Use of compound btd-81 for the preparation of a medicament for the control of magnaporthe grisea

Abstract

The application discloses application of a compound BTD-81 in preparation of a medicine for preventing and treating Pyricularia oryzae, and belongs to the field of plant fungal disease prevention and treatment. The compound BTD-81 has a remarkable effect in preventing and treating rice blast or / and other pathogenic fungi, can effectively inhibit spore pathogenicity of the Pyricularia oryzae in a concentration range of 10-50 ppm, and especially, the compound BTD-81 at 50 ppm can completely prevent and treat the rice blast. The compound BTD-81 can be used as a protective agent, has a better effect when used at an initial stage of the rice blast infection, and under the condition of spraying the compound, the growth state of the rice is good, and there is no obvious adverse effect, indicating that the compound is safe to the rice, and has a practical significance for preventing the rice blast disease.

Description

Technical Field

[0001] This invention belongs to the field of plant fungal disease control, specifically involving the application of compound BTD-81 in the preparation of a drug for controlling rice blast fungus. Background Technology

[0002] Rice, as one of the world's most important food crops, is the main food source for more than half of the population. However, it is susceptible to disease caused by the rice blast fungus (…). Magnaporthe oryzae Rice blast, caused by [a pathogen], is widely recognized as one of the most destructive crop diseases in the world. This pathogen has broad host adaptability and can infect 50 species of grasses, including rice, wheat, and barley. In years of outbreaks, it often causes devastating losses and seriously threatens global food security.

[0003] Rice blast fungus is a haploid ascomycete. Its field spread primarily relies on its conidia, which are dispersed via air currents, rainwater, and insect vectors, ultimately infecting the rice plant surface. Under suitable environmental conditions, the conidia germinate and differentiate into specific dome-shaped infection structures—appendages. Experimental observations have confirmed that during the maturation stage of appendages, a high concentration of glycerol (approximately 3.2 M) can accumulate. This turgor pressure, approximately 8.0 MPa, is generated through osmotic regulation, driving the formation of specific narrow infection pegs at the base, enabling easy invasion of the host plant's leaf tissue.

[0004] Rice blast pathogens are distributed in major rice-producing areas worldwide and exhibit rapid population genetic variation, giving them stronger environmental adaptability and accelerating pathogenicity variations. From a plant pathology perspective, this not only increases the difficulty of disease control technologies but also adds complexity to regional control. Therefore, developing an integrated rice blast control technology system is of significant strategic importance for ensuring food security. Traditional rice blast control mainly employs comprehensive measures such as breeding resistant varieties, pathogen eradication, chemical control, optimized cultivation management, and harmless disposal of diseased plant debris.

[0005] In existing pest control systems, chemical control has become an indispensable key control method due to its stable efficacy and rapid response during the rice blast epidemic season, and it has gained widespread recognition in both scientific research and practical production. However, long-term excessive application of chemical pesticides has had a systemic impact on the agricultural ecological environment. For example, pesticide residues not only alter key physicochemical indicators such as soil pH and organic matter content, but also lead to a decrease in microbial diversity and significantly disrupt the soil microbial balance. In farmland ecosystem monitoring, the populations of pollinating insects and natural enemies have declined significantly, disrupting the biological control function.

[0006] More seriously, the selective pressure resulting from long-term chemical control has significantly accelerated the evolution of pesticide resistance in rice blast fungus populations. Monitoring data from some rice-growing areas indicate that adaptive mutations have occurred at the target sites of many major fungicides in recent years, leading to significant pesticide resistance in rice blast fungus strains in some regions. This resistance not only weakens the effectiveness of chemical pesticides in the field but also poses a serious threat to existing control strategies.

[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 BTD-81 in the preparation of a drug for controlling rice blast fungus. This invention solves the problem of adaptive mutations in existing pesticides at target sites. The drug of this invention acts on MoPex19, effectively inhibiting the pathogenicity of rice blast fungus spores, showing significant control over rice blast, and is non-toxic to crops.

[0011] To achieve the above objectives, this invention provides compound BTD-81 in the preparation of a fungicide for controlling rice blast fungus (… Magnaporthe oryzaeThe chemical structural formula of compound BTD-81 is shown below for use in drugs targeting or / and other pathogenic fungi:

[0012] .

[0013] The other pathogenic fungi include: *Botrytis cinerea* (… Botrytis cinerea ), Fusarium oxysporum ( Fusarium fujikuroi Anthracnose of melons ( ) Colletotrichum orbiculare Alternaria ( Alternaria alternata Fusarium graminearum ( ), Fusarium graminearum ) 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 BTD-81 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*, *Fusarium graminearum*, and *Anthracis acutissima*.

[0015] Preferably, the compound BTD-81 can reduce the turgor pressure of rice blast fungus appressorium and / or slow down the transport of lipid droplets from rice blast fungus conidia to appressorium.

[0016] Preferably, the concentration of compound BTD-81 is 10 to 50 ppm. In this invention, compound BTD-81 can effectively inhibit the pathogenicity of rice blast fungus spores within a concentration range of 10 to 50 ppm.

[0017] More preferably, the concentration of compound BTD-81 is 30-50 ppm. When the concentration of compound BTD-81 is 30-50 ppm, it has a significant inhibitory effect on the pathogenicity of isolated barley.

[0018] Most preferably, the concentration of compound BTD-81 is 50 ppm. When the concentration of compound BTD-81 is 50 ppm, it 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 BTD-81; wherein the other pathogenic fungi include any one or more of the following: Botrytis cinerea, Fusarium oxysporum, Anthracnose cucurbita, Alternaria alternata, Fusarium graminearum, and Anthracnose oxysporum.

[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, Fusarium graminearum, and Anthracnose fusiforme.

[0022] Preferably, the concentration of compound BTD-81 is 10 to 50 ppm.

[0023] More preferably, the concentration of compound BTD-81 is 30 to 50 ppm.

[0024] The application of compound BTD-81 of the present invention in the preparation of a drug for controlling rice blast fungus solves the problem of rice blast fungus populations developing resistance to existing agents, and has the following advantages:

[0025] (1) This invention first discovered that compound BTD-81 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 BTD-81 at 50 ppm can completely prevent and control rice blast disease.

[0026] (2) The compound BTD-81 used in this invention has a significantly better control effect on detached barley leaves than detached rice leaves at the same treatment concentration.

[0027] (3) The compound BTD-81 used in this invention, when mixed with rice blast fungus spore liquid and sprayed at 0 h, has a certain control effect, indicating that the compound BTD-81 of this 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 disease.

[0028] (4) The present invention found that compound BTD-81 can inhibit the growth of rice blast fungus mycelium, increase the appressorium collapse rate, significantly reduce the turgor pressure of rice blast fungus appressorium, and slow down the transport of lipid droplets from rice blast fungus conidia to appressorium. Attached Figure Description

[0029] Figure 1 Predicted binding sites for MoPex19 and compound BTD-81.

[0030] Figure 2 The inhibition of different concentrations of compound BTD-81 on plate growth of rice blast fungus.

[0031] Figure 3 The diameter of rice blast fungus colonies on plates treated with different concentrations of compound BTD-81 ( ) Figure 3 A) Colony growth hyphae inhibition rate ( Figure 3 (B) Comparison plot; In the plot, significant differences in the inhibition rate of compound BTD-81 at different concentrations are represented by different letters a, b, c, d, e, or f; p < 0.05; The data in the plot were obtained by one-way ANOVA.

[0032] Figure 4 The effects of adding different concentrations of compound BTD-81 to rice blast fungus spore suspension on detached barley leaves ( Figure 4 A) Rice leaves ( Figure 4 The incidence of disease in B).

[0033] Figure 5 The study investigated the disease incidence of live rice leaves after spraying with 50 ppm compound BTD-81 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 BTD-81 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 BTD-81 was added exogenously to the spore liquid of rice blast fungus.

[0036] Figure 8 Comparison of attachment formation rates at different time points induced by exogenous addition of 50 ppm compound BTD-81 to rice blast fungus spore liquid.

[0037] Figure 9 The collapse of appressorium under two different concentrations of glycerol when 50 ppm of compound BTD-81 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 BTD-81 was added exogenously to rice blast fungus spore liquid.

[0039] Figure 11 The effects of exogenous addition of 50 ppm of compound BTD-81 to rice blast fungus spore suspension on glycogen transport and degradation at different time points induced by appressorium ( Figure 11 (A); Comparison of glycogen content in conidia at different induction times (Figure A). Figure 11 (B in the figure) and a comparison of the proportion of glycogen in the appressorium () Figure 11 (C in the middle).

[0040] Figure 12 The lipid droplet transport and degradation at different time points induced by appressorium when 50 ppm of compound BTD-81 was added exogenously to the spore suspension of rice blast fungus (BTD-81) Figure 12 (A); Comparison of the proportion of lipid droplets in conidia induced at different times (A); Figure 12 (B in the figure) and a comparison of the proportion of lipid droplets in the appressorium ( Figure 12 (C in the middle).

[0041] Figure 13 This shows the inhibitory effect of compound BTD-81 on the plate growth of different plant pathogenic fungi.

[0042] Figure 14 The graph shows the diameter of plate colonies of different plant pathogenic fungi after treatment with compound BTD-81. The black bars represent the diameter of normal original fungi colonies without BTD-81 (control), while the white bars represent the diameter of pathogenic fungi colonies after BTD-81 treatment.

[0043] Figure 15 The effect of different concentrations of compound BTD-81 on rice growth is shown in the figure, 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 BTD-81 through high-throughput screening of a large number of compounds. To investigate the specific binding of BTD-81 to this target, AlphaFold3 was used to perform structural simulations of the interaction site between BTD-81 and the MoPex19 protein. The predicted results are as follows: Figure 1 As shown in the figure. Analysis indicates that the binding site of BTD-81 is located at the ILE-238 residue of the MoPex19 protein.

[0051] Further research on the control of rice blast fungus by applying compound BTD-81 was conducted. The experiments in the following examples demonstrated the effectiveness of compound BTD-81 in controlling rice blast fungus, laying the foundation for the development of new green pesticides.

[0052] The bacteria and reagents used in the following examples 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, https: / / www.atcc.org / products / 201236).

[0054] 2. The culture medium for laboratory cultivation of rice blast fungus 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 para-aminobenzoic acid, 0.1 mg vitamin B, 0.1 mg thiamine, 0.1 mg niacin, 0.1 mg riboflavin, 0.1 mg biotin, 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. g of agar powder, pH adjusted to 6.5 with NaOH, and brought to a final volume of 1 L with ddH2O, were then autoclaved (121℃, 20 min).

[0055] 3. Compound BTD-81 was purchased from Shanghai Taoshu Biotechnology Co., Ltd., catalog number HIT ID: HIT102381227. Its full chemical name is (5Z)-3-[(4-bromophenyl)methyl]-5-{[5-(morpholin-4-yl)furan-2-yl]methylene}-1,3-thiazolyl-2,4-dione. The molecular structure of compound BTD-81 is as follows: ;

[0056] 4. Preparation and storage of the stock solution of compound BTD-81: 10 mg of compound BTD-81 was diluted to 4 mL of DMSO, resulting in a final concentration of 2500 ppm. This reagent should be stored at -20°C for short periods and aliquoted into small centrifuge tubes and stored at -80°C for long-term storage.

[0057] Example 1: Virulence test and results of compound BTD-81 against rice blast fungus.

[0058] The virulence of compound BTD-81 against *Bacillus oryzae* was determined using the mycelial growth rate method, as follows: *Bacillus oryzae* Guy11 was inoculated onto 7 cm CM plates and cultured for 7 days at 25°C (16 h light, 8 h dark cycle). Different concentrations (0, 10, 20, 30, 40, and 50 ppm) of compound BTD-81 were added to solid CM medium to prepare agar plates containing different drug concentrations. 3 mL of sterile ddH2O was added to the 7-day-old *Bacillus oryzae* Guy11 plates. Conidia were gently scraped off the plates with a spreader, filtered through sterile three-layer filter paper into 1.5 mL centrifuge tubes, and the number of *Bacillus oryzae* conidia was determined under an optical microscope using a hemocytometer. The conidia were then diluted with sterile ddH2O to a final concentration of 1 × 10⁻⁶. 5 spores / mL. Use a pipette to add 5 μL of diluted conidial solution to the center of the drug-containing plate; repeat each concentration treatment 3 times; use CM medium containing an equal concentration of DMSO as a blank control; place the petri dishes in an incubator at 25℃ (16 h light / 8 h dark cycle) for 3 days, and observe the mycelial growth of rice blast fungus in the experimental group.

[0059] The mycelial diameter was measured using the cross-cross method, and combined with the mycelial growth rate method, the inhibition rate of compound BTD-81 on rice blast mycelial growth at different concentration gradients was calculated. The calculation formula is as follows:

[0060] 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.

[0061] Using DPS statistical software, a virulence regression equation was established and the half-maximal effective concentration (EC50) of compound BTD-81 against rice blast fungus was calculated. 50 The virulence level of compound BTD-81 against rice blast fungus was evaluated by using its correlation coefficient (r).

[0062] Table 1. Indoor toxicity of compound BTD-81 at different concentrations against rice blast fungus.

[0063]

[0064] Results Analysis: The effects of different concentrations of compound BTD-81 on the virulence of rice blast fungus were analyzed. Figure 3 As shown in B, all five concentration gradient treatments of compound BTD-81 showed some inhibitory effect on the mycelial growth of rice blast fungus. With increasing concentration of compound BTD-81, the average colony diameter decreased ( Figure 3 A and Figure 2 Increased mycelial growth inhibition rate ( Figure 3(B in the text). Within this concentration gradient range, compound BTD-81 exhibited an inhibition rate of 10%–70% against rice blast fungus; the half-maximum effective concentration (EC50) of compound BTD-81 against rice blast fungus mycelial growth was calculated. 50 The concentration of BTD-81 was 35.98651 ppm (Table 1). The compound BTD-81 exhibited the most significant inhibitory effect against rice blast fungus at a concentration of 50 ppm, with a relative inhibition rate of 62.103% (Table 1).

[0065] Example 2: Determination and results of the pathogenicity of compound BTD-81 against rice blast fungus.

[0066] The pathogenicity of compound BTD-81 against rice blast fungus was determined using two methods: in vitro inoculation and live rice spraying. Details are as follows:

[0067] 1. In vitro inoculation method

[0068] (1) In vitro barley inoculation

[0069] Guy11 strain, stored at 4℃, was activated and inoculated onto 7 cm quantitative CM plates. After incubation at 28℃ for 7 days (16 h light, 8 h dark), 3 mL ddH2O was added to the CM plates. Conidia were gently scraped off using a sterile spreader. The spore solution was filtered through sterile three-layer filter paper into 1.5 mL centrifuge tubes and centrifuged for 2 min (7500 rpm / min). The supernatant was discarded, and the spores were resuspended in 1 mL ddH2O to obtain the spore stock solution. The spore count was diluted to a concentration of 5 × 10⁻⁶ using a hemocytometer under a microscope. 4 spores / mL. The spore solution was uniformly mixed with compound BTD-81 to prepare drug-containing spore solutions at concentrations of 0, 10, 20, 30, 40, and 50 ppm. The dosage difference of compound BTD-81 between different treatment groups was compensated for using DMSO solvent to ensure a consistent DMSO content in the final concentration. A 10 × 10 cm square petri dish was prepared. Absorbent paper was cut to an appropriate size and placed in the dish. The absorbent paper was evenly moistened with an appropriate amount of water and then covered with a filter paper of the same size. Barley leaves, about 5–7 cm long, were cut from leaves planted 8–10 days prior and arranged sequentially 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 was repeated three times. The spore solution for the control group contained the same concentration of DMSO. The petri dishes containing the leaves were placed in a 28°C incubator (16 h light, 8 h dark cycle). After 4 days of incubation, the disease incidence was observed and recorded by photograph.

[0070] Results Analysis: This experiment investigated the inhibitory effect of graded concentrations of compound BTD-81 on rice blast disease in detached barley leaves through inoculation experiments. Figure 4As shown in A, within the concentration range of 10 to 50 ppm, treatment with compound BTD-81 showed a significant inhibitory effect on the infection of detached barley leaves by rice blast fungus; among them, 10 ppm of compound BTD-81 was sufficient to inhibit the pathogenicity of rice blast fungus spores on detached barley leaves, and high concentrations of compound BTD-81 did not show a stronger inhibitory effect, indicating that the minimum inhibitory concentration may be 10 ppm.

[0071] (2) In vitro rice inoculation

[0072] 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 in vitro inoculation is the same as that of inoculating barley.

[0073] Results Analysis: This experiment tested the inhibitory effect of compound BTD-81 on rice blast disease through inoculation experiments on detached leaves. Figure 4 As shown in Figure B, the five concentration gradients of compound BTD-81, within the range of 10 to 50 ppm, all showed some inhibitory effect on detached rice blast disease. Among them, the concentration of compound BTD-81 at 30 to 50 ppm showed a significant inhibitory effect on detached rice blast disease. The concentration of compound BTD-81 at 30 ppm could completely inhibit the infection of rice blast disease on detached rice leaves, and higher concentrations did not show a stronger inhibitory effect.

[0074] like Figure 4 As shown in A and B, at the same treatment concentration, compound BTD-81 showed significantly better control of rice blast disease on detached barley leaves than on detached rice leaves.

[0075] 2. Live rice spraying method

[0076] Oryza sativa CO39 rice was germinated and sown (30 seeds per pot), and cultured outdoors in a well-lit and well-ventilated area for two weeks. After culturing the rice blast fungus on CM medium for 7-9 days, conidia were gently scraped off with a spreader, filtered, 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 prepare a 0.4% gelatin stock solution. Compound BTD-81, the gelatin solution, and the spore solution were mixed to prepare a mixed spore solution containing the drug and gelatin (final spore concentration 5×10⁻⁶). 4The concentration of compound BTD-81 was 50 ppm (spores / mL, gelatin final concentration 0.2%). The dose 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 BTD-81 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 on the rice leaves was observed and photographed, and the lesion area and lesion rate were calculated using ImageJ software.

[0077] Lesion rate (%) = Lesion area / Leaf area × 100%

[0078] The specific optimization experimental steps are as follows: The concentration of compound BTD-81 that inhibits rice blast disease was selected as 50 ppm. Compound BTD-81 was applied at different time points at which the rice blast fungus spores were inoculated, and three experimental groups were set up:

[0079] (1) -24 h: Spray compound BTD-81 24 hours before inoculating with rice blast fungus spore liquid;

[0080] (2) 0 h: Apply compound BTD-81 at the same time as inoculating rice blast fungus spore liquid, that is, mix compound BTD-81 with rice blast fungus spore liquid and spray for inoculation;

[0081] (3) +24 h: Spray compound BTD-81 24 hours after inoculation with rice blast fungus spores. Each treatment was replicated using 3 pots of rice, and a blank control and DMSO solvent control were set up.

[0082] Results Analysis: The efficacy of compound BTD-81 in controlling rice blast disease in live rice was tested by spraying at different time points. Compared to the control group without BTD-81, both the -24h and +24h treatment groups showed some inhibition of rice blast disease in live rice. Figure 5 and Figure 6 This has practical significance in preventing rice blast. Surprisingly, at 0 h, spraying a mixture of compound BTD-81 and rice blast fungus spores showed a certain control effect, indicating that compound BTD-81 can be used as a protective agent with better efficacy in the early stages of rice blast infection. Simultaneously, it was observed that the rice in the control group, which was sprayed only with the same concentration of DMSO or the same concentration of compound BTD-81, showed good growth and no significant adverse effects, suggesting that this compound may be safe for rice. Figure 5This has practical significance in preventing rice blast disease.

[0083] Example 3: Determination and results of the effect of compound BTD-81 on appressorium development of rice blast fungus.

[0084] 1. Method for determining the appressorium formation rate

[0085] Guy11 was inoculated onto 7 cm² agar plates and incubated at 28°C for 7 days (16 h light, 8 h dark cycle). 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. After filtration through three layers of filter paper, the mixture was 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 The spore solution was prepared by mixing the spore solution with compound BTD-81 to form a drug-containing spore solution. The final concentration of compound BTD-81 was 50 ppm, with the dose difference made up with DMSO to ensure a consistent final concentration; a control group containing the same concentration of DMSO was used. Artificial hydrophobic membranes were washed with 70% ethanol solution and placed on glass slides. Three artificial hydrophobic membranes were placed on each slide, and three drops of 20 μL of diluted spore solution were added to each membrane. The prepared slides were transferred to a humidification box (containing an appropriate amount of sterile water) and placed in a dark incubator at 22°C for induction. The appressorium formation rate was recorded at 4 h, 8 h, 16 h, and 24 h after induction, and photographs were taken. Each experiment was repeated three times.

[0086] Results analysis: such as Figure 7 , Figure 8 As shown, compound BTD-81 does not affect the formation of rice blast fungus appressoria at a concentration of 50 ppm.

[0087] 2. Method for measuring the turgor pressure of appressorium

[0088] The process of inducing appressorium was essentially the same as that of appressorium formation assay. Glycerol solutions of 2 M and 3 M concentrations were prepared. After incubation at 28°C for 24 h (16 h light, 8 h dark), the hydrophobic membrane of the induced appressorium was removed, and surface moisture was gently absorbed with a pipette or absorbent paper. An equal volume of the corresponding concentration of glycerol solution was quickly added, and the mixture was allowed to stand at room temperature for 5 min. Immediately afterwards, the appressorium collapse rate (the proportion of collapsed appressoriums to the total number of observed appressoriums) was observed and calculated under an optical microscope. Each strain was replicated three times at each glycerol concentration.

[0089] Results analysis: At two different glycerol concentrations, 50 ppm of compound BTD-81 affected the turgor pressure of appressorium. For example... Figure 9 , Figure 10As shown, at two different concentrations of glycerol, 50 ppm of compound BTD-81 significantly affected the turgor pressure of rice blast fungus appressoriums. At 1 M and 2 M glycerol concentrations, exogenous addition of 50 ppm of compound BTD-81 significantly increased the appressorium collapse rate and significantly reduced the turgor pressure of rice blast fungus appressoriums.

[0090] 3. Method for determining KI / I2 glycogen staining of appressorium

[0091] Prepare KI / I2 solutions (60 mg / mL KI, 10 mg / mL I2) for glycogen staining. The procedure for inducing appressorium is the same as in the experiment 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, 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.

[0092] Results Analysis: The effects of 50 ppm of compound BTD-81 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 BTD-81 had no effect on glycogen synthesis. After 8 h of induced appressorium formation, the exogenous addition of 50 ppm of compound BTD-81 slowed down the transport of glycogen from conidia to appressorium. However, after 16 h and 24 h of induced appressorium formation, the lipid droplet transport and degradation rates were basically the same as those of the control group.

[0093] 4. Bodipy glycogen staining method for appressorium

[0094] Before inducing appressorium, 1 μL of 10 μg / μL tricyclazole was added to 1 mL of spore solution to inhibit melanin formation in the appressorium, facilitating fluorescence observation. The lipid droplet dye Bodipy (Boron dipyrromethene) 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 chamber. The surface moisture was gently absorbed with a pipette or absorbent paper, and an equal volume of diluted Bodipy dye was added. A coverslip was then placed on the membrane, and the glycogen staining of conidia and appressorium was recorded under a fluorescence microscope. Each experiment was repeated three times.

[0095] Results Analysis: The effects of 50 ppm compound BTD-81 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 BTD-81 had no effect on lipid droplet synthesis and degradation in rice blast fungus spores and appressoria, but it did have a certain degree of slowing effect on lipid droplet transport.

[0096] Example 4: Inhibition experiment of compound BTD-81 on other pathogens

[0097] This example also tested the toxic effects of compound BTD-81 on other plant pathogens. A concentration of 50 ppm of compound BTD-81 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 Fusarium graminearum ( ), F. graminearum : Fusarium graminearum ), Anthrax bacillus ( C. acutatum : Colletotrichum acutatum (See Table 2).

[0098] Table 2 Plant pathogens

[0099]

[0100] The results are as follows Figure 13 and Figure 14 As shown, compound BTD-81 exhibits inhibitory effects on the plate growth of various plant pathogenic fungi, inhibiting the mycelial growth of the aforementioned six different genera of plant pathogens to varying degrees, particularly *Botrytis cinerea* and *Anthracnose cucurbita*, with significant inhibitory effects. These results demonstrate the broad-spectrum antibacterial activity of BTD-81, which may be due to its specific targeting of the Pex19 protein. Given the high homology of Pex19 proteins in different pathogens, BTD-81 can bind to Pex19 homologous proteins in various pathogens, thereby achieving a significant antibacterial effect.

[0101] Example 5: Safety evaluation of BTD-81

[0102] 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 BTD-81 and gelatin solution were mixed to prepare a drug-containing gelatin solution (final gelatin concentration of 0.2%), with final BTD-81 concentrations of 25, 50, and 100 ppm. The dosage difference of compound BTD-81 was made up with DMSO to ensure a consistent final DMSO concentration. 2 mL of the mixed spore solution containing the compound and gelatin was sprayed evenly onto rice leaves, and 3 pots of rice were 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.

[0103] like Figure 15 As shown, compound BTD-81 showed no significant difference in rice growth compared to the control, and did not show any obvious negative effects on rice. This suggests that while BTD-81 is effective in controlling rice blast, it may also be safe for rice.

[0104] In summary, the compound BTD-81 of this invention possesses broad-spectrum antibacterial activity and can serve as an effective inhibitor of plant pathogens, particularly showing significant effects against rice blast fungus and various other plant pathogens. This not only provides a new strategy for the control of plant diseases but also offers a scientific basis for the development of broad-spectrum and highly effective antifungal agents. Therefore, compound BTD-81 has broad application prospects and significant practical value in the field of agricultural disease control.

[0105] 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 BTD-81 was used in the preparation of a fungicide for controlling 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 BTD-81 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 , Fusarium graminearum Fusarium graminearum and Phaeosphaeria nodorum Colletotrichum acutatum .

2. Use of the compound BTD-81 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, Fusarium graminearum and Colletotrichum acutatum. The chemical structural formula of the compound BTD-81 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 BTD-81 can reduce the turgor pressure of Magnaporthe oryzae appressorium or / and slow down the transport of lipid droplets from Magnaporthe oryzae conidia to appressorium.

4. Use according to claim 1 or 2, wherein the compound is ###0002### The concentration of the compound BTD-81 is 10-50 ppm.

5. Use according to claim 4, characterized in that, The concentration of the compound BTD-81 is 30-50 ppm.

6. A medicine for controlling Magnaporthe grisea or / and other pathogenic fungi, characterized by, The compound BTD-81 is included. The other pathogenic fungi include any one or two or more of Botrytis cinerea, Fusarium fujikuroi, Colletotrichum orbiculare, Alternaria alternata, Fusarium graminearum and Colletotrichum acutatum. The chemical structural formula of the compound BTD-81 is as follows: 。 7. The medicament according to claim 6, characterized in that, It further comprises an agriculturally acceptable carrier.

8. A method for controlling Pyricularia oryzae or / and other pathogenic fungi, characterized by, The medicine for preventing and treating Magnaporthe oryzae or / and other pathogenic fungi as claimed in claim 6 or 7 is sprayed 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, Fusarium graminearum and Colletotrichum acutatum.

9. The method of claim 8, wherein, The concentration of the compound BTD-81 is 10-50 ppm.

10. The method of claim 9, wherein, The concentration of the compound BTD-81 is 30-50 ppm.

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