Application of small molecular substance TH287 in prevention and treatment of rice blast

Abstract

The invention belongs to the field of plant protection, and particularly relates to application of a small molecule substance TH287 in preventing and treating rice blast. The small-molecule inhibitor TH287 provided by the invention is obtained by screening on the basis of a magnaporthe oryzae NUDIX hydrolase structure, and has very strong specificity. The compound can be used for inhibiting the activity of the magnaporthe oryzae NUDIX hydrolase, inhibiting the pathogenicity of the magnaporthe oryzae, enhancing the resistance of rice to the magnaporthe oryzae, preventing and treating the rice blast caused by the magnaporthe oryzae, preparing a bactericide for preventing and treating the rice blast caused by the magnaporthe oryzae and increasing the rice yield. The method has a great application prospect.

Description

Technical Field

[0001] The invention belongs to the field of plant protection, and particularly relates to application of a small molecule substance TH287 in preventing and controlling rice blast. Background Art

[0002] Rice (Oryza sativa) is an important food crop in the world. Its high yield and safe and stable production are directly related to the stability and development of the national economy. Rice blast, caused by Magnaporthe oryzae, is the most devastating rice disease, ranking first among fungal diseases. The global average annual rice yield reduction caused by rice blast is about 10-30%, and in severe cases, the reduction is 40-50%, or even no grain is harvested. Therefore, controlling rice blast is of great significance for increasing rice yield and ensuring food production safety. At present, the prevention and control of this disease is still mainly based on breeding disease-resistant varieties and chemical control. Due to the complex field pathotypes of rice blast and the rapid changes in population composition, disease-resistant varieties lose their disease resistance after 3-5 years of promotion. Chemical control is the fastest and most direct control method, but it also has the greatest harm to the environment and is easy to cause pathogens to develop drug resistance.

[0003] At present, the fungicides used for the prevention and control of rice blast fungus mainly include sterol demethylation inhibitors (DMIs), quinone outside inhibitors (QoIs) and melanin biosynthesis inhibitors (MBIs). However, due to the long-term use of the above fungicides, rice blast fungus has gradually developed resistance. Therefore, it is urgent to develop new targets to develop new, efficient and low-toxic fungicides.

[0004] When plant pathogens interact with their hosts, a complex "arms race" is taking place. When attacked by pathogens, receptors on plant cell membranes recognize the conserved pattern molecules PAMPs (Pathogen-associated Molecular Patterns) secreted by pathogens, stimulating basic disease resistance PTI (PAMP-triggered immunity), accompanied by the accumulation of a large amount of reactive oxygen. In order to inhibit the host's PTI, pathogens secrete a large number of effectors that enter plant cells or extracellular apoplast space through vesicle transport, thereby inhibiting the host's defense response. Vesicle transport is a key process in the secretion of effectors of rice blast fungus. Abnormalities in this process will lead to abnormal secretion of effectors, which will accumulate in the cell and cause stress. Therefore, analyzing the molecular mechanism of effector transport and secretion not only helps to understand the pathogenic mechanism of pathogens, but also has important reference value for the design of new low-toxic and high-efficiency fungicides. Summary of the invention

[0005] In order to overcome at least one problem existing in the prior art, the present invention screened small molecules mediated by protein structure and identified the small molecule TH287 (small molecule inhibitor TH287), which can inhibit the NUDIX hydrolase activity of rice blast fungus, and is of great significance in inhibiting the pathogenicity of the pathogen and increasing rice yield.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention firstly provides the application of NUDIX12 protein in screening drugs for preventing and treating rice blast caused by rice blast fungus.

[0008] Specifically, it is based on the structural model of NUDIX12 protein and the structural model of nucleic acid oxidative damage repair enzyme MTH1 protein, the structural model of the compound to be tested is docked with them, the docking results are analyzed using Auto Dock Tools, and the docking model is visualized using PyMOL software; if the compound to be tested occupies the pocket position of the NUDIX12 enzyme activity pocket, resulting in MTH1 being unable to directly contact the substrate NADH, the candidate compound for preventing and controlling rice blast is determined.

[0009] Optionally, a substrate NADH structural model is also used as a control to confirm that the candidate compound can hinder the correct binding of NADH to the NUDIX12 enzyme activity site.

[0010] Specifically, the PDB ID of the structural model of the NUDIX12 protein is G4NFV7), and the PDB ID of the structural model of the nucleic acid oxidative damage repair enzyme MTH1 protein is P36639.

[0011] Through the above method, the present invention screened out a small molecule substance TH287 (6-(2,3-dichlorophenyl)-N4-methylpyrimidine-2,4-diamine), whose molecular formula is shown in the following formula (I):

[0012]

[0013] This compound is a known compound that has been used as an inhibitor of MTH1 in mammals and has also been reported in cancer treatment. However, there are no reports of its use in preventing and controlling rice blast.

[0014] Therefore, the present invention further provides a method for controlling or preventing rice blast, which comprises applying a compound of formula (I) or an agrochemically active salt thereof, such as an alkali addition salt of an inorganic base or an organic base, more specifically a potassium salt, sodium salt, ammonium salt, dimethylamine salt, or isopropylamine salt thereof, to rice, its planting site, or its propagation material.

[0015] Specifically, the compound of formula (I) is prepared into a drug in the form of one of emulsifiable concentrates, suspensions, wettable powders, dusts, granules, aqueous solutions, poison baits, mother liquids and mother powders; its content in the drug is 1 to 99% by weight, preferably 10 to 80% by weight, preferably 15 to 50% by weight; when used in the drug, the effective dose concentration is 0.1 ug / ml to 100 ug / ml, and the application amount is 10 to 500 g / ha, preferably 50-200 g / ha.

[0016] The present invention also provides the use of the compound of formula (I) or its agrochemically active salt for enhancing the resistance of rice to rice blast fungus, preferably the agrochemically active salt is a base addition salt such as an inorganic base and an organic base, more specifically its potassium salt, sodium salt, ammonium salt, dimethylamine salt, isopropylamine salt.

[0017] The present invention also provides the use of the compound of formula (I) or its agrochemically active salt in the preparation of pesticides for controlling or preventing rice from being infected by rice blast fungi or causing diseases.

[0018] Specifically, the pesticide is a drug in the form of one of emulsifiable concentrate, suspension, wettable powder, dust, granule, aqueous solution, poison bait, mother solution and mother powder, and further comprises auxiliary ingredients acceptable in pesticides.

[0019] Preferably, the pesticide is formulated in a form suitable for spray application.

[0020] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0021] (1) The present invention provides an effective method for screening drugs for preventing and treating rice blast caused by rice blast fungus, wherein the small molecule inhibitor TH287 screened and identified is obtained based on the structure screening of NUDIX hydrolase of rice blast fungus and has strong specificity; (2) The above-mentioned small molecule inhibitor TH287 is obtained by screening the specific NUDIX hydrolase structure of rice blast fungus, and the homologous proteins in other species are very different. Therefore, it has the characteristics of low toxicity and high safety as a fungicide; (3) The small molecule inhibitor TH287 has a strong inhibitory activity on the pathogenicity of rice blast fungus and can increase the yield of rice to a certain extent; (4) The small molecule inhibitor TH287 may also play an important biological function in regulating the conidia production and appressorium formation pathways of the pathogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the binding region of TH287 and NUDIX in one embodiment of the present invention;

[0023] Figure 2This is a schematic diagram of the results of NUDIX12 enzyme activity verification in one embodiment of the present invention; wherein NUDIX12 hydrolase can degrade a variety of dinucleotide substances;

[0024] Figure 3 This is a schematic diagram of the results of the inhibition of NUDIX12 and its rice homologous protease activity by compound TH287 in one embodiment of the present invention; wherein NADH is used as the substrate;

[0025] Figure 4 The figure is a schematic diagram of the results of the interaction verification between NUDIX12 and its rice homologous protein and compound TH287 in one embodiment of the present invention; wherein, the binding ability of NUDIX12 and its rice homologous protein with TH287 was verified in vitro using microscale thermophoresis (MST) technology, and the Kd reaction dissociation constant, the smaller the value, the stronger the binding;

[0026] Figure 5 This is a schematic diagram of the results of the compound TH287 inhibiting the pathogenicity of rice blast fungus indoors in one embodiment of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described below through specific embodiments in order to provide a better understanding of the present invention. The illustrative embodiments of the present invention and their description are only used to explain the present invention and do not constitute a limitation of the present invention.

[0028] Example 1: Screening of compound TH287

[0029] Based on the structure screening of NUDIX hydrolase from rice blast fungus, the small molecule TH287 was identified. The following steps were included: the structural model of MGG_08699 protein (NUDIX12, see: Chen Long et al. "Bioinformatics and expression characteristics of NUDIX hydrolase family from rice blast fungus." Molecular Plant Breeding 14.5 (2016): 7.) (PDB ID: G4NFV7) was derived from the Alpha Fold protein structure database (https: / / alphafold.ebi.ac.uk / ), and the ligand molecule 3D structure model was downloaded from the PubChem website. Auto Dock Tools (version 1.5.6) software was used to add polar hydrogen to the protein structure and calculate the Gasteiger charge. The Auto Dock Tools ligand preparation program was called for batch conversion, and Auto Dock Vina was run for batch docking. Each molecule docking output the top 10 docking models with the highest affinity value from low to high, and the python program written was used to extract the top 10 docking models and corresponding molecular structures with the highest affinity value from low to high in all docking results. The docking results were analyzed using AutoDock Tools, and the docking model was visualized using PyMOL (version 2.6.0a0), from which the small molecule TH287 was screened.

[0030] Screening results such as Figure 1 As shown, Figure 1 Part A and Part B show the binding states of nucleic acid oxidative damage repair enzyme MTH1 protein with substrate NADH and small molecule substance TH187 respectively. Both are bound at the same position, that is, TH287 occupies the position of NADH, which makes MTH1 unable to directly contact substrate NADH. Figure 1 Part C of the image identifies the binding area of ​​NUDIX12 with TH287 and the substrate NADH. TH287 binds to the enzyme pocket of NUDIX12, hindering the binding of NUDIX12 with NADH. This indicates that there are significant differences in the binding sites of TH287 with MTH1 and NUDIX12.

[0031] Example 2: Functional assay of compound TH287 inhibiting the target protein MoNud5 of rice blast fungus

[0032] 1. The coding sequences of NUDIX12-GFP were constructed in pYF11 vectors and transferred into rice blast fungi by protoplast transformation. After the positive transformants were shaken in CM medium for 48 hours, the mycelial balls were collected and pressed dry with absorbent paper. After grinding with liquid nitrogen, they were added to 1 ml of protein lysis buffer (Biyuntian P0013B). The protein was extracted and then purified by GFP beads (ChromoTek GFP-Trap Agrose, GTA-20) to obtain the corresponding protein.

[0033] NUDIX12-HIS protein and HIS protein were constructed in the pET32a vector and transformed into Escherichia coli BL21 to obtain a prokaryotic expression strain. For prokaryotic expression induction conditions, 100 μM of isopropyl-β-D-thiogalactoside (IPTG) was added to 200 ml of bacterial solution, the induction temperature was 25 degrees Celsius, and the induction was performed for 4 hours. The bacteria obtained by centrifugation were added with 1 ml of PBS buffer and ultrasonically broken (power 20%, breaking time 5s, interval time 5s) until clarified, and the supernatant was obtained by centrifugation at 3600rpm for 15 minutes to obtain the protein. The protein was then purified by HIS beads to obtain NUDIX12-HIS protein and HIS protein.

[0034] 2. The function of TH287 in inhibiting NUDIX12 was verified by the following method, which comprises the steps of:

[0035] Nudix hydrolase activity assay method: The assay reaction was carried out in a 50 μL reaction system, which included 50 mM Tris-HCl (pH 8.5), 2 μg NUDIX12-GFP, NUDIX12 ED -GFP protein, GFP protein, NUDIX12-HIS protein, rice homologous protein OsNUDIX12 (OsJ05P0700)-HIS and HIS protein, 5mM MgCl2, 1mM dithiothreitol, 2mM substrate (NADH) and 4U alkaline phosphatase (sigma: P0114-10KU). In the TH287 treatment group, the control group dimethyl sulfoxide (DMSO) was used as the control, and the TH287 concentrations were 1mM, 100μM and 10μM respectively.

[0036] After incubation at 37°C for 30 minutes, 150 μL of 1N HCl was added to terminate the reaction, followed by the addition of 100 μL of water and 700 μL of a mixture of 0.42% molybdate and 10% ascorbic acid (6:1). The reaction tube was placed in a 45°C water bath for 20 minutes to develop color, then cooled to room temperature. The solution was measured at A820 using a spectrophotometer. The results were as follows: Figure 2 and Figure 3 shown.

[0037] The inventors' research shows that the NUDIX12 protein expressed by the rice blast fungus has NUDIX hydrolase activity. Compared with the control protein GFP (Green fluorescent protein), the NUDIX12 protein can more effectively hydrolyze intracellular dinucleotide substances, including reduced nicotinamide adenine dinucleotide (NADH), adenosine diphosphate ribose (ADPR) and flavin adenine dinucleotide (FLAVIN). By deleting the key site of NUDIX12 enzyme activity (amino acids 104 to 111), the NUDIX12 ED ), the enzyme activities of the three substrates were reduced to 55.95%, 54.71% and 38.27% of the original activities respectively ( Figure 2 ).

[0038] The compound TH287 targets the NUDIX12 protein of rice blast fungus, binds to it specifically, and inhibits its hydrolase activity. Compared with the control group of dimethyl sulfoxide (DMSO), after adding 1mM, 100μM, and 10μM TH287, the hydrolase activity of NUDIX12 was significantly reduced, with inhibition rates reaching 88.57%, 62.86%, and 48.51%, respectively. However, there was no significant difference in the hydrolase activity of the rice homologous protein OsNUDIX12 ( Figure 3 ).

[0039] Example 3: Compound TH287 specifically binds to pathogenic bacteria MoNud5

[0040] In order to verify that TH287 specifically binds to and inhibits the activity of NUDIX12 of rice blast fungus, the dissociation constant (Kd) value of TH287 and NUDIX12 and TH287 and OsNUDIX12 was determined by microscale thermophoresis (MST) experiment, wherein OsNUDIX12 is the NIUDIX hydrolase with the highest homology to NUDIX12 in rice. Compound TH287 was purchased from Merck (Catalog No.: HY-16965).

[0041] 1. The coding sequences of NUDIX12-HIS protein and OsNUDIX12-HIS protein were constructed in pET32a vectors and transformed into Escherichia coli BL21 to obtain prokaryotic expression strains. Prokaryotic expression induction conditions: 100 μM of isopropyl-β-D-thiogalactoside (IPTG) was added to 200 ml of bacterial solution, the induction temperature was 25 degrees Celsius, and the induction was performed for 4 hours. The bacteria obtained by centrifugation were added with 1 ml of PBS buffer, and ultrasonically broken (power 20%, breaking time 5s, interval time 5s) to clarify, and centrifuged at 3600rpm for 15 minutes to obtain the supernatant. The protein was then purified by HIS beads (MCE: HY-K0210) to obtain NUDIX12-HIS protein and OsNUDIX12-HIS protein.

[0042] 2. MST experimental method: NUDIX12-HIS protein and OsNUDIX12-HIS protein were expressed and purified in Escherichia coli BL21, and the interaction strength between the compound and NUDIX12 and OsNUDIX12 was determined by microthermophoresis using the Monolith NT.LabelFree (Nano Temper Technologies GMBH) instrument. First, the RED-NHS second-generation fluorescent probe was labeled on NUDIX12-HIS and OsNUDIX12-HIS proteins using the MonolithTM RED-NHS second-generation protein labeling kit (Cat#MO-L011) and stored in a buffer containing 130mM NaHCO3, 50mM NaCl, and pH 8.2–8.3 at room temperature. Compound TH287 was diluted into different concentration gradients (1 mM as the highest concentration, each sample was half of the previous concentration, and a total of 16 concentration gradients were diluted), and incubated with labeled NUDIX12-His and OsNUDIX12-His proteins for 10 minutes; the above samples were added to NT.Label Free labeled capillaries, and the values ​​were measured at 20% LED and 40% MST parameters. The KD fitting function of Nano Temper analysis software (version 1.5.41) was used to draw the curve and calculate the dissociation constant (Kd) value. The smaller the Kd value, the stronger the interaction.

[0043] The results of the MST experiment showed that the Kd value of compound TH287 for NUDIX12 protein was 7.36 μM, which was much lower than the Kd value of TH287 for rice OsNUDIX12, which was 73.86 μM. This showed that compared with the NUDIX hydrolase OsNUDIX12 in rice, TH287 had a stronger binding ability to the pathogen NUDIX12 ( Figure 4 ).

[0044] Example 4: Indoor determination of the activity of inhibiting the pathogenicity of rice blast fungus and the results

[0045] This example verifies the activity of compound TH287 in inhibiting the pathogenicity of rice blast fungus indoors, which includes the steps of:

[0046] The wild-type strain Guy11 of rice blast fungus was used and preserved in our laboratory. The rice blast strain was placed on complete medium (CM) and cultured in the dark at 28°C. The strain was preserved on potato dextrose agar (PDA) at 10°C and transferred every 3 months; the strain was stored on dry filter paper for a long time and stored in a -20°C refrigerator.

[0047] The method of spraying rice leaves with spore liquid of rice blast fungus comprises:

[0048] (1) First, the rice blast fungus was induced to produce conidia. The mycelial block (2 mm × 2 mm) of the rice blast fungus strain Guy11 on the CM medium was inoculated onto the SDC medium and cultured in the dark at 28°C for 4 days. Then, the surface mycelium was scraped off and induced under a black light for 3 days to obtain conidia.

[0049] (2) Rice seedlings cultured in the greenhouse for 14 days were used for the spray inoculation experiment. 4 mL of conidia were collected from the above SDC plates at a concentration of 1×10 5 / mL, and containing gelatin with a total concentration of 0.2% (w / v), and adding different concentrations (final concentration of 1mM, 100μM and 10μM) of compound TH287. Spray it on rice leaves, culture in the dark for 24 hours, and then culture at 25 degrees with alternating light and dark for 5-7 days. Each treatment was repeated three times.

[0050] (3) Count the diseased area and calculate the inhibition rate: control effect (%) = (disease area of ​​control rice - disease area of ​​treated rice) / (disease area of ​​control rice) × 100.

[0051] The experimental results are shown in Figure 5. Figure 5 Part A shows that the pathogenicity of rice blast fungus under TH287 treatment was significantly reduced compared with the DMSO treatment in the control group; Figure 5 The results of analyzing the rice lesion area using Fijiimage J in Part B also showed that TH287 inhibited the pathogenicity of rice blast fungus to rice.

[0052] Preparation method of PDA culture medium: Wash and peel the potatoes, weigh 200g, cut into small pieces, add water and cook until soft (boil for 30 minutes), filter into a beaker with four layers of gauze, add 20g glucose and 15-20g agar powder, add water to 1L, stir to fully dissolve, divide into Erlenmeyer bottles, sterilize at 121℃ for 20 minutes, cool and set aside.

[0053] Preparation method of CM medium: Measure 50 mL of 20× nitrate (120 g sodium nitrate, 10.4 g potassium chloride, 10.4 g magnesium sulfate heptahydrate, 30.4 g potassium dihydrogen phosphate, add distilled water to dissolve to 1 L), 1000× trace elements (2.2 g zinc sulfate heptahydrate, 1.1 g boric acid, 0.5 g manganese chloride tetrahydrate, 0.5 g iron sulfate heptahydrate, 0.17 g cobalt chloride hexahydrate, 0.16 g copper sulfate pentahydrate, 0.15 g sodium manganate dihydrate, 5 g Tetrasodium EDTA, add distilled water to dissolve to 100mL) 1mL, vitamin solution (0.01g biotin, 0.01g vitamin B6, 0.01g vitamin B1, 0.01 riboflavin, 0.01 p-aminobenzoic acid, 0.01 niacin, add distilled water to dissolve to 100mL) 1mL, weigh 10g glucose, 2g peptone, 1g yeast extract, 1g casamino acid, 15g agar powder, add distilled water to quantify to 1L, divide into Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, cool and set aside.

[0054] Preparation method of spore-producing culture medium: prepare it with corn flour and rice straw, weigh 100g rice straw, add 1L water and boil for 30 minutes, then add 40g corn flour and 15g agar powder and boil for 20 minutes, finally make up to 1L with distilled water, divide it into Erlenmeyer bottles, sterilize at 121℃ for 20 minutes, and set aside after cooling.

[0055] Preparation method of SDC culture medium: weigh 120g rice straw, cut it into small pieces, add water and cook it (boil for 60 minutes), filter it into a beaker with double-layer gauze, add 40g corn flour and 15g agar powder, and boil it for 10 minutes, add water to 1L, stir to mix it thoroughly, and then divide it into Erlenmeyer bottles, sterilize it at 121℃ for 20 minutes, and set it aside after cooling.

[0056] Example 5 - Determination and results of the inhibition of conidia production and appressorium formation of Magnaporthe oryzae by compound TH287

[0057] This example verifies that compound TH287 inhibits conidia production and appressorium formation in rice blast fungus, and comprises the steps of:

[0058] First, the rice blast fungus was induced to produce conidia. The mycelial block (2mm×2mm) of the rice blast fungus strain Guy11 on the CM medium was inoculated into the SDC medium, and different concentrations of TH287 (final concentrations of 1mM, 100μM, 10μM and 1μM) were added respectively. The conidia were cultured in the dark at 28°C for 4 days and induced under black light for 3 days to obtain conidia. The collected conidial liquid was divided into two parts on average. One of them was counted under the hemocytometer to directly count the conidia yield under different TH287 treatments. The results showed that TH287 at a concentration of more than 100μM significantly inhibited the production of conidia of rice blast fungus (as shown in Table 1).

[0059] The spore solution was centrifuged at 4000 rpm for 3 minutes, washed 3 times with sterilized ddH2O, and the concentration was adjusted to 2-5×10 4 / ml, add different concentrations of TH287 (final concentration 1mM, 100μM, 10μM and 1μM), draw 20μL and drop it on a hydrophobic glass slide (12-540-A, Fisherbrand, USA), keep it moist, and culture it in the dark in a 28℃ incubator. After 24 hours of culture, observe the results of spore attachment cell formation. Each treatment was repeated three times. The attachment cell formation rate was counted and the inhibition rate was calculated, inhibition rate (%) = (control spore attachment cell formation rate-treatment group attachment cell formation rate) / (control spore attachment cell formation rate) × 100. The experimental results are shown in Table 2: It shows that TH287 at a concentration of more than 10μM significantly inhibited the attachment cell formation of rice blast fungus.

[0060] Table 1 Inhibitory activity of compound TH287 on spore germination of rice blast fungus

[0061]

[0062] Note: Each treatment was repeated three times in the experiment, and the data in the table are the average values ​​of the three repetitions.

[0063] Table 2 Inhibitory activity of compound TH287 on appressorium formation of Magnaporthe oryzae

[0064]

[0065] Note: Each treatment was repeated three times in the experiment, and the data in the table are the average values ​​of the three repetitions.

[0066] From the above implementation, it can be seen that TH287 screened by the present invention has the characteristics of simple structure, convenient preparation, low cost, strong specificity, etc., and exhibits good inhibitory effect on rice blast fungus, and is worthy of further research and development.

Claims

1. Application of NUDIX12 protein in screening drugs for preventing and treating rice blast caused by rice blast fungus.

2. The use according to claim 1, characterized in that It is based on the structural model of NUDIX12 protein and the structural model of nucleic acid oxidative damage repair enzyme MTH1 protein. The structural model of the compound to be tested is docked with them, the docking results are analyzed using Auto DockTools, and the docking model is visualized using PyMOL software; if the compound to be tested occupies the pocket position of the NUDIX12 enzyme activity pocket, which prevents MTH1 from directly contacting the substrate NADH, the candidate compound for preventing and controlling rice blast is determined.

3. The use according to claim 2, characterized in that The structural model of the substrate NADH was also used as a control to determine whether the candidate compounds could hinder the correct binding of NADH and the active site of NUDIX12.

4. The use according to claim 2, characterized in that The PDB ID of the structural model of NUDIX12 protein is G4NFV7), and the PDB ID of the structural model of nucleic acid oxidative damage repair enzyme MTH1 protein is P36639.

5. A method for controlling or preventing rice blast, comprising applying to rice, its planting site, or its propagation material a compound of formula (I), or an agrochemically active salt thereof, such as an alkali addition salt of an inorganic base or an organic base, more specifically a potassium salt, sodium salt, ammonium salt, dimethylamine salt, isopropylamine salt thereof:

6. The method according to claim 5, characterized in that The compound of formula (I) is prepared into a drug in the form of one of emulsifiable concentrates, suspensions, wettable powders, dusts, granules, aqueous solutions, poison baits, mother solutions and mother powders; the content of the compound in the drug is 1 to 99% by weight, preferably 10 to 80% by weight, preferably 15 to 50% by weight; when used in the drug, the effective dosage concentration is 0.1 ug / ml to 100 ug / ml, and the application amount is 10 to 500 g / ha, preferably 50-200 g / ha.

7. Use of the compound of formula (I) or its agrochemically active salt for enhancing the resistance of rice to rice blast fungus, Preferably, the agrochemically active salts are base addition salts such as inorganic bases and organic bases, more specifically potassium salts, sodium salts, ammonium salts, dimethylamine salts, isopropylamine salts thereof.

8. Use of a compound of formula (I) or an agrochemically active salt thereof in the preparation of a pesticide for controlling or preventing rice from being infected by rice blast fungus or causing disease, 9. The use according to claim 8, characterized in that The pesticide is a drug in the form of one of emulsifiable concentrate, suspension, wettable powder, dust, granule, aqueous solution, poison bait, mother solution and mother powder, and further comprises auxiliary ingredients acceptable in pesticides.

10. The use according to claim 9, characterized in that The pesticide is formulated in a manner suitable for spray application.

Images