Application of dichloro-xazole in inhibition of tea tree leaf spot bacteria

Abstract

The invention belongs to the technical field of biological prevention and control, and provides application of 2-(2-chlorophenyl)-1, 2, 4-oxadiazole in inhibition of tea tree leaf spot bacteria, and the 2-(2-chlorophenyl)-1, 2, 4-oxadiazole synergistically exerts an antibacterial effect through multiple mechanisms of destroying plasma membranes and cell wall structures of pathogenic bacteria, blocking an ATP synthesis pathway, interfering glucose metabolism and the like. The problems of aggravated drug resistance of pathogenic bacteria caused by single action target of the existing chemical bactericide and environmental pollution caused by high-toxicity pesticides are solved; as a derivative of a plant source natural product, namely gallic acid thiadiazole, dichlorxazole can completely inhibit mycelial growth of tea tree phyllosticta under the concentration of 15 mg / L, has the characteristics of high efficiency, low toxicity and environment friendliness, and has an antibacterial effect on various plant pathogenic bacteria such as tea anthracnose bacteria, tea physalospora, botrytis cinerea and sclerotinia sclerotiorum; the composition is suitable for green disease prevention and control of crops such as tea, rape and tomato.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology and relates to the inhibition of plant pathogenic fungi, specifically the application of dichlorooxadazole in inhibiting tea leaf spot pathogens. Background Technology

[0002] Plant pathogenic fungi are a major biological factor threatening global food security and agricultural production, often leading to a severe decline in the yield and quality of major food and cash crops. For example, sclerotinia sclerotinia disease in rapeseed caused by *Sclerotinia sclerotiorum*, gray mold in tomato caused by *Botrytis cinerea*, and leaf spot, ring spot, and anthracnose in tea (*Camellia sinensis*) caused by *Camellia leaf spot fungus*, *Camellia pseudobulb*, and *Camellia anthracnose fungus*, respectively, are all representative diseases with significant economic impact.

[0003] Chemical control remains the primary means of controlling the aforementioned fungal diseases in current technologies. However, the long-term and excessive use of fungicides has led to a series of problems, including increased pathogen resistance, environmental residues and pollution, and continuously rising control costs. Therefore, the development of antifungal agents with novel mechanisms of action, high efficiency, and good environmental compatibility has become an urgent need for the green control of plant diseases.

[0004] In recent years, strategies based on structure-function-oriented design and natural product modification have shown promising promise in the development of novel pesticide molecules. For example, some studies have obtained active compounds that can effectively inhibit plant virus replication by optimizing the structure of the plant natural product chromone; other studies have designed small molecule inhibitors that can efficiently inhibit rice blast fungus based on effector protein targets, and these inhibitors have shown good control efficacy in the field. These achievements provide important insights for the creation of novel green pesticides.

[0005] Gallic acid, a naturally occurring secondary metabolite widely found in plants, has been shown to exhibit inhibitory activity against various pathogenic fungi (such as Candida, Alternaria, and Trichophyton rubrum), demonstrating its potential as a lead compound. Dichlorooxadazole (chemical name: 2-(2,4-dichlorophenyl)-5-(methanesulfonyl)-1,3,4-oxadiazole) is a novel oxadiazole compound synthesized through derivatization and modification using gallic acid as the lead structure. It is initially considered to possess high efficiency, low toxicity, and environmental friendliness. However, to date, the specific bioactivity spectrum of this compound against plant pathogenic fungi, especially its antifungal mechanism at the molecular level, remains unclear, severely limiting its further development and application. Summary of the Invention

[0006] The purpose of this invention is to provide the application of dichlorooxamazole in inhibiting leaf spot pathogens of tea leaves. By disrupting glucose biosynthesis and energy metabolism, it aims to solve the problems of increased pathogen resistance caused by the single target of existing chemical fungicides and the environmental pollution caused by traditional highly toxic fungicides.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention provides the application of dichlorooxamazole in inhibiting leaf spot pathogens in tea trees.

[0008] Preferably, dichlorooxadazole is used to prepare products that inhibit leaf spot disease of tea trees.

[0009] Preferably, the product includes the active ingredient dichloroxazole and a pharmaceutical composition used in combination therewith.

[0010] Preferably, the dichlorodichlorodimethoate is a derivative of gallic acid, and the dichlorodichlorodimethoate at a concentration of 15 mg / L has a 100% growth inhibition rate against *Tea Leaf Spot* pathogens. The dichlorodimethoate also exhibits an EC50-95% inhibition rate against *Tea Leaf Spot* pathogens. 50 The value was 10.5 mg / L.

[0011] Preferably, the dichlorooxadazole achieves antibacterial effect by disrupting the plasma membrane or cell wall structure, blocking the ATP synthesis pathway, and interfering with glucose metabolism, and the pathogen is a plant pathogen.

[0012] Preferably, the plant pathogen is at least one of the following: tea anthracnose fungus, tea leaf spot fungus, tea leaf spot fungus, Botrytis cinerea, or Sclerotinia sclerotiorum.

[0013] Preferably, the dichlorooxadazole is effective against the EC50 of *Anthrax thaliana*. 50 The value was 14.02 mg / L, indicating that the EC50 of the diclofenac was effective against *Tea leaf spot*. 50 The value was 13.45 mg / L, and the EC50 of the diclofenac was 13.45 mg / L against Sclerotinia sclerotiorum. 50 The value was 10.19 mg / L, and the EC50 of the diclofenac sodium against Botrytis cinerea was... 50 The value was 14.83 mg / L;

[0014] Preferably, the pharmaceutical composition further includes an aqueous solution, which is one of water, physiological saline, or glucose aqueous solution.

[0015] Preferably, the pharmaceutical composition can also be prepared into a formulation comprising the pharmaceutical composition and a pharmaceutically acceptable excipient selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, anti-adhesives, integrators, permeation enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

[0016] Preferably, the pharmaceutical formulation includes a variety of acceptable dosage forms.

[0017] Preferably, the dosage form of the pharmaceutical preparation is any one of capsules, granules, tablets, powders, ointments, powders, pills, or liquids.

[0018] The beneficial effects of this invention are:

[0019] 1. Effective antibacterial effect: Experiments show that dichlorooxamazole can completely inhibit the leaf spot pathogen of tea at low concentrations. Specifically, when the concentration is 15 mg / L, its inhibition rate on mycelial growth is as high as 100%, and when the concentration is as low as 5 mg / L, it can completely inhibit its spore germination. The above results indicate that the compound has a highly efficient and rapid bactericidal ability, which can significantly reduce the dosage and cost in practical applications.

[0020] 2. Multi-target mechanism of action: This invention reveals that dichlorooxadazole exerts its antibacterial effect through multiple synergistic mechanisms: firstly, it disrupts the integrity of the pathogen's plasma membrane and cell wall structure; secondly, it specifically blocks the ATP synthesis pathway, interfering with energy metabolism; and thirdly, it significantly affects the activity of key enzymes in glucose metabolism. Through this multi-target mechanism of action, it can effectively delay the development of pathogen resistance, providing a new strategy for solving the widespread problem of drug resistance in current plant disease control.

[0021] 3. It possesses broad-spectrum antibacterial properties. In addition to exhibiting specific activity against tea leaf spot pathogens, dichlorooxadixyl also shows inhibitory effects on a variety of important plant pathogenic fungi, including Colletotrichum camelliae, Pseudopestalotiopsis camelliae, Botrytis cinerea, and Sclerotinia sclerotiorum; thus giving it the potential to be developed into a general-purpose fungicide.

[0022] 4. It has environmental compatibility. As a derivative of gallic acid, a plant-derived natural product, dichlorooxadazole inherits the characteristics of low toxicity and easy degradation of natural products. While exerting a highly efficient bactericidal effect, it shows high safety to the environment and non-target organisms, which meets the environmentally friendly requirements of modern agriculture for green pesticides. Attached Figure Description

[0023] Figure 1 This invention relates to the determination of the antibacterial activity of Erlvejunzuo against Didymellasegeticola, the pathogen causing leaf spot disease in tea.

[0024] Figure 2This invention describes the effects of dichlorooxamazole on the morphology and plasma membrane integrity of *D. segeticola* (A shows the results of staining *D. segeticola* hyphae with propidium iodide (PI) after treatment with different concentrations of dichlorooxamazole for 12 hours, observed under a fluorescence microscope, Bar = 10 μm; B shows the morphology of *D. segeticola* hyphae treated with dichlorooxamazole observed under a scanning electron microscope, Bar = 10 μm; C shows the malondialdehyde (MDA) content in *D. segeticola* after treatment with dichlorooxamazole and DMSO for 24 hours; D shows the morphology of *D. segeticola* after treatment with DMSO and 10 mg / L dichlorooxamazole observed under a transmission electron microscope, Bar = 0.5 μm).

[0025] Figure 3 These are the results of the content leakage assay and enzyme activity assay in this invention (A is the result of the nucleotide leakage assay; B is the result of the protein leakage assay; C is the result of the enzyme activity assay).

[0026] Figure 4 This invention presents the transcriptional expression analysis of *D. segeticola* after treatment with diclofenac. (A is the whole transcriptome PCA analysis of *D. segeticola* after 12 and 24 hours of treatment with DMSO and 10 μM diclofenac; B is the scatter plot distribution of differentially expressed genes (DEGs) after 12 and 24 hours of treatment with DMSO and diclofenac; C is the heatmap distribution of differentially expressed genes after 12 and 24 hours of treatment with 10 μM diclofenac; D is the ATP content determination of *D. segeticola* after 16 hours of treatment with 5, 10, and 20 μM diclofenac and DMSO; E is the NAD-MDH enzyme (NAD-malate dehydrogenase) activity determination of *D. segeticola* after 12, 24, 48, and 72 hours of treatment with 5, 10, and 20 μM diclofenac and DMSO; F is the Venn diagram showing D.) Genetic differences induced by *Segeticola* at 12 and 24 hours; G is the growth phenotype of *D. segeticola* in medium supplemented with *Erlvejunzuo* and glucose.

[0027] Figure 5This invention demonstrates the highly effective control of fungal diseases by dichlorooxamazole, exhibiting broad-spectrum antibacterial activity. (A represents the antibacterial activity test of different concentrations of dichlorooxamazole against *Colletotrichum camelliae*, the causal agent of tea anthracnose; B represents the control effect of different concentrations of dichlorooxamazole against anthracnose in tea; C represents the antibacterial activity test of different concentrations of dichlorooxamazole against *Pseudopestalotiopsis camelliae*, the causal agent of tea leaf spot; D represents the control effect of different concentrations of dichlorooxamazole against gray spot disease in tea; E represents the antibacterial activity test of different concentrations of dichlorooxamazole against *Sclerotinia sclerotiorum*; F represents the control effect of different concentrations of dichlorooxamazole against sclerotinia rot in rapeseed; G represents the antibacterial activity test of different concentrations of dichlorooxamazole against *Botrytis cinerea*; H represents the control effect of different concentrations of dichlorooxamazole against gray mold in tomato.) Detailed Implementation

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] It should be noted that the hydrazine hydrate, methyl 2,4-dichlorobenzoate, petroleum ether (PE), ethyl acetate (EA), ethanol, KOH, 2,4-dichlorobenzoylhydrazine, carbon disulfide, tetrahydrofuran (THF), NH4Cl, anhydrous sodium sulfate, hydrogen peroxide, dimethyl sulfone (DMSO), propidium iodide (PI), etc. used in this invention are all commercially available.

[0032] The tested pathogens were: Didymella segeticola (hereinafter referred to as D. segeticola); Colletotrichum camelliae (hereinafter referred to as C. camelliae); Pseudopestalotiopsis camelliae (hereinafter referred to as P. camelliae); Sclerotinias clerotiorum (hereinafter referred to as S. sclerotiorum); and Botrytis cinerea (hereinafter referred to as B. cinerea). All pathogens were cultured on potato dextrose agar (PDA) at 28°C and preserved in the Department of Plant Pathology, College of Agriculture, Guizhou University.

[0033] The tea trees (Camellia sinensis) used in the experiment were grown in the greenhouse of Guizhou University, and the 3rd to 5th mature leaves were selected for pathogen inoculation experiments.

[0034] Tomato plants (Micro Tom) were cultivated in a greenhouse for inoculation with gray mold. Healthy tomato fruits of uniform size and without mechanical damage were selected, disinfected with 75% alcohol for 5 minutes, rinsed three times with sterile distilled water, and drained before use. Rapeseed plants (Brassica napus L.) were grown in a greenhouse for inoculation with Sclerotinia sclerotiorum.

[0035] The following experimental results were statistically analyzed using GraphPad Prism and SPSS software. Tukey test (P<0.05) and t test were used to assess the significance of differences.

[0036] Example 1: Antibacterial effect and mechanism of action

[0037] 1. In vitro antibacterial activity assay

[0038] 1.1 Method

[0039] Different concentrations of diclofenac were prepared by adding DMSO solution to PDA medium, with an equal volume of DMSO as a negative control. Mycelial discs with a diameter of 5 mm were inoculated into the center of the medium and incubated at 28°C. Each concentration was replicated four times. *D. segeticola*, *C. camelliae*, *P. camelliae*, *S. sclerotiorum*, and *B. cinerea* were cultured on PDA plates in the dark at 28°C for 10, 10, 7, 5, and 4 days, respectively.

[0040] 1.2 Results

[0041] The results showed that dichlorodi ... Figure 1 Even at low concentrations (2.5 mg / L), diclofenac can inhibit mycelial growth, while 15 mg / L of diclofenac can completely inhibit the growth of *D. segeticola*. Figure 1 ). Dichlorooxadazole against D. segeticola EC. 50 The value was 10.5 mg / L, and the in vivo experiment results showed the same trend, as detailed in Application Example 1.

[0042] 2. Effects of diclofenac on the integrity of the pathogen's plasma membrane

[0043] 2.1 Methods

[0044] (1) Propidium iodide (PI) staining experiment: Mycelia cultured on PDA for 5 days (28℃, 220 rpm) were treated with DMSO (control group) or different concentrations of dichlorooxazole for 24 hours. After washing the mycelia twice with PBS, they were stained with 40 mg / L propidium iodide (PI) in the dark for 15-40 minutes and observed with a laser confocal microscope (Zeiss LSM880 Airyscan, Carl Zeiss, Oberkochen, Germany).

[0045] (2) Determination of malondialdehyde (MDA) content: After mycelia cultured for 5 days were treated with DMSO (control group) or 10 / 20 mg / L dichlorooxam for 48 hours, they were flash-frozen in liquid nitrogen. The MDA content in 0.1 g of mycelia was determined using an MDA detection kit (AKFA013M, boxbio, China).

[0046] 2.2 Results

[0047] Dichlorooxamazole disrupts the plasma membrane integrity of D. segeticola.

[0048] (1) Propidium iodide (PI) staining experiment: PI binds to nucleic acids through the damaged cell membrane and emits red fluorescence. The results showed that the hyphae in the 10 and 20 mg / L dichlorooxam-treated groups emitted strong and continuous red fluorescence, while the control group showed no fluorescence signal. Figure 2 (A)

[0049] (2) Determination of malondialdehyde (MDA) content: The results showed that the MDA content increased significantly with the increase of dichloroxazole concentration. The MDA content of the 10 and 20 mg / L treatment groups was 1.71 times and 2.18 times that of the control group, respectively. Figure 2 (C)

[0050] 3. Effects of dichlorooxam on mycelial morphology

[0051] 3.1 Method

[0052] Scanning electron microscopy (SEM) observation: 10-day-old mycelial blocks were inoculated onto PDA plates containing DMSO (control group) or 10 mg / L dichlorooxamazole and cultured at 28°C for 9 days before sample preparation.

[0053] 3.2 Results

[0054] The hyphae in the control group had smooth surfaces and normal morphology, while the hyphae in the treatment group lost their linear structure, with protrusions and dense hyphal tips appearing on the surface. Scanning electron microscopy (SEM) further revealed that the hyphae in the treatment group were wrinkled and broken, while the hyphae in the control group remained linear and smooth. Figure 2 (B)

[0055] 4. Effects of dichlorooxamazole on spore germination of pathogens

[0056] 4.1 Methods

[0057] D. segeticola was cultured in oat medium (30g oat flakes, 1L ddH2O) at 26°C in the dark for 14 days. Spore suspensions were prepared and divided into a DMSO control group and treatment groups with 5, 10, and 20 mg / L dichloroisothiazide. After incubation at 26°C, the germination of more than 200 spores in each group was observed.

[0058] 4.2 Results

[0059] The spore germination rate in the control group reached 87.68%, while no spore germination was observed in the 5, 10, and 15 mg / L dichlorooxam treatment groups after 10, 20, and 30 hours, indicating that dichlorooxam effectively inhibited spore germination.

[0060] 5. Effects of diclofenac on the permeability of pathogenic bacterial plasma membranes

[0061] 5.1 Method

[0062] (1) Transmission electron microscopy (TEM) observation: 10-day-old mycelial blocks were inoculated onto PDA plates containing DMSO or 10 mg / L dichlorooxamazole and cultured at 28°C for 9 days before sample preparation.

[0063] (2) Content leakage determination: Five-day-old *D. segeticola* mycelial blocks were inoculated into PDA liquid medium and cultured at 28°C and 220 rpm for 5 days with shaking. After washing the mycelium three times with ddH2O, 1g of mycelium was suspended in DMSO (control group) or 10mg / L and 20mg / L dichlorooxamazole solutions for 1-11 hours. The leakage of nucleic acids and proteins was detected by micro-spectrophotometer at wavelengths of 260 nm and 280 nm, respectively.

[0064] (3) Enzyme activity assay: Mycelia cultured for 5 days in PDA (28℃, 220 rpm) were treated with DMSO (control group) or 10, 20 mg / L dichlorooxamazole for 12-72 hours and then flash-frozen in liquid nitrogen. β-1,3-glucanase activity was measured using a kit (ASKU038M-50S, boxbio, China), detecting the absorbance at 540 nm; NAD-malate dehydrogenase activity was calculated by the change in NADH absorbance at 340 nm using a kit (AKCO005M, boxbio, China).

[0065] 5.2 Results

[0066] (1) Transmission electron microscopy (TEM) showed that the hyphae of the control group were intact, with uniform cell membranes and regular organelle distribution; while the hyphae in the 10 mg / L dichlorooxamazole treatment group had irregular cell walls and cell membranes, and organelles dissolved and disappeared, ultimately leading to cell death. Figure 2 (D).

[0067] (2) Nucleotide and protein leakage experiments showed that the leakage amount in the treatment group was significantly higher than that in the control group. After 12 hours, the leakage amount of nucleotides was 2.77 times and 3.62 times that of the control group, respectively. Figure 3 In the control group (A), the protein leakage was 2.71 times and 3.56 times that of the control group, respectively. Figure 3 (B)

[0068] (3) β-(1,3)-glucan is a major component of fungal cell walls, and its hydrolytic enzyme β-1,3-glucanase plays a crucial role in maintaining cell wall stability. Experiments showed that the β-1,3-glucanase activity in the control group was stable, while the activity in the treatment group significantly decreased after 24, 48, and 72 hours, reaching only 38.50% and 32.76% of the control group at 72 hours. Figure 3 (C)

[0069] In summary, dichlorooxamazole causes fungal cell death by disrupting plasma membrane permeability and cell wall stability.

[0070] 6. Effects of diclofenac on ATP biosynthesis and metabolic pathways in pathogens

[0071] 6.1 Method

[0072] (1) RNA extraction, qPCR, and transcriptome sequencing: Mycelia cultured in PDB for 5 days were treated with 10 mg / L dichloroisothiazide for 12 / 24 hours and then flash-frozen in liquid nitrogen. Total RNA was extracted using TRIzol reagent (Cwbio, China), reverse transcribed using a PrimeScript™ RT kit (RR047A, Takara, Japan), and qPCR was performed on a Bio-Rad iQ5 system using SYBR Master Mix (Q711-03, Vazyme, China). Transcriptome sequencing was performed by Novogene Co., Ltd (Beijing, China).

[0073] (2) ATP content determination: PDB cultured mycelia were placed in a solution containing 10 mL DMSO or 5-20 mg / L dichlorooxamazole and cultured at 28℃ and 220 rpm for 18 hours with shaking. The ATP content of 0.1 g of mycelia was determined using an ATP assay kit (BC0300-50T / 48S, Solarbio, China) according to the instructions.

[0074] 6.2 Results

[0075] (1) Transcriptome analysis of D. segeticola in response to diclofenac: Principal component analysis (PCA) showed significant differences in the transcriptomes of the treatment group and the control group at 12 and 24 hours. Figure 4 (A). Differentially expressed gene (DEG) analysis revealed 4153 and 4384 DEGs detected at 12 and 24 hours, respectively. Figure 4 B, Figure 4 (C). KEGG analysis showed that ATP biosynthesis, metabolism, proton transport-coupled ATP synthesis pathways, and energy metabolism-related pathways were significantly enriched at both 12 and 24 hours. KEGG analysis further showed that pathways involving β-alanine, tyrosine, starch and sucrose metabolism, and secondary metabolite synthesis were significantly enriched after dichloroxazole treatment.

[0076] (2) Dichlorooxadazole inhibits the energy metabolism pathway of *D. segeticola*: GO analysis revealed that the energy metabolism pathway related to ATP biosynthesis was significantly enriched at both 12 and 24 hours. ATP content determination showed that after 18 hours of treatment, ATP content decreased significantly, with the ATP content in the treated groups being 1.54, 1.23, and 0.94 μM / g, respectively, compared to the control group, representing a decrease of 45.09% to 66.43%. Figure 4(D). Furthermore, NAD-malate dehydrogenase (NAD-MDH) activity was completely lost in the treatment group ( Figure 4 (E). The above results indicate that dichlorooxadazole leads to fungal cell death by inhibiting ATP accumulation and energy metabolism.

[0077] (3) Diclofenac inhibits the starch and sucrose metabolic pathways of D. segeticola: Venn diagram analysis showed that 1939 DEGs were activated at both 12 and 24 hours. Figure 4 KEGG enrichment analysis showed that starch and sucrose metabolic pathways were significantly enriched under diclofenac treatment. Specifically, the expression of genes encoding trehalose-6-phosphate synthase (TPS), α,α-trehalase (TREA), glycogen synthase (GBSS), and 1,4-α-glucan branching enzyme (GBE) was downregulated. qPCR validated these gene expression changes, indicating that diclofenac exerts its antifungal effect by inhibiting starch and sucrose metabolism and glucose biosynthesis.

[0078] (4) Diclofenac targeting the glucose biosynthesis and metabolic pathway of *D. segeticola*: To verify whether diclofenac exerts its antifungal activity by inhibiting glucose biosynthesis, glucose was added exogenously to the culture medium. The results showed that 15 and 20 mg / L diclofenac almost completely inhibited the growth of *D. segeticola*, with inhibition rates of 92.26% and 97.61%, respectively; while after the addition of glucose, the inhibition rates decreased to 27.38% and 36.91%, respectively. Figure 4 (G). Glucose content assays further showed that glucose uptake was significantly reduced in the treatment group. These results confirm that diclofenac inhibits fungal growth by disrupting glucose synthesis and metabolic homeostasis.

[0079] Application Example 1

[0080] 1. Method

[0081] Tea tree disease control: For tea leaf spot pathogen (D. segeticola), spray 5-20 mg / L dichlorodi ...

[0082] Sclerotinia rot in rapeseed: Spray rapeseed leaves with 10-20 mg / L dichlorooxam, inoculate with S. sclerotiorum mycelial blocks 24 hours later, incubate in the dark at 22℃ with humidity, and measure the lesion area after 72 hours.

[0083] Tomato gray mold:

[0084] (1) Plant experiment: Spray with 20 mg / L dichlorooxamazole, inoculate with B. cinerea mycelium (5 mm) 24 hours later, incubate at 25℃ with moisture, and record the lesion area after 48 hours;

[0085] (2) Fruit test: After disinfection, tomato fruits were punctured and inoculated with 10 μL of 10-20 mg / L dichlorooxamazole. After natural air drying, the fruits were inoculated with pathogens and cultured at 25℃ for 48 hours to observe the lesions (≥10 fruits / treatment, 3 replicates).

[0086] 2. Results

[0087] (1) Dichlorooxamazole is highly effective in controlling leaf spot disease.

[0088] The efficacy of dichlorodichlorodimethoate against leaf spot disease was tested on live tea leaves. The control group showed typical leaf spot symptoms with large lesions; while the dichlorodimethoate-treated group showed a significant reduction in lesion area, especially the 15 mg / L treatment, which almost completely prevented the occurrence of leaf spot disease. Figure 1 Furthermore, no obvious phytotoxicity symptoms were observed after treatment with high concentrations of dichlorooxam, indicating its broad application prospects in the control of tea tree diseases.

[0089] (2) Dichlorooxamazole is highly effective in controlling anthracnose and gray blight in tea trees.

[0090] Further testing was conducted on the activity of dichlorooxam against other pathogens. Results showed that its EC50 activity against *C. camelliae*, the causal agent of tea anthracnose, and *P. camelliae*, the causal agent of tea leaf spot, was significantly reduced. 50 The values ​​were 14.02 mg / L and 13.45 mg / L, respectively. Figure 5 China A, Figure 5 (C). In vivo experiments showed that diclofenac significantly reduced the lesion area (C). Figure 5 B, Figure 5 (D) confirmed its high efficiency in the prevention and control of tea tree diseases.

[0091] (3) Dichlorooxamazole exhibits broad-spectrum antifungal activity

[0092] Diclofenac has EC50-95% activity against Sclerotinia sclerotiorum and Botrytis cinerea. 50 The values ​​were 10.19 mg / L and 14.83 mg / L, respectively. Figure 5 E, Figure 5 (G). In in vivo experiments on rapeseed and tomato, dichloroisothiazide significantly reduced the lesion area of ​​sclerotinia rot and gray mold (G). Figure 5 China F, Figure 5 The presence of H indicates that it has broad-spectrum antifungal potential.

[0093] In summary, this study demonstrates that the gallic acid thiadiazole derivative dichlorooxadazole exhibits broad-spectrum antibacterial activity against five important plant pathogens: *C. camelliae*, *P. camelliae*, *D. segeticola*, *B. cinerea*, and *S. sclerotiorum*. It exerts its effects through a triple mechanism: (1) disrupting the plasma membrane / cell wall structure; (2) blocking the ATP synthesis pathway; and (3) interfering with glucose metabolism. Its control efficacy (EC50) on tea (leaf spot / anthracnose / ring spot), rapeseed (sclerotinia rot), and tomato (gray mold) is shown in the study. 50 (10.5~14.83 mg / L) This is superior to commercially available fungicides and causes no phytotoxicity. These studies indicate that dichlorooxam is a promising candidate environmentally friendly pesticide, offering a new solution for sustainable agricultural development.

[0094] While previous studies have shown that some antibacterial compounds can inhibit glucose consumption by interfering with starch and sucrose metabolic pathways, related research has largely focused on bacteria or only provided experimental evidence. This invention found that exogenous glucose supplementation can partially reverse the inhibitory effect of diclofenac on *D. segeticola*, significantly reducing its antibacterial rate by 20.84%–64.88%. This result suggests that diclofenac may exert its antifungal effect by targeting key enzymes in the glucose biosynthesis pathway; however, its specific target remains to be further elucidated.

[0095] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. The application of dichlorooxam in inhibiting leaf spot pathogens in tea, characterized by: The dichlorooxadazole is used to prepare products that inhibit leaf spot disease of tea trees.

2. The application according to claim 1, characterized in that: The product includes the active ingredient dichlorooxazole and a pharmaceutical composition used in combination with it.

3. The application according to claim 1, characterized in that: The dichlorodichlorodimethoate at a concentration of 15 mg / L showed a 100% growth inhibition rate against *Tea Leaf Spot* pathogens. The dichlorodimethoate also exhibited an EC50% inhibition rate against *Tea Leaf Spot* pathogens. 50 The value was 10.5 mg / L.

4. The application according to claim 1, characterized in that: The dichlorooxadazole inhibits bacteria by disrupting the plasma membrane or cell wall structure, blocking ATP synthesis pathways, and interfering with glucose metabolism. The pathogen is a plant pathogen.

5. The application according to claim 5, characterized in that: The plant pathogen is at least one of the following: tea anthracnose fungus, tea leaf spot fungus, Botrytis cinerea, or Sclerotinia sclerotiorum.

6. The application according to claim 5, characterized in that: The dichlorooxadazole is effective against EC5 of Bacillus anthracis. 50 The value was 14.02 mg / L, indicating that the EC50 of the diclofenac was effective against *Tea leaf spot*. 50 The value was 13.45 mg / L, and the EC50 of the diclofenac was 13.45 mg / L against Sclerotinia sclerotiorum. 50 The value was 10.19 mg / L, and the EC50 of the diclofenac sodium against Botrytis cinerea was... 50 The value was 14.83 mg / L.

7. The application according to claim 2, characterized in that: The pharmaceutical composition further includes an aqueous solution, which is one of water, physiological saline, or glucose aqueous solution.

8. The application according to claim 2, characterized in that: The pharmaceutical composition can also be prepared into a pharmaceutical formulation, the pharmaceutical formulation comprising the pharmaceutical composition and a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, anti-adhesives, integrators, permeation enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

9. The application according to claim 8, characterized in that: The pharmaceutical formulation includes a variety of acceptable dosage forms.

10. The application according to claim 9, characterized in that: The dosage form of the pharmaceutical preparation is any one of capsules, granules, tablets, powders, ointments, powders, pills, or liquids.

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