Preparation method of ginkgo oligosaccharide and ginkgo oligosaccharide sugar chain plant vaccine

Abstract

The invention relates to a preparation method of ginkgo oligosaccharide and a ginkgo oligosaccharide sugar chain plant vaccine, and relates to the field of plant vaccines. The preparation method of the ginkgo oligosaccharide comprises the step that the ginkgo oligosaccharide is extracted from ginkgo testa through an enhanced extraction method, the enhanced extraction method is an extraction method combining an ultrasonic extraction method and a water extraction and alcohol precipitation method, the purity of the oligosaccharide is improved, the structure of the oligosaccharide is not changed, and the cost is reduced. The ginkgo oligosaccharide is used for preparing the plant vaccine, and the vaccine takes the ginkgo oligosaccharide extracted from ginkgo testa as a main component, and has the function of inducing plants to generate stress resistance such as disease resistance, drought resistance, cold resistance and hot air resistance.

Description

Technical Field

[0001] This application relates to the technical field of plant vaccines, and particularly to a preparation method of ginkgo oligosaccharides and a ginkgo oligosaccharide sugar chain plant vaccine. Background Art

[0002] Ginkgo oligosaccharides are a type of bioactive oligosaccharides extracted from the outer seed coat of ginkgo. Their sugar groups mainly include glucose, fructose, and mannose, and the degree of polymerization is between 2 and 10. In recent years, studies have found that ginkgo oligosaccharides have broad application prospects in the field of plant protection. In particular, they can induce plants to produce stress resistance and enhance the plant's defense ability against pests and diseases. However, how to efficiently extract ginkgo oligosaccharides from the outer seed coat of ginkgo and prepare them into plant vaccines with practical application value has always been an urgent problem to be solved in this field.

[0003] Traditional oligosaccharide extraction methods often result in low oligosaccharide purity, and the operation process is cumbersome, time-consuming, and energy-consuming, which is not conducive to large-scale production. At the same time, the structure of oligosaccharides may be changed during the extraction process, thus affecting their biological activity. Therefore, developing a preparation method of ginkgo oligosaccharides with high purity, simple operation, low cost, and intact oligosaccharide structure is of great significance for promoting the application of ginkgo oligosaccharides in the field of plant protection. Summary of the Invention

[0004] The purpose of this application is to overcome the above technical problems and provide a preparation method of ginkgo oligosaccharides and a ginkgo oligosaccharide sugar chain plant vaccine.

[0005] In the first aspect, a preparation method of ginkgo oligosaccharides provided by this application adopts the following technical scheme: A preparation method of ginkgo oligosaccharides extracts ginkgo oligosaccharides by an enhanced extraction method, and the preparation steps are as follows: a. Crushing: Crush the outer seed coat of ginkgo into coarse powder with a diameter of 1 - 5 mm; b. Moistening: Moisten the coarse powder with water; c. Ultrasonic treatment: Use ultrasonic waves to break the cell wall; d. Water extraction: Add pure water according to the solid-liquid ratio of 1:(5 - 7), and extract at 90°C ± 5°C for 4 min - 60 min to obtain an extract; e. Filtration: Filter the extract with a 120 - 180 mesh filter screen to obtain a filtrate; f. Concentration: Evaporate and concentrate the filtrate at normal pressure at 90 - 100°C or concentrate the filtrate under vacuum at 50 - 60°C into a syrup-like state to obtain a concentrated solution; g. Alcohol precipitation: Add 95% - 100% ethanol to the concentrated solution and stir well; h. Precipitation: Let it stand for 1 - 2 h to fully precipitate the oligosaccharides, filter out the ethanol solution, and obtain ginkgo oligosaccharides.

[0006] By adopting the above technical solution, after ultrasonic treatment, ginkgo oligosaccharides are extracted by the method of water extraction and alcohol precipitation. Alcohol precipitation further reduces impurities and improves the purity of the ginkgo oligosaccharides required in this application. The prior art uses the method of alcohol extraction to extract substances from the outer seed coat of ginkgo, and the substances extracted are the substances discarded in this application's solution. Therefore, the method for extracting ginkgo oligosaccharides in this application is essentially different from the alcohol extraction method.

[0007] The ginkgo oligosaccharides extracted by the extraction method of this application have high purity, simple operation, low cost, and are conducive to large-scale production. At the same time, this preparation method does not change the structure of the oligosaccharides; it is of great significance for promoting the application of ginkgo oligosaccharides in the field of plant vaccines.

[0008] Preferably, in step c, the parameters for ultrasonic cell wall disruption are: using ultrasonic waves of 600 W for 15 - 25 min.

[0009] Preferably, when preparing ginkgo oligosaccharides, first use ultrasonic waves to disrupt the cell wall for about 20 min by the enhanced extraction method, and then extract with hot water at 90°C ± 5°C for 40 min.

[0010] Preferably, the sugar groups of ginkgo oligosaccharides include glucose, fructose, and mannose, the degree of polymerization is 2 - 20, and the purity of ginkgo oligosaccharides is 50% - 70%.

[0011] In the second aspect, a ginkgo oligosaccharide sugar chain plant vaccine provided by this application adopts the following technical solution: A ginkgo oligosaccharide sugar chain plant vaccine contains the following raw materials in weight ratio: 0.5% - 10% of the above-mentioned ginkgo oligosaccharides; 0.5% - 2.5% of a wetting agent; 0.5% - 10% of an antifreeze; 0.5% - 1.2% of a preservative; 0.5% - 1.2% of a thickening agent, and the solvent makes up 100%.

[0012] By adopting the above technical solution, the sugar chain plant vaccine acts through two channels: one is that the sugar chain plant vaccine inhibits the growth of plant pathogens and inactivates the infectivity of viruses. The other is that the vaccine binds to receptors on plant cells, stimulates the production of resistance signal molecules such as NO, H2O2, Ca 2+ , jasmonic acid, etc. Through signal transduction, it stimulates the expression of resistance genes and produces resistance substances such as phytoalexins, chitinase, phenylalanine ammonia-lyase, polyphenol oxidase, peroxidase, catalase, superoxide dismutase, β-1,3-glucanase, etc., so as to achieve the purpose of preventing pests and diseases.

[0013] Preferably, the sugar chain plant vaccine is one of an aqueous solution, a micro-suspension agent, or an emulsion; Preferably, the solvent is pure water, and the concentration of ginkgo oligosaccharides in the aqueous solution of the sugar chain plant vaccine is 2% - 10%.

[0014] Preferably, the antifreeze agent is one or more of propylene glycol, glycerol, and methanol.

[0015] Preferably, the wetting agent is alkylphenol polyoxyethylene ether or fatty alcohol polyoxyethylene ether.

[0016] Preferably, the thickening agent is xanthan gum.

[0017] In a third aspect, a preparation method of a ginkgo oligosaccharide sugar chain plant vaccine provided by the present application adopts the following technical solution: Take the above-mentioned ginkgo oligosaccharide and add auxiliaries to obtain the ginkgo oligosaccharide sugar chain plant vaccine.

[0018] In a fourth aspect, a ginkgo oligosaccharide sugar chain plant vaccine can be used to prevent and control strawberry root rot, leek phytophthora blight, wheat stripe rust, wheat powdery mildew, tomato phytophthora blight, cucumber downy mildew, strawberry gray mold, tobacco virus disease, tomato virus disease, pepper virus disease, cucumber root-knot nematode disease, watermelon root-knot nematode disease, loofah root-knot nematode disease, and potato root-knot nematode disease.

[0019] In summary, the present invention has at least the following beneficial effects: 1. The functions of the ginkgo oligosaccharide sugar chain plant vaccine not only induce plants to produce stress resistances such as disease resistance, drought resistance, cold resistance, and hot wind resistance, and directly inhibit the growth and reproduction of pathogenic bacteria, but also stimulate the growth of crops, increase crop yields, can be widely applied in agricultural production, improve the pest and disease resistance of plants, reduce the use of pesticides, and promote the development of ecological agriculture.

[0020] 2. The enhanced extraction method, that is, the extraction method combining ultrasonic extraction with the traditional water extraction and alcohol precipitation extraction method, takes the respective advantages of ultrasonic extraction and the traditional water extraction and alcohol precipitation extraction method, and makes up for the deficiencies of each other to create a new extraction method; the oligosaccharides extracted by the enhanced extraction method have high purity, do not change the oligosaccharide structure, save time and heat energy, and have low extraction costs. If the traditional water extraction and alcohol precipitation method is used alone, the extraction temperature is 90 °C, the extraction time is 3 - 4 h, and the purity is only about 30%. If ultrasonic extraction is used alone, the temperature is only 30 - 40 °C, the extraction time is 30 min, and the purity is only 10 - 20%. If the extraction time is more than 40 min, the purity can be increased to 30% - 40%, but the structure of some oligosaccharides is damaged. For the enhanced extraction method, first use ultrasonic waves to break the cell wall, so that the oligosaccharides are easily permeated into water, and then use hot water for extraction, and the purity can reach more than 50% without affecting the structure and composition of the oligosaccharides. Since the present invention uses the enhanced extraction method to prepare the ginkgo oligosaccharide sugar chain plant vaccine, it combines the advantages of ultrasonic extraction and water extraction and alcohol precipitation method, improves the purity of ginkgo oligosaccharides, and reduces the production cost.

[0021] 3. Ginkgo oligosaccharides are extracted from the discarded outer seed coat of Ginkgo biloba. The invention of the ginkgo oligosaccharide sugar chain plant vaccine turns the waste outer seed coat of Ginkgo biloba into a valuable resource. So far, the preparation of plant vaccines using ginkgo oligosaccharides is the first case in this invention. Description of the Drawings

[0022] Figure 1 It is the ion chromatogram of the mixed standard in the monosaccharide test of Preparation Example 1 of this application; Figure 2 It is the chromatogram of the test result of the liquid sample in the monosaccharide test of the ginkgo oligosaccharide prepared in Preparation Example 1 of this application; Figure 3 It is the chromatogram of the test result of the solid sample in the monosaccharide test of the ginkgo oligosaccharide prepared in Preparation Example 1 of this application. Detailed Embodiments

[0023] The following further elaborates on this application in combination with the preparation examples, examples and drawings: For those not specified in the following examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from ordinary commercial sources.

[0024] Preparation Example 1 Extract ginkgo oligosaccharides by the enhanced extraction method. The preparation steps are as follows: a. Crushing: Crush the outer seed coat of Ginkgo biloba into coarse powder with a diameter of 1 mm; b. Moistening: Put the coarse powder into the extraction tank equipped with an ultrasonic device and add water to moisten it; c. Ultrasonic disruption of cell walls: At 35 °C, use ultrasonic waves of 600 W for 15 min; d. Water extraction: Add pure water according to the solid-liquid ratio of 1:5 and extract at 90 °C for 4 min to obtain the extract; e. Filtration: Release the extract and filter it with a 180-mesh filter screen to obtain the filtrate; f. Concentration: Evaporate and concentrate the filtrate at normal pressure at 100 °C into a sugar paste to obtain the concentrated solution; g. Alcohol precipitation: Add 95% ethanol to the concentrated solution according to the weight ratio of ethanol to concentrated solution of 3:1 and stir well; h. Precipitation: Let it stand for 1 h to fully precipitate the oligosaccharides, filter out the ethanol solution to obtain ginkgo oligosaccharides.

[0025] Preparation Example 2 Extract ginkgo oligosaccharides by the enhanced extraction method. The preparation steps are as follows: a. Crushing: Crush the outer seed coat of Ginkgo biloba into coarse powder with a diameter of 3 mm; b. Moistening: Put the coarse powder into the extraction tank equipped with an ultrasonic device and add water to moisten it; c. Ultrasonic disruption of cell walls: At 35 °C, use ultrasonic waves of 600 W for 20 min; d. Water extraction: Add pure water according to a material-liquid ratio of 1:6, and extract at 90 °C for 40 min to obtain an extract. e. Filtration: Release the extract and filter it through a 150-mesh filter screen to obtain a filtrate. f. Concentration: Concentrate the filtrate under normal pressure at 95 °C into a syrup-like state to obtain a concentrated solution. g. Alcohol precipitation: Add 98% ethanol to the concentrated solution according to a weight ratio of ethanol to concentrated solution of 3:1, and stir well; h. Precipitation: Let it stand for 1.5 h to fully precipitate the oligosaccharides, filter out the ethanol solution to obtain ginkgo oligosaccharides.

[0026] Preparation Example 3 Extract ginkgo oligosaccharides by enhanced extraction method, and the preparation steps are as follows: a. Crushing: Crush the outer seed coat of ginkgo into coarse powder with a diameter of 5 mm. b. Moistening: Put the coarse powder into an extraction tank equipped with an ultrasonic device and add water to moisten it. c. Ultrasonic cell wall disruption: Use ultrasonic waves of 600 W at 35 °C for 25 min. d. Water extraction: Add pure water according to a material-liquid ratio of 1:7, and extract at 90 °C for 60 min to obtain an extract. e. Filtration: Release the extract and filter it through a 120-mesh filter screen to obtain a filtrate. f. Concentration: Evaporate and concentrate the filtrate under normal pressure at 90 °C into a syrup-like state to obtain a concentrated solution. g. Alcohol precipitation: Add 100% ethanol to the concentrated solution according to a weight ratio of ethanol to concentrated solution of 3:1, and stir well; h. Precipitation: Let it stand for 2 h to fully precipitate the oligosaccharides, filter out the ethanol solution to obtain ginkgo oligosaccharides.

[0027] Preparation Example 4 Extract ginkgo oligosaccharides by water extraction and alcohol precipitation method, and the preparation steps are as follows: a. Crushing: Crush the outer seed coat of ginkgo into coarse powder with a diameter of 3 mm. b. Moistening: Put the coarse powder into an extraction tank equipped with an ultrasonic device and add water to moisten it. c. Water extraction: Add pure water according to a material-liquid ratio of 1:6, and extract at 90 °C for 180 min to obtain an extract. d. Filtration: Release the extract and filter it through a 150-mesh filter screen to obtain a filtrate. e. Concentration: Concentrate the filtrate under vacuum at 55 °C into a syrup-like state to obtain a concentrated solution. f. Alcohol precipitation: Add 98% ethanol to the concentrated solution according to a weight ratio of ethanol to concentrated solution of 3:1, and stir well; g. Precipitation: Let it stand for 1.5 h to fully precipitate the oligosaccharides, filter out the ethanol solution to obtain ginkgo oligosaccharides.

[0028] Preparation Example 5 Extract ginkgo oligosaccharide by the method of water extraction and alcohol precipitation. The preparation steps are as follows: a. Crushing: Crush the outer seed coat of ginkgo into coarse powder with a diameter of 3 mm; b. Moistening: Put the coarse powder into the extraction tank equipped with an ultrasonic device and add water to moisten it; c. Water extraction: Add pure water according to the solid-liquid ratio of 1:6, and extract at 90 °C for 210 min to obtain an extract; d. Filtration: Release the extract and filter it through a 150-mesh filter screen to obtain a filtrate; e. Concentration: Concentrate the filtrate into a sugar paste at 95 °C to obtain a concentrated solution; f. Alcohol precipitation: Add 98% ethanol to the concentrated solution according to the weight ratio of ethanol to concentrated solution of 3:1, and stir well; g. Precipitation: Let it stand for 1.5 h to fully precipitate the oligosaccharide, filter out the ethanol solution to obtain ginkgo oligosaccharide.

[0029] Preparation Example 6 Extract ginkgo oligosaccharide by the method of water extraction and alcohol precipitation. The preparation steps are as follows: a. Crushing: Crush the outer seed coat of ginkgo into coarse powder with a diameter of 3 mm; b. Moistening: Put the coarse powder into the extraction tank equipped with an ultrasonic device and add water to moisten it; c. Water extraction: Add pure water according to the solid-liquid ratio of 1:6, and extract at 90 °C for 240 min to obtain an extract; d. Filtration: Release the extract and filter it through a 150-mesh filter screen to obtain a filtrate; e. Concentration: Concentrate the filtrate into a sugar paste at 95 °C to obtain a concentrated solution; f. Alcohol precipitation: Add 98% ethanol to the concentrated solution according to the weight ratio of ethanol to concentrated solution of 3:1, and stir well; g. Precipitation: Let it stand for 1.5 h to fully precipitate the oligosaccharide, filter out the ethanol solution to obtain ginkgo oligosaccharide.

[0030] Preparation Example 7 Extract ginkgo oligosaccharide by the ultrasonic treatment method. The preparation steps are as follows: a. Crushing: Crush the outer seed coat of ginkgo into coarse powder with a diameter of 3 mm; b. Moistening: Put the coarse powder into the extraction tank equipped with an ultrasonic device and add water to moisten it; c. Ultrasonic disruption of cell walls, at 35 °C, use ultrasonic waves of 600 W for 30 min to obtain ginkgo oligosaccharide.

[0031] Preparation Example 8 Extract ginkgo oligosaccharide by the ultrasonic treatment method. The preparation steps are as follows: a. Crushing: Crush the outer seed coat of ginkgo into coarse powder with a diameter of 3 mm; b. Moistening: Put the coarse powder into an extraction tank equipped with an ultrasonic device and add water for moistening; c. Ultrasonically break the cell wall, and under 35 °C, use 600 W ultrasonic wave for 45 min to obtain ginkgo oligosaccharide.

[0032] Performance detection test Test method: Use an ion chromatograph to measure the monosaccharide composition of the ginkgo oligosaccharide prepared in Preparation Example 1. The test method is as follows: 1. Prepare 6.5 mL of 50% NaOH solution by diluting 250 mM NaOH solution to 500 mL with water; prepare 1.3 mL of 50% NaOH solution and 20.5 g of NaOAc, and dilute to 500 mL with water to obtain 500 mM NaOH & 50 mM NaAc solution.

[0033] 2. Respectively take appropriate amounts of 16 kinds of monosaccharide standards, add 2 mL of 3 M TFA, hydrolyze at 120 °C for 3 h, dry with nitrogen blowing, add deionized water and vortex to mix evenly to prepare a standard stock solution. The monosaccharide standards are fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, D-galactosamine hydrochloride, D-glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid and mannuronic acid.

[0034] 3. Accurately weigh 5 mg of the sample and place it in an ampoule bottle, add 2 mL of 3 M TFA, and hydrolyze at 120 °C for 3 h. Accurately pipette the acid hydrolysis solution and transfer it to a tube, dry with nitrogen blowing, add 5 mL of water and vortex to mix evenly. Pipette 50 μL and add 950 μL of deionized water, centrifuge at 12000 rpm for 5 min. Take the supernatant for IC analysis.

[0035] 4. Chromatographic method: Chromatographic column: Dionex Carbopac TMPA20 (3 * 150 mm); Mobile phase: A: H2O; B: 250 mM NaOH; C: 500 mM NaOH & 50 mM NaAc; Flow rate: 0.3 mL / min; Injection volume: 25 μL; Column temperature: 30 °C; Elution gradient: 0 min, A phase / B phase / C phase (98:2:0, V / V), 23 min, A phase / B phase / C phase (98:2:0, V / V), 23.1 min, A phase / B phase / C phase (80:20:0, V / V), 33 min, A phase / B phase / C phase (80:20:0, V / V), 33.1 min, A phase / B phase / C phase (80:0:20, V / V), 46 min, A phase / B phase / C phase (80:0:20, V / V), 46.1 min, A phase / B phase / C phase (20:0:80, V / V), 66 min, A phase / B phase / C phase (20:0:80, V / V), 66.1 min, A phase / B phase / C phase (98:2:0, V / V), 80 min, A phase / B phase / C phase (98:2:0, V / V). Detector: Electrochemical detector.

[0036] The standard sample sequence refers to Table 1.

[0037] Table 1 Standard sample sequence table The ion chromatogram of the mixed standard can be seen in Figure 1 , and the test results of liquid ginkgo oligosaccharides refer to Figure 2 and Table 2, and the test results of solid ginkgo oligosaccharides refer to Figure 3 and Table 3, where the solvent peaks: the peak of sodium hydroxide is at 2.0 min, and the peak of sodium acetate is at 40.5 min.

[0038] Table 2 Monosaccharide test results of the liquid sample of ginkgo oligosaccharides prepared in Preparation Example 1 Name Peak area RT Molar ratio ug / mg Fucose 0 5.775 0.000 0.000 D-Galactosamine hydrochloride 0 10.675 0.000 0.000 Rhamnose 0 11.234 0.000 0.000 Arabinose 0 11.6 0.000 0.000 D-Glucosamine hydrochloride 0 13.334 0.000 0.000 Galactose 0 14.459 0.000 0.000 Glucose 63.239 16.784 0.552 294.903 Xylose 0 19.375 0.000 0.000 Mannose 0.416 20.675 0.006 3.412 Fructose 0.624 23.467 0.441 235.768 Ribose 0 25.009 0.000 0.000 Galacturonic acid 0 45.35 0.000 0.000 Guluronic acid 0 46.009 0.000 0.000 Glucuronic acid 0 48.625 0.000 0.000 Mannuronic acid 0 51.109 0.000 0.000 Table 3 Monosaccharide test results of the solid sample of ginkgo oligosaccharides prepared in Preparation Example 1 Name Peak area RT Molar ratio ug / mg Fucose 0 5.775 0.000 0.000 D-Galactosamine hydrochloride 0 10.675 0.000 0.000 Rhamnose 0 11.234 0.000 0.000 Arabinose 0 11.6 0.000 0.000 D-Glucosamine hydrochloride 0 13.334 0.000 0.000 Galactose 0 14.459 0.000 0.000 Glucose 63.239 16.784 0.552 294.903 Xylose 0 19.375 0.000 0.000 Mannose 0.416 20.675 0.006 3.412 Fructose 0.624 23.467 0.441 235.768 Ribose 0 25.009 0.000 0.000 Galacturonic acid 0 45.35 0.000 0.000 Guluronic acid 0 46.009 0.000 0.000 Glucuronic acid 0 48.625 0.000 0.000 Mannuronic acid 0 51.109 0.000 0.000 Refer to Figure 1 , Figure 2 As can be seen from

[0039] and Tables 2 and 3, the ginkgo oligosaccharides extracted by the method of this application contain ginkgo oligosaccharides, and the glycosyl groups of the ginkgo oligosaccharides are glucose, mannose, and fructose.

[0040] Table 3 Ginkgo oligosaccharide purity table of Preparation Examples Combined with Preparation Example 1, Preparation Example 2, and Preparation Example 3 and Table 3, the enhanced extraction method of the present application was adopted to adjust the process parameters to improve the purity of ginkgo oligosaccharides.

[0041] Combined with Preparation Example 2 and Preparation Example 4, in Preparation Example 2, the enhanced extraction method was used, and the water extraction time was 40 min; in Preparation Example 4, only the water extraction and alcohol precipitation method was used, and the water extraction time was 180 min. It can be seen from Table 3 that even if the water extraction time is increased by only using the water extraction and alcohol precipitation method, the purity of ginkgo oligosaccharides is still much lower than that of ginkgo oligosaccharides extracted by the enhanced extraction method.

[0042] Combined with Preparation Example 4 to Preparation Example 6 and Table 3, it can be seen that by using the water extraction and alcohol precipitation method and increasing the water extraction time, the purity of ginkgo oligosaccharides increases, but the purity of ginkgo oligosaccharides obtained by water extraction for 240 min is 32.1%, which is much lower than the purity of ginkgo oligosaccharides obtained by ultrasonic treatment for 40 min + water extraction for 40 min.

[0043] Combined with Preparation Example 2 and Preparation Example 7 and Table 3, it can be seen that in Preparation Example 7, only the ultrasonic treatment method was used for 30 min, and the obtained ginkgo oligosaccharides were in paste form, and the purity of ginkgo oligosaccharides was less than 15%, which was much lower than the purity of ginkgo oligosaccharides prepared by the enhanced extraction method used in Preparation Example 2 of the present application.

[0044] Combined with Preparation Example 7 and Preparation Example 8 and Table 3, it can be seen that without combining the water extraction and alcohol precipitation method and only increasing the ultrasonic treatment time, although the purity of ginkgo oligosaccharides prepared in Preparation Example 8 increased, further experiments proved that the structures of some oligosaccharides were damaged.

[0045] Example 1 This example discloses a ginkgo oligosaccharide sugar chain plant vaccine, and the specific preparation steps are as follows: Take 0.5 kg of ginkgo oligosaccharides prepared in Preparation Example 2, 0.5 kg of wetting agent, 5 kg of antifreeze, 1.2 kg of preservative, 1.2 kg of thickener, and 91.6 kg of pure water and mix them evenly to obtain the ginkgo oligosaccharide sugar chain plant vaccine. The antifreeze is propylene glycol; the wetting agent is alkylphenol polyoxyethylene ether; the preservative is the sorbic acid used as an alcohol preservative in this example; the thickener is xanthan gum.

[0046] Example 2 This example discloses a ginkgo oligosaccharide sugar chain plant vaccine, and the specific preparation steps are as follows: Take 2 kg of the ginkgo oligosaccharide prepared in Preparation Example 2, 1 kg of wetting agent, 2.75 kg of antifreeze, 1 kg of preservative, 1 lg of thickening agent and 92.25 kg of pure water, mix them evenly to obtain the ginkgo oligosaccharide sugar chain plant vaccine. The antifreeze is a mixture of glycerol and propylene glycol mixed in a ratio of 1:1 (by weight); the wetting agent is alkylphenol polyoxyethylene ether; the preservative is an alcohol preservative, and in this example, benfluorethanol is used; the thickening agent is xanthan gum.

[0047] Example 3 This example discloses a ginkgo oligosaccharide sugar chain plant vaccine, and the specific preparation steps are as follows: Take 10 kg of the ginkgo oligosaccharide prepared in Preparation Example 2, 1.5 kg of wetting agent, 0.5 kg of antifreeze, 0.8 kg of preservative, 0.5 kg of thickening agent and 86.7 kg of pure water, mix them evenly to obtain the ginkgo oligosaccharide sugar chain plant vaccine. The antifreeze is a mixture of glycerol and propylene glycol mixed in a ratio of 1:1 (by weight); the wetting agent is fatty alcohol polyoxyethylene ether; the preservative is an alcohol preservative, and in this example, benzoic acid is used; the thickening agent is xanthan gum.

[0048] Example 4 Take 2 kg of the ginkgo oligosaccharide prepared in Preparation Example 2, 2.5 kg of wetting agent, 10 kg of antifreeze, 1 kg of preservative, 0.8 kg of thickening agent and 83.7 kg of essential oil, mix them evenly to obtain the ginkgo oligosaccharide sugar chain plant vaccine. The antifreeze is a mixture of glycerol and propylene glycol mixed in a ratio of 1:1 (by weight); the wetting agent is alkylphenol polyoxyethylene ether; the preservative is an alcohol preservative, and in this example, benfluorethanol is used; the thickening agent is xanthan gum.

[0049] Comparative Example 1 The difference from Example 2 is that the ginkgo oligosaccharide prepared in Preparation Example 5 is used.

[0050] Comparative Example 2 The difference from Example 2 is that the ginkgo oligosaccharide prepared in Preparation Example 7 is used.

[0051] Performance Detection Test Test 1: Strawberry Root Rot Potted Plant Experiment 1. Preparation stage: Take the soil from the areas where strawberry root rot occurs frequently and severely, put it into a sterile self-sealing bag and bring it back to the laboratory.

[0052] Prepare 140 flower pots; prepare 140 healthy strawberry seedlings.

[0053] Use the ginkgo oligosaccharide sugar chain plant vaccine prepared in the examples at a dosage of 20 kg / mu, and dilute the ginkgo oligosaccharide sugar chain plant vaccine for later use.

[0054] 2. Test stage: (1) The transplanting soil is prepared by mixing leaf mold, the brought-back soil, and river sand according to a weight ratio of 5:3:2, and the transplanting soil is separately placed into 140 flower pots.

[0055] (2) The strawberry seedlings are implanted into the flower pots, with 1 strawberry seedling implanted in each flower pot, and the voids around the roots are filled with the transplanting soil. The transplanting soil is gently compacted to make the strawberry seedlings in close contact with the soil.

[0056] (3) After transplantation, the 280 pots of strawberry seedlings are randomly divided into 7 groups, with 40 pots in each group. (4) For the first root irrigation, the diluted ginkgo oligosaccharide sugar chain plant vaccine is irrigated onto the strawberry seedlings using a sprayer or a watering can. Each example corresponds to 1 group of strawberry seedlings, that is, each example corresponds to 40 pots of strawberry seedlings. Note that when irrigating the roots, it should be watered slowly and carefully to avoid the water flow impacting the roots of the strawberry seedlings. Two control groups are set up: Control group 1: The conventional combined medicine for strawberry root rot is used, that is, 500-fold solution of 58% metalaxyl-mancozeb wettable powder + 1500-fold solution of 50% iprodione wettable powder + brassinolide; the dosage and irrigation time are the same as those in the examples / comparative examples.

[0057] Control group 2: The roots are irrigated with an equal amount of clear water. (5) For the second root irrigation, one week after transplantation, the surviving seedlings are irrigated once, and the medicament / water for irrigation is the same as the first time. (6) Observe the strawberry seedlings after transplantation. When the strawberry seedlings show that the leaf margins turn yellow and brown, gradually developing towards the heart leaves, and the margins of the old leaves turn purplish red or purple brown, pull out the strawberry seedlings and observe whether the center of the root system at the roots is reddish brown or black brown, rotten, and the newly grown roots are sparse. If the above situations are encountered, it is counted as a diseased seedling; observe until the strawberry harvest period. (7) Calculate the survival rate (%) at the strawberry harvest period, the incidence rate accumulated until 1 month after strawberry transplantation, which is recorded as the short-term incidence rate (%), and the incidence rate accumulated until the strawberry maturity period, which is recorded as the long-term incidence rate (%): Survival rate (%) = number of surviving seedlings / total number of seedlings × 100% Short-term incidence rate (%) = number of diseased seedlings accumulated until 1 month after transplantation / number of surviving seedlings 1 month after transplantation × 100% Long-term incidence rate (%) = number of diseased seedlings accumulated until maturity / total number of surviving seedlings × 100% The test results are referred to Table 4.

[0058] Table 4 Test results table of strawberry root rot Combined with Example 1, Example 2, Example 3 and Table 4, by adjusting the proportions of ginkgo oligosaccharides, wetting agents, antifreeze agents and pure water, as well as the types of wetting agents and antifreeze agents, the optimal sugar chain plant vaccine formulation for strawberry root rot was obtained.

[0059] Combined with Example 2, Comparative Example 1 and Comparative Example 2, the difference among the three lies in the types of ginkgo oligosaccharides used. Example 2 uses the ginkgo oligosaccharides prepared in Preparation Example 2, and its extraction process is the enhanced extraction method with a purity of 70.4%; Example 4 uses the ginkgo oligosaccharides prepared in Preparation Example 5, and its extraction process is the water extraction and alcohol precipitation method with a purity of 29.8%; Example 5 uses the ginkgo oligosaccharides prepared in Preparation Example 7, and its extraction process is the ultrasonic method with a purity of 14.5%. Combined with Table 4, the sugar chain plant vaccine prepared in Example 2 has outstanding effects on the survival rate of strawberries and the inhibition of strawberry disease incidence.

[0060] Combined with Example 2 and Control Group 1, Control Group 1 uses a conventional pesticide composition to treat strawberry root rot. It can be seen that the conventional pesticide composition has excellent short-term effects when inhibiting strawberry root rot; but in terms of long-term effects, the ginkgo oligosaccharide sugar chain plant vaccine prepared in this application has better control efficacy. In promoting the survival rate of strawberries, the conventional pesticide composition is much lower than the ginkgo oligosaccharide sugar chain plant vaccine prepared in this application.

[0061] Combined with Example 2 and Control Group 2, Control Group 2 uses water irrigation for root watering. It can be seen from Table 4 that the ginkgo oligosaccharide sugar chain plant vaccine prepared in this application effectively inhibits strawberry root rot and improves the survival rate of strawberry transplantation.

[0062] Experiment 2: Field experiment on Chinese chive phytophthora blight 1. Preparation stage: Select a test field with deep and loose soil layers, rich in organic matter, strong water retention capacity, and fallow for more than half a year.

[0063] Select Chinese chive roots that have grown for about 2 years, prune the Chinese chive roots, cut the long root hairs, generally leaving about 4 - 5 cm of old roots, remove weak roots, aging roots, and diseased roots, and retain healthy roots.

[0064] Use 200 ml / mu of the ginkgo oligosaccharide sugar chain plant vaccine prepared in the example, and dilute the ginkgo oligosaccharide sugar chain plant vaccine for later use.

[0065] 2. Test stage (1) Water the test field to moisten the soil, level it and make ridges. Apply 4000 kg of decomposed organic fertilizer, 10 kg of urea, and 40 kg of potassium sulfate compound fertilizer per mu of land; (2) Plot design Each plot is 3m × 3m, and 3 plots are used for each example. Set two control groups: Control group 1: Use the conventional 52.5% Yikuaijing diluted 2000 times, with a dosage of 200 ml / mu. The root planting process and spraying time are the same as those in the examples / comparative examples. Control group 2: Spray with an equal amount of clear water. There are a total of 24 plots in the examples, comparative examples, and control groups. An isolation belt is set between adjacent plots, and adjacent plots use different vaccines / pesticides / clear water in the examples / control groups. (3) On October 15, dig small holes for transplanting, with a plant spacing of 4 cm and a row spacing of 10 cm. (4) After root planting, spray once with the corresponding pesticide / clear water in the examples / control groups; after germination, spray once with the corresponding pesticide / clear water in the examples / control groups on the surviving seedlings; during this period, take care in a unified manner according to the conventional method. (5) Refer to the five-point sampling method in field trials. Investigate 12 plants at each point in each plot, count the number of Chinese chive plants in each plot, and calculate the total number of plants surveyed by combining the plots that spray the same vaccine / pesticide / clear water. Observe the leaf surface of Chinese chives at the sampling points after root planting, once every 7 days until the Chinese chives reach the five-leaf stage.

[0066] Observation content: For Chinese chive leaves, the leaves are small and curved, showing dark green water-soaked lesions, and the lesion area accounts for more than 50% of the entire leaf area. The leaves turn yellow, droop, and rot. Combine the plots that spray the same vaccine / pesticide / clear water and count them as diseased plants. Note: Pull out the diseased Chinese chives in time to avoid double counting. (6) Calculate the control effect (%) of Chinese chive phytophthora blight: Diseased plant rate (%) = number of diseased plants / total number of plants surveyed × 100% Control of Chinese chive phytophthora blight (%) = (diseased plant rate of control group 2 - diseased plant rate of treatment area) / (diseased plant rate of control group 2) × 100% Treatment area: The area where the examples / comparative examples / control group 1 apply pesticides The test results are shown in Table 5.

[0067] Table 5 Test results of Chinese chive phytophthora blight Disease incidence rate / % Control effect of Chinese chive Phytophthora blight / % Example 1 11.25 84.30 Example 2 7.08 90.12 Example 3 10.00 86.05 Example 4 9.17 87.21 Comparative example 1 24.17 66.28 Comparative example 2 21.67 69.77 Control group 1 8.75 87.79 Control group 2 71.67 / Combined with Example 1, Example 2, and Example 3 and Table 4, by adjusting the proportions of ginkgo oligosaccharide, wetting agent, antifreeze agent, and pure water, as well as the types of wetting agent and antifreeze agent, the optimal sugar chain plant vaccine formula for Chinese chive phytophthora blight is obtained.

[0068] Combined with Example 2, Comparative Example 1 and Comparative Example 2, the difference among the three lies in the different types of ginkgo oligosaccharides used. In Example 2, the ginkgo oligosaccharides prepared in Preparation Example 2 were used, and its extraction process was the enhanced extraction method, with a purity of 70.4%; in Comparative Example 1, the ginkgo oligosaccharides prepared in Preparation Example 5 were used, and its extraction process was the water extraction and alcohol precipitation method, with a purity of 29.8%; in Comparative Example 2, the ginkgo oligosaccharides prepared in Preparation Example 7 were used, and its extraction process was the ultrasonic method, with a purity of 14.5%. Combined with Table 4, the sugar chain plant vaccine prepared in Example 2 had a better control effect on Phytophthora blight of leeks.

[0069] Combined with Example 2 and Control Group 1, Control Group 1 used a conventional pesticide composition to treat Phytophthora blight of leeks. During the experiment, it was found that in the first 3 observations, the control efficacy of the conventional pesticide composition against Phytophthora blight of leeks could reach 93%, and the effect was better than that of the ginkgo oligosaccharide sugar chain vaccine prepared in this application. However, as time went by, the control efficacy of Control Group 1 began to weaken. Eventually, from root planting to the 5-leaf stage, the cumulative control efficacy of the ginkgo oligosaccharide sugar chain vaccine prepared in this application was better than that of the conventional pesticide composition.

[0070] Combined with Example 2 and Control Group 2, Control Group 2 used water spraying. It can be seen from Table 4 that the sugar chain plant vaccine prepared in this application had a significant control effect on Phytophthora blight of leeks.

[0071] Experiment 3: Field experiment on wheat stripe rust and powdery mildew 1. Preparation stage: Select the experimental field. The experimental field is arranged in Shanghe County, Jinan City, Shandong Province, a severely affected area of wheat stripe rust and powdery mildew. The terrain is flat, the soil properties are the same, the fertility is uniform, and it conforms to local scientific agricultural practices (GAP). If irrigation is required, record the irrigation method, time and water volume.

[0072] The dosage of the ginkgo oligosaccharide sugar chain plant vaccine prepared in the example was 300 ml / mu, and the ginkgo oligosaccharide sugar chain plant vaccine was diluted for later use.

[0073] 2. Experimental stage (1) Level the experimental field. The wheat variety is Yannong 1212. The machine plows to a depth of 25 cm. The bottom of the ditch should be flat, without missing plowing, leaving no clods and hard-packed places, ensuring that the soil is loose, fine and broken. The seeding quantity is 3.6×106 plants / hm 2 ; (2) Plot arrangement Each plot is 10 m×10 m. Each example uses 3 plots, and two control groups are set: Control Group 1, using a conventional 5% hexaconazole suspension at 40 ml / mu. Except for the different spraying drugs, all other operations are the same as those in the example / comparative example; Control Group 2, using equal amounts of water for spraying; There were a total of 24 plots in the examples, comparative examples, and control groups. An isolation row of 0.5 m was set between adjacent plots. Different vaccines / pesticides / water were used in adjacent plots for the examples / control groups / control groups. Protection rows were set around the experimental field, and the field management of each plot was the same; (3) Spraying was divided into two times. The first time was during the wheat green-reverting stage, and the second time was on the 10th day after the first spraying. During this period, the field was managed uniformly in the conventional manner; (4) Referring to the field experiment, the five-point sampling method was used. 25 wheat seedlings were investigated at each sampling point, and the number of wheat seedlings in each plot was counted. The plots sprayed with the same vaccine / pesticide / water were combined and calculated as the total number of investigated plants; Observation of the wheat situation at the sampling points started from the emergence stage after the snow melted in early spring, and it was observed once every 7 days until the wheat matured.

[0074] Observation content: Many bright yellow oval uredinia were formed on the front of the wheat leaves, arranged longitudinally along the leaf veins in a dotted line shape, and often several were combined and grew in patches. A large amount of bright yellow powder was produced in the uredinia; when approaching maturity, short black telia were produced on the leaf sheaths and leaves, buried under the epidermis. After diagnosis, they were counted as wheat plants infected with stripe rust. Note: The plots sprayed with the same vaccine / pesticide / water were combined and calculated. The wheat plants counted as diseased plants were pulled out in time to avoid double counting; Yellow spots initially appeared on the wheat leaves, gradually expanding into round or oval lesions, with a powdery mildew layer on the surface; the mildew layer gradually turned grayish white and finally light brown, with many black dots on it; when there were many lesions, they could merge into patches; after the wheat stems and leaf sheaths were damaged, the plants were prone to lodging, and severely diseased plants usually dwarfed and did not head. After diagnosis, they were counted as wheat plants infected with stripe rust. Note: The plots sprayed with the same vaccine / pesticide / water were combined and calculated. The wheat plants counted as diseased plants were pulled out in time to avoid double counting; (6) Calculate the control efficacy (%): Diseased plant rate (%) = number of diseased plants / total number of investigated plants × 100% Control (%) = (diseased plant rate of control group 2 - diseased plant rate of treatment area) / (diseased plant rate of control group 2) × 100% Treatment area: the area where the examples / comparative examples / control group 1 were sprayed with drugs The test results are shown in Table 6 for reference.

[0075] Table 6 Results of wheat field experiment Combined with Example 1, Example 2, Example 3, and Table 4, by adjusting the proportions of ginkgo oligosaccharide, wetting agent, antifreeze agent, and pure water, as well as the types of wetting agent and antifreeze agent, the optimal sugar-chain plant vaccine formula for wheat stripe rust and powdery mildew was obtained.

[0076] Combined with Example 2, Comparative Example 1 and Comparative Example 2, the difference among the three lies in the types of ginkgo oligosaccharides used. In Example 2, the ginkgo oligosaccharides prepared in Preparation Example 2 were used, and its extraction process was the enhanced extraction method, with a purity of 70.4%; in Comparative Example 1, the ginkgo oligosaccharides prepared in Preparation Example 5 were used, and its extraction process was the water extraction and alcohol precipitation method, with a purity of 29.8%; in Comparative Example 2, the ginkgo oligosaccharides prepared in Preparation Example 7 were used, and its extraction process was the ultrasonic method, with a purity of 14.5%. Combined with Table 4, the sugar chain plant vaccine prepared in Example 2 had better control effects on wheat stripe rust and powdery mildew.

[0077] Combined with Example 2 and Control Group 1, Control Group 1 used a conventional pesticide composition to treat wheat stripe rust and powdery mildew. During the experiment, it was found that in the first 3 observations, the control efficacy of the conventional pesticide composition against wheat stripe rust reached 94%, and the control efficacy of the conventional pesticide composition against wheat powdery mildew reached 92%, with an effect better than the ginkgo oligosaccharide sugar chain vaccine prepared in this application. However, as time passed, the control effect of Control Group 1 began to weaken. Eventually, from emergence to wheat maturity, the cumulative control effect of the ginkgo oligosaccharide sugar chain vaccine prepared in this application was better than that of the conventional pesticide composition.

[0078] Combined with Example 2 and Control Group 2, Control Group 2 used water spraying. It can be seen from Table 4 that the sugar chain plant vaccine of ginkgo oligosaccharides prepared in this application had a significant control effect on wheat stripe rust and powdery mildew.

[0079] Experiment 4: Field experiment on tomato virus disease 1. Preparation stage: Select the experimental field, the Modern Agricultural Industry Science and Technology Demonstration Park in Bole City, Shouguang County, Shandong Province, with medium soil fertility, loam soil, and tomatoes have been planted for many years.

[0080] Experimental basis: National agricultural industry standard, NY / T 1464.8 - 2007, "Pesticide field efficacy test guidelines Part 8: Fungicides for controlling tomato virus disease".

[0081] 2. Experimental stage: (1) Tested tomato variety: Provence (2) Plot design The tested pesticides are shown in Table 7, where Control Group 1 is 1% aqueous lentinan solution and Control Group 2 is water.

[0082] Table 7 Test pesticide dosage table Dosage of active ingredient (g / hectare) Dosage of formulation (mL / mu) Example 2-1 30 100 Example 2-2 36 120 Example 2-3 45 150 Example 4 36 120 Comparative example 1 36 120 Comparative example 2 36 120 Control group 1 30 200 Control group 2 / / Plot arrangement: Plot area 9m × 3.6m = 32.4m 2 , The experiment was set with 8 treatments, 4 plots for each treatment, and the plots were arranged in a randomized block design, with a total of 32 plots.

[0083] Table 8 Plot queuing table (3) Application method: Apply the medicine before the onset of tomato virus disease. Weigh the medicine according to the dosage required for each treatment, add water, and spray it conventionally. It is required that the spraying is uniform. The control group 2 sprays clear water, which is sprayed simultaneously with the medicine.

[0084] This experiment was carried out with 2 applications of medicine. The first application was on October 24, 2024; the second application was on November 3, 2024.

[0085] (4) Application volume: The liquid medicine volume is 50 L / mu.

[0086] (5) Investigation method, time, and frequency Refer to the relevant regulations of the national agricultural industry standard NY / T 1464.8 - 2007, "Rules for field efficacy trials of pesticides - Part 8: Fungicides for controlling tomato virus disease".

[0087] The first investigation: Conduct an investigation before applying the medicine on October 24, 2024. There are no diseased plants in the field, and the disease index is 0.

[0088] The second investigation: On November 13, 2024, that is, 10 days after the second application of medicine, investigate the control effect; record the disease occurrence according to the grading standard.

[0089] (6) Investigation method Randomly sample 5 points in each plot, investigate 6 plants at each point. If the number of plants in the protected plot is less than 30, the whole plot should be investigated. Record the total number of investigated plants and the number of diseased plants at each level in units of plants.

[0090] Grading method (in units of plants) Grade 0: No symptoms.

[0091] Grade 1: Clear veins in the young leaves, mild mosaic.

[0092] Grade 3: Mosaic in the young leaves and middle leaves.

[0093] Grade 5: Mosaic in the young leaves and middle leaves, a few leaves are deformed, wrinkled or the plant is slightly dwarfed.

[0094] Grade 7: Severe mosaic, most leaves are deformed, wrinkled or the plant is dwarfed.

[0095] Grade 9: Severe mosaic, the leaves are obviously deformed, linear, the plant is severely dwarfed or even dead.

[0096] (7) Calculation method of control effect The calculation method and formula of the control effect are as follows: Calculate the disease index and control effect of each plot, calculate the average control effect of each treatment, and use the Duncan's multiple range test method in SPSS17.0 software to analyze the significance of differences between treatments.

[0097] Disease index (%) = ∑(number of diseased plants at each level × relative level value) / total number of plants surveyed × 9 × 100% Control effect (%) = (disease index in control area - disease index in treatment area) / disease index in control area × 100% One day, two days, and three days after pesticide application, observe whether the pesticide causes phytotoxicity to tomato plants.

[0098] During the efficacy investigation, simultaneously investigate whether the pesticide has an impact on non-target organisms (including beneficial insects) and surrounding crops. The results of the control effect investigation are shown in Table 9.

[0099] Table 9 Field efficacy test results of controlling tomato virus disease Combined with Example 2-1, Example 2-2, and Example 2-3 and Table 9, the dosages of the active ingredients of the ginkgo oligosaccharide aqueous solution prepared in this application are 30 g / ha, 36 g / ha, and 45 g / ha; the control effects are 53.02%, 60.49%, and 68.37% respectively. However, for the medicament 1% lentinan aqueous solution used in Control 1, the active ingredient is 30 g / ha and the control effect is 59.10%. It can be seen that under the same content of active ingredients, the control effect of the ginkgo oligosaccharide aqueous solution prepared in this application on tomato virus disease is better than that of the lentinan aqueous solution.

[0100] Combined with Example 2-1, Example 2-2, Example 2-3, and Table 9, analysis using the Duncan's new multiple range method shows that: at the 0.05 level, there is no significant difference in the control effect between the high concentration and the medium concentration of the ginkgo oligosaccharide sugar chain vaccine aqueous solution prepared in this application and that of the control medicament; there is a significant difference in the control effect between the high concentration and the low concentration; there is no significant difference in the control effect between the medium concentration of the ginkgo oligosaccharide sugar chain vaccine aqueous solution prepared in this application and that of other treatments; there is no significant difference in the control effect between the low concentration of the ginkgo oligosaccharide sugar chain vaccine aqueous solution prepared in this application and that of the control medicament. At the 0.01 level, there is no significant difference in the control effect among the three concentrations of the ginkgo oligosaccharide sugar chain vaccine aqueous solution prepared in this application; and there is also no significant difference in the control effect compared with that of the control medicament. According to the market price, the price of the 1% lentinan aqueous solution is about 50 yuan / kg, and the sugar chain plant vaccine involved in this application is sold at about 18 yuan / kg, with a relatively high cost performance.

[0101] The ginkgo oligosaccharide sugar chain vaccine prepared in the example is safe and has no phytotoxicity to crops at three concentrations of 30 g / ha, 36 g / ha, and 45 g / ha for the dosage of the active ingredient, and has no impact on non-target organisms (including beneficial insects) and surrounding crops.

[0102] Experiment Five: Field experiment on pepper virus disease 1. Preparation stage: Select the experimental field, the Modern Agricultural Industry Science and Technology Demonstration Park in Bole City, Shouguang County, Shandong Province. The soil fertility is medium, loam, and peppers have been planted for many years.

[0103] Test basis: National agricultural industry standard, NY / T 1464.9 - 2007, "Guidelines for field efficacy trials of pesticides - Part 9: Fungicides for controlling pepper virus disease".

[0104] 2. Test stage: (1) Tested varieties: Screw peppers (2) Plot design The test agents are shown in Table 10. Among them, Control Group 1 is 5% amino - oligosaccharide aqueous solution, and Control Group 2 is clear water.

[0105] Table 10 Test agent dosage table Dosage of active ingredient (g / hectare) Dosage of formulation (mL / mu) Example 2-1 30 100 Example 2-2 36 120 Example 2-3 45 150 Example 4 36 120 Comparative example 1 36 120 Comparative example 2 36 120 Control group 1 75 100 Control group 2 / / Plot arrangement: The plot area is 9m×3.6m = 32.4m 2 , The test has a total of 8 treatments, 2 plots for each treatment. The plots are arranged in a randomized block design, with a total of 16 plots. The plots are randomly distributed and separated by ridges.

[0106] (3) Application method: Apply the medicine before the occurrence of pepper virus disease. Dilute the medicine according to the required dosage for each treatment with water, and spray it conventionally. The spraying requirements are uniform, without double - spraying or missing - spraying. The blank control is sprayed with clear water.

[0107] Application time and frequency: This test is applied 2 times. The first application is on September 1, 2024; the second application is on September 10, 2024.

[0108] (4) Investigation method, time, and frequency Refer to the relevant procedures of the national agricultural industry standard NY / T 1464.9 - 2007, "Guidelines for field efficacy trials of pesticides - Part 8: Fungicides for controlling pepper virus disease".

[0109] The first investigation: Before applying the medicine on September 1, 2024, there are no diseased plants in the field, and the disease index is 0.

[0110] The second investigation: On September 20, 2024, 10 days after the last application, investigate the control effect and record the disease occurrence according to the grading standard.

[0111] 1. Investigation method Randomly sample 5 points in each plot, investigate 6 plants at each point. If the number of plants in the protected plot is less than 30, the whole plot should be investigated. Record the total number of investigated plants and the number of diseased plants at each level in units of plants.

[0112] Grading method (in units of plants) Grade 0: No symptoms.

[0113] Level 1: Heart leaves with clear veins or light variegated leaves.

[0114] Level 3: The heart leaves and middle leaves are mosaic, and sometimes necrotic spots appear on the leaves.

[0115] Level 5: Most leaves are mosaic, a few leaves are deformed and wrinkled, sometimes necrotic spots appear on leaves or stems, or short streaks appear on the stems.

[0116] Level 7: Most leaves are deformed and slender, or the stems and leaf veins produce systemic necrosis, and the plants are dwarfed.

[0117] Level 9: The plant suffers from severe systemic mosaic, deformity, or sometimes severe systemic necrosis or even death.

[0118] (6) Calculation method of drug efficacy The disease index and control effect of each plot were calculated, the average control effect of each treatment was calculated, and the significance of differences among treatments was analyzed using Duncan's new multiple range method using SPSS17.0 software.

[0119] Disease index (%) = ∑ (number of diseased plants at each level × relative level value) / total number of plants investigated × 9 × 100 Control effect (%) = (disease index of control area - disease index of treated area) / disease index of control area × 100 Direct impact on crops: Observe whether the pesticide causes phytotoxicity to pepper plants 1, 2, and 3 days after application.

[0120] Impact on non-target organisms (including beneficial insects) and surrounding crops: When conducting efficacy investigations, it is also necessary to investigate whether the pesticides have any impact on non-target organisms (including beneficial insects) and surrounding crops.

[0121] The results of the prevention effectiveness survey are shown in Table 11.

[0122] Table 11 Field efficacy test results for controlling pepper virus diseases Combined with Example 2-1, Example 2-2 and Example 2-3 and Table 11, the dosage of the effective ingredient of the ginkgo oligosaccharide aqueous solution prepared by the present application is 30g / hectare, 36g / hectare, and 45g / hectare; the control effects are 54.54%, 60.60%, and 68.33% respectively. The effective ingredient dosage of the control agent 5% amino oligosaccharide aqueous solution is 75g / hectare, and the control effect is 59.04%. It can be seen that at the same effective ingredient content, the ginkgo oligosaccharide aqueous solution prepared by the present application is better than the shiitake polysaccharide aqueous solution for the prevention of pepper virus disease.

[0123] Combined with Example 2-1, Example 2-2, Example 2-3 and Table 11, the analysis by Duncan's new multiple range method shows that: at the 0.05 level, there is no significant difference in the control efficacy between the high concentration and the medium concentration of the aqueous solution of ginkgo oligosaccharide sugar chain vaccine prepared in this application and that of the control agent; there is a significant difference in the control efficacy between the high concentration and the low concentration; there is no significant difference in the control efficacy between the medium concentration of the aqueous solution of ginkgo oligosaccharide sugar chain vaccine prepared in Example 2-2 of this application and that of Control Group 1; there is no significant difference in the control efficacy between the low concentration of the aqueous solution of ginkgo oligosaccharide sugar chain vaccine prepared in Example 2-1 of this application and that of the agent in Control Group 1. At the 0.01 level, there is no significant difference in the control efficacy among the three concentrations of the aqueous solution of ginkgo oligosaccharide sugar chain vaccine prepared in this application; and there is also no significant difference in the control efficacy compared with that of the control agent. However, 5% amino oligosaccharide aqueous solution was used in Control Group 1. According to the market price, the price of 5% amino oligosaccharide aqueous solution is between 45 yuan / kg and 60 yuan / kg, while the sugar chain plant vaccine involved in this application is sold at about 18 yuan / kg. The sugar chain plant vaccine involved in this application has a relatively high cost performance.

[0124] The ginkgo oligosaccharide sugar chain vaccine prepared in the examples is safe for crops without phytotoxicity at three concentrations of 30 g / ha, 36 g / ha, and 45 g / ha of the active ingredient dosage, and has no impact on non-target organisms (including beneficial insects) and surrounding crops.

[0125] The above are all preferred embodiments of this application, and the protection scope of this application is not limited accordingly. For those skilled in the art, equivalent substitutions or deformations can be made according to the above description, and these substitutions and deformations should all be covered within the protection scope of this application. For example, parameters such as extraction time, extraction temperature, and ethanol concentration can be changed to adapt to different production requirements and process conditions. At the same time, ginkgo oligosaccharides can also be compounded with other active ingredients to develop plant protection products with a wider range of application scenarios.

Claims

1. A method for preparing ginkgo oligosaccharide, characterized in that: Ginkgo oligosaccharides were extracted by enhanced extraction method, and the preparation steps were as follows: a. Grinding: Grind the outer seed coat of ginkgo into coarse powder with a diameter of 1 to 5 mm; b. Wetting: moisten the coarse powder with water; c. Ultrasonic treatment: Ultrasonic waves break up the cell walls; d. Water extraction: add pure water at a solid-liquid ratio of 1: (5-7), extract at 90℃±5℃ for 4min-60min, and obtain the extract; e. Filtration: Filter the extract through a 120-180 mesh filter to obtain a filtrate; f. Concentration: Concentrate the filtrate by evaporation at normal pressure at 90-100°C or by vacuum decompression at 50-60°C into a syrupy state to obtain a concentrated solution; g. Alcohol precipitation: add 95% to 100% ethanol to the concentrate and stir thoroughly; h. Precipitation: Let stand for 1 to 2 hours to allow the oligosaccharides to fully precipitate, filter out the ethanol solution to obtain ginkgo oligosaccharides.

2. The method for preparing ginkgo oligosaccharide according to claim 1, characterized in that: In step c, the parameters for ultrasonic wave breaking the cell wall are: using 600W ultrasonic wave for 15 to 25 minutes.

3. The method for preparing ginkgo oligosaccharide according to claim 1, characterized in that: When preparing Ginkgo oligosaccharides, the cell wall was first broken by ultrasonic treatment for 20 minutes through an enhanced extraction method, and then extracted with hot water at 90℃±5℃ for 40 minutes.

4. The method for preparing ginkgo oligosaccharide according to claim 1, characterized in that: The sugar groups of Ginkgo oligosaccharides include glucose, fructose and mannose, and the purity of Ginkgo oligosaccharides is 50% to 70%.

5. A Ginkgo oligosaccharide sugar chain plant vaccine, characterized in that: The invention comprises the following raw materials in weight ratio: 0.5% to 10% of the ginkgo oligosaccharide according to any one of claims 1 to 4; 0.5% to 2.5% of a wetting agent; 0.5% to 10% of an antifreeze agent; 0.5% to 1.2% of a preservative; 0.5% to 1.2% of a thickener, and the solvent is made up to 100%.

6. The ginkgo oligosaccharide sugar chain plant vaccine according to claim 5, characterized in that: The sugar chain plant vaccine is an aqueous solution, a microsuspension or an emulsion.

7. The ginkgo oligosaccharide sugar chain plant vaccine according to claim 6, characterized in that: The solvent is pure water, and the concentration of ginkgo oligosaccharide in the sugar chain plant vaccine aqueous solution is 2% to 10%.

8. The ginkgo oligosaccharide sugar chain plant vaccine according to claim 5, characterized in that: The antifreeze agent is one or more of propylene glycol, glycerol and methanol; the wetting agent is alkylphenol polyoxyethylene ether or fatty alcohol polyoxyethylene ether.

9. A method for preparing the ginkgo oligosaccharide chain plant vaccine according to any one of claims 5 to 8, characterized in that: The ginkgo oligosaccharide sugar chain plant vaccine is prepared by adding an auxiliary agent to the ginkgo oligosaccharide according to any one of claims 1 to 4.

10. A ginkgo oligosaccharide sugar chain plant vaccine is used to prevent and treat strawberry root rot, leek blight, wheat stripe rust, wheat powdery mildew, tomato blight, cucumber downy mildew, strawberry gray mold, tobacco virus disease, tomato virus disease, pepper virus disease, cucumber root knot nematode disease, watermelon root knot nematode disease, loofah root knot nematode disease, and potato root knot nematode disease.

Images