SPIDER MITE POPULATIONS

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to chemicals which enhance the effectiveness of miticides or pesticides against spider mites, and more particularly, to a novel miticidal composition combining a miticide or pesticide and at least one sesquiterpene alcohol or analog thereof, e.g. farnesol and/or nerolidol, in an effective amount for enhancing the effectiveness of the miticide or pesticide in controlling both female and male spider mites in susceptible and resistant populations.

2. Description of the Prior Art

Spider mites, belonging to that family of Acari known scientifically as Tetranvchidae, are a common form of agricultural pest which damage the foliage of agricultural crops, trees and ornamentals. Two species of spider mites which are common are the carmine spider mite (Teiranychus cinnabaήnus) and the two-spotted

spider mite (Tetranychus urticae), both of which can inflict damage and reduce yields in cotton fields and other crops.

Various miticides are presently available to combat infestations of

spider mites by killing such pests. One popular miticide is fenbutatin-oxide (CAS 13356-08-6), commercially available from DuPont Agricultural Products under the trademark "VENDEX" and from Shell International Chemical Co., Ltd. (London) under the trademarks "OSADAN" and "TORQUE". Another popular miticide is

abamectin, commercially available from Merck & Co. , Inc. , under the trademarks

"AVID", "ZEPHR" and "AGRI-MEK". A third type of popular miticide is amitraz (CAS 33089-61-1), commercially available from NOR-AM Chemical Co. ,

Wilmington, Delaware under the trademarks "MITAC" and "ORASYN" . While such miticides are generally effective against spider mites in the short term, spider mites reproduce rapidly, and resistant strains develop an increased tolerance of such miticides over a period of approximately one to three years. It is believed that spider mites must actually come in physical contact with most miticides in order to be killed thereby. However, as compared with other types of agricultural pests, spider mites move relatively little, primarily colonize the undersides of leaves and are therefore

less likely to come in physical contact with applied miticides unless relatively large amounts of such miticides are applied to ensure total coverage. Apart from the significant expense of applying large amounts of such miticides to the foliage of the plants or trees to be protected, it is believed that multiple applications of large doses

of such miticides may accelerate the development of tolerance to such miticides.

Certain chemical compounds are known to enhance the effectiveness of miticides. However, the effectiveness of such combinations are typically unexpected.

For example, Japanese Patent Application No. 61-291501 discloses an acaricide composition containing Fenpropathrin and Acephate as active ingredients. Fenpropathrin is a synthetic pyrethroid-type insecticide and acaricide known as

effective against spider mites. The effect of Acephate on mites is negligible and the

agent is ineffective as an acaricide. This application discloses that the combination

of Fenpropathrin and Acephate as active components shows superior effects in controlling mites such as carmine spider mites, Kanzawa spider mites, citrus red mites and Polyphagotarsonemus latus Banks. These active ingredients may be combined with liquid carriers such as water, alcohols (C alcohols, ethylene glycol and benzyl

alcohol), aromatic hydrocarbons, halogenated hydrocarbons, ethers, ketones, esters, nitriles, sulfoxides, alcohol ethers, aliphatic or alicyclic hydrocarbons, industrial gasoline and petroleum distillation fractions. It is also known that certain chemicals are naturally produced by the female of certain insect species and other pests in order to attract the male. Such naturally occurring sex attractants are known as pheromones. Various types of such sex pheromones have been chemically extracted from the female of such insects. In some instances, such sex pheromones can be synthetically produced. In still other instances, chemicals which are not naturally present in a species of insect or other pest are nonetheless found to attract, repel, or otherwise influence the behavior of the male

and/or female insect or other pest. Such naturally occurring or synthetically produced

chemicals are sometimes collectively referred to herein as behavior modifying

chemicals.

It is known that certain behavior modifying -chemicals, some naturally

produced by spider mites, affect the behavior of spider mites, either by attracting, repelling or arresting the male of the species. Studies reported by Regev and Cone

indicate that female two-spotted spider mites produce a sex pheromone which attracts

the male of the species to a quiescent female deutonymph and serves to retain the

attracted male until the emergence of the adult female, at which time mating normally occurs; extracts of the spider mite female deutonymphs showed the presence of the sesquiterpene alcohol farnesol. The study by Regev and Cone, entitled "Evidence of

Famesol As a Male Sex Attractant of the Two-Spotted Spider Mite, Tetranychus urticae Koch (Acarina: Tetranychidae)", Environmental Entomology, April 1985, Vol. 4, No. 2, pp. 307-311, indicated that certain isomers of synthetically produced farnesol, at particular concentrations, were effective in attracting male spider mites. A further study by Regev and Cone, "Analysis of Pharate Female Two- spotted Spider

Mites for Nerolidol and Geraniol: Evaluation for Sex Attraction of Males", Environmental Entomology, February 1976, Vol. 5, No. 1, pp. 133-138, also revealed that the two-spotted spider mite female deutonymph also contains the sesquiterpene alcohol nerolidol; their study indicated that synthetic nerolidol, at particular concentrations, served to attract male two-spotted spider mites.

The concept of combining volatile sex attractants with insecticides for

controlling populations of higher insects is known. For example, U.S. Patent No.

4, 122, 165, issued to Kinzer et al., and Australian Patent No. 477,526, issued to Thuron Industries, Inc., both describe the combination of the sex pheromone cis-9- tricosene (muscalure) with an insecticide to control house fly (Musca domestica) populations. Japanese Patent Publication No. 41(1966)-19198 discloses an insecticide compounded with fa esol or substances containing this liquid alcohol, for example,

cabreuva oil. The famesol is compounded with insecticides such as organo

phosphates, organo chlorides and natural insecticides at a concentration within a range

of 0.01 to 3% fa esol to offer an effective insecticide. Further, the famesol is disclosed as delaying pupation, reducing the pupation rate and raising the incidence of incompletely developed pupae in house flies. In example 5, there is disclosed an insecticidal composition containing diazinon (5% by weight), cabreuva oil (80% by

weight), Tween 80 (5.0% by weight) and xylol (10% by weight). Cabrueva oil contains about 2 to 3% famesol and about 75 to 80% nerolidol. Before use, the preparation in Example 5 thereof is diluted 100 to 300 times with water. U.S. Patent No. 4,775,534 discloses a miticidal composition adapted to be sprayed onto foliage to control spider mite populations. The miticidal composition is formed by impregnating a controlled release substrate with famesol and/or nerolidol to form a flowable liquid concentrate or wettable powder. The controlled release substrate serves as a carrier to slowly release the behavior modifying chemical after the miticidal composition has been applied to foliage. The nerolidol and fa esol increase the natural, instinctive movement behavior and search activities of the male spider mite. The resulting increased random movement

increases the likelihood of physical contact between such male spider mites and the

miticides incorporated within the miticidal composition and enhances the effect

thereof. The ultimate concentration of famesol within the final spraying mix should be at least 15 parts per million (ppm) but concentrations exceeding 200 ppm are disclosed as not appearing to improve the effectiveness of the miticidal composition.

Nerolidol concentrations in the final spray mix are at least 10 ppm nerolidol; however,

concentrations of nerolidol in the final spraying mix at or exceeding 100 ppm are

indicated as not appearing to derive any beneficial effect. The preferred range of nerolidol content, relative to the final spray mix is 5 ppm to 50 ppm. A preferred

formulation of the miticidal composition includes at least 100 g famesol and 50 mg

nerolidol.

In regard to the use of famesol and/or nerolidol, the prior art has focused primarily on the male spider mite. As noted above, U.S. Patent 4,775,534 is directed to eliciting a behavior modification in male spider mites. Further, the earlier work by Regev and Cone in their article in Environmental Entomology. Vol.

4, No. 2, was primarily directed to evidence of famesol as a male sex attractant for the two-spotted spider mite. It was observed therein that famesol tended to be lethal

to male spider mites at concentrations of 200 ppm and above. (At page 310). A subsequent article by Regev and Cone in Environmental Entomology. Vol. 5, No. 1 , noted that nerolidol, but not geraniol, was a sex attractant for male spider mites. The male spider mites were found not to be attracted to 100 ppm nerolidol. (At page

137).

Though the foregoing indicates that nerolidol and famesol are sex

attractants for the male spider mites, female spider mites are supplied with sufficient sperm during the first insemination to produce both sexes from its eggs until the time she dies. Regev and Cone explain that, if the mother is fertilized, both sexes are produced from those eggs; hence, mates are not far from each other to necessitate an airborne kind of sex attractant. Further, spider mites, which are not true insects,

exhibit parthenogenesis, i.e., female spider mites can reproduce males without first

mating with a male spider mite. In a later study by Regev and Cone, Environmental

Entomology. Vol. 5, No. 3, they discovered that the two-spotted spider mite females treated topically with 200 ppm famesol in 40% ethanol laid more eggs than females treated with only 40% ethanol. They speculated that the increase in number of eggs laid by the farnesol-treated females was due to the gonadotropic effects of famesol. The foregoing illustrates that though 200 ppm famesol is lethal to male spider mites, it is not toxic to female spider mites. In this regard, it is noted that famesol and nerolidol were identified as sex attractants for male spider mites by isolating these

chemicals from female spider mites.

Though U.S. Patent 4,775,534 discloses materials which are effective in attracting the male spider mites to miticides, this is only part of the problem in that there exists a need for enhancing the toxic effects of miticides on female spider mites in both susceptible and resistant populations which would more effectively address the problem of spider mite infestation.

In view of the distinct differences in the mating patterns, operation of

sex attractants, and general behavior as between spider mites and higher insect forms,

the ability of male spider mite behavior modifying chemicals to influence the toxic effectiveness of miticides relative to female spider mites is not readily apparent and could not have been predicted.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a miticidal composition

adapted to be sprayed onto foliage to control female and male spider populations, the miticidal composition comprises a miticide selected from the group consisting of organotins, preferably fenabutatin-oxide; organophosphates, preferably phosalone; diamides, preferably amitraz; sulfite esters, preferably propargite; macrocyclic lactones, preferably abamectin; and elemental sulfur; and an effective amount of a compound selected from the group consisting of sesquiterpene alcohols, analogs thereof and combinations thereof, preferably famesol, nerolidol and combinations thereof, for enhancing the toxic effectiveness of the selected miticide. The effective amount of the sesquiterene alcohol or analog thereof preferably ranges from about 300 ppm to about 1000 ppm in the final spray solution and depends on the companion miticide and whether the targeted spider mite population is considered susceptible or

resistant. Therefore, it is an object of the present invention to provide a miticidal composition which includes a miticide and which provides significantly improved

effectiveness to control both susceptible and resistant female and male spider mite populations as compared with usage of the miticide alone.

It is another object of the present invention to provide such a miticidal composition which allows lesser application rates of conventional miticides than those presently used without reducing the level of control of female and male spider mite

populations.

It is still a further object of the present invention to provide

compositions in the form of solutions, emulsions, dispersions, powders, dusts, granules, pellets and the like adapted to be combined with a conventional miticide in order to easily form such an improved miticidal composition.

Yet another object of the present invention is to provide a method of

controlling female and male spider mite populations in the foliage of agricultural fields, trees, greenhouses, gardens and homes wherever there are plants susceptible to spider mite damage, through the application of such a miticidal composition. These and other objects of the present invention will become more apparent to those skilled in the art as the description thereof proceeds.

DESCRIPTION OF THE INVENTION

The present invention relates to a miticidal composition adapted to be sprayed onto foliage to control female and male spider mite populations, the miticidal composition comprises a miticide selected from the group consisting of organotins,

preferably fenabutatin-oxide; organophosphates, preferably phosalone; diamides,

preferably amitraz; sulfite esters, preferably propargite; macrocyclic lactones,

preferably abamectin; and elemental sulfur; and an effective amount of a sesquiterpene

compound selected from the group consisting of sesquiterpene alcohols, analogs thereof and combinations thereof, preferably famesol, nerolidol and combinations thereof, for enhancing the toxic effectiveness of the selected miticide. The effective

amount of the sesquiterpene compound(s) preferably ranges from about 300 ppm to

about 1000 ppm in the final spray solution and depends on the companion miticide and

whether the targeted spider mite population is considered susceptible or resistant.

Though not wishing to be bound to any particular theory, it is believed

that the effects of the sesquiterpene compounds, e.g. fa esol and nerolidol, are physiological. Specifically, it is believed that when the female and/or male come in

contact with the miticidal composition, the respective spider mite picks up a toxic level of the miticide. Typically, the spider mites immune system then attempts to counteract and neutralize the effect of the toxicant. It is believed that the famesol and/or nerolidol attaches or binds to the spider mite's receptors and in essence renders it "blind" to the presence of the toxicant or unable to respond appropriately or sufficiently to deactivate the toxicant. Accordingly, the spider mite's immune system fails to neutralize the effect of the toxicant.

Famesol and nerolidol are sesquiterpene alcohols. Accordingly, the chemical may be other sesquiterpene alcohols and analogs thereof.

The combination of the sesquiterpene alcohol and the miticide provides a synergistic effect in surprisingly lowering the LC ₅₀ levels of both susceptible and

resistant mites. Similar unexpected, synergistic responses are observed when sesquiterpene alcohols are combined with specific pesticides. Some of these miticides and pesticides include fenbutatin-oxide, elemental sulfur, propargite, amitraz,

phosalone and abamectin. The effective amount of the sesquiterpene alcohol or analog thereof is greater than or equal to 300 parts per million concentration, except for elemental

sulfur wherein the effective amount of the sesquiterpene alcohol or analog thereof is

greater than or equal to 500 parts per million, in the miticidal composition to obtain a statistically significant enhancement of mortality. The sesquiterpene compound not only enhances the level of activity of the particular miticide against susceptible mites, but also against mites which exhibit significant levels of resistance to such miticides. This is a basis for the present belief that the sesquiterpene compound is attaching or binding to the receptors of the mites,

thereby blocking the spider mites cognizance of the presence of the toxicant. Thus, the effect demonstrated appears to be physiological rather than only behavioral.

The active components, i.e. the miticide or pesticide and the sesquiterpene compound, are combined with conventional carriers and adjuvants such as surface active agents, binders and stabilizers; then the mixture is formed into water- dispersible powders, sols (flowable forms), powders, DL (driftless) powders, or particles according to conventional methods.

The content of the active components within the miticidal composition of the present invention range from about 1 to about 95 weight percent for water-

dispersable powders, emulsions, liquids, sols, powders, DL powders, and granules.

The mixing ratio of the miticide and the sesquiterpene compound depends on the

companion miticide and whether the targeted spider mite is considered susceptible or resistant. Examples of such mixing ratios on a weight: weight basis are shown in Table 1.

TABLE 1

1. Weight ratios are of FCI-119 to Active Compound, wherein FCI-119 is a mixture of famesol and nerolidol (famesol (52.5): Nerolidol (42.5): surfactant (5.0) on a weight basis) available from Fermone Corporation.

In Table 1 , fenabutatin-oxide illustrates that effectiveness of the active compound/sesquiterpene combinations on susceptible female spider mites is indicative

of effectiveness of such combinations on resistant strains. Accordingly, it is expected that the toxic effectiveness of abamectin, amitraz, sulfur and propargite on resistant strains would be enhanced by their combination with a sesquiterpene alcohol, e.g. famesol nerolidol, analogs thereof and combinations thereof. In regard to phosalone, the addition of a sesquiterpene alcohol did not enhance the toxic effectiveness of the

phosalone, which was indicative that the susceptible strains tested were truly

susceptible to phosalone and no enhancement was possible by such an addition.

The miticidal composition of the present invention may be in the form of a flowable liquid concentrate, solutions, emulsions, dispersions, powders, dusts, granules, pellets and the like. Those skilled in the art are generally familiar with methods of formulating compositions in such forms for agricultural use. General

teachings as to methods of preparation of such compositions in the form of solutions, emulsions, dispersions, powders, dusts, granules, pellets and the like may be found in Chemicals For Crop Protection And Pest Control by Green, Hartley and West, Pergamon Press, Oxford 1977, the disclosure of which is hereby incorporated by reference. More detailed information concerning the formulation of dusts, wettable powders and granules may be found in the technical article entitled "Formulation of

Pesticidal Dusts, Wettable Powders and Granules", authored by J.A. Polon, appearing in Pesticide Formulations, edited by W. Van Valkenberg, published by Marcel Dekker, New York, N.Y. 1973, pp. 143-234, the disclosure of which is hereby incorporated by reference. Further information concerning the preparation of flowable pesticide formulations may be found in "Flowable Pesticide Formulations:

Development, Process and the Need for Standard Testing Procedures" , authored by

C.G. Halliday, appearing in Pesticide Formulations and Application Systems, edited

by K.G. Seymour, published by American Society of Testing Materials, STP 795, 1983, pp. 45-52, the disclosure of which is hereby incorporated by reference. Optionally, conventional pest control adjuvants, diluents, modifiers, or

conditioning agents may be added thereto, for example, to the aforementioned

flowable liquid concentrate. Examples of such adjuvants include antifoaming agents (such as dimethyl polysiloxane), pH buffering agents (such as dimethyl polysiloxane), pH buffering agents (such as alkylarylpolyethoxyethanal) and compatibility agents (such as alcohol sulfates). Such adjuvants are commercially available from Kalo AG

Chem, Inc. of Overland Park, Kans. An example of a desirable diluent is a spreader sticker agent, such as alkylarylpolyoxyethylene glucose available from Rigo Company of Buckner, Ky.

The carriers that can be used for the miticidal composition of the present invention may be any solids or liquids that are used for agricultural chemicals.

Examples of solid carriers include mineral powders (such as kaolin, bentonite, clay, montmorillonite, talc, diatomaceous earth, mica, quartz sand, ammonium sulfate, and urea), plant powders (such as soybean powder, flour, wood

shavings, tobacco powder, starch, and crystallized cellulose), alumina, silicates, highly dispersed silicates, and waxes.

Examples of liquid carriers include water, alcohols (such as methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, ethylene

glycol and benzyl alcohol); aromatic hydrocarbons (benzene, toluene, xylene, ethylbenzene, chlorobenzene, cumene, and methylnaphthalene); halogenated hydrocarbons (chloroform, carbon tetrachloride, dichloromethane, chloroethylene, trichlorofluoromethane, and dichlorodifluoromethane); ethers (ethylether, ethylene oxide, dioxane, and tetrahydrofuran); ketones, (acetone, methyl ethyl ketone, cyclohexane, and methylisobutylketone); esters (ethyl acetate, butyl acetate, ethylene glycol acetate, and alumina acetate); nitriles (acetonitrile, propionitrile, and acrylonitrile); sulfoxides (dimethylsulfoxide); alcohol ethers (ethylene glycol monomethyl ether and ethylene glycol monoethyl ether); aliphatic or alicyclic hydrocarbons (n-hexane and cyclo hexane); industrial gasoline (petroleum ether and solvent naphtha); and petroleum distillation fractions (paraffin, kerosene, and light oil).

Other conventional miticides and pesticides such as those sold under the trademarks "KELTHANE", "CARZOL" and "CAPTURE" may be incorporated within such a miticidal composition. Though the effectiveness of the aforementioned miticides and pesticides against spider mites presently do not appear to be enhanced when combined with nerolidol and/or famesol, their effectiveness may be enhanced by their use in combination with such pesticides and miticides that are so enhanced and accordingly weaken the resistance of such spider mites.

EXAMPLES

MATERIALS AND METHODS

A miticide susceptible strain and various resistant strains of the two spotted spider mite Tetranychus urticae Koch (TSSM) were used to test the efficacy

of the various miticidal and pesticidal compounds in combination with the sesquiterpene alcohols famesol and nerolidol analogs thereof and combinations

thereof. The susceptible and resistant strains were provided by Plant Sciences, Inc. , Watsonville, CA (PSI). The susceptible strain was originally established by PSI about 30 years ago. The resistant strains were isolated by PSI from commercial strawberry farms located near Watsonville, CA. All resistant strains were challenged regularly by PSI to maintain genetic resistance and were kept in growth chambers with environmental conditions conducive to maximum growth rate. The colonies were maintained on 14" X 19" flats of Henderson bush beans (baby Lima); with the flats being changed every 7-10 days to provide adequate nutritional requirements. The chambers were maintained at 23-25°C (73 +. 3°F) with a 24/0 hour light/dark cycle, i.e., under constant light.

The following bioassay procedures were used for determining LC ₅₀ , LC ₉₅ and/or mortality percentage values for the miticide alone, the sesquiterpene

alcohol(s) alone, or a combination thereof. About 24 hours prior to treatment, from about 12 to about 20 female adult or mix sex immature mites from the respective susceptible or resistant strain were transferred from infested leaves onto excised 15mm

diameter bean leaf discs which had been placed on deionized water moistened cotton balls in 1 oz. plastic cups ("bioassay cups"). The mites were held in a diurnal growth chamber with a 16/8 light/dark cycle (i.e. , 16 hours of light and 8 hours of darkness) at room conditions (i.e., at 23-25 °C) to allow them to acclimate (i.e., begin feeding, webspinning and oviposition) to the new habitat. Each bioassay consisted of four replicate leaf discs for each of the dose rates plus an untreated check. The bioassay

cups were uniquely labeled (in indelible black ink) with the compound name,

treatment number and replicate number assigned to each replicate.

On the day of the application (just prior to spraying), all dead or weak mites were removed and the exact number of healthy mites on each leaf disc was counted and recorded (using a binocular microscope). The serial dilutions of the combination of miticide (at the predetermined LC ₅₀ rate thereof) and sesquiterpene alcohol(s) (at various rates) were prepared from stock solutions to provide lOOmL of each separate spray solution. Each dose rate solution was placed into its own uniquely labeled, 125 mL Erlenmeyer flask containing a stir bar. The dose rate solutions were continuously mixed prior to and during their application by placement onto a magnetic stir plate. The leaf disc and mites of each replicate were treated with the test compound dose rate solution (at 20 psi) using an airbrush spraying device attached to a ring stand positioned approximately 15cm above the target surface. The distance between the leaf disc and sprayer tip was adjusted so that the spray pattern would provide complete coverage of the leaf disc target only. Each replicate (leaf disc) was

sprayed for about 1/2 second, employing the use of a solenoid activated rheostat

timing device.

After being treated, each leaf disc was immediately transferred to a plexiglass cage (modified Munger cell) containing a whole bean leaf (on a moistened gauze pad) which was previously sprayed with the same treatment and dose rate. The

middle piece of the cage (containing a 1" diameter cell in which the treated leaf

disc/mites are placed) was placed over the leaf and the gauze pad after the treatment

spray, but before the transfer of the treated leaf disc. The treated leaf disc was then transferred into the cell and a third piece of plexiglass (with a nylon mesh covered 1 " diameter hole) was then placed on top of the cell, and the entire "sandwich" held together by rubber bands. Each cage was uniquely labeled with the appropriate

compound name and treatment and replicate numbers. The cages were held at 23 - 25 °C (73 JL 3°F) in a 16/8 hour light/dark cycle for up to six days, during which time they were evaluated for mite mortality. The TSSM were evaluated for mortality at 2-day (48-hour), 4-day (96- hour) and/or 6-day (144 hour) post application intervals (PAI). At each evaluation, the number of living, dead and escaped mites were counted and recorded. From these data, a transformed mortality percentage (corrected for the number of escaped mites) was calculated. For the LC ₅₀ and LC ₉₅ tests, the lethal concentrations of the test compounds were then statistically determined from linear regressions calculated from a probit analysis computer program. A two way ANOVA statistical analysis was then

run on these data to determine if there were any statistical differences between

treatments. Data are expressed (generally in tabular form) as average percent mortality per treatment or as ppm active ingredients required to kill 50% (LC ₅₀ ) and 95 % (LC ₅₀ ) of the test population for the respective treatment and corresponding mite type, i.e., susceptible or resistant and adult or immature.

The compounds tested were:

CAPTURE 2EC (emulsifiable concentrate) (Capture)

- common name: bifenthrin (CAS 82657-04-3)

- chemistry: lα, 3α (Z)]-(±)-(2-methyl [1 , 1 '-biρhenyl]-3-yl)methyl 3-

(2-chloro-3 ,3 ,3-trifluoro- 1 -propenyl-2 ,2-dimethylcyclopropane- carboxylate

- synthetic pyrethroid class insecticide/miticide

- produced and sold by FMC Corporation.

ZOLONE EC (emulsifiable concentrate) (Zolone)

- Common name: phosalone (CAS 2310-17-0) - chemistry: S-[(6-chloro-2-oxo-3(2H)-benzoxazoyl) methyl] O,O- diethylphosphorodithioate

- organophosphate class miticide/acaricide, insecticide

- produced/sold by: Rhone Poulenc.

DICOFOL 4EC (emulsifiable concentrate) (Dicofol)

- common name: dicofol (CAS 115-32-2)

- chemistry: 1,1-Bis (chlorophenyl)-2,2,2-trichloroethanol

- chlorinated hydrocarbon class miticide - produced and sold by: Rohm & Haas (as KELTHANE) &

Mahkteshim- Agan .

CARZOL 2EC (emulsifiable concentrate) (Carzol)

- common name: formetanate hydrochloride (CAS 23422-53-9)

- chemistry: N,N-dimethyl-N'[3-[[(methylamino) carbonyl] oxy] phenyl] methanimidamide monohydrochloride

- carbamate class insecticide/miticide

- produced and sold by NOR- AM Chemical Company.

OMITE 30W (wettable powder) (Omite)

- common name: propargite (CAS 2312-35-8) - chemistry: 2-[4-(l , l-dimethylethyl)phenoxy]cyclohexyl-2-propynyl sulfite

- sulfite ester class miticide/acaricide

- produced/sold by: Uniroyal Chemical Co.

AMITRAZ 1.5EC (emulsifiable concentrate) (Amitraz)

- common name: amitraz (CAS 33089-61-1)

- chemistry: N'-(2,4-dimethylphenyl)-N-[[2,4-dimethylphenyl]methyl]- N-methyl methanimidamide - diamide class insecticide/miticide.

- sold by NOR- AM Chemical Company.

THIOLUX 80% a.i. (micronized flowable sulfur) (Thiolux)

- (CAS 7704-34-9)

- miticide/acaricide, fungicide - sold by: Sandoz Ltd.; Sandoz Crop Protection Corp. (U.S.).

AVID 0.15EC (a/k a Abamectin 0.15EC) (emulsifiable concentrate) (Avid)

- common name: abamectin (a macrocyclic lactone)

- chemistry: avermectin B,; a mixture containing a minimum of 80% avermectin B.,a (5-0-demethylavermectin A,a) and a maximum of 20% avermectin B,b (5-0-demethyl-25-de-l-methylpropyl-25-

(1 -methylethyl) avermectin Aja)

- natural product produced by Streptomyces avermitilis: insecticide/miticide

- produced/sold by: Merck & Co., Inc.

VENDEX 50WP - (wettable powder formulation) (Vendex)

- common name: fenbutatin-oxide (CAS 13356-08-6)

- chemistry: hexakis (2-methyl-2-phenylpropyl)-distannoxane

- organotin class miticide/acaricide - produced/sold by: DuPont Agricultural Products

The sesquiterpene alcohols and analogs tested were: FCI-119a(K): famesol (sesquiterpene alcohol) -(CAS 4602-84-0)

- chemistry: 3,7,ll-trimethyl-2,6, 10-dodecatriene-l-ol - available from Fermone Corporation, Phoenix, AZ.

FCI-119b (G): nerolidol (sesquiterpene alcohol) -(CAS 7212-44-4) -chemistry : 3,7,11 -trimethyl- 1,6,10-dodecatriene-3-ol

- available from Fermone Corporation, Phoenix, AZ.

FCI-119: famesol: nerolidol: an anio ic/nonionic surfactant (Armul 33)

(52.5:42:5:5.0 on a weight basis)

- available from Fermone Corporation, Phoenix, AZ.

FME: farnesyl methyl ether (a famesol analog).

EXAMPLE 1: The Effect of FCI-119 on the Activity of Several Miticides on Two-spotted Spider Mite Tetranychus urticae Koch

In this example, the effect of FCI-119 on the activity of several miticides was determined. The miticides evaluated were Capture, Zolone, Kel thane™ (Dicofol), and Carzol on resistant strains of two-spotted spider mite Tetranychus urticae Koch (TSSM). In addition, if the efficacy of these compounds was found to be enhanced by FCI-119, they were then tested in combination with FCI-119 on strains of TSSM susceptible to the miticides. Omite 30W, Amitraz M 1.5EC, Thiolux 80W and Avid 0.15EC were also tested in combination with FCI-119 on a miticide susceptible strain on TSSM (resistant strains were not available). All these tests were run with FCI-119 at 1000 ppm. Thiolux was also tested with FCI-119 at 500 ppm and with nerolidol at 1000 ppm on a susceptible strain of TSSM.

The bioassay s conducted on the pyrethroid, carbamate, and chlorinated hydrocarbon resistant strains with Capture, Carzol, and Kelthane (Dicofol), respectively, showed no statistically significant enhancement activity (i.e., regarding

LC ₅₀ ) with FCI-119 at 1000 ppm. Therefore, no bioassays were conducted with those compounds on the susceptible strain of TSSM. The 4-day or 6-day data from these trials shown in tabular form in Table 4-6.

TABLE 2: The Effect of FCI-1 19 on the LC ₅₀ and LC _9S for

Adult Female Organo Phosphate Resistant Two-Spider Mites Treated with Zolone at 4-days and 6-days After Application

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I a. pa re tests, b. paired tests za

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TABLE 3: The Effect of FCI-1 19 on the LC ₅₀ and LC ₉₅ for Adult Female Susceptible Two-spotted Spider Mites Treated with Zolone at 4-days and 6-days After Application

TABLE 4: The Effect of FCI-1 19 on the LC ₅₀ and LC _9i for

Adult Female Carbamate Resistant Two-spotted Spider Mites Treated with Carzol at 4-days After Application i ...

S ^{' <} ! Λ rv. ^' 5

TABLE 5: The Effect of FCI-1 19 on the LC ₅₀ and LC ₉₅ for

Adult Female Pyrethroid Resistant Two-spotted Spider Mites Treated with Capture at 6-days After Application

TABLE 7: The Effect of FCI- 1 19 on the LC _W and LC ₉₅ for Adult Female Susceptible Two-spotted Spider Mites Treated with Omite at 4-days and 6-days After Application

CO cσ co

CO

TABLE 8: The Effect of FCI-1 19 on the LC ₅₀ and LC ₉₅ for

33 Adult Female Susceptible Two-spotted Spider Mites c: Treated with Amitraz at 4-days and 6-days After Application

TABLE 9: The Effect of FCI-1 19 on the LC ₅₀ and LC ₉₅ for

Adult Female Susceptible Two-spotted Spider Mites

Treated with Abamectin at 4-days and 6-days After Application

CO

<-_

CD

TABLE 10: The Effect of FCI-119 and Nerolidol on the LC ₅₀ and LC ₉₅ for O Adult Female Susceptible Two-spotted Spider Mites Treated with Thiolux at 4-days and 6-days After Application

(\3

As shown in Tables 4-6, FCI-119 at 1000 ppm did not appear to enhance the effect of carbamate (e.g. , Carzol), chlorinated hydrocarbon (e.g., Dicofol), or pyrethroid (e.g. , Capture) miticides on TSSM resistant to those compounds. FCI-119 at 1000 ppm enhanced the efficacy of Zolone 3EC on TSSM

resistant to organophosphates. ("3EC" stands for a 3 pounds Zolone per gallon

emulsifiable concentrate.) (See Table 2) However, FCI-119 did not appear to enhance the effect of Zolone 3EC on susceptible TSSM. (See Table 3) This is indicative of such spider mites being truly susceptible to Zolone. FCI-119 at 1000 ppm had some enhancing effect on Omite 30W in the first 4-days but appeared to have no further toxicity enhancing effect at 6-days PAI on susceptible TSSM. However, quicker killing power is a desired benefit; i.e. 4-day vs. 6-day. (See Table 7) FCI-119 at 1000 ppm had a moderate enhancing effect with Amitraz 1.5 EC treated susceptible TSSM. (See Table 8) FCI-119 at 500 ppm appeared to enhance the effect of Avid 0.15 EC on susceptible TSSM. (See Table 9)

FCI-119 at both 500 and 1000 ppm appeared to enhance the effect of Thiolux on susceptible TSSM. Nerolidol at 1000 ppm also enhanced the effect of Thiolux on susceptible TSSM. (See Table 10)

EX AMPLE 2: The Effect of FCI-119 on the Activity of Abamectin (Avid) on Adult and Immature Susceptible Two-spotted Spider Mite Tetranychus urticae Koch

In this example, the effect of FCI-119 on the activity of Abamectin 0.15EC (a/k/a Avid 0.15EC) was determined when this miticide was applied in combination with eight serial dilution rates of FCI-119, on both adults and immatures

from a miticide susceptible strain of two-spotted spider mites, Tetranychus urticae

Koch. Eight serial dilutions of FCI-119 were combined with the known LC ₅₀ of Abamectin 0.15EC. These dilutions were then applied to female adult and mixed sex

immature two-spotted spider mites (TSSM) to determine if FCI-119 increased the mortality of the exposed mites.

On the day prior to treatment, 12-15 female adult or mix sex immature TSSM from the susceptible strain were transferred from infested leaves onto excised 15mm diameter bean leaf discs which had been placed on deionized water moistened cotton balls in the "bioassay cups".

The LC ₅₀ of Abamectin 0.15EC was determined from independent tests.

The calculated LC ₅₀ value for susceptible adult TSSM was 0.016 ppm and for

immature TSSM 0.029 ppm. The treatments included Abamectin 0.15EC combined

(using the LC ₅₀ value) with FCI-119 at 300, 100, 30, 10, 3, 1, 0.3 and 0.1 parts per

million(ppm), Abamectin 0.15EC alone, and an untreated check.

Two-day (48 hour) and four-day (96 hour) post application interval (PAI) mortality evaluations were made on all tests conducted. The bioassay data are

presented in tabular form as average percent mortality per treatment for each concentration of FCI-119 for adults and immature (Tables 11 & 12).

The result of the bioassay on adult susceptible TSSM indicated that

FCI-119 at 0.1 to 300 ppm had no statistically significant enhancing effect on mortality when applied at any rate tested with Abamectin 0.15EC at the LC ₅₀ concentration. However, Abamectin 0.15EC plus the highest rate of FCI-119 (300

ppm) did provide greater numerical mortality.

The bioassay on immature TSSM indicated that FCI-119 had some enhancing effect on mortality at high concentrations. This effect was not profound in that the 300 ppm, 100 ppm, 10 ppm, 3 ppm, and .3 ppm rates were statistically equal in percent mortality.

TABLE 11 BIOASSAY DOSE/PERCENT MORTALITY DATE FROM SUSCEPTIBLE ADULT TWO-SPOTTED SPIDER MITES SPRAYED WITH ABAMECTIN 0. 15EC AT THE LC ₅₀ LEVEL IN COMBINATION WITH VARIOUS RATES OF FCI-119 AT 4-DAYS AFTER APPLICATION ¹

Means followed by the same letter are not significantly different according to DNMRT (P = 0.05).

TABLE 12 BIOASSAY DOSE/PERCENT MORTALITY DATA FROM SUSCEPTIBLE IMMATURE TWO-SPOTTED SPIDER MITES SPRAYED WITH ABAMECTIN 0.15EC AT THE LC ₅₀ LEVEL IN COMBINATION WITH VARIOUS RATES OF FCI-119 AT 4-DAYS AFTER APPLICATION ²

Means followed by the same letter are not significantly different according to DNMRT (P = 0.05)

EXAMPLE 3: The Effect of FCI-119 at Increased Rates on the Activity of

Abamectin (Avid) on Adult and Immature Susceptible Two- spotted Spider Mite Tetranychus urticae Koch

In this example, the effect of FCI-119 on the activity of Abamectin

0.15EC (a/k a Avid 0.15EC) was determined when this miticide was applied in combination with six serial dilution rates of FCI-119, on both adults and immatures

from a miticide susceptible strain of two-spotted spider mites, Tetranychus urticae

Koch. This example is a continuation of Example 2 in which were tested the FCI- 119/Abamectin combination at lower concentrations of FCI-119. It was determined from in Example 2 that there may be a potential for increased activity at higher concentrations of FCI-119. Six serial dilutions of FCI-119 were combined with the known LC ₅₀ of Abamectin 0.15EC. These dilutions were then applied to adult female and mixed sex immature two-spotted spider mites (TSSM) to determine if FCI-119 increases the mortality of the exposed mites.

LC ₅₀ values for the abamectin 0.15 EC and for the combined abamectin/FCI-119 test were determined. For each replicate in the bioassay procedure on the day prior to treatment, 12-15 female adult or mixed sex immature TSSM from the susceptible strain were transferred from infested leaves onto excised

15 mm diameter bean leaf discs which had been placed on deionized water moistened cotton balls in "bioassay cups".

As in Example 2, the LC^ of Abamectin 0.15 EC was determined from independent bioassay tests. For this test it was decided to use the calculated LC ₅₀

value that was close to the upper fiducial limit (i.e., confidence limit) for both the

adult and immature susceptible TSSM. The LC ₅₀ value used for immature susceptible TSSM was .038 ppm Abamectin and 0.17 ppm was used for the adults susceptible TSSM. The treatments included Abamectin 0.15 EC combined (using the upper

fiducial LC _J0 value) with FCI-119 at 3000, 1000, 300, 100, 30, and 10 ppm,

Abamectin 0.15EC at the LC ₅₀ alone, and an untreated check.

Four days (96 hours) and six day (144 hour) post application interval

(PAI) mortality evaluations were made on all tests conducted. The bioassay data are presented in tabular form as average percent mortality per treatment for each concentration of FCI-119 for adults and immatures (Tables 13 and 14).

The results of the bioassay on adult TSSM indicated that FCI-119 has some

enhancing effect on mortality at the 1000, 300 and 100 ppm rate which were all statistically equivalent to each other and significantly greater in mortality than all the

other treatments. The highest rate abamectin and FCI-119 treatment at 3000 ppm was statistically equivalent to all the low rate treatments (30 ppm and 10 ppm) and the abamectin alone treatment. The lack of significant activity in the 3000 ppm rate on the adult susceptible mites and the numerically lower value in this rate (3000 ppm) in the immature susceptible TSSM indicates that there may be a suppression of activity at this high rate. The untreated check was statistically significantly lower in mortality than all other treatments (Table 13).

The results of the bioassay on the immature susceptible TSSM were very similar to the adult susceptible TSSM bioassay. The 3000, 1000, 300 and 100

ppm rates were all statistically greater in mortality than the low rates, abamectin

alone, and the untreated check. The 3000 ppm rate was numerically lower in mortality than the 1000 ppm and the 300 ppm rate, and was statistically equivalent to the 30 ppm and 10 ppm rate. The untreated check was statistically lower in mortality than all other treatments (Table 14).

The activity of FCI-119 in combination with abamectin does provide a statistically significant enhancing effect. The highest mortality in the adult susceptible TSSM test was 55.5% at the 1000 ppm rate and in the immature susceptible TSSM bioassay the highest mortality was 74.5% at the 300 ppm rate.

TABLE 13. Bioassay dose/percent mortality data from susceptible adult two- spotted spider mites sprayed with abamectin 0.15EC at the LC ₅₀ level in combination with various rates of FCI-119 at 6-Days After Application ³

³ Means followed by the same letter are not significantly different according to DNMRT

(P = 0.005).

TABLE 14. Bioassay dose/mortality data from susceptible immature two-spotted spider mites sprayed with abamectin 0.015EC at the LCso level in combination with various rates of FCI-119 at 6-Day s After Application ⁴

"Means followed by the same letter are not significantly different according to DNMRT

(P = 0.05)

EXAMPLE 4: The Effect of FCI- 119 on the Activity of Abamectin (Avid) on Adult Susceptible Two-spotted Spider Mite Tetranychus urticae Koch

In this example, the effect of FCI- 119 on previously determined LC ₅₀ and LC _9J values for Abamectin 0.15EC on susceptible strains of two-spotted spider mite T\ urticae Koch (TSSM), was determined.

For each replicate in the bioassay procedure, 15-20 female adult TSSM from

the susceptible strain were transferred the day prior to treatment from infested leaves onto excised 15 mm diameter bean leaf discs which had been placed on deionized water moistened cotton balls in "bioassay cups".

The serial dilutions for the FCI-119/abamectin spray were prepared using the same amount of FCI-119 stock solution with each rate of the Abamectin 0.15EC. The FCI- 119/ Abamectin tests were evaluated for mortality on a 4-day (96 hour) post application interval (PAI). The dilutions for the FCI- 119/ Abamectin were as follows; (all dilutions were created with FCI- 119 at 1000 ppm), 0.000005, 0.0000042, 0.0000033, 0.0000025, 0.0000018, 0.000001, 0.0000003 percent active ingredient. The rates used for Abamectin alone were .00001 and .000005, .0000042, .0000033, .0000025, .0000018, .000001, .0000005, .0000003, active ingredient. The results of the Abamectin/FCI-119 test indicate that FCI-119 at the 1000 ppm had little if any enhancing effect on female adult susceptible TSSM treated with

Abamectin. The LC ₅₀ and LC ₉₅ of abamectin alone was .016 ppm and .065 ppm,

respectively. The LC ₅₀ and LC ₉₅ for abamectin with FCI-119 at 1000 ppm was 0.28 ppm and .119 ppm, respectively.

In reviewing the data regarding abamectin in Examples 1-4, the concentration of the sesquiterpene compound, i.e. fa esol and nerolidol in FCI-119, ranges from about 300 ppm to about 1000 ppm. In Example 2 (Table 11), the highest numerical mortality of susceptible spider mites was achieved with FCI- 119 at 300 ppm. In Example 3, the mortality rate was lower for FCI- 119 at 3000 ppm compared to it a

1000 ppm and 300 ppm. In Example 4, the LC ₅₀ and LC ₉₅ of the abamectin alone versus in combination with FCI- 119 at 1000 ppm indicates that the 1000 ppm concentration for the sesquiterpene compound is its upper limit in view of the results of Example 3 and the expected variability of results when such low concentrations o the abamectin are involved. Further, this range is validated by the results in Exampl 1 (Table 9) which show the improvement in both the LC ₅₀ and LC ₉₅ of abamectin with FCI-119 at 500 ppm over that of abamectin alone. The six-day data in Example 3 (Table 14) also demonstrate this range to be applicable to susceptible immature spider mites.

EX AMPLE 5: The Effect of FCI- 119 in Combination with Vendex 50WP on Adult and Immature Susceptible and Organic Tin Resistant Two-spotted Spider Mite Tetranychus urticae Koch

In this example, the effect of FCI-119 on the activity of Vendex 50WP was determined when this miticide was applied in combination with eight serial

dilution rates of FCI-119, on both female adults and mixed sex immatures from a

miticide susceptible strain of two-spotted spider mites, Tetranychus urticae Koch and an organic tin resistant strain of TSSM. The organic tin resistant strain of TSSM utilized was provided by PSI which isolated it from a commercial strawberry farm located near Watsonville, CA. in 1988. Eight serial dilutions of FCI-119 were combined with the previously determined Vendex 50WP LC ₅₀ for both adults and immatures. These dilutions were then applied to adult female and mixed sex immature TSSM of the miticide susceptible and organic tin resistant strain to determine if FCI-119 increases the mortality of the Vendex 50WP exposed mites.

On the day prior to treatment, 12-15 female adult or mixed sex immature TSSM from the susceptible or organic tin resistant strains were transferred from infested leaves onto excised 15 mm diameter bean leaf discs which had been placed on deionized water moistened cotton balls in "bioassay cups". The serial dilutions for the combination Vendex 50WP and FCI-119 spay were prepared using the same amount of Vendex 50WP solution with each rate

of the FCI- 119. The LC ₅₀ of adult and immature susceptible and organic tin resistant

TSSM to Vendex 50WP was determined from an independent bioassay. The calculated LC ₅₀ value for susceptible adult and immature TSSM was 251 and 23.5 ppm respectively; while the organic tin resistant adult and immature TSSM indicated

LC ₅₀ values of 1162 and 324 ppm respectively. For the adult TSSM from the susceptible and organic tin resistant colonies, Vendex 50WP was combined (using the

LC ₅₀ value), with FCI-119 at 3000, 1000, 300, 100, 30, 10, 3 and 1 ppm, plus a

Vendex 50WP alone treatment, and an untreated check. For the immature TSSM

from the susceptible and organic tin resistant colonies, Vendex 50WP was combined

(using the LC ₅₀ value) with FCI-119 at 3000, 1000, 300, 100, 30 and 10 ppm. In addition, 3000 and 1000 ppm FCI-119 alone treatments were included to determine the effect of FCI- 119 by itself. Also included in the immature test was a Vendex 50WP treatment at the LC ₅₀ and an untreated check.

Two-day (48 hour), four-day (96-hour) and six-day (144 hour) post

application interval (PAI) mortality evaluations were conducted on the adult susceptible and organic tin resistant TSSM tests. Four-day (96 hour) and six -day (144 hour) PAI mortality evaluations were conducted on the immature susceptible and organic tin resistant TSSM tests. It was decided that a two-day (48 hour) PAI mortality evaluation on immature TSSM would be inaccurate due to the difficulty in determining the difference between temporary quiescence of molting mites and

mortality. The bioassay data are presented in tabular form as average percent mortality per treatment for adults and immatures.

The results of the bioassay on the female adult TSSM indicated that

FCI- 119 had a profound, statistically significant effect on mortality in both the susceptible and organic tin resistant strains at the three highest rate, i.e., at the 3000, 1000, and 300 ppm rates. The three highest rates of FCI-119 gave mortality statistically greater than the lower rate treatments in combination with Vendex 50WP at the LC ₅₀ value (a summary of these data are contained in Tables 15 and 16).

The results of the bioassays on immature organic tin resistant TSSM indicated that the three highest rates of FCI- 119 combined with Vendex 50WP at the LC ₅₀ value also provided statistically greater percent mortality than the lower rates, but the high rates are not statistically equivalent to each other (Table 17).

The two highest rates (3000 and 1000 ppm) FCI-119 combined with Vendex 50WP) in the miticide susceptible immature bioassays provided statistically equivalent and higher percent mortality than all other treatments. The effect of FCI- 119 alone at the 3000 and 1000 ppm rates was not statistically different than the untreated check (Table 17 and 18). Therefore, the FCI-119 alone had no effect.

Table 15: Bioassay dose/percent mortality data from adult miticide susceptible two- spotted spider mites sprayed with FCI- 119 at various rates combined with Vendex 50VVP at the LC ₅₀ value ⁵

³ Means followed by the same letter are not statistically different from each other when subjected to ANOVA and DΝMRT (P = 0.05)

TABLE 16: Bioassay dose/percent mortality data from adult organic tin resistant two-spotted spider mites sprayed with FCI- 119 at various rates and Vendex 50WP at the LC _5n value ⁶

' Means followed by the same letter are not statistically different from each other when subjected to ANOVA and DNMRT (P = 0.05)

TABLE 17: Bioassay dose/percent mortality data from immature organic tin resistant two-spotted spider mites sprayed with FCI- 119 at various rates combined with Vendex 50WP at the LCj. value ⁷

Means followed by the same letter are not statistically different from each other when subjected to ANOVA and DNMRT (P = 0.05).

TABLE 18: Bioassay dose/percent mortality data from immature susceptible two-spotted spider mites sprayed with FCI- 119 at various rates combined with Vendex 50WP at LCjo value ⁸

⁶ Means followed by the same letter are not statistically different from each other when subjected to ANOVA and DNMRT (T = 0.05).

EXAMPLE 6: A Ratio Test to Determine the Most Effective Ratio of

Nerolidol and Fa esol for the Enhancement of Vendex 50WP on Two-spotted Spider Mite

Tetranychus urticae Koch

In this example, a ratio test was conducted with Vendex 50WP utilizing different ratios of famesol and nerolidol (the sesquiterpene alcohol components of FCI- 119). This test was conducted on both the susceptible and organic tin resistant

strains of TSSM. The ratio test was conducted utilizing various treatments and

combinations of famesol and nerolidol in combination with the LC ₅₀ of Vendex 50WP

(200 ppm) on susceptible TSSM. The organic tin resistant mites were sprayed with

Vendex 50WP at 1000 ppm in combination with farnesol/nerolidol. The ratios used were as follows with fa esol listed first and nerolidol second in parts per million

1000/0, 850/150, 650/350, 550/450, 350/650, 150/850, 0/1000, one treatment of Vendex alone and one untreated check were also included. The bioassay data are presented in tabular form as average percent mortality per treatment.

The results of the ratio test indicated that all the farnesol/nerolidol ratios tested enhanced the activity of Vendex 50WP (Table 19). All ratios of farnesol/nerolidol performed significantly better than Vendex 50WP alone. The ratio

that appeared to perform best was 650/350 farnesol/nerolidol. However, the 150/850 mixture also was very effective. It seemed both famesol and nerolidol had nearly

identical activity.

TABLE 19: THE EFFECT OF VARIOUS RATIOS OF FARNESOL AND NEROLIDOL ON THE MORTALITY OF ADULT SUSCEPTIBLE AND RESISTANT STRAINS OF TWO-SPOTTED SPIDER MITES TREATED WITH VENDEX 50WP ⁹

VENDEX AT 200 PPM SUSCEPTIBLE TSSM

VENDEX AT 1000 PPM ORGANOTIN RESISTANT TSSM

Means followed by the same letter are not statistically different according to DNMRT/P = 0.05

EXAMPLE 7: The Effect of Fa esol and Nerolidol in Combination with Vendex 50WP on the LC ₅₀ and LC ₉₅ of Vendex on Organic Tin Resistant and Susceptible Strains of the, Two-spotted Spider Mite Tetranychus urticae Koch

In this example, the effect of famesol and/or nerolidol on previously

determined LC ₅₀ and LC ₉₅ values for Vendex 50WP on organic tin resistant and

susceptible strains of two-spotted spider mite Tetranychus urticae Koch (TSSM) was

determined. The organic tin resistant strain of TSSM was provided by PSI which

isolated it from a commercial strawberry farm located near Watsonville, CA. in 1988.

This example is a continuation of Example 5 which demonstrated that using FCI- 119 (a combination of famesol and nerolidol) significantly reduced the LC ₅₀ and LC _9J values for Vendex 50WP on both strains of TSSM. In the present example, the components of FCI-119 were tested separately to determine each component's contribution to the reduction in LC values. Eight serial dilutions of Vendex 50WP were combined with famesol and nerolidol. The dilutions were applied to female adult TSSM from organic tin resistant and susceptible strains of TSSM. The test was conducted two times on each strain of TSSM with famesol applied at 550 and 1000 ppm and nerolidol applied at 450 and 1000 ppm, to determine the degree of Vendex

50WP enhancement at these different concentrations. In addition, one bioassay was performed using various ratios of fa esol and nerolidol on susceptible TSSM to determine the effect thereof on Vendex 50WP.

On the day prior to treatment, 15-20 female adult TSSM from the

susceptible or organic tin resistant strains were transferred from infested leaves onto

excised 15mm diameter bean leaf discs which had been placed on deionized water moistened cotton balls in "bioassay cups" . The serial dilutions for the combination Vendex 50WP and famesol or

nerolidol spray were prepared using the same amount of famesol or nerolidol solution

with each rate of the Vendex 50WP. The susceptible mites were sprayed with the following concentrations of Vendex 50WP in combination with famesol or nerolidol, .05, .025, .01 , .0005, .0025, .001, .005 and .00025 percent active ingredient. The organotin resistant TSSM were sprayed with the following percent active ingredients

for the 550 ppm famesol bioassay, for the 450 ppm nerolidol bioassay, and for the 1000 ppm bioassays, .2324, .1162, .0465, .0232, .0116, .0046, .0023 and .0012.

The susceptible TSSM were evaluated for mortality at 4-day (96 hour) post

application intervals (PSI). The organotin resistant TSSM were evaluated at 4 and 6 day PAI due to low mortality at the 4 day PAI. The bioassay data are presented in tabular form as ppm active ingredients required to kill 50% (LC ₅₀ ) and 95 % (LC ₉₅ ) of the test population (Tables 20 and 21).

The ratio test was conducted utilizing various treatments and combinations of

famesol and nerolidol in combination with the LC ₅₀ of Vendex 50WP on susceptible TSSM (194 ppm). The ratios used were as follows with famesol listed first and the

nerolidol second in parts per million (ppm/ppm) 150/150, 100/200, 75/225, 30/270, 200/100, 225/75, 270/30, 120/180, 180/120, 300/0, 0/300 plus an untreated check

and a FCI- 119 treatment at 300 ppm. The test was evaluated at 4-day PAI. The bioassay data are presented in tabular form as average percent mortality per treatment (Table 22).

Both famesol and nerolidol appeared to enhance the effect of Vendex 50WP on susceptible TSSM. On organotin-resistant TSSM, famesol at 550 ppm appeared

to have enhanced the effect of Vendex 50WP and more markedly at 1000 ppm. Nerolidol at 450 ppm seemed to have inhibited the effect of Vendex 50WP and

enhanced the effect at 1000 ppm. A test of ratios of these two materials indicated that ratios higher in famesol (i.e. greater concentrations of famesol) produced more activity with Vendex 50WP on TSSM (higher mortality). The treatment with the greatest mortality was treatment 8 with a 270/30 ppm farnesol/nerolidol ratio (Table 22).

TABLE 20 THE EFFECT OF TWO DIFFERENT RATES OF FARNESOL AND NEROLIDOL ON THE LC50 AND LC95 FOR ADULT FEMALE MITICIDE SUSCEPTIBLE TWO-SPOTTED SPIDER MITES TREATED WITH VENDEX 50WP AT FOUR DAYS AFTER APPLICATION

TABLE 21 THE EFFECT OF TWO DIFFERENT RATES OF FARNESOL AND NEROLIDOL ON THE LC ₅₀ AND LC ₉₅ FOR ADULT ORGANOTIN RESISTANT FEMALE TWO-SPOTTED SPIDER MITES TREATED WITH VENDEX 50WP

TABLE 22 THE EFFECT OF VARIOUS RATIOS OF FARNESOL AND NEROLIDOL ON THE MORTALITY OF ADULT FEMALE TWO- SPOTTED SPIDER MITES TREATED WITH VENDEX 50WP ¹⁰

* en ex app ie at 1 4 ppm

Means followed by the same letter are not statistically different according to DNMRT .05)

EXAMPLE 8: The Effect of FCI- 119 in Combination with

Vendex 50WP on the LC _J0 and LC ₉₅ of Vendex on Organic Tin Resistant and Susceptible Strains of the Two-spotted Spider Mite Tetranychus urticae Koch

The objective of the present Example was to determine to what extent FCI- 119 applied in combination with Vendex 50WP would decrease the previously determined

LC ₅₀ and LC ₉₅ values for Vendex 50WP on organic tin resistant and susceptible strains of two-spotted spider mite T\ urticae Koch (TSSM). The organic tin resistant strain of TSSM was provided by PSI which isolated it from a commercial strawberry farm located near Watsonville, CA. in 1988. It was determined in Example 5 that FCI- 119 significantly increased the efficacy of Vendex 50WP on TSSM. Eight serial dilutions of Vendex 50WP were combined with FCI-119. The dilutions were applied to female adult TSSM from organic tin resistant and susceptible strains of TSSM. The test was conducted three times on each TSSM strain with FCI- 119 applied at 1000, 300, and

100 ppm to determine the degree of Vendex 50WP enhancement at these different concentrations.

On the day prior to treatment, 12-15 female adult TSSM from the susceptible or organic tin resistant strains were transferred from infested leaves onto excised 15 mm diameter bean leaf discs which had been placed on deionized water moistened cotton balls in "bioassay cups".

The serial dilutions for the combination Vendex 50WP and FCI- 119 spray were prepared using the same amount of FCI-119 solution with each rate of the

Vendex 50WP. The susceptible mites were sprayed with the following concentrations of Vendex 50WP in combination with FCI-119 at 1000 ppm: 0.05, 0.025, 0.01,

0.005, 0.0025, 0.001, 0.0005 and 0.00025 percent active ingredient. The organic tin resistant mites were sprayed with 0.2324, 0.1162, 0.0465, 0.0232, 0.0116, 0.0046, 0.0023, and 0.0012 percent active ingredient in combination with FCI-119 at 1000

ppm. FCI-119 at 300 ppm was applied to susceptible mites at the same concentrations

of Vendex 50WP as the 1000 ppm test. The organic tin resistant mites were sprayed

with the following percent active ingredient of Vendex 50WP in combination with 300 ppm FCI-119: 0.581 , 0.2905, 0.1162, 0.0581, 0.02905, 0.01162, 0.00581 , and 0.002905 percent active ingredient. For the FCI-119 at 100 ppm, the following percent active ingredients of Vendex 50WP were combined: 0.1, 0.05, 0.025, 0.01, 0.005, 0.0025, 0.001, and 0.0005 for the susceptible mites; and 1.162, 0.581, 0.2324, 0.1162, 0.0581, 0.02324, 0.01162 and 0.00581 for the organic tin resistant mites. FCI- 119 at 1000 and 300 ppm rate treatments were evaluated for mortality at 4-day (96-hour) and six day (144 hour) post application intervals (PAI). FCI- 119 at 100 ppm treatments were evaluated at 4-day (96-hour) PAI only due to higher mortality in the untreated check at 6-day. The bioassay data are presented in tabular form as ppm active ingredients (move decimal four places to the right to convert percent concentration to ppm) required to kill 50% (LC ₅₀ ) and 95 % (LC ₉₅ ) of the test population (Table 23). For each of the bioassays, an R value of 0.954- (i.e. ,

at least 95 % confidence level) was produced by the probit analysis, indicating that the

regression lines correlated very well with the raw data.

The data indicated that FCI-119 applied at a rate of 300 ppm or 1000 ppm significantly shifted the regression lines resulting in greatly enhanced activity for Vendex 50WP. It appeared that at the rate of 100 ppm FCI-119 did not enhance the

effect of Vendex 50 WP.

TABLE 23 THE EFFECT OF DIFFERENT RATS OF FCI-119 ON THE LC ₅₀ AND LC ₉₅ FOR ADULT FEMALE TWO-SPOTTED SPIDER MITES TREATED WITH VENDEX 50WP AT FOUR DAYS AFTER APPLICATION

. . - " fi t .

EXAMPLE 9: The Effect of Famesyl Methyl Ether on the

Activity of Vendex on Adult Susceptible Two-spotted Spider Mite Tetranychus urticae Koch

In this example, the effect of famesyl methyl ether (FME) on adult susceptible TSSM, when sprayed in combination with Vendex 50WP at the previously determined LC ₅₀ value was determined. Famesyl methyl ether (FME) is an analog

of famesol.

For each replicate in the bioassay procedure on the day prior to treatment, 12-

15 female adult or mixed sex immature TSSM from the susceptible strain were transferred from infested leaves onto excised 15 mm diameter bean leaf discs which had been placed on deionized water moistened cotton balls in "bioassay cups".

The serial dilutions for the FME/Vendex 50WP were prepared by weighing out the required amount of FME plus an anionic/nonionic surfactant (Armul

33 surfactant available from Witco Chemical Corp., Houston, Texas) at 5% of the FME weight.

The rates used for the Vendex/FME test were as follows. Vendex 50WP at 200 ppm was used as the approximate LC ₅₀ value determined from a bioassay. Vendex 50WP at 200 ppm was combined with FME at 300, 3 and 0.3 ppm. FME was also sprayed at 300, 3 and 0.3 ppm alone. Two treatments at 200 ppm of Vendex 50WP alone were also included. In addition, all tests included an untreated check.

The FME/ Vendex test was evaluated at 4-day and 6-day PAI. The

bioassay data are presented in tabular form as average percent mortality per treatment

(Table 24).

FME may have some enhancing effect with Vendex 50WP on susceptible TSSM particularly at 300 ppm. All the FME alone treatments and the untreated check were statistically equivalent (Table 24).

Table 24. The effect of Famesyl methyl ether in combination with Vendex 50WP on the mortality of adult susceptible two-spotted spider mite, T\ urticae Koch. ¹¹

" Means followed by the same letter are not significantly different according to DNMRT (P = 0.05)

Use of the miticidal composition described herein is believed to enhance the level of female and male spider mite control provided by common miticides whether such spider mites are susceptible or resistant to such miticides when treating such crops as alfalfa, clover, cotton, peanuts, sorghum, citrus, beans, blackberries, raspberries, com, cucumbers, melons, pumpkins, squash, eggplant, peppers,

tomatoes, hops, strawberries, omamentals, grapes, as well as fruit tree and nut tree crops.

While the present invention has been described with reference to the preferred embodiments thereof, the description is for illustrative purposes only and is not to be construed as limiting the scope of the invention. Various modifications and changes may be made by those skilled in the art without departing from the true spirit and scope of the invention as defined by the appended claims.