Antifungal compositions and methods of using them

Abstract

The present disclosure provides antifungal compositions and methods for their use. In particular, the compositions include (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide, which enhances or potentiates the activity of certain fungicides. In one embodiment, disclosed herein is a method for treating crops with an apyrase inhibitor having the formula of Compound 1: In one embodiment, the treatment of a crop with an apyrase inhibitor is synergistic with the fungicide.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the earlier filing date of U.S. Provisional Patent Application No. 63 / 419,568, filed October 26, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to compositions and methods of use for the treatment of crops susceptible to fungal pathogens. [Background technology]

[0003] Fungal pathogens plague crops worldwide. Fungi have developed a range of mechanisms to survive by sequestering, excreting, or detoxifying fungicides. Formulations that enhance fungicide efficacy by blocking certain resistance mechanisms are needed. Summary of the Invention [Means for solving the problem]

[0004] Disclosed herein are methods and compositions for use in supporting crop survival and yield, for example, by protecting the crop from fungal pathogens. In one embodiment, disclosed herein are methods and compositions for use in treating a crop with an apyrase inhibitor having the formula of Compound 1: [ka] In one embodiment, the treatment of crops with an apyrase inhibitor is synergistic with the fungicide.

[0005] The foregoing and other objects, features, and advantages of the present invention will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0006] [Figure 1]Asian soybean rust severity is shown at 1, 7, 9, 14, and 16 days after application (DAA) in untreated crops (UTC) and soybeans treated with azoxystrobin, chlorothalonil, tebuconazole, or azoxystrobin and tebuconazole with and without the accelerator compound (TX15). [Figure 2] Soybean leaf spot infection intensity is shown at 4, 11, and 18 days after application (DAA) (2 trials) in untreated crops (UTC) and soybeans treated with azoxystrobin, chlorothalonil, tebuconazole, or azoxystrobin and tebuconazole with and without the accelerator compound (TX15). [Figure 3] Wheat stripe rust leaf infection percentages are shown for untreated crops (UTC) and for wheat plants treated with preventative applications of AMISTAR® (azoxystrobin), Folpet, PROLINE® (prothioconazole), REVYSTAR® (fluxapyroxad and mefentrifluconazole), A20944 (prothioconazole and pydiflumetofen), ELATUS® ERA (prothioconazole and benzovindiflupyr), A21857 (ADEPIDYN®, pydiflumetofen), INATREQ® (fenpicoxamid), with and without the accelerator compound (TX15). [Figure 4] Figure 1 shows the time course of wheat stripe rust leaf infection percentage in untreated crops and in wheat plants that received preventative applications of prothioconazole or a combination of prothioconazole and the accelerator compound (TX15). [Figure 5]Percentage infection of Septoria leaves on wheat is shown for untreated crops (UTC) and for wheat plants treated with preventative applications of AMISTAR® (azoxystrobin), Folpet, PROLINE® (prothioconazole), REVYSTAR® (fluxapyroxad and mefentrifluconazole), A20944 (prothioconazole and pydiflumetofen), ELATUS® ERA (prothioconazole and benzovindiflupyr), A21857 (ADEPIDYN®, pydiflumetofen), INATREQ® (fenpicoxamid), with and without the accelerator compound (TX15). [Figure 6] The percentage infection of wheat Septoria leaves is shown for four winter wheat crops treated with a standard fungicide versus a standard fungicide and the accelerator compound (TX15). [Figure 7] The severity of downy mildew-infested grape crops assessed on clusters and leaves is shown for untreated crops (UTC) and crops treated with Cyazofamid or Cyazofamid and the accelerator compound (TX15). [Figure 8] Figure 1 shows the infection percentage of untreated (UTC) grapevine crops and grapevine crops treated with QUADRIS TOP® (azoxystrobin and difenoconazole) alone or in combination with the accelerator compound (TX15). [Figure 9] 1 shows the percentage infection by Zymoseptoria tritici of winter wheat after treatment with metconazole and a combination of metconazole and the accelerator compound (TX15), and of an untreated crop. DETAILED DESCRIPTION OF THE INVENTION

[0007] I. Terminology The following explanations of terms and methods are provided to more fully describe the present disclosure and to guide those skilled in the art in practicing the present disclosure. The singular forms "a," "an," and "the" refer to one or more, unless the context clearly dictates otherwise. The term "or" refers to any single element of listed alternative elements or a combination of two or more elements, unless the context clearly dictates otherwise. As used herein, "comprises" means "includes." Thus, "including A or B" means "including A, B, or A and B," excluding additional elements. All references cited herein, including patents and patent applications, are incorporated by reference in their entirety unless otherwise specified.

[0008] Unless otherwise indicated, all numbers expressing amounts of ingredients, molecular weights, percentages, temperatures, times, and the like used in the specification or claims should be understood to be modified by the term "about." Thus, unless implicitly or explicitly indicated otherwise, the numerical parameters given are approximations and may depend on the desired properties sought and / or the limits of detection under standard testing conditions / methods. When directly and explicitly distinguishing an embodiment from the prior art discussed, the numbers of the embodiments are not approximations unless the word "about" is explicitly recited.

[0009] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0010] "Administering" refers to any suitable mode of administration for controlling pathogens, such as fungal pathogens, and includes treating existing crops, produce, seeds, soil, or combinations thereof. As used herein, it is intended that administering to a crop can refer to administering to benefit the crop by treating existing crops, produce, seeds, soil, or combinations thereof.

[0011] "In combination with" refers to the administration of the compounds either simultaneously in a single administration, or sequentially in two or more different administrations that may be separated in any way, time, place, or manner.

[0012] "Control," with respect to fungal pathogens, means blocking, inhibiting, and / or eradicating the pathogen and / or preventing the pathogen from damaging the crop. In one embodiment, control refers to reducing one or more fungi to undetectable levels, or reducing or suppressing the fungal pathogen to an acceptable level as determined by one skilled in the art (e.g., a crop grower). Determining an acceptable level of fungal pathogen reduction is based on numerous factors, including the crop, the pathogen, pathogen severity, use limitations, economic thresholds, and other factors known to those skilled in the art.

[0013] As used herein, the terms "accelerator" and "potentiator" refer to a compound(s) disclosed herein that enhances the effectiveness of a fungicide. Without being limited by theory, the accelerator compounds disclosed herein may function by blocking one or more pathways by which fungal pathogens avoid toxicity, such as by detoxifying, sequestering, or excreting the fungicide. In certain embodiments, the accelerator compounds inhibit the enzymatic apyrase activity, which leads to the strengthening, enhancement, or potentiation of the fungicide. For example, when an accelerator or potentiator is used in conjunction with a fungicide, the combination of the accelerator and fungicide enhances the fungicidal effect of the fungicide, and / or, as a result of the accelerator's activity, fungi that have previously been resistant to the fungicide become susceptible to the fungicide. In most cases, the accelerator compounds do not themselves directly inhibit fungal growth or have a detrimental effect on crops that are (or may be) infected with the pathogen.

[0014] As used herein, the term "phytologically acceptable" refers to a composition, diluent, excipient, and / or carrier that is generally applicable for use on any part of a plant during any part of its life cycle, including, but not limited to, a seed, seedling, plant cell, plant, or flower. The composition may be prepared according to procedures, methods, and formulas known to those skilled in the agricultural arts. Armed with the teachings of the present disclosure, those skilled in the agricultural and / or chemical arts can readily prepare a desired composition. Most generally, the compounds of the present invention can be formulated for storage and / or application as is, or as an aqueous or non-aqueous suspension or emulsion prepared from a concentrated formulation of the composition. Alternatively, the compounds of the present invention can be formulated for use in an aerosol-generating device for application to produce stored in a sealed chamber, an application method known as spraying. Water-soluble, water-suspendable, or emulsifiable formulations containing the presently disclosed compounds can also be converted to or formulated as a solid (e.g., a wettable powder), which can then be diluted into a final formulation. In certain formulations, the compositions of the present disclosure may also be provided to growth media such as in vitro media for the growth of plants or other types of cells, laboratory plant growth media, soil, or for spraying onto seeds, seedlings, roots, trunks, stems, leaves, flowers, or whole plants.

[0015] As used herein, the term "treatment" refers to a method used to administer or apply an effective amount of a disclosed compound or a formulation thereof to a target area of ​​a field and / or plant. Treatment methods can include, but are not limited to, aerosol spray, pressure spray, direct watering, chemical solution irrigation, spraying, and immersion. Target areas of a plant can include, but are not limited to, leaves, roots, stems, buds, flowers, fruits, plant seeds, and plant bulbs, including bulbs, corms, rhizomes, tubers, root nodules, and rhizophores. Treatment can include methods in which a plant is treated in one area (e.g., the root zone or foliage) and another area of ​​the plant is protected (e.g., the foliage is treated when the disclosed compound is applied to the root zone or to the shoots when applied to the foliage).

[0016] II. Accelerator Compounds Described herein are compounds having the structure [ka] The present invention relates to a composition comprising (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide (also referred to herein as Compound 1), an accelerator compound having the formula: The compound is referred to herein by its chemical structure and name; however, as will be apparent to one of ordinary skill in the art, given the structure of the accelerator compound, the compound may also be its Z isomer. The existence of tautomers of the exemplified accelerator compounds will also be readily apparent to one of ordinary skill in the art. All such isomers and tautomers of the accelerator compounds are contemplated herein.

[0017] The compounds are referred to herein as accelerators, potentiators, or apyrase inhibitors, depending on their effects. As will be understood by those skilled in the art, the terms accelerator compound, potentiator compound, and apyrase inhibitor all refer to (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide, its (Z)-isomer, and its tautomers.

[0018] Without being limited to any particular theory, the enhancer compound is believed to function by blocking one or more pathways by which fungal pathogens avoid toxicity, such as by detoxifying, sequestering, or excreting the fungicide. In one embodiment, the compound inhibits the enzymatic apyrase activity, which leads to the strengthening, enhancement, or potentiation of the fungicide. For example, when an enhancer or potentiator is used in conjunction with a fungicide, the combination of the potentiator and the fungicide enhances the fungicidal effect of the fungicide, and / or as a result of the activity of the potentiator, fungi that have previously been resistant to the fungicide become susceptible to the fungicide. In most cases, the enhancer does not itself directly inhibit fungi and does not adversely affect seeds, crops, or crop products that are (or may be) infected with fungi.

[0019] III. Combination Treatment In one embodiment, the disclosed apyrase inhibitors are useful for enhancing the fungicidal effects of various fungicides. The inventors have found that certain fungicides are enhanced by the accelerator compounds, but not others. Therefore, fungicides for use in combination with the specific accelerator compounds described herein must be carefully selected. In one embodiment, combined treatment of a selected fungicide with the accelerator compound provides synergistic fungicidal activity against plant pathogenic filamentous fungi. As shown in Figures 1-8, the accelerator compounds dramatically reduce fungal growth in combination with exemplary fungicides. In certain instances, the accelerator compounds differentially enhance the effectiveness of certain fungicides. Certain fungicides are not enhanced by the accelerator compounds.

[0020] Fungicides whose effectiveness is restored or enhanced by the accelerator compounds in accordance with the present methods and compositions include azoxystrobin, difenoconazole, chlorothalonil, tebuconazole, folpet, prothioconazole, fluxapyroxad, metconazole, mefentrifluconazole, pydiflumetofen, benzovindiflupyr, fenpicoxamid, cyazofamid, pyraclostrobin, and combinations thereof.

[0021] In one embodiment, disclosed is a method of inhibiting fungal infection in seeds or plants at risk thereof, the method comprising contacting the seeds or plants with a fungicide selected from the group consisting of azoxystrobin, difenoconazole, chlorothalonil, tebuconazole, folpet, prothioconazole, fluxapyroxad, metconazole, mefentrifluconazole, pydiflumetofen, benzovindiflupyr, fenpicoxamid, cyazofamid, pyraclostrobin, or a combination thereof.

[0022] In one embodiment, the fungicide potentiated by the accelerator compound includes fenpicoxamid.

[0023] In one embodiment, the fungicide potentiated by the accelerator compound comprises pydiflumetofen. In one such embodiment, the fungicide comprises prothioconazole in addition to pydiflumetofen.

[0024] In one embodiment, the fungicide potentiated by the accelerator compound comprises benzovindiflupyr, hi one such embodiment, the fungicide comprises prothioconazole in addition to benzovindiflupyr.

[0025] In one embodiment, the fungicide boosted by the accelerator compound includes fluxapyroxad. In one embodiment, the fungicide boosted by the accelerator compound includes mefentrifluconazole. In one embodiment, the fungicide boosted by the accelerator compound includes fluxapyroxad and mefentrifluconazole.

[0026] In one embodiment, the fungicide enhanced by the accelerator compound includes a strobilurin, such as azoxystrobin, pyraclostrobin, or both. In one embodiment, the fungicide enhanced by the accelerator compound includes a fungicide selected from azoxystrobin, pyraclostrobin, or a combination thereof. In one embodiment, the fungicide enhanced by the accelerator compound includes azoxystrobin. In one embodiment, the fungicide enhanced by the accelerator compound includes pyraclostrobin, and in one such embodiment, the fungicide includes pyraclostrobin and mefentrifluconazole.

[0027] In one embodiment, the fungicides boosted by the accelerator compounds include azoxystrobin, fluxapyroxad, pydiflumetofen, or a combination thereof. In one embodiment, the fungicides boosted by the accelerator compounds include azoxystrobin, pydiflumetofen, or both. In one embodiment, the fungicides boosted by the accelerator compounds include azoxystrobin and pydiflumetofen.

[0028] In one embodiment, the fungicide enhanced by the accelerator compound includes difenoconazole, mefentrifluconazole, prothioconazole, or both. In one embodiment, the fungicide enhanced by the accelerator compound includes mefentrifluconazole, prothioconazole, or both. In one embodiment, the fungicide enhanced by the accelerator compound is mefentrifluconazole. In one embodiment, the fungicide enhanced by the accelerator compound is prothioconazole. In one embodiment, the fungicide enhanced by the accelerator compound is difenoconazole.

[0029] In one embodiment, the fungicides boosted by the accelerator compounds include fluxapyroxad.In one embodiment, the fungicides boosted by the accelerator compounds include mefentrifluconazole and fluxapyroxad.

[0030] In one embodiment, the fungicide potentiated by the accelerator compound includes folpet.

[0031] In one embodiment, the fungicide potentiated by the accelerator compound includes a triazole carboxamide, for example, ethaboxam.

[0032] In one embodiment, the present disclosure provides compositions and methods for treating plants or plant seeds infected or at risk of infection with fungal pathogens. In one embodiment, the composition of the present disclosure comprises a fungicide, such as one or more of the fungicides described above, the enhancer compound, and a botanically acceptable carrier. In another embodiment, the fungicide and the enhancer compound are administered in separate compositions.

[0033] In a further embodiment, agricultural or horticultural fungicides are used in combination with other compounds in addition to the disclosed accelerator compounds. Such other compounds can be administered in the same or separate compositions as the fungicide and / or formulation. Examples of other components include known carriers used to formulate the fungicide. Additional examples include conventionally known herbicides, insecticides / acaricides, nematodes, molluscicides, soil pesticides, plant protection agents, synergists, fertilizers, and soil conditioners. In one embodiment, the inclusion of such other components results in further improved crop growth, and in some embodiments, in synergistic effects on crop growth.

[0034] IV. Target Crops and Their Pathogens The present disclosure provides formulations and methods for use in treating crops, agricultural products, seeds, and soil for fungal pathogens. In one embodiment, the accelerator compound is administered in combination with an agricultural fungicide, such as the fungicides described above in Section III. Crops that can be treated include those affected by fungal pathogens, as known to those skilled in the art of agriculture. For example, agricultural and horticultural crops that can be treated according to the present disclosure include plants, including their harvests, whether genetically modified or not, such as grains; vegetables; root vegetables; potatoes; fruit trees, such as banana trees, tea trees, coffee trees, or cocoa trees; grasses; turf; or cotton.

[0035] The agricultural or horticultural enhancer and fungicide combinations disclosed herein can be applied to each part of a plant, such as leaves, stems, stems, flowers, buds, fruits, seeds, shoots, roots, tubers, tuberous roots, shoots, or stumps. The agricultural or horticultural enhancers according to the present disclosure can also be applied to improved varieties / varieties, cultivars, and mutant, hybrid, and genetically modified embodiments of these plants.

[0036] The agricultural or horticultural treatments described herein can be used to perform seed treatments, foliage applications, soil applications, or water applications to control a variety of diseases occurring in agricultural or horticultural crops, including flowers, turf, and pasture grasses.

[0037] The accelerator compounds are particularly useful for enhancing the effectiveness of fungicides against fungal pathogens of plants. Examples of pathogens that may be treated according to the present methods and compositions include, but are not limited to, Botrytis cinerea, Colletotrichum graminicola, Fusarium oxysporum, Sclerotiana sclerotiorum, Verticillium dahlia, Mycospharella gramincola, and Sphacelotheca reliana.

[0038] Botrytis cinerea is an airborne plant pathogen whose necrotrophic lifestyle affects over 200 crop hosts worldwide. The fungus primarily attacks dicotyledonous plant species, including important protein, oil, fiber, and horticultural crops, grapes, and strawberries; Botrytis also causes soft rot of fruits and vegetables during storage, transport, and marketing. Many classes of fungicides have failed to control Botrytis cinerea due to its genetic plasticity.

[0039] The genus Colletotrichum consists of approximately 600 species that infect over 3,200 species of monocotyledonous and dicotyledonous plants. Colletotrichum graminicola primarily infects corn (Zea mays), causing losses of approximately $1 billion annually in the United States alone (Connell et al., 2012).

[0040] Another pathogen that can be treated according to the present methods and compositions is banana wilt, caused by the soil-borne pathogen Fusarium oxysporum f.sp. cubense, which is a major threat to banana production worldwide. Currently, no fungicides are available to effectively control this disease once plants are infected (Peng J et al., 2014).

[0041] The mildew fungus Sclerotinia sclerotiorum is known to infect over 400 host species and is considered one of the most prolific plant pathogens. Sclerotinia sclerotiorum is another pathogen that can be treated according to the present methods and compositions to improve crop health and yield. The majority of affected crop species are dicotyledonous plants, as well as several agriculturally important monocotyledonous plants. Some important crops affected by S. sclerotiorum include legumes (soybeans), most vegetables, stone fruits, and tobacco.

[0042] The ascomycete Verticillium dahliae is a soil-borne fungal plant pathogen that causes vascular wilt disease in a wide range of dicotyledonous host species. V. dahliae can cause severe yield and quality losses in cotton and other important crops, such as vegetables, fiber, fruit, nuts, trees, and ornamentals.

[0043] The ascomycete fungus Mycospharella grammingola (anamorph: Septoria tritici) is one of the most important foliar diseases of wheat leaves and occurs wherever wheat is grown. Yield losses due to this disease range from 25% to 50%, and are particularly high in Europe, the Mediterranean region, and East Africa. Infection by M. gramincola is initiated by airborne ascospores produced in residues from the previous year's crop. Primary infection usually occurs after seedling emergence in spring or autumn. Mature disease is characterized by necrotic lesions on the leaves and stems of infected plants.

[0044] The basidiomycete fungus Sphacelotheca reliana infects maize (Zea mays) systemically and causes head smut. Yield losses due to the disease are variable and directly depend on the incidence of the disease. The fungus overwinters as diploid teliospores in crop debris or soil. Floral structures transform into sporangia containing powdery teliospore masses, similar to the galls of common smut.

[0045] Examples of crops that may be treated using the presently disclosed methods and compositions, as well as plant diseases (pathogens) that may be controlled, include, but are not limited to, the following: Sugar beet: brown spot disease (Cercospora beticola), black root disease (Aphanomyces cochlioides), root rot disease (Thanaphorus cucumeris), Thanatephorus cuumeris disease (Thanaphorus cuumeris), leaf rot disease (Thanaphorus cucumeris), etc. Peanut: Brown spot (Mycosphaerella arachidis), leaf mold (Ascochyta sp.), rust (Puccinia arachidis), damping-off (Pythium debaryanum), rust spot (Alternaria alternata), stem rot (Sclerotium rolfsii), black rust (Mycosphaerella berkeleyi), etc. Cucumber: Powdery mildew (Sphaerotheca fuliginea), downy mildew (Pseudoperonospora cubensis), vine blight (Mycosphaerella melonis), wilt (Fusarium oxysporum), sclerotinia sclerotiorum, gray mold (Botrytis cinerea), anthracnose (Colletotrichum orbiculare), black spot (Cladosporium cucumerinum), brown spot (Corynespora cassiicola), damping-off (Pythium debaryanum, Rhizoctonia solani Kuhn), Phomopsis root rot (Phomopsis sp.), bacterial spot (Pseudomonas syringae pv. Lechrymans), etc. Tomatoes: Gray mold (Botrytis cinerea), leaf mold (Cladosporium fulvum), leaf blight (Phytophthora infestans), verticillium wilt (Verticillium albo-atrum, Verticillium dahliae), powdery mildew (Oidium neolycopersici), summer blight (Alternaria solani), leaf mold (Pseudocercospora fuligena), etc. Eggplant: Gray mold (Botrytis cinerea), black rot (Corynespora melongenae), powdery mildew (Erysiphe cichoracearum), leaf mold (Mycovellosiella nattrassii), sclerotinia rot (Sclerotinia sclerotiorum), verticillium wilt (Verticillium dahlia), brown spot (Phomopsis vexans), etc. Strawberries: Gray mold (Botrytis cinerea), powdery mildew (Sphaerotheca humuli), anthracnose (Colletotrichum acutatum, Colletotrichum fragariae), Phytophthora rot (Phytophthora cactorum), soft rot (Rhizopus stolonifer), Fusarium wilt (Fusarium oxysporum), and Verticillium dahlia wilt, etc. Onion: Neck dwarf disease (Botrytis allii), gray mold (Botrytis cinerea), leaf blight (Botrytis squamosa), downy mildew (Peronospora destructor), Phytophthora porn, etc. Cabbage: Clubroot (Plasmodiophora brassicae), soft rot (Erwinia carotovora), black rot (Xanthomonas campestris pv. campestris), bacterial black spot (Pseudomonas syringae pv. Maculicola, Pspv. alisalensis), dew fungus (Peronospora parasitica), sclerotinia rot (Sclerotinia sclerotiorum), black spot (Alternaria brassicicola), gray mold (Botrytis cinerea), etc. Common beans: Sclerotinia rot (Sclerotinia sclerotiorum), gray mold (Botrytis cinerea), anthracnose (Colletotrichum lindemuthianum), angular spot disease (Phaeoisariopsis griseola), etc. Apples: Powdery mildew (Podosphaera leucotricha), black spot (Venturia inaequalis), monilinia (Monilinia mali), black spot (Mycosphaerella pomi), canker (Valsa mali), leaf spot (Alternaria mali), rust (Gymnosporangium yamadae), ring rot (Botryosphaeria berengeriana), anthracnose (Glomerella cingulata, Colletotrichum acutatum), leaf rot (Diplocarpon mali), fly speck disease (Zygophiala jamaicensis), sooty blotch (Gloeodes pomigena), purple root rot (Helicobasidium mompa), gray mold (Botrytis cinerea), etc. Plum: Black spot (Cladosporium carpophilum), gray mold (Botrytis cinerea), brown spot (Monilinia mumecola), etc. Persimmon: Powdery mildew (Phyllactinia kakicola), anthracnose (Gloeosporium kaki), angular leaf spot (Cercosporakaki), etc. Peaches: Brown rot (Monilinia fructicola), black spot (Cladosporium carpophilum), Phomopsis root rot (Phomopsis sp.), bacterial bore (Xanthomonas campestris pv.pruni), etc. Almonds: Monilinia taxa, Stigmina carpophila, Cladosporium carpophilum, Polystigma rubrum, Alternaria alternata, Colletotrichum gloeospoides, etc. Yellow peach: Monilinia fructicola, anthracnose (Colletotrichum acutatum), black spot (Alternaria sp.), young fruit sclerotia (Monilinia kusanoi), etc. Grapes: Gray mold (Botrytis cinerea), powdery mildew (Uncinula necator), late rot (Glomerella cingulata, Colletotrichum acutatum), downy mildew (Plasmopara viticola), anthracnose (Elsinoe ampelina), brown spot (Pseudocercospora vitis), black rot (Guignardia bidwellii), white rot (Coniella castaneicola), rust (Phakopsora ampelopsidis), etc. Pear: Common scab (Venturia nashicola), rust (Gymnosporangium asiaticum), black spot (Alternaria kikuchiana), ring rot (Botryosphaeria berengeriana), powdery mildew (Phyllactinia mali), chitospora blight (Phomopsis fukushii), brown spot blotch disease (Stempphylium vesicarium), anthracnose (Glomerella cingulata), etc. Tea: Ring spot (Pestalotiopsis longiseta, P. theae), anthracnose (Colletotrichum theae-sinensis), net blight (Exobasidium reticulatum), etc. Citrus fruits: Common scab (Elsinoe fawcettii), blue mold (Penicillium italicum), green mold (Penicillium digitatum), gray mold (Botrytis cinerea), black spot (Diaporthe citri), canker (Xanthomonas campestris pv. Citri), powdery mildew (Oidium sp.), etc. Wheat: Powdery mildew (Blumeria graminis f.sp. tritici), Fusarium head blight (Gibberella zeae), Red rust (Puccinia recondita), Brown snow rot (Pythium iwayamai), Pink snow rot (Monographella nivalis), Eyespot (Pseudocercosporella herpotrichoides), Leaf scorch (Septoria tritici), Leaf blight (Leptosphaeria nodorum), Small snow rot (Typhula incarnata), Large snow rot (Myriosclerotinia borealis), Damping-off (Gaeumannomyces graminis), Ergot (Claviceps purpurea), Smut (Tilletia caries), Pungent slump (Telletia caries), Naked smut (Ustilago nuda), etc. Barley: Leaf spot disease (Pyrenophora graminea), net spot disease (Pyrenophora teres), leaf blight (Rhynchosporium secalis), naked smut disease (Ustilago tritici, U. nuda), etc. Rice: Blight (Pyricularia oryzae), Sheath blight (Rhizoctonia solani), Bakanae disease (Gibberella fujikuroi), Brown spot disease (Cochliobolus miyabeanus), Damping-off disease (Pythium graminicola), Bacterial leaf blight (Xanthomonas oryzae), Bacterial seedling blight (Burkholderia plantarii), Brown stripe disease (Acidovorax avenae), Bacterial grain rot (Burkholderia glumae), Brown spot disease (Cercospora oryzae), Rice smut (Ustilaginoidea virens), Brown spot disease of rice (Alternaria alternata, Curvularia intermedia), Black rice (Alternaria padwickii), Pink rice grain disease (Epicoccum purpurascens), etc. Tobacco: Sclerotinia rot (Sclerotinia sclerotiorum), powdery mildew (Erysiphe cichoracearum), Phytophthora rot (Phytophthora nicotianae), etc. Tulips: Gray mold (Botrytis cinerea), etc. Sunflower: Dew fungus (Plasmopara halstedii), Sclerotinia rot (Sclerotinia sclerotiorum), etc. Bentgrass: Snow mold (Sclerotinia borealis), large patch (Rhizoctonia solani), brown patch (Rhizoctonia solani), dollar spot (Sclerotinia homoeocarpa), blight (Pyricularia sp.), Pythium wilt (Pythium aphanidermatum), anthracnose (Colletotrichum graminicola), etc. Orchardgrass: Powdery mildew (Erysiphe graminis), etc. Soybean: Purple spot (Cercospora kikuchii), dew fungus (Peronospora manshurica), Phytophthora rot (Phytophthora sojae), rust (Phakopsora pachyrhizi), Sclerotinia rot (Sclerotinia sclerotiorum), anthracnose (Colletotrichum truncatum), gray mold (Botrytis cinerea), black rot (Elsinoe glycines), black spot (Diaporthe phaseolorum var. sojae), etc. Potato: Phytophthora rot (Phytophthora infestans), summer blight (Alternaria solani), bruise (Thanatephorus cucumeris), and verticillium wilt (Verticillium alboatrum, V. dahlia, V. nigrescens, etc.). Banana: Panama disease (Fusarium oxysporum), Sigatoka disease (Mycosphaerella fijiensis, M. musicola), etc. Rapeseed: Sclerotinia rot (Sclerotinia sclerotiorum), root rot (Phoma lingam), black leaf spot (Alternaria brassicae), etc. Coffee: Rust (Hemileia vastatrix), anthracnose (Colletotrichum coffeanum), leaf spot (Cercospora coffeicola), etc. Sugarcane: Brown rust (Puccinia melanocephala), etc. Corn: Ring spot (Gloeocercospora sorghi), rust (Puccinia sorghi), southern rust (Puccinia polysora), ear smut (Ustilago maydis), brown spot (Cochliobolus heterostrophus), sooty leaf spot (Setosphaeria turcica), etc. Cotton: Seedling damping-off (Pythium sp.), rust (Phakopsora gossypii), white mold (Mycosphaerella areola), anthracnose (Glomerella gossypii), etc.

[0046] V. Preparation The present disclosure provides enhancer compounds and formulations thereof that enhance the efficacy of fungicides to effectively limit the growth of pathogenic fungi affecting crops, agricultural products, seeds, and / or soil. In certain non-limiting embodiments, the apyrase inhibitor may be provided in an amount of about 0.01 to about 80% by weight, or about 25% to about 55%, e.g., about 30% to about 50%, or about 35% to about 45%, e.g., about 0.01, 0.05, 0.1, 0.5, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 4.0, 5.0, 7.5, 10, 20, 30, 40, 50, 55, 60, or 80% by weight of the final composition. In one embodiment, the apyrase inhibitor is provided in a liquid form that is about 0.01 to about 50% volume to volume in the final diluted composition, e.g., about 15% to about 50%, about 20% to about 45%, or about 25% to about 40%, e.g., about 0.01, 0.05, 0.1, 0.5, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 4.0, 5.0, 7.5, 10, 15, 20, 30, 40, or 50%. Those skilled in the art will recognize that formulations of pesticides, apyrase inhibitors, or combinations thereof, can be provided in concentrates that can be diluted before use, or in ready-to-use diluted forms.

[0047] The accelerator, the pesticide, and their combinations are not particularly limited by the dosage form. Examples of dosage forms include wettable powders, emulsions, emulsifiable concentrates, oil dispersions, powders, granules, water-soluble agents, suspensions, water-dispersible granules, and tablets. The method for preparing the formulation is not particularly limited, and conventionally known methods can be used depending on the dosage form. [Example]

[0048] Some formulation examples are described below. The preparation formulations shown below are merely examples and may be modified within the scope consistent with the essence of the present disclosure. For example, additional active and inactive ingredients may be added to the formulations below. "Parts" means "parts by weight" unless otherwise specified.

[0049] Formulation Example 1 Wettable powder 40 parts of an accelerator compound disclosed herein, 53 parts of diatomaceous earth, 4 parts of an ethoxylated higher alcohol sulfate, and 3 parts of an alkyl naphthalene sulfonate in combination with a suitable solid carrier such as magnesium sulfate are uniformly mixed and then pulverized to obtain a wettable powder containing 40 parts by weight of the accelerator.

[0050] Formulation Example 2 Aqueous Suspension Concentrate 2.5 g of accelerator compound is placed in a 100 mL glass beaker along with 0.5 g of dispersant Tamol SN, 1 g of propylene glycol, 0.006 g (10 mM) of boric acid powder buffer, 5 g of water, optionally 0.05 g of low molecular weight surfactant Surfonic L24-7, and optionally 0.01 g of antifoam agent SAG 1572. 30 g of 2 mm diameter glass beads are added, and the suspension is milled using a mechanical stirrer to a median diameter of less than 1.5 microns as measured with a Malvern Mastersizer 3000. 0.5 g of a pregel containing 2% xanthan polysaccharide and 2% biocide Acticide B20 is added to the suspension concentrate, and the mechanical stirrer is operated for an additional 10 minutes. The pH is adjusted to 9.0 using 2% phosphoric acid or 1 M sodium hydroxide, as needed. Water is added as needed to achieve a final concentration of 25% by weight of Compound B. The suspension concentrate is collected by sieving through glass beads.

[0051] Formulation Example 3 Aqueous Suspension Concentrate An aqueous suspension concentrate was prepared with the following composition: 30 wt% Compound 1, 2.5 wt% tristyrylphenol ethoxylate surfactant, 2.0 wt% ethylene oxide-propylene oxide block copolymer dispersant, 5.0 wt% propylene glycol cryoprotectant, 0.1 wt% silicone oil antifoamant, and 52.4 wt% distilled water. After 2 hours of grinding, 8.0 wt% viscosity modifier gel containing 2.0% xanthan and 1.0% biocide in water was added. Aliquots of this suspension were adjusted to pH 8 with 10 wt% sulfuric acid, pH 10 borate buffer, and 10 wt% sodium hydroxide, respectively.

[0052] VI. Methods for assessing activity The presently disclosed compounds exhibit activity against a variety of pathogens. Their activity is assessed, in part, according to the following assays:

[0053] Method 1: In vitro apyrase assay: Apyrase inhibitory activity was assessed using an in vitro assay, as follows: The method of Windsor, BioTechniques 33:1024-1030 (November 2002) was used as follows: Screening of apyrase inhibitors A 96-well plate was used for the assay: (Greiner bio-one: REF-655901-96 well, PS, F-bottom, clear, non-binding) Buffer: Reaction buffer: 60 mM Hepes, 3 mM MgCl, 3 mM CaCl, and 3 mM ATP (pH 6.5) Developing buffer A: 2% ammonium molybdate aqueous solution Developing buffer B: 11% ascorbic acid in 37.5% TCA aqueous solution Stop buffer C: 2% trisodium citrate in 2% aqueous acetic acid Add 100 μl of reaction buffer to each well. Add 10 μl of DMSO (control) or inhibitor / compound or compounds such as N1915 or orthovanadate to each well (inhibitor concentration 1 mM; 2 mM orthovanadate and 1 mM N1915 were used). Add 10 μl of apyrase (concentration based on optimization - dilute 1 U / μl enzyme to different concentrations such as 0.1 U, 0.05 U, 0.0025, 0.001 U, 0.0005 U - to find a good range) Incubate the plate at room temperature for 1 hour. Mix Developing Buffer A and Developing Buffer B in a ratio of 1:1.5 (just before use). Add 50ul of A:B mix to each well (incubate for 2 minutes) Add 50ul of C to each well. Measure / read the plate absorbance at 630nm

[0054] Compound 1 inhibited apyrase by approximately 60% in this assay.

[0055] Method 2: Greenhouse crop testing In this method, compound 1 was evaluated for its ability to enhance the activity of one of four fungicides, Amistar (azoxystrobin), Intrex (fluxapyroxad), Proline (prothioconazole), or Balaya (mefentrifluconazole and pyraclostrobin), in combination under controlled greenhouse conditions against plant pathogenic fungi: Zymoseptoria tritici in wheat, Botrytis cinerea in tomato, Asian soybean rust (Phakopsora pachyrhizi) in soybean, and brown rust (Puccinia recondita) in wheat. Soybean cultivar Siverka, tomato (Money maker), and wheat plants (JB Diego) were used in these studies. Seeds were sown 1-2 cm deep in 9 cm diameter pots using Petersfield horticultural compost (75% medium peat, 12% sieved sterilized loam, 3% medium vermiculite, 10% granular (sieved to 5 mm, lime-free), 1.5 kg PG mix per m³, lime added to pH 5.5-6.0, and a wetting agent (Vitax Ultrawet, 200 ml per m³). Germinated and grown at 23°C under 16 hours of light per day and 8 hours of light overnight. Plants were treated 2-3 weeks after sowing, when they were at the BBCH11 growth stage (the first pair of true leaves (single leaves) had developed). Using a track sprayer, plants were treated with a mixture of commercial fungicide and test compound at a water volume of 200 L / ha. 24 hours after treatment, plants were inoculated with the appropriate fungus (pathogen). The fungal pathogen was Botrytis The fungicides tested were P. cinerea (gray mold on tomato plants), Zymoseptoria tritici (Septoria leaf blight on wheat plants), Puccinia triticina (brown rust on wheat plants), and Phakopsora pachyrhyzi (Asian soybean rust on soybean plants). Four replicates were used for each combination of fungicide, pathogen, and test compound. When disease symptoms were fully developed, 7–20 days (depending on the pathogen), each plant was evaluated for % disease control. Appropriate controls, including "inoculation checks," were used in all experiments; plants were inoculated with the specific pathogen and assessed for disease levels.Each commercially available fungicide was also tested alone as part of each treatment, and this was the basis for evaluating combinations of Compound 1 and fungicides. Exemplary combinations demonstrated enhanced disease control compared to the disease control observed with the fungicide alone. That is, the compounds are not fungicidal by themselves, but they enhance the activity of the fungicides.

[0056] In these studies, fungicides were applied at the following rates: [Table 1]

[0057] Compound 1 was applied at either 15 ppm or 30 ppm.

[0058] At 15 ppm, the results showed further disease control as shown in the table below. [Table 2]

[0059] At 30 ppm, the results showed further disease control as shown in the table below. [Table 3]

[0060] In the above tables, the symbol "--" indicates that no results were obtained or that there was no observable activity. In some tests, the lack of activity was due to the fungicide application rate being too high, resulting in no observed activity compared to the control. The results in the above tables demonstrate that Compound 1 exhibits significant synergy with Amister, Intrex, and Proline against Z. tritici, Botrytis, and Asian soybean rust. The compound also exhibits significant synergy in combination with Balaya against Asian soybean rust and brown rust. Compound 1 also exhibits significant synergy in combination with Amister and Intrex against brown rust.

[0061] Method 3: Greenhouse crop testing of compound 1 and metconazole To exclude possible bacterial contamination, 2019 field isolates of Zymoseptoria tritici (Septoria tritici) were grown for 6 days at 20°C on potato dextrose agar (PDA) amended with penicillin and streptomycin, allowing for comparison of fungicide activity against current strains of the pathogen harboring recent insusceptibility (resistance) mutations.

[0062] Spore suspensions were made by filling plates with sterile distilled water and gently scraping. The spore suspension was determined by hemocytometry and appropriate dilutions to 10 6 Conidia mL -1 After adjusting to 1.5gL -1 of gelatin and 0.5gL -1 The cells were resuspended in potato dextrose broth amended with sodium oleate.

[0063] Septoria-susceptible winter wheat cultivar Trinity was planted in Levington M3 compost. Seeds (12–15 seeds per pot) were sown in 9 cm pots and grown to growing season 12. Plants were housed in a glasshouse heated to 20°C during the day, ventilated to 22°C, and heated to maintain 15°C at night. 6 spores ml -1 Plants were inoculated with Z. tritici by spraying a spore suspension of Z. tritici until just before runoff. Three replicate pots were used per treatment. Plants were placed in sealed, transparent propagators for 72 hours to maintain high relative humidity and ensure free water remained on the leaves. A shade was provided to prevent the plants from overheating in the propagator. Preventative fungicide sprays were made one day before inoculation. Treatments were randomized within the glasshouse. All fungicides were applied at an equal rate of 200 L of water per hectare using a pressurized, handheld sprayer with a measuring scale. This was done by placing the treated plants between 0.5 m 2This was achieved by placing 10 mL of fungicide spray over an area of ​​1000 m². The results are shown in Figure 9, demonstrating the synergistic effect of the combination of Compound 1 (designated as TX15 in Figure 9) and metconazole in treating winter wheat infected with Z. tritici.

[0064] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be construed as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. The inventors therefore claim as their invention all that comes within the spirit and scope of these claims.

Claims

1. 1. A method for inhibiting fungal infection in seeds or plants at risk of fungal infection, comprising treating the seeds or plants with a fungicide comprising azoxystrobin, difenoconazole, chlorothalonil, tebuconazole, folpet, prothioconazole, fluxapyroxad, metconazole, mefentrifluconazole, pydiflumetofen, benzovindiflupyr, fenpicoxamide, cyazofamid, pyraclostrobin, or a combination thereof; and the method comprising contacting the compound with an apyrase inhibitor, (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide.

2. 10. The method of claim 1, wherein the fungicide comprises metconazole.

3. 10. The method of claim 1, wherein the fungicide comprises mefentrifluconazole.

4. 10. The method of claim 1, wherein the fungicide comprises pydiflumetofen.

5. 10. The method of claim 1, wherein the disinfectant comprises benzovindiflupyr.

6. 10. The method of claim 1, wherein the fungicide comprises fluxapyroxad.

7. 5. The method of claim 4, further comprising contacting the seed or plant with azoxystrobin.

8. 10. The method of claim 1, wherein the fungicide comprises prothioconazole.

9. 10. The method of claim 1 or claim 6, wherein the fungicide comprises mefentrifluconazole.

10. 10. The method of claim 1, wherein the fungicide comprises fenpicoxamid.

11. 10. The method of claim 1, wherein the fungicide comprises difenoconazole.

12. 12. The method of claim 11, wherein the fungicide further comprises azoxystrobin.

13. 10. The method of claim 1, wherein the fungicide is selected from azoxystrobin, pyraclostrobin, or a combination thereof.

14. The method of claim 1 , wherein the fungicide comprises folpet.

15. 10. The method of claim 1, wherein the disinfectant comprises chlorothalonil.

16. 10. The method of claim 1 or claim 9, wherein the fungicide comprises pyraclostrobin.

17. 10. The method of claim 1, wherein the fungicide comprises cyazofamid.

18. 1. A method for inhibiting fungal infection in seeds or plants at risk of fungal infection, comprising contacting the seeds or plants with azoxystrobin, difenoconazole, chlorothalonil, tebuconazole, folpet, prothioconazole, fluxapyroxad, metconazole, mefentrifluconazole, pydiflumetofen, benzovindiflupyr, fenpicoxamide, cyazofamid, pyraclostrobin, and the apyrase inhibitor (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide, wherein the fungal infection comprises Asian soybean rust (Phakopsora pachyrhizi), soybean leaf spot (Cercospora sojina), stripe rust (Puccinia striiformis), downy mildew, or a combination thereof.

19. 20. The method of claim 1 or claim 18, wherein the combination of the apyrase inhibitor and the fungicide has a synergistic effect against fungal infections.

20. A synergistically effective antifungal composition comprising a fungicide selected from the group consisting of fungicides comprising azoxystrobin, difenoconazole, chlorothalonil, tebuconazole, folpet, prothioconazole, fluxapyroxad, metconazole, mefentrifluconazole, pydiflumetofen, benzovindiflupyr, fenpicoxamide, cyazofamid, pyraclostrobin, or combinations thereof, and the apyrase inhibitor (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide.

21. 21. The synergistically effective antifungal composition of claim 20, wherein the fungicide comprises a compound selected from the group consisting of pydiflumetofen, benzovindiflupyr, fluxapyroxad, azoxystrobin, folpet, prothioconazole, mefentrifluconazole, fenpicoxamide, difenoconazole, and combinations thereof.

22. 21. The synergistically effective antifungal composition of claim 20, wherein the fungicide comprises azoxystrobin.

23. 21. The synergistically effective antifungal composition of claim 20, which is synergistically effective against Asian soybean rust (Phakopsora pachyrhizi), soybean leaf spot (Cercospora sojina), stripe rust (Puccinia striiformis), downy mildew, or a combination thereof.

24. 1. A method for inhibiting infection of Z. tritici, Botrytis, Asian soybean rust, or a combination thereof in a seed or plant at risk of infection with said infection, said method comprising contacting said seed or plant with azoxystrobin, fluxapyroxad, prothioconazole, or a combination thereof, and (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide.

25. 1. A method for inhibiting Asian soybean rust, brown rust, or both infection in seeds or plants at risk of infection with said disease, the method comprising contacting said seeds or plants with mefentrifluconazole, pyraclostrobin, and (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide.

26. 1. A method for inhibiting brown rust infection in seeds or plants at risk of infection, the method comprising contacting the seeds or plants with azoxystrobin, fluxapyroxad, or both, and (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide.

27. 1. A method for inhibiting Asian soybean rust infection in seeds or plants at risk of infection, the method comprising contacting the seeds or plants with a fungicide comprising (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide and azoxystrobin and tebuconazole.

28. 28. The method of claim 27, wherein the disinfectant further comprises chlorothalonil.

29. 1. A method for inhibiting soybean spot infection in seeds or plants at risk of infection, the method comprising contacting the seeds or plants with a fungicide comprising (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide and azoxystrobin and tebuconazole.

30. 30. The method of claim 29, wherein the disinfectant further comprises chlorothalonil.

31. 1. A method for inhibiting stripe rust infection in seeds or plants at risk of infection, comprising contacting the seeds or plants with a fungicide comprising (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide and azoxystrobin, folpet, prothioconazole, fluxapyroxad, mefentrifluconazole, pydiflumetofen, benzovindiflupyr, fenpicoxamid, or a combination thereof.

32. 32. The method of claim 31 , wherein the fungicide comprises prothioconazole.

33. 1. A method for inhibiting Septoria infection in seeds or plants at risk of infection, comprising contacting the seeds or plants with a fungicide comprising (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide and azoxystrobin, folpet, prothioconazole, fluxapyroxad, mefentrifluconazole, pydiflumetofen, benzovindiflupyr, fenpicoxamid, or a combination thereof.

34. 1. A method for inhibiting downy mildew infection in seeds or plants at risk of said infection, the method comprising contacting said seeds or plants with a fungicide comprising (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide and cyazofamid.

35. 1. A method for inhibiting downy mildew infection in seeds or plants at risk of said infection, said method comprising contacting said seeds or plants with a fungicide comprising (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide and azoxystrobin, difenoconazole, or both.

36. 1. A method for inhibiting infection by Z. tritici, comprising contacting seeds or plants with a fungicide comprising (E)-3-methyl-N'-(1-(naphthalen-2-yl)ethylidene)benzohydrazide and metconazole.

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