WO1996028022A2

WO1996028022A2 - Fatty acid based compositions and methods for the control of plant infections and pests

Info

Publication number: WO1996028022A2
Application number: PCT/US1996/002599
Authority: WO
Inventors: Steven D. Savage; Steven L. Evans; Robert A. Haywood; Paul S. Zorner; Keith Jones
Original assignee: Mycogen Corporation
Priority date: 1995-03-08
Filing date: 1996-02-26
Publication date: 1996-09-19
Also published as: AU5174596A; WO1996028022A3

Abstract

The invention described here concerns the unique utility of fatty acids and their derivatives to eradicate existing fungal and bacterial infections in plants. Also, described herein are combination treatments whereby fatty acids are used to enhance or augment the activity of fungicides, bactericides, and biological control agents.

Description

DESCRIPTION

FATTY ACID BASED COMPOSITIONS AND METHODS FOR THE CONTROL OF PLANT INFECTIONS AND PESTS

Cross-Reference to a Related Application This is a continuation-in-part of copending application Serial No. 08/237,080, filed May 3, 1994; which is a division of Serial No. 07/871,511, filed April 23, 1992, now U.S. Patent No. 5-366,995; which is a continuation-in-part of Serial No. 07/694,193, filed May 1, 1991, now abandoned.

Field of the Invention This invention pertains generally to the fields of insect pest control and the control of bacterial and fungal plant infections. Fatty add compositions are provided for each of these applications.

Background of the Invention A. Control of fungal and bacterial plant infections. The protection of desirable plants and their produce from fungal and bacterial pathogen infection has traditionally required preventative applications of fungiddal or bacteriddal agents. Fungiddal and baαeriddal compounds have long been used to increase yields and extend agricultural production capabilities into new areas. They have also been extremely important tools for ameliorating season-to-season differences in yield and quality caused by weather-driven variations in disease pressure.

The future role of fungiddes and bacteriddes in agriculture is increasingly threatened by several factors including; the development of pest resistance, increasing concerns about food safety, and environmental accumulation of toxic compounds. As older fungiddes and bacteriddes are removed from the market due to regulatory changes, and new fungiddes and bacteriddes are becoming increasingly expensive to register, there is an increasing need to find ways to more wisely use the remaining, safest fungiddes. This is particularly true for the many crop/disease combinations which do not represent large enough markets to pay for the cost of new compound registration. Wiser fungidde and bacteridde use will include ways to reduce application rates (and thus potential residues), finding ways to extend registrations to new crops, and identifying new fungiddal and bacteriddal compositions and treatments to combat the development of pest resistance. Chemical fungiddes and bacteriddes have provided an effective method of control; however, the public lias become concerned about the amount of residual chemicals which might be found in food, ground water and the environment. Stringent new restrictions on the use of chemicals and the elimination of some effective pestiddes from the market place could limit economical and effective options for controlling fungi and bacteria.

It is well recognized by those skilled in this art that there is a clear distinction between preventative microbiddal (fungiddal and bacteriddal) activity and curative activity. Compositions and methods which may be effective to prevent microbial growth may have very little or no impact on established infections. Of course, it is often desirable to prevent infections altogether, however, this is not always possible and there is a great need for compositions which have the unique ability to arrest the growth of established infections. This is particularly true in the control of infections which become established on agricultural products after harvest Curative fungiddal activity has been observed when some biological agents are used for disease control (eg., strain of Bacillus subtilis) and this activity can usually be attributed to the production of antibiotic compounds by the biocontrol organism. Because expensive toxicological screening and residue/metabolite monitoring may be required for such an antibiotic, the normal registration-cost advantage of these non-chemical agents is diminished. Biological control agents which do not make antibiotics would be much easier to register, but they tend to have only preventive control.

The commercialization of disease biocontrol agents has also been hampered by inconsistent field performance. Organisms which show biocontrol potential in laboratory and greenhouse experiments often fail to compete with the existing microflora when applied outdoors and are thus unable to express their biocontrol potential, regardless of mode of action.

Specifically there is a need for disease control methods which are more compatible with the need for affordable and effective disease control, a high degree of food safety, and minimal environmental impact

One example of the need to control post-harvest spoilage of agriculture products pertains to green and blue molds of dtrus fruits caused by PenicLULum digitatum and P. italicum. These molds cause severe damage during storage and shipping. The existing fresh-market industry relies completely on a combination of several chemical treatments to deliver sound fruit to distant markets over substantial periods of time without excessive damage caused by these molds. Unfortunately, there are increasing concerns about the safety of the chemicals currently used to control these fungal pathogens. Also, there are increasing problems with fungal strains with resistance to the most effective compounds.

In another example, powdery mildew of grapes caused by Uncinula necator can cause severe damage even in dry areas such as California. Traditionally this disease was controlled with applications of elemental sulfur, but this necessitates frequent, high volume applications of an irritating material. The introduction of egosterol biosynthesis inhibiting fungiddes (primarily triazoles) greatly simplifies control, but also selects for tolerant strains. Some of these compounds are also known to have potential teratogenic effects and very long soil residuals. In these and other examples, alternative control methods are in great demand-particularly methods which are safer or more environmentally benign.

Fatty adds are a class of natural compounds which occur abundantly in nature and which have interesting and valuable biological activities. The in vitro activity of fatty adds against many medically important fungi and bacteria is well known; however, their in vivo antifungal activity is often very limited and it is difficult to predict on the basis of in vitro experiments. There is a much smaller body of literature concerning the activity of fatty adds and their derivatives against pathogens on agricultural crops. Ahmed et al (Ahmed, S.M., F. Ahmad, S.M. Osman [1985] JAOCS 62:1578-1580) report in vitro inhibition of radial growth of several fungal genera with plant pathogenic representatives. Recently there has been an expanding use of "insectiddal soaps" in agriculture which are salts of certain fatty adds. This has resulted in a few observations of impact on fungal disease. For instance, Chase et al (Chase, A.R., L.S. Osborne [1983] Plant Disease 67:1021-1023) observed that applications of an 18:1 fatty add salt "insectiddal soap" gave moderate preventive control of two foliage plant diseases and actually exacerbated two other diseases. Puritch et al described the effect of fatty add salts on fungi in vitro (Puritch, G.S., W.C.

Tan, J.C. Hopkins [1981] Canadian Journal of Botany 59(4):491-494). Nickel and silver salts of fatty adds have been used to control pathogens on plants. GB Patent Nos.907,842 and 1,219,077. In U.S. Patent No. 3,983,214, Misato et al claim a fungiddal composition containing a sucrose fatty add ester. Misato et al emphasize the preventative activity of their composition. Similarly, in U.S. Patent No. 4,771,571, Obrero et al describe a method of preventing infections of pineapple by treating the fruit, while on the bush, with a surfactant. In U.S. Patent No.4,002,775, Kabara et al claim microbiddal food additives comprising 1 or 2-mono-laurin polyol ester. Kabara's work is also described in: Chapter 14 of Ecology and Metabolism of Plant Lipids, American Chemical Sodety (1987); "Fatty Adds and Derivatives as Antimicrobial Agents," In: Antimicrobial Agents and Chemotherapy, American Sodety for Microbiology (1972), pp. 23-28;

"Antimicrobial Agents Derived from Fatty Adds," (1984) JAOCS 61(2):397-403; and "Antimicrobial Lipids: Natural and Synthetic Fatty Adds and Monoglycerides," Lipids 12(9):753- 759. Also, the use of fatty add esters and alcohols for the control of powdery mildew on apple buds has been described (Frick, E.L., R.T. Burchill [1972] Plant Disease Reporter 56:770-772; U.S. Patent No. 3,931,413). In the '413 patent, Frick et al emphasize the phytotoxidty of fatty adds and state that the add or salt form should only be used on dormant plant tissue. The phytotoxidty of fatty adds and their salts is well documented and has long been believed to be a barrier to the use of these compositions on living plants. See U.S. Patent No. 5,246,716.

Most in vitro tests for antimicrobial activity involve monitoring the germination and growth of pathogen propagules in a liquid or solid format in which there is exposure to the chemical agent. These assays are directly analogous to preventive applications in an agricultural setting applications which are made prior to the time when the pathogen initiates an infection. The primary screening process for synthetic chemicals in industrial settings is almost exdusively based on in vitro and preventive in vivo testing. Thus, compounds without significant preventive activity are rejected. There are no reports of fatty adds acting in a curative mode (applied after fungal infection is established). B. Pest control. Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.

Chemical pestiddes have provided an effective method of pest control; however, the public has become concerned about the amount of residual chemicals that might be found in food, ground water, and the environment Stringent new restrictions on the use of pestiddes and the elimination of some effective pestiddes from the market place could limit economical and effective options for controlling costly pests. Thus, there is an urgent need to identify pest control methods and compositions which are not harmful to the environment Various pestiddal compositions having a fatty add component, or a fatty add derivative as a component, are well known to those skilled in the art See, for example, U.S. Patent No. 5,192-546; DE Patent 3342.529; Australian Patent No. AU-B1-35-221/78; and U.S. Patent Nos. 4,774,234; 4,826,678; 4,904,645; 4,870,102; 4,861,762; 4,707,496; 3,954,977; 5,093,124; and 4,891385. None of these patents described pestidde activity for amide derivatives of fatty adds.

Brief Summary of the Invention The subject invention concerns materials and methods for the control of fungal and bacterial plant pathogens and for the control of pests such as insect pests. The materials and methods of the subject invention utilize fatty add compositions which are both highly effective and non-hazardous to the environment As described more fully herein, the invention can be thought of in terms of three primary embodiments.

In a first embodiment, the subject invention pertains to the discovery that fatty adds, their salts and derivatives, when used at the appropriate concentration range and timing, are useful for the eradication of established fungal and bacterial infections in or on plant tissues. Thus, the subject invention provides parameters of application which allow the useful application of these agents for the control of plant disease. In a specific example of this embodiment of the subject invention, the fatty add component is applied to non-dormant plant tissue.

According to this first embodiment of the subject invention, established fungal and bacterial infections are effectively controlled by compositions comprising one or more substituted (or unsubsτituted) saturated (or unsaturated) fatty adds (or their salts or derivatives). The fatty acids of the subject invention can be from about C7 to about C20 and can be, for example, in the epoxide, lactone, cyclopropane, methylated, or hydroxylated forms. Specifically exemplified herein are saturated and mono-unsaturated fatty adds of length C9, C12, and C18. The use of the compositions described here, when used in the proportions and application rates set forth more fully hereinafter, results in an unexpected control of established fungal infections. The lack of preventive activity of these compositions makes this discovery highly unexpected. This invention demonstrates that the same fatty adds which kick preventive activity for disease control exhibit advantageous curative control This activity is most advantageous over a range of concentrations between low doses which are ineffective and higher doses which are phytotoxic to the host plant This critical range varies with the form of the add (free add, salt, formulation) and the host/pathogen system under consideration, but can be determined by a person skilled in this art using the teachings of the subject invention.

The discovery of curative activity for fatty adds has further significance because that utility along with the non-phytotoxic properties of these compounds make them extremely useful for combinations with other disease control agents. Thus, fatty adds can be advantageously combined with other disease control chemicals. In one example, fatty adds can be combined with preventative antifungal or antibacterial agents. Specifically exemplified herein is the use of fatty add compounds in conjunction with formulations containing elemental sulfur which are widely used for preventative antifungal treatment

A second major embodiment of the subject invention pertains to combination treatments whereby fatty adds (or their salts or derivatives) are combined with biological control agents. An important limitation of live biological control agents is their inability to compete with resident microflora. This problem can be overcome in accordance with the teachings of the subject invention. The application of a fatty add composition to a plant surface in accordance with the teachings of the subject invention can be used to substantially disrupt the existing balance of microorganisms. This provides an opportunity for appropriately selected, Live biological control agents to become established on the plant surface. When these "disrupted microbial niches" are re-colonized a microorganism which is particularly adapted to surviving that disruption event is more reliably established during subsequent colonization episodes.

In the case of bacterial disease episodes, many of which involve an epiphytic build-up phase, the fatty add can be used to reduce the pathogen population and simultaneously open the way for subsequent colonization by desirable microorganisms. Colonization by desirable microbes can be even further enhanced by applying a fatty add, an enrichment agent (eg., a particular nutrient source such as starch, cellulose or other macromolecular foodbase) and an organism particularly suited to survival and growth in that specific regime of negative and positive selection agents. Also, the fatty add itself or its breakdown products can provide the foodbase which favors colonization by a certain organism. Alternatively, the food base can consist of the addition of another agent in the formulation. Thus, a second major embodiment of the subject invention is the use of fatty adds, their salts, or derivatives, as "niche-clearing" agents.

In the third major embodiment of the subject invention, certain amide derivatives of fatty adds have been discovered which have excellent pestiddal activity. Specifically exemplified are fatty add compositions, such as cocoate, which has been reacted with diethanolamine to produce cocoamide DEA. As is known in the art, cocoate is a mixture of free fatty adds derived from a

"cracking" of coconut oil. The mixture consists primarily of C12 and C14 saturated fatty adds.

The fatty adds of the subject invention and their derivatives are highly advantageous for pestiddal use because they occur commonly in nature, have little mammalian toxidty, are compatible with other biological control strategies and are readily broken down to innocuous components.

Thus, among the advantages and the embodiments of the subject invention are the following:

■*• the use of a fatty add as the active ingredient in a composition to eradicate existing pbytopathogens such as fungal infections or bacterial colonization;

*■ the use of a fatty add to improve or compliment the activity of other fungiddal and bacteriddal chemicals; » the combination of curative fatty adds with preventive biological control agents to provide an enhanced scope of protection; ► the use of a curative fatty add to provide control and to also perturb the plant surface microflora to enhance the subsequent colonization of that surface by a compatible biological control agent;

*^■ the combined use of a fatty add, an enrichment agent and a biological control agent which is tolerant to the fatty add and favored by the enrichment agent; and ► the combined use of a fatty add with or without an enrichment agent to disrupt microbial colonization and enhance subsequent colonization by a biocontrol agent *■ the combined use of a fatty add and a prophylactic antimicrobial to provide both curative and preventive control of microbial plant infections. ► the use of nitrogen-containing fatty add derivatives, both for control of microbial infections and for pestiddal use.

Detailed Description of the Invention

The subject invention concerns unique materials and methods for the effective and environmentally safe control of insect pests and microbial plant pathogens. In each embodiment of the subject invention, fatty add compositions constitute a critical component of the control strategy. As described in detail below, the fatty add component may be in the add form or may be a salt or derivative. As used herein, reference to "fatty add" should be understood to include salts and derivatives thereof as described herein unless the context dearly pertains only to the add form. For certain applications, the use of a particular fatty add derivative is a critical aspeα of the invention. For example, diethanolamine amides of C12 fatty adds have been discovered to have particular surprising activity against insects. For the control of fungi and bacteria, it is possible to use a variety of fatty adds and their salts and derivatives. However, even for antifungal and antibacterial applications, certain fatty adds, their salts, or their derivatives are preferred as described herein.

The common theme to the invention described herein is the use of environmentally friendly fatty add compositions in methods to control pests or microbes. The invention can best be understood by reference to three primary embodiments. First, the subjeα invention pertains to direct curative control of fungal or bacterial plant pathogens with fatty add compositions. In this embodiment, the activity of the fatty add compositions can be enhanced or supplemented by using the fatty add component in conjunction with chemical or biological agents to achieve the desired control. In one specific embodiment, a fatty add derivative is used in conjunction with a sulfur compound to achieve excellent antimicrobial activity without phytotoxidty.

In a second major embodiment of the subjeα invention, fatty add compositions are used to dear away existing microorganisms on a plant or in another natural setting, and desired microbes are then introduced with a greatly enhanced opportunity to colonize the site. The desired microbes may be, for example, inseαiddal, or they may express insectiddal proteins. This embodiment of the invention is referred to herein as "niche-clearing."

In a third major embodiment, specific fatty add derivatives have been discovered which have highly advantageous pestiddal activity. Specifically exemplified are nitrogen-containing amide derivatives of C12 fatty adds. Each of these three major embodiments is discussed in detail below. Before detailing the three major embodiments, a general description of the fatty add compositions useful according to the subjeα invention is provided.

I. Fatty Add Compositions Fatty add compositions useful according to the subjeα invention can be unsubstituted, or substituted, saturated, or unsaturated, fatty adds (or their salts or derivatives), of about C7 to about C20. Specifically exemplified are fatty adds of length C9, C12, C14, and C18 typified by, but not limited to, pelargonic add, Laurie add, myristic add, oleic add, and various salts, amides, or esters of these acids. The fatty add component of the subjeα invention may be a single fatty acid or salt, or a mixture of two or more fatty adds or salts thereof. The fatty acid salts which can be used according to the subjeα invention include, but are not limited to, sodium, potassium, and nitrogen-containing salts including isopropylamine salts. Also, various esters and amides are useful according to the subjeα invention, including monoethylene glycol ester and DEA amides. The fatty add amide derivatives described herein have been discovered to have both antimiαobial and pestiddal activity.

The fatty add compounds useful according to the subjeα application can be represented by the following formula: o

II RιYιY₂CZ_% I

in the case of curative control of established microbial infections,

Z = O, N, or S R_j = C5 to C19 saturated or unsaturated hydrocarbon, or an epoxide, or cyclopropane thereof Υ₁ = H, C^-C_ζ hydrocarbon, or hydroxyl at any position along R Y₂ = H, C_j-C₅ hydrocarbon, or hydroxyl at any position along R_x

R₂ = CJ-CK, saturated or unsaturated, branched or unbranched, hydrocarbon having at least one hydroxyl group at any position on R₂; salt; or H. The fatty add compounds claimed according to the subjeα invention for use in combination with live biocontrol agents, as "niche-clearing" agents, or as a pestidde, can also be represented by Formula I wherein:

Z = O, N, or S

R = C5 to C19 saturated or unsaturated hydrocarbon, or an epoxide, or cyclopropane thereof Y_x - H, CJ-CJ hydrocarbon, or hydroxyl at any position along R_x Y₂ = H, CJ-CJ hydrocarbon, or hydroxyl at any position along R_α

R₂ = CJ-CJO saturated or unsaturated, branched or unbranched, hydrocarbon which may have one or more hydroxyl groups at any position on R₂; carbohydrate; salt; or H.

In a preferred embodiment of the invention, R₂ is selected from the group consisting of aliphatic amines which form cationic aliphatic ammonium compounds; K⁺; Na⁺; and H⁺. Oleic, Linoleic, linolenic, lauric, capric, myristic, palmitic, and pelargonic adds and their salts and esters are particularly useful according to the subject invention. We have also found that the monoethylene glycol ester of fatty adds is particularly useful according to the subjeα invention. As those skilled in the art would readily recognize, when Z=N, there will be two R groups attached to N. Thus, in this specific case of Formula 1, the formula can be represented as follows:

wherein

R_j = C5 to C19 saturated or unsaturated hydrocarbon, or an epoxide, or cyclopropane thereof

Y_j = H, C_j-C₅ hydrocarbon, or hydroxyl at any position along R_χ Y₂ -= H, C_J-C5 hydrocarbon, or hydroxyl at any position along R_j R₂ = C^-C^_Q saturated or unsaturated, branched or unbranched, hydrocarbon which may have one or more hydroxyl groups at any position on R₂; carbohydrate; salt; or H;

R₃ = CJ-CJ_Q saturated or unsaturated, branched or unbranched, hydrocarbon which may have one or more hydroxyl groups at any position on R₂; carbohydrate; salt; or H.

As described more fully herein, compounds of Formula IA are useful both as fungiddes and pestiddes. Of particular interest are certain diethanolamine (DEA) fatty add compounds.

It is well known in the art that DEA fatty add compounds can be prepared by reacting fatty adds with diethanolamine. These compounds can be readily prepared from fatty add compositions including fatty arid-containing natural products derived from coconut oil. Specific examples of such products include, but are not limited to, cocoamide DEA, non-ionic coconut amides, coconut diethanolamides, and fatty diethanolamides. These products are available under a variety of commercial names including the "AMIDEX" series, Chemron Corp.; "INCROMIDE CA," Croda

Inc.; "AMDMOL," Finetex, Inc.; and "T-TERGAMIDE," Harαos Chemicals, Inc. A preferred embodiment is cocoamide DEA, which surprisingly shows excellent pestiddal activity. Also, DEA fatty adds can be readily prepared by reacting DEA with a fatty add or fatty acid-containing composition. The fatty add composition may be, for example, a coconut fatty add composition.

DEA can be obtained from Ashland Chemical, Inc. The coconut fatty add starting material can be obtained from Henkel Corporation. DEAs can be made using standard procedures as described, for example, in Fatty Acids, E.H. Pryde, ed., 1979, The American Oil Chemists' Sodety.

Sources of fatty adds other than coconut oil can also be utilized. Examples of other fatty add sources include soy, palmitic, stearic, and tallow fatty acids. Preferably, the source of fatty adds would comprise fatty add compositions with about C9 to about C18 chain lengths. The following table provides an analysis of a typical coconut fatty add composition. Table 1.

Fatty Add Length %

Laurie 12 48

Myristic 14 20 Palmitic 16 10

Oleic 18:1 10

Capric 10 5

Caprylic 8 4

Linoleic 18:2 1

Tank mixes of fatty adds can be prepared according to procedures which are well known to those skilled in the art For example, a ratty add spray oil can be prepared using a solvent solution or emulsion of the fatty add, a surfactant, and suffident water to dilute the mixture to the desired concentration. Salts of fatty adds are readily dispersable or soluble in water. The surfactants which may be used to emulsify the fatty add in the aqueous formulations can be any of the non-phytotoxic surfactants, which are customarily used in preparing formulations for use on agricultural αops. The composition of the subjeα invention may also be combined with a spray oil as described in U.S. Patent No. 4,560,677.

Fatty adds which can be used according to the subjeα invention are widely available and are sold under a variety of tradenames induding "M-PEDE," "SCYTHE," "SHARPSHOOTER,'

"DE MOSS," and "SAFER" Insectidde Concentrate (SIC). As used herein, the term "SHARPSHOOTER" refers to an 80% "SHARPSHOOTER" formulation which consists of 80% pelargonic add, 2% emulsifier (such as Dowfax 32B) and 18% surfactant (such as Stepfac 8170). Also, fatty adds are readily available as components of natural products. For example, commonly available compositions such as dtrus seed extracts and coconut oil can be used to supply the fatty add component for use according to the subjeα invention. II. Control of Fungal or Bacterial Plant Pathogens

In the first of three major embodiments, the subjeα invention concerns the in situ use of fatty adds and their salts or derivatives for the control of fungal and baαerial plant diseases. This mode of action is very compatible with other chemical and biological control approaches and fits well into the alternative pest control strategy which sodety is demanding.

The compositions and methods described herein can be used to control a broad range of fungal and baαerial targets. These targets include, but are not limited to spedes of Penicillium (Le., expansum, digitatum, itaiicum), Botrytis sp., MonUinia sp., Altemaria sp., AspergUlus sp., Rhizopus sp., members of the Erisyphales (powdery mildews Sphaerotheca sp., Erisyphe sp.,

Uncinula sp., Podosphaera sp.), members of the Peronosporales (downy mildews, Phytoptiiora sp., Pythium sp., Peronospora sp.) Hemibasidiomycetes (rusts and smuts), Venturia sp., Cercospora sp., Pseudocercosporella sp., Cercospora sp., Cercosporidium sp., Fusarium sp., Ophiostoma sp. and other wood staining fungi, and Diplodia sp., other targets include Erwinia sp., Pseudomonas sp., and Xanthomonas sp. These targets can be controlled on seeds, conns, bulbs, flowers, stems, leaves exposed roots and fruits of plants including but not limited to grapes, pears, apples, peaches, nectarines, grapefruit, cherries, apricots, lemons, oranges, mangos, bananas, tangerines, potatoes, tomatoes, cucumbers, lettuce, rice, wheat, rye and other cereals, flower αops, and almonds. As used herein, the term "produce" includes, but is not limited to, any of the plant surfaces listed above. Also, as used herein, the term dtrus refers to fruits such as oranges, lemons, limes, grapefruit, and the like. The compositions can also be applied to surfaces such as freshly cut lumber for the control of fungal or baαerial targets. The fatty add compositions of the subjeα invention can be used to control microbial plant disease on both dormant and non- dormant plant tissue. As known by those skilled in the art, non-dormant tissue includes growing vegetation and fruits (pre- and post-harvest). Control of microbial plant pathogens on non- dormant tissue without phytotoxidty is particularly surprising and advantageous.

In one specific example, it has been discovered that pelargonic add and its salts or derivatives, in a concentration of about 0.25 to about 3% w/v, have excellent curative activity against established fungal infections of produce (Examples 2 through 5). If produce is wounded and infeαed with the pathogen and then 18-24 hours later it is treated, disease does not develop. Disease control is not observed if the fatty adds are applied at the same time the fungus is inoculated or prior to that inoculation. Similarly, applications of about 2% "M-PEDE" (mainly salts of cl8 fatty adds) or about 0.5% pelargonic add or about 0.75-1% potassium cocoate can dramatically reduce further sporulation when applied to plants which are already severely infected with powdery mildew pathogens. Again, application of the same fatty adds to the plants prior to infeαion (preventative) are ineffective.

Thus, the unexpeαed antifungal and antibaαerial activity of fatty adds which we have now observed pertains specifically to their ability to eradicate existing infections. As is shown in Examples 2 through 5, fatty adds are capable of arresting disease development in Penicillium inoculated lemons. This is a wound pathogen, and by the time dtrus fruit reaches the packing house, infections of harvesting wounds are typically well established (12-24 hours) and require therapeutic action.

The utility of fatty adds and their derivatives for therapeutic control is further documented in Examples 6 and 7 where pelargonic add shows curative activity against Botrytis cinerea infeαion of pear, Monilinia fructicola infection of nectarine, and Penicillium infection of lemons and oranges.

In these cases where fatty acids are useful for eradication of existing infections of fruit, the further protection of that fruit from subsequent infections can be achieved by the simultaneous or subsequent application of a fungidde, baαeridde, or a biological control organism in a dip or spray application. This application can also be made along with the application of various waxes or finishes which are commonly used with fruit The formulation of such applications can also include nutrients which will benefit the establishment of the biocontrol organism.

Fatty adds are also active against obligate parasites such as powdery mildews. Attempts to control these diseases currently involve rigorous, preventive control programs based on either sulfur products or synthetic fungiddes which inhibit ergosterol biosynthesis. If a mildew epidemic becomes too advanced, it is extremely difficult to use those same products to halt its further spread. As shown in Example 8 through 21, fatty add compositions which lack the ability to prevent mildew infection are capable of killing severe, established infections. As such, they are highly advantageous as "rescue treatments" in the event of severe mildew infestations.

One element of this invention concerns the concentration range for the efficadous use of fatty add compositions. At very low concentrations there is no activity, at an intermediate range there is desirable activity, but at higher concentrations the host plant can be damaged and this can aαually enhance infection (e.g., in Example 3 where concentrations of pelargonic add of about 0.5% and higher were more severely infected than the water control). In the case of powdery mildew control with pelargonic add (Example 8), concentrations of about 1% and above can become highly phytotoxic as this fatty add is used commercially as an herbidde. The safety margin between antimiαobial aαrvϊty and phytotoxidty can be widened by the formulation of the fatty add. In particular, certain salts are much less phytotoxic and only slightly less fungiddal than the parent add (Example 4). Appropriate formulations and concentrations can be readily ascertained by those skilled in this art using the teachings of the subjeα invention.

A. Combination of fatty adds with other agents. The fatty adds of the subjeα invention do not show preventive activity; however, one aspeα of the first embodiment of the subjeα invention is the combination of the potent, therapeutic activity of fatty add compositions with the preventive action of chemical fungiddes, baαeriddes, or the exclusionary and/or competitive capabilities of biological control agents. The benefits of these combinations fall into two main categories: fungidde and baαeridde rate reductions, and enhanced aαivity against pathogens of interest

The potent, curative activity of the fatty add compositions of the subjeα invention combined with other fungiddes or baαeriddes makes it possible to achieve the same level of control while using a smaller quantity of the non-fatty add fungidde or baαeridde component of the mixture. We have discovered that the compositions of the present invention can comprise a mixture of components wherein said mixture is sufficiently aαive so that application of the composition enables utilization of reduced amounts of each of the aαive ingredients while still providing effective activity. This is significant because lower use rates lead to lower residues on the αop or in the environment, lower costs of application, an expansion of the margin between crop safety and efficacy for fungiddes which can be phytotoxic (thus enhancing their safety or expanding the αops, varieties or timings for their use), and lower total "market basket" exposure for a multi-use fungidde or baαeridde. We have discovered that combinations of other fungiddes or baαeriddes with fatty adds offer additional advantages because of the particular mode of action of these materials. One such advantage is a reduction in selection pressure for resistant forms. It is often difficult to find appropriate resistance mixing partners for systemic curative fungiddes since materials which have a different mode of action and which are also curative are rare. Mixtures of curative and non- curative fungiddes are considered to be less desirable for resistance management Use rates of fungiddes can also be lowered in cases where their current use rate is high to provide control partially tolerant pathogen strains.

Many fungiddes or baαeriddes have excellent preventive efficacy, but are ineffective for the eradication of existing infections. Used alone, these compounds must be continually reapplied to maintain a constant, protective cover over the αop tissues. Combinations of such material with a curative, fatty add product increases overall efficacy of the disease management strategy, allows less frequent use of the protectant, and extends to new αops or regions a control program which uses the fungidde or baαeridde in question.

Chemical control agents which can be combined with fatty adds according to the subjeα invention include, but are not limited to benomyl, borax, captafol, captan, chlorothalonil, various formulations containing copper; various formulations containing zinc, dichlone, dicloran, iodine, various ergosterol biosynthesis inhibiting fungiddes including but not limited to fenarimol, imazalil, myclobutanil, propiconazole, prochloraz, terbutrazole, flusilazole, triadimefon, and tebuconazole; folpet, iprodione, mancozeb, maneb, metalaxyl, oxycarboxin, oxytetracycline, PCNB, pentachlorophenol, quinomethionate, sodium arsenite, sodium DNOC, sodium hypochlorite, sodium phenylphenate, streptomycin, sulfur, thiabendazole, thiophanate-methyl, triforine, vinclozolin, zineb, ziram, tricyclazole, cymoxanil, blastiridin, validimycin. The fatty adds can also be combined with various spray oils.

Specifically exemplified herein is the combination of a fatty add component with a sulfur compound. In a preferred embodiment, the fatty add component is a potassium cocoate which is combined with sulfur. Alternatively, salts of about C9 to about C12 fatty adds can be used with sulfur. Surprisingly, this combination results in excellent fungiddal activity (preventative and curative) without phytotoxidty.

III. Use of Fatty Add Compositions in Niche-Clearing Methods

The broad spectrum antimiαobial activity of fatty acids are particularly advantageous for a unique strategy which combines fatty acid compositions with various biocontrol agents. A common limitation of biological controls has been the inability of the desired live control agents to colonize treated surfaces. The existing miαoflora of that surface, if well developed, can predude the establishment of the applied organism. The fatty add compositions of the subjeα invention are useful for disrupting existing miαoflora (pathogens and saprophytes), making it more likely that a biocontrol organism will successfully colonize the surface if it is applied at the same time or soon after the fatty add composition. This is a particularly attractive possibility with fatty add compositions because they do not persist on the plant at effective concentrations. A further extension of this concept involves the addition of a nutrient or other enrichment agent to the fatty add plus biological strategy. It is within the skill of a person trained in this art to use the teachings presented herein to devise the appropriate compositions of fatty adds, biologicals and enrichment agents.

The biological control agents that can be used according to the subjeα invention include but are not limited to Bacillus sp., Pseudomonas sp., Trichoderma sp., Erwinia sp., Pichia sp., Candida sp., Cryptococcus sp., Takaromyces sp., P. fumosoreus, B. bassiana, Chaetomium sp., Gliocladium sp., Aureobasidium sp., Dabaryomyces sp., Exophilia sp., Ampelomyces sp., and

Mariannaea sp. One embodiment of the subjeα invention is to seleα for biological agents which are tolerant to and thus well suited to a use in combination with fatty add compositions.

Spray oils (also known as agricultural spray oils) which can be used as negative selection agents according to the subjeα invention include, but are not limited to, paraffin oils such as 6N, 7N, 9N, and UN sold by the Sun Oil Co. of Philadelphia, PA. Other negative selection agents include, but are not limited to, carbonate salts, alcohols, inorganic metals and combinations of these various agents. Positive selection agents which can be used according to the subjeα invention include, but are not limited to, yeast ghosts, baαerial ghosts, algal ghosts, complex carbohydrates, simple carbohydrates, organic nitrogen, or combinations of these agents.

IV. Pestiddal Use of Fatty Acid Compositions

The subject invention further concerns the discovery of fatty add derivatives with excellent pestiddal activity. As used herein, pests include, but are not limited to, insects and mites. Specifically, fatty acid amides have been found to have particularly advantageous pestiddal properties. Thus, compounds having the Formula IA can be used in pestiddal applications according to this embodiment of the invention. Specifically disclosed herein is the excellent pestiddal activity of diethanolamine amide derivatives of fatty adds. One preferred embodiment is cocoamide DEA, which can be prepared by reacting coconut fatty add preparations with diethanolamine. As shown in Example 15, these compounds are highly effective in controlling pests. Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 — Activity of Seleαed Fatty Adds Against Plant Pathogens

Various concentrations of pelargonic add in one of two forms was added to molten potato dextrose agar (PDA) and poured into small disposable petri plates. The add was added either as an emulsified free add or as the potassium salt of the pure add. Control plates were made by adding comparable concentrations of the surfactants in the pelargonic add formulation or by adding water. To these plates were added suspensions of the spores of Penicillium digitatum oτBotrytis cinerea. These plates were observed 3 days later for fungal germination and growth and the results are shown in Table 2.

Table 2.

Penicillium Botrytis

Treatment %a.i. digitatum cinerea

80% formulation 2.0 — —

(emulsified add) 0.5 — —

0.125 — —

0.03 — —

80% formulation 2.0 + + surfactant blank 0.5 + + (equivalent dilutions) 0.125 + +

0.3 + +

Potassium salt of 2.0 — — pelargonic add 0.5 — —

0.125 — —

0.03 + —

Media control — + +

+ = germination — = no germination

Example 2 — A Comparison of Preventive and Curative Activity of Pelargonic Add for the Control of Green Mold Infections of Lemons

Lemons grown without the application of synthetic chemicals were harvested and inoculated at 5 marked locations with spores of Penicillium digitatum (10⁷ conidia/ml) by pricking to a depth of 2 mm with an 18-gauge needle dipped in a spore suspension. After inoculation the lemons were held in closed plastic boxes over wet paper towel at 22°C. The lemons were either not-dipped or dipped in a 1% a.i. pelargonic add suspension at 0, 5, 16 or 24 hours after inoculation. Infection was rated 4 days after inoculation based on the number of wounds which became infected (Table 3).

Table 3.

Application Timing Percent Infection

Treatment (hours after inoculation) 4 days after inoculation

None — 84 l% a.i 0 100 5 58 16 2 24 4

Example 3 — Dose Response Charaαeristics of Pelargonic Acid for the Control of Green Mold of Lemons

Lemons grown without the application of synthetic chemicals were harvested and surface disinfeαed by washing in a 1:10 dilution of household bleach. They were inoculated at 5 marked locations with spores of Penicillium digitatum (10⁵ conidia per ml) by pricking to a depth of 2 mm with an 18-gauge needle dipped into the spore suspension. They were incubated for 18 hours at 22°C in closed, plastic boxes on trays above wet paper towels. At that time they were removed and immersed for 15 seconds in dilutions of pelargonic add or in water. The lemons were allowed to drain-off and then returned to the boxes to incubate at 22°C for 13 days. Disease was rated 13 days later based on the percentage of wound sites which became infected (Table 4).

Table 4.

10⁵ conidia/ml %

Treatment a.ι. infection 13 DAT

80% formulation 1.0 80 (emulsified suspension of 0.5 50 pelargonic add)

0.25 0

0.125 0

0.063 0

Water control 30

Example 4 — Efficacy of Pelargonic Add in Various Forms for the Control of Green Mold of Lemons Lemons grown without the application of synthetic chemicals were harvested and surface disinfeαed by washing in a 1:10 dilution of household bleach. They were inoculated at 5 marked locations with spores of Penicillium digitatum (10° conidia per ml) by pricking to a depth of 2 mm with an 18-gauge needle dipped into the spore suspension. They were incubated for 18 hours at 22°C in closed, plastic boxes on trays above wet paper towels. At that time they were removed and immersed for 15 seconds in dilutions of an emulsified suspension of pelargonic add in a surfactant blank without the fatty add, or in pelargonic add which was converted to the potassium salt by titration to pH 7 with 10 N KOH. The lemons were allowed to drain-off and then returned to the boxes to incubate at 22°C. As in the other examples, the spore concentrations and the incubation temperature used constitutes a severe test of the ability of an agent to provide control of this disease. Either a delay in disease onset or in the highest disease level realized constitutes an indication of useful control under aαual storage conditions. Disease was rated 5 days later based on the percentage of wound sites which became infeαed (Table 5).

Table 5.

% infection 5 days after

Treatment a.ι. inoculation

Pelargonic add 2.0 8

0.5 12

0.125 10

Pelargonic add 2.0 0 converted to 0.5 8 potassium salt

0.125 18

Surfactant blank 2.0 70

0.5 64

0.125 90

Example 5 — The Activity of Various Salts and Esters of Pelargonic Add for the Control of

Lemon Green Mold

Lemons were surface disinfeαed in 10% bleach and dried. These were stab-inoculated with a 3 mm long, 18-gauge needle dipped into a spore suspension containing 10° spores/ml of

Penicillium digitatum. Five injuries were made in each fruit along a diagonal mark. The lemons were incubated at 22°C for 18 hours at high humidity. The fruit was then treated with various salts or esters of pelargonic add.

To synthesize ethylene glycol monopelargonate, 51.5 g pelargonic add and 51 g ethylene glycol were dissolved in 200 ml of dichloromethane, and 20 drops of H₂S0₄ were added to the mixture. This mixture was stored at room temperature for 6 days. After 6 days, 150 ml of 0.1 N NaOH was added to the reaction mixture which was then vigorously shaken. The dichloromethane layer (lower layer) was collected and washed with saturated NaCl solution.

After drying on Na₂S0₄, the chloroform layer was evaporated. Remaining oil (38 g) was subjected to vacuum distillation yielding 34.8 g (yield 53.8%) of ethylene glycol monopelargonate

(b.p. 135-137°C (7 mm Hg)). A ready-to use aqueous formulation of the isopropylamine salt of pelargonic (nonanoic) add was prepared. The pelargonic add was obtained as "EMERY 1202" from Quantum Chemical Corporation, Cindnnati, Ohio, and is a mixture of normal fatty adds of chain length 8, 9, and 10, with C9 being predominant Various aqueous formulations were prepared with up to 20% active ingredient as the fatty add and up to 6% isopropylamine, with the balance being water. The requisite amount of pelargonic add was dispensed into an appropriate mixing vessel and the mixing initiated. The requisite amount of water was added to the add and the add dispersed into the water by mixing, thus forming a cloudy, unstable dispersion. Isopropylamine (Aldrich Chemical Company, Milwaukee, WI) was added slowly, with continuous mixing, in suffident quantity to bring the pH of the formulation to approximately 7.4-7.8. At this approximate pH the cloudy dispersion became translucent as the fatty add isopropylamine salt became water soluble.

The 2% treatments were applied with a cotton swab, the lower concentrations were applied by dipping the fruit in the test solution. The fruit was incubated in the same conditions for an additional 72 hours, after which the infeαion was rated based on the number of injury sites exhibiting characteristic softening and sporulation. The percent disease control was calculated by comparing the level of infection to that in the untreated check (83% of injuries infeαed).

Table 6.

Treatment concentration applied percent disease control

Potassium salt 2.0 83

0.5 90

0.125 86

Isopropyl amine salt 2.0 71

0.5 95

0.125 81

Mono-ethylene glycol ester 2.0 56

0.5 76

0.125 54

Ethyl ester 2.0 16

0.5 16

0.125 0

Pelargonic add emulsion 2.0 69

Ammonium salt 2.0 98

Example 6 — Treatment of Citrus with Fatty Add Compositions

All fruits were washed with 100 ppm chlorine followed by a fresh water rinse. The pathogens (Penicillium digitatum or Penicillium italicum) were inoculated by painting a scratch injury (20-30 mm long by 1.5-2 mm deep) with a cotton swab dipped into a spore suspension of 1° conidia ml. 20-24 hours after inoculation, fruit was treated by dipping or, in some cases, using a commercial style washer waxer machine. The fruit was then stored at 70°F at 80-90% RH for 7 days or when the check treatment had become 100% infeαed. For each treatment there were three replicates of 15-20 fruits. Percent decay was calculated by the ratio of diseased to sound fruit regardless of the severity of the disease lesion. Table 7 shows the results on lemons. The treatments were applied in packout wax. Table 8 shows the results on oranges with dip treatment Table 9 shows the results on lemons dipped for 3 minutes at 105°F, then rinsed. Table 10 shows results on lemons dipped and then treated in the storage wax. Table 11 shows dip treatment of early and late Valenda oranges.

Table 7.

Rate (ppm aαive) Percent Decay wax only - 55

Potassium pelargonate 0.5% 63

0.75% 40

1.0% 21

Thiabendazole 5000 ppm 8

Table 8.

Treatment Concentration Fruit Injury Percent Decay

Check no 100

SOPP 5000 ppm no 18

Potassium 0.5% no 42 pelargonate

1.0% no 25

1.5% no 25

2.0% no 22

2.5% no 22

Pelargonic add 0.5% no 63

1.0% yes 40

1.5% yes 40

2.0% yes 20

2.5% yes 22 Table 9.

Treatment Concentration Percent Decay

Check - 100

Potassium pelargonate 1.0% 90

2.0% 15

Soda Ash 3.0% 21

Table 10.

Dip Treatment Wax Treatment Percent Decay at 7 Percent Decay at

Days 22 Days

Check Check 100 100

3% Soda Ash 2000 ppm lmazilil 0 9

5% Borax 2000 ppm lmazilil 0 0

2% Potassium 2000 ppm lmazilil 0 22 pelargonate

2% Potassium 0.5% Potassium 12 42 pelargonate pelargonate

2% Potassium 1.0% Potassium 3 25 pelargonate pelargonate

Table 11.

Treatment Percent Decay Late Percent Decay Early Valenda Valencia

Check 100 100

1% Potassium pelargonate, 40 8 rinsed

2% Potassium pelargonate, 22 4 rinsed

1% Potassium pelargonate, 30 0 not rinsed

2% Potassium pelargonate, 2 0 not rinsed

0.5% SOPP, rinsed 0 Example 7 — Efficacy of Pelargonic Add and its Salt for the Control of Botrvtis cinerea Infection of Pears and of Monilinia fructicola Infeαion of Nectarines

Undamaged apples were prick inoculated with spores of Botrytis cinerea (10° conidia ml) by dipping an 18 gauge needle in the spore suspension and using it to make a 2 mm deep wound at 5 locations on each of 4 fruits. Nectarines were similarly inoculated with Monilinia fructicola (10⁶ cfu/ml). The fruits were then placed in closed, plastic boxes on trays above wet paper towels. After 18 hours of incubation at 22°C, the fruits were removed and dipped for 15 seconds in water, in dilutions of an emulsified suspension of pelargonic add or in pelargonic add converted to its potassium salt by titration to pH 7 with 10 N KOH. They were returned to the boxes and allowed to incubate for 7 or 14 days at 22°C at which time they were rated for percent infection based on the number of wounds which developed decay typical of the disease in question (Table 12).

Table 12.

% Infection % Infection % Infection

Botrytis Monilinia Monilinia

Pears Nectarines Nectarines

Treatment % a.i. 7 DAT 7 DAT 14 DAT

Emulsified add 1.0 35 0 33 0.25 35 0 33 0.063 70 45 100

Pelargonic add 1.0 15 0 25 converted to potassium 0.25 20 25 80 salt 0.063 30 70 100

Water control 55 50 85

Example 8 — Dose-Response Effects of Various Fatty Adds for the Control of Powdery Mildew on Kentucky Blueerass

Kentucky Bluegrass plants were grown in 6-cell Jiffy strips for four weeks, cut to 5 cm and transplanted cell-by-cell into a 4 inch plastic pot with Promix putting medium. One half of the pots were allowed to become naturally infested with powdery mildew (Erysiphe graminis) so that 85-100 percent of the leaf area was covered with sporulating colonies of the fungus. The other half of the pots were grown without exposure to powdery mildew. Both types of plants were treated with water (as a control), with dilutions of "M-PEDE" (mainly potassium salts of cl8:0 fatty adds), or with dilutions of emulsified pelargonic add. The already infected plants thus received a "curative" treatment One day after the treatment, the as-yet uninfected plants ("preventive treatment") were inoculated by shaking heavily infected plants over the pots. All plants were then incubated in a greenhouse and at different intervals were evaluated for percent coverage of the leaf surface by powdery mildew. These results are expressed as percent control relative to the water check for the curative treatments and percent infection for the preventive treatments (Table 13).

Table 13.

Treatment Curative %

% a.L Control 15 DAT

"M-PEDE" (potassium salts of 1.0 90 prindpally cl8 fatty adds) 0.5 80 25 57 .125 20

Emulsified pelargonic add 0.8 97 ' 0.4 82 0.2 73 0.1 43

Water control 0

Example 9 — Control of Mildew on Roses

Roses (cv 'Samantha') in a commercial greenhouse were treated five times on a weekly basis for control of powdery mildew. Applications were made with a backpack sprayer at 100 gpa. No plant or flower injury occurred in any of these treatments. Because this was a commercial greenhouse, it was not possible to leave an untreated check. Under these conditions, untreated roses would have extremely severe mildew infection. What this data shows is that the fatty adds can be applied many times without injury and that the mildew is kept under control, decreasing over time. The results are shown in Table 14.

Table 14.

Treatment Concentration Mildew colonies per Mildew colonies per (percent active) plot after 3 weeks of plot after 4 weeks of treatment treatment

M-PEDE 0.75 162 30 (Potassium oleate) 0.5 162 81

Potassium cocoate 0.6 73 16 0.4 55 36

Example 10 — Control of Powdery Mildew with Fatty Adds and Various Derivatives

Wheat var. Newton was grown for one week in a 17°C growth chamber. After one week wheat powdery mildew (Erysiphe graminis) was applied to seedlings by shaking infeαed plants over the top. After 5 days treatments were applied with an airbrush. Materials were applied to wet the leaves but without excessive runoff. This approximated 200 to 300 gallons per aαe. Infeαed plants were placed in sαeen cages in a 17°C growth chamber. There were 6 replicate pots per treatment with three plants in each pot Plants were rated for visible mildew colonies 5-7 days after inoculation. The top 1/3 of the first true leaf was rated for percent coverage with mildew. Results shown in Table 15 are expressed as percentage control as compared to either water or no treatment

Table 15.

Compound Rate (ai) % Control

"M-PEDE" 0.8 87

"M-PEDE" 0.4 58

"M-PEDE" 0.2 12

Soyamide DEA 0.2 76

Soyamide DEA 0.1 58

Potassium Cocoate 0.6 90

Potassium Cocoate 0.3 90

Potassium Cocoate 0.15 56

Cocoamide DEA 0.1 82

Cocoamide DEA 0.05 76

Example 11

Ripe lemons were marked with a line using a felt-tip pen. Five three-millimeter deep stab injuries were made along the line using an 18-gauge stainless steel needle. A cotton swab was used to apply a spore suspension of Penicillium italicum to those injuries (3x10° spores/ml). The lemons were then incubated for 24 hours at 22°C in a closed container. After this incubation, lemons were immersed in various test solutions for a few seconds and then returned to a closed, humid box for 22°C incubation for 10 days. At the end of that time, the lemons were rated for "blue mold" decay with the following results: Table 16.

Test Solution Percent "Blue Mold"-Damaged Lemons

Water 69

Pelargonic add - 0.15% &.L, oil/water 35 emulsion

Cocoamide DEA - 0.4% in water 50

Because the lemons used in this test were ripe and because the inoculum rate was high, even the incomplete confrol observed with the chemical freatments was significant

Example 12 — Control of Mildew on Grape Plants

In this grape test, both potassium cocoate and potassium oleate were tested at low concentrations mixed with sulfur. The safety with the cocoate is excellent

Applications of the sulfur and fatty add composition mixtures were made to a commercial vineyard (cv. 'Chardonnay') using a "mist blower" sprayer applying 100 gallons of spray per aαe. The expanding leaves were rated 4 days later for any damage from the earlier spray. The form of sulfur used in this experiment is the product "Thiolux," which is a wettable 80% sulfiir formulation. Results are shown in Table 17.

Table 17.

Treatment Concentration^) Observations of expanding (% aαive ingredient) leaves

Potassium cocoate 0.3% no injury 0.5% no injury

Potassium cocoate plus 03%, 0.48% no injury Thiolux 0.5%, 0.48% no injury

Potassium oleate 0.75% no injury

Potassium oleate plus 0.75%, 0.48% many expanding leaves Thiolux severely burned

Thiolux 0.48% no injury

-Example 13 — Evaluation of Phytotoxidty of Fatty Acid Component Combined with Sulfur

An experiment was conduαed on 2-year old grape vines growing outdoors in 3-gallon containers. The vines had new shoot growth of 3 to 12 inches and were growing rapidly. Test materials were applied to these using a syringe hand sprayer which makes a spray through a normal agricultural flat fan nozzle. The application was "high volume," consisting of 40 ml per pot an amount which led to runoff from the treated leaves. Following the application the vines were moved inside overnight to a lighted growth chamber with 28°C daytime temperature and 25°C nighttime temperature. After spending approximately 16 hours in this chamber, the plants were removed for phytotoxidty evaluation expressed as % leaf damage on expanding leaves. The test materials included various fatty adds, fatty add derivatives, wettable sulfur ("THIOLUX"), and mixtures of the former materials with sulfur. Each material was tested at the desired, fungiddal use rate for the field. The results are presented in Table 18.

Table 18. Phytotoxidty ratings of grape shoots

Test material Concentration % leaf injury on % a.i. expanding leaves

"M-PEDE" (potassium oleate) 0.75 v/v none

"M-PEDE" 0.75 v/v 80-90% plus sulfur 0.48 v/v

Potassium cocoate 0.4 v/v none

Potassium cocoate 0.4 v v 10-20% plus sulfur 0.48 w v

Potassium cocoate 0.375 v/v none

Potassium cocoate 0.375 v/v 0-5% plus sulfur 0.48 w v

Cocoamide-DEA 0.25 w v none

Cocoamide-DEA 0.25 w/v 70-80% plus sulfur 0.48 w/v

Cocoamide-DEA 0.15 w/v none

Cocoamide-DEA 0.15 w v 40-50% plus sulfur 0.48 w/v

Example 14 — The Use of Various Agents Including Fatty Acids for the Disruption of Peanut Leaf Surface Miαoflora

Peanut plants were grown in the greenhouse for three weeks, after which time they were sprayed with a leaf-washing suspension from local landscape plants. This provided a charge of potential leaf surface-colonizing miαobes. These plants were then held each night in a 22°C dew chamber and placed outdoors in full sun each day. This step provides realistic selection pressure for normal leaf surface miαoflora. After three days in this regime, the plants were freated each day with various agents with the potential to exercise selective pressure on the population of miαobes and thus to enrich for organisms tolerant to or favored by the applied agent. The agents consisted of various combinations of potassium carbonate (0.05%), the potassium salt of pelargonic add (0.5%), and yeast ghosts (10⁸ cells ml, Baker's yeast killed by boiling and washed extensively by centrifugation). The potassium carbonate and pelargonic add were considered "negative" selection agents and the yeast ghosts were considered a "positive" selection agent These agents were applied on each of three days, during which time the plants continued to cycle between the dew chamber and sun exposure. At the end of this freatment period, individual leaves were harvested and washed to recover surface colonizing organisms. These washings were dilution-plated on both nutrient agar and potato dextrose agar, and the mean populations recovered are listed in Table 19.

Table 19. Mean colony forming units recovered per peanut leaf following enrichment with various agents cfu/leaf on cfu/leaf on potato

Selection treatment applied nutrient agar dextrose agar

1. water 1.9 x 10⁵ 4.4 x 10⁵

2. yeast ghosts 1.9 x 10⁶ 8.3 x 10^s

3. 0.5% K⁺ salt of pelargonic add 2.9 x 10⁴ 1.1 x 10⁴ plus 0.05% potassium carbonate

4. a combination of the components 3.2 x 10⁴ 3.3 x 10⁴ of treatments 2 and 3 above

In addition to effects on the total surface populations recovered, these treatments also had evident effects on the composition of the miαoflora (based on colony size, morphology, color, and growth on different media). Following this enrichment procedure, individual colonies can be isolated. Microorganisms thus isolated can then serve as hosts for heterologous genes which may be transformed into said host. Advantageously, these heterologous genes could code for a protein which is toxic to a plant pest Such toxins are widely known in the art as are the genes which code for these toxins. For example, it is well known that many Bacillus thuringiensis express proteins which are toxic to plant pests. B.ts may be applied to plants, according to the subjeα invention, in conjunction with fatty add treatment or, alternatively, Bx genes coding for toxins may be placed into, and expressed in, other hosts which are particularly adapted to growth and persistence on plants, especially in the presence of fatty adds. Methods for inserting these genes into an appropriate host are also well known. See, for example, published European Patent Application 0200344. The transformed microorganism can then be applied to appropriate plants in need of protection from pests. The plants may first be freated with a fatty add composition to clear away competing miαobes and to control baαerial and fungal infection, if necessary. Once transformed miαobes are applied to the plants, fatty adds may subsequently be applied to clear away competing or undesirable miαobes. Applications of fatty add may be accompanied by enrichment agents to assist the colonization of the desired microbes. Also, the desired miαobes may be further transformed with additional gene(s) which make these miαobes particularly adapted to selective enrichment

Example 15 — Application of Microfloral Disruption Agents in a Field Enrichment Protocol

Agents with the potential to disrupt leaf and flower surface miαoflora were applied on 4 days during a 1 week period to tomato plants in a commercial production field. After this period, leaf washings from 15-20 separate leaflets or flowers were dilution-plated for each freatment and the number of colonies (yeasts and bacteria) were determined as reported in Table 20.

Table 20. Mean colony forming units recovered per leaflet or flower following a field selection protocol.

Recovery on Nutrient Agar Recovery on Potato Dextrose Agar

Selection treatment Flowers Leaves Flowers Leaves

1. water 16,250 5,200 1,175 1,488

2. yeast ghosts 48300 12,940 51,700 10,740

3. K⁺ salt of 8,275 6,141 1,225 741 pelargonic add

4. 2 and 3 7325 2-520 2,160 1,158

As in Example 12, these agents were able to alter both the density and composition of the miαoflora on leaves and flowers. As described in Example 12, miαobes isolated after application of selection treatments can be used as excellent plant colonizers for application of recombinant toxin-producing miαobes.

Example 16 — Insecticidal Aαivitv of Fatty Adds and Various Derivatives

A colony of green peach aphids was maintained on lettuce at 17°C in the growth chamber. For testing purposes, 10-20 aphids were transferred to detached lettuce leaves, which were placed in petri plates containing a moistened filter paper. Test solutions were applied at a rate of 1.5 ml per plate (200-300 gallons per aαe). The covers were placed on the petri plates. The number of dead aphids was counted after 1 hour. Experience has shown that material efficacy under this test system is 8-10 time greater than real world conditions. For example, the effective insecticidal rates of "M-PEDE" in the field is 0.75-1.0% ai, whereas in this described test system, the lowest effeαive concentration is 0.13%. This relationship holds for other inseαiridally active materials. Results are shown in Table 21. The compounds of the subjeα invention can be used as pestiddes at concentrations from about 0.05% to about 1% or more. Table 21.

Compound Rate (ai) % Control

"M-PEDE" 0-5 91

"M-PEDE" 0.25 87

"M-PEDE' 0.13 73

"M-PEDE" 0.06 45

"M-PEDE" 0.03 25

Cocoamide DEA 0.05 100

Cocoamide DEA 0.03 88

Cocoamide DEA 0.01 73

Potassium Cocoate 0.2 15

Potassium Cocoate 0.1 4

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Claims

1. A method for the control of established fungal or baαerial plant disease, said method comprising the application of a fungiddal or baαeriddal amount of a fatty add, its salt or derivative, or mixture thereof; to the situs of said plant disease.

2. The method, according to claim 1, wherein said fatty add, its salt or derivative, is a compound which can be represented by Formula I or Formula IA, wherein Formula I is as follows:

wherein Z = O, N, or S R_j = C5 to C19 saturated or unsaturated hydrocarbon, or an epoxide, or cyclopropane thereof Y_j = H, Ca_j hydrocarbon, or hydroxyl at any position along R_j Y₂ = H, C_j-C₅ hydrocarbon, or hydroxyl at any position along R_α R₂ -= C--C₁₀ saturated or unsaturated, branched or unbranched, hydrocarbon having at least one hydroxyl group at any position on R₂; salts; or H; and Formula IA is as follows:

ft R_ R.YιY₂CN^ IA R$ wherein R_j = C5 to C19 saturated or unsaturated hydrocarbon, or an epoxide, or cyclopropane thereof Y_x = H, C-^-C_ζ hydrocarbon, or hydroxyl at any position along R_χ Y₂ = H, C_j-C₅ hydrocarbon, or hydroxyl at any position along R_x R₂ = J-CJO saturated or unsaturated, branched or unbranched, hydrocarbon which may have one or more hydroxyl groups at any position on R₂; carbohydrate; salt; or H; R₃ = CJ-CJ_Q saturated or unsaturated, branched or unbranched, hydrocarbon which may have one or more hydroxyl groups at any position on R₂; carbohydrate; salt; or H.

3. The method, according to claim 2, wherein Z = O, and R₂ is seleαed from the group consisting of: aliphatic amines which form cationic aliphatic ammonium compounds; K; Na; and H.

4. The method, according to claim 3, wherein R₂ is isopropyl amine.

5. The method, according to claim 2, wherein said compound is a mono-glycol ester.

6. The method, according to claim 2, wherein said fatty add, or mixture thereof, is selected from the group consisting of oleic add, linoleic add, linolenic add, pelargonic add, lauric add, myristic add, their salts and esters.

7. The method, according to claim 2, wherein said fatty add, its salt or derivative, is contained in a natural product which is formulated for the purpose of application to αops, produce, or wood products.

8. The method, according to claim 2, wherein said compound is a diethanolamine fatty add compound.

9. The method, according to claim 8, wherein said compound is seleαed from the group consisting of cocoamide DEA, non-ionic coconut amides, coconut diethanolamides, and fatty diethanolamides.

10. The method, according to claim 2, which further comprises the application of a synthetic or inorganic fungidde or baαeridde.

11. The method, according to claim 10, wherein said synthetic or inorganic fungidde or baαeridde is seleαed from the group consisting of benomyl, borax, captafol, captan, chlorothalonil, various forms of copper and zinc, dichlone, dicloran, iodine, fenarimol, ima alil, myclobutanil, propiconazole, prochloraz, terbutrazole, flusilazole, triadimefon, tebuconazole, folpet, iprodione, mancozeb, maneb, metalaxyl, oxycarboxin, oxytetracycline, PCNB, pentachlorophenol, quinomethionate, sodium arsenite, sodium DNOC, sodium hypochlorite, sodium phenylphenate, streptomycin, sulfur, thiabendazole, thiophanate-methyl, triforine, vindozolin, zineb, ziram, tricyclazole, cymoxanil, blastiddin and validimycin.

12. The method, according to claim 2, wherein said plant disease is caused by a pathogen seleαed from the group consisting of Penicillium sp., Botrytis sp., Monilinia sp., Alternaria sp., Aspergβus sp., Mucor sp., Rhizopus sp., Geotrichum sp., Diplodia sp., CoUetotrichum sp., members of the orders Erisyphales, Peronosporales, Hemiascomycetes, Venturia sp., Cercospora sp., Cercosporidium sp., Pseudocercosporella sp., Myrothecium sp., Fusarium sp., Ophiostoma sp., Erwinia sp., Pseudomonas sp., and Xanthomonas sp.

13. The method, according to claim 12, wherein said pathogen is selected from the group consisting of Penicillium sp., Botrytis cinera, Monilinia ftucticola, and powdery mildew fungi in the order Erisyphales.

14. The method, according to claim 2, wherein said fungal plant disease is affecting produce seleαed from the group consisting of dtrus, bananas, mangos, tomatoes, pears, grapes, apples, peaches, cherries, apricots, and nectarines.

15. The method, according to claim 2, wherein said fatty add is applied to foliage or produce before harvest

16. The method, according to claim 2, wherein said fatty add is applied to foliage or produce after harvest

17. The method, according to claim 2, which further comprises the administration of a biocontrol agent

18. A method for the control of pests which comprises applying to said pests a pestiddal amount of a fatty add amide.

19. The method, according to claim 18, wherein said pest is an insect or a mite and wherein said fatty add amide has the following formula:

ft Rz RjYjYzCN IA wherein * & R_j -> C5 to C19 saturated or unsaturated hydrocarbon, or an epoxide, or cyclopropane thereof Y_j = H, C,-C₅ hydrocarbon, or hydroxyl at any position along R_x Y₂ = H, <- -C₅ hydrocarbon, or hydroxyl at any position along R- 32 R₂ = Cγ-C^_Q saturated or unsaturated, branched or unbranched, hydrocarbon which may have one or more hydroxyl groups at any position on R_j carbohydrate; salt; or H; R₃ = CJ-CJO saturated or unsaturated, branched or unbranched, hydrocarbon which may have one or more hydroxyl groups at any position on R^ carbohydrate; salt; or H.

20. The method, according to claim 19, wherein said fatty add amide is a diethanolamine fatty add compound.

21. The method, according to claim 20, wherein said diethanolamine compound is seleαed from the group consisting of cocoamide DEA, non-ionic coconut amides, coconut diethanolamides, and fatty diethanolamides.

22. The method, according to claim 19, wherein said fatty add amide is a compound produced when diethanolamine is reaαed with a composition comprising a fatty add or salt thereof.

23. The method, according to claim 22, wherein said fatty add composition is coconut oil fatty adds.

24. The method, according to claim 19, which comprises applying cocoamide DEA to said inseα or mite pest.

25. A method for manipulating the miαobial population of a wood, plant, or harvested produce surface, said method comprising the application of a negative selection agent to said wood, plant, or harvested produce surface to disrupt the resident surface miαoflora and allow enhanced colonization of that surface by microorganisms favored by the application of said selection agent

26. The method, according to claim 25, wherein said negative selection agent is chosen from the group consisting of fatty adds, and their salts and derivatives; natural products containing fatty adds, and their salts and derivatives; carbonate salts; agricultural spray oils derived from petroleum based products; alcohols; inorganic metals; and combinations thereof.

27. The method, according to claim 26, wherein said fatty add, its salt or derivative, is a compound which can be represented by Formula I or Formula LA, wherein Formula I is as follows:

wherein Z = O, N, or S R_j = C5 to C19 saturated or unsaturated hydrocarbon, or an epoxide, or cyclopropane thereof Y_j = H, C_j-C₅ hydrocarbon, or hydroxyl at any position along R_α Y₂ = H, C_j-C₅ hydrocarbon, or hydroxyl at any position along R_j R₂ = C_j-C_j0 saturated or unsaturated, branched or unbranched, hydrocarbon having at least one hydroxyl group at any position on R₂; salts; or H; and Formula IA is as follows:

wherein R_j = C5 to C19 saturated or unsaturated hydrocarbon, or an epoxide, or cyclopropane thereof Y_j = H, C_j-C₅ hydrocarbon, or hydroxyl at any position along R_j Y₂ = H, C_j-C₃ hydrocarbon, or hydroxyl at any position along R_j R₂ = C_j-C_j0 saturated or unsaturated, branched or unbranched, hydrocarbon which may have one or more hydroxyl groups at any position on R₂; carbohydrate; salt; or H; R₃ = CJ-CJ_Q saturated or unsaturated, branched or unbranched, hydrocarbon which may have one or more hydroxyl groups at any position on R^ carbohydrate; salt; or H.

28. The method, according to claim 27, wherein Z = O, and R₂ is seleαed from the group consisting of: aliphatic amines which form cationic aliphatic ammonium compounds; K; Na; and H.

29. The method, according to claim 26, wherein said agricultural spray oil is chosen from the group consisting of SunSpray oils 6N, 7N, 8N, 9N, and UN.

30. The method, according to claim 26, which further comprises the addition of a positive selection agent in combination with the negative selection agent and wherein said positive selection agent is chosen from the group consisting of yeast ghosts, bacterial ghosts, algal ghosts, complex carbohydrates, simple carbohydrates, organic nitrogen, and combinations thereot

31. The method, according to claim 26, wherein said method is used to promote the establishment and growth of a desired miαoorganism on a plant surface, said method comprising applying a composition comprising a fatty add, its salt or derivative, as defined in claim 32, to said plant, wherein said method further comprises applying said desired miαoorganism to said plant

32. The method, according to claim 31, wherein said desired miαoorganism is seleαed from the group consisting of baαeria, filamentous fungi, yeasts, and actinomycetes.

33. The method, according to claim 31, wherein said desired miαoorganism has been seleαed or engineered for tolerance to fatty adds.

34. The method, according to claim 31, wherein said desired miαoorganism is selected or engineered for resistance to fatty adds.

35. The method, according to claim 31, wherein said desired miσoorganism expresses a toxin which is toxic to a plant pest

36. The method, according to claim 35, wherein said miαoorganism is a Bacillus thuringiensis.

37. The method, according to claim 35, wherein said miαoorganism comprises a heterologous gene wherein said gene expresses said toxin.

38. The method, according to claim 37, wherein said heterologous gene is a Bacillus thuringiensis gene.

39. The method, according to claim 26, wherein said method is used to isolate microorganisms resistant to fatty adds, their salts or derivatives, said method comprising applying a composition comprising a fatty add, its salt or derivative, to said plant and culturing the remaining miαobes to isolate miαoorganisms which have survived said application of fatty add, its salt or derivative.