Blastocin, its application and preparation method, pesticide agent and method for preventing and controlling rice blast

Abstract

The present application relates to the technical field of biological pesticides, in particular to a mycospocin, application and preparation method thereof, a pesticide and a method for preventing and treating rice blast. The mycospocin is selected from at least one of the compounds shown in the following structural formula: wherein R is selected from hydroxyl or amino substituted ester group. The mycospocin has special and strong inhibitory effect on rice blast fungus and can be used for preventing and treating rice blast.

Description

Technical Field

[0001] The present invention relates to the technical field of biological pesticides, in particular to blastin, its application and preparation method, a pesticide agent and a method for preventing and controlling rice blast. Background Art

[0002] Rice blast is a devastating disease of rice caused by the rice blast fungus. Currently, the pesticides used to prevent and control rice blast mainly include chemical synthetic pesticides such as organophosphorus, triazoles and amides. Among them, blastifungin and blastamide are the two most widely used pesticides.

[0003] Specifically, blasticide is an organophosphorus fungicide that works by inhibiting lipid metabolism in pathogens. Field trials have shown that it can achieve a 75%-85% efficacy against rice blast. This fungicide boasts strong systemic activity and a long-lasting effect (approximately 20 days). However, long-term use has led to the emergence of resistant strains in many areas. Furthermore, blasticide is typically synthesized organically, and the synthesis process generates large amounts of phosphorus-containing wastewater, which can easily cause water pollution.

[0004] Paddy blastamide belongs to the methoxyacrylate class of fungicides, which works by blocking the mitochondrial electron transport chain. It has an excellent control efficacy of 85%-90% and possesses both therapeutic and protective properties. However, it is somewhat toxic to aquatic organisms. Paddy blastamide also has a complex organic synthesis process involving 7-8 steps, including nitration, reduction, and etherification. The synthesis requires the use of metal catalysts, resulting in high production costs.

[0005] At the same time, the average carbon emission intensity of these organic synthetic antibacterial agents or pesticides is dozens of times that of biosynthetic pesticides. Harmful substances are also easily left in the synthesis process, which may pose certain environmental and health risks. Therefore, it is of great significance to develop biosynthetic pesticides that are lower in cost, have good antibacterial effects, are green, mild, and easy to prepare.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The present invention aims to provide blastocin, its application, its preparation method, pesticide formulation, and method for controlling rice blast. The present invention provides a blastocin that has a unique and strong inhibitory effect on rice blast fungi and can be used to control rice blast. Furthermore, the blastocin is prepared using a biological method, not organic synthesis, and thus does not introduce harmful substances.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a pesticidal agent, which is selected from at least one compound represented by the following structural formula:

[0010] , wherein R is selected from a hydroxyl or amino substituted ester group.

[0011] In an optional embodiment, it includes compounds represented by the following formula 1 and formula 2:

[0012] Formula 1 and Formula 2, wherein the mass ratio of the compound represented by Formula 1 to the compound represented by Formula 2 is (1-9):(9-1).

[0013] In a second aspect, the present invention provides the use of the blastin described in the aforementioned embodiment in the preparation of the following agents: (1) inhibiting rice blast fungus;

[0014] (2) Prevent and control rice blast.

[0015] In a third aspect, the present invention provides a pesticide formulation comprising the pesticidal agent described in the aforementioned embodiment.

[0016] In an optional embodiment, the pesticide agent meets at least one of the following requirements: (1) the mass concentration of scutellarin in the pesticide agent is 0.1-10%;

[0017] (2) The pesticide formulation further comprises at least one of a solvent, a cosolvent, a wetting agent, an adhesive and a penetrant.

[0018] In a fourth aspect, the present invention provides a method for preventing and controlling rice blast, comprising: applying the blastin described in the aforementioned embodiment or the pesticide described in the aforementioned embodiment to the leaf surface of vegetation.

[0019] In an optional embodiment, the effective concentration of the pesticidal agent acting on the vegetation leaf surface is 7-220 mg / L.

[0020] In a fifth aspect, the present invention provides a method for preparing the pesticidal agent described in the aforementioned embodiment, comprising: fermenting Streptomyces, and then subjecting the fermentation product formed by the fermentation to column chromatography.

[0021] In an optional embodiment, the preparation method comprises any one of the following steps:

[0022] (1) including: column chromatography followed by recrystallization;

[0023] (2) Includes: column chromatography followed by recrystallization and liquid phase purification.

[0024] In an optional embodiment, the conditions in the preparation method meet the following requirements:

[0025] (1) Column chromatography includes: using a mixed solvent of an ether solvent and an ester solvent for isocratic elution;

[0026] (2) Recrystallization comprises: mixing and dissolving a C1-C3 monohydric alcohol solvent and a material formed by column chromatography under heating conditions to form a saturated solution, then mixing and filtering the saturated solution with an adsorbent to form a filtrate, and gradient cooling the filtrate;

[0027] (3) Liquid phase purification: gradient elution using a mixed solvent of nitrile solvent and water.

[0028] The present invention has the following beneficial effects: Examples of the present invention provide a novel compound, named blasticidin, which exhibits a unique and potent inhibitory effect against the rice blast pathogen, significantly outperforming existing pesticides for controlling rice blast. Blasticillin is also produced by fermentation rather than chemical organic synthesis, making the preparation method easy to implement, without introducing new harmful substances, and resulting in low production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 This is the appearance of the pesticidal prepared in Example 1;

[0031] Figure 2 This is the high-pressure liquid chromatography spectrum of pesticidal prepared in Example 1;

[0032] Figure 3 This is a correlation diagram of punacin A prepared in Example 2;

[0033] Figure 4 This is a correlation diagram of punicolide B prepared in Example 2;

[0034] Figure 5 The mass spectrum of punicolin A prepared in Example 2 is shown;

[0035] Figure 6 shows the hydrogen spectrum of punicolin A prepared in Example 2;

[0036] Figure 7 The carbon spectrum of punicolin A prepared in Example 2 is shown;

[0037] Figure 8 HSQC spectrum of punicolin A prepared in Example 2 is shown;

[0038] Figure 9The peccaryin A prepared in Example 2 is shown in FIG. 1 H- 1 H COSY spectrum;

[0039] Figure 10 The HMBC spectrum of punicolin A prepared in Example 2 is shown;

[0040] Figure 11 shows the mass spectrum of punicolin B prepared in Example 2;

[0041] Figure 12 1H spectrum of peccolicin B prepared in Example 2;

[0042] Figure 13 The carbon spectrum of punicolin B prepared in Example 2 is shown;

[0043] Figure 14 HSQC spectrum of punicolin B prepared in Example 2 is shown;

[0044] Figure 15 The scutellarin B prepared in Example 2 1 H- 1 H COSY spectrum;

[0045] Figure 16 The HMBC spectrum of punicolin B prepared in Example 2 is shown;

[0046] Figure 17 The graph showing the antibacterial effects of blasticidin A, blasticidin B, blastifungin, and blastamide on the rice blast pathogen provided in Test Example 1;

[0047] Figure 18 The antibacterial results of different column volume eluents provided for Optimization Example 1;

[0048] Figure 19 High-pressure liquid chromatography spectra of different content ratios of scopolamine A and scopolamine B provided for optimization example 2;

[0049] Figure 20 This is the potted plant test result diagram of the control of rice blast by blasticide provided in Test Example 3. DETAILED DESCRIPTION

[0050] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0051] In a first aspect, an embodiment of the present invention provides a pesticidal agent selected from at least one of the compounds represented by the following structural formulas:

[0052] , wherein R is selected from a hydroxyl group or an amino substituted ester group. The blasticide has a significant inhibitory effect on rice blast fungi and can be used to treat rice blast.

[0053] In the embodiments of the present invention, the punicotin may be a single compound or a combination of two compounds. Specifically, the punicotin may be any one of the compounds represented by the following formula 1 and formula 2; or may contain the compounds represented by formula 1 and formula 2 at the same time:

[0054] Formula 1 and Formula 2.

[0055] It should be noted that, for the convenience of description, the compound shown in Formula 1 is recorded as punicolin A, and the compound shown in Formula 2 is recorded as punicolin B.

[0056] Experiments have confirmed that both pure blasticidin A and pure blasticidin B have inhibitory effects against rice blast fungi. Therefore, both can be used independently as active ingredients in pesticides targeting rice blast. They can also be mixed to form a composition as an active ingredient in a pesticide targeting rice blast. In this case, blasticidin A and blasticidin B can be used in any ratio and still have inhibitory effects against rice blast fungi. However, in the present embodiments, the preferred mass ratio of blasticidin A to pure blasticidin B is (1-9):(9-1), for example, 1:9, 2:8, 3:7, 4:6, 1:1, 6:4, 7:3, 8:2, 9:1, or any value between (1-9):(9-1).

[0057] In a second aspect, embodiments of the present invention provide a method for preparing the aforementioned scopolamine, comprising fermenting Streptomyces and then subjecting the fermentation product formed therefrom to column chromatography. The fermentation process employed in the present invention to prepare the aforementioned scopolamine avoids the formation of harmful substances during organic synthesis, is easy to implement, and has low production costs.

[0058] The specific preparation process is as follows:

[0059] S1. Fermentation:

[0060] The Streptomyces is inoculated into a culture medium, such as LB culture medium, and the culture liquid is cultured at room temperature for 1-3 days to prepare a seed liquid. The Streptomyces can be an existing Streptomyces, such as Streptomyces ( Streptomyces sp. )D828 (deposit number CGMCC No.28241).

[0061] The seed liquid is inoculated into a fermentation medium for fermentation at an inoculum size of 1%. The fermentation medium can be a conventional culture medium for fermenting Streptomyces. The culture medium of the embodiment of the present invention comprises, per liter or per kg, 35 grams of glucose, 25 grams of peanut meal, 2 grams of sodium chloride, 0.2 grams of magnesium sulfate heptahydrate, 0.2 grams of potassium hydrogen phosphate trihydrate, and 0.5 milliliters of a defoamer, with a pH of 7.0-7.2.

[0062] The fermentation medium was fermented in a 200-liter bioreactor at a liquid fill ratio of 70-75%. The fermentation tank parameters were: a height-to-diameter ratio of 2.0-2.5:1; and a three-layer impeller system consisting of, from top to bottom, a six-blade turbine impeller, a four-blade axial flow impeller, and a six-blade turbine impeller. Fermentation process parameters were: a tank pressure of 0.05-0.06 MPa, an air flow of 50-75 L / min, an agitator speed of 350-450 rpm, a temperature of 28-30°C, and a fermentation cycle of 4-5 days.

[0063] S2, separation:

[0064] After the fermentation is completed, the fungus residue is collected by tubular centrifugation; the obtained fungus residue is thoroughly mixed with anhydrous ethanol in a mass ratio of 1:1-2, and the extraction is repeated 3 times. The extracts obtained from the 3 times are mixed and placed at 55-60℃ under vacuum and dried until they become a paste.

[0065] S3, column chromatography;

[0066] Mix the paste obtained in S2 with silica gel powder in a 1:1 mass ratio and load onto a 1 L silica gel column using a dry method. Perform isocratic elution using an eluent, such as a mixture of an ether solvent and an ester solvent, such as petroleum ether 60-90 and ethyl acetate in a 2:1 volume ratio.

[0067] After elution, 3 to 12 column volumes of eluate are collected, preferably 6 to 9 column volumes of eluate are collected, and the collected eluate is then dried to obtain a white powder.

[0068] The white powder contains blasticidin A and blasticidin B, is capable of inhibiting rice blast fungi, and can be used as an active ingredient of pesticide for treating rice blast.

[0069] In a preferred embodiment of the present invention, the white powder is further post-processed to obtain peccaryin with modified purity or pure peccaryin A or pure peccaryin B.

[0070] Specifically, S4, post-processing:

[0071] S4.1, recrystallization;

[0072] A C1-C3 monohydric alcohol solvent, such as a 60-80% methanol aqueous solution, and the white powder formed by S3 are mixed and dissolved under heating conditions (e.g., 60-80°C) to form a saturated solution. An adsorbent, such as 0.3-0.8% activated carbon, is added to the saturated solution and stirred thoroughly. The solution is then filtered to remove insoluble matter to obtain a filtrate. The filtrate is then gradually cooled to room temperature, for example, at a cooling rate of 1-2°C / min, and the precipitated white crystals are collected.

[0073] After testing, it was found that the white crystals were a mixture of blastocin A and blastocin B, wherein the mass ratio of blastocin A to blastocin B was (6-7):(4-3).

[0074] S4.2 Liquid phase purification;

[0075] If pure pesticin A and pesticin B are desired, the white crystals obtained in S4.1 can be further purified by liquid phase.

[0076] Specifically, semi-preparative liquid phase purification was used for separation and purification. Liquid phase purification conditions included isocratic elution using a mixture of a nitrile solvent and water. For example, isocratic elution was performed using any ratio of acetonitrile:water (70:30, 71:29, 72:28, 73:27, 74:26, 75:25, or any ratio between (70-75):(25-30). The flow rate was 7 mL / min, the wavelength was 200 nm, and the column temperature was 30-40°C.

[0077] It should be noted that, since the contents of pecctalcin A and pecctalcin B in the white crystals obtained in S4.1 are relatively fixed, if the ratio of pecctalcin A and pecctalcin B needs to be adjusted, the pecctalcin A and pecctalcin B obtained in S4.2 can be mixed in the desired ratio.

[0078] In a third aspect, an embodiment of the present invention provides a pesticide formulation comprising the aforementioned pesticidal agent and existing auxiliary materials capable of preparing the pesticide formulation, including but not limited to solvents, cosolvents, wetting agents, adhesives, and penetrants.

[0079] Specific solvents include, but are not limited to, ethanol, xylene, methanol, cyclohexanone, toluene, DMF, acetonitrile, or DMAC. Cosolvents include, but are not limited to, NMP, PBO, APEO, biodiesel, methyl oleate, DMSO, and propylene glycol. Wetting agents include, but are not limited to, sodium butylnaphthalenesulfonate, sodium lauryl sulfate, fatty alcohol ethylene oxide condensate, sulfosuccinates, nonylphenol polyoxyethylene ether, or T-1004. Adhesives include, but are not limited to, starch paste, gelatin, mineral oil, molasses, gum, CMC, and PVA. Penetrants include, but are not limited to, polyether-modified trisiloxanes, fatty alcohol polyoxyethylene ethers (such as penetrant JFC), polyether-modified heptamethyl trisiloxanes, alkylphenol polyoxyethylene ethers (such as NP-10), water-soluble azones (laurozone), and penetrant T (dioctyl sulfosuccinate).

[0080] Furthermore, the mass concentration of scopolamine in the pesticide formulation is 0.1-10%, for example, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any value between 0.1-10%. It will be understood that the mass concentration of scopolamine in the pesticide formulation provided in the embodiments of the present invention is merely exemplary, and the mass concentration of scopolamine in the pesticide formulation can be any concentration. If the concentration is too high, it can be appropriately diluted before use.

[0081] Furthermore, the dosage form of the pesticide includes but is not limited to solid dosage forms such as powder, wettable powder, granule or water granule, or liquid dosage forms such as emulsifiable concentrate, suspension, aqueous solution or microemulsion, or special dosage forms such as smoke agent and seed coating agent.

[0082] In a fourth aspect, an embodiment of the present invention provides a method for preventing and controlling rice blast, comprising: applying the above-mentioned blasticide or pesticide agent to the leaves of vegetation.

[0083] The effective concentration of scopolamine on plant leaves is 7-220 mg / L. This means that when using pesticides, scopolamine or pesticides can be diluted to achieve the above effective concentration.

[0084] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0085] Example 1

[0086] An embodiment of the present invention provides a method for preparing peccoli spp. (a mixture of peccoli spp. A and peccoli spp. B), comprising:

[0087] 1) Pick Streptomyces ( Streptomyces sp. ) D828 (deposit number CGMCC No. 28241) strain was inoculated into LB culture medium and cultured at 30℃ with shaking for 3 days to prepare seed solution.

[0088] 2) Inoculate the fermentation medium with the seed solution at a 1% ratio. Per liter or per kg of medium, the following ingredients are present: 35 g glucose, 25 g peanut meal, 2 g sodium chloride, 0.2 g magnesium sulfate heptahydrate, 0.2 g potassium hydrogen phosphate trihydrate, 0.5 ml defoamer, pH 7.0-7.2.

[0089] 3) The fermentation medium was fermented in a 200 L bioreactor at a 70% liquid fill rate. The fermentor parameters were: a height-to-diameter ratio of 2.0:1; and a three-layer impeller configuration consisting of, from top to bottom, a six-blade turbine impeller, a four-blade axial flow impeller, and a six-blade turbine impeller. The fermentation process parameters were: a tank pressure of 0.055 MPa, an air flow of 60 L / min, an agitator speed of 400 rpm, a temperature of 28°C, and a fermentation period of 4 days.

[0090] 4) After fermentation, the residue was collected by tubular centrifugation. The residue was thoroughly mixed with anhydrous ethanol in a 1:1 mass ratio, and the extraction was repeated three times. The three extracts were combined and vacuum-dried at 60°C until a paste was formed.

[0091] 5) Mix the resulting paste with silica gel powder in a 1:1 mass ratio and load onto a 1 L silica gel column using a dry method. Using an elution solvent (petroleum ether 60-90:ethyl acetate = 2:1), collect the eluate from the 6th to 9th column volume and vacuum dry at 60°C until a white powder is obtained.

[0092] 6) Dissolve the white powder and 70% methanol in water at 75°C to form a saturated solution. Add 0.5% activated carbon and stir thoroughly. Remove the insoluble matter by filtration. Cool the filtrate to 25°C at a rate of 1°C / min. Collect the white crystals precipitated during the cooling period ( Figure 1 ).

[0093] Representation 1

[0094] The above white crystals were dissolved in an appropriate amount of methanol and the content was tested using analytical liquid chromatography. The specific liquid chromatography detection method was: mobile phase (acetonitrile: water = 75:25), flow rate 0.8-1 mL / min, wavelength 200 nm, chromatographic column C18 4.6um*250mm, column temperature 30-40℃. Figure 2 .

[0095] according to Figure 2 It can be seen that the white crystals contain two substances, named peccotrin A and peccotrin B. The peak time of peccotrin A is 9.8-9.9 min, and its peak area accounts for about 65%; while the peak time of peccotrin B is 9.0-9.1 min, and its peak area accounts for about 35%.

[0096] Example 2

[0097] The present invention provides a method for preparing peccotrin (peccotrin A and peccotrin B), comprising:

[0098] The white crystals formed in Example 1 were finely separated and purified using a semi-preparative liquid phase. The specific liquid phase purification method was as follows: mobile phase (acetonitrile:water = 70:30), flow rate 7 mL / min, wavelength 200 nm, chromatographic column, column temperature 40°C.

[0099] The peak A eluate collected at 9.5-10.0 min was concentrated and dried at 60°C to a white powder to obtain a standard of 99% purity of peccaryin A. The HPLC spectrum and sample pictures are shown in Figure 3 .

[0100] The B peak eluate collected at 8.5-9.5 min was concentrated and dried at 60°C to a white powder to obtain a 99% purity ≥ peccaryin B standard. The HPLC spectrum and sample images are shown in Figure 4 .

[0101] Representation 2

[0102] The characterization of the pesticidalin A prepared in Example 2 was carried out. Figure 5-10 ,in, Figure 5 The mass spectrum of punicolin A prepared in Example 2 is shown; Figure 6 shows the hydrogen spectrum of punicolin A prepared in Example 2; Figure 7 The carbon spectrum of punicolin A prepared in Example 2 is shown; Figure 8 HSQC spectrum of punicolin A prepared in Example 2 is shown; Figure 9 The scutellarin A prepared in Example 2 is shown in FIG. 1 H- 1 H COSY spectrum; Figure 10 The HMBC spectrum of punicolin A prepared in Example 2 is shown.

[0103] The mass spectrum shows that it contains 4 adduct ion peaks, namely: [MH] - m / z 748.46387, [M+Cl] - m / z 784.44049, [M+HCOO] - m / z 794.46942 and [M+CH3COO] - m / z 808.48511. Five fragmentation ion peaks were found: m / z 85.02811, m / z 123.04392, m / z 601.37305 and m / z 619.38330.

[0104] The H NMR spectrum showed that it contained 67 proton signals, of which 5 were active hydrogens (δ 6.47 m, 5.48 brs, 5.03 brs, 4.59 brs, 4.47 brs) and 27 were methyl hydrogens (δ 0.89 s, δ 1.14 d, 0.89 d, 0.78d, 0.81 d, 0.70 d, 0.84 d, 1.41 s, 1.36 s).

[0105] 13 The C NMR-HSQC spectrum showed that it contained 41 carbon signals, including 9 primary carbons (δ 18.91, 10.93, 18.02,19.44, 12.83, 7.43, 15.83, 10.78, 13.38), 9 secondary carbons (δ 34.26, 26.86, 25.47, 34.77, 41.31, 43.64, 37.51, 36.22, 35.42), and 17 tertiary carbons (δ 117.47, 79.24, 129.14, 80.80, 129.85, 137.43, 34.60, 31.08, 82.34, 97.59, 72.71, 73.52, 71.75,31.53, 31.20, 76.35, 48.96), 6 quaternary carbons (δ 93.44, 131.81, 134.11, 171.91, 156.45,214.51); further, 1 acetal group, 1 ketal group, 6 alkenyl groups, 1 amide group, 1 lactone group, 1 ketone group, and 8 tertiary carbons were found in an oxidized state (δ 71.75-97.59).

[0106] Combine 1 H- 1 The structural relationship of punicolin A can be obtained based on H COSY-HMBC spectrum and mass spectrometry data.

[0107] Representation 3

[0108] The characterization of the pesticidalin B prepared in Example 2 was carried out. Figures 11-16 . Figure 11 shows the mass spectrum of punicolin B prepared in Example 2; Figure 12 1H spectrum of peccolicin B prepared in Example 2; Figure 13 The carbon spectrum of punicolin B prepared in Example 2 is shown; Figure 14 HSQC spectrum of punicolin B prepared in Example 2 is shown; Figure 15 The scutellarin B prepared in Example 2 1 H- 1H COSY spectrum; Figure 16 The HMBC spectrum of punicolin B prepared in Example 2 is shown.

[0109] The mass spectrometry results showed that it contained three adduct ion peaks, namely: [M+NH4] + m / z 724.49774, [M+Na] + m / z 729.45398 and [M+K] + m / z 745.42700, and four fragmentation ion peaks were found, namely: m / z 671.45474, m / z 599.40132, m / z 541.38794 and m / z 523.37706.

[0110] The H NMR spectrum showed that it contained 66 proton signals, of which 4 were active hydrogens (δ 5.47 brs, 4.84 brs, 4.78 brs, 4.57 brs) and 27 were methyl hydrogens (δ 0.90 s, 0.89 d, 1.12 d, 0.82 d, 0.85 d,0.79 d, 0.71 d, 1.41 t, 1.37 t).

[0111] 13 The C NMR-HSQC spectrum showed that it contained 40 carbon signals, including 9 primary carbons, 9 secondary carbons, 17 tertiary carbons, and 5 quaternary carbons. Furthermore, it was found that 7 tertiary carbons contained oxygen, 4 tertiary carbons contained olefins, 1 tertiary carbon contained acetal, 1 quaternary carbon contained ketone, 1 quaternary carbon contained ester, 1 quaternary carbon contained ketal, and 2 quaternary carbons contained olefins.

[0112] Combine 1 H- 1 The structural relationship of punicolin B can be obtained from the H COSY-HMBC spectrum and mass spectrometry data.

[0113] Test Example 1 Antibacterial Effect Test

[0114] The MIC values ​​of blasticidin A, blasticidin B, blastifungin and blastamide against the rice blast pathogen were tested by plate dilution method. The groups are as follows:

[0115] Group 1: Pure ramectin A was dissolved in a small amount of methanol and then added to the culture medium to achieve final concentrations of 0.01, 0.03, 0.15, 0.75, 1.55, 3.12, 6.25, 12.50, and 25.00 mg / L.

[0116] Group 2: Pure ramectin B was dissolved in a small amount of methanol and then added to the culture medium to achieve final concentrations of 0.01, 0.03, 0.15, 0.75, 1.55, 3.12, 6.25, 12.50, and 25.00 mg / L.

[0117] The third group: Blastoflurane, a common pesticide for preventing and controlling rice blast, was selected and added to the culture medium to achieve final concentrations of 7.80, 15.60, 31.20, 62.50, and 125.00 mg / L.

[0118] Group 4: Blastamide, a commonly used pesticide for preventing and controlling rice blast, was selected and added to the culture medium to a final concentration of 100, 200, and 400 mg / L.

[0119] All treatment groups were cultured to the 6th day, and their MIC values ​​against rice blast pathogen were determined.

[0120] Results see Figure 17 ,according to Figure 17 It can be seen that the MIC values ​​of blasticidin A and blasticidin B provided in the examples of the present invention against the rice blast pathogen are 0.15 mg / L, respectively. The MIC value of blasticillin against the rice blast pathogen is 60 mg / L, which is 1 / 400 of the efficacy of blasticidin A or B. The MIC value of the pesticide blasticillamide against the rice blast pathogen is 200 mg / L, which is 1 / 1333 of the efficacy of blasticidin A or B. These results show that blasticidin A and blasticidin B have a unique and strong inhibitory effect on the rice blast pathogen, significantly outperforming existing pesticides for controlling rice blast.

[0121] Optimization Example 1

[0122] The white powder formed in 5) of Example 1 was obtained by referring to the preparation method provided in Example 1. The only difference was that different column volumes of eluent were collected. In this optimized example, 1-3 column volumes of eluent, 3-6 column volumes of eluent, and 9-12 column volumes of eluent were collected. The white powder formed by the above eluents and the white powder of Example 1 were added dropwise to a 1×10 6 cfu / mL of blast fungus spore culture medium, and after culturing at 26℃ for 7 days, the antibacterial activity was observed. Figure 18 .

[0123] according to Figure 18 It can be seen that the 3rd to 12th column volumes have an antibacterial effect on rice blast fungus, and the 6th to 9th column volumes have a more excellent antibacterial effect on rice blast fungus.

[0124] Optimization Example 2

[0125] This optimization example provides a series of pesticidal compounds and their preparation methods, which are mixtures of pesticidal compound A and pesticidal compound B, as follows:

[0126] (1) According to the preparation method of Example 1, the eluate of the 3rd to 6th column volumes was collected to prepare pesticidal. After liquid phase testing, the content ratio of pesticidal A to pesticidal B in the pesticidal was 70%:30% (see Figure 19 Middle A).

[0127] (2) The pesticidalin A and pesticidalin B prepared in Example 2 were mixed in a mass ratio of 2.5:7.5 to form pesticidalin. After liquid phase testing, the content ratio of pesticidalin A to pesticidalin B in the pesticidalin was 25%:75% (see Figure 19 Middle B).

[0128] (3) The pesticidalin A and pesticidalin B prepared in Example 2 were mixed in a mass ratio of 5.5:4.5 to form pesticidalin. After liquid phase testing, the content ratio of pesticidalin A to pesticidalin B in the pesticidalin was 55%:45% (see Figure 19 Middle C).

[0129] It can be seen that if pecctin is a mixture of pecctin A and pecctin B, it can be prepared directly by fermentation, or by mixing high-purity pecctin A and pecctin B.

[0130] Test Example 3

[0131] The blastocin provided in Optimization Example 2 (the content ratio of blastocin A and blastocin B is 70%:30%) was dissolved in a small amount of ethanol to prepare a 100 mg / L aqueous solution, which was evenly sprayed on the surface of rice leaves pre-inoculated with the rice blast pathogen.

[0132] Results see Figure 20 ,according to Figure 20 It can be seen that the blastocin provided by the embodiment of the present invention can effectively prevent and control rice blast, and there is no obvious difference between the rice leaves and healthy leaves.

[0133] Test Example 4 Field Control Effectiveness Test

[0134] Different amounts of punicolin (with a 70%:30% ratio of punicolin A to punicolin B) were weighed and dissolved in a solution containing water, ethanol, and DMSO (cosolvent). Appropriate amounts of sodium lauryl sulfate (wetting agent), gelatin (adhesive), and fatty alcohol polyoxyethylene ether (penetrant) were then added to the solution. The resulting liquid pesticide formulations had concentrations of 0.3125%, 0.625%, 1.25%, 2.5%, 5%, and 10%, respectively. These liquid pesticide formulations were then applied according to the following grouping and method.

[0135] Control group: 100 mL of mixed solvent without blasticidin was used as the control, diluted 450 times with water, and sprayed on rice leaves in the early stage of rice blast. The control effect was investigated 14 days after application.

[0136] Group 1: Dilute 100 mL of 0.3125% liquid pesticide 450 times with water and spray it on rice leaves in the early stage of rice blast. The control effect was evaluated 14 days after spraying.

[0137] Group 2: Dilute 100 mL of 0.625% liquid pesticide 450 times with water and spray it on rice leaves in the early stage of rice blast. The control effect was evaluated 14 days after spraying.

[0138] Group 3: Dilute 100 mL of 1.25% liquid pesticide 450 times with water and spray it on rice leaves in the early stage of rice blast. The control effect was evaluated 14 days after spraying.

[0139] Group 4: Dilute 100 mL of 2.5% liquid pesticide 450 times with water and spray it on rice leaves in the early stage of rice blast. The control effect was evaluated 14 days after spraying.

[0140] Group 5: Dilute 100 mL of 5% liquid pesticide 450 times with water and spray it on rice leaves in the early stage of rice blast. The control effect was evaluated 14 days after spraying.

[0141] Group 6: Dilute 100 mL of 10% liquid pesticide 450 times with water and spray it on rice leaves in the early stage of rice blast. The control effect was evaluated 14 days after spraying.

[0142] The results are shown in the table below.

[0143]

[0144] Substituting the above results into the Probit curve for calculation, it can be seen that when the effective amount of blasticidin reaches 4.5 grams per mu, the control effect on rice blast can reach more than 90%.

[0145] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A pesticidal agent, characterized in that It is composed of the compounds shown in the following formula 1 and formula 2: Formula 1 and Formula 2, wherein the mass ratio of the compound represented by Formula 1 to the compound represented by Formula 2 is 70%:30%; The preparation method of the pesticidal is as follows: fermenting Streptomyces, and then performing column chromatography on the fermentation product formed by the fermentation, wherein the eluent used in the column chromatography is a mixture of petroleum ether 60-90 and ethyl acetate in a volume ratio of 2:1 to form an eluent; After elution, collect 6-9 column volumes of eluate.

2. The pesticidal agent according to claim 1, characterized in that The preparation method Include any of the following steps: (1) Comprising: column chromatography followed by recrystallization; (2) Includes: column chromatography followed by recrystallization and liquid phase purification.

3. The pesticidal agent according to claim 2, characterized in that The conditions in the preparation method meet the following requirements: (1) Recrystallization comprises: mixing and dissolving a C1-C3 monohydric alcohol solvent and a material formed by column chromatography under heating conditions to form a saturated solution, then mixing the saturated solution with an adsorbent and filtering to form a filtrate, and gradient cooling the filtrate; (2) Liquid phase purification: gradient elution using a mixed solvent of nitrile solvent and water.

4. A pesticide agent, characterized in that: It comprises the pesticidal agent according to claim 1.

5. The pesticide according to claim 4, characterized in that The pesticide agent meets at least one of the following requirements: (1) the mass concentration of scutellarin in the pesticide agent is 0.1-10%; (2) The pesticide formulation further comprises at least one of a solvent, a cosolvent, a wetting agent, an adhesive and a penetrant.

6. A method for preventing and controlling rice blast, characterized in that: include: The pesticidal agent according to claim 1 or the pesticide according to claim 4 or 5 is applied to the leaves of vegetation.

7. The method for preventing and controlling rice blast according to claim 6, characterized in that: The effective concentration of the pesticidal agent acting on the vegetation leaf surface is 7-220 mg / L.

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