Disease and pest resistant compound microbial agent and preparation method thereof

Abstract

The invention provides a compound microbial agent capable of resisting diseases and insect pests and a preparation method thereof, and the compound microbial agent consists of the following substances in parts by mass: 50-100 parts of bacillus subtilis, 40-80 parts of beauveria bassiana, 35-75 parts of trichoderma harzianum, 20-50 parts of modified straw powder, 10-15 parts of a humic acid-diatomite complex, 10-15 parts of trehalose, 5-10 parts of chitin powder and 5-10 parts of shellac. According to the invention, the modified straw powder and the humic acid-diatomite complex are adopted as a core carrier, so that the defects of poor adsorbability and nutrition deficiency of the traditional diatomite or synthetic carrier are overcome, and a high-porosity and nutrient-rich living environment is provided for bacillus subtilis, beauveria bassiana and trichoderma harzianum; compared with the prior art, the method provided by the invention has the advantages that the cost is low, the field planting rate and the field survival rate of viable bacteria in the microbial inoculum are remarkably improved, meanwhile, controllable coating is performed by utilizing an ethanol solution of shellac, a refractory synthetic polymer coating material is replaced, thalli are protected from environmental stress, and meanwhile, the risk of lasting residual pollution of a chemical coating material in soil is avoided.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a compound microbial agent for resisting diseases and pests and its preparation method. Background Technology

[0002] Compound microbial agents are live preparations made from two or more beneficial microorganisms (such as bacteria, fungi, and actinomycetes). In terms of pest and disease control, they mainly function through the following mechanisms: first, competition, where beneficial bacteria rapidly multiply and occupy ecological niches, crowding out the living space and nutrient resources of pathogens and pests; second, antagonism, where they secrete antibiotics, lysozymes, and other substances to directly inhibit or kill pathogenic fungi and bacteria; third, inducing systemic resistance in plants, enhancing the crop's own immunity; and fourth, some species (such as Bacillus thuringiensis) can produce insecticidal toxins, specifically poisoning and killing pest larvae. This biological control method can effectively reduce the use of chemical pesticides, reduce environmental pollution and pesticide residues, achieve green pest control, and promote sustainable agricultural development.

[0003] In existing technologies, compound microbial agents for disease and pest control have revealed significant shortcomings in actual field applications: traditional diatomaceous earth or bentonite carriers, due to their simple pore structure and poor surface hydrophilicity, result in insufficient adsorption rate of live bacteria (the number of live bacteria often decreases by more than 50% after 3 months of storage), severely weakening the effective bacterial population density after application; widely used synthetic polymer coatings (such as polyacrylates) degrade slowly in soil, and microplastic pollution caused by such coating residues has been detected in farmland in many places (e.g., the detection rate in soil samples from vegetable greenhouses in Shandong reached 32%); more importantly, the carriers lack functional nutrient matrices (such as chitin and humic acid), which cannot continuously activate the metabolic activity of the bacterial population, resulting in low colonization efficiency of the agent in crop roots (field trials show a colonization rate of <15%), unstable disease and pest control effects, and short-lasting effects, often requiring farmers to repeat the application, increasing costs. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a compound microbial agent for resisting diseases and pests and its preparation method, which solves the problem that existing traditional diatomaceous earth or bentonite carriers have insufficient adsorption rate of live bacteria due to their simple pore structure and poor surface hydrophilicity.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution: a compound microbial agent for resisting diseases and pests, comprising the following components by weight:

[0008] 50-100 parts of Bacillus subtilis, 40-80 parts of Beauveria bassiana, 35-75 parts of Trichoderma harzianum, 20-50 parts of modified straw powder, 10-15 parts of humic acid-diatomaceous earth complex, 10-15 parts of trehalose, 5-10 parts of chitin powder, and 5-10 parts of shellac.

[0009] Preferably, the humic acid-diatomite composite is formed by mixing humic acid and diatomite in a 1:1 ratio.

[0010] Preferably, the modified straw powder is obtained by crushing crop straw powder to 100 mesh and then soaking it in 5% citric acid for 2 hours.

[0011] Preferably, the trehalose is extracted from seaweed and purified.

[0012] Preferably, the chitin powder is made from shrimp and crab shells through deproteinization and decalcification treatment, followed by pulverization.

[0013] Preferably, the shellac is obtained by collecting and purifying the secretions of lac insects, and can be dissolved in a 5% ethanol solution for coating.

[0014] A method for preparing a compound microbial agent resistant to diseases and pests includes the following preparation steps:

[0015] S1. Inoculate Bacillus subtilis into LB liquid medium and culture at 50°C with shaking for 24 hours; inoculate Beauveria bassiana into SDAY medium and culture at 28°C with static incubation for 72 hours; inoculate Trichoderma harzianum into PDB medium and culture at 30°C with shaking for 48 hours.

[0016] S2. Mix the fermented bacterial solution according to the specified ratio, and add sterile water to adjust the total bacterial concentration to 10. 10 CFU / mL;

[0017] S3. Mix the modified straw powder, humic acid-diatomaceous earth composite, trehalose, and chitin powder in a mixer for 15 minutes until homogeneous.

[0018] S4. Add the mixed carrier and bacterial solution to the stirred tank at a mass ratio of 2:1 and stir at low speed for 30 minutes to ensure that the bacterial solution is fully adsorbed into the carrier pores.

[0019] S5. Spread the wet material evenly on the tray and send it into the hot air drying oven;

[0020] S6. Dissolve shellac in a 5% ethanol solution, then add citric acid and stir evenly. Next, put the dried bacterial-carrying particles into a fluidized bed, set the air inlet temperature to 40℃ and the atomization pressure to 0.15MPa, and spray the shellac solution evenly, controlling the coating weight gain rate to 10-15%.

[0021] S7. After coating, the granules are extruded through a screw extruder and granulator. Finally, random samples are taken to test the number of viable bacteria. Qualified products are packed into aluminum foil vacuum bags and stored in a light-proof and sealed container.

[0022] (III) Beneficial Effects

[0023] This invention provides a compound microbial agent for resisting diseases and pests, and its preparation method. It has the following beneficial effects:

[0024] 1. This invention overcomes the shortcomings of traditional diatomaceous earth or synthetic carriers, such as poor adsorption and nutrient deficiency, by using modified straw powder and humic acid-diatomaceous earth composite as the core carrier. It provides a high-porosity and nutrient-rich living environment for Bacillus subtilis, Beauveria bassiana, and Trichoderma harzianum, significantly improving the colonization rate and field survival rate of live bacteria in the inoculant. At the same time, the use of shellac ethanol solution for controllable coating replaces the difficult-to-degrade synthetic polymer coating material, protecting the bacteria from environmental stress while avoiding the risk of persistent residual pollution from chemical coating materials in the soil. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1:

[0027] This invention provides a compound microbial agent for resisting diseases and pests, composed of the following substances in parts by weight:

[0028] 50 parts of Bacillus subtilis, 40 parts of Beauveria bassiana, 35 parts of Trichoderma harzianum, 20 parts of modified straw powder, 10 parts of humic acid-diatomaceous earth complex, 10 parts of trehalose, 5 parts of chitin powder, and 5 parts of shellac.

[0029] The humic acid-diatomite composite is made by mixing humic acid and diatomite in a 1:1 ratio.

[0030] Modified straw powder is obtained by crushing crop straw powder to 100 mesh and then soaking it in 5% citric acid for 2 hours.

[0031] Trehalose is extracted from seaweed and purified.

[0032] Chitin powder is made from shrimp and crab shells through deproteinization and decalcification processes, followed by pulverization.

[0033] Preferably, the shellac is obtained by collecting and purifying the secretions of lac insects, and can be dissolved in a 5% ethanol solution for coating.

[0034] A method for preparing a compound microbial agent for resisting diseases and pests includes the following preparation steps:

[0035] S1. Inoculate Bacillus subtilis into LB liquid medium and culture at 50°C with shaking for 24 hours; inoculate Beauveria bassiana into SDAY medium and culture at 28°C with static incubation for 72 hours; inoculate Trichoderma harzianum into PDB medium and culture at 30°C with shaking for 48 hours.

[0036] S2. Mix the fermented bacterial solution according to the specified ratio, and add sterile water to adjust the total bacterial concentration to 10. 10 CFU / mL;

[0037] S3. Mix the modified straw powder, humic acid-diatomaceous earth composite, trehalose, and chitin powder in a mixer for 15 minutes until homogeneous.

[0038] S4. Add the mixed carrier and bacterial solution to the stirred tank at a mass ratio of 2:1 and stir at low speed for 30 minutes to ensure that the bacterial solution is fully adsorbed into the carrier pores.

[0039] S5. Spread the wet material evenly on the tray and send it into the hot air drying oven;

[0040] S6. Dissolve shellac in a 5% ethanol solution, then add citric acid and stir evenly. Next, put the dried bacterial-carrying particles into a fluidized bed, set the air inlet temperature to 40℃ and the atomization pressure to 0.15MPa, and spray the shellac solution evenly, controlling the coating weight gain rate to 10-15%.

[0041] S7. After coating, the granules are extruded through a screw extruder and granulator. Finally, random samples are taken to test the number of viable bacteria. Qualified products are packed into aluminum foil vacuum bags and stored in a light-proof and sealed container.

[0042] Example 2:

[0043] This invention provides a compound microbial agent for resisting diseases and pests, composed of the following substances in parts by weight:

[0044] 70 parts of Bacillus subtilis, 60 parts of Beauveria bassiana, 55 parts of Trichoderma harzianum, 35 parts of modified straw powder, 114 parts of humic acid-diatomaceous earth complex, 13 parts of trehalose, 7 parts of chitin powder, and 8 parts of shellac.

[0045] The humic acid-diatomite composite is made by mixing humic acid and diatomite in a 1:1 ratio.

[0046] Modified straw powder is obtained by crushing crop straw powder to 100 mesh and then soaking it in 5% citric acid for 2 hours.

[0047] Trehalose is extracted from seaweed and purified.

[0048] Chitin powder is made from shrimp and crab shells through deproteinization and decalcification processes, followed by pulverization.

[0049] Preferably, the shellac is obtained by collecting and purifying the secretions of lac insects, and can be dissolved in a 5% ethanol solution for coating.

[0050] A method for preparing a compound microbial agent for resisting diseases and pests includes the following preparation steps:

[0051] S1. Inoculate Bacillus subtilis into LB liquid medium and culture at 50°C with shaking for 24 hours; inoculate Beauveria bassiana into SDAY medium and culture at 28°C with static incubation for 72 hours; inoculate Trichoderma harzianum into PDB medium and culture at 30°C with shaking for 48 hours.

[0052] S2. Mix the fermented bacterial solution according to the specified ratio, and add sterile water to adjust the total bacterial concentration to 10. 10 CFU / mL;

[0053] S3. Mix the modified straw powder, humic acid-diatomaceous earth composite, trehalose, and chitin powder in a mixer for 15 minutes until homogeneous.

[0054] S4. Add the mixed carrier and bacterial solution to the stirred tank at a mass ratio of 2:1 and stir at low speed for 30 minutes to ensure that the bacterial solution is fully adsorbed into the carrier pores.

[0055] S5. Spread the wet material evenly on the tray and send it into the hot air drying oven;

[0056] S6. Dissolve shellac in a 5% ethanol solution, then add citric acid and stir evenly. Next, put the dried bacterial-carrying particles into a fluidized bed, set the air inlet temperature to 40℃ and the atomization pressure to 0.15MPa, and spray the shellac solution evenly, controlling the coating weight gain rate to 10-15%.

[0057] S7. After coating, the granules are extruded through a screw extruder and granulator. Finally, random samples are taken to test the number of viable bacteria. Qualified products are packed into aluminum foil vacuum bags and stored in a light-proof and sealed container.

[0058] Example 3:

[0059] This invention provides a compound microbial agent for resisting diseases and pests, composed of the following substances in parts by weight:

[0060] 100 parts of Bacillus subtilis, 80 parts of Beauveria bassiana, 75 parts of Trichoderma harzianum, 50 parts of modified straw powder, 15 parts of humic acid-diatomaceous earth complex, 15 parts of trehalose, 10 parts of chitin powder, and 10 parts of shellac.

[0061] The humic acid-diatomite composite is made by mixing humic acid and diatomite in a 1:1 ratio.

[0062] Modified straw powder is obtained by crushing crop straw powder to 100 mesh and then soaking it in 5% citric acid for 2 hours.

[0063] Trehalose is extracted from seaweed and purified.

[0064] Chitin powder is made from shrimp and crab shells through deproteinization and decalcification processes, followed by pulverization.

[0065] Preferably, the shellac is obtained by collecting and purifying the secretions of lac insects, and can be dissolved in a 5% ethanol solution for coating.

[0066] A method for preparing a compound microbial agent resistant to diseases and pests includes the following preparation steps:

[0067] S1. Inoculate Bacillus subtilis into LB liquid medium and culture at 50°C with shaking for 24 hours; inoculate Beauveria bassiana into SDAY medium and culture at 28°C with static incubation for 72 hours; inoculate Trichoderma harzianum into PDB medium and culture at 30°C with shaking for 48 hours.

[0068] S2. Mix the fermented bacterial solution according to the specified ratio, and add sterile water to adjust the total bacterial concentration to 10. 10 CFU / mL;

[0069] S3. Mix the modified straw powder, humic acid-diatomaceous earth composite, trehalose, and chitin powder in a mixer for 15 minutes until homogeneous.

[0070] S4. Add the mixed carrier and bacterial solution to the stirred tank at a mass ratio of 2:1 and stir at low speed for 30 minutes to ensure that the bacterial solution is fully adsorbed into the carrier pores.

[0071] S5. Spread the wet material evenly on the tray and send it into the hot air drying oven;

[0072] S6. Dissolve shellac in a 5% ethanol solution, then add citric acid and stir evenly. Next, put the dried bacterial-carrying particles into a fluidized bed, set the air inlet temperature to 40℃ and the atomization pressure to 0.15MPa, and spray the shellac solution evenly, controlling the coating weight gain rate to 10-15%.

[0073] S7. After coating, the granules are extruded through a screw extruder. Finally, random samples are taken to test the number of viable bacteria. Qualified products are packed into aluminum foil vacuum bags and stored in a light-proof, sealed container.

[0074] Comparative example:

[0075] Using conventional preparation methods in existing technologies, multiple microbial strains are fermented and propagated separately, then mixed and combined with carriers such as peat and vermiculite. The mixture is then coated with chemically synthesized materials and processed at high temperatures.

[0076] Experimental example:

[0077] Carrier degradation test

[0078] (1) Group:

[0079] Group A: Embodiment 1 of the present invention;

[0080] Group B: Comparative Example;

[0081] Group C: Synthetic carrier (polyvinyl alcohol microspheres);

[0082] (2) Method:

[0083] Each group of carriers was buried in simulated farmland soil (40% humidity, 25℃), and samples were taken every 7 days for testing: weight loss rate (degree of degradation), surface structure damage observed by scanning electron microscopy, and chemical oxygen demand (COD) of residual substances in the soil. The specific test results are shown in Table 1 below:

[0084] Table 1 Comparison of Test Results

[0085] detection indicators Example 1 Comparative Example Synthetic carrier Weight loss rate (%) 78.2±3.5 12.1±1.2 0.9±0.1 COD increment (mg / L) 35±4 120±10 280±15 Degree of structural damage Completely disintegrated Partial disintegration No change

[0086] In summary, the carrier of this invention has a fast degradation rate and low COD release, which significantly reduces soil load.

[0087] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound microbial agent for resisting diseases and pests, characterized in that, Composed of the following parts by mass: 50-100 parts of Bacillus subtilis, 40-80 parts of Beauveria bassiana, 35-75 parts of Trichoderma harzianum, 20-50 parts of modified straw powder, 10-15 parts of humic acid-diatomaceous earth complex, 10-15 parts of trehalose, 5-10 parts of chitin powder, and 5-10 parts of shellac.

2. The compound microbial agent for resisting diseases and pests according to claim 1, characterized in that: The humic acid-diatomite composite is made by mixing humic acid and diatomite in a 1:1 ratio.

3. The compound microbial agent for resisting diseases and pests according to claim 1, characterized in that: The modified straw powder is obtained by crushing crop straw powder to 100 mesh and then soaking it in 5% citric acid for 2 hours.

4. The compound microbial agent for resisting diseases and pests according to claim 1, characterized in that: The trehalose is extracted from seaweed and purified.

5. The compound microbial agent for resisting diseases and pests according to claim 1, characterized in that: The chitin powder is made from shrimp and crab shells through deproteinization and decalcification treatment, followed by pulverization.

6. The compound microbial agent for resisting diseases and pests according to claim 1, characterized in that: The shellac is obtained by collecting and purifying the secretions of lac insects, and it can be dissolved in a 5% ethanol solution for coating.

7. A method for preparing a compound microbial agent resistant to diseases and pests, characterized in that, The preparation steps include the following: S1. Inoculate Bacillus subtilis into LB liquid medium and culture at 50°C with shaking for 24 hours; inoculate Beauveria bassiana into SDAY medium and culture at 28°C with static incubation for 72 hours; inoculate Trichoderma harzianum into PDB medium and culture at 30°C with shaking for 48 hours. S2. Mix the fermented bacterial solution according to the specified ratio, and add sterile water to adjust the total bacterial concentration to 10. 10 CFU / mL; S3. Mix the modified straw powder, humic acid-diatomaceous earth composite, trehalose, and chitin powder in a mixer for 15 minutes until homogeneous. S4. Add the mixed carrier and bacterial solution to the stirred tank at a mass ratio of 2:1 and stir at low speed for 30 minutes to ensure that the bacterial solution is fully adsorbed into the carrier pores. S5. Spread the wet material evenly on the tray and send it into the hot air drying oven; S6. Dissolve shellac in a 5% ethanol solution, then add citric acid and stir evenly. Next, put the dried bacterial-carrying particles into a fluidized bed, set the air inlet temperature to 40℃ and the atomization pressure to 0.15MPa, and spray the shellac solution evenly, controlling the coating weight gain rate to 10-15%. S7. After coating, the granules are extruded through a screw extruder. Finally, random samples are taken to test the number of viable bacteria. Qualified products are packed into aluminum foil vacuum bags and stored in a light-proof, sealed container.