Composite biological bacterial fertilizer for promoting yield increase of kidney beans and preparation method of composite biological bacterial fertilizer

Abstract

The invention relates to the field of agricultural microbial fertilizers, and discloses a composite biological bacterial fertilizer for promoting yield increase of kidney beans, which is prepared from chitosan modified zeolite, salt-tolerant growth-promoting bacteria, potassium-solubilizing and phosphate-solubilizing bacteria, plant signal bacteria and humic acid. The modified zeolite loads potassium ions, adsorbs redundant Na < + >, slowly releases potassium nutrients and adjusts the K < + > / Na < + > ratio of rhizosphere; the flora synergistically acts to secrete betaine, IAA and other substances, salt stress is relieved, and root development is promoted; the humic acid improves the soil structure and improves the water and fertilizer retention capability. Through a freeze-drying immobilization technology, the activity and durability of the flora are enhanced. The granular design of the bacterial fertilizer realizes slow release of nutrients and microorganisms, and improves the fertilizer efficiency and the application convenience. The saline-alkali soil conditioner is suitable for saline-alkali soil agricultural production, and has the effects of improving soil, relieving salt stress and promoting crop yield increase.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microbial fertilizers, and specifically to a compound biological bacterial fertilizer for promoting the yield increase of kidney beans and a preparation method thereof. Background Art

[0002] Saline-alkali land is one of the major limiting factors in global agricultural production. Its main characteristics are that the soil contains high concentrations of soluble salts (such as Na + ), as well as a relatively high alkaline environment (high pH value), which poses a serious threat to the normal growth of plants and the yield and quality of crops. The formation of saline-alkali soil not only deteriorates the physical structure of the soil, reduces the aggregates, and decreases the permeability, but also significantly inhibits the absorption of nutrients by plants. Especially when there is a large accumulation of Na + ions, the K + / Na + ratio in the rhizosphere soil is unbalanced, which hinders the absorption of essential nutrients such as potassium ions (K + ) by plant roots, thus further exacerbating the salt stress and growth obstacles of plants. Therefore, the treatment of saline-alkali land and the sustainable utilization of agriculture have become hot issues of global concern.

[0003] Currently, in order to address the problem of saline-alkali land, traditional improvement techniques mainly focus on physical and chemical methods. Physical improvement methods include deep soil tillage, salt leaching, and salt drainage. However, these methods are usually costly, have a long cycle, and have a high demand for groundwater resources, especially their application is restricted in areas with water shortages. Chemical improvement techniques replace Na + ions in the soil by applying chemical amendments such as gypsum and calcium and magnesium ions. Although they can improve the ion balance in the soil in a short time, the cost of chemical amendments is relatively high, and they are prone to loss, which may cause secondary pollution to the environment. At the same time, these methods have limited effects in improving the physical structure of saline-alkali soil and enhancing its fertility, and it is difficult to meet the requirements of long-term sustainable utilization.

[0004] In recent years, microbial bacterial fertilizers, as an environmentally friendly and efficient soil improvement means, have been widely used in agricultural production. Bacterial fertilizers regulate the soil microecological environment through the metabolic activities of microorganisms, release nutrients required by plants, and enhance the stress resistance of plants, especially showing good potential in the improvement of saline-alkali soil. However, the application of existing microbial bacterial fertilizers in saline-alkali environments still has the following technical problems: Insufficient activity and persistence of microbial flora: The high-salt and high-osmotic pressure environment in saline-alkali soil poses a serious threat to the survival of the flora. The activity of microorganisms rapidly decreases after being applied to the soil, and the survival time is short, making it difficult to maintain the fertilizer effect for a long time. In addition, traditional bacterial fertilizers are mostly applied in liquid or powder form, and the flora is easy to diffuse and lose, further reducing their application effect.

[0005] Single function and insufficient microbial community cooperation: Most existing bio-fertilizers use single-functional strains, such as potassium-solubilizing bacteria, nitrogen-fixing bacteria, or plant growth-regulating bacteria. Although these strains can promote plant growth in one aspect, in the complex stress environment of saline-alkali land, single-functional strains cannot simultaneously alleviate salt stress, regulate the rhizosphere microenvironment, and promote plant nutrient absorption, resulting in insignificant comprehensive improvement effects.

[0006] Poor combination of bio-fertilizer and soil environment: Bio-fertilizers lack the ability to improve the physical properties of soil in the saline-alkali land environment. For example, they cannot effectively increase the content of soil organic matter, the proportion of aggregates, and the water and fertilizer retention capacity, thus restricting the absorption of nutrients by plant roots. At the same time, the microbial community does not form a stable rhizosphere ecosystem in the soil and cannot play a long-term role.

[0007] Therefore, the present invention proposes a compound bio-fertilizer for promoting the yield increase of kidney beans and its preparation method to solve the deficiencies of the prior art. Summary of the Invention

[0008] Aiming at the problems of insufficient activity and persistence, single function, and limited improvement effect of bio-fertilizers in saline-alkali soil in the prior art, the present invention provides a compound bio-fertilizer capable of significantly alleviating saline-alkali stress, improving the yield and quality of kidney beans, and its preparation method. The technical solution of the present invention realizes the high efficiency and persistence of bio-fertilizers through the loading technology of chitosan-modified zeolite, the freeze-drying immobilization process, the multi-microbial community synergistic effect, and the humic acid granulation design, overcoming the limitations of traditional bio-fertilizers in the saline-alkali land environment.

[0009] To achieve the above objectives, the present invention is realized through the following technical solutions: A compound bio-fertilizer for promoting the yield increase of kidney beans, including the following components in a weight percentage range: Modified zeolite: 60%–80% Modified zeolite is used as the main carrier and soil conditioner. Zeolite has a high cation exchange capacity (CEC), which can effectively adsorb excessive sodium ions (Na + +) in the soil, reducing the sodium toxicity of saline-alkali soil to plants. The particle size range is 0.5–2 mm, ensuring that it has a large specific surface area and suitable soil permeability, and improving the ion exchange efficiency. The CEC is 150–200 cmol(+) / kg, and it has a high ion exchange capacity and can continuously regulate the rhizosphere ion balance.

[0010] Chitosan: 5%–10% Chitosan is used for surface modification of zeolite. With a deacetylation degree of 80%–90% and a molecular weight of 10–100 kDa, chitosan has a large number of amino and hydroxyl groups, endowing it with positive charge and good biocompatibility. The addition of chitosan improves the adsorption and immobilization ability of zeolite for microbial flora, and at the same time, it has antibacterial effects itself, which can inhibit harmful microorganisms and improve the soil microecological environment.

[0011] Microbial flora: 10%–20%, including: Salt-tolerant growth-promoting bacteria (Halomonas sp.): 5%–10% Halomonas sp. belongs to salt-tolerant bacteria and can survive in high-salt environments. They can synthesize and secrete osmoregulatory substances (such as betaine, proline) to help plant cells maintain osmotic balance and enhance the salt tolerance of plants. In addition, these strains can also produce auxin (IAA) to promote the root growth of kidney beans and enhance the absorption ability of nutrients and water. Potassium-solubilizing and phosphorus-dissolving bacteria (Bacillus subtilis): 3%–8% Bacillus subtilis has the ability to dissolve phosphorus and potassium, and can convert insoluble phosphorus and potassium elements in the soil into forms that can be utilized by plants, improving soil fertility. By increasing the content of available nutrients in the rhizosphere environment, it promotes the growth and development of kidney beans, especially in saline-alkali soils where the nutrient availability is low, and the role of potassium-solubilizing and phosphorus-dissolving bacteria is particularly important.

[0012] Plant signal bacteria (Pseudomonas sp): 2%–5% Pseudomonas sp. can produce a variety of secondary metabolites (such as siderophore, antibiotics, etc.) to inhibit the growth of pathogenic microorganisms and induce the systemic resistance of plants. At the same time, they can also secrete plant growth regulatory substances such as IAA to promote the growth of kidney beans and improve stress resistance.

[0013] Humic acid: 5%–15% Humic acid is derived from natural humus raw materials such as weathered coal, peat or lignite, and its molecular weight ranges from 500–5000 Da. Humic acid can improve soil structure, increase the organic matter content, and enhance the water and fertilizer retention capacity of the soil. It can also chelate trace elements in the soil and enhance the absorption efficiency of plants for nutrients.

[0014] Preferably, the particle size range of the modified zeolite is 0.5–2 mm, and the cation exchange capacity is 150–200 cmol(+) / kg.

[0015] The modified zeolite with a particle size of 0.5–2 mm has a large specific surface area, which is beneficial to ion exchange and the attachment of microorganisms. With a high CEC (150–200 cmol(+) / kg), it can effectively adsorb Na + , release K⁺, and regulate the K + / Na + ratio in the soil, reducing the impact of salt stress on plants.

[0016] Preferably, the deacetylation degree of the chitosan is 80%–90%, and the molecular weight is 10–100 kDa.

[0017] When the deacetylation degree of chitosan is 80%–90%, it contains a large number of free amino groups, increasing its positive charge and facilitating the adsorption of negatively charged microbial cell walls. With a molecular weight of 10–100 kDa, it ensures that chitosan has good solubility and film-forming properties, facilitating the uniform modification of zeolite and improving the immobilization efficiency of microorganisms.

[0018] Preferably, the salt-tolerant growth-promoting bacterium is Halomonas sp., the potassium-dissolving and phosphorus-solubilizing bacterium is Bacillus subtilis, and the plant signaling bacterium is Pseudomonas sp.

[0019] Halomonas sp. (salt-tolerant growth-promoting bacterium): Can survive in a high-salt environment and help plants resist salt stress.

[0020] Bacillus subtilis (potassium-dissolving and phosphorus-solubilizing bacterium): Improves the availability of potassium and phosphorus in the soil to meet the growth needs of plants.

[0021] Pseudomonas sp. (plant signaling bacterium): Promotes plant growth and enhances disease resistance.

[0022] The synergistic effect of the three strains comprehensively improves the growth performance of kidney beans in saline-alkali soil.

[0023] Preferably, the compound bio-fertilizer is granular, and its particle diameter is 2–5 mm.

[0024] Making the compound bio-fertilizer granular with a particle diameter of 2–5 mm facilitates field application. The granular form can slow-release nutrients and microorganisms, extend the fertilizer efficiency period, and ensure the uniform distribution of nutrients and beneficial microbial populations in the soil.

[0025] Preferably, a preparation method of a compound bio-fertilizer for promoting the yield increase of kidney beans includes the following steps: Preparation of modified zeolite: Wash and dry the zeolite Wash and remove impurities and dust on the surface of zeolite, and dry it (60–80 °C) to remove moisture to prevent affecting the adsorption of chitosan during subsequent modification; Soak the zeolite in the chitosan solution at a reaction temperature of 40–60 °C for 4–8 hours, and then dry it Chitosan adsorbs on the surface of zeolite under warm conditions to form a layer of chitosan film, endowing the zeolite surface with positive charge and enhancing the immobilization ability for microorganisms. At the same time, the presence of chitosan increases the cation exchange sites of zeolite and improves the adsorption ability for Na + ; Soak the modified zeolite in 1–2 M KCl solution and let it stand for 12–24 hours to obtain potassium ion-loaded modified zeolite Through ion exchange, K + replaces Na + and other cations in zeolite, and the zeolite is saturated with K + . In this way, after the zeolite is applied to the soil, it can release K + for plants to absorb, and at the same time adsorb excessive Na + in the soil to improve the rhizosphere ion environment; Preparation and immobilization of microbial flora: Cultivate salt-tolerant growth-promoting bacteria, potassium-dissolving phosphorus-dissolving bacteria and plant signal bacteria separately to a bacterial solution with an OD600 value of 1.0–1.5 Ensure that there are enough viable bacteria in the bacterial solution to improve the immobilization effect and the activity of subsequent biological bacterial fertilizers; Mix the bacterial solution with the modified zeolite at a ratio of 1:2–1:3, let it stand and adsorb at 37 °C for 2–4 hours, and then freeze-dry to obtain an immobilized microbial carrier The surface of chitosan-modified zeolite is positively charged and tightly binds to the negatively charged microbial cell wall through electrostatic interaction. 37 °C is the suitable temperature for microbial growth and adsorption. Freeze-drying (-40 to -60 °C, 24–48 hours) can effectively remove moisture, maintain the activity of microorganisms, and extend the shelf life of bacterial fertilizers; Preparation of finished product of compound bacterial fertilizer: Mix the immobilized microbial carrier with humic acid in proportion to prepare a compound bacterial fertilizer in granular form The addition of humic acid further increases the soil organic matter content and enhances the microbial activity. Making it into granular form is convenient for application, and the components in the granules can be released synergistically and play a continuous role.

[0026] Preferably, the concentration of the chitosan solution is 2%–5% (w / v).

[0027] Within this concentration range, the chitosan solution has appropriate viscosity and film-forming property, which is conducive to uniformly coating the zeolite surface and ensuring the modification effect.

[0028] Preferably, after the bacterial liquid is mixed with the modified zeolite, it is dried by a freeze-drying process. The freezing temperature is -40 to -60 °C, and the drying time is 24–48 hours.

[0029] Freeze-drying is carried out at a low temperature to avoid the destruction of microbial activity by high temperature, fully remove moisture, and prevent the inactivation of microorganisms during storage.

[0030] Preferably, the source of the humic acid is natural humus raw materials, selected from weathered coal, peat or lignite, and prepared by alkali dissolution and acid precipitation treatment. Its molecular weight range is 500–5000 Da.

[0031] Natural humus raw materials are selected and treated by alkali dissolution and acid precipitation to obtain highly active humic acid. Humic acid with a molecular weight of 500–5000 Da has good water solubility and biological activity, can promote plant growth, and improve soil structure.

[0032] Preferably, the application of the compound biological bacterial fertilizer in improving the soil structure of saline-alkali land.

[0033] Regulate the ion balance in the rhizosphere: The modified zeolite adsorbs Na + and releases K + , increasing the K + / Na + ratio in the rhizosphere soil and reducing the impact of salt stress on kidney beans.

[0034] Enhance the salt tolerance of plants: The salt-tolerant and growth-promoting bacteria secrete osmoregulatory substances to improve the osmoregulatory ability of plant cells. The plant signal bacteria induce the expression of stress-resistant genes in plants, enhancing the salt tolerance.

[0035] Improve soil fertility: The potassium-solubilizing and phosphorus-dissolving bacteria convert the insoluble potassium and phosphorus in the soil into available forms, increasing the soil nutrient supply. Humic acid improves the soil structure, increases the organic matter content, and enhances the water and fertilizer retention capacity.

[0036] Improve the soil microecological environment: Under the action of chitosan and beneficial bacterial communities, the growth of harmful microorganisms is inhibited, the reproduction of beneficial bacterial communities is promoted, and the soil microbial community structure is optimized.

[0037] Through the above multiple mechanisms, the compound biological bacterial fertilizer has played a significant role in improving the soil structure of saline-alkali land, increasing the yield and quality of kidney beans.

[0038] The present invention provides a compound biological bacterial fertilizer for promoting the yield increase of kidney beans and its preparation method. It has the following beneficial effects: 1. The present invention adopts a technical solution of synergistically regulating modified zeolite loaded with potassium ions and microbial flora. By providing high-efficient cation exchange capacity through modified zeolite, it accurately adsorbs excessive sodium ions (Na + +) in the rhizosphere environment, and simultaneously slowly releases potassium ions (K + +). Combined with the osmotic adjustment substances secreted by salt-tolerant and growth-promoting flora, it realizes the dynamic balance of rhizosphere ion concentration and the alleviation effect of soil salt stress. Compared with the prior art in which the regulation of salt by relying solely on chemical adsorbents or microbial fertilizers has limited and unsustainable effects, it solves the problems in the prior art that it is difficult to maintain the K + / Na + ratio in saline-alkali soil for a long time, local ion imbalance in the rhizosphere, and the continuous aggravation of salt stress on plants.

[0039] 2. The present invention combines chitosan-modified zeolite with freeze-drying immobilized microorganism technology. By using the modification effect of chitosan on the surface of modified zeolite, it enhances its microbial adsorption capacity, forms a stable immobilized carrier structure, and effectively maintains the microbial activity through the freeze-drying process, thereby significantly improving the environmental adaptability of the flora and the duration of fertilizer efficiency. This technology realizes the long-acting and stable performance of compound microbial fertilizers in high-salt and high-osmotic pressure environments. Compared with the prior art in which the bacterial liquid or bacterial powder is directly prepared into fertilizer, but the survival rate of the flora in saline-alkali soil is low and the fertilizer efficiency period is short, it solves the problems that the activity of strains is lost in saline-alkali stress environment and the growth-promoting function cannot be released persistently.

[0040] 3. The present invention adopts a multi-flora synergistic technical solution of salt-tolerant and growth-promoting bacteria, potassium-dissolving and phosphorus-dissolving bacteria, and plant signal bacteria. The salt-tolerant and growth-promoting bacteria secrete osmotic adjustment substances such as betaine to help plants resist salt stress. The potassium-dissolving and phosphorus-dissolving bacteria convert the insoluble potassium and phosphorus elements in the soil into available nutrients that can be absorbed by plants. The plant signal bacteria further promote root development by secreting growth hormones (such as IAA) and regulating the root microenvironment, enhancing the plant's ability to absorb nutrients and water, and realizing the synergistic effect of multi-flora. Compared with the prior art in which a single strain has a single function and only provides a growth-promoting effect in a certain aspect, it solves the defects that the function of strains is single in the complex environment of saline-alkali land and the comprehensive regulation ability of plant stress resistance and nutrient absorption is insufficient.

[0041] 4. Through the granulation design technical solution of humic acid and compound bacterial fertilizer, the present invention uses highly active natural humic acid (such as derived from weathered coal or peat) and a compound bacterial fertilizer carrier to make granular fertilizer. The humic acid further improves the soil structure, increases the water and fertilizer retention capacity, chelates trace elements in the soil, makes the rhizosphere environment more suitable for the growth of plant roots. At the same time, the granular design realizes the slow release of nutrients and microorganisms, improves the application convenience and the sustainability of fertilizer efficiency. Compared with the prior art which mainly uses liquid bacterial fertilizer or powder bacterial fertilizer and cannot improve the physical structure of saline-alkali land and is easy to lose during field application, it solves the problems of poor combination of bacterial fertilizer with the soil environment and limited ability to improve soil physical and chemical properties. Detailed implementation mode

[0042] The following will clearly and completely describe in conjunction with the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0043] Example 1: Specific steps: Preparation of modified zeolite Wash natural zeolite with a particle size of 1–2 mm three times with deionized water to remove surface impurities and dust.

[0044] Place the washed zeolite in an oven at 60°C and dry for 12 hours until completely dehydrated.

[0045] Prepare a chitosan solution with a concentration of 3% (w / v), dissolve chitosan in 1% acetic acid solution, and stir for 30 minutes until completely dissolved; Add the dried zeolite to the chitosan solution and react at 40°C for 5 hours; take it out and dry at 70°C for 12 hours to obtain chitosan-modified zeolite; Soak the modified zeolite in 1.5 M KCl solution, take it out after standing for 20 hours, rinse the surface residual KCl solution with deionized water, and dry it again at 60°C for 12 hours to obtain potassium ion-loaded modified zeolite.

[0046] Preparation and immobilization of microbial flora Respectively inoculate Halomonas sp. (salt-tolerant growth-promoting bacteria), Bacillus subtilis (potassium-dissolving and phosphorus-dissolving bacteria), and Pseudomonas sp. (plant signal bacteria) into LB medium, and shake culture at 28°C for 12 hours until the OD600 value reaches 1.2; Prepare a mixed bacterial solution and mix it according to the volume ratio of Halomonas sp.: Bacillus subtilis: Pseudomonas sp. = 2:1.5:1; Mix the mixed bacterial solution and the modified zeolite in a ratio of 1:2, and let it stand and adsorb at 37 °C for 3 hours; Place the adsorbed carrier in a freeze-drying device and freeze it at -50 °C for 24 hours to obtain an immobilized microbial carrier.

[0047] Preparation of compound bacterial fertilizer Mix the immobilized microbial carrier and humic acid (derived from peat, molecular weight 1000–2000 Da) in a mass ratio of 8:2; Granulate the mixture with a granulator to form particles with a diameter of 3–5 mm, and finally obtain the compound bacterial fertilizer.

[0048] Application Conduct a kidney bean planting experiment in slightly saline-alkali soil (conductivity 2 dS / m, pH 8.2); Apply the granular bacterial fertilizer into the planting ditch at a rate of 30 kg of compound bacterial fertilizer per mu of land, cover the soil, and then plant kidney beans; Regularly detect the K + / Na + ratio and the growth of kidney beans during the growth period.

[0049] Example 2: Specific steps: Preparation of modified carrier Select natural zeolite with a particle size of 0.5–1 mm, wash it 3 times with distilled water, and dry it at 80 °C for 16 hours; Prepare a 2.5% (w / v) chitosan solution, heat the chitosan solution to 50 °C and stir to dissolve; Soak the dried zeolite in the chitosan solution, react at 50 °C for 6 hours, and dry it for later use.

[0050] Cultivation of bacterial flora Inoculate Halomonas sp., Bacillus subtilis and Pseudomonas sp. into the inorganic salt medium respectively, and shake-culture at 28 °C for 16 hours until the OD600 value reaches 1.5; Prepare a mixed bacterial solution and mix it according to the volume ratio of Halomonas sp.: Bacillus subtilis: Pseudomonas sp. = 1:1:1; Mix the mixed bacterial solution and the modified zeolite in a ratio of 1:3, and let it stand and adsorb at 37 °C for 4 hours; Freeze-dry at -40 °C for 48 hours to obtain an immobilized carrier.

[0051] Granulation of Compound Bacterial Fertilizer Mix the immobilized carrier with humic acid derived from lignite (molecular weight 3000–4000 Da) in a ratio of 7:3; Add 2% moisture and use a granulator to press into particles with a diameter of 2–4 mm.

[0052] Application Conduct a saline-alkali soil improvement test in moderately saline-alkali soil (conductivity 4 dS / m, pH 8.5); Apply 40 kg of bacterial fertilizer per mu, evenly spread it and then plow it into the soil. After 3 months, detect the physical and chemical properties of the soil.

[0053] Example 3: Specific steps: Carrier Preparation Use zeolite with a particle size of 1–2 mm and carry out chitosan modification and potassium ion loading according to the method of Example 1.

[0054] Strain Preparation Culture Halomonas sp. alone until the OD600 value reaches 1.2; Mix Bacillus subtilis and Pseudomonas sp. in a ratio of 1:1 and culture until the OD600 value reaches 1.5.

[0055] Immobilization and Granulation Mix and immobilize according to the ratio of bacterial liquid to carrier of 1:2, and after freeze-drying, mix and granulate with peat humic acid in a ratio of 9:1.

[0056] Application Plant kidney beans under saline-alkali land conditions and apply 35 kg of compound bacterial fertilizer per mu; Monitor the root growth indexes (root length, root activity) of kidney beans and the leaf nutrients (K, P content).

[0057] Example 4: Specific steps Carrier and Bacterial Flora Preparation Prepare the carrier according to the modified carrier and immobilization process of Example 2; Mix the bacterial liquids of Halomonas sp. and Bacillus subtilis (in a ratio of 2:1).

[0058] Compound Bacterial Fertilizer Preparation and Application Mix the immobilized carrier with humic acid (derived from lignite, molecular weight 500–1000 Da) in a mass ratio of 8:2 and make particles with a diameter of 3–5 mm.

[0059] Apply 50 kg of microbial fertilizer per mu, and apply it continuously for 2 years, twice a year.

[0060] Monitoring indicators Regularly monitor the physical and chemical indicators of the soil (K + / Na + ratio, conductivity, pH value) and changes in microbial diversity.

[0061] Control example: Control example 1: Unoptimized preparation of modified zeolite Specific process: Carrier preparation Use natural zeolite with a particle size of 1–2 mm. After washing it 3 times with deionized water, dry it at 60 °C for 12 hours; Skip the chitosan modification step. Directly soak the dried zeolite in 1.5 M KCl solution, take it out after standing for 20 hours, rinse the residual KCl solution on the surface with deionized water and then dry it to obtain unmodified potassium ion-loaded zeolite.

[0062] Preparation and immobilization of microbial flora Prepare the mixed bacterial solution by the same method as in Example 1; Mix the bacterial solution with unmodified potassium-loaded zeolite in a ratio of 1:2 and let it stand and adsorb at 37 °C for 3 hours; After freeze-drying, obtain the immobilized microbial carrier.

[0063] Preparation of compound microbial fertilizer Mix the immobilized microbial carrier and peat humic acid in a mass ratio of 8:2 and granulate to obtain granular compound microbial fertilizer.

[0064] Differences from Example 1 Zeolite was not surface-modified with chitosan, which reduced the microbial adsorption capacity and cation exchange efficiency of the carrier.

[0065] Control example 2: Preparation of microbial fertilizer without using the immobilization process Specific process: Carrier preparation Select natural zeolite with a particle size of 0.5–1 mm and carry out chitosan surface modification and potassium loading treatment according to the modification method of Example 2.

[0066] Preparation of microbial flora Inoculate Halomonas sp., Bacillus subtilis, and Pseudomonas sp. into the inorganic salt medium respectively and shake-culture at 28 °C for 16 hours until the OD600 value reaches 1.5; Prepare a mixed bacterial solution by mixing Halomonas sp., Bacillus subtilis, and Pseudomonas sp. at a volume ratio of 1:1:1.

[0067] Preparation of bacterial fertilizer Skip the immobilization process of the bacterial solution and directly mix the mixed bacterial solution with modified zeolite at a volume ratio of 1:5, and simply stir evenly. After mixing with lignite humic acid at a ratio of 7:3, directly granulate to obtain granular bacterial fertilizer.

[0068] Differences from Example 2 The freeze-drying immobilization technology is not adopted, and the bacterial solution is directly mixed, which may lead to a significant reduction in the activity of the bacteria during storage and in the saline-alkali land environment.

[0069] Comparative Example 3: Preparation of bacterial fertilizer with a single strain Specific process: Carrier preparation Use zeolite with a particle size of 1–2 mm and perform chitosan modification and potassium ion loading according to the method of Example 3.

[0070] Preparation of microbial strains Use Bacillus subtilis (potassium-dissolving and phosphorus-dissolving bacteria) alone and culture it until the OD600 value reaches 1.5 as the only functional bacterial solution. Skip the culture and mixing of the bacterial solutions of Halomonas sp. and Pseudomonas sp.

[0071] Immobilization and granulation Mix the Bacillus subtilis bacterial solution with the modified carrier at a ratio of 1:2 and let it stand and adsorb at 37°C for 3 hours. After freeze-drying, mix it with peat humic acid at a ratio of 9:1 and granulate to obtain compound bacterial fertilizer.

[0072] Differences from Example 3 Only a single strain is used, lacking the synergistic effect of multiple strains, which may reduce the comprehensive ability to cope with salt stress.

[0073] Comparative Example 4: Preparation of bacterial fertilizer without adding humic acid Specific process: Preparation of carrier and bacterial community Use zeolite with a particle size of 0.5–1 mm and perform chitosan modification and potassium ion loading treatment according to the method of Example 2. Mix and culture the bacterial solution of Halomonas sp. and Bacillus subtilis at a ratio of 2:1 and immobilize it on the surface of the modified zeolite.

[0074] Preparation of Compound Bacterial Fertilizer Without adding humic acid, the immobilized carrier is directly applied to the soil in granular form.

[0075] Differences from Example 4 Without adding humic acid may lead to a significant decline in the effect of bacterial fertilizer in improving soil physical and chemical properties and water and fertilizer retention capacity.

[0076] Control Example 5: Preparation of Bacterial Fertilizer with Simplified Carrier Process Specific process: Carrier preparation Natural zeolite with a particle size of 1–2 mm is used, washed and directly subjected to potassium loading treatment (skipping the chitosan modification step), treated with 1.5 M KCl solution for 20 hours and then dried to obtain potassium-loaded zeolite.

[0077] Immobilization of Bacterial Community and Preparation of Bacterial Fertilizer The cultivation, adsorption and freeze-drying immobilization of the mixed bacterial community are carried out in the same way as in Example 1; Finally, it is mixed and granulated with peat humic acid in a ratio of 8:2 to obtain granular compound bacterial fertilizer.

[0078] Differences from Example 1 The carrier is not modified with chitosan, which may lead to a decrease in the binding efficiency between the carrier and the bacterial community.

[0079] Test experiment: Experiment 1: Specific experimental description of the K+ / Na+ ratio regulation ability Experimental steps: Test plot preparation: A lightly saline-alkali land (conductivity 2 dS / m, pH 8.2) is selected as the experimental site, divided into 5 test plots, each plot has an area of 1 mu, and they are randomly arranged to ensure the repeatability of the experiment.

[0080] Soil samples are collected before fertilization for baseline data analysis, including K+ / Na+ ratio, conductivity and pH value.

[0081] Treatment group setting: Example 1: Apply 30 kg of compound bacterial fertilizer per mu.

[0082] Example 2: Apply 40 kg of compound bacterial fertilizer per mu.

[0083] Control Example 1: Apply 30 kg of bacterial fertilizer prepared with unoptimized and modified zeolite per mu.

[0084] Control Example 5: Apply 30 kg of bacterial fertilizer with a carrier not modified with chitosan per mu.

[0085] Blank group: Apply the same amount of ordinary chemical fertilizers (potassium fertilizer and nitrogen-phosphorus fertilizer) per mu.

[0086] Each treatment group was repeated 3 times, and data were recorded separately.

[0087] Fertilization operation: According to the experimental design, apply the bio-fertilizer or ordinary chemical fertilizer into the planting furrow, mix well with the surface soil, and then cover it.

[0088] Data collection and analysis: During the planting cycle, collect rhizosphere soil samples every two weeks and analyze the following indicators: K+ / Na+ ratio: Determine the contents of K+ and Na+ in the soil with an atomic absorption spectrometer and calculate the ratio.

[0089] Soil conductivity and pH value: Use a multi-parameter soil tester.

[0090] Plant growth indicators: Record plant height and number of leaves.

[0091] Endpoint determination: At the end of the kidney bean growth period, collect the data on the change of K+ / Na+ ratio during the whole cycle, and conduct a comparative analysis in combination with the plant growth data.

[0092] Experimental data: Data on the change of soil K+ / Na+ ratio and plant growth in the kidney bean planting experiment In this experiment, by comparing the change of K + / Na + ratio and the plant growth performance of different treatment groups, the significant advantage of the synergistic regulation mechanism of the present invention in alleviating salt stress was revealed. The results showed that the K + / Na + ratio of the treatments in Example 1 and Example 2 was significantly higher than that of the comparative example and the blank group during the whole growth cycle, and the plant growth performance was also more excellent, further verifying the technical effectiveness of the present invention.

[0093] The high efficient cation exchange capacity of the modified zeolite is the key to improving the K + / Na + ratio. After being modified by chitosan, the surface active sites of the zeolite increased significantly, making its adsorption capacity for sodium ions (Na + ) increase greatly, and the loaded potassium ions (K + ) are continuously supplied to the plant roots in a slow-release manner to maintain the ion balance in the rhizosphere. In the control groups (Comparative Example 1 and Comparative Example 5), due to the lack of optimization of the carrier preparation process, the cation exchange capacity was insufficient, and the K + / Na +The ratio remains at a low level, further leading to a decline in the salt tolerance and growth ability of the plants.

[0094] In addition, the present invention combines the effects of salt-tolerant and growth-promoting bacteria to synergistically alleviate salt stress. In the experiment, Examples 1 and 2 significantly increased the K + / Na + ratio, which is not only due to the cation exchange function of the modified zeolite but also closely related to the osmoregulatory substances secreted by the salt-tolerant and growth-promoting bacteria. Substances such as betaine secreted by Halomonas sp. help plant cells maintain osmotic balance. Bacillus subtilis enhances the availability of potassium ions through potassium solubilization, and IAA secreted by Pseudomonas sp. further promotes root development, enhancing the absorption ability and stress tolerance of plants.

[0095] This experiment verified the technical advantages of the examples. Through the dual regulation of "modified zeolite + microbial flora", the dynamic balance of rhizosphere ion concentration in saline-alkali soil was achieved, significantly increasing the K + / Na + ratio and the salt tolerance of plants. Compared with the comparative examples, the synergistic effect of the present invention shows great potential in improving the soil environment of saline-alkali land and promoting the growth of kidney beans, further demonstrating its application value in saline-alkali land agriculture.

[0096] Experiment 2: Comparison test of microbial flora activity and persistence Experimental steps: Preparation of experimental materials: The immobilized carrier of Example 1 (lyophilized, chitosan-modified).

[0097] The carrier of Example 3 (modified zeolite, different proportions of microbial flora).

[0098] Comparative Example 2 (non-immobilized process, carrier simply adsorbing bacterial liquid).

[0099] Comparative Example 3 (immobilized carrier of single strain, only containing Bacillus subtilis).

[0100] Prepare a salt stress simulation medium with a NaCl concentration of 0.5% (w / v).

[0101] Inoculation of microbial flora: Add the microbial flora carriers of different treatments to 50 mL of salt stress medium respectively, with an initial inoculation concentration of 1 × 10 7 CFU / mL.

[0102] Each treatment is repeated 5 times, placed randomly, and cultured with constant shaking at 28°C.

[0103] Determination of microbial flora activity: Samples were taken every 5 days, the culture medium solution was diluted, and the viable count of the bacterial community was detected using the streak plate method (counted in CFU).

[0104] The concentrations of osmoregulatory substances (betaine, IAA) secreted in the culture medium supernatant were recorded.

[0105] The turbidimetric method (OD600 value) was used to monitor the change in the bacterial solution concentration in real time.

[0106] Endpoint determination: After 30 days of culture, the final survival rate, activity retention time, and secretion amount of osmoregulatory substances of the bacterial communities in each treatment were counted.

[0107] Combined with the data on the activity changes of the bacterial community, the synergistic effect of the immobilization process and the multi-bacterial community combination was analyzed.

[0108] Experimental data: Changes in the activity of the bacterial community and the secretion amount of betaine under salt stress culture conditions In this experiment, by comparing the activities and betaine secretion amounts of different treated bacterial communities, the improvement effect of the freeze-drying immobilization process of the present invention on the persistence and growth-promoting function of the bacterial community was revealed. The experimental results showed that the bacterial communities treated in Example 1 and Example 3 showed significantly longer activity maintenance time and higher betaine secretion amount in the salt stress environment, while in Comparative Example 2 (non-immobilization process) and Comparative Example 3 (single strain), the activity decreased significantly in the early stage and could not continuously release growth-promoting substances.

[0109] The freeze-drying immobilization technology is the core to enhance the environmental adaptability of the bacterial community. In Example 1, the chitosan-modified zeolite carrier provided a stable microenvironment for the bacterial cells through its porosity and biocompatibility, and at the same time avoided the damage to the bacterial cells caused by direct contact with the high osmotic pressure environment under salt stress. In contrast, the non-immobilized bacterial solution in Comparative Example 2 quickly lost its activity in the salt stress environment and was difficult to play a role for a long time.

[0110] In addition, the multi-bacterial community synergistic effect was also verified in the experiment. Example 1 combined salt-tolerant growth-promoting bacteria (Halomonas sp.), potassium-dissolving and phosphorus-solubilizing bacteria (Bacillus subtilis), and plant signal bacteria (Pseudomonas sp.). By secreting osmoregulatory and growth-promoting substances such as betaine and IAA, it effectively helped plants maintain the cell osmotic pressure balance and healthy root development under salt stress conditions. In Comparative Example 3, due to the single bacterial strain, the bacterial community had a single function, resulting in the betaine secretion amount being only about half of that in Example 1.

[0111] The immobilized carrier and freeze-drying technology of the present invention also significantly extend the maintenance time of the microbial community activity. Experimental data show that the activity retention time of the microbial community in Example 1 can reach more than 30 days under salt stress environment, while in Comparative Example 2, the microbial community basically loses its activity after 15 days, further illustrating the importance of the immobilization process for the persistence of the microbial community. This improvement in persistence also enables the microbial community to continuously release growth-promoting substances, enhancing the salt tolerance and nutrient absorption ability of plants.

[0112] Generally speaking, this experiment verified the combined effects of the immobilization process, multi-microbial community cooperation, and growth-promoting substance secretion, providing strong support for the application of bio-fertilizer in saline-alkali soil. Through the protection of the modified carrier and the cooperation of the microbial community, the present invention effectively solves the problem of activity loss of the microbial community in the high-salt stress environment, providing technical guarantee for the long-term effectiveness of the compound bio-fertilizer.

[0113] Experiment 3: Comparative experiment on improvement of soil physical and chemical properties Experimental steps: Test plot preparation: Select moderately saline-alkali soil (electrical conductivity 4 dS / m, pH 8.5) as the experimental site, which is divided into 5 test plots, and the area of each plot is 1 mu.

[0114] Before the experiment, soil samples are collected and the basic physical and chemical properties (organic matter content, aggregate ratio, electrical conductivity, etc.) are recorded.

[0115] Treatment group setting: Example 2: Compound bio-fertilizer with chitosan-modified zeolite carrier immobilized microbial community and humic acid, applied at 40 kg per mu.

[0116] Example 4: Compound bio-fertilizer of immobilized microbial community and humic acid derived from lignite, applied at 50 kg per mu.

[0117] Comparative Example 4: Bio-fertilizer without added humic acid, applied at 40 kg per mu.

[0118] Comparative Example 5: Bio-fertilizer with simplified carrier process (not chitosan-modified), applied at 40 kg per mu.

[0119] Blank group: No bio-fertilizer is applied, only the same amount of ordinary chemical fertilizers (potassium fertilizer and nitrogen-phosphorus fertilizer) are applied.

[0120] Fertilization and management: The bio-fertilizer or ordinary chemical fertilizer is evenly spread and then plowed into the soil, and kidney beans are planted.

[0121] During the experiment, conventional farmland management is maintained, but no other fertilizers are applied additionally.

[0122] Data collection: Soil samples are collected monthly to measure the following key indicators: Soil organic matter content: Determined by the potassium dichromate oxidation method.

[0123] Soil aggregate proportion: Separate large aggregates and small aggregates using the wet sieving method.

[0124] Soil conductivity: Determined by a conductivity meter.

[0125] Soil microbial diversity: Analyze the changes in microbial diversity using high-throughput sequencing.

[0126] Endpoint determination: After the end of the growing season, collect soil samples again and summarize and analyze the changes in physical and chemical properties.

[0127] Combine the data with the growth performance (plant height, yield) of kidney beans to evaluate the comprehensive improvement effect.

[0128] Experimental data: Improvement effects of physical and chemical properties of saline-alkali soil under different treatments In this experiment, by comparing the improvement effects of different treatments on the physical and chemical properties of saline-alkali soil, the superiority of the compound microbial fertilizer in the examples in enhancing soil fertility, improving physical structure and promoting microbial diversity was verified. The results showed that Example 2 and Example 4 were significantly superior to the control treatment group and the blank group in improving soil organic matter content and aggregate proportion, while significantly reducing soil conductivity and increasing the microbial diversity index.

[0129] The chitosan-modified zeolite carrier in the present invention provides a stable immobilization environment for the microbial community, significantly enhancing the activity and persistence of the microbial community. After being applied to the soil, the immobilized microbial community can continuously secrete osmotic adjustment substances (such as betaine) in the rhizosphere. While alleviating salt stress, it promotes the decomposition and transformation of organic matter through metabolic activities, increasing the accumulation of active organic matter in the soil. In addition, the addition of humic acid further enhances the water and fertilizer retention capacity of the soil and aggregate formation, effectively improving the physical structure of the soil. In Comparative Example 4, the treatment group without the addition of humic acid showed a lower aggregate proportion, proving the key role of humic acid in soil improvement.

[0130] In addition, the multi-bacterial community synergy mechanism of the compound bacterial fertilizer has played a significant role in soil improvement. The salt-tolerant and growth-promoting bacteria (Halomonas sp.) alleviate salt stress by regulating the rhizosphere ion balance; the potassium-dissolving and phosphorus-solubilizing bacteria (Bacillus subtilis) convert insoluble potassium and phosphorus in the soil into available nutrients, further increasing the content of elements that can be absorbed by plants; the plant signaling bacteria (Pseudomonas sp.) promote root growth by secreting IAA, enhancing the binding ability between roots and soil. This synergistic effect was fully demonstrated in the example group, while in Comparative Example 5, due to the unoptimized carrier, the activity and persistence of the bacterial community were significantly reduced, resulting in poor soil improvement effects.

[0131] The experimental results also showed that a significant increase in soil microbial diversity is another important effect of the compound bacterial fertilizer. In the Example 2 group, the immobilized bacterial community was gradually released in the soil, forming a stable interaction relationship with indigenous microorganisms, significantly enhancing the diversity of the microbial community, while the microbial diversity indices of the blank group and the comparative example group were relatively low. This further verified the synergistic effect of the multi-bacterial community combination and the immobilization process, providing a new idea for the ecological restoration of saline-alkali soil.

[0132] In summary, Examples 2 and 4 demonstrated excellent performance in saline-alkali soil improvement through the triple synergy of "chitosan-modified carrier + immobilized bacterial community + humic acid". Compared with the comparative example group, the present invention significantly increased the organic matter accumulation, aggregate ratio, and microbial diversity of the soil, reduced soil salt stress, and provided technical support for agricultural production in saline-alkali land.

[0133] Experiment 4: Comparative test on the yield and quality of kidney beans Experimental steps: Selection of test plots: Test areas were set up in slightly saline-alkali soil (electrical conductivity 2 dS / m, pH 8.2) and moderately saline-alkali soil (electrical conductivity 4 dS / m, pH 8.5) respectively.

[0134] Each test area was divided into 5 treatment plots, each plot with an area of 1 mu, for the comparison of different fertilizer treatments.

[0135] Setting of treatment groups: Slightly saline-alkali soil: Example 1: Apply 30 kg of compound bacterial fertilizer per mu.

[0136] Comparative Example 3: Treatment with single-strain bacterial fertilizer, apply 30 kg per mu.

[0137] Blank group: Apply the same amount of ordinary chemical fertilizers (potassium fertilizer and nitrogen-phosphorus fertilizer) per mu.

[0138] Moderately saline-alkali soil: Example 2: Apply 40 kg of compound bacterial fertilizer per mu.

[0139] Control Example 4: Treatment with bacterial fertilizer without added humic acid, apply 40 kg per mu.

[0140] Blank group: Apply an equal amount of ordinary chemical fertilizers (potassium fertilizer and nitrogen and phosphorus fertilizers) per mu.

[0141] Fertilization and planting: According to the experimental design, evenly spread the fertilizer and then plow it into the soil, plant kidney bean seeds, and keep the planting density uniform.

[0142] During the planting process, conduct unified irrigation and weeding management, without additional fertilization.

[0143] Data collection: During the growth period, regularly record the growth indicators of kidney beans: Germination rate: Count the germination rate one week after planting.

[0144] Growth rate: Measure the plant height and leaf area every two weeks.

[0145] Flowering rate: Count the proportion of flowering plants during the flowering stage.

[0146] Measurement at harvest: Yield: The total harvest of kidney beans per mu.

[0147] Quality: Protein content (Kjeldahl method) and sugar content (spectrophotometry) of kidney bean grains.

[0148] Final measurement: After harvest, count all the data and conduct comparative analysis on the differences in yield and quality.

[0149] Experimental data: Effects of different treatments on the yield and quality of kidney beans in saline-alkali land The experimental results show that the treatments of Example 1 and Example 2 significantly improved the germination rate, plant height, flowering rate, and final yield of kidney beans, and were also superior to the control examples and the blank group in terms of quality indicators such as protein and sugar content. This indicates that the compound bacterial fertilizer can not only improve the growth performance of plants in the saline-alkali land environment, but also enhance the quality of agricultural products.

[0150] The multiple synergistic mechanisms of compound bacterial fertilizer are the key to improving the yield and quality of kidney beans. In Example 1 and Example 2, the chitosan-modified zeolite carrier effectively regulated the rhizosphere ion concentration by slowly releasing potassium ions and adsorbing excess sodium ions, significantly improving the nutrient utilization efficiency of plants under saline-alkali stress. Osmotic regulating substances such as betaine secreted by salt-tolerant growth-promoting bacteria (Halomonas sp.) further alleviated the osmotic pressure damage of salt stress to cells, helping kidney beans to maintain healthy growth in a saline-alkali environment.

[0151] The addition of humic acid also plays an important role in promoting plant nutrient absorption and improving soil environment. The humic acid in the embodiment provides a more suitable microenvironment for plant roots by improving soil structure and enhancing water and fertilizer retention capacity. In addition, humic acid can also chelate trace elements in the soil, further improving the nutrient absorption capacity of plants. This mechanism is significant in the embodiment group, while the growth index and yield of kidney beans in the treatment group without humic acid addition in Comparative Example 4 are lower than those in the embodiment group.

[0152] The synergistic effect of multiple bacterial communities is also an important mechanism of the present invention. Halomonas sp. and Bacillus subtilis work together to help plants resist salt stress and provide more available nutrients for plants through potassium and phosphorus dissolving functions. At the same time, Pseudomonas sp. promotes root development by secreting plant hormones such as IAA, further enhancing the plant's ability to absorb nutrients and water. The comprehensive function of this multi-bacteria community is fully reflected in the examples, while the yield and quality of kidney beans in Comparative Example 3, which lacks synergistic effects, are poor.

[0153] In general, the combination of "chitosan modified carrier + immobilized bacterial community + humic acid" in the embodiment significantly improved soil conditions in saline-alkali land environment and enhanced the salt tolerance and nutrient absorption capacity of plants. Compared with the comparative example, the present invention achieves a comprehensive improvement in kidney bean yield and quality through multiple synergistic effects, providing an efficient and environmentally friendly solution for saline-alkali land agricultural production.

[0154] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composite biological fertilizer for promoting bean production, characterized in that: The following components are included in the range of weight percentage: Modified zeolite: 60%–80%, Chitosan: 5%–10%, Microbiome: 10%–20%, including: Salt-tolerant growth-promoting bacteria: 5%–10%, Potassium-dissolving and phosphate-dissolving bacteria: 3%–8%, Plant signal bacteria: 2%–5%, Humic acid: 5%–15%.

2. The composite biological fertilizer for promoting bean production increase according to claim 1, characterized in that: The modified zeolite has a particle size range of 0.5-2 mm and a cation exchange capacity of 150-200 cmol(+) / kg.

3. The composite biological fertilizer for promoting bean production increase according to claim 1, characterized in that: The chitosan has a deacetylation degree of 80%-90% and a molecular weight of 10-100 kDa.

4. The composite biological fertilizer for promoting bean production increase according to claim 1, characterized in that: The salt-tolerant growth-promoting bacteria are Halomonas sp., the potassium-solubilizing and phosphate-dissolving bacteria are Bacillus subtilis, and the plant signal bacteria are Pseudomonas sp.

5. The composite biological fertilizer for promoting bean production increase according to claim 1, characterized in that: The composite biological fertilizer is in granular form, and the particle diameter is 2-5 mm.

6. A method for preparing a composite biological fertilizer for promoting bean production according to any one of claims 1 to 5, characterized in that: The following steps are involved: Modified zeolite preparation: washing and drying the zeolite; The zeolite is immersed in the chitosan solution at a reaction temperature of 40–60°C for 4–8 hours and then dried; The modified zeolite is immersed in a 1–2 M KCl solution and allowed to stand for 12–24 hours to obtain a potassium ion-loaded modified zeolite; Preparation and immobilization of microbial flora: The salt-tolerant growth-promoting bacteria, potassium-solubilizing and phosphate-dissolving bacteria, and plant signaling bacteria were cultured to a bacterial solution with an OD600 value of 1.0–1.5; The bacterial solution was mixed with modified zeolite in a ratio of 1:2–1:3, and allowed to stand at 37°C for 2–4 hours for adsorption, followed by freeze-drying to obtain an immobilized microbial carrier; Preparation of finished compound fertilizer: The immobilized microbial carrier and humic acid are mixed in proportion to prepare a granular composite bacterial fertilizer.

7. The method for preparing a composite biological fertilizer for increasing bean production according to claim 6, characterized in that: The concentration of the chitosan solution is 2%-5% (w / v).

8. The method for preparing a composite biological fertilizer for promoting bean production increase according to claim 6, characterized in that: After the bacterial liquid is mixed with the modified zeolite, it is dried by a freeze-drying process, the freezing temperature is -40 to -60°C, and the drying time is 24-48 hours.

9. The method for preparing a composite biological fertilizer for promoting bean production increase according to claim 6, characterized in that: The humic acid is sourced from natural humus raw materials selected from weathered coal, peat or lignite, and is prepared by alkali dissolution and acid precipitation, and has a molecular weight range of 500-5000 Da.

10. Use of the composite biological fertilizer prepared according to any one of claims 6 to 9 in improving the soil structure of saline-alkali land.

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