CN113980849A - Leek disease control composite bacterium and preparation method thereof - Google Patents
Leek disease control composite bacterium and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention provides a leek disease control composite bacterium, which comprises the following components in parts by mass: 6.5-7.5 parts of paecilomyces lilacinus dry powder, 3.5-4.5 parts of bacillus licheniformis dry powder, 1.8-2.2 parts of diketene grafted sodium carboxymethylcellulose, 0.8-1.2 parts of chain polyethylene glycol, 1.8-2.2 parts of sodium borate buffer solution, 0.8-1.2 parts of alginic acid, 0.8-1.2 parts of sodium alginate, 0.8-1.2 parts of sucrose and 43-47 parts of water. The invention also provides a preparation method of the leek disease control composite bacterium, which comprises the steps of preparing diketene grafted sodium carboxymethylcellulose, preparing chain polyethylene glycol and preparing the composite bacterium. The composite bacteria prepared by the invention has stable prevention and treatment effects, and has small effect difference among multiple batches of application batches.
Description
Technical Field
The invention relates to a leek disease control composite bacterium and a preparation method thereof, belonging to the technical field of crop disease control.
Background
Leek (A. tuber stem rot. ex Spreng.) belongs to perennial herb plants of liliaceae, has special strong smell, is cultivated in all parts of China, is one of fragrant and pungent leaf vegetables with the widest production range in China, is rich in nutrition, contains various vitamins and inorganic salts such as calcium, phosphorus, iron and the like, and has more crude fiber and protein content. The leek has aromatic smell, is not only suitable for stir-frying, making stuffing and seasoning, but also has good medical function and is deeply loved by people.
The diseases of the Chinese chives mainly comprise Chinese chive gray mold, Chinese chive white fungus, Chinese chive white spot and the like, the main control method in the prior art is chemical drug control, and the problems of environmental pollution, drug resistance and the like caused by chemical drugs are increasingly prominent.
CN111990402A discloses a new application of lipopeptide or lipopeptide extract, wherein the control effect is achieved by a single strain through the bacillus subtilis and the extract thereof, and the control effect is single because the type of the disease is only leek gray mold.
At present, no composite bacteria for preventing and treating various diseases of Chinese chives exist on the market, the comprehensive prevention and treatment effect can be achieved by preparing the composite bacteria, the liquid composite bacteria is more convenient to use than the solid composite bacteria, but the prevention and treatment effect is unstable, and the storage time of the liquid composite bacteria is short, so that the content of effective microorganisms is reduced after the liquid composite bacteria are stored for a long time, and the prevention and treatment effect is reduced.
CN1331534C discloses a compound bacterin for preventing and treating diseases of aquaculture animals, which limits the propagation of mixed bacteria by adding a certain amount of antiseptic benzyl alcohol to prolong the storage date, but simultaneously causes the reduction of effective bacteria activity.
In conclusion, the existing composite bacteria have the following disadvantages:
(1) the prevention and treatment effect of the liquid composite bacteria in the prior art is unstable, and the effect difference between multiple batches of liquid composite bacteria is large;
(2) the preservative is used for ensuring the storage date of the compound bacteria and limiting the propagation of the mixed bacteria, but the control effect of the effective bacteria is reduced.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art, and the following aims are achieved by adding the grafted sodium carboxymethyl cellulose and the chained polyethylene glycol to prepare the composite bacteria:
(1) the liquid compound bacterium has stable control effect, and the effect difference between multiple batches of application batches is small;
(2) the storage date of the compound bacteria is ensured without using a preservative, and the reduction of the control effect of effective bacteria is avoided while the propagation of mixed bacteria is limited.
In order to solve the technical problems, the invention adopts the following technical scheme:
the composite bacterium for preventing and treating the disease of the Chinese chives comprises the following components in parts by mass: 6.5-7.5 parts of paecilomyces lilacinus dry powder, 3.5-4.5 parts of bacillus licheniformis dry powder, 1.8-2.2 parts of diketene grafted sodium carboxymethylcellulose, 0.8-1.2 parts of chain polyethylene glycol, 1.8-2.2 parts of sodium borate buffer solution, 0.8-1.2 parts of alginic acid, 0.8-1.2 parts of sodium alginate, 0.8-1.2 parts of sucrose and 43-47 parts of water.
The paecilomyces lilacinus dry powder is prepared by culturing paecilomyces lilacinus by a conventional method and preparing into dry powder, wherein the number of viable bacteria is 4.1-4.3 x 1011 CFU /g；
The Bacillus licheniformis dry powder is prepared by culturing Bacillus licheniformis by conventional method, and the viable count is 2.4-2.6 x 1011 CFU /g。
The Paecilomyces lilacinus (Paecilomyces lilacinus) is obtained by market with the strain deposit number of ACCC 30636;
the Bacillus licheniformis (Bacillus licheniformis) is deposited under the accession number ACCC 19747 and is obtained by market purchase.
The following is a further improvement of the above technical solution:
the preparation method comprises the steps of preparing the diketene-grafted sodium carboxymethyl cellulose, preparing the chain polyethylene glycol and preparing the composite bacteria.
The preparation method comprises the steps of preparing diketene-grafted sodium carboxymethylcellulose, mixing sodium carboxymethylcellulose with deionized water, heating to 70-75 ℃, stirring for 3-4h at 1200r/min under the heat preservation state until the sodium carboxymethylcellulose is completely dissolved to obtain sodium carboxymethylcellulose solution, cooling to room temperature after complete dissolution, adjusting the pH to 7.1-7.3 by using hydrochloric acid solution with the mass concentration of 4-6%, then putting the sodium carboxymethylcellulose solution into a condensation reflux device, carrying out ultrasonic treatment for 25-35min under the power of 50-70W, introducing nitrogen for protection during the period, keeping the temperature at 48-52 ℃ under the ultrasonic treatment, then adding potassium permanganate solution and sulfuric acid solution, slowly adding the potassium permanganate solution and the sulfuric acid solution within 4-6min, stirring for 15-30min under the power of 600 + 1000r/min after the addition is finished, adding monomer diketene after the stirring is finished, reacting for 5-7h at the temperature of 350r/min under 250-52 ℃, keeping the temperature at 48-52 ℃ during the reaction, cooling to room temperature after the reaction is finished, washing with ethanol, filtering, washing with deionized water, and drying in vacuum to constant weight to obtain the diketene-grafted sodium carboxymethyl cellulose.
The molecular weight of the sodium carboxymethyl cellulose is 80000-90000;
the mass ratio of the sodium carboxymethyl cellulose to the water in the sodium carboxymethyl cellulose solution is 1: 75-85;
the concentration of the potassium permanganate solution is 0.08-0.12 mol/L;
the mass concentration of the sulfuric acid solution is 8-12%;
the mass ratio of the potassium permanganate solution to the sulfuric acid solution to the sodium methyl cellulose solution is 4-6: 1-2: 1850-;
the mass ratio of the monomer diketene to the sodium methyl cellulose solution is 1: 480-520.
The preparation method comprises the steps of mixing tetramethylpiperidine oxynitride, sodium bromide and deionized water, stirring at the speed of 500-700r/min until the mixture is completely dissolved, adding polyethylene glycol and sodium hypochlorite solution to react at the speed of 250-350r/min for 20-30min after the dissolution is finished, adding ethanol solution, adjusting the pH value to 1.6-1.8 by using hydrochloric acid solution, extracting by using dichloromethane for three times, mixing the lower layer clear liquid after the three-time extraction, dissolving in hot ethanol, keeping at the temperature of-5-15 ℃ for 14-20h, taking out, and performing vacuum drying at the temperature of 38-42 ℃ to obtain the chain-shaped polyethylene glycol.
8. The method for preparing leek disease control composite bacteria according to claim 7, wherein the method comprises the following steps:
the mass ratio of the tetramethylpiperidine oxynitride to the sodium bromide to the deionized water is 1:1: 480-520;
the effective chlorine content of the sodium hypochlorite solution is 10.2%;
the mass ratio of the added polyethylene glycol to the sodium hypochlorite solution to the deionized water is 1:1: 4.5-5.5;
the mass concentration of the ethanol solution is 99.5%;
the mass ratio of the ethanol solution to the deionized water is 1: 4.5-5.5.
The preparation method of the composite bacteria comprises the step of mixing paecilomyces lilacinus dry powder, bacillus licheniformis dry powder, diketene grafted sodium carboxymethylcellulose, chain polyethylene glycol, a sodium borate buffer solution, alginic acid, sodium alginate, sucrose and water to obtain the composite bacteria.
The effective microorganism content of the compound bacteria is 2.9-3.1 x 1010CFU/mL。
Compared with the prior art, the invention has the following beneficial effects:
the composite bacteria prepared by the invention has stable control effect, the effect difference between multiple batches of use batches is small, the Chinese chives are grown and transplanted in different plots of the same soil environment in batches, the total amount of the Chinese chives is five mu, each mu is a batch and is divided into A, B, C, D, E batches, the composite bacteria are diluted to 300 times and are sprayed on leaves in the seventh day after the Chinese chives are transplanted, the composite bacteria are used for 300 times of liquid 5kg in each mu, the liquid is sprayed once every ten days, the liquid is sprayed for 4 times in total, the rest normal field fertilization management is carried out, and the disease incidence of the Chinese chives in the A batch is as follows: 1.89-1.99% of leek gray mold, 2.31-2.35% of leek white fungus disease, 2.17-2.21% of leek leukoderma, and the incidence of leeks in batch B is as follows: 1.91-1.85% of leek gray mold, 2.22-2.30% of leek white fungus disease, 2.23-2.28% of leek leukoderma, and the incidence of leeks in batch C is as follows: 1.81-1.89% of leek gray mold, 2.29-2.39% of leek white fungus disease, 2.25-2.33% of leek leukoderma, and the morbidity of the leeks in batch D is as follows: 1.95-2.02% of leek gray mold, 2.22-2.31% of leek white fungus disease, 2.17-2.20% of leek leukoderma, and the morbidity of leeks in batch E is as follows: 2.06-2.11% of leek gray mold, 2.25-2.35% of leek white fungus and 2.27-2.35% of leek leukoderma;
the composite bacteria prepared by the invention can still maintain the control effect after being stored for a long time, and the effective microorganism content is 2.2-2.3 x 10 after being stored for 6 months at normal temperature10CFU/mL, effective microorganism content of 89.8-90.2%, and applying to folium Allii tuberosi with incidence rate of 2.15-2.22%, 2.41-2.52%, and 2.33-2.44%, wherein the effective microorganism content is 1.8-1.9 × 10 after 12 months of storage at room temperature10CFU/mL, the effective microorganism content is 73.6-74.2%, and the disease rate of the Chinese chives is 3.28-3.41% of the gray mold of the Chinese chives, 3.56-3.67% of the white fungus disease of the Chinese chives and 3.37-3.42% of the white spot disease of the Chinese chives.
Detailed Description
Example 1
(1) Preparation of diketene-grafted sodium carboxymethylcellulose
Mixing sodium carboxymethylcellulose with deionized water, heating to 72 ℃, stirring for 3.5h at 1000r/min under a heat preservation state until the sodium carboxymethylcellulose is completely dissolved to obtain a sodium carboxymethylcellulose solution, cooling to room temperature after complete dissolution, adjusting the pH to 7.2 by using a hydrochloric acid solution with the mass concentration of 5%, then putting the sodium carboxymethylcellulose solution into a condensation reflux device, performing ultrasonic treatment at the power of 60W for 30min, introducing nitrogen for protection, maintaining the temperature at 50 ℃ under ultrasonic treatment, then adding a potassium permanganate solution and a sulfuric acid solution, slowly adding the potassium permanganate solution and the sulfuric acid solution within 5min, stirring for 20min at the speed of 800r/min after the addition is completed, adding monomer diketene after the stirring is completed, reacting for 6h at the speed of 300r/min, maintaining the temperature at 50 ℃ during the reaction, cooling to room temperature after the reaction is completed, washing with ethanol, performing suction filtration, finally washing with deionized water, performing vacuum drying to constant weight, obtaining the sodium carboxymethyl cellulose grafted by ketene dimer;
the molecular weight of the sodium carboxymethyl cellulose is 85000;
the mass ratio of the sodium carboxymethyl cellulose to the water in the sodium carboxymethyl cellulose solution is 1: 80;
the concentration of the potassium permanganate solution is 0.1 mol/L;
the mass concentration of the sulfuric acid solution is 10 percent;
the mass ratio of the potassium permanganate solution to the sulfuric acid solution to the sodium methyl cellulose solution is 5: 1: 2000;
the mass ratio of the monomer diketene to the sodium methyl cellulose solution is 1: 500.
(2) Preparation of chain polyethylene glycol
Mixing tetramethylpiperidine nitrogen oxide, sodium bromide and deionized water, stirring at 600r/min until the mixture is completely dissolved, adding polyethylene glycol and sodium hypochlorite solution to react for 25min at 300r/min after the dissolution is finished, adding ethanol solution to stop oxidation, adjusting the pH to 1.7 by using hydrochloric acid solution, extracting by using dichloromethane, repeating for three times, mixing the lower layer clear liquid after the three-time extraction, dissolving in hot ethanol, keeping the hot ethanol at-10 ℃ for 16h, taking out, and performing vacuum drying at 40 ℃ to obtain chain-shaped polyethylene glycol;
the mass ratio of the tetramethylpiperidine oxynitride to the sodium bromide to the deionized water is 1:1: 500;
the effective chlorine content of the sodium hypochlorite solution is 10.2%;
the mass ratio of the added polyethylene glycol to the sodium hypochlorite solution to the deionized water is 1:1: 5;
the mass concentration of the ethanol solution is 99.5%;
the mass ratio of the ethanol solution to the deionized water is 1:5.
(3) Preparation of composite bacteria
The composite bacteria comprise the following components in parts by mass: 7 parts of paecilomyces lilacinus dry powder, 4 parts of bacillus licheniformis dry powder, 2 parts of diketene grafted sodium carboxymethylcellulose, 1 part of chain polyethylene glycol, 2 parts of sodium borate buffer solution, 1 part of alginic acid, 1 part of sodium alginate, 1 part of sucrose and 45 parts of water;
the preparation method of the composite bacteria comprises the steps of mixing paecilomyces lilacinus dry powder, bacillus licheniformis dry powder, diketene grafted sodium carboxymethylcellulose, chain polyethylene glycol, a sodium borate buffer solution, alginic acid, sodium alginate, sucrose and water to obtain the composite bacteria;
the paecilomyces lilacinus dry powder is prepared by using paecilomyces lilacinusCulturing by conventional method, and making into dry powder with viable bacteria count of 4.2 x 1011 CFU /g；
The bacillus licheniformis dry powder is prepared by culturing bacillus licheniformis by a conventional method and preparing into dry powder, wherein the number of viable bacteria is 2.5 x 1011 CFU /g；
The Paecilomyces lilacinus (Paecilomyces lilacinus) is obtained by market with the strain deposit number of ACCC 30636;
the Bacillus licheniformis (Bacillus licheniformis) is obtained by the market with the strain deposit number of ACCC 19747;
the effective microorganism content of the compound bacteria is 3.1 x 1010CFU/mL；
The compound bacteria prepared in the embodiment 1 has stable control effect, the effect difference between multiple batches of the compound bacteria is small, the Chinese chives are grown, the Chinese chives are transplanted in different plots of the same soil environment in batches, the total amount is five acres, each acre is a batch, the Chinese chives are divided into A, B, C, D, E batches, the seventh day after the Chinese chives are transplanted, the compound bacteria are diluted to 300 times and are sprayed on leaves, the compound bacteria are used for 300 times of liquid 5kg per acre, the compound bacteria are sprayed once every ten days, the compound bacteria are sprayed for 4 times in total, the rest normal field fertilization management is carried out, and the disease incidence of the Chinese chives in A batch is as follows: 1.89% of leek gray mold, 2.35% of leek white fungus and 2.17% of leek leukoderma, wherein the incidence of leeks in batch B is as follows: 1.92% of grey mould disease of leeks, 2.29% of white fungus disease of leeks and 2.23% of white spot disease of leeks, wherein the morbidity of leeks in batch C is as follows: 1.81% of gray mold of Chinese chives, 2.39% of white fungus disease of the Chinese chives and 2.25% of white spot disease of the Chinese chives, wherein the disease rate of the Chinese chives in batch D is as follows: 1.95% of gray mold of leeks, 2.31% of white fungus diseases of leeks and 2.19% of white spot diseases of leeks, wherein the morbidity of leeks in batch E is as follows: 2.06% of gray mold of Chinese chives, 2.25% of white fungus disease of Chinese chives and 2.31% of white spot disease of Chinese chives;
the composite bacteria prepared in example 1 can still maintain the control effect after being stored for a long time, and the effective microorganism content is 2.3 x 10 after being stored for 6 months at normal temperature10CFU/mL, effective microorganism content of 90.2%, applied to folium Allii tuberosi with incidence rate of 2.15% of gray mold, 2.41% of white fungus and 2.33% of white spot, and effective microorganism content of 1.9 x 10 after 12 months at room temperature10CFU/mL, the effective microorganism content is 74.2%, and the application is applied to the Chinese chives, and the Chinese chives have the disease rateComprises 3.28% of gray mold of Chinese chives, 3.56% of white fungus disease of Chinese chives and 3.37% of white spot disease of Chinese chives.
Example 2
(1) Preparation of diketene-grafted sodium carboxymethylcellulose
Mixing sodium carboxymethylcellulose with deionized water, heating to 70 ℃, stirring for 4h at 900r/min under a heat preservation state until the sodium carboxymethylcellulose is completely dissolved to obtain a sodium carboxymethylcellulose solution, cooling to room temperature after complete dissolution, adjusting the pH to 7.1 by using a hydrochloric acid solution with the mass concentration of 5%, then putting the sodium carboxymethylcellulose solution into a condensation reflux device, performing ultrasonic treatment at the power of 50W for 35min, introducing nitrogen for protection, maintaining the temperature at 48 ℃ under ultrasonic treatment, then adding a potassium permanganate solution and a sulfuric acid solution, slowly adding the potassium permanganate solution and the sulfuric acid solution within 4min, stirring for 30min at 600r/min after the addition is completed, adding monomer diketene after the stirring is completed, reacting for 7h at 250r/min, maintaining the temperature at 48 ℃ during the period, cooling to room temperature after the reaction is completed, washing with ethanol, performing suction filtration, finally washing with deionized water, and performing vacuum drying to constant weight, obtaining the sodium carboxymethyl cellulose grafted by ketene dimer;
the molecular weight of the sodium carboxymethyl cellulose is 80000;
the mass ratio of the sodium carboxymethyl cellulose to the water in the sodium carboxymethyl cellulose solution is 1: 75;
the concentration of the potassium permanganate solution is 0.08 mol/L;
the mass concentration of the sulfuric acid solution is 8%;
the mass ratio of the potassium permanganate solution to the sulfuric acid solution to the sodium methyl cellulose solution is 4: 1: 1850;
the mass ratio of the monomer diketene to the sodium methyl cellulose solution is 1: 480.
(2) Preparation of chain polyethylene glycol
Mixing tetramethylpiperidine nitrogen oxide, sodium bromide and deionized water, stirring at 500r/min until the mixture is completely dissolved, adding polyethylene glycol and sodium hypochlorite solution to react at 250r/min for 30min after the dissolution is finished, adding ethanol solution to stop oxidation, adjusting the pH to 1.8 by using hydrochloric acid solution, extracting by using dichloromethane, repeating for three times, mixing the lower layer clear liquid after the three-time extraction, dissolving in hot ethanol, keeping at-15 ℃ for 14h, taking out, and performing vacuum drying at 38 ℃ to obtain chain-shaped polyethylene glycol;
the mass ratio of the tetramethylpiperidine oxynitride to the sodium bromide to the deionized water is 1:1: 480;
the effective chlorine content of the sodium hypochlorite solution is 10.2%;
the mass ratio of the added polyethylene glycol to the sodium hypochlorite solution to the deionized water is 1:1: 4.5;
the mass concentration of the ethanol solution is 99.5%;
the mass ratio of the ethanol solution to the deionized water is 1: 4.5.
(3) Preparation of composite bacteria
The composite bacteria comprise the following components in parts by mass: 6.5 parts of paecilomyces lilacinus dry powder, 3.5 parts of bacillus licheniformis dry powder, 1.8 parts of diketene grafted sodium carboxymethylcellulose, 0.8 part of chain polyethylene glycol, 1.8 parts of sodium borate buffer solution, 0.8 part of alginic acid, 0.8 part of sodium alginate, 0.8 part of cane sugar and 43 parts of water;
the preparation method of the composite bacteria comprises the steps of mixing paecilomyces lilacinus dry powder, bacillus licheniformis dry powder, diketene grafted sodium carboxymethylcellulose, chain polyethylene glycol, a sodium borate buffer solution, alginic acid, sodium alginate, sucrose and water to obtain the composite bacteria;
the paecilomyces lilacinus dry powder is prepared by culturing paecilomyces lilacinus by a conventional method and preparing into dry powder, wherein the number of viable bacteria is 4.3 to 1011 CFU /g；
The bacillus licheniformis dry powder is prepared by culturing bacillus licheniformis by a conventional method and preparing into dry powder, wherein the number of viable bacteria is 2.6 x 1011 CFU /g；
The Paecilomyces lilacinus (Paecilomyces lilacinus) is obtained by market with the strain deposit number of ACCC 30636;
the Bacillus licheniformis (Bacillus licheniformis) is obtained by the market with the strain deposit number of ACCC 19747;
the effective microorganism content of the compound bacteria is 2.9 x 1010CFU/mL；
The compound bacteria prepared in the embodiment 2 has stable control effect, the effect difference between multiple batches of the compound bacteria is small, the Chinese chives are grown, the Chinese chives are transplanted in different plots of the same soil environment in batches, the total amount is five acres, each acre is a batch, the Chinese chives are divided into A, B, C, D, E batches, the seventh day after the Chinese chives are transplanted, the compound bacteria are diluted to 300 times and are sprayed on leaves, the compound bacteria are used for 300 times of liquid 5kg per acre, the compound bacteria are sprayed once every ten days, the compound bacteria are sprayed for 4 times in total, the rest normal field fertilization management is carried out, and the disease incidence of the Chinese chives in A batch is as follows: 1.92% of grey mould disease of leeks, 2.33% of white fungus disease of leeks and 2.21% of white spot disease of leeks, wherein the incidence rate of the leeks in batch B is as follows: 1.91% of leek gray mold, 2.22% of leek white fungus and 2.28% of leek leukoderma, wherein the incidence of leeks in batch C is as follows: 1.89% of grey mould disease of leeks, 2.35% of white fungus disease of leeks and 2.33% of white spot disease of leeks, wherein the disease rate of the leeks in batch D is as follows: 1.97% of grey mould disease of leeks, 2.38% of white fungus disease of leeks and 2.20% of white spot disease of leeks, wherein the morbidity of leeks in batch E is as follows: 2.08 percent of gray mold of Chinese chives, 2.27 percent of white fungus disease of the Chinese chives and 2.27 percent of white spot disease of the Chinese chives;
the composite bacteria prepared in example 2 can still maintain the control effect after being stored for a long time, and the effective microorganism content is 2.2 x 10 after being stored for 6 months at normal temperature10CFU/mL, effective microorganism content of 89.8%, applied to folium Allii tuberosi with incidence rate of 2.22% of gray mold, 2.49% of white fungus and 2.41% of white spot, and effective microorganism content of 1.8 × 10 after 12 months at room temperature10CFU/mL, the effective microorganism content is 73.7%, and the application is applied to the leeks, wherein the morbidity of the leeks is 3.35% of leek gray mold, 3.67% of leek white fungus and 3.42% of leek white spot.
Example 3
(1) Preparation of diketene-grafted sodium carboxymethylcellulose
Mixing sodium carboxymethylcellulose with deionized water, heating to 75 ℃, stirring for 3h at 1200r/min under a heat preservation state until the sodium carboxymethylcellulose is completely dissolved to obtain a sodium carboxymethylcellulose solution, cooling to room temperature after complete dissolution, adjusting the pH to 7.3 by using a hydrochloric acid solution with the mass concentration of 5%, then putting the sodium carboxymethylcellulose solution into a condensation reflux device, performing ultrasonic treatment at the power of 70W for 25min, introducing nitrogen for protection, maintaining the temperature at 52 ℃ under ultrasonic treatment, then adding a potassium permanganate solution and a sulfuric acid solution, slowly adding the potassium permanganate solution and the sulfuric acid solution within 6min, stirring for 15min at 1000r/min after the addition is completed, adding monomer diketene after the stirring is completed, reacting for 5h at 350r/min, maintaining the temperature at 52 ℃ during the period, cooling to room temperature after the reaction is completed, washing with ethanol, performing suction filtration, finally washing with deionized water, and performing vacuum drying to constant weight, obtaining the sodium carboxymethyl cellulose grafted by ketene dimer;
the molecular weight of the sodium carboxymethyl cellulose is 90000;
the mass ratio of the sodium carboxymethyl cellulose to the water in the sodium carboxymethyl cellulose solution is 1: 85;
the concentration of the potassium permanganate solution is 0.12 mol/L;
the mass concentration of the sulfuric acid solution is 12%;
the mass ratio of the potassium permanganate solution to the sulfuric acid solution to the sodium methyl cellulose solution is 6: 2: 2150;
the mass ratio of the monomer diketene to the sodium methyl cellulose solution is 1: 520.
(2) Preparation of chain polyethylene glycol
Mixing tetramethylpiperidine nitrogen oxide, sodium bromide and deionized water, stirring at 700r/min until the mixture is completely dissolved, adding polyethylene glycol and sodium hypochlorite solution to react for 20min at 350r/min after the dissolution is finished, adding ethanol solution to stop oxidation, adjusting the pH to 1.6 by using hydrochloric acid solution, extracting by using dichloromethane, repeating for three times, mixing the lower layer clear liquid after the three-time extraction, dissolving in hot ethanol, keeping the hot ethanol at the temperature of-5 ℃ for 20h, taking out, and performing vacuum drying at the temperature of 42 ℃ to obtain chain-shaped polyethylene glycol;
the mass ratio of the tetramethylpiperidine oxynitride to the sodium bromide to the deionized water is 1:1: 520;
the effective chlorine content of the sodium hypochlorite solution is 10.2%;
the mass ratio of the added polyethylene glycol to the sodium hypochlorite solution to the deionized water is 1:1: 5.5;
the mass concentration of the ethanol solution is 99.5%;
the mass ratio of the ethanol solution to the deionized water is 1: 5.5.
(3) Preparation of composite bacteria
The composite bacteria comprise the following components in parts by mass: 7.5 parts of paecilomyces lilacinus dry powder, 4.5 parts of bacillus licheniformis dry powder, 2.2 parts of diketene grafted sodium carboxymethylcellulose, 1.2 parts of chain polyethylene glycol, 2.2 parts of sodium borate buffer solution, 1.2 parts of alginic acid, 1.2 parts of sodium alginate, 1.2 parts of sucrose and 47 parts of water;
the preparation method of the composite bacteria comprises the steps of mixing paecilomyces lilacinus dry powder, bacillus licheniformis dry powder, diketene grafted sodium carboxymethylcellulose, chain polyethylene glycol, a sodium borate buffer solution, alginic acid, sodium alginate, sucrose and water to obtain the composite bacteria;
the paecilomyces lilacinus dry powder is prepared by culturing paecilomyces lilacinus by a conventional method and preparing into dry powder, wherein the number of viable bacteria is 4.1 to 1011 CFU /g；
The bacillus licheniformis dry powder is prepared by culturing bacillus licheniformis by a conventional method and preparing into dry powder, wherein the number of viable bacteria is 2.4 x 1011 CFU /g；
The Paecilomyces lilacinus (Paecilomyces lilacinus) is obtained by market with the strain deposit number of ACCC 30636;
the Bacillus licheniformis (Bacillus licheniformis) is obtained by the market with the strain deposit number of ACCC 19747;
the effective microorganism content of the compound bacteria is 3.0 x 1010CFU/mL；
The compound bacteria prepared in the embodiment 3 has stable control effect, the effect difference between multiple batches of the compound bacteria is small, the Chinese chives are grown, the Chinese chives are transplanted in different plots of the same soil environment in batches, the total amount is five acres, each acre is a batch, the Chinese chives are divided into A, B, C, D, E batches, the seventh day after the Chinese chives are transplanted, the compound bacteria are diluted to 300 times and are sprayed on leaves, the compound bacteria are used for 300 times of liquid 5kg per acre, the compound bacteria are sprayed once every ten days, the compound bacteria are sprayed for 4 times in total, the rest normal field fertilization management is carried out, and the disease incidence of the Chinese chives in A batch is as follows: 1.99% of grey mould disease of leeks, 2.31% of white fungus disease of leeks and 2.19% of white spot disease of leeks, wherein the incidence rate of the leeks in batch B is as follows: 1.95% of gray mold of leeks, 2.30% of white fungus diseases of leeks and 2.25% of white spot diseases of leeks, wherein the morbidity of leeks in batch C is as follows: 1.87% of gray mold of leeks, 2.29% of white fungus diseases of leeks and 2.29% of white spot diseases of leeks, wherein the morbidity of the leeks in batch D is as follows: 2.02% of gray mold of leeks, 2.22% of white fungus diseases of leeks and 2.17% of white spot diseases of leeks, wherein the morbidity of leeks in batch E is as follows: 2.11 percent of gray mold of Chinese chives, 2.35 percent of white fungus disease of the Chinese chives and 2.35 percent of white spot disease of the Chinese chives;
the composite bacteria prepared in example 3 can still maintain the control effect after being stored for a long time, and the effective microorganism content is 2.3 x 10 after being stored for 6 months at normal temperature10CFU/mL, effective microorganism content of 90.1%, applied to folium Allii tuberosi with incidence rate of 2.19%, 2.52% and 2.44%, and after 12 months at room temperature, effective microorganism content of 1.9 × 1010CFU/mL, the effective microorganism content is 73.6% of the original, and the application is applied to the leeks, and the morbidity of the leeks is 3.41% of leek gray mold, 3.62% of leek white fungus and 3.41% of leek white spot.
Claims (10)
1. The leek disease control composite bacterium is characterized by comprising the following components in parts by mass: 6.5-7.5 parts of paecilomyces lilacinus dry powder, 3.5-4.5 parts of bacillus licheniformis dry powder, 1.8-2.2 parts of diketene grafted sodium carboxymethylcellulose, 0.8-1.2 parts of chain polyethylene glycol, 1.8-2.2 parts of sodium borate buffer solution, 0.8-1.2 parts of alginic acid, 0.8-1.2 parts of sodium alginate, 0.8-1.2 parts of sucrose and 43-47 parts of water.
2. The leek disease control composite bacterium according to claim 1, which is characterized in that:
the paecilomyces lilacinus dry powder is prepared by culturing paecilomyces lilacinus by a conventional method and preparing into dry powder, wherein the number of viable bacteria is 4.1-4.3 x 1011 CFU /g；
The Bacillus licheniformis dry powder is prepared by culturing Bacillus licheniformis by conventional method, and the viable count is 2.4-2.6 x 1011 CFU /g。
3. The leek disease control composite bacterium according to claim 2, wherein the leek disease control composite bacterium comprises:
the paecilomyces lilacinus is obtained by the market, and the strain preservation number is ACCC 30636;
the bacillus licheniformis is obtained by the market with the strain preservation number of ACCC 19747.
4. The preparation method of the leek disease control composite bacterium is characterized by comprising the steps of preparing diketene-grafted sodium carboxymethylcellulose, preparing chain polyethylene glycol and preparing the composite bacterium.
5. The method for preparing leek disease control composite bacteria according to claim 4, wherein the method comprises the following steps:
the preparation method comprises the steps of preparing diketene-grafted sodium carboxymethylcellulose, mixing sodium carboxymethylcellulose with deionized water, heating to 70-75 ℃, stirring for 3-4h at 1200r/min under the heat preservation state until the sodium carboxymethylcellulose is completely dissolved to obtain a sodium carboxymethylcellulose solution, cooling to room temperature, adjusting the pH to 7.1-7.3 by using a hydrochloric acid solution with the mass concentration of 4-6%, then putting the sodium carboxymethylcellulose solution into a condensation reflux device, carrying out ultrasonic treatment for 25-35min under the power of 50-70W, introducing nitrogen for protection during the ultrasonic treatment, keeping the temperature at 48-52 ℃ under the ultrasonic treatment, then adding a potassium permanganate solution and a sulfuric acid solution, slowly adding the potassium permanganate solution and the sulfuric acid solution within 4-6min, stirring for 15-30min at 1000r/min under the power of 600 + and adding diketene after the stirring is finished, adding monomer diketene after the stirring is finished, reacting for 5-7h at the temperature of 350r/min under 250-52 ℃, keeping the temperature at 48-52 ℃ during the reaction, cooling to room temperature after the reaction is finished, washing with ethanol, filtering, washing with deionized water, and drying in vacuum to constant weight to obtain the diketene-grafted sodium carboxymethyl cellulose.
6. The method for preparing leek disease control composite bacteria according to claim 5, wherein the method comprises the following steps:
the molecular weight of the sodium carboxymethyl cellulose is 80000-90000;
the mass ratio of the sodium carboxymethyl cellulose to the water in the sodium carboxymethyl cellulose solution is 1: 75-85;
the concentration of the potassium permanganate solution is 0.08-0.12 mol/L;
the mass concentration of the sulfuric acid solution is 8-12%;
the mass ratio of the potassium permanganate solution to the sulfuric acid solution to the sodium methyl cellulose solution is 4-6: 1-2: 1850-;
the mass ratio of the monomer diketene to the sodium methyl cellulose solution is 1: 480-520.
7. The method for preparing leek disease control composite bacteria according to claim 4, wherein the method comprises the following steps:
the preparation method comprises the steps of mixing tetramethylpiperidine oxynitride, sodium bromide and deionized water, stirring at the speed of 500-700r/min until the mixture is completely dissolved, adding polyethylene glycol and sodium hypochlorite solution to react at the speed of 250-350r/min for 20-30min after the dissolution is finished, adding ethanol solution, adjusting the pH value to 1.6-1.8 by using hydrochloric acid solution, extracting by using dichloromethane for three times, mixing the lower layer clear liquid after the three-time extraction, dissolving in hot ethanol, keeping at the temperature of-5-15 ℃ for 14-20h, taking out, and performing vacuum drying at the temperature of 38-42 ℃ to obtain the chain-shaped polyethylene glycol.
8. The method for preparing leek disease control composite bacteria according to claim 7, wherein the method comprises the following steps:
the mass ratio of the tetramethylpiperidine oxynitride to the sodium bromide to the deionized water is 1:1: 480-520;
the effective chlorine content of the sodium hypochlorite solution is 10.2%;
the mass ratio of the added polyethylene glycol to the sodium hypochlorite solution to the deionized water is 1:1: 4.5-5.5;
the mass concentration of the ethanol solution is 99.5%;
the mass ratio of the ethanol solution to the deionized water is 1: 4.5-5.5.
9. The method for preparing leek disease control composite bacteria according to claim 4, wherein the method comprises the following steps:
the preparation method of the composite bacteria comprises the step of mixing paecilomyces lilacinus dry powder, bacillus licheniformis dry powder, diketene grafted sodium carboxymethylcellulose, chain polyethylene glycol, a sodium borate buffer solution, alginic acid, sodium alginate, sucrose and water to obtain the composite bacteria.
10. The method for preparing a leek disease control composite bacterium according to claim 9, which is characterized in that:
the effective microorganism content of the compound bacteria is 2.9-3.1 x 1010CFU/mL。
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