CA2196972A1 - Repellent compositions containing flavonoid 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

~ w096~9827 2 1 9 6 9 7 2 PCT~S95/170S0 REPELLENTCOMrOS~ONSCONTA~NG
FLAVONOIDALDE~YDES

~ODUC~QN

TfY~hnirsll Fj.~lA
SThis invention relates to flavonoid aldehydes as pest repellents. The invention is by the use of cinnamic aldehyde or alpha hexyl cinnamic aldehyde as a mosquito repellent and for repelling a~Tir~lh~l pests such as aphids and thrips.

r- ~C.
In many counhies today, diseases such as malaria, vector-bome h ~ fevers, 10cu~hu~l.~ allergies, filth fleas, bubonic plague, ticks viruses, rickettsiae, spirochate bacteria, snails-c ~ and sand fly fever are still .. ,~ ;I,lr for serious illnesses and numerous deaths among ' ' The ever-growing concern for the protection of~ species and the downward hend in availability of the broad spectrum pesticides for public health are forcing scientists to look for other . ' means of providing 15 protection from vectors of discase. Moreover, the cost of pesticidcs often is too high for many of the less developed nations and the increasing resistance to these ~ by vector ~,~ is a growing problem.
The use of repellents is an excellent altemate means of providing relief when other CU~I~I " ' Yector conhol methods are not feasible. For ' vector targets, 20 repellents properly applied to the skin and/or clothing are an ;"~ , and practical means of reducing the biting activity of I ,' ~ arthropods and for the prevention of vector disease i ~ Repellents are effective agLunst a wide range of disease vectors, whereas a separate vaccine must be developed for each disease. Moreover, only a few vaccines are effective against vechor-bome diseases. Diseases spread by vectors also affect ''S plants. For example, Dutch Elm Disease has destroyed millions of elm hees across the United States. The disease is caused by a fungus which is spread from tree to tree by a palt; ' '~, species of insects athacted to the elms. Current methods of l~vA~h~ have had _ _ only limited success. A need exists for an effective ~J~U~AI,~I lAi l for this destructive plant disease.
R.stT~rtirn~ of the use of repellents in vector control and disease prevention usually center on questions of safety and cost. For example, one of the more eA~ 'y usedS repellents was the ~2-2 repellent which contained dimethyl phthalate, ethyl l- - ' -l, and Indalone in the proportion 6:2:2. Dimethyl phthalate and Indalone are still in limited use, but in 1991, the U.S. L,~ ' Protection Agency canceled all 1~ Li~ of ethyl hexanediol at the request of the ' concerned. This action was taken because of new ' on possible adverse effects on fetal .IC~ ~IU~ L.
As another example, one of the most effective mosquito repellents is DEET (N.N-diethyl-1.3-.n~Ll,ylh. ~ ). 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 pPrlnethnn, a synthetic derivative of pyrethrum, has largely replaced DEET for use on clothing and other fabric items. As a repellent, DEET is highly 15 effective, but it may also cause allergic and toxic effects, especially when used on the skin repeatedly in high ~- - Repellent 'c ~ containing 90-99% DEET are considered high s, whereas repellent ' ~ containing 50% or less DEEiT
are considered as effective as a ~ of 100%. A of 33% DEET is effective in providing 10-14 hour protection. However, for products containing even low 20 ...~ . ,~ ..c of DEET, it is .~ L .I that the skin be cleaned with mild soap and rinsed with water as soon as the repellent is no longer needed, in order to minimize possible adverse reactions. These 1. ' frequently are impractical in third world countries and for military use.
Repellent ~ - exist for topical application to a mammal, as well as to repel 25 insects from entering a dwelling or other area. However, the safety of many of the topical , ~ has boen .. ' Moreover, many of the topical cc. ~ f - are of limited crr~i~l,.l~,, especiaDy in areas of severe infestation with insects. Treatment for external insect; ~ . c of a mammal, such as lice or crabs, often involves topical application of harsh toxic ~ - ' ' ~c ~ ;r c to skin or scalp. Irritation often develops, 30 and adverse health effects from long-term use are also known. A need exists for a safe, effective topical repellent c A ~' for a mammal. Repellent ~ for the prevention of entry of insects are similarly ineffective. Many of the known such repellent ~ also are not safe for use in enclosed spaces due to their high toxicity, especially ~ wo 96/39827 2 1 9 6 9 7 2 F~ l/lJ:~y~
where children and pcts may come into contact with them. A need exists for a non-irritating, non-toxic, effcctive repellent ~ , Insect . ~f~ -' of trees and other woody plants destroy millions of nrr~ anda~nr~ ~l trees every year. Current treatments are only partially successful, and may 5 render a crop of ~, ' ' products worthless due to their persistent toxicity. Thus, a need also exists for a relatively non-persistent, effective means for repelling insect ;i~ 8...c of trees and woody shrubs.

RP1~VSII1t L
USPN 5,093,326 discloses repellent . . which include an ozonized 1 ' L.1J.u~bon, including terpenes. USPN 5,365,017 discloses ~ ion of a transgenic plant having increased levels of ~ CIU~L.UL F~l,L~dliu.l~ relating to repellant include Reifenrath et al. (1989) J. Am. Mosqulto Control Arro~~inhon 5: 45-61 and Reifenrath (1995) Cosmedcs ~ Toiletries Magazine 110: 85-93.

SUMMARY OF m~.~NVF.l~TlON
The present invention provides repellent, , which contain flavonoid aldehydes and methods of using these , The repellent , contain a compound which has a formula shown in (I) below:

R4 (1) wherein R represents -CH2OH or -CHO; n is an integer from 0 to 3; and each Rl ly represents OH or an organic substituent containing from I to 10 carbon atoms~20 and from 0 to 5 h~L.I s, wherein the total number of carbon and k~ udtu~ in all Rl of said compound is no more than 15; and R4 represents hydrogen or an organic constituent containing from I to 10 carbon atoms. These . , ' include natural such as cinnamic aldehyde. Also of interest are alpha substituted aldehydes, such as alpka hexyl cinnamic aldehyde (HCA). In use, the . , ' are applied to a~25 surface, such as skin, clothing, bark, habitat . , and the li~e, from wkich it is ~ . . .

wo sG/3ssz7 2 1 9 6 9 7 2 PCT/US95117050 desirable to repel msects and other pests. The invention finds use, for example, in the prevention of disease and infection which can result from conhct with a disease-carlying insect vector or other pest vector.

DE~ PI ION OF splir~FIc FMRO~ Ts Methods and . , are provided for obtaining and/or ~ ~ an area ~ - lly free of insects and other pests. Mammals, birds, fish and their habitats, as well as seeds, seedlings, plants, and plant parts such as fruit ' 'Iy free of pathogenic organisms such as fungi, insects and other pests, as well as viruses, bacteria, spirochetes, and other disease-causing organisms, and sap-sucking insects are provided together with a method to repel pests and disease-causing organisms. A surface of interest is contacted with flavonoid aldehyde in an amount sufficient to repel an insect or other pest. The amount of repellent that is applied will depend in part upon the nature of the surface, and to some extent upon the r ~ " and the specifc . , ' l" used and, therefore, must be empirically~' ' for best results with a particular insect or other pest.
The . , and methods of the subject invention offer several advantages over existing ~ , and methods. A major advantage is that the formula , are generally regarded as safe (GRAS) and approved for food use. For example, a number of the aromatic aldehydes which may find use in the subject invention, such as IY-hexyl cinnamic aldehyde (HCA), ' ' ' tJ~, and vanillin are GRAS synthetic flavoring agents (21 CFR
~172.515). HCA was in public use before the 1950's and today is widely used in consumer (soaps, detergents, creams, lotions, perfumes) (Mnn~-grArhc on fragrances raw materials. Food Cosnlet. Toxicol. 12: suppl., 915, 1974). HCA was granted GRAS status by FEMA (Flavoring E~ctract r ~ Association. Survey of flavoring ingredient usage levels. No. 2569. Fd. Technol., (~ , 19: (part 2) 155, 1965) in 1965 and is approved by the US FDA for use in food (21CFR121.1164). The Council of Europe (1970) (Council of Europe. Natural and Artificial Flavouring Substances. Partial Agreement in the Social and Public Health Field. Str~cho~g, List A(l), Series 1, no. 129, p. 55, 1970) included HCA in the list of admissible artificial fiavoring substances at a level of 1 ppm.
Surfactants which can be used as emulsifiers in the subject r.", ~ . such as theTweens (~IJ ' ' also already are used as food additives, as is saponin (which also has GRAS status). In addition, r ~ ~ residuality can be managed. 1rhis is of great beneSt for imtegrated pest programs with beneficial insects because short term residuals wo 96~59827 2 1 9 6 9 7 2 PCT/US95/17050 can be obtained. The long term control of pathogenic organisms results in a healthier plant and an improved yield of produce by the host plant as compared to , plants.The aromatic aldehydes in particular have positive Ull, ', "- and olefactory properties which in some cases can improve the flavor and/or smell of treated products and eliminate S the unpleasant odor associated with many pest repellants. The odor of a-hexyl cinnamic aldehyde (HCA), for example, is described as floral or jasmine-like with some herbaceous character (Technical Data Sheet).
For plants, the active ingredients can be made by the plant following ih~ ' of the gene(s) .. ~ 1r for synthesis of the various aldehydes into the plant cell genome. The use of transgenic plants rather than topical application of repellent to the plant decreases the likelihood of any adverse side effects to field workers, or to animals, fish or fowl which ingest the tissues or parts of the plants, since many of the, , ' of formula (1), in particular those of formulas (3) and (4), are GRAS food additives. In addition, the subject invention overcomes the failure of current pesticides to L. l Ic , for example, to roots for treatment of phylloxera. Also, prevention of infestation by repelling the vector which carries diseases, or damages the target for the pest, ~, 'y decreases the number of target animals or plants which 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 animals, including humans, the subject c~ ~ - are non-toxic and non-irritating to the skin at the ~ ~ n~ used. For example, ~Y-hexyl Phyde (HCA) has an oral LD50 of 3.1 g/kg in rats and a dermal LD50 of greater than 3 glkg (Moreno,-O.M. Report to RIFM, March 24, 1971). HCA was found to be , irritating when the neat compound was applieo to intact or abraded rabbit skin for 24 hours under occlusion (Moreno). When tested at 12% in ~e~ ' HCA produced no irritation after a 48 hour closed-patch test on human subjects and produced no ,- - ';~ in a test carried out on 25 human subjects (ICligman (1966) J. Invest. Dermatol.
47: 393). HCA at 20% in di~ ' ' ' produced no positive reactions in a repeated insult patch test conducted on 100 human subjects. Jimbo tabulated ~ ;ly data found in the literature for 18 fragrance , ' While cinnamic aldehyde had a positive reaction from the human test, HCA was negative in the test. Patch test results of 2%
HCA on 100 eczema and dermatitis patients were negative (0 out of 100). Of 4 patients sensitive to 2% ~ ' byJ~" none were found to cross react with 2% HCA. The skin ~ ~ = = = = = = = = = = ~ . .. ..

wo 96/39827 2 ~ 9 6 q 7 2 PCT/US95/17050 ~

fn~quently reported for c ' ' ' ~.lu is probably initiated by reaction of amino groups on proteins with the aldehyde functional group. SUbctl~ nn of bulky alkyl groups in the alpha position (e.g. the hexyl group of HCA) relative to the aldehyde group can rcduce this reactivity by creating steric hindrance as well as reducing the ~ u~h~ ;Iy of~ the aldehydic carbon. A';' s ' ' ~ ' ' '.1J~s, to which skin is react very slowly or not at all with amines in ~o-- r ~ ,.. with ~ hyde.
In studies using the ~ ~ test in guinea pigs, Senma and coworkers report a tendency that as the numoer of hydl~u~ of alkyl groups replacing the alpha-hydrogen in ~t' .~J-, increased, the rate of ~ h~.. reaction declined.
The subject ' ' also provide for effective control of multiple organisms, such as both fungi and insects. The ~ , ' also have been reported to have inhibitory activity against C. bot~in~n spore ~ (Bowles and Miller, G. Food Protechon (1993) 56:
788-794). This multi-target efficacy reduces the need for application of multiple agents to a plant or animal to be protected, and ' 'Iy eliminates the need for application of 15 pesticides. In particular situations, such as where an insect damages an animal or a plant part or tissue and a secondary fungal or bacterial disease develops, this aspect of the invention is l~culi~.~l~ly a~ Lb~
The general f ' is as shown in formula (I) above. A preferred c( - is shown in formula (2) below: R~
~ R

wherein R~ l CHO, R2 l~. OH or an organic substituent containing from I to 10 carbon atoms, R3 represents a methoxy group or organic substituent containing from I to 10 carbon atoms, and R~ represents a hydrogen or an organic substituent containing from I to 10 carbon atoms. Of particular interest are flavonoid aldehydes, ~ uLuly aromatic aldehydes. E~amples of aromatic aldehydes of use in the present invention are 25 cinnamic aldehyde ((3) below): C 1-10 ~3) ,~
~ 6 and coniferyl aldehyde ((4) below):
(~
C~3~.~ c ~o ~ .YO
Other . . _ - ' of interest include analogs of the compound of formula (1) such as substituted at the alpha position with an alkyl, such as a hexyl group, or a branched allcyl group such as an amyl group. Generally the group at the alpha position is S from C-5 to C-10. Such: , ' include a-hexyl ~ d- and ~Y-amyl s ' ' ' ~1. . The chemical structure of c~-hexyl cinnamic aldehyde (HCA) is shown in (S) below.

CH~

(CH2)5 ¢~CUO

The Chemical Abstracts Service (CAS) name of HCA is 2-(~he..r' ;L...e) octanal and the CAS Registry Number is [101-8~0]. The compound is also described by the chemical name 10 of 2-hexyl-3-phenyl-2-propenal. The r ~ 'S formula is C~ oO and molecular weight is 216.3. ElCA 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 minor e is the self aldol ' product of octanal (1-1.5% (Personal C- 1, June Burkhardt, Firmenich, Plainsboro, New lersey).
The , ' can be used either alone or in with other active or inactive substances and can be applied by spraying, pouring, dipping, injecting, in the form of ' liquids, solutions, ., powders and the lii~e, containing such .. . . .. .. . .. . . .... ........ ...

of the active compound as is most suited for a particular purpose at hand.
They can be applied, for example, in the form of dilute solution, in a suitable solvent directly to the ~ Jh~,le either as part of am irrigation schedule or as a separate arplirqtinn For use as a foliar spray, although the aldehyde can be formulated alone, it can be 5 rendered substantive by including an emulsifier such as Tween 80. Other detergents which can be used include anionic detergents such as those described in U.S. Patent Application No. 4,978,686. Other ~ l, ' which can be used alone or in . ; with detergents include saponins from any of a variety of sources, p~.~uld Iy saponin from Yucca sch~digera or Yucca valida. Generally, detergents and other agents used in the r~ do not 10 detract from the repellent properties of the flavonoid aldehydes but do increase the substantive properties of the fiu~ ulaliOIl (see for example, U.S. Patent No. 4,477,361) and may improve the pesticide properties, including fungicide properties (see below). Additional ~ such as an aqueous preparation of a salt of a polyprotic acid such as sodium l~ ' , sodium sulfate, sodium phosphate or sodium ' r' ~ ' can be included in the 15 r ~ '' , to increase the antifungal properties of the rw---ùhti~,--. The resulting emulsion is diluted to an n~ ' ' for use.
In a preferred ' ' t~ the r ~ includes a-hexyl cinnamic aidehyde, cinnamic aldehyde and/or coniferyl aldehyde in a fl,l ' containing Tween 80 or saponin as an emulsifier and may include sodium 1.;~ The preferred ~t ~ for 20 repelling flies, ... ~_ , fleas, tici~s, lice, ~;o~hw.,h~,s, two-spotted spider mites, silverleaf white flies, aphids, l. -n..~ thrips and ants is 10-50r~ ppm; for ticks, 100-2500 ppm.
Generaliy, the total amount of aidehyde(s) present in the r~ ti" ~ is 2% or less. The r l are effective without the use of - ~ , particular aldehydes may have inherent q-ltinYiA~t properties, for example, coniferyl aldehyde. Alcohols, such as glycols, 25 including propylene glycol, are likewise not required for efficacy of the r ' - and in fact may be harmful to the piant.
Stabiiity of the c~ ~ can be evaiuated by a variety of methods, including ' tests in which a r.,., ~ ~;n" of interest is exposed to elevated tl,n.~.dl.~ over a set time. Samples of the r~ ~ ~ are taken at regular intervais and anaiyzed chemicaliy 30 by methods known to those skilled in the art to determine the rate and nature of ~
For example, HCA can be anaiyzed by Gas Liquid Ci,. O ~ ' .y (GLC), using a 30 meter non-polar ~Iyd~ lhy' ' capillary column (e.g. HP-1, Hewlett-Packard, or SPB-1, Supelco) and a flame-ionization detector (Personai r~ ~ ). Using helium as a carrier gas (8 ml/min.) ~nd a column i , of ~ 'y 240~C, the (E)-cis isomer (major , - t) bas a retention time of ~ 'y 6.0 minutes and the (Z)-trans isomer (minor , ~., has a retention time of a~ 'y 6.3 minutes.
The most effective amount for ~ including r ' of formula (3) S and/or formula (4) and/or formula (5) as well as the amount of other .-r-l, ' of formula (l) wbich find use can be determined using protocols known to those of skill in the art for evaluating repellent efficacy of , ' Examples of such protocols follow. These protocols also can be used to optimize each r~ nn for specific pathogens using any of the c -r~ ~ I by formula (l) or formula (5) as well as for specific ~
10 to minimize plant yb ~ UlU~.il,;ly or skin sensitivity and other side effects for animals while ~ ~ ~ ng the ~ effect of the r~ u.~"
In some instances, the efficacy of the r~ caln be increased by adding one or more other , i.e., a compound other than a compound of formula (I) or (5), to the rul ~ where it is desirable to alter particular aspects of the Ç~ ;.." As an 15 example, it may be desirable for certain plant ~ c if there is an u..d~;l~l,le amount of ph~tu~ y to decrease the IJh~tulu~b,;~y effect t~Jh~tutu~ y rating of 2 or less, witb 1 or less preferred, see below) or to increase the repellent effect of the r~ ' or both. It is preferable that the additional , '~s) minimize any side effects to plants or animals while increasing the repellent effect of the rul ' Of particular interest is the use of a 20 ~ , tts~ which is a synergist to increase repellency while ~ ~, any side effects as related to a particular r ' '- By "synergist" is intended a component which, by virtue of its presence, increases the desired effect by more than an additive amount. The of one or more of the other ' ' ingredients can be modified while preserving or enhancing the desired repellent effect of the r~ ;.,.. Of particular interest is the addition of , to a ' ' to allow for a reduction in the, of one or more other ingredients in a given r~ - while substantially lP. efficacy of the ' ' C ' of such a component with other ingredients of the can be ,' ' ' in one or more steps at any suitable stage of mixing and/or application of the . 1, Preferred additional , include saponins, as they can be substituted for sùrfactants as; ' ~ , agents, and additionally on at least same plants have a growth promotant effect at the ~ used. Generally, the use of saponin does not interferewith the repeUant properties of the r~ Saponins are a class of s~ , ' each consisting of a sapogenin portion and a sugar moiety. The sapogenin may be a steroid or a triterpene and the sugar moietv may be glucose, galactose, a pentose, or a ' ~', S.
Budavari, ed., 7he Merck Index, 11th ed., Merck & Co., Inc., Rahway, N.J., 1990,p. 1328. The saponins for use in the present invention can be produced and/or isolated from S various plant parts including fruit, leaf, seed and/or root, using means known in the art, from a variety of 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 with the present invention are preferably non-toxic to humans and higher animals.
Most preferably the saponin for use in the present invention is non-toxic food grade, the 10 source being from yucca plants. Even more preferred are the saponins from Yucca schldigera or Y. valida and their ~4u;. ' The saponins are generally prepared by a cold press extraction process and the 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 0.01 to 0.1 % and most preferably about 0.01 % v/v aqueous 15 solution of 10~ brix saponin extract.
A variety of structurally related saponins are known, the most variable structural feature being the ~;ly~,O~yl~liul~ pattern. Saponins also may contain additional rn~ifi~tinnc~
such as r , ~ which are saponins with a steroid attached, and saponin structure can be modified by any number of enzymatic, chemical and/or ' ' means known in the art.~0 Saponins from Yucca schidigera contain steroidal saponins with the major sapogenins being ~ g ~ and tigogenin. The: r ~ yields on hydrolysis, s~ pn,~ -(Sa~ 5-beta, 20-betaF, 22-deltaF, 25-betaF; also known as spirostan-3-beta-01 and parigenin), glucose and galactose. The ~ c- -- has a molecular formula of C~ 44O3.
Nobel, Park S., Agaues, Oxford Univ. Press, New York, 1994. A variety of ~I.u.,tul,llly 25 related saponins are known, the most variable structural feature being the ~lywlyl~liO"
pattern. S. Budavari, ed., The Merck Index, 11th ed., Merck & Co., Inc., Rahway, N.J., 1990, p. 1328. Saponins also may contain additional . ~ fi~,.c such as the which are saponins with a steroid attached, and saponin structure can be modified by any number of enzymatic, chemical and/or mechanical means known in the art. Generally, an 30 effective amount of saponin is of the range of about 0.01 to 39'o and most preferably about 0.25% v/v aqueous solution of 10~ brix saponin extract. 10~ brix is a terms of art in sugar chemistry. The brix degrees equals the percent by weight of sugar in the solution. Hawley, /o ~ w096/39827 ; 2 1 96972 1.~ lS75/li .
ed., lhe Condensed Chemical Dictionary, 10th ed., Van Nostrand Reinhold, New York, 1981, p. 149.
For p~ where the r~ ~ is to be used to prepare the ground or other growth substrate for planting of host plants susceptible to particular pathogens, particularly S where the growth substrate is already infested, the ,r ~ " of the subject invention can be added directly to the '- .' or the substrate or they can be bound to a solid support or; , ' ' in a time release material to repel l ' ' ' insects and other pests. Where a solid carrier is used, materials which can lead to oxidation of the active aldehydes should be avoided. Examples of delivery systems which can be used include starch-dextran, and the 10 like. See Yuan et al., F ' ' and Applied Toxicology (1993) 20: 83-87 for other examples of ~ vl materials.
In addition to the specific ~ . h of the formulas (1), (2), (3), (4) and (5) above, precursors of any of these ~ , ' that produce a compound of the formulas identified above upon action of a biological system on the precursors are considered to be equivalent to 15 . . ' of the invention. Thus application of precursor . , ' to plant parts ortissues would be equivalent to the practice of the present invention. Biological conversion of precursor ~ ' into flavonoid aldehydes is described in, for example, U.S. PatentApplication No. 5,149,715 and references cited therein. See also Casey and Dobb En~me Microb. TechnoL (1992) 14: 739-747.
The method of the present invention is carried out by i.~lludu~;.. g onto a surface of interest a sufficient amount of an repellent agent to repel the insect or other pest.
Al~ll~dfiYIy, where the surface of interest is skin, fur, hair, clothing and the like, the application can be by ~way ûf contacting the surface of interest with a r ~ ~ that has been rendered substantive for the surface of interest so that a repellant amount of the 25 ' ' remains on the surface so treated and is released at a rate sufficient to repel susceptible insect or other pest. A rul,l..,laliu.. containing the repellent agent generally is introduced by topical application to a surface. For example, the l'wll,ul~liun is sprayed on, as a wet or dry r ~ " , the surface and/or underside as applicable to the surface of interest. Among the r -, ~ suitable for application are sprays, sticks, and repellent oils 30 or ointments. In some instances, the surface of interest can be , , ' with the repellent ~ ~ by absorption into the surface. Alternately, the r.- ... lA~ can be applied wet or dry to the ' . ' where it can vaporize in the vicinity of the roots and associated pathogenic organisms which colonize the roots at a rate sufficient to repel a susceptible insect W O 96139827 2 1 9 6 9 7 2 PCT~US95/17050 ~
or pest. In some instances, air can be introduced into the . h " ~1 h ~ to increase the ti"n process. To prevent an ingress of insects into an area, the ~ u l-u~ of theinvention can be applied to surfaces within and/or ~ . u ~1 ~d .~g the area, for example, the ~ al~ m~ - can be applied to doors, windows and other openings of a building and/or to 5 surfaces that surround these openings.
Where the surface of interest is a plant or plant part, the presence of the repellent agent can be a result of topical ~ ; for example, the , can be aerially applied to crops, or it can be by elaboration from the host plant as a result of genetic "' of the host plant.
The aromatic and aliphatic aldehydes 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 Cllemistry: Reactions, ~lfif~' . , and Structure, 2nd Ed., McGraw-Hill, New York, 1977. C~ hyde can be prepared synthetically, for example, by oxidation of cinnamyl alcohol (Traynelis et al., J. Am. Chem. Soc. (1964) 86:298) or by r~. ~L .. ~ n of styrene with rul-.. yl.n~LI.~l~.iline (Brit. patent 504,125). The subject aldehydes also can be obtained by isolation from natural sources. For example, ' ' ' yd~ can be isolated from ~.~k ~ fungus, Stereum r~ Fi~ r ~
ct al., Biochem. J. (1957) 66:188, and a hexyl cinnamic aldehyde (HCA) can be obtained from rice, or ~ ' as described in USPN 5,û55,621.
A preferred method for producing a desired component of the present r.. , ~ in a plant host is through 1~ ' DNA means, ~ , by modifying the level of at least one compound of interest of the formula (l), (2), (3), (4), or (5) in plant tissues of interest through eu..~Ul~,Liùll of transgenic plants using .. ' techniques known in the art. The methods involve i ~Jllllil.t, a plant cell of interest with an expression cassette 25 functional in a plant cell comprising as operably linked . . in the 5' to 3' direction of I , , a ~ and ~ initiation regulatory region, joined in reading frame S' to a DNA sequence encoding and capable of modulating the production and/or required to produce the compound of interest, and; ' ~ and h ~
~ regions. Expression of an enzyme required to produce the compound of interest 30 provides for an increase in production of the compound as a result of altered .~. -- - n~
of the enzymes involved in the ~ r ' ~ " ' Of particular interest is the sdective control of cinnamic and/or coniferyl aldehyde and/or HCA production in plant tissues such as leaves, roots, fruits and seeds. One or more . , ' of the present 1~

~ WO 96139827 2 1 9 6 9 7 2 PCT/13S95117050 can be produced by modulating the expression of one or more genes or a gene encoding or inore enzymes or an enzyme pathway or cluster required to control the level of the compound of interest in a plant, plant part, plant cell, specific plant tissue and/or associated with a particular stage of plant growth.
The enzyme or enzymes can be in a b;u~ lic pathway o} a ~ ;. pathway and the regulation will be up or down ~~li~ , i.e., to modulate expression of anindigenous or an . ~ - plant gene an indigenous plant gene is one which is native to the genome of the host plant. An . 1..~" .,.~ plant gene is one that is present in the genome of the plant host of interest, and may be an indigenous gene or a gene that is present as a result of infection of the plant (e.g., a viral gene), or otherwise naturally ill~UI~ ' ~ into the plant genome. The host plant also can be modified by, ~ I means or by traditional plant breeding methods to introduce one or more genes exogenous to the host plant which encode enzymes which control the level of the compound of interest andlor are in the synthetic pathway for one or more , ' of formula (1), (2), (3), (4) or (5). By " ' ' of gene expression" is intended control of production of a gene product ofinterest at the level of i - translation and/or post translation. The level of the compound of interest is controlled by modulating the expression of one or more ~genes or transgenes encoding one or more enzymes required to synthesize the compound of interest.
Methods for modulating gene expression in plants are ~nown in the art. Vai iation in growth conditions or exogenous application of . ' to a plant can affect gene p~c~i,.n At the molecular level, gene expression depends ~ ~Iy on the A, tianslation and i control regions which regulate expression of a structural gene coding region. By exploiting the plant signals which regulate these control regions or by the direct ' ~, ' ~ of the control regions, expression of a gene encoding an enzyme required to control the level of cinnamic aldehyde, for example, can be modulated. For use in a transgene supplied e~ùO_r.~ Jly to a plant host, the transgene will include control regions that are selected and designed to achieve the desired tissue and/or level and timing of gene expression. As a~JIuA ~ , the control regions may be hUIIIOIUOJ
(native) or non-l -' O (non-native) to the gene of interest. By "1- -' O " it ismeant that the control region(s) is from or substantially similar to a control region normally associated with the gene of interest. By ~non-hu ~ it is meant that the control region(s) originates from a different nucleoade source or sequence or is substantially different l~

wo 96/39827 2 1 9 6 9 7 2 PCT/US95117050 ~
from the control region(s) normally associated with the gene of interest. For example, if the enzyme coding sequence is nua ' ' ~ in source as compared to the control regions, in order to have expression of the gene in a plant cell of interest, I"-~e. .;l,l;,. I and ' initiation regulatory regions or promoters functional in these plant cells must be provided operably linlced to the coding sequence. T.,~ , \ and translation initiation signals functional in plant cells include those from genes which are present in the plant host or other plant species, and direct constitutive or selective expression in a plant host.
Of particular interest are the gene control regions that selectively regulate structural gene expression in a plant, plant part, plant cell, specific plant tissue and/or are associated 10 with a particular stage of plant growth. Preferred are those control regions, that are known in the art, and in particular, ~ l control regions or promoters, that can be used to modulate the expression of a gene encoding an enzyme required to control the level of a compound of formula (1), (2), (3), (4) andlor (5) in a plant, plant part, plant cell, or specific 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 4,943,674 and USPN 5,175,095; seed in USPN 5,315,001; and in rapidly developing tissues and tender shocts in USPN 5,177,011.
For selective control of ! J ' ~ of cinnamic and/or coniferyl aldehyde and/or HCA in a plant tissue of interest, plant cells are l~ ru....~ with an expression cassette 20 comprising DNA encoding a structural gene for one or more enzymes required to synthesize cinnamic and/or coniferyl aldehyde and/or HCA and capable of increasing the amount of these aldehydes in the tissue of interest. Of particular interest are those genes encoding one or more enzymes capable of ,-- ~ l;,;.,g a precursor compound required for the b;o~
of cinnamic and/or coniferyl aldehyde and/or HCA from substrates normally found in a plant 25 cell, more ~li.,ukul~ 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 for integration of the expression cassette into the genome of a plant host. Integration can be A- ~ ~ using n,, r " ", ~ systems known in the art such as A,~ ~lt, 30 cl~l.u~u.~liu,. or high-velocity ~ u~liclc mediated; c( Depending upon the ~plir~ti~n, saponin or one of the other ~ . ' of interest can be ~ f~,., "y expressed in a tissue of interest and/or a particular organelle. Tissue specificity is ~ u ~ ~ by the use of i ', ' regulatory regions having tbe desired expression profile. T ' ~

~ wo 96/39827 2 1 9 6 9 7 2 PCT/US95/i7050 of the enzyme to a particular organelle is ~~ hl ~.1 by the use of an ~
'- peptide. Methods for tissue and organelle specific expression of DNA
constructs have been described are Imown in the art.
To verify regulation and expression of the gene of interest, various techniques exist S for :1~ 1,, whether the desired DNA sequences present in the plant cell are integrated into the genome and are being ~..ct~nh~ Techniques such ~ the Northern blot can be employed for detecting messenger RNA which codes for the desired enzyme. Expression can further be detected by ~saying for enzyme activity or y for the protein product.Most preferably the level of the compound of interest present in a plant host is measured using methods known in the art. A desired phenotype, for example, is increased HCA
content in a plant tissue of interest ~ measured by expression of the gene of interest and/or tbe level of HCA present in the plant host as compared to a control plant.
For il~LIudu~liùll of one or more , ' of the present Ç ' to the target organism, a plant host expressing a gene encoding an enzyme required to control the level of the compound of interest results in the exposure of a target organism to at least one component of the repellent Ç~ ;"" At least one component of the repellent Çull~ul~liu~
can be expressed by the plant host and optionally other , of the repellent are ~ ~b' -'J -~y applied to the plant host so that the . ' elicits the desired repellent effect.
Transgenic plants having an increased ability to - ' flavonoid aldehydes such as '' ' ~d., and coniferyl aldehyde and HCA to provide self-protection against plant pests or be used as a natural source of flavonoid aldehydes for extraction and subsequent use ~ a repellant can be prepared.
~ ' of flavonoid aldehydes can be achieved by ~ g the expression of specific plant genes that encode enzymes which either cause further ' ' of the desired aldehydes or divert metabolic " away from the desired aldehydes.
In the case of ' ' ' Jd " for example, this involves d~, .. .,,~,~,ul~.L,.g the expression of cinnamate 4-hrJl~ l~ (CA4H) and cinnamic alcohol d~hyJlv~ ~ (CAD). CA4H
ordinarily diverts some cinnamic acid away from f ~ hyJ~, to produce p-coumaric 30 acid, itself a metabolic " Reducing CA4H activity alone is not sufficient to cause P ~ of '' ' ~du because CAD can rapidly convert . '' ' yJ~ to chmarnyl alcohol, wbich then becomes incoll ' into lignin or _ ~ ~ glycosides 'y reducing both CA4H and CAD activities results in increased metabolic flux , _ _ . .

WO 96/39877 2 1 9 6 9 7 2 PCTIUS95/17050 ~

from cinnamic acid into ~ d., and decreased conversion of . ~ " ' Jde into cinnamyl alcohol. Some ~ ' ' ' 1d-, becomes incull ' into lignin but lA~hyde (either free or as glycosides) also - ' to above-normal levels, p~Li~,ul~ly at times when the l,;v~.~ ' of cinnamic acid is elevated. This occurs when 5 the level of L ~ ~ unmonia Iyase (PAL; the first and rate-limiting step in general P~ - , Hahlbrock and Scheel (1989) Annu. Rev. Plant PhysfoL PlantMol. Biol. 40:347-369) activity is high, a situation that naturally occurs in plants in response to a wide r mge of stimuli including invasion by fungal pathogens and rnechanical damage associated with wounding and insect feeding.
Inhibiting CAD activity in transgenic plants has been proposed as a method of reducing lignin synthesis in plants and thereby improving the d;6~lib;1ily of fodder crops (WO 93/05159). These ~ suggested that lignin b;~J..Lh.,~;s had been altered u~liL~Li~ , but not necessarily ~u~LiL~Livcly, but did not ~'- )r or appreciate the desirability of ~ ' h ~ rd~i as a method of increasing insect and otner pest 15 repellancy.
A number of plant CA4H and CAD genes have been cloned and their sequences are available from GenBank. Portions of these genes that include nucleotide sequences that are conserved between different plant species can be used directly in a plant expression vector (antisense or sense ~ ) to suppress the expression of the ~ g ~ lr~" ....
20 genes (e.g., Pear, e~ al., The Plant Cell Antisense Res. and Develop. (1993) 3:181-190, Napoli, et a~., The Plant Cell (1990) 2:279-289. More preferably, these conserved gene sequences are used to isolate CA4H and CAD cDNA clones from a cDNA library of the plant species that is to be modified. The resulting cDNA clones, or portions thereof, are then introduced into a plant expression vector (antisense or sense) and used to transform the 25 plant(s) of interest. DNA constructs according to the invention preferably comprise a sequence of at least 50 bases which is h .A- ~k,c", to the P~ IuL,f ~ CA4H or CAD genes.
A .~ ' DNA molecule can be produced by u~.~Li~ly linking a vector to a useful DNA segment to forrn a plasmid that can be used for plant I- r ~'r~ A vector capable of directing the expression of RNA from a cloned portion of a gene is referred to 30 herein as an "expression vector. ~ Such expression vectors contain expression control elements including a promoter. Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from tne Ti plasmid ûf A~v~c, , ~r described by Rogers et al., Methods in Enzymology (1987) ~ W096139827 2~96972 PCT/~IS95/17050 153:253-277. A common promoter that is used to provide strong ~ expression of an intrwiuced gene is the ~ '' . mosaic virus (C~V) 35 S promoter (available from Pharmacia, Piscataway, NJ). Either ~;ur.JLiLuLivc~ promoters (such as CaMV 35S) or inducible or .1.,..1~, "y regulated promoters (such as the promoter from a PAL gene or theS ~ "4L. ~"~ CA4H or CAD genes) can be used. Use of a constitutive promoter will tend to affect functions in all parts of the plant, while use of an inducible or du~ r l~y regulated promoter has the advantage that the antisense or sense RNA is only produced in the tissue and under the conditions it is required. The use of du~ " ~lly regulated promoters is preferred in the use of this invention because the down-regulation of 10 l ' .~I,ul~ . ' ' yl.tl.~s;, is known to be capable of producing ~ I.Ir side-effects on the ie~lul~uu.~ of transgenic plants containing a l.~tululogou~ PAL gene (Elkind, Y. et al., 199~J Proc. Nat. Acad Sci. (1990) 87:9057-9061.
A number of different i ~u ~ Liun methods are available for the routine of a wide range of plant species. One method that is ~ u Liuukuly efficient for 15 the transfer of DNA into dh~uL~ Aiunuus plants involves the use of A~ ~tc, In this method the gene of interest is inserted between the borders of the T-DNA region that have been spliced into a small ' plasmid with a selectable marker gene (for example encoding neomycin ~ r Il or ' ~t~' r ), The plasmid is then introduced into an A~j,u~aL~, host by I r ~ or 20 triparental mating. The A,~v~u~tt~ . strain cartying the gene(s) of interest is then usecl to transform plant tissue by co-culturing the bacteria with an a~ , ' plant tissue (e.g., leaf disc). T r ~ celIs are selected in tissue culture using the ~ , selection agent and plants are then ~~ ' (see Horsch, R. B. et al., Science (1985) 227:1229-1231.
Other methods that have been used in the j r " of plant cells, and in particular the 25 more l~ ' crop plants, include biolistics and el~LIu~l..Lio" (for detailed protocols, see Sar~ond, et al., (1993) Met~lods in F, ~C~5y 217:483-509; and Potter, (1993) Metiwds in Er~ ~1~ 217:461-478.
Once transgenic plants have been produced, cu,,~ lLiv,,al enzyme assays for CA4Hand CAD are used to determine the level of ~ - of enzyme activity achieved in 30 different i r ' It is likely that only a small fraction of the lld~rullll~ulb produced will have a ~urrl~ tly low residual enzyme activity to cause the q~ ' of flavonoid aldehydes without also producing some . --1. ~ side-effects on plant d~lu~ ,,l. For tbis reason, a preferrec method of producing the desired j r ' with both CA4H and ~7 WO 96/398~7 PCT/US95/17050 ~
CAD suppressed is to introduce the two genes separately into different i r ' and then combine them by stdndard sexual crosses. This permits a larger number of, ' ~ of level of gene ~ to be evaluated at the same time.
An alternative to u.~ h g flavonoid aldehydes in transgenic plants is to use the5 plant genes to confer on a microbial host the capability of ~h~,~;L-g specific flavonoid aldehydes. The resulting microbes can be used either to produce the flavonoid aldehydes in a ' '- system or as a natural delivery system of the flavonoid aldehydes in viable or non-viable microbial ~)~C~dliUII>~ Yeasts, especially Sa~,',o,l , cerevisiae, are preferred organisms for this purpose because they have already been engineered for high-level expression of PAL (Faulkener e~ al. (1994) Gene 143: 13020) and a plant cinnamate 4-hydlu~yla~c has been shown to function in yeast (Urban e~ al. (1994) Eur. J. Biochem.
222:843-850).
The expression of PAL introduces the capability to produce cinnammic acid from phenylalanine. Two additional enzymic steps are required to produce ~ d~, from ~h~ ' ' In plants, these steps are catalyzed by the enzymes ~ CoA ligase (CL) and cinnamoylCoA reductase (CCoAR), but as 4-co~ rl~ ligase (4CL) can also use cinnamic acid as substance (Knobloch, and E~ahlbrock (1977) ~rch. Biochem. Biophys.
184:237-248), 4CL can be used instead of CL. More than 20 cloned PAL genes and more than 6 4CL genes have bcen described in sufficient detail (GenBank) to facilitate their use in practicing the current invention. A gene for a CCoAR is obtained by applying standard gene cloning techniques to isolate a cDNA clone using as a probe sequence derived from the amino acid sequence of the N t~,~ or peptide fragments, of the purified protein.CCoAR has been purified and partially ~ -1 from soybean cultures (~ , J~ e~
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 e~ al.
(1984) Eur. J. Biochem, 139:259-265) and I:rF . .a;~ g xylem of Eucalyp~us gunnii (Goffner e~ al. (1994) Plant Physiol. 106:625-632). The preferred method of ~ ~~ is that of Goffner e~ al. (supra) because it results in a single protein band on SDS-IJ~ ~ ' gels that an be used for protein l . g The cloned genes are introduced into standard expression vectors and used to transform a microbial host, preferably yeast, by standard L..m ,rul~u~.~iu~l techniques such as rl- I~ Y~ (Becker and Guarante (1991) Methods in Enzymol. 194:182-187). Standard enzyme assays are used to confirm the functional expression of the engineered genes and /~

~ WO 96/39827 2 t 9 6 9 72 PCTIUS9~/17050 assays for flavonoid aldehydes are used to select strains with maximal ,u ~ h '' 13ecause flavonoid aldehydes have ' ~ ' ' ' properties it is preferred to use expression vectors that will cause expression of the introduced genes only late in the growth cycle or in response to a chemical inducer. It may also be desirable to grow the engineered microbial host in an 5 ' ' " ' whole cell reactor (e.g. Evans et al. (1987) P;~ s ~cg~ and Bio~ .,s:. .. ;~.~$
30:1067-1072) to prevent the aldehydes from ~ ' ' g 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 secretes nutrients required by the fungus.
10 Examples of fungi and the plant parts which they colonize are as follows. Black spot on fruit;
Fusariurn sp. on flowers roots and leaves; and Fusariurn spp. and Aspergillus on roots and leaves. Fusarium causes vascular wilts of annual vegetables and flowers, herbaceous perennial ~ '5, plantation crops and the mimosa tree. Different plants are attacked by special forms or races of the fungus. Vemculum (V. albo-atriurn and V. dahlise) cause 15 vascular wilts and colonize roots, flowers and leaves. In addition the following also constitute target organisms: r.l" ~ ~ spp; l~iF'c-~cp rosae; S~h~c~u~ a tannosa;Oibiapsis sicula; rAJ ~ r ~ 1 U taraesitica; hJ ~ r ~ a infestans Puccinia spp; Alternaria spp; Susaiun spp; Bofrytis cinera; Sclero~inia ~ -- .~ Tricophyton ~ h~, Dutch Elm disease (Ceratocystis ulmr~ and oak wilt (C J; ~ ~ ). Ceratocysns causes 20 vascular wilts, mainly of trees. Also included are blue-green algae (C~ ). The vectors for these diseases which can be repelled by the subject r~ include beetles and wasps Target organisms also include insects which damage the plants which they colonize, ~i' ~ ~,'~those of the orders Orthoptera; IhJ r-'~ ~ which includes water weevil and thrips; and ~r r '~ ~ which include aphids such as root aphid and leaf aphid, 25 l. r~ white flies, mealy bugs, thrips, cicadas, caterpillar, such as velvet bean caterpillar, codling moth, leaf roller, and scale insects. Other target organisms include arachnids (~i~,ulafl~ spider mites), flies (Musca domestica) ~o~hu~.~h~s, gastropods, moths, and bed bugs (Cimex lecn~laris) and its close relatives (poultry bug (H- r I
indorus Duges), the European pigeon bug (Cimex r~ ' ' ~~ Jerjus), and the swallow bug 30 (Oeciains vicarius Hrovath)).
Also of particular interest is prevention of phylloxera infestation in grapes by repelling the phylloxera. For this arr~ nn, it is necessary to deliver the rul l~tion to the roots of the plants which are the usual habit for phylloxera. When used in a solid form or WO 96/39827 2 1 9 6 9 7 2 PCT/US9S/l70s0 7 the dosage used is typically on the order of 1% to 35 % on a w/w basis, the maximum loading to be determined as a function of shell material selected. Analytical chemical techniques are used to determine and optimize rate of release. For qualitative purposes, GC techniques can be used to determine the amount of aldehyde released. The 5 samples of ~ (pelletized) product are mixed with the soil types selected and sampled at different time periods to measure release. Al ~ , the volatile gases released from the r~ can also be analyzed. For measuring the activity of foliar and drip irrigation c~ .n~ the stability of the f, lqfinnc over time can also be evaluated using the GC . ~ using methods known to those skilled in the art. Methanol or 10 alcohol extractions of the ' ~ also can be prepared for HPLC analysis. The preferred method of repelling phylloxera and other root dwelling pests, however, is to provide for a systemic response to, for example, a foliar application of the ' ' which is then i ~ ' to the root. The timing of such ~ ;.- will need to be determined empirically for particular plants as the flow of water from the leaves to the roots is required 15 for i '- Generally, such flow is greatest at cooler i . c.g. during the evening hours, at night, or in the early morning hours, and pre or post fruit or vegetable d~
The subject ' ' i, in particular those containing HCA, are also useful for treating: grape to repel pests such as thrips, nematodes, and leaf roller; roses to repel thrips 20 and melon aphids; cattle to repel soft ticks; humans to repel mosquitos; apple to repel codling moth; animals 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 subject ' ' - to repel insects and other pests which attack the seeds and/or which act as 25 vectors for disease organisms. The seeds can be dusted with a powder preparation (see U.S.
Patent Application No. 4,978,686 for examples of inorganic materials to which the can be adsorhed) or admixed in a plant substrate such as ~.,. ' Seedlings grown under sterile conditions from treated seeds are free of susceptible fungi and insects.
AdditionaUy, seedlings also can be treated with the subject ' ' In some instances it 30 may be necessary to adjust the treatment ' ' so as to reduce any p~ d~;Ly associated with the treatment as tender young shoots are more likely to exhibit phyl~tv~.h,;ly symptoms. The treatment ' ' are also useful for controlling the time of pollination of flowering plants. For example, to prevent or delay pollination the ' ' are ~o ~ WO 96/39827 2 1 9 6 9 72 PCT/US95/17050 applied in an amount sufficient to repel bees and other pollinating insects. By adjusting the residuality of tbe c~ ~ one can -control the length of time during which pollination is inhibited. On the other hand, for plants whether cross-pollination is required for fertilization, application of the ' ' during this period should be avoided if the pollinating insect is S repelled by the ' ' ' In order to oetermine the , ' li~y of patticular insects to repellency by the claimed , , in vitro and in vivo tests which compare the behavior of the target pest towards, for example, dy~ , a "bait" food in the presence and absence of the test are used. The ~rf~Li~ of the ' ' over time can be evaluated by 10 extending the time period of Ub..~l ~diull until few of the test insects (less than about 50%) are repelled from the vicinity of the bait. For pathogen vector insects, a 90% or greater repellency is usually in order. For the common nuisance pest, reduction in the magnitude of 80% is suitable (e.g., in garden and food areas). The r~ also need to be evaluated for 1' ytutu~ i~;Ly for use on plants and for dermal sensitivity, p~Li~ul~ly for use on skin 15 and/or clothing of humans; contact dermatitis and olfactory sensitivity are monitored using tests for dermal sensitivity known to those of skill in the art. Likewise, l!hytuAi~,;Ly testing can be done using methods l~nown to those of skill in the art. Ph~tvLu~ iLy can be rated as follows in order of increasing severity of toxicity: 0-plants without any symptoms; 1-very slightly browning of hypocotyl (no other sy : ~, 2-some wilting of plant, dying of lower 20 leaves, some browning of vascular system; 3-wilting of entire plant, leaves dying, hypocotyl with external and internal symptoms; 4-necrosis of stem, plant dying. lt is preferable that the '- used have a yL.~utuAi~lty rating of 2 or less, more preferably I or less.
The ~ , ~ of a ' ' " to be used for a particular application can be ~' ~ ' by evaluating first the . range over which a given component has no 25 activity to where it provides maximum activity (a dose response curve) and then evaluating this component separately and in o."~ ;-, with other . , of interest for a givenThe repellent andlor phytotoxic and/or dermal effects of a particular ~ '- on a given insect or other pest and the host is then measured for each formula and component with or without a serial diluent of any additional component of interest.
30 Optimal dose-ranges are calculated in vitro and in viW using techniques known to those of ordinary skill in the art. r. are identified which provide: repellency of 90%, and/or a yhjtutu~ rating of 2 or less for plants, with I or less being optimum, and ~ "~, free of contact dermatitis for animals and fowl.
~J

WO 96139827 ' 2 1 q 6 q 7 2 PCTIUS95/170S0 ~
The following examples are offered by way of illustration and not by way of limitation.

F~AMP~ FC
The following products were used in the example protocols set forth below: (1) 5 cinnamic aldehyde from Spectrum Chemical Co., New Jersey, USA; (2) coniferyl aldehyde from ADIN Chemical Co., VF; (3) sodium l,iwl and Tween 80 from Spectrum Chemical Co., New Jersey, USA.

~amvle I
FliPC ,~
The purpose of this experiment is to evaluate the repellency activity of cinnamic ald_hyde and ~-hexyl cinnamic aldehyde against flies (Musca domesnca). Twenty 2-3 day old female flies are released in a 62 x 62 x 34 cm cage with 325 mesh roof screening to permit air circulation (Carolina Biological Supplies). Bait made of sweet milk (Carnation) (90%) plus glucose (10%) and a dye ~ ~i' ~' blue 0.01%) with I ml of r ~ '- is placed in a 3.5 cm petri dish and set inside a pine cage (Carolina Biological Supplies) with a 1 cm inch diameter hole drilled through the top to permit access to the cage containing the bait. A 3.5 cm petri dish with 5 ml H,O is placed in the cage for water. After 24 hours, flies are remov_d and crushed on filter paper to check for the presence of dye which would 20 indicate feeding activity. Entry of more than 10% of the flies is taken as an indication of lack of formula repellent activity.

~xample 2 BIatella ~enr~jç) The aim of this experiment is to evaluate the repellancy activity of cinnamic aldehyde 25 formula against ~u~h~.,h~,s (Blatella germaruc). Fifty nymphs and adults 1.5 cm to 3.5 cm in length are released in a 62 x 62 x 34 cm cage with 325 mesh roof screening to permit air circulation. The inner surface of the cage walls from 5 cm to 10 cm from the floor are treated with a mixture of mineral oil and petroleum jelly (2:3) to prevent escape of the co ~ The cc~h~h_. are fed on dog chow (Purina), milk powder and water for 30 48 hours for a~ A Two Whatma~ filter papers "C" (4 x 4 cm) are folded twice, and stapled and wet with 1 ml of formula each. Filter papers are allowed to air dry. After 2~ 96972 air drying, a filter paper is placed inside of one of two 4 cm x 4 cm x 4 cm cubes, each with a single 0.75 cm door at floor (base) level for entry. The two shelter boxes are placed on the bottom fioor of the cage 14 cm apart. After twenty hours, the shelters are removed and the number of shelter V,~L.~.- h ' are removed and counted. An entry of more than 10% of 5 ~C~ L,..-- h ~ into the shelter boxes is regarded as an indication of loss of formula repellency.

Examvle 3 ~hid ~phidfabae) The purpose of this experiment is to determine the repellancy activity of a cinnamic aldehyde r ~ '- against black bean aphids. Sugar beet plants ~eta vulgarisJ are grown 10 im 7.5 mm pots in potting soil in a g ' When plants reach the tbree leaf stage, eight plants are selected at random. In separate trials, four plants are treated with: cinnamic aldehyde at S0 ppm; S0 ppm cinnamic aldehyde formula ~NaHCO3 + Tween 80); NaHCO3;
Tween 80; and formula blanlc. Treatment is a foliar application of S ml of material sprayed as fine mist by a hand sprayer (Gilmour). Four plants are untreated and one sprayed with lS water only. 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 pernutting air ' At 4, 8 and 24 hours, the number of aphids on treated and untreated plants, rows A and B, are counted and recorded.

~xam~le 4 - ,~ily~rl~o~f Wl- t~ Fly ~ trar~h~ urticac) The purpose of this experiment is to determine tbe repellency of cinnamic aldehyde ag~unst siiver leaf white fly. In a ~l~ ' , cotton piants 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 aidehyde at S0 ppm; 50 ppm cinnamic 25 aldehyde formuia (NaHCO3 + Tween 80); NaHCO3; Tween 80; and formula blank.
Treatment is a foiiar application of 5 mi of material sprayed as fine mist by a hand sprayer (Giimour). Four untreated plants receive a foliar spraying of 5 ml water. Tbe treated and untreated plants are placed in two rows, A or B, treated and untreated, ~l~o~ti~ly~ in a 60x 60 x 30 cm cage with a 325 mesh wire screen roof ailowing air, . ~ At 4, 8 and 24 30 hours, the number of silverleaf white flies are counted for presence on plants in rows A and ~3 wo B (treated and untreated). At 48 hours, the number of eggs on plants in each row are counted and recorded.

Example 5 T P~fhn~r~Pr5 ~ n~
The purpose of this experiment is to determine the repellency of cinnamic aldehyde against ~ fl~n~r.c In a 6 - cotton plants are grown in 7.5 mm pots in potting soil.
When plants reach the three leaf stage, eight plants are selected at andom. In separate trials, four plants are treated with: cinnamic aldehyde at S0 ppm; 50 ppm cinnamic aldehyde formula (NaHCO3 + Tween 80); NaHCO3; Tween 80; and formula blank. Treatment is afoliar application of S ml of material spayed as fine mist by a hand spayer (Gilmour). Four untreated plants receive a foliar spay of S ml of H2O. The treated and untreated plants are placed in two rows, A or B, treated and untreated, ~ y~li~,l.y, in a 60 x 60 x 30 cm box cage with a 325 mesh wire screen roof allowing air circulation. At 4, 8 and 24 hours, the number of l. n..y~ are counted for presence on plants in rows A and B (treated and 15 untreated). At 48 hours, the number of eggs on plants in each of the rows is counted and recorded.

~mpl~ 6 Thrips (~hv.,u,.v~
The purpose of this experiment is to determine the repellency of cinnamic aldehyde 20 against thrips. In a 6 tomato plants are grown in 7.5 mm pots in potting soil.
When plants reach the three leaf stage, eight plants are selected at andom. In separate trials, four plants are treated with: cinnamic aldehyde at 50 ppm; 50 ppm cinnamic aldehyde formula (NaHCO3 + Tween 80); NaHCO3; Tween 80; and formula blank. Treatment is afoliar application of 5 ml of material spayed as fine mist by a hand spayer (Gilmour). Four 25 untreated plants receive a foliar spay of 5 ml of water. The treated and untreated plants are placed in two rows, A and 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 thrips are counted for presence on plants in rows A and B (treated and untreated). At 48 hours, the number of eggs (in leaf slits) on plants in each row are counted and recorded.

~4 ~ WO 96/39827 2 1 9 6 9 7 2 PCT/US95/17050 l~a,mplQ7 , ~
Tw~ ~tt~A S3?i~1Pr Mit~ ' u~rr~.DJ
The aim of this experiment is to evaluate the repellency of cinnamic aldehyde ontwospotted spider mites. Cotton plants are grown in 7.5 mm pots in potting soil in a S 1;'' ' When plants reach the three leaf stage, eight plants are selected at random. In separate trials, four plants are treated with: cinnamic aldehyde at 50 ppm; 50 ppm cinnamic aldehyde formula (NaHCO3 + Tween 80); NaHCO3; Tween 80; and formula blank Treatment is a foliar application of S ml of material sprayed as fine mist by a hand sprayer (Gilmour). Four untreated plants each receive a foliar spray of S ml of water. The treated 10 and untreated plants are placed in two rows, A or B, treated or untreated, l~L~,ly, in a 60 x 60 x 30 cm cage with a 325 mesh wire screen roof allowing air ~irr.lloti~n 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.

Example 8 ~ ~AD~D~ ~D~V~tZ) y T.-ct E~ ~ In Viho The purpose of this experiment is to evaluate the repellency of cinnamic aldehyde against mosquitos. Twenty unblooded adult female mosquitoes .~ Iy 4 days of age are introduced into test chambers. Four ml of a test rul ~ is pipetted onto a 16 cm ~2 20 Whatman filter paper circle and air dried. The treated filter paper is placed on the vent intake chamber. CO~-is bubbled through water at the vent intake end of a wind tuMel olr t - ~ t~ cbamber; the lowest rpm fan setting is used. The trap chamber is opened for 5 minutes, then closed and the number of mosquitoes counted and recorded. DEFT at 23 % is used as a positive control.

25 ~Pll~n~-y Test P F;ol~1 Tri~l The purpose of this experiment is to bioassay the activity of cinnamic aldehyde as a mosquito repellent. Two circles 18 cm in diameter and two 16 cm in diameter were cut from I mm mesh nylon mosquito cage bolt of screen material. The treatment circle (16 cm) was soaked in l ml '~ ~ cinnamic aldehyde (2%) in 2~ Tween 8û and 6% NaHCO3, 30 then allowed to air dry for 2 hours. Ten unfed female ACdeS aD.~ptz mosquitoes (5-7 days _ _ _,, _, ,, . . . ~ . . .

wo 96/39827 2 1 9 6 9 7 2 PCT/US9S/170s0 ~
old) from the Kearney Agriculture Center, Mosquito Control Research Laboratory, were introduced into 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 scre~ns and sealed with a rim from the pint carton, the lid section having been removed. An 5 adult male volunteer placed the treated 16 mm circle on one of his legs (which had been washed and rinsed with soap and water) and the untreated 16 mm circle on the other leg (washed and rinsed with soap and water). The one pint mosquito cartons were put in flush contact with the mesh screen side to the leg screen patches for 5 minutes. l~,f. , did not come in direct contact with compound. After S minutes, the number of blooded/well-10 gorged insects out of 10 were counted. The results ~ shown in Table 1, below. Out of atotal of thirty insects evaluated, only two were not repelled by the cinnamic aldehyde ~ " , as compared to 19 in the control (untreated) group.

Mnqlni~ R~ y IS(~ of blooded/well-gorged insects/10 insects) Trial I Trial 2 Trial 3 Sum Cinnamic aldehyde 0/10 2110 0/10 2/30 ~ ,- I
Control (untreated) 5/10 7/10 7/10 19/30 ~ Cinnamic aldehyde (2%) with 2% tween 80, and 6% NaHCO3 in H2O.
A protocol similar to that described above is used to test ~Y-hexyl cinnamic aldehyde.

xample 9 oviposirior~l Repellency The purpose of this experiment is to determine the repellency of cinnamic aldehyde ag~unst 13eet Armyworm adult moths. An apparatus is built that forces an airstream over treated and non-treated potted plants in a flight cage. Five tomato plants at the three leaf stage are treated with S ml of various of ~6 ~, wo s6/3s827 2 1 9 6 q 7 2 P~
chemical formula and, , and then placed in the cage. Five tomato plants are sprayed with 5 ml of H2O as control plants and then placed in the cage. Forty egg laying ready Beet Armyworm (~p~ ', . u exlgna) adults are released in the cage. The exhaust fan on the apparatus is turned on and a low velocity linear flow of S air is allowed to flow through the cage as plumelets of air c ~ g chemical formula. After 24 hours, oviposition is determined on treated plants, non-treated plants, and cage walls.

'- 10 Phy]1~YPrq - Va,~nr Test for RP~PllPnf y The purpose of this experiment is to evaluate the vapor repellency of cinnamic aldehyde to phylloxera. Root pieces of grape stock with viable phylloxera eggs (n-30) is placed in 50 x 9 mm dishes that have been treated on inner surfaces with 400 ml of known product ~ The chernical is not placed directly on the root, so that absorption or ~ ", by the root is not a factor. The dishes are shut and sealed with tape. After 7 days, the dishes are opened, and a ~' is made as to whether insects are able to establish on the roots and develop, orwhether the newly hatched msects avoid the roots and die.

FYqmrl~
ln. u~,,, of P!qvnn~
Al~ y i~c in Triqnc~,oni~ plqnlc Twenty llg of polyA RNA is prepared and cDNA synthesized. Part of this is cloned into lambda-ZAP 11 vector (a ~;ally available cloning vecvor). At least 500,000 ' ~ are screened using an ~'I;C~ ~ 1;1 probe designed from conserved sequences of cloned CA4H and CAD genes obtained from GenBank, or designed from peptide sequence of purified protein from the intended host plant.Strongly 1,.~' ~ ' ~ ~ clones are selected and used to rescreen the cDNA library. The resulting clones are sequenced to enable the h,LIu.lu~.lion of ~ , gene sequences into a plant expression cassette in either antisense or sense ~lri~n~q~ic)n The an~dsense and sense constructs are introduced mto A,~/v~lc~ sf LBA4404 by direct u - - f ~ following published procedures. Tobacco (N.
ta~aclun, variety Samsun) leaf discs are ~ r, using well estabished published ~t7 Wo 96/39827 2 1 9 6 9 7 2 PCT/USgS/170s0 ~
procedures (EIorsch ct al. (1985) Sciencc 227:1229-1231. Plants containing either CA4H or CAD constructs are identified by PCR and selected for further analysis.
Plant material from both ~. , r-, d and ~ r ' control plants is used for J~t~l of CA4H and CAD enzyme activity using well established S published assays. Plants in which the activity of CA4H or CAD has been reduced to less than 20% of that seen in control plants are selected for further analysis. Selected plants with low CA4H activity are crossed with plants with low CAD activity and progeny inheriting both gene constructs are selected by PCR. Plants with suppressed CA4H and suppressed CAD activity are analyzed for flavonoid aldehyde production using stimdard published procedures. Those plants that produce flavonoid aldehydes are then tested for efficacy of repelling insects or other pests using any a~lulexample, e.g. E~umple 3 to test transgenic cotton plants for their capacity to repel aphids.

F~AMPT F 12 i~ of FI~Y~mnirl ~ pllydes jn h/liPrnhi~l S~vsfPmr~
A cDNA library is generated using RNA extracted from six week old tobacco stems. 20~g of polyA RNA is prepared and cDNA ~ Lh~;~l. Part of this is cloned into lambda-ZAP Il vector (a commercially available cloning vector). At least 500,000 .~ ' are screened using an ~ 'ig -l-'lti~iP probe designed from peptide sequence sequences of CCoAr protein purified from six week old tobacco stem tissue using the protocol of Goffner et al. (1994) Plant Physiol. 106:625.
Strongly l.ylJIlfJi~h.D clones are selected and used to rescreen the cDNA library. The resulting clones are sequenced to enable the j~ of full-length cDNA inserts and the il-tl~,du~,Lion of a~yl~, ' CCoAR gene sequences into yeast expression vector pMTL8110 (Faulkner el al. (1994) ~ene 143: 13-20. The coding sequences for Rhcd~ ,v, 'i, toruloides pLIl.~' ' ' ammonia Iyase (PAL; GenBank locus R~IDPAL) and a parsley 4-. CoAI ligase (4CL; GenBank locus PC4CLIAA) are similarly introduced into equivalent yeast expression vectors. The PAL,4CL and CCoAR constructs are used to transform SllcJk~...yccs cerevisiae strains by I~LIurJlaLi~ using established published procedures (Becker, and Guarente, Methods in Enzymology 194:182-187, 1991; Simon (1993) Methods in Enzymol 217:478-483. T r ~ are selected on minimal medium lacking leucine.
~8 ~ WO 96139827 2 1 9 6 9 7 2 PCT/US95/17050 T ' strains carrying all three gene constructs are identified by PCR and selecter for further analysis.
Extracts from both ll - f ~ ''l and u, ~ - r ,.. ~.1 control strains are used for ;' of PAL, 4CL and CCoAR enzyme activities using well established published assays. Strains in which the activity of PAL, 4CL and CCoAR is 'y greater than the l,~Ljl~ ' activity detected in control strains are selected for further analysis. Selected sttains are analyzed for flavonoid aldehyde production using standard published procedures and tbose producing significant arnounts of e ~ ,d-, are selected for a~ ~ of ' conditions.
The resulting products are then tested for their efficacy in repelling insects and other tests using any of the described methods.

EY~ P 13 FTl~A Antivity a~ In~t pf~ t The purpose of this experiment is to deter nine whether ~Y-hexyl cinnamic lS aldehyde is an effective insect repellant. Following negative skin-irritation tests on rabbits at the FDA, KCA was evaluated on tbe skin of 2 to 4 male human subjects.One ml of the compound was rubbed over one forearm. A glove was worn to protect the untreated hand while the treated forearm was exposed in a cage containing a high number (2,000-4,000) of unfed mosquitoes for 3 minutes at intervals of ~ ly 30 minutes until two bites were received (t vo bites in one test period or one bite in each of two WII~U~ . test periods). The time interval between application and when two bites were received was defined as the "protection time.~
Against the yellow fever mosquito (Aedes aegyptJ (L)), HCA was rated as 3 (121-180 minutes duration). Against the malaria mosquito (Anopheles 5 ' ~i' '' Say), HCA was rated as 2 (31-oO minutes).
HCA was also evaluated on treated cloth against the yellow fever mosquito.
In these tests, women's mercerized-cotton stockings were used. A measured section above the ankle was " ~ with HCA at a rate equivalent to 3.3 g/ft2. The stocking was spread on a rack to dry and then hung indoors on a line. The first tests were done 24 hours after treatment. The stocking was drawn over the arm, with the treated portion midway on the forearm. The untreated hand was protected with a glove and the stocking-covered arm was eYposed for one minute in a test cage. If 5 ~q wo 96~9827 2 1 9 6 9 7 2 PCT/US95/17050 ~

bites were received, the treatment was considered ineffective. If less than 5 bites were received, the exposures were continued daily until the 14th day and at wee~y 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.

S These exarnples d~-- that the subject cirmamic aldehyde and ct-hexyl cinnamic aldehyde r ~ " ~ are effective repellents against mosquitos.

All ~ ' ' and patent ~ - mentioned in this ~ are indicative of the level of skill of those skilled in the art to which this invention pertains. All ~ ' I and patent ~l.L~ are herein ;II~,UI~ldtCd by reference to the saune extent as if each individual publication or patent application was specifically and individually indicated to be hl~,ull ' by reference.
The invention now having been fully described, it will be apparent to one of ordinary skill in the art that many changes and ~ " can be made thereto without departing from the spirit or scope of the appended claims.

3~

Claims

AMENDED CLAIMS
1. A composition for repelling pests, said repellent composition comprising at least one flavonoid aldehyde at a concentration sufficient to provide an aroma which repels pests, wherein said concentration is 10-5000 ppm of the total composition.

2. The composition according to claim 1, wherein said flavonoid aldehyde is cinnamic aldehyde, .alpha.-hexyl cinnamic aldehyde, and/or coniferyl aldehyde.

4. The composition according to any one of claims 1, 2, and 20, wherein said further comprises saponin.

5. 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 the claims 1-2, 4, and 18-20 in an amount which does not cause dermal irritation.

6. The method according to claim 5, wherein said composition is in a form selected from the group consisting of a spray, a stick, a repellent oil and an ointment.

7. The method according to claim 5 or claim 6, wherein said mammal is selected from the group consisting of a human, a bovine, and an ovine.

8. The method according to any one of the claims 5-7, wherein said mammal is a human and said pests are fleas or mosquitoes.

9. The method according to any one of the claims 5-7, wherein said mammal is a bovine and said pests are soft ticks.

10. A method of repelling pests from the vicinity of a plant, said method comprising application of a composition according to any one of the claims 1-2, 4, and 18-20, wherein said plant is selected from the group consisting of grape, rose, apple, and corn, said composition has a phytotoxicity rating of 2 or less.

11. The method according to claim 10, wherein said plant is a grape plant and said pests are selected from the group consisting of thrips, nernatodes, phylloxera and leaf rollers.

12. The method according to claim 10, wherein said plant is a rose plant and said pests are thrips or melon aphids.

13. The method according to claim 10, wherein said plant is an apple tree and said pests are coddling moths.

14. The method according to claim 10, wherein said plant is a corn plant and said pests are root aphids.

15. A method of preventing an ingress of insects into an area through the application of a composition according to any one of the claims 1-2, 4, and 18-20 to the vicinity of said ingress.

16. The method according to claim 15, wherein said insects are cockroaches.

17. 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 1-2, 4, and 18-21.

18. A composition for repelling pests, said repellent composition comprising saponin and at least one flavonoid aldehyde at a concentration sufficient to provide an aroma which repels pests.

19. A composition according to claim 18,wherein said flavonoid aldehyde is selected from the group consisting of cinnamic aldehyde, .alpha.-hexyl cinnamic aldehyde, and coniferyl aldehyde.

20. A composition for repelling pests, said repellant composition comprising at least one of .alpha.-hexyl cinnamic aldehyde and coniferyl aldehyde, at a sufficient to provide an aroma which repels pests.

21. A method of preventing infestations of a tree by pests, said method comprising: contacting the trunk of said tree with a composition comprising at least one flavonoid aldehyde at a concentration sufficient to provide an aroma which repels pests.