Plant disease control fungicide and preparation method thereof

Abstract

The invention relates to a preparation method of a plant disease control fungicide, which comprises the following steps: taking traditional Chinese medicines with an antibacterial effect as raw materials, and carrying out first-step fermentation by adopting Trichoderma harzianum; beauveria bassiana and Penicillium baylii are subjected to second-step fermentation, bacillus velezensis is subjected to third-step fermentation, fermentation liquor is collected, a filter cake is further subjected to reflux extraction, an extracting solution and the fermentation liquor are collected, combined and concentrated into clear paste, an ethanol solution is added, standing is carried out, supernate is taken, borneol, gelatin and cellulose acetate are added to prepare mixed liquor, ultrafiltration is carried out, ultrafiltrate is collected, and freeze-dried powder is obtained. The microbial inoculum required by the invention is obtained. According to the invention, specific traditional Chinese medicines are adopted, an excellent broad-spectrum antibacterial system is formed through specific microbial fermentation, and the antibacterial system is compounded with borneol, gelatin, cellulose acetate and other components to form the fungicide, so that the effectiveness duration and the effect taking rate of the fungicide on the surface of a plant are effectively improved, and efficient prevention and treatment on various diseases such as fungi, bacteria and the like are formed.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and particularly relates to a plant disease control bactericide and a preparation method thereof. Background Art

[0002] Garden plants refer to the general term of plants that have been artificially selected, cultivated and configured for landscaping, landscape construction, ecological restoration and other scenarios. They usually have the following characteristics: mainly for ornamental and functional purposes, relying on pruning and maintenance to maintain the landscape effect, with economic value, practical value or ornamental value as the core. However, since most garden plants are artificially selected varieties with the main goal of ornamentality, this will sacrifice some disease resistance, and the varieties in a certain area are seriously single and homogeneous during planting, resulting in easier large-scale spread of diseases. And due to dense planting, frequent pruning, and improper management of fertilization and watering, which damage the natural resistance of plants, diseases are also likely to occur.

[0003] Diseases of garden plants are mainly caused by three types of pathogens: fungi, bacteria and viruses. Among them, fungal diseases are the most common and most prevalent type of diseases. They are diseases caused by plant pathogenic fungi, accounting for about 70-80% of plant diseases. Some fungi have weak parasitism in the north and do not cause diseases, but can cause serious diseases in the tropics and subtropics. Several or even dozens of fungal diseases can be found on one plant. The most common ones include powdery mildew, black spot, gray mold, leaf spot, anthracnose, root rot, rust, etc. According to different disease parts, they can be divided into leaf diseases, fruit diseases, root and stem diseases, etc. For example, in leaf diseases, after being infected with powdery mildew, the leaf surface is covered with white powdery substances (hyphae and conidia), and the powder layer turns grayish-brown in the later stage, the leaves turn yellow and distorted, the disease spots expand, resulting in the distortion of young shoots and the non-opening of flower buds; rust diseases will cause yellowish-brown or orange-red rusty spore masses to appear on the leaves or stems, and rusty powder will be emitted after rupture; downy mildew will cause a grayish-white or purple frost-like mildew layer to appear on the back of the leaf, and chlorotic yellow spots to appear on the leaf surface, and the leaves will be scorched when it is serious. Fruit diseases such as anthracnose will show round sunken brown spots on the fruit surface, with small black dots (conidiophores) in the center, and pink sticky substances will overflow when it is wet, and gray mold will cause the fruit or flower organs to rot, with a gray mold layer covering the surface. Root and stem diseases such as root rot cause the roots to rot and turn black, the plants wilt and turn yellow, and are easy to pull out. Sclerotinia sclerotiorum forms white flocculent hyphae and black mouse dung-like sclerotia inside the stem base or fruit, and the plants will lodge. In addition, common bacterial diseases such as crown gall and leaf blight, and viral diseases such as mosaic disease are also relatively common diseases in garden plants.

[0004] Currently, the common disease prevention and control measures mainly include agricultural control and chemical control. Among them, agricultural control mainly prevents diseases through reasonable seed selection, removing pathogen-infected plants, and specific fertilization, but the control effect is not good. For chemical control, chemical control agents are mainly selected, such as lime sulfur mixture and spraying sulfur powder. On the one hand, it pollutes the environment and has strong drug resistance. On the other hand, there will also be residues, which affect the ornamental and edible properties of plants. Summary of the Invention

[0005] The object of the present invention is to provide a composite biological agent for preventing and controlling multiple plant diseases.

[0006] Another object of the present invention is to provide a preparation method of a bactericide for preventing and controlling horticultural plant diseases. By fermenting traditional Chinese medicine with specific microbial agents, the present invention effectively improves the control effect of the bactericide on different fungal and bacterial pathogenic diseases, and then combines with components such as borneol, gelatin, and cellulose acetate to form a bactericide, which improves the adhesion ability of the bactericide on the plant surface and promotes the action of effective bactericidal and antibacterial components on pathogenic bacteria, further improving the control effect on diseases.

[0007] The object of the present invention is achieved through the following technical solutions: A preparation method of a bactericide for preventing and controlling plant diseases, characterized in that it is carried out according to the following steps: (1) Take traditional Chinese medicine with antibacterial efficacy, crush it and pass through a 30-mesh sieve to obtain traditional Chinese medicine powder, then add corn starch, glucose, peptone, and soybean meal, mix evenly, and sterilize to obtain a mixed powder; (2) Add purified water and Trichoderma harzianum ( Trichoderma harzianum ) to the mixed powder for the first-step fermentation. After the fermentation is completed, collect fermentation broth 1 and filter cake 1; (3) Add Beauveria bassiana ( Beauveria bassiana ), Penicillium bilaiae ( Penicillium bilaiae ) and purified water to filter cake 1 for the second-step fermentation. After the fermentation is completed, collect fermentation broth 2 and filter cake 2; (4) Add Bacillus velezensis ( Bacillus velezensis ) and purified water to filter cake 2, mix evenly, and carry out the third-step fermentation. After the fermentation is completed, collect fermentation broth 3 and filter cake 3; (5) Take filter cake 3, add ethanol solution, reflux and extract. After the extraction is completed, discard the medicinal residues and collect the extract as filtrate 4; (6) Combine filtrate 1, filtrate 2, filtrate 3 and filtrate 4 to obtain a fermentation extract, and concentrate it under reduced pressure to obtain a clear paste; (7) Add ethanol solution to the clear paste, let it stand for 24 - 36 h, take the supernatant, add borneol, gelatin, and cellulose acetate to make a mixed solution, and then carry out ultrafiltration to collect the ultrafiltrate, which is the bactericide required by the present invention.

[0008] Furthermore, the raw materials in step (1) are composed of Gleditsia sinensis Lam. spine, Smilax glabra Roxb., Phellodendron amurense Rupr. bark, and Cnidium monnieri (L.) Cuss. By weight, there are 20 - 30 parts of Gleditsia sinensis Lam. spine, 15 - 20 parts of Smilax glabra Roxb., 8 - 12 parts of Phellodendron amurense Rupr. bark, 8 - 10 parts of Cnidium monnieri (L.) Cuss., 20 - 30 parts of corn flour, 10 - 15 parts of soybean meal, 15 - 20 parts of glucose, 15 - 20 parts of corn starch, and 6 - 8 parts of peptone.

[0009] Furthermore, in step (2), the temperature is 30 - 35°C, fermentation lasts for 12 - 15 days, and the air flow rate in the fermentation tank is maintained at 1 - 1.5 m 3 / h. The mass ratio of the mixed powder, Trichoderma harzianum, and purified water is 100:0.2 - 0.3:50 - 60.

[0010] Furthermore, in step (3), the fermentation temperature is 25 - 30°C, fermentation lasts for 8 - 10 days, and the air flow rate in the fermentation tank is maintained at 1 - 1.5 m 3 / h. The mass ratio of filter cake 1, Beauveria bassiana, Paecilomyces byssinus, and purified water is 100:0.1 - 0.2:0.2 - 0.3:30 - 40.

[0011] Furthermore, in step (4), the fermentation temperature is 30 - 35°C, fermentation lasts for 7 - 9 days, and the air flow rate in the fermentation tank is maintained at 1.5 - 2 m 3 / h. The mass ratio of filter cake 2, Bacillus velezensis mold, and purified water is 100:0.1 - 0.2:30 - 40.

[0012] Furthermore, in step (5), take filter cake 3, add an ethanol solution with a volume fraction of 50% - 60% according to the mass ratio of filter cake 3 to the ethanol solution of 1:10 - 15, reflux and extract 2 times. The first extraction lasts for 2 - 2.5 h, and the second extraction lasts for 1.5 - 2 h. After the extraction is completed, discard the medicinal residues, combine the two extraction solutions, and obtain filtrate 4.

[0013] Furthermore, in step (6), the vacuum degree is -0.05 - -0.08 MPa, the temperature is 45 - 50°C, and it is concentrated under reduced pressure to a clear paste with a relative density of 1.10 - 1.15.

[0014] Furthermore, in step (7), add an ethanol solution with a volume fraction of 70% - 75% to the clear paste, and the mass ratio of the clear paste to the ethanol is 1:20 - 25.

[0015] Furthermore, add 12 - 15 parts of borneol, 6 - 8 parts of gelatin, and 2 - 5 parts of cellulose acetate in the formula amount to form a mixed solution. Heat the temperature to 40 - 45 °C, stir and disperse evenly, then cool it to 20 - 35 °C. Then place it in a ceramic membrane with a molecular retention volume of 10,000 - 20,000 for ultrafiltration treatment. The ultrafiltration pressure is 0.3 - 0.5 MPa, and the temperature is 25 - 35 °C. After the ultrafiltration is completed, collect the ultrafiltrate to obtain the bacterial agent required by the present invention.

[0016] Further preferably, add cetyltrimethylammonium bromide accounting for 0.1% - 0.2% of the mass of the mixed solution before heating.

[0017] In the present invention, by using Gleditsia sinensis Lam., Smilax glabra Roxb., Phellodendron amurense Rupr., and Cnidium monnieri (L.) Cuss. containing antibacterial active ingredients as raw materials, through specific microbial combination fermentation and extraction, the obtained fermentation extract has excellent antibacterial effects. However, after being sprayed on the plant surface, due to the volatilization of the active ingredients and being affected by factors such as wind blowing and rain washing in the natural environment, the persistence of the control effect is poor, and the spores of many pathogenic microorganisms cannot be killed, making it easy to relapse.

[0018] In the present invention, by adding borneol to the fermentation mixture, the attachment stability of the bacterial agent on the plant surface is improved, and it also takes effect quickly. When the bacterial agent is sprayed on the plant surface, the low surface tension of borneol can reduce the resistance of the plant surface wax layer. The rapid volatilization of borneol and ethanol, due to the overall low pH of the fermentation extract in the bacterial agent, being acidic, gelatin forms polycations in this system, improving the composite membrane with stable structure formed by electrostatic interaction with cellulose acetate, helping the microorganisms in the bacterial agent to better attach and colonize on the surface of branches and leaves, thereby prolonging the control efficacy of antibacterial and bacteriostatic effects. In addition, under the regulation of CTAB, the biocontrol bacteria carried preferentially aggregate on the surface of negatively charged pathogens, targeting the pathogens, effectively improving the control effect. At the same time, the formed membrane layer also effectively isolates the attachment and colonization of new pathogenic bacteria on the plant surface, reducing the infection rate.

[0019] During the use process, since the outer layer of the fungal cell wall is rich in negatively charged mannan and phosphorylated proteins, it forms an obstacle to the lysozyme in the bacterial agent, reducing the disease control efficacy of the bacterial agent. The cationic property of CTAB used in the present invention can neutralize the negative charge on the cell wall surface and play its hydrophobic role to destroy the structure of mannan and phosphorylated proteins, reducing the contact resistance between the lysozyme and the cell wall, and promoting the adsorption of enzyme molecules on the cell wall surface, acting efficiently on the fungal cell wall.

[0020] Most specifically, a preparation method of a plant disease control bacterial agent is characterized by including the following steps: Formula: Take 20 - 30 parts of Gleditsiae Spina, 15 - 20 parts of Smilax Glabra, 10 - 15 parts of soybean meal, 20 - 30 parts of corn flour, 8 - 12 parts of Phellodendron Bark, 15 - 20 parts of glucose, 8 - 10 parts of Cnidii Fructus, 15 - 20 parts of corn starch, 6 - 8 parts of peptone, 12 - 15 parts of borneol, 6 - 8 parts of gelatin, 2 - 5 parts of cellulose acetate according to weight parts; (1) Take Gleditsiae Spina, Smilax Glabra, Phellodendron Bark, and Cnidii Fructus, mix them, place them in a universal grinder, pulverize and pass through a 30 - mesh sieve to obtain medicinal powder. Add corn flour, glucose, peptone, and soybean meal to the medicinal powder, mix evenly, sterilize at 121 °C for 15 min to obtain a mixed powder; (2) In a sterile environment, add the mixed powder prepared in step (1) to a fermentation tank, add purified water, mix evenly, add Trichoderma harzianum, stir evenly, keep the temperature at 30 - 35 °C, ferment for 12 - 15 days, and maintain the air flow rate of the fermentation tank at 1 - 1.5 m 3 / h. After fermentation, sterilize, and after sterilization, filter. Collect filtrate 1 and filter cake 1 respectively. The mass ratio of the mixed powder, Trichoderma harzianum, and purified water is 100:0.2 - 0.3:50 - 60; (3) In a sterile environment, add Beauveria bassiana, Penicillium bilaiae, and purified water to filter cake 1, mix evenly, keep the fermentation temperature at 25 - 30 °C, ferment for 8 - 10 days, and maintain the air flow rate of the fermentation tank at 1 - 1.5 m 3 / h. After fermentation, sterilize, and after sterilization, filter. Collect filtrate 2 and filter cake 2 respectively. The mass ratio of filter cake 1, Beauveria bassiana, Penicillium bilaiae, and purified water is 100:0.1 - 0.2:0.2 - 0.3:30 - 40; (4) In a sterile environment, add Bacillus velezensis and purified water to filter cake 2, mix evenly, place them together in a fermentation tank, keep the fermentation temperature at 30 - 35 °C, ferment for 7 - 9 days, and maintain the air flow rate of the fermentation tank at 1.5 - 2 m 3 / h. After fermentation, sterilize, and after sterilization, filter. Collect filter cake 3 and filtrate 3 respectively. The mass ratio of filter cake 2, Bacillus velezensis, and purified water is 100:0.1 - 0.2:30 - 40; (5) Take filter cake 3, add an ethanol solution with a volume fraction of 50% - 60% according to the mass ratio of filter cake 3 to the ethanol solution of 1:10 - 15, reflux and extract 2 times. The first extraction is for 2 - 2.5 h, and the second extraction is for 1.5 - 2 h. After extraction, discard the medicinal residues, and combine the two extraction liquids to obtain filtrate 4; (6) Combine filtrate 1, filtrate 2, filtrate 3, and filtrate 4 to obtain a fermentation mixture. Place it in a vacuum concentration tank, set the vacuum degree at - 0.05 - - 0.08 MPa, and the temperature at 45 - 50 °C, and concentrate under reduced pressure to obtain a clear extract with a relative density of 1.10 - 1.15; (7) Add an ethanol solution with a volume fraction of 70% - 75% to the clear extract. The mass ratio of the clear extract to ethanol is 1:20 - 25. Stir evenly, let it stand for 24 - 36 h, take the supernatant, add the formulated borneol, gelatin, and cellulose acetate to form a mixed solution, add cetyltrimethylammonium bromide accounting for 0.1% - 0.2% of the mass of the mixed solution, heat up to 40 - 45 °C, stir to make it evenly dispersed, cool down to 20 - 35 °C, and then place it in a ceramic membrane with a molecular cut-off of 10000 - 20000 for ultrafiltration treatment. The ultrafiltration pressure is 0.3 - 0.5 MPa, and the temperature is 25 - 35 °C. After ultrafiltration, collect the ultrafiltrate to obtain the target antibacterial agent for prevention and treatment.

[0021] A composite biological agent for preventing and treating multiple plant diseases, characterized in that: the antibacterial agent for prevention and treatment uses Chinese herbal medicine raw materials such as Gleditsia sinensis thorn, Smilax glabra, Phellodendron amurense bark, and Cnidium monnieri, obtains a fermentation broth through fermentation with a composite bacterial agent, further extracts the filter cake after fermentation and filtration to obtain an extract, mixes and concentrates the fermentation broth and the extract into a clear extract, adds ethanol, borneol, gelatin, and cellulose acetate to form a mixed solution, and obtains the antibacterial agent for prevention and treatment through ultrafiltration.

[0022] Furthermore, in the fermentation, add corn flour, glucose, corn starch, peptone, and soybean meal to the Chinese herbal medicine raw materials, mix evenly to obtain a mixed powder. Add Trichoderma harzianum and purified water to the sterilized mixed powder for the first-step fermentation. After the fermentation is completed, filter the fermentation broth 1 and the filter cake 1. After sterilizing the filter cake 1, add Beauveria bassiana and purified water for the second-step fermentation. After the fermentation is completed, collect the fermentation broth 2 and the filter cake 2. After sterilizing the filter cake 2, add Bacillus velezensis and Penicillium bilaiae for the third-step fermentation. After the fermentation is completed, collect the fermentation broth 3 and the filter cake 3. After sterilizing the filter cake 3, add ethanol for reflux extraction, and then filter to obtain the filtrate 4. Mix the fermentation broth 1, fermentation broth 2, fermentation broth 3, and filtrate 4 to obtain a fermentation extract.

[0023] Furthermore, by weight, the Gleditsia sinensis thorn is 20 - 30 parts, the Smilax glabra is 15 - 20 parts, the Phellodendron amurense bark is 8 - 12 parts, the Cnidium monnieri is 8 - 10 parts, the corn flour is 20 - 30 parts, the soybean meal is 10 - 15 parts, the glucose is 15 - 20 parts, the corn starch is 15 - 20 parts, the peptone is 6 - 8 parts, the borneol is 12 - 15 parts, the gelatin is 6 - 8 parts, and the cellulose acetate is 2 - 5 parts.

[0024] Furthermore, in the first-step fermentation, the mass ratio of the mixed powder, Trichoderma harzianum, and purified water is 100:0.2 - 0.3:50 - 60.

[0025] Furthermore, the fermentation temperature of the first-step fermentation is 30 - 35 °C, the fermentation lasts for 12 - 15 days, and the air flow rate in the fermentation tank is maintained at 1 - 1.5 m 3 / h.

[0026] Further, in the second - step fermentation, the mass ratio of the filter cake 1, Beauveria bassiana, Penicillium bilaiae, and purified water is 100:0.1 - 0.2:0.2 - 0.3:30 - 40.

[0027] Further, the fermentation temperature of the second - step fermentation is 25 - 30 °C, the fermentation lasts for 8 - 10 days, and the air flow rate in the fermenter is maintained at 1 - 1.5 m 3 / h.

[0028] Further, in the second - step fermentation, the mass ratio of the filter cake 2, Bacillus velezensis, and purified water is 100:0.1 - 0.2:30 - 40.

[0029] Further, the fermentation temperature of the third - step fermentation is 30 - 35 °C, the fermentation lasts for 7 - 9 days, and the air flow rate in the fermenter is maintained at 1.5 - 2 m 3 / h.

[0030] Further, in the reflux extraction, according to the mass ratio of the filter cake 3 to the ethanol solution of 1:10 - 15, an ethanol solution with a volume fraction of 50% - 60% is added to the filter cake 3 for reflux extraction, and the extract collected is the filtrate 4.

[0031] Further, in the vacuum concentration, the fermentation extract composed of the filtrate 1, filtrate 2, filtrate 3, and filtrate 4 is concentrated under a vacuum degree of - 0.05 - - 0.08 MPa and a temperature of 45 - 50 °C to a clear paste with a relative density of 1.10 - 1.15.

[0032] Further, for the mixed solution, according to the mass ratio of the clear paste to the ethanol solution of 1:20 - 25, an ethanol solution with a volume fraction of 70% - 75% is added to the clear paste. After standing for 24 - 36 h, the supernatant is taken, and borneol, gelatin, and cellulose acetate are added to form a mixed solution. The temperature is raised to 40 - 45 °C and stirred evenly to obtain the mixed solution.

[0033] Further preferably, cetyltrimethylammonium bromide with 0.1% - 0.2% of the mass of the mixed solution is added before heating.

[0034] Further, for the ultrafiltration, it is carried out using a ceramic membrane with a molecular cut - off of 10000 - 20000. The ultrafiltration pressure is 0.3 - 0.5 MPa, the temperature is 25 - 35 °C. After ultrafiltration, the ultrafiltrate is collected to obtain the antibacterial agent.

[0035] The present invention has the following technical effects: The present invention uses traditional Chinese medicines with antibacterial active ingredients such as Gleditsiae Spina, Phellodendri Cortex, Smilacis Glabrae Rhizoma, and Cnidii Fructus as fermentation raw materials, and forms an excellent broad-spectrum antibacterial system through specific microbial fermentation, which can effectively prevent and control various fungal and bacterial disease infections. In addition, by compounding this antibacterial system with components such as borneol, gelatin, and cellulose acetate, a bacterial agent is formed, effectively improving the efficacy duration and onset rate of the bacterial agent on the plant surface, and further enhancing the control effect of the bacterial agent on diseases. Detailed Embodiments

[0036] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above content of the present invention.

[0037] The microorganisms used in the present invention are all purchased from the Guangdong Provincial Microbial Culture Collection Center (GDMCC), and the preservation numbers of each microorganism are as follows: Trichoderma harzianum ( Trichoderma harzianum ) GDMCC NO.3.442; Penicillium bilaiae ( Penicillium bilaiae ) GDMCC NO.3.631; Beauveria bassiana ( Beauveria bassiana ) GDMCC NO.3.428; Bacillus velezensis ( Bacillus velezensis ) GDMCC NO.1.402.

[0038] Example 1 A preparation method of a bacterial agent for preventing and controlling horticultural plant diseases, characterized by comprising the following steps: Formulation: Take 25 parts of Gleditsiae Spina, 18 parts of Smilacis Glabrae Rhizoma, 12 parts of soybean meal, 25 parts of corn flour, 10 parts of Phellodendri Cortex, 18 parts of glucose, 9 parts of Cnidii Fructus, 18 parts of corn starch, 7 parts of peptone, 14 parts of borneol, 7 parts of gelatin, and 4 parts of cellulose acetate according to weight; (1) Take Gleditsiae Spina, Smilacis Glabrae Rhizoma, Phellodendri Cortex, and Cnidii Fructus and mix them. Place them in a universal pulverizer, pulverize and pass through a 30-mesh sieve to obtain medicinal powder. Add corn flour, glucose, peptone, and soybean meal to the medicinal powder, mix evenly, and sterilize at 121 °C for 15 min to obtain a mixed powder; (2) In a sterile environment, add the mixed powder prepared in step (1) to a fermentation tank, add purified water in the formulated amount, mix evenly, add Trichoderma harzianum, stir evenly, keep the temperature at 30-35 °C, ferment for 14 days, and keep the air flow rate of the fermentation tank at 1-1.5 m 3 / h. After fermentation, place it in a moist heat sterilizer, set the temperature to 121 °C, and the sterilization time to 15 min. After sterilization, filter, and collect filtrate 1 and filter cake 1 respectively. The mass ratio of the mixed powder, Trichoderma harzianum, and purified water is 100:0.5:55; (3) In a sterile environment, add Beauveria bassiana, Penicillium bilaiae and purified water to the filter cake 1 and mix evenly. The fermentation temperature is 25-30 °C, and the fermentation lasts for 9 days. Keep the air flow rate in the fermenter at 1-1.5 m 3 / h. After fermentation, place it in a moist heat sterilizer, set the temperature to 121 °C, and the sterilization time to 15 min. After sterilization, filter and collect filtrate 2 and filter cake 2 respectively. The mass ratio of filter cake 1, Beauveria bassiana, Penicillium bilaiae and purified water is 100:0.5:0.25:35; (4) In a sterile environment, add Bacillus velezensis and purified water to the filter cake 2 and mix evenly. Then place them together in a fermenter. The fermentation temperature is 30-35 °C, and the fermentation lasts for 8 days. Keep the air flow rate in the fermenter at 1.5-2 m 3 / h. After fermentation, place it in a moist heat sterilizer, set the temperature to 121 °C, and the sterilization time to 15 min. After sterilization, filter and collect filter cake 3 and filtrate 3 respectively. The mass ratio of filter cake 2, Bacillus velezensis and purified water is 100:0.5:35; (5) According to the mass ratio of filter cake 3 to ethanol solution of 1:12, add ethanol solution with a volume fraction of 50%-60% to filter cake 3, and reflux and extract 2 times. The first extraction is for 2 h, and the second extraction is for 2 h. After extraction, discard the medicinal residues, combine the two extraction liquids to obtain filtrate 4; (6) Combine filtrate 1, filtrate 2, filtrate 3 and filtrate 4 to obtain a fermentation mixture. Place it in a vacuum concentration tank, set the vacuum degree to -0.05 to -0.08 MPa, and the temperature to 45 °C, and concentrate under reduced pressure to a clear paste with a relative density of 1.10-1.15; (7) According to the mass ratio of clear paste to ethanol of 1:24, add ethanol solution with a volume fraction of 75% to the clear paste, stir evenly, let it stand for 30 h, take the supernatant, add the formulated amount of borneol, gelatin and cellulose acetate to form a mixture, add cetyltrimethylammonium bromide (CTAB) accounting for 0.15% of the mass of the mixture, heat up to 42 °C, stir to make it evenly dispersed, cool to 30 °C, and then place it in a ceramic membrane with a molecular cut-off of 15000 for ultrafiltration treatment. The ultrafiltration pressure is 0.4 MPa, and the temperature is 30 °C. After ultrafiltration, collect the ultrafiltrate to obtain the target control fungicide.

[0039] Comparative Example 1 Compared with Example 1, the difference lies in step (7). During the further preparation of the control fungicide from the clear paste, the operations are as follows: (1) Compared with Example 1, CTAB was not added, and the prepared fungicide is denoted as D1-1; (2) Compared with Example 1, dodecyltrimethylammonium chloride (DTAC) was used instead of CTAB to participate in the preparation of the fungicide, and the prepared fungicide is denoted as D1-2; (3) Compared with Example 1, borneol was not added during the mixing process, and the prepared microbial agent was denoted as D1-3.

[0040] Verification Experiment 1: Take the leaves of 5 groups of kiwifruits with consistent growth traits, trim them into equal-area sizes and fix them. Spray equal amounts of the control microbial agents prepared in Example 1 and the comparative examples on 4 groups of leaves respectively, and leave 1 group untreated. After standing for 24 h, spray water on the 5 groups of leaves for 5 min to simulate the leaves being washed by rain (during the spraying process, the same water spraying amount, spraying angle, and spraying rate are maintained for each group). Filter and detect some active ingredients (astilbin, berberine, osthole) in the water. The results are shown in Table 1.

[0041] Table 1:

[0042] As can be seen from the above table, during the cotton swab wiping process of the control microbial agent prepared in Example 1 of the present invention, the loss amount of the active ingredients from the surface of the plant leaves is relatively low, indicating that the control microbial agent is effectively attached to the leaf surface and is not easily lost from the leaf surface, resulting in the failure of the microbial agent. After spraying the several active ingredients tested in each microbial agent in Comparative Example 1, the loss amount is higher. This shows that compared with Example 1, the attachment efficiency of each microbial agent in Comparative Example 1 on the leaf surface is poor, resulting in a greater loss during the spraying process, which will cause the microbial agent to be more susceptible to the environment during use and make the control persistence effect worse.

[0043] Comparative Example 2 Compared with Example 1, the difference lies in that during the fermentation process, the fermentation steps of the fermentation microbial agent are adjusted as follows: (1) Compared with Example 1, Trichoderma harzianum was used for the first-step fermentation, Bacillus velezensis and Penicillium bilaiae were mixed for the second-step fermentation, and Beauveria bassiana was used for the third-step fermentation during fermentation. The prepared microbial agent was denoted as D2-1.

[0044] (2) Compared with Example 1, Trichoderma harzianum was used for the first-step fermentation, Penicillium bilaiae was used for the second-step fermentation, and Bacillus velezensis and Beauveria bassiana were mixed for the third-step fermentation during fermentation. The prepared microbial agent was denoted as D2-2.

[0045] (3) Compared with Example 1, during the second-step fermentation process, single Beauveria bassiana was used for fermentation, and the other fermentation steps were the same as those in Example 1. The prepared microbial agent was denoted as D2-3.

[0046] Due to the differences in the fermentation conditions of different biocontrol bacteria, different fermentation steps will also have obvious effects on the types and contents of the active ingredients in the fermentation extract prepared by fermentation. During the experiment, attempts were also made to perform one-step fermentation by mixing Trichoderma harzianum, Beauveria bassiana, Bacillus velezensis, and Penicillium bilaiae. It was found that obvious antagonistic phenomena occurred. Among them, the hyphal expansion of Trichoderma harzianum was severely inhibited, the degradation efficiency of lignocellulose in traditional Chinese medicine raw materials decreased, and the subsequent fermentation effect was extremely poor, resulting in unsatisfactory types and contents of antibacterial active ingredients in the fermentation broth.

[0047] Verification experiment two: The strawberry planting areas that have simultaneously suffered from powdery mildew, leaf spot, gray mold, and crown gall are equally divided into 8 groups for strawberry planting. Among them, 7 groups are respectively sprayed with the biocontrol agent prepared in Example 1, the agents D1-1, D1-2, and D1-3 prepared in Comparative Example 1, and the agents D2-1, D2-1, and D2-1 prepared in Comparative Example 2. Equal amounts are sprayed at intervals of 2 months. The 8th group is not subjected to any control treatment as a blank group. After planting for 12 months under the same conditions, the average infection rates of various diseases in each group of strawberry plants are detected, and the results are shown in Table 2.

[0048] Table 2:

[0049] As can be seen from the table, in the blank group without spraying the biocontrol agent, the infection rates of strawberries for powdery mildew, leaf spot, gray mold, and crown gall are all at relatively high levels. After applying the agents in Example 1, Comparative Example 1, and Comparative Example 2 respectively, the infection rates of various diseases in strawberries all decreased to varying degrees. However, compared with Example 1, the overall disease control effect is relatively poor. Among them, the pH values of the agents in Comparative Example 2 showed obvious fluctuations compared with those in Example 1.

[0050] Example 2 A preparation method of a biocontrol agent for horticultural plant diseases, characterized by comprising the following steps: Formulation: Take 30 parts of Gleditsia sinensis thorns, 20 parts of Smilax glabra, 15 parts of soybean meal, 30 parts of corn flour, 12 parts of Phellodendron amurense bark, 20 parts of glucose, 10 parts of Cnidium monnieri, 20 parts of corn starch, 8 parts of peptone, 15 parts of borneol, 8 parts of gelatin, and 5 parts of cellulose acetate according to weight; (1) Take Gleditsia sinensis thorns, Smilax glabra, Phellodendron amurense bark, and Cnidium monnieri, mix them, place them in a universal grinder, pulverize them through a 30-mesh sieve to obtain medicinal powder, add corn flour, glucose, peptone, and soybean meal to the medicinal powder, mix evenly, and sterilize at 121 °C for 15 min to obtain a mixed powder; (2) In a sterile environment, add the mixed powder prepared in step (1) into a fermentation tank, add purified water, mix evenly, add Trichoderma harzianum, stir evenly, keep the temperature at 30 - 35 °C, ferment for 15 days, maintain the air flow rate of the fermentation tank at 1 - 1.5 m 3 / h. After fermentation, place it in a moist heat sterilizer, set the temperature at 121 °C, and the sterilization time at 15 min. After sterilization, filter and collect filtrate 1 and filter cake 1 respectively. The mass ratio of the mixed powder, Trichoderma harzianum, and purified water is 100:0.3:60; (3) In a sterile environment, add Beauveria bassiana, Penicillium bilaiae, and purified water to filter cake 1 and mix evenly. Ferment at a temperature of 25 - 30 °C for 10 days, maintain the air flow rate of the fermentation tank at 1 - 1.5 m 3 / h. After fermentation, place it in a moist heat sterilizer, set the temperature at 121 °C, and the sterilization time at 15 min. After sterilization, filter and collect filtrate 2 and filter cake 2 respectively. The mass ratio of filter cake 1, Beauveria bassiana, Penicillium bilaiae, and purified water is 100:0.2:0.3:40; (4) In a sterile environment, add Bacillus velezensis and purified water to filter cake 2 and mix evenly. Then place them together in a fermentation tank. Ferment at a temperature of 30 - 35 °C for 9 days, maintain the air flow rate of the fermentation tank at 1.5 - 2 m 3 / h. After fermentation, place it in a moist heat sterilizer, set the temperature at 121 °C, and the sterilization time at 15 min. After sterilization, filter and collect filter cake 3 and filtrate 3 respectively. The mass ratio of filter cake 2, Bacillus velezensis, and purified water is 100:0.2:40; (5) Take filter cake 3, add ethanol solution with a volume fraction of 60% according to the mass ratio of filter cake 3 to ethanol solution of 1:15, and reflux extract twice. The first extraction is for 2.5 h, and the second extraction is for 1.5 h. After extraction, discard the medicinal residues, combine the two extraction solutions to obtain filtrate 4; (6) Combine filtrate 1, filtrate 2, filtrate 3, and filtrate 4 to obtain a fermentation mixture. Place it in a vacuum concentration tank, set the vacuum degree at -0.05 - -0.08 MPa, and the temperature at 50 °C, and concentrate under reduced pressure to a clear paste with a relative density of 1.10 - 1.15; (7) Add ethanol solution with a volume fraction of 70% to the clear paste according to the mass ratio of clear paste to ethanol of 1:25, stir evenly, let it stand for 36 h, take the supernatant, add the formulated amount of borneol, gelatin, and cellulose acetate to form a mixture, add cetyltrimethylammonium bromide (CTAB) at 0.1% of the mass of the mixture, heat up to 45 °C, stir to make it evenly dispersed, cool down to 35 °C, and then place it in a ceramic membrane with a molecular cut-off of 20000 for ultrafiltration treatment. The ultrafiltration pressure is 0.5 MPa, and the temperature is 25 °C. After ultrafiltration, collect the ultrafiltrate to obtain the required target control bactericide.

[0051] Example 3 A preparation method of a bactericide for controlling horticultural plant diseases, characterized by comprising the following steps: Formulation: Take 20 parts of Chinese honey locust thorns, 15 parts of glabrous greenbrier rhizome, 10 parts of soybean meal, 20 parts of corn flour, 8 parts of phellodendron bark, 15 parts of glucose, 8 parts of cnidium fruit, 15 parts of corn starch, 6 parts of peptone, 12 parts of borneol, 6 parts of gelatin, and 2 parts of cellulose acetate according to weight; (1) Take Chinese honey locust thorns, glabrous greenbrier rhizome, phellodendron bark, and cnidium fruit and mix them. Place them in a universal grinder, pulverize and pass through a 30-mesh sieve to obtain medicinal powder. Add corn flour, glucose, peptone, and soybean meal to the medicinal powder, mix evenly, and sterilize at 121 °C for 15 min to obtain a mixed powder; (2) In a sterile environment, add the mixed powder prepared in step (1) to a fermenter, add purified water, mix evenly, add Trichoderma harzianum, stir evenly, keep the temperature at 30 - 35 °C, ferment for 12 days, and keep the air flow rate of the fermenter at 1 - 1.5 m 3 / h. After fermentation, place it in a moist heat sterilizer, set the temperature at 121 °C, and the sterilization time at 15 min. After sterilization, filter and collect filtrate 1 and filter cake 1 respectively. The mass ratio of the mixed powder, Trichoderma harzianum, and purified water is 100:0.2:50; (3) In a sterile environment, add Beauveria bassiana, Penicillium bilaiae, and purified water to filter cake 1 and mix evenly. The fermentation temperature is 25 - 30 °C, ferment for 8 days, and keep the air flow rate of the fermenter at 1 - 1.5 m 3 / h. After fermentation, sterilize it in a moist heat sterilizer, set the temperature at 121 °C, and the sterilization time at 15 min. After sterilization, filter and collect filtrate 2 and filter cake 2 respectively. The mass ratio of filter cake 1, Beauveria bassiana, Penicillium bilaiae, and purified water is 100:0.1:0.2:30; (4) In a sterile environment, add Bacillus velezensis and purified water to filter cake 2 and mix evenly. Place them together in a fermenter. The fermentation temperature is 30 - 35 °C, ferment for 7 days, and keep the air flow rate of the fermenter at 1.5 - 2 m 3 / h. After fermentation, place it in a moist heat sterilizer, set the temperature at 121 °C, and the sterilization time at 15 min. After sterilization, filter and collect filter cake 3 and filtrate 3 respectively. The mass ratio of filter cake 2, Bacillus velezensis, and purified water is 100:0.1:30; (5) Take filter cake 3, add it to an ethanol solution with a volume fraction of 50% according to the mass ratio of filter cake 3 to the ethanol solution of 1:10, reflux and extract twice, extract for 2 h for the first time and 2 h for the second time. After extraction, discard the medicinal residues, combine the two extraction solutions to obtain filtrate 4; (6) Combine filtrate 1, filtrate 2, filtrate 3 and filtrate 4 to obtain a fermentation mixture, place it in a vacuum concentration tank, set the vacuum degree to -0.05~-0.08 MPa and the temperature to 45 °C, and concentrate under reduced pressure to obtain a clear paste with a relative density of 1.10~1.15; (7) According to the mass ratio of the clear paste to ethanol being 1:20, add an ethanol solution with a volume fraction of 70% to the clear paste, stir evenly, let stand for 24 h, take the supernatant, add the formulated borneol, gelatin, and cellulose acetate to form a mixture, add 0.1%~0.2% of cetyltrimethylammonium bromide (CTAB) based on the mass of the mixture, heat up to 40 °C, stir to make it evenly dispersed, cool to 20 °C, and then place it in a ceramic membrane with a molecular cut-off of 10,000 for ultrafiltration treatment. The ultrafiltration pressure is 0.3 MPa and the temperature is 35 °C. After the ultrafiltration is completed, collect the ultrafiltrate to obtain the target control bactericide.

[0052] Spray the control bactericides prepared in Example 1, Example 2 and Example 3 on tomato, kiwifruit and grape plants. Use the plants without any control as the blank group for comparison. After 12 months of use, count the infection rate of the main diseases. The control effects on the main easily infected diseases are shown in Table 3.

[0053] Table 3:

[0054] It can be seen that the control bactericide prepared by the present invention has excellent control effects on various diseases such as fungi and bacteria.

Claims

1. A preparation method of a plant disease control bacterial agent, characterized in that, The steps are as follows: (1) Take traditional Chinese medicine with antibacterial effect as raw material, crush it through a 30-mesh sieve to obtain traditional Chinese medicine powder, then add corn flour, glucose, peptone, and soybean meal, mix evenly, and sterilize to obtain a mixed powder; (2) Add purified water and Trichoderma harzianum Trichoderma harzianum ) to the mixed powder for the first fermentation. After the fermentation is completed, collect the fermentation broth 1 and the filter cake 1; (3) Add Beauveria bassiana ( Beauveria bassiana ), Penicillium bilaiae ( Penicillium bilaiae ) and purified water to the filter cake 1 for the second fermentation. After the fermentation is completed, collect the fermentation broth 2 and the filter cake 2; (4) Add Bacillus velezensis ( Bacillus velezensis ) and purified water to the filter cake 2, mix evenly, and carry out the third fermentation. After the fermentation is completed, collect the fermentation broth 3 and the filter cake 3; (5) Take Filter Cake 3, add an ethanol solution, reflux and extract. After the extraction is completed, discard the medicinal residues, and collect the extract as Filtrate 4; (6) Combine Filtrate 1, Filtrate 2, Filtrate 3, and Filtrate 4 to obtain a fermentation extract, and concentrate it under reduced pressure to obtain a clear paste; (7) Add an ethanol solution to the clear paste, let it stand for 24 - 36 h, take the supernatant, add borneol, gelatin, and cellulose acetate to form a mixed solution, then perform ultrafiltration, and collect the ultrafiltrate to obtain the bacterial agent required by the present invention.

2. The preparation method of a plant disease control bacterial agent as described in claim 1, characterized in that: In the step (2), the temperature is 30~35°C, fermentation lasts for 12~15 days, the air flow rate of the fermentation tank is maintained at 1~1.5 m 3 / h, and the mass ratio of the mixed powder, Trichoderma harzianum, and purified water is 100:0.2~0.3:50~60.

3. The preparation method of a plant disease control bacterial agent according to claim 1 or 2, characterized in that: In the step (3), the fermentation temperature is 30-35°C, the fermentation lasts for 8-10 days, and the air flow rate in the fermentation tank is maintained at 1-1.5 m 3 / h. The mass ratio of filter cake 1, Beauveria bassiana, Beauveria brongniartii, and purified water is 100:0.1-0.2:0.2-0.3:30-40.

4. The preparation method of a plant disease control bacterial agent according to any one of claims 1-3, characterized in that: In the step (4), the fermentation temperature is 25-30°C, the fermentation lasts for 7-9 days, and the air flow rate in the fermentation tank is maintained at 1.5-2 m 3 / h. The mass ratio of filter cake 2, Bacillus velezensis mold, and purified water is 100:0.1-0.2:30-40.

5. The preparation method of a plant disease control bactericide according to claim 4, characterized in that: In step (5), take Filter Cake 3, add an ethanol solution with a volume fraction of 50% - 60% according to the mass ratio of Filter Cake 3 to the ethanol solution of 1:10 - 15, reflux and extract twice. The first extraction is for 2 - 2.5 h, and the second extraction is for 1.5 - 2 h. After the extraction is completed, discard the medicinal residues, combine the two extraction solutions to obtain Filtrate 4.

6. The preparation method of a plant disease control bacterial agent according to claim 5, characterized in that: In step (6), the vacuum degree is -0.05 to -0.08 MPa, the temperature is 45 - 50 °C, and it is concentrated under reduced pressure to a clear paste with a relative density of 1.10 - 1.

15.

7. The preparation method of a plant disease control bacterial agent according to claim 5 or 6, characterized in that: In step (7), add an ethanol solution with a volume fraction of 70% - 75% to the clear paste, and the mass ratio of the clear paste to the ethanol is 1:20 - 25.

8. A preparation method of a plant disease control bacterial agent according to any one of claims 5-7, characterized in that: Add 12 - 15 parts by weight of borneol, 6 - 8 parts by weight of gelatin, and 2 - 5 parts by weight of cellulose acetate in formula amounts to form a mixed solution, heat it to 40 - 45 °C, stir and disperse evenly, cool it to 20 - 35 °C, then place it in a ceramic membrane with a molecular cut-off of 10,000 - 20,000 for ultrafiltration treatment. The ultrafiltration pressure is 0.3 - 0.5 MPa, the temperature is 25 - 35 °C. After the ultrafiltration is completed, collect the ultrafiltrate to obtain the bacterial agent required by the present invention.

9. A preparation method of a plant disease control bacterial agent, characterized in that, It includes the following steps: Formula: Take 20 - 30 parts by weight of Gleditsia sinensis Thorn, 15 - 20 parts by weight of Smilax glabra, 10 - 15 parts by weight of soybean meal, 20 - 30 parts by weight of corn flour, 8 - 12 parts by weight of Phellodendron amurense bark, 15 - 20 parts by weight of glucose, 8 - 10 parts by weight of Cnidium monnieri, 15 - 20 parts by weight of corn starch, 6 - 8 parts by weight of peptone, 12 - 15 parts by weight of borneol, 6 - 8 parts by weight of gelatin, and 2 - 5 parts by weight of cellulose acetate; (1) Take Gleditsia sinensis Thorn, Smilax glabra, Phellodendron amurense bark, and Cnidium monnieri, mix them, place them in a universal grinder, crush them through a 30-mesh sieve to obtain medicinal powder, add corn flour, glucose, peptone, and soybean meal to the medicinal powder, mix evenly, and sterilize at 121 °C for 15 min to obtain a mixed powder; (2)In a sterile environment, add the mixed powder prepared in step (1) into a fermenter, add purified water, mix evenly, add Trichoderma harzianum, stir evenly, keep the temperature at 30 - 35 °C, ferment for 12 - 15 days, maintain the air flow rate in the fermenter at 1 - 1.5 m 3 / h. After fermentation is completed, sterilize, filter after sterilization, and separately collect filtrate 1 and filter cake 1. The mass ratio of the mixed powder, Trichoderma harzianum, and purified water is 100:0.2 - 0.3:50 - 60; (3)In a sterile environment, Beauveria bassiana, Penicillium bilaiae and purified water are added to the filter cake 1 and mixed evenly. The fermentation temperature is 30-35°C, and the fermentation lasts for 8-10 days. The air flow rate in the fermentation tank is maintained at 1-1.5 m 3 / h. After the fermentation is completed, sterilization is carried out. After sterilization, filtration is performed, and the filtrate 2 and the filter cake 2 are collected separately. The mass ratio of the filter cake 1, Beauveria bassiana, Penicillium bilaiae and purified water is 100:0.1-0.2:0.2-0.3:30-40; (4)In a sterile environment, add Bacillus velezensis and purified water to the filter cake 2 and mix evenly. Then place them together in a fermenter. The fermentation temperature is 25~30 °C, and the fermentation lasts for 7~9 days. Keep the air flow rate in the fermenter at 1.5~2 m 3 / h. After fermentation, sterilize, and then filter. Collect filter cake 3 and filtrate 3 respectively. The mass ratio of the filter cake 2, Bacillus velezensis, and purified water is 100:0.1~0.2:30~40; (5) Take Filter Cake 3, add an ethanol solution with a volume fraction of 50% - 60% according to the mass ratio of Filter Cake 3 to the ethanol solution of 1:10 - 15, reflux and extract twice. The first extraction is for 2 - 2.5 h, and the second extraction is for 1.5 - 2 h. After the extraction is completed, discard the medicinal residues, combine the two extraction solutions to obtain Filtrate 4; (6) Combine filtrate 1, filtrate 2, filtrate 3 and filtrate 4 to obtain a fermentation mixture, place it in a vacuum concentration tank, set the vacuum degree to -0.05~-0.08 MPa and the temperature to 45~50 °C, and concentrate it under reduced pressure to a clear paste with a relative density of 1.10~1.15; (7) Add an ethanol solution with a volume fraction of 70%~75% to the clear paste, with a mass ratio of the clear paste to ethanol of 1:20~25. Stir evenly, let it stand for 24~36 h, take the supernatant, add the formula amounts of borneol, gelatin and cellulose acetate to form a mixture, add cetyltrimethylammonium bromide accounting for 0.1%~0.2% of the mass of the mixture, heat up to 40~45 °C, stir to make it evenly dispersed, cool it to 20~35 °C, and then place it in a ceramic membrane with a molecular cut-off of 10000~20000 for ultrafiltration treatment. The ultrafiltration pressure is 0.3~0.5 MPa and the temperature is 25~35 °C. After ultrafiltration is completed, collect the ultrafiltrate to obtain the target control bactericide.

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