A pyrroline dione compound, a preparation method thereof and a bactericide application

Abstract

The application provides a pyrroline dione compound and a preparation method and fungicide application thereof. The pyrroline dione compound is named 1-(2,6-diethyl-4-methylphenyl)-1H-pyrrole-2,5-dione, and a structural formula is shown as formula I: the pyrroline dione compound and an agricultural fungicide with 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, good in safety, has broad-spectrum and high-efficiency antibacterial activity, has no cross resistance with other existing fungicides, can be used for preventing and treating plant diseases of vegetables, fruits, food crops, oil crops, economic crops, garden crops and Chinese medicinal materials, and has good chemical protection and internal absorption treatment effects on resistant crop diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agrochemical technology, in particular to a pyrroline dione compound, a preparation method thereof and a fungicide application. BACKGROUND

[0002] Safe and efficient selective pesticides are indispensable production materials for modern agricultural production. However, due to the single action site of selective pesticides, the genetic variation of harmful organisms is prone to drug resistance problems. Many existing pesticides are known to have the same mode of action, and there is cross-resistance between them. Like plant pathogenic microorganisms with high reproductive rate and rapid variation, they are particularly prone to drug resistance problems under the selection pressure of pesticides, causing the failure of crop disease control, and there are few alternative pesticides. Therefore, discovering new targets for pesticides and developing new types of selective pesticides with different modes of action is a major demand for the sustainable control of harmful organisms and the prevention of drug resistance.

[0003] The inventors of the present patent technology have a deep understanding of the specific and precise interaction between ligand small molecule compounds and receptor proteins through decades of basic research on the toxicity and resistance of fungicides. That is, small molecule compounds can only bind to a specific protein molecule at a certain structural site, and the binding force with the site depends on the fine structure of the ligand and the receptor. The single amino acid difference in the drug-sensitive site of the target protein of different species determines the selectivity of the ligand small molecule compound, and the subtle changes 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 spectrum and biological activity.

[0004] The inventors of the present patent found, through toxicological research of drug-target protein interaction, that there is a transmembrane motor protein with important life function in the important plant pathogenic fungus Fusarium graminearum, and the key amino acid site for small molecule binding is significantly differentiated in animals, plants and microorganisms, providing patentability for creating safe and efficient selective targeted inhibitors. The inventors of the present patent found that pyrroline dione small molecule compounds can interact with the functional site of the protein through computer simulation screening based on the structural characteristics of the protein drug sensitivity site of different plant pathogenic fungi. By substituting H at positions 1, 3 and 4 of pyrrole with phenyl, alkyl and halogen alone or in different combinations, it was found that the introduction of phenyl at position 1 can improve the antibacterial activity. Further introducing -Cl, -Br, -F, -CF3, -CH3, -C2H5, -n-C3H7, -i-C3H7, -NH2, -NO2, CN and other groups on the benzene ring, structure optimization and antibacterial spectrum and biological activity determination were carried out, and it was found that the compound with ethyl introduced at positions 2 and 6 and methyl introduced at position 4 of the benzene ring has good lipophilicity and water solubility, which is beneficial to the absorption of the compound by fungi, oomycetes and bacteria, the antibacterial spectrum is maximized, and the antibacterial activity is significantly higher than that of other derivatives. A pyrroline dione compound 1-(2,6-diethyl-4-methylbenzene)-1H-pyrrole-2,5-dione (hereinafter referred to as "a pyrroline dione compound") with novel molecular structure and strong broad-spectrum bactericidal activity against plant pathogenic bacteria, fungi and oomycetes was invented.

[0005] The present inventors have determined the antibacterial spectrum and antibacterial activity of the above pyrroline dione compound using 11 kinds of pathogenic fungi, including Fusarium graminearum, which causes bunt of wheat and the like, Magnaporthe grisea, which causes rice blast, F. moniliforme, which causes sheath blight of rice, Setosphaeria turcica, which causes large spot of corn, Bipolaris madis, which causes small spot of corn, Botrytis cinerea, which causes gray mold of fruits and the like, Glomerella cingulata, which causes anthracnose, Alternaria solani, which causes early blight, Sclerotinia sclerotiorum, which causes sclerotinia, Didymella bryoniae, which causes gummy stem blight of watermelon, Cercospora arachidicola, which causes brown spot of peanut, 8 kinds of plant pathogenic oomycetes, including Phytophthora infestans, which causes late blight of potato and tomato, Phytophthora soja, which causes soybean blight, Phytophthora capsici, which causes pepper blight, Phytophthora parasitica, which causes black shank of tobacco, Peronophthora litchii, which causes litchi blight, Plasmopara viticola, which causes downy mildew of grape, Pseudoperonospora cubensis, which causes cucumber downy mildew, and Pythium sp., which causes seedling blight of rice, 7 kinds of plant pathogenic bacteria, including Xanthomonas oryzae, which causes bacterial leaf blight of rice, X. oryzicola, which causes bacterial leaf streak of rice, Pseudomonas solanacearum, which causes bacterial wilt of tomato, X. citri, which causes canker of citrus, Erwinia carotovora, which causes soft rot of Chinese cabbage, Erwinia amylovora, which causes fire blight of pear, and Acidovorax citrulli, which causes fruit spot, and the like, and have completed the present invention by conducting a control test of different plant diseases using the compound processed into a fungicide preparation such as a suspension concentrate, a water dispersible granule, and an emulsion. SUMMARY

[0006] An object of the present invention is to provide a pyrroline dione compound.

[0007] Another object of the present invention is to provide a method for preparing the above pyrroline dione compound.

[0008] Another object of the present invention is to provide an agricultural fungicide having the above pyrroline dione compound as an effective ingredient.

[0009] Another object of the present application is to provide the application of the above-mentioned pyrroline dione compound or the agricultural fungicide with the above-mentioned pyrroline dione compound as the effective component in the prevention and treatment of plant diseases.

[0010] In order to achieve the above-mentioned objects, the present application provides the following technical solutions.

[0011] The present application provides a pyrroline dione compound, which has the structure shown in formula I.

[0012]

[0013] The present application provides a preparation method of the above-mentioned pyrroline dione compound, which is characterized by comprising the following steps.

[0014] The compound II, maleic anhydride, a catalyst and an organic solvent are mixed and then reacted to obtain a pyrroline dione compound having the structure shown in formula (I), wherein the compound II has the 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 methyl sulfonic acid, p-toluenesulfonic acid or benzene sulfonic acid; and the organic solvent is selected from one or more of benzene, toluene, xylene and cyclohexane.

[0017] The compound II, maleic anhydride, a catalyst and an organic solvent are mixed and then reacted, specifically by heating and refluxing to perform a dehydration reaction, and the reaction is completed when the amount of dehydration reaches the theoretical value; then the product system is cooled, and then washed and dried in sequence to obtain a crude product. In the present application, the washing liquid used for washing is preferably saturated NaHCO3 solution and distilled water in sequence; the number of times of washing with the saturated NaHCO3 solution is preferably 2-4 times, and the main function is to remove the residual dehydration catalyst; and the number of times of washing with distilled water is preferably based on the washing until neutral. In the present application, the drying agent used for drying is anhydrous Na2SO4 or anhydrous MgSO4, and preferably anhydrous Na2SO4; after drying, the present application preferably filters the obtained material, collects the liquid and removes the solvent by vacuum distillation to obtain a crude product.

[0018] After obtaining the crude product, the present application purifies the crude product by recrystallization or column chromatography to obtain a pyrroline dione compound having the structure shown in Formula I. The crude product in the present application is specifically a solid crude product, which can be recrystallized or subjected to column chromatography. In the present application, the recrystallization reagent used in the recrystallization is preferably a mixed reagent prepared by mixing petroleum ether and ethyl acetate in a volume ratio of 100:0-100; and the recrystallization preferably further includes filtration, and drying of the filter cake to obtain a pyrroline dione compound having the structure shown in Formula I. In the present application, the eluent used in the column chromatography is preferably a mixed reagent prepared by mixing petroleum ether and ethyl acetate in a volume ratio of 100:0-100. In the present application, the column chromatography preferably further includes: removing the solvent from the eluate obtained by vacuum distillation to obtain a pyrroline dione compound having the structure shown in Formula I. The pyrroline dione compound provided in the present application is a yellow, light yellow or white solid.

[0019] The present application provides an agricultural fungicide composed of an effective component and an auxiliary material; the effective component refers to the pyrroline dione 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 thickening agent, a stabilizer, an antifoaming agent, an antifreezing agent, and a synergist.

[0020] The content of the effective component of the agricultural fungicide described in the above technical solution is 3-95% by weight.

[0021] The dosage form of the agricultural fungicide described in the above technical solution is at least one selected from the group consisting of an emulsifiable concentrate, a suspension concentrate, a suspoemulsion, a water agent, an aqueous emulsion, a microemulsion, a powder, a wettable powder, a soluble powder, a granule, a water dispersible granule, an oil suspension, a smoke agent, and a bait. The processing of the dosage form of the agricultural fungicide described in the above technical solution is prepared according to the methods described in Shen Jinliang, ed., Pesticide Dosage Form Processing and Management, China Agriculture Press, Beijing (2002), and Wang Kaiyun, ed., Pesticide Formulation Science, China Agriculture Press, Beijing (2002).

[0022] The present application provides the use of the pyrroline dione compound or the agricultural fungicide described in the above technical solution in the prevention and treatment of 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 disease is a disease caused by pathogenic fungi such as Fusarium, Magnaporthe, Rhizoctonia, Setosphaeria, Bipolaris, Botrytis, Glomerella, Alternaria, Sclerotinia, Didymella, Cercospora, Puccinia, Uromyces, Phakopsora, and the like; a disease caused by pathogenic oomycetes such as Phytophthora, Sclerophthora, Sclerospora, Plasmopara, Peronophythora, Pseudoperonospora, Peronospora, Bremia, Paraperonospora, and Albugo, Pythium, and the like; a plant disease caused by pathogenic bacteria such as Xanthomonas, Pseudomonas, Erwinia, and the like. For example, but not limited to, wheat scab, wheat rust, wheat powdery mildew, wheat foot rot, rice blast, rice sheath blight, rice sheath blight, rice seedling bacterial wilt, rice bacterial leaf blight, rice bacterial leaf streak, corn southern blight, corn northern leaf blight, corn rust, corn foot rot, citrus and kiwi canker, fruit and vegetable gray mold, cruciferous vegetable sclerotinia, peanut brown spot, watermelon gummy stem blight, potato and tomato late / early blight, pepper late blight, Chinese cabbage soft rot, soybean bacterial blight, soybean rust, litchi bacterial blight, cucumber downy mildew, cucumber angular leaf spot, vegetable downy mildew, grape downy mildew, and various fruit and vegetable crops anthracnose and powdery mildew, and the like.

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

[0026] The present application provides a pyrroline dione compound or an agricultural fungicide with the antibacterial spectrum and antibacterial activity determination technical solution of the above technical solution.

[0027] Specifically, using the plant pathogenic material preserved and temporarily collected in the laboratory, the pyrroline dione compound or the agricultural fungicide was tested for the inhibition activity on the conidium germination of 11 kinds of pathogenic fungi, including Fusarium graminearum, Magnaporthe grisea, F. moniliforme, Setosphaeria turcica, Bipolaris madis, Botrytis cinerea, Glomerella cingulata, Alternaria solani, Sclerotinia sclerotiorum, Didymella bryoniae and Cercospora arachidicola; the inhibition activity on the sporangium germination of 8 kinds of plant pathogenic oomycetes, including Phytophthora infestans, Phytophthora soja, Phytophthora capsici, Phytophthora parasitica, Peronophthora litchii, Plasmopara viticola, Pseudoperonospora cubensis and Pythium sp.; and the inhibition activity on the growth of 7 kinds of plant pathogenic bacteria, including Xanthomonas oryzae, X. oryzicola, Pseudomonas solanacearum, X. citri, Erwinia carotovora, Erwinia amylovora and Acidovorax citrulli.

[0028] Specifically, the biological activity of the pyrroline dione compound or the agricultural fungicide on plant pathogenic fungi and oomycetes is determined by the classical spore germination method. The pyrroline dione compound or the agricultural fungicide is dissolved in dimethyl sulfoxide (DMSO) or methanol to obtain a mother liquor of the compound at 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 contain 20.0, 6.0, 2.0, 0.6, 0.2, 0.06, 0 (CK) μg / mL of the drug. The above prepared liquid medicine is mixed with the fungal conidium suspension or oomycete sporangium suspension at a ratio of 1:1, so that the final drug concentration is 10.0, 3.0, 1.0, 0.3, 0.1, 0.03, 0 (CK) μg / mL. 10 mL of the mixture of the liquid medicine and the spores is added to each Petri dish (φ 90 mm), and incubated at different suitable temperatures for 5-8 hours. When the spore germination rate of the control treatment reaches 95%, the minimum concentration of the drug that completely inhibits spore / sporangium germination (MIC) is investigated under a microscope.

[0029] The biological activity of the pyrroline dione compound or the agricultural fungicide on plant pathogenic bacteria is determined by the turbidity method. That is, the above prepared liquid medicine is mixed with the bacterial suspension with a turbidity of 50 at 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 determined. Subsequently, the bacterial growth is completely inhibited at 28°C for 72 hours, and the minimum concentration of the drug that completely inhibits bacterial growth (MIC) is determined by a turbidimeter.

[0030] The preparation of the pathogenic fungus inoculum is to culture the pathogenic fungi on PDA solid medium plates or in liquid medium according to the relevant plant pathology literature, collect conidia, and adjust the conidium suspension to 1×10 5 / mL for spore germination inhibition activity determination and inoculation test on plants.

[0031] The preparation of the pathogenic oomycete inoculum is to wash the collected grape or cucumber downy mildew diseased leaves with tap water, place them on wet filter paper in a 15 cm diameter glass Petri dish, cover and incubate overnight at 18°C, then wash the sporangia with 4°C tap water, adjust the sporangium suspension to 10 5 / mL; or cultivate P. infestans and P. capsici on CMA medium at 20°C, respectively, after 3 days, pick the mycelium from the edge of the colony to inoculate in a Petri liquid culture medium and shake cultivate for 2-3 days, filter with gauze and centrifuge at 1000 r / min for 5 minutes, collect the sporangia and resuspend them with sterilized water, adjust the sporangium concentration to 1×10 5 / mL sporangium suspension for sporangium germination inhibition activity determination and inoculation test on plants.

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

[0033] The antibacterial activity determination of the pyrroline dione compound or the agricultural fungicide according to the technical solution is referenced from Shen Jinliang, editor, Pesticide Bioassay (China Agricultural Press, 2013, Beijing) and other documents, and the antibacterial activity on other plant pathogenic bacteria can also be determined according to these documents.

[0034] The application provides an evaluation scheme of the application effect of the agricultural fungicide in preventing and treating plant diseases. Specifically, the prevention and treatment of rice seedling bacterial leaf blight is that the fungicide containing the pyrroline dione compound is diluted to 250 mg / L, 500 mg / L and 800 mg / L of the pesticide solution by water, 750 L of the pesticide solution is sprayed per hectare at the booting stage, the second pesticide is sprayed after 7 days, and the prevention and treatment effect is investigated 10 days after the two pesticides.

[0035] Specifically, the prevention and treatment of rice seedling bacterial leaf blight is that the fungicide containing the pyrroline dione compound is diluted to 250 mg / L, 500 mg / L and 800 mg / L of the pesticide solution by water, 750 L of the pesticide solution is sprayed per hectare at the booting stage, the second pesticide is sprayed after 7 days, and the prevention and treatment effect is investigated 10 days after the two pesticides.

[0036] Specifically, the prevention and treatment of rice seedling bacterial leaf blight is that the fungicide containing the pyrroline dione compound is diluted to 250 mg / L, 500 mg / L and 800 mg / L of the pesticide solution by water, 750 L of the pesticide solution is sprayed per hectare at the booting stage, the second pesticide is sprayed after 7 days, and the prevention and treatment effect is investigated 10 days after the two pesticides.

[0037] Specifically, the prevention and treatment of rice seedling bacterial leaf blight is that the fungicide containing the pyrroline dione compound is diluted to 250 mg / L, 500 mg / L and 800 mg / L of the pesticide solution by water, 750 L of the pesticide solution is sprayed per hectare at the booting stage, the second pesticide is sprayed after 7 days, and the prevention and treatment effect is investigated 10 days after the two pesticides.

[0038] Specifically, the prevention and treatment of rice seedling bacterial leaf blight is that the fungicide containing the pyrroline dione compound is diluted to 250 mg / L, 500 mg / L and 800 mg / L of the pesticide solution by water, 750 L of the pesticide solution is sprayed per hectare at the booting stage, the second pesticide is sprayed after 7 days, and the prevention and treatment effect is investigated 10 days after the two pesticides.

[0039] Specifically, the prevention and treatment of grape downy mildew is to dilute the suspension of the pyrroline dione compound to 3.125, 6.25, 12.5, 25, 50 mg / L of liquid medicine with water, spray the back of grape leaves until the liquid medicine flows off (750-1000 L of liquid medicine is sprayed per hectare), dry, and spray spore cyst suspension of downy mildew after 24 hours, move to 20-25℃, and cultivate alternately under light / dark (12 hours), and investigate the control effect after 7 days.

[0040] Specifically, the prevention and treatment of potato late blight and pepper blight is to dilute the water dispersible granules of the pyrroline dione compound to 125, 250, 500 mg / L of liquid medicine with water, spray the leaves until the liquid medicine flows off, dry, and drop the spore cyst suspension of the pathogen after 24 hours, cultivate in a greenhouse (20-30℃) after keeping wet overnight, and investigate the control effect after 7 days.

[0041] The application can also be used to determine the prevention and treatment effect on other plant diseases by using known pesticide treatment and pathogen inoculation methods.

[0042] The application provides an interactive resistance determination scheme of the pyrroline dione compound or the agricultural fungicide with existing fungicides.

[0043] Specifically, the wild sensitive strain of Botrytis cinerea and the multi-resistant strain of benzimidazole, dioxime, aminopyrimidine, benzopyrrole, QoI and SDHI are inoculated and cultured on the medium plate containing 20 μg / mL of the pyrroline dione compound or the effective component of the agricultural fungicide, or the wild sensitive strain of pepper Phytophthora capsici and the strain resistant to benzamide, dimethomorph, QoI and oxysterol binding protein inhibitor are inoculated and cultured, and whether the resistance is shown is judged according to whether the growth is possible.

[0044] The pyrroline dione compound or the agricultural fungicide with the pyrroline dione compound as the effective component has ideal broad-spectrum antibacterial activity on plant pathogenic fungi, oomycetes and bacteria, is safe to plants, has selectivity, is suitable for the environment, meets the development direction of original fungicides with safety, high efficiency, low toxicity, low residue and environmental compatibility, meets the requirement of protecting the ecological safety of the environment, has no interactive resistance with existing fungicides, can be combined with various fungicides to prevent and treat resistant diseases, can solve the problem that no drug can be used to treat multiple resistant diseases, has high antibacterial activity, long effective period, low unit area dosage, low use cost, long suitable use time, high input-output ratio and good economic benefits, and meets the requirement of promoting the high-quality green development of "three agricultures". DETAILED DESCRIPTION

[0045] The present application can be better understood by the following examples.

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

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

[0048] 0.02 mol of the compound of Formula II, 0.0202 mol of maleic anhydride, and 0.0005 mol of methyl sulfonic acid were uniformly mixed in 40 mL of toluene, and heated to reflux under stirring using a heating reflux water trap; when the amount of water reached 0.02 mol, the reaction was stopped, the obtained reaction solution was cooled, and saturated NaHCO3 solution was added to the reaction solution for washing according to the volume ratio of the reaction solution to saturated NaHCO3 solution of 1:1.2, a total of 2 times, and then washed with distilled water in the same way until neutral, the obtained neutral combined reaction solution was dried with anhydrous Na2SO4, and 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 according to the volume ratio of 100:10, then filtered, and dried using an oven at a temperature of 50°C to obtain a light yellow solid pyrroline dione compound as shown in Formula I, with a melting point of 126.0-127.5°C, a yield of 98%, and the following nuclear magnetic resonance and high resolution mass spectrometry data:

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

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

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

[0052] The compound of formula II, 0.10 mol of maleic anhydride, and 0.002 mol of p-methylbenzenesulfonic acid are mixed uniformly in 100 mL of benzene, and heated to reflux under stirring using a heating reflux water trap; when the amount of water reflux reaches 0.10 mol, the reaction is ended, the obtained reaction solution is cooled, and saturated NaHCO3 solution is added to the reaction solution for washing in a volume ratio of 1:1, and the washing is performed for 3 times, and then the obtained neutral reaction solution is washed with distilled water in the same way until neutral, and the obtained neutral reaction solution is dried with anhydrous Na2SO4, and the filtrate is collected after filtration, and distilled under reduced pressure using a rotary evaporator to obtain a solid crude product; the solid crude product is recrystallized in a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 100:8, and then filtered, and dried using an oven at a temperature of 55°C to obtain a white solid pyrroline dione compound, with a melting point of 126.2-127.1°C, and a yield of 99%, and the nuclear magnetic resonance and high resolution mass spectrometry data are the same as the results measured in Example 1.

[0053] Example 3: Determination of antibacterial spectrum and bactericidal activity of a pyrroline dione compound

[0054] According to the technical scheme, the biological activity of a pyrroline dione compound on plant pathogenic fungi and oomycetes is determined by using a classical spore germination method. The pyrroline dione compound provided by the application is dissolved in dimethyl sulfoxide (DMSO) or methanol to obtain a medicament mother liquor of the compound with a concentration of 10 mg / mL or 20 mg / mL, which is 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 medicament. The prepared medicament solution is mixed with the fungal conidial suspension or oomycete sporangial suspension at a ratio of 1:1, so that the final concentration of the medicament is 10.0, 3.0, 1.0, 0.3, 0.1, 0.03, 0 (CK) μg / mL. 10 mL of the mixture of the medicament solution and the spores is added to each culture dish (φ 90 mm), and incubated at a suitable temperature for spore germination of different pathogenic fungi for 5-8 hours. When the spore germination rate of the control treatment reaches 95%, the minimum concentration of the medicament that completely inhibits spore / sporangium germination (MIC) is investigated under a microscope.

[0055] The results show that the pyrroline dione compound has a 100% minimum inhibitory concentration (MIC) of 1.0 μg / mL for Fusarium graminearum, F. moniliforme, Magnaporthe grisea, Botrytis cinerea, Glomerella cingulata, Sclerotinia sclerotiorum and Didymella bryoniae conidial germination and for Phytophthora infestans, Phytophthora capsici, Phytophthora parasitica, Peronophthora litchii and Plasmopara viticola sporangial germination; a MIC of 3.0 μg / mL for Cercospora arachidicola conidial germination, for Phytophthora soja, Pythium sp. and Pseudoperonospora cubensis sporangial germination; and a MIC of 10.0 μg / mL for Setosphaeria turcica, Bipolaris madis and Alternaria solani conidial germination. At a treatment of 0.03 μg / mL, the conidial germination rate and sporangial germination rate of other fungi tested are less than 50%, except for Setosphaeria turcica, Bipolaris madis and Alternaria solani conidial germination rate, which is higher than 50%, i.e. the compound has an LD 50 less than 0.03 μg / mL.

[0056] A bacterial suspension with a turbidity of 50 is mixed with different concentrations of the liquid medicine 1:1 to have a final drug concentration of 10.0, 3.0, 1.0, 0.3, 0.1, 0.03, 0 (CK) μg / mL. The mixture is placed on a shaking bed (180 r / min) for 3 days, and the minimum inhibitory concentration (MIC) is determined by turbidity. The results show that the pyrroline dione compound has a minimum inhibitory concentration (MIC) of 1 μg / mL for the growth of Xanthomonas oryzae, X. oryzicola, Pseudomonas solanacearum, X. citri, Erwinia carotovora, Erwinia amylovora and Acidovorax citrulli, and has a strong bactericidal effect.

[0057] The determination results of the antibacterial activity of the pyrroline dione compound according to the present application show that the compound has strong broad-spectrum bactericidal activity on 26 representative plant pathogenic fungi, oomycetes and bacteria tested, indicating that the target of the compound is conservative in different pathogenic microorganisms. The pyrroline dione compound according to the present application is processed into a bactericide preparation according to the method described in the specification, and an agricultural bactericide with the pyrroline dione compound as the effective ingredient is obtained. Further, the prevention and treatment effect of the bactericide on some common diseases on plant tissues or plant seedlings is determined, or the efficacy test is carried out in the field, and the safety to plants is determined by comparison with the currently commonly used bactericides.

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

[0059] According to the effective content of the preparation to be processed, 20% by weight of the pyrroline dione compound (particle size 300 mesh), 1% wetting agent (specifically sodium dodecyl sulfonate), 3% dispersing agent (specifically NNO), 0.2% tackifier (specifically xanthan gum), 5% stabilizer (specifically bentonite), 3% antifreeze (specifically polyethylene glycol 400), and 0.1% defoamer (specifically lauric acid) are weighed, and water is added to 100% to obtain a mixture for preparing a suspension concentrate with an effective ingredient content of 20%, i.e. 20% SC.

[0060] According to the effective content of the preparation to be processed, 20% by weight of the pyrroline dione compound (particle size 300 mesh), 1% wetting agent (specifically sodium dodecyl sulfonate), 3% dispersing agent (specifically NNO), 0.2% tackifier (specifically xanthan gum), 5% stabilizer (specifically bentonite), 3% antifreeze (specifically polyethylene glycol 400), and 0.1% defoamer (specifically lauric acid) are weighed, and water is added to 100% to obtain a mixture for preparing a suspension concentrate with an effective ingredient content of 20%, i.e. 20% SC.

[0061] The above two mixtures are respectively mixed and formulated into slurries, and then ground by a sand mill for 3-4 times. Finally, the pH value, fluidity and wettability and other technical indicators are adjusted to obtain bactericide preparations with an effective ingredient content of 20% SC or 50% SC, respectively.

[0062] The sample quality is detected according to the standard method of NY / T 4014-2021, and the results show that the relative error of the effective ingredient content is ±0.1-0.5%, there is no stratification and precipitation after being placed in the laboratory for 30 days, and it can maintain a stable suspended state after being diluted with water, and the effective suspension rate is greater than 90% after being static for 1 h.

[0063] Example 5: Preparation of a bactericide water dispersible granule (10% WG or 25% WG)

[0064] According to the effective content of the preparation to be processed, 10% by weight of a pyrroline dione compound, 10% of an adjuvant (specifically, talc), 10% of a disintegrating agent (specifically, 4:1 ammonium sulfate + sodium chloride), and 70% of a carrier (specifically, white carbon black) were weighed, respectively, to obtain a mixture for preparing a water dispersible granule with an effective ingredient content of 10%, i.e., 10 wt% WG;

[0065] According to the effective content of the preparation to be processed, 10% by weight of a pyrroline dione compound, 10% of an adjuvant (specifically, talc), 10% of a disintegrating agent (specifically, 4:1 ammonium sulfate + sodium chloride), and 70% of a carrier (specifically, white carbon black) were weighed, respectively, to obtain a mixture for preparing a water dispersible granule with an effective ingredient content of 10%, i.e., 10 wt% WG;

[0066] The above two mixtures were respectively subjected to air flow crushing, and the mixtures with a particle size of 10 pm or less were collected and then subjected to extrusion granulation and drying, respectively, to obtain fungicide preparations with an effective ingredient content of 10 wt% WG or 25 wt% WG.

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

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

[0069] A pyrroline dione compound was weighed at 10% by volume weight of the dosage form and dissolved in about 2 / 3 of the total volume of 100# solvent oil, 10% of an emulsifier w / v (a mixture of 5% of Farm Emulsion 600 and 5% of Tween 40) was added, and then 100# solvent oil was added to the set volume, to obtain a fungicide preparation of 10% EC (w / v).

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

[0071] Example 7: Test of an agricultural fungicide for preventing rice bakanae disease (Fusarium fujikuroi)

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

[0073] The pyrroline dione compound described in the present application was processed into 20% SC; the control agent, kresoxim-methyl 25% SC (provided by Jiangsu Pesticide Research Co., Ltd.).

[0074] Test conditions: greenhouse pot culture test;

[0075] Test method: The above test formulation samples were diluted with tap water to active ingredient content of 100 mg a.i. / L, 200 mg a.i. / L and 400 mg a.i. / L of liquid medicine, respectively, and 150 L of liquid medicine (15-60 g a.i. / ha) was used to soak the seed with fungus per hectare. After soaking the seed at room temperature (20-30°C) for 48 hours, the seed was germinated at 25-30°C for 48 hours, and then sown in plastic pots and cultured in a greenhouse for 21 days, and then the seedling morbidity was checked.

[0076] % of control effect = [1-(seedling morbidity of treatment ÷ seedling morbidity of blank control)] x 100.

[0077] Test results: After the seedlings soaked in water were sown for 21 days, the cumulative disease rate of seedlings with bakanae disease was 16.4%. The seed soaked in 100, 200 and 400 mg / L of 20% SC of the agricultural fungicide with one pyrroline dione compound as the active ingredient had a control effect on bakanae disease of 86.7%, 93.9% and 98.8%, respectively, which was significantly higher than the control effect of 80.4%, 84.8% and 88.1% of the same concentration of the control agent. In addition, the germination and emergence of the seed soaked in all treatments were normal, and no phytotoxicity was observed.

[0078] Example 8: Test of the agricultural fungicide for controlling rice seedling bacterial wilt

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

[0080] The pyrroline dione compound described in the present application was processed into 10% WG; the control agent was 4% metalaxyl-M + fludioxonil + azoxystrobin SC (produced by Anhui Fengle Agricultural Chemical Co., Ltd., purchased online).

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

[0082] Test method: The above test formulation 10% WG sample was prepared into "poison soil" by mixing with 1000, 750, 500 g a.i. per hectare, and the control agent was prepared into "poison soil" by mixing with 750 g of active ingredient per hectare, and then 45 g of poison soil was applied per square meter of seedbed when the seedlings had two leaves and one heart. After 10 days, the dead seedling rate was investigated and the control effect was calculated.

[0083] % of control effect = [1-(seedling morbidity of treatment ÷ seedling morbidity of blank control)] x 100.

[0084] Test results: the seedling mortality rate of the control of scattering non-pesticide fine soil is 14.34%. The prevention and treatment effects of the 1000, 750 and 500 g a.i. / ha soil treatment of the 10% WG of the agricultural fungicide with one pyrroline dione compound as the effective component on the bacterial wilt are 96.4%, 89.6% and 80.3% respectively, which are significantly higher than the 73.3% prevention and treatment effect of the 750 g a.i. / ha soil treatment of the control pesticide. The seedlings of all soil treatments grow normally and no adverse effects of pesticide are observed.

[0085] Example 9: Test of the prevention and treatment of rice blast by the agricultural fungicide

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

[0087] The test is carried out on the prevention and treatment of rice blast by the 10% and 25% fungicide water dispersible granules with one pyrroline dione compound as the effective component according to the present application, and the control pesticide is the 40% isoprothiolane emulsion produced by Shandong Tianrong Biological Technology Co., Ltd. (purchased on the network).

[0088] Test conditions: greenhouse pot culture test;

[0089] Test method: the above fungicide samples are weighed respectively, and the effective content is diluted to 400 mg / L, 200 mg / L and 100 mg / L of water, and the control pesticide isoprothiolane is diluted to 500 mg / L and 250 mg / L of water. The above pesticide solutions are sprayed on 4 pots of 3-leaf stage paddy rice seedlings (paddy rice variety Sujing 4699 with medium susceptibility to rice blast) respectively, 24 hours later, the rice blast Magnaporthe grisea conidial suspension is sprayed on the leaves, and then the seedlings are placed in a dark environment at 25℃ for 12 hours, and then moved to a greenhouse (20-30℃) for culture. The effect of the compound on the prevention and treatment of rice blast is determined. After 10 days of inoculation, the number of rice blast spots on the surfaces of the 2nd and 3rd leaves from the bottom is investigated, and the control effect is calculated compared with the control.

[0090] The control effect % = [1-(the average number of spots on the treated leaves ÷ the average number of spots on the blank control leaves)] x 100.

[0091] Test results: the results are shown in Table 1.

[0092] Table 1: Prevention and treatment effect of the agricultural fungicide on rice seedling stage rice blast

[0093]

[0094] The results show that the agricultural fungicide with one pyrroline dione compound as the effective component according to the present application has a more excellent prevention and treatment effect on rice blast than the control pesticide, and the effect is positively correlated with the effective dose of the treatment and is irrelevant to the formulation content. No adverse effects on the growth of seedlings are observed in each treatment.

[0095] Example 10: Test of the agricultural fungicide for controlling bacterial leaf streak of rice

[0096] The test example is implemented as follows:

[0097] A pyrroline dione compound of the present application is processed into 10% EC according to the specification, and a control agent 20% zinc thiazole SC (provided by Zhejiang Xinong Co., Ltd.).

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

[0099] Test method: The above test preparation samples are respectively diluted with tap water to active ingredient contents of 250 mg a.i. / L, 500 mg a.i. / L and 800 mg a.i. / L of liquid medicine, 750 L of liquid medicine per hectare is sprayed, i.e. 187.5-600 g a.i. / ha, and the control is sprayed with clean water. The second spraying is performed after 7 days. The leaf disease rate of the flag leaf and the second leaf from the bottom is checked after 10 days, and the control effect calculation method is:

[0100] % of control effect = [1-(treated leaf disease rate ÷ blank control leaf disease rate)] x 100.

[0101] The test result investigation shows that 7 days after the first spraying, about 15% of the leaves in the control begin to be diseased, and 10 days after the second spraying, the disease leaf rate of the control reaches 78.54%, and the disease leaf rates of the agricultural fungicide of the present application at 250 mg a.i. / L, 500 mg a.i. / L and 800 mg a.i. / L are respectively 7.68%, 2.45% and 0.24%. The control effects are respectively 90.2%, 96.9% and 99.7%. In addition, it is noted that the number of disease spots on the leaves in the treatment area is significantly less than that on the leaves in the control area.

[0102] Example 11: Test of the agricultural fungicide for controlling grape downy mildew

[0103] The test example is implemented as follows:

[0104] A, test compound:

[0105] The 20% pyrroline dione fungicide suspension concentrate (SC) prepared in Example 3 of the present application is diluted with water to 3.125, 6.25, 12.5, 25.0, 50.0, 500 mg / L of liquid medicine.

[0106] The control agent is 10% omethoate win green OD (produced by DuPont Company). Diluted with water to 3.125, 6.25, 12.5, 25.0 mg / L of liquid medicine.

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

[0108] B, test method and condition:

[0109] Take the 3rd-7th healthy leaves of greenhouse potted grape (variety: Muscat Hamburg) vines, rinse with tap water and dry, then spray different concentrations of test pesticide solution on the back or front of the leaves until the leaf surface is completely wet (equivalent to spraying 1500 kg of pesticide solution per hectare or 4.69 g a.i.~750 g a.i. / ha). After 24 hours, spray the back of the leaves with grape downy mildew spore suspension; move to the artificial climate room under the conditions of light / dark (12h), temperature 20~25℃, relative humidity RH 90%~95% for 7 days, then investigate the disease incidence, and calculate the control effect according to the ratio of leaf disease area (spore area).

[0110] Control effect % = [1-(treated average disease spot area ratio ÷ blank control average disease spot area ratio)] x 100.

[0111] C, test results:

[0112] The test results are listed in Table 2

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

[0114]

[0115] The results show that the agricultural fungicide containing the pyrroline dione compound as the effective ingredient has a good preventive effect on grape downy mildew. All the leaves treated with 25, 50 and 500 mg / L of pesticide solution are not diseased. Moreover, the treatment of the front of the leaves has a high control effect on the back inoculation, indicating that the active ingredient can be conducted from the front to the back of the leaves.

[0116] Example 12: Test of agricultural fungicide for controlling potato late blight and pepper bacterial wilt

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

[0118] A, test agent

[0119] The 20% and 50% pyrroline dione compound suspending agent (SC) fungicide preparation prepared in Example 3 of the present application.

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

[0121] B, test conditions in greenhouse

[0122] 5-leaf stage potato seedlings and pepper seedlings, 2 plants per pot; incubated in a glass sunlight greenhouse at a temperature of 20-30°C.

[0123] C. Test method

[0124] I. Preparation of test liquid

[0125] The above-mentioned compounds were weighed and diluted with water to obtain a concentration of 500 mg / L, 250 mg / L and 125 mg / L of active ingredient, respectively, to prepare three concentrations of test liquid.

[0126] II. Test operation method

[0127] The test liquid at three concentrations of each compound was sprayed on 3 pots of potato seedlings and pepper seedlings using a throat sprayer until the leaves were dripping with the test liquid. After 24 hours, the prepared spore cyst suspension of Phytophthora infestans and P. capsici was inoculated on 10 μl per inoculation point, a total of 24 leaflets (8 leaflets per pot of seedlings). After drying, the pots were placed in the greenhouse and covered with black plastic cloth to keep them moist overnight, then the plastic cloth was removed and the incubation was continued for 7 days. The number of lesions and lesion length were investigated, and the control effect was calculated according to the following formula:

[0128] Control effect % = [1-(average lesion length of treatment ÷ average lesion length of blank control)] x 100.

[0129] D. Test results

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

[0131] Table 3: Test results of the agricultural fungicide for controlling potato late blight and pepper blight

[0132]

[0133] The test results show that the agricultural fungicide of the present application has an ideal preventive effect on potato late blight and pepper blight, and the control effect is proportional to the effective ingredient dose of the test and is independent of the content of the prepared preparation. The control effect of the agricultural fungicide of the present application, which contains a pyrroline dione compound as the effective ingredient, on the two diseases is obviously better than that of the control fungicide.

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

[0135] Test agent sample: a pyrroline dione compound of the present application, and laboratory-stored fungicides of different mechanisms of action, such as boscalid, carbendazim, fenhexamid, procymidone, azoxystrobin, fludioxonil, metalaxyl, dimethomorph, and flutriafol technical agent.

[0136] Test method: a pyrroline dione compound of the present application and a commonly used fungicide technical material were dissolved with methanol and carbendazim was dissolved with 0.1 mol hydrochloric acid, to prepare a 10 mg / mL mother liquor, which was then diluted with sterile water to 200 μg / mL, and then diluted with PDA medium to prepare a final concentration of 20 μg / mL drug-containing medium plate.

[0137] The strains used for the test were the laboratory-preserved multi-resistant strain B5 of Botrytis cinerea against dicyclanil, carbendazim, probenazole, procymidone, azoxystrobin and fludioxonil, and the wild sensitive strain B1, the multi-resistant strain PcR of Phytophthora capsici against azoxystrobin, metalaxyl, dimethomorph and flutriafol, and the wild sensitive strain PcS. Botrytis cinerea and Phytophthora capsici were respectively cultured on drug-free PDA and lima bean medium plates, and 5 mm diameter bacterial discs were prepared at the near edge of the colony, which were respectively inoculated on each drug-containing plate and cultured at 25°C for 5 days. Growth was recorded as "+" and non-growth as "-". The results are shown in Table 4. From the results of whether the growth on the drug-containing medium, it can be seen that the pyrroline dione compound of the present application has no cross-resistance with the existing fungicides, indicating that it has a new mechanism of action.

[0138] Table 4 Cross-resistance mode of a pyrroline dione compound with existing fungicides of different action modes

[0139]

[0140] The above description is only a preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification, equivalent change and modification of the above embodiments without departing from the technical solution content of the present application, according to the technical essence of the present application, all belong to the scope of the technical solution of the present application.

Claims

1. A pyrroline dione compound, characterized by, The pyrroline dione compound has a structure shown in formula I: 。 2. The method for preparing a pyrrolidone compound according to claim 1, characterized in that, The method comprises the following steps: The compound II, maleic anhydride, a catalyst and an organic solvent are mixed and reacted to obtain a pyrroline dione compound having a structure shown in formula I, wherein the compound II has a structure shown in formula II: ； 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 methyl sulfonic acid, p-methyl benzene sulfonic acid and benzene sulfonic acid; The organic solvent is selected from one or more of benzene, toluene, xylene and cyclohexane.

3. An agricultural fungicide, characterized by comprising: The effective component is the pyrroline dione compound of claim 1.

4. The pyrroline dione compound of claim 1 or the agricultural fungicide of claim 3 is applied to the prevention and treatment of plant diseases.

5. Use according to claim 4, characterized in that, The plant diseases include diseases caused by one or more of pathogenic fungi, pathogenic oomycetes and pathogenic bacteria.

6. Use according to claim 4 or 5, characterized in that, 15-1000 g of the effective component of the pyrroline dione compound of claim 1 or the agricultural fungicide of claim 3 is used per hectare.

7. Use according to any one of claims 4 to 6, characterized in that, The seed and soil treatment or spraying method is used.

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