Preparation method and application of anti-cropping microbial fertilizer

Abstract

The application discloses a preparation method of anti-continuous cropping microbial fertilizer and application thereof, and aims to solve the problems of crop disease and pest intensification and yield and quality reduction caused by continuous cropping planting. The preparation steps are as follows: Bacillus subtilis and Bacillus licheniformis are activated and uniformly mixed, alpha-lactalbumin solution and calcium chloride solution are added to prepare bacillus spore aggregates; after adding polyhydroxyamylase solution and bentonite, the mixture is ultrasonically treated with humic acid solution to prepare modified polyhydroxyamylase-bentonite compound; bacillus spore aggregates, bacillus subtilis and bacillus licheniformis are uniformly mixed, and wall material solution is added to prepare composite microbial microcapsule preparation; finally, the microcapsules, corn straw biochar and chitosan are mixed to obtain the finished product. The calcium ion induction and the embedding modification of humic acid can improve the stability and the number of viable bacteria of the strains, can activate soil organic matter, nitrogen, phosphorus and potassium nutrients, can inhibit pathogenic bacteria, can improve the rhizosphere environment of crops, and can effectively improve the growth indexes and quality of red kidney beans and potatoes, and is suitable for anti-continuous cropping planting of crops.

Description

Technical Field

[0001] This invention belongs to the field of microbial fertilizer processing technology, specifically relating to a method for preparing a microbial fertilizer resistant to continuous cropping and its application. Background Technology

[0002] Continuous cropping can easily lead to replanting obstacles, exacerbating crop diseases and pests, and significantly reducing yield and quality. Replanting obstacles are soil-borne diseases, primarily caused by years of continuous cropping leading to nutrient imbalances in the rhizosphere soil, deficiencies in certain nutrients, changes in soil physicochemical properties, decreased soil enzyme activity, and an imbalance in the rhizosphere microbial community. Pathogenic microorganisms such as fungi, bacteria, nematodes, and actinomycetes gradually increase in both type and quantity, while beneficial microorganisms decrease. Furthermore, continuous planting causes crop roots to secrete autotoxic substances that inhibit crop growth (such as phenolic acids, organic acids, and flavonoids), which accumulate with increasing planting years. Continuous cropping obstacles mainly harm the crop roots, hindering their access to the nutrients needed for growth, affecting crop growth, and ultimately leading to reduced yield and quality.

[0003] Currently, there is a lack of effective control measures for continuous cropping obstacles. While chemical control is fast-acting, long-term and excessive use of chemical pesticides leads to environmental pollution and easily generates resistance, reducing control effectiveness. Furthermore, some existing anti-continuation products mainly rely on supplementing chemical elements, especially micronutrients, or applying bio-organic fertilizers and anti-continuation microbial agents. These methods are limited in function, slow in action, short-lived, and have poor control effects. These products are primarily applied as basal fertilizers, which is inconvenient during the crop's growth period, and the addition of chemical elements can easily cause environmental and soil problems, potentially exacerbating disease occurrence. In addition, the microbial strains in bio-organic fertilizers are mostly applied directly to the planting soil. This not only leads to competition between beneficial microorganisms and dominant pathogens in the soil, producing an inhibitory effect, but also makes it difficult for beneficial microorganisms to effectively colonize and exert their effects at the crop root system. Summary of the Invention

[0004] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to disclose a method for preparing and applying a microbial fertilizer resistant to continuous cropping, aiming to solve the problems of increased crop diseases and pests, and decreased yield and quality caused by continuous cropping. The preparation steps are as follows: Bacillus subtilis and Bacillus licheniformis are activated and mixed, and α-lactalbumin solution and calcium chloride solution are added to prepare Bacillus aggregates; a polytrehalose solution is mixed with bentonite and then ultrasonically treated with humic acid solution to prepare a modified polytrehalose-bentonite complex; *Trichoderma harzianum*, *Trichoderma harzianum*, and Bacillus aggregates are mixed, and a wall material solution is added to prepare a composite microcapsule formulation; finally, the microcapsules are mixed with corn straw biochar, chitosan oligosaccharide, etc., to obtain the finished product. This invention, through calcium ion induction and humic acid modification and encapsulation, improves the stability and viable count of the strain, activates soil organic matter, nitrogen, phosphorus, and potassium nutrients, inhibits pathogens, improves the rhizosphere environment of crops, and effectively improves the growth indicators and quality of red kidney beans and potatoes, making it suitable for crops resistant to continuous cropping.

[0005] Technical solution: A method for preparing a microbial fertilizer resistant to continuous cropping, comprising the following steps: S1. After activating Bacillus subtilis and Bacillus licheniformis, mix them at a volume ratio of 1:(2-4) to prepare a mixed Bacillus bacterial solution. Then, mix the mixed Bacillus bacterial solution with an α-lactalbumin solution at a volume ratio of 10-30 mg / mL at a volume ratio of 1:(1-3). Add a calcium chloride solution at a concentration of 1.7-5 mM and stir at 25-37℃ and 100-200 rpm for 30-90 min. Centrifuge, wash and dry to obtain Bacillus aggregates. S2. Add bentonite to a 2-5 wt% polytrehalose solution and stir at 180-220 rpm for 30-60 min to prepare a polytrehalose-bentonite suspension; mix the polytrehalose-bentonite suspension and a 1-2 wt% humic acid solution at a volume ratio of 1:(5-10) and treat with an ultrasonic power of 300-500W for 15-20 min, then dry to obtain the modified polytrehalose-bentonite composite. S3. After activating Trichoderma harzianum and Bacillus aggregates, a compound bacterial suspension was prepared by mixing the compound bacterial suspension with Bacillus aggregates. The compound bacterial suspension and a modified polytrehalose-bentonite complex with a concentration of 10-15 wt% were then mixed at a volume ratio of (1-3):1 and stirred at 25-30℃ and 120-200 rpm for 20-40 min. The mixture was then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. Weigh 10-20 parts of compound microbial microcapsule preparation, 20-30 parts of corn straw biochar, 2-4 parts of chitosan oligosaccharide, 4.5-6.5 parts of mineral-derived potassium humate, and 1-2 parts of trace element chelation preparation, mix them evenly to prepare anti-replanting microbial fertilizer.

[0006] Preferably, the concentration of Bacillus subtilis in S1 is (0.5-2)×10⁻⁶.9 CFU / mL; Bacillus licheniformis bacterial concentration was (1-3.5)×10⁻⁶. 9 CFU / mL; the concentration of the mixed Bacillus bacterial suspension was (1-3)×10⁻⁶. 9 CFU / mL.

[0007] Preferably, the amount of calcium chloride solution added in S1 is 1 / 10 to 1 / 5 of the total volume of the mixed Bacillus bacterial solution and α-lactalbumin solution.

[0008] Preferably, the amount of bentonite added in S2 is 1-3 wt%, calculated as a polytrehalose solution.

[0009] Preferably, the concentration of *Gastrodia elata* in the S3 solution is (1-3) × 10⁻⁶. 8 The concentration of *Trichoderma harzianum* in the culture was (2.5-4) × 10⁻⁶ CFU / mL. 8 CFU / mL.

[0010] Preferably, the ratio of Trichoderma harzianum liquid, Trichoderma harzianum liquid and Bacillus aggregate in S3 is (0.5-2) mL: (1-3) mL: 0.1 g.

[0011] The anti-replanting microbial fertilizer prepared by any of the above methods.

[0012] Preferably, the application of the anti-replanting microbial fertilizer in crop cultivation. Beneficial effects

[0013] 1. This invention uses calcium ions to induce the adsorption of lactalbumin nanotubes onto the surfaces of Bacillus subtilis and Bacillus licheniformis, forming Bacillus aggregates and avoiding the influence of differences in the characteristics of different bacterial species. Calcium ions specifically electrostatically bind to the negatively charged groups of the lactalbumin molecular chain, causing the protein molecular chain to transition from a folded state to an extended state, exposing more hydrophobic regions and active sites. Calcium ions also act as a "bridging agent," connecting dispersed proteins into linear polymer chains, which then coil and helix to form closed nanotube structures. The amino and carboxyl groups on the surface of the lactalbumin nanotubes initially bind to the amino and teichoic acid phosphate groups of peptidoglycan in the Bacillus cell wall through electrostatic interactions, followed by enhanced binding through hydrogen bonds, hydrophobic interactions, and van der Waals forces. This allows a single Bacillus to bind multiple lactalbumin nanotubes, aggregating into Bacillus aggregates through a "bacterium-nanotube-bacterium" bridging effect, ensuring good stability of both Bacillus species.

[0014] 2. This invention utilizes a humic acid-modified polytrehalose-bentonite gel network to simultaneously encapsulate *Trichoderma harzianum*, *Bacillus*, and *Bacillus* polymers, producing a composite microcapsule formulation that avoids the impact of dominant pathogens in continuously cropped soils on the survival of the microorganisms. The polytrehalose polysaccharide molecular chains insert into the layered structure of bentonite, and its hydroxyl groups form hydrogen bonds with the hydroxyl groups of the bentonite layers. Molecular self-association forms cross-linking points, constituting a porous three-dimensional gel network that provides binding sites for the microorganisms. The carboxyl groups of humic acid form electrostatic salt bonds with exchangeable cations between the bentonite layers, and its carboxyl and phenolic hydroxyl groups form hydrogen bonds with the hydroxyl groups of the gel network, binding to the gel network, enhancing water absorption and retention capacity, increasing active adsorption sites, and strengthening physical strength, thus creating conditions for isolating pathogen interference. The carboxyl groups of the gel network are electrostatically attracted to the amino groups on the surface of the bacteria, and the hydroxyl groups form electrostatic interactions with the phosphate groups and carboxyl groups on the surface of the bacteria, achieving rapid and synchronous anchoring. Hydrogen bonds are also formed between the network and the bacteria, and hydrophobic interactions occur in the hydrophobic regions to enhance the binding, which is conducive to the formation of a stable embedding structure and reduces the threat of pathogens to the survival of the bacteria.

[0015] 3. The composite microcapsule formulation prepared in this invention is used in soils where crops are continuously planted. On the one hand, the aggregate formed by Bacillus subtilis and Bacillus licheniformis occupies the ecological niche of the red kidney bean root system and surrounding soil due to its structural advantage, competing for carbon, nitrogen, and oxygen, reducing the nutrient intake of pathogens. At the same time, both produce antibacterial substances that destroy the cell walls of pathogens. Trichoderma harzianum acts on pathogens in a parasitic manner, releasing hydrolytic enzymes to degrade virulence, and Aureobasidium globosum metabolizes to produce antioxidant active substances, reducing the infection channels of pathogens, thereby comprehensively improving the stress resistance of crop roots. On the other hand, Bacillus subtilis secretes phosphatase to decompose insoluble organic phosphorus in the planting soil for crop absorption, Bacillus licheniformis synthesizes transport proteins to promote the release of readily available potassium from insoluble potassium minerals, and Aureobasidium globosum activates the transport of trace elements to the roots. The humic acid in the gel network chelates soil nutrients to prevent loss, trehalose decomposes to promote the proliferation of composite strains, and bentonite improves soil aggregate structure, which is conducive to the efficient absorption of nutrients and root growth by crops, thereby improving crop yield and quality. Detailed Implementation

[0016] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: The bacterial strains used in this invention are: Bacillus subtilis CICC22109, Bacillus licheniformis CICC22069, Trichoderma harzianum CICC2652, and Trichoderma harzianum CICC13010, all of which were purchased from the China Industrial Microbial Culture Collection Center.

[0017] The activation process of the strain described in this invention is as follows: ① Activation of Bacillus subtilis: Take one loopful of Bacillus subtilis slant culture and inoculate it into LB liquid medium. Incubate at 37℃ and 180 rpm for 24 h with shaking to obtain Bacillus subtilis seed culture. Transfer the seed culture to LB liquid medium at a 2% inoculation rate and continue to incubate for 24 h. Centrifuge at 4000 rpm for 10 min to obtain Bacillus subtilis cells. Resuspend the cells in sterile water to obtain Bacillus subtilis bacterial culture for later use. ② Activation of Bacillus licheniformis: Take one loopful of Bacillus licheniformis slant culture and inoculate it into LB liquid medium. Incubate at 35℃ and 200 rpm for 24 h with shaking to obtain Bacillus licheniformis seed culture. Transfer the seed culture to fresh LB medium at an inoculation rate of 2.5% and continue incubation for 24 h. Centrifuge at 4000 rpm for 10 min to obtain Bacillus licheniformis cells. Resuspend the cells in sterile water to obtain Bacillus licheniformis bacterial culture for later use. ③ Activation of *Anomala*: Take one loopful of *Anomala* slant culture and inoculate it into PDA liquid medium. Incubate at 25℃ and 150 rpm for 72 h with shaking to obtain *Anomala* seed culture. Transfer the seed culture to fresh PDA liquid medium at a 5% inoculation rate and continue culturing for 48 h. Centrifuge at 3500 rpm for 10 min to obtain *Anomala* cells. Resuspend the cells in sterile water to obtain *Anomala* liquid for later use. ④ Trichoderma harzianum activation: Take one loopful of Trichoderma harzianum slant culture and inoculate it into PDA liquid medium. Incubate at 30℃ and 180 rpm for 48 h with shaking to obtain Trichoderma harzianum seed culture. Transfer the seed culture to fresh PDA liquid medium at an inoculation rate of 3.5% and continue to incubate for 48 h. Centrifuge at 3500 rpm for 10 min to obtain Trichoderma harzianum cells. Resuspend the cells in sterile water to obtain Trichoderma harzianum liquid for later use. Example 1

[0018] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1×10⁻⁶) 9 CFU / mL) and 60 mL of Bacillus licheniformis bacterial solution (bacterial concentration of 2×10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with a bacterial concentration of 1.75 × 10⁻⁶ CFU / mL. 9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 60 mL of α-lactalbumin solution with a concentration of 20 mg / mL, and slowly add 18 mL of 2 mM calcium chloride solution. Stir at 30 °C and 150 rpm for 60 min, centrifuge at 4000 rpm for 8 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; mix 10mL of the trehalose-bentonite suspension with 70mL of a 1.5% (w / w) humic acid solution and treat with an ultrasonic power of 300W for 15min, then dry to obtain the modified trehalose-bentonite composite. S3. Add 30 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8 The CFU / mL) was mixed with 1g of Bacillus aggregate to prepare a compound bacterial suspension; then 40mL of the compound bacterial suspension and 20mL of a 12wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30℃ and 150rpm for 30min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Example 2

[0019] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 2 × 10⁻⁶) 9 CFU / mL) and 60 mL of Bacillus licheniformis bacterial solution (strain concentration of 1×10⁻⁶ CFU / mL) 9 Prepare a mixed Bacillus bacterial suspension (CFU / mL) by mixing thoroughly (strain concentration of 1.25 × 10⁻⁶ CFU / mL). 9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 60 mL of α-lactalbumin solution with a concentration of 20 mg / mL, and slowly add 18 mL of 4 mM calcium chloride solution. Stir at 35 °C and 150 rpm for 60 min, centrifuge at 3500 rpm for 10 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; mix 10mL of the trehalose-bentonite suspension with 70mL of a 1.5% (w / w) humic acid solution and treat with an ultrasonic power of 300W for 15min, then dry to obtain the modified trehalose-bentonite composite. S3. Add 30 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8 The CFU / mL) was mixed with 1g of Bacillus aggregate to prepare a compound bacterial suspension; then 40mL of the compound bacterial suspension and 20mL of a 12wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30℃ and 150rpm for 30min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Example 3

[0020] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1.5 × 10⁻⁶). 9 CFU / mL) and 40 mL of Bacillus licheniformis bacterial suspension (strain concentration of 2.5 × 10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with the bacterial concentration of 2.17 × 10⁻⁶ CFU / mL. 9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 60 mL of α-lactalbumin solution with a concentration of 20 mg / mL, and slowly add 13 mL of 2 mM calcium chloride solution. Stir at 30 °C and 150 rpm for 60 min, centrifuge at 4000 rpm for 8 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; mix 10mL of the trehalose-bentonite suspension with 70mL of a 1.5% (w / w) humic acid solution and treat with an ultrasonic power of 300W for 15min, then dry to obtain the modified trehalose-bentonite composite. S3. Add 30 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8The CFU / mL) was mixed with 1g of Bacillus aggregate to prepare a compound bacterial suspension; then 40mL of the compound bacterial suspension and 20mL of a 12wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30℃ and 150rpm for 30min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Example 4

[0021] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1×10⁻⁶) 9 CFU / mL) and 60 mL of Bacillus licheniformis bacterial solution (bacterial concentration of 2×10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with a bacterial concentration of 1.75 × 10⁻⁶ CFU / mL. 9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 90 mL of α-lactalbumin solution with a concentration of 10 mg / mL, and slowly add 12 mL of 2 mM calcium chloride solution. Stir at 30 °C and 150 rpm for 60 min, centrifuge at 3500 rpm for 10 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; mix 10mL of the trehalose-bentonite suspension with 70mL of a 1.5% (w / w) humic acid solution and treat with an ultrasonic power of 400W for 15min, then dry to obtain the modified trehalose-bentonite composite. S3. Add 30 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8 The CFU / mL) was mixed with 1g of Bacillus aggregate to prepare a compound bacterial suspension; then 40mL of the compound bacterial suspension and 20mL of a 12wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30℃ and 150rpm for 30min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Example 5

[0022] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1.5 × 10⁻⁶). 9 CFU / mL) and 40 mL of Bacillus licheniformis bacterial suspension (strain concentration of 2.5 × 10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with the bacterial concentration of 2.17 × 10⁻⁶ CFU / mL. 9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 60 mL of α-lactalbumin solution with a concentration of 20 mg / mL, and slowly add 13 mL of 2 mM calcium chloride solution. Stir at 30 °C and 150 rpm for 60 min, centrifuge at 4000 rpm for 8 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of 2% polytrehalose solution and stir at 200rpm for 30min to prepare polytrehalose-bentonite suspension; mix 10mL of polytrehalose-bentonite suspension with 70mL of 2% humic acid solution and treat with ultrasonic power of 400W for 15min, and dry to obtain modified polytrehalose-bentonite composite. S3. Add 30 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8 The CFU / mL) was mixed with 1g of Bacillus aggregate to prepare a compound bacterial suspension; then 40mL of the compound bacterial suspension and 20mL of a 12wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30℃ and 150rpm for 30min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Example 6

[0023] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1.5 × 10⁻⁶). 9 CFU / mL) and 40 mL of Bacillus licheniformis bacterial suspension (strain concentration of 2.5 × 10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with the bacterial concentration of 2.17 × 10⁻⁶ CFU / mL. 9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 60 mL of α-lactalbumin solution with a concentration of 20 mg / mL, and slowly add 13 mL of 2 mM calcium chloride solution. Stir at 30 °C and 150 rpm for 60 min, centrifuge at 4000 rpm for 8 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of 5% polytrehalose solution and stir at 200rpm for 30min to prepare a polytrehalose-bentonite suspension; mix 10mL of polytrehalose-bentonite suspension with 50mL of 1.5% humic acid solution and treat with ultrasonic power of 300W for 15min, then dry to obtain the modified polytrehalose-bentonite composite. S3. Add 30 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 30 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8 The CFU / mL) was mixed with 1g of Bacillus aggregate to prepare a compound bacterial suspension; then 40mL of the compound bacterial suspension and 20mL of a 12wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30℃ and 150rpm for 30min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Example 7

[0024] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1.5 × 10⁻⁶). 9 CFU / mL) and 40 mL of Bacillus licheniformis bacterial suspension (strain concentration of 2.5 × 10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with the bacterial concentration of 2.17 × 10⁻⁶ CFU / mL.9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 60 mL of α-lactalbumin solution with a concentration of 20 mg / mL, and slowly add 13 mL of 2 mM calcium chloride solution. Stir at 35℃ and 150 rpm for 60 min, centrifuge at 3500 rpm for 10 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; mix 10mL of the trehalose-bentonite suspension with 70mL of a 1.5% (w / w) humic acid solution and treat with an ultrasonic power of 400W for 15min, then dry to obtain the modified trehalose-bentonite composite. S3. Add 30 mL of *Agropyron cristatum* solution (inoculum concentration of 1.5 × 10⁻⁶). 8 CFU / mL), 30 mL of Trichoderma harzianum (inoculum concentration of 4×10⁻⁶ CFU / mL), 8 The CFU / mL) was mixed with 1g of Bacillus aggregate to prepare a compound bacterial suspension; then 40mL of the compound bacterial suspension and 20mL of a 12wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30℃ and 150rpm for 30min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Example 8

[0025] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1.5 × 10⁻⁶). 9 CFU / mL) and 40 mL of Bacillus licheniformis bacterial suspension (strain concentration of 2.5 × 10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with the bacterial concentration of 2.17 × 10⁻⁶ CFU / mL. 9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 60 mL of α-lactalbumin solution with a concentration of 20 mg / mL, and slowly add 13 mL of 2 mM calcium chloride solution. Stir at 30 °C and 150 rpm for 60 min, centrifuge at 4000 rpm for 8 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; mix 10mL of the trehalose-bentonite suspension with 70mL of a 1.5% (w / w) humic acid solution and treat with an ultrasonic power of 300W for 15min, then dry to obtain the modified trehalose-bentonite composite. S3. Add 20 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8 The CFU / mL) was mixed with 1g of Bacillus aggregate to prepare a compound bacterial suspension; then 40mL of the compound bacterial suspension and 20mL of a 12wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30℃ and 150rpm for 30min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Example 9

[0026] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1.5 × 10⁻⁶). 9 CFU / mL) and 40 mL of Bacillus licheniformis bacterial suspension (strain concentration of 2.5 × 10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with the bacterial concentration of 2.17 × 10⁻⁶ CFU / mL. 9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 60 mL of α-lactalbumin solution with a concentration of 20 mg / mL, and slowly add 13 mL of 2 mM calcium chloride solution. Stir at 30 °C and 150 rpm for 60 min, centrifuge at 4000 rpm for 8 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; mix 10mL of the trehalose-bentonite suspension with 70mL of a 1.5% (w / w) humic acid solution and treat with an ultrasonic power of 300W for 15min, then dry to obtain the modified trehalose-bentonite composite. S3. Add 20 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8 The CFU / mL) was mixed with 1g of Bacillus aggregate to prepare a compound bacterial suspension; then 40mL of the compound bacterial suspension and 20mL of a 12wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30℃ and 150rpm for 30min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh 40g of compound microbial microcapsule preparation, 50g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation; then put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Then add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping.

[0027] Comparative Example 1

[0028] The difference between this comparative example and Example 8 is that α-lactalbumin is replaced with chitosan.

[0029] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1.5 × 10⁻⁶). 9 CFU / mL) and 40 mL of Bacillus licheniformis bacterial suspension (strain concentration of 2.5 × 10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with the bacterial concentration of 2.17 × 10⁻⁶ CFU / mL. 9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 60 mL of chitosan solution with a concentration of 20 mg / mL, and slowly add 13 mL of 2 mM calcium chloride solution. Stir at 30 °C and 150 rpm for 60 min, centrifuge at 4000 rpm for 8 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; mix 10mL of the trehalose-bentonite suspension with 70mL of a 1.5% (w / w) humic acid solution and treat with an ultrasonic power of 300W for 15min, then dry to obtain the modified trehalose-bentonite composite. S3. Add 20 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8The CFU / mL) was mixed with 1g of Bacillus aggregate to prepare a compound bacterial suspension; then 40mL of the compound bacterial suspension and 20mL of a 12wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30℃ and 150rpm for 30min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Comparative Example 2

[0030] The difference between this comparative example and Example 8 is that α-lactalbumin and calcium chloride are not added.

[0031] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1.5 × 10⁻⁶). 9 CFU / mL) and 40 mL of Bacillus licheniformis bacterial suspension (strain concentration of 2.5 × 10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with the bacterial concentration of 2.17 × 10⁻⁶ CFU / mL. 9 (CFU / mL) S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; mix 10mL of the trehalose-bentonite suspension with 70mL of a 1.5% (w / w) humic acid solution and treat with an ultrasonic power of 300W for 15min, then dry to obtain the modified trehalose-bentonite composite. S3. Add 20 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8 The CFU / mL solution was mixed with 20 mL of mixed Bacillus bacterial solution to prepare a compound bacterial mixture; then 40 mL of the compound bacterial mixture and 20 mL of 12 wt% modified polytrehalose-bentonite complex solution were mixed and stirred at 30 °C and 150 rpm for 30 min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Comparative Example 3

[0032] The difference between this comparative example and Example 8 is that humic acid modification was not used.

[0033] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1.5 × 10⁻⁶). 9 CFU / mL) and 40 mL of Bacillus licheniformis bacterial suspension (strain concentration of 2.5 × 10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with the bacterial concentration of 2.17 × 10⁻⁶ CFU / mL. 9 (CFU / mL), then mix 30 mL of mixed Bacillus bacterial culture and 60 mL of α-lactalbumin solution with a concentration of 20 mg / mL, and slowly add 13 mL of 2 mM calcium chloride solution. Stir at 30 °C and 150 rpm for 60 min, centrifuge at 4000 rpm for 8 min, wash 3 times with sterile physiological saline, and dry to obtain Bacillus aggregates. S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; S3. Add 20 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8 A compound bacterial suspension was prepared by mixing 1 g of Bacillus aggregate with 40 mL of the compound bacterial suspension and 20 mL of polytrehalose-bentonite suspension at 30 °C and 150 rpm for 30 min, and then freeze-dried to obtain the compound bacterial microcapsule formulation. S4. First, weigh out 30g of compound microbial microcapsule preparation, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating preparation. Then, put the compound microbial microcapsule preparation and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15 minutes. Next, add chitosan oligosaccharide, mineral-derived potassium humate, and trace element chelating preparation and mix well. Package the mixture to make a microbial fertilizer resistant to continuous cropping. Comparative Example 4

[0034] The difference between this comparative example and Example 8 is that only the compound bacterial mixture was used to prepare the anti-replanting microbial fertilizer.

[0035] A method for preparing a microbial fertilizer resistant to continuous cropping includes the following steps: S1. Add 20 mL of Bacillus subtilis bacterial suspension (bacterial concentration of 1.5 × 10⁻⁶). 9 CFU / mL) and 40 mL of Bacillus licheniformis bacterial suspension (strain concentration of 2.5 × 10⁻⁶ CFU / mL) 9 Mix well to prepare a mixed Bacillus bacterial suspension (CFU / mL) with the bacterial concentration of 2.17 × 10⁻⁶ CFU / mL. 9 (CFU / mL) S2. Add 2g of bentonite to 100mL of a 3.5% (w / w) trehalose solution and stir at 200rpm for 30min to prepare a trehalose-bentonite suspension; mix 10mL of the trehalose-bentonite suspension with 70mL of a 1.5% (w / w) humic acid solution and treat with an ultrasonic power of 300W for 15min, then dry to obtain the modified trehalose-bentonite composite. S3. Add 20 mL of *Agrostis spp.* solution (strain concentration 2 × 10⁻⁶) to the solution. 8 CFU / mL), 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL), and 20 mL of Trichoderma harzianum (inoculum concentration of 3×10⁻⁶ CFU / mL). 8 A compound bacterial mixture was prepared by mixing (CFU / mL) with 20 mL of mixed Bacillus bacterial solution. S4. First, take 30mL of compound bacterial mixture, 40g of corn straw biochar, 6g of chitosan oligosaccharide, 10g of mineral-derived potassium humate, and 3g of trace element chelating agent; then put the compound bacterial mixture and corn straw biochar into a horizontal ribbon mixer and stir at low speed for 15min. Then add chitosan oligosaccharide, mineral-derived potassium humate and trace element chelating agent and mix well. Package to make anti-replanting microbial fertilizer.

[0036] Performance testing Effective viable bacteria count, moisture content, pH and fineness According to the "Inspection Procedures for Microbial Fertilizer Products" (NY / T2321-2013), the effective viable bacteria count of microbial fertilizers is determined using the plate count method.

[0037] Moisture content According to the "Inspection Procedures for Microbial Fertilizer Products" (NY / T2321-2013), the moisture content of microbial fertilizers is determined by the direct drying method.

[0038] pH According to the "NY / T2321-2013 Inspection Procedure for Microbial Fertilizer Products", the pH of microbial fertilizers is determined using a pH meter.

[0039] Fineness According to the "Inspection Procedures for Microbial Fertilizer Products" (NY / T2321-2013), the fineness of microbial fertilizers is determined by the dry sieving method.

[0040] Table 1. Effective viable count, moisture content, pH, and fineness of Examples 1-8 and Comparative Examples 1-4

[0041] As shown in Table 1, the effective viable count, moisture content, pH, and fineness of Examples 1-9 were all superior to those of Comparative Examples 1-4. This indicates that the use of calcium chloride to induce α-lactalbumin to form nanotubes through calcium ion bridging, which then form electrostatic bonds and hydrogen bonds with the Bacillus cell wall, reducing interference from differences in characteristics between strains. The humic acid-modified trehalose-bentonite encapsulation to construct a porous three-dimensional gel network isolates the strains from external conditions, ensuring good survival stability and thus guaranteeing the quality stability of the microbial fertilizer. In contrast, Comparative Examples 1-4, due to the substitution of raw materials, omission of key steps, or simplification of processes, compromised strain protection and product uniformity, resulting in microbial fertilizers of lower quality than those of Examples 1-9. Example 10

[0042] Experiment on the Application Effect of Anti-Continuous Cropping Microbial Fertilizer in Red Kidney Bean Cultivation A red kidney bean planting experiment was conducted using a scheme that "for every 100 kg of red kidney bean seeds produced, the required nitrogen (N) is approximately 5.49 kg, the required phosphorus (P₂O₅) is approximately 1.33 kg, and the required potassium (K₂O) is approximately 4.90 kg". 30 mu (approximately 2 hectares) of soil from continuous red kidney bean planting was randomly divided into six treatment groups: a blank control group, Example 9 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, and Comparative Example 4 group. Each treatment covered an area of ​​0.33 hectares. 2 (5 mu), the planting density of red kidney beans was 185,000 plants / hm. 2 The row spacing is 50cm, the plant spacing is 30cm, and the fertilization treatment plan is shown in Table 2.

[0043] Table 2 Fertilization treatment schemes for each group in the red kidney bean planting experiment.

[0044] Note: Phosphorus nitrate (N≥26.5%, P2O5≥11.5%), urea (N≥46%).

[0045] After the red kidney bean planting experiment was completed, the soil physicochemical indicators, red kidney bean growth indicators, and quality indicators were analyzed to evaluate the effect of the anti-replanting microbial fertilizer.

[0046] (1) Soil nutrient determination Soil samples from the top 0-20cm layer were taken before and after the experiment using the serpentine marking method. The samples were thoroughly mixed, air-dried, and then used for testing. The contents of organic matter, available nitrogen, available phosphorus, and available potassium in the soil were then determined.

[0047] Table 3. Contents of organic matter, available nitrogen, available phosphorus, and available potassium in the soil after application of microbial fertilizer.

[0048] Table 3 shows that the soil nutrient content of the anti-replanting microbial fertilizer prepared in Example 9 was higher than that of the blank control group and comparative groups 1-4. This indicates that the microbial fertilizer can significantly increase the content of organic matter, available nitrogen, available phosphorus, and available potassium in the soil, effectively improving the nutrient status of soils subjected to continuous cropping. Compared with other treatment groups, the Example 9 group performed best in all indicators, indicating that the synergistic effect of functional bacteria and organic carriers in its formula is more conducive to nutrient activation and retention, creating a favorable soil environment for the growth of red kidney beans.

[0049] (2) Growth indicators During the maturity period of red kidney beans, 20 uniform and representative red kidney bean plants were selected from each treatment, and their plant height and pod length were measured with a measuring tape.

[0050] Protein and fat For each treatment, 200g of red kidney bean sample was weighed to determine its protein and fat content. Protein was determined using the fully automated Kjeldahl nitrogen determination method, and fat was determined using the Soxhlet extraction method.

[0051] Table 4. Plant height, pod length, protein and fat content of red kidney beans.

[0052] Table 4 shows that the red kidney beans in Example 9 group were significantly higher than those in other groups in terms of plant height, pod length, protein content, and fat content. Specifically, plant height increased by 24.4% compared to the control group, pod length increased by 24.2%, protein content increased by 14.0%, and fat content increased by 20.6%. This indicates that applying this microbial fertilizer not only improves soil nutrients but also effectively promotes the growth and development of red kidney bean plants and enhances the nutritional quality of the seeds. While the comparative groups showed some improvement, their effects were not as significant as those in Example 9 group, further validating the superiority and stability of this method. Example 11

[0053] Experiment on the Application Effect of Anti-Continuous Cropping Microbial Fertilizer in Potato Planting Thirty mu (approximately 6.7 hectares) of soil from continuous cropping was randomly divided into six treatment groups: a blank control group, Example 9 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, and Comparative Example 4 group. The experimental area for each treatment was 0.33 hectares. 2(5 mu), row spacing 0.6m, plant spacing 0.3m, planting density is 60,000 plants / hm. 2 From the date of planting, compound fertilizer (N:P2O5:K2O=11:17:22) and anti-continuous cropping microbial fertilizer were applied to the experimental field once before sowing according to the experimental design dosage (Table 5). Topdressing was applied to the five treatment groups in a timely manner during the potato tuber enlargement period. All other cultivation, irrigation and field management measures were carried out uniformly and consistently according to conventional methods.

[0054] Table 5. Fertilization treatment schemes for each group in the potato planting experiment.

[0055] After the potato planting trial, the growth and quality indicators of the potatoes were analyzed to evaluate the effect of the anti-replanting microbial fertilizer.

[0056] (1) Growth indicators Potato plant height and emergence rate were measured, with a sampling area of ​​0.5m × 2m. Plant height was measured using a ruler, from the base of the plant stem to the growing point. Emergence rate was calculated based on the number of seedlings in the sampling area.

[0057] (2) Quality Indicators The dry matter content of potatoes was determined by the dry weight method; the protein content was determined by the Kjeldahl nitrogen determination method; the starch content was determined by the anthrone method; the vitamin C content was determined by the 2,6-dichlorophenolindophenol titration method; and the reducing sugar content was determined by the 3,5-dinitrosalicylic acid colorimetric method (DNS).

[0058] Table 6. Growth and quality indicators of potatoes

[0059] Table 6 shows that the plant height, emergence rate, and quality indicators of Group 9 were superior to other groups. The plant height reached 98.65 cm, and the emergence rate was 92.56%, significantly higher than the blank control group and other comparative groups. The protein content was 2.85 g / 100 g, the vitamin C content was 45.83 mg / 100 g, and the reducing sugar content was the lowest at 0.21 g / 100 g, indicating that the anti-replanting microbial fertilizer effectively promoted potato growth and improved its nutritional quality. Meanwhile, the dry matter content of Group 9 was 20.85 g / 100 g, slightly higher than the control group and other comparative groups, indicating that the fertilizer also had a positive effect on improving the accumulation of nutrients in potato tubers. The indicators of each comparative group were close to those of the blank control group, indicating that a single component or the absence of key microbial species significantly weakened the fertilizer effect. In summary, the anti-replanting microbial fertilizer effectively improved potato growth vigor and nutritional quality by optimizing the rhizosphere microecology, and has significant application value.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing a microbial fertilizer resistant to continuous cropping, characterized in that, Includes the following steps: S1. Activated Bacillus subtilis and Bacillus licheniformis were mixed at a volume ratio of 1:(1.5-4) to prepare a mixed Bacillus bacterial suspension. The mixed Bacillus bacterial suspension and an α-lactalbumin solution with a concentration of 10-30 mg / mL were then mixed at a volume ratio of 1:(1-3). A calcium chloride solution with a concentration of 1.7-5 mM was added, and the mixture was stirred at 25-37℃ and 100-200 rpm for 30-90 min. After centrifugation, washing, and drying, Bacillus aggregates were obtained. The concentration of the Bacillus subtilis bacterial suspension was (0.5-3) × 10⁻⁶. 9 CFU / mL; Bacillus licheniformis bacterial concentration was (1-3.5)×10⁻⁶. 9 CFU / mL; the concentration of the mixed Bacillus bacterial suspension was (1-3)×10⁻⁶. 9 CFU / mL; The amount of calcium chloride solution added in S1 is 1 / 10 to 1 / 5 of the total volume of the mixed Bacillus bacterial solution and α-lactalbumin solution; S2. Add 1-3.5 wt% bentonite to a 2-5 wt% polytrehalose solution and stir at 180-220 rpm for 30-60 min to prepare a polytrehalose-bentonite suspension; mix the polytrehalose-bentonite suspension and a 1-2 wt% humic acid solution at a volume ratio of 1:(5-10) and treat with an ultrasonic power of 300-500W for 15-20 min, and dry to obtain the modified polytrehalose-bentonite composite. S3. After activating Trichoderma harzianum and mixing it with Bacillus aggregates, a compound bacterial suspension is prepared. The compound bacterial suspension and a modified polytrehalose-bentonite complex solution with a concentration of 10-15wt% are then mixed at a volume ratio of (1-3):1 and stirred at 25-30℃ and 120-200rpm for 20-40min. The mixture is then freeze-dried to obtain a compound bacterial microcapsule formulation. S4. Weigh 10-20 parts of compound microbial microcapsule preparation, 20-30 parts of corn straw biochar, 2-4 parts of chitosan oligosaccharide, 4.5-6.5 parts of mineral-derived potassium humate, and 1-2 parts of trace element chelation preparation, mix them evenly to prepare anti-replanting microbial fertilizer.

2. The method for preparing a replant-resistant microbial fertilizer according to claim 1, characterized in that: The concentration of *Gastrodia elata* in the S3 culture was (1-3)×10⁻⁶. 8 The concentration of *Trichoderma harzianum* in the culture was (2.5-4) × 10⁻⁶ CFU / mL. 8 CFU / mL.

3. The method for preparing a replant-resistant microbial fertilizer according to claim 1, characterized in that: The ratio of *Trichoderma harzianum* solution, *Trichoderma harzianum* solution, and *Bacillus* aggregate in S3 is (0.5-2) mL: (1-3) mL: 0.1 g.

4. The anti-replanting microbial fertilizer prepared according to any one of claims 1-3.

5. The application of the anti-replanting microbial fertilizer according to claim 4 in crop cultivation.

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