Use of compound MSP-10 in the preparation of a drug for preventing and treating Magnaporthe grisea

Abstract

The application discloses application of a compound MSP-10 in preparation of a medicine for preventing and treating Magnaporthe oryzae, belongs to the field of plant fungal disease prevention, and the compound MSP-10 can prevent and treat Magnaporthe oryzae ( Magnaporthe oryzae ) or / and other pathogenic fungi. The compound MSP-10 of the application has certain prevention and treatment effect when being mixed with spore liquid of Magnaporthe oryzae and sprayed at 0 h, indicating that the compound MSP-10 can be used as a protective agent at the initial stage of Magnaporthe oryzae infection and has better effect; moreover, the rice sprayed with the compound has a good growth trend and has no obvious adverse effect, indicating that the compound is safe to the rice and has practical significance for preventing the Magnaporthe oryzae disease.

Description

Technical Field

[0001] This invention belongs to the field of plant fungal disease control, specifically involving the application of compound MSP-10 in the preparation of drugs for controlling rice blast fungus. Background Technology

[0002] Rice is one of the world's most important food crops. However, plant diseases pose a significant threat to food crop production, among which rice blast fungus (… Magnaporthe oryzae Rice blast, caused by [unspecified pathogen], is one of the most devastating crop diseases in the world, posing a significant threat to global rice production. Rice blast has a wide host range; different strains can infect at least 50 species of grasses, including core food crops such as rice, wheat, barley, and oats.

[0003] The rice blast fungus primarily infects rice in the field through conidia. These conidia are dispersed via air currents, water flow, raindrops, and insects. Conidia landing on rice leaves germinate under suitable conditions, producing specific dome-shaped appressorium structures. During development, these appressoriums accumulate a high concentration of glycerol (approximately 3.2 M), generating a turgor pressure as high as 8.0 MPa, which promotes the formation of narrow infection spikes at the base of the appressorium, infecting the host leaf. Notably, the rice blast fungus exhibits wide geographical distribution, strong environmental adaptability, and rapid genetic variation. These characteristics not only increase the difficulty of disease control but also make rice blast a hot and challenging issue in plant pathology research and disease management practice. Therefore, the development and implementation of integrated pest management strategies for rice blast are of great significance for ensuring food crop production security and maintaining global food supply stability.

[0004] Traditional disease control strategies encompass measures such as breeding disease-resistant and high-yielding crop varieties, eliminating pathogen sources, implementing chemical control, strengthening field management, optimizing water and fertilizer management, and properly disposing of infected grains and straw. Among these, chemical control, as a crucial component, has garnered significant attention and favor from agricultural researchers and practitioners due to its remarkable control effects even during periods of severe crop disease. However, in recent decades, the excessive and frequent use of chemical pesticides has led to a rapid degradation of farmland soil properties and a continuous decline in the health of farmland ecosystems. Simultaneously, long-term pesticide selection pressure has prompted the rice blast pathogen to evolve numerous pesticide-resistant physiological races, undoubtedly increasing the complexity and challenge of rice blast control.

[0005] In response to the aforementioned serious problems, there is an urgent need to accelerate the research and development of environmentally friendly and green pesticides by conducting in-depth research on the potential pathogenic targets of rice blast fungus. This initiative aims to effectively resist rice blast infection, protect rice crops from severe damage, promote sustainable agricultural development, and ensure that rice yield and quality are not affected, thereby achieving the ideal effect of pesticide control.

[0006] Peroxisomes are key organelles in fungal cells, performing multiple core metabolic functions, including fatty acid β-oxidation, reactive oxygen species (ROS) scavenging, and the glyoxylate cycle. They are often referred to as the "metabolic center" of the cell. Their biogenesis depends on a class of proteins—peroxosin—encoded by the PEX gene. Currently, 37 peroxosin species have been identified in fungi, plants, and animals. MoPex19 is one such soluble molecular chaperone that specifically recognizes and binds to newly generated peroxisomal membrane proteins (PMPs), guiding their directional transport to the peroxisomal membrane and thus promoting membrane assembly. Without MoPex19, most PMPs cannot correctly locate and integrate into the membrane, leading to peroxisomal assembly failure and complete loss of function, indicating that this protein plays an upstream role in the biogenesis pathway and has a core regulatory function.

[0007] MoPex19 is the rice blast fungus ( Magnaporthe oryzae Key proteins in the transport and maintenance of peroxisome membrane proteins (PMPs) in peroxisomes. MoPex19 The deletion mutant exhibits various abnormalities in fungal development and pathogenicity-related morphogenesis, completely losing its pathogenicity to the host. However, there are currently no publicly available pesticides targeting MoPex19. Therefore, there is a need to develop innovative pesticides targeting MoPex19 to effectively combat rice blast fungus infection and ensure the safe production of core food crops such as rice.

[0008] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to provide the application of compound MSP-10 in the preparation of drugs for the control of rice blast fungus, which solves the problem of drug resistance to pathogen-related targets of existing drugs. This compound has advantages such as significant effect, long duration of action, safety and simple application method in the control of rice blast.

[0010] To achieve the above objectives, this invention provides compound MSP-10 in the preparation of a fungicide for controlling rice blast fungus (… Magnaporthe oryzaeThe compound MSP-10 has the following chemical structural formula and is used in drugs for the treatment of or / and other pathogenic fungi:

[0011] ;

[0012] The other pathogenic fungi include: *Botrytis cinerea* (… Botrytis cinerea ), Fusarium oxysporum ( Fusarium fujikuroi Anthracnose of melons ( ) Colletotrichum orbiculare Alternaria ( Alternaria alternata ) and Anthrax bacillus fusiforme ( Colletotrichum acutatum Any one or more of the following.

[0013] A second objective of this invention is to provide the use of compound MSP-10 in the preparation of a medicament for inhibiting the hyphal growth and / or spore germination and / or appressorium development of *Strombus haematobium* or / and other pathogenic fungi, wherein the other pathogenic fungi include any one or more of *Botrytis cinerea*, *Fusarium oxysporum*, *Anthracis cucurbita*, *Alternaria alternata*, and *Anthracis acutissima*.

[0014] Preferably, the compound MSP-10 can reduce the turgor pressure of the appressorium of *Strombus oryzae*, and / or slow down the transport of glycogen from *Strombus oryzae* conidia to the appressorium, and / or slow down the rate of glycogen degradation in the appressorium.

[0015] Preferably, the concentration of compound MSP-10 is 10 to 50 ppm. In this invention, compound MSP-10 can effectively inhibit the pathogenicity of rice blast fungus spores within a concentration range of 10 to 50 ppm.

[0016] More preferably, the concentration of compound MSP-10 is 30-50 ppm. In this invention, a concentration of MSP-10 of 30-50 ppm exhibits a significant inhibitory effect on isolated barley blast disease.

[0017] More preferably, the concentration of compound MSP-10 is 50 ppm, at which concentration MSP-10 can completely inhibit isolated barley blast disease.

[0018] A third objective of this invention is to provide a drug for controlling rice blast fungus and / or other pathogenic fungi, comprising compound MSP-10; wherein the other pathogenic fungi include any one or more of the following: Botrytis cinerea, Fusarium oxysporum, Anthracnose cucurbita, Alternaria alternata, and Anthracnose fusiforme.

[0019] Preferably, it also includes an agriculturally acceptable carrier.

[0020] A fourth objective of this invention is to provide a method for controlling rice blast fungus and / or other pathogenic fungi, wherein the drug for controlling rice blast fungus and / or other pathogenic fungi is sprayed onto the leaves of plants; the other pathogenic fungi include any one or more of the following: Botrytis cinerea, Fusarium oxysporum, Anthracnose cucurbita, Alternaria alternata, and Anthracnose fusiforme.

[0021] Preferably, the concentration of the compound MSP-10 is 10 to 50 ppm.

[0022] More preferably, the concentration of the compound MSP-10 is 30 to 50 ppm.

[0023] The application of compound MSP-10 of the present invention in the preparation of drugs for controlling rice blast fungus solves the problem of drug resistance to pathogen-related targets in existing drugs, and has the following advantages:

[0024] (1) This invention first discovered that compound MSP-10 has a significant effect in inhibiting rice blast fungus and preventing rice blast disease. It can effectively inhibit the pathogenicity of rice blast fungus spores in the concentration range of 10 to 50 ppm. In particular, compound MSP-10 at 50 ppm can completely prevent and control rice blast disease, and the application method is simple.

[0025] (2) The compound MSP-10 used in this invention, under the same treatment concentration, has a significantly better control effect on rice blast disease in detached rice leaves than on detached barley leaves.

[0026] (3) The compound MSP-10 of the present invention, when mixed with rice blast fungus spore liquid and sprayed at 0 h, has a certain control effect, indicating that the compound MSP-10 of the present invention can be used as a protective agent in the early stage of rice blast infection with better effect; moreover, the rice growing condition is good after spraying the compound and there are no obvious adverse effects, indicating that the compound is safe for rice and has practical significance in preventing rice blast.

[0027] (4) The present invention found that compound MSP-10 can inhibit the mycelial growth of rice blast fungus, increase the appressorium collapse rate, significantly reduce the turgor pressure of rice blast fungus appressorium, and slow down the transport of glycogen from rice blast fungus conidia to appressorium, thus slowing down the rate of glycogen degradation in appressorium.

[0028] (5) The compound MSP-10 of the present invention can not only inhibit rice blast fungus, but also has a significant inhibitory effect on a variety of other pathogenic fungi, showing its broad-spectrum antifungal activity. In agricultural production practice, this means that the use of compound MSP-10 can simultaneously address multiple disease problems caused by different pathogenic fungi, reduce the types and frequency of pesticide use, reduce agricultural production costs, and also benefit environmental protection and the maintenance of ecological balance. It is less likely to cause environmental pollution and ecological damage, and meets the needs of modern agriculture for green and environmentally friendly pesticides. Attached Figure Description

[0029] Figure 1 This is a predicted diagram of the binding sites of compounds MSP-10 and MoPex19 in this invention.

[0030] Figure 2 The inhibition of different concentrations of compound MSP-10 on plate growth of rice blast fungus.

[0031] Figure 3 The figure shows a comparison of the diameter of rice blast fungus on plate (A) and the inhibition rate of mycelium growth (B) under different concentrations of compound MSP-10. In the figure, the different letters a, b, c, d, e or f indicate significant differences in the inhibition rate of different concentrations of compound MSP-10; p < 0.05; the data in the figure were obtained by one-way ANOVA.

[0032] Figure 4 The disease incidence of detached barley leaves (A) and detached rice leaves (B) was observed when different concentrations of the compound MSP-10 were added to the spore liquid of rice blast fungus.

[0033] Figure 5 The study investigated the disease incidence in live rice leaves after spraying with 50 ppm of compound MSP-10 at different times following inoculation with rice blast fungus spores.

[0034] Figure 6 A comparison of the relative lesion area of ​​live rice leaves after spraying with 50 ppm compound MSP-10 at different times after inoculation with rice blast fungus spore liquid.

[0035] Figure 7 The formation of appressorium at different time points when 50 ppm of compound MSP-10 was added exogenously to rice blast fungus spore liquid.

[0036] Figure 8 A comparison of the attachment formation rate induced at different time points when 50 ppm of compound MSP-10 was added exogenously to rice blast fungus spore liquid.

[0037] Figure 9The collapse of appressorium under two different concentrations of glycerol when 50 ppm of compound MSP-10 was added exogenously to rice blast fungus spore liquid.

[0038] Figure 10 Comparison of appressorium collapse rate at two different concentrations of glycerol when 50 ppm of compound MSP-10 was added exogenously to rice blast fungus spore liquid.

[0039] Figure 11 When 50 ppm of compound MSP-10 was added exogenously to the spore liquid of rice blast fungus, the glycogen transport and degradation at different time points induced by appressorium were observed (A); a comparison of the proportion of glycogen in conidia at different induction times was shown (B); and a comparison of the proportion of glycogen in appressorium was shown (C).

[0040] Figure 12 When 50 ppm of compound MSP-10 was added exogenously to the spore liquid of rice blast fungus, the lipid droplet transport and degradation at different time points induced by appressorium were observed (A); a comparison of the proportion of lipid droplets in conidia at different induction times was shown (B); and a comparison of the proportion of lipid droplets in appressorium was shown (C).

[0041] Figure 13 The inhibitory effect of 50 ppm compound MSP-10 on the growth of different plant pathogenic fungi on plate.

[0042] Figure 14 The graph shows the statistical distribution of colony diameters of different plant pathogenic fungi after treatment with 50 ppm of compound MSP-10. The black bars represent the colony diameters of pathogenic fungi that grew normally without MSP-10 (control), while the white bars represent the colony diameters of pathogenic fungi treated with MSP-10. n = 3.

[0043] Figure 15 The effect of different concentrations of compound MSP-10 on rice growth is shown; where the scale bar = 2 cm.

[0044] Note: In the figure, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] It should be noted that: Unless otherwise specified in the examples, conditions should be followed according to standard conditions or the manufacturer's recommendations. Instruments whose manufacturers are not specified are all commercially available products. Raw materials and reagents whose manufacturers are not specified are all commercially available goods or can be prepared using known methods.

[0047] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0048] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0049] MoPex19 is the rice blast fungus ( Magnaporthe oryzae) Key proteins for the transport and maintenance of peroxisome membrane proteins (PMPs) in the middle peroxisome. MoPEX19 The deletion mutant exhibited various abnormalities in fungal development and pathogenicity-related morphogenesis, completely losing its pathogenicity to the host. These results indicate that MoPex19 plays a crucial role in maintaining peroxisomal and peroxisomal-derived structures, and is essential for the metabolism, development, and pathogenicity of rice blast fungus (MoPex19, which Is Essential for Maintenance of Peroxisomal Structure and Woronin Bodies, Is Required for Metabolism and Development in the Rice BlastFungus, PLoS One. 2014 Jan 14;9(1):e85252). However, there are currently no reports on drugs targeting MoPex19 for the control of rice blast.

[0050] The inventors of this invention used MoPex19 (MoPex19 gene information available in NCBI, MGG_00971) as a target and obtained the lead compound MSP-10 through high-throughput screening of a large number of compounds. To investigate the specific binding of MSP-10 to this target, AlphaFold3 was used to perform structural simulations of the interaction sites between MSP-10 and the MoPex19 protein. The predicted results are as follows: Figure 1As shown. Analysis indicates that the binding sites of compound MSP-10 in the MoPex19 protein are TYR-269, ARG-268, ASP-265, THR-261, and SER-314.

[0051] Further research on the control of rice blast fungus was conducted by applying compound MSP-10. The following examples demonstrate the effectiveness of compound MSP-10 in controlling rice blast fungus.

[0052] The experimental materials used in the following embodiments of the present invention are as follows:

[0053] 1. The rice blast fungus used in the embodiments of the present invention is the wild-type strain Guy11 preserved in the laboratory (ATCC Strain Center, USA).

[0054] 2. The culture medium for culturing rice blast fungus in the laboratory is a complete medium (CM). The CM medium formula is as follows: 10 g glucose, 2 g peptone-140, 1 g casein amino acids, 1 g yeast extract, 6 g NaNO3, 1.52 g KH2PO4, 0.52 g KCl, 0.52 g MgSO4·7H2O, 0.1 mg biotin, 0.1 mg vitamin B, 0.1 mg thiamine, 0.1 mg riboflavin, 0.1 mg niacin, 0.1 mg para-aminobenzoic acid, 1.5 mg Na2MoO4·5H2O, 1.6 mg CuSO4·5H2O, 1.7 mg CoCl2·6H2O, 5 mg MnCl2·4H2O, 5 mg FeSO4·7H2O, 11 mg H3BO3, 22 mg ZnSO4·7H2O, 50 mg Na4EDTA·2H2O, and 15 mg MgSO4·7H2O. g agar powder, diluted to 1 L with deionized water, pH adjusted to 6.5 with NaOH, and autoclaved at 121℃ for 15 min;

[0055] 3. Compound MSP-10, chemically named 4-[(5Z)-5-[(4-methoxyphenyl)methylene]-4-oxo-2-thio-1,3-thiazolidin-3-yl]-N-phenylbutyramide, was purchased from Shanghai Taoshu Biotechnology Co., Ltd., catalog number HIT ID: HIT 103730647. The molecular structure of compound MSP-10 is as follows: ;

[0056] 4. Preparation and storage of the stock solution of compound MSP-10: Dilute 10 mg of compound MSP-10 to 4 mL of DMSO to achieve a final concentration of 2500 ppm. This reagent can be stored at -20°C for short periods and at -80°C for long-term aliquot storage.

[0057] Example 1: Virulence determination and results of compound MSP-10 against rice blast fungus.

[0058] The virulence of compound MSP-10 against rice blast fungus was determined using the mycelial growth rate method, as follows:

[0059] The rice blast fungus Guy11 was inoculated onto 7 cm CM plates and incubated for 7 days at 25°C under a 16-h light-8-h dark incubator. Different doses (0, 10, 20, 30, 40, and 50 ppm) of compound MSP-10 were added to solid CM medium cooled to 40–50°C to prepare drug-containing plates. Under sterile conditions, 3 mL of sterile water was added to the 7-day-old inoculated Guy11 plates. Conidia were washed off using a sterile and cooled spreader, filtered through three layers of filter paper into 1.5 mL centrifuge tubes, and the number of conidia was counted under a light microscope using a hemocytometer. The conidial solution was diluted to 1×10⁻⁶. 5 5 μL of diluted conidial solution was added to the center of the drug-containing plate using a 10 μL pipette at spores / mL; each concentration treatment was repeated 3 times; CM medium containing the same concentration of DMSO was used as a control; after incubating the petri dishes upside down in an incubator at 25℃ for 16 h of light and 8 h of darkness for 3 days, the growth of rice blast fungus mycelium in the experimental group was observed.

[0060] The mycelial diameter was measured using the cross-cross method, and the mycelial growth rate method was used to calculate the inhibition rate of rice blast mycelial growth under different gradient concentrations of compound MSP-10. The calculation formula is as follows:

[0061] Mycelial growth inhibition rate (%) = (average diameter of colonies in control group - average diameter of colonies in treatment group) / (average diameter of colonies in control group - 0.5) × 100.

[0062] Statistical analysis was performed using DPS software to establish a virulence regression equation and calculate the EC50 of compound MSP-10 against rice blast fungus. 50 The correlation coefficient r was used to evaluate the virulence level of compound MSP-10 against rice blast fungus.

[0063] Table 1. Indoor toxicity of different concentrations of compound MSP-10 against rice blast fungus.

[0064]

[0065] Results Analysis: The toxicity of different concentrations of compound MSP-10 against rice blast fungus was tested. Figure 2 As shown, the five concentrations of compound MSP-10 in the experiment all had a certain inhibitory effect on the mycelial growth of rice blast fungus. The higher the concentration, the smaller the average colony diameter and the higher the mycelial growth inhibition rate. Figure 3Within this concentration gradient range, compound MSP-10 exhibited inhibition rates against rice blast fungus ranging from 10% to 80%, indicating high accuracy and reliability of the experimental data. Compound MSP-10 also showed an effect on the EC50 of rice blast fungus mycelial growth. 50 The value was 34.7035 ppm (Table 1). When the concentration of compound MSP-10 was 50 ppm, it had the most significant inhibitory effect on rice blast fungus, with a relative inhibition rate of 76.40% (Table 1).

[0066] Example 2: Determination and results of the pathogenicity of compound MSP-10 against rice blast fungus.

[0067] The pathogenicity of compound MSP-10 against rice blast fungus was determined using two methods: in vitro inoculation and live rice spraying. Details are as follows:

[0068] 1. In vitro inoculation method

[0069] (1) In vitro barley inoculation

[0070] Guy11 strain was inoculated onto 7 cm quantitative CM plates and incubated for 7 days in an incubator (28℃, 16 h light, 8 h dark). Then, 3 mL of ddH2O was added to the CM plates, and conidia were washed off using a sterile and cooled spreader. The conidia were filtered through three layers of filter paper into 1.5 mL centrifuge tubes. MSP-10 was mixed with the conidia to prepare conidia with different drug concentrations (0, 10, 20, 30, 40, and 50 ppm), with a final concentration of 5 × 10⁻⁶. 4 The concentration of MSP-10 in different treatment groups was adjusted using DMSO to ensure a consistent final concentration. A 10 cm square petri dish was prepared. Absorbent paper was cut to the size of the dish and placed inside. A suitable amount of water was added to moisten the paper, and then a piece of filter paper of the same size was placed on top. Barley leaves (5-7 cm) were cut from the barley plants 8-10 days after planting and placed in the dishes. Three drops (20 μL) of spore solution were added to each leaf. Each treatment was repeated three times. The control group's spore solution contained the same concentration of DMSO. The petri dishes containing the leaves were carefully placed in an incubator (28℃, 16 h light, 8 h dark). After 4 days of incubation, the disease incidence was observed and photographed.

[0071] Results Analysis: The inhibitory effects of gradient concentrations of compound MSP-10 on rice blast disease in detached barley leaves were tested. Figure 4As shown in (A), treatment with compound MSP-10 at concentrations of 10 to 50 ppm showed a certain inhibitory effect on the infection of detached barley leaves by rice blast fungus; and within this range, the higher the concentration tested, the stronger the inhibitory effect; among them, the concentration of compound MSP-10 at 40 to 50 ppm had a significant inhibitory effect on rice blast fungus spores on detached barley leaves; the concentration of compound MSP-10 at 50 ppm could completely inhibit the occurrence of rice blast in detached barley leaves.

[0072] (2) In vitro rice inoculation

[0073] Take the healthy second leaf of a three-leaf stage rice plant (grown by the China National Rice Research Institute, variety: Oryza sativa CO39) (cultivated for about 15 days). The specific operation method of in vitro inoculation is the same as that of in vitro barley inoculation.

[0074] Results Analysis: The inhibitory effects of gradient concentrations of compound MSP-10 on rice blast disease in vitro were tested. Figure 4 As shown in (B), among the five concentration treatments of compound MSP-10 in the experiment, compound MSP-10 at concentrations of 10 to 50 ppm all showed a certain inhibitory effect on detached rice blast disease; and within this range, the higher the concentration, the stronger the inhibitory effect; among them, compound MSP-10 at concentrations of 20 to 50 ppm had a significant inhibitory effect on detached rice leaf blast disease; and compound MSP-10 at a concentration of 40 ppm could completely inhibit the occurrence of detached rice blast disease in detached rice leaves.

[0075] like Figure 4 As shown in (A) and (B), at the same concentration of the compound, compound MSP-10 is more effective in controlling rice blast disease in detached rice leaves than in detached barley leaves.

[0076] 2. Live rice spraying method

[0077] First, Oryza sativa CO39 rice was sown (30 seeds per pot) and cultured outdoors under full sunlight for two weeks. The rice blast pathogen was then cultured on CM medium for 7-9 days to obtain spores. The compound MSP-10, gelatin solution, and spores were mixed to prepare a spore solution containing the drug and gelatin (final spore concentration 5 × 10⁻⁶). 4The concentration of MSP-10 was determined by mixing spores per mL (with a final gelatin concentration of 0.2%) to achieve a final concentration of 50 ppm. The dosage difference of MSP-10 was compensated with DMSO to ensure a consistent final DMSO concentration. Two mL of the mixed spore solution containing the compound and gelatin was evenly sprayed onto rice leaves, with three rice plants inoculated in each treatment group. Controls were provided for toxicity testing, including spraying with spore solutions containing the same concentration of DMSO and gelatin, and spraying with a gelatin solution containing 50 ppm of MSP-10 but without spores. The inoculated rice plants were then cultured in the dark at 22°C for 48 h, followed by incubation at 25°C with a light / dark cycle of 16 h / 8 h for 3-4 days. Disease incidence was observed and photographed on the rice leaves, and the lesion area and lesion rate were calculated using ImageJ software.

[0078] Lesion rate (%) = Lesion area / Leaf area × 100

[0079] The specific optimization experimental steps are as follows: The concentration of compound MSP-10 that inhibits rice blast disease was selected as 50 ppm. Compound MSP-10 was applied at different time points after inoculation with rice blast fungus spores, and three experimental groups were set up:

[0080] (1) -24 h: Spray compound MSP-10 24 hours before inoculating with rice blast fungus spore liquid;

[0081] (2) 0 h: Apply compound MSP-10 at the same time as inoculating rice blast fungus spore liquid, that is, mix compound MSP-10 with rice blast fungus spore liquid and spray for inoculation;

[0082] (3) +24 h: Spray compound MSP-10 24 hours after inoculation with rice blast fungus spore liquid.

[0083] Each treatment used 3 pots of rice as replicates, and included a blank control and a DMSO solvent control.

[0084] Results Analysis: This study tested the efficacy of compound MSP-10 at different time points in controlling rice blast disease in live rice. Compared to the control group without MSP-10, the -24h, 0h, and +24h treatment groups all significantly inhibited rice blast disease in live rice. Figure 5 and Figure 6 This has practical significance in preventing rice blast. Meanwhile, it was observed that rice in the control group treated with only the same concentration of DMSO or the same concentration of compound MSP-10 showed good growth with no significant adverse effects, indicating that this compound may be safe for rice. Figure 5 This has practical significance in preventing rice blast disease.

[0085] Example 3: Determination and results of the effect of compound MSP-10 on appressorium development of rice blast fungus.

[0086] 1. Method for determining the appressorium formation rate

[0087] Guy11 was inoculated onto 7 cm² agar plates and grown for 7 days in an incubator with 28°C, 16 h light, and 8 h dark. Then, 3 mL of ddH₂O was added to the plates, and the conidia attached to the aerial mycelia were gently scraped off with a spreader. The resulting spore stock solution was obtained by filtering through three layers of filter paper and diluted with ddH₂O to a final concentration of 5 × 10⁻⁶. 4 Spores / mL spore solution: MSP-10 and spore solution were mixed to prepare drug-containing spore solution, so that the final concentration of MSP-10 was 50 ppm. The dosage difference of MSP-10 was made up with DMSO to make the final concentration of DMSO consistent. A control containing the same concentration of DMSO was used. The artificial hydrophobic membrane was washed with 70% ethanol solution and placed on a glass slide. Three artificial hydrophobic membranes were placed on each glass slide. 20 μL of diluted spore solution was dropped onto each hydrophobic membrane. The prepared glass slides were transferred to a humidification box (with an appropriate amount of sterile water added) and placed in a dark incubator at 22℃ for induction. The appressorium formation rate was counted at 4 h, 8 h, 16 h and 24 h of induction and photographed. Each group of experiments was repeated 3 times.

[0088] Results Analysis: The effect of 50 ppm compound MSP-10 on the appressorium formation rate of rice blast fungus at different time points was tested. Figure 7 and Figure 8 As shown, 50 ppm of compound MSP-10 inhibited appressorium formation at all four different time points in the experiment. At different stages of appressorium development (4 h, 8 h, 16 h, and 24 h), exogenous addition of 50 ppm of compound MSP-10 significantly reduced the appressorium formation rate.

[0089] 2. Method for measuring the turgor pressure of appressorium

[0090] The process of inducing appressorium is basically the same as that of appressorium formation assay. Glycerol solutions with concentrations of 2 M and 3 M were prepared. After 24 h of induction, the hydrophobic membrane was removed, and the surface moisture was gently absorbed with a pipette or absorbent paper. An equal volume of glycerol solution was quickly added, and the membrane was allowed to stand at room temperature for 5 min. Then, the collapse rate of the appressorium, i.e. the proportion of collapsed appressorium to all appressorium, was quickly calculated. Each strain was repeated 3 times at each glycerol concentration.

[0091] Results Analysis: The effect of 50 ppm compound MSP-10 on appressorium turgor pressure at two different glycerol concentrations was tested. Figure 9 and Figure 10As shown, 50 ppm of compound MSP-10 had a certain effect on the appressorium turgor pressure at two different glycerol concentrations in the experiment. At 2 M and 3 M glycerol concentrations, the exogenous addition of 50 ppm of compound MSP-10 significantly increased the appressorium collapse rate. These results indicate that the exogenous addition of 50 ppm of compound MSP-10 significantly reduced the appressorium turgor pressure of rice blast fungus.

[0092] 3. Method for determining KI / I2 glycogen staining of appressorium

[0093] Prepare KI / I2 solutions (60 mg / mL KI, 10 mg / mL I2). The process for inducing appressoria is the same as described above. After induction for 0 h, 8 h, 16 h, and 24 h, remove the hydrophobic membrane containing the spore solution from the humidification box. Gently absorb the surface moisture with a pipette or absorbent paper. Add an equal volume of KI / I2 solution with a pipette, cover with a coverslip, and count the glycogen staining of conidia and appressoria under an optical microscope. Take photos and record the results. Each experiment is repeated 3 times.

[0094] Results Analysis: The effects of 50 ppm compound MSP-10 on glycogen transport and degradation in *Strombus haematococcus* at different time points were tested. Figure 11 As shown, the exogenous addition of 50 ppm of compound MSP-10 had no effect on glycogen synthesis, but significantly slowed down the transport of glycogen from conidia to appressoria. After 16 h of hydrophobic membrane inoculation, the exogenous addition of 50 ppm of compound MSP-10 significantly slowed down the rate of glycogen degradation in appressoria. After 24 h of hydrophobic membrane inoculation, glycogen degradation in appressoria returned to normal.

[0095] 4. Method for determining Bodipy lipid droplet staining in appressoria

[0096] Before inducing appressorium, tricyclazole was added to 1 mL of spore solution at a ratio of 1 μL of 10 μg / μL. The purpose of this was to inhibit the formation of melanin in the appressorium and facilitate fluorescence observation. Bodipy (Boron dipyrromethene) was diluted 1:1000. After induction for 0 h, 4 h, 8 h, and 24 h, the hydrophobic membrane containing the spore solution was removed from the humidification box. The surface moisture was gently absorbed by pipette or absorbent paper. An equal volume of diluted Bodipy dye was added by pipette, and a coverslip was placed on top. The staining of glycogen in conidia and appressorium was recorded under a fluorescence microscope and photographed. Each experiment was repeated 3 times.

[0097] Results Analysis: The effects of 50 ppm compound MSP-10 on lipid droplet transport and degradation of rice blast fungus at different time points were tested. Figure 12As shown, the exogenous addition of 50 ppm of compound MSP-10 had no significant effect on lipid droplet synthesis, lipid droplet transport, and lipid droplet degradation.

[0098] Example 4: Inhibition experiment of compound MSP-10 on other pathogens

[0099] This example also tested the toxic effects of compound MSP-10 on other plant pathogens. A concentration of 50 ppm of compound MSP-10 was used, and the experimental procedures were the same as in Example 1. Other plant pathogens included: *Botrytis cinerea* (…). B. cinerea : Botrytis cinerea ), Fusarium oxysporum ( F. fujikuroi : Fusarium fujikuroi Anthracnose of melons ( ) C. orbiculare : Colletotrichum orbiculare Alternaria ( A. alternata : Alternaria alternata ) and Anthrax bacillus fusiforme ( C. acutatum : Colletotrichum acutatum (See Table 2).

[0100] Table 2 Plant pathogens

[0101]

[0102] The results are as follows Figure 13 and Figure 14 As shown, compound MSP-10 exhibits inhibitory effects on the plate growth of various plant pathogenic fungi, inhibiting the mycelial growth of the five different genera of plant pathogens to varying degrees, especially *Botrytis cinerea* and *Anthracnose cucurbita*, with significant inhibitory effects. These results demonstrate the broad-spectrum antibacterial activity of MSP-10, which may be due to its specific targeting of the Pex19 protein. Given the high homology of Pex19 proteins in different pathogens, MSP-10 can bind to Pex19 homologous proteins in various pathogens, thereby achieving a significant antibacterial effect.

[0103] Example 5

[0104] Oryza sativa CO39 rice seeds were placed in a humidified container at 37°C for 48 hours to germinate, and then sown in culture pots (30 seeds / pot) and cultured outdoors in a netted room for 14 days. Compound MSP-10 and gelatin solution were mixed to prepare a drug-containing gelatin solution (final gelatin concentration 0.2%), resulting in final MSP-10 concentrations of 25, 50, and 100 ppm. The dosage difference of MSP-10 was compensated with DMSO to ensure a consistent final DMSO concentration. 2 mL of the mixed spore solution containing the compound and gelatin was evenly sprayed onto rice leaves, with 3 pots of rice inoculated for each treatment group. A gelatin solution containing the same volume of ddH2O and a gelatin solution containing 100 ppm DMSO were used as negative controls; a gelatin solution containing 100 ppm tricyclazole was used as a positive control. After spraying, the live rice plants were placed at 25°C and cultured under a light-dark cycle of 16 h / 8 h for 6-7 days; the growth of the rice was observed and photographed.

[0105] like Figure 15 As shown, compound MSP-10 showed no significant difference in rice growth compared to the control, and did not exhibit any obvious negative effects on rice. This suggests that MSP-10 may be safe for rice while effectively controlling rice blast.

[0106] In summary, the compound MSP-10 of this invention possesses broad-spectrum antibacterial activity and can play an important role in agricultural production as an effective inhibitor of plant pathogens. It not only exhibits significant control effects against rice blast fungus but also demonstrates good inhibitory effects against various other plant pathogens, without producing significant negative effects on rice, showing good biosafety. Therefore, compound MSP-10 shows promise as a highly efficient and safe new option for the control of plant pathogens, providing strong support for the sustainable development of agricultural production.

[0107] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Compound MSP-10 was used in the preparation of a fungicide for the control of rice blast fungus. Magnaporthe oryzae Its use in drugs containing ) or / and other pathogenic fungi is characterized by, The chemical structure of the compound MSP-10 is shown as follows: ， The other pathogenic fungi include any one or two or more of Botrytis cinerea Botrytis cinerea , Fusarium fujikuroi Fusarium fujikuroi , Guignardia bidwellii Colletotrichum orbiculare , Alternaria alternata Alternaria alternata , and Phoma exigua Colletotrichum acutatum .

2. Use of the compound MSP-10 for the manufacture of a medicament for inhibiting mycelial growth or / and spore germination or / and appressorium development of Magnaporthe grisea or / and other pathogenic fungi, characterized in that, The other pathogenic fungi include any one or two or more of Botrytis cinerea, Fusarium fujikuroi, Colletotrichum orbiculare, Alternaria alternata and Colletotrichum acutatum; The chemical structure of the compound MSP-10 is shown as follows: 。 3. Use according to claim 1 or 2, wherein the compound is of formula (I) ###0001### (I) or a pharmaceutically acceptable salt thereof. The compound MSP-10 can reduce the turgor pressure of the appressorium of Magnaporthe oryzae, or / and slow down the transport of glycogen from conidium to appressorium, or / and slow down the rate of glycogen degradation in appressorium.

4. Use according to claim 1 or 2, wherein the compound is ###0002### The concentration of the compound MSP-10 is 10 ~ 50 ppm.

5. Use according to claim 4, characterized in that, The concentration of the compound MSP-10 is 30 ~ 50 ppm.

6. A method for controlling Pyricularia oryzae or / and other pathogenic fungi, characterized by, Spraying the medicine for preventing and treating Magnaporthe oryzae or / and other pathogenic fungi on the leaves of plants; The other pathogenic fungi include any one or two or more of Botrytis cinerea, Fusarium fujikuroi, Colletotrichum orbiculare, Alternaria alternata and Colletotrichum acutatum; The medicine for preventing and treating Magnaporthe oryzae or / and other pathogenic fungi includes the compound MSP-10, and the chemical structure of the compound MSP-10 is shown as follows: 。 7. The method of claim 6, wherein, The concentration of the compound MSP-10 is 10 ~ 50 ppm.

8. The method of claim 7, wherein, The concentration of the compound MSP-10 is 30 ~ 50 ppm.

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