Use of compound cma-80 in the preparation of a medicament for controlling pyricularia oryzae

Abstract

The application discloses application of a compound CMA-80 in preparation of a medicine for preventing and treating Magnaporthe oryzae, and belongs to the field of plant fungal disease prevention and treatment. The application finds that the compound CMA-80 has a remarkable effect in preventing and treating rice blast or / and other pathogenic fungi, can effectively inhibit spore pathogenicity of Magnaporthe oryzae in a concentration range of 3-50 ppm, and especially, the compound CMA-80 at 50 ppm can completely prevent and treat rice blast. The compound CMA-80 adopted in the application has a prevention and treatment effect on rice blast of an in-vitro rice leaf which is remarkably better than that on rice blast of an in-vitro barley leaf at the same treatment concentration. Moreover, the compound CMA-80 of the application can be used as a protective agent at an early stage of Magnaporthe oryzae infection and has a better effect.

Description

Technical Field

[0001] This invention belongs to the field of plant fungal disease control, specifically involving the application of compound CMA-80 in the preparation of drugs for controlling rice blast fungus. Background Technology

[0002] Rice blast fungus ( Magnaporthe oryzae Rice blast, caused by [unspecified pathogen], is one of the most serious diseases affecting rice. This pathogen has broad host adaptability, infecting over 50 species of grasses, including rice, wheat, barley, and oats. In years of epidemic spread, it can cause total crop failure, seriously threatening global food security.

[0003] Rice blast fungus primarily spreads and infects through conidia. These conidia are dispersed via air currents, water currents, and insects. When environmental conditions are suitable, they germinate on the surface of rice and form specialized infection structures—appendage cells. Studies have shown that appendage cells generate enormous turgor pressure (8.0 MPa) by accumulating high concentrations of glycerol (3.2 M), driving the infection pinnae to penetrate the plant leaves. This highly efficient mechanical infection mechanism is key to its pathogenicity.

[0004] It is worth noting that the rice blast pathogen is characterized by its wide distribution and rapid genetic variation, enabling it to quickly adapt to different environmental conditions. This high degree of adaptability not only increases the technical difficulty of disease control but also provides the possibility for the continuous evolution of the pathogen. Therefore, establishing an effective integrated prevention and control system is crucial for ensuring food security.

[0005] Traditional control measures mainly include disease-resistant variety selection, chemical control, and cultivation management optimization. Among them, chemical control plays a key role in disease control due to its rapid and efficient characteristics. However, long-term excessive use of chemical pesticides has caused multiple ecological and environmental problems: (1) Pesticide residues not only change the physical and chemical properties of the soil, but also lead to a decline in soil biodiversity and an imbalance in the structure of soil microbial communities. (2) Biodiversity in farmland ecosystems shows a continuous decline trend, especially significantly affecting pollinating insects and natural enemy populations, thereby weakening the self-regulating function of the ecosystem.

[0006] The more serious issue is that the selective pressure from long-term chemical control is accelerating the formation of pesticide-resistant populations of rice blast fungus. Monitoring in recent years in some rice-growing areas has revealed that field strains have developed varying degrees of resistance to major fungicides (such as tricyclazole). This evolution of pesticide resistance in rice blast fungus strains not only significantly weakens existing field application of pesticides but also increases the risk of traditional chemical control strategies failing, posing new challenges to the integrated management of rice blast fungus.

[0007] Peroxisomes are crucial organelles in fungal cells, responsible for executing multiple core metabolic processes, such as fatty acid β-oxidation, reactive oxygen species (ROS) scavenging, and the glyoxylate cycle, thus widely regarded as the "metabolic center" of the cell. The formation of these organelles depends on a class of proteins called peroxisomals, encoded by the PEX gene. To date, 37 peroxisomals 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 promoting membrane assembly. Without MoPex19, most PMPs cannot properly locate and integrate into the membrane, leading to peroxisomal assembly failure and complete loss of function, indicating that this protein plays a crucial upstream role in the biogenesis pathway and possesses a core regulatory function.

[0008] 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 novel, green pesticides for the control of rice blast.

[0009] 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

[0010] The purpose of this invention is to provide the application of compound CMA-80 in the preparation of a drug for controlling rice blast fungus, which solves the problem of pesticide resistance in existing pathogens. The drug of this invention acts on MoPex19, can effectively inhibit the pathogenicity of rice blast fungus spores, has a significant effect on controlling rice blast, and is non-toxic to crops.

[0011] To achieve the above objectives, the present invention provides the use of compound CMA-80 in the preparation of a medicament for controlling rice blast fungus and / or other pathogenic fungi, the chemical structural formula of which is shown below:

[0012] ;

[0013] 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.

[0014] A second objective of this invention is to provide the use of compound CMA-80 in the preparation of a medicament for inhibiting the mycelial 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*.

[0015] Preferably, the compound CMA-80 can reduce the turgor pressure of rice blast fungus appressorium and / or slow down the transport of lipid droplets and / or glycogen from rice blast fungus conidia to appressorium.

[0016] Preferably, the concentration of compound CMA-80 is 3 to 50 ppm. Within this concentration range, compound CMA-80 can effectively inhibit the pathogenicity of rice blast fungus spores.

[0017] More preferably, the concentration of compound CMA-80 is 12.5~50 ppm, and within this concentration range, compound CMA-80 has a significant inhibitory effect on isolated barley blast disease.

[0018] More preferably, the concentration of compound CMA-80 is 50 ppm, at which concentration compound CMA-80 can completely inhibit detached barley blast disease.

[0019] A third objective of this invention is to provide a drug for controlling rice blast fungus and / or other pathogenic fungi, comprising compound CMA-80; wherein the other pathogenic fungi include any one or more of the following: Botrytis cinerea, Fusarium oxysporum, Anthracnose cucurbita, Alternaria alternata, and Anthracnose fusiforme.

[0020] Preferably, it also includes an agriculturally acceptable carrier.

[0021] 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.

[0022] Preferably, the concentration of the compound CMA-80 is 3 to 50 ppm.

[0023] More preferably, the concentration of the compound CMA-80 is 12.5 to 50 ppm.

[0024] The application of compound CMA-80 of the present invention in the preparation of a drug for controlling rice blast fungus solves the problem of pesticide resistance in existing pathogens and has the following advantages:

[0025] (1) This invention first discovered that compound CMA-80 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 3 to 50 ppm. In particular, compound CMA-80 at 50 ppm can completely prevent and control rice blast disease, and the application method is simple.

[0026] (2) The compound CMA-80 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.

[0027] (3) The compound CMA-80 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 CMA-80 of the present invention can be used as a protective agent in the early stage of rice blast infection and has a 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.

[0028] (4) The present invention found that compound CMA-80 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 lipid droplets and / or glycogen in rice blast fungus conidia to appressorium. Attached Figure Description

[0029] Figure 1 Predicted binding sites for MoPex19 and compound CMA-80.

[0030] Figure 2 The inhibition of different concentrations of compound CMA-80 on plate growth of rice blast fungus.

[0031] Figure 3 A comparison of colony growth diameter (A) and mycelial inhibition rate (B) of rice blast fungus treated with different concentrations of compound CMA-80. In the figure, significant differences in inhibition rate of different concentrations of compound CMA-80 are represented by different letters a, b, c, d, e or f; 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 CMA-80 were added to the spore liquid of rice blast fungus.

[0033] Figure 5 The study investigated the disease incidence of live rice leaves after spraying with 50 ppm compound CMA-80 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 CMA-80 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 CMA-80 was added exogenously to rice blast fungus spore liquid.

[0036] Figure 8 Comparison of appressorium formation rates at different time points when 50 ppm of compound CMA-80 was added exogenously to rice blast fungus spore liquid.

[0037] Figure 9 The collapse of appressoria at two different concentrations of glycerol when 50 ppm of compound CMA-80 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 CMA-80 was added exogenously to rice blast fungus spore liquid.

[0039] Figure 11 When 50 ppm of compound CMA-80 was added exogenously to the rice blast fungus spore liquid, 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 in (B) and (C).

[0040] Figure 12 The lipid droplet transport and degradation at different time points induced by appressorium when 50 ppm of compound CMA-80 was added exogenously to the spore liquid of rice blast fungus (A); the comparison of the proportion of lipid droplets in conidia at different induction times (B) and the comparison of the proportion of lipid droplets in appressorium (C).

[0041] Figure 13 The inhibition of different plant pathogenic fungi on plate growth by compound CMA-80.

[0042] Figure 14 This is a statistical graph showing the growth diameter of different plant pathogenic fungi on agar plates after treatment with compound CMA-80; the black bars represent the diameter of normal original fungi colonies without CMA-80 (control), and the white bars represent the diameter of pathogenic fungi colonies after CMA-80 treatment.

[0043] Figure 15 The effect of different concentrations of compound CMA-80 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 in the transport and maintenance of peroxisome membrane proteins (PMPs) in peroxisomes. MoPex19The 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 CMA-80 through high-throughput screening of a large number of compounds. To verify the specific binding of the CMA-80 molecule to this target, this experiment used AlphaFold3 to predict the structure of the interaction site between CMA-80 and the MoPex19 protein. The results are as follows: Figure 1 As shown. Prediction indicates that the binding site of CMA-80 on the MoPex19 protein is GLU-270.

[0051] Further research on the control of rice blast fungus by applying compound CMA-80 was conducted. The experiments in the following examples demonstrated the effectiveness of compound CMA-80 in controlling rice blast fungus, laying the foundation for the development of new green pesticides.

[0052] The materials used in the following embodiments 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 (1L) is as follows: 10 g anhydrous glucose, 2 g peptone-140, 1 g casein amino acids, 1 g yeast extract, 1.52 g KH2PO4, 6 g NaNO3, 0.52 g KCl, 0.52 g MgSO4·7H2O, 0.1 mg biotin, 0.1 mg vitamin B, 0.1 mg riboflavin, 0.1 mg thiamine, 0.1 mg para-aminobenzoic acid, 0.1 mg niacin, 1.5 mg Na2MoO4·5H2O, 1.6 mg CuSO4·5H2O, 1.7 mg CoCl2·6H2O, 5 mg FeSO4·7H2O, 5 mg MnCl2·4H2O, 11 mg H3BO3, 22 mg ZnSO4·7H2O, 50 mg Na4EDTA·2H2O, and 15 mg H3BO3. Add g of agar powder and NaOH to adjust the pH to 6.5. Then, bring the volume to 1 L with deionized water and sterilize by autoclaving (121℃, 15 min).

[0055] 3. Compound CMA-80, full name 2-{[4-(4-chlorophenyl)-1-(4-methylphenyl)-1H-imidazol-2-yl]thio}-N-[(tetrahydrofuran-2-yl)methyl]acetamide, was purchased from Shanghai Taoshu Biotechnology Co., Ltd., catalog number HITID: HIT 100091729. The molecular structure of compound CMA-80 is as follows:

[0056] ;

[0057] 4. Preparation and storage of CMA-80 stock solution: Dilute 10 mg of CMA-80 to 4 mL of DMSO to a final concentration of 2500 ppm. This reagent should be stored at -20°C for short periods and aliquoted and stored at -80°C for long periods.

[0058] Example 1: Determination and results of the toxicity of compound CMA-80 against rice blast fungus.

[0059] The virulence of compound CMA-80 against rice blast fungus was determined using the mycelial growth rate method, as detailed below:

[0060] The rice blast fungus Guy11 was inoculated onto 7 cm CM plates and cultured for 7 days at 25°C (16 h light / 8 h dark). Different doses (0, 3.125, 6.25, 12.5, 25, and 50 ppm) of compound CMA-80 were added to solid CM medium cooled to 50–55°C to prepare drug-treated agar plates. In a sterile laminar flow hood, 3 mL of sterile ddH2O was added to the 7-day-old Guy11 inoculated plates. Conidia were gently scraped off with a sterile spreader, filtered through three layers of filter paper into 1.5 mL centrifuge tubes, and the number of conidia was determined under an optical microscope using a hemocytometer. The plates were then diluted to a final concentration of 1 × 10⁻⁶. 5 spores / mL. Using a 10 μL pipette, 5 μL of diluted conidial solution was added to the center of the drug-containing plate; each concentration treatment was repeated 3 times; CM medium containing an equal concentration of DMSO was used as a blank control. After incubating the petri dishes upside down in a 25℃ incubator (16 h light / 8 h dark) for 3 days, the growth of rice blast fungus mycelium in the experimental group was observed.

[0061] The change in hyphal diameter was measured using the cross-cross method, and combined with the hyphal growth rate method, the inhibition rate of compound CMA-80 on rice blast mycelial growth at different concentration gradients was calculated. The formula for calculating the hyphal growth inhibition rate is as follows:

[0062] 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.

[0063] Using DPS statistical software, a virulence regression equation was established to calculate the half-maximal effective concentration (EC50) of compound CMA-80 against rice blast fungus. 50 The virulence level of compound CMA-80 against rice blast fungus was evaluated by using its correlation coefficient (r).

[0064] Table 1. Indoor toxicity of different concentrations of compound CMA-80 against rice blast fungus.

[0065]

[0066] Results Analysis: The effects of different concentrations of compound CMA-80 on the virulence of rice blast fungus were tested. Figure 1 As shown, all five concentrations of compound CMA-80 exhibited inhibitory effects on the mycelial growth of *Strombus oryzae*. Higher concentrations resulted in smaller average colony diameters and higher mycelial growth inhibition rates. Figure 3Within this concentration gradient range, compound CMA-80 exhibited an inhibition rate of 20%–80% against rice blast fungus, with good data reproducibility and reliable results. The half-maximum effective concentration (EC50) of compound CMA-80 against rice blast fungus mycelial growth was calculated. 50 The concentration of CMA-80 was 10.328 ppm (Table 1). The compound CMA-80 exhibited the most significant inhibitory effect against rice blast fungus at a concentration of 50 ppm, with a relative inhibition rate of 77.06% (Table 1).

[0067] Example 2: Determination and results of the pathogenicity of compound CMA-80 against rice blast fungus.

[0068] The pathogenicity of compound CMA-80 against rice blast fungus was determined using two methods: in vitro inoculation and live rice spraying. Details are as follows:

[0069] 1. In vitro inoculation method

[0070] (1) In vitro barley inoculation

[0071] Guy11 strain was inoculated onto 7 cm quantitative CM plates and incubated at 28°C for 7 days (16 h light, 8 h dark). Then, 3 mL of ddH2O was added to the CM plates, and conidia were scraped off using a sterile spreader. The conidia were filtered through sterile three-layer filter paper into 1.5 mL centrifuge tubes, centrifuged at 7500 rpm for 2 min, the supernatant was discarded, and the plates were resuspended in ddH2O to obtain the spore stock solution. The spore count was adjusted to a concentration of 5 × 10⁻⁶ using a hemocytometer under a microscope. 4 spores / mL. The spore solution was mixed thoroughly with compound CMA-80 to prepare spore solutions with different drug concentrations (0, 3.125, 6.25, 12.5, 25, and 50 ppm). The dosage difference of compound CMA-80 between different treatment groups was made up with DMSO to ensure a consistent final DMSO concentration. A 10 cm square petri dish was prepared. Absorbent paper was cut to an appropriate size and placed in the dish. A suitable amount of water was added to moisten the paper, and then a piece of filter paper of the same size as the absorbent paper was placed on top. Barley leaves, about 5-7 cm long, were cut from leaves planted 8-10 days prior and placed orderly in the dish. Three sites were inoculated at equal intervals on each leaf, and 20 μL of spore solution was added to each site. Each leaf treatment was repeated three times. The spore solution in the control group contained the same concentration of DMSO. The petri dishes containing the leaves were placed in a 28℃ incubator (16 h light, 8 h dark). After 4 days of incubation, the disease development was observed and recorded by photograph.

[0072] Results Analysis: This experiment tested the inhibitory effect of gradient concentrations of compound CMA-80 on barley leaves against rice blast through inoculation experiments on detached leaves. Figure 4As shown in (A), within the concentration range of 3.125 to 50 ppm, the treatment with compound CMA-80 showed a significant inhibitory effect on the infection of detached barley leaves by *Strombus oryzae*. Furthermore, within this range, the higher the tested concentration, the stronger the inhibitory effect. Specifically, the concentrations of compound CMA-80 at 12.5 to 50 ppm showed a significant inhibitory effect on *Strombus oryzae* spores on detached barley leaves. Although lesion formation was still observed in the 50 ppm treatment group, the lesion area was significantly smaller than that in the 3.125 ppm treatment group, indicating that high-concentration treatment can effectively inhibit pathogen infection.

[0073] (2) In vitro rice inoculation

[0074] 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 14 days). The specific operation method of the in vitro inoculation method is the same as that of in vitro barley inoculation.

[0075] Results Analysis: This experiment tested the inhibitory effects of gradient concentrations of compound CMA-80 on rice blast disease through inoculation experiments on detached leaves. Figure 4 As shown in (B), the five concentration gradients of compound CMA-80, within the range of 3.125 to 50 ppm, all showed a certain inhibitory effect on detached rice blast disease. Within this range, the higher the tested concentration, the stronger the inhibitory effect. Among them, compound CMA-80 with a concentration of 6.25 to 50 ppm had a significant inhibitory effect on detached rice blast disease. Compound CMA-80 with a concentration of 12.5 ppm could completely inhibit the infection of rice blast disease on detached rice leaves.

[0076] like Figure 4 As shown in (A) and (B), at the same treatment concentration, compound CMA-80 showed significantly better control of rice blast disease on detached rice leaves than on detached barley leaves.

[0077] 2. Live rice spraying method

[0078] Oryza sativa CO39 rice was germinated and sown (30 seeds per pot), and cultured outdoors under full sunlight for two weeks. The rice blast fungus was cultured on CM medium for 7-9 days. Conidia were gently scraped off with a sterile spreader, filtered through three layers of filter paper, mixed with ddH2O, and resuspended to obtain a spore solution. 0.4g of solid gelatin was dissolved in 100mL of ddH2O, stirred in a water bath, and cooled to room temperature to prepare a 0.4% gelatin stock solution. Compound CMA-80, the gelatin solution, and the spore solution were mixed to prepare a spore solution containing the drug and gelatin (final spore concentration 5×10⁻⁶). 4The final concentration of the compound CMA-80 was 50 ppm, with spores / mL and a final gelatin concentration of 0.2%. The dosage difference in the mixed spore solution was made up 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. A toxicity control was used, consisting of a spore solution containing the same concentration of DMSO and gelatin, and a gelatin solution containing 50 ppm of compound CMA-80 but without spores. The inoculated live rice plants were cultured in the dark at 22°C for 48 h, then at 25°C with a light / dark cycle of 16 h / 8 h for 3-4 days. Disease incidence on the rice leaves was observed and photographed, and the lesion area and lesion rate were calculated using ImageJ software.

[0079] Lesion rate (%) = Lesion area / Leaf area × 100

[0080] The specific optimization experimental steps are as follows: The concentration of compound CMA-80 that inhibits rice blast disease was selected as 50 ppm. Compound CMA-80 was applied at different time points at which the rice blast fungus spores were inoculated, and three experimental groups were set up:

[0081] (1) -24 h: Spray compound CMA-80 24 hours before inoculating with rice blast fungus spore liquid;

[0082] (2) 0 h: Apply compound CMA-80 at the same time as inoculating with rice blast fungus spore liquid, that is, mix compound CMA-80 with rice blast fungus spore liquid and spray for inoculation;

[0083] (3) +24 h: Spray compound CMA-80 24 hours after inoculation with rice blast fungus spore liquid.

[0084] Each treatment used 3 pots of rice as replicates, and included a blank control and a DMSO solvent control.

[0085] Results Analysis: The efficacy of CMA-80 at different time points in controlling rice blast disease in live rice plants was tested. Compared with the control group without CMA-80, the -24h, 0h, and +24h treatment groups all significantly inhibited rice blast disease in live rice plants. Figure 5 and Figure 6 This has practical significance for the prevention of rice blast. Meanwhile, it was observed that rice in the control group sprayed with only the same concentration of solvent DMSO or the same concentration of compound CMA-80 showed good growth and no significant adverse effects, indicating that this compound may be safe for rice. Figure 5 This has practical significance for preventing rice blast disease.

[0086] Example 3: Determination and results of the effect of compound CMA-80 on appressorium development of rice blast fungus.

[0087] 1. Method for determining the appressorium formation rate

[0088] Guy11 was inoculated onto 7 cm² agar plates and incubated at 28°C for 7 days (16 h light, 8 h dark). Then, 3 mL of ddH₂O was added to the rice blast fungus plates. Conidia attached to the aerial mycelia were scraped off with a spreader, filtered through three layers of filter paper, centrifuged at 7500 rpm for 2 min, the supernatant was discarded, and the mixture was resuspended in ddH₂O to obtain the spore stock solution. This stock solution was then diluted with ddH₂O to a final concentration of 5 × 10⁻⁶. 4 Spore suspension at spores / mL. The spore suspension was mixed with compound CMA-80 to prepare a drug-containing spore suspension. A final concentration of 50 ppm of compound CMA-80 was used, with the dosage difference made up using DMSO solvent to ensure a consistent final DMSO concentration; a control group containing the same concentration of DMSO was used. Artificial hydrophobic membranes were washed with 75% ethanol solution. Three artificial hydrophobic membranes were placed on each slide, and three drops of 20 μL of diluted spore suspension were added to each membrane. The prepared slides were transferred to a humidity chamber (containing sterile water) and placed in a dark incubator at 22℃ to induce appressorium formation. The appressorium formation rate was recorded after 4 h, 8 h, 16 h, and 24 h of induction, and photographs were taken. Each experiment was repeated three times.

[0089] Results analysis: Compound CMA-80 at a concentration of 50 ppm affected the appressorium formation rate of rice blast fungus at different time points. For example... Figure 7 and Figure 8 As shown, 50 ppm of compound CMA-80 inhibited the formation of rice blast fungus appressorium at four different time points in the experiment. At different stages of rice blast fungus appressorium development (4 h, 8 h, 16 h and 24 h), exogenous addition of 50 ppm of compound CMA-80 significantly reduced the appressorium formation rate.

[0090] 2. Method for measuring the turgor pressure of appressorium

[0091] The process of inducing appressorium is basically the same as that of appressorium formation assay. Glycerol solutions with concentrations of 1 M and 2 M were prepared. After 24 h of induction, the hydrophobic membrane of the induced appressorium 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 mixture was allowed to stand at room temperature for 5 min. The collapse rate of the appressorium, i.e., the proportion of collapsed appressorium to the total number of appressorium observed, was then immediately observed and counted. Each strain was repeated 3 times at each glycerol concentration.

[0092] Results analysis: At two different glycerol concentrations, 50 ppm of compound CMA-80 affected the turgor pressure of appressorium. For example... Figure 9 and Figure 10 As shown, at two different glycerol concentrations, 50 ppm of compound CMA-80 had a certain effect on appressorium turgor pressure. At 1 M and 2 M glycerol concentrations, exogenous addition of 50 ppm of compound CMA-80 significantly increased the appressorium collapse rate. These results indicate that exogenous addition of 50 ppm of compound CMA-80 significantly reduced appressorium turgor pressure of rice blast fungus.

[0093] 3. Method for determining KI / I2 glycogen staining of appressorium

[0094] Prepare KI / I2 solutions (60 mg / mL KI, 10 mg / mL I2). The induction of appressorium is the same as described above. After 0 h, 8 h, 16 h, and 24 h of induction, 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, cover with a coverslip, and statistically analyze the glycogen staining of conidia and appressorium under an optical microscope, and record the results by photograph. Each experiment was repeated three times.

[0095] Results Analysis: The effects of 50 ppm of compound CMA-80 on glycogen transport and degradation in *Bacillus oryzae* at different time points were tested. Figure 11 As shown, the exogenous addition of 50 ppm of compound CMA-80 had no effect on glycogen synthesis and glycogen degradation in appressoria, but it significantly slowed down the transport of glycogen from conidia to appressoria.

[0096] 4. Method for determining Bodipy lipid droplet staining of appressorium

[0097] Before inducing appressorium, 1 μL of 10 μg / μL tricyclazole was added to 1 mL of spore solution to inhibit melanin formation in appressorium and facilitate fluorescence observation. The lipid droplet dye Bodipy (Borondipyrromethene) was diluted 1:1000. After inducing appressorium at 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 with a pipette or absorbent paper. An equal volume of diluted Bodipy dye was added using a pipette, and a coverslip was placed on top. The staining of conidia and appressorium lipid droplets was recorded under a fluorescence microscope and photographed. Each experiment was repeated three times.

[0098] Results Analysis: The effects of 50 ppm compound CMA-80 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 CMA-80 had no significant effect on lipid droplet synthesis and degradation in appressoria of rice blast fungus spores and appressoria, but it significantly slowed down the transport of lipid droplets from conidia to appressoria.

[0099] Example 4: Inhibition experiment of compound CMA-80 on other pathogens

[0100] This example also tested the toxic effects of compound CMA-80 on other plant pathogens. A concentration of 50 ppm of compound CMA-80 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).

[0101] Table 2 Plant pathogens

[0102]

[0103] The results are as follows Figure 13 and Figure 14 As shown, compound CMA-80 exhibits a certain degree of inhibition 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 CMA-80, which may be due to the specific targeting of the Pex19 protein by CMA-80. Given the high homology of Pex19 proteins in different pathogens, CMA-80 can bind to Pex19 homologous proteins in various pathogens, thereby achieving a significant antibacterial effect.

[0104] Example 5

[0105] 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 CMA-80 and gelatin solution were mixed to prepare a drug-containing gelatin solution (final gelatin concentration 0.2%), resulting in final CMA-80 concentrations of 25, 50, and 100 ppm. The dosage difference of CMA-80 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.

[0106] like Figure 15 As shown, compound CMA-80 showed no significant difference in rice growth compared to the control, and did not show any obvious negative effects on rice. This suggests that CMA-80 may be safe for rice while effectively controlling rice blast.

[0107] In summary, the compound CMA-80 of this invention has broad-spectrum antibacterial activity and can serve as an effective inhibitor of plant pathogens, especially showing significant effects against rice blast fungus and other plant pathogens. At the same time, it has no obvious adverse effects on rice growth and demonstrates good biosafety. This discovery provides strong support for the development of novel, environmentally friendly, and efficient plant disease control agents.

[0108] 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 CMA-80 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 structural formula of the compound CMA-80 is 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 CMA-80 for the preparation 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 structural formula of the compound CMA-80 is 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 CMA-80 can reduce the turgor pressure of the appressorium of Magnaporthe oryzae or / and slow down the transport of lipid droplets and / or glycogen in the conidium of Magnaporthe oryzae to the appressorium.

4. Use according to claim 1 or 2, wherein the compound is ###0002### The concentration of the compound CMA-80 is 3-50 ppm.

5. Use according to claim 4, characterized in that, The concentration of the compound CMA-80 is 12.5-50 ppm.

6. A medicine for controlling Magnaporthe grisea or / and other pathogenic fungi, characterized by, The compound CMA-80; 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 structural formula of the compound CMA-80 is as follows: 。 7. The medicament according to claim 6, characterized in that, Further comprising an agriculturally acceptable carrier.

8. 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 as claimed in claim 6 or 7 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.

9. The method of claim 8, wherein, The concentration of the compound CMA-80 is 3-50 ppm.

10. The method of claim 9, wherein, The concentration of the compound CMA-80 is 12.5-50 ppm.

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