Control method for plant diseases caused by bacteria having reduced sensitivity to agricultural chemicals due to enhancement of efflux pumps

Abstract

The present disclosure provides a control method for plant diseases caused by bacteria that have reduced sensitivity to agricultural chemicals due to the enhancement of efflux pumps. The method involves applying a composition that includes, as active ingredients, (A) fluazinam and (B) at least one bactericide selected from the group consisting of cyazofamid, amisulbrom, valifenalate, mandipropamid, oxathiapiprolin, zoxamide, and ethaboxam to a plant or to soil in which a plant is cultivated.

Description

Method for controlling plant diseases caused by fungi with reduced agrochemical sensitivity due to enhanced drug efflux pumps

[0001] The present invention relates to a method for controlling plant diseases caused by fungi with reduced agrochemical sensitivity due to enhanced drug efflux pumps.

[0002] When agrochemicals are used continuously for many years, organisms with reduced drug sensitivity emerge and pose problems due to factors such as reduced drug affinity accompanying genetic mutations in target sites, drug inactivation by modifying enzymes or degrading enzymes, reduced drug permeability from changes in structures surrounding cell membranes, and active drug efflux caused by enhanced drug efflux pumps. In particular, organisms with reduced drug sensitivity due to enhanced drug efflux pumps are a major problem because they are capable of effluxing multiple drugs by their nature.

[0003] Patent Documents 1, 2 and 3 describe that wheat leaf blight and gray mold of grape caused by fungi that have acquired efflux-type multidrug resistance derived from ABC transporters or MFS transporters can be controlled by pyridazine compounds, pyrazole compounds and dithiinetetracarboximide compounds.

[0004] Fluazinam is a fungicide with a broad fungicidal spectrum covering diseases such as gray mold, sclerotinia rot, and clubroot, and is widely used especially as a specific remedy for potato late blight. The mechanism of action of fluazinam against plant pathogenic fungi is known to be "uncoupling of oxidative phosphorylation", which is classified under FRAC code C5.

[0005] In general, in mitochondria, hydrogen ions are pumped into the intermembrane space by the function of the electron transport chain to form a proton gradient, and oxidative phosphorylation that produces ATP using this gradient is coupled. Uncoupling refers to inhibiting the coupling between the proton gradient formed by electron transport in oxidative phosphorylation and ATP production. As an uncoupler, fluazinam eliminates the proton gradient formed across the inner mitochondrial membrane, thereby inhibiting ATP production.

[0006] The diarylamine fungicide SYP-14288, developed using fluazinam as a model, is the same uncoupling agent as fluazinam and is active against oomycetes and other organisms. It has been confirmed that strains of Phytophora capsici resistant to SYP-14288 excrete SYP-14288 through enhanced (overexpression) of the ABC transporter, and similarly excrete fluazinam, chlorothalonil, and oxathiapiprolin. Furthermore, since susceptibility was restored when amitriptyline, an ABC transporter inhibitor, was added, it is clear that resistance was acquired through enhanced ABC transporter activity (Non-Patent Literature 1).

[0007] International Publication No. 2018 / 203560, International Publication No. 2018 / 212328, International Publication No. 2018 / 212329

[0008] Pest Management Science 2022;78,2240-2249

[0009] The present invention aims to provide a method for controlling plant diseases caused by fungi whose susceptibility to pesticides has been reduced due to the enhancement of the pesticide discharge pump.

[0010] In order to solve the above problems, the inventors conducted diligent research and found that a composition containing (A) fluazinam and (B) at least one fungicide selected from the group consisting of cyazofamide, amisulbrom, valifenarate, mandipropamide, oxathiapiproline, zoxamide, and etaboxam as active ingredients has excellent efficacy against plant diseases caused by fungi whose susceptibility to pesticides has decreased due to enhanced drug efflux pumps (hereinafter also referred to as "pesticide-decreased susceptibility fungi"), thus completing the present invention.

[0011] In other words, the present invention relates to a method for controlling plant diseases caused by fungi whose susceptibility to pesticides has been reduced due to enhanced drug discharge pumps, including the following embodiments, and includes applying a composition containing (A) fluazinam and (B) at least one fungicide selected from the group consisting of cyazofamide, amisulbrom, valifenarate, mandipropamide, oxathiapiproline, zoxamide, and etaboxam as active ingredients to plants or the soil in which plants are cultivated.

[0012] This disclosure relates to the following embodiments: [1] A method for controlling plant diseases caused by fungi whose susceptibility to pesticides has been reduced due to enhanced drug efflux pumps, comprising applying a composition containing (A) fluazinam and (B) at least one fungicide selected from the group consisting of cyazofamide, amisulbrom, valifenarate, mandipropamide, oxathiapiproline, zoxamide, and etaboxam as active ingredients to a plant or the soil in which the plant is cultivated. [2] The method according to [1], wherein the drug efflux pump is at least one selected from the group consisting of ABC transporters and MFS transporters. [3] The method according to [1] or [2], wherein the drug efflux pump is an ABC transporter. [4] The method according to any one of [1] to [3], wherein the plant disease is potato blight. [5] The method according to any one of [1] to [4], wherein the pesticide-sensitive fungus is a potato blight fungus in which susceptibility to fluazinam has decreased. [6] Use of a composition for controlling plant diseases caused by fungi in which susceptibility to pesticides has decreased due to enhanced drug efflux pumps, comprising (A) fluazinam and (B) at least one fungicide selected from the group consisting of cyazofamide, amisulbrom, valifenarate, mandipropamide, oxathiapiproline, zoxamide, and etaboxam as active ingredients.

[0013] According to the present invention, plant diseases caused by fungi whose susceptibility to pesticides has been reduced by enhancing the drug discharge pump can be controlled.

[0014] The plant disease control method according to the present invention will be described in detail below, but the scope of the present invention is not limited to this description, and modifications may be made to the extent that the spirit of the present invention is not impaired, in addition to the examples given below.

[0015] In this disclosure, the mixing ratio of (A) fluazinam (hereinafter also referred to as component (A)) and (B) at least one bactericide selected from the group consisting of cyazofamide, amisulbrom, valifenarate, mandipropamide, oxathiapiproline, zoxamide, and etaboxam (hereinafter also referred to as component (B)) is 1:1,000 to 1,000:1 by mass ratio, preferably 1:100 to 100:1, and more preferably 1:5 to 20:1.

[0016] The compositions disclosed herein can be formulated in various forms such as emulsions, powders, granules, wettable powders, granular wettable powders, aqueous suspensions, oily suspensions, liquids, aerosols, and pastes by mixing them with various formulation auxiliary components, as in the case of conventional pesticide formulations. However, they can be formulated in any form commonly used in the field, as long as they are suitable for the purposes of this disclosure. Furthermore, the compositions disclosed herein may be formulated by combining the active ingredients (A) and (B) (hereinafter also simply referred to as "active ingredients") together and applying them, or each active ingredient may be formulated separately and mixed at the time of application. The order in which components (A) and (B) are applied sequentially is not specified. The typical proportion of the formulation to the total formulation is 1 to 99.999 parts by mass of the formulation auxiliary component to 0.001 to 99 parts by mass of the active ingredient, preferably 5 to 99.99 parts by mass of the formulation auxiliary component to 0.01 to 95 parts by mass of the active ingredient, and more preferably 20 to 99.99 parts by mass of the formulation auxiliary component to 0.01 to 80 parts by mass of the active ingredient. When actually using these formulations, they can be used as is or diluted to a predetermined concentration with a diluent such as water.

[0017] The formulation auxiliary components include: plant or animal powders such as starch, activated carbon, soy flour, wheat flour, wood flour, fish meal, and milk powder; mineral powders such as talc, kaolin, bentonite, calcium carbonate, zeolite, diatomaceous earth, white carbon, clay, and alumina; sulfur powder; anhydrous sodium sulfate; solid carriers such as water; alcohols such as methyl alcohol and ethylene glycol; ketones such as acetone, methyl ethyl ketone, and N-methyl-2-pyrrolidone; ethers such as dioxane and tetrahydrofuran; aliphatic hydrocarbons such as kerosene and lamp oil; xylene, trimeth Examples of liquid carriers include aromatic hydrocarbons such as chlorobenzene, tetramethylbenzene, cyclohexane, and solvent naphtha; halogenated hydrocarbons such as chloroform and chlorobenzene; acid amides such as dimethylformamide; esters such as ethyl acetate and fatty acid glycerol esters; nitriles such as acetonitrile; sulfur-containing compounds such as dimethyl sulfoxide; and vegetable oils such as soybean oil and corn oil. Emulsifiers, suspending agents, dispersants, spreading agents, penetrating agents, wetting agents, thickeners, defoaming agents, stabilizers, and antifreeze agents can be added as needed.

[0018] The compositions disclosed herein can be applied by spraying (e.g., misting, atomizing, granulation, water surface application, etc.), soil application (mixing, drenching, etc.), surface application (coating, powdering, covering, etc.), etc. They can also be applied using aerial devices such as drones or unmanned helicopters, or by so-called ultra-low volume application. When applying, it is possible to arbitrarily choose to apply to plants and to their cultivation soil (either before or after the onset of the plant disease).

[0019] Furthermore, when applying it to plants, it may be used on any part of the plant, such as the leaves, stems, stalks, flowers, buds, fruits, seeds, roots, tubers, or bulbs.

[0020] The application rate of the composition disclosed herein varies depending on weather conditions, target crops, method of use, formulation form, etc., and cannot be specified in general terms. However, for example, when applied to the stems and leaves of plants, the total concentration of the active ingredients of component (A) and component (B) is usually 0.1 to 10,000 ppm, preferably 1 to 4,000 ppm, and more preferably 1 to 2,000 ppm, relative to the stems and leaves of plants. When applied to soil, the total amount of the active ingredients of component (A) and component (B) is usually 0.1 to 10,000 g / ha, preferably 1 to 2,000 g / ha. When applied to seeds, the total amount of the active ingredients of component (A) and component (B) is usually 0.25 to 500 g / 100 kg of seeds, preferably 10 to 200 g / 100 kg of seeds.

[0021] The compositions disclosed herein can be mixed or used in combination with other pesticides, fertilizers, phytotoxicity reducers, etc., and in this case, they may exhibit even better efficacy and action. Examples of other pesticides include herbicides, fungicides, antiviral agents, attractants, plant hormones, plant growth regulators, and biopesticides.

[0022] The compositions of this disclosure include grasses such as rice, wheat, barley, rye, oats, sorghum, and maize; legumes such as soybeans, peanuts, broad beans, peas, and chickpeas; cucurbits such as cucumbers, pumpkins, watermelons, melons, and zucchini; nightshades such as tomatoes, eggplants, bell peppers, potatoes, and chili peppers; brassicas such as cabbage, Chinese cabbage, radishes, turnips, broccoli, cauliflower, and rapeseed; daisies such as burdock and lettuce; lilies such as leeks, onions, and asparagus; carrots and parsley; and mints. It can be used on the following plants: flowers such as roses, chrysanthemums, carnations, and gerberas; fruit trees such as grapes, apples, pears, citrus fruits, peaches, plums, cherries, blueberries, and persimmons; trees such as tea, coffee, cocoa, olives, and almonds; pasture grasses such as orchardgrass, Italian ryegrass, alfalfa, and clover; turfgrass such as bentgrass and Korean grass; and other plants such as okra, cotton, bananas, ginger, strawberries, and sweet potatoes.

[0023] Furthermore, the aforementioned plants also include genetically modified plants created using genetic engineering technology. Examples of genetically modified plants include herbicide-resistant plants, insect-resistant plants, plants with specific plant components, and plants with plant pathogen resistance. In addition, stacked varieties that combine multiple useful traits are also included.

[0024] While research on drug efflux pumps has focused primarily on multidrug-resistant bacteria, in this specification, the term refers to a pump that expels drugs from inside a cell to the outside. Increased activity of this pump can lower the concentration of drugs inside the cell, leading to decreased drug sensitivity. Generally, membrane proteins that transport ions and small molecules across the cell membrane are called transporters. Drug efflux pumps are a type of transporter that has a structure with gaps through which substances such as drugs can pass, and they can expel multiple drugs with different mechanisms of action. Examples of drug efflux pumps include, but are not limited to, transporter families such as ABC transporters (ATP-binding cassette superfamily), MFS transporters (Major facilitator superfamily), SMR transporters (Small multidrug resistance family), RND transporters (Resistance-nodulation-cell division family), and MATE transporters (Multidrug and toxic compound extraction family).

[0025] To determine whether the drug susceptibility of target organisms is reduced due to the enhancement of drug efflux pumps, consider the following known inhibitors of drug efflux pumps: L-norepinephrine monohydrate, berberine chloride hydrate, ellagic acid dihydrate, (-)-epigallocatechin gallate hydrate, quinine hydrochloride dihydrate, L-adrenaline, and hydrogen tartrate. Methods include administering L-adrenaline, artesunate, berberine sulfate, biochanin A, cholecalciferol, celecoxib, amitriptyline, berberine, chalcone, farnesol, ibuprofen, benztropin, nisoxetine, curcumin, phenothiazine, propafenone, isocyanide, cerurenin, milbemycin, tacrolimus, enyatin, beuverisin, chlorgiline, etc., and measuring changes in drug sensitivity; measuring drugs excreted from cells using HPLC, LC-TOF / MS, or LC / Q-TOF / MS, etc.; measuring the excretion of fluorescent markers such as rhodamine 6G to indicate the enhancement of ABC transporter-related molecules such as P-glycoprotein; and measuring the expression of excretion-related and metabolism-related genes. More specific methods are described in Patent Documents 1-3 and Non-Patent Document 1.

[0026] Examples of plant diseases that can be controlled by the compositions disclosed herein, which are caused by fungi whose susceptibility to pesticides has been reduced due to enhanced drug discharge pumps, include plant diseases caused by plant pathogenic fungi belonging to the following groups: Oomycetes, Zygomycota, Ascomycetes, Basidiomycetes, and Deuteromycetes.

[0027] Specific examples of the aforementioned plant diseases include, but are not limited to, the following.

[0028] As oomycetes, diseases caused by Phytophthora species such as potato or tomato blight (Phytophthora infestans) and tomato gray blight (Phytophthora capsici); diseases caused by Pseudoperonospora species such as cucumber downy mildew (Pseudoperonospora cubensis); diseases caused by Plasmopara species such as grape downy mildew (Plasmopara viticola); diseases caused by Pythium species such as rice seedling blight (Pythium graminicola) and wheat brown snow mold (Pythium iwayamai); radish root constriction (Aphanomyces raphani) and spinach root rot (Aphanomyces Examples include diseases caused by Aphanomyces species (such as cochlioides) and diseases caused by Albugo species (such as Albugo macrospora), including spinach white rust (Albugo occidentalis).

[0029] Ascomycetes include diseases caused by Erysiphe species such as wheat powdery mildew (Erysiphe graminis); diseases caused by Sphaerotheca species such as cucumber powdery mildew (Sphaerotheca fuliginea) and strawberry powdery mildew (Sphaerotheca humuli); diseases caused by Uncinula species such as grape powdery mildew (Uncinula necator); diseases caused by Podosphaera species such as apple powdery mildew (Podosphaera leucotricha); pea brown spot (Mycosphaerella pinodes), apple black spot (Mycosphaerella pomi), banana black sigatoka disease (Mycosphaerella musicola), persimmon leaf spot (Mycosphaerella nawae), and strawberry eye disease (Mycosphaerella Diseases caused by Mycosphaerella species such as fragariae; diseases caused by Venturia species such as apple scab (Venturia inaequalis) and pear scab (Venturia nashicola); diseases caused by Pyrenophora species such as barley net spot (Pyrenophora teres) and barley leaf spot (Pyrenophora graminea); various sclerotinia diseases such as bean sclerotinia, cucumber sclerotinia, cabbage sclerotinia, Chinese cabbage sclerotinia, pepper sclerotinia, bell pepper sclerotinia or onion sclerotinia (Sclerotinia sclerotiorum); wheat snow mold large grain sclerotinia (Sclerotinia borealis); tomato small grain sclerotinia (Sclerotinia minor); alfalfa sclerotinia (Sclerotinia Diseases caused by fungi of the genus Sclerotinia, such as trifoliorum; diseases caused by fungi of the genus Botryolinia, such as peanut sclerotinia (Botryolinia arachidis);Diseases caused by fungi of the genus Cochliobolus, such as rice leaf spot (Cochliobolus miyabeanus); diseases caused by fungi of the genus Didymella, such as cucumber vine blight (Didymella bryoniae); diseases caused by fungi of the genus Gibberella, such as rice bakanae disease (Gibberella fujikuroi); diseases caused by fungi of the genus Elsinoe, such as grape black pox (Elsinoe ampelina) and citrus scab (Elsinoe fawcettii); diseases caused by fungi of the genus Diaporthe, such as citrus black spot (Diaporthe citri) and grape branch swelling (Diaporthe sp.); diseases caused by fungi of the genus Monilinia, such as apple monilinia (Monilinia mali) and peach gray mold (Monilinia fructicola); and grape late blight (Glomerella). Examples include diseases caused by Glomerella species such as Glomerella cingulata.

[0030] Examples of basidiomycetes include diseases caused by Rhizoctonia species, such as rice sheath blight (Rhizoctonia solani); diseases caused by Ustilago species, such as wheat smut (Ustilago nuda); diseases caused by Puccinia species, such as oat crown rust (Puccinia coronata), wheat red rust (Puccinia recondita), and wheat yellow rust (Puccinia striiformis); diseases caused by Phakopsora species, such as soybean rust (Phakopsora pachyrhizi); and diseases caused by Typhula species, such as wheat or barley snow mold (Typhula incarnata, Typhula ishikariensis).

[0031] As imperfect fungi, diseases originating from Septoria species such as wheat blight (Septoria nodorum) and wheat leaf blight (Septoria tritici); various gray mold diseases such as grape gray mold, citrus gray mold, cucumber gray mold, tomato gray mold, strawberry gray mold, eggplant gray mold, green bean gray mold, adzuki bean gray mold, pea gray mold, peanut gray mold, chili pepper gray mold, bell pepper gray mold, lettuce gray mold, onion gray mold, statice gray mold, carnation gray mold, rose gray mold, pansy gray mold, or sunflower gray mold (Botrytis cinerea); and onion gray rot (Botrytis Diseases caused by Botrytis species, such as onion leaf blight (Botrytis squamosa, Botrytis byssoidea, Botrytis tulipae); diseases caused by Fusarium species, such as wheat red mold (Fusarium graminearum) and cucumber wilt (Fusarium oxysporum); diseases caused by Pyricularia species, such as rice blast and wheat blast (Pyricularia oryzae); diseases caused by Cercospora species, such as sugar beet brown spot (Cercospora beticola) and persimmon corn spot (Cercospora kakivola); diseases caused by Colletotrichum species, such as cucumber anthracnose (Colletotrichum orbiculare);Diseases caused by Alteraria species such as apple leaf spot (Alternaria alternata apple pathotype), pear black spot (Alternaria alternata Japanese pear pathotype), potato summer blight or tomato ring spot (Alternaria solani), cabbage or Chinese cabbage black spot (Alternaria brassicae), cabbage sooty mold (Alternaria brassicola), onion or leek black spot (Alternaria porri); plant diseases caused by Phoma species such as cabbage root rot (Phoma lingam); diseases caused by Pseudocercosporella species such as wheat eye spot (Pseudocercosporella herpotrichoides); diseases caused by Pseudocercospora species such as grape brown spot (Pseudocercospora vitis); barley cloud spot (Rhynchosporium Examples include diseases caused by fungi of the genus Rhynchosporium (such as secalis); diseases caused by fungi of the genus Cladosporium (such as peach scab / Cladosporium carpophilum); diseases caused by fungi of the genus Phomopsis (such as peach rot / Phomopsis sp.); diseases caused by fungi of the genus Gloeosporium (such as persimmon anthracnose / Gloeosporium kaki); diseases caused by fungi of the genus Fulvia (such as tomato leaf mold / Fulvia fulva); and diseases caused by fungi of the genus Corynespora (such as cucumber brown spot / Corynespora cassiicola).

[0032] Below, to provide a more detailed explanation of the plant disease control methods related to this disclosure, examples of formulations and test examples are shown.

[0033] Examples of formulations of this disclosure are described below, but the amounts, dosage forms, etc. of formulations in this disclosure are not limited to those described. Formulation Example 1 (1) Kaolin 78 parts by weight (2) β-Naphthalenesulfonic acid sodium formalin condensate 2 parts by weight (3) Polyoxyethylene alkylaryl sulfate 5 parts by weight (4) Hydrated amorphous silicon dioxide A wettable powder can be obtained by mixing and grinding a mixture of each component in an 8:1:1 weight ratio with component (A) and component (B).

[0034] Formulation Example 2 (1) Component (A) 0.5 parts by weight (2) Component (B) 0.5 parts by weight (3) Bentonite 20 parts by weight (4) Kaolin 74 parts by weight (5) Sodium ligninsulfonate 5 parts by weight or more of each component are crushed and mixed, an appropriate amount of water is added and kneaded, then granulated and dried to obtain granules.

[0035] Formulation Example 3 (1) Component (A) 2 parts by weight (2) Component (B) 3 parts by weight (3) Talc A powder is obtained by uniformly grinding and mixing each component in an amount of 95 parts by weight or more.

[0036] Formulation Example 4 (1) Component (A) 10 parts by weight (2) Component (B) 10 parts by weight (3) N,N'-dimethylacetamide 20 parts by weight (4) Polyoxyethylene alkylphenyl ether 10 parts by weight (5) Xylene 50 parts by weight or more of each component is uniformly mixed and dissolved to obtain an emulsion.

[0037] Formulation Example 5 (1) Component (A) 25 parts by weight (2) Component (B) 25 parts by weight (3) Polyoxyethylene alkylphenyl ether phosphate triethanolamine salt 2 parts by weight (4) Silicone 0.2 parts by weight (5) Water 47.8 parts by weight Each of the above components is uniformly mixed and pulverized to obtain a stock solution, to which (6) Sodium polycarboxylate 5 parts by weight and (7) Anhydrous sodium sulfate 42.8 parts by weight are added and uniformly mixed, granulated and dried to obtain a granular wettable powder.

[0038] Formulation Example 6 (1) Component (A) 10 parts by weight (2) Component (B) 10 parts by weight (3) Polyoxyethylene alkylphenyl ether phosphate triethanolamine salt 2 parts by weight (4) Silicone 0.2 parts by weight (5) Xanthan gum 0.1 parts by weight (6) Ethylene glycol 5 parts by weight (7) Water 72.7 parts by weight By uniformly mixing and grinding each of the above components, an aqueous suspension can be obtained.

[0039] Test Example 1 Method for testing the efficacy of Phytophthora infestans on potato blight with reduced susceptibility to fluazinum due to enhanced drug excretion pumps Test organism: Phytophthora infestans 18BE02 strain (Genotype: EU37_A2, strain with reduced susceptibility to fluazinum) 18FR07 strain (Genotype: EU13_A2, strain susceptible to fluazinum) Test agent: Phytophthora infestans Potato leaf discs (φ20 mm) were placed with the underside of the leaves facing up in a plastic petri dish (8 cm in diameter, manufactured by Fursteri) lined with moistened filter paper. A predetermined concentration of the chemical solution was sprayed using an airbrush (model HP-PB, manufactured by Anest Iwata Corporation) (spray volume 1,000 L / ha), and then placed in a rectangular petri dish (24.5 cm square, manufactured by Corning). Rye A agar medium (rye seed extract, 2% sucrose, 1.5% agar) or V8 juice agar medium (V8 juice, 1.5% agar) was dispensed into plastic petri dishes (8.6 cm in diameter, manufactured by Fursteri). Each strain was cultured for 7 days at 18°C ​​in the dark to induce zoosporangia formation. After that, tap water (5 mL) was added, and the zoosporangia were scraped off with a brush and collected in a test tube to obtain a zoosporangia suspension. 10 μL of the zoosporangia suspension was inoculated onto the underside of potato leaves and cultured for 3 days at 20°C in the dark. Then, zoosporangia formation was induced for 3 days at 18°C ​​(16 hours light, 8 hours dark). Potato leaves with formed zoosporangia were placed in a centrifuge tube (50 mL capacity, Corning), and 20-25 mL of germination solution (0.2 mM calcium chloride, 0.05 mM magnesium sulfate, 0.05 mM potassium dihydrogen phosphate, 0.5 mM sodium bicarbonate) was added and stirred to separate the zoosporangia from the potato leaves. After washing and filtering 2-3 times using a cell strainer (5 μm), zoospore release was induced in a 4°C environment for 1 hour. The resulting zoospore suspension was sprayed onto the leaves with an airbrush one day after treatment (5 mL / square petri dish) and cultured on a plant growing shelf (model RS4-15060, Kitagawa Electric Co., Ltd.) (16 hours of light, 8 hours of darkness).

[0040] Five days after vaccination, the disease lesion area percentage was investigated, and the control efficacy was calculated using the following formula.

[0041] Control value = (1 - percentage of diseased area in treated area / percentage of diseased area in untreated area) × 100

[0042]

[0043] The numbers in the table represent control values

[0044] Fluazinam exhibited high control effect against the 18FR07 strain, but showed low control effect against the 18BE02 strain, so the 18BE02 strain was considered to be a strain with reduced sensitivity to fluazinam.

[0045] Test Example 2 Effect of fluazinam on the drug efflux pump of Phytophthora infestans Test strains: Phytophthora infestans 18BE02 strain (Genotype: EU37_A2, strain with reduced sensitivity to fluazinam) 18FR07 strain (Genotype: EU13_A2, fluazinam-sensitive strain) The zoosporangia obtained by the same method as in Test Example 1 were allowed to stand at 4°C for 2.5 hours for the 18BE02 strain and 1.5 hours for the 18FR07 strain, then zoospores were released under an environment of 18°C for 15 minutes. After filtration through a cell strainer (10 μm), the filtrate was agitated (Vortex Mixer, Model No. OK-0500-902, manufactured by Scientific Industries, Inc.) to encyst the zoospores, and a cystospore suspension was obtained.

[0046] The cystospore suspension was mixed with a mixture of fluazinam at a predetermined concentration (final concentration 100 ppb to 400 ppb) and a rhodamine 6G solution (final concentration 0.05 μM). After standing at room temperature for 30 minutes, 10 μL of the mixture was dropped onto a slide glass, and fluorescence observation (excitation 500 nm / fluorescence 540 nm, shutter speed 50 ms) and bright-field observation were performed under a microscope. The fluorescence accumulation amount of the fluorescence image was quantified using ImageJ.

[0047]

[0048] The fluazinam-sensitive strain showed a high fluorescence accumulation amount regardless of the presence or absence of fluazinam, while the fluazinam low-sensitivity strain showed a low fluorescence accumulation amount regardless of the presence or absence of fluazinam, which was considered to be due to the enhanced activity of the drug efflux pump excreting the fluorescent substance.

[0049] Test Example 3 Efficacy of drug efflux pump inhibitors against potato blight fungus Test organism: Potato blight fungus (Phytophthora infestans) 18BE02 strain (Genotype: EU37_A2, strain with reduced susceptibility to fluazinam) 18FR07 strain (Genotype: EU13_A2, strain susceptible to fluazinam) Test agent: Chlorgiline Zoospore release was induced using the same method as in Test Example 1, and zoospores were released in an environment of 18°C ​​for 15 minutes. After filtration with a cell strainer (10 μm), the filtrate was stirred (vortex mixer, model 0K-0500-902, manufactured by Scientific Industries) to encapsulate the zoospores and obtain an encapsulated spore suspension.

[0050] To the encapsulated spore suspension 3 to 5 minutes after the above treatment, a drug solution containing chlorgiline at a predetermined concentration was added and allowed to stand at room temperature for 30 minutes. To assess the effect of the encapsulated spore inhibitor, the fluorescence accumulation amount of rhodamine 6G solution (0.05 μM) was analyzed by flow cytometry (model BD FACSVerse® cell analyzer, manufactured by BD Biosciences). Analysis conditions: FSC: 170 (threshold 2,000), SSC: 300 (threshold 2,000), PE-A: 400 (threshold 5,000) (2,500 cells each).

[0051]

[0052] When chlorgiline, a drug efflux pump inhibitor, was added, fluorescence accumulation increased in both strains. Since the fluorescence accumulation of strain 18BE02 (fluazinam-reduced susceptibility strain) was lower than that of strain 18FR07 (fluazinam-susceptible strain), it was considered that strain 18BE02 (fluazinam-reduced susceptibility strain) had a stronger enhancement of the drug efflux pump than the susceptible strain, which is thought to be one of the causes of reduced susceptibility to fluazinam.

[0053] Test Example 4 Effect on Potato Late Blight Fungus that acquired reduced susceptibility due to enhanced drug efflux pump Test organisms: Potato Late Blight Fungus (Phytophthora infestans) 18BE02 strain (Genotype: EU37_A2, strain with reduced susceptibility to fluazinam) 18FR07 strain (Genotype: EU13_A2, strain susceptible to fluazinam) Test agents: Cyazofamide, Amisulbrom, Valifenarate, Mandipropamide, Oxatiapiproline, Zoxamide, Etaboxam Zoospore release was induced using the same method as in Test Example 1, and zoospores were released in an environment of 18°C ​​for 15 minutes. After filtration with a cell strainer (10 μm), the filtrate was stirred (vortex mixer, model 0K-0500-902, manufactured by Scientific Industries) to encapsulate the zoospores and obtain an encapsulated spore suspension.

[0054] Three to five minutes after the above treatment, a drug solution containing each drug at a predetermined concentration was added to the encapsulated spore suspension and left to stand at room temperature for 30 minutes. The "treatment concentration" listed in Tables 4-1 to 10-2 indicates the concentration of the drug in the encapsulated spore suspension during the 30 minutes of standing at room temperature. For living cells, the amount of fluorescence accumulation of rhodamine 6G solution (0.05 μM) was measured, and for dead cells, counting was performed by propidium iodide staining (final concentration 1 μg / mL).

[0055] The fluorescence accumulation levels of living cells and the determination of dead cells were analyzed by flow cytometry (the analysis conditions were the same as in Experiment 2).

[0056] The fluorescence retention effect of the test reagents was evaluated by the amount of fluorescence accumulation (fluorescence intensity) in cells treated with rhodamine 6G and the fluorescence retention rate. The amount of fluorescence accumulation (fluorescence intensity) of rhodamine 6G was measured as the geometric mean (Geomean). Since rhodamine 6G also stains dead cells, the fluorescence retention rate was calculated based on the dead cell rate detected after staining with propidium iodide. In addition, a corrected dead cell rate was calculated based on the dead cell rate of the untreated group. Corrected dead cell rate = [(Dead cell rate in the drug-treated group - Dead cell rate in the untreated group) / (100 - Dead cell rate in the untreated group)] × 100 Corrected fluorescence retention rate = Fluorescence retention rate - Corrected dead cell rate The results are shown in Tables 4-1 to 10-2.

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] When various test agents, not known to inhibit drug efflux pumps, were added to strains 18BE02 (fluazinam-decreased susceptibility strain) and 18FR07 (fluazinam-susceptible strain), fluorescence accumulation increased in all test bacteria. Although rhodamine 6G also fluoresces in dead cells, the corrected dead cell rate was low or the corrected fluorescence-retaining cell rate was high under all conditions, indicating that the increase in fluorescence accumulation was not due to cell death but rather a result of inhibition of drug efflux pumps.

[0072] From the above, it was revealed that each test agent, which is not known to have a drug efflux pump inhibitory effect, has the effect of inhibiting the function of the drug efflux pump in strain 18BE02 (fluazinam-reduced susceptibility strain) and strain 18FR07 (fluazinam-susceptible strain).

[0073] Test Example 5 Test method for the efficacy of mixing with fluazinam against potato blight fungus: Same as Test Example 4. Test fungus: Potato blight fungus (18BE02 strain (Genotype: EU37_A2, fluazinam-decreased susceptibility strain)) Test agent: Fluazinam, the agent used to mix with fluazinam is the same as in Test Example 4. The results are shown in Tables 11 to 17. When fluazinam is mixed with other test agents, in Tables 11 to 17, the concentration of fluazinam is shown to the left of "+", and the concentration of the agent used to mix with fluazinam is shown to the right of "+". Based on the obtained corrected fluorescence-retaining cell rate, theoretical values ​​were calculated using Colby's formula. The theoretical values ​​calculated using Colby's formula are shown in parentheses in Tables 11 to 17.

[0074] Colby's formula = (X + Y) - XY / 100 X: Corrected fluorescence-retaining cell rate for component (A) alone Y: Corrected fluorescence-retaining cell rate for component (B) alone

[0075]

[0076] The numbers in parentheses for the corrected fluorescence-retaining cell rate are theoretical values.

[0077]

[0078] The numbers in parentheses for the corrected fluorescence-retaining cell rate are theoretical values.

[0079]

[0080] The numbers in parentheses for the corrected fluorescence-retaining cell rate are theoretical values.

[0081]

[0082] The numbers in parentheses for the corrected fluorescence-retaining cell rate are theoretical values.

[0083]

[0084] The numbers in parentheses for the corrected fluorescence-retaining cell rate are theoretical values.

[0085]

[0086] The numbers in parentheses for the corrected fluorescence-retaining cell rate are theoretical values.

[0087]

[0088] The numbers in parentheses for the corrected fluorescence-retaining cell rate are theoretical values.

[0089] Mixing fluazinam with the tested drug increased intracellular fluorescence accumulation, resulting in a significantly higher percentage of fluorescence-retaining cells compared to treatment with each drug alone. These compositions inhibited the enhancement of drug efflux pumps, restoring drug sensitivity.

[0090] Test Example 6: Test method for the combined effect of fluazinam against potato blight: Same as Test Example 1. However, the spraying water volume for the combined test of oxathiapiproline and zoxamide was set to 2,000 L / ha.

[0091] Test fungus: Potato blight fungus (Phytophthora infestans) 18BE02 strain (Genotype: EU37_A2, strain with reduced susceptibility to fluazinam) Test agents: Fluazinam, cyazofamide, valifenarate, oxathiapiproline, zoxamide Based on the obtained control values, theoretical values ​​were calculated using Colby's formula. The theoretical values ​​calculated using Colby's formula are shown in parentheses in Tables 18 to 21.

[0092] Colby's formula = (X + Y) - XY / 100 X: Control value of component (A) alone Y: Control value of component (B) alone

[0093]

[0094] The numbers in the table represent the control efficacy, and the values ​​in parentheses are theoretical values.

[0095]

[0096] The numbers in the table represent the control efficacy, and the values ​​in parentheses are theoretical values.

[0097]

[0098] The numbers in the table represent the control efficacy, and the values ​​in parentheses are theoretical values.

[0099]

[0100] The numbers in the table represent the control efficacy, and the values ​​in parentheses are theoretical values.

[0101] When fluazinam was mixed with cyazofamide, valifenarate, oxathiapiproline, or zoxamide, its susceptibility was restored, and all of these showed high control efficacy, with each exhibiting a synergistic effect.

[0102] This disclosure provides a method for controlling plant diseases caused by fungi whose susceptibility to pesticides has been reduced due to enhanced pesticide discharge pumps, and has industrial applicability.

[0103] It is clear to those skilled in the art that various modifications or alterations can be conceived within the scope of the claims, and these will naturally fall within the technical scope of the present invention. Furthermore, the components of the above embodiments may be combined in any way without departing from the spirit of the invention.

[0104] This application is based on a Japanese patent application (Patent Application No. 2025-066430) filed on April 14, 2025, the contents of which are incorporated herein by reference.

Claims

1. A method for controlling plant diseases caused by fungi whose susceptibility to pesticides has been reduced due to an enhanced drug discharge pump (hereinafter also referred to as "pesticide-reduced susceptibility fungi"), comprising applying a composition containing (A) fluazinam and (B) at least one fungicide selected from the group consisting of cyazofamide, amisulbrom, valifenarate, mandipropamide, oxathiapiproline, zoxamide, and etaboxam as active ingredients to a plant or the soil in which the plant is cultivated.

2. The method according to claim 1, wherein the drug discharge pump is at least one selected from the group consisting of ABC transporters and MFS transporters.

3. The method according to claim 1, wherein the drug discharge pump is an ABC transporter.

4. The method according to claim 1 or 2, wherein the plant disease is potato blight.

5. The method according to claim 1 or 2, wherein the pesticide-impaired bacterium is a potato blight bacterium in which susceptibility to fluazinum has been reduced.

6. Use of a composition containing (A) fluazinam and (B) at least one fungicide selected from the group consisting of cyazofamide, amisulbrom, valifenarate, mandipropamide, oxathiapiproline, zoxamide, and etaboxam as active ingredients to control plant diseases caused by fungi whose susceptibility to pesticides has been reduced due to enhanced drug discharge pumps.

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