A saline-alkali resistant microbial agent and a preparation method thereof

Abstract

The application discloses a kind of anti-saline-alkali microbial inoculants and preparation method thereof, belong to the field of microbial technology.The microbial inoculants is made of composite microbial inoculants 100-150 parts, fish bone hydrolysate 10-30 parts, nano silicon sol 1-3 parts.The composite microbial inoculants is made of Trichoderma reesei, Pseudomonas plecoglossicida, Acinetobacter baumannii.The anti-saline-alkali microbial inoculants has remarkable effect on saline-alkali soil improvement, greatly reduces soil acidity, salt content and bulk density, and the yield of saline-alkali soil wheat planting is obviously improved.The soil organic matter content and soil enzyme activity are significantly improved after use, the soil quality and rhizosphere microecological environment are improved, and the enzyme activity in wheat is improved, the stress resistance is improved, the yield reduction caused by saline-alkali stress is reduced, and the use of microbial inoculants can also reduce the amount of chemical fertilizer and pesticide, reduce production cost, and further improve the economic benefit of wheat planting.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a salt-alkali resistant microbial agent and its preparation method. Background Technology

[0002] Soil salinization is a phenomenon caused by the accumulation of soluble salts in the topsoil of arid, semi-arid, and semi-humid regions due to natural or anthropogenic factors. In recent years, with the impact of climate change and human activities, soil salinization has intensified, and the area affected by it is constantly increasing. Soil salinization not only hinders crop growth but also often results in excessive levels of heavy metals, leading to crops grown in salinized soils containing excessive levels of heavy metals, seriously endangering animal and human health. Currently, soil salinization continues to rise globally, affecting more than 100 countries, with saline-alkali land areas reaching 1.1 × 10⁻⁶. 9 hm 2 This severely restricts global crop yields and ecological security, causing serious economic losses and ecological crises. While saline-alkali arable land remains a major source of low- and medium-yield arable land in my country, high concentrations of exchangeable sodium in the soil cause soil compaction, leading to poor soil structure and decreased fertility. Furthermore, soil salinity reduces plant nutrient acquisition and transport, resulting in nutrient imbalances, decreased productivity, and degradation of the surrounding ecological environment. These problems seriously hinder the improvement of local land use efficiency and ecological security.

[0003] Currently, methods for improving saline-alkali land mainly include physical and hydraulic engineering regulation, biological improvement, and chemical conditioning. Hydraulic engineering measures reduce the soluble salt content of the topsoil through "salt leaching," but this also removes essential minerals for plants, hindering soil fertility. Physical measures primarily alter soil structure or reduce soil moisture evaporation to change water-salt movement, but these require significant time and equipment investment. Chemical measures enhance cation exchange capacity and promote aggregate formation, thereby increasing soil permeability and accelerating desalination, but they carry the risk of introducing new harmful ions and heavy metals, potentially causing secondary pollution of soil salt ions and jeopardizing food security. Biological improvement measures can improve saline-alkali land through the cultivation of salt-tolerant plants and the development of salt-tolerant microorganisms. In recent years, with the continuous development of microbial technology, its application in saline-alkali land management has gradually demonstrated unique advantages. However, single microbial strains are less effective at improving saline-alkali soil and have poor stability and long-term effects on the ecosystem. Applying compound microbial agents containing multiple strains can not only increase the number of microorganisms in the soil and enhance its biological activity, but also promote nutrient cycling, thereby improving crop resistance and yield. Therefore, there is an urgent need for a high-efficiency, stable, and long-lasting compound microbial agent to improve the saline-alkali soil environment and increase crop yields. Summary of the Invention

[0004] The purpose of this invention is to provide a salt-alkali resistant compound microbial agent, which improves the rhizosphere microecological environment, reduces the damage to plants caused by salt and alkali stress, and promotes plant growth by screening three superior strains and applying them in combination in the soil.

[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A salt-alkali resistant microbial agent is made from the following raw materials in parts by weight: 100-150 parts of compound microbial inoculum, 10-30 parts of fish bone hydrolysate, and 1-3 parts of nano-silica sol; the compound microbial inoculum is made from *Trichoderma*, *Pseudomonas*, and *Acinetobacter*; *Trichoderma* strain number CCTCC DF 20081027, deposited on October 11, 1994, purchased from the China Center for Type Culture Collection; *Pseudomonas* strain number CCTCC AB 2014015, deposited on January 8, 2014, purchased from the China Center for Type Culture Collection; and *Acinetobacter* strain number CCTCC AB 205294, deposited on November 12, 2005, purchased from the China Center for Type Culture Collection. The three strains of Trichoderma synergae, Pseudomonas ayuensis, and Acinetobacter benzii used in this invention can all be purchased through the open catalog of the depositary center, without the need for repeated deposit.

[0006] Preferably, the fish bone hydrolysate is prepared by the following method: (1) Clean the fish bones, freeze-dry them at low temperature, and make them into fish bone powder; (2) Mix fish bone powder and water at a ratio of 1g:10ml, steam at 120℃ for 20-30 minutes, remove the upper layer of fat, and homogenize to obtain a slurry; (3) Add 1.5wt% neutral protease to the slurry and enzymatically hydrolyze it at 35-40℃ for 8-10h. After inactivation of the enzyme, the first enzymatic hydrolysate is obtained. (4) Add the compound hydrolytic enzyme solution to the first enzymatic hydrolysate at a volume ratio of 1:0.8-1.2 and continue enzymatic hydrolysis at 38-45℃ for 10-12h. After inactivation of the enzyme, the second enzymatic hydrolysate is obtained. (5) Centrifuge the second enzymatic hydrolysate in a centrifuge at 4000-6000 r / min for 3-5 min, remove the solids and take the filtrate as fish bone hydrolysate.

[0007] Preferably, the neutral protease activity is 100,000 u / g.

[0008] Preferably, the complex enzyme solution contains 20-30 g / L of papain and 15-25 g / L of trypsin; the papain activity is 500,000 u / g and the trypsin activity is 100,000 u / g.

[0009] Preferably, the composite microbial culture solution is prepared by the following method: (a) Trichoderma koningii, Pseudomonas ayuensis, and Acinetobacter benzini were activated and cultured in LB liquid seed medium to obtain a concentration of 2×10⁻⁶. 8cfu / mL ~4×10 8 Seed culture with cfu / mL; (b) Mix the crushed plant straw and wheat bran in a 1:1 mass ratio, then add 2-3 times the weight of the solid mixture of water, sterilize, and obtain a mixed solution; inoculate the mixed solution with Trichoderma ts., and ferment naturally at 28-30℃ for 10 days to obtain a primary fermentation mixture; then add supplementary substrate and stir evenly, adjust the stirring speed to 150-200 r / min, the ventilation rate to 1.0-1.5 vvm, and the fermentation temperature to 30-35℃ and continue fermentation for 8 days to obtain a secondary fermentation broth, and separate the solid and liquid to obtain the Trichoderma ts. (c) The seed cultures of *Pseudomonas aeruginosa* and *Acinetobacter benzi* strains were inoculated into 200 mL of fermentation liquid culture medium at an inoculation rate of 10%, and cultured at 30 °C and 200 r / min for 72 h to obtain two fermentation broths. (d) The fermentation broth of Trichoderma ts., Pseudomonas aeruginosa, and Acinetobacter benzie were mixed in a volume ratio of 2:1:1 to obtain the composite microbial culture.

[0010] Preferably, in step (b), the amount of Trichoderma syn. seed liquid inoculated is 8-10% of the volume of the mixed solution.

[0011] Preferably, in step (b), the amount of supplementary substrate added is 5-8% of the volume of the primary fermentation mixture; the supplementary substrate contains 10-15 g / L glucose, 15-20 g / L peptone, 1-2 g / L inositol phospholipids, 5-10 g / L corn flour, 0.5-2 g / L ammonium sulfate, 0.2-0.5 g / L ferrous sulfate, and 2-5 g / L potassium dihydrogen phosphate.

[0012] Preferably, the fermentation liquid culture medium in step (c) consists of: yeast extract 10-15 g / L, starch hydrolysate 15-20 g / L, corn steep liquor 15-20 g / L, sodium chloride 5-10 g / L, magnesium sulfate 1-3 g / L, and potassium dihydrogen phosphate 2-5 g / L.

[0013] Preferably, the nano-silica sol contains 30% nano-silica, has a particle size of 55-65 nm, and a pH of 7.0-7.5.

[0014] The present invention also provides a method for preparing the above-mentioned salt-alkali resistant microbial agent, comprising the following steps: S1: Prepare fish bone hydrolysate and mix it evenly with nano silica sol. Adjust the pH of the mixture to 7.0±0.1 and then sonicate for 5-10 min to obtain a first mixture. S2: Mix the primary mixture with the prepared composite microbial inoculum in a specific ratio to obtain the final product.

[0015] The salt-alkali resistant compound microbial agent of the present invention can be applied by irrigation, drip irrigation or direct spraying, with a dosage of 5.0 kg-6.0 kg / mu.

[0016] The composite microbial inoculum of this invention includes products fermented and cultured by *Trichoderma spp.*, *Pseudomonas ayutae*, and *Acinetobacter bengal*, respectively. *Trichoderma spp.*, under the conditions described in this invention, can produce a large amount of biochemical humic acid during fermentation. This humic acid contains various active groups such as carboxyl, hydroxyl, and methoxy groups, exhibiting chelating, complexing, and adsorption properties. When applied to the soil, it can react with harmful salt ions, increasing soil aggregate structure, strengthening the soil's buffering capacity against salinity and alkali, improving water and fertilizer retention, promoting wheat growth and development, and enhancing wheat's stress resistance. *Pseudomonas ayutae* and *Acinetobacter bengal* have excellent phosphorus-solubilizing abilities, converting insoluble phosphorus in the soil into soluble phosphorus for wheat absorption and utilization, accelerating wheat photosynthesis, improving fertilizer utilization, and contributing to improved growth and development of wheat in saline-alkali land, thus enhancing wheat's salt and alkali resistance. When used in combination, the three strains significantly improved the soil rhizosphere microbial environment. The synergistic effect of multiple microorganisms promoted each other, and their metabolites included a variety of active substances such as organic acids, polysaccharides, and growth hormones. These substances could participate in chemical reactions in the soil, regulate soil pH and salinity, help improve the soil microenvironment, make it more conducive to wheat growth, and enhance the stress resistance of wheat.

[0017] The fish bone enzymatic hydrolysate prepared in this invention contains active small molecule peptides, abundant amino acids, and mineral elements. On one hand, it can serve as a carbon and nitrogen source for microorganisms, promoting the growth and reproduction of beneficial microorganisms in the soil and enhancing soil microbial activity. On the other hand, it can be directly absorbed and utilized by wheat, promoting its growth and development, enhancing its stress resistance, and helping crops better adapt to saline-alkali environments. Synergistically, it can achieve even better results in improving saline-alkali soil when used with compound microbial inoculants.

[0018] The high specific surface area and tunable pore size of nano-silica sol provide more attachment sites and transport channels for the small molecule active peptides contained in the fish bone enzymatic hydrolysate obtained in this invention, thereby enhancing the bioactivity and utilization rate of the active peptides. The highly active small molecule polypeptides also promote the growth and metabolism of microbial agents, further improving their effect on improving saline-alkali soil environments. Simultaneously, nano-silica can also be directly absorbed and utilized by wheat. After entering the wheat body, it can enhance wheat's tolerance to heavy metal, salt, and drought stress by reducing ROS production or regulating hormone levels, thus improving wheat's stress resistance.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The salt-alkali resistant microbial agent prepared by the present invention has a significant effect on improving saline-alkali land, greatly reducing soil pH, salt content and bulk density, and significantly increasing wheat yield in saline-alkali land. After use, the soil organic matter content and soil enzyme activity are significantly increased, improving soil quality and rhizosphere micro-ecological environment. At the same time, it enhances the activity of various stress-resistant enzymes in wheat, improves its stress resistance, and reduces the damage to wheat yield caused by salt-alkali stress. In addition, the use of the microbial agent of the present invention can also reduce the amount of chemical fertilizers and pesticides used, reduce production costs, and further improve the economic benefits of wheat planting. Attached Figure Description

[0020] Figure 1 This is a comparison image of mature wheat ears after the application of salt-alkali resistant microbial agents and before the application of salt-alkali resistant microbial agents in wheat planting trials of the present invention; Figure 2 The dry matter accumulation of wheat in different treatment groups according to the present invention; Figure 3 The total chlorophyll content of wheat leaves in different treatment groups according to the present invention; Figure 4 This is a comparison image of wheat seedlings grown using salt-tolerant microbial agents and those not using salt-tolerant microbial agents in the wheat planting experiment of this invention. Figure 5 The results of SOD activity testing in wheat leaves of different treatment groups in this invention; Figure 6 These are the CAT activity test results in wheat leaves of different treatment groups according to the present invention; Figure 7 The results of POD activity testing in wheat leaves of different treatment groups in this invention are shown. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to specific embodiments, but is not limited thereto. Unless otherwise specified, all raw materials and reagents used in the present invention are commercially available. Specifically, *Trichoderma ts.* strain number CCTCCDF 20081027, deposited on October 11, 1994, was purchased from the China Center for Type Culture Collection (CCTCC); *Pseudomonas ayuensis* strain number CCTCC AB 2014015, deposited on January 8, 2014, was purchased from CCTCC; and *Acinetobacter bengal* strain number CCTCC AB 205294, deposited on November 12, 2005, was purchased from CCTCC. The three strains of *Trichoderma ts.*, *Pseudomonas ayuensis*, and *Acinetobacter bengal* used in the present invention can all be purchased through the open catalog of the collection centers, without the need for duplicate deposit.

[0022] Example 1 A salt-alkali resistant microbial agent is made from the following raw materials in parts by weight: 100 kg of compound microbial liquid, 10 kg of fish bone hydrolysate, and 1 kg of nano-silica sol; the compound microbial liquid is made from Trichoderma synergum, Pseudomonas aeruginosa, and Acinetobacter bengal; the nano-silica sol contains 30% nano-silica, has a particle size of 55 nm, and a pH of 7.0.

[0023] The fish bone hydrolysate was prepared using the following method: (1) Clean the fish bones, freeze-dry them at low temperature, and make them into fish bone powder; (2) Mix fish bone powder and water at a ratio of 1g:10ml, steam at 120℃ for 20 minutes, remove the upper layer of fat, and homogenize to obtain a slurry; (3) Add 1.5wt% of neutral protease to the slurry, and enzymatically hydrolyze at 35℃ for 8h. After enzyme inactivation, the first enzymatic hydrolysate is obtained; the neutral protease activity is 100,000 u / g.

[0024] (4) Add the compound hydrolytic enzyme solution to the first enzymatic hydrolysate at a volume ratio of 1:0.8 and continue enzymatic hydrolysis at 38°C for 10 h. After inactivation of the enzyme, the second enzymatic hydrolysate is obtained. The compound enzyme solution contains 20 g / L papain and 15 g / L trypsin. The papain activity is 500,000 u / g and the trypsin activity is 100,000 u / g.

[0025] (5) Centrifuge the second enzymatic hydrolysate at 4000 r / min for 3 to 5 min, remove the solids and take the filtrate as fish bone hydrolysate.

[0026] The composite microbial culture was prepared using the following method: (a) Trichoderma koningii, Pseudomonas ayuensis, and Acinetobacter benzini were activated and cultured in LB liquid seed medium to obtain a concentration of 2×10⁻⁶. 8 Seed culture with cfu / mL; (b) The crushed plant straw and wheat bran were mixed in a 1:1 mass ratio, and then water with a weight of 2 times that of the solid mixture was added. The mixture was sterilized to obtain a mixed solution. 8% Trichoderma tumefaciens seed solution was inoculated into the mixed solution and fermented naturally at 28°C for 10 days to obtain a primary fermentation mixture. Then, a supplementary substrate was added and stirred evenly. During fermentation, the stirring speed was adjusted to 150 r / min, the ventilation rate was 1.0 vvm, and the fermentation temperature was 30°C. Fermentation continued for another 8 days to obtain a secondary fermentation broth. The solid and liquid were separated and the filtrate was used to obtain the Trichoderma tumefaciens fermentation broth. The amount of supplementary substrate added was 5% of the volume of the primary fermentation mixture. The supplementary substrate contained 10 g / L glucose, 15 g / L peptone, 1 g / L inositol phospholipids, 5 g / L corn flour, 0.5 g / L ammonium sulfate, 0.2 g / L ferrous sulfate, and 2 g / L potassium dihydrogen phosphate.

[0027] (c) Seed cultures of *Pseudomonas aeruginosa* and *Acinetobacter benzi* strains were inoculated into 200 mL of fermentation liquid culture medium at a 10% inoculation rate and cultured at 30 °C and 200 r / min for 72 h to obtain two fermentation broths. The fermentation liquid culture medium consisted of: yeast extract 10 g / L, starch hydrolysate 15 g / L, corn steep liquor 15 g / L, sodium chloride 5 g / L, magnesium sulfate 1 g / L, and potassium dihydrogen phosphate 2 g / L.

[0028] (d) The fermentation broth of Trichoderma ts., Pseudomonas aeruginosa, and Acinetobacter benzie were mixed in a volume ratio of 2:1:1 to obtain the composite microbial culture.

[0029] The preparation method of the above-mentioned salt-alkali resistant microbial agent includes the following steps: S1: Prepare fish bone hydrolysate and mix it evenly with nano silica sol. Adjust the pH of the mixture to 7.0±0.1 and then sonicate for 5-10 min to obtain a first mixture. S2: Mix the primary mixture with the prepared composite microbial inoculum in a specific ratio to obtain the final product.

[0030] Example 2 A salt-alkali resistant microbial agent is made from the following raw materials in parts by weight: 150 kg of compound microbial liquid, 30 kg of fish bone hydrolysate, and 3 kg of nano-silica sol; the compound microbial liquid is made from Trichoderma synergum, Pseudomonas aeruginosa, and Acinetobacter bengal; the nano-silica sol contains 30% nano-silica, has a particle size of 65 nm, and a pH of 7.5.

[0031] The fish bone hydrolysate was prepared using the following method: (1) Clean the fish bones, freeze-dry them at low temperature, and make them into fish bone powder; (2) Mix fish bone powder and water at a ratio of 1g:10ml, steam at 120℃ for 30 minutes, remove the upper layer of fat, and homogenize to obtain a slurry; (3) Add 1.5wt% of neutral protease to the slurry, and enzymatically hydrolyze it at 40℃ for 10h. After inactivation of the enzyme, the first enzymatic hydrolysate is obtained; the activity of the neutral protease is 100,000 u / g.

[0032] (4) Add the compound hydrolytic enzyme solution to the first enzymatic hydrolysate at a volume ratio of 1:1.2 and continue enzymatic hydrolysis at 45°C for 12 hours. After enzyme inactivation, the second enzymatic hydrolysate is obtained. The compound enzyme solution contains 30 g / L papain and 25 g / L trypsin. The papain activity is 500,000 u / g and the trypsin activity is 100,000 u / g.

[0033] (5) Centrifuge the second enzymatic hydrolysate at 6000 r / min for 3 to 5 min, remove the solids and take the filtrate as fish bone hydrolysate.

[0034] The composite microbial culture was prepared using the following method: (a) Trichoderma koningii, Pseudomonas aeruginosa, and Acinetobacter benziae were activated and cultured in LB liquid seed medium to obtain a concentration of 4 × 10⁻⁶. 8 Seed culture with cfu / mL; (b) The crushed plant straw and wheat bran were mixed in a 1:1 mass ratio, and then 3 times the weight of the solid mixture of water was added. The mixture was sterilized to obtain a mixed solution. 10% Trichoderma tumefaciens seed liquid was inoculated into the mixed solution and fermented naturally at 30°C for 10 days to obtain a primary fermentation mixture. Then, a supplementary substrate was added and stirred evenly. During fermentation, the stirring speed was adjusted to 200 r / min, the ventilation rate was 1.5 vvm, and the fermentation temperature was 35°C. Fermentation was continued for another 8 days to obtain a secondary fermentation broth. The solid and liquid were separated and the filtrate was used to obtain the Trichoderma tumefaciens fermentation broth. The amount of supplementary substrate added was 8% of the volume of the primary fermentation mixture. The supplementary substrate contained 15 g / L glucose, 20 g / L peptone, 2 g / L inositol phospholipids, 10 g / L corn flour, 2 g / L ammonium sulfate, 0.5 g / L ferrous sulfate, and 5 g / L potassium dihydrogen phosphate.

[0035] (c) Seed cultures of *Pseudomonas aeruginosa* and *Acinetobacter benzi* strains were inoculated into 200 mL of fermentation liquid culture medium at a 10% inoculation rate and cultured at 30 °C and 200 r / min for 72 h to obtain two fermentation broths. The fermentation liquid culture medium consisted of: yeast extract 15 g / L, starch hydrolysate 20 g / L, corn steep liquor 20 g / L, sodium chloride 10 g / L, magnesium sulfate 3 g / L, and potassium dihydrogen phosphate 5 g / L.

[0036] (d) The fermentation broth of Trichoderma ts., Pseudomonas aeruginosa, and Acinetobacter benzie were mixed in a volume ratio of 2:1:1 to obtain the composite microbial culture.

[0037] The preparation method of the above-mentioned salt-alkali resistant microbial agent includes the following steps: S1: Prepare fish bone hydrolysate and mix it evenly with nano silica sol. Adjust the pH of the mixture to 7.0±0.1 and then sonicate for 5-10 min to obtain a first mixture. S2: Mix the primary mixture with the prepared composite microbial inoculum in a specific ratio to obtain the final product.

[0038] Example 3 A salt-alkali resistant microbial agent is made from the following raw materials in parts by weight: 130 kg of compound microbial liquid, 20 kg of fish bone hydrolysate, and 2 kg of nano-silica sol; the compound microbial liquid is made from Trichoderma synergum, Pseudomonas aeruginosa, and Acinetobacter bengal; the nano-silica sol contains 30% nano-silica, has a particle size of 60 nm, and a pH of 7.2.

[0039] The fish bone hydrolysate was prepared using the following method: (1) Clean the fish bones, freeze-dry them at low temperature, and make them into fish bone powder; (2) Mix fish bone powder and water at a ratio of 1g:10ml, steam at 120℃ for 30 minutes, remove the upper layer of fat, and homogenize to obtain a slurry; (3) Add 1.5wt% of neutral protease to the slurry, and enzymatically hydrolyze at 37℃ for 9h. After inactivation of the enzyme, the first enzymatic hydrolysate is obtained; the neutral protease activity is 100,000 u / g.

[0040] (4) Add the compound hydrolytic enzyme solution to the first enzymatic hydrolysate at a volume ratio of 1:1 and continue enzymatic hydrolysis at 40°C for 11 hours. After inactivation of the enzyme, the second enzymatic hydrolysate is obtained. The compound enzyme solution contains 25 g / L papain and 20 g / L trypsin. The papain activity is 500,000 u / g and the trypsin activity is 100,000 u / g.

[0041] (5) Centrifuge the second enzymatic hydrolysate at 5000 r / min for 3 to 5 min, remove the solids and take the filtrate as fish bone hydrolysate.

[0042] The composite microbial culture was prepared using the following method: (a) Trichoderma koningii, Pseudomonas ayuensis, and Acinetobacter benzini were activated and cultured in LB liquid seed medium to obtain a concentration of 3 × 10⁻⁶. 8 Seed culture with cfu / mL; (b) The crushed plant straw and wheat bran were mixed in a 1:1 mass ratio, and then 3 times the weight of the solid mixture of water was added. The mixture was sterilized to obtain a mixed solution. 10% Trichoderma synergae seed liquid was inoculated into the mixed solution and fermented naturally at 30°C for 10 days to obtain a primary fermentation mixture. Then, a supplementary substrate was added and stirred evenly. During fermentation, the stirring speed was adjusted to 180 r / min, the ventilation rate was 1.3 vvm, and the fermentation temperature was 33°C. Fermentation was continued for another 8 days to obtain a secondary fermentation broth. The solid and liquid were separated and the filtrate was used to obtain the Trichoderma synergae fermentation broth. The amount of supplementary substrate added was 6% of the volume of the primary fermentation mixture. The supplementary substrate contained 12 g / L glucose, 18 g / L peptone, 2 g / L inositol phospholipids, 10 g / L corn flour, 1 g / L ammonium sulfate, 0.3 g / L ferrous sulfate, and 3 g / L potassium dihydrogen phosphate.

[0043] (c) Seed cultures of *Pseudomonas aeruginosa* and *Acinetobacter benzi* strains were inoculated into 200 mL of fermentation liquid culture medium at a 10% inoculation rate and cultured at 30 °C and 200 r / min for 72 h to obtain two fermentation broths. The fermentation liquid culture medium consisted of: yeast extract 13 g / L, starch hydrolysate 15 g / L, corn steep liquor 20 g / L, sodium chloride 7 g / L, magnesium sulfate 2 g / L, and potassium dihydrogen phosphate 3 g / L.

[0044] (d) The fermentation broth of Trichoderma ts., Pseudomonas aeruginosa, and Acinetobacter benzie were mixed in a volume ratio of 2:1:1 to obtain the composite microbial culture.

[0045] The preparation method of the above-mentioned salt-alkali resistant microbial agent includes the following steps: S1: Prepare fish bone hydrolysate and mix it evenly with nano silica sol. Adjust the pH of the mixture to 7.0±0.1 and then sonicate for 5-10 min to obtain a first mixture. S2: Mix the primary mixture with the prepared composite microbial inoculum in a specific ratio to obtain the final product.

[0046] Comparative Example 1 A salt-alkali resistant microbial agent is made from the following raw materials in parts by weight: 130 kg of compound microbial liquid, 20 kg of fish bone hydrolysate, and 2 kg of nano-silica sol; the compound microbial liquid is made from *Pseudomonas aeruginosa* and *Acinetobacter bengal*; the nano-silica sol contains 30% nano-silica, has a particle size of 60 nm, and a pH of 7.2.

[0047] The fish bone hydrolysate was prepared using the following method: (1) Clean the fish bones, freeze-dry them at low temperature, and make them into fish bone powder; (2) Mix fish bone powder and water at a ratio of 1g:10ml, steam at 120℃ for 30 minutes, remove the upper layer of fat, and homogenize to obtain a slurry; (3) Add 1.5wt% of neutral protease to the slurry, and enzymatically hydrolyze at 37℃ for 9h. After inactivation of the enzyme, the first enzymatic hydrolysate is obtained; the neutral protease activity is 100,000 u / g.

[0048] (4) Add the compound hydrolytic enzyme solution to the first enzymatic hydrolysate at a volume ratio of 1:1 and continue enzymatic hydrolysis at 40°C for 11 hours. After inactivation of the enzyme, the second enzymatic hydrolysate is obtained. The compound enzyme solution contains 25 g / L papain and 20 g / L trypsin. The papain activity is 500,000 u / g and the trypsin activity is 100,000 u / g.

[0049] (5) Centrifuge the second enzymatic hydrolysate at 5000 r / min for 3 to 5 min, remove the solids and take the filtrate as fish bone hydrolysate.

[0050] The composite microbial culture was prepared using the following method: (a) *Pseudomonas aeruginosa* and *Acinetobacter bengal* were activated and cultured in LB liquid seed medium to obtain a concentration of 3 × 10⁻⁶. 8 Seed culture with cfu / mL; (b) Seed cultures of *Pseudomonas aeruginosa* and *Acinetobacter benzi* strains were inoculated into 200 mL of fermentation liquid culture medium at a 10% inoculation rate and cultured at 30 °C and 200 r / min for 72 h to obtain two fermentation broths. The fermentation liquid culture medium consisted of: yeast extract 13 g / L, starch hydrolysate 15 g / L, corn steep liquor 20 g / L, sodium chloride 7 g / L, magnesium sulfate 2 g / L, and potassium dihydrogen phosphate 3 g / L.

[0051] (c) The fermentation broth of Pseudomonas aeruginosa and the fermentation broth of Acinetobacter benziae are mixed in a volume ratio of 1:1 to obtain the compound microbial culture.

[0052] The preparation method of the above-mentioned salt-alkali resistant microbial agent includes the following steps: S1: Prepare fish bone hydrolysate and mix it evenly with nano silica sol. Adjust the pH of the mixture to 7.0±0.1 and then sonicate for 5-10 min to obtain a first mixture. S2: Mix the primary mixture with the prepared composite microbial inoculum in a specific ratio to obtain the final product.

[0053] This comparative example is basically the same as Example 3, except that the composite microbial liquid does not contain the fermentation broth of Trichoderma kangaroo and the steps of its preparation method.

[0054] Comparative Example 2 A salt-alkali resistant microbial agent is made from the following raw materials in parts by weight: 130 kg of compound microbial liquid, 20 kg of fish bone hydrolysate, and 2 kg of nano-silica sol; the compound microbial liquid is made from Trichoderma synergum and Acinetobacter bengal; the nano-silica sol contains 30% nano-silica, has a particle size of 60 nm, and a pH of 7.2.

[0055] The fish bone hydrolysate was prepared using the following method: (1) Clean the fish bones, freeze-dry them at low temperature, and make them into fish bone powder; (2) Mix fish bone powder and water at a ratio of 1g:10ml, steam at 120℃ for 30 minutes, remove the upper layer of fat, and homogenize to obtain a slurry; (3) Add 1.5wt% of neutral protease to the slurry, and enzymatically hydrolyze at 37℃ for 9h. After inactivation of the enzyme, the first enzymatic hydrolysate is obtained; the neutral protease activity is 100,000 u / g.

[0056] (4) Add the compound hydrolytic enzyme solution to the first enzymatic hydrolysate at a volume ratio of 1:1 and continue enzymatic hydrolysis at 40°C for 11 hours. After inactivation of the enzyme, the second enzymatic hydrolysate is obtained. The compound enzyme solution contains 25 g / L papain and 20 g / L trypsin. The papain activity is 500,000 u / g and the trypsin activity is 100,000 u / g.

[0057] (5) Centrifuge the second enzymatic hydrolysate at 5000 r / min for 3 to 5 min, remove the solids and take the filtrate as fish bone hydrolysate.

[0058] The composite microbial culture was prepared using the following method: (a) Trichoderma koningii and Acinetobacter benzi were activated and cultured in LB liquid seed medium to obtain a concentration of 3×10⁻⁶. 8 Seed culture with cfu / mL; (b) The crushed plant straw and wheat bran were mixed in a 1:1 mass ratio, and then 3 times the weight of the solid mixture of water was added. The mixture was sterilized to obtain a mixed solution. 10% Trichoderma synergae seed liquid was inoculated into the mixed solution and fermented naturally at 30°C for 10 days to obtain a primary fermentation mixture. Then, a supplementary substrate was added and stirred evenly. During fermentation, the stirring speed was adjusted to 180 r / min, the ventilation rate was 1.3 vvm, and the fermentation temperature was 33°C. Fermentation was continued for another 8 days to obtain a secondary fermentation broth. The solid and liquid were separated and the filtrate was used to obtain the Trichoderma synergae fermentation broth. The amount of supplementary substrate added was 6% of the volume of the primary fermentation mixture. The supplementary substrate contained 12 g / L glucose, 18 g / L peptone, 2 g / L inositol phospholipids, 10 g / L corn flour, 1 g / L ammonium sulfate, 0.3 g / L ferrous sulfate, and 3 g / L potassium dihydrogen phosphate.

[0059] (c) The seed culture of Acinetobacter benzi was inoculated into 200 mL of fermentation liquid culture medium at an inoculation rate of 10%, and cultured at 30 °C and 200 r / min for 72 h to obtain fermentation broth; the fermentation liquid culture medium consisted of: yeast extract 13 g / L, starch hydrolysate 15 g / L, corn steep liquor 20 g / L, sodium chloride 7 g / L, magnesium sulfate 2 g / L, and potassium dihydrogen phosphate 3 g / L.

[0060] (d) The fermentation broth of Trichoderma synergae and the fermentation broth of Acinetobacter benziae are mixed at a volume ratio of 2:1 to obtain the composite microbial culture.

[0061] The preparation method of the above-mentioned salt-alkali resistant microbial agent includes the following steps: S1: Prepare fish bone hydrolysate and mix it evenly with nano silica sol. Adjust the pH of the mixture to 7.0±0.1 and then sonicate for 5-10 min to obtain a first mixture. S2: Mix the primary mixture with the prepared composite microbial inoculum in a specific ratio to obtain the final product.

[0062] This comparative example is basically the same as Example 3, except that the compound microbial liquid does not contain the fermentation broth of *Pseudomonas ayuensis* and its corresponding preparation method.

[0063] Comparative Example 3 A salt-alkali resistant microbial agent is made from the following raw materials in parts by weight: 130 kg of compound microbial liquid, 20 kg of fish bone hydrolysate, and 2 kg of nano-silica sol; the compound microbial liquid is made from Trichoderma synergum and Pseudomonas aeruginosa; the nano-silica sol contains 30% nano-silica, has a particle size of 60 nm, and a pH of 7.2.

[0064] The fish bone hydrolysate was prepared using the following method: (1) Clean the fish bones, freeze-dry them at low temperature, and make them into fish bone powder; (2) Mix fish bone powder and water at a ratio of 1g:10ml, steam at 120℃ for 30 minutes, remove the upper layer of fat, and homogenize to obtain a slurry; (3) Add 1.5wt% of neutral protease to the slurry, and enzymatically hydrolyze at 37℃ for 9h. After inactivation of the enzyme, the first enzymatic hydrolysate is obtained; the neutral protease activity is 100,000 u / g.

[0065] (4) Add the compound hydrolytic enzyme solution to the first enzymatic hydrolysate at a volume ratio of 1:1 and continue enzymatic hydrolysis at 40°C for 11 hours. After inactivation of the enzyme, the second enzymatic hydrolysate is obtained. The compound enzyme solution contains 25 g / L papain and 20 g / L trypsin. The papain activity is 500,000 u / g and the trypsin activity is 100,000 u / g.

[0066] (5) Centrifuge the second enzymatic hydrolysate at 5000 r / min for 3 to 5 min, remove the solids and take the filtrate as fish bone hydrolysate.

[0067] The composite microbial culture was prepared using the following method: (a) Trichoderma kangaroo and Pseudomonas ayuensis were activated and cultured in LB liquid seed medium to obtain a concentration of 3×10⁻⁶. 8 Seed culture with cfu / mL; (b) The crushed plant straw and wheat bran were mixed in a 1:1 mass ratio, and then 3 times the weight of the solid mixture of water was added. The mixture was sterilized to obtain a mixed solution. 10% Trichoderma synergae seed liquid was inoculated into the mixed solution and fermented naturally at 30°C for 10 days to obtain a primary fermentation mixture. Then, a supplementary substrate was added and stirred evenly. During fermentation, the stirring speed was adjusted to 180 r / min, the ventilation rate was 1.3 vvm, and the fermentation temperature was 33°C. Fermentation was continued for another 8 days to obtain a secondary fermentation broth. The solid and liquid were separated and the filtrate was used to obtain the Trichoderma synergae fermentation broth. The amount of supplementary substrate added was 6% of the volume of the primary fermentation mixture. The supplementary substrate contained 12 g / L glucose, 18 g / L peptone, 2 g / L inositol phospholipids, 10 g / L corn flour, 1 g / L ammonium sulfate, 0.3 g / L ferrous sulfate, and 3 g / L potassium dihydrogen phosphate.

[0068] (c) The seed culture of *Pseudomonas aeruginosa* strain was inoculated into 200 mL of fermentation liquid culture medium at an inoculation rate of 10%, and cultured at 30 °C and 200 r / min for 72 h to obtain fermentation broth. The fermentation liquid culture medium consisted of: yeast extract 13 g / L, starch hydrolysate 15 g / L, corn steep liquor 20 g / L, sodium chloride 7 g / L, magnesium sulfate 2 g / L, and potassium dihydrogen phosphate 3 g / L.

[0069] (d) The fermentation broth of Trichoderma synergae and the fermentation broth of Acinetobacter benziae are mixed at a volume ratio of 2:1 to obtain the composite microbial culture.

[0070] The preparation method of the above-mentioned salt-alkali resistant microbial agent includes the following steps: S1: Prepare fish bone hydrolysate and mix it evenly with nano silica sol. Adjust the pH of the mixture to 7.0±0.1 and then sonicate for 5-10 min to obtain a first mixture. S2: Mix the primary mixture with the prepared composite microbial inoculum in a specific ratio to obtain the final product.

[0071] This comparative example is basically the same as Example 3, except that the compound microbial culture does not contain Acinetobacter bengal fermentation broth and the corresponding preparation method.

[0072] Comparative Example 4 A salt-alkali resistant microbial agent is made from the following raw materials in parts by weight: 130 kg of compound microbial liquid and 20 kg of fish bone hydrolysate; the compound microbial liquid is made from Trichoderma synergum, Pseudomonas aeruginosa, and Acinetobacter bengal.

[0073] The fish bone hydrolysate was prepared using the following method: (1) Clean the fish bones, freeze-dry them at low temperature, and make them into fish bone powder; (2) Mix fish bone powder and water at a ratio of 1g:10ml, steam at 120℃ for 30 minutes, remove the upper layer of fat, and homogenize to obtain a slurry; (3) Add 1.5wt% of neutral protease to the slurry, and enzymatically hydrolyze at 37℃ for 9h. After inactivation of the enzyme, the first enzymatic hydrolysate is obtained; the neutral protease activity is 100,000 u / g.

[0074] (4) Add the compound hydrolytic enzyme solution to the first enzymatic hydrolysate at a volume ratio of 1:1 and continue enzymatic hydrolysis at 40°C for 11 hours. After inactivation of the enzyme, the second enzymatic hydrolysate is obtained. The compound enzyme solution contains 25 g / L papain and 20 g / L trypsin. The papain activity is 500,000 u / g and the trypsin activity is 100,000 u / g.

[0075] (5) Centrifuge the second enzymatic hydrolysate at 5000 r / min for 3 to 5 min, remove the solids and take the filtrate as fish bone hydrolysate.

[0076] The composite microbial culture was prepared using the following method: (a) Trichoderma koningii, Pseudomonas ayuensis, and Acinetobacter benzini were activated and cultured in LB liquid seed medium to obtain a concentration of 3 × 10⁻⁶. 8 Seed culture with cfu / mL; (b) The crushed plant straw and wheat bran were mixed in a 1:1 mass ratio, and then 3 times the weight of the solid mixture of water was added. The mixture was sterilized to obtain a mixed solution. 10% Trichoderma synergae seed liquid was inoculated into the mixed solution and fermented naturally at 30°C for 10 days to obtain a primary fermentation mixture. Then, a supplementary substrate was added and stirred evenly. During fermentation, the stirring speed was adjusted to 180 r / min, the ventilation rate was 1.3 vvm, and the fermentation temperature was 33°C. Fermentation was continued for another 8 days to obtain a secondary fermentation broth. The solid and liquid were separated and the filtrate was used to obtain the Trichoderma synergae fermentation broth. The amount of supplementary substrate added was 6% of the volume of the primary fermentation mixture. The supplementary substrate contained 12 g / L glucose, 18 g / L peptone, 2 g / L inositol phospholipids, 10 g / L corn flour, 1 g / L ammonium sulfate, 0.3 g / L ferrous sulfate, and 3 g / L potassium dihydrogen phosphate.

[0077] (c) Seed cultures of *Pseudomonas aeruginosa* and *Acinetobacter benzi* strains were inoculated into 200 mL of fermentation liquid culture medium at a 10% inoculation rate and cultured at 30 °C and 200 r / min for 72 h to obtain two fermentation broths. The fermentation liquid culture medium consisted of: yeast extract 13 g / L, starch hydrolysate 15 g / L, corn steep liquor 20 g / L, sodium chloride 7 g / L, magnesium sulfate 2 g / L, and potassium dihydrogen phosphate 3 g / L.

[0078] (d) The fermentation broth of Trichoderma ts., Pseudomonas aeruginosa, and Acinetobacter benzie were mixed in a volume ratio of 2:1:1 to obtain the composite microbial culture.

[0079] The preparation method of the above-mentioned salt-alkali resistant microbial agent includes the following steps: S1: Preparation of fish bone hydrolysate; S2: Mix the fish bone hydrolysate with the prepared composite microbial inoculum in a certain proportion to obtain the final product.

[0080] This comparative example is basically the same as Example 3, except that it does not contain nano-silica sol.

[0081] Comparative Example 5 A salt-alkali resistant microbial agent is made from the following raw materials in parts by weight: 130 kg of compound microbial liquid and 2 kg of nano silica sol; the compound microbial liquid is made from Trichoderma synergum, Pseudomonas aeruginosa, and Acinetobacter bengal; the nano silica sol has a nano silica content of 30%, a particle size of 60 nm, and a pH of 7.2.

[0082] The composite microbial culture was prepared using the following method: (a) Trichoderma koningii, Pseudomonas ayuensis, and Acinetobacter benzini were activated and cultured in LB liquid seed medium to obtain a concentration of 3 × 10⁻⁶. 8 Seed culture with cfu / mL; (b) The crushed plant straw and wheat bran were mixed in a 1:1 mass ratio, and then 3 times the weight of the solid mixture of water was added. The mixture was sterilized to obtain a mixed solution. 10% Trichoderma synergae seed liquid was inoculated into the mixed solution and fermented naturally at 30°C for 10 days to obtain a primary fermentation mixture. Then, a supplementary substrate was added and stirred evenly. During fermentation, the stirring speed was adjusted to 180 r / min, the ventilation rate was 1.3 vvm, and the fermentation temperature was 33°C. Fermentation was continued for another 8 days to obtain a secondary fermentation broth. The solid and liquid were separated and the filtrate was used to obtain the Trichoderma synergae fermentation broth. The amount of supplementary substrate added was 6% of the volume of the primary fermentation mixture. The supplementary substrate contained 12 g / L glucose, 18 g / L peptone, 2 g / L inositol phospholipids, 10 g / L corn flour, 1 g / L ammonium sulfate, 0.3 g / L ferrous sulfate, and 3 g / L potassium dihydrogen phosphate.

[0083] (c) Seed cultures of *Pseudomonas aeruginosa* and *Acinetobacter benzi* strains were inoculated into 200 mL of fermentation liquid culture medium at a 10% inoculation rate and cultured at 30 °C and 200 r / min for 72 h to obtain two fermentation broths. The fermentation liquid culture medium consisted of: yeast extract 13 g / L, starch hydrolysate 15 g / L, corn steep liquor 20 g / L, sodium chloride 7 g / L, magnesium sulfate 2 g / L, and potassium dihydrogen phosphate 3 g / L.

[0084] (d) The fermentation broth of Trichoderma ts., Pseudomonas aeruginosa, and Acinetobacter benzie were mixed in a volume ratio of 2:1:1 to obtain the composite microbial culture.

[0085] The preparation method of the above-mentioned salt-alkali resistant microbial agent includes the following steps: S1: Preparation of composite microbial inoculum solution; S2: Mix the nano-silica sol with the prepared composite microbial inoculum in a certain proportion to obtain the final product.

[0086] This comparative example is basically the same as Example 3, except that it does not contain fish bone hydrolysate and the corresponding preparation method.

[0087] Comparative Example 6 A salt-alkali resistant microbial agent, which is made from Trichoderma synergum, Pseudomonas aeruginosa, and Acinetobacter benzii.

[0088] The salt-alkali resistant microbial agent is prepared by the following method: (a) Trichoderma koningii, Pseudomonas ayuensis, and Acinetobacter benzini were activated and cultured in LB liquid seed medium to obtain a concentration of 3 × 10⁻⁶. 8 Seed culture with cfu / mL; (b) The crushed plant straw and wheat bran were mixed in a 1:1 mass ratio, and then 3 times the weight of the solid mixture of water was added. The mixture was sterilized to obtain a mixed solution. 10% Trichoderma synergae seed liquid was inoculated into the mixed solution and fermented naturally at 30°C for 10 days to obtain a primary fermentation mixture. Then, a supplementary substrate was added and stirred evenly. During fermentation, the stirring speed was adjusted to 180 r / min, the ventilation rate was 1.3 vvm, and the fermentation temperature was 33°C. Fermentation was continued for another 8 days to obtain a secondary fermentation broth. The solid and liquid were separated and the filtrate was used to obtain the Trichoderma synergae fermentation broth. The amount of supplementary substrate added was 6% of the volume of the primary fermentation mixture. The supplementary substrate contained 12 g / L glucose, 18 g / L peptone, 2 g / L inositol phospholipids, 10 g / L corn flour, 1 g / L ammonium sulfate, 0.3 g / L ferrous sulfate, and 3 g / L potassium dihydrogen phosphate.

[0089] (c) Seed cultures of *Pseudomonas aeruginosa* and *Acinetobacter benzi* strains were inoculated into 200 mL of fermentation liquid culture medium at a 10% inoculation rate and cultured at 30 °C and 200 r / min for 72 h to obtain two fermentation broths. The fermentation liquid culture medium consisted of: yeast extract 13 g / L, starch hydrolysate 15 g / L, corn steep liquor 20 g / L, sodium chloride 7 g / L, magnesium sulfate 2 g / L, and potassium dihydrogen phosphate 3 g / L.

[0090] (d) The fermentation broth of Trichoderma ts., the fermentation broth of Pseudomonas aeruginosa, and the fermentation broth of Acinetobacter benziae are mixed in a volume ratio of 2:1:1 to obtain the salt-alkali resistant microbial agent.

[0091] Field application trials Basic information about the experimental site: A saline-alkali soil area located in Guantai Village, Yangkou Town, Shouguang City, Shandong Province was used as the experimental field. The soil type was yellow-brown soil. Soil samples were collected from the 0-30 cm soil layer of the experimental field. After collection, the soil was placed in a cool place to air dry, crushed, and sieved through a 2 mm sieve to remove soil stones and other impurities. The basic physicochemical properties of the soil were measured and are shown in Table 1.

[0092] Table 1 Basic physical and chemical properties of soil The crop and variety tested: wheat, "Jimai 22".

[0093] Experimental method: A total of 12 treatment groups were set up in the experiment, with each treatment group having an area of ​​100m². 2 Groups 1-3 were treated with conventional fertilizer plus the salt-alkali resistant microbial agents prepared in Examples 1-3; groups 4-9 were treated with conventional fertilizer plus the salt-alkali resistant microbial agents prepared in Comparative Examples 1-6; group 10 was treated with conventional fertilizer plus fish bone hydrolysate; group 11 was treated with conventional fertilizer plus nano-silica sol; and group 12 was a blank control group. The conventional fertilizer used was (N:P2O5:K2O=15-15-15, total nutrients ≥45%). Fertilizer was applied to the soil before planting in all treatment groups. Conventional fertilizer was applied at 30 kg / mu evenly on the surface and then tilled; microbial agents (or other comparative raw materials) were applied at 5 kg / mu by spraying or flushing with water into the soil. All other field management measures for each treatment group were routine. At maturity, 15 ears of wheat were randomly selected from each plot, and the average number of grains per ear was measured. 1000 grains were randomly selected from each plot, with three replicates. The difference between replicates was less than 0.5 g, and the thousand-grain weight was measured. All ears of wheat in each treatment group were harvested to calculate the yield, and the yield per acre was calculated based on the area. The specific results are shown in Table 2.

[0094] Table 2 Results of wheat planting experiment As can be seen from the data in Table 2 above, after using the salt-alkali resistant microbial agents prepared in Examples 1-3 of this invention, wheat yield, thousand-grain weight, and average number of grains per ear were significantly increased, with a yield increase of more than 38.2% compared with the blank control group; Figure 1As shown, the wheat ear traits are significantly different from the blank control. The wheat ears treated with the microbial agent of this invention have fuller and more compact grains, while the wheat ears in the blank control are loosely arranged. The wheat traits and yield increase effects of the treatment groups in Comparative Examples 1-6, the single fish bone hydrolysate treatment group, and the nano-silica sol treatment group are significantly lower than those in the treatment groups in Examples 1-3. This is because the various raw material components of this invention interact and promote each other, reducing soil salinity and alkalinity on the one hand, and improving wheat's resistance to saline-alkali environments on the other, thus reducing the damage to wheat growth caused by soil salinity and alkalinity stress and increasing wheat yield. Changing the type and proportion of any of the raw materials weakens the effect.

[0095] At the wheat maturity stage, 30 uniformly growing wheat stalks were selected from each treatment group, and the above-ground parts were collected and divided into stems, leaves, rachis, glumes, and grains. All samples were blanched at 105℃ for 30 min and dried at 75℃ to constant weight. The dry matter accumulation was measured, and the specific results are as follows: Figure 2 As shown. Wheat dry matter is the source of wheat grain yield, and increasing wheat dry matter accumulation can increase wheat yield. The results of this experiment show that, compared with the blank control group, the application of the salt-alkali resistant microbial agent of this invention can increase the dry matter accumulation of wheat at maturity, and the increase in dry matter accumulation at maturity varies among different microbial agent formulations. Because the microbial agent of this invention produces a large amount of biochemical humic acid and polysaccharides, when applied to the soil, it can react with harmful salt ions in the soil, and the resulting insoluble salts help reduce soil salinity; while a large number of beneficial microorganisms promote the decomposition of soil organic matter during their growth, thereby increasing the total amount of soil organic matter, improving the soil physical structure, enhancing the soil's water and nutrient retention capacity, promoting the absorption and utilization of nutrients by wheat, and thus increasing the dry matter accumulation.

[0096] For each treatment group, take fresh flag leaves from wheat at the flowering stage (using the five-point sampling method, 10 flag leaves at each sampling point), rinse and wipe away impurities from the tissue surface, cut into small pieces (remove the midrib), and mix well; weigh 0.2 g of the cut fresh sample, a total of 3 portions, put them in a mortar, add a small amount of quartz sand and calcium carbonate powder and 2-3 mL of 95% ethanol, grind into a homogeneous paste, then add 10 mL of ethanol and continue grinding until the tissue turns white. Let stand for 3-5 min; take a filter paper, place it in a funnel, moisten it with ethanol, and pour the extract into the funnel along the glass rod, filter into a 25 mL brown volumetric flask, rinse the mortar, grinding rod and residue several times with a small amount of ethanol, and finally pour it into the funnel along with the residue; use a dropper to draw ethanol to wash all the chloroplast pigments on the filter paper into the volumetric flask. Continue until there is no green in the filter paper and residue. Finally, make up to 25 mL with ethanol and shake well; pour the chloroplast pigment extract into a 1 cm diameter cuvette. The total chlorophyll content of each treatment group was determined using spectrophotometry. Specific results are as follows: Figure 3As shown, compared with the blank control group, the total chlorophyll content increased by more than 39.5%, indicating that the salt-alkali resistant microbial agent of the present invention promoted photosynthesis in wheat planting. Enhanced photosynthesis increases the photosynthetic rate of wheat leaves, increases the accumulation of photosynthetic products, provides wheat with necessary energy and nutrients, and also promotes wheat growth and development. Figure 4 It can be clearly seen that the wheat seedlings treated with the microbial agent of this invention are emerald green, while the wheat seedlings in the blank control group are yellowish.

[0097] Superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) in wheat are important components of the antioxidant enzyme system. They can scavenge reactive oxygen species (ROS) generated under stress, thereby protecting wheat cells from oxidative damage. This invention also determined the activity of these defensive enzymes in wheat leaves from different treatment groups. The specific procedure was as follows: Before tillering, one wheat plant was randomly selected from each treatment group, and 0.1 g of wheat leaves were weighed. 3 ml of PBS (pH 7.8) was added, and the mixture was homogenized in an ice bath. Then, 2 mL of PBS was added to rinse the mortar, and the mixture was poured into centrifuge tubes and centrifuged at 10,000 r / min for 20 min at 4°C. The supernatant was the crude enzyme solution, which could be used for enzyme activity determination. The contents of SOD, CAT, and POD in the crude enzyme extract were determined separately. SOD was determined using the nitroblue tetrazolium photoreduction method; CAT was determined using the ultraviolet absorption method; and POD was determined using the guaiacol method. Specific results are as follows: Figure 5-7 As shown.

[0098] from Figure 5-7The data shows that after using the salt-resistant microbial agent prepared in the embodiments of this invention, the activities of three enzymes in wheat were significantly higher than those in other comparative examples. Under normal growth conditions, the production of ROS in plants plays a crucial role in plant growth and development, and the ROS in the plant is in dynamic equilibrium. However, when plants are subjected to salt stress, electron transport on the cell surface is hindered, leading to the accumulation of a large amount of ROS in the cells that cannot be cleared in time. This causes peroxidation of the plant cell membrane, severely damaging the plant's normal physiological metabolic capacity. SOD, POD, and CAT are antioxidant enzymes in plants that respond to external stress. When plants are subjected to salt stress, they increase the activity of these enzymes out of self-defense needs, thereby improving their own stress resistance. In this invention, the activities of SOD, POD, and CAT enzymes in the treatment groups of Examples 1-3 all increased significantly. This indicates that when wheat is stimulated by external salt stress, the microbial agent of this invention can increase the activity of these enzymes through multiple actions to cope with the damage caused by salt stress and improve the stress resistance of wheat. Under saline-alkali stress, highly active defense enzymes can help wheat plants better adapt to and resist these stresses, reduce the infection of pests and diseases, and promote the growth and development of wheat. By protecting wheat cells from stress damage, they provide wheat with more growth space and opportunities, thereby improving wheat yield and quality.

[0099] Topsoil samples (0-30cm) were collected after wheat harvest. Soil samples were collected from each treatment group using the S-shaped sampling method. During sampling, the soil auger was drilled to a depth of 30cm in a single pass. Three replicates were collected from each treatment group, and the three replicates from the same treatment group were combined into one sample. Visible debris such as gravel and plant roots were removed, and the samples were brought back to the laboratory for determination of basic soil physicochemical properties, total soil salinity, and Na+. + Quantity. See Table 3 for details.

[0100] Table 3. Measurement results of basic soil properties after planting As can be seen from Table 3 above, after using the salt-resistant microbial agent of the present invention, the soil pH, bulk density, total salt content, and Na+ content decreased. + The amount of pollutants decreased significantly, while the soil organic matter content increased. The microbial agent of this invention is also rich in active small-molecule peptides and nano-silica particles. The high specific surface area and controllable pore size of the nano-silica sol provide more attachment sites and transport channels for the small-molecule active peptides contained in the fish bone enzymatic hydrolysate obtained in this invention, thereby enhancing the biological activity and utilization rate of the active peptides. The highly active small-molecule peptides also promote the growth and metabolism of the microbial agent, further improving its effect on improving the saline-alkali land environment, significantly improving the soil physicochemical environment, and correspondingly increasing the activities of urease, catalase, acid phosphatase, and sucrase in the soil.

[0101] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A salt-alkali resistant microbial agent, characterized in that, It is made from the following raw materials in parts by weight: 100-150 parts of compound microbial inoculum, 10-30 parts of fish bone hydrolysate, and 1-3 parts of nano-silica sol; the compound microbial inoculum is composed of *Trichoderma ts.* Trichoderma ), Pseudomonas aeruginosa ( Pseudomonas Acinetobacter bellii ( ) Acinetobacter The strains were prepared as follows: *Trichoderma koningii*, strain number CCTCC DF 20081027, deposited on October 11, 1994, purchased from the China Center for Type Culture Collection; *Pseudomonas ayuensis*, strain number CCTCC AB 2014015, deposited on January 8, 2014, purchased from the China Center for Type Culture Collection; and *Acinetobacter behnkeni*, strain number CCTCC AB 205294, deposited on November 12, 2005, purchased from the China Center for Type Culture Collection.

2. The salt-alkali resistant microbial agent according to claim 1, characterized in that, The fish bone hydrolysate was prepared using the following method: (1) Clean the fish bones, freeze-dry them at low temperature, and make them into fish bone powder; (2) Mix fish bone powder and water at a ratio of 1g:10ml, steam at 120℃ for 20-30 minutes, remove the upper layer of fat, and homogenize to obtain a slurry; (3) Add 1.5wt% neutral protease to the slurry and enzymatically hydrolyze it at 35-40℃ for 8-10h. After inactivation of the enzyme, the first enzymatic hydrolysate is obtained. (4) Add the compound hydrolytic enzyme solution to the first enzymatic hydrolysate at a volume ratio of 1:0.8-1.2 and continue enzymatic hydrolysis at 38-45℃ for 10-12h. After inactivation of the enzyme, the second enzymatic hydrolysate is obtained. (5) Centrifuge the second enzymatic hydrolysate in a centrifuge at 4000-6000 r / min for 3-5 min, remove the solids and take the filtrate as fish bone hydrolysate.

3. The salt-alkali resistant microbial agent according to claim 2, characterized in that, The neutral protease activity is 100,000 u / g.

4. The salt-alkali resistant microbial agent according to claim 2, characterized in that, The complex enzyme solution contains 20-30 g / L of papain and 15-25 g / L of trypsin; the papain activity is 500,000 u / g and the trypsin activity is 100,000 u / g.

5. The salt-alkali resistant microbial agent according to claim 1, characterized in that, The composite microbial culture solution is prepared using the following method: (a) Trichoderma koningii, Pseudomonas ayuensis, and Acinetobacter benzini were activated and cultured in LB liquid seed medium to obtain a concentration of 2×10⁻⁶. 8 cfu / mL ~4×10 8 Seed culture with cfu / mL; (b) Mix the crushed plant straw and wheat bran in a 1:1 mass ratio, then add 2-3 times the weight of the solid mixture of water, sterilize, and obtain a mixed solution; inoculate the mixed solution with Trichoderma ts., and ferment naturally at 28-30℃ for 10 days to obtain a primary fermentation mixture; then add supplementary substrate and stir evenly, adjust the stirring speed to 150-200 r / min, the ventilation rate to 1.0-1.5 vvm, and the fermentation temperature to 30-35℃ and continue fermentation for 8 days to obtain a secondary fermentation broth, separate the solid and liquid and take the filtrate to obtain the Trichoderma ts. (c) The seed cultures of *Pseudomonas aeruginosa* and *Acinetobacter benzi* strains were inoculated into 200 mL of fermentation liquid culture medium at an inoculation rate of 10%, and cultured at 30 °C and 200 r / min for 72 h to obtain two fermentation broths. (d) The fermentation broth of Trichoderma ts., Pseudomonas aeruginosa, and Acinetobacter benzie were mixed in a volume ratio of 2:1:1 to obtain the composite microbial culture.

6. The salt-alkali resistant microbial agent according to claim 5, characterized in that, In step (b), the inoculation amount of Trichoderma syn. seed solution is 8-10% of the volume of the mixed solution.

7. The salt-alkali resistant microbial agent according to claim 5, characterized in that, In step (b), the amount of supplementary substrate added is 5-8% of the volume of the primary fermentation mixture; the supplementary substrate contains 10-15 g / L glucose, 15-20 g / L peptone, 1-2 g / L inositol phospholipids, 5-10 g / L corn flour, 0.5-2 g / L ammonium sulfate, 0.2-0.5 g / L ferrous sulfate, and 2-5 g / L potassium dihydrogen phosphate.

8. The salt-alkali resistant microbial agent according to claim 5, characterized in that, The fermentation liquid culture medium in step (c) consists of: yeast extract 10-15 g / L, starch hydrolysate 15-20 g / L, corn steep liquor 15-20 g / L, sodium chloride 5-10 g / L, magnesium sulfate 1-3 g / L, and potassium dihydrogen phosphate 2-5 g / L.

9. The salt-alkali resistant microbial agent according to claim 1, characterized in that, The nano-silica sol contains 30% nano-silica, has a particle size of 55-65 nm, and a pH of 7.0-7.

5.

10. A method for preparing a salt-alkali resistant microbial agent according to any one of claims 1-9, characterized in that, Includes the following steps: S1: Prepare fish bone hydrolysate and mix it evenly with nano silica sol. Adjust the pH of the mixture to 7.0±0.1 and then sonicate for 5-10 min to obtain a first mixture. S2: Mix the primary mixture with the prepared composite microbial inoculum in a specific ratio to obtain the final product.

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