Application of thymol in prevention and treatment of soil-borne diseases caused by fusarium oxysporum

Abstract

The invention discloses a novel application of thymol for preventing and treating soil-borne diseases caused by fusarium oxysporum, and belongs to the technical field of biopesticides. According to the embodiment of the invention, the action mechanism of the thymol on the fusarium oxysporum is analyzed by measuring the sensitivity of the thymol on the fusarium oxysporum and the influence of treatment with different concentrations on physiological and biochemical indexes such as hyphae, spores, proteins, DNA (Deoxyribonucleic Acid) and ergosterol contents of pathogenic bacteria and the yield of the oxytoxin; and a theoretical basis is provided for research and development of a novel biopesticide dosage form, namely thymol.

Description

Technical Field

[0001] This invention relates to the field of biopesticide technology, and more specifically to the application of thymol in the control of soil-borne diseases caused by Fusarium oxysporum. Background Technology

[0002] Stem rot caused by Fusarium oxysporum is a difficult-to-control soil-borne disease. Currently, control mainly relies on chemical pesticides for soil disinfection or root irrigation. However, the continuous use and accumulation of chemical pesticides inevitably brings a series of negative problems to soil and the environment. For example, crops absorbing long-lasting chemical pesticides can accumulate through the food chain, ultimately causing significant harm to humans or animals; beneficial organisms in the soil, such as earthworms, will die in large numbers after ingesting pesticides; the application of chemical pesticides greatly reduces the types and numbers of beneficial microorganisms in the soil, causing an imbalance in the soil microecology; and residual chemical pesticides in the soil entering water bodies can cause mass deaths of fish and aquatic plants. Therefore, while controlling crop diseases, it is also essential to actively protect the soil environment.

[0003] Currently, some highly toxic, bioaccumulated, and heavily polluting pesticides are being gradually banned, while environmentally compatible biopesticides are being developed and used more and more frequently. Thymol, a plant-derived fungicide, is a key component of plant essential oils and is safe for both humans and the environment. Recent studies have shown that this compound exhibits high inhibitory activity against various bacteria, fungi, and viruses. Filing documents from the US National Institutes of Health (NIH) and the NCBIPubChem Project indicate that thymol is widely used in the medical and food fields as an antibacterial disinfectant and antibacterial additive. This compound has also been proven to have good control efficacy against Fusarium wilt. Furthermore, the European Commission issued Regulation (EU) N0568 / 2013, approving the market launch of thymol as a plant protection product. Some studies have reported that thymol and carvacrol exert antioxidant activity through Schiff bases, thereby causing damage to pathogens. However, whether thymol has an inhibitory effect on the pathogenic fungus *Fusarium oxysporum* has not yet been reported.

[0004] Therefore, elucidating the mechanism of action of thymol against Fusarium oxysporum is of great significance for the development of this novel biological pesticide formulation. Summary of the Invention

[0005] To address the aforementioned problems, this invention applies thymol to the prevention and control of soil-borne diseases caused by Fusarium oxysporum and elucidates the mechanism of action of thymol against Fusarium oxysporum.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Application of thymol in the control of soil-borne diseases caused by Fusarium oxysporum.

[0008] Another object of the present invention is to provide the use of thymol in the preparation of drugs against Fusarium oxysporum.

[0009] Preferably, the above-mentioned drugs are targeted at Fusarium oxysporum.

[0010] Another object of the present invention is to provide an antifungal drug for Fusarium oxysporum, comprising thymol.

[0011] Preferably, the above-mentioned drugs also include other active ingredients and / or excipients.

[0012] Preferably, thymol exerts its antifungal effect against Fusarium oxysporum by inhibiting the growth of Fusarium hyphae and spores.

[0013] Preferably, thymol exerts its antifungal effect against Fusarium oxysporum by inhibiting the synthesis of soluble proteins, DNA, and ergosterol in the mycelium of Fusarium oxysporum.

[0014] Preferably, thymol can inhibit the secretion of oxytocin by Fusarium oxysporum.

[0015] As can be seen from the above technical solution, this invention provides a new application of thymol—controlling soil-borne diseases caused by Fusarium oxysporum. The embodiments of this invention, by determining the sensitivity of thymol to Fusarium oxysporum and the effects of different concentrations on physiological and biochemical indicators such as mycelium, spores, proteins, DNA, ergosterol content, and phytotoxicant yield of the pathogen, elucidate the mechanism of action of thymol against Fusarium oxysporum, providing a theoretical basis for the development of this novel biological pesticide formulation. Attached Figure Description

[0016] Figure 1 Results of antibacterial assay of thymol against Fusarium oxysporum;

[0017] Figure 2 Results of the effects of different final concentrations of thymol on the mycelial morphology of Fusarium oxysporum;

[0018] Figure 3 Effects of different final concentrations of thymol on the germination rate of Fusarium oxysporum spores;

[0019] Figure 4 Growth of Fusarium oxysporum strains after 7 days of liquid culture following treatment with different final concentrations of thymol.

[0020] Figure 5 Standard curve of soluble protein BSA content;

[0021] Figure 6Standard curve of ergosterol content corresponding to peak area;

[0022] Figure 7 Results of the effect of different final concentrations of thymol on the ergosterol content of Fusarium oxysporum mycelium;

[0023] Figure 8 : The toxicity regression curve of thymol in inhibiting ergosterol content in Fusarium oxysporum mycelium;

[0024] Figure 9 Results of the effect of different final concentrations of thymol on the content of Fusarium oxysporum toxin;

[0025] Figure 10 The toxicity regression curve of thymol on the content of Fusarium oxysporum toxin. Detailed Implementation

[0026] 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.

[0027] All reagents used in the embodiments of this invention were purchased from commercial channels. Methods not mentioned are conventional experimental methods and will not be described in detail here.

[0028] 1. Materials:

[0029] Thymol (purity 99.5%, German product, imported and repackaged by Nanjing Hengxin Chemical Co., Ltd.) was dissolved in anhydrous ethanol to prepare a 100 mg / mL stock solution.

[0030] PDA medium: 200g fresh peeled potatoes, 20g glucose, 16g agar powder, 1000mL distilled water.

[0031] YEPD liquid culture medium: 10g yeast extract, 20g peptone, 20g glucose, 1000mL distilled water.

[0032] Richard's liquid medium: sucrose 30g, K₂HPO₄ 1g, MgSO₄ 4. 0.5g 7H2O, 0.5g KCl, 0.01g FeSO4, 3g NaNO3, 1000mL distilled water.

[0033] The tested pathogen, *Fusarium oxysporum* strain, was provided by the Institute of Plant Protection, Fujian Academy of Agricultural Sciences.

[0034] Column-type fungal DNA extraction kit (Beijing Solarbio Science & Technology Co., Ltd.)

[0035] Use a UV-Vis spectrophotometer (UV-1800PC, Shanghai Meipuda Instrument Co., Ltd.).

[0036] 2. Method:

[0037] (1) Determination of the susceptibility of Fusarium oxysporum to thymol

[0038] The effective inhibitory concentration (EC) of thymol against Fusarium oxysporum was determined using the colony growth diameter method. 50 (μg / mL). Cultured *Fusarium oxysporum* were inoculated into the center of PDA plates containing 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL thymol. PDA plates without thymol served as controls. The plates were incubated at 25°C for 7 days, with each treatment repeated three times. Colony diameter was determined using the cross-hatching method (see example). Figure 1 ).

[0039] The inhibition rate (%) was calculated using the formula: (Control colony diameter - Treated colony diameter / Control colony diameter - Inoculated mycelium diameter) × 100. Using DPS data processing software, the virulence regression curve equation was calculated based on the logarithm of the drug concentration (X) and the inhibition rate (Y), thus yielding its EC50. 50 The concentration was 51.02 μg / mL, and the toxicity regression curve equation was Y = 1.3574 + 3.7225x, with a correlation coefficient of 0.9687.

[0040] Depend on Figure 1 It can be seen that thymol has a significant inhibitory effect on Fusarium oxysporum.

[0041] (2) Effects of thymol on the mycelial growth and morphology of Fusarium oxysporum

[0042] Take five Erlenmeyer flasks containing 100 mL of YEPD medium. Collect five 5 mm mycelial discs from the edge of a well-cultured *Fusarium oxysporum* colony and transfer them into the medium. Incubate on a shaker at 175 rpm and 25°C for one day. Then, add thymol to each flask to achieve final concentrations of 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL, respectively. Use a blank control (without thymol) as the control. Continue incubation on a shaker at 175 rpm and 25°C for another 6 days. Filter the mycelium through double-layered gauze and observe its morphology under a microscope (see schematic diagram). Figure 2After drying, the mycelia were placed in a 25℃ oven for 24 hours, and their dry weight was measured. Each treatment was repeated three times. The mycelial growth inhibition rate (%) was calculated according to the formula: (control mycelial dry weight - treated mycelial dry weight / control mycelial dry weight) × 100. Using DPS data processing software, the regression curve equation of the inhibition rate of thymol on the mycelial growth of the pathogen was calculated based on the logarithm of the agent concentration (X) and the mycelial growth inhibition rate (Y), and the EC was obtained. 50 .

[0043] EC 50 The concentration was 57.30 μg / mL, and the toxicity regression curve equation was Y = 0.3743 + 3.0586x, with a correlation coefficient of 0.9860, indicating that thymol can effectively inhibit the growth of Fusarium oxysporum mycelium.

[0044] like Figure 2 As shown, after liquid culture, the CK group exhibited vigorous hyphal growth, exhibiting linear growth with uniform distribution of mycelial cell protoplasm and a large number of free and germinating conidia in the culture medium. When the concentration of thymol was 25 μg / mL, hyphal growth was inhibited, conidia production was suppressed, and the number of conidia in the culture medium decreased significantly. With the increase of thymol concentration, hyphal growth slowed down, the hyphae gradually became shorter and thicker, the vacuoles inside the hyphae swelled, and the branching decreased. At a concentration of 100 μg / mL, hyphal growth almost stopped, and some hyphae broke.

[0045] (3) Effects of thymol on spore production and germination of Fusarium oxysporum

[0046] ① Take 5 Erlenmeyer flasks, each containing 100 mL of YEPD medium. Take 5 5 mm mycelial cakes from the edge of the cultured Fusarium oxysporum colony and transfer them into the medium. After culturing on a shaker at 175 r / min and 25℃ for 1 day, add thymol to each flask to make the final concentrations 25 μg / mL, 50 μg / mL, 75 μg / mL and 100 μg / mL. Use no thymol as a blank control. Continue culturing on a shaker at 175 r / min and 25℃ for 7 days. Collect spores by filtering with filter paper and make up to 100 mL with sterile water to prepare a spore suspension. Observe the number of spores produced under a microscope using a hemocytometer. Each experiment is repeated 3 times.

[0047] The spore production inhibition rate (%) was calculated using the formula: (Number of control spores - Number of treated spores / Number of control spores) × 100. Using DPS data processing software, the regression curve equation for thymol's effect on pathogen spore production was calculated based on the logarithm of the agent concentration (X) and the spore production inhibition rate (Y). The virulence regression equation was obtained as Y = 0.4158 + 3.3949x, with a correlation coefficient of 0.9567. The effective median concentration (EC) for inhibiting spore production was then calculated. 50It was 39.37 μg / mL.

[0048] ② Take 5 Erlenmeyer flasks, each containing 100mL of sterile water, and add Fusarium oxysporum spores to the sterile water to prepare 1×10 5 Spore suspensions of cfu / mL were prepared and thymol was added to each suspension to achieve final concentrations of 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL, with a control without thymol. The suspensions were incubated at 28℃. Germination counts were recorded at 2h, 4h, 6h, 8h, 10h, 12h, and 14h, and changes in the spore suspension were observed at 14h. Each treatment was repeated in triplicate.

[0049] The relative inhibition rate of spore germination (%) is calculated using the following formulas: spore germination rate (%) = (total number of germinating spores / total number of spores surveyed) × 100, and relative inhibition rate of spore germination (%) = (control spore germination rate - treatment spore germination rate / control spore germination rate) × 100.

[0050] Using DPS data processing software, regression curve equations were calculated for the inhibition rate of thymol on the spore germination of pathogenic fungi, based on the logarithm of the agent concentration (X) and the spore germination inhibition rate (Y). The virulence regression equation was Y = 1.6037 + 3.8198x, with a correlation coefficient of 0.9968. The effective medium concentration (EC) for inhibiting spore germination was calculated. 50 It was 53.55 μg / mL.

[0051] The germination count at different time points under different concentrations of thymol treatment is shown in the figure. Figure 3 .like Figure 3 As shown, the spore germination rate slowed down significantly with increasing thymol concentration. When the treatment concentrations were 25 μg / mL, 50 μg / mL, 75 μg / mL and 100 μg / mL, the germination rates of conidia after 14 h were 89%, 56%, 32% and 13%, respectively.

[0052] After 7 days of continued liquid culture, the growth of the strains in each Erlenmeyer flask is shown in the figure. Figure 4 ,Depend on Figure 4 It was observed that only the blank control group and the 25 μg / mL treatment group turned pink, while the other treatment groups did not change color. This indicates that thymol has an inhibitory effect on the germination of Fusarium oxysporum spores, thereby affecting their expression characteristics.

[0053] (4) Effect of thymol on soluble proteins in Fusarium oxysporum mycelium

[0054] ① Sample preparation

[0055] Take five Erlenmeyer flasks containing 100 mL of YEPD medium. Take five 5 mm mycelial discs from the edge of the cultured Fusarium oxysporum colony and transfer them into the medium. After culturing on a shaker at 175 r / min and 25℃ for 1 day, add thymol to make the final concentrations 25 μg / mL, 50 μg / mL, 75 μg / mL and 100 μg / mL. Use no thymol as a blank control. Continue to culture on a shaker for 6 days. Filter with double-layer gauze and collect the mycelia for later use.

[0056] After grinding the bacteria into powder with liquid nitrogen, weigh 0.5g of the powder into a 50mL centrifuge tube, add 10mL of phosphate buffer (0.2mol / L, pH 7.5), centrifuge at 4℃ and 8000r / min for 15min, and store the supernatant in a -20℃ refrigerator for later use.

[0057] ②Preparation of test reagents

[0058] Standard protein solution (100 μg / mg bovine serum albumin): Weigh 25 mg of bovine serum albumin (BSA), dissolve it in distilled water, and bring the volume to 100 mL. Take 40 mL of this solution and dilute it to 100 mL with distilled water. Coomassie Brilliant Reagent: Weigh 20 mg of Coomassie Brilliant Blue G-250, dissolve it in 10 mL of 95% ethanol solution, add 20 mL of 85% H3PO4 solution, dilute with distilled water to 200 mL, and filter through filter paper.

[0059] ③ Plotting the standard curve

[0060] Take six 10mL glass test tubes and number them 0-5. Add 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of standard protein solution (100μg / mL BSA solution) sequentially in ascending order of number. Then add 1.0mL, 0.8mL, 0.6mL, 0.4mL, 0.2mL, and 0mL of distilled water sequentially in ascending order of number. Mix well, then add 5mL of Coomassie Brilliant reagent to each tube. After adding each tube, mix thoroughly and let stand for 5 minutes. Use test tube 0 as a blank control and measure the absorbance at a wavelength of 595nm. The results are shown in Table 1.

[0061]

[0062] Plot a standard curve with BSA content on the ordinate and absorbance on the abscissa (see...). Figure 5 The linear regression equation Y = 342.92x - 1.1903 was obtained, with a correlation coefficient of 0.9945.

[0063] ④ Determination of soluble protein

[0064] Take 1 mL of sample supernatant, add 5 mL of Coomassie Brilliant reagent, mix thoroughly, let stand for 5 min, and measure the absorbance value according to the standard curve determination method. Repeat the experiment 3 times.

[0065] The results of the mycelial protein content determination are shown in Table 2.

[0066] Table 2. Effects of different concentrations of thymol on the protein content of Fusarium oxysporum mycelium.

[0067]

[0068] As shown in Table 2, the protein content of Fusarium oxysporum mycelium decreased after treatment with thymol compared with the blank control. The inhibitory effect was not obvious when the concentration of thymol was 25 μg / mL. As the concentration increased, the soluble protein content of the mycelium was affected. When the concentration was 100 μg / mL, the inhibition rate was 36.73%, indicating that thymol has an inhibitory effect on the synthesis of soluble protein in the mycelium.

[0069] (5) Effect of thymol on DNA content in Fusarium oxysporum mycelium

[0070] Mycelia were collected under culture conditions with final thymol concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL using the method shown in (4)①. DNA was extracted using a column-based fungal DNA extraction kit according to the instructions. The purity and concentration of *Fusarium oxysporum* DNA were then determined using a UV-Vis spectrophotometer. Detection standard: DNA should be within OD... 260 There is an absorption peak, OD 260 A value of 1 corresponds to 50 μg / mL. OD 260 / OD 280 A ratio between 1.7 and 1.9 indicates that the DNA purity is acceptable. The results are shown in Table 3.

[0071] Table 3. Effects of different concentrations of thymol on DNA content in Fusarium oxysporum mycelium.

[0072]

[0073] As shown in Table 3, the mycelial DNA content of Fusarium oxysporum was significantly reduced after treatment with thymol compared with the blank control. The inhibitory effect was not obvious when the concentration of thymol was 25 μg / mL. As the concentration increased, the DNA synthesis of mycelium was severely affected. When the concentration of thymol was 100 μg / mL, the inhibition rate was 47.29%, indicating that thymol can inhibit the DNA synthesis in the mycelium of Fusarium oxysporum.

[0074] (6) Effect of thymol on ergosterol content in Fusarium oxysporum mycelium

[0075] Ergosterol is an important component of fungal cell membranes. It is structurally stable and highly specific, and is crucial for maintaining the integrity and function of the cell membrane. Fungal biomass can be measured by detecting the content of ergosterol. To clarify the effect of different concentrations of thymol on ergosterol in Fusarium oxysporum, the following procedures were performed.

[0076] ① Plotting the standard curve

[0077] Ergosterol standard was dissolved in methanol to prepare a stock solution for later use. The ergosterol stock solution was then diluted to prepare a series of standard solutions with concentrations of 320 g / mL, 160 g / mL, 80 g / mL, 40 g / mL, 20 g / mL, 10 g / mL, 5 g / mL, and 2.5 g / mL. The solutions were then analyzed using HPLC with the external standard method. Detection conditions: XB-C18 stationary column (250 mm × 4.6 mm); 100% methanol (chromatographic grade); column temperature 25℃; detection wavelength 282 nm; flow rate 1 mL / min. -1 Injection volume: 20 μL; Plot a standard curve with peak area on the ordinate and ergosterol content (ng / μL) on the abscissa (see...). Figure 6 The linear regression equation Y = 26.031x + 66.258 was obtained, with a correlation coefficient of 0.9996.

[0078] ② Sample preparation

[0079] Mycelia with final thymol concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL were collected using the method shown in (4)①. After grinding with liquid nitrogen, 0.5 g of mycelial powder was weighed into a 50 mL glass tube, and 10 mL of a 3:1 mixture of methanol and chloroform was added. The tube was left to stand overnight at room temperature. The next day, 10 mL each of water, chloroform, and 0.5 mol / L phosphate buffer containing 2.0 mol / L KCl were added sequentially. After extraction, the chloroform phase was dried at 60 °C using a nitrogen blower. 10 mL of a 4:1 mixture of methanol and ethanol containing 1.4 mol / L KOH was added, and the tube was saponified at 60 °C for 1 h. 10 mL of petroleum ether was added, and the layers were extracted and separated. The petroleum ether layer was dried on a nitrogen blower, dissolved in anhydrous ethanol, and brought to a final volume of 10 mL. The mixture was then filtered through a 0.45 μm filter membrane for later use.

[0080] ③ Determination of soluble protein

[0081] The ergosterol concentration in each sample was determined by high performance liquid chromatography using the external standard method. A histogram was constructed with the final thymol concentration as the X-axis and the ergosterol content (μg / g) as the Y-axis (see [link to histogram]). Figure 7 ).

[0082] Depend on Figure 7 It was found that, compared with the untreated Fusarium oxysporum, the ergosterol content in the mycelium of Fusarium oxysporum decreased with increasing thymol concentration. At a treatment concentration of 25 μg / mL, the content decreased by 17.85% compared with the control; at a concentration of 50 μg / mL, the content decreased by 19.71% compared with the control; at a concentration of 75 μg / mL, the content decreased by 41.58%; and at a treatment concentration of 100 μg / mL, the content decreased by 48.11% compared with the control.

[0083] By establishing the toxicity regression equation Y = 1.8067 + 1.5405x ( Figure 8 The EC50 of thymol inhibiting ergosterol content in Fusarium oxysporum mycelia was measured. 50 The concentration was 118.28 μg / mL. This result indicates that thymol effectively inhibits the production of ergosterol within the mycelium of *Fusarium oxysporum*, thereby achieving its inhibitory effect on *Fusarium oxysporum*.

[0084] (7) Effect of thymol on the content of Fusarium oxysporum toxin

[0085] Fusarium wilt toxins are important pathogenic factors of pathogenic strains of Fusarium oxysporum, which can cause wilt disease in a variety of plants, including flowers, watermelons, cucumbers, tomatoes, beans, and cotton.

[0086] Five Erlenmeyer flasks containing 100 mL of YEPD medium were used. Five 5 mm mycelial discs were collected from the edge of the cultured *Fusarium oxysporum* colonies and transferred to the medium. After culturing on a shaker at 175 rpm and 25°C for 24 h, thymol was added to each flask to achieve final concentrations of 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL, respectively. A blank control was used without thymol. Culturing continued on a shaker at 175 rpm and 25°C for 15 days. The flasks were then filtered through double-layered gauze, and the filtrate was collected. Layer extraction was performed three times at a ratio of filtrate:ethyl acetate = 100 mL: 35 mL. The extract was concentrated to obtain a pale yellow crude extract, which was then redissolved in 5 mL of ethyl acetate. The absorbance was measured at 268 nm using a UV-Vis spectrophotometer. The fusarium toxin content was calculated using the formula Y = 0.045x - 0.02 (where X is the concentration and Y is the absorbance). The results are shown in the figure. Figure 9 .

[0087] Depend on Figure 9 It was found that, compared with the control, the content of thymol in Fusarium oxysporum mycelium decreased with the increase of thymol concentration. When the treatment concentration was 25 μg / mL, it decreased by 28.93% compared with the control; when the concentration was 50 μg / mL, it decreased by 47.93% compared with the control; when the concentration was 75 μg / mL, it decreased by 69.42%; and when the treatment concentration reached 100 μg / mL, it decreased by 76.86% compared with the control.

[0088] By establishing the toxicity regression equation Y = 1.3253 + 2.1993x ( Figure 10 ), and the EC of thymol inhibiting atrophic toxin was measured. 50 The concentration was 46.86 μg / mL. This indicates that thymol can effectively inhibit the production of phytotoxic toxins within the mycelium of Fusarium oxysporum, thereby achieving the effect of inhibiting Fusarium oxysporum.

[0089] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0090] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of thymol in the control of soil-borne diseases caused by Fusarium oxysporum.

2. Application of thymol in the preparation of drugs against Fusarium oxysporum.

3. The application according to claim 2, characterized in that, The drug is targeted at Fusarium oxysporum.

4. An antifungal drug against Fusarium oxysporum, characterized in that, Including thymol.

5. The antifungal drug for Fusarium oxysporum according to claim 4, characterized in that, It also includes other active ingredients and / or excipients.

6. The application of thymol according to claim 1 or 2, characterized in that, Thymol exerts its antifungal effect against Fusarium oxysporum by inhibiting the growth of its hyphae and spores.

7. The application of thymol according to claim 1 or 2, characterized in that, Thymol exerts its antifungal effect against Fusarium oxysporum by inhibiting the synthesis of soluble proteins, DNA, and ergosterol in the mycelium of Fusarium oxysporum.

8. The application of thymol according to claim 1 or 2, characterized in that, Thymol can inhibit the secretion of oxytocin by Fusarium oxysporum.

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