Biological source bacteriostatic agent for preventing and treating tea anthracnose and application of biological source bacteriostatic agent

Abstract

The invention discloses a biological source bacteriostatic agent for preventing and treating tea anthracnose and application thereof.According to the biological source bacteriostatic agent, the inhibition effect of thymol and osthole on tea anthracnose pathogens under the indoor condition is evaluated through an indoor toxicity determination test, and the result shows that both thymol and osthole can effectively inhibit growth of the tea anthracnose pathogens; the compounding mass ratio is 3: 7, so that a synergistic inhibition result can be achieved. Natural products thymol and cnidium lactone are compounded to be used for preventing and treating the tea tree anthracnose, the potential of improving the resistance of tea trees to the tea anthracnose is achieved, and the compound can be used as a reference agent for green prevention and treatment of the tea anthracnose.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease and insect pest prevention and control, and relates to a biogenic antibacterial agent for preventing and controlling tea anthracnose and application thereof. Background Art

[0002] Tea, rich in nutrients such as flavonoids, purine alkaloids, and 1-theanine ketone, is widely consumed and is one of the world's most popular non-alcoholic beverages. While warm and humid environments favor the growth of tea plants, they also create favorable conditions for the development of tea diseases. Hundreds of tea plant pathogens have been documented both domestically and internationally, with fungal pathogens causing a significant number of diseases, including tea anthracnose. Anthracnose is a major leaf disease of tea plants, damaging the leaves and severely impacting yield and quality, resulting in significant economic losses for tea production.

[0003] Currently, tea anthracnose is controlled by planting resistant varieties and using fungicides. However, planting resistant varieties is more sporadic, requires a longer timeframe, and is difficult to commercialize, making fungicides the primary treatment. Commonly used fungicides include mancozeb, thiophanate-methyl, and chlorothalonil. However, long-term use of these common fungicides has led to pathogens developing varying degrees of resistance, decreasing their effectiveness and even causing problems such as excessive pesticide residues in tea leaves. Therefore, a new, highly effective, bio-based agent for controlling tea anthracnose is urgently needed. Summary of the Invention

[0004] The purpose of the present invention is to provide a bio-source antibacterial agent for preventing and treating tea anthracnose and its application. By using it in combination, the antibacterial effect on the pathogen of tea anthracnose is enhanced, and it can effectively prevent and treat tea anthracnose. It can be used as a reference agent for green prevention and treatment of tea anthracnose.

[0005] The technical solution of the present invention is a biogenic antibacterial agent for preventing and treating tea anthracnose. The active components of the antibacterial agent include thymol and osthole. The mass ratio of the thymol to the osthole is 1-9:1-9.

[0006] The mass ratio of the thymol to osthole is 2-4:6-8.

[0007] Specifically, the mass ratio of thymol to osthole is 3:7.

[0008] The preparation method of the aforementioned biogenic antibacterial agent for preventing and treating tea anthracnose is to first weigh thymol and osthole according to the formula ratio, dissolve them in anhydrous ethanol, and then dilute them with sterile water, and mix them to obtain the biogenic antibacterial agent.

[0009] The usage amount of the above-mentioned anhydrous ethanol is 5% of the total mass of thymol and osthole.

[0010] The total concentration of the aforementioned thymol and osthole is 10-100 μg / mL.

[0011] Specifically, the total concentration of thymol and osthole is 100 μg / mL.

[0012] The aforementioned antibacterial agent is used to prevent and inhibit the growth of tea tree anthracnose pathogens.

[0013] The aforementioned tea tree anthracnose pathogen is Colletotrichum camellia.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. Thymol and osthole can effectively inhibit the growth of C.camellia after compounding. Its EC 50 Reaching 24.3560μg / mL, which is higher than the EC of thymol alone 50 37.5766μg / mL and osthole EC 50 The highest inhibition rate was 97.69±0.65% when the mass ratio of thymol to osthole was 3:7 and the reagent concentration was 100 μg / mL.

[0016] 2. This invention proposes, for the first time, a formulation combining the natural products thymol and osthole for the prevention and treatment of tea anthracnose, effectively improving tea plants' resistance to the disease. To date, no such biological agents have been reported to improve tea plant resistance to anthracnose. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 :Inhibitory effects of natural products on tea anthracnose pathogen gys03-1;

[0018] Figure 2 : Rescreened antibacterial effects of natural products against tea anthracnose pathogens (Note: The concentrations of thymol, osthole, and licoricechalcone are marked in the lower left corner of each panel, in μg / mL);

[0019] Figure 3 : Inhibitory effect of thymol and osthole complex on tea anthracnose pathogen. DETAILED DESCRIPTION

[0020] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0021] Example 1:

[0022] Thymol and osthole were weighed, dissolved in 5% anhydrous ethanol respectively, and then prepared with sterile water to prepare 100 μg / mL thymol solution and 100 μg / mL osthole solution respectively. Then 3 mL of thymol solution and 7 mL of osthole solution were taken and mixed to obtain a biogenic antibacterial agent.

[0023] Example 2:

[0024] Weigh thymol and osthole, dissolve them respectively with 5% anhydrous ethanol, and then prepare 10 μg / mL thymol solution and 10 μg / mL osthole solution with sterile water, then take 9 mL of thymol solution and 1 mL of osthole solution, mix them, and then obtain a biogenic antibacterial agent.

[0025] Example 3:

[0026] Weigh thymol and osthole, dissolve them respectively with 5% anhydrous ethanol, and then prepare 100 μg / mL thymol solution and 100 μg / mL osthole solution with sterile water, then take 2 mL of thymol solution and 8 mL of osthole solution, mix them, and then obtain a biogenic antibacterial agent.

[0027] Example 4:

[0028] Weigh thymol and osthole, dissolve them respectively with 5% anhydrous ethanol, and then prepare 80 μg / mL thymol solution and 80 μg / mL osthole solution respectively with sterile water. Then take 1 mL of thymol solution and 9 mL of osthole solution, mix them, and obtain a biogenic antibacterial agent.

[0029] Example 5:

[0030] Weigh thymol and osthole, dissolve them respectively with 5% anhydrous ethanol, and then prepare 100 μg / mL thymol solution and 100 μg / mL osthole solution with sterile water, then take 5 mL of thymol solution and 5 mL of osthole solution, mix them, and then obtain a biogenic antibacterial agent.

[0031] Example 6:

[0032] Weigh thymol and osthole, dissolve them respectively with 5% anhydrous ethanol, and then prepare 20 μg / mL thymol solution and 20 μg / mL osthole solution with sterile water, then take 7 mL of thymol solution and 3 mL of osthole solution, mix them, and obtain a biogenic antibacterial agent.

[0033] Example 7:

[0034] Weigh thymol and osthole, dissolve them respectively with 5% anhydrous ethanol, and then prepare 60 μg / mL thymol solution and 60 μg / mL osthole solution with sterile water, then take 5 mL of thymol solution and 5 mL of osthole solution, mix them, and then obtain a biogenic antibacterial agent.

[0035] Example 8:

[0036] Weigh thymol and osthole, dissolve them respectively with 5% anhydrous ethanol, and then prepare 40 μg / mL thymol solution and 40 μg / mL osthole solution respectively with sterile water. Then take 8 mL of thymol solution and 2 mL of osthole solution, mix them, and obtain a biogenic antibacterial agent.

[0037] The present invention has carried out corresponding experimental research to prove the antibacterial effect of the antibacterial agent. The following are the results of the experimental research of the present invention:

[0038] 1 Materials and Methods

[0039] 1.1 Pathogens

[0040] The pathogen of tea anthracnose, Colletotrichum camellia ygs03-1, was obtained from the Guizhou Institute of Biotechnology.

[0041] 1.2 Test natural products and culture medium

[0042] Coumarin (98% purity, standard product), paolimycin B (70% purity, standard product), γ-aminobutyric acid (standard product), licorice chalcone (99% purity, standard product), osthole (99% purity, analytical grade), and thymol (98% purity, analytical grade).

[0043] PDA is potato dextrose medium (OXOID)

[0044] 1.3 Preliminary screening of the antibacterial effect of natural products on tea anthracnose pathogens

[0045] Coumarin, paococcin B, γ-aminobutyric acid, licorice chalcone, osthole, and thymol were dissolved in a small amount of anhydrous ethanol to prepare a 500 μg / mL solution. Sterile water containing an equal amount of anhydrous ethanol was used as a control. The solution was then mixed with PDA at a ratio of 1:9 to prepare a drug-containing plate at a concentration of 50 μg / mL. A 0.6 cm diameter cake of the tea anthracnose pathogen was inoculated and incubated at 25°C for 7 days. The inhibitory rate of the natural products against ygs03-1 was measured and calculated.

[0046] Inhibition rate = (colony diameter of control group - colony diameter of test group) / (colony diameter of control group - 0.6 cm) × 100%

[0047] 1.4 Indoor toxicity determination of natural products against tea anthracnose pathogens

[0048] Select natural products with high antibacterial rates, dissolve them in anhydrous ethanol, and prepare 10mL solutions with concentrations of 50μg / mL, 40μg / mL, 30μg / mL, 20μg / mL, and 10μg / mL with sterile water. Mix them with PDA at a ratio of 1:9 to prepare drug-containing plates. Use sterile water containing an equal amount of anhydrous ethanol as a control. Inoculate tea anthracnose pathogen cakes with a diameter of 0.6cm and culture them at 25℃ for 7 days. Measure and calculate the antibacterial rate of natural products against ygs03-1. Use the logarithm value of the concentration to the base 10 as the independent variable x and the probability value corresponding to the mycelial growth inhibition rate as the dependent variable y to obtain the linear regression equation for the natural product against the pathogen and calculate the EC 50 and the correlation coefficient r.

[0049] 1.5 Indoor toxicity test of two natural products against tea anthracnose pathogen

[0050] The two natural products with the best inhibitory effect on tea anthracnose pathogens were dissolved in anhydrous ethanol and prepared with sterile water to a concentration of 1000 μg / mL stock solution. The stock solution was diluted to prepare five concentration gradient solutions, each with an ethanol content of 5%. (The concentration settings of each compound ratio are shown in Table 1). The stock solution was then mixed at a ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. The stock solution was then mixed with PDA at a ratio of 1:9 to prepare drug-containing plates. Sterile water containing an equal amount of anhydrous ethanol was used as a control. A 0.6 cm diameter tea anthracnose pathogen cake was inoculated and cultured at 25°C for 7 days. The inhibition rate of the natural products against ygs03-1 was measured and calculated. The logarithm value of the concentration to the base 10 was used as the independent variable x, and the probability value corresponding to the mycelial growth inhibition rate was used as the dependent variable y. The linear regression equation for the compound agent against the pathogen was obtained, and the EC was calculated. 50 And the correlation coefficient r. Then the Wadley method is used to calculate the synergistic coefficient (SR) of the compound agent. The calculation formula is as follows:

[0051] EC 50 (th)=(a+b) / (a / EC 50 (A)+b / EC 50 (B)

[0052] SR=EC 50 (th) / EC 50 (ob)

[0053] Where th is the theoretical effective median concentration of the compound, ob is the measured effective median concentration of the compound, A and B are the individual fungicides 1 and 2, respectively; a and b are the mass ratios of fungicides 1 and 2 in the compound. When SR>1.5, the compound has a synergistic effect; when 0.5≤SR≤1.5, the compound has an additive effect; and when SR<0.5, the compound has an antagonistic effect.

[0054] Table 1 Concentration gradient setting of thymol and osthole compound

[0055]

[0056] 1.6 Data Statistics and Analysis

[0057] WPS Excel was used to perform statistics and analysis on the experimental data.

[0058] 2 Results and Analysis

[0059] 2.1 Preliminary screening results of the antibacterial effects of the tested natural products on ygs03-1 Colletotrichum camellia

[0060] The results are as follows Figure 1 As shown in Table 1, licorice chalcone, osthole, and thymol at a concentration of 50 μg / mL showed high inhibition rates against the pathogen ygs03-1, with average inhibition rates of 37.94%, 46.37%, and 33.63%, respectively. Coumarin and paociclomycin B showed lower inhibition rates against ygs03-1, with average inhibition rates of 19.04% and 10.04%, respectively. γ-Aminobutyric acid had no significant inhibitory effect on ygs03-1.

[0061] Table 2 Inhibition rate of natural products against tea anthracnose pathogen ygs03-1

[0062]

[0063] 2.2 Results of indoor toxicity tests of natural products against ygs03-1 Colletotrichum camellia

[0064] The results are as follows Figure 2 As shown in Table 2, thymol, osthole and licorice chalcone all had inhibitory effects on ygs03-1 at five concentration gradients. Thymol had the best inhibitory effect on ygs03-1, with EC 50 The second most common osthole is 37.5766 μg / mL. 50 The toxicity of licorice chalcone to ygs03-1 was relatively low, and the EC 50 It is 83.9920μg / mL.

[0065] Table 3 Results of indoor toxicity test of natural products against tea anthracnose pathogens

[0066]

[0067] 2.3 Results of biological activity assay of thymol and osthole complexes on ygs03-1 Colletotrichum camellia

[0068] Table 4 Inhibition rate of thymol and osthole complex on C. camellia

[0069]

[0070]

[0071] As shown in Table 4, when the mass ratio of thymol to osthole was 3:7 and the reagent concentration was 100 μg / mL, the inhibition rate was the highest, reaching 97.69±0.65%.

[0072] Table 5 Results of indoor toxicity test of thymol and osthole complex on C. camellia

[0073]

[0074] like Figure 3 As shown in Table 5, when the ratio of thymol to osthole was 3:7, the toxicity to the pathogen ygs03-1 was the highest, EC 50 The concentration of the compound was 24.3560 μg / mL, with a synergistic coefficient of 1.63, indicating synergistic activity. Compounds with ratios of 1:9, 4:6, and 6:4 exhibited higher toxicity against ygs03-1, with synergistic coefficients of 0.77, 0.88, and 0.59, respectively, indicating an additive effect. Compounds with ratios of 2:8, 5:5, 7:3, 8:2, and 1:9 exhibited lower toxicity against ygs03-1, with synergistic coefficients of 0.22, 0.21, 0.34, 0.22, and 0.12, respectively, indicating a subtractive effect.

[0075] 3 Conclusion

[0076] In this study, the indoor antibacterial activity of six natural products against the pathogen of tea anthracnose, C. camellia, was determined. Thymol and osthole, which had higher toxicity to C. camellia, were selected through primary and secondary screening tests. 50The concentrations of thymol and osthole were 37.5766 μg / mL and 40.5523 μg / mL, respectively. Combination experiments determined their synergistic mass ratio against C. camellia to be 3:7. This study validated the potential of two natural products, thymol and osthole, to control tea anthracnose. Osthole, a commercialized biofungicide, can be combined with thymol to enhance the antibacterial effects of both against the tea anthracnose pathogen, making it a promising reference agent for the green control of tea anthracnose.

Claims

1. A biogenic antibacterial agent for preventing and treating tea anthracnose, characterized in that: The active components of the antibacterial agent include thymol and osthole, and the mass ratio of the thymol to the osthole is 1-9:1-9.

2. The biogenic antibacterial agent for preventing and treating tea anthracnose according to claim 1, characterized in that: The mass ratio of thymol to osthole is 2-4:6-8.

3. The biogenic antibacterial agent for preventing and treating tea anthracnose according to claim 2, characterized in that: The mass ratio of thymol to osthole is 3:

7.

4. The method for preparing the biogenic antibacterial agent for preventing and treating tea anthracnose according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: firstly weighing thymol and osthole according to the proportion of the formula, dissolving them with anhydrous ethanol, and then diluting them with sterile water, and mixing them to obtain the biogenic antibacterial agent.

5. The method for preparing the biogenic antibacterial agent for preventing and treating tea anthracnose according to claim 4, characterized in that: The usage amount of the anhydrous ethanol is 5% of the total mass of thymol and osthole.

6. The method for preparing the biogenic antibacterial agent for preventing and treating tea anthracnose according to claim 4, characterized in that: The total concentration of thymol and osthole is 10-100 μg / mL.

7. The method for preparing the biogenic antibacterial agent for preventing and treating tea anthracnose according to claim 6, characterized in that: The total concentration of thymol and osthole is 100 μg / mL.

8. Use of the biogenic antibacterial agent for preventing and treating tea anthracnose according to any one of claims 1 to 7, characterized in that: The antibacterial agent is used for preventing and inhibiting the growth of tea tree anthracnose pathogens.

9. The use of the biogenic antibacterial agent for preventing and treating tea anthracnose according to claim 8, characterized in that: The tea tree anthracnose pathogen is Colletotrichum camellia .

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