A method for rapid colonization of soil microorganisms on newly cultivated land in dryland areas

Abstract

This application relates to the field of agricultural microbial technology, specifically disclosing a method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas. The method includes the following steps: spraying a modified sodium alginate solution onto the surface soil of the newly reclaimed farmland, simultaneously applying a calcium source and a pH buffer to prepare a three-layered microbial agent; forming a sowing furrow in the rotary-tilled soil; applying the three-layered microbial agent to the sowing furrow; sowing seeds in the furrow after applying the three-layered microbial agent; and then irrigating. The irrigation is divided into a first stage and a second stage, with the irrigation water in the first stage being acidic and the irrigation water in the second stage being neutral or weakly alkaline. This application utilizes an in-situ ionic cross-linking reaction to bind loose soil particles into water-stable microaggregates. A slow-release carbon source material in the core provides continuous nutrient supply to the microbial community. Electrostatic adsorption allows the microbial agent to spatially match the microaggregates, achieving anchoring of the microbial agent in the soil.

Description

Technical Field

[0001] This application relates to the field of agricultural microbiology, and more specifically, to a method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas. Background Technology

[0002] Dryland farming areas refer to regions that rely on natural rainfall for agricultural production. In my country, they are widely distributed in the Northwest, North China, and western Northeast. Newly reclaimed arable land refers to wasteland, abandoned land, or reclaimed land that is being used for agricultural production for the first time or again. These types of soils usually have problems such as low organic matter content, loose soil structure, and a small number of microorganisms. Soil microorganisms play a key role in soil nutrient cycling, organic matter decomposition, and crop nutrient supply. Therefore, rapidly establishing beneficial soil microbial communities on newly reclaimed arable land is of great significance for improving soil quality and promoting crop growth.

[0003] Currently, the common method for establishing soil microbial communities on newly reclaimed farmland is to apply exogenous microbial agents to the soil at the time of sowing. Liquid agents or solid powders containing single or multifunctional strains are physically mixed with organic carriers such as well-rotted organic fertilizer, biochar, or peat soil, and then applied to the soil by dipping roots, mixing seeds, or fertigation, in the hope that the exogenous microorganisms can survive and function in the soil. However, the soil in newly reclaimed farmland lacks a stable aggregate structure, and the soil particles are in a loose state. Exogenous microorganisms are lost through deep seepage or surface runoff with irrigation water or rainfall, and cannot effectively reside in the topsoil. Moreover, exogenous microorganisms are directly exposed to the barren and arid soil environment, lacking a continuous carbon source supply and physical protection. In addition, the soil moisture content in dryland areas fluctuates drastically, and a large number of microorganisms die under the stress of alternating dry and wet conditions, resulting in low microbial colonization efficiency. Summary of the Invention

[0004] To address the problem of low colonization efficiency in existing rapid colonization methods for soil microorganisms on newly reclaimed farmland in dryland areas, this application provides a rapid colonization method for soil microorganisms on newly reclaimed farmland in dryland areas.

[0005] This application provides a method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas, which adopts the following technical solution: A method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas includes the following steps: S1. Spray modified sodium alginate solution onto the surface soil of newly cultivated land, while simultaneously applying calcium source and pH buffer, and perform rotary tillage to cause in-situ ionic cross-linking reaction on the surface of soil particles, forming water-stable micro-aggregates, and assigning charges to the surface of the micro-aggregates. S2. Prepare a three-layered bacterial agent, wherein the three-layered bacterial agent consists of a core layer, a shell layer and a canopy layer from the inside out. The core layer includes a slow-release carbon source material, the shell layer includes a composite functional bacterial community and a gel material, and the canopy layer includes a water-retaining material and microbial signaling molecules. S3. A sowing furrow is formed in the soil after rotary tillage. The three-layer structured microbial agent is applied to the sowing furrow. The surface of the three-layer structured microbial agent has a charge opposite to that on the surface of the micro-aggregates. The microbial agent is attached to the surface of the micro-aggregates through electrostatic adsorption. S4. After sowing in the sowing furrow after applying the three-layer structure microbial agent, irrigation is carried out. The irrigation is divided into a first stage and a second stage. The irrigation water in the first stage is acidic, and the irrigation water in the second stage is neutral or weakly alkaline.

[0006] By adopting the above technical solution, the loose soil particles are bonded into water-stable micro-aggregates through in-situ ionic cross-linking reaction, thus constructing a stable physical structure for newly reclaimed farmland soil. Simultaneously, the surface of the micro-aggregates is charged, providing binding sites for the electrostatic adsorption and anchoring of the microbial agent. The slow-release carbon source material in the core provides continuous nutrient supply to the microbial community, the gel material in the shell provides physical protection, and the water-retaining material in the canopy alleviates water stress. The microbial signaling molecules in the canopy can activate the metabolic activity and chemotaxis of the microbial community. Through electrostatic adsorption, the microbial agent and micro-aggregates form a spatial match, achieving anchoring of the microbial agent in the soil and preventing its loss with water. The release of the microbial agent is triggered by acidic irrigation water in the first stage, and the micro-aggregate structure is protected by weakly alkaline irrigation water in the second stage, achieving a temporal match between the release of the microbial agent and soil moisture conditions. This solves the problem of low microbial colonization efficiency caused by existing rapid colonization methods for newly reclaimed farmland in dryland areas.

[0007] Preferably, in step S1, the modified sodium alginate is a blend of sodium alginate and polyethylene glycol, the mass ratio of sodium alginate to polyethylene glycol is 4-6:1, the concentration of the modified sodium alginate solution is 0.8%-1.2%, the calcium source is calcium gluconate, the application rate is 10-15 g / m², the pH buffer is calcium carbonate micro powder with a particle size of 10-30 μm, the application rate is 5-8 g / m², the rotary tillage depth is 8-10 cm, and the surface charge of the micro-aggregates is positively charged, which is achieved by adding chitosan quaternary ammonium salt to the modified sodium alginate solution, the concentration of the chitosan quaternary ammonium salt is 0.05%-0.1%.

[0008] By adopting the above technical solution, the blending modification of polyethylene glycol with sodium alginate increases the flexibility and anti-cracking properties of the gel layer, enabling the micro-aggregates to maintain structural stability under alternating dry and wet conditions in dryland areas. Calcium gluconate provides calcium ions to trigger in-situ cross-linking reactions, calcium carbonate micropowder acts as a pH buffer to provide neutralization reserves for subsequent weakly acidic irrigation, and chitosan quaternary ammonium salt imparts a positive charge to the surface of the micro-aggregates, providing binding sites for the electrostatic adsorption of the microbial agent. Through the synergistic effect of each component, the soil micro-aggregate structure remains intact after irrigation, while providing a stable charged interface for the anchoring of the microbial agent.

[0009] Preferably, in step S2, the method for preparing the core layer is as follows: soluble starch, humic acid and microcrystalline cellulose are mixed in a mass ratio of 3-5:2-4:2-4, water is added to make a slurry with a solid content of 25%-35%, and the spherical core with a particle size of 150-250μm is made by extrusion and sphericalization process, and then dried to a moisture content of <8%.

[0010] By adopting the above technical solution, soluble starch is used as a fast-acting carbon source, humic acid as a slow-acting carbon source, and microcrystalline cellulose as a structural framework. The three are combined in proportion to form a dense spherical core. The extrusion and spheroidization process gives the core high compressive strength and a dense structure, which can achieve a slow-release carbon source supply, provide continuous energy substances for the microbial community, and avoid the decline of the microbial community due to the depletion of carbon source.

[0011] Preferably, in step S2, the shell layer is prepared by mixing the composite functional bacterial group with sodium alginate solution, adding calcium citrate microcrystals and mixing evenly, coating it onto the core surface through a spray coating process to form a shell layer with a thickness of 30-50 μm, and then allowing it to stand and solidify.

[0012] By adopting the above technical solution, the sodium alginate gel layer provides a protective barrier for the microbial community. Calcium citrate microcrystals are uniformly dispersed in the gel layer, releasing calcium ions at the interface between the shell and the canopy to form local cross-links and enhance the interfacial bonding strength. At the same time, under weakly acidic irrigation conditions, the calcium citrate microcrystals accelerate the release of calcium ions and synergistically trigger the release of the microbial agent, so that the microbial community remains active during storage and application, and is released as needed when triggered by irrigation.

[0013] Preferably, the composite functional microbial community includes Bacillus amyloliquefaciens, Bacillus mucilaginosus, and Pseudomonas fluorescens, each with a bacterial concentration of 10. 9The bacterial culture is mixed at a volume ratio of 0.8-1.2:0.8-1.2:0.8-1.2, with a sodium alginate solution concentration of 1.2%-1.8% and a volume ratio of 1:3-5 between the bacterial culture and the sodium alginate solution. The calcium citrate microcrystals have a particle size of <10μm and are added at a rate of 0.1%-0.3% of the total mass of the mixture of the compound functional bacterial culture and the sodium alginate solution. The static curing temperature is 25-30℃, the relative humidity is 55%-65%, and the time is 1.5-2.5 hours.

[0014] By adopting the above technical solution, three functional bacteria, namely Bacillus amyloliquefaciens, Bacillus colloidis, and Pseudomonas fluorescens, are combined to form a synergistic bacterial community with multiple functions such as phosphorus solubilization, potassium solubilization, nitrogen fixation, and extracellular polysaccharide production. The concentration and mixing volume ratio of sodium alginate give the coating slurry suitable viscosity and film-forming properties. The particle size and addition amount of calcium citrate microcrystals are controlled to ensure their uniform distribution in the gel layer. Static curing causes moderate cross-linking of the gel layer, which protects the bacterial community and maintains release responsiveness, so that the shell layer can ensure the survival of the bacterial community while having good triggering release performance.

[0015] Preferably, in step S2, the canopy is prepared by spraying a composite functional solution onto the surface of the shell to form a canopy with a thickness of 5-10 μm. The spraying amount is 0.5-1 mL / g of bacterial agent. The composite functional solution includes 0.15%-0.25% γ-polyglutamic acid, 8-12 mM γ-aminobutyric acid, 15-25 mM xylose, 4-6 mM sodium lactate, and 0.05%-0.15% sodium alginate.

[0016] By adopting the above technical solution, γ-polyglutamic acid, as a highly absorbent and water-retaining material, forms a local water film around the bacterial agent to alleviate drought stress. γ-aminobutyric acid, as a chemotactic signaling molecule, induces the bacterial community to migrate to the rhizosphere. Xylose and sodium lactate, as initiating carbon sources, activate the metabolic activity of the bacterial community. Sodium alginate imparts a negative charge to the canopy, providing conditions for the electrostatic adsorption of the bacterial agent and positively charged micro-aggregates. Through the synergy of each component in the canopy, the bacterial agent can complete signal activation and water retention protection after being applied to the soil, laying the foundation for subsequent colonization.

[0017] Preferably, in step S3, the application amount of the three-layer structure microbial agent is 20-30 g / m², and the application depth is 5-8 cm in the sowing furrow. The surface of the three-layer structure microbial agent carries a negative charge, which is achieved by adding sodium alginate to the composite functional solution of the canopy.

[0018] By adopting the above technical solution, the amount and depth of application of microbial agent ensure that the microbial agent is concentrated in the rhizosphere area of ​​the crop. Sodium alginate in the canopy imparts a negative charge to the surface of the microbial agent, which forms an electrostatic adsorption pair with the positive charge on the surface of the micro-aggregates, thereby achieving precise anchoring of the microbial agent on the surface of the micro-aggregates. This makes the microbial agent less likely to be washed away by water under irrigation and rainfall conditions, and the colonization location highly overlaps with the crop rhizosphere.

[0019] Preferably, in step S4, the irrigation is a one-time micro-sprinkler irrigation with a total irrigation volume of 12-15 mm. The irrigation time in the first stage is 0-2 hours, the pH of the irrigation water is 5.0-5.5, and the irrigation water volume accounts for 30%-40% of the total irrigation volume. The irrigation time in the second stage is 2-6 hours, the pH of the irrigation water is 6.5-7.0, and the irrigation water volume accounts for 60%-70% of the total irrigation volume.

[0020] By adopting the above technical solution, the first stage of acidic irrigation water triggers the release of calcium ions from calcium citrate microcrystals in the bacterial agent shell layer, accelerates the dissociation of calcium alginate gel surface, and initiates the release of bacterial communities. The second stage of weakly alkaline irrigation water, combined with pre-applied calcium carbonate micropowder, neutralizes soil acidity and protects the micro-aggregate structure from excessive dissociation. The synergistic effect of water distribution and pH control in the two stages achieves the time-series control of acid-triggered release followed by neutralization and protection of the structure.

[0021] Preferably, in step S4, the pH of the irrigation water in the first stage is adjusted by adding citric acid, and the amount of citric acid added is 0.1-0.2 g / L. In the second stage of irrigation, the calcium carbonate powder reacts with the residual hydrogen ions in the soil, so that the soil pH returns to neutral.

[0022] By adopting the above technical solution, citric acid, as a weak organic acid, can adjust the pH of irrigation water to the trigger range without causing toxicity to the soil and microorganisms. Calcium carbonate micropowder slowly dissolves under weak acid conditions and reacts with hydrogen ions to generate carbon dioxide and water, causing the soil pH to automatically rise back to the neutral range. This not only meets the functional requirement of triggering the release of microbial agents, but also ensures the long-term stability of the micro-aggregate structure.

[0023] Preferably, in step S4, the soil covering depth after sowing is 2-3 cm, and the soil moisture content of the topsoil after irrigation is 70%-80% of field capacity.

[0024] By adopting the above technical solutions, the soil covering depth is controlled to ensure that the inoculant and seeds are in the same soil layer, which facilitates the migration of the microbial community to the rhizosphere. The control of water content after irrigation ensures that the topsoil reaches the appropriate moisture conditions for microbial activity and crop emergence, while avoiding excessive irrigation that could cause deep leakage of the inoculant, thus ensuring the synchronicity of inoculant release, microbial colonization and crop emergence.

[0025] In summary, this application has the following beneficial effects: 1. This application utilizes in-situ ionic cross-linking to bind loose soil particles into water-stable micro-aggregates, thus constructing a stable physical structure for newly reclaimed farmland soil. Simultaneously, it imparts charges to the surface of the micro-aggregates, providing binding sites for the electrostatic adsorption and anchoring of the microbial agent. The slow-release carbon source material in the core provides continuous nutrient supply to the microbial community, the gel material in the shell provides physical protection, and the water-retaining material in the canopy alleviates water stress. The microbial signaling molecules in the canopy activate the metabolic activity and chemotaxis of the microbial community. Through electrostatic adsorption, the microbial agent spatially matches the micro-aggregates, achieving anchoring of the agent in the soil and preventing its loss with water. The first stage of acidic irrigation water triggers agent release, while the second stage of weakly alkaline irrigation water protects the micro-aggregate structure, achieving a temporal match between agent release and soil moisture conditions. This solves the problem of low microbial colonization efficiency caused by existing rapid colonization methods for newly reclaimed farmland in dryland areas.

[0026] 2. In this application, polyethylene glycol-modified sodium alginate is used to improve the flexibility and anti-cracking properties of the gel layer. Chitosan quaternary ammonium salt is used to impart a positive charge to the surface of the micro-aggregates. Calcium carbonate micropowder is used as a pH buffer to maintain the structural stability of the micro-aggregates under alternating wet and dry conditions, while providing a stable charged interface for the electrostatic adsorption of the bacterial agent. The core is prepared by compounding soluble starch, humic acid and microcrystalline cellulose in a certain proportion, and a dense spherical structure is formed by extrusion and spheroidization process, which realizes the slow-release carbon source supply and avoids the death of the bacterial community due to carbon source depletion.

[0027] 3. This application uses sodium alginate gel layer to embed bacterial communities and adds calcium citrate microcrystals, which not only provides physical protection for the bacterial communities, but also realizes the functions of bacterial community protection and triggered release through the cross-linking enhancement effect and acid response release characteristics of calcium citrate microcrystals at the shell and canopy interface. The canopy is prepared by compounding γ-polyglutamic acid, γ-aminobutyric acid, xylose and sodium lactate. Through the synergistic effect of water-retaining materials to alleviate drought stress, signal molecules to activate chemotaxis, and carbon source to activate metabolic activity, the bacterial agent can quickly complete the colonization preparation after being applied to the soil. Attached Figure Description

[0028] Figure 1 This is a flowchart of a method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas, as provided in this application. Detailed Implementation

[0029] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0030] Technical concept: Newly reclaimed farmland in dryland areas generally suffers from problems such as low organic matter content, loose soil structure, and scarce microorganisms. Existing technologies aim to establish beneficial microbial communities by directly applying exogenous microbial agents into the soil. However, newly reclaimed farmland soil lacks a stable aggregate structure, and exogenous microorganisms are lost through deep seepage or surface runoff with irrigation water, making it impossible for them to effectively reside in the topsoil. At the same time, exogenous microorganisms are directly exposed to the barren and arid soil environment, lacking a continuous carbon source supply and physical protection. In addition, the soil moisture content in dryland areas fluctuates drastically, and a large number of microorganisms die under the stress of alternating dry and wet conditions, resulting in low colonization efficiency.

[0031] Based on the above findings, this application binds loose soil particles into water-stable micro-aggregates and imparts surface charge through in-situ ionic cross-linking reaction. This constructs a stable physical framework for newly reclaimed farmland soil and provides electrostatic binding sites for the microbial agent. On this basis, a three-layer structure microbial agent is formed by preparing a slow-release carbon source material as the core, a composite functional microbial community and gel material as the shell, and a water-retaining material and microbial signaling molecules as the canopy. This structure enables continuous nutrient supply, physical protection, and colonization activation of the microbial community. Through electrostatic adsorption, the microbial agent and micro-aggregates form a spatial match, enabling precise anchoring of the microbial agent in the soil. Through staged pH gradient irrigation, the release of the microbial agent is triggered in the acidic stage, and the structure of the micro-aggregates is protected in the neutral stage. This achieves a temporal match between the release of the microbial agent and the soil moisture conditions, solving the problem of low microbial colonization efficiency caused by existing rapid colonization methods for soil microorganisms in newly reclaimed farmland in dryland areas.

[0032] Unless otherwise specified, all experimental methods used below are conventional methods. All materials, reagents, methods, and instruments used, unless otherwise specified, are conventional materials, reagents, methods, and instruments in this field, which can be obtained commercially or prepared according to literature methods by those skilled in the art.

[0033] Preparation Example 1: Preparation of Modified Sodium Alginate Solution The modified sodium alginate solution is prepared by blending sodium alginate with polyethylene glycol PEG-400. The specific preparation method is as follows: Weigh 50g of sodium alginate and 10g of polyethylene glycol PEG-400, add 5.94L of deionized water, stir at 300-500r / min at 25-30℃ until completely dissolved, and bring the volume to 6L to obtain a 1.0% modified sodium alginate solution.

[0034] Preparation Example 2: Preparation of Complex Functional Microbial Community The composite functional bacterial community is composed of a mixture of three bacterial suspensions: Bacillus amyloliquefaciens, Bacillus mucilaginosus, and Pseudomonas fluorescens. The specific preparation method is as follows: Preparation of Bacillus amyloliquefaciens bacterial culture: A slant culture of Bacillus amyloliquefaciens was inoculated into LB liquid medium and cultured with shaking at 30±1℃ and 180 r / min for 24-36 h until the bacterial concentration reached 10⁻⁶. 9 CFU / mL was used to obtain Bacillus amyloliquefaciens bacterial culture. LB liquid medium consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with a pH of 7.0-7.2.

[0035] Preparation of Bacillus sclerotiorum culture: Take Bacillus sclerotiorum slant culture, inoculate it into nitrogen-free medium, and incubate with shaking at 30±1℃ and 150r / min for 48-60h until the bacterial concentration reaches 10. 9 CFU / mL was used to obtain a gelatinous Bacillus spore suspension. The nitrogen-free culture medium included 10 g / L sucrose, 0.5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.2 g / L sodium chloride, and 1 g / L calcium carbonate, with a pH of 7.0-7.2.

[0036] Preparation of *Pseudomonas fluorescens* bacterial suspension: *Pseudomonas fluorescens* slant culture was inoculated into KB liquid medium and cultured with shaking at 28±1℃ and 180 r / min for 24-30 h until the bacterial concentration reached 10⁻⁶. 9 CFU / mL was used to obtain fluorescent Pseudomonas bacterial suspension. KB liquid medium included 20 g / L peptone, 10 mL / L glycerol, 1.5 g / L dipotassium hydrogen phosphate, and 1.5 g / L magnesium sulfate, with a pH of 7.0-7.2.

[0037] Preparation of the mixed solution of compound functional bacteria: Take 100 mL of the above-prepared Bacillus amyloliquefaciens bacterial solution, 100 mL of Bacillus spp. gelatinous bacterial solution, and 100 mL of Pseudomonas fluorescens bacterial solution, and mix them evenly under aseptic conditions to obtain 300 mL of mixed solution of compound functional bacteria.

[0038] Preparation Example 3: Preparation of Multifunctional Solution The composite functional solution is used for canopy spraying, and the specific preparation method is as follows: Weigh 2.0 g of γ-polyglutamic acid and 1.0 g of sodium alginate, add them to 800 mL of deionized water, and stir at 200-300 r / min at 25-30℃ until completely dissolved. Weigh 1.03 g (10 mM) of γ-aminobutyric acid and 3 g (20 mM) of xylose, and measure 0.56 g (5 mM) of sodium lactate, add them to the above solution, continue stirring until completely dissolved, and bring the volume to 1 L to obtain the composite functional solution.

[0039] Preparation Example 4: Formulation of Coating Slurry The coating slurry is used for shell coating, and the specific preparation method is as follows: Take 100 mL of the above-prepared compound functional microbial mixture, add 400 mL of 1.5% sodium alginate solution, add 1 g of calcium citrate microcrystals, and stir at 100-200 r / min for 5-10 min until uniform to obtain 500 mL of coating slurry.

[0040] To better understand the above technical solutions, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0041] The following is a further description with reference to the embodiments: Example 1: Please refer to the appendix Figure 1 A method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas includes the following steps: S1. Spray modified sodium alginate solution onto the surface soil of newly cultivated land, while simultaneously applying calcium source and pH buffer, and perform rotary tillage to cause in-situ ionic cross-linking reaction on the surface of soil particles, forming water-stable micro-aggregates, and assigning charges to the surface of the micro-aggregates. The modified sodium alginate is a blend of sodium alginate and polyethylene glycol, with a mass ratio of sodium alginate to polyethylene glycol of 5:1. In this embodiment, 50g of sodium alginate and 10g of polyethylene glycol are used. The concentration of the modified sodium alginate solution is 1%. The calcium source is calcium gluconate, with an application rate of 12.5g / m². The pH buffer is calcium carbonate micropowder with a particle size of 20μm, with an application rate of 6.5g / m². The rotary tillage depth is 9cm. The surface charge of the micro-aggregates is positive, achieved by adding chitosan quaternary ammonium salt to the modified sodium alginate solution. The concentration of chitosan quaternary ammonium salt is 0.075%.

[0042] S2. Prepare a three-layered bacterial agent. The three-layered bacterial agent consists of an inner core layer, a shell layer, and a canopy layer from the inside out. The inner core layer includes a slow-release carbon source material, the shell layer includes a composite functional bacterial community and a gel material, and the canopy layer includes a water-retaining material and microbial signaling molecules. The core layer is prepared as follows: soluble starch, humic acid, and microcrystalline cellulose are mixed in a mass ratio of 4:3:3. In this embodiment, soluble starch is 40g, humic acid is 30g, and microcrystalline cellulose is 30g. Water is added to make a slurry with a solid content of 30%, which is then extruded and spherically rolled to form spherical cores with a particle size of 200μm. The cores are then dried until the moisture content is <8%. The shell layer is prepared as follows: a composite functional bacterial group is mixed with a sodium alginate solution, calcium citrate microcrystals are added and mixed evenly, and then coated onto the surface of the core layer using a spray coating process to form a shell layer with a thickness of 40μm. The mixture is then allowed to stand and solidify. The composite functional bacterial group includes *Bacillus amyloliquefaciens*, *Bacillus colloidea*, and *Pseudomonas fluorescens*, all with a bacterial concentration of 10%. 9CFU / mL, mixed at a volume ratio of 1:1:1. In this embodiment, 100mL of Bacillus amyloliquefaciens bacterial solution, 100mL of Bacillus lentigines bacterial solution, and 100mL of Pseudomonas fluorescens bacterial solution were taken and mixed evenly to obtain 300mL of composite functional bacterial mixture. The concentration of sodium alginate solution was 1.5%, and the mixing volume ratio of bacterial solution to sodium alginate solution was 1:4. In this embodiment, 100mL of composite functional bacterial mixture was taken and 400mL of sodium alginate solution was added and mixed evenly to obtain 500mL of coating slurry. The particle size of calcium citrate microcrystals was <10μm, and the addition amount was 0.2% of the total mass of composite functional bacterial mixture and sodium alginate solution. The static curing temperature was 27.5℃, the relative humidity was 60%, and the time was 2 hours. In step S2, the canopy is prepared by spraying a composite functional solution onto the surface of the shell to form a canopy with a thickness of 7.5 μm. The spraying amount is 0.75 mL / g of bacterial agent. The composite functional solution includes 0.2% γ-polyglutamic acid, 10 mM γ-aminobutyric acid, 20 mM xylose, 5 mM sodium lactate, and 0.1% sodium alginate.

[0043] S3. Form a sowing furrow in the soil after rotary tillage, and apply the three-layer structured microbial agent into the sowing furrow. The surface of the three-layer structured microbial agent has a charge opposite to that on the surface of the micro-aggregates, and the microbial agent is attached to the surface of the micro-aggregates through electrostatic adsorption. The application rate of the three-layer structure microbial agent is 25g / m², and the application depth is 6.5cm in the sowing furrow. The surface of the three-layer structure microbial agent carries a negative charge, which is achieved by adding sodium alginate to the composite functional solution of the canopy.

[0044] S4. After sowing in the sowing furrow after applying the three-layer structure microbial agent, irrigate. Irrigation is divided into a first stage and a second stage. The irrigation water in the first stage is acidic, and the irrigation water in the second stage is neutral or weakly alkaline.

[0045] The irrigation was a single-stage micro-sprinkler irrigation, with a total irrigation volume of 13.5 mm. The first stage of irrigation lasted 0-2 hours, with the irrigation water having a pH of 5.25 and accounting for 35% of the total irrigation volume. The second stage of irrigation lasted 2-6 hours, with the irrigation water having a pH of 6.75 and accounting for 65% of the total irrigation volume. In the first stage, the pH of the irrigation water was adjusted by adding citric acid at a concentration of 0.15 g / L. During the second stage of irrigation, the calcium carbonate powder reacted with residual hydrogen ions in the soil, causing the soil pH to return to neutral. The sowing depth was 2.5 cm, and the topsoil moisture content after irrigation was 75% of field capacity.

[0046] Example 2: This example differs from Example 1 above in that: A method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas includes the following steps: S1. Spray modified sodium alginate solution onto the surface soil of newly cultivated land, while simultaneously applying calcium source and pH buffer, and perform rotary tillage to cause in-situ ionic cross-linking reaction on the surface of soil particles, forming water-stable micro-aggregates, and assigning charges to the surface of the micro-aggregates. The modified sodium alginate is a blend of sodium alginate and polyethylene glycol, with a mass ratio of sodium alginate to polyethylene glycol of 4:1. In this embodiment, 40g of sodium alginate and 10g of polyethylene glycol are used. The concentration of the modified sodium alginate solution is 0.8%. The calcium source is calcium gluconate, with an application rate of 10g / m². The pH buffer is calcium carbonate micropowder with a particle size of 10μm and an application rate of 5g / m². The rotary tillage depth is 8cm. The surface charge of the micro-aggregates is positive, which is achieved by adding chitosan quaternary ammonium salt to the modified sodium alginate solution. The concentration of chitosan quaternary ammonium salt is 0.05%.

[0047] S2. Prepare a three-layered bacterial agent. The three-layered bacterial agent consists of an inner core layer, a shell layer, and a canopy layer from the inside out. The inner core layer includes a slow-release carbon source material, the shell layer includes a composite functional bacterial community and a gel material, and the canopy layer includes a water-retaining material and microbial signaling molecules. The core layer is prepared as follows: soluble starch, humic acid, and microcrystalline cellulose are mixed in a mass ratio of 3:2:2. In this embodiment, the soluble starch is 30g, the humic acid is 30g, and the microcrystalline cellulose is 30g. Water is added to make a slurry with a solid content of 25%, which is then extruded and spherically rolled to form spherical cores with a particle size of 150μm, and dried until the moisture content is <8%. The shell layer is prepared as follows: the composite functional bacterial flora is mixed with sodium alginate solution, calcium citrate microcrystals are added and mixed evenly, and then coated onto the surface of the core layer by a spray coating process to form a shell layer with a thickness of 30μm, which is then allowed to solidify. The composite functional bacterial flora includes Bacillus amyloliquefaciens, Bacillus colloidis, and Pseudomonas fluorescens, all with a bacterial concentration of 10%. 9CFU / mL, mixed at a volume ratio of 0.8:0.8:0.8. In this embodiment, 80mL of Bacillus amyloliquefaciens bacterial solution, 80mL of Bacillus lentigines bacterial solution, and 80mL of Pseudomonas fluorescens bacterial solution were taken and mixed evenly to obtain 240mL of composite functional bacterial mixture. The concentration of sodium alginate solution was 1.2%, and the mixing volume ratio of bacterial solution to sodium alginate solution was 1:3. In this embodiment, 100mL of composite functional bacterial mixture was taken and 300mL of sodium alginate solution was added and mixed evenly to obtain 400mL of coating slurry. The particle size of calcium citrate microcrystals was <10μm, and the addition amount was 0.1% of the total mass of composite functional bacterial mixture and sodium alginate solution. The static curing temperature was 25℃, the relative humidity was 55%, and the time was 1.5 hours. The canopy was prepared by spraying a composite functional solution onto the surface of the shell to form a canopy with a thickness of 5 μm. The spraying amount was 0.5 mL / g of bacterial agent. The composite functional solution included 0.15% γ-polyglutamic acid, 8 mM γ-aminobutyric acid, 15 mM xylose, 4 mM sodium lactate, and 0.05% sodium alginate.

[0048] S3. Form a sowing furrow in the soil after rotary tillage, and apply the three-layer structured microbial agent into the sowing furrow. The surface of the three-layer structured microbial agent has a charge opposite to that on the surface of the micro-aggregates, and the microbial agent is attached to the surface of the micro-aggregates through electrostatic adsorption. The application rate of the three-layer structure microbial agent is 20g / m², and the application depth is 5cm in the sowing furrow. The surface of the three-layer structure microbial agent has a negative charge, which is achieved by adding sodium alginate to the composite functional solution of the canopy.

[0049] S4. After sowing in the sowing furrow after applying the three-layer structure microbial agent, irrigate. Irrigation is divided into a first stage and a second stage. The irrigation water in the first stage is acidic, and the irrigation water in the second stage is neutral or weakly alkaline.

[0050] The irrigation method is a single-stage micro-sprinkler irrigation, with a total irrigation volume of 12-15 mm. The first stage irrigation lasts 0-2 hours, with the irrigation water pH at 5.0, accounting for 30% of the total irrigation volume. The second stage irrigation lasts 2-6 hours, with the irrigation water pH at 6.5, accounting for 60% of the total irrigation volume. In the first stage, the pH of the irrigation water is adjusted by adding citric acid at a concentration of 0.1 g / L. During the second stage irrigation, the calcium carbonate powder reacts with residual hydrogen ions in the soil, causing the soil pH to return to neutral. The sowing depth is 2 cm, and the topsoil moisture content after irrigation is 70% of field capacity.

[0051] Example 3: This example differs from Example 1 above in that: A method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas includes the following steps: S1. Spray modified sodium alginate solution onto the surface soil of newly cultivated land, while simultaneously applying calcium source and pH buffer, and perform rotary tillage to cause in-situ ionic cross-linking reaction on the surface of soil particles, forming water-stable micro-aggregates, and assigning charges to the surface of the micro-aggregates. The modified sodium alginate is a blend of sodium alginate and polyethylene glycol, with a mass ratio of sodium alginate to polyethylene glycol of 6:1. In this embodiment, 60g of sodium alginate and 10g of polyethylene glycol are used. The concentration of the modified sodium alginate solution is 1.2%. The calcium source is calcium gluconate, applied at a rate of 15g / m². The pH buffer is calcium carbonate micropowder with a particle size of 30μm, applied at a rate of 8g / m². The rotary tillage depth is 10cm. The surface charge of the micro-aggregates is positive, achieved by adding chitosan quaternary ammonium salt to the modified sodium alginate solution. The concentration of chitosan quaternary ammonium salt is 0.1%.

[0052] S2. Prepare a three-layered bacterial agent. The three-layered bacterial agent consists of an inner core layer, a shell layer, and a canopy layer from the inside out. The inner core layer includes a slow-release carbon source material, the shell layer includes a composite functional bacterial community and a gel material, and the canopy layer includes a water-retaining material and microbial signaling molecules. The core layer is prepared as follows: soluble starch, humic acid, and microcrystalline cellulose are mixed in a mass ratio of 5:4:4. In this embodiment, the soluble starch content is 50g, the humic acid content is 40g, and the microcrystalline cellulose content is 40g. Water is added to form a slurry with a solid content of 35%. This slurry is then extruded and spherically rounded to form spherical cores with a particle size of 250μm, and dried until the moisture content is <8%. The shell layer is prepared as follows: a composite functional bacterial group is mixed with a sodium alginate solution, calcium citrate microcrystals are added and mixed evenly, and then coated onto the surface of the core layer using a spray coating process to form a shell layer with a thickness of 50μm. The mixture is then allowed to stand and solidify. The composite functional bacterial group includes *Bacillus amyloliquefaciens*, *Bacillus colloidea*, and *Pseudomonas fluorescens*, all with a bacterial concentration of 10%. 9CFU / mL, mixed at a volume ratio of 1.2:1.2:1.2. In this embodiment, 120mL of Bacillus amyloliquefaciens bacterial solution, 120mL of Bacillus lentigines bacterial solution, and 120mL of Pseudomonas fluorescens bacterial solution were taken and mixed evenly to obtain 360mL of composite functional bacterial mixture. The concentration of sodium alginate solution was 1.8%, and the mixing volume ratio of bacterial solution to sodium alginate solution was 1:5. In this embodiment, 100mL of composite functional bacterial mixture was taken and 500mL of sodium alginate solution was added and mixed evenly to obtain 600mL of coating slurry. The particle size of calcium citrate microcrystals was <10μm, and the addition amount was 0.3% of the total mass of composite functional bacterial mixture and sodium alginate solution. The static curing temperature was 30℃, the relative humidity was 65%, and the time was 2.5 hours. The canopy was prepared by spraying a composite functional solution onto the surface of the shell to form a canopy with a thickness of 10 μm. The spraying amount was 1 mL / g of bacterial agent. The composite functional solution included 0.25% γ-polyglutamic acid, 12 mM γ-aminobutyric acid, 25 mM xylose, 6 mM sodium lactate, and 0.15% sodium alginate.

[0053] S3. Form a sowing furrow in the soil after rotary tillage, and apply the three-layer structured microbial agent into the sowing furrow. The surface of the three-layer structured microbial agent has a charge opposite to that on the surface of the micro-aggregates, and the microbial agent is attached to the surface of the micro-aggregates through electrostatic adsorption. The application rate of the three-layer structure microbial agent is 20-30 g / m², and the application depth is 5-8 cm in the sowing furrow. The surface of the three-layer structure microbial agent carries a negative charge, which is achieved by adding sodium alginate to the composite functional solution of the canopy.

[0054] S4. After sowing in the sowing furrow after applying the three-layer structure microbial agent, irrigate. Irrigation is divided into a first stage and a second stage. The irrigation water in the first stage is acidic, and the irrigation water in the second stage is neutral or weakly alkaline.

[0055] The irrigation was a single-stage micro-sprinkler irrigation, with a total irrigation volume of 15 mm. The first stage of irrigation lasted 0-2 hours, with the irrigation water having a pH of 5.5 and accounting for 40% of the total irrigation volume. The second stage of irrigation lasted 2-6 hours, with the irrigation water having a pH of 7.0 and accounting for 70% of the total irrigation volume. In the first stage, the pH of the irrigation water was adjusted by adding citric acid at a concentration of 0.2 g / L. During the second stage of irrigation, the calcium carbonate powder reacted with residual hydrogen ions in the soil, causing the soil pH to return to neutral. The sowing depth was 3 cm, and the topsoil moisture content after irrigation was 80% of field capacity.

[0056] Comparative Example 1: Method for soil microbial colonization on newly reclaimed farmland in dryland areas, including the following steps: Before sowing, the bacterial solution concentration was 10. 8Bacillus subtilis inoculum of CFU / mL was mixed with well-rotted cow manure at a mass ratio of 1:10 to make microbial fertilizer. When sowing, mix the microbial fertilizer with fine dry soil at a mass ratio of 1:5, and then spread it evenly in the sowing furrow. The application rate is 50g / m² of microbial fertilizer. After sowing, cover with soil and then flood irrigate with a water volume of 25 mm to bring the soil moisture content of the topsoil to 80% of field capacity.

[0057] Comparative Example 2: This comparative example differs from Example 1 above in that: In step S1, no chitosan quaternary ammonium salt is added to the modified sodium alginate solution, and no positive charge is imparted to the surface of the micro-aggregates.

[0058] The rest is the same as in Example 1.

[0059] Comparative Example 3: This comparative example differs from Example 1 above in that: In step S2, instead of preparing a three-layered bacterial agent, the composite functional bacterial group is physically mixed with soluble starch and humic acid at a mass ratio of 1:2:2 and then directly applied as a bacterial agent. The composite functional bacterial group consists of Bacillus amyloliquefaciens, Bacillus colloidis, and Pseudomonas fluorescens, with a bacterial concentration of 10%. 9 CFU / mL, mixed at a volume ratio of 1:1:1.

[0060] The rest is the same as in Example 1.

[0061] Comparative Example 4: This comparative example differs from Example 1 above in that: In step S4, irrigation is carried out without stages, and a one-time micro-sprinkler irrigation is performed directly. The total irrigation volume is 13.5 mm, the pH of the irrigation water is 7.0, and no citric acid is added to adjust the pH.

[0062] The rest is the same as in Example 1.

[0063] Comparative Example 5: This comparative example differs from Example 1 above in that: In step S2, the composite functional solution of the canopy does not contain γ-aminobutyric acid, xylose and sodium lactate, but only contains 0.2% γ-polyglutamic acid and 0.1% sodium alginate.

[0064] The rest is the same as in Example 1.

[0065] Performance testing: The following performance tests were conducted on the rapid colonization methods for soil microorganisms in newly reclaimed dryland farmland prepared in Examples 1-3 and Comparative Examples 1-5 of this application: Soil microbial colonization rate determination: Rhizosphere soil samples (0-20cm) were collected at 7, 30 and 60 days after treatment. The gene copy number of Bacillus amyloliquefaciens, Bacillus mucilaginosus and Pseudomonas fluorescens in the soil was detected by qPCR. The colonization rate (%) was expressed as the percentage of the target bacterial group to the initial bacterial group. Soil aggregate stability determination: Soil samples (0-15cm) were collected 60 days after treatment. The average weight diameter (MWD) of water-stable soil aggregates was determined by wet sieving. The higher the MWD value, the higher the aggregate stability. Crop emergence rate and biomass determination: Crop emergence rate was recorded 15 days after sowing; aboveground parts of plants were collected 60 days after sowing, dried at 80℃ to constant weight, and aboveground biomass (g / plant) was determined. The test results are shown in Table 1.

[0066] Table 1

[0067] As can be seen from Examples 1 to 3 and Comparative Examples 1 to 5, and Table 1, this application constructs a stable microenvironment for exogenous microorganisms through the synergistic effect of in-situ soil cross-linking granulation, three-layer structured microbial agents, electrostatic adsorption anchoring, and staged pH gradient irrigation. This enables exogenous microorganisms to quickly establish dominant populations in newly reclaimed farmland in dryland areas, while promoting the formation of soil aggregates and crop growth.

[0068] As can be seen from Example 1 and Comparative Example 2, and Table 1, the charge imparting on the surface of the micro-aggregates enables the microbial agent to achieve spatial matching with the soil. Through electrostatic adsorption, the microbial agent is firmly attached to the surface of the micro-aggregates, preventing the microbial agent from being lost with water and allowing the microbial agent to continue to play a role at the colony site.

[0069] As can be seen from Example 1 and Comparative Example 3, and Table 1, the synergistic design of the three-layered bacterial agent—with its core slow-release carbon source, shell gel protection, canopy water retention, and signal induction—provides continuous nutrient supply and physical protection for the bacterial community, enabling it to survive and maintain its activity for a long time under alternating dry and wet stress.

[0070] As can be seen from Example 1 and Comparative Example 4, and Table 1, the phased pH gradient irrigation achieves the timing matching of microbial agent release with soil moisture conditions. The acidic phase triggers the dissociation of the microbial agent shell and initiates the release of microbial communities, while the neutral phase protects the micro-aggregate structure, so that the peak of microbial agent release is synchronized with the soil moisture saturation period.

[0071] As can be seen from Example 1 and Comparative Example 5, and Table 1, the introduction of signaling molecules in the canopy can activate the colonization ability of the microbial community. By inducing the chemotaxis and metabolic activity of the microbial community, the microbial community can quickly migrate to the rhizosphere and occupy the ecological niche in the early stage of colonization.

[0072] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas, characterized in that, Includes the following steps: S1. Spray modified sodium alginate solution onto the surface soil of newly cultivated land, while simultaneously applying calcium source and pH buffer, and perform rotary tillage to cause in-situ ionic cross-linking reaction on the surface of soil particles, forming water-stable micro-aggregates, and assigning charges to the surface of the micro-aggregates. S2. Prepare a three-layered bacterial agent, wherein the three-layered bacterial agent consists of a core layer, a shell layer and a canopy layer from the inside out. The core layer includes a slow-release carbon source material, the shell layer includes a composite functional bacterial community and a gel material, and the canopy layer includes a water-retaining material and microbial signaling molecules. S3. A sowing furrow is formed in the soil after rotary tillage. The three-layer structured microbial agent is applied to the sowing furrow. The surface of the three-layer structured microbial agent has a charge opposite to that on the surface of the micro-aggregates. The microbial agent is attached to the surface of the micro-aggregates through electrostatic adsorption. S4. After sowing in the sowing furrow after applying the three-layer structure microbial agent, irrigation is carried out. The irrigation is divided into a first stage and a second stage. The irrigation water in the first stage is acidic, and the irrigation water in the second stage is neutral or weakly alkaline.

2. The method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas according to claim 1, characterized in that: In step S1, the modified sodium alginate is a blend of sodium alginate and polyethylene glycol, with a mass ratio of sodium alginate to polyethylene glycol of 4-6:

1. The concentration of the modified sodium alginate solution is 0.8%-1.2%. The calcium source is calcium gluconate, applied at a rate of 10-15 g / m². The pH buffer is calcium carbonate micropowder with a particle size of 10-30 μm, applied at a rate of 5-8 g / m². The rotary tillage depth is 8-10 cm. The surface charge of the micro-aggregates is positive, achieved by adding chitosan quaternary ammonium salt to the modified sodium alginate solution, with a concentration of 0.05%-0.1%.

3. The method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas according to claim 1, characterized in that: In step S2, the preparation method of the core layer is as follows: soluble starch, humic acid and microcrystalline cellulose are mixed in a mass ratio of 3-5:2-4:2-4, water is added to make a slurry with a solid content of 25%-35%, and spherical cores with a particle size of 150-250μm are made by extrusion and sphericalization process, and dried to a moisture content of <8%.

4. The method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas according to claim 1, characterized in that: In step S2, the shell layer is prepared by mixing the composite functional bacterial group with sodium alginate solution, adding calcium citrate microcrystals and mixing evenly, coating it onto the core surface through a spray coating process to form a shell layer with a thickness of 30-50 μm, and then allowing it to stand and solidify.

5. A method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas according to claim 4, characterized in that: The composite functional bacterial community includes *Bacillus amyloliquefaciens*, *Bacillus mucilaginosus*, and *Pseudomonas fluorescens*, all with a bacterial concentration of 10%. 9 The bacterial culture is mixed at a volume ratio of 0.8-1.2:0.8-1.2:0.8-1.2, with a sodium alginate solution concentration of 1.2%-1.8% and a volume ratio of 1:3-5 between the bacterial culture and the sodium alginate solution. The calcium citrate microcrystals have a particle size of <10μm and are added at a rate of 0.1%-0.3% of the total mass of the mixture of the compound functional bacterial culture and the sodium alginate solution. The static curing temperature is 25-30℃, the relative humidity is 55%-65%, and the time is 1.5-2.5 hours.

6. The method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas according to claim 1, characterized in that: In step S2, the canopy is prepared by spraying a composite functional solution onto the surface of the shell to form a canopy with a thickness of 5-10 μm. The spraying amount is 0.5-1 mL / g of bacterial agent. The composite functional solution includes 0.15%-0.25% γ-polyglutamic acid, 8-12 mM γ-aminobutyric acid, 15-25 mM xylose, 4-6 mM sodium lactate, and 0.05%-0.15% sodium alginate.

7. The method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas according to claim 1, characterized in that: In step S3, the application rate of the three-layer structure microbial agent is 20-30 g / m², and the application depth is 5-8 cm in the sowing furrow. The surface of the three-layer structure microbial agent carries a negative charge, which is achieved by adding sodium alginate to the composite functional solution of the canopy.

8. A method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas according to claim 1, characterized in that: In step S4, the irrigation is a one-time micro-sprinkler irrigation with a total irrigation volume of 12-15 mm. The irrigation time in the first stage is 0-2 hours, the pH of the irrigation water is 5.0-5.5, and the irrigation water volume accounts for 30%-40% of the total irrigation volume. The irrigation time in the second stage is 2-6 hours, the pH of the irrigation water is 6.5-7.0, and the irrigation water volume accounts for 60%-70% of the total irrigation volume.

9. A method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas according to claim 1, characterized in that: In step S4, the pH of the irrigation water in the first stage is adjusted by adding citric acid, and the amount of citric acid added is 0.1-0.2 g / L. In the second stage of irrigation, the calcium carbonate powder reacts with the residual hydrogen ions in the soil, so that the soil pH returns to neutral.

10. A method for rapid colonization of soil microorganisms on newly reclaimed farmland in dryland areas according to claim 1, characterized in that: In step S4, the soil covering depth after sowing is 2-3 cm, and the soil moisture content of the topsoil after irrigation is 70%-80% of field capacity.

Images