Application of holly essential oil

Abstract

The invention discloses an application of holly essential oil, and particularly relates to an application of the holly essential oil in preparation of a product for preventing and treating phytophthora nicotianae, and the product is a pesticide. The dosage form of the product is microemulsion, aerosol or emulsion in water. The holly essential oil has obvious influence on the form of phytophthora nicotianae hypha, so that the hypha surface is not smooth any more, hypha bending is increased, germination on hypha is increased, and hypha branches are obviously increased. When the concentration of the essential oil is maximum, most of the mycelia are expanded, almost no obvious complete mycelia and sporophytes are found, and the mycelia are broken and distorted more seriously. The holly essential oil has the advantages of high efficiency, environment friendliness, low cytotoxicity, low residue, easiness in degradation and the like, and is expected to become a novel environment-friendly pesticide for preventing and treating phytophthora nicotianae.

Description

Technical Field

[0001] The invention belongs to the technical field of biological applications, further belongs to the technical field of biological pesticides, and specifically relates to an application of wintergreen essential oil. Background Art

[0002] Tobacco Phytophthora parasitica var. nicotianae Tucker is a soil-borne pathogen that mainly parasitizes on plants of the Solanaceae, Araceae and Passifloraceae families. It can cause rhizome rot, leaf wilt and other diseases in plants such as tobacco, konjac, pineapple, cherry and passion fruit, especially in Yunnan tobacco-growing areas, causing serious economic losses to tobacco farmers. Tobacco Phytophthora parasitica var. nicotianae Tucker can cause root and stem rot and leaf wilt, and can infect and cause disease in all growth stages of tobacco growth. At present, the prevention and control of this disease mainly adopts the method of continuous and large-scale use of chemical pesticides, which leads to the generation of pathogenic bacteria resistant physiological subspecies, the destruction of soil microbial communities, the destruction of farmland ecological environment and the excessive pesticide residues in agricultural products, which harms human health. Therefore, it is extremely important to develop safe, effective and environmentally friendly bio-source pesticides to replace chemical pesticides.

[0003] Plant essential oils are secondary metabolites derived from different parts of plants. In recent years, with the increasing demand for green and safe agricultural products, the application of plant essential oils as a substitute for traditional chemical pesticides has become a hot topic in the study of agricultural diseases. Holly (Ilex chinensis.) is an evergreen tree of the Aquifoliaceae family and the genus Ilex. Holly is distributed in Japan, China and Vietnam worldwide, and is mainly distributed in central, southern and southeastern China. The main chemical component of holly essential oil is methyl salicylate. Existing studies have found that it has stimulating, anti-inflammatory, analgesic and other effects, and can promote local blood circulation. Holly essential oil has a significant killing effect on pests, and also has a certain inhibitory effect on Bacillus subtilis and some plant fungi, but whether holly essential oil has the activity of inhibiting tobacco phytophthora has not been reported. Summary of the invention

[0004] The object of the present invention is to provide an application of wintergreen essential oil to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions: an application of wintergreen essential oil, and an application of the wintergreen essential oil in the preparation of a product for preventing and controlling tobacco blight.

[0006] Preferably, the product is a pesticide.

[0007] Preferably, the dosage form of the product is a microemulsion, an aerosol or an aqueous emulsion.

[0008] Preferably, the product is in the form of a fungicide for controlling tobacco blight, and the fungicide includes wintergreen essential oil, an organic solvent or an emulsifier.

[0009] Preferably, the volume ratio of the wintergreen essential oil to the organic solvent or emulsifier is 1:(1-10).

[0010] Preferably, the concentration of the wintergreen essential oil is above 193.5 μg / mL.

[0011] Preferably, the organic solvent is acetone or ethanol, and the emulsifier is Tween-20 or Tween-80.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: in order to achieve green prevention and control of crop diseases, efficient bactericidal active ingredients are screened from medicinal plants, and the antibacterial activity of wintergreen essential oil on tobacco phytophthora is determined by the mycelium growth rate method. The results show that wintergreen essential oil has a significant inhibitory effect on the growth of tobacco phytophthora hyphae: under direct contact, the efficiency of wintergreen essential oil in inhibiting tobacco phytophthora is 8.19-70.49%. Wintergreen essential oil has a significant effect on the morphology of tobacco phytophthora hyphae, making the mycelium surface no longer smooth, the mycelium bending increases, and the germination on the mycelium increases, and the mycelium branches increase significantly. When the essential oil concentration is the highest, most of the mycelium shows swelling, and there is almost no obvious complete mycelium and spore body, and the mycelium fracture and distortion phenomenon are more serious. The present invention finds that wintergreen essential oil has many advantages such as high efficiency, environmental protection, low cytotoxicity, low residue and easy degradation, and is expected to become a new environmentally friendly pesticide for preventing and controlling tobacco phytophthora. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The effect of wintergreen essential oil on the colony size of tobacco phytophthora.

[0014] Figure 2 The inhibition efficiency of wintergreen essential oil at different concentrations on tobacco phytophthora.

[0015] Figure 3 The effect of holly essential oil on the hyphae morphology of tobacco Phytophthora. DETAILED DESCRIPTION

[0016] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] See also Figure 1-Figure 3The present invention provides a technical solution: the application of the wintergreen essential oil is the application of the wintergreen essential oil in the preparation of products for preventing and controlling tobacco blight.

[0018] The product described is a pesticide.

[0019] The product is a microemulsion, an aerosol or an aqueous emulsion.

[0020] The tobacco bactericide for preventing and controlling tobacco phytophthora is composed of holly essential oil, organic solvent or emulsifier.

[0021] The volume ratio of the wintergreen essential oil to the organic solvent or emulsifier is 1: (1-10).

[0022] The concentration of the wintergreen essential oil is above 53.05 μg / mL.

[0023] The organic solvent is acetone or ethanol, and the emulsifier is Tween-20 or Tween-80.

[0024] The wintergreen essential oil of the present invention comprises the steps of preparing the essential oil agent, using the wintergreen essential oil agent, and observing the disease-resistant morphology of the wintergreen essential oil agent when preparing the fungicide for preventing and controlling tobacco phytophthora. Specifically, the steps include:

[0025] 1. Preparation of wintergreen essential oil: Mix wintergreen essential oil with Tween-20 or Tween-80 or acetone or ethanol in a ratio of 1:1 to 10 times, mix thoroughly and set aside;

[0026] 2. Essential oil pharmaceutical treatment

[0027] Under sterile conditions, first dissolve different volumes of essential oils of medicinal plants in organic solvents or emulsifiers, add 20 mL of sterilized rye solid culture medium, mix thoroughly, pour into a culture dish, and wait for it to cool naturally. Use a hole puncher to take a 5 mm diameter tobacco phytophthora cake under sterile conditions and insert it into the center of the culture medium. After sealing with a sealing film, place it at 25°C for about 4 days, observe the size and shape of tobacco phytophthora plaques, measure the colony diameter, and calculate the antibacterial rate and effective medium concentration (EC50 value).

[0028] 3. Effect of holly essential oil on mycelial morphological growth

[0029] The mycelial morphology after wintergreen essential oil treatment was observed using a scanning electron microscope. The bacterial cake after 5 days of culture was inoculated on rye culture medium with / without wintergreen essential oil, and cultured for about 4 days until the mycelium of the control group approached the edge of the culture dish. The sample was placed under a scanning electron microscope to observe the mycelial morphology.

[0030] The present invention is further described below with specific implementation cases:

[0031] Example 1

[0032] Preparation of wintergreen essential oil preparation: Mix wintergreen essential oil with Tween-20 at a ratio of 1:10, mix thoroughly, and then dilute with distilled water to make the final concentration of wintergreen essential oil 10 μg / mL.

[0033] Example 2

[0034] Preparation of wintergreen essential oil preparation: Mix wintergreen essential oil with Tween-80 in a ratio of 1:7, mix thoroughly, and then dilute with distilled water to make the final concentration of wintergreen essential oil 25 μg / mL.

[0035] Example 3

[0036] Preparation of wintergreen essential oil preparation: Mix wintergreen essential oil with acetone in a ratio of 1:5, mix thoroughly, and then dilute with distilled water to make the final concentration of wintergreen essential oil 50 μg / mL.

[0037] Example 4

[0038] Preparation of wintergreen essential oil preparation: Mix wintergreen essential oil with ethanol in a ratio of 1:1, mix thoroughly, and then dilute with distilled water to make the final concentration of wintergreen essential oil 250 μg / mL.

[0039] Example 5

[0040] Calculation of wintergreen essential oil concentration: Take 1, 2, 3, 4 and 5 μL of wintergreen essential oil, mix thoroughly with an appropriate amount of organic solvent or emulsifier, and add them to 20 mL of rye solid culture medium. Based on the density of wintergreen essential oil of 1.1606 g / mL, the final concentrations of wintergreen essential oil in the culture medium are 53.05 μg / mL, 106.10 μg / mL, 159.15 μg / mL, 212.20 μg / mL and 265.25 μg / mL, respectively.

[0041] Example 6

[0042] Observation of tobacco phytophthora plaques treated with essential oil agents: Under sterile conditions, first dissolve different volumes of medicinal plant essential oils in 0.5% Tween 80 solution, add 20 mL of sterilized rye solid culture medium, mix thoroughly, pour into a culture dish (90 mm in diameter), and wait for it to cool naturally. Use a hole puncher to take a 5 mm diameter tobacco phytophthora cake under sterile conditions and insert it into the center of the culture medium. After sealing with a sealing film, place it at 25°C for about 4 days, and observe the size and shape of tobacco phytophthora plaques.

[0043] The results are as follows Figure 1 As shown: Wintergreen essential oil at different concentrations had a significant inhibitory effect on tobacco phytophthora, inhibiting the growth of tobacco phytophthora hyphae. Compared with the blank control group, wintergreen essential oil treatment caused the tobacco phytophthora colony to become significantly smaller, and the higher the concentration, the more significant the inhibitory effect.

[0044] Example 7

[0045] The inhibitory effect of different concentrations of wintergreen essential oil on tobacco phytophthora: Use a hole puncher to take a 5mm diameter tobacco phytophthora cake under sterile conditions and inoculate it into the center of the rye culture medium containing different essential oil concentrations (0, 1, 2, 3, 4 and 5 μL). After sealing with a sealing film, place it at 25°C and culture it for about 5 days. The colony diameter is measured and the inhibition rate is calculated. Each treatment is repeated 3 times. The inhibition rate is calculated according to the following formula: Inhibition rate (%) = (dC-dT) / (dC-5)×100. In the formula: dC is the control colony diameter; dT is the colony diameter under different treatment conditions. For the antibacterial activity of the above essential oils of different mass concentrations against tobacco phytophthora, the regression equation y = a + bx and the effective median concentration (EC50 value) are calculated. 70.49%, 8.19%.

[0046] The results are as follows Figure 2 As shown in the figure, by calculating the mycelium growth inhibition rate, it was found that wintergreen essential oil had a significant inhibitory effect on the mycelium growth of tobacco phytophthora, and it was concentration-dependent. With the increase of concentration, its inhibitory effect increased. At a higher concentration (265.25 μg / mL), the inhibition efficiency of wintergreen essential oil reached 70.49%. At a lower concentration (53.05 μg / mL), its inhibition rate on tobacco phytophthora was 8.19%. According to the inhibition rate of different concentrations, the regression equation was obtained as Y=0.3162*X-11.17(R 2 =0.9796), the EC50 value of wintergreen essential oil against tobacco phytophthora was 193.5 μg / mL, indicating that it has a good inhibitory effect on tobacco phytophthora.

[0047] Example 8

[0048] Effect of wintergreen essential oil on hyphae morphology of tobacco phytophthora: The hyphae morphology after wintergreen essential oil treatment was observed using a scanning electron microscope. The 5-day-old bacterial cake was inoculated on rye culture medium with / without wintergreen essential oil, and cultured for about 4 days until the hyphae of the control group approached the edge of the culture dish. The samples were placed under a scanning electron microscope to observe the hyphae morphology.

[0049] The results are as follows Figure 3It shows that wintergreen essential oil has a great influence on the growth state of tobacco phytophthora hyphae. The hyphae of the blank control group are uniformly filamentous, with a smooth surface, uniform distribution of internal substances, longer hyphae, fewer branches, no obvious abnormal swelling, and no obvious conidia. The surface of tobacco phytophthora hyphae treated with wintergreen essential oil is no longer smooth, the mycelium is more curved, and the number of germinations on the mycelium increases, and the number of mycelium branches increases significantly. When the essential oil concentration is the highest, most of the mycelium shows swelling, and almost no obvious complete mycelium and spores are seen, and the mycelium is broken and deformed more seriously. Vacuoles appear inside some mycelium, which may be caused by the destruction of the internal structure of the mycelial cells, the damage of the cell membrane, and the release of endolysates to the outside of the cells after the wintergreen essential oil treatment.

[0050] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An application of wintergreen essential oil, characterized in that, The application of the wintergreen essential oil in preparing products for preventing and controlling tobacco blight.

2. The use of wintergreen essential oil according to claim 1, characterized in that The product described is a pesticide.

3. The use of wintergreen essential oil according to any one of claims 1 or 2, characterized in that: The dosage form of the product is microemulsion, aerosol or aqueous emulsion.

4. The use of wintergreen essential oil according to any one of claims 1 or 2, characterized in that: The formulation of the product is a fungicide for preventing and controlling tobacco blight, and the fungicide comprises wintergreen essential oil, an organic solvent or an emulsifier.

5. The use of wintergreen essential oil according to claim 4, characterized in that: The volume ratio of the wintergreen essential oil to the organic solvent or emulsifier is 1:(1-10).

6. The use of wintergreen essential oil according to claim 5, characterized in that: The concentration of the wintergreen essential oil is above 193.5 μg / mL.

7. The use of wintergreen essential oil according to claim 6, characterized in that: The organic solvent is acetone or ethanol, and the emulsifier is Tween-20 or Tween-80.

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