Application of thymol in preparation of medicine for inhibiting growth of botryosphaeria dothidea and synthesizing glycerol

Abstract

The invention specifically discloses an application of thymol in preparation of a medicine for inhibiting botryosphaeria dothidea growth and glycerol synthesis, which comprises the following steps: dissolving thymol with the purity of 99% in 40% ethanol to prepare a stock solution with the concentration of 20 mg / mL; the minimum inhibitory concentration of thymol to botryosphaeria dothidea is determined to be 0.25 mg / mL through a double dilution method; thymol inhibits glycerol synthesis of botryosphaeria dothidea hyphae in a concentration-dependent manner. The thymol has the beneficial effects that the thymol is revealed for the first time to not only effectively inhibit the growth of botryosphaeria dothidea hyphae, but also remarkably inhibit the glycerol synthesis of the botryosphaeria dothidea hyphae, destroy the steady state of germ cells and achieve the purpose of efficient prevention and control; thymol has the characteristics of safety, low toxicity and environmental friendliness, has wide application potential in the field of pathogenic bacterium prevention and control, and particularly provides a new green prevention and control means and technical support in the aspect of corrosion prevention and fresh keeping of fruits such as kiwi fruits and the like.

Description

Technical Field

[0001] The invention belongs to the field of microbial control preparations, and specifically relates to an application of thymol in the preparation of a drug for inhibiting the growth of Botrytis cinerea and glycerol synthesis. Background Art

[0002] Kiwifruit is a very popular fruit, a typical respiratory climacteric fruit, with sweet fruit and high nutritional value. However, most kiwifruit varieties are prone to softening and rotting after harvest, which leads to loss of flavor and edible value. In particular, soft rot caused by infection of Botrytis cinerea has caused serious economic losses in the storage, transportation and sales of kiwifruit. Therefore, it is particularly important to implement effective antiseptic and fresh-keeping measures after harvest to maintain the quality of the fruit.

[0003] As a broad-spectrum pathogenic ascomycete, Botrytis cinerea can cause the degradation of various forest wood materials and reduce fruit tree yields. The epidermis of the fruit infected by Botrytis cinerea collapses and becomes soft, showing round or oval water-soaked lesions. At present, the prevention and control measures for Botrytis cinerea mainly rely on chemical control, but the use of chemical fungicides often leads to the residue of toxic and harmful substances, causing serious safety hazards. Therefore, it is of great significance to develop new green and low-toxic antibacterial agents as control agents for Botrytis cinerea.

[0004] Plant-derived thymol is an environmentally friendly natural antibacterial agent, also known as thymol. It belongs to the monoterpenoid class of compounds and is in the form of white crystals or powder at room temperature. It exists in plants such as Ranunculaceae, Verbenaceae, and Apiaceae. Thymol can bind to the hydrophobic region of proteins, causing the loss of integrity of the cell wall membrane of pathogens, resulting in a weakened ability to regulate external osmotic pressure. In recent years, it has attracted widespread attention due to its strong antibacterial and antifungal properties in the field of food preservation. Summary of the invention

[0005] In view of the above requirements, the present invention provides the use of thymol in preparing drugs for inhibiting the growth of Botrytis cinerea and synthesizing glycerol.

[0006] The present invention is achieved through the following technical solutions: Application of thymol in the preparation of drugs for inhibiting the growth of Botrytis cinerea.

[0007] Application of thymol in the preparation of drugs for inhibiting glycerol synthesis in Botrytis cinerea.

[0008] The strain number of the Botrytis cinerea is ACCC 38020.

[0009] The purity of thymol is 99%, and its structural formula is as follows .

[0010] The specific method of using thymol is as follows: thymol is dissolved in 40% ethanol to prepare a 20 mg / mL thymol stock solution.

[0011] The minimum inhibitory concentration of thymol against Botrytis cinerea is 0.25 mg / mL The thymol inhibits the glycerol synthesis of Botrytis cinerea hyphae in a concentration-dependent manner.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The invention discloses for the first time that thymol can inhibit the growth of Botrytis cinerea hyphae and discloses the MIC of thymol for inhibiting the growth of Botrytis cinerea hyphae.

[0013] The present invention discovers for the first time that thymol can inhibit the glycerol synthesis of Botrytis cinerea hyphae, thereby effectively destroying the cell homeostasis of Botrytis cinerea and achieving the purpose of prevention and control.

[0014] Thymol has the advantages of being safe, low-toxic and environmentally friendly, so it has good application prospects in the prevention and control of pathogens and can be used as an antibacterial agent for Botrytis cinerea. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The inhibition of thymol on the growth of Botrytis cinerea mycelium, fresh weight, dry weight and water retention rate of Botrytis cinerea mycelium at different concentrations.

[0016] Figure 2 The hyphae morphology of Botrytis cinerea in the treatment groups with different concentrations of thymol and the control group Figure 3 The inhibition of thymol on glycerol synthesis of Botrytis cinerea and the glycerol content of Botrytis cinerea mycelium at different concentrations are shown. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The embodiments are intended to explain the present invention rather than to limit it.

[0018] The present invention has found through experiments that thymol has the effect of inhibiting the synthesis of Botrytis cinerea and glycerol Preferably, the Botryosphaeria dothidea is deposited in the China Agricultural Microbiological Culture Collection Center with the strain number ACCC 38020.

[0019] Preferably, the mycelium of Botrytis cinerea is picked in the center of potato dextrose agar (PDA) medium, placed in a 25°C incubator for static culture for 5-7 days, and a Botrytis cinerea cake with a diameter of 6 mm is cut at the edge of the mycelium with a sterilized puncher for standby use.

[0020] Preferably, the minimum inhibitory concentration (MIC) of six plant extracts, including thymol, cinnamaldehyde, ferulic acid, chlorogenic acid, vanillin, and vanillic acid, against Botrytis cinerea was determined by the two-fold dilution method, and it was found that thymol had the best antibacterial effect against Botrytis cinerea, with a MIC value of 0.25 mg / mL.

[0021] Preferably, the Botrytis cinerea is cultured in a potato dextrose liquid medium (PDB) at a speed of 180 r / min and a constant temperature shaker at 25°C for 3 days, and then thymol is added to make the thymol concentration reach 0 (control), 1 / 4MIC, 1 / 2MIC and MIC, and the culture is continued for 1 day.

[0022] Preferably, by measuring the fresh weight, dry weight and water retention rate of Botrytis cinerea hyphae in the thymol treatment groups with different concentrations and the control group, it was found that thymol had a significant inhibitory effect on the growth of Botrytis cinerea hyphae.

[0023] Preferably, by measuring the glycerol content of Botrytis cinerea hyphae in groups treated with different concentrations of thymol and in the control group, it is found that thymol can affect the homeostasis of Botrytis cinerea cells.

[0024] Example 1 Determination of the minimum inhibitory concentration of different plant extracts against Botrytis cinerea 1. Experimental materials: (1) The experimental strain Botrytis cinerea (ACCC 38020) was purchased from the China Agricultural Microbiological Culture Collection and stored in slant tubes at -4°C for a long time.

[0025] (2) The plant extracts used in this study were purchased from Shanghai Yuanye Biotechnology Co., Ltd., of which thymol had a purity of 99% and a CAS number of 89-83-8. Potato dextrose agar (PDA) was purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0026] 2. Experimental operation: (1) Strain activation culture conditions: Under sterile conditions, use a sterile inoculation stick to pick up the mycelium of Botrytis cinerea and place it in the center of potato dextrose agar (PDA) medium. Place it in a 25°C incubator and culture it for 5-7 days. Use a sterile cork puncher to cut a Botrytis cinerea cake with a diameter of 6 mm at the edge of the mycelium and set it aside.

[0027] (2) Preparation of thymol stock solution: Dissolve thymol in 40% ethanol to prepare a 20 mg / mL thymol stock solution and store it at room temperature for future use.

[0028] (3) MIC determination: The six plant extracts were dissolved in anhydrous ethanol to prepare a 50 mg / ml stock solution. Then different volumes of the stock solution were added to the melted PDA medium. The final concentrations were adjusted to 0 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL by the two-fold dilution method to prepare the drug-containing culture medium. A 6 mm diameter activated bacterial cake was placed in the center of the culture medium with the mycelium side facing down. The culture medium was then incubated at 25°C for 7 days to observe the growth of the bacteria. 3. Experimental results: The MIC of cinnamaldehyde and other 6 different plant extracts against Botrytis cinerea was determined by double dilution method. The results are shown in Table 2-4. Among them, the MIC value of thymol against Botrytis cinerea is 0.25 mg / mL, while the MIC of cinnamaldehyde against Botrytis cinerea is 0.5 mg / mL, the MIC of ferulic acid, vanillin and vanillic acid against Botrytis cinerea is 2.0 mg / mL, and the MIC of other substances against Botrytis cinerea is 4.0 mg / mL. It can be seen that thymol has the best antibacterial effect against Botrytis cinerea.

[0029] Table 1 MIC of different plant extracts against Botrytis cinerea Antibacterial Agents 0 0.03125 0.0625 0.0125 0.25 0.5 1.0 2.0 4.0 MIC Cinnamaldehyde + + + + + - - - - 0.5 Thymol + + + + - - - - - 0.25 Ferulic acid + + + + + + + - - 1.0 Chlorogenic acid + + + + + + + + - 4.0 Vanillin + + + + + + + - - 2.0 Vanillic acid + + + + + + + - - 2.0 Solvent Control + + + + + + + + - 4.0 Note: +: growth and proliferation, -: no growth.

[0030] Example 2 Inhibitory effect of thymol on the mycelial growth of Botrytis cinerea 1. Experimental operation: (1) The effect of thymol on the growth of Botrytis cinerea was determined by the dry weight method. Botrytis cinerea cakes with a diameter of 6 mm were transferred to potato dextrose liquid medium (PDB) and cultured in a constant temperature shaker at 180 r / min and 25°C for 3 days. Thymol was added to make the thymol concentration reach 0 (control), 1 / 4MIC, 1 / 2MIC and MIC, and the culture was continued for 1 day. The mycelium was collected by filtering through sterilized eight-layer gauze, rinsed with distilled water 2-3 times, and the collected mycelium was placed in a 45°C oven to constant weight. The fresh mycelium and the dry mycelium were weighed using an electronic balance. Each treatment was repeated three times, and the dry weight inhibition rate and water retention rate were calculated.

[0031] (2) Use scanning electron microscopy (SEM) to observe the morphology of Botrytis ciliata hyphae. Collect the mycelium and completely immerse it in 2.5% glutaraldehyde fixative and let it stand for 1 day. After discarding the fixative, wash it with distilled water 3 times, with an interval of 10 minutes. Then use a concentration gradient of 30%, 50%, 70%, 90%, and 100% ethanol for gradient dehydration, once every 10 minutes. Then immerse the sample in isoamyl acetate for replacement for 30 minutes. After natural drying in a fume hood, stick the sample, spray gold coating, and place it under a desktop scanning electron microscope to observe the microscopic morphology of the hyphae surface.

[0032] 2. Experimental results: Figure 1 The results showed that the increase in thymol treatment concentration reduced the mycelial growth (fresh weight, dry weight) of Botrytis cinerea, and also reduced the water retention rate of mycelium. Under a scanning electron microscope, the mycelium of the control group was uniform in thickness, smooth in surface, and round and plump. After treatment with 1 / 4MIC thymol, structural abnormalities began to appear on the surface of the mycelium. The mycelium treated with 1 / 2MIC thymol showed partial shrinkage and depression on the surface. The mycelium treated with MIC concentration thymol showed more severe shrinkage and depression on the surface, and appeared shriveled and irregularly twisted, such as Figure 2 The higher the concentration of thymol, the greater the damage to the mycelium, indicating that thymol destroys the integrity and normal physiological metabolic function of the Botrytis ciliata mycelium and hinders the normal growth and development of the mycelium.

[0033] Example 3 Inhibitory effect of thymol on glycerol synthesis in Botrytis cinerea 1. Experimental operation: The glycerol content in mycelium was determined by the glycerate copper colorimetric method. After collecting the mycelium and quenching it with liquid nitrogen, 0.1 g of mycelium was accurately weighed and resuspended in 4 mL of sterile water, incubated in a water bath at 80 ° C for 15 min, centrifuged at 8500 rpm for 10 min, and the supernatant was collected. 2 mL of the supernatant was mixed with 0.2 mL of copper sulfate solution and 0.7 mL of sodium hydroxide solution, shaken at 100 rpm for 15 min, filtered, and the absorbance at A630 was measured.

[0034] 2. Experimental results: Figure 3 The results showed that the glycerol synthesis of Botrytis cinerea could be reduced under the action of thymol treatment at the control group, 1 / 2MIC, 1 / 4MIC and MIC concentrations, and the glycerol content in the mycelium of Botrytis cinerea gradually decreased with the increase of thymol treatment concentration. After SPSS software analysis, there were significant differences in glycerol content among the treatment groups, indicating that thymol treatment had a significant inhibitory effect on the mycelium's ability to regulate external osmotic pressure.

Claims

1. The application of thymol in the preparation of drugs for inhibiting the growth of Botrytis cinerea.

2. Application of thymol in the preparation of drugs for inhibiting glycerol synthesis in Botrytis cinerea.

3. The use according to claim 1 or 2, characterized in that: The strain number of the Botrytis cinerea is ACCC38020.

4. The use according to claim 3, characterized in that: The purity of thymol is 99%, and its structural formula is as follows 。 5. The use according to claim 4, characterized in that: The specific method of using thymol is as follows: thymol is dissolved in 40% ethanol to prepare a 20 mg / mL thymol stock solution.

6. The use according to claim 1, characterized in that: The minimum inhibitory concentration of thymol to Botrytis cinerea is 0.25 mg / mL.

7. The use according to claim 2, characterized in that: The thymol inhibits the glycerol synthesis of Botrytis cinerea hyphae in a concentration-dependent manner.

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