Straw biochar load bacillus amyloliquefaciens preparation and preparation method thereof

Abstract

This invention relates to the field of microbial preparation technology, specifically disclosing a Bacillus amyloliquefaciens preparation supported on straw biochar and its preparation method. The preparation comprises a modified straw biochar carrier and a Bacillus amyloliquefaciens bacterial solution loaded thereon. The preparation method includes: modifying the straw biochar with citric acid and chitosan to construct a positively charged functional layer; loading the Bacillus amyloliquefaciens bacterial solution containing sodium alginate into the pores of the biochar under negative pressure, forming a gel network through electrostatic complexation; subsequently, gradient addition of calcium ion solution to induce ionic cross-linking, constructing a stable interpenetrating network structure within the pores; and finally, gentle drying with trehalose glassy protection. This invention, through the synergistic effect of chemical modification and multi-level cross-linking, firmly anchors the bacteria within the carrier, solving the problems of weak loading and easy loss in traditional physical adsorption methods, significantly extending the effective period, and improving the control effect against tomato wilt disease.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology, and relates to a straw biochar-supported Bacillus amyloliquefaciens preparation and its preparation method. Background Technology

[0002] Fusarium wilt of tomato is a devastating soil-borne disease caused by Fusarium oxysporum tomato-specific strain, seriously threatening tomato yield and quality. Utilizing biocontrol microorganisms, such as Bacillus amyloliquefaciens, is currently recognized as a green and environmentally friendly control strategy. Bacillus amyloliquefaciens can inhibit the pathogen by producing antimicrobial lipopeptides, competing for ecological niches, and inducing systemic resistance in plants.

[0003] However, direct application of Bacillus amyloliquefaciens bacterial solution has problems such as low bacterial survival rate, easy wash-off by rainwater or irrigation, poor rhizosphere colonization ability, and short effective action period. To overcome these defects, existing technologies often use biochar as a carrier. The porous structure and huge specific surface area of ​​biochar can provide a physical "shelter" for microorganisms, but its adsorption mainly relies on van der Waals forces. The binding force between the bacterial cells and the carrier is weak, and they are easy to desorb and fall off. In addition, simple physical adsorption of bacterial cells by biochar is easy for the bacterial cells to fall off, and a large number of bacterial cells die during the drying process, resulting in a low survival rate.

[0004] Therefore, there is an urgent need to develop a formulation and its preparation method that can significantly improve the survival rate, colonization ability and control effect of Bacillus amyloliquefaciens. Summary of the Invention

[0005] In view of the shortcomings of existing technologies, this invention provides a Bacillus amyloliquefaciens preparation supported on straw biochar and its preparation method. The core innovation of this invention lies in: constructing a positively charged functional layer on the surface of straw biochar by sequentially citric acid esterification and chitosan modification; preparing a bacterial solution by blending Bacillus amyloliquefaciens with sodium alginate, and forcibly loading it into the pores of the biochar using negative pressure; forming a primary gel network through in-situ electrostatic complexation of chitosan and sodium alginate; subsequently, initiating ionic cross-linking of the "egg-box" structure of sodium alginate by gradient dropwise addition, and coordinating with the carboxyl groups of citric acid residues, thereby constructing a stable "biochar-interpenetrating network gel-bacterial cell" composite structure within the biochar. This design fundamentally solves the technical problems of weak bacterial cell loading, easy loss, and poor environmental tolerance inherent in traditional physical adsorption methods.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a preparation of *Bacillus amyloliquefaciens* supported on straw biochar. The preparation comprises a modified straw biochar carrier and a *Bacillus amyloliquefaciens* bacterial solution supported thereon. The mass ratio of the modified straw biochar carrier to the *Bacillus amyloliquefaciens* bacterial solution used for loading is 1:5-10. The *Bacillus amyloliquefaciens* bacterial solution is a mixed solution of *Bacillus amyloliquefaciens* and sodium alginate aqueous solution, wherein the final concentration of *Bacillus amyloliquefaciens* is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

[0008] Further, the preparation method of the modified straw biochar carrier is as follows: (a) Take straw, wash and dry it, crush it, and place it in a vacuum atmosphere furnace. Under nitrogen protection, pyrolyze it at 500℃ for 2 hours. After cooling, grind it through a 200-mesh sieve to obtain primary biochar; (b) Disperse the primary biochar in a 5% (w / w) citric acid aqueous solution, add it to the system at a solid-liquid ratio of 1g:15mL, and add 1% (w / w) of sodium hypophosphite as an esterification catalyst. Stir the reaction at 85℃ for 2 hours. (c) Then, a 2% (w / w) chitosan acetate solution was added dropwise to the system to adjust the pH to 5.0. The reaction was continued at 85°C for 1-2 hours to ionize the remaining two free carboxyl groups on the citric acid and make them negatively charged. These groups then interacted with the protonated amino groups on the chitosan molecular chain through strong electrostatic attraction and hydrogen bonding. After the reaction was completed, the mixture was filtered, washed with deionized water until neutral, and freeze-dried to obtain the modified straw biochar carrier.

[0009] Furthermore, the straw is any one of corn straw, wheat straw, and rice straw, with corn straw being preferred.

[0010] Further, the preparation method of the *Bacillus amyloliquefaciens* bacterial suspension is as follows: *Bacillus amyloliquefaciens* is inoculated into LB liquid medium and cultured with shaking at 30°C and 160 rpm until the late logarithmic growth stage. After centrifugation at 5000 rpm for 5 minutes, the supernatant is discarded, and the bacterial precipitate is resuspended twice with sterile physiological saline. The precipitate is collected by centrifugation and then resuspended in sterile sodium alginate aqueous solution to adjust the final bacterial concentration to 1 × 10⁻⁶. 8 -1×10 9 The CFU / mL concentration yields the Bacillus amyloliquefaciens bacterial solution.

[0011] This invention also provides a method for preparing a Bacillus amyloliquefaciens preparation supported on straw biochar, comprising the following steps:

[0012] S1. Negative pressure preloading and in-situ polyelectrolyte complexation: The modified straw biochar carrier is placed in a vacuum dryer, and Bacillus amyloliquefaciens bacterial solution is added until it is completely submerged. The vacuum is drawn to -0.09 MPa and maintained for 10-15 minutes, so that the bacterial solution is forced into the micropores of the biochar under negative pressure. After restoring normal pressure, it is shaken at 25°C and 80 rpm for 30 minutes. During this process, sodium alginate (negatively charged) in the bacterial solution and chitosan molecules (positively charged) on the surface of the modified carrier undergo in-situ electrostatic complexation in the biochar pores to form polyelectrolyte complex gel microspheres with an interpenetrating network structure. Excess free bacterial solution is drained to obtain preloaded biochar.

[0013] S2. Gradient ion crosslinking and anchoring: Place the pre-loaded biochar obtained in step S1 in a container, and slowly add 0.1 mol / L calcium chloride solution at a rate of 1-2 mL / min, so that Ca... 2+ Gradient diffusion into the pores of biochar, Ca 2+ Not only does it undergo "egg-box" ionic cross-linking with the uncomplexed guluronic acid segments in the sodium alginate within the pores, further strengthening the gel framework, but trace amounts of Ca also... 2+ It can also coordinate with the remaining free carboxyl groups on citric acid to form a multi-linked network. After the addition is complete, let it stand at 20-25℃ for another hour to crosslink.

[0014] S3. Gentle solid-liquid separation: After cross-linking, filter with sterile gauze, collect the solid filter residue, and gently rinse the surface with a small amount of sterile physiological saline to remove uncross-linked free gel and bacterial cells;

[0015] S4. Low-temperature protective drying: The washed filter residue is spread evenly on a sterile tray in the ultra-clean workbench, and a 5% trehalose solution is sprayed evenly. It is then gently dried in a clean airflow at 22℃ and humidity <30% until the moisture content is 10% to obtain a solid material. In this process, trehalose replaces water to form a glassy protective film, maintaining the integrity of the bacterial cell membrane.

[0016] S5. Formulation: The solid material obtained in step S4 is directly packaged to obtain the straw biochar-supported Bacillus amyloliquefaciens formulation.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] The straw biochar loading formulation prepared in this invention constructs a stable biochar framework enriched with positive charges on its surface through citric acid esterification bridging and chitosan modification; sodium alginate-containing bacterial solution is penetrated deep into micropores under negative pressure, forming a primary flexible gel network through in-situ electrostatic complexation; a gradient introduction of Ca... 2+This further induces ionic cross-linking of the "egg box" structure and coordination with the free carboxyl groups of citric acid, firmly anchoring the gel network chemically within the pores. This multi-force synergistic system completely overcomes the limitations of weak binding forces in traditional physical adsorption. Even after three sterile water rinses, the bacterial cell retention rate remains above 85%, effectively overcoming the defect of easy detachment and loss of bacteria under rainfall or irrigation conditions.

[0019] Building upon this foundation, the dense interpenetrating network gel provides a physical barrier for the bacteria, buffering against external moisture, pH, and mechanical shock. Trehalose sprayed during the drying stage forms a glassy protective film, maintaining the cell membrane integrity of the bacteria in a dehydrated state. This synergistic "gel buffering-sugar protection" mechanism ensures that the viable bacterial survival rate remains above 60% after 6 months of room temperature storage. Furthermore, this invention eliminates conventional mechanical pulverization and post-processing, maintaining the porous aggregated state for direct packaging, avoiding physical damage to the fragile internal gel network and bacteria from shear forces. This in-situ constructed multi-level interpenetrating network system slowly degrades with the soil environment, achieving controlled slow release and long-term colonization of the bacteria. Pot experiments showed that 60 days after application, the control efficacy against tomato wilt remained above 85%, an improvement of over 70% compared to conventional physical adsorption agents, achieving long-lasting, highly effective, and green control. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0021] Figure 1 This is a comparison chart of tomato growth in the potted plant efficacy experiment of the present invention, where (a) represents the tomatoes in the Example 1 group and (b) represents the tomatoes in the blank control group. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0024] Unless otherwise specified, all materials used in the following implementation are new materials purchased from the market.

[0025] Example 1: This example provides a straw biochar-supported Bacillus amyloliquefaciens preparation and its preparation method. The preparation includes modified straw biochar and a Bacillus amyloliquefaciens bacterial solution supported thereon. The specific steps of the preparation method are as follows:

[0026] S1. Preparation of modified straw biochar carrier: (a) Weigh 5.0g of washed and dried corn straw pulverized material and place it in a vacuum atmosphere furnace. Under nitrogen protection, pyrolyze at 500℃ for 2 hours. After cooling, grind through a 200-mesh sieve to obtain primary biochar; (b) Disperse 5.0g of primary biochar in 75.0mL of 5% citric acid aqueous solution, add 0.80g of sodium hypophosphite, and react at 85℃ with magnetic stirring for 2 hours; (c) Slowly add 30.0mL of 2% chitosan acetate solution to the system. During the addition, adjust the pH of the system to 5.0 with 0.1mol / L NaOH solution. Continue to react at 85℃ for 1.5 hours. After the reaction, filter, wash the filter cake with deionized water until the washing liquid is neutral, and dry it in a freeze dryer to constant weight to obtain modified straw biochar carrier;

[0027] S2. Preparation of Bacillus amyloliquefaciens bacterial suspension: Bacillus amyloliquefaciens was inoculated into LB liquid medium and cultured at 30℃ and 160 rpm with shaking until the late logarithmic growth phase (OD600=0.8). The culture was centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and the bacterial cells were resuspended twice with sterile physiological saline. The bacterial precipitate was collected by centrifugation and resuspended in 40.0 mL of 0.5% sterile sodium alginate aqueous solution, adjusting the final bacterial concentration to 5 × 10⁻⁶. 8 CFU / mL yields Bacillus amyloliquefaciens bacterial solution;

[0028] S3. Negative pressure preloading and in-situ polyelectrolyte complexation: Weigh 5.0g of modified straw biochar carrier and place it in a vacuum desiccator. Add 40.0mL of the above Bacillus amyloliquefaciens bacterial solution until completely submerged. Turn on the vacuum pump to evacuate to -0.09MPa and maintain for 12 minutes to force the bacterial solution into the micropores of the biochar under negative pressure. After restoring normal pressure, transfer the material to an Erlenmeyer flask and shake at 25℃ and 80rpm for 30 minutes. Then, use sterile gauze to drain off the excess free bacterial solution to obtain preloaded biochar.

[0029] S4. Gradient ion crosslinking and anchoring: The preloaded biochar obtained in step S3 was spread evenly in a petri dish, and 20.0 mL of sterile calcium chloride solution with a concentration of 0.1 mol / L was slowly added dropwise at a rate of 1.5 mL / min using a peristaltic pump. After the addition was completed, the mixture was allowed to stand at 22°C for 1 hour for crosslinking.

[0030] S5. Gentle solid-liquid separation and low-temperature protective drying: After cross-linking, the solid filter residue is collected by filtering with sterile gauze, and the surface is gently rinsed with 10.0 mL of sterile physiological saline. The washed filter residue is spread evenly on a sterile tray in the ultra-clean workbench, and 2.0 mL of 5% trehalose solution is sprayed evenly. The material is then gently dried in a clean airflow at 22°C and 28% humidity until the moisture content of the material is 10%, thus obtaining a solid material.

[0031] S6. Formulation: The solid material obtained in step S5 is packaged into aluminum foil bags and sealed to obtain the straw biochar-supported Bacillus amyloliquefaciens formulation.

[0032] Example 2: This example provides a straw biochar-supported Bacillus amyloliquefaciens preparation and its preparation method. The preparation includes modified straw biochar and a Bacillus amyloliquefaciens bacterial solution supported thereon. The specific steps of the preparation method are as follows:

[0033] S1. Preparation of modified straw biochar carrier: (a) Weigh 5.0g of washed and dried wheat straw pulverized material and place it in a vacuum atmosphere furnace. Under nitrogen protection, pyrolyze at 500℃ for 2 hours. After cooling, grind through a 200-mesh sieve to obtain primary biochar; (b) Disperse 5.0g of primary biochar in 75.0mL of 5% citric acid aqueous solution, add 0.80g of sodium hypophosphite, and react at 85℃ with magnetic stirring for 2 hours; (c) Slowly add 30.0mL of 2% chitosan acetate solution to the system. During the addition, adjust the pH of the system to 5.0 with 0.1mol / L NaOH solution. Continue to react at 85℃ for 1 hour. After the reaction is completed, filter, wash the filter cake with deionized water until the washing liquid is neutral, and dry it in a freeze dryer to constant weight to obtain modified straw biochar carrier;

[0034] S2. Preparation of Bacillus amyloliquefaciens bacterial suspension: Bacillus amyloliquefaciens was inoculated into LB liquid medium and cultured at 30℃ and 160 rpm with shaking until the late logarithmic growth phase (OD600=0.8). The culture was centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and the bacterial cells were resuspended twice with sterile physiological saline. The bacterial precipitate was collected by centrifugation and resuspended in 25.0 mL of 0.5% sterile sodium alginate aqueous solution to adjust the final bacterial concentration to 1×10⁻⁶. 8 CFU / mL yields Bacillus amyloliquefaciens bacterial solution;

[0035] S3. Negative pressure preloading and in-situ polyelectrolyte complexation: Weigh 5.0g of modified straw biochar carrier and place it in a vacuum desiccator. Add 25.0mL of the above Bacillus amyloliquefaciens bacterial solution until completely submerged. Turn on the vacuum pump to evacuate to -0.09MPa and maintain for 10 minutes to force the bacterial solution into the micropores of the biochar under negative pressure. After restoring normal pressure, transfer the material to an Erlenmeyer flask and shake at 25℃ and 80rpm for 30 minutes. Then, use sterile gauze to drain off the excess free bacterial solution to obtain preloaded biochar.

[0036] S4. Gradient ion crosslinking and anchoring: The preloaded biochar obtained in step S3 is spread evenly in a petri dish, and 12.5 mL of sterile calcium chloride solution with a concentration of 0.1 mol / L is slowly added dropwise at a rate of 1.0 mL / min using a peristaltic pump. After the addition is completed, the mixture is allowed to stand at 20°C for 1 hour for crosslinking.

[0037] S5. Gentle solid-liquid separation and low-temperature protective drying: After cross-linking, the solid filter residue is collected by filtering with sterile gauze, and the surface is gently rinsed with 10.0 mL of sterile physiological saline. The washed filter residue is spread evenly on a sterile tray in the ultra-clean workbench, and 2.0 mL of 5% trehalose solution is sprayed evenly. The material is then gently dried in a clean airflow at 22℃ and 25% humidity until the moisture content of the material is 10%, thus obtaining a solid material.

[0038] S6. Formulation: The solid material obtained in step S5 is packaged into aluminum foil bags and sealed to obtain the straw biochar-supported Bacillus amyloliquefaciens formulation.

[0039] Example 3: This example provides a straw biochar-supported Bacillus amyloliquefaciens preparation and its preparation method. The preparation includes modified straw biochar and a Bacillus amyloliquefaciens bacterial solution supported thereon. The specific steps of the preparation method are as follows:

[0040] S1. Preparation of modified straw biochar carrier: (a) Weigh 5.0g of washed and dried rice straw pulverized material and place it in a vacuum atmosphere furnace. Under nitrogen protection, pyrolyze at 500℃ for 2 hours. After cooling, grind through a 200-mesh sieve to obtain primary biochar; (b) Disperse 5.0g of primary biochar in 75.0mL of 5% citric acid aqueous solution, add 0.80g of sodium hypophosphite, and react at 85℃ with magnetic stirring for 2 hours; (c) Slowly add 30.0mL of 2% chitosan acetate solution to the system. During the addition, adjust the pH of the system to 5.0 with 0.1mol / L NaOH solution. Continue to react at 85℃ for 2 hours. After the reaction is completed, filter, wash the filter cake with deionized water until the washing liquid is neutral, and dry it in a freeze dryer to constant weight to obtain modified straw biochar carrier;

[0041] S2. Preparation of Bacillus amyloliquefaciens bacterial suspension: Bacillus amyloliquefaciens was inoculated into LB liquid medium and cultured at 30℃ and 160 rpm with shaking until the late logarithmic growth phase (OD600=0.8). The culture was centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and the bacterial cells were resuspended twice with sterile physiological saline. The bacterial precipitate was collected by centrifugation and resuspended in 50.0 mL of 0.5% sterile sodium alginate aqueous solution to adjust the final bacterial concentration to 1×10⁻⁶. 9 CFU / mL yields Bacillus amyloliquefaciens bacterial solution;

[0042] S3. Negative pressure preloading and in-situ polyelectrolyte complexation: Weigh 5.0g of modified straw biochar carrier and place it in a vacuum desiccator. Add 50.0mL of the above Bacillus amyloliquefaciens bacterial solution until completely submerged. Turn on the vacuum pump to evacuate to -0.09MPa and maintain for 15 minutes to force the bacterial solution into the micropores of the biochar under negative pressure. After restoring normal pressure, transfer the material to an Erlenmeyer flask and shake at 25℃ and 80rpm for 30 minutes. Then, use sterile gauze to drain off the excess free bacterial solution to obtain preloaded biochar.

[0043] S4. Gradient ion crosslinking and anchoring: The preloaded biochar obtained in step S3 was spread evenly in a petri dish, and 25.0 mL of sterile calcium chloride solution with a concentration of 0.1 mol / L was slowly added dropwise at a rate of 2.0 mL / min using a peristaltic pump. After the addition was completed, the mixture was allowed to stand at 25°C for 1 hour for crosslinking.

[0044] S5. Gentle solid-liquid separation and low-temperature protective drying: After cross-linking, the solid filter residue is collected by filtering with sterile gauze, and the surface is gently rinsed with 10.0 mL of sterile physiological saline. The washed filter residue is spread evenly on a sterile tray in the ultra-clean workbench, and 2.0 mL of 5% trehalose solution is sprayed evenly. The material is then gently dried in a clean airflow at 22°C and 20% humidity until the moisture content of the material is 10%, thus obtaining a solid material.

[0045] S6. Formulation: The solid material obtained in step S5 is packaged into aluminum foil bags and sealed to obtain the straw biochar-supported Bacillus amyloliquefaciens formulation.

[0046] Comparative Example 1: This comparative example provides a common biochar-supported Bacillus amyloliquefaciens preparation and its preparation method. The difference from Example 1 is that unmodified primary biochar is used, and the citric acid and chitosan modification steps are omitted. That is, the specific steps of the preparation method are modified as follows:

[0047] S1. Preparation of primary biochar: Weigh 5.0g of washed and dried corn stalk pulverized material and place it in a vacuum atmosphere furnace. Purge with nitrogen for protection and pyrolyze at 500℃ for 2 hours. After cooling, grind through a 200-mesh sieve to obtain primary biochar.

[0048] S2. Preparation of Bacillus amyloliquefaciens suspension: Bacillus amyloliquefaciens was inoculated into LB liquid medium and cultured at 30℃ and 160 rpm with shaking until the late logarithmic growth phase (OD600=0.8). The culture was centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and the culture was resuspended twice with sterile physiological saline. The bacterial pellet was collected by centrifugation and resuspended in 25.0 mL of sterile physiological saline, adjusting the final bacterial concentration to 5 × 10⁻⁶. 8 CFU / mL, which yields a suspension of Bacillus amyloliquefaciens;

[0049] S3. Pre-loading under negative pressure: Weigh 5.0g of the initial biochar and place it in a vacuum desiccator. Add 40.0mL of Bacillus amyloliquefaciens suspension until completely submerged. Turn on the vacuum pump to evacuate to -0.09MPa and maintain for 12 minutes. After restoring to normal pressure, transfer the material to an Erlenmeyer flask and shake at 25℃ and 80rpm for 30 minutes. Then, use sterile gauze to drain off the excess free bacterial solution to obtain pre-loaded biochar.

[0050] S4. Gradient ion crosslinking and anchoring: The preloaded biochar obtained in step S3 was spread evenly in a petri dish, and 20.0 mL of sterile calcium chloride solution with a concentration of 0.1 mol / L was slowly added dropwise at a rate of 1.5 mL / min using a peristaltic pump. After the addition was completed, the mixture was allowed to stand at 22°C for 1 hour for crosslinking.

[0051] S5. Gentle solid-liquid separation and low-temperature protective drying: After cross-linking, the solid filter residue is collected by filtering with sterile gauze, and the surface is gently rinsed with 10.0 mL of sterile physiological saline. The washed filter residue is spread evenly on a sterile tray in the ultra-clean workbench, and 2.0 mL of 5% trehalose solution is sprayed evenly. The material is then gently dried in a clean airflow at 22°C and 28% humidity until the moisture content of the material is 10%, thus obtaining a solid material.

[0052] S6. Formulation: The solid material obtained in step S5 is packaged into aluminum foil bags and sealed to obtain the ordinary biochar-supported Bacillus amyloliquefaciens formulation.

[0053] Comparative Example 2: This comparative example provides a biochar-supported Bacillus amyloliquefaciens preparation without sodium alginate encapsulation and its preparation method. The difference from Example 1 is that sodium alginate is not used in the preparation of the bacterial suspension. Specifically, step S2 of the preparation method is modified as follows: Preparation of Bacillus amyloliquefaciens suspension: Bacillus amyloliquefaciens is inoculated into LB liquid medium and cultured at 30°C and 160 rpm with shaking until the late logarithmic growth stage (OD600=0.8). After centrifugation at 5000 rpm for 5 minutes, the supernatant is discarded, and the bacterial cells are resuspended and washed twice with sterile physiological saline. The bacterial precipitate is collected by centrifugation and directly resuspended in 40.0 mL of sterile physiological saline to adjust the final bacterial concentration to 5 × 10⁻⁶. 8CFU / mL yields a suspension of Bacillus amyloliquefaciens; step S4 is omitted, and the remaining steps are the same as in Example 1.

[0054] Comparative Example 3: This comparative example discloses a biochar-supported Bacillus amyloliquefaciens preparation without gradient ion crosslinking and its preparation method. The difference from Example 1 is that the calcium chloride ion crosslinking step is omitted. The preparation method step S4 is modified as follows: mild solid-liquid separation: the pre-loaded biochar obtained in step S3 is directly filtered with sterile gauze, the solid filter residue is collected, and the surface is gently rinsed with 10.0 mL of sterile physiological saline to remove uncomplexed free bacterial liquid; the remaining steps are the same as in Example 1.

[0055] Experimental methods:

[0056] To verify the advantages of the technical solution of the present invention, performance tests were conducted on the formulations prepared in the above embodiments and comparative examples:

[0057] Loading strength test: Accurately weigh 1.0 g of the preparations prepared in Examples 1-3 and Comparative Examples 1-3, respectively, and place them in an Erlenmeyer flask containing 50 mL of sterile physiological saline. Wash vigorously at 200 rpm for 10 minutes. After washing, filter and collect the solid residue. Transfer the residue to 50 mL of physiological saline containing sterile glass beads and shake vigorously at 200 rpm for 30 minutes to fully elute and release the loaded bacteria. Use the tenfold serial dilution plate counting method to determine the number of viable bacteria retained after washing and calculate the loading strength. Loading strength (%) = (number of viable bacteria after washing / number of viable bacteria before washing) × 100%.

[0058] Storage stability test: Each formulation sample was sealed in an aluminum foil bag and stored in a refrigerator at 4°C in the dark. At 0 days and 90 days of storage, three parallel samples were taken, each weighed accurately as 1.00 g. The sample was placed in an Erlenmeyer flask containing 50 mL of sterile physiological saline and several glass beads. The flask was shaken at 150 rpm for 20 minutes to fully disperse the bacteria. Then, 1 mL of the suspension was serially diluted 10 times. The viable count was determined by the plate spread method, and the survival rate (%) was calculated.

[0059] Potted plant efficacy experiment: Tomato seedlings of uniform growth were selected and transplanted into soil containing the pathogen of tomato wilt. The above-mentioned preparation with an equal amount of live bacteria was applied to each seedling, with water treatment serving as a blank control. Each treatment was replicated in triplicate, with 10 seedlings per replicate. After 30 days of cultivation, the incidence rate was investigated according to relevant disease grading standards. The comparison of tomato growth between Example 1 group and the blank control group is shown in the figure below. Figure 1 As shown.

[0060] Table 1. Results of performance tests and potted plant efficacy trials for each formulation.

[0061]

[0062] As can be seen from the data in Table 1, the formulations prepared in Examples 1-3 of this invention exhibit significantly superior performance compared to the comparative examples in terms of loading stability, storage stability, and control efficacy against tomato wilt. The loading stability of the formulations prepared in Examples 1-3 is significantly higher than that of Comparative Examples 1 and 2, which directly proves that the negative pressure pre-filling and in-situ polyelectrolyte complexation technology used in this invention can firmly anchor *Bacillus amyloliquefaciens* within the micropores of the biochar carrier, rather than simply physically adsorbing it onto the surface. Furthermore, the loading survival rate of Comparative Example 3 is lower than that of the examples, which fully demonstrates that the gradient calcium ion crosslinking in step S4 plays a crucial role in further securing the bacterial cells and resisting water erosion. Furthermore, the formulations prepared in Examples 1-3 exhibited superior stability compared to the comparative examples. This high storage stability is attributed to: trehalose forming a glassy protective film during drying, replacing water molecules to form hydrogen bonds with phospholipids and proteins in the bacterial cell membrane, preventing membrane phase transitions and protein denaturation; the porous structure of the modified biochar providing a physical barrier, reducing oxygen and free radical damage; and the polyelectrolyte complex gel maintaining suitable microenvironmental humidity. Pot experiment results showed that, compared to the control group, the formulations prepared in this invention significantly inhibited tomato wilt, reduced the incidence rate, and combined with… Figure 1 It can be seen that the tomatoes in Example 1 group grew vigorously, which indicates that the modified biochar carrier and slow-release microecological environment of the present invention can not only effectively prevent Fusarium wilt but also synergistically promote the development of tomato roots, achieving the dual beneficial effects of "disease prevention + growth promotion".

[0063] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0064] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A straw biochar-supported Bacillus amyloliquefaciens preparation, characterized in that, The formulation comprises a modified straw biochar carrier and a Bacillus amyloliquefaciens liquid loaded thereon; the mass ratio of the modified straw biochar carrier to the Bacillus amyloliquefaciens liquid used for loading is 1:5-10; the Bacillus amyloliquefaciens liquid is a mixed solution of Bacillus amyloliquefaciens and sodium alginate aqueous solution.

2. The straw biochar-supported Bacillus amyloliquefaciens preparation according to claim 1, characterized in that, The modified straw biochar carrier is prepared as follows: (a) Take straw, wash it, crush it, and then pyrolyze it. After cooling, grind it, and sieve it to obtain primary biochar; (b) Disperse the primary biochar in an aqueous citric acid solution and add sodium hypophosphite as an esterification catalyst and stir the reaction; (c) Then add chitosan acetate solution to the system dropwise, adjust the pH of the system, and continue the reaction. After the reaction is completed, filter it, wash it with deionized water until neutral, and freeze dry it to obtain the modified straw biochar carrier.

3. The straw biochar-supported Bacillus amyloliquefaciens preparation according to claim 2, characterized in that, The straw is any one of corn straw, wheat straw, rice straw or cotton straw.

4. The straw biochar-supported Bacillus amyloliquefaciens preparation according to claim 2, characterized in that, The solid-liquid ratio of the prepared biochar to the citric acid aqueous solution is 1g:15mL.

5. The straw biochar-supported Bacillus amyloliquefaciens preparation according to claim 1, characterized in that, The preparation method of the Bacillus amyloliquefaciens bacterial solution is as follows: Bacillus amyloliquefaciens is inoculated into LB liquid medium, cultured to the late logarithmic growth stage, centrifuged and the supernatant is discarded, the bacterial precipitate is collected after washing, and then resuspended in sterile sodium alginate aqueous solution to obtain the Bacillus amyloliquefaciens bacterial solution.

6. The straw biochar-supported Bacillus amyloliquefaciens preparation according to claim 5, characterized in that, The bacterial concentration of the Bacillus amyloliquefaciens culture was 1×10⁻⁶. 8 -1×10 9 CFU / mL.

7. A method for preparing a Bacillus amyloliquefaciens-supported preparation from straw biochar according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Negative pressure pre-filling and in-situ polyelectrolyte complexation: The modified straw biochar carrier is added to the Bacillus amyloliquefaciens solution until it is completely submerged, vacuum is applied and pressure is applied, and after restoring normal pressure, it is shaken to obtain pre-loaded biochar; S2. Gradient ion crosslinking anchoring: Place the preloaded biochar in a container and slowly add calcium chloride solution dropwise. After the addition is complete, allow it to stand for crosslinking. S3. Gentle solid-liquid separation: After cross-linking, filter with sterile gauze, collect the solid filter residue, and then wash; S4. Low-temperature protective drying: Spread the washed filter residue evenly, then spray it with trehalose solution evenly, and then dry it to obtain a solid material; S5. Formulation: The solid material is packaged to obtain the straw biochar-supported Bacillus amyloliquefaciens formulation.

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