Use of an aminoisoxazole compound in the prevention and treatment of ralstonia solanacearum

Abstract

The application discloses application of an amino isoxazole compound in preventing and treating Ralstonia solanacearum, and belongs to the technical field of plant bacterial wilt prevention and treatment; the amino isoxazole compound is 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine; it is found for the first time that 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine has strong inhibitory activity on Ralstonia solanacearum; experiments show that the MIC of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine on Ralstonia solanacearum is 6 mu g / mL at 24 h, is equivalent to that of benzio carb, and can be used for preventing and treating plant bacterial wilt, thereby providing a new candidate compound for development of a bactericide for Ralstonia solanacearum.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant bacterial wilt prevention and treatment, and particularly relates to application of an aminoisoxazole compound in prevention and treatment of bacterial wilt. BACKGROUND

[0002] Ralstonia solanacearum is a soil-borne bacterial pathogen that widely harms solanaceous plants such as tomatoes, eggplants, peppers, potatoes and other plants, and is one of the main pathogenic bacteria of plant bacterial wilt. Ralstonia solanacearum R. solanacearum can be transmitted through irrigation water, agricultural tools, diseased residues and soil carrying bacteria, invade the plant roots and rapidly spread through the vascular system, causing vascular blockage, leaf wilting and plant death. R. solanacearum has strong adaptability and invasion ability and rapidly spreads in a high-temperature and high-humidity environment, which greatly harms agricultural production.

[0003] Due to strong survival ability, wide host range, strong variability, hidden invasion and rapid transmission, there are multiple limitations in the prevention and treatment of R. solanacearum. At present, the chemical agents for preventing and treating R. solanacearum are mostly broad-spectrum fungicides, which lack specificity, have general effects on R. solanacearum and have great environmental protection pressure. Therefore, in-depth research on high-efficiency and low-toxicity compounds for preventing and treating R. solanacearum and the use of the compounds as lead compounds for new pesticide creation have become an important content of the research on the prevention and treatment of bacterial wilt. SUMMARY

[0004] The first object of the present application is to provide application of an aminoisoxazole compound in prevention and treatment of R. solanacearum, and the second object is to provide a preparation for preventing and treating plant bacterial wilt.

[0005] The first object of the present application is achieved by the application of the aminoisoxazole compound in prevention and treatment of R. solanacearum, wherein the aminoisoxazole compound is 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine, and the chemical structural formula is as follows:

[0006] ;

[0007] The 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine has a MIC of 6 μg / mL for R. solanacearum at 24 h.

[0008] The second object of the present application is achieved by the bactericide for preventing and treating R. solanacearum, wherein the 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine is used as a main active ingredient or one of active ingredients.

[0009] The 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine described in the application is derived from Shanghai Taoshu Biological Technology Co., Ltd., and it is found for the first time that the 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine has strong inhibitory activity on Ralstonia solanacearum; tests show that the MIC of the 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine on Ralstonia solanacearum is 6 μg / mL in 24 hours, which is equivalent to the MIC (5 μg / mL) of bupirimate on Ralstonia solanacearum, and the 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine shows strong inhibitory activity on Ralstonia solanacearum, and can be used for plant bacterial wilt prevention and treatment, and provides a new candidate compound for Ralstonia solanacearum fungicide development. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The chemical structural formula of the compound 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine in the application;

[0011] Figure 2 The calculation results of the antibacterial activity determination of the 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine on Ralstonia solanacearum in the test example;

[0012] Figure 3 The calculation results of the antibacterial activity determination of bupirimate on Ralstonia solanacearum in the test example. DETAILED DESCRIPTION

[0013] The application will be described in more detail below in combination with test examples and examples, however, the application is not limited thereto, and any transformation or improvement based on the application should be covered within the protection scope of the application.

[0014] The application of the amino isoxazole compound in the prevention and treatment of Ralstonia solanacearum, the amino isoxazole compound is 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine, and the chemical structural formula is as follows:

[0015] ;

[0016] The MIC of the 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine on Ralstonia solanacearum is 6 μg / mL in 24 hours.

[0017] Ralstonia solanacearum is the pathogen of plant bacterial wilt, and the plants include but are not limited to tomato, tobacco, peanut, soybean and cowpea.

[0018] The fungicide for preventing and treating Ralstonia solanacearum described in the application is mainly active ingredient or one of active ingredients of the 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine.

[0019] Further, the effective concentration of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine applied in the fungicide is ≥ 6 μg / mL.

[0020] Further, the dosage form of the fungicide is aqueous agent, emulsion in water, wettable powder, soluble powder, soluble granule, suspension agent, tablet or effervescent granule.

[0021] Further, the solvent of the aqueous agent or emulsion in water is a fatty alcohol aqueous solution with a concentration ≤ 5% by mass percentage.

[0022] Further, the fatty alcohol is methanol, ethanol or propanol.

[0023] The 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine described in the present application is derived from a drug-like diversity compound library containing 50,000 compounds developed by Shanghai Taoshu Biological Technology Co., Ltd., with a LibraryID of F3379-0307, a CAS Number of 698975-77-8, a relative molecular mass of 124.1405, and a chemical structural formula as shown in Figure 1 .

[0024] The present application carries out the following tests on 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine and the positive control drug thiabendazole:

[0025] Test Example 1: Detection of the bacteriostatic activity of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine and thiabendazole on Ralstonia solanacearum

[0026] 1. Preparation of test drug solution

[0027] 1-1. Test drug solution for test group I: 1.0-3.0 mg of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine (hereinafter referred to as the original drug) was dissolved in DMSO, and water was added to dilute it into test drug solutions with different concentrations of the original drug. The concentration of the original drug in each test drug solution was 10 μg / mL, 30 μg / mL, 50 μg / mL, 100 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, and 1000 μg / mL, and the content of DMSO in each test drug solution was 0.4% (mass percentage);

[0028] 1-2, Test group II test drug solution: 1.0-3.0 mg of thiamethoxam (hereinafter referred to as positive drug) was dissolved in DMSO, and water was used to dilute the test drug solution to different positive drug concentrations. The positive drug concentrations in each test drug solution were 10 μg / mL, 30 μg / mL, 50 μg / mL, 100 μg / mL, 300 μg / mL, 500 μg / mL, 1000 μg / mL, and 1500 μg / mL, respectively. The content of DMSO in each test drug solution was 0.4% (mass fraction).

[0029] 2, Test bacteria solution preparation

[0030] 2-1, The single colony of Ralstonia solanacearum cultured on NA solid medium was inoculated into NB liquid medium and cultured at 30°C, 200 rpm in a constant temperature shaker to the logarithmic growth phase to obtain the Ralstonia solanacearum stock solution;

[0031] The formula of NA solid medium: peptone 5 g, beef extract 3 g, sodium chloride 5 g, agar 15 g, distilled water 1 L, pH 7.0;

[0032] The formula of NB liquid medium: peptone 5 g, beef extract 3 g, sodium chloride 5 g, distilled water 1 L;

[0033] 2-2, The Ralstonia solanacearum stock solution was diluted with NB liquid medium to a concentration of OD=0.1 (the concentration of Ralstonia solanacearum was about 1.5×10 8 CFU / mL), to obtain the test bacteria solution.

[0034] 3, Test method

[0035] 3-1, Test group I: multiple groups of 99 μL test bacteria solution were taken and placed in the well plate, and each group was added with 1 μL of test group I test drug solution with different original drug concentrations, and cultured at 30°C, 200 rpm in a constant temperature shaker for 24 h. The OD 600 value was measured by an enzyme marker, and the bacteriostatic effect was observed. At this time, the final concentration of the original drug in each well of the plate was 0.1 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 1 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, and 10 μg / mL, respectively.

[0036] Test group II: multiple groups of 99 μL test bacteria solution were taken and placed in the well plate, and each group was added with 1 μL of test group II test drug solution with different positive drug concentrations, and cultured at 30°C, 200 rpm in a constant temperature shaker for 24 h. The OD 600The values ​​were used to observe the antibacterial effect; at this time, the final concentrations of the positive control drug in each well were 0.1 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 1 μg / mL, 3 μg / mL, 5 μg / mL, 10 μg / mL, and 15 μg / mL, respectively.

[0037] For both experimental groups I and II, the lowest concentration at which no Ralstonia solanacearum growth was observed was defined as the minimum inhibitory concentration (MIC) against Ralstonia solanacearum.

[0038] 3-2. Negative control group: 99 μL of the test bacterial culture was placed in a well plate, and 1 μL of water containing 0.4% (w / v) DMSO was added. The plate was incubated at 30℃ for 24 h in a constant temperature shaker at 200 rpm. The OD was measured using a microplate reader. 600 value;

[0039] 3-3. Blank control group: Place 99 μL of NB liquid culture medium in a well plate, add 1 μL of water containing 0.4% (w / v) DMSO, and measure the OD using a microplate reader. 600 value;

[0040] 3-4. The entire experiment was repeated three times, and the OD values ​​were taken three times. 600 The average value is used to calculate the corrected inhibition rate using the following formula:

[0041] Corrected inhibition rate % = [(negative control group OD)] 600 -Experimental group OD 600 ) / (Negative control group OD 600 - Blank control group OD 600 )]×100.

[0042] 4. Test Results

[0043] The antibacterial activities of 5,6-dihydro-4H-cyclopentadien[c]isoxazole-3-amine and thiamethoxam against Ralstonia solanacearum are shown in Table 1 and 2, respectively. Figure 2 Table 2 and Figure 3 As shown; the OD of the negative control group was measured. 600 The value was 0.561±0.001, and the OD value of the blank control group was... 600 The value was 0.298±0.001; combined with the OD values ​​of experimental groups I and II in Tables 1 and 2. 600 The corrected inhibition rate (%) corresponding to the concentration of the test drug solution in each experimental group is obtained from the value and the formula for calculating the corrected inhibition rate in Tables 1 and 2.

[0044] Table 1. Antibacterial activity of 5,6-dihydro-4H-cyclopentadiene[c]isoxazole-3-amine against Ralstonia solanacearum

[0045]

[0046] Table 2 Inhibition activity of thiophameton on Ralstonia solanacearum

[0047]

[0048] In combination with the determination, the MIC of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine on Ralstonia solanacearum is 6 μg / mL at 24 h; it can be seen that the inhibition activity of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine on Ralstonia solanacearum is equivalent to that of thiophameton, and 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine exhibits very strong inhibition activity on Ralstonia solanacearum and can be used for the prevention and treatment of plant bacterial wilt. Figure 3 Figure 2 Example 1 Preparation of bactericidal water agent for preventing and treating Ralstonia solanacearum

[0049] The effective concentration of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine in the bactericidal water agent is 30 μg / mL, the solvent is 4% (mass fraction) methanol aqueous solution, and other conventional preparation methods are used for preparation.

[0050] Example 2 Preparation of bactericidal water emulsion for preventing and treating Ralstonia solanacearum

[0051] The effective concentration of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine in the bactericidal water emulsion is 20 μg / mL, the solvent is 3% (mass fraction) ethanol aqueous solution, and other conventional preparation methods are used for preparation.

[0052] Example 3 Preparation of bactericidal wettable powder for preventing and treating Ralstonia solanacearum

[0053] The effective concentration of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine in the bactericidal wettable powder is 6 μg / mL, and other conventional preparation methods are used for preparation.

[0054] The soluble powder, soluble granule, suspension agent, tablet, effervescent granule disclosed by the application use 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine as an active ingredient (effective concentration ≥ 6 μg / mL), and other conventional preparation methods are used for preparation.

[0055] The soluble powder, soluble granule, suspension agent, tablet, effervescent granule disclosed by the application use 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine as an active ingredient (effective concentration ≥ 6 μg / mL), and other conventional preparation methods are used for preparation.

[0056] ​In addition, the MIC of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine obtained based on the test example 1 for Ralstonia solanacearum is 6 μg / mL at 24 h, and when the effective concentration of 5,6-dihydro-4H-cyclopenta[c]isoxazol-3-amine in the bactericide is ≥ 6 μg / mL, the bactericide can achieve efficient inhibition (close to 100%) of Ralstonia solanacearum; in the production of bactericide products, a large amount of active ingredients is usually selected, and the bactericide can be diluted to an appropriate concentration for application, which is convenient for storage and transportation.

Claims

1. The application of an aminoisoxazole compound in the control of Ralstonia solanacearum, characterized in that, The aminoisoxazole compound is 5,6-dihydro-4H-cyclopentadieno[c]isoxazole-3-amine, and its chemical structural formula is as follows: ； The MIC of the 5,6-dihydro-4H-cyclopentadiene[c]isoxazole-3-amine against Ralstonia solanacearum was 6 μg / mL at 24 h.

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