Use of compound dbp-64 in the preparation of a drug for preventing and treating pyricularia oryzae

Abstract

The application discloses application of a compound DBP-64 in preparation of a medicine for preventing and treating Magnaporthe oryzae, and belongs to the field of plant fungal disease prevention and treatment. It is found that the compound DBP-64 has a remarkable effect in inhibiting Magnaporthe oryzae or / and other pathogenic fungi, can effectively inhibit the pathogenicity of Magnaporthe oryzae spores in a concentration range of 3-50 ppm, especially, the compound DBP-64 at 50 ppm can completely prevent and treat rice blast, and the application method is simple. The compound DBP-64 has a certain prevention and treatment effect when mixed with Magnaporthe oryzae spore liquid and sprayed at 0 h, indicating that the compound DBP-64 can be used as a protective agent at the initial stage of rice blast infection and has better effect; moreover, the sprayed compound has a good growth trend of rice and has no obvious adverse effect, indicating that the compound is safe to rice and has practical significance in preventing rice blast.

Description

Technical Field

[0001] This invention belongs to the field of plant fungal disease control, specifically involving the application of compound DBP-64 in the preparation of a drug for controlling rice blast fungus. Background Technology

[0002] Rice is one of the main food sources for humankind. However, rice faces various biological stresses during its growth, among which rice blast fungus (… Magnaporthe oryzae Rice blast, caused by a fungus, is one of the most devastating diseases. It is a broad-spectrum fungal disease that can infect the leaves, stems, panicles, and grains of rice. Typical symptoms include spindle-shaped grayish-white lesions, stem rot, and panicle death.

[0003] The field infection process of rice blast fungus mainly relies on its conidia as a transmission vector. The pathogen spreads conidia through multiple pathways, including airborne dispersal, hydrodynamic diffusion, and insect-mediated transmission. After conidia settle on rice leaves, they germinate under suitable environmental conditions (temperature 25-28℃, relative humidity ≥90%), forming germ tubes. The tips of these germ tubes swell to form melanized appressoriums, where osmotic regulators such as glycerol accumulate, generating a turgor pressure as high as 8 MPa. This pressure drives the infection pinnae to penetrate the rice epidermal cells mechanically. Rice blast fungus exhibits strong environmental adaptability, wide geographical distribution, and high genetic diversity. These characteristics significantly increase the risk of failure in field control systems, making this disease a core challenge for the development of sustainable control technologies. Therefore, it is crucial to systematically develop comprehensive control programs that combine pathogen targeting with ecological compatibility, and to achieve technological integration through scientific implementation pathways.

[0004] Traditional disease control systems mainly include the following strategies: screening disease-resistant and high-yielding crop varieties, cutting off pathogen transmission chains, implementing chemical control, strengthening cultivation management, optimizing water and fertilizer regulation, and standardizing the treatment of infected plants. Among these measures, chemical control has always occupied a core position and received significant attention from academia and industry due to its ability to effectively control disease during periods of high incidence. However, it is important to be wary that the over-reliance on chemical pesticides over the past half-century has triggered a series of ecological problems: on the one hand, it has led to the deterioration of the physical and chemical properties of farmland soil and the degradation of ecosystem functions; on the other hand, it has led to the screening of multiple drug-resistant physiological races in the rice blast pathogen population, forming a vicious cycle of "pesticide application - resistance development - increased dosage," making disease control increasingly difficult and technically challenging.

[0005] To address these challenges, deciphering the molecular mechanisms of pathogenicity and identifying key effector proteins have become urgent priorities. This necessitates accelerating the research and development of green pesticides based on targeted regulation. Such innovative pesticides need to control diseases by precisely interfering with key pathogen infection pathways. This synergistic model of "precision control-ecological conservation" can not only overcome the existing predicament of pesticide resistance but also reconstruct a healthy farmland ecosystem, ultimately forming a sustainable governance paradigm that prioritizes both crop protection and ecological security.

[0006] 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.

[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, it is necessary to develop pesticides targeting MoPex19 for the control of rice blast to address the issue of pesticide resistance.

[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 DBP-64 in the preparation of drugs for the control of rice blast fungus. It solves the problem of drug resistance to the target of existing pesticides, can effectively inhibit the pathogenicity of rice blast fungus spores, has a significant effect on the control of rice blast, and is non-toxic to crops.

[0010] To achieve the above objectives, the present invention provides the use of compound DBP-64 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:

[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 DBP-64 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 DBP-64 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.

[0015] Preferably, the concentration of compound DBP-64 is 3 to 50 ppm. Within this concentration range, compound DBP-64 can effectively inhibit the pathogenicity of rice blast fungus spores.

[0016] More preferably, the concentration of compound DBP-64 is 12.5~50 ppm, and within this concentration range, compound DBP-64 has a significant inhibitory effect on isolated barley blast disease.

[0017] Most preferably, the concentration of compound DBP-64 is 50 ppm, at which concentration compound DBP-64 can completely inhibit detached 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 DBP-64; 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 DBP-64 is 3 to 50 ppm.

[0022] More preferably, the concentration of the compound DBP-64 is 12.5 to 50 ppm.

[0023] The application of compound DBP-64 of the present invention in the preparation of a drug for controlling rice blast fungus solves the problem of resistance to the target site in existing pesticides, and has the following advantages:

[0024] (1) This invention first discovered that compound DBP-64 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 DBP-64 at 50 ppm can completely prevent and control rice blast disease, and the application method is simple.

[0025] (2) The compound DBP-64 used in this invention has a significantly better control effect on rice blast disease in detached rice leaves than in detached barley leaves at the same treatment concentration.

[0026] (3) The compound DBP-64 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 DBP-64 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.

[0027] (4) The present invention found that compound DBP-64 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.

[0028] (5) The compound DBP-64 of the present invention can not only target rice blast fungus, but also has a certain inhibitory effect on other pathogenic fungi (Botrytis cinerea, Fusarium oxysporum, Anthracnose of cucurbita, Alternaria alternata, Fusarium graminearum and Anthracnose of acetic acid). This makes compound DBP-64 have a wider range of applications in agricultural production, and can effectively deal with a variety of plant diseases caused by different pathogenic fungi, reduce the need to use a variety of pesticides due to different diseases, and reduce agricultural production costs and environmental pressure. Attached Figure Description

[0029] Figure 1 This is a predicted diagram of the binding sites of compounds DBP-64 and MoPex19 of the present invention.

[0030] Figure 2 The inhibition of different concentrations of compound DBP-64 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 under different concentrations of compound DBP-64. In the figure, significant differences in inhibition rate of different concentrations of compound DBP-64 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 DBP-64 were added to the spore liquid of rice blast fungus.

[0033] Figure 5 The study investigated the disease incidence in living rice leaves after spraying with 50 ppm compound DBP-64 at different times following inoculation with rice blast fungus spores.

[0034] Figure 6 A comparison of the relative lesion area of ​​living rice leaves after spraying with 50 ppm compound DBP-64 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 DBP-64 was added exogenously to the spore liquid of rice blast fungus.

[0036] Figure 8 Comparison of appressorium formation rates at different time points induced by exogenous addition of 50 ppm compound DBP-64 to rice blast fungus spore liquid.

[0037] Figure 9The collapse of appressoria at two different concentrations of glycerol when 50 ppm of compound DBP-64 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 DBP-64 was added exogenously to rice blast fungus spore liquid.

[0039] Figure 11 When 50 ppm of compound DBP-64 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 (B); and a comparison of the proportion of glycogen in appressorium was shown (C).

[0040] Figure 12 When 50 ppm of compound DBP-64 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 DBP-64 on the growth of different plant pathogenic fungi on plate.

[0042] Figure 14 Statistical graph of colony growth diameter of different plant pathogenic fungi after treatment with 50 ppm compound DBP-64; where the black bars are the colony diameter of pathogens that grow normally without DBP-64 (control), and the white bars are the colony diameter of pathogens treated with DBP-64, n = 3.

[0043] Figure 15 The effect of different concentrations of compound DBP-64 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. 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 DBP-64 through high-throughput screening of a large number of compounds. To further verify the targeting specificity of the DBP-64 molecule, AlphaFold3 was used to predict the binding site between the DBP-64 molecule and the MoPex19 protein. The prediction results are as follows: Figure 1As shown in the figure. The prediction results indicate that the binding sites of the DBP-64 compound in the MoPex19 protein are TYR-269, ARG-268, THR-261, and PRO-312.

[0051] Further research on the control of rice blast fungus was conducted by applying compound DBP-64. The experiments in the following examples demonstrated the effectiveness of compound DBP-64 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 culture medium (CM). The CM culture medium formula is as follows: 2 g peptone, 10 g glucose, 1 g casein amino acids, 1 g yeast extract, 50 mL 20× Nitrate salt, 1 mL 1000× Vitamin solution, 1 mL 1000× Trace elements and 15 g agar powder. The volume is adjusted to 1 L with deionized water, the pH is adjusted to 6.5 with NaOH, and the medium is autoclaved at 121℃ for 15 min.

[0055] The preparation of 20× Nitrate salt is as follows: 120 g NaNO3, 10 g KCl, 10 g MgSO4·7H2O, 30 g KH2PO4, diluted with pure water to 1 L, and stored in a refrigerator at 4℃.

[0056] The 1000×Vitamin solution was prepared as follows: 0.1 g Pyridoxin, 0.1 g Riboflavin, 0.1 g Biotin, 0.1 g Nicotinic acid, 0.1 g Thiamine, 0.1 g P-aminobenzoic acid, and diluted with pure water to 1 L. It was then stored at 4°C.

[0057] The 1000×Trace elements were prepared as follows: 1.6 g CuSO4·5H2O, 1.2 g Na2MoO4·2H2O, 5 g MnCl2·4H2O, 1.7 g CoCl2·6H2O, 0.183 g FeSO4·7H2O, 5 g ZnSO4·7H2O, 48.7 g Na4EDTA·2H2O, 11 g H3BO3, and diluted to 1 L with pure water. The mixture was then stored at 4°C.

[0058] 3. Compound DBP-64, chemically named 2-(dimethylamino)ethyl 3-[(5Z)-5-(2H-1,3-benzodioxolane-5-ylmethylene)-4-oxo-2-thio-1,3-thiazolidin-3-yl]propionate, was purchased from Shanghai Taoshu Biotechnology Co., Ltd., catalog number HIT ID: HIT100862994. The molecular structure of compound DBP-64 is as follows:

[0059] ;

[0060] 4. Preparation and storage of the stock solution of compound DBP-64: Dilute 10 mg of compound DBP-64 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.

[0061] Example 1: Virulence determination and results of compound DBP-64 against rice blast fungus.

[0062] The virulence of compound DBP-64 against rice blast fungus was determined using the mycelial growth rate method, as follows:

[0063] The rice blast pathogen strain Guy11 was inoculated onto 7 cm diameter CM agar plates and pre-cultured for 7 days in a 16-8 h light-dark cycle incubator at 25°C. After cooling the sterilized solid CM agar to 50±5°C, DBP-64 solution was added at final concentrations of 0 (blank control), 3.125, 6.25, 12.5, 25, and 50 ppm, with an equal volume of DMSO as a control. Under aseptic conditions in a laminar flow hood, 3 mL of sterile water was added to the surface of the 7-day-old Guy11 agar plates. Conidia were gently scraped using a disposable sterile applicator, filtered through three layers of sterile filter paper, and collected in 1.5 mL centrifuge tubes. Spore density was calibrated using a hemocytometer under an optical microscope and finally adjusted to 1×10⁻⁶. 5 Standard concentration: spores / mL; use a pipette to draw 5 μL of 1×10⁻⁶ solution. 5 Apply conidial solution of spores / mL to the center of the drug-containing plate; repeat each concentration treatment 3 times; use CM medium containing the same concentration of DMSO as a control; incubate the inoculated plate upside down in an incubator at 25℃ with a light-dark cycle of 16 h / 8 h for 3 days, and then observe the growth of rice blast fungus mycelium in the experimental group.

[0064] The hyphal diameter was measured using the cross-cross method, and the hyphal growth rate inhibition rate of rice blast mycelium under different gradient concentrations of compound DBP-64 was calculated using the hyphal growth rate method. The calculation formula is as follows:

[0065] 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.

[0066] Using DPS statistical software, a virulence regression equation was established to calculate the half-maximal effective concentration (EC50) of compound DBP-64 against rice blast fungus. 50 The virulence level of compound DBP-64 against rice blast fungus was evaluated by using its correlation coefficient (r).

[0067] Table 1. Indoor toxicity of different concentrations of compound DBP-64 against rice blast fungus.

[0068]

[0069] Results Analysis: The toxicity of different concentrations of compound DBP-64 against rice blast fungus was tested. Figure 2 As shown, the DBP-64 treatment group exhibited a significant inhibitory effect on mycelial growth at all five concentration gradients. With increasing treatment concentration from 3.125 ppm to 50 ppm, the average colony diameter tended to decrease, while the mycelial growth inhibition rate increased. Figure 3 Within this concentration gradient range, compound DBP-64 exhibited an inhibition rate of 4.5%–60% against rice blast fungus, indicating high accuracy and reliability of the experimental data. Compound DBP-64 also showed an inhibitory effect on the EC50 of rice blast fungus mycelial growth. 50 The value was 28.4118 ppm (Table 1). When the concentration of compound DBP-64 was 50 ppm, it had the most significant inhibitory effect on rice blast fungus, with a relative inhibition rate of 60.24% (Table 1).

[0070] Example 2: Determination and results of the pathogenicity of compound DBP-64 against rice blast fungus.

[0071] The pathogenicity of compound DBP-64 against rice blast fungus was determined using two methods: in vitro inoculation and live rice spraying. Details are as follows:

[0072] 1. In vitro inoculation method

[0073] (1) In vitro barley inoculation

[0074] The rice blast pathogen strain Guy11 was inoculated onto 15 ml CM agar plates (7 cm in diameter) and incubated for 7 days at 28°C with a 16 h / 8 h light / dark cycle. Under aseptic conditions in a laminar flow hood, 3 mL of sterile water was added to the surface of the 7-day-old Guy11 agar plates. Conidia were gently scraped using a disposable sterile spreader, filtered through three layers of sterile filter paper, and collected in 1.5 mL centrifuge tubes. Compound DBP-64 was mixed with the spore suspension to prepare spore suspensions with different concentrations (0, 3.125, 6.25, 12.5, 25, and 50 ppm), with a final concentration of 5 × 10⁻⁶. 4 The concentration of spores / mL was adjusted by DMSO to compensate for the difference in the dosage of compound DBP-64 in different treatment groups, ensuring a consistent final concentration of DMSO. Two layers of 9 cm × 9 cm absorbent paper and one sheet of filter paper were prepared. The absorbent paper was placed in a 10 cm square petri dish, moistened with an appropriate amount of water, and then covered with a layer of filter paper. Barley leaves (5-7 cm) planted for 8-10 days were cut and placed in the square dish in an orderly manner. Three drops (20 μL) of spore solution were inoculated onto each leaf. Each treatment was repeated three times. The spore solution of the control group contained the same concentration of DMSO. The petri dishes containing the leaves were carefully placed in an incubator (temperature 25℃, light / dark cycle 16 h / 8 h). After 4 days of incubation, the disease incidence was observed and photographed.

[0075] Results analysis: The gradient concentrations of the experimental compound DBP-64 showed an inhibitory effect on rice blast disease in detached barley leaves. (For example...) Figure 4 As shown in (A), compound DBP-64 has a certain inhibitory effect on the infection of detached barley leaves by rice blast fungus in the concentration range of 3.125 ~ 50 ppm, and the inhibition rate of lesion area increases with the increase of concentration; among them, the inhibition rate of lesions on detached barley leaves is most obvious when the concentration of compound DBP-64 is 25 ~ 50 ppm; compound DBP-64 at a concentration of 50 ppm can completely inhibit the occurrence of rice blast on detached barley leaves.

[0076] (2) In vitro rice inoculation

[0077] Select the second leaf of healthy three-leaf stage rice seedlings (grown by China National Rice Research Institute, variety: Oryza sativa CO39) that have been cultured for 15 days. The specific operation method of in vitro inoculation is the same as that of in vitro barley inoculation.

[0078] Results Analysis: The experiment investigated the inhibitory effects of compound DBP-64 at varying concentrations on rice blast in vitro. Figure 4As shown in (B), compound DBP-64 exhibits a certain inhibitory effect on detached rice blast in the concentration range of 3.125 ~ 50 ppm; and the inhibition rate of lesion area increases with increasing concentration; among them, the inhibition rate of compound DBP-64 on detached rice blast is most obvious when the concentration is 6.25 ~ 50 ppm; and the concentration of compound DBP-64 at 50 ppm has the best inhibitory effect on detached rice blast.

[0079] 2. Live rice spraying method

[0080] Oryza sativa CO39 rice seeds were placed in a humidified container at 37℃ for 48 hours to germinate, then sown in culture pots (30 seeds / pot) and cultured outdoors in a netted room for 14 days. Rice blast strain Guy11 was inoculated onto 9 cm diameter CM agar plates and pre-cultured at 25℃ for 7 days under a 16 h / 8 h light / dark cycle to obtain spores. Compound DBP-64, gelatin solution, and spores were mixed to prepare a spore solution containing the drug and gelatin (final spore concentration 5 × 10⁻⁶). 4 The concentration of DBP-64 was adjusted to 50 ppm (spores / mL, final gelatin concentration of 0.2%), with the dosage difference of DBP-64 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. A control group was prepared by spraying spore solutions containing the same concentration of DMSO and gelatin, and a control group was prepared by spraying a gelatin solution containing 50 ppm of DBP-64 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.

[0081] Lesion rate (%) = Lesion area / Leaf area × 100

[0082] The specific optimization experimental steps are as follows: The concentration of compound DBP-64 that inhibits rice blast disease was selected as 50 ppm. Compound DBP-64 was applied at different time points after inoculation with rice blast fungus spores, and three experimental groups were set up:

[0083] (1) -24 h: Spray compound DBP-64 24 hours before inoculating with rice blast fungus spore liquid;

[0084] (2) 0 h: Apply compound DBP-64 at the same time as inoculating rice blast fungus spore liquid, that is, mix compound DBP-64 with rice blast fungus spore liquid and spray for inoculation;

[0085] (3) +24 h: Spray compound DBP-64 24 hours after inoculation with rice blast fungus spore liquid.

[0086] Each treatment used 3 pots of rice as replicates, and included a blank control and a DMSO solvent control.

[0087] Results Analysis: This study tested the control efficacy of compound DBP-64 against rice blast disease in live rice at different time points. Compared with the control group, DBP-64 showed significant control effects in three key window periods: 24 hours before pathogen inoculation (preventive treatment), at inoculation (simultaneous treatment), and 24 hours after inoculation (treatment treatment). Figure 5 and Figure 6 This has practical significance in preventing rice blast. Rice in the control group, treated with the same concentration of solvent DMSO or the same concentration of compound DBP-64, showed good growth and no obvious phytotoxicity symptoms, indicating that this compound may be safe for rice. Figure 5 This has practical significance in preventing rice blast disease.

[0088] Example 3: Determination and results of the effect of compound DBP-64 on appressorium development of rice blast fungus.

[0089] 1. Method for determining the appressorium formation rate

[0090] The rice blast fungus Guy11 was inoculated onto 7 cm² CM plates and cultured at 28℃ with alternating light and dark conditions for 16 h / 8 h for 7 days. 3 mL of ddH₂O was added to the plates, and conidia attached to the aerial mycelia were gently scraped off with a spreader. The resulting spore stock solution was obtained by filtration through three layers of filter paper and diluted with ddH₂O to a final concentration of 5 × 10⁻⁶. 4 spores / mL spore solution; mix compound DBP-64 with the spore solution to prepare a drug-containing spore solution (final spore concentration of 5 × 10⁻⁶). 4 The concentration of compound DBP-64 was adjusted to 50 ppm by adding DMSO to make up the difference in DBP-64 concentration. DMSO of the same concentration was used as a control. The artificial hydrophobic membranes were cleaned in 70% ethanol. Three artificial hydrophobic membranes were placed on each slide, and 20 μL of diluted spore solution was added to each membrane. The slides were placed in a humidification box (with an appropriate amount of sterile water) and incubated at 22°C in the dark. The appressorium formation rate was recorded at 4 h, 8 h, 16 h, and 24 h. Each experiment was repeated three times.

[0091] Results Analysis: The effect of compound DBP-64 at a concentration of 50 ppm 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 DBP-64 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 DBP-64 significantly reduced the appressorium formation rate.

[0092] 2. Method for measuring the turgor pressure of appressorium

[0093] The process of inducing appressorium was consistent with the appressorium formation assay. Glycerol solutions with concentrations of 1 M and 2 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. 20 μL of glycerol solution was quickly added, and after standing at room temperature for 5 min, the number of collapsed appressoriums was quickly counted. The proportion of collapsed appressoriums to all appressoriums was calculated. Each strain was repeated 3 times at each glycerol concentration.

[0094] Results Analysis: The effect of 50 ppm compound DBP-64 on appressorium turgor pressure was tested at two different glycerol concentrations. Figure 9 and Figure 10 As shown, the concentration of compound DBP-64 at 50 ppm affected the turgor pressure of appressoriums at different glycerol concentrations. At 1 M and 2 M glycerol concentrations, the exogenous addition of 50 ppm compound DBP-64 significantly increased the appressorium collapse rate. The results indicate that the exogenous addition of 50 ppm compound DBP-64 reduced the turgor pressure of appressoriums of *Strombus rice*.

[0095] 3. Method for determining KI / I2 glycogen staining of appressorium

[0096] The preparation of KI / I2 solution (60 mg / mL KI, 10 mg / mL I2) and the induction of appressorium were the same as described above. After induction for 0 h, 8 h, 16 h, and 24 h, the water on the hydrophobic membrane surface containing the spore solution was removed with a pipette or absorbent paper, 20 μL of KI / I2 solution was added, a coverslip was placed, and the glycogen staining of conidia and appressorium was recorded under a color microscope. Each experiment was repeated three times.

[0097] Results Analysis: The effects of 50 ppm compound DBP-64 on glycogen transport and degradation in rice blast fungus at different time points were tested. Figure 11 As shown, the exogenous addition of 50 ppm of compound DBP-64 did not affect glycogen synthesis, but slowed down the transport of glycogen from conidia to appressoria; after 16 h and 24 h of hydrophobic membrane inoculation, the exogenous addition of 50 ppm of compound DBP-64 significantly slowed down the degradation rate of glycogen in appressoria.

[0098] 4. Method for determining Bodipy lipid droplet staining in appressoria

[0099] Add 1 μL of 10 μg / μL tricyclazole (which inhibits the formation of melanin in appressorium) to 1 mL of diluted spore solution. Dilute the lipid drop dye Bodipy (Boron dipyrromethene) at a ratio of 1:1000. After induction for 0 h, 4 h, 8 h, and 24 h, remove the moisture from the hydrophobic membrane surface containing the spore solution using a pipette or absorbent paper. Add 20 μL of diluted Bodipy dye, cover with a coverslip, and count the staining of conidia and appressorium lipid droplets under a fluorescence microscope. Take photos and record the results. Each experiment was repeated 3 times.

[0100] Results Analysis: The effects of 50 ppm compound DBP-64 on lipid droplet transport and degradation of rice blast fungus at different time points were tested. Figure 12 As shown, the exogenous addition of 50 ppm of compound DBP-64 had no significant effect on lipid droplet synthesis and degradation, but slowed down the lipid droplet transport rate in conidia after 24 hours.

[0101] Example 4: Inhibition experiment of compound DBP-64 on other pathogens

[0102] This example also tested the toxic effects of compound DBP-64 on other plant pathogens. A concentration of 50 ppm of compound DBP-64 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).

[0103] Table 2 Plant pathogens

[0104]

[0105] The results are as follows Figure 13 and Figure 14As shown, compound DBP-64 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 DBP-64, which may be due to the specific targeting of the Pex19 protein by the DBP-64 molecule. Given the high homology of Pex19 proteins in different pathogens, DBP-64 can bind to Pex19 homologous proteins in various pathogens, thereby achieving a significant antibacterial effect.

[0106] Example 5

[0107] 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 DBP-64 and gelatin solution were mixed to prepare a drug-containing gelatin solution (final gelatin concentration 0.2%), resulting in final DBP-64 concentrations of 25, 50, and 100 ppm. The dosage difference of DBP-64 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.

[0108] like Figure 15 As shown, compound DBP-64 showed no significant difference in rice growth compared to the control, and did not show any obvious negative effects on rice. This suggests that DBP-64 may be safe for rice while effectively controlling rice blast.

[0109] In summary, the compound DBP-64 of this invention exhibits significant efficacy in controlling rice blast fungus. It effectively inhibits the growth and reproduction of the fungus in both in vitro inoculation and in vivo rice spraying experiments. Particularly in in vivo rice spraying experiments, DBP-64 demonstrated significant control effects during the three key window periods of pre-inoculation, inoculation, and post-inoculation, without any obvious negative effects on rice growth, demonstrating its great potential as a drug for controlling rice blast. Furthermore, DBP-64 also exhibits inhibitory effects on various other plant pathogens, proving its broad-spectrum antibacterial activity. These findings provide strong scientific evidence for the application of DBP-64 in agricultural disease control and foreshadow its broad prospects in future plant protection.

[0110] 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 DBP-64 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 DBP-64 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 DBP-64 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 structure of the compound DBP-64 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 DBP-64 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 DBP-64 is 3-50 ppm.

5. Use according to claim 4, characterized in that, The concentration of the compound DBP-64 is 12.5-50 ppm.

6. A method for controlling Pyricularia oryzae or / and other pathogenic fungi, characterized by, Spray 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 DBP-64, and the chemical structure of the compound DBP-64 is shown as follows: 。 7. The method of claim 6, wherein, The concentration of the compound DBP-64 is 3-50 ppm.

8. The method of claim 7, wherein, The concentration of the compound DBP-64 is 12.5-50 ppm.

Images