A complex microbial agent for promoting growth and activating rhizosphere phosphorus of rice and application thereof

Abstract

This invention relates to the field of microbial fermentation engineering technology, specifically to a compound microbial agent for promoting rice growth and activating rhizosphere phosphorus, and its application. This agent is a microcapsule solid formulation composed of Bacillus phosphate-solubilizing spore powder, concentrated freeze-dried Pseudomonas alginate, a cross-linked wall material with modified starch, and a carrier filler. The technical solution involves using the core components of rice root exudates as a limiting carbon source to induce Bacillus to form spores and express chemotactic receptors. This is then compounded with microfiltration-concentrated freeze-dried Pseudomonas alginate, encapsulated in the cross-linked wall material, and spray-dried at low temperature. This solution overcomes the problems of low survival rate and inability to directionally colonize existing liquid agents in flooded and oxygen-deficient environments. It utilizes the spore state combined with a microcapsule barrier to improve survival rate and relies on induced receptors to achieve active chemotactic colonization in the rhizosphere.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation engineering technology, specifically to a compound microbial agent for promoting rice growth and activating rhizosphere phosphorus, and its application. Background Technology

[0002] Currently, compound microbial preparations for promoting rice growth and activating rhizosphere phosphorus are mostly prepared using liquid fermentation technology. The conventional process involves inoculating *Bacillus phosphate-solubilizing* and *Pseudomonas aeruginosa* in the same fermenter containing conventional carbon and nitrogen sources for mixed liquid fermentation. After fermentation, no morphological transformation or drying is performed; the fermentation broth containing live bacteria is directly packaged as a liquid inoculant. In field application, this liquid inoculant is directly sprinkled onto the surface of the paddy field or applied to the soil with irrigation water.

[0003] When conventional liquid bacterial agents are directly applied to rice paddies, the long-term flooded and oxygen-deficient environment during the rice growth cycle causes drastic changes in osmotic pressure and extreme hypoxia in the nutrient-containing microorganisms within the liquid fermentation system, leading to cell rupture and death. The conventional carbon and nitrogen sources used in the liquid fermentation process cannot induce microorganisms to develop stress-resistant structures to cope with flooded environments. Furthermore, the microorganisms, without root-specific induction, lack the mechanism to recognize and respond to signals from the rice rhizosphere. Consequently, the applied live bacteria cannot move directionally towards the rice roots and can only passively suspend in the water layer, being lost during drainage or infiltration, thus failing to establish a stable live bacterial community in the rice rhizosphere. Summary of the Invention

[0004] To address the shortcomings of existing liquid microbial agents for promoting rice growth and activating rhizosphere phosphorus, such as poor bacterial resistance, inability to colonize the rice rhizosphere, low survival rate of live bacteria, and easy loss with water, this invention provides a composite microbial agent for promoting rice growth and activating rhizosphere phosphorus, and its application.

[0005] To address the aforementioned technical problems, this invention provides a composite microbial agent for promoting rice growth and activating rhizosphere phosphorus. The composite microbial agent is a microcapsule solid preparation, composed of the following components by weight percentage: 10%-20% Bacillus phosphate-solubilizing spore powder, 5%-10% freeze-dried concentrated Pseudomonas aeruginosa cell powder, 10%-20% sodium alginate and modified starch cross-linked wall material, and 50%-75% carrier filler. The Bacillus phosphate-solubilizing spore powder is a dried spore-forming material formed after induction culture of core components from rice root exudates. The freeze-dried concentrated Pseudomonas aeruginosa cell powder is a dried cell material after microfiltration concentration treatment. In the sodium alginate and modified starch cross-linked wall material, the mass ratio of sodium alginate to modified starch is 1:2 to 1:4. The carrier filler is a mixture of diatomaceous earth, rice husk powder, and bentonite, with a mass ratio of diatomaceous earth, rice husk powder, and bentonite of 2:1:1.

[0006] In practice, this composite microbial agent employs a microcapsule structure, encapsulating Bacillus phosphate-solubilizing spores and Pseudomonas aeruginosa within a three-dimensional gel network formed by the cross-linking of sodium alginate and modified starch. Bacillus phosphate-solubilizing spores induced by rice root exudates possess a thick, dense peptidoglycan outer wall and extremely low metabolic activity, enabling them to withstand osmotic pressure fluctuations and hypoxia stress in flooded environments. After microfiltration concentration and freeze-drying, the Pseudomonas aeruginosa retains its intact cell structure and remains dormant. When the microcapsule particles are applied to the paddy field, the wall material slowly swells and degrades under the influence of soil moisture and root exudates, gradually releasing the internal bacteria. The released Bacillus phosphate-solubilizing spores can recognize signaling substances secreted by rice roots, migrate directionally to the rhizosphere through chemotaxis, and germinate into vegetative cells, secreting organic acids to dissolve insoluble phosphates in the soil. Pseudomonas aeruginosa, after colonizing the rhizosphere, produces auxins, gibberellins, and other plant growth regulators, promoting rice root development and nutrient absorption.

[0007] Furthermore, in the above technical solution, the core components of the rice root exudate include organic acids and glycosides, wherein the organic acids are a mixture of tartaric acid and malic acid, and the glycosides are sophorose; in the preparation process of the Bacillus phosphate-solubilizing spore powder, the core components of the rice root exudate are added as a limiting carbon source to the second-stage fermentation broth of Bacillus phosphate-solubilizing, and the total mass concentration of the organic acids and glycosides in the second-stage fermentation broth is controlled to be 0.5 g / L-2 g / L.

[0008] In practice, adding the core components of rice root exudates as a limiting carbon source to the second-stage fermentation broth causes carbon starvation stress in Bacillus phosphate-solubilizing, triggering a cascade regulatory system for sporulation and promoting the transformation of the bacteria from the vegetative growth stage to the sporulation stage. Simultaneously, these root exudate components can induce Bacillus phosphate-solubilizing to express specific chemokine receptor proteins, enabling the bacteria to recognize and respond to rice rhizosphere signals, thus allowing them to actively migrate and colonize the rice root system after field application.

[0009] Furthermore, in the above technical solution, the modified starch is cross-linked starch that has been cross-linked with propylene oxide and has a degree of substitution of 0.1-0.2; the preparation process of the sodium alginate and modified starch cross-linked wall material is as follows: after mixing the sodium alginate aqueous solution and the modified starch gelatinized liquid, calcium chloride solution is added to carry out calcium ion cross-linking reaction to form a gel network, the mass concentration of the calcium chloride solution is 1%-3%, the temperature of the calcium ion cross-linking reaction is 40℃-50℃, and the time of the calcium ion cross-linking reaction is 30min-60min.

[0010] In practice, modified starch with a degree of substitution of 0.1-0.2, after being cross-linked with propylene oxide, exhibits good film-forming properties and mechanical strength. When combined with sodium alginate, calcium ions undergo an ionic cross-linking reaction with guluronic acid residues on the sodium alginate molecular chain, forming a three-dimensional gel network with an egg-box structure. This network structure combines the biocompatibility and sustained-release properties of sodium alginate with the water resistance and mechanical stability of modified starch, effectively protecting the internal bacterial cells from external environmental damage while controlling the release rate of the cells.

[0011] Furthermore, in the above technical solution, in the preparation process of the concentrated Pseudomonas aeruginosa lyophilized powder, a ceramic microfiltration membrane with a pore size of 0.1μm-0.2μm is used to concentrate and retain the Pseudomonas aeruginosa fermentation broth. The operating pressure of the ceramic microfiltration membrane is 0.1MPa-0.3MPa, the temperature of the fermentation broth is maintained at 4℃-10℃ during the concentration and retention process, and the volume concentration factor of the fermentation broth at the end of the concentration is 8-12 times. The concentrated retentate is then freeze-dried under vacuum to obtain the concentrated Pseudomonas aeruginosa lyophilized powder.

[0012] In practice, ceramic microfiltration membranes with pore sizes of 0.1μm-0.2μm are used to efficiently retain *Pseudomonas aeruginosa* cells while allowing small-molecule metabolic waste and culture medium components in the fermentation broth to pass through, thus achieving cell concentration and purification. Concentration is performed at low temperatures of 4℃-10℃ to reduce the metabolic activity of the cells and minimize cell damage; vacuum freeze-drying removes water from the cells through sublimation, putting the cells into a dormant state and maximizing their biological activity.

[0013] Furthermore, in the above technical solution, the microcapsule solid formulation is prepared by a low-temperature spray drying process. The inlet air temperature of the low-temperature spray drying is 100℃-120℃, the outlet air temperature is 45℃-55℃, the feed rate is 5mL / min-10mL / min, and the atomizer frequency of the low-temperature spray drying is 20Hz-30Hz. Before the low-temperature spray drying, the Bacillus spore powder, the concentrated freeze-dried Pseudomonas alginate, and the aqueous solution of sodium alginate and modified starch crosslinking wall material are mixed to form a homogeneous suspension with a total solids concentration of 15%-20%.

[0014] In practice, the low-temperature spray drying process rapidly removes moisture from the suspension at a lower temperature, avoiding the damage to bacterial activity caused by high temperatures. By controlling parameters such as inlet air temperature, outlet air temperature, feed rate, and atomizer frequency, microcapsule particles with uniform particle size and low moisture content can be prepared. Pre-mixing the bacterial powder with the wall material aqueous solution to form a homogeneous suspension ensures that the bacteria are uniformly dispersed in the wall material network, avoiding excessively high or low local bacterial concentrations and guaranteeing the stability of the microcapsule product quality.

[0015] Furthermore, in the above technical solution, in the first stage fermentation for preparing the Bacillus spore powder, the first stage fermentation medium contains, by mass-volume ratio: 1%-2% soluble starch, 0.5%-1% yeast extract, 0.5%-1% peptone, 0.02%-0.05% magnesium sulfate, and 0.1%-0.2% dipotassium hydrogen phosphate. The initial pH of the first stage fermentation medium is 7.0-7.2, the fermentation temperature is 30℃-32℃, the stirring speed is 200rpm-250rpm, and the fermentation time is 18h-24h.

[0016] In practice, the first stage of fermentation uses a nutrient-rich culture medium to provide sufficient carbon, nitrogen, and inorganic salts for the rapid growth and reproduction of Bacillus phosphate-solubilizing, enabling the cells to reach a high biomass in a short time. Controlling appropriate pH, temperature, stirring speed, and culture time creates optimal conditions for the vegetative growth of Bacillus phosphate-solubilizing, ensuring the cells are in the late logarithmic growth phase, thus preparing for the second stage of spore induction.

[0017] Furthermore, in the above technical solution, the diatomaceous earth in the carrier filler undergoes acid washing and high-temperature calcination pretreatment. The acid washing involves soaking in a 5% hydrochloric acid solution for 2 hours and then washing with water until neutral. The high-temperature calcination temperature is 400℃-500℃, and the calcination time is 2-3 hours. The rice husk powder has a particle size passing through an 80-100 mesh standard sieve. The bentonite is sodium-based bentonite. Before participating in the preparation of the microcapsule solid formulation, the carrier filler is premixed with an aqueous solution of sodium alginate and modified starch crosslinking wall material and subjected to ultrasonic emulsification treatment. The ultrasonic emulsification power is 300W-500W, and the ultrasonic time is 10-15 minutes.

[0018] In practice, acid washing and high-temperature calcination pretreatment remove impurities and organic matter from diatomaceous earth, increasing its porosity and specific surface area, and improving its adsorption performance. Rice husk powder (80-100 mesh) exhibits good dispersibility and adsorption properties, while sodium-based bentonite possesses excellent suspension and binding properties. Mixing these three substances in a specific ratio as a carrier filler allows for adjustment of the microcapsule bulk density and improved dispersibility in soil and water. Ultrasonic emulsification ensures the carrier filler is uniformly dispersed in the wall material aqueous solution, preventing particle agglomeration and guaranteeing the uniformity of the microcapsule structure.

[0019] Furthermore, in the above technical solution, before forming the microcapsule solid formulation, the Bacillus spore powder and the Pseudomonas aeruginosa concentrated lyophilized bacterial powder are compounded at a live bacteria ratio of 2:1 to 3:1, and skim milk powder is added as a lyophilization protectant during the compounding process. The mass fraction of skim milk powder in the compounding system is 3%-5%. The compounded mixed bacterial powder and the dry weight of the sodium alginate and modified starch crosslinked wall material are then encapsulated a second time at a mass ratio of 1:1 to 1:1.5.

[0020] In practice, a live bacteria ratio of 2:1 to 3:1 is used to combine Bacillus phosphate-solubilizing and Pseudomonas aeruginosa, enabling the two bacteria to work synergistically in the rhizosphere. Bacillus phosphate-solubilizing is responsible for activating soil phosphorus, while Pseudomonas aeruginosa promotes rice growth. Skim milk powder, acting as a freeze-drying protectant, forms a protective film on the bacterial surface, preventing damage to cell structure from ice crystals during freeze-drying. The secondary encapsulation process further enhances the protective effect of the microcapsules on the bacteria, improving their survival rate during storage, transportation, and application.

[0021] Furthermore, in the above technical solution, the particle size distribution of the microcapsule solid formulation is 50μm-150μm, the moisture content of the microcapsule solid formulation is controlled below 5%, and the bulk density of the microcapsule solid formulation is 0.4g / mL-0.6g / mL; a hydrophobic isolation layer formed of magnesium stearate is coated on the surface of the microcapsule solid formulation particles, and the coating amount of magnesium stearate is 0.5%-1% of the total weight of the microcapsule solid formulation.

[0022] In practice, controlling the particle size of the microcapsule particles within the range of 50μm-150μm ensures good dispersibility and suspension in soil and water; controlling the moisture content below 5% inhibits microbial metabolic activity and extends the product's shelf life; and maintaining a bulk density of 0.4g / mL-0.6g / mL facilitates product transportation and application. Coating the surface of the microcapsule particles with a hydrophobic magnesium stearate layer blocks external moisture from penetrating into the microcapsules, preventing premature bacterial activation, while also improving the flowability of the microcapsule particles for easier mechanical application.

[0023] Furthermore, this invention also provides an application of a compound microbial agent for promoting rice growth and activating rhizosphere phosphorus. The compound microbial agent described above is applied as basal fertilizer in rice transplanted fields or as topdressing during the tillering stage. Specifically, when applying basal fertilizer 7-10 days before rice transplanting, the compound microbial agent is mixed evenly with fine soil at a mass ratio of 1:50 and then spread on the surface of the cultivated layer of the rice field. The field is then tilled. The application rate of the compound microbial agent is 2-4 kg / mu. When applying topdressing during the rice tillering stage, the compound microbial agent is dissolved in the paddy field water and sprayed evenly on the leaves and water surface using a drone. The mass concentration of the compound microbial agent in the spray solution is 0.5%-1%, and the spray solution volume per mu of rice field is 30-50 L. The topdressing is applied within 10-15 days after rice transplanting.

[0024] In practice, when applying base fertilizer, the microbial agent is mixed with fine soil, spread, and then tilled. This ensures the agent is evenly distributed in the tillage layer where rice roots are mainly distributed, creating a favorable soil environment for the colonization and reproduction of the microorganisms. Topdressing during the tillering stage utilizes drone-based aerial spraying, improving application efficiency and uniformity, allowing the agent to act on both rice leaves and the water surface simultaneously, promoting tillering and root growth. Applying the agent at two key times—7-10 days before transplanting and 10-15 days after transplanting—maximizes its growth-promoting and phosphorus-activating effects during these critical growth stages.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention employs a two-stage fermentation system. After the propagation of Bacillus phosphate-solubilizing bacteria, the core components of root exudates are added as a limiting carbon source, inducing the bacteria to form a spore state with stress resistance and express specific chemotactic receptors. Simultaneously, the Pseudomonas fermentation broth is microfiltered and concentrated, and then encapsulated together with the induced spore powder in a wall material network formed by cross-linking sodium alginate and modified starch. This is then spray-dried at low temperature to produce a microcapsule solid formulation. This formulation structure protects the bacteria from the direct impact of osmotic pressure changes when in contact with flooded layers. The spore morphology, combined with the physical barrier of the microcapsules, resists the anoxic environment of flooded conditions, improving survival rates. The induced chemotactic receptors enable Bacillus to actively recognize and approach rice root exudates in field water, achieving colonization in the rhizosphere.

[0026] 2. In the preparation of the wall material, this invention utilizes cross-linked starch with a specific degree of substitution and sodium alginate to undergo a calcium ion cross-linking reaction to form a dense gel network. Combined with secondary encapsulation by a skim milk powder freeze-drying protectant and a hydrophobic magnesium stearate barrier layer on the particle surface, this reduces the water content of the microcapsule particles, preventing external moisture penetration and maintaining bacterial activity during storage and transportation. The carrier filler undergoes acid washing and calcination pretreatment and ultrasonic emulsification dispersion in the wall material aqueous solution. This improves the suspension uniformity of the microcapsules during soil mixing with base fertilizer and dissolution during tillering and aerial spraying, preventing particle sedimentation and aggregation, and ensuring uniform distribution and effective release of live bacteria in the application medium. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments. Those skilled in the art can reproduce the technical solution of the present invention and achieve its claimed technical effects based on the content disclosed in this specification. It should be noted that the following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. Any non-substantial improvements and adjustments made based on the core concept of the present invention should fall within the scope of protection of the present invention.

[0028] Example 1: This example provides a compound microbial agent for promoting rice growth and activating rhizosphere phosphorus, which is composed of the following components by weight percentage: 15% Bacillus phosphate-solubilizing spore powder, 7.5% concentrated freeze-dried Pseudomonas aeruginosa bacterial concentrate, 15% sodium alginate and modified starch cross-linked wall material, and 62.5% carrier filler.

[0029] The preparation method is as follows: Preparation of Bacillus phosphate-solubilizing spore powder 1.1 First-stage fermentation: Prepare the first-stage fermentation medium, which contains 1.5% soluble starch, 0.75% yeast extract, 0.75% peptone, 0.035% magnesium sulfate, and 0.15% dipotassium hydrogen phosphate by weight-volume ratio, and adjust the initial pH to 7.1. Inoculate the Bacillus phosphate-solubilizing seed culture into the above medium at an inoculation rate of 5%, and incubate at 31℃ with a stirring speed of 225 rpm for 21 hours.

[0030] 1.2 Second-stage induced fermentation: Rice root exudate core components, composed of tartaric acid, malic acid, and sophorose in a mass ratio of 1:1:1, were added to the first-stage fermentation broth as a limiting carbon source. The total mass concentration of these components in the second-stage fermentation broth was controlled at 1.25 g / L. The mixture was cultured for another 12 hours until the spore formation rate reached over 95%.

[0031] 1.3 Collection and drying: The fermentation broth was centrifuged to collect the spores, washed twice with sterile physiological saline, and dried with hot air until the moisture content was less than 5% to obtain Bacillus phosphate-solubilizing spore powder.

[0032] Preparation of concentrated freeze-dried Pseudomonas aeruginosa cells The fermentation broth of *Pseudomonas aeruginosa* was concentrated and retained using a ceramic microfiltration membrane with a pore size of 0.15 μm. The operating pressure was 0.2 MPa, and the fermentation broth temperature was maintained at 7 °C until the volume concentration factor reached 10 times. The concentrated retentate was then freeze-dried under vacuum to obtain concentrated freeze-dried *Pseudomonas aeruginosa* cell powder.

[0033] Preparation of cross-linked wall material of sodium alginate and modified starch Cross-linked starch with a degree of substitution of 0.15 and treated with propylene oxide was used. Sodium alginate and modified starch were prepared into a 2% aqueous solution and a 5% gelatinized solution at a mass ratio of 1:3. After being mixed evenly, a 2% calcium chloride solution was added, and a calcium ion cross-linking reaction was carried out at 45°C for 45 min to form a gel network wall material aqueous solution.

[0034] Pretreatment and dispersion of carrier fillers The carrier filler is composed of diatomaceous earth, rice husk powder, and sodium bentonite in a mass ratio of 2:1:1. The diatomaceous earth is soaked in a 5% hydrochloric acid solution for 2 hours, washed with water until neutral, and then calcined at 450℃ for 2.5 hours. The rice husk powder is passed through a 90-mesh standard sieve. The pretreated carrier filler is added to the above-mentioned wall material aqueous solution and ultrasonically emulsified at 400W power for 12 minutes to form a uniform dispersion.

[0035] Microbial powder compounding and secondary coating Bacillus spore powder and concentrated lyophilized Pseudomonas aeruginosa bacterial cells were compounded at a live cell ratio of 2.5:1, and 4% (w / w) of skim milk powder was added as a lyophilization protectant. The mixture was then thoroughly mixed. The compounded bacterial powder and the dry weight of the wall material were added to the above dispersion at a mass ratio of 1:1.25, and stirred until a homogeneous suspension was formed. The total solids concentration was controlled at 17.5%.

[0036] Low-temperature spray drying granulation The homogeneous suspension was subjected to low-temperature spray drying with an inlet air temperature of 110°C, an outlet air temperature of 50°C, a feed rate of 7.5 mL / min, and an atomizer frequency of 25 Hz to obtain microcapsule particles.

[0037] Surface hydrophobic coating Add 0.75% magnesium stearate by total weight to the above microcapsule particles and mix in a three-dimensional motion mixer for 10 minutes to uniformly coat the particle surface with magnesium stearate, thus obtaining the final composite microbial agent.

[0038] The obtained bacterial agent particles had a particle size distribution of 80μm-120μm, a moisture content of 3.2%, and a bulk density of 0.5g / mL.

[0039] Example 2: The composite microbial agent of this example consists of the following components by weight percentage: 10% Bacillus phosphate-solubilizing spore powder, 5% concentrated freeze-dried Pseudomonas aeruginosa cell powder, 20% sodium alginate and modified starch cross-linked wall material, and 65% carrier filler.

[0040] The remaining conditions are the same as in Example 1.

[0041] Example 3: The composite microbial agent of this example consists of the following components by weight percentage: 20% Bacillus phosphate-solubilizing spore powder, 10% concentrated freeze-dried Pseudomonas aeruginosa cell powder, 10% sodium alginate and modified starch cross-linked wall material, and 60% carrier filler.

[0042] The remaining conditions are the same as in Example 1.

[0043] Example 4: In this example, the mass ratio of sodium alginate to modified starch is 1:2.

[0044] The remaining conditions are the same as in Example 1.

[0045] Example 5: In this example, the mass ratio of sodium alginate to modified starch is 1:4.

[0046] The remaining conditions are the same as in Example 1.

[0047] Example 6: In this example, the total mass concentration of the core components of rice root exudates in the second-stage fermentation broth is 0.5 g / L.

[0048] The remaining conditions are the same as in Example 1.

[0049] Example 7: In this example, the degree of substitution of the modified starch is 0.1.

[0050] The remaining conditions are the same as in Example 1.

[0051] Example 8: In this example, the volume concentration factor of the *Pseudomonas aeruginosa* fermentation broth is 8 times.

[0052] The remaining conditions are the same as in Example 1.

[0053] Example 9: In this example, the amount of magnesium stearate coating is 0.5% of the total weight of the compound microbial agent.

[0054] The remaining conditions are the same as in Example 1.

[0055] Example 10: In this example, the inlet air temperature of the low-temperature spray dryer is 100°C and the outlet air temperature is 45°C.

[0056] The remaining conditions are the same as in Example 1.

[0057] Example 11: In this example, the temperature of the calcium ion crosslinking reaction is 40°C and the reaction time is 60 min.

[0058] The remaining conditions are the same as in Example 1.

[0059] Example 12: In this example, the power of ultrasonic emulsification is 300W and the ultrasonic time is 15min.

[0060] The remaining conditions are the same as in Example 1.

[0061] Comparative Example 1: In this comparative example, Bacillus phosphate-solubilizing was not subjected to a second stage of induced fermentation. Instead, the vegetative cells after the first stage of fermentation were directly collected and dried with hot air to obtain bacterial powder, which replaced the Bacillus phosphate-solubilizing spore powder in Example 1.

[0062] The remaining conditions are the same as in Example 1.

[0063] Comparative Example 2: Preparation of microbial agent using the conventional liquid fermentation process described in the background art: *Bacillus phosphate-solubilizing* and *Pseudomonas aeruginosa* were inoculated into the same fermenter at a live cell ratio of 2.5:1, using the first-stage fermentation medium from Example 1, and cultured at 31°C and 225 rpm for 33 hours. After fermentation, no treatment was performed, and the fermentation broth containing live bacteria was directly used as the liquid microbial agent.

[0064] Comparative Example 3: In this comparative example, the inlet air temperature of the low-temperature spray dryer is 150℃ and the outlet air temperature is 70℃.

[0065] The remaining conditions are the same as in Example 1.

[0066] Comparative Example 4: Step 7 in Example 1 is omitted in this comparative example, and the magnesium stearate surface hydrophobic coating is not performed.

[0067] The remaining conditions are the same as in Example 1.

[0068] Test method: Storage and transportation stability test: The bacterial agents of each example and comparative example were stored at 25°C and 60% relative humidity for 3 months. The number of viable bacteria before and after storage was determined by plate count method, and the viable bacteria survival rate was calculated.

[0069] Rhizosphere colonization capacity test: Rice seedlings with uniform growth were selected and transplanted into pots containing sterilized soil. Each pot was treated with an inoculant equivalent to 2 kg / mu. Fourteen days after application, the number of colonies of the target strain in the rice rhizosphere soil was determined using the rhizosphere soil dilution plate method, and the rhizosphere colonization capacity was calculated.

[0070] Rhizosphere phosphorus activation capacity test: In the above pot experiment, the available phosphorus content in the soil was determined by sodium bicarbonate extraction-molybdenum antimony colorimetric method 30 days after application.

[0071] Rice growth-promoting effect test: In the above pot experiment, the plant height and fresh weight of the above-ground parts of rice were measured 30 days after application.

[0072] Test results:

[0073]

[0074] Results analysis: After 3 months of storage, the viable bacterial survival rate of all the bacterial agents in the embodiments remained above 85%, with Example 1 reaching 92.3%. The survival rate of the liquid bacterial agent in Comparative Example 2 was only 12.5%, indicating that the microcapsule solid formulation structure of the present invention can significantly improve the storage and transportation stability of the bacteria. In Comparative Example 3, due to the spray drying temperature exceeding the range defined by the present invention, a large number of bacteria were inactivated due to the high temperature, resulting in a significant decrease in the survival rate to 35.6%. Comparative Example 4 lacked a magnesium stearate hydrophobic barrier layer, allowing external moisture to easily penetrate into the microcapsule, leading to premature activation of the bacteria and a survival rate dropping to 68.9%, demonstrating that the hydrophobic barrier layer plays an important role in maintaining bacterial activity.

[0075] The rhizosphere colonization rate reached 7.2 × 10⁻⁶ in all embodiments. 6 The concentration of CFU / g root soil was above 1, while in Comparative Example 1 it was only 1.2 × 10⁻⁶. 6 CFU / g root soil. This result fully demonstrates that Bacillus phosphate-solubilizing bacteria induced by the core components of rice root exudates can express specific chemotactic receptors, actively recognize them, and move directionally towards the rice rhizosphere, thereby achieving efficient colonization. Comparative Example 2, due to its extremely low viable bacterial survival rate, had a rhizosphere colonization rate of only 0.3 × 10⁻⁶. 6 The CFU / g root soil test further verified the synergistic advantages of the present invention in improving bacterial survival rate and colonization ability.

[0076] The available phosphorus content in the soil of all embodiments reached over 32.5 mg / kg, significantly higher than that of the comparative examples. Comparative Example 1, due to its low rhizosphere colonization, had limited phosphorus solubilization, resulting in a soil available phosphorus content of only 15.6 mg / kg. Comparative Example 2 had a soil available phosphorus content of only 8.9 mg / kg, showing no significant difference from the blank control. This indicates that the compound microbial agent of the present invention can establish a stable viable bacterial community in the rice rhizosphere, continuously secreting organic acids to dissolve insoluble phosphates in the soil, significantly improving soil phosphorus availability.

[0077] The rice plant height and aboveground fresh weight in all embodiments were significantly higher than those in the comparative examples. In Example 1, the rice plant height reached 32.5 cm, and the aboveground fresh weight reached 12.8 g / plant, representing increases of 67.5% and 120.7% respectively compared to Comparative Example 2. This result indicates that the compound microbial agent of the present invention can significantly promote rice growth and development through the synergistic effect of Bacillus phosphate-solubilizing activating soil phosphorus and the production of plant growth regulators by Pseudomonas aeruginosa.

[0078] In summary, this invention, through the use of techniques such as inducing spore formation with rice root exudates, microfiltration to concentrate the bacterial cells, sodium alginate-modified starch cross-linked microcapsule encapsulation, and surface hydrophobic coating, successfully solves the technical problems of poor bacterial resistance, weak rhizosphere colonization ability, and low survival rate of live bacteria in existing liquid microbial agents. It has achieved unexpected technical effects and has significant inventiveness and practicality.

Claims

1. A compound microbial agent for promoting rice growth and activating rhizosphere phosphorus, wherein the compound microbial agent is a microcapsule solid preparation, and is composed of the following components by weight percentage: 10%-20% Bacillus phosphate-solubilizing spore powder, 5%-10% concentrated freeze-dried Pseudomonas aeruginosa bacterial cell powder, 10%-20% sodium alginate and modified starch cross-linked wall material, and 50%-75% carrier filler; The phosphate-solubilizing Bacillus spore powder is a dried spore-formed bacterial cell induced by the core components of rice root exudate. The concentrated lyophilized Pseudomonas aeruginosa cell powder is a dried cell product after microfiltration concentration treatment. In the cross-linked wall material of sodium alginate and modified starch, the mass ratio of sodium alginate to modified starch is 1:2 to 1:

4. The carrier filler is a mixture of diatomaceous earth, rice husk powder and bentonite, and the mass ratio of diatomaceous earth, rice husk powder and bentonite is 2:1:

1.

2. The compound microbial agent for promoting rice growth and activating rhizosphere phosphorus according to claim 1, wherein the core components of the rice root exudate include organic acids and glycosides, wherein the organic acids are a mixture of tartaric acid and malic acid, and the glycosides are sophorose. In the preparation of the Bacillus phosphate-solubilizing spore powder, the core components of rice root exudates are added as a limiting carbon source to the second-stage fermentation broth of Bacillus phosphate-solubilizing, and the total mass concentration of organic acids and glycosides in the second-stage fermentation broth is controlled to be 0.5 g / L-2 g / L.

3. The composite microbial agent for promoting rice growth and activating rhizosphere phosphorus according to claim 1, wherein the modified starch is cross-linked starch that has been cross-linked with propylene oxide and has a degree of substitution of 0.1-0.2; The preparation process of the sodium alginate and modified starch crosslinked wall material is as follows: Sodium alginate aqueous solution was mixed with modified starch gelatinized liquid, and then calcium chloride solution was added to carry out calcium ion crosslinking reaction to form a gel network. The mass concentration of the calcium chloride solution was 1%-3%, the temperature of the calcium ion crosslinking reaction was 40℃-50℃, and the time of the calcium ion crosslinking reaction was 30min-60min.

4. In the preparation process of the concentrated Pseudomonas aeruginosa lyophilized powder according to claim 1, a ceramic microfiltration membrane with a pore size of 0.1μm-0.2μm is used to concentrate and retain the Pseudomonas aeruginosa fermentation broth. The operating pressure of the ceramic microfiltration membrane is 0.1MPa-0.3MPa. The temperature of the fermentation broth is maintained at 4℃-10℃ during the concentration and retention process. The volume concentration factor of the fermentation broth at the end of the concentration is 8-12 times. The concentrated retentate is then freeze-dried under vacuum to obtain the concentrated Pseudomonas aeruginosa lyophilized powder.

5. The compound microbial agent for promoting rice growth and activating rhizosphere phosphorus according to claim 1, wherein the microcapsule solid preparation is prepared by a low-temperature spray drying process, wherein the inlet air temperature of the low-temperature spray drying is 100℃-120℃, the outlet air temperature is 45℃-55℃, the feeding rate is 5mL / min-10mL / min, and the atomizer frequency of the low-temperature spray drying is 20Hz-30Hz; Before performing the low-temperature spray drying, the spore powder of Bacillus phosphate-solubilizing, the freeze-dried concentrated bacterial powder of Pseudomonas alginate, and the aqueous solution of sodium alginate and modified starch crosslinked wall material are mixed to form a homogeneous suspension with a total solids concentration of 15%-20%.

6. The composite microbial agent for promoting rice growth and activating rhizosphere phosphorus according to claim 2, wherein in the first stage fermentation of the preparation of the Bacillus phosphate-solubilizing spore powder, the first stage fermentation medium comprises, by mass-volume ratio: The fermentation medium contains 1%-2% soluble starch, 0.5%-1% yeast extract, 0.5%-1% peptone, 0.02%-0.05% magnesium sulfate, and 0.1%-0.2% dipotassium hydrogen phosphate. The initial pH of the fermentation medium in the first stage is 7.0-7.2, the fermentation temperature in the first stage is 30℃-32℃, the stirring speed is 200rpm-250rpm, and the fermentation time is 18h-24h.

7. The composite microbial agent for promoting rice growth and activating rhizosphere phosphorus according to claim 1, wherein the diatomaceous earth in the carrier filler is pretreated by acid washing and high-temperature calcination, wherein the acid washing is performed by soaking in a 5% hydrochloric acid solution for 2 hours and then washing with water until neutral, and the high-temperature calcination temperature is 400℃-500℃ and the calcination time is 2h-3h. The rice husk powder has a particle size that passes through a standard sieve of 80-100 mesh. The bentonite is sodium-based bentonite; Before participating in the preparation of the microcapsule solid formulation, the carrier filler is premixed with an aqueous solution of sodium alginate and modified starch crosslinked wall material and subjected to ultrasonic emulsification treatment. The ultrasonic emulsification power is 300W-500W and the ultrasonic time is 10min-15min.

8. A compound microbial agent for promoting rice growth and activating rhizosphere phosphorus according to claim 4 or 5, wherein before forming the microcapsule solid preparation, the phosphate-solidifying Bacillus spore powder and the concentrated freeze-dried Pseudomonas aeruginosa bacterial cell powder are compounded at a live cell ratio of 2:1 to 3:1, and skim milk powder is added as a freeze-drying protectant during the compounding process, wherein the mass fraction of skim milk powder in the compounding system is 3%-5%, and the compounded mixed bacterial powder and the dry weight of the sodium alginate and modified starch cross-linked wall material are secondary-encapsulated at a mass ratio of 1:1 to 1:1.

5.

9. The compound microbial agent for promoting rice growth and activating rhizosphere phosphorus according to claim 5, wherein the particle size distribution of the microcapsule solid preparation is 50μm-150μm, the moisture content of the microcapsule solid preparation is controlled below 5%, and the bulk density of the microcapsule solid preparation is 0.4g / mL-0.6g / mL. The surface of the microcapsule solid formulation particles is coated with a hydrophobic barrier layer formed of magnesium stearate, and the amount of magnesium stearate coating is 0.5%-1% of the total weight of the microcapsule solid formulation.

10. Application of a compound microbial agent for promoting rice growth and activating rhizosphere phosphorus, wherein the compound microbial agent for promoting rice growth and activating rhizosphere phosphorus as described in any one of claims 1 to 9 is applied as basal fertilizer or topdressing during the tillering stage in rice transplanted fields, and the specific application process is as follows: When applying base fertilizer 7-10 days before rice transplanting, mix the compound microbial agent with fine soil at a mass ratio of 1:50 and spread it evenly on the surface of the cultivated layer of the paddy field. Then, plow and prepare the land. The application rate of the compound microbial agent is 2 kg / mu-4 kg / mu. When applying topdressing fertilizer during the tillering stage of rice, the compound microbial agent is dissolved in the paddy field water and sprayed evenly on the leaves and water surface using a drone. The mass concentration of the compound microbial agent in the spray solution is 0.5%-1%, and the amount of spray solution used per acre of paddy field is 30L-50L. The topdressing fertilizer is applied within 10-15 days after rice transplanting.

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