A microbial preparation containing Bacillus laterosporus and preparation method thereof

Abstract

The present application relates to the field of biocontrol agents, and specifically discloses a microbial preparation containing Bacillus laterosporus and a preparation method thereof. A preparation method of a microbial preparation containing Bacillus laterosporus comprises the following steps: adding the clay to a liquid culture medium after sterilization, then adding Bacillus laterosporus, mixing evenly, maturing, centrifuging, washing, adding ultrapure water, and preparing a modified clay suspension; by weight, 30-50 parts of the modified clay suspension are mixed with 10-15 parts of concentrated biogas slurry, 1-2 parts of glycerol, 2-4 parts of γ-aminobutyric acid, and 3-7 parts of selenium nanomaterials to prepare a microbial agent. The microbial preparation of the present application has the advantages of promoting seed germination and crop growth.

Description

Technical Field

[0001] The present application relates to the technical field of biocontrol preparations, and more specifically, to a microbial preparation containing Bacillus laterosporus and a preparation method thereof. Background Art

[0002] Soil-borne diseases cause significant economic losses to agricultural production every year. Common and devastating soil-borne diseases include wilt, soft rot, sheath blight and bacterial wilt. Biological control has become a hot topic in the prevention and control of soil-borne plant diseases at home and abroad due to its low cost, environmental friendliness and no drug residue. Bacillus is widely distributed in nature and has the advantages of strong stress resistance, high temperature resistance and good biocontrol effect. It has become an ideal biocontrol bacteria resource for soil-borne diseases. At the same time, Bacillus and its metabolites can effectively improve soil fertility, improve soil environment, enhance crop quality and enhance plant disease resistance. Bacillus colonizes in the rhizosphere, body surface or body of plants, competes with pathogens for nutrients around the plants, secretes antibacterial substances to inhibit the growth of pathogens, and induces the plant defense system to resist the invasion of pathogens, thereby achieving the purpose of biocontrol.

[0003] The germination and growth of plant seeds are the basis of crop growth and are also the sensitive period for plants to respond to the outside world. Due to the dormancy characteristics, thick seed coat, and poor water and air permeability of plant seeds, their natural germination efficiency is low, which in turn affects plant growth and development. At present, there are few studies on the effects of Bacillus laterosporus on plant seed germination. Summary of the invention

[0004] In order to promote the germination of plant seeds and improve the germination rate, the present application provides a microbial preparation containing Bacillus laterosporus and a preparation method thereof.

[0005] In a first aspect, the present application provides a method for preparing a microbial preparation containing Bacillus laterosporus, using the following technical solution:

[0006] A method for preparing a microbial preparation containing Bacillus laterosporus comprises the following steps:

[0007] Sterilize the clay and add it to the liquid culture medium until the clay concentration is 25-30 g / L, then add 50-55 g / L of Bacillus laterosporus, mix well, mature at 28-30° C. for 22-24 hours, centrifuge, wash, and add ultrapure water to obtain a modified clay suspension;

[0008] By weight, 30-50 parts of modified clay suspension, 10-15 parts of concentrated biogas slurry, 1-2 parts of glycerol, 2-4 parts of gamma-aminobutyric acid and 3-7 parts of selenium nanomaterials are mixed to prepare a microbial agent.

[0009] Seed germination is the starting point of plant life activities. At the beginning of germination, the swelling effect promotes water and oxygen to enter the seed, and a series of material metabolism in the germination process is started. Respiration is the core of seed life activities. The entire germination process is inseparable from the nutrients and energy provided by respiration. By adopting the above technical solution, Bacillus laterosporus is cultured in clay. The clay can locally enrich Bacillus laterosporus, and the surface of Bacillus laterosporus has a negative charge. The clay also has a negative charge. Therefore, Bacillus laterosporus forms a cationic bond bridge through extracellular cationic secretions and electrolytes and adheres to the clay surface with the help of flagella. In addition, clay promotes the growth of Bacillus laterosporus. As the maturation time increases, the density of Bacillus laterosporus increases, thereby weakening the growth and reproduction of pathogens and promoting plants. Growth and development; γ-aminobutyric acid is a non-protein amino acid and an endogenous substance in plants. It has the functions of signal transduction, increasing stress resistance and regulating plant growth and development in plants. It is synthesized by the polyamine metabolic pathway or the glutamate decarboxylation pathway, and is metabolized by γ-aminobutyric acid transaminase to enter the tricarboxylic acid cycle. It can regulate the balance of carbon and nitrogen nutrients and connect the tricarboxylic acid cycle with amino acid metabolism. The tricarboxylic acid cycle is an important part of plant respiration, and it itself is an alkaline substance. It can affect plant germination and respiratory metabolism through other channels such as affecting the internal environment of plant cells, thereby improving the germination index, vitality index and respiratory rate of seeds to a certain extent; with the development of poultry and livestock farming and biogas projects, the discharge of biogas slurry has increased dramatically, and its utilization has also become a hot topic in the fields of environmental protection and agriculture. Biogas slurry contains a large amount of nutrients such as nitrogen, phosphorus and potassium, as well as active substances such as amino acids, humic acid, vitamins and growth hormones. It can be used as a good liquid organic fertilizer and also has disease-suppressing function. However, biogas slurry has a high water content, and the direct application of fertilizer is not high. In addition, the transportation cost is high and commercial utilization is difficult, which seriously restricts the promotion and application of biogas slurry fertilizer.

[0010] The compounding of concentrated biogas slurry and Bacillus laterosporus has the advantages of both organic fertilizer and microbial fertilizer. It not only contains a large amount of organic matter and nutrients, but also contains a large amount of biocontrol microorganisms and their metabolites, which can enhance the disease resistance of plants. Glycerol is added as a protective agent to prevent the high EC and alkaline pH in the biogas slurry from affecting the activity of Bacillus laterosporus. The active substances such as gibberellins and organic acids contained in the biogas slurry can promote seed germination and plant growth. After adding Bacillus laterosporus, it has a synergistic effect on growth promotion and germination, which can significantly promote seed germination and growth. If the biogas slurry concentration is too high, the excessive ammonium nitrogen in the biogas slurry will cause ammonium toxicity to crops and inhibit crop growth. Gibberellin in concentrated biogas slurry is an important growth-promoting plant hormone that promotes seed germination, seedling growth, flowering and leaf extension. Gibberellin in biogas slurry has a synergistic effect with γ-aminobutyric acid; organic acid components and gibberellin contained in biogas slurry have a significant inhibitory effect on pathogens, and can significantly inhibit the germination of plant pathogen spores in vivo, the growth of mycelium in vitro, and the formation of conidia. Nano-selenium materials can improve the germination potential, germination rate and vitality index of seeds, promote the growth of young roots and young shoots of plants, and promote the absorption of nutrients by higher plants, thereby increasing the content of chlorophyll in leaves, which helps to promote light deterioration and growth and development. Therefore, the microbial preparation prepared by the above method can not only improve the germination and germination rate of seeds, but also promote plant growth.

[0011] Optionally, the clay comprises silica microspheres and montmorillonite in a mass ratio of 1:0.2-0.4.

[0012] By adopting the above technical scheme, montmorillonite is a layered structure, flaky crystal silicate clay mineral, which is a monoclinic crystal system. There are cations or water molecules between the structural layers. It can adsorb proteins in the extracellular polymers secreted by Bacillus laterosporus through electrostatic action, thereby improving the adsorption effect on Bacillus laterosporus. Silica microspheres can reduce primary diseases of plants and improve the inhibitory effect on pathogenic bacteria.

[0013] Optionally, the preparation method of the silica microspheres is as follows:

[0014] By weight, 1.3-2 parts of silicon dioxide powder are dispersed in 200-300 parts of deionized water to prepare a dispersion;

[0015] Mix 1.7-2 parts of copper nitrate trihydrate, 300-350 parts of deionized water and 50-60 parts of aqueous ammonia, add the dispersion, heat to 140-150° C., react for 20-24 hours, filter and wash to obtain an intermediate product;

[0016] 1-2 parts of the intermediate product are reacted with 240-320 parts of hydrochloric acid at 50-55°C for 3-4 hours, centrifuged, washed, and vacuum dried.

[0017] By adopting the above technical scheme, the copper ions that can be complexed with ammonia are mixed with ammonia water and silica powder for heating reaction, a core-shell intermediate is produced, and the residual silica core is slowly dissolved in an alkaline solution at 140°C, and finally the copper in the shell is removed with hydrochloric acid to form sea urchin-shaped silica microspheres. Its large specific surface area provides more adsorption sites for Bacillus laterosporus, thereby increasing the loading capacity of Bacillus laterosporus, and has super strong adhesion and adhesion to seeds. The silica microspheres with a large number of nano-tubular structures are tightly adhered to the surface of the seeds, so that the microbial preparation is more conducive to the loading and release of Bacillus laterosporus when treating seeds.

[0018] Optionally, the silica microspheres are pretreated as follows:

[0019] The silica microspheres are dispersed in deionized water to form a suspension, and ammonium chloride is added. After stirring evenly, the suspension is centrifuged and vacuum dried. The mass ratio of the silica microspheres to the ammonium chloride is 1:0.15-0.2.

[0020] By adopting the above technical solution, the silica microspheres can adsorb and load a certain amount of ammonium chloride, thereby preparing silica microspheres containing nitrogen fertilizer, improving the growth ability of the root system and promoting the growth of crops.

[0021] Optionally, the preparation method of the nano-selenium material is as follows:

[0022] Mix nickel nitrate hexahydrate, ethylenediamine and distilled water, stir evenly, add sodium selenite, perform ultrasonication for 20-30 minutes, heat to 180-200°C for reaction for 20-24 hours, centrifuge, wash and vacuum dry to obtain an intermediate, wherein the mass ratio of nickel nitrate hexahydrate to sodium selenite is 1:1.8-2;

[0023] The intermediate is added to an ethanol solution, zirconium oxide is added, stirred for 20-24 hours, filtered, washed, and dried. The mass ratio of the intermediate to zirconium oxide is 0.04-0.05:1.

[0024] By adopting the above technical scheme, nickel and selenium are combined to form an intermediate containing nickel and selenium, thereby promoting the growth of the root system and increasing the growth rate of crops. Zirconium oxide is also added. When the radicle of the seed breaks through the seed coat, the root system is in direct contact with the microbial preparation solution. During the growth and development process, the microbial preparation solution can enter the seedling through the root cells and transfer to various parts of the plant for continuous accumulation, which can improve seed germination and increase the vitality index of the seed, while promoting the growth of the seedling root system and seedling stem.

[0025] Optionally, the concentrated biogas slurry is prepared by the following method: filtering the biogas slurry using an ultrafiltration membrane with a pore size of 1-100 nm to obtain a filtrate, adding 30-50 wt % of graphene oxide based on the mass of the filtrate, and mixing to obtain the concentrated biogas slurry.

[0026] By adopting the above technical scheme, the ultrafiltration membrane removes a large amount of suspended matter, excess water and impurities in the biogas slurry. The surface of graphene oxide carries a large number of hydrophilic acidic functional groups. Its SP3-SP2 blended structure enables its surface functional group structure (SP3) to enrich water in the environment and transmit it quickly. It has good wettability and surface activity. It is acidic under normal circumstances, while the biogas slurry is alkaline. Mixing graphene oxide with the filtrate can reduce the alkalinity of the biogas slurry and reduce the effect of the alkalinity of the biogas slurry on the activity of Bacillus laterosporus. In addition, graphene oxide can also promote seed development and improve plant growth rate.

[0027] Optionally, the concentration of the modified clay suspension is 0.1-0.5 g / L.

[0028] In a second aspect, the present application provides a microbial preparation containing Bacillus laterosporus, using the following technical solution:

[0029] A microbial preparation containing Bacillus laterosporus is prepared by adopting the preparation method.

[0030] 1. Since the present application uses clay to first culture Bacillus laterosporus, increase the density of Bacillus laterosporus, and mix it with concentrated biogas slurry, γ-aminobutyric acid and nano-selenium material, the prepared microbial preparation can not only increase seed germination, but also improve the growth rate of plants.

[0031] 2. In the present application, specific silica microspheres and montmorillonite are preferably used as clay, which can increase the enrichment of Bacillus laterosporus and promote the growth of Bacillus laterosporus. In addition, the silica microspheres are pretreated and nitrogen fertilizer is loaded on their surface to enhance the growth-promoting effect of the microbial preparation.

[0032] 3. In the present application, it is preferred to add graphene oxide after filtering the biogas slurry. Graphene oxide can reduce the effect of the alkalinity of the biogas slurry on the activity of Bacillus laterosporus, and can further increase the effect of promoting germination and growth of the microbial preparation. DETAILED DESCRIPTION

[0033] The following examples further illustrate the present application in detail.

[0034] Preparation Example of Silica Microspheres

[0035] Preparation Example 1: (1) 1.3 g of silicon dioxide powder was dispersed in 200 g of deionized water to prepare a dispersion, wherein the silicon dioxide powder was selected from Guangzhou Meigu Chemical, model ML-100;

[0036] (2) 1.7 g of copper nitrate trihydrate, 300 g of deionized water and 50 g of ammonia water were mixed, added to the dispersion, mixed evenly and then transferred to a reaction kettle, heated to 140° C., reacted for 24 h, filtered, and the reaction product was washed with anhydrous ethanol and deionized water to obtain an intermediate product, wherein the concentration of ammonia water was 25 wt %;

[0037] (3) 1 g of the intermediate product was reacted with 240 g of hydrochloric acid (0.5 M) at 50° C. for 4 h, centrifuged, washed, vacuum dried at 60° C. for 10 h, and crushed to obtain silica microspheres with a particle size of 500 nm.

[0038] Preparation Example 2: (1) 2 g of silicon dioxide powder was dispersed in 300 g of deionized water to prepare a dispersion, wherein the silicon dioxide powder was selected from Guangzhou Meigu Chemical, model ML-100;

[0039] (2) 2 g of copper nitrate trihydrate, 350 g of deionized water and 60 g of ammonia water were mixed, added to the dispersion, mixed evenly and then transferred to a reaction kettle, heated to 150° C., reacted for 20 h, filtered, and the reaction product was washed with anhydrous ethanol and deionized water to obtain an intermediate product, wherein the ammonia concentration was 25 wt %;

[0040] (3) 2 g of the intermediate product was reacted with 320 g of hydrochloric acid (0.5 M) at 55° C. for 3 h, centrifuged, washed, vacuum dried at 80° C. for 12 h, and crushed to obtain silica microspheres with a particle size of 500 nm.

[0041] Preparation example of nano-selenium material

[0042] Preparation Example 3: 3.7 g of nickel nitrate hexahydrate, 26 g of ethylenediamine and 400 g of distilled water were mixed, and sodium selenite was added after stirring evenly. The mixture was ultrasonicated for 30 min, heated to 180° C. for reaction for 24 h, centrifuged, washed with deionized water and ethanol for 3 times respectively, and vacuum dried at 80° C. for 24 h to obtain an intermediate. The mass ratio of nickel nitrate hexahydrate to sodium selenite was 1:2.

[0043] The intermediate was added to 100 g of ethanol solution, and 6.16 g of zirconium oxide was added. The mixture was stirred for 24 h, filtered, washed, and dried. The mass ratio of the intermediate to zirconium oxide was 0.05:1.

[0044] Preparation Example 4: 3.7 g of nickel nitrate hexahydrate, 26 g of ethylenediamine and 400 g of distilled water were mixed, and sodium selenite was added after stirring evenly. The mixture was ultrasonicated for 20 min, heated to 200° C. for reaction for 20 h, centrifuged, washed with deionized water and ethanol for 3 times respectively, and vacuum dried at 80° C. for 24 h to obtain an intermediate. The mass ratio of nickel nitrate hexahydrate to sodium selenite was 1:1.8.

[0045] The intermediate was added to 100 g of ethanol solution, and 6.16 g of zirconium oxide was added. The mixture was stirred for 20 h, filtered, washed, and dried. The mass ratio of the intermediate to zirconium oxide was 0.04:1.

[0046] Preparation Example 5: Mix 3.7 g of nickel nitrate hexahydrate, 26 g of ethylenediamine and 400 g of distilled water, stir well, add sodium selenite, ultrasonicate for 30 min, heat to 180°C for reaction for 24 h, centrifuge, wash with deionized water and ethanol three times respectively, and vacuum dry at 80°C for 24 h to obtain nano-selenium material. The mass ratio of nickel nitrate hexahydrate to sodium selenite is 1:2.

[0047] Example

[0048] In the following examples, Bacillus spporidia HP-2 is deposited in China Center for Type Culture Collection with the deposit number CCTCC. NO: M20221789, the preservation date is November 14, 2022, the preservation address is Wuhan University, Wuhan, China, the LB liquid culture medium formula is: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH value is 7.4, the mass concentration of organic matter in the sludge is 4.5g / L, the mass concentration of humic acid is 1.9g / L, the mass concentration of total nitrogen is 5g / L, the mass concentration of total phosphorus is 2.4g / L, the mass concentration of total potassium is 5.6g / L, the mass concentration of calcium is 864mg / L, the mass concentration of iron is 104mg / L, the mass concentration of magnesium is 25mg / L, the mass concentration of manganese is 16mg / L, the mass concentration of zinc is 16mg / L, and the mass concentration of copper is 11mg / L.

[0049] Embodiment 1: A microbial preparation containing Bacillus laterosporus, and a preparation method thereof, comprising the following steps:

[0050] S1, sterilize 30g of clay in a high-temperature steam cooker at 121°C for 30min, then sterilize under an ultraviolet lamp for 20min, cool to room temperature, add to LB liquid culture medium sterilized at 121°C for 20min, until the clay concentration is 30g / L, then add 50g / L of Bacillus laterosporus, mix evenly, mature at 28°C for 24h, centrifuge, wash, add ultrapure water, and prepare a modified clay suspension with a concentration of 0.5g / L, the clay includes silica microspheres and montmorillonite in a mass ratio of 1:0.2, the silica microspheres are prepared by Preparation Example 1, and the montmorillonite is made of Lingshou County Longchuan drilling plugging material, model LD-E;

[0051] S2. 50 g of modified clay suspension is mixed with 15 g of concentrated biogas slurry, 2 g of glycerol, 4 g of γ-aminobutyric acid and 7 g of selenium nanomaterial to prepare a microbial agent. The concentrated biogas slurry is prepared by filtering the biogas slurry through an inorganic ceramic ultrafiltration membrane with a pore size of 100 nm. The nano-selenium material is sodium selenite.

[0052] Embodiment 2: A microbial preparation containing Bacillus laterosporus, and a preparation method thereof, comprising the following steps:

[0053] S1, sterilize 25g of clay in a high-temperature steam cooker at 121°C for 30min, then sterilize under an ultraviolet lamp for 20min, cool to room temperature and add to LB liquid culture medium sterilized at 121°C for 20min, until the clay concentration is 25g / L, then add 55g / L of Bacillus laterosporus, mix evenly, mature at 30°C for 22h, centrifuge, wash, add ultrapure water, and prepare a modified clay suspension with a concentration of 0.1g / L, the clay includes silica microspheres and montmorillonite in a mass ratio of 1:0.2, the silica microspheres are prepared by Preparation Example 1, and the montmorillonite is made of Lingshou County Longchuan drilling plugging material, model LD-E;

[0054] S2. 30 g of modified clay suspension is mixed with 10 g of concentrated biogas slurry, 1 g of glycerol, 2 g of γ-aminobutyric acid and 3 g of selenium nanomaterial to prepare a microbial agent. The concentrated biogas slurry is prepared by filtering the biogas slurry through an inorganic ceramic ultrafiltration membrane with a pore size of 50 nm. The nano-selenium material is sodium selenite.

[0055] Embodiment 3: A microbial preparation containing Bacillus laterosporus, and a preparation method thereof, comprising the following steps:

[0056] S1, the clay was sterilized in a high temperature steam cooker at 121 ° C for 30 minutes, and then sterilized under an ultraviolet lamp for 20 minutes, cooled to room temperature, added to the LB liquid culture medium sterilized at 121 ° C for 20 minutes, and then added with 50 g / L of Bacillus laterosporus, mixed evenly, aged at 28 ° C for 24 hours, centrifuged, washed, and added with ultrapure water to obtain a modified clay suspension with a concentration of 0.3 g / L, the clay included silica microspheres and montmorillonite in a mass ratio of 1: 0.2, the silica microspheres were prepared by Preparation Example 1, and the montmorillonite was made of Lingshou County Longchuan Drilling Plug Material, model LD-E;

[0057] S2. 40 g of modified clay suspension is mixed with 13 g of concentrated biogas slurry, 1.5 g of glycerol, 3 g of γ-aminobutyric acid and 5 g of selenium nanomaterial to prepare a microbial agent. The concentrated biogas slurry is prepared by filtering the biogas slurry through an inorganic ceramic ultrafiltration membrane with a pore size of 1 nm. The nano-selenium material is sodium selenite.

[0058] Example 4: A method for preparing a microbial preparation containing Bacillus laterosporus, which is different from Example 1 in that the clay includes silica microspheres and montmorillonite in a ratio of 1:0.4 in Preparation Example, and the silica microspheres are prepared in Preparation Example 2.

[0059] Example 5: A method for preparing a microbial preparation containing Bacillus laterosporus. The difference from Example 1 is that the silica microspheres are commercially available solid silica microspheres selected from Beijing Deke Island, model DKSIO000500, and particle size of 500 nm.

[0060] Example 6: A method for preparing a microbial preparation containing Bacillus laterosporus, which is different from Example 1 in that an equal amount of calcium carbonate is used to replace silica microspheres.

[0061] Example 7: A method for preparing a microbial preparation containing Bacillus laterosporus, which is different from Example 1 in that an equal amount of silica microspheres prepared in Preparation Example 1 are used in clay to replace montmorillonite.

[0062] Example 8: A method for preparing a microbial preparation containing Bacillus laterosporus, which differs from Example 1 in that the silica microspheres are pretreated as follows: 25 g of silica microspheres are dispersed in 1.5 times their mass of deionized water to form a suspension, ammonium chloride is added, magnetic stirring is performed for 6 hours, centrifuged, and dried at 80°C for 5 hours. The mass ratio of silica microspheres to ammonium chloride is 1:0.15.

[0063] Example 9: A method for preparing a microbial preparation containing Bacillus laterosporus, which differs from Example 1 in that the silica microspheres are pretreated as follows: 25 g of silica microspheres are dispersed in deionized water twice their mass to form a suspension, ammonium chloride is added, magnetic stirring is performed for 6 hours, centrifuged, and dried at 60°C for 6 hours. The mass ratio of silica microspheres to ammonium chloride is 1:0.2.

[0064] Example 10: A method for preparing a microbial preparation containing Bacillus laterosporus, which is different from Example 9 in that the nano-selenium material is prepared by Preparation Example 3.

[0065] Example 11: A method for preparing a microbial preparation containing Bacillus laterosporus, which is different from Example 9 in that the nano-selenium material is prepared by Preparation Example 4.

[0066] Example 12: A method for preparing a microbial preparation containing Bacillus laterosporus, which is different from Example 9 in that the nano-selenium material is prepared according to Preparation Example 5.

[0067] Example 13: A method for preparing a microbial preparation containing Bacillus laterosporus. The difference from Example 11 is that the method for preparing concentrated biogas slurry is: filtering the biogas slurry through an inorganic ceramic ultrafiltration membrane with a pore size of 100 nm to obtain a filtrate, adding 50 wt% of graphene oxide by mass of the filtrate to the filtrate, and mixing. The graphene oxide is selected from Suzhou Andy Nanotechnology Co., Ltd. and the model is MGOG4.

[0068] Example 14: A method for preparing a microbial preparation containing Bacillus laterosporus. The difference from Example 11 is that the method for preparing concentrated biogas slurry is: filtering the biogas slurry through an inorganic ceramic ultrafiltration membrane with a pore size of 100 nm to obtain a filtrate, adding 30 wt% of graphene oxide by mass of the filtrate to the filtrate, and mixing. The graphene oxide is selected from Suzhou Andy Nanotechnology Co., Ltd. and the model is MGOG4.

[0069] Comparative Example

[0070] Comparative Example 1: A method for preparing a microbial preparation containing Bacillus laterosporus, which is different from Example 1 in that no nano-selenium material is added.

[0071] Comparative Example 2: A method for preparing a microbial preparation containing Bacillus laterosporus, which differs from Example 1 in that an equal amount of γ-aminobutyric acid is used to replace concentrated biogas slurry.

[0072] Comparative Example 3: A method for preparing a microbial preparation containing Bacillus laterosporus, which differs from Example 11 in that, in step S1, no clay is added, and Bacillus laterosporus is added to a liquid culture medium at a concentration of 50 g / L, matured at 28°C for 24 hours, centrifuged, washed, and ultrapure water is added to obtain a culture solution with a concentration of 0.5 g / L; in step S2, 50 g of the culture solution is mixed with 15 g of concentrated biogas slurry, 2 g of glycerol, 4 g of γ-aminobutyric acid and 7 g of selenium nanomaterial to obtain a microbial agent, the concentrated biogas slurry is obtained by filtering the biogas slurry through an inorganic ceramic ultrafiltration membrane with a pore size of 100 nm, and the nano-selenium material is sodium selenite.

[0073] Performance testing

[0074] 1. Determination of seed germination rate Test objects: Solanaceae (tomato and pepper), Gramineae (wheat and rice) and Cruciferae (Chinese cabbage and Chinese cabbage);

[0075] Detection method: 50 seeds of uniform size and full grains were repeatedly selected for each plant, germinated in 55°C clean water for 30 minutes, left to stand overnight, and washed the seeds and placed in a culture dish covered with filter paper. 50 mL of clean water was added as a control group. The microbial preparation prepared by the method in the embodiment and the comparative example was taken to prepare a 400-fold dilution, 5 mL was drawn and placed in a culture dish, and each treatment was repeated 3 times. According to the inspection procedures for crop seeds, germination was recorded when the radicle length exceeded 1 / 2 of the seed length. The number of seeds that germinated was recorded every day, and the germination rate was counted after 5 days. The germination rate (%) = the total number of seeds germinated within 5 days / the total number of seeds tested × 100%. The test results are recorded in Table 1.

[0076] 2. Determination of seed rooting

[0077] The indoor agar rooting method was used to germinate the seeds of tomato, rice and Chinese cabbage using the above-mentioned seed germination rate determination method. When the germination length was 2 mm, the seeds were transplanted onto the solidified agar medium and placed in a fully intelligent artificial climate plant box together with the medium for cultivation. When the main root of the plant seed touched the bottom of the beaker, the main root length (L1) and stem length (L2) of the rice seeds, the root length (L3) and stem diameter (L4) of the tomato seeds, the plant height (L5) and stem diameter (L6) of the Chinese cabbage seeds were measured with a caliper. At the same time, the main root length (L1) of the rice seeds in the control group treated with clear water was measured. 1CK ) and stem height (L 2CK ), taproot length of tomato seeds in the water control group (L 3CK ) and stem diameter (L 4CK ), the height of Chinese cabbage seeds in the water control group (L 5CK ) and stem diameter (L 6CK ), each treatment was repeated 3 times, the results were averaged, and the rice promotion rate was calculated according to the following formula: main root growth promotion rate = (L1-L 1CK ) / L 1CK × 100%, stem length promotion rate = (L3-L 3CK ) / L 3CK × 100%, tomato growth promotion rate: main root growth promotion rate = (L3-L 3CK ) / L 3CK × 100%, stem thickness promotion rate = (L4-L 4CK ) / L 4CK × 100%, vegetable heart growth promotion rate: plant height promotion rate = (L5-L 5CK ) / L 5CK × 100%, stem thickness promotion rate = (L6-L 6CK ) / L 6CK × 100%, where the L of rice seeds in the water control group was measured 1CK 54.8mm, L 2CKThe L of tomato seeds in the water control group was 24.6 mm. 3CK 9.3mm, L 4CK The L of Chinese cabbage seeds in the water control group was 4.4 mm. 5CK 23.6mm, L 6CK The test results are recorded in Table 2.

[0078] Table 1

[0079]

[0080] It can be seen from the data in Table 1 that in Examples 1-4, specific clay was used to culture Bacillus laterosporus and mixed with concentrated biogas slurry and other raw materials. After the microbial preparation was diluted a certain multiple, it had a good effect on promoting seed germination, whether it was Solanaceae plants, Gramineae plants, or Cruciferae plants.

[0081] Example 5 uses commercially available solid silica microspheres, while in Example 6, calcium carbonate is used instead of silica microspheres, and in Example 7, silica microspheres are used instead of montmorillonite. It can be seen from the data in Table 1 that the microbial preparation prepared in Example 7 has a better sprout-promoting effect, followed by Example 5, and the worst is Example 6. Compared with Example 1, the germination effects of Examples 5-7 are reduced, indicating that the silica microspheres and montmorillonite prepared in the present application are combined as clay to cultivate Bacillus laterosporus, and a microbial preparation with a better sprout-promoting effect can be obtained.

[0082] Compared with Example 1, Examples 8 and 9 further pre-treated the silica microspheres. The data in Table 1 show that, regardless of Solanaceae, Gramineae and Cruciferae, after the seeds were treated with the microbial preparations prepared in Examples 8 and 9, the germination rate of the seeds increased and the germination effect was improved.

[0083] In Example 10 and Example 11, the special nano-selenium materials prepared in Preparation Example 3 and Preparation Example 4 were used respectively. Compared with Example 9, after the plant seeds were pretreated in Example 10 and Example 11, the germination rate of the seeds increased.

[0084] In Example 12, the nano-selenium material prepared in Preparation Example 5 was used. Compared with Examples 10 and 11, the germination rate of seeds treated with the microbial preparation prepared in Example 12 was reduced, indicating that the addition of zirconium oxide can improve the seed vitality index and the germination rate.

[0085] Compared with Example 11, Example 13 and Example 14 further added graphene oxide to the concentrated biogas slurry, and the microbial preparations prepared thereby further increased the germination rate of seeds.

[0086] In Comparative Example 1, no nano-selenium material was added. Compared with Example 1, the germination rate of seeds treated with Comparative Example 1 decreased and the germination effect was weakened.

[0087] In Comparative Example 2, an equal amount of γ-aminobutyric acid was used to replace concentrated biogas slurry. Compared with Example 1, the germination rate of seeds treated with the microbial preparation decreased.

[0088] In Comparative Example 3, no clay was used. Compared with Example 1, the germination rate of various seeds in Comparative Example 3 decreased.

[0089] Table 2

[0090]

[0091]

[0092] It can be seen from the data in Table 1 that in Examples 1-4, specific clay was used to culture Bacillus laterosporus and mixed with raw materials such as concentrated biogas slurry. The prepared microbial preparation was diluted a certain multiple and pre-treated on the seeds of Solanaceae, Gramineae and Cruciferae, which can significantly promote the growth and development of plant seeds and accelerate rooting and germination. No nano-selenium material was added in Comparative Example 1, an equal amount of γ-aminobutyric acid was used instead of concentrated biogas slurry in Comparative Example 2, and no clay was used in Comparative Example 3. The data in Table 2 show that the growth-promoting effect of the microbial preparations prepared in Comparative Examples 1-3 decreased.

[0093] In Example 5, commercially available solid silica microspheres were used, while in Example 6, calcium carbonate was used instead of silica microspheres, and in Example 7, silica microspheres were used instead of montmorillonite. It can be seen from the data in Table 2 that the main root growth rate of rice, tomato and Chinese cabbage was reduced, and the growth rate was slower than that of Example 1, indicating that the silica microspheres and montmorillonite prepared in the present application are combined as clay to cultivate Bacillus laterosporus, and a microbial preparation that promotes plant growth better can be obtained.

[0094] Compared with Example 1, Examples 8 and 9 further pre-treated the silica microspheres. The data in Table 2 showed that the growth rates of rice, tomatoes and Chinese cabbage were accelerated.

[0095] In Example 10 and Example 11, the special nano-selenium materials prepared in Preparation Example 3 and Preparation Example 4 were used respectively. Compared with Example 9, after the plant seeds were pretreated in Example 10 and Example 11, the promotion rate of the root system and main stem of tomato, rice and Chinese cabbage was increased.

[0096] In Example 12, the nano-selenium material prepared in Preparation Example 5 was used. Compared with Examples 10 and 11, the root growth rate of the seeds treated with the microbial preparation prepared in Example 12 was slowed down, indicating that zirconium oxide can improve the growth rate of seeds.

[0097] Compared with Example 11, Example 13 and Example 14 further added graphene oxide to the concentrated biogas slurry, so that the microbial preparations prepared thereby further enhanced the growth-promoting effect on seeds.

[0098] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing a microbial preparation containing Bacillus laterosporus, characterized in that: The following steps are involved: Sterilize the clay and add it to the liquid culture medium until the clay concentration is 25-30 g / L, then add Bacillus spporidia at 50-55 g / L, mix evenly, mature at 28-30° C. for 22-24 hours, centrifuge, wash, and add ultrapure water to prepare a modified clay suspension, wherein the clay includes silica microspheres and montmorillonite at a mass ratio of 1:0.2-0.4, and the Bacillus spporidia HP-2 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20221789; By weight, 30-50 parts of modified clay suspension, 10-15 parts of concentrated biogas slurry, 1-2 parts of glycerol, 2-4 parts of gamma-aminobutyric acid and 3-7 parts of selenium nanomaterials are mixed to prepare a microbial agent; The preparation method of the nano selenium material is as follows: Mix nickel nitrate hexahydrate, ethylenediamine and distilled water, stir evenly, add sodium selenite, perform ultrasonication for 20-30 minutes, heat to 180-200°C for reaction for 20-24 hours, centrifuge, wash and vacuum dry to obtain an intermediate, wherein the mass ratio of nickel nitrate hexahydrate to sodium selenite is 1:1.8-2; The intermediate is added to an ethanol solution, zirconium oxide is added, stirred for 20-24 hours, filtered, washed, and dried. The mass ratio of the intermediate to zirconium oxide is 0.04-0.05:

1.

2. The method for preparing a microbial preparation containing Bacillus laterosporus according to claim 1, characterized in that: The preparation method of the silica microspheres is as follows: By weight, 1.3-2 parts of silicon dioxide powder are dispersed in 200-300 parts of deionized water to prepare a dispersion; Mix 1.7-2 parts of copper nitrate trihydrate, 300-350 parts of deionized water and 50-60 parts of aqueous ammonia, add the dispersion, heat to 140-150° C., react for 20-24 hours, filter and wash to obtain an intermediate product; 1-2 parts of the intermediate product are reacted with 240-320 parts of hydrochloric acid at 50-55°C for 3-4 hours, centrifuged, washed, and vacuum dried.

3. The method for preparing a microbial preparation containing Bacillus laterosporus according to claim 2, characterized in that: The silica microspheres are pretreated as follows: The silica microspheres are dispersed in deionized water to form a suspension, and ammonium chloride is added. After stirring evenly, the suspension is centrifuged and vacuum dried. The mass ratio of the silica microspheres to the ammonium chloride is 1:0.15-0.

2.

4. The method for preparing a microbial preparation containing Bacillus laterosporus according to claim 1, characterized in that: The concentrated biogas slurry is prepared by the following method: filtering the biogas slurry with an ultrafiltration membrane with a pore size of 1-100 nm to obtain a filtrate, adding 30-50 wt % of graphene oxide by weight of the filtrate, and mixing to obtain the concentrated biogas slurry.

5. The method for preparing a microbial preparation containing Bacillus laterosporus according to claim 1, characterized in that: The concentration of the modified clay suspension is 0.1-0.5 g / L.

6. A microbial preparation containing Bacillus laterosporus, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 5.

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