WO2001001977A1 - Methods for controlling fungal pathogens - Google Patents

Abstract

The invention is directed to methods and compositions for controlling fungal pathogens. The compositions comprise a compound having fungicidal activity and one of the formulas: (I), (II), (III), (IV).

Description

METHODS FOR CONTROLLING FUNGAL PATHOGENS

BACKGROUND Technical Field This invention relates to a method of controlling fungal pathogens. More particularly, this invention relates to fungicides comprising naturally-occurring compounds or compounds derived from naturally-occurring compounds.

Background Fungal pathogens are detrimental to man, his food and fibers. Such pathogens infest plants and cause economic loss of plant crops, and are also capable of infesting animals, including man. Methods for controlling fungal pathogens on plants include spraying plants with fungicides on a 3-21 day schedule when environmental conditions favor disease development. Typical fungicides used include compounds of heavy metals such as copper and arsenic, as well as organosulfur and organochlorine compounds. These compounds are often not satisfactory because of their potential for accumulating in the environment to levels considered to be unsafe, in particular contaminating natural resources such as drinking water and polluting the soils. Further, state of the art fungicides have strong physiological effects on the plants, may produce residues that are toxicity to food crops, can have high animal toxicity, and are potentially hazardous to workers using them.

Biorational fungicides, i.e., those that are naturally-occurring, also are used in controlling fungal pathogens. Such biorational fungicides include phosphates and sodium bicarbonate. Unfortunately, excessive use of phosphates produces agricultural runoffs that can cause water pollution and bicarbonate salts are problematic in that they are fungicidal only at pH 8.6 and are non-fungicidal at pH 6.0, along with possibly being phytotoxic as a function of environmental conditions at the time of treatment. Anti-transpirants are also used for the control of fungi in the field. These chemicals are applied directly to a plant and reduce the rate of transpiration or water loss by the plant. Anti-transpirants form a film on a plant surface that acts as a barrier against invading fungi. The anti-transpirant formulations are reported to have low mammalian toxicity, but, because the anti-transpirant controls fungal infestations through the formation of a protective barrier, it is useful only as a means of prevention rather than as a treatment of an already infected plant.

Therefore, it is of interest to identify and develop compositions and methods for controlling the growth of fungal pathogens which use formulations derived from natural products or are known to have lower environmental toxicity than the formulations currently in use, while remaining effective in controlling fungi without damaging a treated plant and/or plant part or a host tissue. It also is of interest to develop a new composition which is effective against more than one kind of fungal pathogen so as to decrease the need for application of multiple control agents, is obtainable from a readily available source or is easy to manufacture, and is relatively inexpensive.

Eutypine is a toxin produced by Eutypa lata, a fungus that attacks grapevines and apricots, causing localized necrosis in the wood, stunting, and ultimately, death of limbs or cordons. Research has been conducted on E. lata in an attempt to identify a means of combating this disease due to its serious impact on the wine industry. See for example,

Deswarte, et al., J. Plant. Physiol. (149):336-342 (1996). Much of this research has focused on disease prevention by developing resistant plant varietals. See for example, Mauro, et al., Am. J. Enol. Vitic. 39(3):200-204 (1988). Research has also focused on learning more about eutypine, its routes of biosynthesis and its metabolites, in an attempt to understand how the disease works. Siccayne, another compound produced by Eutypa lata, has been suggested to have marginal antifungal activity when tested against human fungal pathogens in the classes Ascomycetes and Basidiomycetes. Reported Pyricularia activity was noted but was not supported or quantified (Kupka, et al., The Journal of Antibiotics 34(3):298-304 (1981)). Eutypine and its other related compounds have never been evaluated for antifungal properties and it is a surprising discovery that these compounds, produced by the E. lata fungus, are themselves useful as fungicides.

Objects of the Invention One object of this invention is to provide a new family of fungicides based on easily obtainable compounds. Another object of this invention is to provide a new family of fungicides using naturally-occurring compounds or compounds derived therefrom.

Yet another object of this invention is to provide a new family of fungicides having low toxicity to ornamental and agricultural plants, domesticated animals and wildlife, and humans when used at a fungicidally effective level. Other objects may be apparent to one of skill in the art upon reading the following specification.

SUMMARY OF THE INVENTION The present invention is directed to a method for controlling a fungal pathogen, such as by eliminating or deterring the growth of the fungal pathogen, using a composition containing a fungicidally effective amount of a compound of the invention. More particularly, this invention relates to fungicides comprising naturally-occurring compounds or compounds derived therefrom. The method finds use in controlling fungal pathogens and in preventing infestation of a host with a fungal pathogen, where the fungal pathogen is a pathogenic fungus that infests plants and/or animals. The method of the invention includes the step of contacting the fungal pathogen with a fungicidally effective amount of a composition comprising a compound of the invention in combination with an agriculturally or pharmaceutically-acceptable carrier. The invention is also directed to a method for preventing infestation of a host with a fungus by contacting the host, such as by spraying, with a composition comprising a compound of the invention in combination with an agriculturally-acceptable carrier in an amount sufficient to prevent infestation.

The invention is also directed to a method for treating a fungal infestation of an animal host by contacting the host, such as by an inhaled aerosol formulation, with a composition comprising a compound of the invention in combination with a pharmaceutically-acceptable carrier in an amount sufficient to eradicate such infestation.

The invention also provides a composition comprising a suitable carrier in combination with a fungicidally active ingredient of the invention. The present invention is also directed to uses for such compositions in methods for controlling a fungal population such as by preventing infestation of a host with a fungus, eradicating fungal infestation on a plant or animal, and treating a fungal infestation of an agricultural or ornamental plant in need thereof.

The invention also pertains to an article of manufacture comprising a container in association with instructions and/or a label indicating that the subject composition can be used to control fungal pathogen, i.e., used as a fungicide and holding a composition comprising an agriculturally- or pharmaceutically-acceptable carrier and a fungicidally active ingredient of the invention.

DETAILED DESCRIPTION Methods and compositions for controlling a fungal infestation of a plant or an animal, particularly a mammal, are provided. The composition is a fungicide and is either non- phytotoxic or not dermally toxic at the recommended dosage, if the intended application is to the tissue of the host. The compounds include eutypine, intermediates found in the natural pathway of its biosynthesis, and its major metabolites, along with analogues of such compounds. The preferred compounds are naturally-occurring. The fungal infestation is a pathogenic organism population which spreads disease and/or damages the host, and includes pathogenic fungi that infest plants and/or animals.

The present invention provides very efficacious fungicides which, in its preferred aspect, are designated as biorational. A biorational fungicide is a chemical substance of natural origin that can be synthesized. The preferred fungicides of the present invention have an inhibitory effect on specific fungal targets, when administered at the recommended dosage. Unlike the bulk of currently available fungicides on the market, the preferred compositions have active ingredients that are expected to be substantially non-toxic to man and domestic animals and which have minimal adverse effects on wildlife and the environment.

The compounds useful as fungicides in the methods and compositions of the invention relate to, and include, the acetylenic phenol, 4-hydroxy-3-(3-methyl-3-buten-l-ynyl)- benzaldehyde (CAS 121007-17-8), which is commonly referred to as eutypine (1):

Eutypine (1) is one of the numerous compounds produced by the Eutypa lata fungus. Tsoupras, et al., Bioact. Mol. 7:93-100 (1988) has proposed a biogenetic pathway of secondary metabolites isolated from a E. lata filtrate culture medium, which includes the following Schemes A-C:

SCHEME A

SCHEME B

SCHEME C

Accordingly, in one aspect of the invention, compounds useful as fungicides in the methods and compositions described herein, have the Formula (I) and exhibit anti-fungal activity:

where R¹ is selected from the group consisting of H, alkyl, alkylcarbonyl, benzoyl and C_1-6alkyloxyCι__₆alkyl; and R is selected from the group consisting of -OH, -CHO, -CH OH, -COOH, -C(O)CH₃ and C_1-6alkoxy; with the proviso that when R¹ is H, R² is not -OH.

Another aspect of the invention relates to compounds useful as fungicides in the methods and compositions described herein, having the Formula (II) and exhibiting antifungal activity:

where R is selected from the group consisting of H, alkyl, alkylcarbonyl, benzoyl and C_1-6alkyloxyC_1-6alkyl; and R⁴ is selected from the group consisting of -OH, -CHO, -CH₂OH, -COOH, -C(O)CH₃ and C_1- alkoxy; and R⁵ is selected from the group consisting of H, Cι- alkyl, C _1-6alkoxy, C _.₆alkenyl, C_2-6alkynyl and halogen, where the C_1-6alkyl, C_2-6alkenyl and C_2-6alkynyl groups are optionally substituted with 1-3 hydroxyl groups.

In another aspect of the invention, compounds useful as fungicides in the methods and compositions described herein, have the Formula (III) and exhibit anti-fungal activity:

where R is selected from the group consisting of H, Cι_-6alkyl, C_2-6alkenyl, C₂.₆alkynyl, and a straight or branched chain having 4-6 carbon atoms, a double and a triple bond; where the Cj-₆alkyl, C_2-6alkenyl, C_2-6alkynyl and chain are optionally substituted with 1-3 hydroxyl groups.

In another aspect of the invention, compounds useful as fungicides in the methods and compositions described herein, have the Formula (IV) and exhibit anti-fungal activity:

where R⁷ is selected from the group consisting of -OH, -CHO, -CH₂OH, -COOH, -C(O)CH₃ and C_1- alkoxy; and R⁸ is selected from the group consisting of H, C_1- alkyl, C - alkenyl and C_2- alkynyl; where C_1- alkyl, C_2- alkenyl and C_2-6alkynyl are optionally substituted with 1-3 hydroxyl groups.

As used herein, the term "alkyl" means a straight (unbranched) or branched saturated monovalent hydrocarbon radical containing 1 to 12 carbon atoms, such as methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-hexyl, n-octyl and the like, unless otherwise indicated. Preferably the alkyl group is a lower alkyl (branched or unbranched saturated monovalent hydrocarbon radical) having 1 to 6 carbon atoms (C]- alkyl), such as methyl, ethyl, tert-butyl, and the like.

The term "C_2-6alkenyl" refers to a straight (unbranched) or branched unsaturated divalent hydrocarbon radical containing about 1 to 3 double bonds and 2 to 6 carbon atoms, such as ethene, 1-propene, 1-butene, 3-methylbut-l-ene, 1-pentene, and the like.

The term "C_2-6alkynyl" refers to a straight (unbranched) or branched unsaturated trivalent hydrocarbon radical containing about 1 to 3 triple bonds and 2 to 6 carbon atoms, such as ethyne, 1-propyne, 1-butyne, 3-methylbut-l-yne, 1-pentyne, and the like. The term "Cι_-6alkoxy" refers to the group -O-Cj-_όalkyl where C_1-6alkyl is as herein defined.

The term "Cι_-6alkyloxyC_1-6alkyl" refers to the group Cι_-6alkyl-O-C_1-6alkyl where Cι_-6alkyl is as herein defined.

The term "alkylcarbonyl" refers to an alkyl group, attached through a carbonyl, - C(O)- group, and includes by way of example, methyl carbonyl and ethyl carbonyl and phenyl carbonyl. Preferably the alkylcarbonyl group is a Cι_-5alkylcarbonyl group.

The term "benzoyl" refers to the group -C(O)-phenyl, and is also known as phenyl carbonyl.

The term "halogen" refers to chloride (-C1), bromide (-Br), iodide (-1), or fluoride (-F). "Optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, "C_2-6alkenyl optionally substituted with 1-3 hydroxyl groups means that the C . alkenyl group may or may not be substituted with one, two or three hydroxyl groups and that the description includes both unsubstituted C_2-6alkenyl and substituted C _-6alkenyl.

Compounds useful in the methods and compositions of this invention are readily obtained by biosynthetic processes or are synthesized by techniques as are well known in the art. The compounds of the invention can be prepared by cultivating an organism which forms the compounds of Formulas (I), (II), (III) and (IV) and is of the species Eutypa lata, and then extracting the compounds from the mycelium and/or culture medium. Eutypa lata is available from known repositories. Using a simple small scale test, it is within the skill in the art to select from these microorganisms those which form the fungicidal compounds of the invention.

Suitable nutrient media for the cultivation of organisms are the conventional ones which contain carbon sources, nitrogen sources, inorganic salts and, if needed, small amounts of trace elements and vitamins. Suitable nitrogen sources include inorganic and organic nitrogen compounds or materials which contain these compounds, such as ammonium salts, corn steep liquor, brewer's yeast autolysate, soybean meal hydrolysate, wheat gluten, yeast extract, yeast, urea and potato protein. Suitable carbon sources include sugars, such as D- glucose, mannose or galactose, polyalcohols, such as mannitol, and alcohols such as ethanol. Exemplary inorganic salts are salts of calcium, magnesium, manganese, potassium, zinc, copper, iron and other metals. Growth factors such as pantothenic acid, p-aminobenzoic acid and thiamine, may also be added to the nutrient medium. The mixing ratio of the stated nutrients will depend upon the type of fermentation and is determined on a case by case basis. The compounds of Formulas (I), (II), (III) and (IV) are isolated from the mycelium or culture medium after the fermentation and purified by standard techniques. However, the methods of the invention may also be practiced by direct application of the fermentation broth itself, which may contain one or more compounds of Formulas (I) to (IV).

There are several methods known in the art for the chemical synthesis of compounds of Formulas (I) to (IV). Defranq, et al., Helvetica Chimica Acta 76(l):425-430 (1993) first described the chemical synthesis of eutypine. Bates, et al., Tetrahedrons 51 :8199-8212 (1995) and Amatore, et al., J. Org. Chem. 60:6829-6839 (1995) also describe suitable synthetic methods. All of the aforementioned descriptions being incorporated herein by reference.

The Defranq synthesis is summarized in Scheme D, by which compounds of Formula (I), such as eutypine (1) can be synthesized using commercially available 4- hydroxy benzaldehye (8) (Aldrich) as a starting material. (10)

(8) (9) DMF CH₃

Cu' ^^ CH₂

SCHEME D

10

Scheme E illustrates one method by which compounds of Formula (II) such as compounds (16), (19) and (20), can be synthesized:

₂CH(CH₃)₂

BrCH₂CHC(CH₃)₂ (17) NaBH, A1₂0₃ 25°C

T

SCHEME E

Scheme F illustrates yet another method by which compounds of Formula (II) such as compounds (21) and (22), can be synthesized, where Pd TPPTS is a palladium triphenylphosphine catalyst:

OCOCH₃ ^CH3 OCOCH₃ CH₃

^ ^" OH

CH₃ I O OHi CH₃

Pd TPPTS (21)

CHO CHO

Scheme G illustrates another method by which compounds of Formula (III) such as compound (24), can be synthesized:

SCHEME G

Scheme H illustrates another method by which compounds of Formula (IV) such as compound (25), can be synthesized:

SCHEME H

The compounds of the invention may exist as isomers, stereoisomers, enantiomers or diastereoisomers. A "stereoisomer" is an isomer (different compounds that have the same molecular formula) that differs only in the way the atoms are arranged in space.

"Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other, and a 1 : 1 mixture of a pair of enantiomers is referred to as a "racemic" mixture. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The compounds of Formulas (I) to (IV) may also be used in the methods and compositions of the invention in their salt form, as long as they retain the fungicidal effectiveness and properties of the compounds of Formulas (I) to (IV), and are not agriculturally, horticulturally, environmentally or otherwise undesirable. Such salts may be derived from bases. Exemplary salts include, by way of illustration and not limitation, metal salts such as sodium, potassium, calcium and magnesium salts; ammonium salts such as isopropyl ammonium salts; and trialkylsulfonium salts such as triethylsulfonium salts. The compounds of Formulas (I), (II), (III) and (IV) may also be used in the methods and compositions of the invention in their acidic form, and such forms are intended to be encompassed by the invention. For example, the compounds of the invention may be packaged in a buffer formulation and will thus be the basic form of the compounds of Formulas (I) to (IV).

Compounds of particular interest include those listed in Tables I-IV below, where the "R" groups correspond to the "R" groups defined for Formulas (I) to (IV). It is to be understood that the compounds shown are merely representative and not exhaustive. Others will be apparent to those of skill in the art, given this disclosure.

Compounds of Formula (I) have the structure:

The R substituent can be H, alkyl, alkylcarbonyl, benzoyl or -ealkyloxyCi-_δalkyl. Examples of R¹ substituents include, by way of illustration and not limitation, H, C_1-6alkyl, C_1-5alkylcarbonyl, benzoyl and Cι.₆alkyloxyCι- alkyl. The R substituent can be -OH, -CHO, -CH₂OH, -COOH, -C(O)CH or Cι- alkoxy. Examples of R² substituents include, by way of illustration and not limitation, -CHO, -COOH and -C(O)CH₃. Noting, however, that when R¹ is H, R is not -OH. Exemplar ryy ccoommppoouunnddss ooff FFoorrmmuullaa ((II)) aarree iilllustrated in Table I

Table I - Formula (I) Compounds

Compounds of Formula (I) of particular interest include eutypine. Compounds of Formula (II) have the structure:

The R³ substituent can be H, alkyl, alkylcarbonyl, benzoyl or

Examples of R³ substituents include, by way of illustration and not limitation, H, C_1-6alkyl, C_1-5alkylcarbonyl, benzoyl and Cι_.₆alkyloxyC_1-6alkyl. The R⁴ substituent can be -OH, -CHO, -CH OH, -COOH, -C(O)CH₃ or -Cι_-6alkoxy. Examples of R⁴ substituents include, by way of illustration and not limitation, -CHO, -COOH and -C(O)CH₃. The R⁵ substituent can be H, Cι_-6alkyl, C_1- alkoxy, C_2-6alkenyl, C _-6alkynyl or halogen. The C_1- alkyl, C _-6alkenyl and C _-6alkynyl groups may be substituted with 1-3 hydroxyl groups. Examples of R⁵ substituents include, by way of illustration and not limitation, -C≡C-C(CH₃)(CH₂OH)(OH), -C≡C-CH(CH₃)(OH), -C≡C-CH₃, -CH₂CH=C(CH₃)₂, -CH(OH)-CH₂-CH(CH₃)₂, -CH=CHCH(CH₃) , -OCH₃ and I. Exemplary compounds of Formula (II) are illustrated in Table II.

Compounds of Formula (III) have the structure:

The R⁶ substituent can be H, C]- alkyl, C₂.₆alkenyl, C_2-6alkynyl or a straight or branched chain having 4-6 carbon atoms, a double and a triple bond. The Ci^alkyl, C_2-6alkenyl, C _-6alkynyl and chain can be substituted with 1-3 hydroxyl groups. Examples of R⁶ substituents include, by way of illustration and not limitation, -C≡C-C(CH₃χCH₂),-C≡C-C(CH₃χCH₂OH)(OH), -C≡C-CH(CH₃)(OH), -C≡C-CH₃, -CH₂CH=C(CH₃)₂, -CH(OH)-CH₂-CH(CH₃)₂ and -CH=CHCH(CH₃)₂. Exemplary compounds of Formula (III) are illustrated in Table III.

Compounds of Formula (IV) have the structure:

The R⁷ substituent can be -OH, -CHO, -CH₂OH, -COOH, -C(O)CH₃ or Cι_-6alkoxy. Examples of R⁷ substituents include, by way of illustration and not limitation, -CHO. The R⁸ substituent can be H, C_1-6alkyl, C _-6alkenyl or C_2-6alkynyl. The

C₂-₆alkenyl, C .₆alkynyl groups may be substituted with 1-3 hydroxyl groups. Examples of R⁸ substituents include, by way of illustration and not limitation, -C≡C-C(CH₃)(CH OH)(OH), -C≡C-CH(CH₃)(OH), -C≡C-CH₃, -CH₂CH=C(CH₃)₂, -CH(OH)-CH₂-CH(CH₃)₂ and -CH=CHCH(CH ) . Exemplary compounds of Formula (IV) are illustrated in Table IV. Table IV - Formula (IV) Compounds

One aspect of the present invention is a method for controlling a fungal population, such as by eliminating or deterring the growth of the fungal pathogen. The method comprises contacting the fungal population with a composition comprising a carrier and a fungicidally effective amount of a compound or compounds selected from the group consisting of Formula (I), (II), (III) and (IV). The carrier is an agriculturally- or a pharmaceutically- acceptable carrier, depending upon whether the intended use is to treat plants or animals.

As used herein the term "fungi" refers to pathogenic fungi that negatively affect plants or animals by colonizing, attacking or infecting them. This includes fungi that spread disease and/or damage the host and/or compete for host nutrients. The compounds of the invention have fungicidal activity against one or more fungi. However, it is understood that certain compounds may be more effective on some fungi than others, and may even be ineffective against some fungi. That does not in any way detract from their value as fungicides since the invention contemplates using some of these compounds, while others have utility as specific or selective fungicides. However, due to the nature of the fungal genera, it is expected that the compounds of the invention are more likely to be useful in specific applications against one or a select group of fungi, rather than as broader, general acting fungicides, although the latter is contemplated as part of the invention.

The term "fungi" includes, by way of illustration, and not limitation: Cladosporium spp., Botrytis spp., ringworm, rhizoctonia and pythium. These fungi further include the broad species of the classes Ascomycetes, Basidiomycetes, Deuteromycetes and Oomycetes. More specifically, the term "fungi" is intended to include, by way of illustration and not limitation, specific members the following species: Botrytis species such as B. cinerea; Aspergillis species such as A. niger; Monilinia species such as M.fructicola; Colletotrichum species such as C. accutatum; Venturia species such as V. inaequalis; Fusarium species such as F. culmorum, F. nivale and F. oxysporum; Cercospora species such as C. canescens, C. viticola, C. beticola, C. arachidicola, C. personata and C. apii.; Alternaria species such as A. brassicae and A. alter nata; Corynespora species such as C. melongenae; Mycosphaerella species such as M. pomi; Marssonina species such as M. mali; Sclerotinia species such as S. sclerotiorum and S. cinerea; Gloeosporium species such as G. kaki; Cladosporium species such as C. cucumerin, C.fulvum and C. carpophilum; Glomerella species such as G. cingulata; Diplocarpon species such as D. rosae; and so forth. Fungi of particular interest that are plant pathogens include, by way of illustration and not limitation, M.fructicola, Cladosporium sp., A. alternata, B. cinerea, and C. accutatum.

The term "fungi" also includes those that are animal pathogens. Such fungi of particular interest include, by way of illustration and not limitation, Candida species such as C. albicans, and Cryptococcus species such as C. neoformans.

The efficacy of the methods and compositions described herein, along with the active ingredients used in the methods and contained in the compositions, is monitored by determining the mortality of or damage to the fungal population, i.e., by determining its adverse effect upon treated fungi. This includes damage to the fungus, inhibition or modulation of fungal growth, inhibition of fungal reproduction by slowing or arresting its proliferation, or complete destruction/death (eradication) of the fungus, all of which are encompassed by the term "controlling". The term "fungicidally effective amount" is an amount of the compound of the invention, or a composition containing the compound, that has an adverse affect on at least 25% of the fungi treated, more preferably at least 50%, most preferably at least 70% or greater. Preferably, an "effective fungal-inhibiting amount" is an amount of the compound of the invention, or a composition containing the compound, where 25% or greater mortality against fungi is achieved, preferably 50% or greater, more preferably 70% or greater mortality. Similarly, an "effective fungal-growth modulating amount" is preferably one where 25% or greater fungal-growth modulation is achieved, preferably 50% or greater, more preferably 70% of greater. The term "amount sufficient to prevent infestation" is also used herein and is intended to mean an amount that is sufficient to deter all but an insignificant sized fungi population so that a disease or infected state is prevented.

The actual value of a fungicidally effective amount for a given compound is preferably determined by routine screening procedures employed to evaluate fungicidal activity and efficacy, such as are well known by those skilled in the art and as are described in the Examples. It is expected that compounds of the invention having a higher level of fungicidal activity can be used in smaller amounts and concentrations, while those having a lower level of activity may require larger amounts or concentrations in order to achieve the same fungicidal effect. Efficacy is also monitored by phytotoxicity to the plants that are infested with the fungi, tissue damage to the host infected with the fungi and any adverse effects that might be experienced by a human user who is applying the composition to an infested plant or animal. Accordingly, the amount of composition or active ingredient used in the methods of the invention, meets the mortality, modulation or prevention criteria above, and preferably has minimal or no adverse effect on ornamental and agricultural plants (such as phytotoxicity), wildlife and humans that may come into contact with such compound. The Examples set forth below illustrate methods by which the fungicidal activity of the compounds or compositions described herein may be readily ascertained.

The compositions of the invention offer several advantages over currently used fungicides. First, the preferred compounds used in the composition of the invention are naturally-occurring compounds, therefore qualifying for the US EPA's Biorational Pesticide Program for Reduced Risk Pesticides. Consequently, when used for treating plant pathogenic fungi, food crops can be treated using the composition up to and immediately before the harvesting period, a practice that generally is avoided when using conventional methods of fungi control. The composition also can be used to control the growth of fungal organisms on harvested crops. The harvested food can be used directly as food for animals or humans. By using the subject compositions, the environmental and health hazards involved in fungal control are minimized by reducing the toxicity of the chemical compounds. Because of the low toxicity, when necessary, the composition can be used as a preventative on a repeated basis and, thus, can be integrated into integrated pest management (IPM) programs. The composition can be applied to skin or to objects such as clothing, fur, feathers, or hair which come into contact with skin when used to treat fungi that infest animals. The active ingredient of the fungicidal compositions of the present invention are believed to be biorational chemicals that qualify for the US EPA biorational program.

Another advantage of the compositions is that the compounds used have not previously been used as a fungicide against fungal organisms, and therefore, fungal pathogens have not acquired resistance to them. Resistance to chemicals occurs commonly in fungi. A new fungicide often becomes noticeably less effective against a particular pathogen after several growing seasons. As fungicides become more specific for pathogens, the likelihood of resistance increases. This can be attributed to the narrow mode of action of certain fungicides, which disrupt only one metabolic process of the fungal organism. The result is the appearance of resistant populations by selection of resistant individuals in a population. Generally, the more specific the site and mode of a fungicidal action, the greater the likelihood for a fungal population to develop resistance to that chemical. A new composition will address the pathogen resistance problem. To avoid developing future disease resistance in fungi, different chemicals should be alternated for treatment with the methods of the invention.

The subject methods offer several advantages over existing methods of fungal control. The long term control of fungi results in a healthier plant and an improved quality and yield of agricultural produce by the host plant as compared with untreated plants. The low concentration of anti-fungal agents decreases the likelihood of damage to the plant and/or its crop, and decreases the likelihood of adverse side effects to workers applying the fungicide, or to animals, fish or fowl which ingest the tissues of treated animals or parts of treated plants. The composition may be solid (i.e., in a powdered form) or liquid depending on the carrier and the needs of the user. If the composition is solid, suitable carriers include various known, agriculturally-useful powders that are generally used for this purpose. If the composition is liquid, it may be aqueous or non-aqueous and may be a solution, suspension, or emulsion, depending on the needs of the user applying the fungicidal composition. Generally, a composition of this invention will be prepared as a concentrate for industrial application and further dilution or as a fully diluted ready-to-apply composition. Preferably, the composition is applied as a liquid, whether aqueous or non-aqueous, but preferably the former. The concentrate, if solid, will be formulated to be mixed to form an appropriate non-aqueous or aqueous composition. Thus, the composition will generally contain the active ingredient along with a surfactant carrier to effect miscibility or suspendability of the composition in a liquid.

The composition of the invention is either non-phytotoxic or non-dermal sensitive if the intended application at the recommended dosage is to the tissue of a plant or animal host, respectively. The preferred compositions comprise a fungicidally effective amount of a compound of the invention. In general, the percentage by weight of the active ingredient will be about 0.1% to 50wt%. The preferred amount is determined using bioassays on a fungi-by- fungi or host-tissue by host-tissue basis. The higher concentrations are usually preferred for purposes of manufacture, shipment, and storage. For example, as a concentrate for use by crop-protection professionals, the percentage will be at least about 10wt%, preferably about 25 to 50% by weight. Prior to use, the high concentration composition is diluted in a solvent to an appropriate concentration for the intended use of the composition. When fully diluted for consumer use as a "ready for use" product, the composition will be typically be about 0.5% to 10wt%, more preferably 1 to 5wt%.

The subject composition can include an antioxidant at a level sufficient to increase the product shelf life, inhibit decomposition of the active ingredient in the fungicidal composition, or improve the stability of the controlling effect when the composition is applied to hosts infested with the targeted fungi. Suitable antioxidants, include, but are not limited to, ascorbyl palmitate, anoxomer, benzoic acid, benzlkonium chloride, benzethonium chloride, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, dehydroacetic acid, ethylenediamine, ferulic acid, potassium benzoate, potassium metabisulfite, potassium sorbate, n-propyl gallate BP, propylparaben, sassafras oil, sodium benzoate, sodium bisulfite, sodium metabisulfite, sorbic acid, vitamin E, eugenol, α- tocopherol, and the like. Particularly suitable antioxidants include sodium benzoate, vitamin E and α-tocopherol. Antioxidants can be included in the composition so long as the formulation remains active without damage if applied to a host. The amount of antioxidant used is in general about 0.01-10% by weight, but generally no more than about lwt%. A preferred amount can be determined by a shelf-life stability trial in accordance with an EPA standard protocol. A minimal amount of antioxidant which increases shelf life and/or maintains product stability is selected to reduce manufacturing costs.

The subject composition can be an aqueous composition using water as the solvent or an organic composition using an organic solvent, such as ether, ketone, kerosene, or alcohol where the application is not directly to a host tissue, or in a concentration of organic solvent that will not harm a host tissue if applied to a host tissue. It is preferred to use water as the solvent because it mimics nature (biorational), is environmentally safe, is non-phytotoxic or non-dermal sensitive, and also costs little. The compositions of this invention, particularly liquids and soluble powders, preferably contain, as a conditioning agent, one or more surface- active agents in amounts sufficient to render a given composition readily dispersible in water or in an organic solvent. The incorporation of a surfactant into the compositions greatly enhances their efficiency. The water, organic solvent, or surfactant (alone or in combination with a solvent) functions as the agriculturally-acceptable carrier. By the term "surfactant" it is understood that wetting agents, surface-active agents or surfactants, dispersing agents, suspending agents, emulsifying agents, and combinations thereof, are included therein. Anionic, cationic and non-ionic surface-active agents can be used, although non-ionic agents are preferred. The non-ionic surface-active agents include allinol, nonoxynol, octoxynol, oxycastrol, oxysorbic (for example, polyoxyethylated sorbitol fatty-acid esters (TWEEN^®), thalestol, and polyethylene glycol octylphenol ether

(TRITON^®). The anionic type of agents include fatty-acid salts, higher alcohol sulfiiric esters and alkylallylsulfonates. The cationic type of agents include aliphatic amino salts, quaternary ammonium salts and alkylpyridinium salts. Particularly suitable surfactants include, by way of illustration and not limitation, TWEEN^®20 (polyoxyethylene sorbitan monolaurate), TWEEN^®40, TWEEN^®80, TRITON^®SP150, TRITON^®SP180 and TRITON^®SP190; the most preferred being TWEEN^®80 and TRITON^®SP190. Usually, the amount of surfactant used is the minimum amount required to get the compound into solution/emulsion, and will generally be 0.5 to 10% by weight, more typically 0.5 to 1%.

The common and chemical names of other generally available adjuvants include, but are not limited to, the following list, in which the first name is the common name used in the industry, the second name is the general chemical name, the third name is the class of the compound, the fourth name is the type of surfactant, and the trade name is last.

Albenate: Alkyl(Cι₈C₂₄)benzene sulfonic acid and its salts; Alkylaryl sulfonate; Anionic surfactant; Nacconol 88SA, Calsoft F-90, DDBSA, Santomerse No. 3. Alfos: α-Alkyl(C₁₀-Ci₆)-ω-hydroxypoly(oxyethylene) mixture of dihydrogen phosphates esters; polyoxyethylene alkyl phosphate ester; Anionic; Emcol PS-131.

Allinate: α-Lauryl-ω-hydroxypoly(oxy ethylene) sulfate; lauryl polyoxyethylene sulfate salts; Anionic; Sipon ES.

Allinol: α-Alkyl(Cn-Ci₅)-ω-hydroxypoly(oxyethylene); Cπds linear primary alcohol ethoxylate; Nonionic; Neodol 25-3, Alfonic 1014-40 and other alfonic materials.

Diocusate: Sodium dioctyl-sulfosuccinate; Dioctyl sodium sulfosuccinate; Anionic; TRITON GR-5, Aerosol OT.

Dooxynol : α-(p-Dodecy 1-pheny l)-ω -hydroxypoly (oxy ethylene) ; dodecylphenol condensation with ethylene oxide; Nonionic; Igepal RC-630, Tergitol 12-P-9, Sterox D Series.

Ligsolate: Lignosulfonate, NH₄, Ca, Mg, K, Na, and Zn salts; Salts of lignosulfonic acids; Anionic; Marasperse N-22, Polyfon O. Nofenate: α-(p-Nonylphenyl)-ω-hydroxypoly(oxyethelene) sulfate, NLLj, Ca, Mg, K,

Na, Zn salts, Nonyl group is a propylene trimer isomer; Salts of sulfate ester of nonylphynoxypoly(ethyleneoxy) ethanol; Anionic; Alipal CO Series

Nonfoster: α-(p-Nonylphenyl)-ω-hydroxypoly(oxyethylene); mixture of dihydrogen phosphate and nonophosphate esters; Polyoxyethylene nonylphenol phosphate esters; Anionic; Gafac RM 510.

Nonoxynol : α-(p-Nony lphenyl)-ω -hy droxypoly (oxy ethylene) ; poly oxy alky lene nonylphenol; Nonionic; Sterox N Series, Makon 6, Igepal CO Series TRITON N Series, T- DET N.

Octoxynol: α-[p-l,l,3,3-Tetramethyl butyl phenyl]-ω-hydroxypoly(oxyethylene); polyoxyethylene octyl phenol; Nonionic; Igepal CA-630, TRITON X-100.

Oxycastol: Castor oil polyoxyethylated; Ethoxylated castor oil; Nonionic; Emulphor EL-719, Emulphor EL-620, Trylox CO-40, T-DET C-40

Oxysorbic: Polyoxyethylated sorbitol fatty acid esters (nonosterate, monoleate etc); Polyoxyethylated sorbitol fatty acid esters; Nonionic; Atlox 1045, Drewmulse POE-STS, TWEEN Series G- 1045.

Tall oil: Tall oil, fatty acids not less than 58%, rosin acids not greater than 44%, unsapolifiables not greater than 8%; Tall oil; Anionic; Ariz. S.A. Agent 305.

Thalestol: Polyglyceryl phthalate ester of coconut oil fatty acid; Modified phthalic glycerol alkyl resin; Nonionic; TRITON B-1956. The composition can include other active or inactive substances. In some instances, the efficacy of the formulation can be increased by adding one or more other components to the formulation. It is preferable that the additional component(s) minimize toxicity to hosts such as plants or mammals while increasing the anti-fungal effect of the formulation. Especially preferred is the use of a synergist, which is a component that, by virtue of its presence, increases the desired effect by more than an additive amount. Of particular interest is the addition of components to a formulation to allow for a reduction in the concentration of one or more active ingredients in a given formulation while substantially maintaining efficacy of the formulation. The subject composition may be prepared by simply mixing together the requisite amount of at least one compound of the invention and at least one agriculturally acceptable carrier such as a surfactant, alone or with a solvent. Other additives, such as saponins and antioxidants, may be included prior to mixing. The composition can be encapsulated or microencapsulated. It can also be produced as dust, powder, etc. A preferred pH of the composition is between 6.0 and 8.0 with an optimal range of 6.5-7.5. A neutralized composition is preferred to lower the risk of harm caused by alkalinity. Water-dispersible powder, capsule, or pellet compositions can be made containing one or more compounds of the invention, an inert solid extender, and one or more wetting and dispersing agents. The inert solid extenders are usually of mineral origin, such as natural clays, diatomaceous earth and synthetic minerals derived from silica and the like. Examples of such extenders include kaolinites, attapulgite clay and synthetic magnesium silicate. The water-dispersible powders can also include fatty-acid esters and antioxidants. For controlling the growth of fungi on a plant or a plant part (such as foliage/leaves, trunk, stems, branches or roots and so forth), the method of the invention can be carried out by applying a fungicidally effective amount of the subject composition to a plant host or to the substrate in which it is growing or is to be grown. For controlling fungi on other than a plant or a plant part, a method is provided to obtain and/or maintain an area substantially free of fungi, using the subject compositions. The composition is provided to fungi to eliminate them, to deter their growth, and/or to prevent infestation of a host for the fungi. The method of introduction of the subject fungicide into the target fungi can be by contacting the fungi or by feeding a target fungi a nutrient-providing, organic matter treated with the fungicide. In some instances, the fungicide is absorbed by the fungi, particularly where the formulation, for example, a detergent formulation, provides for uptake by the outer surface fungi. In some instances, the outer surface of the target fungi is substantially dissolved by contact with the formulation.

The method of use of the compounds and compositions of the invention will depend at least in part upon the fungi to be treated and its feeding habits, as well as breeding habits. While very minor dosage rates of the novel compositions will have an adverse effect on fungi, adequate control usually involves the application of a sufficient amount to either eliminate fungi entirely or significantly deter their growth and/or rate of proliferation. Dosage rates required for efficacy vary depending on the target fungi and maturity, i.e., stage of growth. Dose response experiments using different dilutions (for example, 1 :1000, 1:100, 1 :10 and 1 :3) of the fungicidally active ingredient on target organisms and on plants are performed to determine the optimal concentration of the active ingredient that shows a fungicidal activity without phytotoxicity or dermal sensitivity.

Infestation of target fungi can be treated using a solid support that has been treated with a composition of the invention, or treated directly with a powder or detergent formulation. The composition also can be sprayed on as a wet or dry composition on the surface of material infested with a target fungus, or material susceptible to infestation with a target fungus. Alternately, the composition can be applied wet or dry to an area of infestation where it can contact the target fungus. The composition can be used as a fumigant to mix with pre-plant soil for crops such as tomatoes, strawberries, cucumbers, watermelons and pumpkins, to kill fungi. The composition also can be used as a fungicide to protect agricultural produce during storage and transportation. The amount of fungicidal agent that is applied either to the plant itself or to the soil will depend upon the degree of infestation and to some extent, upon the formulation of the composition used, and therefore is empirically determined for best results.

When the area of infestation is a plant or a plant part, a composition containing the fungicidally active ingredient of the invention is provided to a plant tissue or a plant part either pre- or post-harvest. Methods of application include spraying, pouring, dipping, injecting, fogging, fumigation or the like, along with applying the composition by means of power dusters, boom and hand sprayers and spray dusters. The compositions can also be applied from airplanes as a dust or a spray because of their effectiveness at low dosages. The active ingredient can be in the form of a concentrated liquid, solution, suspension, powder or the like. For example, the composition can be sprayed on as a wet or dry composition to the surface and/or underside of the leaves or other plant tissue or part of a plant infected with a plant fungus, or of a plant susceptible to infestation with a plant fungus, preferably to- the point of run off when a wet formulation is used. The plants can be sprayed prior to or after infestation, preferably prior to infestation. The formulation also can be applied wet or dry, either as part of an irrigation schedule or as a separate application, to the rhizosphere where it can contact the roots of the plant and associated fungal organisms which colonize the roots. In some instances, time-release formulations may find use, particularly for applications to the rhizosphere, or to post harvest materials.

As noted above, in one preferred method of the invention, plants are treated prior to fungal infestation. Accordingly, one aspect of the invention is directed to a method for preventing infestation of a host with a fungus by contacting the host, such as by spraying, with a composition comprising a compound selected from the group consisting of Formula (I), (II), (III) and (IV) in combination with an agriculturally-acceptable carrier in an amount sufficient to prevent infestation. For controlling the growth of fungi on a plant or a plant part, a composition comprising a fungicidally effective amount of the compounds described herein is applied to the plant or plant part. The composition is provided in a non-phytotoxic solvent to minimize damaging the plant. The phytotoxicity of the formulation can be evaluated by applying the composition on living plants and determining the toxicity of the composition to the plants. As stated above, a non-organic aqueous solvent or emulsion is in general preferable for its non-phytotoxicity, and a fatty-acid ester is sometimes included in the formulation to increase the fungicidal activity of the formulation, thus reducing the necessary amount of the active ingredient(s). The efficacy of treatment is monitored by determining the mortality of fungi and phytotoxicity, with absence of phytotoxicity being desirable. Plants suitable for treatment are those of agricultural and/or horticultural importance, such as agricultural crops, including food crops and fruit trees; and horticultural crops such as ornamental plants and flowers. These include by way of illustration and not limitation, lettuce varietals, grains and cereals, root crops such as potatoes and carrots, and fruit bearing plants and trees including grape vines, strawberry plants, apple, pear, citrus and other fruit trees, tomato plants, cucumbers; ornamental plants and trees such as roses and miniroses, carnations, tulips, herbs, rhododendron, magnolia, primroses, orchids, chrysanthemums and poinsettias; and other agricultural crops such as cotton.

It is also expected that the methods described herein can employ the compositions of this invention, along with sequential treatments with herbicides, phytotoxicants, fertilizers, and the like for maximum effect. For example, a field could be sprayed with a composition of this invention either before or after being treated with materials such as fertilizers, herbicides, phytotoxicants, and the like. The compositions of this invention can also be admixed such materials and be applied in a single application.

These methods are best illustrated by the preferred embodiments set forth below. One aspect of this invention is a method of controlling fungi, for example by eliminating or deterring the growth of the fungal population, which involves applying a composition of this invention to a fungi or a site of fungal infestation (such as a plant, plant part, or animal) at a fungicidally effective level. As noted above, application methods include ground, aerial, chemigation, surface, soil incorporation, preplant, preemergent, postemergent, spraying, brushing, dipping, and the like, depending on the conditions of the weather, the type of fungi, the type of plant or animal being treated, the time of year, and other factors known to those of skill in the art. An exemplary method for controlling fungi comprises applying (such as by spraying) to a fungus or site of fungal infestation, a fungicidally effective amount of a composition comprising an agriculturally-acceptable carrier in combination with a compound of Formula (I), (II) or (III). Preferably, the composition is applied in an amount sufficient to prevent infestation of the host and the composition does not damage the host's tissue. Of particular interest is use of the fungicide compositions of the invention in treating fungal infestations of plants. By treatment of a diseased plant with the composition of the invention in an amount sufficient to treat such a fungal infestation, fungi can be controlled or eliminated, thus restoring the plant to a healthy state.

The compositions of the invention also find utility when employed as a protectant to be combined with a material to protect such material from fungal attack or infestation. Such materials are typically industrial materials that can be damaged by fungi, and include, by way of illustration and not limitation, raw materials such as paper, textile and raw lumber; liquid products such as glue, sizing, paint and lubricants; solid products comprised of paper, wood, textile, leather and plastic. The compositions of the invention can be applied to the surface of the raw materials or incoφorated into the manufacture process of a solid or liquid product. Although the methods and compositions of the invention find particular applicability in the agricultural industry, pharmaceutical applications are also contemplated. One suitable method comprises contacting the fungal population with a composition comprising a carrier and a fungicidally effective amount of a compound selected from the group consisting of Formula (I), (II), (III) and (IV), said carrier being a pharmaceutically-acceptable carrier. For controlling pathogenic organisms on animals the compositions can be applied to the area infested or subject to infestation by fungal organisms. As appropriate, for example, when the composition is to be applied to the skin of a mammal or to objects or materials which can come into contact with the skin of a mammal, the composition is evaluated for dermato logical effects. It may also be important for at least one evaluation of the toxicity of the composition be conducted on animal hosts for the target fungi or on animals which may come in contact with a treated surface so that the dermatological effects can be evaluated for the dosage of fungicide used. Such dermatological sensitivity tests can be conducted using methods known to those skilled in the art (see Kligman (1966) J. Invest. Deπnatol., 47:393). In some instances it may be necessary to adjust the treatment composition so as to reduce any dermatological effects associated with the formulation of the composition. When applied to animals, including humans, the subject composition is provided in a carrier which is non- toxic-and non-irritating to the skin, typically referred to as a pharmaceutically-acceptable carrier. Animals to be treated include humans, companion animals (e.g., feline and canine), and agriculturally bred animals, including those raised for human consumption, such as bovine and poultry (e.g. avian).

It is well known that fungal spores often attack immune-compromised species of animals. Since these airborne spores attack the lungs, the preferred means of applying the fungal population with a fungicidally effective amount of a composition of the invention is by aerosol delivery such by nasal or bronchial inhalation, such as is well known in the art. Accordingly, one aspect of the invention is directed to a method for treating a fungal infestation of an animal host by contacting the host, such as by treating the host with an inhaled aerosol formulation, with a composition comprising a compound selected from the group consisting of Formula (I), (II) and (III) in combination with a pharmaceutically- acceptable carrier in an amount sufficient to eradicate such infestation.

There are several pharmaceutical inhalation devices that are suited for use in the aerosol delivery of the compositions of the invention, such as nebulizer inhalers, metered- dose inhalers and dry powder inhalers. Nebulizer devices produce a stream of high velocity air that would allow the fungicidal composition to spray as a mist which would then be transported into a patient's respiratory tract. The composition would be packaged with a compressed gas if a metered-dose inhaler was to be used. Upon actuation, the metered dose inhaler releases a measured amount of the fungicidal composition by compressed gas. Dry powder inhalers would administer the fungicidal composition in the form of a free flowing powder formulated with an excipient, such as lactose, and the composition would be dispersed in a patient's air-stream during breathing by the device.

However, any other pharmaceutically acceptable mode of administration can be used, such as by any accepted systemic or local route, including oral, parenteral, topical or transdermal, which includes transmucosal, across buccal and mucosal membranes, body tissues, and skin, including epithelium and stratum corneum, in the form of solid, semi-solid or liquid or aerosol dosage forms, such as, for example, tablets, pills, capsules, powders, liquids, lotions, solutions, emulsion, injectables, suspensions, suppositories, aerosols or the like. The formulation of the invention can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, pills, transdermal (including patches, electrotransport devices, and vaginal and rectal suppositories), and the like, for the prolonged administration of the fungicide at a predetermined rate. The formulations will include a conventional pharmaceutical carrier or excipient and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc.

Oral compositions include liquids such as syrups, suspensions and emulsions, tablets, capsules and lozenges. A liquid composition will generally consist of a suspension or solution of the fungicide or pharmaceutically acceptable salt in a suitable liquid carrier(s), for example ethanol, glycerine, sorbitol, non-aqueous solvent such as polyethylene glycol, oils or water, with a suspending agent, preservative, surfactant, wetting agent, flavoring or coloring agent. Tablets can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid compositions, such as magnesium stearate, starch, lactose, sucrose, microcrystalline cellulose and binders such as polyvinylpyrrolidone. Capsules can be prepared using routine encapsulation procedures, for example by incorporation of the fungicide and excipients into a hard gelatin capsule. Alternatively, a semi-solid matrix of fungicide and high molecular weight polyethylene glycol can be prepared and filled into a hard gelatin capsule; or a solution of fungicide in polyethylene glycol or a suspension in edible oil, for example liquid paraffin or fractionated coconut oil can be prepared and filled into a soft gelatin capsule. Parenteral compositions can be formulated for intramuscular or intravenous administration. Intramuscular formulations typically consist of a suspension or solution of the fungicide in an oil, for example arachis oil or sesame oil, while a composition for intravenous administration will normally consist of a sterile isotonic aqueous solution containing, for example the fungicide, dextrose, sodium chloride, a co-solvent, for example polyethylene glycol and, optionally, a chelating agent, for example ethylenediamine tetracetic acid and an anti-oxidant, for example, sodium metabisulphite. Transdermal compositions may include, for example, a backing, active compound reservoir, a control membrane, liner and contact adhesive. A typical suppository formulation will generally consist of the fungicide with a binding and/or lubricating agent such as a gelatin or cocoa butter or other low melting vegetable or synthetic wax or fat. Topical formulations are similar in composition to the liquid formulations described herein that are useful for treatment of plants.

Use of fungicides is regulated in the United States by the Environmental Protection Agency (EPA) under authority of the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). Tolerance for residues of fungicides in agricultural commodities are established by the (EPA) and enforced by the Food and Drug Administration (FDA) under authority of the Federal Food, Drug and Cosmetic Act (FD&C Act).

This regulatory environment leads to another aspect of this invention, which is an article of manufacture. As indicated above, the invention provides for a composition comprising a fungicidally active compound selected from the group consisting of Formula (I), (II), (III) and (IV) with a suitable carrier, useful in methods for preventing infestation of a host with a fungus, for eradicating fungal infestation on a plant or animal, and for treating a fungal infestation of an agricultural or ornamental plant in need thereof. Accordingly, in another aspect of the invention, an article of manufacture comprises a fungicidally active compound selected from the group consisting of Formula (I), (II), (III) and (IV), combined with an agriculturally-or pharmaceutically acceptable carrier in a container that will be suitable for storing the composition for its shelf life. The container may have printed instructions and/or a printed label indicating that the composition contained therein can be used to control fungal pathogens, i.e., used as a fungicide, and providing instructions for using the composition for fungicidal purposes in accordance with the treatment methods set forth herein. The container may have associated with it a delivery device that allows the composition to be applied to the fungal population or to the area to be treated. For liquid compositions this is generally a hand-operated, motorized or pressurized pressure-driven sprayer. The container may be made of any suitable material such as a polymer, glass, metal, or the like. Usually, the labeling is associated with the container by being adhered to the container, or accompanying the container in a package sold to the user. Such label may indicate that the composition is approved for use as a fungicide. The instructions will spell out the type of fungi for which the fungicidal composition is to be used, the application method, the rate of application, dilution requirements, use precautions, and the like.

The following examples are presented as illustrations, not limitations.

EXAMPLES Abbreviations Al Active Ingredient

HPLC High Performance Liquid Chromatography

PDA Potato-Dextrose-Agar medium (Merck)

T20 TWEEN^®20 TLC Thin Layer Chromatography

T-SP 190 TRITON^®SP 190 (Union Carbide)

Example 1 Biosynthesis Protocol Eutypa Culture and Fermentation:

Eutypa lata isolates are cultured. A suitable liquid culture medium is then prepared

(See for example, Pezet, R. (1983) Rapport interne. Station Federale de Changins, Suisse).

The medium is then seeded with 5 mm discs of mycelium-bearing PDA, and the E. lata is incubated in 1 L flasks, each containing 400 mL of medium, over a period of 3-6 weeks at 21-25°C under normal light (16 hrs/day and 12 W/m ) and with continuous gyratory shaking at 100 rpm.

Isolation of Eutypine:

Eutypine was isolated in the manner described in Mauro, et al., Am. J. Enol.

Viticulture 39:200-204 (1988), which is reiterated herein. The mycelial mat is centrifuged and filtered off, and the culture medium is adjusted to a pH of 4.5-5.0. If desired, an equivalent volume (400 mL) of ethanol can be added at this point. The precipitated material is removed by filtration and the alcohol evaporated from the filtrate, the latter step being optional. The filtrate is extracted in a separatory funnel with three consecutive 120 mL portions of diethyl ether per flask (400 mL). The lower phase is drained out and the organic (upper) phase dried over about 20 g of anhydrous sodium sulfate overnight. The solution is decanted and the solid sulfate is rinsed with 50 mL of ether that is then combined with the main portion (Extract A).

The ether is evaporated or distilled off and the residue is subjected to column chromatography on powdered silica gel, using a gradient of methanol in chloroform at a rate of 20 mL/hr. An alternate procedure involves sequential elution with methylene chloride, methylene chloride-methanol (18:1), methylene chloride-methanol (9:1) and finally water.

About 200 mL of organic solvent will be required. Fractions of 15 mL are collected, each is analyzed by TLC on a silica gel plate with chloroform-ethyl acetate (5:1) as the solvent.

Spots are visualized under 254 nm UV radiation. Fractions showing a spot corresponding to eutypine at R_f 0.58 (in chloroform-ethyl acetate 5: 1) are combined and the solvent evaporated. This will provide sufficient purity for most uses contemplated by the invention. Alternately, the dried and concentrated Extract A can be streaked in a narrow band across several silica gel TLC plates, chromatographed in one of the aforementioned solvents, the UV-absorbing area corresponding to eutypine scraped off and the compound dissolved in silicic acid with ethanol.

Additional purification can be accomplished by semipreparative-scale HPLC with an

80 x 115 mm reversed-phase C-18 column (Lichrosorb RP18) and a linear solvent gradient program of 40 to 80% aq. Methanol at 1 mL/min, 254 nm UV detector. Fractions are collected that represent the major HPLC peaks. Eutypine is found in the fraction showing maximum UV absorption (Tey-Ruhl, et al., Phvtochemistry 30:471-473 (1991). This additional purification step is useful for structure identification but is not necessary for use of the materials in the methods of the invention. A yield of 2 mg of eutypine per 400 mL of medium is expected, i.e., about 5 mg/L. Based upon this, 20 L of medium is expected to yield 100 mg of eutypine.

Example 2 Anti-fungal activity against M. fructicola and A. niser

Eutypine was synthesized by the methods described by Defranq (1993), supra. A 10 μL and a 25 μL aliquot of eutypine was spotted out onto filter paper (#1 qualitative, 4.25 cm round, Whatman International Ltd., Maidstone, England), allowed to air- dry, then oversprayed with a spore suspension of a Monilinia fructicola, isolated from peach, in a nutrient solution for on-chromatogram inhibition assays, as described for Cladosporium species (Homans, et al., J. Chromatog. 51 :327-329 (1970)). Spores were suspended by washing with water from the surface of a plate culture on PDA through a paper towel to filter large debris. The spores were sprayed by atomizer. Non-treated filter papers were also oversprayed. These moist filter papers were then placed on plastic supports in high humidity conditions (plastic crisper with water in the bottom) to incubate.

M.fructicola fungus did not grow on the filter papers. Airborne contaminant A. niger did grow, but revealed distinct zones of inhibition surrounding the eutypine drops after 7-10 days. No other fungal contaminants had colonized the spots by day 33, despite favorable growth conditions.

In a similar experiment, the same results were noted for the airborne contaminant fungi Cladosporium sp. and Alternaria alternata. Example 3

Anti-fungal activity against B. cinerea. C. accutatum and Cladosporium sp.

Eutypine was synthesized by the methods described by Defranq (1993), supra.

A 1 μL and a 10 μL aliquot of eutypine was spotted out onto filter paper (#1 qualitative, 4.25 cm round, Whatman International Ltd., Maidstone, England), allowed to air- dry, then oversprayed with spore suspensions of Botrytis cinerea, Colletotrichum accutatum and Cladosporium sp., in a nutrient solution for on-chromatogram inhibition assays, as described for Cladosporium species (Homans (1970) supra). Spores were suspended by washing with water from the surface of a plate culture on PDA through a paper towel to filter large debris. The spores were sprayed by atomizer. Non-treated filter papers were also oversprayed. These moist filter papers were then placed on wire supports in high humidity conditions (plastic crisper with water in the bottom) to incubate.

At day 2, inhibition of Cladosporium sp. by eutypine at both amounts was visible as zones of inhibition. At day 3, inhibition of B. cinerea and C. accutatum was also apparent. No fungal contaminants had colonized the spots by day 21, despite favorable growth conditions.

Example 4 Phytotoxicity Protocol This example provides a protocol for laboratory bioassays evaluating the compatibility of candidate materials for effects on agricultural and ornamental plants.

Geometric dilutions of candidate materials are compared to an adjuvant only and water only control treatments. Degree of plant injury resulting from all treatment applications is assessed using a 1-5 injury rating with "1" indicating no injury and "5" indicating severe injury.

At least 3 replicates of each plant type are compared for each treatment tested. Each replicate consists of one plant. Plants are matched for similar growth stage and health. Applications are made using a hand held atomizer "to drip" (applied until material drips from the leaves). Plants are allowed to dry and placed in the greenhouse. Plants are observed for a suitable period at which time injury from necrotic damage and/or growth distortion will be assigned to each plant in the trial.

Candidate materials are assessed at dosages ranging between the minimum inhibitory concentration to the limit of concentrate solubility. Each candidate material is evaluated against plant varieties of interest. Example 5

Dose Response Experiment Against Target Fungi

The compositions of the inventions at different dose ranges are assessed for fungicidal activity by fungal mortality. Geometric dilutions of test material are compared with formula blank and water only control treatments. Fungal mortality at each concentration is calculated. For each target fungus, at least 3 dosages of the composition are tested against the appropriate control. The target fungi include plant and animal fungal pathogens. The experiment is conducted in an assay chamber which is supplied with adequate nutrition to support a test fungal population during the course of the experiment. Sufficient CFUs ("colony forming units") of the target fungi are placed in each container. The Al is applied at a controlled volume sufficient to insure good contact with the target fungi using a calibrated spray tower. Filter paper placed at the bottom assay chambers is used to absorb excess material. After treatment application, the fungi is left undisturbed for at least 24 hours. The container is then be examined under dissection microscopes and the number of dead and living fungal organisms are recorded. A minimum of four replicates

(each container constitutes a replicate) is treated for each dosage inducting a water plus adjuvant control. Results will establish the dose-mortality relationships and analyzed using probit analysis to determine the optimal concentrations for controlling 90% or more of the fungal populations. Each experiment is repeated twice for each of the fungi tested. Each trial consists of

20 replicates (4 replicates for each treatment, 5 treatments total). The analysis estimates the optimal dosage that results in 50 and 90% kill of the fungi. These are standard parameters that can be used to provide the best dosage for conducting efficacy trials and suggest the label use rates for the label recommendations for commercial use. Four concentrations of Al at a single concentration of T20 are compared on each plant type as follows:

T20: determined by the results from the phytotoxicity trial.

Al: 0.1 , 0.2, 0.4 and 0.6 percent concentration by volume

Controls: water plus T20 only Numerous other suitable protocols have been established by the American

Phytopathological Society's Committee on Standardization of Fungicidal Tests

(Phytopathology 33:627-632 (1943)).

Example 6 Protocols for glasshouse testing The following protocol is a procedure for evaluating new products for the control of

Botrytis cinerea on Lycopersicon esculentum.

(1) Test provided product at highest concentration for potential phytotoxicity as evidenced by leaf necrosis, flower blasting, killing, etc, usually apparent by 48 hours. (2) If phytotoxic, run dilution series and retest to determine threshold. Focus in by repeat test in range of phenotype change from toxic to not, by geometric dilution.

(3) Determine minimum inhibitory concentration ("MIC") to fungi by running dilution series of product from phytotoxic threshold on down, focusing as for determining phytotoxic threshold.

(4) After determination of MIC, run eradicant protectant residual testing at MIC.

This is a lengthy process, requiring application of the product, usually at a range of times from 2 weeks prior to 2 weeks following an inoculation event. After results are analyzed, closer focus is sometimes required with repeat testing. Example 7

Protocol for Field Testing of Fungicidal Efficacy

A randomized complete block design is placed in crop production fields to compare different fungicidal treatments against a selected fungal infestation, with from 30 to 100 melon plants used for each treatment; i.e., a minimum of 3 replications of 10 plants up to a manageable size of 5 replications of 20 plants are used to evaluate treatments. Actual number used at each field trial is dependent upon space availability. Bed design is either 2 or 4 rows per bed; bed spacing ranges from about 1.3 to 1.7 m on center; plant spacing ranges from about 30 to 45 cm between plants depending upon variety. Standard farming practices are used, which might include methyl bromide fumigation of the field, pre-plant slow release fertilizer, irrigation and insect control, but no other foliar fungicidal treatment for control of Botrytis rot due to interference with the evaluation of treatment efficacy.

Application of foliar fungicidal treatments is made with a composition of the invention at an optimal concentration which produces sufficient fungal mortality and minimal phytotoxicity to plants. Compositions comprising 100 ppm to 2000 ppm of the Al are evaluated.

The composition is applied with a backpack sprayer or by securing a standard wide spray nozzle to the formulae bottles, and spraying the designated areas with assigned bottles at intervals between 3-14 days. Treatments are sprayed separately, one trial plot at a time. Diseases severity evaluation is done at least once during any field trial, but usually more often. Example 8 Evaluation of Compounds

Table V - Anti-Fungal Activity (as evidenced by fungal growth over treated area)

^* Did not show efficacy against the target fungus at tested concentrations.

The technique used to obtain the values in Table V was similar to the one described in Example 2. An aliquot of the compound to be tested was spotted out onto filter paper (#1 qualitative, 4.25 cm round, Whatman International Ltd., Maidstone, England), allowed to air- dry, then oversprayed with a spore suspension of the target fungus. As controls, non-treated filter papers were also oversprayed. These moist filter papers were then placed on wire supports in high humidity conditions (plastic crisper with water in the bottom) to incubate. At regular intervals, the filter papers were tested for zones of inhibition.

In addition to the target fungi in Table V, eutypine was effective against Colletotrichum accutatum and Monilinia fructicola at a concentration of 71 ppm. At the tested concentrations, the compounds were also evaluated for their phytotoxicity on strawberry and tobacco plants. Most of the tested compounds did not exhibit any phytotoxic effects.

Example 9

Table VI -Efficacy against Plant Pathogenic Fungi in-vitro

Table VIA - Fungicidal Activity

Table VIB - Fungistatic Activity

A dried spot transfer assay was used to obtain the fungicidal and fungistatic values in Tables VIA and VIB. A dried spot of the compound being tested was prepared. To this dried spot was added a spore suspension of the target fungus. The compound and spore suspension were kept in a humid chamber and monitored for germination of the spore. After 48 hours, the compound and spore suspension were removed to a nutrient media. These were monitored for spore germination. At this stage, if no growth was observed the tested compound was considered to be fungicidal and the LD_{J OO} value was computed. If spore germination was observed, the tested compound was considered to be fungistatic and the EC₅₀ or ECioo values were computed.

In addition, eutypine was efficacious in greenhouse and field tests.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incoφorated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference.

The invention now having been fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

CLAIMS What is claimed 1. A method for controlling a fungal population, said method comprises contacting the fungal population with a composition comprising a carrier and a fungicidally effective amount of a compound having the formula:

where R¹ is selected from the group consisting of H, alkyl, alkylcarbonyl, benzoyl and Cι_-6alkyloxyCι_-6alkyl; and R is selected from the group consisting of -OH, -CHO, -CH₂OH, -COOH, -C(O)CH₃ and C_1-6alkoxy; with the proviso that when R¹ is H, R² is not -OH.

2. The method of Claim 1 wherein R¹ is selected from the group consisting of H, C^ alkyl, C]_-5alkylcarbonyl, benzoyl and C_1-6alkyloxyC_1- alkyl.

3. The method of Claim 1 R is selected from the group consisting of -CHO, -COOH and -C(O)CH₃.

4. The method of Claim 1 wherein said compound is eutypine, eutypine acetate and O- methyl eutypine.

5. The method of Claim 1 wherein said fungal population is a plant fungal population, and said composition is non-phytotoxic to a plant infested with said fungus.

6. The method of Claim 5 wherein said composition is applied in the vicinity of said plant.

7. The method of Claim 5 wherein said composition is applied to said plant by spraying foliage to run off.

8. The method of Claim 5 wherein said fungal population is selected from the group consisting of Aspergillius niger, Botrytis cinerea, Penicillium sp. , Cladosporium sp. ,

Rhizopus stolinofer, Verticillium dahliae, Monilinia fructicola, Alternaria alter nata and Colletotrichum accutatum. 9. A method for controlling a fungal population, said method comprises contacting the fungal population with a composition comprising a carrier and a fungicidally effective amount of a compound having the formula:

where R >3 is selected from the group consisting of H, alkyl, alkylcarbonyl, benzoyl and

Cι_-6alkyloxyC_1-6alkyl; and R >4 is selected from the group consisting of -OH, -CHO, -CH₂OH, -COOH, -C(O)CH₃ and -Cι_-6alkoxy; and R⁵ is selected from the group consisting of H, Cι_-6alkyl, C ι- alkoxy, C _-6alkenyl, C_2-6alkynyl and halogen, where the Cι_-6alkyl, C_2-6alkenyl and C_2-6alkynyl groups are optionally substituted with 1-3 hydroxyl groups.

10. The method of Claim 9 wherein R³ is selected from the group consisting of H, Cj.

₆alkyl, C_1-5alkylcarbonyl, benzoyl and C_1-6alkyloxyC_1-6alkyl.

11. The method of Claim 9 wherein R⁴ is selected from the group consisting of -CHO,

-COOH and -C(O)CH₃.

12. The method of Claim 9 wherein R⁵ is selected from the group consisting of

-C≡C-C(CH₃)(CH₂OH)(OH), -C≡C-CH(CH₃)(OH), -C≡C-CH₃, -CH₂CH=C(CH₃)₂,

-CH(OH)-CH₂-CH(CH₃)₂, -CH=CHCH(CH₃)₂, -OCH₃ and I.

13. The method of Claim 9 wherein said compound is selected from the group consisting of 3-methyl- ?-anisaldehyde, 4-acetoxy-3-(3'methyl-3'-hydroxy- -butynyl) benzaldehyde, 4-hydroxy-3-(3'-methyl-2'-butenyl) benzaldehyde, 4-hydroxy-3- iodobenzaldehyde, 4-hydroxy-3-methyl-benzaldehyde, 4-hydroxy-benzaldehyde, para-anisaldehyde, vanillin acetate and 4-acetoxy-3-iodobenzaldehyde.

14. The method of Claim 9 wherein said fungal population is a plant fungal population, and said composition is non-phytotoxic to a plant infested with said fungus.

15. The method of Claim 14 wherein said composition is applied in the vicinity of said plant.

16. The method of Claim 14 wherein said composition is applied to said plant by spraying foliage to run off.

17. The method of Claim 14 wherein said fungal population is selected from the group consisting of Aspergillius niger, Botrytis cinerea, Penicillium sp. , Cladosporium sp. , Rhizopus stolinofer, Verticillium dahliae, Monilinia fructicola, Alternaria alternata and Colletotrichum accutatum.

18. A method for controlling a fungal population, said method comprises contacting the fungal population with a composition comprising a carrier and a fungicidally effective amount of a compound having the formula:

where R is selected from the group consisting of H, C_1-6alkyl, C _-6alkenyl, C _-6alkynyl, and a straight or branched chain having 4-6 carbon atoms, a double and a triple bond; where the Cι-₆alkyl, C .₆alkenyl, C_2-6alkynyl and chain are optionally substituted with 1 -3 hydroxyl groups.

19. The method of Claim 18 wherein R⁶ is selected from the group consisting of -C≡C-C(CH₃)(CH₂); -C≡C-C(CH₃)(CH₂OH)(OH), -C≡C-CH(CH₃)(OH), -C≡C-CH₃, -CH₂CH=C(CH₃)₂, -CH(OH)-CH₂-CH(CH₃)₂ and -CH=CHCH(CH₃)₂. 20. The method of Claim 18 wherein said fungal population is a plant fungal population, and said composition is non-phytotoxic to a plant infested with said fungus.

21. The method of Claim 20 wherein said composition is applied in the vicinity of said plant.

22. The method of Claim 20 wherein said composition is applied to said plant by spraying foliage to run off.

23. The method of Claim 20 wherein said fungal population is selected from the group consisting of Aspergillius niger, Botrytis cinerea, Penicillium sp. , Cladosporium sp. , Rhizopus stolinofer, Verticillium dahliae, Monilinia fructicola, Alternaria alternata and Colletotrichum accutatum. 24. A method for controlling a fungal population, said method comprises contacting the fungal population with a composition comprising a carrier and a fungicidally effective amount of a compound having the formula:

where R⁷ is selected from the group consisting of -OH, -CHO, -CH OH, -COOH, -C(O)CH₃ and C_1-6alkoxy; and R⁸ is selected from the group consisting of H,

C_1-6alkyl, C_2-6alkenyl and C_2-6alkynyl; where Ci^alkyl, C_2-6alkenyl and C_2-6alkynyl are optionally substituted with 1-3 hydroxyl groups.

25. The method of Claim 24 wherein R⁷ is -CHO.

26. The method of Claim 24 wherein R⁸ is selected from the group consisting of -C≡C-C(CH₃)(CH₂OH)(OH), -C≡C-CH(CH₃)(OH), -C≡C-CH₃, -CH₂CH=C(CH₃)₂,

-CH(OH)-CH₂-CH(CH₃)₂ and -CH=CHCH(CH₃)₂.

27. The method of Claim 24 wherein said compound is eutypine benzofuran.

28. The method of Claim 24 wherein said fungal population is a plant fungal population, and said composition is non-phytotoxic to a plant infested with said fungus. 29. The method of Claim 28 wherein said composition is applied in the vicinity of said plant.

30. The method of Claim 28 wherein said composition is applied to said plant by spraying foliage to run off.

31. The method of Claim 28 wherein said fungal population is selected from the group consisting of Aspergillius niger, Botrytis cinerea, Penicillium sp., Cladosporium sp.,

Rhizopus stolinofer, Verticillium dahliae, Monilinia fructicola, Alternaria alternata and Colletotrichum accutatum.

32. A method for preventing infestation of a host with a fungus, said method comprises contacting the plant with a composition comprising a carrier and a compound of Claim 1, 9, 18 or 24, in an amount sufficient to prevent infestation of said host.

33. A composition for controlling a fungal population, comprising a carrier in combination with a fungicidally active compound of Claim 1, 9, 18 or 24.

34. An article of manufacture that comprises a container in association with instructions for controlling fungi and holding a composition comprising a carrier and a fungicidally active compound of Claim 1, 9, 18 or 24.

WO 01/01977 PCTYUSOO/18225

AMENDED CLAIMS

[received by the International Bureau on 24 November 2000 (24.11.00); original claims 9, 11, 18 and 24 amended; remaining claims unchanged (3 pages)] and Colletotrichum accutatum. 9. A method for controlling a fungal population, said method comprises contacting the fungal population with a composition comprising a carrier and a fungicidally effective amount of a compound having the formula:

where R is selected from the group consisting of H, alkyl, alkylcarbonyl, benzoyl and Cι_-6alkyloxyC_1-6alkyl; and R⁴ is selected from the group consisting of -OH, -CHO, -CH₂OH, -COOH and -C_1- alkoxy; and R⁵ is selected from the group consisting of H, C_1-6alkyl, C ι_-6alkoxy, C_2-6alkenyl, C_2-6alkynyl and halogen, where the C_1-6alkyl, C_{2- 6}alkenyl and C₂-₆alkynyl groups are optionally substituted with 1-3 hydroxyl groups.

10. The method of Claim 9 wherein R is selected from the group consisting of H, . ₆alkyl, C_1-5alkylcarbonyl, benzoyl and

11. The method of Claim 9 wherein R⁴ is selected from the group consisting of -CHO and -COOH

12. The method of Claim 9 wherein R⁵ is selected from the group consisting of -C=C-C(CH₃)(CH₂OH)(OH), -C≡C-CH(CH₃)(OH), -C≡C-CH₃, -CH₂CH=C(CH₃)₂, -CH(OH)-CH₂-CH(CH₃)₂, -CH=CHCH(CH₃)₂, -OCH₃ and I.

13. The method of Claim 9 wherein said compound is selected from the group consisting of 3-methyl-p-anisaldehyde, 4-acetoxy-3-(3'methyl-3'-hydroxy-l'-butynyl) benzaldehyde, 4-hydroxy-3-(3'-methyl-2'-butenyl) benzaldehyde, 4-hydroxy-3- iodobenzaldehyde, 4-hydroxy-3-methyl-benzaldehyde, 4-hydroxy-benzaldehyde, para-anisaldehyde, vanillin acetate and 4-acetoxy-3-iodobenzaldehyde.

14. The method of Claim 9 wherein said fungal population is a plant fungal population, and said composition is non-phytotoxic to a plant infested with said fungus.

15. The method of Claim 14 wherein said composition is applied in the vicinity of said plant.

16. The method of Claim 14 wherein said composition is applied to said plant by spraying foliage to run off.

17. The method of Claim 14 wherein said fungal population is selected from the group consisting of Aspergillius niger, Botrytis cinerea, Penicillium sp., Cladosporium sp.,

Rhizopus stolinofer, Verticillium dahliae, Monilinia fructicola, Alternaria alternata and Colletotrichum accutatum.

18. A method for controlling a fungal population, said method comprises contacting the fungal population with a composition comprising a carrier and a fungicidally effective amount of a compound having the formula:

where R⁶ is selected from the group consisting of C_1-6alkyl, C_2-6alkenyl, C_2- alkynyl, and a straight or branched chain having 4-6 carbon atoms, a double and a triple bond; where the Cι_-6alkyl, C _-6alkenyl, C_2-6alkynyl and chain are optionally substituted with

1-3 hydroxyl groups.

19. The method of Claim 18 wherein R⁶ is selected from the group consisting of

-C≡C-C(CH₃)(CH₂); -C≡C-C(CH₃)(CH₂OH)(OH), -C≡C-CH(CH₃)(OH), -C≡C-CH₃, -CH₂CH=C(CH₃)₂, -CH(OH)-CH₂-CH(CH₃)₂ and -CH=CHCH(CH₃)₂.

20. The method of Claim 18 wherein said fungal population is a plant fungal population, and said composition is non-phytotoxic to a plant infested with said fungus.

21. The method of Claim 20 wherein said composition is applied in the vicinity of said plant.

22. The method of Claim 20 wherein said composition is applied to said plant by spraying foliage to run off.

23. The method of Claim 20 wherein said fungal population is selected from the group consisting of Aspergillius niger, Botrytis cinerea, Penicillium sp. , Cladosporium sp. , Rhizopus stolinofer, Verticillium dahliae, Monilinia fructicola, Alternaria alternata and Colletotrichum accutatum.

24. A method for controlling a fungal population, said method comprises contacting the fungal population with a composition comprising a carrier and a fungicidally effective amount of a compound having the formula:

where R⁷ is selected from the group consisting of -OH, -CHO, -CH₂OH, -COOH, -C(O)CH₃ and Cι- alkoxy; and R is selected from the group consisting of H,

C _-6alkyl, C _-6alkenyl and C_2-6alkynyl; where C_1-6alkyl, C_2- alkenyl and C_2-6alkynyl are optionally substituted with 1-3 hydroxyl groups.

25. The method of Claim 24 wherein R⁷ is -CHO.

26. The method of Claim 24 wherein R⁸ is selected from the group consisting of -C≡C-C(CH₃)(CH₂OH)(OH), -C≡C-CH(CH₃)(OH), -C≡C-CH₃, -CH₂CH=C(CH₃)₂,

-CH(OH)-CH₂-CH(CH₃)₂ and -CH=CHCH(CH₃)₂.

27. The method of Claim 24 wherein said compound is eutypine benzofuran.

28. The method of Claim 24 wherein said fungal population is a plant fungal population, and said composition is non-phytotoxic to a plant infested with said fungus.

29. The method of Claim 28 wherein said composition is applied in the vicinity of said plant.

30. The method of Claim 28 wherein said composition is applied to said plant by spraying foliage to run off.

31. The method of Claim 28 wherein said fungal population is selected from the group consisting of Aspergillius niger, Botrytis cinerea, Penicillium sp. , Cladosporium sp. ,

Rhizopus stolinofer, Verticillium dahliae, Monilinia fructicola, Alternaria alternata