheterologous PAL gene (Elkind, Y. et al., 1990) Proc. Nat. Acad. Sci. (1990) 87:9057-9061. A number of different transformation methods are available for the routinetransformation of a wide range of plant species. One method that is particularly efficient forthe transfer of DNA into dicotyledonous plants involves the use of Agrobacterium. In thismethod the gene of interest is inserted between the borders of the T-DNA région that hâvebeen spliced into a small recombinant plasmid with a selectable marker gene (for exampleencoding neomycin phosphotransferase II or phosphinothricin acetyltransferase). Therecombinant plasmid is then introduced into an Agrobacterium host by transformation ortriparental mating. The Agrobacterium strain carrying the gene(s) of interest is then used totransform plant tissue by co-culturing the bacteria with an appropriate plant tissue {e.g., leafdise). Transformed cells are selected in tissue culture using the appropriate sélection agent andplants are then regenerated (see Horsch, R. B. et al., Science (1985) 227:1229-1231. Othermethods that hâve been used in the transformation of plant cells, and in particular the morerécalcitrant crop plants, include biolistics and electroporation (for detailed protocols, seeSanford, et al., (1993) Methods in Enzymology 217:483-509; and Potter, (1993) Methods inEnzymology 217:461-478.

Once transgenic plants hâve been produced, conventional enzyme assays for CA4H andCAD are used to détermine the level of suppression of enzyme activity achieved in differenttransformants. It is likely that only a small fraction of the transformants produced will hâve asufficiently low residual enzyme activity to cause the accumulation of aromatic aldéhydeswithout also producing some undesirable side-effects on plant development. For this reason, apreferred method of producing the desired transformants with both CA4H and CADsuppressed is to introduce the two genes separately into different transformants and thencombine them by standard sexual crosses. This permits a larger number of combinations oflevel of gene suppression to be evaluated at the same time.

An alternative to overproducing aromatic aldéhydes in transgenic plants is to use theplant genes to confer on a microbial host the capability of synthesizing spécifie aromaticaldéhydes. The resulting microbes can be used either to produce the aromatic aldéhydes in afermentation System or as a natural delivery System of the aromatic aldéhydes in viable or non· 010731 viable microbial préparations. Yeasts, especially Saachoromyces cerevisiae, are preferred organisms for this purpose because they hâve already been engineered for high-level expression of PAL (Faulkener et al. (1994) Gene 143:13020) and a plant cinnamate 4- hydroxylase has been shown to function in yeast (Urban et al. (1994) Eur. J. Biochem. 222:843-850).

The expression of PAL introduces the capability to produce cinnammic acid fromphenylalanine. Two additional enzymic steps are required to produce cinnamaldéhyde fromphenylalanine. In plants, these steps are catalyzed by the enzymes cinnamate:CoA ligase (CL)and cinnamoylCoA reductase (CCoAR), but as 4-coumarateCoA ligase (4CL) can also usecinnamic acid as substance (Knobloch, and Hahlbrock (1977) Arch. Biochem. Biophys.184:237-248), 4CL can be used instead of CL. More than 20 cloned PAL genes and morethan 6 4CL genes hâve been described in sufficient detail (GenBank) to facilitate their use inpracticing the current invention. A gene for a CCoAR is obtained by applying standard genecloning techniques to isolate a cDNA clone using as a probe sequence derived from the aminoacid sequence of the N-terminus, or peptide fragments, of the purified protein. CCoAR hasbeen purified and partially characterized from soybean cultures (Wengenmayer et al. (1976)Eur. J. Biochem, 65:529-536; Luderitz and Grisebach (1981) Eur. J. Biochem, 119:115-124),spruce cambial sap (Luderitz and Grisebach, supra), poplar xylem (Sarni et al. (1984) Eur. J.Biochem, 139:259-265) and differentiating xylem of Eucalyptus gunnii (Goffner et al. (1994)Plant Physiol. 106:625-632). The preferred method of purification is that of Goffner et al.{supra) because it results in a single protein band on SDS-polyacrylamide gels that an be usedfor protein sequencing.

The cloned genes are introduced into standard expression vectors and used to transforma microbial host, preferably yeast, by standard transformation techniques such aselectroporation (Becker and Guarante (1991) Methods in Enzymol. 194:182-187). Standardenzyme assays are used to confirm the functional expression of the engineered genes andassays for aromatic aldéhydes are used to select strains with maximal production. Becausearomatic aldéhydes hâve antimicrobial properties it is preferred to use expression vectors thatwill cause expression of the introduced genes only late in the growth cycle or in response to aChemical inducer. It may also be désirable to grow the engineered microbial host in animmobilized whole cell reactor (e.g., Evans et al. (1987) Biotechnology and Bioengineering 010731 30:1067-1072) to prevent the aldéhydes from accumulating in the culture medium.

The target insects and other pests include those which are vectors for disease organisms such as fungi which colonize a surface of a part of a plant which is an elicitor for the fungus.

By elicitor is intended that the plant sécrétés nutrients required by the fungus. Examples offungi and the plant parts which they colonize are as follows. Black spot on fruit; Fusarium sp.on flowers roots and leaves; and Fusarium spp. and Aspergillus on roots and leaves. Fusariumcauses vascular wilts of annual vegetables and flowers, herbaceous perennial ornamentals,plantation crops and the mimosa tree. Different plants are attacked by spécial forms or racesof the fungus. Verticulum (V. albo-atrium and V. dahlise) cause vascular wilts and colonizeroots, flowers and leaves. In addition the following also constitute target organisms:Phragmidium spp-, Diplocaopan rosae; Sphaerotheca tannosa; Oibiapsis sicula; Phytophthorataraesitica; Phytophthora infestons, Puccinia spp-, Alternaria spp; Susaiun spp; Botrytis cinera;Scierotinia homoeocarca; Tricophyton mentagrophpytes; Dutch Elm disease (Ceratocystis ulmï)and oak wilt (C. fagacearum). Ceratocystis causes vascular wilts, mainly of trees. Alsoincluded are blue-green algae (Cyanobacteria). The vectors for these diseases which can berepelled by the subject formulations include beetles and wasps. Target organisms also includeinsects which damage the plants which they colonize, particularly those of the ordersOrthoptera; Thysanoptera which includes water weevil and thrips; and Homoptera whichinclude aphids such as root aphid and leaf aphid, leafhoppers, white flies, mealy bugs, thrips,cicadas, Caterpillar, such as velvetbean Caterpillar, codling moth, leaf roller, and scale insects.

Other target organisms include arachnids (particularly spider mites), flies (Musca domestica),cockroaches, gastropods, moths, and bed bugs (Cimex lectularis) and its close relatives(poultry bug (Haematosiphon indorus Duges), the European pigeon bug (Cimex columbariusJerjus), and the swallow bug (Oeciains vicarius Hrovath)).

Also of particular interest is prévention of phylloxéra infestation in grapes by repellingthe phylloxéra. For this application, it is necessary to deliver the formulation to the roots ofthe plants which are the usual habit for phylloxéra. When used in a solid form ormicroencapsulated, the dosage used is typically on the order of 1% to 35% on a w/w basis, themaximum loading to be determined as a function of shell material selected. AnalyticalChemical techniques are used to détermine and optimize rate of release. For qualitativepurposes, GC techniques can be used to détermine the amount of aldéhyde released. The 21 010731 samples of encapsulated (pelletized) product are mixed with the soil types selected and sampledat different time periods to measure release. Alternatively, the volatile gases released from theformulation can also be analyzed. For measuring the activity of foliar and drip irrigationapplications the stability of the formulations over time can also be. evaluated using the GCmethodology using methods known to those skilled in the art. Methanol or alcohol extractionsof the formulations also can be prepared for HPLC analysis. The preferred method ofrepelling phylloxéra and other root dwelling pests, however, is to provide for a systemicresponse to, for example, a foliar application of the formulation which is then translocated tothe root. The timing of such applications will need to be determined empirically for particularplants as the flow of water from the leaves to the roots is required for translocation.

Generally, such flow is greatest at cooler températures e.g. during the evening hours, at night,or in the early morning hours, and pre or post fruit or vegetable development.

The subject formulations, in particular those containing HCA, are also useful fortreating: grape to repel pests such as thrips, nematodes, and leaf roller; roses to repel thripsand melon aphids; cattle to repel soft ticks; humans to repel mosquitos; apple to repel codlingmoth; animais to repel fleas; cockroach habitats to prevent or eliminate cockroach infestation;and corn to repel root aphid.

In addition to treating a host plant, seeds can also be treated using the subjectformulations to repel insects and other pests which attack the seeds and/or which act as vectorsfor disease organisms. The seeds can be dusted with a powder préparation {see U.S. PatentApplication No. 4,978,686 for examples of inorganic materials to which the formulations canbe adsorbed) or admixed in a plant substrate such as vermiculite. Seedlings grown under stérileconditions from treated seeds are free of susceptible fungi and insects. Additionally, seedlingsalso can be treated with the subject formulations. In some instances it may be necessary toadjust the treatment formulation so as to reduce any phytotoxicity associated with the treatmentas tender young shoots are more likely to exhibit phytotoxicity symptoms. The treatmentformulations are also useful for controlling the time of pollination of flowering plants. Forexample, to prevent or delay pollination the formulations are applied in an amount sufficient torepel bees and other pollinating insects. By adjusting the residuality of the formulation, onecan control the length of time during which pollination is inhibited. On the other hand, forplants whether cross-pollination is required for fertilization, application of the formulation 22 010731 during this period should be avoided if the pollinating insect is repelled by the formulation.

In order to détermine the susceptibility of particular insects to repellency by the claimedcompositions, in vitro and in vivo tests which compare the behavior of the target pest towards,for example, approaching a "bait" food in the presence and absence of the test formulation areused. The effectiveness of the formulation over time can be evaluated by extending the timeperiod of observation until few of the test insects (less than about 50%) are repelled from thevicinity of the bait. For pathogen vector insects, a 90% or greater repellency is usually inorder. For the common nuisance pest, réduction in the magnitude of 80% is suitable (e.g., ingarden and food areas). The formulations also need to be evaluated for phytotoxicity for useon plants and for dermal sensitivity, particularly for use on skin and/or clothing of humans;contact dermatitis and olfactory sensitivity are monitored using tests for dermal sensitivityknown to those of skill in the art. Likewise, phytoxicity testing can be done using methodsknown to those of skill in the art. Phytotoxicity can be rated as follows in order of increasingseverity of toxicity: 0-plants without any symptoms; 1-very slightly browning of hypocotyl(no other symptoms); 2-some wilting of plant, dying of lower leaves, some browning ofvascular System; 3-wilting of entire plant, leaves dying, hypocotyl with external and internaisymptoms; 4-necrosis of stem, plant dying. It is préférable that the formulation used hâve aphytotoxicity rating of 2 or less, more preferably 1 or less.

The components of a formulation to be used for a particular application can bedetermined by evaluating first the concentration range over which a given component has noactivity to where it provides maximum activity (a dose response curve) and then evaluating thiscomponent separately and in combination with other components of interest for a givenformulation. The repellent and/or phytotoxic and/or dermal effects of a particular formulationon a given insect or other pest and the host is then measured for each formula and componentwith or without a serial diluent of any additional component of interest. Optimal dose-rangesare calculated in vitro and in vivo using techniques known to those of ordinary skill in the art.Formulations are identified which provide: repellency of 90%, and/or a phytotoxicity rating of2 or less for plants, with 1 or less being optimum, and substantially free of contact dermatitisfor animais and fowl.

The following examples are offered by way of illustration and not by way of limitation. 23 010731

EXAMPLFS

The following products were used in the example protocols set forth below: (1)cinnamic aldéhyde from Spectrum Chemical Co., New Jersey, USA; (2) coniferyl aldéhyde 5 from AD IN Chemical Co., VF; (3) sodium bicarbonate and Tween 80 from SpectrumChemical Co., New Jersey, USA.

Example 1

Flies (Musca domestica) 10 The purpose of this experiment is to evaluate the repellency activity of cinnamic aldéhyde and a-hexyl cinnamic aldéhyde against flies (Musca domestica). Twenty 2-3 day oldfemale flies are released in a 62 x 62 x 34 cm cage with 325 mesh roof screening to permit aircirculation (Carolina Biological Supplies). Bait made of sweet milk (Carnation) (90%) plusglucose (10%) and a dye (bromophenol blue 0.01%) with 1 ml of formulation is placed in a

15 3.5 cm pétri dish and set inside a pine cage (Carolina Biological Supplies) with a 1 cm inchdiameter hole drilled through the top to permit access to the cage containing the bait. A 3.5 cm pétri dish with 5 ml H2O is placed in the cage for water. After 24 hours, Aies areremoved and crushed on filter paper to check for the presence of dye which would indicatefeeding activity. Entry of more than 10% of the flies is taken as an indication of lack of 2 0 formula repellent activity.

Example 2 0 1 073 1

Cockroaches (Blatella germanic)

The aim of this experiment is to evaluate the repellancy activity of cinnamic aldéhydeformula against cockroaches (Blatella germanic). Fifty nymphs and adults 1.5 cm to 3.5 cm inlength are released in a 62 x 62 x 34 cm cage with 325 mesh roof screening to permit aircirculation. The inner surface of the cage walls from 5 cm to 10 cm from the floor are treatedwith a mixture of minerai oil and petroleum jelly (2:3) to prevent escape of the cockroaches.The cockroaches are fed on dog chow (Purina), milk powder and water for 48 hours foracclimatization. Two Whatman filter papers "C" (4x4 cm) are folded twice, and stapled andwet with 1 ml of formula each. Filter papers are allowed to air dry. After air drying, a filterpaper is placed inside of one of two 4 cm x 4 cm x 4 cm cubes, each with a single 0.75 cmdoor at floor (base) level for entry. The two shelter boxes are placed on the bottom floor ofthe cage 14 cm apart. After twenty hours, the shelters are removed and the number of sheltercockroaches are removed and counted. An entry of more than 10% of cockroaches into theshelter boxes is regarded as an indication of loss of formula repellency.

Example 3Aphid (Aphidfabae}

The purpose of this experiment is to détermine the repellancy activity of a cinnamicaldéhyde formulation against black bean aphids. Sugar beet plants (Beta vulgaris) are grownin 7.5 mm pots in potting soil in a greenhouse. When plants reach the three leaf stage, eightplants are selected at random. In separate trials, four plants are treated with: cinnamicaldéhyde at 50 ppm; 50 ppm cinnamic aldéhyde formula (NaHCO3 + Tween 80); NaHCO3;Tween 80; and formula blank. Treatment is a foliar application of 5 ml of material sprayed asfine mist by a hand sprayer (Gilmour). Four plants are untreated and one sprayed with wateronly. The treated and untreated plants are placed in two rows, A or B, treated or untreated,respectively, in a 60 x 60 x 30 cm box cage with a 325 mesh screen roof permitting aircirculation. At 4, 8 and 24 hours, the number of aphids on treated and untreated plants,rows A and B, are counted and recorded. 25

Example 4 0 1 0 731

Silverleaf White Fly (Tetranychus urticae)

The purpose of this experiment is to détermine the repellency of cinnamic aldéhydeagainst silver leaf white fly. In a greenhouse, cotton plants are grown in 7.5 mm pots in 5 potting soil. When plants reach the three leaf stage, eight plants are selected at random. Inseparate trials, four plants are treated with: cinnamic aldéhyde at 50 ppm; 50 ppm cinnamicaldéhyde formula (NaHCO3 + Tween 80); NaHCO3; Tween 80; and formula blank.

Treatment is a foliar application of 5 ml of material sprayed as fine mist by a hand sprayer(Gilmour). Four untreated plants receive a foliar spraying of 5 ml water. The treated and 10 untreated plants are placed in two rows, A or B, treated and untreated, respectively, in a 60x60 x 30 cm cage with a 325 mesh wire screen roof allowing air circulation. At 4, 8 and 24hours, the number of silverleaf white flies are counted for présence on plants in rows A and B(treated and untreated). At 48 hours, the number of eggs on plants in each row are countedand recorded. 15

Example 5

Leafhoppers (Cicadellidae)

The purpose of this experiment is to détermine the repellency of cinnamic aldéhydeagainst leafhoppers. In a greenhouse, cotton plants are grown in 7.5 mm pots in potting soil. 2 0 When plants reach the three leaf stage, eight plants are selected at random. In separate trials,four plants are treated with: cinnamic aldéhyde at 50 ppm; 50 ppm cinnamic aldéhyde formula(NaHCO3 + Tween 80); NaHCO3; Tween 80; and formula blank. Treatment is a foliarapplication of 5 ml of material sprayed as fine mist by a hand sprayer (Gilmour). Fouruntreated plants receive a foliar spray of 5 ml of H2O. The treated and untreated plants are 25 placed in two rows, A or B, treated and untreated, respectively, in a 60 x 60 x 30 cm box cagewith a 325 mesh wire screen roof allowing air circulation. At 4, 8 and 24 hours, the numberof leafhoppers are counted for presence on plants in rows A and B (treated and untreated). At48 hours, the number of eggs on plants in each of the rows is counted and recorded. 010731

Example 6

Thrips (Thysanoptera)

The purpose of this experiment is to détermine the repellency of cinnamic aldéhyde 5 against thrips. In a greenhouse, tomato plants are grown in 7.5 mm pots in potting soil.

When plants reach the three leaf stage, eight plants are selected at random. In separate trials,four plants are treated with: cinnamic aldéhyde at 50 ppm; 50 ppm cinnamic aldéhyde formula(NaHCO3 + Tween 80); NaHCO3; Tween 80; and formula blank. Treatment is a foliarapplication of 5 ml of material sprayed as fine mist by a hand sprayer (Gilmour). Four 10 untreated plants receive a foliar spray of 5 ml of water. The treated and untreated plants areplaced in two rows, A and B, treated or untreated, respectively, in a 60 x 60 x 30 cm cagewith a 325 mesh wire screen roof allowing air circulation. At 4, 8 and 24 hours, the numberof thrips are counted for presence on plants in rows A and B (treated and untreated). At 48hours, the number of eggs (in leaf slits) on plants in each row are counted and recorded. 15

Example 7

Twospotted Spider Mite (Tetranychus urticae)

The aim of this experiment is to evaluate the repellency of cinnamic aldéhyde on twospotted spider mites. Cotton plants are grown in 7.5 mm pots in potting soil in a 2 0 greenhouse. When plants reach the three leaf stage, eight plants are selected at random. Inseparate trials, four plants are treated with: cinnamic aldéhyde at 50 ppm; 50 ppm cinnamicaldéhyde formula (NaHCO3 + Tween 80); NaHCO3; Tween 80; and formula blank.

Treatment is a foliar application of 5 ml of material sprayed as fine mist by a hand sprayer(Gilmour). Four untreated plants each receive a foliar spray of 5 ml of water. The treated and 2 5 untreated plants are placed in two rows, A or B, treated or untreated, respectively, in a 60 x 60 x 30 cm cage with a 325 mesh wire screen roof allowing air circulation. At 4, 8 and 24 hours, the number of spider mites are counted for presence on plants in rows A and B. At 48 hours, the number of eggs on plants in each row are counted and recorded. 27

Example 8

Mosquito (Aedes aegypti) 010731

Repellencv Test Procedure In Vitro

The purpose of this experiment is to evaluate the repellency of cinnamic aldéhydeagainst mosquitos. Twenty unblooded adult female mosquitoes approximately 4 days of âgeare introduced into test chambers. Four ml of a test formulation is pipetted onto a 16 cm #2Whatman filter paper circle and air dried. The treated filter paper is placed on the vent intakechamber. CO2 is bubbled through water at the vent intake end of a wind tunnel olfactometerchamber; the lowest rpm fan setting is used. The trap chamber is opened for 5 minutes, thenclosed and the number of mosquitoes counted and recorded. DEET at 23% is used as apositive control.

Repellency Test Procedure. Field Trial

The purpose of this experiment is to bioassay the activity of cinnamic aldéhyde as amosquito repellent. Two circles 18 cm in diameter and two 16 cm in diameter were eut from1 mm mesh nylon mosquito cage boit of screen material. The treatment circle (16 cm) wassoaked in 1 ml formulation: cinnamic aldéhyde (2%) in 2% Tween 80 and 6% NaHCO3, thenallowed to air dry for 2 hours. Ten unfed female Aedes aegypti mosquitoes (5-7 days old)from the Kearney Agriculture Center, Mosquito Control Research Laboratory, were introducedinto each of two Kearney (Fischer) one pint mosquito cartons (control and treatment cartons).Each carton was covered with one of the untreated 18 cm circle mesh screens and sealed with arim from the pint carton, the lid section having been removed. An adult male volunteer placedthe treated 16 mm circle on one of his legs (which had been washed and rinsed with soap andwater) and the untreated 16 mm circle on the other leg (washed and rinsed with soap andwater). The one pint mosquito cartons were put in flush contact with the mesh screen side tothe leg screen patches for 5 minutes. Mosquitoes did not corne in direct contact withcompound. After 5 minutes, the number of blooded/well-gorged insects out of 10 werecounted. The results are shown in Table I, below. Out of a total of thirty insects evaluated,only two were not repelled by the cinnamic aldéhyde formulation, as compared to 19 in thecontrol (untreated) group. 2δ

Table I 010731

Mosquito Repellancv (# of blooded/well-gorged insects/10 insects)

Trial 1 Trial 2 Trial 3 Sum Cinnamic aldéhyde 0/10 2/10 0/10 2/30 formulation1 Control (untreated) 5/10 7/10 7/10 19/30 1 Cinnamic aldéhyde (2%) with 2% tween 80, and 6% NaHCO3 in5 H2O. A protocol similar to that described above is used to test a-hexyl cinnamicaldéhyde. io Example 9

Lepidopteran ovipositional Repellencv

The purpose of this experiment is to détermine the repellency of cinnamic aldéhydeagainst Beet Armyworm adult moths. An apparatus is built that forces an airstream overtreated and non-treated potted plants in a flight cage. Five tomato plants at the three leaf stage 15 are treated with 5 ml of various concentrations of Chemical formula and components and thenplaced in the cage. Five tomato plants are sprayed with 5 ml of H2O as control plants and thenplaced in the cage. Forty egg laying ready Beet Armyworm (Spodoptera exigna) adults arereleased in the cage. The exhaust fan on the apparatus is turned on and a low velocity linearflow of air is allowed to flow through the cage as plumelets of air evaporating Chemical 2 0 formula. After 24 hours, oviposition is determined on treated plants, non-treated plants, andcage walls. 010731

Example 10

Phylloxéra - Vapor Test for Repellencv

The purpose of this experiment is to evaluate the vapor repellency of cinnamic aldéhydeto phylloxéra. Root pièces of grape stock with viable phylloxéra eggs (n-30) is placed in 50 x9 mm dishes that hâve been treated on inner surfaces with 400 ml of known productconcentration. The Chemical is not placed directly on the root, so that absorption ormetabolism by the root is not a factor. The dishes are shut and sealed with tape. After 7 days,the dishes are opened, and a détermination is made as to whether insects are able to establishon the roots and develop, or whether the newly hatched insects avoid the roots and die.

Example 11

Overproduction of Aromatic

Aldéhydes in Transgénie Plants

Twenty mg of polyA RNA is prepared and cDNA synthesized. Part of this is clonedinto lambda-ZAP II vector (a commercially available cloning vector). At least 500,000recombinants are screened using an oligonucleotide probe designed from conserved sequencesof cloned CA4H and CAD genes obtained from GenBank, or designed from peptide sequenceof purified protein from the intended host plant. Strongly hybridizing clones are selected andused to rescreen the cDNA library. The resulting clones are sequenced to enable theintroduction of appropriate gene sequences into a plant expression cassette in either antisenseor sense orientation. The antisense and sense constructs are introduced into Agrobacteriumtumefaciens LBA4404 by direct transformation following published procedures. Tobacco (N.tabacum, variety Samsun) leaf dises are transformed using well estabished publishedprocedures (Horsch et al. (1985) Science 227:1229-1231. Plants containing either CA4H orCAD constructs are identified by PCR and selected for further analysis.

Plant material from both transformed and untransformed control plants is used fordéterminations of CA4H and CAD enzyme activity using well established published assays.Plants in which the activity of CA4H or CAD has been reduced to less than 20% of that seenin control plants are selected for further analysis. Selected plants with low CA4H activity arecrossed with plants with low CAD activity and progeny inheriting both gene constructs are 010731 selected by PCR. Plants with suppressed CA4H and suppressed CAD activity are analyzed for aromatic aldéhyde production using standard published procedures. Those plants that produce aromatic aldéhydes are then tested for efficacy of repelling insects or other pests using any appropriate example, e.g. Example 3 to test transgenic cotton plants for their capacity to repel aphids. 30 EXAMPLE 12

Production of Aromatic Aldéhydes in Microbial Systems A cDNA library is generated using RNA extracted from six week old tobacco stems.20mg of polyA RNA is prepared and cDNA synthesized. Part of this is cloned into lambda-ZAP II vector (a commercially available cloning vector). At least 500,000 recombinants arescreened using an oligonucleotide probe designed from peptide sequence sequences of CCoArproie in purified from six week old tobacco stem tissue using the protocol of Goffner et al.(1994) Plant Physiol. 106:625. Strongly hybridizing clones are selected and used to rescreenthe cDNA library. The resulting clones are sequenced to enable the identification of full-length cDNA inserts and the introduction of appropriate CCoAR gene sequences into yeastexpression vector pMTL8110 (Faulkner et al. (1994) Gene 143:13-20. The coding sequencesfor Rhodosporidium toruloides phenylalanine ammonia lyase (PAL; GenBank locus RHDPAL)and a parsley 4-coumarate:CoAl ligase (4CL; GenBank locus PC4CL1AA) are similarlyintroduced into équivalent yeast expression vectors. The PAL,4CL and CCoAR constructs areused to transform Saccharomyces cerevisiae strains by electroporation using establishedpublished procedures (Becker, and Guarente, Methods in Enzymology 194:182-187, 1991;Simon (1993) Methods in Enzymol 217:478-483. Transformants are selected on minimalmedium lacking leucine. Transformant strains carrying ail three gene constructs are identifiedby PCR and selecter for further analysis.

Extracts from both transformed and untransformed control strains are used fordéterminations of PAL, 4CL and CCoAR enzyme activities using well established publishedassays. Strains in which the activity of PAL, 4CL and CCoAR is significantly greater than thebackground activity detected in control strains are selected for further analysis. Selectedstrains are analyzed for aromatic aldéhyde production using standard published procedures andthose producing significant amounts of cinnamaldéhyde are selected for optimization of 31 010731 fermentation conditions. The resulting products are then tested for their efficacy in repellinginsects and other tests using any of the described methods.

Example 13 HCA Activity as Insect Repellant

The purpose of this experiment is to détermine whether a-hexyl cinnamic aldéhyde is aneffective insect repellant. Following négative skin-irritation tests on rabbits at the FDA, HCAwas evaluated on the skin of 2 to 4 male human subjects. One ml of the compound was rubbedover one forearm. A glove was worn to protect the untreated hand while the treated forearmwas exposed in a cage containing a high number (2,000-4,000) of unfed mosquitoes for 3minutes at intervals of approximately 30 minutes until two bites were received (two bites inone test period or one bite in each of two consecutive test periods). The time interval betweenapplication and when two bites were received was defined as the "protection time." Againstthe yellow fever mosquito (Aedes aegypti (L)), HCA was rated as 3 (121-180 minutesduration). Against the malaria mosquito (Anopheles quadrimaculatus Say), HCA was rated as2 (31-60 minutes). HCA was also evaluated on treated cloth against the yellow fever mosquito. In thesetests, women's mercerized-cotton stockings were used. A measured section above the anklewas impregnated with HCA at a rate équivalent to 3.3 g/ft2. The stocking was spread on arack to dry and then hung indoors on a line. The first tests were done 24 hours aftertreatment. The stocking was drawn over the arm, with the treated portion midway on theforearm. The untreated hand was protected with a glove and the stocking-covered arm wasexposed for one minute in a test cage. If 5 bites were received, the treatment was consideredineffective. If less than 5 bites were received, the exposures were continued daily until the14th day and at weekly or biweekly intervals thereafter. HCA received a grade of 4 in this test(effective for more than 10 days), the same value as reported for DEET.

These examples demonstrate that the subject cinnamic aldéhyde and a-hexyl cinnamicaldéhyde formulations are effective repellents against mosquitos.

Ail publications and patent applications mentioned in this spécification are indicative of 32 01 073 î the level of skill of those skilled in the art to which this invention pertains. Ail publicationsand patent applications are herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically and individually indicated to beincorporated by reference. 5 The invention now having been fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departingfrom the spirit or scope of the appended daims.

Claims (19)

1. 33 010731 CLAIMS

   1. A composition for repelling pests, said repellent composition comprising at leastone aromatic aldéhyde at a concentration sufficient to provide an aroma which repels pests, B wherein said concentration is 10-5000 ppm of the total composition.
2. 2. The composition according to claim 1, wherein said aromatic aldéhyde iscinnamic aldéhyde, a-hexyl cinnamic aldéhyde, and/or coniferyl aldéhyde.
3. 3. The composition according to any one of daims 1, 2, and 19, wherein said composition further comprises saponin.
4. 4. A method of repelling pests, e.g. insects, from the vicinity of the body of amammal; said method comprising topical application of a composition according to any one of 15 the daims 1-3, and 17-19 in an amount which does not cause dermal irritation.
5. 5. The method according to claim 4, wherein said composition is in a formselected from the group consisting of a spray, a stick, a repellent oil and an ointment. 2 0 6. The method according to claim 4 or claim 5, wherein said mammal is selected from the group consisting of a human, a bovine, and an ovine.
6. 7. The method according to any one of the daims 4-6, wherein said mammal is ahuman and said pests are fleas or mosquitoes. 25
7. 8. The method according to any one of the daims 4-6, wherein said mammal is abovine and said pests are soft ticks.
8. 9. A method of repelling pests from the vicinity of a plant, said method comprising 3 0 application of a composition according to any one of the daims 1-3, and 17-19, wherein said plant is selected from the group consisting of grape, rose, apple, and corn, said composition 010731 has a phytotoxicity rating of 2 or less.
9. 10. The method according to claim 9, wherein said plant is a grape plant and saidpests are selected from the group consisting of thrips, nematodes, phylloxéra and leaf rollers.
10. 11. The method according to claim 9, wherein said plant is a rose plant and saidpests are thrips or melon aphids.
11. 12. The method according to claim 9, wherein said plant is an apple tree and saidpests are coddling moths.
12. 13. The method according to claim 9, wherein said plant is a corn plant and saidpests are root aphids.
13. 14. A method of preventing an ingress of insects into an area through the applicationof a composition according to any one of the claims 1-2, 3, and 17-19 to the vicinity of saidingress.
14. 15. The method according to claim 14, wherein said insects are cockroaches.
15. 16. A method of preventing infestations of a tree by pests, said method comprising:contacting the trunk of said tree with a composition according to any one of the claims 1-3 and17-20.
16. 17. A composition for repelling pests, said repellent composition comprisingsaponin and at least one aromatic aldéhyde at a concentration sufficient to provide an aromawhich repels pests.
17. 18. A composition according to claim 17, wherein said aromatic aldéhyde is selectedfrom the group consisting of cinnamic aldéhyde, a-hexyl cinnamic aldéhyde, and coniferylaldéhyde. 010731 -35- »
18. 19 - A composition for repelling pests, said repellant composition comprising at least » one of a-hexyl cinnamic aldéhyde and coniferyl aldéhyde, at a concentration sufficient to provide an aroma which repels pests.
19. 20 - A method of preventing infestations of a tree by pests, said method comprising : 5 contacting the trunk of said tree with a composition comprising at least one aromatic aldéhyde at a concentration sufficient to provide an aroma which repels pests.