A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation and its preparation method and application

Abstract

The present invention discloses a nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation and its preparation method and application, and belongs to the technical field of microbial materials. The nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation of the present invention comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, microbial protective agent and microbial nutrient carrier, and the mass ratio is 1: (1-2): (0.3-0.7): (50-80). After the microbial preparation of the present invention is applied to the soil, the interaction of each component can effectively play the nitrogen-fixing and growth-promoting effect, especially for corn planting, it can effectively improve the resistance of crops, enhance its environmental adaptability, and have a significant growth-promoting effect, and the dosage form is solid and easy to use.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial materials, and in particular relates to a nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation, a preparation method and an application thereof. Background Art

[0002] Nitrogen and phosphorus are essential mineral elements for plants. They are important components of various structural and functional substances such as proteins, nucleic acids, phospholipids, enzymes, and plant hormones in plant living cells. They play an important role in plant energy metabolism, organic matter synthesis and metabolism, material transportation, gene expression and regulation, and cell signal transduction. The lack of nitrogen and phosphorus will inhibit the growth and development of plants, cause some deficiency diseases, and even prevent them from completing their life history. The long-term and large-scale application of chemical fertilizers not only increases the cost of agricultural production, but also causes the selective accumulation of certain elements in the soil and the deterioration of the physical and chemical properties of the soil, causing pollution to the environment. How to replace or partially replace chemical fertilizers with microbial fertilizers is a common concern of many researchers.

[0003] Microbial agents mainly refer to microorganisms with specific functions that are artificially screened or cultivated, and processed into highly active live bacterial preparations to help humans improve soil and degrade harmful substances. Nitrogen-fixing bacterial powder mainly refers to microbial fertilizers developed by bacteria that can fix nitrogen. There are many types of nitrogen-fixing bacterial powders, which can be classified according to different compositions and types. According to the types of nitrogen-fixing bacteria in the agents, they can be divided into rhizobia agents, symbiotic nitrogen-fixing bacteria agents, and autogenic nitrogen-fixing bacteria agents; according to the target of action, they can be divided into corn nitrogen-fixing bacterial powder, wheat nitrogen-fixing bacterial powder, soybean nitrogen-fixing bacterial powder, rapeseed nitrogen-fixing bacterial powder, etc.; according to the number of bacterial species contained, they can be divided into single nitrogen-fixing bacterial powder and composite nitrogen-fixing bacterial powder.

[0004] At present, there are many studies on composite microbial nitrogen-fixing powder, which mainly consists of mixing multiple nitrogen-fixing bacteria or combining nitrogen-fixing bacteria with plant growth-promoting bacteria (PGPB). While stabilizing the nitrogen-fixing effect, other strains can also play a beneficial role on plants.

[0005] For example, Liu Huawei et al. inoculated wheat under osmotic stress with two nitrogen-fixing bacteria, Azorhizobium caulinodans 'ORS571' and Azospirillum brasilense 'Yu62', and found that inoculation with nitrogen-fixing bacteria could significantly increase the proline and soluble protein content of wheat seedlings (Liu Huawei, Lin Xiaojun, Sun Chao, et al., Inoculation with two nitrogen-fixing bacteria enhances the resistance of wheat seedlings to osmotic stress and growth ability [J]. Journal of Plant Ecology, 2013, 37(01): 70-79.).

[0006] However, the existing technology basically combines nitrogen-fixing bacteria with multiple different functions, which have single functions, low overall activity and poor adaptability, making it difficult for the bacteria to function effectively and sustainably.

[0007] Therefore, how to develop an efficient nitrogen-fixing and growth-promoting bacterial agent product is a technical problem that needs to be solved urgently. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a highly efficient composite microbial agent, which has both nitrogen fixation and growth promotion functions, can enhance the stress resistance of corn, improve the adaptability of corn, and has a solid dosage form and is easy to use.

[0009] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0010] A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, a microbial protective agent and a microbial nutrient carrier.

[0011] Preferably, the mass ratio of the nitrogen-fixing and growth-promoting bacterial powder, the auxiliary bacterial powder, the microbial activity stimulator and the microbial nutrient carrier is 1:(1-2):(0.3-0.7):(50-80).

[0012] Preferably, the nitrogen-fixing and growth-promoting bacterial powder is Pseudomonas fluorescens and Sphingomonas zeicaulis, the strain number of Pseudomonas fluorescens is CGMCC No.1.4522, purchased from the General Microbiology Center of China National Microbiological Culture Collection (CGMCC); the strain number of Sphingomonas zeicaulis is CGMCC No.1.15008, purchased from the General Microbiology Center of China National Microbiological Culture Collection (CGMCC).

[0013] The two strains selected in the present invention can be publicly purchased through the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration, without the need for repeated biological preservation.

[0014] More preferably, the preparation method of the nitrogen-fixing and growth-promoting bacterial powder is as follows: after thawing and activating Pseudomonas fluorescens and Sphingomonas maydis, two rings of bacteria are scraped and inoculated into LB liquid culture medium respectively, and cultured in a shaker at a speed of 180-200 rpm and a temperature of 28-30°C for 22-26 hours to obtain seed liquid of the two bacteria; then, the seed liquids of the two bacteria are mixed in a mass ratio of 1:1, inoculated into the fermentation medium at an inoculation rate of 5%, and cultured in a shaker at a speed of 160-180 rpm and a temperature of 28-30°C for 22-26 hours for seed expansion culture, and the total number of effective live bacteria is controlled to be 2.5-3.0×108 CFU / mL, and finally the fermented bacterial solution is added into the spray drying equipment for spray drying to obtain nitrogen-fixing and growth-promoting bacterial powder.

[0015] More preferably, the activation method of Pseudomonas fluorescens and Sphingomonas zeae is: transferring the frozen bacteria to a slant activation medium, culturing at a constant temperature of 28-30°C for 24-26h to complete the activation. The components of the slant activation medium are as follows: 10g / L tryptone, 5g / L yeast extract, 8g / L sodium chloride, 8g / L agar powder, adjusting the pH value to 7.0 with 1M NaOH, and sterilizing by high pressure at 121°C for 20min.

[0016] More preferably, the fermentation medium comprises: 13 g / L soluble starch, 11 g / L tryptone, 8 g / L CaCl2, the pH value is adjusted to 7.0 with 1 M NaOH, and the medium is sterilized by high pressure at 121° C. for 20 min.

[0017] Preferably, the auxiliary bacterial powder comprises Bacillus subtilis and Bacillus amyloliquefaciens in a mass ratio of 1:1.

[0018] More preferably, the Bacillus subtilis and Bacillus amyloliquefaciens are commercially available products, and the effective viable cell count is selected to be 1.0-3.0×10 8 Commercial bacterial powder with CFU / mL can be used.

[0019] Preferably, the microbial protective agent is obtained by mixing rhamnolipid, trehalose, sodium glutamate and starch in a mass ratio of 0.5:(1-3):(0.1-0.5):(5-10).

[0020] Preferably, the microbial nutrient carrier is a mixture of corn flour, diatomaceous earth, polyvinyl alcohol and brown algae powder in a mass ratio of 10:50:3:5.

[0021] A method for preparing a nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises the following preparation steps:

[0022] (1) preparing nitrogen-fixing and growth-promoting bacterial powder;

[0023] (2) After the nitrogen-fixing growth-promoting bacterial powder and the auxiliary bacterial powder are mixed evenly in proportion, the microbial nutrient carrier is added and mixed and stirred evenly, and then water is added to immerse the solids, and after standing and adsorbing for 1-2 hours, a microbial protective agent is added, and after stirring, it is allowed to stand for 20-30 minutes again, and then dried and crushed to obtain a mixed bacterial powder;

[0024] (3) The mixed bacterial powder is transported to a granulator to form granules, which are then dried and packaged into finished products for storage.

[0025] Preferably, the particle size of the particles formed in step (3) is 2-3 mm.

[0026] The invention discloses an application of a nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation, which is used in corn planting to promote corn growth and enhance corn stress resistance.

[0027] The method for using the nitrogen-fixing and growth-promoting fluorescent Pseudomonas composite preparation of the present invention is as follows: first, the soil is plowed, and the fluorescent Pseudomonas composite preparation of the present invention is applied, and corn can be sown after mixing, wherein the plowing depth is 30-50 cm, and the application amount of the composite preparation is 30 kg per mu.

[0028] Beneficial Effects

[0029] (1) The present invention selects fluorescent Pseudomonas and corn sphingomonas to form core functional microorganisms. Fluorescent Pseudomonas has efficient nitrogen fixation and growth promotion ability, and can metabolize nitrogen through various pathways, including nitrification, denitrification, and ammonification. These pathways enable fluorescent Pseudomonas to effectively utilize and transform nitrogen under different environmental conditions. At the same time, after fluorescent Pseudomonas is inoculated into the soil, it can promote the growth of corn plants and root development, and enhance the activity of stress-resistant enzymes and crop resistance. At the same time, an equal amount of corn sphingomonas is added. Corn sphingomonas has phosphate solubilization activity and autogenous nitrogen fixation activity, which can alleviate the obstacles of continuous cropping of crops. At the same time, it can produce a variety of active substances, regulate plant disease resistance, participate in the regulation of a variety of physiological behaviors, and help plants resist diseases. More importantly, when Sphingomonas cornii and Pseudomonas fluorescens are mixed in equal proportions, Sphingomonas cornii can secrete surfactant, which promotes the colonization of Pseudomonas fluorescens around the root system, allowing the beneficial bacteria to quickly become the dominant bacteria in the plant rhizosphere. The synergistic effect between the bacteria is obvious, and the nitrogen fixation and growth promotion effect is doubled.

[0030] (2) Secondly, the present invention uses Bacillus subtilis and Bacillus amyloliquefaciens as auxiliary microorganisms. In addition to their own disease-resistant and growth-promoting effects, more importantly, they can also enhance the stability of the overall ecosystem by improving the soil environment and promoting the growth of functional microorganisms and other beneficial bacteria, thereby improving the stability and effectiveness of the bacterial agent as a whole.

[0031] (3) Finally, the present invention also uses a microbial protective agent composed of rhamnolipid, trehalose, sodium glutamate and starch, and a microbial nutrient carrier composed of corn flour, diatomaceous earth, polyvinyl alcohol and brown algae powder. Adding a small amount of rhamnolipid to the soil can effectively reduce the salinity of rhizosphere soil, alleviate salt stress, and change the composition of rhizosphere microbial community, increase the number of plant growth-promoting bacteria, and reduce the abundance of plant pathogens. In addition, rhamnolipid can also increase the expression of functional genes related to carbon, nitrogen, sulfur and phosphorus cycles, drive nutrient cycles, enhance microecological functions, and thus improve the photosynthesis process and growth yield of plants. Trehalose can help microorganisms and plants tolerate harsh environmental conditions and improve their ecological adaptability and survival ability. The microbial carrier composed of corn flour, diatomaceous earth, polyvinyl alcohol and brown algae powder, diatomaceous earth has a large specific surface area and good chemical stability, and can effectively fix microorganisms; polyvinyl alcohol has the advantages of high strength, good chemical stability, strong anti-microbial decomposition performance, non-toxic to microorganisms, and low price. It can fix microorganisms by forming a gel, and this immobilization method can significantly improve the loading capacity and biological activity of microorganisms; ingredients such as corn flour and brown algae powder, as part of the nutrient materials, can fully absorb water and adsorb bacterial liquid. At the same time, brown algae powder contains a variety of nutrients needed to maintain microbial activity, which is beneficial to the maintenance of microbial activity.

[0032] (4) In summary, after the microbial preparation of the present invention is applied to the soil, the interaction of various components can effectively play the role of nitrogen fixation and growth promotion. Especially for corn planting, it can effectively improve the resistance of crops, enhance their ability to adapt to the environment, and have a significant growth-promoting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The planting experiment results (plant height) of the embodiments of the present invention and the comparative examples are shown below;

[0034] Figure 2 The figures are the planting experiment results (stem thickness) of the embodiments of the present invention and the comparative examples;

[0035] Figure 3 The planting experiment results (SPAD) of the embodiments of the present invention and the comparative examples are shown in FIG.

[0036] Figure 4 The nitrogen fixation ability experimental diagram of the bacterial solution of Example 1 and Comparative Examples 1-4 of the present invention, wherein A is Example 1, B is Comparative Example 1, D is Comparative Example 2, C is Comparative Example 3, and D is Comparative Example 4;

[0037] Figure 5 The nitrogen content of soil microbial biomass after planting in Example 1 of the present invention and Comparative Examples 1-4;

[0038] Figure 6 This is a graph of soil urease activity after planting in Example 1 of the present invention and Comparative Examples 1-4. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0040] Example 1

[0041] A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, a microbial protective agent and a microbial nutrient carrier.

[0042] The mass ratio of the nitrogen-fixing and growth-promoting bacterial powder, the auxiliary bacterial powder, the microbial activity stimulator and the microbial nutrient carrier is 1:2:0.3:80.

[0043] The nitrogen-fixing and growth-promoting bacterial powders are Pseudomonas fluorescens and Sphingomonas zeicaulis. The strain number of Pseudomonas fluorescens is CGMCC No.1.4522, which is purchased from the General Microbiological Center of China National Microbiological Culture Collection (CGMCC); the strain number of Sphingomonas zeicaulis is CGMCC No.1.15008, which is purchased from the General Microbiological Center of China National Microbiological Culture Collection (CGMCC).

[0044] The preparation method of the nitrogen-fixing and growth-promoting bacterial powder is as follows: after thawing and activating Pseudomonas fluorescens and Sphingomonas maydis, two rings of bacterial strains are scraped and inoculated into LB liquid culture medium respectively, and the mixture is cultured in a shaking incubator at a rotation speed of 180 rpm and a temperature of 28-30° C. for 22-26 hours to obtain seed liquids of the two bacteria; the seed liquids of the two bacteria are then mixed in a mass ratio of 1:1, inoculated into a fermentation medium at an inoculation rate of 5%, and cultured in a shaking incubator at a rotation speed of 180 rpm and a temperature of 28-30° C. for 22-26 hours to perform seed expansion culture, and the total number of effective live bacteria is controlled to be 2.5-3.0×10 8 CFU / mL, and finally the fermented bacterial solution is added into the spray drying equipment for spray drying to obtain nitrogen-fixing and growth-promoting bacterial powder.

[0045] The activation method of Pseudomonas fluorescens and Sphingomonas zeae is as follows: transfer the frozen bacteria to a slant activation medium, culture at a constant temperature of 28-30°C for 24 hours to complete the activation. The components of the slant activation medium are as follows: 10 g / L tryptone, 5 g / L yeast extract, 8 g / L sodium chloride, 8 g / L agar powder, adjust the pH value to 7.0 with 1M NaOH, and sterilize at 121°C for 20 minutes.

[0046] The fermentation medium is composed of: 13 g / L soluble starch, 11 g / L tryptone, 8 g / L CaCl2, the pH value is adjusted to 7.0 with 1 M NaOH, and the medium is sterilized by high pressure at 121° C. for 20 min.

[0047] The auxiliary bacterial powder comprises Bacillus subtilis and Bacillus amyloliquefaciens, and the mass ratio of the two is 1:1.

[0048] The Bacillus subtilis and Bacillus amyloliquefaciens are commercially available products, and the effective viable cell count is selected to be 1.0-3.0×10 8 Commercial bacterial powder with CFU / mL can be used.

[0049] The microbial protective agent is obtained by mixing rhamnolipid, trehalose, sodium glutamate and starch in a mass ratio of 0.5:1:0.5:5.

[0050] The microbial nutrient carrier is obtained by mixing corn flour, diatomaceous earth, polyvinyl alcohol and brown algae powder in a mass ratio of 10:50:3:5.

[0051] A method for preparing a nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises the following preparation steps:

[0052] (1) preparing nitrogen-fixing and growth-promoting bacterial powder;

[0053] (2) After the nitrogen-fixing growth-promoting bacterial powder and the auxiliary bacterial powder are mixed evenly in proportion, the microbial nutrient carrier is added and mixed and stirred evenly, and then water is added to immerse the solid. After standing and adsorbing for 1 hour, a microbial protective agent is added, and after stirring, it is allowed to stand for another 20 minutes, and then dried and crushed to obtain a mixed bacterial powder;

[0054] (3) The mixed bacterial powder is transported to a granulator to form granules, which are then dried and packaged into finished products for storage.

[0055] The particle size of the particles formed in step (3) is 2-3 mm.

[0056] Example 2

[0057] A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, a microbial protective agent and a microbial nutrient carrier.

[0058] The mass ratio of the nitrogen-fixing and growth-promoting bacterial powder, the auxiliary bacterial powder, the microbial activity stimulator and the microbial nutrient carrier is 1:1:0.7:65.

[0059] The nitrogen-fixing and growth-promoting bacterial powders are Pseudomonas fluorescens and Sphingomonas zeicaulis. The strain number of Pseudomonas fluorescens is CGMCC No.1.4522, which is purchased from the General Microbiological Center of China National Microbiological Culture Collection (CGMCC); the strain number of Sphingomonas zeicaulis is CGMCC No.1.15008, which is purchased from the General Microbiological Center of China National Microbiological Culture Collection (CGMCC).

[0060] The two strains (Pseudomonas fluorescens and Sphingomonas maydis) selected in all the embodiments of the present invention can be purchased publicly through the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration, without the need for repeated biological preservation.

[0061] The preparation method of the nitrogen-fixing and growth-promoting bacterial powder is as follows: after thawing and activating Pseudomonas fluorescens and Sphingomonas maydis, two rings of bacterial strains are scraped and inoculated into LB liquid culture medium respectively, and the mixture is cultured in a shaking incubator at a rotation speed of 200 rpm and a temperature of 28-30° C. for 22-26 hours to obtain seed liquids of the two bacteria; the seed liquids of the two bacteria are then mixed in a mass ratio of 1:1, inoculated into a fermentation medium at an inoculation rate of 5%, and cultured in a shaking incubator at a rotation speed of 160 rpm and a temperature of 28-30° C. for 22-26 hours to perform seed expansion culture, and the total number of effective live bacteria is controlled to be 2.5-3.0×10 8 CFU / mL, and finally the fermented bacterial solution is added into the spray drying equipment for spray drying to obtain nitrogen-fixing and growth-promoting bacterial powder.

[0062] The activation method of Pseudomonas fluorescens and Sphingomonas zeae is as follows: transfer the frozen bacteria to a slant activation medium, culture at a constant temperature of 28-30°C for 26 hours to complete the activation. The components of the slant activation medium are as follows: 10 g / L tryptone, 5 g / L yeast extract, 8 g / L sodium chloride, 8 g / L agar powder, adjust the pH value to 7.0 with 1M NaOH, and sterilize at 121°C for 20 minutes.

[0063] The fermentation medium is composed of: 13 g / L soluble starch, 11 g / L tryptone, 8 g / L CaCl2, the pH value is adjusted to 7.0 with 1 M NaOH, and the medium is sterilized by high pressure at 121° C. for 20 min.

[0064] The auxiliary bacterial powder comprises Bacillus subtilis and Bacillus amyloliquefaciens, and the mass ratio of the two is 1:1.

[0065] The Bacillus subtilis and Bacillus amyloliquefaciens are commercially available products, and the effective viable cell count is selected to be 1.0-3.0×10 8Commercial bacterial powder with CFU / mL can be used.

[0066] The Bacillus subtilis and Bacillus amyloliquefaciens used in the present invention were purchased from Weifang Yihao Biotechnology Co., Ltd., and the effective viable count was 1.0-3.0×10 8 CFU / mL.

[0067] The microbial protective agent is obtained by mixing rhamnolipid, trehalose, sodium glutamate and starch in a mass ratio of 0.5:3:0.1:10.

[0068] The microbial nutrient carrier is obtained by mixing corn flour, diatomaceous earth, polyvinyl alcohol and brown algae powder in a mass ratio of 10:50:3:5.

[0069] A method for preparing a nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises the following preparation steps:

[0070] (1) preparing nitrogen-fixing and growth-promoting bacterial powder;

[0071] (2) After the nitrogen-fixing growth-promoting bacterial powder and the auxiliary bacterial powder are mixed evenly in proportion, the microbial nutrient carrier is added and mixed and stirred evenly, and then water is added to immerse the solid. After standing and adsorbing for 1.5 hours, a microbial protective agent is added, and after stirring, it is allowed to stand for another 20 minutes, and then dried and crushed to obtain a mixed bacterial powder;

[0072] (3) The mixed bacterial powder is transported to a granulator to form granules, which are then dried and packaged into finished products for storage.

[0073] The particle size of the particles formed in step (3) is 2-3 mm.

[0074] The invention discloses an application of a nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation, which is used in corn planting to promote corn growth and enhance corn stress resistance.

[0075] The method for using the nitrogen-fixing and growth-promoting fluorescent Pseudomonas composite preparation is as follows: first, the soil is plowed, and the fluorescent Pseudomonas composite preparation of the present invention is applied, and corn can be sown after mixing, wherein the plowing depth is 30-50 cm, and the application amount of the composite preparation is 30 kg per mu.

[0076] Example 3

[0077] A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, a microbial protective agent and a microbial nutrient carrier.

[0078] The mass ratio of the nitrogen-fixing and growth-promoting bacterial powder, the auxiliary bacterial powder, the microbial activity stimulator and the microbial nutrient carrier is 1:1.5:0.5:50.

[0079] The nitrogen-fixing and growth-promoting bacterial powders are Pseudomonas fluorescens and Sphingomonas zeicaulis. The strain number of Pseudomonas fluorescens is CGMCC No.1.4522, which is purchased from the General Microbiological Center of China National Microbiological Culture Collection (CGMCC); the strain number of Sphingomonas zeicaulis is CGMCC No.1.15008, which is purchased from the General Microbiological Center of China National Microbiological Culture Collection (CGMCC).

[0080] The preparation method of the nitrogen-fixing and growth-promoting bacterial powder is as follows: after thawing and activating Pseudomonas fluorescens and Sphingomonas maydis, two rings of bacterial strains are scraped and inoculated into LB liquid culture medium respectively, and the mixture is cultured in a shaking incubator at a rotation speed of 200 rpm and a temperature of 28-30° C. for 22-26 hours to obtain seed liquids of the two bacteria; the seed liquids of the two bacteria are then mixed in a mass ratio of 1:1, inoculated into a fermentation medium at an inoculation rate of 5%, and cultured in a shaking incubator at a rotation speed of 160 rpm and a temperature of 28-30° C. for 22-26 hours to perform seed expansion culture, and the total number of effective live bacteria is controlled to be 2.5-3.0×10 8 CFU / mL, and finally the fermented bacterial solution is added into the spray drying equipment for spray drying to obtain nitrogen-fixing and growth-promoting bacterial powder.

[0081] The activation method of Pseudomonas fluorescens and Sphingomonas zeae is as follows: transfer the frozen bacteria to a slant activation medium, culture at a constant temperature of 28-30°C for 24-26 hours to complete the activation. The components of the slant activation medium are as follows: 10 g / L tryptone, 5 g / L yeast extract, 8 g / L sodium chloride, 8 g / L agar powder, adjust the pH value to 7.0 with 1M NaOH, and sterilize at 121°C for 20 minutes.

[0082] The fermentation medium is composed of: 13 g / L soluble starch, 11 g / L tryptone, 8 g / L CaCl2, the pH value is adjusted to 7.0 with 1 M NaOH, and the medium is sterilized by high pressure at 121° C. for 20 min.

[0083] The auxiliary bacterial powder comprises Bacillus subtilis and Bacillus amyloliquefaciens, and the mass ratio of the two is 1:1.

[0084] The Bacillus subtilis and Bacillus amyloliquefaciens are commercially available products, and the effective viable cell count is selected to be 1.0-3.0×10 8 Commercial bacterial powder with CFU / mL can be used.

[0085] The microbial protective agent is obtained by mixing rhamnolipid, trehalose, sodium glutamate and starch in a mass ratio of 0.5:2:0.3:7.

[0086] The microbial nutrient carrier is obtained by mixing corn flour, diatomaceous earth, polyvinyl alcohol and brown algae powder in a mass ratio of 10:50:3:5.

[0087] A method for preparing a nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises the following preparation steps:

[0088] (1) preparing nitrogen-fixing and growth-promoting bacterial powder;

[0089] (2) After the nitrogen-fixing growth-promoting bacterial powder and the auxiliary bacterial powder are mixed evenly in proportion, the microbial nutrient carrier is added and mixed and stirred evenly, and then water is added to immerse the solid. After standing and adsorbing for 2 hours, a microbial protective agent is added, and after stirring, it is allowed to stand for another 30 minutes, and then dried and crushed to obtain a mixed bacterial powder;

[0090] (3) The mixed bacterial powder is transported to a granulator to form granules, which are then dried and packaged into finished products for storage.

[0091] The particle size of the particles formed in step (3) is 2-3 mm.

[0092] Comparative Example 1

[0093] A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, a microbial protective agent and a microbial nutrient carrier.

[0094] The mass ratio of the nitrogen-fixing and growth-promoting bacterial powder, the auxiliary bacterial powder, the microbial activity stimulator and the microbial nutrient carrier is 1:2:0.3:80.

[0095] The nitrogen-fixing and growth-promoting bacterial powder is Pseudomonas fluorescens, the strain number of Pseudomonas fluorescens is CGMCC No.1.4522, and it is purchased from the General Microbiology Center of China National Microbiological Culture Collection Committee (CGMCC).

[0096] The preparation method of the nitrogen-fixing and growth-promoting bacterial powder is as follows: after thawing and activating Pseudomonas fluorescens, two rings of bacterial strains are scraped and inoculated into LB liquid culture medium, and the culture is carried out in a shaking incubator with a rotation speed of 180 rpm and a temperature of 28-30° C. for 22-26 hours to obtain seed liquid; then, the seed liquid is inoculated into a fermentation culture medium at an inoculation rate of 5%, and the culture is carried out in a shaking incubator with a rotation speed of 180 rpm and a temperature of 28-30° C. for 22-26 hours to perform seed expansion culture, and the total number of effective live bacteria is controlled to be 2.5-3.0×10 8 CFU / mL, and finally the fermented bacterial solution is added into the spray drying equipment for spray drying to obtain nitrogen-fixing and growth-promoting bacterial powder.

[0097] In this comparative example, except that only a single strain of Pseudomonas fluorescens was used in the preparation of the nitrogen-fixing and growth-promoting bacterial powder, the other raw materials and preparation methods were the same as those in Example 1.

[0098] Comparative Example 2

[0099] A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, a microbial protective agent and a microbial nutrient carrier.

[0100] The mass ratio of the nitrogen-fixing and growth-promoting bacterial powder, the auxiliary bacterial powder, the microbial activity stimulator and the microbial nutrient carrier is 1:2:0.3:80.

[0101] The nitrogen-fixing and growth-promoting bacterial powder is Sphingomonas maydis, the strain number of the Sphingomonas maydis is CGMCC No.1.15008, and it is purchased from the General Microbiology Center of China National Microbiological Culture Collection Committee (CGMCC).

[0102] The preparation method of the nitrogen-fixing growth-promoting bacterial powder comprises the following steps: thawing and activating corn sphingomonas, scraping two rings of bacteria, inoculating them into LB liquid culture medium, and culturing them in a shaking incubator with a rotation speed of 180 rpm and a temperature of 28-30° C. for 22-26 hours to obtain seed liquid; then inoculating the seed liquid into a fermentation culture medium at an inoculation rate of 5%, culturing them in a shaking incubator with a rotation speed of 180 rpm and a temperature of 28-30° C. for 22-26 hours to perform seed expansion culture, and controlling the total number of effective live bacteria to be 2.5-3.0×10 8 CFU / mL, and finally the fermented bacterial solution is added into the spray drying equipment for spray drying to obtain nitrogen-fixing and growth-promoting bacterial powder.

[0103] In this comparative example, except that only a single strain of Sphingomonas maydis was used in the preparation of the nitrogen-fixing and growth-promoting bacterial powder, the other raw materials and preparation methods were the same as those in Example 1.

[0104] Comparative Example 3

[0105] A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, a microbial protective agent and a microbial nutrient carrier.

[0106] The mass ratio of the nitrogen-fixing and growth-promoting bacterial powder, the auxiliary bacterial powder, the microbial activity stimulator and the microbial nutrient carrier is 1:2:0.3:80.

[0107] The nitrogen-fixing and growth-promoting bacterial powders are Pseudomonas fluorescens and Sphingomonas zeicaulis. The strain number of Pseudomonas fluorescens is CGMCC No.1.4522, which is purchased from the General Microbiological Center of China National Microbiological Culture Collection (CGMCC); the strain number of Sphingomonas zeicaulis is CGMCC No.1.15008, which is purchased from the General Microbiological Center of China National Microbiological Culture Collection (CGMCC).

[0108] The preparation method of the nitrogen-fixing and growth-promoting bacterial powder is as follows: after thawing and activating Pseudomonas fluorescens and Sphingomonas maydis, two rings of bacterial strains are scraped and inoculated into LB liquid culture medium respectively, and the mixture is cultured in a shaking incubator at a rotation speed of 180 rpm and a temperature of 28-30° C. for 22-26 hours to obtain seed liquids of the two bacteria; the seed liquids of the two bacteria are then mixed in a mass ratio of 2:1, inoculated into a fermentation medium at an inoculation rate of 5%, and cultured in a shaking incubator at a rotation speed of 180 rpm and a temperature of 28-30° C. for 22-26 hours for seed expansion culture, and the total number of effective live bacteria is controlled to be 2.5-3.0×10 8 CFU / mL, and finally the fermented bacterial solution is added into the spray drying equipment for spray drying to obtain nitrogen-fixing and growth-promoting bacterial powder.

[0109] In this comparative example, except that only the dosage ratio of Pseudomonas fluorescens and Sphingomonas maydis was changed in the preparation of nitrogen-fixing and growth-promoting bacterial powder, the other raw materials and preparation method were the same as those in Example 1.

[0110] Comparative Example 4

[0111] A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, a microbial protective agent and a microbial nutrient carrier.

[0112] The mass ratio of the nitrogen-fixing and growth-promoting bacterial powder, the auxiliary bacterial powder, the microbial activity stimulator and the microbial nutrient carrier is 1:2:0.3:80.

[0113] The nitrogen-fixing and growth-promoting bacterial powders are Pseudomonas fluorescens and Sphingomonas zeicaulis. The strain number of Pseudomonas fluorescens is CGMCC No.1.4522, which is purchased from the General Microbiological Center of China National Microbiological Culture Collection (CGMCC); the strain number of Sphingomonas zeicaulis is CGMCC No.1.15008, which is purchased from the General Microbiological Center of China National Microbiological Culture Collection (CGMCC).

[0114] The preparation method of the nitrogen-fixing and growth-promoting bacterial powder is as follows: after thawing and activating Pseudomonas fluorescens and Sphingomonas maydis, two rings of bacterial strains are scraped and inoculated into LB liquid culture medium respectively, and the mixture is cultured in a shaking incubator at a speed of 180 rpm and a temperature of 28-30° C. for 22-26 hours to obtain seed liquids of the two bacteria; the seed liquids of the two bacteria are then mixed in a mass ratio of 1:2, inoculated into a fermentation medium at an inoculation rate of 5%, and cultured in a shaking incubator at a speed of 180 rpm and a temperature of 28-30° C. for 22-26 hours to perform seed expansion culture, and the total number of effective live bacteria is controlled to be 2.5-3.0×10 8CFU / mL, and finally the fermented bacterial solution is added into the spray drying equipment for spray drying to obtain nitrogen-fixing and growth-promoting bacterial powder.

[0115] In this comparative example, except that only the dosage ratio of Pseudomonas fluorescens and Sphingomonas maydis was changed in the preparation of nitrogen-fixing and growth-promoting bacterial powder, the other raw materials and preparation method were the same as those in Example 1.

[0116] Comparative Example 5

[0117] A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, a microbial protective agent and a microbial nutrient carrier.

[0118] The mass ratio of the nitrogen-fixing and growth-promoting bacterial powder, the auxiliary bacterial powder, the microbial activity stimulator and the microbial nutrient carrier is 1:2:0.3:80.

[0119] The microbial protective agent is obtained by mixing sodium glutamate and starch in a mass ratio of 0.5:5.

[0120] In this comparative example, except that rhamnolipid and trehalose are not added to the microorganism protective agent, the other raw materials and preparation method are the same as those in Example 1.

[0121] Comparative Example 6

[0122] A nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation comprises nitrogen-fixing and growth-promoting bacterial powder, auxiliary bacterial powder, a microbial protective agent and a microbial nutrient carrier.

[0123] The mass ratio of the nitrogen-fixing and growth-promoting bacterial powder, the auxiliary bacterial powder, the microbial activity stimulator and the microbial nutrient carrier is 1:2:0.3:80.

[0124] The microbial nutrient carrier is obtained by mixing corn flour and diatomaceous earth in a mass ratio of 10:50.

[0125] In this comparative example, except that polyvinyl alcohol and brown algae powder are not added to the microbial nutrient carrier, the other raw materials and preparation method are the same as those in Example 1.

[0126] Performance Testing

[0127] Nitrogen fixation and growth promotion performance test: The fermented bacterial liquid of Examples 1-3 and Comparative Examples 1-4 before spray drying was tested for nitrogen fixation, phosphorus solubilization, IAA secretion and other abilities. Each group of experiments was repeated three times, and the results were averaged. The test method is as follows:

[0128] Nitrogen fixation ability: Take 10 μL of the bacterial solution to be tested and drop it in the center of the Axubei solid culture medium plate. Incubate it upside down at 28-30℃ for 3 days to observe whether the colony can grow normally.

[0129] Nitrogenase activity: 200 μL of bacterial solution was inoculated into semi-solid NFM medium, and the uninoculated medium was used as the control. After culturing at 28-30℃ for 48 h, the cotton plug was replaced with a rubber plug, 1 mL of gas was drawn out with a sterile syringe and 1 mL of acetylene was injected. After culturing at 28-30℃ for 48 h, the C2H4 peak time and peak area percentage were determined using a gas chromatograph (Agilent7890A, USA), and the nitrogenase activity was calculated.

[0130] Phosphorus dissolving capacity: inoculate the prepared bacterial solution into a conical flask containing 20 mL of Montana inorganic phosphorus medium and place it at 28-30℃ and 180r·min. -1 After culturing for 12 days under the same conditions, the pH was measured, and the mixture was centrifuged. 1 mL of the supernatant was mixed with 19 mL of NaHCO3 solution and shaken on a shaker for 30 min. 5 mL of the filtrate was accurately drawn and 5 mL of molybdenum antimony colorimetric solution was added to a 50 mL volumetric flask. The OD was measured using a UV-visible spectrophotometer. 700 , calculate the dissolved phosphorus amount by referring to the standard curve.

[0131] Siderophore production ability: The CAS detection liquid method was used to determine the strain's ability to synthesize siderophores (Ge Miaomiao, Bo Yonglin, Liu Chen, et al., Screening of soil siderophore-producing bacteria and their activation and utilization of iron oxides [J]. Bulletin of Microbiology, 2023, 50(3): 1062-1072).

[0132] Ability of bacterial agent to secrete IAA: Using the Salkowski colorimetric method, the bacterial solution to be tested was inoculated into a conical flask containing 20 mL of liquid culture medium. After shaking culture for 10 days, 10 mL was aspirated and centrifuged. 5 mL of the supernatant was added with 5 mL of Salkowski colorimetric solution. After dark treatment for 30 minutes, the absorbance at 530 nm was quickly measured with a spectrophotometer, and the IAA concentration of the test solution was found on the standard curve.

[0133] Table 1 Growth-promoting ability test

[0134]

[0135] From the data in the table, we can see that the nitrogen-fixing and growth-promoting bacterial liquid of the embodiment of the present invention has a nitrogen-fixing and growth-promoting ability that is significantly better than that of a single strain (Comparative Examples 1-2). At the same time, the growth-promoting ability of the mixture of the two in equal proportions is also significantly better than that of Comparative Examples 3-4.

[0136] Pot experiment:

[0137] Select seeds of full size and uniformity (the tested corn variety is 'Bai Nuo 158'), rinse with clean water, and soak overnight. Sow 2 seeds in each pot. In order to more realistically restore the saline-alkali soil state under natural conditions, the soil used in the potted test is sieved and mixed with vermiculite in a volume ratio of 1:1. The soil is soaked with a 9g / kg mixed salt concentration solution, and 15kg of soil is placed in each pot. The fertilizer application rate is N0.1g / kg, N / P=2:1, and the test fertilizers are urea (N46%) and potassium dihydrogen phosphate (P2O5: 52%, K2O: 34%). The amount of compound preparation added to the treatment group is 5g. Ten parallel tests are set up for each group, and the results are averaged. Water regularly and quantitatively during the growth of crops to maintain appropriate moisture. The experimental groups are:

[0138] S1: Example 1;

[0139] S2: Example 2;

[0140] S3: Example 3;

[0141] S4: Comparative Example 1;

[0142] S5: Comparative Example 2;

[0143] S6: Comparative Example 3;

[0144] S7: Comparative Example 4;

[0145] S8: Comparative Example 5;

[0146] S9: Comparative Example 6;

[0147] CK: No microbial preparations are added.

[0148] Determination indicators and methods:

[0149] Determination of growth indicators: plant height was measured with a tape measure, and stem diameter was measured with a vernier caliper.

[0150] Physiological index determination: The chlorophyll content of corn was determined using a SPAD-502Plus portable chlorophyll meter. The superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium method, the malondialdehyde (MDA) activity was determined using the thiobarbituric acid method, the peroxidase (POD) activity was determined using the guaiacol method, and the catalase (CAT) activity was determined using ultraviolet spectrophotometry.

[0151] The specific results of crop shape are as follows Figure 1-3 The physiological indicators of stress resistance are shown in Table 2:

[0152] Table 2 Effects of different treatment groups on maize stress resistance physiological indicators

[0153] CAT activity u / g SOD activity u / g POD activity u / g Malondialdehyde umo / g S1 65.2 320.5 5100 4.5 S2 66.9 325.6 5210 4.3 S3 68.5 330.2 5300 4.0 S4 55.4 250.1 4500 5.2 S5 56.2 241.5 4450 5.5 S6 58.9 265.4 4800 5.2 S7 59.7 270.1 4750 5.1 S8 60.1 263.4 4850 4.9 S9 58.1 264.8 4810 4.8 CK 35.2 150.1 3560 7.5

[0154] From the data in Table 2, it can be seen that the use of the microbial preparation product of the embodiment of the present invention not only improves the SOD, POD and CAT activities of corn leaves under saline-alkali stress, but also significantly reduces the MDA content of corn leaves, indicating that the use of microorganisms enhances the plant's defense against reactive oxygen damage, slows down cell membrane lipid peroxidation, and enhances crop resistance. However, in Comparative Examples 1-4 with changed microbial composition and Comparative Examples 5-6 with changed microbial protective agent and carrier component composition, the protective and promoting effects on microorganisms are weakened, thus resulting in a decrease in the effect. It can be seen that the strain of the present invention and the microbial protective agent and carrier component composition are the key to achieving efficient nitrogen fixation and growth promotion effects, and the lack of one will result in a weak effect.

[0155] To further test the soil environment after planting, the test method is as follows:

[0156] Soil microbial biomass nitrogen (SMBN) was determined by first treating the soil sample with chloroform fumigation and then extracting it with 0.5 mol / L K2SO4 solution. The nitrogen in the extract was determined by the Kjeldahl method. The calculation formula is as follows:

[0157] Soil microbial biomass nitrogen (mg / kg) = (Ec-Ec0) / 0.54.

[0158] Where, Ec is the amount of organic nitrogen in the fumigated soil extract; Ec0 is the amount of organic nitrogen in the non-fumigated soil extract; and 0.54 is the correction factor.

[0159] Determination of soil urease activity: Urease activity was determined using the indigo blue colorimetric method. The result was expressed as milligrams of NH3-N in 1g of soil after 24 hours.

[0160] Soil microbial biomass nitrogen (MBN) is an important component of active organic nitrogen in the soil and plays a vital role in soil nitrogen cycle and crop nitrogen supply. Soil microbial biomass nitrogen plays a key role in the soil nitrogen transformation process, and its content can reflect the soil nitrogen supply capacity.

[0161] Soil urease is an important active substance involved in the soil nitrogen cycle. Urease activity reflects the ability of soil organic nitrogen to convert into effective nitrogen and the supply capacity of soil inorganic nitrogen. It is an important indicator for evaluating soil quality.

[0162] From the soil test results ( Figure 5-6 ) It can also be seen that in the experimental group where microorganisms were applied, soil activity was significantly improved.

[0163] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. An application of a nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation, characterized in that: Used for corn planting, promoting corn growth, and improving corn stress resistance; the composite preparation comprises nitrogen-fixing growth-promoting bacterial powder, auxiliary bacterial powder, microbial protective agent and microbial nutrient carrier; the mass ratio of the nitrogen-fixing growth-promoting bacterial powder, auxiliary bacterial powder, microbial protective agent and microbial nutrient carrier is 1: (1-2): (0.3-0.7): (50-80); the nitrogen-fixing growth-promoting bacterial powder is Pseudomonas fluorescens ( Pseudomonas fluorescens ) and Sphingomonas zeae ( Sphingomonas zeicaulis ), the strain number of the fluorescent Pseudomonas is CGMCC No.1.4522, which was purchased from the General Microbiology Center of China National Microbiological Culture Collection Committee (CGMCC); the strain number of the corn sphingomonas is CGMCC No.1.15008, which was purchased from the General Microbiology Center of China National Microbiological Culture Collection Committee (CGMCC); the preparation method of the nitrogen-fixing and growth-promoting bacterial powder is as follows: after thawing and activating the fluorescent Pseudomonas and corn sphingomonas, two rings of bacteria are scraped and inoculated into LB liquid culture medium respectively, and cultured in a shaker with a speed of 180-200 rpm and a temperature of 28-30°C for 22-26 hours to obtain seed liquid of the two bacteria; then the seed liquids of the two bacteria are mixed in a mass ratio of 1:1, inoculated into the fermentation medium at an inoculation rate of 5%, and cultured in a shaker with a speed of 160-180 rpm and a temperature of 28-30°C for 22-26 hours for seed expansion culture, and the total number of effective viable bacteria is controlled to be 2.5-3.0×10 8 CFU / mL, and finally adding the fermented bacterial liquid into a spray drying device for spray drying to obtain nitrogen-fixing and growth-promoting bacterial powder; the auxiliary bacterial powder comprises Bacillus subtilis and Bacillus amyloliquefaciens, and the mass ratio of the two is 1:1; the microbial protective agent is obtained by mixing rhamnolipid, trehalose, sodium glutamate and starch in a mass ratio of 0.5: (1-3): (0.1-0.5): (5-10); the microbial nutrient carrier is obtained by mixing corn flour, diatomaceous earth, polyvinyl alcohol and brown algae powder in a mass ratio of 10:50:3:

5.

2. The use of the nitrogen-fixing and growth-promoting Pseudomonas fluorescens composite preparation according to claim 1, characterized in that: The preparation method of the composite preparation comprises the following preparation steps: (1) preparing nitrogen-fixing and growth-promoting bacterial powder; (2) After the nitrogen-fixing growth-promoting bacterial powder and the auxiliary bacterial powder are mixed evenly in proportion, the microbial nutrient carrier is added and mixed and stirred evenly, and then water is added to immerse the solids, and after standing and adsorbing for 1-2 hours, a microbial protective agent is added, and after stirring, it is allowed to stand for 20-30 minutes again, and then dried and crushed to obtain a mixed bacterial powder; (3) The mixed bacterial powder is transported to a granulator to form granules, which are then dried and packaged into finished products for storage.

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