Inhibition and dispersion of biofilms in plants with imidazole-triazole derivatives

Disclosure is provided for methods of preventing, removing or inhibiting microbial biofilm formation or microbial infection in a plant or plant part thereof, including applying thereto a treatment effective amount of an active compound as described herein, or an agriculturally acceptable salt thereof. Methods of enhancing a microbicide (e.g., including a copper, antibiotic, bacteriophage, etc.) and/or plant defense activator are also provided, including applying an active compound as described herein. Compositions comprising an active compound as described herein in an agriculturally acceptable carrier are also provided, and in some embodiments the compositions further include a microbicide (e.g., including copper, antibiotic, bacteriophage, etc.) and/or plant defense activator.

FIELD OF THE INVENTION

This invention relates to compositions and methods useful for controlling biofilms and microorganisms in plants, particularly vascular plants.

BACKGROUND OF THE INVENTION

New approaches are urgently needed to improve agricultural production, given the steadily growing global population that is predicted to reach 6-9 billion persons by mid-century, the continual strain on existing and finite agricultural lands, and the recent diversion of valuable agricultural land from production of crops to production of biomass for fuels. Here we describe new approaches to increase agricultural production by controlling the adverse effects of microorganisms on plants.

The five main crops on which modern societies depend most heavily include corn, cotton, rice, soybeans, and wheat. All of these crops are affected in a deleterious manner by biofilm formation. In addition, other valuable plants such as those producing fruits and vegetables are similarly affected. Plants grown for biomass stand to increase as a valuable crop, albeit not for food, and also can benefit from protection from biofilm formation. Forestry crops and ornamentals also suffer from biofilms.

SUMMARY OF THE INVENTION

The present invention is a method of preventing, removing or inhibiting microbial biofilm formation or microbial infection in a plant or plant part thereof, comprising applying to the plant or plant part a treatment effective amount of a compound selected from the group consisting of compounds of Formula (I), Formula (I)(a)(1), Formula (I)(b)(1), Formula (I)(a)(2), Formula (I)(b)(2), Formula (I)(i), Formula (I)(i)(a), Formula (II), Formula (II)(a), Formula (II)(i), Formula (II)(i)(a), Formula (III), Formula (III)(a), Formula (III)(b), Formula (III)(b)(i), Formula (III)(b)(ii), Formula (IV), Formula (IV)(a), Formula (IV)(i), Formula (IV)(i)(a), Formula (V), Formula (V)(a), Formula (V)(i), Formula (V)(i)(a), Formula (VI), Formula (VI)(a), Formula (VI)(i) and Formula (VI)(i)(a) as described herein, or an agriculturally acceptable salt thereof.

In some embodiments, the plant is a fruit or a vegetable crop plant.

In some embodiments, the plant is a citrus tree, and the compound is applied in an amount effective to treat or control a bacterial disease selected from the group consisting of canker, bacterial spot, Black Pit (fruit), Blast, citrus variegated chlorosis, and Citrus Huanglongbing. In some embodiments, the citrus tree is selected from the group consisting of orange, grapefruit, Mandarin, lemon, lime and Kumquat.

In some embodiments, the plant is a pome fruit, and the compound is applied in an amount effective to treat or control a bacterial disease selected from the group consisting of Fire Blight, Crown Gall, Blister spot and Hairy root. In some embodiments, the pome fruit is selected from the group consisting of apple, pear, quince, Asian pear, and loquats.

In some embodiments, the plant is aMusaspecies such as a banana, and the compound is applied in an amount effective to treat or controlRalstonia solanacearum.

In some embodiments, the plant is a cole (Brassicaceae) such as cabbage or broccoli, and the compound is applied in an amount effective to treat or control black rot (Xanthononas campestris).

In some embodiments, the plant is a winegrape, and the compound is applied in an amount effective to treat or control for Pierce's disease (Xylella fastidosa) or crown gall (Agrobacterium vitas, A. tumefaciens).

In some embodiments, the plant is a stone fruit or nut (e.g., peaches, nectarines, plums, almonds, walnuts), and the compound is applied in an amount effective to treat or control bacterial spot and/or blight caused byXanthomonas arboricola; blight caused byPseudomonas syringae); crown gall caused byAgrobacterium tumefaciens; phony peach and plum; or almond leaf scorch caused byXylella fastidosa.

In some embodiments, the plant is a landscape and/or shade tree (e.g., oak, maple, birch, etc.) for bacterial leaf scorch disease (e.g., cause byXylella fastidosa).

In some embodiments, the plant is a potato, and the compound is applied in an amount effective to treat or control soft rot or black leg (Erwinia, Pectobacterium).

In some embodiments, the plant is a pepper plant, and the compound is applied in an amount effective to treat or control a bacterial disease selected from the group consisting of Bacterial Spot, Bacterial wilt, Bacterial canker, and Syringae seedling blight and leaf spot.

In some embodiments, the plant is a tomato plant, and the compound is applied in an amount effective to treat or control a bacterial disease selected from the group consisting of: bacterial canker, bacterial speck, bacterial spot, bacterial stem rot and fruit rot, Bacterial wilt, Pith necrosis, and Syringae leaf spot.

In some embodiments, the plant is a soybean plant, and the compound is applied in an amount effective to treat or control a bacterial disease selected from the group consisting of Bacterial blight, Bacterial pustules, Bacterial wilt, Bacterial crinkle leaf, Bacterial tan spot, and Wildfire.

In some embodiments, the plant is corn, and the compound is applied in an amount effective to treat or control a bacterial disease selected from the group consisting of: Bacterial leaf blight, stalk rot, bacterial stripe, chocolate spot,holcusspot all causes byPseudomonasspecies, Bacterial leaf spot caused byXanthomomasspecies, Bacterial stalk rot, top rot and Stewart's disease caused byErwiniaspecies, seed rot-seedling blight caused byBacillusspecies, Purple leaf sheath caused byHemiparasiticbacteria, Corn stunt caused bySpriroplasma kunkelii, Goss's bacterial wilt and blight caused byClivibacter michiganensis.

In some embodiments, the plant is cotton, and the compound is applied in an amount effective to treat or control a bacterial disease selected from the group consisting of Bacterial blight caused byXanthomonasspecies, and Crown gall caused byAgrobacteriumspecies and Lint degradation caused byErwiniaspecies.

In some embodiments, the plant is wheat, and the compound is applied in an amount effective to treat or control a bacterial disease selected from the group consisting of Bacterial leaf blight, bacterial sheath rot and Basal glume rot caused byPseudomonasspecies, Bacterial mosaic and Spike blight caused byClavibacterspecies, Black chaff caused byXanthomonasspecies, and Pink seed caused byErwinia(Pantoea) species.

In some embodiments, the plant is rice, and the compound is applied in an amount effective to treat or control a bacterial disease selected from the group consisting of bacterial blight and leaf streak caused byXanthomonasspecies, Foot rot caused byErwiniaspecies, Grain rot caused byBurkholderiaspecies, and Sheath brown rot caused byPseudomonasspecies.

In some embodiments, the plant is pineapple, and the compound is applied in an amount effective to treat or control a bacterial disease selected from the group consisting of bacterial heart rot, fruit collapse, bacterial fruitlet brown rot, marbled fruit, pink fruit and soft rot caused byErwiniaspecies, and Acetic souring caused by Acetic acid bacteria.

In some embodiments, the microbial biofilm formation or microbial infection is caused by a fungi. In some embodiments, the compound is applied to the plant in an amount effective to treat or control a fungal disease selected from the group consisting of rots, leaf molds, blights, wilts, damping-off, spot, root rot, stem rot, mildew, brown spot, gummosis, melanose, post-bloom fruit drop, scab,alternaria, canker, flyspeck, fruit blotch, dieback, downy mildews, ear rots, anthracnose bunts, smut, rust, eyespot and pecky rice.

In some embodiments, the plant is citrus, and the compound is applied in an amount effective to treat or control a fungal disease selected from the group consisting of:Alternariabrown spot caused byAlternaria alternaria, Brown rot caused byPhytophtora citricola, Greasy spot and Greasy spot rind blotch caused byMycosphaerella citri, Melanose caused byDiaporthe citri, Phytophthorafoot rot, gummosis and root rot caused byPhytophthora citrophthora, Phytophthora palmivora, Phytophthora syringaeand otherPhytophthoraspp, Post bloom fruit drop caused byColletotrichum acutatum, and Scab caused byElsinoe fawcettii.

In some embodiments, the plant is Pome fruit, and the compound is applied in an amount effective to treat or control a fungal disease selected from the group consisting of: Apple scab caused byVenturia inaequalis, Bitter rot caused byColletotrichum gloeosporioides, Diplodiacanker caused byDilpodia mutila, Phytophthoracrown, collar, root and fruit rot caused byPhytophthoraspp., Powdery mildew caused byPodosphaera leucotricha, Pacific Coast pear rust, Cedar apple rust, Quince rust caused byGymnosporangiumspp., and Flyspeck caused bySchizothyrium pomi.

In some embodiments, the plant is Peppers, and the compound is applied in an amount effective to treat or control a fungal disease selected from the group consisting of: Anthracnose caused byColletotrichumspp., Damping-off and root rot caused byRhizoctonia solani, Phytophthoraspp.,Fusariumspp., andPythiumspp.,Phytophthorablight caused byPhytophthora capsici, andVerticilliumwilt caused byVerticillium albo-atrium.

In some embodiments, the plant is Tomato, and the compound is applied in an amount effective to treat or control a fungal disease selected from the group consisting of:Alternariastem canker caused byAlternaria alternaria, Anthracnose caused byColletotrichumspp.,Fusariumcrown, root rot and wilt caused byFusarium oxysporum, Gray mold caused byBotrytis cinerea, Late blight caused byphytophthora infestans, Pythiumdamping-off and fruit rot caused byPythiumspp.,Rhizoctoniadamping-off and fruit rot caused byRhizoctonia solani, Septorialeaf spot caused bySeptoria lycopersici, Verticilliumwilt caused byVerticillium albo-atrum, and White mold caused bySclerotinia sclerotiorum.

In some embodiments, the plant is Soybean, and the compound is applied in an amount effective to treat or control a fungal disease selected from the group consisting of:Phytophthoraroot and stem rot caused byPhytophthora sojae, Pythiumroot rot, damping-off and seed decay caused byPythiumspp., Brown stem rot caused byPhialophora gregata, Rhizoctoniaroot and stem rot caused byRhizoctonia solani, Stem canker, pod and stem blight caused byDiaporthe phaseolorum, Phomopsisseed decay caused byPhomopsis longicolla, Charcoal rot caused byMacrophomina phaseolina, Sclerotiniastem rot caused bySclerotinia sclerotiorum, Sudden death syndrome caused byFusarium solani, and Soybean Rust caused byPhakopsora pachyrhizi.

In some embodiments, the plant is Grape, and the compound is applied in an amount effective to treat or control a fungal disease selected from the group consisting of:Alternariarot caused byAlternaria alternaria, Angular leaf spot caused byMycosphaerella angulata, Botrytisbunch rot and blight caused byBotrytis cinerea, Diplodiacane dieback and bunch rot caused byDiplodia natalensis, Downy mildew caused byPlasmopara viticola, Phytophthoracrown and root rot caused byPhytophthoraspp., Powdery mildew caused byUncinula necator, Ripe rot caused byGlomerella cingulata, Septorialeaf spot caused bySeptoria ampelopsidis, andVerticilliumwilt caused byVerticillium dahliae.

In some embodiments, the plant is Potato, and the compound is applied in an amount effective to treat or control a fungal disease selected from the group consisting of: Brown spot, Black pit and Early blight caused byAlternariaspp.,Fusariumdry rot and wilt caused byFusariumspp., Gangrene caused byPhomaspp., Late blight and Pink rot caused byPhytophthoraspp.,Rhizoctoniacanker and black scurf caused byRhizoctonia solani, Roselliniablack rot caused byRoselliniaspp.,Septorialeaf spot caused bySeptoria lycopersici, Stem rot caused bySclerotium rolfsii, Verticilliumwilt caused byVerticillium albo-atrum, and White mold caused bySclerotinia sclerotiorum.

In some embodiments, the plant is Pineapple, and the compound is applied in an amount effective to treat or control a fungal disease selected from the group consisting of: Anthracnose caused byColletotrichum ananas, Butt rot and White leaf spot caused byChalara paradoxa, Leaf spot caused byCurvularia eragrostidis, Phytophthoraheart rot caused byPhytophthora cinnamomiandPhytophthora parasitica, Root rot and Seedling blight caused byPythiumspp., and Leaking brown ring caused byTofflieadis dimenationa.

In some embodiments, the plant is Corn, and the compound is applied in an amount effective to treat or control a fungal disease selected from the group consisting of: Anthracnose caused byColletotrichum graminicola, Aspergillusear and kernel rot caused byAspergillus flavus, Banded leaf, sheath spot, root rot and stalk rot caused byRhizoctonia solani, Brown spot, Black spot and Stalk rot caused byPhysoderma maydis, Curvularialeaf spot caused byCurvularia clavata, Diplodiaear rot, stalk rot, seed rot and seedling blight caused byDilpodiaspp., Downey mildews caused bySclerophthoraspp. orPeronosclerosporaspp., Ear rots caused byAlternaria alternaria, Ergot caused byClaviceps gigantea, Fusariumear, stalk, kernel, root, seed rot, seedling blight caused byFusariumspp.,Cercosporaleaf spot caused byCercospora zeae-maydis, Helminthosporiumear rot caused byHelminthosporium carbonum, Pythiumroot rot and stalk rot caused byPythiumspp.,Rhizoctoniaear rot caused byRhizoctonia zeae, Common corn rust and Southern corn rust caused byPucciniaspp., Southern blight caused byAthelia rolfsii, Common smut caused byUstilago zeae, Southern corn leaf blight and stalk rot caused byCochliobolus heterostrophus, and storage rots caused byAspergillusspp. andPenicilliumspp.

In some embodiments, the plant is Rice, and the compound is applied in an amount effective to treat or control a fungal disease selected from the group consisting of: Black kernel caused byCurvularia lunata, Blast caused byPyricularia oryzae, Brown spot caused byCochliobolus miyabeanus, Downy mildew caused bySclerophthora macrospora, False smut caused byUstilaginoidea virens, Narrow brown leaf spot caused byCercospora janseana, Pecky rice caused byFusariumspp.,Microdochium oryzae, orSarocladium oryzae, Root rot caused byFusariumspp, orPythiumspp., Seedling blight caused by fungi (e.g.,Cochliobolus miyabeanus, Curvulariaspp.,Fusariumspp.,Rhizoctonia solani, Sclerotium rolfsiiandAthelia rolfsii), Stackburn caused byAlternaria padwickii, Stem rot caused byMagnaporthe salvinii, Water-mold (seed-rot and seedling disease) caused byAchlyaspp.,Fusariumspp., orPythiumspp.

A further aspect of the present invention is an agricultural composition comprising: (a) an agriculturally acceptable carrier (e.g., an aqueous carrier or a solid particulate carrier); and (b) an antimicrobial or biofilm preventing, removing or inhibiting compound selected from the group consisting of compounds of Formula (I), Formula (I)(a)(1), Formula (I)(b)(1), Formula (I)(a)(2), Formula (I)(b)(2), Formula (I)(i), Formula (I)(i)(a), Formula (II), Formula (II)(a), Formula (II)(i), Formula (II)(i)(a), Formula (III), Formula (III)(a), Formula (III)(b), Formula (III)(b)(i), Formula (III)(b)(ii), Formula (IV), Formula (IV)(a), Formula (IV)(i), Formula (IV)(i)(a), Formula (V), Formula (V)(a), Formula (V)(i), Formula (V)(i)(a), Formula (VI), Formula (VI)(a), Formula (VI)(i) and Formula (VI)(i)(a) as described herein, or an agriculturally acceptable salt thereof. In some embodiments, the composition further includes a microbicide. In some embodiments, the microbicide comprises copper (e.g., copper hydroxide). In some embodiments, the microbicide comprises an antibiotic or a bacteriophage. In some embodiments, the composition further includes a plant defense activator. In some embodiments, the composition further includes both a plant defense activator and a microbicide. In some embodiments, the compound is a compound of Formula (II)(a)(5)(D):

or an agriculturally acceptable salt thereof.

Further provided are methods of enhancing the effects of a microbicide comprising applying an active compound selected from the group consisting of compounds of Formula (I), Formula (I)(a)(1), Formula (I)(b)(1), Formula (I)(a)(2), Formula (I)(b)(2), Formula (I)(i), Formula (I)(i)(a), Formula (II), Formula (II)(a), Formula (II)(i), Formula (II)(i)(a), Formula (III), Formula (III)(a), Formula (III)(b), Formula (III)(b)(i), Formula (III)(b)(ii), Formula (IV), Formula (IV)(a), Formula (IV)(i), Formula (IV)(i)(a), Formula (V), Formula (V)(a), Formula (V)(i), Formula (V)(i)(a), Formula (VI), Formula (VI)(a), Formula (VI)(i) and Formula (VI)(i)(a) as described herein, in combination with said microbicide. In some embodiments, the microbicide comprises copper (e.g., copper hydroxide). In some embodiments, the microbicide is an antibiotic or a bacteriophage. In some embodiments, the applying step is carried out by applying the active compound and the microbicide simultaneously. In some embodiments, the applying step is carried out by applying the active compound and the microbicide sequentially. In some embodiments, the compound is a compound of Formula (II)(a)(5)(D):

or an agriculturally acceptable salt thereof.

Also provided are methods of enhancing the effects of a plant defense activator comprising applying an active compound selected from the group consisting of compounds of Formula (I), Formula (I)(a)(1), Formula (I)(b)(1), Formula (I)(a)(2), Formula (I)(b)(2), Formula (I)(i), Formula (I)(i)(a), Formula (II), Formula (II)(a), Formula (II)(i), Formula (II)(i)(a), Formula (III), Formula (III)(a), Formula (III)(b), Formula (III)(b)(i), Formula (III)(b)(ii), Formula (IV), Formula (IV)(a), Formula (IV)(i), Formula (IV)(i)(a), Formula (V), Formula (V)(a), Formula (V)(i), Formula (V)(i)(a), Formula (VI), Formula (VI)(a), Formula (VI)(i) and Formula (VI)(i)(a) as described herein, in combination with said plant defense activator. In some embodiments, the applying step is carried out by applying the active compound and the microbicide simultaneously. In some embodiments, the applying step is carried out by applying the active compound and the microbicide sequentially. In some embodiments, the compound is a compound of Formula (II)(a)(5)(D):

or an agriculturally acceptable salt thereof.

A further aspect of the present invention is an antimicrobial or biofilm preventing, removing or inhibiting compound selected from the group consisting of compounds of Formula (I), Formula (I)(a)(1), Formula (I)(b)(1), Formula (I)(a)(2), Formula (I)(b)(2), Formula (I)(i), Formula (I)(i)(a), Formula (II), Formula (II)(a), Formula (II)(i), Formula (II)(i)(a), Formula (III), Formula (III)(a), Formula (III)(b), Formula (III)(b)(i), Formula (III)(b)(ii), Formula (IV), Formula (IV)(a), Formula (IV)(i), Formula (IV)(i)(a), Formula (V), Formula (V)(a), Formula (V)(i), Formula (V)(i)(a), Formula (VI), Formula (VI)(a), Formula (VI)(i) and Formula (VI)(i)(a) as described herein, for use in treating or preventing a bacterial or fungal infection in a plant or plant part as described above and below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is further described below. All patent references referred to in this patent application are hereby incorporated by reference in their entirety as if set forth fully herein.

“Active compound” as used herein refers to the various embodiments of compounds described in Section B (triazole derivatives) set forth below.

“Plant” as used herein includes all members of the plant kingdom, including higher (or “vascular”) plants and lower (“non-vascular”) plants, and particularly including all plants in the divisions Filicinae, Gymnospermae (or “gymnosperm”), and Angiospermae (or “Angiosperm”). Nonvascular plants of the present invention include, but are not limited to, bryophytes.

“Angiosperm” as used herein includes, but is not limited to, plants of the sub-classes Monocotyledoneae (or monocots) and Dicotyledoneae (or dicots).

Gymnospermae (or “Gymnosperms”) as used herein includes but is not limited to conifers.

“Plant part” as used herein refers to seeds, roots, leaves, shoots, fruits (e.g., apples, pineapples, citrus fruit, etc.), vegetables, tubers, flowers (e.g., cut flowers such as roses, as well as the reproductive parts of plants), petals, stem, trunk, etc., harvested or collected from a plant as described herein. The plant part of a vascular plant may be a non-vascular part, such as a seed or meristem (growing tip of a shoot).

“Applying” as described herein can be carried out directly or indirectly by any suitable technique, including topically applying to the plant or plant part, applying to the media in which the plant or plant part is grown, stored, displayed or maintained (e.g., adding to water in which the stems of cut flowers are placed), etc. Note that the plant may be grown in any suitable media, including but not limited to soil, potting soil, soilless media such as sand and hydroponic media (including solution culture, medium culture, and deep water culture), etc.

“Triazole” refers to the commonly known structures:

“Imidazole” refers to the commonly known structure:

“H” refers to a hydrogen atom. “C” refers to a carbon atom. “N” refers to a nitrogen atom. “O” refers to an oxygen atom. “Halo” refers to F, Cl, Br or I. The term “hydroxy,” as used herein, refers to an —OH moiety. “Br” refers to a bromine atom. “Cl” refers to a chlorine atom, “I” refers to an iodine atom. “F” refers to a fluorine atom.

An “acyl group” is intended to mean a group —C(O)—R, where R is a suitable substituent (for example, an acetyl group, a propionyl group, a butyroyl group, a benzoyl group, or an alkylbenzoyl group).

As generally understood by those of ordinary skill in the art, “saturation” refers to the state in which all available valence bonds of an atom (e.g., carbon) are attached to other atoms. Similarly, “unsaturation” refers to the state in which not all the available valence bonds are attached to other atoms; in such compounds the extra bonds usually take the form of double or triple bonds (usually with carbon). For example, a carbon chain is “saturated” when there are no double or triple bonds present along the chain or directly connected to the chain (e.g., a carbonyl), and is “unsaturated” when at least one double or triple bond is present along the chain or directly connected to the chain (e.g., a carbonyl). Further, the presence or absence of a substituent depending upon chain saturation will be understood by those of ordinary skill in the art to depend upon the valence requirement of the atom or atoms to which the substituent binds (e.g., carbon).

The term “optionally substituted” indicates that the specified group is either unsubstituted, or substituted by one or more suitable substituents. A “substituent” is an atom or atoms substituted in place of a hydrogen atom on the parent chain or cycle of an organic molecule, for example, H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide.

“Alkenyl,” as used herein, refers to a straight or branched chain hydrocarbon containing from 1 or 2 to 10 or 20 or more carbons, and containing at least one carbon-carbon double bond, formed structurally, for example, by the replacement of two hydrogens. Representative examples of “alkenyl” include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl and the like. In some embodiments, alkenyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide.

“Alkynyl,” as used herein, refers to a straight or branched chain hydrocarbon group containing from 1 or 2 to 10 or 20 or more carbon atoms, and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, 1-butynyl and the like. In some embodiments, alkynyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide.

“Heteroaryl” means a cyclic, aromatic hydrocarbon in which one or more carbon atoms have been replaced with heteroatoms. If the heteroaryl group contains more than one heteroatom, the heteroatoms may be the same or different. Examples of heteroaryl groups include pyridyl, pyrimidinyl, imidazolyl, thienyl, furyl, pyrazinyl, pyrrolyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, indolyl, isoindolyl, indolizinyl, triazolyl, pyridazinyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, isothiazolyl, and benzo[b]thienyl. Preferred heteroaryl groups are five and six membered rings and contain from one to three heteroatoms independently selected from the group consisting of: O, N, and S. The heteroaryl group, including each heteroatom, can be unsubstituted or substituted with from 1 to 4 suitable substituents, as chemically feasible. For example, the heteroatom S may be substituted with one or two oxo groups, which may be shown as ═O. In some embodiments, heteroaryl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide.

An “amine” or “amino” is intended to mean the group —NH2. “Optionally substituted” amines refers to —NH2groups wherein none, one or two of the hydrogens is replaced by a suitable substituent as described herein, such as alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, carbonyl, carboxy, etc. In some embodiments, one or two of the hydrogens are optionally substituted with independently selected, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide. Disubstituted amines may have substituents that are bridging, i.e., form a heterocyclic ring structure that includes the amine nitrogen.

An “amide” as used herein refers to an organic functional group having a carbonyl group (C═O) linked to a nitrogen atom (N), or a compound that contains this group, generally depicted as:

A “thiol” or “mercapto” refers to an —SH group or to its tautomer ═S.

A “sulfone” as used herein refers to a sulfonyl functional group, generally depicted as:

A “sulfoxide” as used herein refers to a sulfinyl functional group, generally depicted as:

The term “oxo,” as used herein, refers to a ═O moiety. The term “oxy,” as used herein, refers to a —O— moiety.

“Nitro” refers to the organic compound functional group —NO2.

“Carbonyl” is a functional group having a carbon atom double-bonded to an oxygen atom (—C═O). “Carboxy” as used herein refers to a —COOH functional group, also written as —(C═O)—OH.

“Amino acid sidechain” as used herein refers to any of the 20 commonly known groups associated with naturally-occurring amino acids, or any natural or synthetic homologue thereof. An “amino acid” includes the sidechain group and the amino group, alpha-carbon atom, and carboxy groups, as commonly described in the art. Examples of amino acids include glycine, and glycine that is substituted with a suitable substituent as described herein, such as alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, carbonyl, carboxy, etc., or an agriculturally acceptable salt thereof. For example, “Histidine” is one of the 20 most commonly known amino acids found naturally in proteins. It contains an imidazole side chain substituent. Other examples of naturally-occurring amino acids include lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, cryptophan, and cysteine. Also included in the definitions of “amino acid sidechain” and “amino acid” is proline, which is commonly included in the definition of an amino acid, but is technically an imino acid. As used in this application, both the naturally-occurring L-, and the non-natural D-amino acid enantiomers are included. The single letter code for amino acids is A (Ala), C (Cys), D (Asp), E (Glu), F (Phe), G (Gly), H(His), I (Ile), K (Lys), L (Leu), M (Met), N (Asn), P (Pro), Q (Gln), R (Arg), S (Ser), T (Thr), V (Val), W (Trp), and Y (Tyr). A “peptide” is a linear chain of amino acids covalently linked together, typically through an amide linkage, and contains from 1 or 2 to 10 or 20 or more amino acids, and is also optionally substituted and/or branched.

The term “optionally substituted” indicates that the specified group is either unsubstituted, or substituted by one or more suitable substituents. A “substituent” is an atom or atoms substituted in place of a hydrogen atom on the parent chain or cycle of an organic molecule.

B. Active Compounds

Active compounds are provided below. In some of the embodiments provided in the present invention, active compounds are derivatives of triazole. In some embodiments, active compounds include imidazole-triazole conjugates. In some embodiments, active compounds include 2-aminoimidazole-triazole conjugates (“2-AIT”). Active compounds as described herein can be prepared as detailed below or in accordance with known procedures or variations thereof that will be apparent to those skilled in the art.

As will be appreciated by those of skill in the art, the active compounds of the various formulas disclosed herein may contain chiral centers, e.g. asymmetric carbon atoms. Thus, the present invention is concerned with the synthesis of both: (i) racemic mixtures of the active compounds, and (ii) enantiomeric forms of the active compounds. The resolution of racemates into enantiomeric forms can be done in accordance with known procedures in the art. For example, the racemate may be converted with an optically active reagent into a diastereomeric pair, and the diastereomeric pair subsequently separated into the enantiomeric forms.

Geometric isomers of double bonds and the like may also be present in the compounds disclosed herein, and all such stable isomers are included within the present invention unless otherwise specified. Also included in active compounds of the invention are tautomers (e.g., tautomers of triazole and/or imidazole) and rotamers.

All chains defined by the formulas herein which include three or more carbons may be saturated or unsaturated unless otherwise indicated.

Carbons or other atoms along a chain identified by the Formulas herein may be identified by number, and when identified by number shall be numbered from left to right. For example:

To illustrate where there are two or more discrete chains, for Formula (II)(i)(a) described below, wherein R2, R3, R4, R5, R7and R8=H, and R6=phenyl:

the exemplary structure below shows n=5, saturated, one of either Rxor Ry=methyl at C4; m=3, unsaturated, Ru=methyl at C2 (Rvis absent at C2); and Rx, Ry, Ruand Rv=H at all other occurrences:

Active compounds for carrying out the present invention include compounds of Formula (I):

A, B, D, E, F, G and H are each independently selected from carbon, N, S and O, wherein at least one of D, E, F, G and H is carbon; and

or an agriculturally acceptable salt thereof.

As will be appreciated by those of skill in the art, a given substituent (R1-R8) may be present or absent depending upon the valence requirement of the atom or atoms to which the substituent binds (e.g., carbon versus nitrogen).

In some embodiments of Formula (I), R1is a substituted amino, A, B, F, G and H are each N, and D and E are each carbon, generally depicted by Formulas (I)(a)(1)-(I)(b)(1):

or an agriculturally acceptable salt thereof.

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (I), R1is a substituted amino, A, B, F, G and D are each N, and D and E are each carbon, generally depicted by Formula (I)(b)(2):

or an agriculturally acceptable salt thereof.

Active compounds further include compounds of Formula (I)(i):

A, B, D, E, F, G and H are each independently selected from carbon, N, S and O, wherein at least one of D, E, F, G and H is carbon; and

or an agriculturally acceptable salt thereof.

As will be appreciated by those of skill in the art, a given substituent (R1-R8) may be present or absent depending upon the valence requirement of the atom or atoms to which the substituent binds (e.g., carbon versus nitrogen).

In some embodiments of Formula (I)(i), R1is a substituted amino; R2, R3, R4, R5, R7and R8=H; A, B, F, G and H are each N, and D and E are each carbon, generally depicted by Formula (I)(i)(a):

or an agriculturally acceptable salt thereof.

Active compounds for carrying out the present invention include compounds of Formula (II):

A, B, D, E, F, G and H are each independently selected from carbon, N, S and O, wherein at least one of D, E, F, G and H is carbon; and

n=0 to 20; and

or an agriculturally acceptable salt thereof.

As will be appreciated by those of skill in the art, a given substituent (R1-R8) may be present or absent depending upon the valence requirement of the atom or atoms to which the substituent binds (e.g., carbon versus nitrogen).

In some embodiments of Formula (II), R1is a substituted amino, A, B, F, G and H are each N, and D and E are each carbon, generally depicted by Formula (II)(a):

n=0 to 20; and

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (II)(a), R1a, R1b, R2, R3and R5are each H and R6is phenyl, examples of which include, but are not limited to, the following exemplary Formulas. Each occurrence of Rx, Ry, Ruand Rvpresent is H unless otherwise indicated.

Active compounds further include compounds of Formula (II)(i):

A, B, D, E, F, G and H are each independently selected from carbon, N, S and O, wherein at least one of D, E, F, G and H is carbon;

n=0 to 20, saturated or unsaturated; and

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (II)(i), R1is a substituted amino, A, B, F, G and H are each N, and D and E are each carbon, generally depicted by Formula (II)(i)(a):

n=0 to 20, saturated or unsaturated; and

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (II)(i)(a), R1aand R1bare each H, and R6is heteroaryl, examples of which include, but are not limited to, the following exemplary Formulas:

Also provided are compounds of Formula (III):

A, B, D, E and F are each independently selected from carbon, N, S and O, wherein at least one of A, B, D, E and F is carbon;

or an agriculturally acceptable salt thereof.

As will be appreciated by those of skill in the art, a given substituent (R5-R9) may be present or absent depending upon the valence requirement of the atom or atoms to which the substituent binds (e.g., carbon versus nitrogen).

In some embodiments of Formula (III), B, D and E are each N, and A and F are each carbon, generally depicted as Formula (III)(a):

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (III), A, D and E are each N, and B and F are each carbon, generally depicted as Formula (III)(b):

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (III)(b), R6and R9are each H, generally depicted by Formula (III)(b)(i):

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (III)(b), R5and R6are each H, generally depicted by Formula (III)(b)(ii):

or an agriculturally acceptable salt thereof.

Also provided are compounds of Formula (IV):

A, B, D, E, F, G and H are each independently selected from carbon, N, S and O, wherein at least one of D, E, F, G and H is carbon; and

m=0 to 20; and

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (IV), R1is a substituted amino, A, B, F, G and H are each N, and D and E are each carbon, generally depicted by Formula (IV)(a):

m=0 to 20; and

or an agriculturally acceptable salt thereof.

In some embodiments, R6is a group:

X, Y and Z are each independently selected from the group consisting of: H, methyl, Br and Cl.

In some embodiments, R6is a group:

Further provided are compounds of Formula (IV)(i):

A, B, D, E, F, G and H are each independently selected from carbon, N, S and O, wherein at least one of D, E, F, G and H is carbon;

m=0 to 20, saturated or unsaturated; and

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (IV)(i), R1is a substituted amino, A, B, F, G and H are each N, and D and E are each carbon, generally depicted by Formula (IV)(i)(a):

m=0 to 20, saturated or unsaturated; and

or an agriculturally acceptable salt thereof.

Also provided are compounds of Formula (V):

A, B, D, E, F, G and H are each independently selected from carbon, N, S and O, wherein at least one of D, E, F, G and H is carbon; and

m=0 to 20; and

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (V), R1is a substituted amino, A, B, F, G and H are each N, and D and E are each carbon, generally depicted by Formula (V)(a):

m=0 to 20; and

or an agriculturally acceptable salt thereof.

Further provided are compounds of Formula (V)(i):

A, B, D, E, F, G and H are each independently selected from carbon, N, S and O, wherein at least one of D, E, F, G and H is carbon;

m=0 to 20, saturated or unsaturated; and

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (V)(i), R1is a substituted amino, A, B, F, G and H are each N, and D and E are each carbon, generally depicted by Formula (V)(i)(a):

m=0 to 20, saturated or unsaturated; and

or an agriculturally acceptable salt thereof.

Also provided are compounds of Formula (VI):

A, B, D, E, F, G and H are each independently selected from carbon, N, S and O, wherein at least one of D, E, F, G and H is carbon; and

m=0 to 20; and

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (VI), R1is a substituted amino, A, B, F, G and H are each N, and D and E are each carbon, generally depicted by Formula (VI)(a):

m=0 to 20; and

or an agriculturally acceptable salt thereof.

Further provided are compounds of Formula (VI)(i):

A, B, D, E, F, G and H are each independently selected from carbon, N, S and O, wherein at least one of D, E, F, G and H is carbon;

m=0 to 20, saturated or unsaturated; and

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (VI)(i), R1is a substituted amino, A, B, F, G and H are each N, and D and E are each carbon, generally depicted by Formula (VI)(i)(a):

m=0 to 20, saturated or unsaturated; and

or an agriculturally acceptable salt thereof.

In some embodiments of Formula (VI)(i)(a), R1aand R1bare each H, and R6is aryl or heteroaryl.

C. Microbicides and Plant Defense Activators

In some embodiments, an active compound described herein is applied in combination with a microbicide. “Microbicide” as used herein refers to a substance with the ability to kill or to inhibit the growth of microorganisms (e.g., bacteria, fungal cells, protozoa, etc.), which microbicide is not an active compound in the group herein disclosed of triazole derivatives. Common microbicides used for microbial control in plants include copper compounds. Examples of copper compounds include, but are not limited to, Bordeaux mixture, copper hydroxide, copper oxychloride, copper sulfate, cuprous oxide, mancopper or oxine-copper. However, microorganisms (e.g., bacteria such asXanthomonasandPseudomonas) may become resistant to treatment with copper.

In some embodiments, resistant microorganisms (e.g., copper-resistant bacteria) are rendered more susceptible to a microbicides and/or the effectiveness of treatment with a microbicides is enhanced upon application in combination with an active compound described herein (e.g., fruit or vegetable yield is increased as compared to diseased plant producing the fruit or vegetable that is untreated or treated only with the microbicide).

An “antibiotic” as used herein is a type of “microbicide.” Common antibiotics include aminoglycosides, carbacephems (e.g., loracarbef), carbapenems, cephalosporins, glycopeptides (e.g., teicoplanin and vancomycin), macrolides, monobactams (e.g., aztreonam) penicillins, polypeptides (e.g., bacitracin, colistin, polymyxin B), quinolones, sulfonamides, tetracyclines, etc. Antibiotics treat infections by either killing or preventing the growth of microorganisms. Many act to inhibit cell wall synthesis or other vital protein synthesis of the microorganisms.

Aminoglycosides are commonly used to treat infections caused by Gram-negative bacteria. Examples of aminoglycosides include, but are not limited to amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, and paromomycin.

Other microbicides that may be used in combination with the active compounds of the present invention include bacteriophages (bacterial viruses) such asBacillus. Examples of bacteriophage microbicides include, but are not limited to, AgriPhage™ (OmniLytics, Inc., Salt Lake City, Utah) and Serenade® (AgraQuest, Davis, Calif.). See, e.g., U.S. Pat. Nos. 5,919,447 and 6,077,506 to Marrone et al.; U.S. Pat. No. 6,103,228 to Heins et al.; and U.S. Patent Application Publication 20080152684.

In some embodiments, an active compound described herein is applied in combination with a plant defense activator. A “plant defense activator” as used herein is a compound that improves disease resistance by activating a plant's natural defense mechanisms, e.g., induces the plant to produce disease-fighting compounds. Examples of plant defense activators include, but are not limited to, prohexadione-calcium (Apogee), Cropset (plant booster element complex), probenazole, potassium phosphate (e.g., ProPhyt®, Helena Chemical Company), harpin protein (e.g., Messenger®, Eden Biosciences Ltd, Bothell, Wash.), acibenzolar or acibenzolar-S-methyl (e.g., Actigard™, Syngenta Crop Production, Inc, Greensboro, N.C.), streptomycin sulfate, reynoutria sachalinensis extract (reysa), etc.

Active compounds of the present invention can be used to prepare agrochemical compositions in like manner as other antimicrobial compounds. See, e.g., U.S. Pat. Application 2006/0094739; see also U.S. Pat. Nos. 6,617,330; 6,616,952; 6,569,875; 6,541,500, and 6,506,794.

Active compounds described herein can be used for protecting plants against diseases that are caused by microorganisms, including biofilm-forming microorganisms. The active compounds can be used in the agricultural sector and related fields as active ingredients for controlling plant pests. The active compounds can be used to inhibit or destroy the pests that occur on plants or parts of plants (fruit, blossoms, leaves, stems, tubers, roots) of different crops of useful plants, optionally while at the same time protecting also those parts of the plants that grow later e.g. from phytopathogenic microorganisms.

Active compounds may be used as dressing agents for the treatment of plant propagation material, in particular of seeds (fruit, tubers, grains) and plant cuttings (e.g. rice), for the protection against fungal infections as well as against phytopathogenic fungi occurring in the soil.

The active compounds can be used in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession with further compounds. These further compounds can be e.g. fertilizers or micronutrient donors or other preparations which influence the growth of plants. They can also be selective herbicides as well as insecticides, fungicides, bactericides, nematicides, molluscicides, plant growth regulators, plant activators or mixtures of several of these preparations, if desired together with further carriers, surfactants or application promoting adjuvants customarily employed in the art of formulation.

Suitable carriers and adjuvants can be solid or liquid and are substances useful in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binders or fertilizers.

The active compounds are used in unmodified form or, preferably, together with the adjuvants conventionally employed in the art of formulation. To this end they are conveniently formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations e.g. in polymeric substances. As with the type of the compositions, the methods of application, such as spraying, atomizing, dusting, scattering, coating or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.

The formulation, i.e. the compositions containing the active compound and, if desired, a solid or liquid adjuvant, are prepared in known manner, typically by intimately mixing and/or grinding the compound with extenders, e.g. solvents, solid carriers and, optionally, surface active compounds (surfactants).

Suitable carriers and adjuvants may be solid or liquid and correspond to the substances ordinarily employed in formulation technology, such as, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binding agents or fertilizers. Such carriers are for example described in WO 97/33890.

Further surfactants customarily employed in the art of formulation are known to the expert or can be found in the relevant literature.

The agrochemical formulations will usually contain from 0.1 to 99% by weight, preferably from 0.1 to 95% by weight, of a compound described herein, 99.9 to 1% by weight, preferably 99.8 to 5% by weight, of a solid or liquid adjuvant, and from 0 to 25% by weight, preferably from 0.1 to 25% by weight, of a surfactant.

Whereas it is preferred to formulate commercial products as concentrates, the end user will normally use dilute formulations.

The compositions may also contain further adjuvants such as stabilizers, antifoams, viscosity regulators, binders or tackifiers as well as fertilizers, micronutrient donors or other formulations for obtaining special effects.

E. Methods of Use

The methods, active compounds and compositions can be used to treat bacterial infections in a variety of plants, with specific examples including but not limited to those set forth below.

In citrus trees (including orange, lemon, lime, and grapefruit) active compounds and compositions as described herein can be used to treat or control a variety of microbial diseases, including but not limited to canker (caused byXanthomonas campestrisorXanthomonas axonopodisinfection), bacterial spot (caused byXanthomonas campestrispv.Citrumeloinfection); Black Pit (fruit) (caused byPseudomonas syringaeinfection); Blast (caused byPseudomonas syringaeinfection) citrus variegated chlorosis (caused byXylella fastidiosainfection), and Citrus Huanglongbing (HLB) caused byCandidatusLiberibacter asiaticus.

In pome fruits (including apple, pear, quince, Asian pear, and loquat), active compounds and compositions as described herein can be used to treat or control a variety of microbial infections, including but not limited to Fire Blight (caused byErwinia amylovorainfection), Crown Gall (caused byAgrobacterium tumefaciensinfection); Blister spot (caused byPseudomonas syringaeinfection) and Hairy root (caused byAgrobacterium rhizogenesinfection).

In pepper plants, active compounds and compositions as described herein can be used to treat or control a variety of microbial infections, including but not limited to: Bacterial Spot (caused byXanthomonas campestrispv.vesicatoriainfection); Bacterial wilt (caused byRalstonia solanacearuminfection), and Syringae seedling blight and leaf spot (caused byPseudomonas sryingaeinfection).

In tomato plants, active compounds and compositions as described herein can be used to treat or control a variety of microbial infections, including but not limited to: Bacterial canker (caused byClavibacter michiganesis), Bacterial speck (caused byPseudomonas syringae), Bacterial spot (caused byXanthomonas campestrisvesicatoria), Bacterial stem rot and fruit rot (caused byErwinia carotovora), Bacterial wilt (caused byRalstonia solanacearum), Pith necrosis (caused byPseudomonas corrugate), and Syringae leaf spot (caused byPseudomonas syringae).

In soybeans, active compounds and compositions as described herein can be used to treat or control a variety of microbial infections, including but not limited to: Bacterial blight (caused byPseudomonas amygdale), Bacterial pustules (caused byXanthomonas axonopodispv.Glycines), and Bacterial wilt (caused byRalstonia solanacearumorCurtobacterium flaccumfaciens).

In corn, cotton, wheat and rice, active compounds and compositions as described herein can be used to treat or control a variety of microbial infections, including but not limited to: bacterial blights, leaf spots and leaf streak caused byXanthomonasspecies; bacterial sheath rot, stripe and spot caused byPseudomonasspecies; and to bacterial stalk and top rot, wilt, foot rot, pink seed and lint degradation caused byErwiniaspecies.

In pineapple, active compounds and compositions as described herein can be used to treat or control a variety of microbial infections, including but not limited to: Bacterial heart rot and Fruit collapse (caused byErwinia chrysanthemi), Bacterial fruitlet brown rot (caused byErwinia ananas), Marbled fruit and Pink fruit (caused byErwinia herbicola), Soft rot (caused byErwinia carotovora), and Acetic souring (caused by Acetic acid bacteria).

The above listing is but a sampling, and active compounds and compositions as described herein may also be used to treat or control bacteria (some of which are named above) in a variety of plants. For example, the bacteriaXylella fastidiosainfects citrus trees as noted above (citrus variegated chlorosis), and also infects grapevines (Pierce's disease). Other plant hosts ofXylella fastidiosainclude, but are not limited to, ornamentals, oleander (leaf scorch), almond, coffee, maple, mulberry, elm, sycamore, alfalfa, etc. Similarly,Ralstonia solanacearuminfects soybeans (bacterial wilt) as well as banana (Moko disease), tobacco (Granville wilt), geranium (southern bacterial wilt), potato (brown rot) and a wide variety of other plants, including ginger and mulberry.

In addition to treating or controlling bacterial infections, active compounds and compositions as described herein can be used to treat or control fungal infections such as rots, leaf molds, blights, wilts, damping-off, spot, root rot, stem rot, mildew, brown spot, gummosis, melanose, post-bloom fruit drop, scab,alternaria, canker, flyspeck, fruit blotch, dieback, downy mildews, ear rots, anthracnose bunts, smut, rust, eyespot and pecky rice. Genera of plant-pathogenic fungi that can be treated or controlled by the active compounds, compositions, and methods described herein include but are not limited to:Pythiumspp.,Fusariumspp.,Rhizoctoniaspp.,Cercosporaspp.,Alternariaspp.,Colletotrichumspp.,Ustilagospp.,Phomaspp.,Gibberellaspp.Penicilliumspp.,Glomerellaspp.Diplodiaspp.,Curvulariaspp.,Sclerosporaspp.,Peronosclerosporaspp.,Cercosporaspp.,Pucciniaspp.,Ustilagospp.,Aspergillusspp.,Phomopsisspp.,Diaporthespp.,Botrytisspp.,Verticilliumspp.,Phytophthorsspp.

Particular fungal infections that can be treated or controlled by the methods, compounds and compositions described herein, in vegetables and greenhouse crops, includePhytophthorablight (caused byPhytophthora capsici) andPythiumdamping-off (caused byPythiumspp).

Note thatPhytophthoraalso has adverse effects on crops ranging from pineapples to cotton. It can kill woody citrus seedlings and young citrus trees (oranges, grapefruits, lemons, limes). In the greenhouse, germinating seed and seedlings are very susceptible to damping-off caused byPhytophthora, Pythium, SclerotinaandRhizoctoniaspecies. The cost to the grower to lose his crop to any of these fungi is substantial. The loss can happen at transplant time or when the crop is ready to be harvested.

The problems of fungi are not restricted to traditional crops but also extend to forestry products and have worldwide scope.Phytophthora cinnamomiis a soil-borne water mould that leads to a condition in plants called “root rot” or “dieback.”P. cinnamomicauses root rot affecting woody ornamentals including azalea, dogwood, forsythia, Fraser fir, hemlock, Japanese holly, juniper, rhododendron, white pine, and American chestnut.P. cinnamomiis responsible for the destruction of the elegant American chestnut tree. In Australia,P. cinnamomihas spread through the forests of western Australia, and into coastal forests of Victoria, where entire plant ecosystems are being obliterated. Given thatP. cinnamomiis a soil-borne water mould that infects the roots, almost the entire action takes place below ground. This problem highlights the importance of developing new compounds to counter fungal infections, even those that directly affect only the roots of the plant rather than the more visible effects on fruits or vegetables.

Active compounds of the invention can be applied to plants or plant loci in accordance with known techniques. The compound(s) can be tank mixed with other agricultural, turf, ornamental nursery, forestry and all other plant-labeled compatible pesticides. The compound(s) can be applied to seed. The compound(s) can be applied to edible and non-edible crops. The compound(s) can be applied to roots and all other parts of all plants. The compound(s) can be applied in greenhouses. The compound(s) can be applied and used in food-processing facilities. The compound(s) can be applied to plastic food bags and containers. The compound(s) can be applied as a solid, as its free base, or as a salt. The salts can include, but are not limited to, HI, HCl, HBr, H2SO4, acetic acid, and trifluoroacetic acid. The compound(s) can applied as a solution from 0.0001% to 99.9%. The compound(s) can be applied as a solid or solution with copper-based cidal compounds. The compound(s) can be applied with specific additional active agents, including but not limited to bactericides, fungicides, pesticides, biological insecticides and microbial insecticides.

Application can be carried out with any suitable equipment or technique, such as: Aerial—Fixed wing and Helicopter; Ground Broadcast Spray—Boom or boomless system, pull-type sprayer, floaters, pick-up sprayers, spray coupes, speed sprayers, and other broadcast equipment, water wagons and water bags; Low pressure boom sprayers, High pressure sprayers; Air blast sprayers; Low volume air sprayers (mist blowers); Ultra-low volume sprayers (ULV); Aerosol Generators (foggers); Dusters; Soil Injector; Hand-Held or High-Volume Spray Equipment—knapsack and backpack sprayers, pump-up pressure sprayers, hand guns, motorized spray equipment; Selective Equipment—Recirculating sprayers, shielded and hooded sprayers; Controlled droplet applicator (CDA) hand-held or boom-mounted applicators that produce a spray consisting of a narrow range of droplet size; Any and all greenhouse sprayers; Micro-sprinkler or drip irrigation systems; Chemigation.

One method of applying an active compound of the invention, or an agrochemical composition which contains at least one of said compounds, is foliar application. The frequency of application and the rate of application will depend on the risk of infestation by the corresponding pathogen. However, the active compounds can also penetrate the plant through the roots via the soil (systemic action) by drenching the locus of the plant with a liquid formulation, or by applying the compounds in solid form to the soil, e.g. in granular form (soil application). In crops of water such as rice, such granulates can be applied to the flooded rice field. The active compounds may also be applied to seeds (coating) by impregnating the seeds or tubers either with a liquid formulation of the fungicide or coating them with a solid formulation.

The term locus as used herein is intended to embrace the fields on which the treated crop plants are growing, or where the seeds of cultivated plants are sown, or the place where the seed will be placed into the soil. The term seed is intended to embrace plant propagating material such as cuttings, seedlings, seeds, and germinated or soaked seeds.

Advantageous rates of application are normally from 5 g to 2 kg of active ingredient (a.i.) per hectare (ha), preferably from 10 g to 1 kg a.i./ha, most preferably from 20 g to 600 g a.i./ha. When used as seed drenching agent, convenient dosages are from 10 mg to 1 g of active substance per kg of seeds.

F. Combination Treatments

In some embodiments, methods of enhancing the effects of a microbicide (such as a microbicide comprising copper, e.g., Kocide® 2000 or Kocide® 3000 (DuPont™, with active ingredient copper hydroxide) are disclosed, comprising the step of applying an active compound in combination with a microbicide, the active compound being applied in an amount effective to enhance the effects of the microbicide.

In some embodiments, methods of enhancing the effects of a plant defense activator are disclosed, comprising the step of applying an active compound in combination with a plant defense activator, the active compound being applied in an amount effective to enhance the effects of the plant defense activator.

“Enhancing” the effects of a microbicide by applying an active compound in combination with the microbicide refers to increasing the effectiveness of the microbicide, such that the microorganism killing and/or growth inhibition is higher at a certain concentration of the microbicide applied in combination with the active compound than without. In some embodiments, a bacteria or other microorganism is “sensitized” to the effects of a microbicide, such that the bacteria or other microorganism that was resistant to the microbicide prior to applying the active compound (e.g., little to none, or less than 20, 10, 5 or 1% are killed upon application) is rendered vulnerable to that microbicide upon or after applying the active compound (e.g., greater than 20, 30, 40, 50, 60, 70, 80, 90, or 95% or more are killed).

Similarly, “enhancing” the effects of a plant defense activator by applying an active compound in combination with the plant defense activator refers to increasing the effectiveness of the plant defense activator, such that the microorganism killing and/or growth inhibition is higher at a certain concentration of the plant defense activator applied in combination with the active compound than without. In some embodiments, a bacteria or other microorganism is “sensitized” to the effects of a plant defense activator, such that the bacteria or other microorganism that was resistant to the effects of the plant defense activator prior to applying the active compound (e.g., little to none, or less than 20, 10, 5 or 1% are killed upon application) is rendered vulnerable to the effects of that plant defense activator upon or after applying the active compound (e.g., greater than 20, 30, 40, 50, 60, 70, 80, 90, or 95% or more are killed).

As used herein, the application of two or more compounds (inclusive of active compounds and microbicides) “in combination” means that the two compounds are applied closely enough in time that the application of or presence of one alters the biological effects of the other. The two compounds may be applied simultaneously (concurrently) or sequentially.

Simultaneous application of the compounds may be carried out by mixing the compounds prior to application, or by applying the compounds at the same point in time but at different sites of the plant or using different types of applications, or applied at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are applied at the same point in time.

Sequential application of the compounds may be carried out by applying, e.g., an active compound at some point in time prior to application of a microbicide, such that the prior application of active compound enhances the effects of the microbicide (e.g., percentage of microorganisms killed and/or slowing the growth of microorganisms). In some embodiments, an active compound is applied at some point in time prior to the initial application of a microbicide. Alternatively, the microbicide may be applied at some point in time prior to the application of an active compound, and optionally, applied again at some point in time after the application of an active compound.

EXAMPLES

Synthesis of 2-Aminoimidazole-Triazole (2-AIT) Chemical Library

There is a paucity of reactions that have been reported to be compatible with 2-aminoimidazoles. To test the applicability of the Cu(I)-catalyzed [3+2] alkyne/azide cycloaddition (Click reaction, see Kolb et al.,Angewandte Chemie-International Edition2001, 11, 2004-2021; Rodionov et al.,Angewandte Chemie-International Edition2005, 15, 2210-2215), we synthesized the alkyne derived 2-aminoimidzole 1 and tested its ability to participate in a Cu(I)-catalyzed [3+2] cycloaddition with benzyl azide.

The alkyne derived 2-aminoimidazole (2-AI) was synthesized as outlined in Scheme 1. Amino acid 2 (Kotha et al.,Tetrahedron2002, 45, 9203-9208) was subjected to small scale Akabori reduction (Akabori,Berichte Der Deutschen Chemischen Gesellschaft1933, 66, 151-158), which, followed by condensation with cyanamide (Xu Yz et al.,J Org Chem1997, 3, 456-464) delivered the target alkyne 2-AI 1 in 88% yield. With 1 in hand, we explored various conditions to elicit the Cu-catalyzed [3+2] cycloaddition between 1 and benzyl azide (Table 1).

Reactions in THF using Cu(I) yielded no reaction and only returned starting material. We then switched to using CuSO4and sodium ascorbate in a 1:1 solvent mixture of H2O/EtOH. Again, no reaction was noted. However, when the reaction was heated to 40° C. we noted clean conversion to the desired 2-AIT conjugate 3 in 86% yield. Unfortunately, when the reaction was scaled up, we observed a significant amount of decomposition. Room temperature click reactions have been noted when a 1:1:1 solvent mixture of H2O/EtOH/CH2Cl2(Lee et al.,Tetrahedron Letters2006, 29, 5105-5109) is employed instead of the 1:1 H2O/EtOH mixture. When these reaction conditions were tested, we observed conversion to 3 in 93% yield.

With the methodology established to access 2-AIT conjugates, we employed the synthetic approach outlined in Scheme 1 to synthesize 2-AI alkynes 4 and 5 in which we systematically extended the methylene space between the alkyne and the 2-AI. The click reaction was then performed between each of the 2-AI alkynes and 12 azides to yield an initial 2-AIT library (shown below). Each compound was characterized (1H NMR,13C NMR, HRMS).

In conclusion, we have developed a synthetic approach to access 2-aminoimidazole/triazole (2-AIT) conjugates that is underpinned by the Cu(I)-catalyzed [3+2] alkyne/azide cycloaddition. Using this chemistry we have assembled a focused library of 2-AIT conjugates.

1. Experimental Protocols for 2-AIT Conjugate Synthesis

All reagents used for chemical synthesis were purchased from commercially available sources and used without further purification. Chromatography was performed using 60 Å mesh standard grade silica gel from Sorbtech (Sorbent Technologies, Inc., Atlanta, Ga.). NMR solvents were obtained from Cambridge Isotope Laboratories, Inc. (Andover, Mass.) and used as received.1H NMR (300 MHz or 400 MHz) and13C NMR (75 MHz or 100 MHz) spectra were recorded at 25° C. on Varian Mercury spectrometers. Chemical shifts (δ) are given in ppm relative to tetramethylsilane or the respective NMR solvent; coupling constants (J) are in hertz (Hz). Abbreviations used are s=singlet, bs=broad singlet, d=doublet, dd=doublet of doublets, t=triplet, dt=doublet of triplets, bt=broad triplet, qt=quartet, m=multiplet, bm=broad multiplet and br=broad. High and low resolution mass spectra were obtained at the North Carolina State Mass Spectrometry Laboratory for Biotechnology. FAB experiments were carried out with a JOEL HX110HF mass spectrometer while ESI experiments were carried out on an Agilent LC-TOF mass spectrometers.

To a 100 mL round-bottomed flask equipped with a magnetic stir was added trans-2-methyl-3-phenyl-2-propen-1-ol (2.00 g, 13.5 mmol) and 75 mL of methylene chloride. The solution was then cooled to 0° C. while stirring. Then, triethylamine (2.75 g, 27.0 mmol) is added followed by a dropwise addition of methanesulfonyl chloride (2.34 g, 20.4 mmol) and a two hour stir period. The reaction mixture is washed with water (2×75 mL), dried with sodium sulfate and then concentrated de vacuo. The crude mixture is then dissolved in 75 mL of DMF and then stirred via magnetic stir bar. To this mixture, sodium azide (1.76 g, 27.0 mmol) is added. The reaction mixture is then heated to 80° C. and allowed to stir for two hours. At this time, volatiles are concentrated de vacuo and the resulting residue is purified via column chromatography (1:9 ethyl acetate/hexane) providing (3-Azido-2-methyl propenyl)-benzene (2.08 g, 89%) as a colorless oil.1H NMR (300 MHz, CDCl3) δ 7.36-7.25 (m, 5H), δ 6.53 (s, 1H), δ 3.87 (s, 2H) ppm;13C NMR (75 MHz, CDCl3) δ 129.4, 129.2, 128.9, 128.6, 128.4, 127.1, 59.9, 52.0, 22.3, 16.5 ppm; LRMS (EI) calcd for C10H11N3(M+) 173. found 173.

General Procedure for Click Reactions:

The terminal alkyne (1.0 equiv.) was dissolved in a 1:1:1 mixture of tert-butyl alcohol, water and methylene chloride (ca. 10 mL per 0.300 g of terminal alkyne). To this solution, the appropriate azide (1.2 equiv.) was added while stirring vigorously at room temperature. Copper (II) sulfate pentahydrate (15 mol %) and sodium ascorbate (45 mol %) were then added sequentially to the solution. Reaction mixtures were allowed to stir until completion via TLC analysis (12-24 hrs). The solvents were then removed de vacuo in which the resulting residue was dissolved in methanol and purified by flash chromatography (10-20% ammonia saturated methanol:methylene chloride). The resulting fractions were evaporated under reduced pressure followed by a 24 hr high vacuum treatment to remove all ammonia traces. Methanol saturated with HCl is then added to the purified product in which all volatiles are then removed under reduced pressure.

Activity Testing of 2-AIT Library Members

A Standard Crystal Violet reporter assay is employed to assess the effect of compounds from the 2-amino on the formation of biofilms. Among others, the following strains are tested:

Xanthomonasis a Gram-negative rod-shaped bacterium that is a common plant pathogen.Xanthomonasbacteria grow almost exclusively in plants.Xanthomonasspecies testing includesX. vesicatoria(crop=tomato),X. euvesicatoria(crop=pepper),X. campestris(crop=crucifers, particularly cabbage),X. zinniae(crop=zinnia), andX. fragariae(crop=strawberry).Ralstonia solanacearumis a Gram-negative bacterium that is found in soil.

Bacteria are allowed to form biofilms in a multi-well plate in the absence or presence of one or more compounds. Planktonic (or free growing) bacteria are then removed, wells washed vigorously, and crystal violet added. Crystal violet stains the remaining bacteria which, following ethanol solubilization, is quantitated by spectrophotometry (A540). Time-dependent and concentration-dependent analysis of each compound are performed.

Activity Testing of 2-AIT Library Members onXanthomonas

Biofilm formation on PVC microtiter wells was accomplished usingXanthomonasstrains Xcv 135 (known to infect peppers and tomatoes) and Xcv 5 (known to infect tomatoes but not peppers) as models. The Starting Optical Density (OD at 600 nm) for biofilm attachment assay was 0.55, the temperature for this assay was 28° C., the duration of incubation was 6 hours under, and the assay was static.

Biofilm inhibition results are as follows for screens with Xcv 135. The Xcv 5 strain is tested in the same manner.

Activity Testing in Pepper Plants Inoculated withXanthomonas euvesicatoria(Bacterial Spot)

To test the effects of the triazole derivatives for plant biofilm inhibition activity, the compound of Formula (II)(a)(5)(D) was used as an exemplary compound (the “biofilm inhibitor/disperser” or “BFI” in the text hereinafter of Example 4).

The compound was evaluated under field conditions to determine whether it controls or enhances control of bacterial spot (caused by the bacteriumXanthomonas euvesicatoria) of pepper when applied alone or in tank-mixtures with a copper (Kocide 3000), an antibiotic (GWN-9350, gentamicin), or a putative plant defense activator (Prophyt). Tests were performed April-July 2008 at the North Carolina Agricultural Research Service Sandhills Research Station, Montgomery County.

EXPERIMENTAL DESIGN and TREATMENT APPLICATIONS: Each treatment consisted of two 7-plant rows running east-west replicated in a four-block completely randomized design. Each 7-plant row was 10.5 ft×2 ft. This area was used to calculate the quantity of spray material extrapolated for a per acre basis. Plants of bell pepper cultivar ‘X3R-Camelot’ were transplanted to the field on Apr. 24, 2008. On May 6, two plants in the south row of each plot of each treatment were inoculated with a suspension of copper-resistant strains of the bacterial pathogen. These plants were destined to serve as the inoculum source for each plot. Each of the spray test materials was mixed in 1.5 liters of water and applied using a backpack sprayer by making a single pass on each side of the plant row. Treatments were applied weekly starting immediately after the plants had been inoculated May 6 followed by 7 additional weekly sprays for a total of 8 applications through June 24.

DATA: Ratings of foliar disease were started May 22, when symptoms were observed on the inoculated plants and continued weekly through June 25. Disease was most severe on the inoculated plants and then spread to the non-inoculated plants. Data from these two groups (inoculated plants and the non-inoculated) were evaluated separately. The results are expressed as disease progress over time using the calculated Area Under the Disease Progress Curve (AUDPC). Two fruit harvests were done; July 1 and 8. Because there had been plant loss in some plots, the number of plants per plot was counted and yields (number of fruit and weight) were calculated and are reported on a per plant basis per treatment. Results are presented in Table 2 below.

Activity Testing in Fungus

The compound of Formula (II)(a)(5)(D) was used as an exemplary compound to evaluate the ability of 2-AIT compounds to inhibit the formation ofCandida albicansbiofilms.

It was screened at 100 μM for its ability to inhibit to the formation ofC. albicansbiofilms using a crystal violet reporter assay. Briefly, biofilms were allowed to form for 24 hours in a 96-well microtiter plate in the absence or presence of 100 μM of the compound. The wells were subsequently washed thoroughly with water to remove free-floating and loosely adherent fungus, and then treated with crystal violet. Crystal violet stains the remaining surface attached fungus (i.e. the biofilm), which following solublization, can be quantified by spectrophotometry (A540).

From this initial screen, we determined that the exemplary compound was able to inhibitC. albicansformation by 12% at 100□μM. Follow up growth curves at 100 μM demonstrated that this anti-biofilm activity was non-fungicidal (data not shown).

With this initial success of inhibiting fungal biofilms with a 2-AI derivative, we asked the question whether analogue synthesis could deliver alternative 2-AIT derivatives with enhanced anti-biofilm activity in the context of fungal biofilms. Previous work in our lab has demonstrated that 2-AI-based inhibitors of biofilms can be sub-divided into three separate sections: 1) the 2-AI head, 2) the linker region, and 3) the tail region. Structure activity relationship (SAR) data indicates that selectivity and activity can be tuned/enhanced by modification of the tail region.

Based upon this data, we synthesized a new pilot library of 2-AI derivatives for anti-biofilm testing in which diversity could be rapidly assembled via substituents off the triazole ring through commercially available carboxylic acids.

The synthetic approach to this library is outlined in Scheme 2. In our previous synthesis of 2-AIT conjugates, we employed the alkyne-derived 2-AI as a precursor to the CuI-mediated [3+2] alkyne/azide cycloaddition (click reaction). Although this reaction worked well, purification of the resulting product was cumbersome due to the use of copious amounts of ammonia saturated methanol for column chromatography. Therefore, we decided to revise the route by employing a boc-protected 2-AI alkyne that would allow more traditional means of purification (i.e. methanol/dichloromethane columns). The boc-protected scaffold was synthesized from 7-octynoic acid by treatment with oxayl chloride followed by diazomethane and quenching the resulting α-diazo ketone with HBr to generate the intermediate α-bromo ketone. Cyclization with boc-guanidine then delivered the target 2-AI alkyne 5.

Once 5 had been synthesized, we assembled a diverse array of azido amides to employ in the click reaction to create our pilot library of 2-AIT conjugates. Briefly, 2-bromo-ethylamine was treated with sodium azide to deliver 2-azido-ethylamine, which following acylation (via the respective acid chloride) generated the azido amides for elaboration into the 2-AIT pilot library. Each azido amide was then subjected to the click reaction with the 2-AI 5. Boc-deprotection (TFA/CH2Cl2) followed by counterion exchange (trifluoroacetate for chloride) delivered the target 2-AIT library for anti-biofilm screening.

Each member of the pilot library was assayed at 100 μM for its ability to inhibit the formation ofC. albicansbiofilms using the crystal violet reporter assay. From this assay, 2-AIT derivatives 7f and 7m were determined to be the most potent. Subsequent dose response studies revealed that 7f had an IC50of 2.9 μM while 7m had an IC50of 3.3 μM (Table 3). Growth curve and colony count analysis of 7f and 7m at respective IC50values demonstrated their antibiofilm activity to be non-fungicidal (data not shown).

Next, we addressed whether 7f and 7m could disperse pre-formedC. albicansbiofilms.C. albicanswas allowed to establish biofilms in 96-well microtiter plate for 24 hours. Plates were then washed to remove any free floating or loosely adherent fungus. The appropriate 2-AIT (7f and 7m) was then added to each well at 75 μM and the plate was allowed to incubate at 37° C. for 24 hours. Wells were then washed with water and stained with CV to quantify any remaining biofilms. In comparison to biofilms treated with media only, compound 7f dispersed 56% while 7m dispersed 62% of the pre-formed biofilm. Once we had established that both compounds could disperse pre-formed biofilms, we quantified this effect by determining 7f and 7 m's EC50value against pre-formedC. albicansbiofilms. Here, EC50is defined as the concentration at which the compound will disperse 50% of a pre-formed biofilm. Dose response studies revealed EC50's of 37.2 μM and 24.7 μM for 7f and 7m respectively (Table 1). From a medical perspective, molecules that simply inhibit the formation of a biofilm could be used in a prophylactic sense; however, given that a majority of patients already have an established biofilm infection when they seek medical intervention, molecules that are effective against a pre-formed biofilm are more clinically significant.

Once we had established that these next generation 2-AIT conjugates had the ability to inhibit and disperseC. albicansbiofilms, we addressed whether members of this library would also inhibit and disperse biofilms fromCryptococcus neoformans, an opportunistic fungal strain known to infect immunosuppressed patients, especially those with HIV infections. Initial screening of our library showed that 7e and 7m had potent anti-biofilm activity againstC. neoformans. Follow up dose response studies revealed IC50's of 1.3 μM and 8.0 μM (Table 3). Comparison of fungal growth in the presence or absence of either compound indicated that each compound was not fungicidal. Unfortunately, neither of these compounds was able to disperse pre-formedC. neoformansbiofilms at the concentrations tested.

All reagents used for chemical synthesis were purchased from commercially available sources and used without further purification. Chromatography was performed using 60 {acute over (Å)} mesh standard grade silica gel from Sorbtech. NMR solvents were obtained from Cambridge Isotope Labs and used as is.1H NMR (300 MHz or 400 MHz) and13C NMR (75 MHz or 100 MHz) spectra were recorded at 25° C. on Varian Mercury spectrometers. Chemical shifts (δ) are given in ppm relative to tetramethylsilane or the respective NMR solvent; coupling constants (J) are in hertz (Hz). Abbreviations used are s=singlet, bs=broad singlet, d=doublet, dd=doublet of doublets, t=triplet, dt=doublet of triplets, bt=broad triplet, qt=quartet, m=multiplet, bm=broad multiplet and br=broad.

General Procedure for Click Reactions and Subsequent Boc Deprotection:

The terminal alkyne (1.0 equiv.) was dissolved in a 1:1:1 mixture of ethanol, water and methylene chloride (ca. 9 mL per 0.300 g of terminal alkyne). To this solution, the appropriate azide (1.0 equiv.) was added while stirring vigorously at room temperature. Copper (II) sulfate (15 mol %) and sodium ascorbate (45 mol %) were then added sequentially to the solution. Reaction mixtures were allowed to stir until completion via TLC analysis (12-24 hrs). The solvents were then removed de vacuo in which the resulting residue was dissolved in dichloromethane and purified via silica gel column chromatography (1:40 methanol:dichloromethane to 1:10 methanol:dichloromethane). To remove the Boc protecting group, the resulting product was then dissolved in a 1:4 trifluoroacetic acid:dichloromethane mixture and allowed to stir for 5 hr. Upon completion, the reaction mixture was concentrated de vacuo and then left on a high vacuum overnight. Then, methanol supplemented with HCl was added to the product forming the HCl salt of the deprotected product and then was concentrated de vacuo. The resulting residue was washed with diethyl ether and then placed on a high vacuum overnight.

Procedure to Determine the Inhibitory Effect of Test Compounds onC. albicans, C. neoformansand a MixedS. epidermidis/C. albicansBiofilm Formation:

Inhibition assays were performed by taking an overnight culture of yeast or yeast/bacteria strain and subculturing it at an OD600of 0.05 into YPD (Yeast extract, peptone and dextrose (BD 242820)) media for the yeast alone or tryptic soy broth for theS. epidermidis/C. albicans. Stock solutions of predetermined concentrations of the test compound were then made in the necessary media. These stock solutions were aliquoted (100 μL) into the wells of the 96-well PVC microtiter plate. Sample plates were then wrapped in GLAD Press n' Seal® followed by an incubation under stationary conditions for 24 h at 37° C. After incubation, the media was discarded from the wells and the plates were washed thoroughly with water. Plates were then stained with 100 μL of 0.1% solution of crystal violet (CV) and then incubated at ambient temperature for 30 min. Plates were washed with water again and the remaining stain was solubilized with 200 μL of 95% ethanol. A sample of 125 μL of solubilized CV stain from each well was transferred to the corresponding wells of a polystyrene microtiter dish. Biofilm inhibition was quantitated by measuring the OD540of each well in which a negative control lane wherein no biofilm was formed served as a background and was subtracted out.

Procedure to Determine the Dispersal Effect of Test Compounds onC. albicansandC. neoformansPreformed Biofilms:

Dispersion assays were performed by taking an overnight culture of bacterial strain and subculturing it at an OD600of 0.01 into Yeast extract, peptone and dextrose (BD 242820) media. The resulting bacterial suspension was aliquoted (100 μL) into the wells of a 96-well PVC microtiter plate. Plates were then wrapped in GLAD Press n' Seal® followed by an incubation under stationary conditions at ambient temperature to establish the biofilms. After 24 h, the media was discarded from the wells and the plates were washed thoroughly with water. Stock solutions of predetermined concentrations of the test compound were then made in the necessary media. These stock solutions were aliquoted (100 μL) into the wells of the 96-well PVC microtiter plate with the established biofilms. Media alone was added to a subset of the wells to serve as a control. Sample Plates were then incubated for 24 h at 37° C. After incubation, the media was discarded from the wells and the plates were washed thoroughly with water. Plates were then stained with 100 μL of 0.1% solution of crystal violet (CV) and then incubated at ambient temperature for 30 min. Plates were washed with water again and the remaining stain was solubilized with 200 μL, of 95% ethanol. A sample of 125 μL of solubilized CV stain from each well was transferred to the corresponding wells of a polystyrene microtiter dish. Biofilm dispersion was quantitated by measuring the OD540of each well in which a negative control lane wherein no biofilm was formed served as a background and was subtracted out.