Application of anethole in preparation of ralstonia solanacearum control product
By using the fennel brain, the growth and occurrence of the leer's leering bacteria has been effectively inhibited, and the prevention and treatment of tobacco leering bacteria has been solved, and new drug active ingredients are provided for tobacco disease prevention and control.
Patent Information
- Application Number
- CN202510104265.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-22
- Publication Date
- 2025-07-08
AI Technical Summary
The prior art lacks effective methods to prevent and control the tobacco germ, especially the long-standing diseases caused tobacco by the germ of Rael's germ of Rael's germ of tobacco.
The methanol extract of fennel brain or fennel seeds is used as the active ingredient, and is prepared into a chlorophyllium control product by dissolving it in DMSO solvent, and sprayed on the leaves of tobacco to inhibit the growth and biofilm formation of the chlorophyllium.
Fennel brain significantly inhibits the growth of Rheer's Cyperus, reduces its incidence on tobacco, destroys its cell membrane, and interferes with the generation of biofilms, providing new drug-active ingredients for tobacco disease prevention and control.
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Figure CN120266867A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of agriculture, and particularly to the application of anethole in the preparation of products for controlling Ralstonia solanacearum. Background Art
[0002] Ralstonia solanacearum can enter the soil together with the diseased plant residues and form an infection source after long-term survival. Soil moisture has a great impact on its survival in the soil. In alluvial soil with high humidity, it can survive for up to 2 - 3 years, while in dry soil, it can only survive for a few days. Ralstonia solanacearum does not survive in a dormant state in the soil, but multiplies in the rhizosphere of the above-mentioned diseased plants or certain weeds. Ralstonia solanacearum existing in the soil mainly infects plants through wounds caused during operation or wounds caused by root pests such as root-knot nematodes and larvae of Popillia japonica Newman, and diseases occur in the stem conduit part and roots. Sometimes it also infects plants through non-wounded fine roots and causes diseases.
[0003] Therefore, it is necessary to develop a product for controlling Ralstonia solanacearum. Summary of the Invention
[0004] The object of the present invention is to provide the application of anethole in the preparation of products for controlling Ralstonia solanacearum. The present invention discovers for the first time that the organic component anethole has a significant inhibitory effect on Ralstonia solanacearum of tobacco, providing a new drug active ingredient for the prevention and control of tobacco diseases.
[0005] To achieve the above object, the present invention adopts the following technical solutions:
[0006] In the first aspect of the present invention, there is provided the application of anethole or a methanol extract of fennel seeds in the preparation of products for controlling Ralstonia solanacearum.
[0007] Further, the Ralstonia solanacearum is Ralstonia solanacearum.
[0008] Further, in the above application, the anethole is dissolved in the solvent DMSO, and the concentration of the anethole is 1.56 - 25 ml / L.
[0009] Further, the source of the anethole includes organic synthesis or extraction from fennel fruits.
[0010] Further, the method for obtaining the methanol extract of fennel seeds includes:
[0011] Take 1 g of fennel seeds, wash and dry them, then put them into a mortar. Add 5 ml of anhydrous methanol until the seeds are covered. Use a pestle to slowly crush the seeds, and dropwise add anhydrous methanol in small amounts multiple times during the crushing process until 8 ml is reached. After the fennel seeds are completely crushed, use a pipette to aspirate the liquid and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 1500 rpm for 5 min, and aspirate the supernatant to obtain the methanol extract of fennel seeds.
[0012] Further, the conditions in the HPLC separation include: setting the chromatographic conditions, using octadecylsilane-bonded silica gel as the filler; designing mobile phase A as 0.1% formic acid water, mobile phase B as acetonitrile, the detection wavelength as 254 nm, and the column temperature as 35°C; setting the elution program, adopting gradient elution, setting the flow rate as 0.5 mL / min, and collecting the substances in the range of 3 - 5 min.
[0013] In the second aspect of the present invention, a reagent for preventing or inhibiting Ralstonia solanacearum is provided, and the active ingredient of the reagent is anethole.
[0014] In the third aspect of the present invention, the application of anethole or the methanol extract of fennel seeds in the preparation of a product for preventing tobacco bacterial wilt is provided.
[0015] In the fourth aspect of the present invention, a method for preventing tobacco bacterial wilt is provided, and the method includes:
[0016] Spray the antibacterial solution of Ralstonia solanacearum prepared by mixing anethole and DMSO or the methanol extract of fennel seeds on the tobacco leaves.
[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
[0018] The application of anethole provided by the present invention in the preparation of a reagent for preventing or inhibiting Ralstonia solanacearum. The present invention first discovers that the organic component anethole has a significant inhibitory effect on tobacco Ralstonia solanacearum, providing a new drug active ingredient for tobacco disease prevention and control. Anethole has great application prospects in the preparation of products for preventing Ralstonia solanacearum. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.
[0020] Figure 1 It is the size of the antagonistic circle. A is the size of the antagonistic circle of the fennel extract against Ralstonia solanacearum, and B is the size of the antagonistic circle of an equal amount of deionized water against Ralstonia solanacearum.
[0021] Figure 2 Results of purification by high performance liquid chromatography analysis
[0022] Figure 3 1H-NMR, 13C NMR, HSQC spectrum and HMBC spectrum of monomer compound A. (A) is the 1 1H NMR (400 MHz, CDCl3) of monomer compound A, (B) is the 13C NMR (100 MHz, CDCl3) of monomer compound A, (C) is the HSQC spectrum of monomer compound A, and (D) is the HBMC spectrum of monomer compound A
[0023] Figure 4 Planar structure of monomer compound A
[0024] Figure 5 Growth curve of Ralstonia solanacearum growth under treatment with bacteriostatic solutions of different concentrations of Ralstonia solanacearum
[0025] Figure 6 Number of Ralstonia solanacearum in capillaries under the action of bacteriostatic solutions of different concentrations of Ralstonia solanacearum
[0026] Figure 7 Effect of bacteriostatic solutions of different concentrations of Ralstonia solanacearum on the formation of biofilm of Ralstonia solanacearum
[0027] Figure 8 Incidence of tobacco bacterial wilt under treatment with bacteriostatic solutions of different concentrations of Ralstonia solanacearum Detailed implementation manners
[0028] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than to limit the present invention
[0029] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail
[0030] Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or can be obtained by existing methods
[0031] The present application will be described in detail below in combination with examples and experimental data
[0032] Example 1: Extraction of anethole and antibacterial experiment
[0033] 1. Biological materials and instruments
[0034] Ralstonia solanacearum was purchased from Shanghai Merck Chemical Technology Co., Ltd. All reagents used in this study were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The instruments used in this experiment included a Spectra MaxiD3 microplate reader; an Easysep-3030 high-performance liquid chromatograph; a German LABCONCO cryogenic concentrator.
[0035] 2. Fennel seed extract
[0036] Take 1 g of fennel seeds, wash and dry them, put them into a mortar, add 5 ml of anhydrous methanol until the seeds are covered, use a pestle to slowly crush the seeds, and add anhydrous methanol drop by drop in small amounts multiple times until 8 ml. After the fennel seeds are completely crushed, use a pipette to aspirate the liquid and transfer it to a 1.5 mL centrifuge tube, centrifuge at 1500 rpm for 5 min, and aspirate the supernatant to obtain the methanol extract of fennel seeds.
[0037] 3. Identification of antagonistic ability
[0038] Pour 30 ml of NA solid medium into a petri dish, aspirate 200 μL of the Ralstonia solanacearum suspension (OD600 ≈ 1.0) and spread it evenly on the surface of the solid medium. After using a sterilized pipette tip to punch holes (the pore size is 2.5 mm), vertically add 20 μL of the extract (using equal amounts of deionized water as the control group). Calculate the antibacterial rate of the extract according to the following formula:
[0039]
[0040] In the formula, r0 is the colony radius of the extract group, and r is the colony radius of the control group.
[0041] The antibacterial effect of the fennel extract is as Figure 1 shown. There is no obvious antibacterial zone in the control group, and the antibacterial zone radius of the treatment group is 10.76 mm (6.52, 12.59, 13.18), indicating that deionized water has no antibacterial effect, while the fennel extract has an obvious antibacterial effect and can effectively inhibit the growth of Ralstonia solanacearum.
[0042] 4. Isolation and identification of antibacterial substances
[0043] The fennel extract was separated by high-performance liquid chromatography (HPLC). The results showed that within the first 8 min, a total of 4 chromatographic peaks ( Figure 2 ) were identified. After separating each peak individually and verifying their respective antibacterial effects, it was found that the monomeric compound A solution at 4.71 min had a strong antibacterial effect, and the antibacterial zone radius reached 13.76 mm (15.232, 10.37, 14.71 mm).
[0044] The 1H-NMR, 13C-NMR, HSQC and HMBC spectra of pure compound A ( Figure 3 ) were comprehensively analyzed to determine and verify the compound structure.
[0045] In the 1H-NMR spectrum of this compound, ( Figure 3 A) there are 4 groups of a total of 6 proton signals in the low field region at 6 - 8 ppm: 7.24 (d, J = 8.5 Hz, 2H, 1-H), 6.81 (d, J = 8.5 Hz, 2H, 3-H), 6.33 (d, J = 15.8 Hz, 1H, 6-H), 6.08 (dq, J = 15.8, 6.6 Hz, 1H, 7-H), and there are 2 groups of methyl hydrogen signals in the high field region: 3.77 (s, 3H, 5-H), 1.84 (d, J = 6.6 Hz, 3H, 8-H). The area ratio of the 4 groups of hydrogen peaks in the low field region is 2:2:1:1. Judging from the coupling constant and chemical shift values, the two groups of hydrogen at 7.24 ppm and 6.81 ppm show coupling splitting, the two groups of hydrogen at 6.33 ppm and 6.08 ppm show coupling splitting, and the proton at 6.08 ppm shows coupling splitting with the methyl hydrogen at 1.84 ppm in the high field region. The two groups of hydrogen at 7.24 ppm and 6.81 ppm conform to the structural characteristics of 1,4-disubstituted benzene, the two groups of hydrogen at 6.33 ppm and 6.08 ppm conform to the coupling characteristics of trans double bond hydrogen. From the coupling characteristics between the protons at 6.08 ppm and 1.84 ppm, it can be determined that the hydrogen at 1.84 ppm is allylic methyl hydrogen.
[0046] According to the 13C-NMR spectrum of the compound ( Figure 3 B), the carbon signal at 158.52 ppm conforms to the basic characteristics of the benzene ring carbon connected to the methoxy group, and the carbon signals at 130.30 ppm and 113.84 ppm conform to the structural characteristics of 1,4-disubstituted benzene; while the carbon signal at 55.17 ppm conforms to the basic characteristics of methoxy carbon. Thus, it is inferred that the structure of compound A is as Figure 4 shown.
[0047] Through the HSQC spectrum ( Figure 3 C), it is determined that the carbon signals corresponding to the hydrogens at each position are 158.52 (4-C), 130.75 (6-C), 130.30 (2-C), 126.82 (1-C), 123.39 (7-C), 113.84 (3-C), 55.17 (5-C), 18.36 (8-C). The HSQC spectrum is consistent with the structure of compound A. And from the HMBC spectrum ( Figure 3 D), it can also be seen that 5-H is correlated with 4-C, 6-H is correlated with 1-C, 8-C is correlated with 6-H and 7-H, 8-H is correlated with 6-C and 7-C, which is consistent with the structure of compound A.
[0048] In summary, through the analysis of 1H-NMR, 13C NMR, HSQC spectrum and HMBC spectrum, it is speculated that this substance is trans-anethole. Liquid phase analysis of the trans-anethole standard product found that under the conditions of the same program, the peak emergence time of the anethole standard product was consistent with that of compound A. Therefore, we determined that the monomer compound A is trans-anethole (E)-1-methoxy-4-(prop-1-en-1-yl)benzene. Based on this, the present invention identified that the antibacterial component in the methanol extract of fennel seeds is anethole. Next, antibacterial solutions of different concentrations of Ralstonia solanacearum were prepared with anethole standard product and solvent DMSO for in vitro antibacterial experiments, chemotaxis assays, biofilm formation ability assays, and experiments on the effect of antibacterial solutions of different concentrations of Ralstonia solanacearum on the disease incidence of tobacco.
[0049] Example 2: In vitro antibacterial experiment of anethole
[0050] 1. Effect of antibacterial solutions of different concentrations on the growth of Ralstonia solanacearum
[0051] Take a sterile 96-well plate and place it on the ultra-clean workbench. Add 100 μL of bacterial suspension and 100 μL of antibacterial solution of different concentrations of Ralstonia solanacearum to each well in the sample test group, add 100 μL of bacterial suspension and 100 μL of DMSO solution to each well in the positive control group, and add 200 μL of NB liquid medium to the blank control group. Seal the 96-well plate with a sealing film and incubate it at 28 °C for 24 h. Set the microplate reader to measure the absorbance values of each well at different time intervals at 600 nm.
[0052] Table 1: Analysis of variance (ANOVA)
[0053]
[0054] The effects of antibacterial solutions of different concentrations of Ralstonia solanacearum on the growth of Ralstonia solanacearum are shown in Table 1 and Figure 5 as follows. As the culture time extended, the absorbance values (OD600) of each well showed a downward trend, indicating that antibacterial solutions of different concentrations of Ralstonia solanacearum all had inhibitory effects on the growth of Ralstonia solanacearum. Especially after 2 hours of culture, the antibacterial solution significantly inhibited the growth of Ralstonia solanacearum (ANOVA, P < 0.01). After 12 h of culture, the absorbance values of each treatment group and the control group had extremely significant differences statistically (P < 0.01), further verifying the effect of the antibacterial solution of Ralstonia solanacearum on the growth of Ralstonia solanacearum.
[0055] 2. Determination of the minimum inhibitory concentration
[0056] Pre-add 100 μL of Ralstonia solanacearum bacterial solution with a concentration of 1×CFU / mL in a 96-well culture plate. Add 100 μL of Ralstonia solanacearum bacteriostatic solutions with different concentrations (prepared from anethole standard + DMSO) to the experimental groups, so that the final concentrations in each well are 25 mL / L, 12.5 mL / L, 6.25 mL / L, 3.13 mL / L, and 1.56 mL / L in sequence. Set up a blank control (50 μL of Ralstonia solanacearum bacterial suspension + 150 μL of nutrient broth) and a negative control (50 μL of Ralstonia solanacearum bacterial solution + 50 μL of DMSO + 100 μL of nutrient broth), culture at 37 °C for 24 h, and measure the OD600 value using an enzyme-labeling instrument. Calculate the bacteriostatic rate according to the following formula:
[0057]
[0058] According to the calculation formula of the bacteriostatic rate, calculate the inhibitory effects of the bacteriostatic solutions at different concentrations respectively. The results are shown in Table 2 below. Among them, when the concentration of anethole is 25 mL / L, the maximum bacteriostatic rate is 61.91%.
[0059] Use GraphPad Prism 8 software to fit the bacteriostatic effect of anethole and calculate the IC50 (the drug concentration when the bacterial growth inhibition rate is 50%). The equation fitted by the software is as follows:
[0060] y = 6.69ln(x) + 41.06
[0061] where x is the drug concentration (mL / L) and y is the bacteriostatic rate (%).
[0062] Table 2: Bacteriostatic rates at different anethole concentrations
[0063]
[0064] Calculated by GraphPad Prism 8 software, the IC50 = 3.86 mL / L. When the concentration of the bacteriostatic solution reaches 3.86 mL / L, the inhibitory effect of the drug on Ralstonia solanacearum reaches 50%.
[0065] Example 3: Chemotaxis determination
[0066] On the basis of Yang Shanshan et al., the chemotaxis experiment protocol was modified. The Ralstonia solanacearum was cultured in NB liquid medium at 28 °C and 180 rpm for 12 h, and the cells were collected by centrifugation at 6000 rpm for 5 min and resuspended in an equal volume of phosphate buffer (100 mmol / L, pH 7.0). Then, different concentrations (0 ml / L, 25 mL / L, 12.5 mL / L, 6.25 mL / L, 3.13 mL / L, 1.56 mL / L) of the antibacterial solution of Ralstonia solanacearum (prepared from anethole standard + DMSO) were aspirated into a sterilized capillary with an inner diameter of 1 mm and vertically placed into a petri dish containing the Ralstonia solanacearum solution. After standing for 40 min, the liquid in the capillary was removed with a syringe and diluted to 1×10 -5 and then spread on an NA solid plate containing red tetrazolium and counted after culturing at 30 °C for 18 h. Each treatment was repeated 3 times, and DMSO was used as the control.
[0067] Bacteria will tend to favorable chemicals and avoid harmful chemicals. Effective drug inhibitors can not only inhibit the growth of Ralstonia solanacearum, but also change the distribution of R. solanacearum, affecting the community composition and spatio-temporal distribution of the microecosystem. The effects of antibacterial solutions of different concentrations of Ralstonia solanacearum on the chemotaxis of Ralstonia solanacearum are as Figure 6 shown.
[0068] It can be Figure 6 seen that the number of colonies in the capillary is inversely proportional to the concentration of the antibacterial solution, and there are significant differences in the number of colonies between each treatment group and the control group statistically (ANOVA, P < 0.05). Compared with the control group, the number of Ralstonia solanacearum in the capillary of each treatment group showed a downward trend, and when the concentration reached 25 mL / L, the number of bacteria reached the lowest. The effect of the drug on the chemotaxis of R. solanacearum indicates that the antibacterial solution may affect the composition and structure of the microecosystem, thus inhibiting the Ralstonia solanacearum pathogen in many aspects.
[0069] Example 4: Determination of biofilm formation ability
[0070] The concentration of 1×0 8The Ralstonia solanacearum bacterial solution at CFU / mL was inoculated into the NB liquid medium containing the Ralstonia solanacearum bacteriostatic solution at a ratio of 1%. The concentrations of the Ralstonia solanacearum bacteriostatic solution (prepared from anethole standard + DMSO) were 25 mL / L, 12.5 mL / L, 6.25 mL / L, 3.13 mL / L, and 1.56 mL / L in sequence. At the same time, a blank control group was set as a negative control. After thorough mixing, 200 μL of the mixed solution was pipetted into a sterile 96-well culture plate, and 3 replicate wells were set for each group. The plate was placed in an incubator at 37 °C for 24 h. After the incubation ended, the liquid in the wells was aspirated clean, slowly washed 3 times with sterile PBS, and then left to dry in the incubator. After drying, 200 μL of methanol solution was added to each well to fix for 20 min, the methanol was aspirated off, and it was left to dry at room temperature for 15 min. After drying, 200 μL of 1% crystal violet staining solution was added to each well, stained for 30 min, the staining solution was discarded, washed 3 times with sterile water, 200 μL per well, and after aspirating off the double-distilled water, the culture plate was gently flicked until it was dry. After complete drying, 200 μL of 33% glacial acetic acid solution was added to each well and thoroughly mixed. After standing for 10 min, the OD490 was measured using an enzyme-linked immunosorbent assay (ELISA) reader.
[0071] The results are as Figure 7 shown. Compared with the control group, the bacteriostatic solution inhibited the formation of biofilm to varying degrees at each concentration. Specifically, after treatment with the active substance, the amount of biofilm formed by Ralstonia solanacearum decreased significantly, indicating that the bacteriostatic solution can effectively interfere with the biofilm formation process of Ralstonia solanacearum.
[0072] Example 5. Effects of Ralstonia solanacearum bacteriostatic solutions at different concentrations on the disease incidence of tobacco
[0073] The experimental protocol of HanST et al. was further optimized. Tobacco seedlings of Yunyan 87 grown for 50 days were taken, and the leaves were sprayed with Ralstonia solanacearum bacteriostatic solutions at 25 ml / L, 12.5 ml / L, 6.25 ml / L, 3.13 ml / L, and 1.56 ml / L (prepared from anethole standard + DMSO), 3 mL was sprayed per plant, and 3 replicates were set for each concentration treatment, with 5 tobacco seedlings in each replicate. After the first spraying, the bacteriostatic solution was sprayed again after an interval of 3 days; spraying an equal amount of deionized water was used as the control group. 2 days after the second spraying, 1 × 10 8 CFU / mL of Ralstonia solanacearum suspension was inoculated by root irrigation without damaging the roots, 15 mL was inoculated per plant. The tobacco seedlings were placed in a constant temperature incubator at 28 °C for cultivation, with natural light and humidity. Starting from the second day after inoculation, the disease incidence of tobacco was investigated at the same time every day, the number of diseased plants was recorded, and the incidence rate was calculated according to the following formula:
[0074]
[0075] The results are as Figure 8As shown, 16 days after inoculation with Ralstonia solanacearum, the incidence rates of tobacco treated with bacteriostatic solutions at 25 ml / L, 12.5 ml / L, 6.25 ml / L, 3.13 ml / L, and 1.56 ml / L were 60%, 80%, 67%, 87%, and 87% respectively, while the incidence rate of tobacco in the control group was as high as 93%. Compared with the control, when the concentration of the bacteriostatic solution reached 6.25 ml / L, it could significantly inhibit the incidence rate of tobacco bacterial wilt, and the incidence rate of tobacco treated with 25 ml / L bacteriostatic solution was significantly lower than that of other concentration treatments.
[0076] In summary, the bacteriostatic solution may inhibit the growth and biofilm formation of Ralstonia solanacearum by damaging its cell membrane. This mechanism may be closely related to the interference of active substances with the integrity, permeability, and membrane-related physiological processes of the cell membrane.
[0077] Finally, it should also be noted that the term "comprising", "including", or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or device.
[0078] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention. Obviously, those skilled in the art can make various changes and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.
Claims
1. Use of the methanol extract of anethole or fennel seeds in the preparation of a reagent for preventing or inhibiting Ralstonia solanacearum.
2. The application according to claim 1, wherein The Ralstonia solanacearum is Ralstonia solanacearum.
3. The application according to claim 2, wherein In the said use, the anethole is dissolved in the solvent DMSO, and the concentration of the anethole is 1.56 - 25 ml / L.
4. The application according to claim 2, wherein The method for obtaining the methanol extract of the fennel seeds includes: Wash the fennel seeds, dry them in the air, add anhydrous methanol and crush them. After the fennel seeds are completely crushed, transfer them to a centrifuge tube, centrifuge, and aspirate the supernatant to obtain the methanol extract of the fennel seeds.
5. The application according to claim 4, characterized in that, The conditions in the HPLC separation include: setting the chromatographic conditions, using octadecylsilane-bonded silica gel as the filler; designing mobile phase A as 0.1% formic acid water, mobile phase B as acetonitrile, the detection wavelength is 254 nm, and the column temperature is 35°C; setting the elution program, adopting gradient elution, setting the flow rate as 0.5 mL / min, and collecting the substances for 3 - 5 min.
6. A reagent for preventing or inhibiting Ralstonia solanacearum, characterized in that, The active ingredient of the said reagent is anethole.
7. Use of the methanol extract of anethole or fennel seeds in the preparation of a product for preventing tobacco bacterial wilt.
8. A method for preventing and controlling tobacco bacterial wilt, characterized in that, The said method includes: Spraying the bacteriostatic solution of Ralstonia solanacearum prepared from anethole and DMSO or the methanol extract of fennel seeds on the tobacco leaf surface.
9. The method according to claim 8, characterized in that The said method specifically includes: Spraying the bacteriostatic solution of Ralstonia solanacearum prepared from anethole and DMSO or the methanol extract of fennel seeds on the tobacco leaf surface for the first time, and then spraying the second time after an interval of 3 days.