Pyrroline diketone compound as well as preparation method and bactericide application thereof

Abstract

The invention provides a pyrroline diketone compound as well as a preparation method and a bactericide application thereof. The name of the pyrroline diketone compound is 1-(2, 6-diethyl-4-methylphenyl)-1H-pyrrole-2, 5-diketone, and the structural formula of the pyrroline diketone compound is shown as a formula I. The pyrroline diketone compound and an agricultural bactericide taking the compound as an effective component are used for preventing and treating various plant diseases caused by plant pathogenic fungi, oomycetes and bacteria. The compound is novel in structure and action mechanism, convenient in synthesis method and good in safety, has broad-spectrum and efficient antibacterial activity, does not have cross resistance with other existing bactericides, can be used for preventing and treating plant diseases of vegetables, fruits, food crops, oil crops, commercial crops, garden crops, traditional Chinese medicinal materials and the like, and has broad application prospects. And particularly, the composition has good chemical protection effect and systemic treatment effect on drug-resistant crop diseases.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural chemistry, and in particular to a pyrrolinedione compound, a preparation method thereof and a fungicide application thereof. Background Art

[0002] Safe and efficient selective pesticides are indispensable means of production for modern agricultural production. However, due to the single action site of selective pesticides, genetic variation of pests is very likely to cause resistance problems. It is known that many existing pesticides have the same mode of action, and there is cross-resistance between them. Plant pathogenic microorganisms with high reproduction rates and rapid mutations are particularly prone to resistance problems under the selective pressure of pesticides, resulting in failure in the prevention and control of crop diseases, and there are few alternative pesticides. Therefore, discovering new pesticide targets and developing selective pesticides with new modes of action are major needs for managing resistance and ensuring sustainable prevention and control of pests.

[0003] Through decades of basic research on fungicide toxicology and resistance, the inventors of this patented technology have deeply realized that the interaction between ligand small molecule compounds and receptor proteins is specific and precise. That is, small molecule compounds can only bind to sites of certain structures of specific protein molecules, and the binding force with the site depends on the fine structure of the ligand and receptor. The single amino acid difference in the drug sensitivity site of the target protein of different species determines the selectivity of the ligand small molecule compound, and the slight change or optimization of the structure of the small molecule compound determines its binding mode and binding force with the receptor protein and the rate of cell absorption. Therefore, the fine structure of the small molecule compound determines its antibiotic spectrum and biological activity.

[0004] The inventor of this patent technology has found through toxicological research on drug-target protein interaction that there is a transmembrane motor protein with important life functions in the important plant pathogenic fungus Fusarium graminearum, and the key site amino acids that bind to small molecules are significantly differentiated in animals, plants and microorganisms, providing patentability for the creation of safe and efficient selective targeted inhibitors. The inventor of this patent has found through computer simulation screening that pyrrolinedione small molecule compounds can interact with the functional sites of proteins based on the structural characteristics of different plant pathogenic protein drug sensitivity sites. By replacing the H at the 1, 3, and 4 positions of pyrrole with phenyl, alkyl and halogen alone or in different combinations, it was found that the introduction of phenyl at the 1 position can improve the antibacterial activity. Further, groups such as -Cl, -Br, -F, -CF3, -CH3, -C2H5, -n-C3H7, -i-C3H7, -NH2, -NO2, and CN were introduced on the benzene ring to optimize the structure and measure the antibacterial spectrum and biological activity. Compounds with ethyl groups introduced at the 2nd and 6th positions of the benzene ring and methyl groups introduced at the 4th position were found. Their lipidic and water-soluble properties are conducive to the absorption of compounds by fungi, oomycetes, and bacteria, and the antibacterial spectrum is maximized. The antibacterial activity is significantly higher than that of other derivatives. A pyrrolinedione compound 1-(2,6-diethyl-4-methylbenzene)-1H-pyrrole-2,5-dione (hereinafter referred to as "a pyrrolinedione compound") with a novel molecular structure and strong broad-spectrum fungicidal activity against plant pathogenic bacteria, fungi, and oomycetes was invented.

[0005] The inventor of this patented technology used 11 pathogenic fungi, including Fusarium graminearum, which causes fusarium graminearum of wheat and other crops, Magnaporthe grisea of ​​rice blast, F. moniliforme of rice seedling disease, Setosphaeriaturcica of corn leaf spot, Bipolaris madis of corn leaf spot, Botrytis cinerea of ​​gray mold of fruits and vegetables, Glomerella cingulata of anthracnose, Alternaria solani of early blight, Sclerotiniasclerotiorum of sclerotinia, Didymella bryoniae of watermelon vine blight, Cercosporaarachidicola of peanut brown spot, Phytophthora infestans, which causes late blight of potato and tomato, Phytophthora soja of soybean, Phytophthora capsici of pepper, Phytophthora parasitica of tobacco black shank, Peronophthora litchii, Plasmopara viticola of grape downy mildew, Pseudoperonospora cubensis of cucumber downy mildew and Pythium sp. of rice seedling rot, and seven plant pathogenic bacteria, including Xanthomonas oryzae that causes rice white leaf blight, X.oryzicola of rice bacterial leaf streak, Pseudomonas solanacearum of tomato bacterial wilt, X.citri of citrus canker, Erwinia carotovora of Chinese cabbage soft rot, Erwinia amylovora of amylovora and Acidovorax citrulli of fruit spot, were used as materials to determine the antibacterial spectrum and antibacterial activity of the above-mentioned pyrrolinedione compounds, and the compounds were processed into fungicide preparations such as suspension concentrate, water-dispersible granules and emulsifiable concentrates, which were used in the prevention and control experiments of different plant diseases, thereby completing the technical invention. Summary of the invention

[0006] The purpose of the present invention is to provide a pyrrolinedione compound.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned pyrrolinedione compound.

[0008] Another object of the present invention is to provide an agricultural fungicide containing the above-mentioned pyrrolinedione compound as an effective ingredient.

[0009] Another object of the present invention is to provide the use of the above-mentioned pyrrolinedione compound or an agricultural fungicide with the above-mentioned pyrrolinedione compound as an effective ingredient in preventing and controlling plant diseases.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a pyrrolinedione compound, wherein the pyrrolinedione compound has a structure shown in Formula I:

[0012]

[0013] The present invention provides a method for preparing a pyrrolinedione compound according to the above technical solution, characterized in that it comprises the following steps:

[0014] Compound II, maleic anhydride, a catalyst and an organic solvent are mixed and reacted to obtain a pyrrolinedione compound having a structure shown in formula (I), wherein the compound II has a structure shown in formula II:

[0015]

[0016] Preferably, the molar ratio of the compound II to maleic anhydride is 1:0.95-1.20; the catalyst is selected from one or more of methanesulfonic acid, p-toluenesulfonic acid or benzenesulfonic acid; and the organic solvent is selected from one or more of benzene, toluene, xylene and cyclohexane.

[0017] The above technical solution is described in that compound II, maleic anhydride, a catalyst and an organic solvent are mixed and reacted, specifically, a dehydration reaction is carried out by heating and refluxing, and the reaction is completed when the amount of dehydration reaches the theoretical value, and the product system is cooled, and then washed and dried in sequence to obtain a crude product. In the present invention, the washing liquid used for the washing is preferably a saturated NaHCO3 solution and distilled water in sequence; the number of washings with a saturated NaHCO3 solution is preferably 2 to 4 times, and the main function is to remove the residual dehydration catalyst; the number of washings with distilled water is preferably based on washing to neutrality. In the present invention, the desiccant used for the drying is anhydrous Na2SO4 or anhydrous MgSO4, preferably anhydrous Na2SO4; after the drying, the present invention preferably filters the obtained material, collects the feed liquid, and performs reduced pressure distillation to remove the solvent to obtain a crude product.

[0018] After obtaining the crude product, the present invention performs recrystallization or column chromatography separation and purification on the crude product to obtain a pyrrolinedione compound having a structure shown in Formula I. The crude product of the present invention is specifically a solid crude product, and the solid crude product can be recrystallized or separated by column chromatography. In the present invention, the recrystallization reagent used for the recrystallization is preferably a mixed reagent prepared by petroleum ether and ethyl acetate in a volume ratio of 100:0 to 100; after the recrystallization, it is preferably further included to filter, collect the filter cake and dry it, so as to obtain a pyrrolinedione compound having a structure shown in Formula I. In the present invention, the eluent used for column chromatography separation is preferably a mixed reagent prepared by petroleum ether and ethyl acetate in a volume ratio of 100:0 to 100. In the present invention, after the column chromatography separation, it is preferably further included to: remove the solvent from the obtained eluent by reduced pressure distillation to obtain a pyrrolinedione compound having a structure shown in Formula I; the pyrrolinedione compound provided by the present invention is a yellow, light yellow or white solid.

[0019] The present invention provides an agricultural fungicide, which is composed of an active ingredient and an auxiliary material; the active ingredient is a pyrrolinedione compound described in the above technical solution. The auxiliary material is preferably an auxiliary material acceptable in agricultural production, and more preferably includes one or more of a carrier, a solvent, a cosolvent, a surfactant, a dispersant, an emulsifier, a wetting agent, a suspending agent, a thickener, a stabilizer, a defoamer, an antifreeze agent, and a synergist.

[0020] The content of the active ingredient in the agricultural fungicide described in the above technical solution ranges from 3 to 95% (by weight).

[0021] The agricultural fungicide described in the above technical solution has a dosage form selected from at least one of emulsifiable concentrates, suspensions, suspoemulsions, aqueous solutions, aqueous emulsions, microemulsions, powders, wettable powders, soluble powders, granules, water-dispersible granules, oil suspensions, smoke agents, and baits. The agricultural fungicide described in the above technical solution is prepared according to the method described in Shen Jinliang, editor-in-chief, "Pesticide Formulation Processing and Management", China Agricultural Press, Beijing (2002), and Wang Kaiyun, editor-in-chief, "Pesticide Formulation", China Agricultural Press, Beijing (2002).

[0022] The present invention provides the use of a pyrrolinedione compound or an agricultural fungicide described in the above technical solution in preventing and controlling plant diseases.

[0023] Preferably, the plant diseases include diseases caused by one or more of pathogenic fungi, pathogenic oomycetes and pathogenic bacteria.

[0024] Preferably, the plant diseases are caused by pathogenic fungi such as Fusarium, Magnaporthe, Rhizoctonia, Setosphaeria, Bipolaris, Botrytis, Glomerella, Alternaria, Sclerotinia, Didymella, Cercospora, Puccinia, Uromyces, Phakopsora, etc.; diseases caused by pathogenic fungi such as Phytophthora. Diseases caused by pathogenic oomycetes such as Phytophthora, Sclerophthora, Sclerospora, Plasmopara, Peronophythora, Pseudoperonospora, Peronospora, Bremia, Paraperonosporora, Albugo, Pythium; plant diseases caused by pathogenic bacteria such as Xanthomonas, Pseudomonas, Erwinia. For example, but not limited to, wheat ergot, wheat rust, wheat powdery mildew, wheat stem base rot, rice blast, rice seedling blight, rice sheath blight, seedling bacterial wilt, rice white leaf blight, rice bacterial streak, corn major blight, corn minor blight, corn rust, corn stem base rot, citrus and kiwifruit canker, fruit and vegetable gray mold, cruciferous crop sclerotinia, peanut brown spot, watermelon vine blight, potato and tomato late / early blight, pepper blight, Chinese cabbage soft rot, soybean blight, soybean rust, litchi blight, cucumber downy mildew, cucumber angular leaf spot, vegetable downy mildew, grape downy mildew and anthracnose and powdery mildew of various fruit and vegetable crops, etc.

[0025] Preferably, the plants include one or more of vegetables, fruits, grain crops, oil crops, garden crops, Chinese medicinal materials, lawns and other plants.

[0026] The present invention provides a technical solution for determining the antibacterial spectrum and antibacterial activity of a pyrrolinedione compound or an agricultural fungicide as described in the above technical solution.

[0027] Specifically, using the plant pathogenic fungus materials stored and temporarily collected in this laboratory, the germination inhibition activity of the pyrrolinedione compound or the agricultural fungicide on the conidia of 11 pathogenic fungi, including wheat fusarium graminearum, rice blast fungus Magnaporthe grisea, rice seedling pathogen F. moniliforme, corn leaf blight fungus Setosphaeriaturcica, corn leaf blight fungus Bipolaris madis, gray mold fungus Botrytis cinerea of ​​fruits and vegetables, anthracnose fungus Glomerella cingulata, early blight fungus Alternaria solani, sclerotinia sclerotiorum, watermelon vine blight fungus Didymella bryoniae, and peanut brown spot fungus Cercosporaarachidicola, was determined; the inhibition activity of the pyrrolinedione compound or the agricultural fungicide on the conidia of potato and tomato late blight fungus Phytophthora infestans, soybean blight fungus Phytophthora soja, pepper blight fungus Phytophthora capsici, tobacco black shank fungus Phytophthoraparasitica, litchi blight fungus Peronophthora litchii, grape downy mildew fungus Plasmopara viticola, cucumber downy mildew fungus Pseudoperonospora cubensis and rice seedling rot fungus Pythium sp.; the inhibitory activity of a pyrrolinedione compound or an agricultural fungicide on the growth of seven plant pathogenic bacteria, including rice white leaf blight fungus Xanthomonas oryzae, rice bacterial leaf streak fungus X.oryzicola, tomato bacterial wilt fungus Pseudomonas solanacearum, citrus canker fungus X.citri, Chinese cabbage soft rot fungus Erwiniacarotovora, amylovora Erwinia amylovora, and fruit spot fungus Acidovorax citrulli was determined.

[0028] Specifically, the biological activity of the pyrrolinedione compound or agricultural fungicide against plant pathogenic fungi and oomycetes is determined by the classical spore germination method. The pyrrolinedione compound or agricultural fungicide is dissolved in dimethyl sulfoxide (DMSO) or methanol to obtain a mother solution of the compound with a concentration of 10 mg / mL or 20 mg / mL, which is stored at 4°C for a short period of time. When used, it is diluted with tap water containing 1% orange juice to a drug content of 20.0, 6.0, 2.0, 0.6, 0.2, 0.06, 0 (CK) μg / mL. The prepared drug solution is mixed with a fungal conidia suspension or an oomycete sporangium suspension in a ratio of 1:1 to make the final drug concentration 10.0, 3.0, 1.0, 0.3, 0.1, 0.03, 0 (CK) μg / mL. Add 10 mL of a mixture of the drug solution and spores to each culture dish (φ90 mm), and culture them statically for 5-8 hours at a temperature suitable for spore germination of different pathogens. When the spore germination rate of the control treatment reaches 95%, investigate the minimum drug concentration (MIC) that completely inhibits spore / sporangium germination under a microscope.

[0029] The biological activity of the pyrrolinedione compound or agricultural fungicide on plant pathogenic bacteria is determined by turbidity method. The prepared drug solution is mixed with a bacterial suspension with a turbidity of 50 in a ratio of 1:1 to a final concentration of 10.0, 3.0, 1.0, 0.3, 0.1, 0.03, 0 (CK) μg / mL, and the initial turbidity is measured. Subsequently, the mixture is shaken at 28°C for 72 hours, and the minimum concentration (MIC) of the drug that completely inhibits bacterial growth is measured by a turbidometer.

[0030] The preparation of pathogen inoculum is to culture the pathogenic fungus on PDA solid medium plate or liquid medium according to relevant plant pathology literature, collect conidia, and prepare 1×10 5 / mL conidia suspension was used for spore germination inhibition activity assay and inoculation test on plants.

[0031] The preparation of pathogenic oomycete inoculum is to collect grape or cucumber downy mildew diseased leaves, rinse them with tap water, place them on wet filter paper in a 15 cm diameter glass culture dish, cover them and culture them at 18°C ​​overnight, then wash the sporangium with 4°C tap water and prepare 10 5 / mL of sporangium suspension; or culture potato infestation pathogens and pepper phytophthora on CMA medium at 20°C, pick mycelium from the edge of the colony and inoculate it in Petri liquid culture medium for 2-3 days, filter with gauze and centrifuge at 1000r / min for 5 minutes, collect sporangium and resuspend it with sterile water, and adjust the sporangium concentration to 1×10 5 The suspension was prepared by mixing 100 μg / mL of sporangium for determination of sporangium germination inhibition activity and inoculation test on plants.

[0032] The preparation of pathogenic bacterial inoculum is to streak the bacteria on NA (beef juice agar) slant, wash off the pus with sterile water, and prepare a bacterial suspension with a turbidity of 50 for growth inhibition activity determination.

[0033] The antibacterial activity determination of a pyrrolinedione compound or an agricultural fungicide described in the above technical scheme refers to the literature such as "Pesticide Bioassay" edited by Shen Jinliang (China Agriculture Press, 2013, Beijing), and the antibacterial activity against other plant pathogens can also be determined according to these literatures.

[0034] The present invention provides an evaluation scheme for the application effect of the agricultural fungicide described in the above technical scheme in preventing and controlling plant diseases. The effective ingredient of the agricultural fungicide is 15-1000g per hectare, and the control effect of the agricultural fungicide on rice seedling blight, rice seedling bacterial wilt, rice blast, rice bacterial leaf streak, potato late blight, pepper blight and grape downy mildew is measured by seed treatment, soil treatment and spraying.

[0035] Specifically, the method for preventing and controlling rice seedling blight is to dilute the agricultural fungicide suspension containing the pyrrolidinedione compound with water to a solution containing 100, 200, or 400 mg / L of the drug, and use 150 L of the solution per hectare to soak the infected rice seeds. Clean water soaking is used as a control. After stirring, the seeds are soaked under natural conditions of 20 to 30°C for 48 hours, the rice seeds are taken out and sown in plastic pots, and cultured in a greenhouse for 21 days. The diseased plant rate and prevention effect of each treatment are investigated.

[0036] Specifically, for preventing and controlling bacterial wilt of rice seedlings, water-dispersible granules containing a pyrrolinedione compound are mixed with 225 kg of fine soil at an effective ingredient of 1000, 750, or 500 g per hectare, and applied when the seedlings have two leaves and one bud. After 10 days, the seedling mortality rate on each treatment seedbed is investigated and the prevention effect is calculated.

[0037] Specifically, the method for controlling rice blast is to dilute the water-dispersible granules of the fungicide containing the pyrrolinedione compound with water to 100, 200, or 400 mg / L of liquid, spray 750 L of liquid per hectare on the 3-leaf seedlings, spray and inoculate with a spore suspension of rice blast fungus 24 hours later, move to a greenhouse for cultivation under dark and moisturizing conditions overnight, and investigate the prevention effect after 10 days.

[0038] Specifically, the control of rice bacterial leaf streak disease is to dilute the fungicide emulsifiable concentrate containing the pyrrolidinedione compound with water to 250, 500, 800 mg / L solution, spray it at the booting stage at a rate of 750L solution per hectare, spray the second time after an interval of 7 days, and investigate the control effect 10 days after the second spray.

[0039] Specifically, the control of grape downy mildew is to dilute the suspension of the pyrrolinedione compound with water to 3.125, 6.25, 12.5, 25, 50 mg / L of liquid, spray the back of the grape leaves until the liquid is lost (750-1000 L of liquid sprayed per hectare), dry and spray inoculate downy mildew fungus spore suspension after 24 hours, move to 20-25 ° C to keep moist, light / dark (12 hours) alternating culture, and investigate the prevention effect after 7 days.

[0040] Specifically, for preventing and controlling potato late blight and pepper blight, the water dispersible granules of the pyrrolinedione compound are diluted with water to 125, 250, or 500 mg / L of liquid medicine, sprayed until the liquid is lost from the leaves, dried, and dripped with a suspension of pathogenic spores after 24 hours. After moisturizing overnight, the leaves are moved to a greenhouse (20-30° C.) for cultivation, and the control effect is investigated after 7 days.

[0041] The present invention can also be used to measure the control or treatment effects on other plant diseases using known drug treatment and pathogen inoculation methods.

[0042] The present invention provides a cross-resistance determination scheme between a pyrrolinedione compound or an agricultural fungicide and an existing fungicide as described in the above technical scheme.

[0043] Specifically, a wild strain of Botrytis cinerea that is sensitive to 20 μg / mL of a pyrrolinedione compound or an agricultural fungicide active ingredient is inoculated and cultured on a culture medium plate containing 20 μg / mL of the pyrrolidone compound or an agricultural fungicide active ingredient described in the above technical solution; or a wild strain of Phytophthora capsici that is sensitive to 20 μg / mL of a pyrrolidone compound or an agricultural fungicide active ingredient is inoculated and cultured and a strain that is multi-resistant to benzimidazoles, dicarboximides, aminopyrimidines, phenylpyrroles, QoIs and SDHIs is inoculated and cultured; and whether the drug resistance is exhibited is determined based on whether the drug resistance can be achieved.

[0044] The beneficial effects of the present invention are as follows: the pyrrolinedione compound or an agricultural fungicide with the pyrrolinedione compound as an effective ingredient described in the present invention has ideal broad-spectrum antibacterial activity against plant pathogenic fungi, oomycetes and bacteria, is safe for plants, is selective, and has adaptability to the environment, conforms to the development direction of original fungicides that are safe, efficient, low-toxic, low-residue, and compatible with the environment, and meets the requirements for ensuring environmental ecological safety; it has no cross-resistance with existing fungicides, and can be used in combination with a variety of fungicides to prevent and control drug-resistant diseases, and can especially solve the problem that there is no drug to treat multiple drug-resistant diseases; it has the advantages of high antibacterial activity, long effective period, small dosage per unit area, low use cost, long suitable medication time, high input-output ratio, good economic benefits, etc., and conforms to the requirements for promoting high-quality green development of the "three rural issues". DETAILED DESCRIPTION

[0045] The present invention will be better understood through the following examples.

[0046] Example 1: Preparation of a pyrrolinedione compound (Formula I)

[0047] The implementation steps of this embodiment are as follows:

[0048] 0.02 mol of the compound of formula II, 0.0202 mol of maleic anhydride and 0.0005 mol of methanesulfonic acid were mixed uniformly in 40 mL of toluene, and heated and refluxed under stirring to separate water using a heating and reflux water separation device; when the amount of water separation reached 0.02 mol, the reaction was terminated, and the obtained reaction solution was cooled, and saturated NaHCO3 solution was added to the reaction solution for washing at a volume ratio of 1:1.2 between the reaction solution and saturated NaHCO3 solution, and washed twice in total, and then washed with distilled water in the same manner. The reaction mixture was washed until neutral, and the neutral combined reaction solution was dried over anhydrous Na2SO4. The filtrate was collected after filtration and distilled under reduced pressure using a rotary evaporator to obtain a solid crude product. The solid crude product was recrystallized in a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 100:10, and then filtered and dried in an oven at a temperature of 50°C to obtain a light yellow solid pyrrolinedione compound as shown in Formula I, with a melting point of 126.0-127.5°C and a yield of 98%. Its nuclear magnetic resonance and high-resolution mass spectrum data are as follows:

[0049] 1 H-NMR (400MHz, CDCl3) δ: 7.01 (s, 2H, ArH), 6.87 (s, 2H, COCH=CHCO), 2.36 (q, J=7.6Hz, 4H, 2CH2), 2.35 (s, 3H, ArCH3), 1.12 (t, J=7.6Hz, 6H, 2CH3); HRMS (ESI): m / z calcdfor C 15 H 18 NO2([M+H] + ): 244.1332, found: 244.1332.

[0050] Example 2: Preparation of a pyrrolinedione compound (Formula I)

[0051] The implementation steps of this embodiment are as follows:

[0052] 0.10 mol of the compound of formula II, 0.10 mol of maleic anhydride and 0.002 mol of p-toluenesulfonic acid were mixed uniformly in 100 mL of benzene, and heated reflux water separation device was used to heat and reflux water separation under stirring conditions; when the amount of water separation reached 0.10 mol, the reaction was terminated, and the obtained reaction solution was cooled, and saturated NaHCO3 solution was added to the reaction solution in a volume ratio of 1:1 to saturated NaHCO3 solution for washing, and washed 3 times in total, and then washed with distilled water in the same manner. The reaction mixture was washed to neutrality, and the neutral combined reaction solution was dried over anhydrous Na2SO4. The filtrate was collected after filtration and distilled under reduced pressure using a rotary evaporator to obtain a solid crude product. The solid crude product was recrystallized in a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 100:8, and then filtered and dried in an oven at a temperature of 55°C to obtain a white solid pyrrolinedione compound with a melting point of 126.2-127.1°C and a yield of 99%. Its nuclear magnetic resonance and high-resolution mass spectrometry data were the same as the results measured in Example 1.

[0053] Example 3: Determination of the antibacterial spectrum and bactericidal activity of a pyrrolinedione compound

[0054] According to the above technical scheme, the biological activity of a pyrrolinedione compound against plant pathogenic fungi and oomycetes is determined by the classic spore germination method. A pyrrolinedione compound provided by the present invention is dissolved in dimethyl sulfoxide (DMSO) or methanol to obtain a pharmaceutical mother solution with a concentration of 10 mg / mL or 20 mg / mL of the compound, and diluted with tap water containing 1% orange juice to contain 20.0, 6.0, 2.0, 0.6, 0.2, 0.06, 0 (CK) μg / mL of the drug. The prepared drug solution is mixed with a fungal conidia suspension or an oomycete sporangium suspension in a ratio of 1:1 to make the final drug concentration 10.0, 3.0, 1.0, 0.3, 0.1, 0.03, 0 (CK) μg / mL. Add 10 mL of a mixture of the drug solution and spores to each culture dish (φ90 mm), and culture them statically for 5-8 hours at a temperature suitable for spore germination of different pathogens. When the spore germination rate of the control treatment reaches 95%, investigate the minimum drug concentration (MIC) that completely inhibits spore / sporangium germination under a microscope.

[0055] The results showed that the pyrrolinedione compound had a minimum concentration (MIC) of 100% inhibition rate on the conidia germination of Fusarium graminearum, Fmoniliforme, Magnaporthe grisea, Botrytis cinerea, Glomerella cingulata, Sclerotinia sclerotiorum and Didymella bryoniae and on the sporangium germination of Phytophthorainfestans, Phytophthora capsici, Phytophthoraparasitica, Peronophthora litchii and Plasmopara viticola; the MIC value of the conidia germination of Cercospora arachidicola, the sporangium germination of Phytophthora soja, Pythium sp. and Pseudoperonospora cubensis was 3.0 μg / mL; the MIC value of the conidia germination of Cercospora arachidicola, the sporangium germination of Phytophthora soja, Pythium sp. and Pseudoperonospora cubensis was 3.0 μg / mL; the MIC value of the sporangium germination of Setosphaeria turcica, Bipolaris madis and Alternaria The MIC of conidia germination of solani was 10.0 μg / mL. Under the treatment of 0.03 μg / mL, except for Setosphaeria turcica, Bipolaris madis and Alternaria solani, the conidia germination rates of other fungi and sporangium germination rates of oomycetes were all lower than 50%, that is, the LD of this compound against most pathogenic fungi and oomycetes was 50 Less than 0.03μg / mL.

[0056] The bacterial suspension with a turbidity of 50 was mixed with different concentrations of drug solution in a ratio of 1:1, so that the final drug concentration was 10.0, 3.0, 1.0, 0.3, 0.1, 0.03, 0 (CK) μg / mL. The suspension was placed on a shaker (180 r / min) and cultured for 3 days, and the minimum inhibitory concentration (MIC) was determined by turbidity. The results showed that the minimum inhibitory concentration MIC value of the pyrrolinedione compound described in the present invention on the growth of 7 plant pathogenic bacteria such as Xanthomonasoryzae, X.oryzicola, Pseudomonas solanacearum, X.citri, Erwinia carotovora, Erwinia amylovora, and Acidovorax citrulli was 1 μg / mL, showing a strong bactericidal effect.

[0057] The antibacterial activity test results of a pyrrolinedione compound described in the present invention show that the compound has strong broad-spectrum fungicidal activity against 26 representative plant pathogenic fungi, oomycetes and bacteria tested, indicating that the target of the compound is conservative among different pathogenic microorganisms. A pyrrolinedione compound described in the present invention is processed into a fungicide preparation according to the method described in this specification, that is, an agricultural fungicide with the pyrrolinedione compound as an active ingredient is obtained, and the control effect on some common diseases is further measured on plant tissues or plant seedlings, or the efficacy test is carried out in the field, and compared with the currently commonly used fungicides, and the safety to plants is determined at the same time.

[0058] Example 4: Preparation of fungicide suspension (20% SC or 50% SC)

[0059] According to the effective content of the preparation to be processed, based on the weight of the effective ingredient, weigh a pyrrolidinedione compound (with a particle size of 300 mesh), 1% of a wetting agent (specifically sodium dodecyl sulfate), 3% of a dispersing suspending agent (specifically NNO), 0.2% of a viscosity increasing agent (specifically xanthan gum), 5% of a stabilizer (specifically bentonite), 3% of an antifreeze agent (specifically polyethylene glycol 400), and 0.1% of a defoaming agent (specifically lauric acid), and add water to 100% to obtain a suspension concentrate with an effective ingredient content of 20%, i.e., a mixture of 20wt% SC;

[0060] Weigh 50% of a pyrrolidinedione compound (with a particle size of 300 mesh), 1.5% of a water-soluble wetting agent (specifically Nongru 100), 5% of a dispersing suspending agent (specifically sodium salt of ethylenediaminetetraacetic acid), 0.5% of a viscosity increasing agent (specifically gum arabic), 15% of a stabilizer (specifically bentonite), 8% of an antifreeze agent (specifically urea), and 0.2% of a defoaming agent (specifically sulfonamide), and add water to 100% to obtain a suspension concentrate with an active ingredient content of 50%, i.e., a mixture of 50wt% SC;

[0061] The above two mixtures are mixed and prepared into slurry respectively, and then ground by sand mill for 3-4 times, and finally the technical indicators such as pH value, fluidity and wettability are adjusted to obtain fungicide preparations with active ingredient content of 20wt% SC or 50wt% SC respectively.

[0062] The sample quality was tested according to the NY / T 4014-2021 standard method. The results showed that the relative error of the active ingredient content was ±0.1~0.5%, there was no stratification and precipitation after being placed indoors for 30 days, and it could maintain a stable suspension state when diluted with water. The effective suspension rate after standing still for 1 hour was greater than 90%.

[0063] Example 5: Preparation of fungicide water dispersible granules (10% WG or 25% WG)

[0064] According to the effective content of the preparation to be processed, based on the weight of the effective ingredient, 10% of a pyrrolinedione compound, 10% of an auxiliary agent (specifically, pull-opening powder), 10% of a disintegrant (specifically, 4:1 ammonium sulfate + sodium chloride), and 70% of a carrier (specifically, white carbon black) are weighed to prepare a water-dispersible granule with an effective ingredient content of 10%, i.e., a mixture of 10wt% WG;

[0065] Weighing 25% of a pyrrolidinedione compound, 10% of an auxiliary agent (a mixture of 5% of a naphthalenesulfonate dispersant GY-D10, 2% of sodium butylbenzenesulfonate, and 3% of urea), 20% of a disintegrant (specifically ammonium sulfate and sucrose in a mass ratio of 3:1), and 45% of a carrier (specifically white carbon black) to obtain a mixture of 25% water-dispersible granules containing an active ingredient, i.e., 25wt% WG;

[0066] The above two mixtures were respectively pulverized by air flow, and the mixtures with particle sizes below 10 pm were collected and successively granulated by extrusion and dried to obtain fungicide preparations with active ingredient contents of 10 wt% WG or 25 wt% WG, respectively.

[0067] The sample quality was tested according to the HG / T 4463-2012 standard method. The results showed that the above two types of 10wt% WG and 25wt% WG met the quality requirements.

[0068] Example 6: Preparation of fungicide emulsifiable concentrate (10% EC w / v)

[0069] Weigh 10% of a pyrrolidinedione compound by weight of the dosage form and dissolve it in about 2 / 3 of the total volume of No. 100 solvent oil, add 10% w / v of an emulsifier (a mixture of 5% Nongru 600 and 5% Tween 40), and then make up to the set volume with No. 100 solvent oil to obtain a 10% EC (w / v) fungicide preparation.

[0070] The sample quality was tested according to the GB / T 1540-2002 standard method, and the results showed that the above 10% EC w / v met the quality requirements.

[0071] Example 7: Experiment on the Control of Rice Bakanae Disease (Fusarium fujikuroi) by Agricultural Bactericides

[0072] The implementation method of this test example is as follows:

[0073] The pyrrolinedione compound of the present invention is processed into 20% SC; the control agent cyproconazole 25% SC (provided by Jiangsu Pesticide Research Institute Co., Ltd.)

[0074] Experimental conditions: greenhouse pot test;

[0075] Test method: The above test preparation samples were diluted with tap water to an active ingredient content of 100 mg ai. / L, 200 mg ai / L and 400 mg ai / L respectively, and 150 L of solution (15-60 g ai / ha) was used per hectare to soak the infected rice seeds. After soaking the seeds at room temperature (20-30°C) for 48 hours, they were germinated at 25-30°C for 48 hours, sown in plastic pots, and placed in a greenhouse for further cultivation for 21 days. The incidence of the seedlings was checked.

[0076] Prevention effect % = [1-(incidence rate of treated seedlings ÷incidence rate of blank control seedlings)] × 100.

[0077] Test results: 21 days after seeding, the cumulative disease rate of seedlings with bak-nai disease reached 16.4%. The 100, 200 and 400 mg / L solution of 20% SC, a pyrrolidine dione compound, was tested for seed soaking. The control effect of bak-nai disease was 86.7%, 93.9% and 98.8%, respectively, which was significantly higher than the 80.4%, 84.8% and 88.1% control effect of the control agent at the same concentration. In addition, all seeds treated with seed soaking germinated and emerged normally, and no phytotoxicity was observed.

[0078] Example 8: Experiment on the Control of Rice Seedling Bacterial Wilt by Agricultural Bactericides

[0079] The implementation method of this test example is as follows:

[0080] The pyrrolinedione compound of the present invention is processed into 10% WG; the control agent is 4% metalaxyl-M, fludioxonil, and azoxystrobin SC (produced by Anhui Fengle Agrochemical Co., Ltd., purchased online).

[0081] Experimental conditions: field seedbed test; rice variety "Yinliangyou 836".

[0082] Test method: 10% WG samples of the test preparation were used at 1000, 750, and 500 g ai per hectare, and the control agent was used at a dosage of 750 g of active ingredient per hectare, respectively, and mixed with 450 kg of fine soil to prepare "poisoned soil". When the seedlings had two leaves and one heart, 45 g of poisoned soil was spread per square meter of the seedling bed. After 10 days, the mortality rate of seedlings was investigated and the control effect was calculated.

[0083] Prevention effect % = [1-(death rate of treated seedlings ÷ death rate of blank control seedlings)] × 100.

[0084] Test results: The seedling death rate of the control group of the unfertilized fine soil was 14.34%. The control effects of 1000, 750 and 500g ai / ha soil treatments of 10% WG of a pyrrolidine dione compound as an active ingredient on bacterial wilt were 96.4%, 89.6% and 80.3%, respectively, which were significantly higher than the 73.3% control effect of the control agent 750g ai / ha soil treatment. The seedlings of all soil treatments grew normally without any pesticide damage.

[0085] Example 9: Experiment on the Control of Rice Blast by Agricultural Fungicide

[0086] The implementation method of this test example is as follows:

[0087] The test was conducted on rice blast control using 10% and 25% fungicide water dispersible granules containing a pyrrolinedione compound as an active ingredient and 40% rice blast-in-one emulsifiable concentrate (purchased online) produced by Shandong Tianrong Biotechnology Co., Ltd. as a control agent.

[0088] Experimental conditions: Greenhouse pot test;

[0089] Test method: Weigh the above-mentioned fungicide samples and dilute them to 400mg / L, 200mg / L, and 100mg / L of liquid according to the effective content. Dilute the control agent, pyrochlorein, to 500mg / L and 250mg / L of water. Spray the above-mentioned liquid on 4 pots of rice seedlings (Sujing 4699, a rice variety susceptible to rice blast) at the 3-leaf stage. After 24 hours, spray the conidia of Magnaporthe grisea on the leaves. After 12 hours at 25℃, keep moist and dark conditions, move to a greenhouse (20-30℃) for culture, and determine the effect of the compound on preventing and controlling rice blast. 10 days after inoculation, investigate the number of rice blast spots on the second and third leaves from the bottom, and calculate the control effect compared with the control.

[0090] Protection efficiency % = [1-(average number of lesions on treated leaves ÷ average number of lesions on blank control leaves)] × 100.

[0091] Test results: See Table 1 for the results.

[0092] Table 1: Effects of agricultural fungicides on rice blast at the seedling stage

[0093]

[0094] The results show that the agricultural fungicide with the pyrrolinedione compound as the active ingredient has a better preventive effect on rice blast than the control agent, and its effect is positively correlated with the effective dose of the treatment and has nothing to do with the content of the preparation. No adverse effects were observed on the growth of the seedlings in each treatment.

[0095] Example 10: Experiment on the Control of Rice Bacterial Leaf Streak Disease by Agricultural Fungicides

[0096] The implementation method of this test example is as follows:

[0097] A pyrrolinedione compound of the present invention was processed into 10% emulsifiable concentrate (EC) according to the specification, and a control agent 20% zinc thiazole SC (provided by Zhejiang Xinnong Co., Ltd.) was used.

[0098] Experimental conditions: field; rice variety: Yinliangyou 836; growth period: booting stage

[0099] Test method: Dilute the above test preparation samples with tap water to the active ingredient content of 250mg ai. / L, 500mg ai / L and 800mg ai / L, spray 750L of solution per hectare, i.e. 187.5-600g ai / ha, and spray water for control. Spray the second time 7 days later. Check the leaf disease incidence of the sword leaf and the second leaf after 10 days. The control effect calculation method is:

[0100] Prevention effect % = [1-(disease incidence rate of treated leaves ÷ disease incidence rate of blank control leaves)] × 100.

[0101] The test results show that 7 days after the first application, about 15% of the leaves in the control group began to become diseased. Ten days after the second application, the diseased leaf rate in the control treatment area reached 78.54%, and the diseased leaf rates in the treatment areas of 250mg ai / L, 500mg ai / L and 800mg ai / L of the agricultural fungicide of the present invention were 7.68%, 2.45% and 0.24%, respectively. The control effects were 90.2%, 96.9% and 99.7%, respectively. In addition, it was noted that the number of lesions on the diseased leaves in the treatment area was significantly less than that in the control area.

[0102] Example 11: Experiment on the Control of Grape Downy Mildew by Agricultural Fungicides

[0103] The implementation method of this test example is as follows:

[0104] A. Test compound:

[0105] The 20% pyrrolinedione fungicide suspension (SC) prepared in Example 3 of the present invention was diluted with water to 3.125, 6.25, 12.5, 25.0, 50.0, and 500 mg / L of liquid.

[0106] The control agent was 10% Zengweiying Green OD (produced by DuPont). It was diluted with water to 3.125, 6.25, 12.5, and 25.0 mg / L.

[0107] 50% methacryloyl morpholine WP (produced by Tianjin Hanbang Plant Protection Agent Co., Ltd., purchased online), 25.0, 50.0, 100.0, 200.0 mg / L solution.

[0108] B. Test methods and conditions:

[0109] Take the 3rd to 7th healthy leaves from the last leaf of the greenhouse potted grape vine (variety: Rose Fragrance), rinse with tap water and dry, then spray different concentrations of test solution on the back or front until the leaf surface is completely wet (equivalent to spraying 1500kg solution or 4.69g ai~750g ai / ha per hectare). After 24 hours, spray the back of the leaf with a suspension of sporangium of grape downy mildew; move to an artificial climate room and culture for 7 days under the conditions of light / dark (12h), temperature 20~25℃, and relative humidity RH90%~95%, investigate the disease situation, and calculate the prevention effect according to the proportion of leaf disease area (sporangium production area).

[0110] Protection efficiency % = [1-(average lesion area percentage of treatment ÷ average lesion area percentage of blank control)] × 100.

[0111] C. Test results:

[0112] The test results are listed in Table 2

[0113] Table 2: Test results of agricultural fungicides for controlling grape downy mildew

[0114]

[0115] The results show that the agricultural fungicide with a pyrrolinedione compound as an active ingredient has a good preventive effect on grape downy mildew, and all leaves treated with 25, 50 and 500 mg / L liquid medicines are free from disease. Moreover, the treatment on the front side of the leaf also has a high preventive effect on the inoculation on the back side, indicating that it has the activity of spreading from the front side of the leaf to the back side.

[0116] Example 12: Experiment on the Control of Potato Late Blight and Pepper Blight by Agricultural Fungicides

[0117] The implementation method of this test example is as follows:

[0118] A. Test Agents

[0119] 20% and 50% pyrrolinedione compound suspension (SC) fungicide preparations prepared in Example 3 of the present invention.

[0120] The control agent was 50% oxadiazine WP (produced by Tianjin Hanbang Plant Protection Agent Co., Ltd., purchased online).

[0121] B. Greenhouse potted plant test conditions

[0122] Potato and pepper seedlings at the 5-leaf stage, 2 plants per pot; cultured in a glass solar greenhouse at a temperature of 20-30℃.

[0123] C. Test methods

[0124] I. Preparation of test solution:

[0125] The suspensions of the above compounds were weighed separately, diluted with water to active ingredient concentrations of 500 mg / L, 250 mg / L and 125 mg / L, to prepare drug solutions of three treatment concentrations.

[0126] II. Test operation method:

[0127] Spray 3 pots of potato seedlings and pepper seedlings with 3 treatment concentrations of each compound using a throat sprayer until the leaves have the liquid flowing. After 24 hours, the prepared sporangium suspension of potato late blight (Phytophthorainfestans) and pepper phytophthora capsici (P. capsici) was drip-inoculated at 10 μl / each inoculation point, and a total of 24 leaflets were inoculated (8 leaves were inoculated per pot of seedlings). After drying, the pots were placed in a greenhouse and covered with black plastic cloth to keep them moist overnight, and then the plastic cloth was removed and continued to be cultured for 7 days. The number and length of lesions were investigated, and the control effect was calculated by the following formula:

[0128] Protection effect % = [1-(average lesion length of the treated group ÷ average lesion length of the blank control group)] × 100.

[0129] D. Test results:

[0130] The test results are listed in Table 3.

[0131] Table 3: Test results of agricultural fungicides against potato late blight and pepper blight

[0132]

[0133] The test results show that the agricultural fungicide of the present invention has an ideal preventive effect on potato late blight and pepper blight, and the control effect is proportional to the effective ingredient dosage of the test and has nothing to do with the content of the processed preparation. The agricultural fungicide of the present invention with a pyrrolidinedione compound as an effective ingredient has a significantly better control effect on the two diseases than the control agent.

[0134] Example 13: Determination of cross-resistance between a pyrrolidinedione compound and existing fungicides

[0135] Test agent samples: a pyrrolinedione compound of the present invention, and the original drugs of fungicides with different action mechanisms, including boscalid, carbendazim, pyrimethanil, procymidone, myclobutanil, fludioxonil, metalaxyl, dimethomorph, and fluthiazolinone, which are stored in the laboratory.

[0136] Test method: A pyrrolinedione compound of the present invention and a commonly used fungicide original drug are dissolved in methanol and carbendazim is dissolved in 0.1 mol hydrochloric acid to prepare a 10 mg / mL stock solution, which is then diluted to 200 μg / mL with sterile water, and then diluted with PDA culture medium to prepare a drug-containing culture medium plate with a final concentration of 20 μg / mL.

[0137] The strains used for the determination are the laboratory-preserved multi-resistant strain B5 of Botrytis cinerea resistant to boscalid, carbendazim, pyrimethanil, procymidone, azoxystrobin, and fludioxonil, and the wild sensitive strain B1, and the multi-resistant strain PcR of Phytophthora capsici resistant to azoxystrobin, metalaxyl, dimethomorph, and fluthiazolinone, and the wild sensitive strain PcS. Botrytis cinerea and Phytophthora capsici were cultured on drug-free PDA and lima bean culture plates, respectively. A 5mm diameter bacterial disk was prepared near the edge of the colony, and it was inoculated on each drug-containing plate and cultured at 25°C for 5 days. Those that can grow are recorded as "+", those that cannot grow are recorded as "-", and those that are not measured are recorded as " / ". The results are shown in Table 4. From the results of whether it can grow on the drug-containing culture medium, it can be seen that a pyrrolinedione compound of the present invention has no cross-resistance with existing fungicides, indicating that it has a new mechanism of action.

[0138] Table 4 Cross-resistance pattern between a pyrrolinedione compound and existing fungicides with different modes of action

[0139]

[0140] The above description is only a preferred implementation case of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of the technical solution of the present invention.

Claims

1. A pyrrolinedione compound, characterized in that: The pyrrolinedione compound has a structure shown in formula (I):

2. The method for preparing a pyrrolinedione compound according to claim 1, characterized in that: The following steps are involved: Compound II, maleic anhydride, a catalyst and an organic solvent are mixed and reacted to obtain a pyrrolinedione compound having a structure shown in formula (I), wherein the compound II has a structure shown in formula II:

3. The method for preparing a pyrrolinedione compound according to claim 2, characterized in that: The molar ratio of the compound II to maleic anhydride is 1:0.95-1.20; The catalyst is selected from one or more of methanesulfonic acid, p-toluenesulfonic acid or benzenesulfonic acid; The organic solvent is selected from one or more of benzene, toluene, xylene and cyclohexane.

4. An agricultural fungicide, characterized in that: The invention is composed of an effective ingredient and excipients, wherein the effective ingredient is a pyrrolinedione compound as claimed in claim 1.

5. Use of a pyrrolinedione compound according to claim 1 or an agricultural fungicide according to claim 4 in preventing and controlling plant diseases.

6. The use according to claim 5, characterized in that: The plant diseases include diseases caused by one or more of pathogenic fungi, pathogenic oomycetes and pathogenic bacteria.

7. The use according to claim 5 or 6, characterized in that: 15 to 1000 g of the active ingredient of the pyrrolinedione compound according to claim 1 or the agricultural fungicide according to claim 4 is used per hectare.

8. The use according to any one of claims 5 to 7, characterized in that: It can be applied as seed and soil treatment or spray.

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