Microemulsion containing 3% ginkgolic acid and application thereof

Abstract

The invention discloses a 3% ginkgolic acid microemulsion and application thereof, and belongs to the technical field of biological control. The ginkgolic acid microemulsion is prepared from the following raw materials in percentage by mass: 3% of ginkgolic acid, 1% of ethanol, 1% of n-butyl alcohol, 5% of alkylphenol polyoxyethylene (OP10), 10% of calcium dodecyl benzene sulfonate (DBS-Ca) and 80% of distilled water. The potting experiment result shows that the best prevention and treatment effect of the 3% ginkgolic acid microemulsion on the plant anthracnose is that the 3% ginkgolic acid microemulsion is diluted by 12.5 times and then diluted by 50 times, the relative prevention effects are 75.87% and 71.96% respectively, and the 3% ginkgolic acid microemulsion is equivalent to that of a 20% dicyano.pyrazole ester suspending agent; the invention also discloses a field control effect result, after the 3% ginkgolic acid microemulsion is diluted by 50 times for treatment, the plant anthracnose morbidity is 3.03%, the disease index is 2.25, and the relative control effect is 58.95%.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and particularly relates to a 3% ginkgolic acid microemulsion and application thereof. Background Art

[0002] Plant anthracnose is a widespread fungal disease that poses a serious threat to agricultural production and plant resources. Colletotrichum is a fungus of the phylum Ascomycota. It is one of the most important plant pathogens in the world. It is a common endophyte, epiphyte and saprophyte. Different species of anthracnose can cause diseases in the same plant, and the same species of anthracnose can cause diseases in different plants. It has strong host adaptability. The diseases caused on plants are collectively referred to as anthracnose. At present, a variety of control measures such as chemical, biological and agricultural can be used for anthracnose. The use of chemical agents is still one of the most effective methods for the prevention and control of anthracnose. However, studies have shown that the effectiveness of chemical fungicides on anthracnose varies greatly, and it is easy to cause pathogens to develop drug resistance, drug tolerance, and pollute the environment. Nowadays, new species of anthracnose are constantly being discovered, and their resistance to chemical pesticides is also constantly changing. Therefore, it is necessary to combine agriculture, green control and other means to prevent and control the disease in an efficient and environmentally friendly way. Therefore, finding a new, efficient and environmentally friendly method to prevent and control plant anthracnose has become a current research hotspot.

[0003] In recent years, with the deepening of research on botanical pesticides, ginkgo acid, as a natural product, has gradually revealed its potential in plant disease prevention and control. Ginkgo acid is derived from the outer seed coat and leaves of ginkgo biloba, has a unique chemical structure and biological activity, and has antiviral, selective bactericidal, and antibacterial effects. Studies have reported that ginkgo acid has a good antibacterial effect on a variety of plant pathogenic fungi, and the most significant effect is on the genus Anthrax, but there are no relevant research reports on the in-depth antibacterial mechanism of ginkgo acid on the genus Anthrax. As a natural biological pesticide, ginkgo acid not only has low toxicity and environmental friendliness, but its unique biological activity mechanism helps to overcome the drug resistance of pathogens.

[0004] In order to further exert the advantages of ginkgo acid in the prevention and control of plant anthracnose, in addition to the exploration of the inhibitory mechanism of ginkgo acid on anthracnose, the development of microemulsion technology has become the key. Microemulsion is a transparent or translucent liquid preparation formed by dispersing poorly water-soluble pesticides into the aqueous phase through emulsification technology. It has the advantages of improving the stability of pesticides, increasing their biological activity, and reducing environmental pollution. Ginkgo acid is used as a plant extract for plant disease prevention and control, and its biggest advantages are green, safe and efficient. However, ginkgo acid has problems such as poor water solubility and low utilization rate, which limits its application in disease prevention and control in actual production. By preparing ginkgo acid into a microemulsion, not only can its solubility problem in water be solved, but also its bioavailability and prevention and control effect can be improved. Summary of the invention

[0005] The present invention aims to explore the inhibitory mechanism of ginkgolic acid on anthrax, and through the development of microemulsion technology, to provide a new type of highly efficient and environmentally friendly botanical pesticide preparation for agricultural production, which is of great significance for the prevention and treatment of plant diseases.

[0006] The invention provides a 3% ginkgolic acid microemulsion, which is prepared from the following raw materials in percentage by mass: 3% ginkgolic acid, 1% ethanol, 1% n-butanol, 5% OP10 (alkylphenol polyoxyethylene ether), 10% DBS-Ca (calcium dodecylbenzene sulfonate), and 80% distilled water.

[0007] Preferably, the preparation method of the 3% ginkgolic acid microemulsion is: ginkgolic acid is first dissolved in ethanol and n-butanol, then OP10 and DBS-Ca are added, and finally distilled water is added.

[0008] The present invention also provides application of the 3% ginkgolic acid microemulsion in preventing and treating plant anthracnose.

[0009] Preferably, the plant is at least one of coptis chinensis, azalea, pomegranate, hollyhock, pepper, kidney bean, banana, tobacco, sorghum, plum, bayberry, peanut, grapefruit, walnut and citrus.

[0010] The invention also provides a product for preventing and treating plant anthracnose, wherein the product contains the 3% ginkgolic acid microemulsion.

[0011] The present invention also provides a method for preventing and controlling plant anthracnose using the 3% ginkgolic acid microemulsion, the method comprising the following steps:

[0012] (1) diluting 3% ginkgolic acid microemulsion;

[0013] (2) Spray the diluted 3% ginkgolic acid microemulsion prepared in step (1) on the leaves of the plants.

[0014] Preferably, the dilution factor of the 3% ginkgolic acid microemulsion in step (1) is 12.5 or 50.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention preliminarily explores the antibacterial mechanism of ginkgolic acid on plant anthrax, and finds that ginkgolic acid has a certain influence on the mycelial growth rate, conidia germination, cell membrane permeability, soluble protein and chitin content of various plant anthrax fungi, indicating that ginkgolic acid has a good inhibitory effect on such fungi. The present invention successfully prepares a 3% ginkgolic acid microemulsion system (3% ginkgolic acid, 1% anhydrous ethanol, 1% n-butanol, 5% OP10, 10% DBS-Ca, 80% distilled water), and through indoor prevention effect experiments, it is concluded that the best effect of 3% ginkgolic acid microemulsion is diluted 12.5 times, followed by 50 times, and field plot experiments find that after 3% ginkgolic acid microemulsion is diluted 50 times, the incidence rate is 3.03%, the disease index is 2.25, and the relative prevention effect is 58.95%, thereby verifying that it has good activity in killing anthrax. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The inhibitory effect of ginkgolic acid on 5 plant anthrax fungi in Example 1;

[0018] Figure 2 The optimal inhibitory concentration of ginkgolic acid against 18 plant anthracnose fungi in Example 1;

[0019] Figure 3 The effect of ginkgolic acid on the mycelial morphology of C. truncatum observed by optical microscope in Example 1 (100 times magnification);

[0020] Figure 4 The effect of ginkgolic acid on the mycelial morphology of C. musae observed by optical microscope in Example 1 (100 times magnification);

[0021] Figure 5 The inhibitory effect of ginkgolic acid on the mycelial growth of four plant anthrax fungi in Example 1;

[0022] Figures 6-1 to 6-6 The inhibitory effect of ginkgolic acid on spore germination of 6 plant anthrax pathogens in Example 1;

[0023] Fig. 7A The effect of ginkgolic acid on the cell membrane permeability of C. truncatum GULJ01 in Example 1;

[0024] Figure 7B The effect of ginkgo acid on the cell membrane permeability of C. musae GUXJ in Example 1;

[0025] Fig. 8A The effect of ginkgolic acid on the soluble protein content of C. truncatum GULJ01 in Example 1;

[0026] Figure 8B The effect of ginkgolic acid on the soluble protein content of C. musae GUXJ in Example 1;

[0027] Fig. 9 The transparent temperature range of the ginkgo acid microemulsion in Example 2;

[0028] Fig.10 is the pH value of the ginkgo acid microemulsion in Example 2;

[0029] Fig.11 is the sustained foaming volume of the ginkgo acid microemulsion in Example 2;

[0030] Fig.12 The ginkgo acid microemulsion prepared in Example 2;

[0031] Fig.13 The control effect of 3% ginkgolic acid microemulsion on pepper anthracnose in Example 3 is shown. DETAILED DESCRIPTION

[0032] Example 1 Preliminary Study on the Inhibitory Mechanism of Ginkgo Biloba Acid on Plant Anthrax

[0033] 1.1 Test materials

[0034] As shown in Table 1, 18 species of plant anthracnose fungi.

[0035] Table 1 Information on strains used in the present invention

[0036]

[0037] 1.2 Test agents

[0038] (1) Ginkgo acid: provided by the team of Professor He Jun from Guizhou Biochemical Engineering Center; the preparation method of ginkgo acid is as follows: take dried ginkgo seed coat, extract with methanol at 40°C for one and a half hours, extract twice, combine the two extracts after extraction, filter the extract into a concentration tank, concentrate to extract, add the extract into an extraction tank, extract twice with ethyl acetate, combine the ethyl acetate layers and concentrate to extract, dissolve the extract with 70% ethanol and apply HPD-5000 resin, remove impurities with 2BV 70% ethanol solution, elute with 4BV 90% ethanol, collect the 90% ethanol eluate, concentrate to extract, transfer to a 60°C vacuum drying oven until constant weight, and obtain ginkgo acid extract.

[0039] (2) Anhydrous ethanol (produced by Chongqing Chuandong Chemical (Group) Co., Ltd.): dissolves ginkgolic acid;

[0040] (3) Coomassie Brilliant Blue (produced by Shanghai MacLean Biochemical Technology Co., Ltd.): Determine the soluble protein content;

[0041] (4) Bovine serum albumin (produced by Beijing Solebow Technology Co., Ltd.): for determination of soluble protein content;

[0042] (5) Potassium hydroxide (KOH) (produced by Chongqing Chuandong Chemical (Group) Co., Ltd.): for determining chitin content;

[0043] (6) Glycerol (produced by Biosharp): for determining chitin content;

[0044] (7) 85% phosphoric acid (produced by Shanghai MacLean Biochemical Technology Co., Ltd.): Determine the soluble protein content.

[0045] 1.3 Test culture medium

[0046] (1) PDA medium (potato dextrose solid medium): 200.0 g / L potato, 20 g / L glucose, 15-20 g / L agar powder, distilled water to 1 L, sterilized at 121°C for 15 min;

[0047] (2) PDB medium (potato glucose liquid medium): 200.0 g / L potato, 20 g / L glucose, 1 L distilled water, sterilized at 121°C for 15 min.

[0048] 1.4 Effects of Ginkgo Biloba on the Growth Rate of Mycelium of Plant Pathogens

[0049] The effect of ginkgolic acid on the growth rate of 18 plant anthrax fungi was determined by the mycelial growth rate method. Five geometric gradient concentrations of ginkgolic acid were set. 1 mL of each concentration of ginkgolic acid was added to 9 mL of PDA medium to make a drug-containing plate. Three replicates were set, with sterile water as the blank control and an equal amount of anhydrous ethanol as the negative control. A 6 mm diameter cake was taken from the edge of the activated fungal colony and inoculated onto the drug-containing plate. Cultured at 28 ° C, when the blank control colony grew more than 2 / 3 of the entire plate, the colony diameter was measured by the cross method, and the inhibition rate was calculated according to the following formula:

[0050] Inhibition rate (%) = (colony diameter of control group – colony diameter of treatment group) / colony diameter of control group × 100%

[0051] 1.5 Effects of ginkgolic acid on the morphology and weight of mycelium of plant anthracnose fungi

[0052] Observe the mycelial morphology of plant anthracnose fungi after treatment with ginkgolic acid under an optical microscope. Pipette 1mL of ginkgolic acid of corresponding concentration and add it to 9mL of PDA to make a drug-containing plate, with sterile water as blank control and anhydrous ethanol as negative control, and set up 3 replicates. Inoculate the activated plant anthracnose fungi strains into the drug-containing culture medium, and after culturing for 2 days, observe the difference in mycelial morphology between the control group and the treatment group under an optical microscope.

[0053] Take fresh plant anthrax cake and place it in PDB, add the prepared ginkgolic acid to make the final concentration of 0.19, 0.38, and 0.76 mg mL -1 Sterile water was used as the blank control and anhydrous ethanol as the negative control. Three replicates were set up and cultured at 28°C for 3 days with shaking. The culture was washed with sterile water three times, and the mycelium was freeze-dried using a freeze dryer and weighed on an electronic balance.

[0054] 1.6 Effects of ginkgolic acid on conidia germination of plant anthracnose fungi

[0055] In a sterile operating table, rinse the plant anthracnose plate with sterile water, blow the plate with a 1 mL syringe (without a needle), filter the mycelium with a 1 mL syringe (with a needle), and obtain 1×10 6 CFU mL -1 The spore suspension was prepared by setting the concentration of ginkgolic acid to 0.011, 0.023, 0.047, 0.095, 0.19, 0.38, 0.76, and 1.52 mg mL -1 8 concentrations were set, and blank and negative controls were set. 10 μL of the prepared ginkgolic acid was added to 90 μL of the spore suspension, mixed and dropped onto a cover glass, placed in a sterile culture dish and cultured at 28°C until the germination of the blank control exceeded 80%, then photographed and counted, and the inhibitory effect of ginkgolic acid on the germination of spores of the six plant anthracnose fungi was calculated.

[0056] 1.7 Effects of ginkgolic acid on cell membrane permeability of plant anthrax

[0057] The conductivity method was used to determine the permeability of the plant anthrax cell membrane. 3-5 6mm fresh plant anthrax cakes were placed in PDB and heated at 28℃ and 120r·min. -1 After 3 days of shaking culture, 1 g of plant anthracnose mycelium was placed in 20 mL of ddH2O, and ginkgolic acid was added at concentrations of 0.19, 0.38, and 0.76 mg mL -1 , respectively, the conductivity of the suspension was measured at 0, 1, 2, 3, 4, 5, and 6 hours. After 6 hours of measurement, the suspension was boiled in boiling water, and the conductivity was measured after cooling. A blank control and a negative control were set, and each treatment was repeated 3 times. The relative permeability was calculated by the following formula:

[0058]

[0059] p: relative permeability; a: conductivity at each time period (1–6 h); b: conductivity at 0 h; c: conductivity measured after boiling and cooling.

[0060] 1.8 Effect of ginkgolic acid on soluble protein content of plant anthracnose fungi

[0061] Sample preparation: Use a 6 mm cork puncher to punch out fresh plant anthrax fungus cakes, place them in PDB medium, shake and culture at 28°C for 3 days, add the prepared ginkgolic acid to make the final concentrations of 0.095, 0.19, 0.38, and 0.76 mg mL -1 , set up blank and negative controls at the same time, extract the hyphae of each treatment at 0, 2, 4, 6, and 12 hours after adding ginkgo acid, wash with sterile water 3 times, centrifuge at 4℃, 8500 (×g) for 30 minutes, add Tris-HCL buffer at 1g / 5mL to obtain mycelium, grind after quick freezing in liquid nitrogen, centrifuge at 4℃, 15000 (×g) for 30min, take the supernatant and place it in a -20℃ refrigerator for later use. Refer to the G-250 (Coomassie Brilliant Blue) method to determine the effect of ginkgo acid on the soluble protein content of plant anthracnose fungi. Take 100μL of the supernatant, add 1.9mL ddH2O and 5mL G-250, and detect the absorbance at a wavelength of 595nm.

[0062] 1.9 Effect of ginkgolic acid on chitin content in plant anthrax

[0063] The plant anthrax fungus cake was inoculated on PDA and cultured for 7 days. A 6 mm fresh cake was inoculated into PDB and cultured at 28°C, 120 rpm, with shaking for 2 days. The culture was washed 2-3 times under sterile operation and filtered. 20 g of mycelium was weighed and placed in PDB. Different concentrations of ginkgolic acid were added to make the final concentrations 0.19, 0.38, and 0.76 mg·mL -1 , with sterile water as blank control and anhydrous ethanol as negative control, samples were taken at 0, 1, 3, 6, and 12 hours, washed 2-3 times with water under sterile operation, filtered and dried to constant weight, accurately weighed 3g (W1), and 3 replicates were set for each treatment. The weighed mycelium was added to a digestive tube containing a saturated KOH solution and placed in a glycerol bath (160°C, 20min). The final product was transparent, poured onto filter paper, slowly rinsed with running water, dehydrated with 95% and 100% alcohol, and weighed (W2). The chitin content was calculated according to the following formula:

[0064] Calculation formula: Chitin content (%) = W2 / W1×1.26*×100 (*conversion coefficient, generally 1.0)

[0065] The results showed that the effect of ginkgolic acid on the mycelial growth rate of 18 plant anthrax fungi was determined by the mycelial growth rate method. The results showed that ginkgolic acid had a better effect on plant anthrax fungi at a concentration of 0.46 mg·mL- 1 The inhibition rate of Ginkgo biloba against C. musaeGUXJ can reach 63.03%. The EC50 of ginkgolic acid against five plant anthracnose fungi (Table 2, Figure 1 ), the EC50 of ginkgolic acid against C. musae GUXJ and Colletotrichum sp. GUGJ were 0.2938 and 1.3075 mg·mL -1 The optimal inhibitory concentration of ginkgolic acid against 18 plant anthrax fungi was determined, among which the optimal inhibitory concentration of ginkgolic acid against 7 plant anthrax fungi, including C. kahawae GULJ03, C. musae GUXJ, and C. truncatum GULJ01, was 0.38 mg·mL -1 ( Figure 2 ); observed by optical microscope ( Figure 3 , Figure 4 ), 0.38 mg·mL -1 Two days after the treatment of C. truncatum GULJ01 and C. musae GUXJ with ginkgolic acid, the hyphae showed abnormal shapes such as swelling, twisting and deformation. By treating the four plant anthrax fungi with different concentrations of ginkgolic acid, it was found that the hyphae weight was significantly reduced compared with the control ( Figure 5 ); The conidia germination experiment found that, compared with the control, different concentrations of ginkgolic acid had a certain inhibitory effect on the six plant anthrax fungi ( Figures 6-1 to 6-6 ); and had a certain effect on the cell membrane permeability and soluble protein of two plant anthrax fungi, C. truncatum GULJ01 and C. musae GUXJ ( Fig. 7A , 7B ); Through the detection of chitin content, it was found that the different concentrations of ginkgolic acid on C. truncatum GULJ01 and C. kahawae GULJ03 decreased sharply in the first 6 hours, and then tended to stabilize ( Fig. 8A , 8B ).

[0066] Table 2 Determination of the antibacterial activity against 5 different plant anthracnose fungi

[0067]

[0068] Example 2 Preparation of a Ginkgo Acid Microemulsion

[0069] 2.1 Experimental agents

[0070] Methanol, ethyl acetate, anhydrous ethanol, dimethylformamide, dimethyl sulfoxide, n-butanol, xylene, isopropanol, n-hexane, glycerol, n-propanol, acetonitrile, ether, acetone, calcium dodecylbenzenesulfonate (DBS-Ca), styrylphenol polyoxyethylene ether (Nongru 600#), castor oil polyoxyethylene ether (BY-110), alkylphenol polyoxyethylene ether (OP-7), alkylphenol polyoxyethylene ether (OP-9), alkylphenol polyoxyethylene ether (OP-10), alkylphenol polyoxyethylene ether (OP-15), phenethylphenol polyoxyethylene ether (601#), phenethylphenol polyoxyethylene ether (602#), phenethylphenol polyoxyethylene ether (603#), phenethylphenol polyoxyethylene polyoxypropylene ether (1602#), alcohol ether glycoside, trifluoroacetic acid, etc.

[0071] 2.2 Experimental instruments

[0072] Analytical balance; digital display constant temperature water bath (HH-6); CNC ultrasonic cleaner (JOYN-22600A); high-speed refrigerated centrifuge (D1524R).

[0073] 2.3 Microemulsion preparation method (10 mL system)

[0074] 2.3.1 Solvent screening

[0075] According to the property that ginkgolic acid is insoluble in water but soluble in organic solvents, methanol, ethyl acetate, anhydrous ethanol, dimethylformamide, dimethyl sulfoxide, n-butanol, xylene, isopropanol, n-hexane, glycerol, n-propanol, acetonitrile, ether and acetone were selected as alternative solvents. 0.3 g of ginkgolic acid was added to a 10 mL centrifuge tube, and the solvent was added in small amounts and multiple times until the ginkgolic acid in the tube was completely dissolved. At this time, the solution was clear and transparent.

[0076] 2.3.2 Microemulsion system and transparent temperature range screening

[0077] Microemulsions cannot lack nonionic or anionic surfactants for emulsification and stabilization of emulsions. Dodecylbenzenesulfonate calcium (DBS-Ca), styrylphenol polyoxyethylene ether (Agricultural milk 600#), castor oil polyoxyethylene ether (BY-110), alkylphenol polyoxyethylene ether (OP-7), alkylphenol polyoxyethylene ether (OP-9), alkylphenol polyoxyethylene ether (OP-10), alkylphenol polyoxyethylene ether (OP-15), phenethylphenol polyoxyethylene ether (601#), phenethylphenol polyoxyethylene ether (602#), phenethylphenol polyoxyethylene ether (603#), phenethylphenol polyoxyethylene polyoxypropylene ether (1602#), alcohol ether glycosides, etc. were used as alternative emulsifiers to prepare microemulsions containing more ginkgo acid active ingredients. According to the ratio requirements between the components of the microemulsion, the emulsifier should not exceed 20%. Weigh 0.3g of ginkgolic acid and dissolve it in a solvent, then add 0.5, 1.0, and 1.5g of DBS-Ca, and then add various non-ionic surfactants to mix. After stirring, observe that the whole system is clear and transparent. The clear and transparent system is further screened and its state at different temperatures is observed. Generally speaking, the wider the transparency range of the emulsion, the better.

[0078] 2.3.3 Cosolvent Screening

[0079] Cosolvents can reduce the amount of solvent used. Using low-cost cosolvents is beneficial to saving the cost of emulsion development. Several commonly used cosolvents, such as anhydrous ethanol, acetone, methanol, n-butanol, and isopropanol, were used to explore whether the same dissolution effect could be achieved. Weigh 0.3 g of ginkgolic acid into a centrifuge tube, add cosolvents at 100, 200, and 300 μL, and then add cosolvents in small amounts multiple times, with the total amount not exceeding the total amount of the original solvent used.

[0080] 2.3.4 Water quality screening

[0081] Water is an important component of microemulsions. Different water qualities will affect the microemulsion system. Therefore, the determination of water quality is also crucial in the research and development of microemulsions, and has important theoretical guidance for subsequent actual production. Deionized water, tap water, distilled water, and hard water are used for water quality screening. These four different water qualities are slowly added to the clear and transparent emulsion. The water that can still maintain clear and transparent emulsion in the end is selected for the preparation of the microemulsion.

[0082] 2.3.5 National Standard Test for Microemulsion

[0083] For stability determination, refer to "Method for Determination of Stability of Pesticide Emulsions" GB / T 1603-2001; for low temperature stability determination, refer to "Specification for the Preparation of Pesticide Emulsion Product Standards" HG / T 2467.2-2003; for hot storage stability determination, refer to "Specification for the Preparation of Pesticide Emulsion Product Standards" HG / T 2467.2-2003; for pH value determination, refer to "Method for Determination of Pesticide pH" GB / T 1601-93; for continuous foaming volume, refer to "Specification for the Preparation of Pesticide Product Standards" HG / T 2467.5-2003; for transparent temperature range, refer to "Specification for the Preparation of Pesticide Product Standards" HG / T 2467.10-2003.

[0084] The results show that: as shown in Table 3, ethyl acetate, anhydrous ethanol, n-hexane and acetone have the best solubility. Considering safety, economy and sustainability, anhydrous ethanol is selected as the main solvent of the ginkgo acid microemulsion. According to the results of Tables 3, 4, 5, 6 and 7, it is finally determined that 3% ginkgo acid, 1% ethanol, 1% n-butanol, 5% alkylphenol polyoxyethylene ether (OP10), 10% calcium dodecylbenzene sulfonate (DBS-Ca), 80% distilled water are used to prepare a 3% ginkgo acid microemulsion (such as Fig.12 The stability of 3% ginkgolic acid microemulsion was tested with reference to the GB / T 1603 method. As shown in Table 8, there was some precipitation when the 3% ginkgolic acid microemulsion was diluted 50 times, but no precipitation when it was diluted 100 times and 200 times. Fig. 9 As shown in Table 9, the transparent temperature range of 3% ginkgolic acid microemulsion is 0-81°C. The refrigerated and hot storage stability tests of 3% ginkgolic acid microemulsion were carried out, as shown in Table 9 and Table 10. The results are in line with the expected assumptions. The pH value of ginkgolic acid microemulsion was measured. The difference of the data of three parallel measurements was not greater than 0.1, so the pH value of 3% ginkgolic acid microemulsion was 5.79 ( Fig.10 The foaming volume of ginkgolic acid microemulsion was measured, and the foaming volume of 3% ginkgolic acid was 29.88mL / 180mL ( Fig.11 ).

[0085] Table 3 Screening results of solvent system

[0086]

[0087] Table 4 Screening test results of emulsifiers

[0088]

[0089]

[0090] Table 5 Temperature screening test results of emulsifiers

[0091]

[0092] Table 6 Screening of cosolvents

[0093]

[0094]

[0095] Table 7 Water quality screening of ginkgo acid microemulsion

[0096]

[0097] Table 8 Stability determination of ginkgo acid microemulsion

[0098]

[0099] Table 9 Physical properties of cold storage (0℃, 7d)

[0100]

[0101] Table 10 Physical properties of hot storage (54±2℃, 14d)

[0102]

[0103] Example 3 Application of Ginkgo acid microemulsion in preventing and controlling plant diseases

[0104] 3.1 Test materials

[0105] The pepper varieties tested were Guishu Jiamanfeng (registration number: GPD Pepper (2021) 520756); 3% original drug content of ginkgolic acid microemulsion, 20% dicyandiamide (Mingdelida Mingshun), and 1% osthole microemulsion (Yunnan Nanbao Biotechnology Co., Ltd.).

[0106] 3.2 Indoor protection test

[0107] (1) Pepper seedling cultivation

[0108] After sterilizing the seedling medium at high temperature, put it into the seedling pot, evenly sow the precious vegetables, keep the medium moist, and place it in a greenhouse (temperature 25℃, light-dark ratio 2:1, relative humidity 60%) for cultivation. Transplant it when it is a seedling, and wait until the seedling grows to the fruiting period for use;

[0109] (2) Preparation of anthrax spore suspension

[0110] The test strain GULJ01 was inoculated on a PDA plate and cultured at 28°C with 12 h light / 12 h dark for 7-14 days. The conidia were washed with sterile water and prepared with a hemocytometer to a concentration of 1×10 6 CFU / mL spore suspension is ready for use.

[0111] (3) Anthrax inoculation treatment

[0112] Use a 500mL small spray bottle to evenly inoculate the spore suspension onto healthy pepper fruits in the form of spray, and spray an equal amount of water as a blank control. The temperature is 28°C, the relative humidity is 60%, and the light / dark period is 12h. The symptoms of disease are observed every day. When sporadic lesions appear on the inoculated fruits, the next step of experimental treatment is carried out.

[0113] (4) Ginkgo acid treatment settings

[0114] When pepper plants enter the fruiting period, dilute the ginkgolic acid microemulsion at different times (3% ginkgolic acid microemulsion dilution multiples: 12.5, 25, 50, 100, 200 times) to the leaves and spray until water drips (apply once every 7 days in the early stage of the disease, apply once every 3 days during the peak period, and apply 3 times in succession). Spray an equal amount of clean water as a blank control, spray the botanical pesticide 1% osthole microemulsion (each 30 ml is diluted with 15 kg of water, and apply every 7 days in the early stage of the disease). 1 time, apply once every 3 days during the peak period, and apply it three times in succession) and 20% dicyandiamide·pyraclostrobin suspension chemical pesticide (each 15 grams is diluted with 15 kg of water, and the agent is sprayed immediately after the disease occurs, and it is applied once every 7-10 days, and sprayed twice in succession) as positive controls, 10 plants are treated each time, and repeated 3 times; the manufacturer of the above-mentioned 1% osthole microemulsion is Yunnan Nanbao Biotechnology Co., Ltd., and the manufacturer of 20% dicyandiamide·pyraclostrobin suspension is Mingdelida Mingshun.

[0115] (5) Disease investigation and control effect investigation

[0116] Disease investigation, classification standards and efficacy investigation calculation methods were carried out in accordance with the "Guidelines for Field Efficacy Tests of Pesticides (1)" (Compiled by the Pesticide Testing Laboratory of the Ministry of Agriculture and Rural Affairs of the People's Republic of China, 2000) and the "Guizhou Provincial Local Standard DB52 / T 1501.14-2020" Technical Specifications for Identification of Disease Resistance of Crops Part 14: Pepper Anthracnose Resistance. Data were analyzed for variance using Excel spreadsheets and the Duncan new multiple range method of the DPS7.05 data processing system.

[0117] Incidence rate (%) = (number of diseased plants / total number of plants investigated) × 100;

[0118] Disease index (DI) = ∑ (number of diseased plants at each level × disease level value) / [(total number of plants surveyed × highest disease level) × 100]

[0119] Relative prevention effect (%) = [(CK disease index - treatment disease index) / CK disease index] × 100

[0120] 3.3 Field plot test

[0121] The developed ginkgo acid microemulsion was tested for field efficacy in Jiulong Village, Yangchang Township, Pingba District, Anshun City. Pepper anthracnose was the main field control target, and pepper planting plots with uniform fertility and anthracnose in previous years were selected. The cultivation conditions of all test plots, such as soil type, fertilization, variety, and plant spacing, were consistent; 3 treatments were set, and 4 replicates were set for each treatment. There were 80 plants in each plot, and random block groups were used to keep the original conditions of each plot consistent as much as possible. The experiment had 3 treatments and 1 control.

[0122] The 3 treatments are as follows:

[0123] Treatment 1: Chemical agent, 20% dicyandiamide suspension (15g diluted with 15kg of water), pay close attention to the occurrence of diseases in the test field, and immediately spray the pepper field with the agent when anthrax occurs, once every 7-10 days, and use it twice in succession;

[0124] Treatment 2: Botanical pesticide, 1% osthole microemulsion (15 kg per 30 ml of water), pay close attention to the occurrence of diseases in the test field, and immediately spray the pesticide when anthrax occurs in the pepper field. Apply once every 7 days in the early stage of the disease, once every 3 days during the high-incidence period, and apply it three times in succession;

[0125] Treatment 3: 3% ginkgolic acid microemulsion, the treatment method is the same as treatment 2;

[0126] The blank control was based on no spraying of pesticides for preventing and controlling pepper leaf diseases and normal water and fertilizer management. Other field management measures were implemented in accordance with local production plans.

[0127] Disease investigation and prevention effectiveness statistics: refer to 3.2 for investigation and calculation.

[0128] The results show that: (1) Indoor protection test, from Table 11 and Fig.13 It can be seen that the best control effect of 3% ginkgolic acid microemulsion is 12.5 times diluted, followed by 50 times diluted, with relative control effects of 75.87% and 71.96% respectively, which is equivalent to 20% dicyandiamide suspension. (2) Field plot test, as shown in Table 12, after 3% ginkgolic acid microemulsion was diluted 50 times, the incidence rate was 3.03%, the disease index was 2.25, and the relative control effect was 58.95%.

[0129] Table 11 The control effect of ginkgo acid microemulsion on pepper anthracnose

[0130]

[0131] Table 12 Field control effectiveness test

[0132]

[0133] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A 3% ginkgolic acid microemulsion, characterized in that: The 3% ginkgolic acid microemulsion is prepared from the following raw materials in percentage by mass: 3% ginkgolic acid, 1% ethanol, 1% n-butanol, 5% OP10 (alkylphenol polyoxyethylene ether), 10% DBS-Ca (calcium dodecylbenzene sulfonate), and 80% distilled water.

2. The 3% ginkgolic acid microemulsion according to claim 1, characterized in that: The preparation method of the 3% ginkgolic acid microemulsion is as follows: ginkgolic acid is first dissolved in ethanol and n-butanol, then OP10 and DBS-Ca are added, and finally distilled water is added.

3. Use of the 3% ginkgolic acid microemulsion according to claim 1 or 2 in preventing and controlling plant anthracnose.

4. The use according to claim 3, characterized in that: The plant is at least one of coptis root, azalea, pomegranate, cyperus rotundus, pepper, kidney bean, banana, tobacco, sorghum, plum, bayberry, peanut, grapefruit, walnut and citrus.

5. A product for preventing and treating plant anthracnose, characterized in that: The product contains the 3% ginkgolic acid microemulsion described in claim 1.

6. A method for controlling plant anthracnose using the 3% ginkgolic acid microemulsion according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) diluting 3% ginkgolic acid microemulsion; (2) Spray the 3% ginkgolic acid microemulsion diluted in step (1) on the leaves of the plants.

7. The method according to claim 6, characterized in that The dilution multiple of the 3% ginkgolic acid microemulsion in step (1) is 12.5 or 50.

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