Acid and alkali resistant rhizosphere growth promoting megaspora cyphella and preparation method thereof

Abstract

This invention relates to the field of microbial agriculture technology, specifically to an acid- and alkali-resistant rhizosphere growth-promoting *Sordaria macrospora* and its preparation method. Based on the total mass of the preparation, it is composed of the following raw materials in the following percentages by mass: *Sordaria macrospora* bacterial solution 50%, carrier 40%, protectant 6%, and growth-promoting adjuvant 4%. The strain of the *Sordaria macrospora* bacterial solution is *Sordaria macrospora* XJ-01, with the strain preservation number CGMCCNO.29081. This strain can grow normally in an environment with a pH of 2.0 to 12.0 and a viable count of not less than 1.0 × 10⁻⁶. 9 CFU / mL. The acid- and alkali-resistant rhizosphere growth-promoting macrosporum and its preparation method provided by this invention effectively solve the technical problems of existing rhizosphere growth-promoting bacteria having difficulty surviving, poor colonization, and unsatisfactory growth-promoting effects in extremely acidic and alkaline soils.

Description

Technical Field

[0001] This invention relates to the field of microbial agriculture technology, specifically to an acid- and alkali-resistant rhizosphere growth-promoting macrosporum foetida and its preparation method. Background Technology

[0002] Rhizosphere growth-promoting bacteria, as a core application material in microbial agriculture, can promote crop growth through nitrogen fixation, phosphorus solubilization, and secretion of plant growth regulators, while improving soil nutrient status. They have broad application prospects in the fields of improving agricultural quality and efficiency and soil ecological restoration.

[0003] However, existing rhizosphere growth-promoting bacteria have significant technical shortcomings, making it difficult to adapt to the production needs of extremely acidic and alkaline soils. This has become a key issue restricting their widespread application in low-yield soils such as saline-alkali land and acidic red soil. Existing strains have a narrow range of acid and alkali tolerance, are easily inactivated in extremely acidic and alkaline environments, and cannot stably colonize, thus failing to exert their growth-promoting function. Although some strains have undergone simple modifications, their acid and alkali tolerance stability and growth-promoting ability have only been improved to a limited extent. Moreover, most of these methods rely on single domestication or mutagenesis techniques and lack synergistic improvement designs.

[0004] Meanwhile, existing culture systems often use single carbon and nitrogen sources and simple buffers, which cannot provide comprehensive nutrition for bacterial growth. This leads to pH fluctuations during culture, resulting in slow bacterial proliferation and low secretion of growth-promoting metabolites. In addition, existing bacterial agent preparation processes lack targeted protection and carrier design, resulting in high inactivation rates and poor storage stability of the strains during storage. After being applied to the soil, they are difficult to colonize in the rhizosphere, further reducing their effectiveness in field applications.

[0005] Currently, commercially available conventional rhizosphere growth-promoting bacteria preparations are more suitable for neutral soils, and their activity drops significantly in acidic and alkaline soils, failing to meet the needs of agricultural production for adaptability to extreme soil environments. Therefore, the development of a rhizosphere growth-promoting bacteria with strong acid and alkali resistance, excellent growth-promoting effect, stable storage and good field colonization, as well as its preparation method, has become an urgent need in the field of microbial agriculture. Summary of the Invention

[0006] The primary objective of this invention is to provide an acid- and alkali-resistant rhizosphere growth-promoting macrosporum foetida and its preparation method.

[0007] A further objective of this invention is to provide an acid- and alkali-resistant rhizosphere growth-promoting Sordaria macrospora preparation, which, based on the total mass of the preparation, comprises the following raw materials in the following percentages by mass: 50% Sordaria macrospora bacterial solution, 40% carrier, 6% protectant, and 4% growth-promoting adjuvant; the strain of the Sordaria macrospora bacterial solution is Sordaria macrospora, with the strain preservation number CGMCC NO. 29081, deposited at the China General Microbiological Culture Collection Center on November 21, 2023. This strain can grow normally in an environment with a pH of 2.0 to 12.0 and a viable count of not less than 1.0 × 10⁻⁶. 9 The strain, with a concentration of CFU / mL, possesses rhizosphere growth-promoting functions including nitrogen fixation, phosphorus solubilization, and indoleacetic acid secretion. The carrier is a mixture of diatomaceous earth and corn straw powder at a mass ratio of 3:1; the protectant is a mixture of sodium alginate and chitosan at a mass ratio of 2:1; and the growth-promoting adjuvant is a mixture of humic acid and potassium dihydrogen phosphate at a mass ratio of 5:1. After 12 months of storage, the viable bacterial count of this preparation is no less than 1 × 10⁻⁶. 9 CFU / g.

[0008] Preferably, the *Fasciola heliotropium* bacterial solution is obtained after gradient acclimatization, ultraviolet mutagenesis, culture in a compound carbon and nitrogen source medium, and staged feeding and stress induction. The bacterial solution has an indoleacetic acid content of not less than 37.9 mg / L, a phosphorus solubility of not less than 29.9 mg / L, and a nitrogenase activity of not less than 18.9 nmol / (mL·h).

[0009] Preferably, the formulation is in granular form with a particle size of 1.2 mm and a moisture content of 12%.

[0010] A method for preparing the acid- and alkali-resistant rhizosphere growth-promoting macrosporum preparation includes the following steps: (1) Concentration of bacterial culture: The bacterial culture of *Fasciola heliotropium* was concentrated by centrifugation at a speed of 7000 r / min for 15 minutes until the viable count was not less than 5 × 10⁻⁶. 9 CFU / mL was used to obtain a concentrated bacterial solution. (2) Mixing and stirring: Add the concentrated bacterial solution and the protectant to a sterile mixing tank and stir evenly. Let it stand for 30 minutes, then add the carrier and growth promoter and continue stirring to form a uniform mixture; (3) Molding and drying: After granulation, the mixture is placed in a constant temperature drying oven at 35℃ and dried until the moisture content of the mixture is 12% to obtain granular preparation; (4) Packaging and storage: After aseptic packaging, the granular preparations are stored in a cool and dry place.

[0011] Preferably, in step (2), the stirring conditions for the concentrated bacterial solution and the protectant are 25°C and 100r / min; the stirring conditions after adding the carrier and the growth promoter are 28°C and 120r / min, and the stirring time is 30 minutes.

[0012] Preferably, in step (3), the particle size of the granulated mixture is 1.2 mm.

[0013] Preferably, the preparation of the *Macrobrachium cladosporum* bacterial solution includes the following steps: (1) Strain screening: wheat rhizosphere soil from the boundary area between saline-alkali land and acidic red soil was collected to prepare the inoculum. After being serially diluted and spread on screening medium with pH 2.0 to 12.0, the inoculum was purified and screened to obtain Sordaria macrospora, which meets the requirements for growth and viable number of bacteria. (2) Synergistic improvement: The selected strains were subjected to gradient domestication and ultraviolet mutagenesis synergistic treatment to obtain target strains with improved acid and alkali resistance and growth promotion ability; (3) Optimized culture: The target strain was inoculated into an optimized culture medium containing a compound carbon and nitrogen source and a compound buffer system. The culture was carried out by a combination of staged feeding culture and stress induction to obtain the culture liquid of *Cytomyces coccidioides*.

[0014] Preferably, in step (2), the initial culture medium pH for gradient acclimatization is 7.0. The pH is decreased by 1.0 for acidity and increased by 1.0 for alkalinity, gradually adjusted to pH 2.0 and pH 12.0. Each pH gradient is cultured for 3 generations, with each generation cultured for 72 hours. The culture conditions are 25℃ and 150r / min shaking culture. For ultraviolet induction, the bacterial solution of the acclimatized strain is adjusted to an OD600 value of 0.6 and inducing mutation for 45 seconds with an ultraviolet lamp at a distance of 30 cm and a power of 20 watts. After mutation, it is kept in the dark for 1 hour. The target strain is obtained by primary screening and secondary screening for growth promotion ability.

[0015] Preferably, in step (3), the carbon source of the optimized culture medium is a composite carbon source of 3% corn flour, 3% wheat bran and 2% glucose, the nitrogen source is a composite nitrogen source of 2% soybean meal, 1% yeast extract and 0.5% urea, the buffer is a composite buffer system of 1.5% potassium dihydrogen phosphate-dipotassium hydrogen phosphate and 0.5% sodium bicarbonate, and 0.01% magnesium sulfate and 0.005% ferrous sulfate are added at the same time; all percentages are based on the total mass of the optimized culture medium.

[0016] Preferably, in step (3), the staged feeding culture is as follows: during the 0-48 hour proliferation period, a carbon source mixture of corn flour and glucose in a mass ratio of 1:1 is added at 24 hours, with the addition amount being 30% of the total mass of the initial culture medium carbon source; during the 48-96 hour metabolite secretion period, a nitrogen source mixture of soybean meal and yeast extract in a mass ratio of 2:1 is added at 60 hours, with the addition amount being 25% of the total mass of the initial culture medium nitrogen source, and 0.2% proline is added simultaneously; during the stress induction period, the pH is 7.0 from 0 to 24 hours, and from 24 to 48 hours, the pH is adjusted by 1.0 pH unit every 6 hours to pH 2.0 or pH 12.0, and from 48 to 96 hours, the pH is maintained at pH 2.0 or pH 12.0; the percentage of proline is based on the total mass of the culture medium after feeding.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. The acid- and alkali-resistant rhizosphere growth-promoting macrosporum and its preparation method provided by the present invention effectively solve the technical problems of existing rhizosphere growth-promoting bacteria having difficulty surviving, poor colonization, and poor growth-promoting effect in extremely acidic and alkaline soils.

[0018] 2. The *Coprinus macrosporus* strain obtained by screening and synergistic improvement in this invention has a significantly broadened range of acid and alkali tolerance, and can grow normally in extreme acid and alkali environments. At the same time, it has comprehensive rhizosphere growth-promoting functions such as nitrogen fixation, phosphorus solubilization, and secretion of indoleacetic acid. It can stably colonize in acidic and alkaline soils, laying a core foundation for field application.

[0019] 3. The synergistic treatment of gradient domestication and ultraviolet mutagenesis in this invention enhances the acid and alkali resistance and growth-promoting ability of the strain at the genetic level, with effects far superior to single improvement methods. The optimized composite carbon and nitrogen source and composite buffer system of this invention can meet the nutritional needs of the strain at different growth stages, stabilize the pH of the culture environment, and significantly improve the strain's proliferation rate and the secretion of growth-promoting metabolites. The combination of staged fed culture and stress induction further enhances the strain's stress resistance and promotes the secretion of acid and alkali resistance-related enzymes and growth-promoting substances.

[0020] 4. The formulation of the microbial agent of this invention is scientific. The carrier, protectant and growth promoter work synergistically to improve the storage stability of the strain, extend the shelf life, and increase the rhizosphere colonization rate of the strain in the soil, thus ensuring the field application effect.

[0021] 5. When applied in the field, the strains and preparations of this invention can not only significantly promote crop growth and increase crop yield and quality, but also effectively improve soil nutrient status, achieving the dual effect of promoting growth and improving soil. They can be widely used in agricultural production in low-yield soils such as saline-alkali land and acidic red soil, providing a new and effective solution for the promotion and application of microbial agriculture in extreme soil environments. At the same time, the technical solution of this invention has a wide range of raw material selection, and the cultivation and preparation process is easy to realize industrial production, thus having good industrialization prospects. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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] Example 1:

[0024] (1) Strain screening: Rhizosphere soil samples from wheat plants in the boundary area between saline-alkali and acidic red soil were collected. Stones, weeds, and other impurities were removed. 10 grams of soil sample were added to 90 ml of 0.85% sterile physiological saline and placed in a constant-temperature shaker at 25°C and 150 rpm for 30 minutes. After removal, the sample was allowed to settle for 30 minutes, and the clear supernatant was used as the inoculum. A gradient dilution plating method was used to sequentially dilute the inoculum to 10-7 times. 0.1 ml of each dilution was evenly spread onto screening media at different pH values. The screening media used corn flour and sucrose as the basic carbon source and peptone as the basic nitrogen source. The pH was adjusted with 0.5% potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, setting five gradients: pH 2.0, pH 3.0, pH 7.0, pH 11.0, and pH 12.0. 0.1% rare earth solution was added as a selector to promote the growth of the target strain. The screening media were autoclaved at 121°C for 20 minutes and cooled to 30°C before use.

[0025] The spread culture medium was statically incubated at 25℃ for 120 hours. Colony morphology was observed, and single colonies with regular morphology, vigorous growth, and no contamination were selected and inoculated into slant culture media of corresponding pH values. The cultures were subcultured three times at 25℃ for 72 hours each time to obtain purified single-line strains. The purified strains were then inoculated into liquid culture media of different pH values ​​and cultured at 25℃ with shaking at 150 rpm for 72 hours. The viable cell count was determined using the plate count method, and strains that could grow normally within the pH range of 2.0 to 12.0 with a viable cell count of not less than 1.0 × 10⁻⁶ were selected. 9 The strain with CFU / mL was identified as Sordaria macrospora by morphological identification and 16S rRNA gene sequencing. The strain has the preservation number CGMCCNO.29081. It has rhizosphere growth-promoting functions such as nitrogen fixation, phosphorus solubilization, and indoleacetic acid production. It can stably colonize in extremely acidic and alkaline soils, significantly promote crop growth and improve soil nutrient status.

[0026] (2) Gradient acclimatization and ultraviolet mutagenesis: To enhance the acid and alkali resistance and growth-promoting ability of the strain, the selected *Fasciola heliotropium* was subjected to a combined gradient acclimatization and ultraviolet mutagenesis treatment. Gradient acclimatization involved gradually adjusting the pH of the culture medium. The initial pH was 7.0, and the pH was adjusted after each generation, decreasing by 1.0 for acidic conditions and increasing by 1.0 for alkaline conditions, gradually adjusting to pH 2.0 and pH 12.0. Each pH gradient was cultured for three generations, with each generation lasting 72 hours. The culture conditions were 25℃ and 150 rpm with shaking, resulting in acid and alkali acclimatized strains. The acclimatized bacterial culture was adjusted to an OD600 value of 0.6 by ultraviolet mutagenesis treatment. 10 ml of the culture was placed in a sterile petri dish, spread into a thin layer, and placed under an ultraviolet lamp at a distance of 30 cm, a power of 20 watts, for 45 seconds. After mutagenesis, the culture was kept in the dark for 1 hour. The culture was initially screened on pH 2.0 acidic medium and pH 12.0 alkaline medium. Single colonies with good growth were selected and then screened again for growth promotion ability. Finally, the target strain with significantly improved acid and alkali resistance and growth promotion ability was obtained.

[0027] (3) Basic training: The basal culture medium was prepared with the following composition: carbon source 2% corn flour and 2% sucrose, nitrogen source 1% peptone and 0.5% yeast extract, buffer 0.5% potassium dihydrogen phosphate-dipotassium hydrogen phosphate, trace elements 0.01% magnesium sulfate and 0.005% ferrous sulfate, and the remainder being sterile water. All raw materials were analytical grade or conventional agricultural grade. After thorough mixing, the pH of the medium was adjusted to 7.0, and the medium was autoclaved at 121℃ for 20 minutes. After cooling to 30℃, the target bacterial strain obtained above was inoculated at a rate of 5%. The medium was incubated at 25℃ with shaking at 150 rpm for 72 hours to obtain the basal culture. The viable count was determined using the plate count method to be 1.2 × 10⁻⁶. 9 The concentration of indoleacetic acid was determined to be 25.3 mg / L by CFU / mL using high performance liquid chromatography, the amount of phosphorus dissolved was determined to be 20.5 mg / L by molybdenum-antimony colorimetric method, and the nitrogenase activity was determined to be 13.2 nmol / (mL·h) by acetylene reduction method.

[0028] Example 2:

[0029] This embodiment, based on the basic culture in Example 1, optimizes the carbon and nitrogen source composition of the culture medium and the type and amount of buffer added to improve the acid and alkali resistance and proliferation rate of the strain, verify the feasibility of different raw material ratios, and expand the protection range of raw material selection. All operating steps are consistent with Example 1, only the composition of the culture medium is adjusted to ensure the reproducibility of the experiment.

[0030] (1) Optimized culture medium formulation: The carbon source used is a composite carbon source consisting of 3% corn flour, 3% wheat bran, and 2% glucose. Compared to the carbon source combination in Example 1, this better meets the nutritional needs of the strain at different growth stages and enhances the strain's stress resistance. The nitrogen source used is a composite nitrogen source consisting of 2% soybean meal, 1% yeast extract, and 0.5% urea. Soybean meal is an agricultural byproduct, which can reduce cultivation costs. It also works synergistically with yeast extract and urea to increase the secretion of growth-promoting metabolites by the strain. The buffer used is a composite buffer system consisting of 1.5% potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer and 0.5% sodium bicarbonate. This system can stabilize the pH of the culture medium within a range of 2.0 to 12.0, preventing pH fluctuations from affecting the strain's growth during cultivation. The composition and amount of trace elements are maintained as in Example 1, with the remainder being sterile water.

[0031] (2) Optimized cultivation: The optimized culture medium was adjusted to pH 3.0 (acidic), 7.0 (neutral), and 11.0 (alkaline) conditions, and cultured separately. After autoclaving at 121℃ for 20 minutes and cooling to 30℃, the target bacterial strain obtained in Example 1 was inoculated at a rate of 6%, cultured at 28℃, with a shaking speed of 180 r / min, for 72 hours. After culture, the viable bacterial count and the content of growth-promoting metabolites were determined using corresponding detection methods under different pH conditions. The viable bacterial count under acidic conditions was 1.8 × 10⁻⁶. 9 CFU / mL, indoleacetic acid content 30.1 mg / L, phosphorus solubility 25.7 mg / L, nitrogenase activity 15.6 nmol / (mL·h); viable count under neutral conditions 2.5 × 10⁻⁶. 9 CFU / mL, indoleacetic acid content 35.2 mg / L, phosphorus solubility 28.6 mg / L, nitrogenase activity 18.3 nmol / (mL·h); viable count under alkaline conditions 1.6 × 10⁻⁶. 9 The concentrations of CFU / mL, indoleacetic acid (IAA) content (28.9 mg / L), phosphorus solubility (24.3 mg / L), and nitrogenase activity (14.8 nmol / (mL·h)) were significantly increased compared to Example 1. Furthermore, the growth stability under extreme acid-base conditions was also enhanced.

[0032] Example 3:

[0033] This embodiment further optimizes the culture process based on the optimized culture medium in Example 2. It employs a combination of staged fed-batch culture and stress induction to enhance the strain's acid and alkali tolerance limits and the secretion of growth-promoting metabolites, verifying the feasibility of different process parameters and expanding the protection range of these parameters. The operation steps are clear and explicit, ensuring that different personnel can achieve the same results by following these steps.

[0034] (1) Fed culture program: A two-stage feeding method was adopted. The first stage was the bacterial proliferation period from 0 to 48 hours. At 24 hours of culture, a carbon source mixture was added, consisting of corn flour and glucose in a 1:1 mass ratio, at a level of 30% of the total initial carbon source mass. After addition, the culture was continued with shaking. The second stage was the metabolite secretion period from 48 to 96 hours. At 60 hours of culture, a nitrogen source mixture was added, consisting of soybean meal and yeast extract in a 2:1 mass ratio, at a level of 25% of the total initial nitrogen source mass. At the same time, 0.2% proline was added as a stress resistant agent to improve the metabolic stability of the strain under extreme acid and alkaline conditions. After addition, the culture was continued until the end of the culture period.

[0035] (2) Stress-induced protocol: During the cultivation process, the pH of the culture medium was adjusted in stages to induce stress. From 0 to 24 hours, the pH was adjusted to 7.0 to promote rapid bacterial proliferation. From 24 to 48 hours, the pH was gradually adjusted to 2.0 (acidic stress) or 12.0 (alkaline stress), with an adjustment rate of 1.0 pH unit every 6 hours to avoid damage to the bacterial strain from sudden pH changes. From 48 to 96 hours, the pH was maintained at 2.0 or 12.0 to induce the bacterial strain to secrete acid- and alkali-tolerant enzymes and growth-promoting metabolites. Sterile hydrochloric acid or sodium hydroxide solution was used for pH adjustment, added slowly and stirred thoroughly to ensure pH stability.

[0036] (3) Optimize the cultivation process: Using the optimized culture medium from Example 2, the final pH was set for the acid stress group (2.0), the alkaline stress group (12.0), and the neutral control group (7.0). The inoculum size was 7%, the culture temperature was 26℃, and the shaking speed was 170 r / min. The culture was carried out according to the above feeding and stress induction protocols for 84 hours. After culture, the viable cell count and the content of growth-promoting metabolites were determined using corresponding detection methods. The viable cell count in the acid stress group was 1.5 × 10⁻⁶. 9 CFU / mL, indoleacetic acid content 35.2 mg / L, phosphorus solubility 28.6 mg / L, nitrogenase activity 18.3 nmol / (mL·h); viable count in the alkaline stress group was 1.4 × 10⁻⁶. 9 CFU / mL, indoleacetic acid content 33.8 mg / L, phosphorus solubility 27.3 mg / L, nitrogenase activity 17.6 nmol / (mL·h); viable count in the neutral control group 2.8 × 10⁻⁶. 9 The concentrations of CFU / mL, indoleacetic acid (IAA) were 40.5 mg / L, phosphorus solubility was 32.1 mg / L, and nitrogenase activity was 20.1 nmol / (mL·h). Compared to Example 2, the secretion of probiotic metabolites was significantly increased, and the activity remained high even under extreme conditions of pH 2.0 and pH 12.0.

[0037] Example 4:

[0038] This embodiment, based on the optimized culture process of Example 3, prepares a *Tetranychus macrocephala* formulation, optimizes the formulation and preparation process, improves the stability, storage tolerance, and field application efficacy of the formulation, verifies the feasibility of formulation preparation, and expands the protection scope of the formulation raw materials. The preparation steps are clearly defined, and the raw material specifications are clearly defined, ensuring the feasibility of industrial-scale production.

[0039] (1) Composition of raw materials for the formulation: The core component is the *Coprinus macrocephala* bacterial culture obtained in Example 3, added at 50%. The carrier is diatomaceous earth plus corn straw powder, added at 40%. The carrier adsorbs the bacterial strain, enhancing its colonization ability in the soil, while the corn straw powder serves as a slow-release nutrient source for the strain. The protectant is sodium alginate plus chitosan, added at 5%. Sodium alginate and chitosan form an encapsulation system, protecting the strain from damage caused by extreme soil acidity and alkalinity, and extending the strain's survival time. The growth-promoting agent is humic acid plus potassium dihydrogen phosphate, added at 3%, which works synergistically with the strain to further enhance rhizosphere growth promotion. All raw materials are agricultural or food-grade conventional raw materials, free of toxic and harmful components.

[0040] (2) Formulation preparation method: The first step was bacterial concentration. The bacterial culture of *Coprinus coccidioides* obtained in Example 3 was concentrated by centrifugation at 7000 rpm for 15 minutes until the viable count was not less than 5 × 10⁻⁶. 9 The concentration of bacterial culture was increased to CFU / mL to obtain a concentrated bacterial solution. The second step involved mixing and stirring the concentrated bacterial solution and the protectant in a sterile mixing tank. The mixture was stirred at 25°C and 100 rpm until homogeneous, then allowed to stand for 30 minutes to allow the protectant to fully coat the bacterial strains. The carrier and growth promoter were then added, and the mixture was stirred at 28°C and 120 rpm for 30 minutes to form a homogeneous mixture. The third step involved granulation and drying. The mixture was granulated using a granulator to a particle size of 1.2 mm, and then dried in a constant temperature drying oven at 35°C until the moisture content reached 12%, obtaining a granular formulation. The fourth step involved aseptic packaging and storage of the dried granules in a cool, dry place at 15°C. The shelf life was over 12 months, and the viable count remained at least 1×10⁻⁶ after storage. 9 CFU / g.

[0041] Example 5:

[0042] This embodiment, based on Embodiments 1 to 4 above, uses different specific raw material ratios and process parameters to prepare bacterial cultures and formulations, verifying the wide applicability of the technical solution of the present invention, further expanding the scope of protection, and proving that the present invention can achieve excellent technical effects under different specific conditions. All verification schemes follow the same operating procedures to ensure the authenticity and reliability of the verification results.

[0043] (1) Verification scheme: Scheme 1 uses 10% wheat bran as the carbon source, 5% soybean meal as the nitrogen source, and 3% sodium bicarbonate as the buffer. The culture temperature is 35℃, the shaking speed is 220 r / min, the inoculum size is 10%, and the supplementation amount is 50%. The bacterial culture and formulation are prepared according to the culture process of Example 3 and the formulation preparation method of Example 4. Scheme 2 uses 5% sucrose and 5% glucose as the carbon source, 2% peptone and 3% yeast extract as the nitrogen source, and 1% citric acid-sodium citrate and 1% sodium bicarbonate as the buffer. The culture temperature is 15℃, the shaking speed is 120 r / min, the inoculum size is 3%, and the supplementation amount is 20%. The bacterial culture and formulation are prepared according to the culture process of Example 3 and the formulation preparation method of Example 4. Scheme 3 uses 2% corn flour as the carbon source, 1% urea as the nitrogen source, and 0.5% potassium dihydrogen phosphate as the buffer. The culture temperature is 25℃, the shaking speed is 150 r / min, the inoculum size is 5%, and the supplementation amount is 30%. The bacterial culture and formulation are prepared according to the culture process of Example 3 and the formulation preparation method of Example 4.

[0044] (2) Verification results: In all three schemes, the bacterial strains grew normally within a pH range of 2.0 to 12.0. Scheme 1 showed a viable bacterial count of 1.3 × 10⁻⁶. 9 The viable count of the formulation after 12 months of storage was 9.2 × 10⁸ CFU / g, with an indoleacetic acid content of 32.7 mg / L, a phosphorus solubility of 26.5 mg / L, and a nitrogenase activity of 16.8 nmol / (mL·h). The viable count of the second formulation was 1.1 × 10⁸ CFU / g. 9 CFU / mL, viable count of the formulation after 12 months of storage: 8.5 × 10⁻⁶ 8 CFU / g, indoleacetic acid content 30.5 mg / L, phosphorus solubility 25.2 mg / L, nitrogenase activity 15.3 nmol / (mL·h). Scheme 3 bacterial culture viable count 1.0 × 10⁻⁶. 9 CFU / mL, viable count of the formulation after 12 months of storage: 8.0 × 10⁻⁶ 8 The concentrations of CFU / g, indoleacetic acid (IAA) content (30.1 mg / L), phosphorus solubility (25.0 mg / L), and nitrogenase activity (15.0 nmol / (mL·h)) were all satisfactory. All schemes met the requirements for acid- and alkali-resistant rhizosphere growth promotion, indicating that the raw material ratios and process parameters of this invention have a wide protection range and can achieve the technical effects of this invention under different specific conditions.

[0045] Comparative Example 1: Using the original *Coprinus macrosporus* strain from Example 1, which had not undergone gradient acclimatization or UV mutagenesis, a formulation was prepared according to the culture medium formulation, culture process, and formulation preparation method of Example 4. Its acid and alkali resistance and growth-promoting effect were then determined. The results showed that this strain could only grow in the pH range of 4.0 to 9.0, and could not survive at pH 2.0 and pH 12.0. After 6 months of storage, the viable cell count of the formulation decreased to 0.8 × 10⁸. 8 The CFU / g, indoleacetic acid content of 18.5 mg / L, phosphorus solubility of 15.2 mg / L, and nitrogenase activity of 8.7 nmol / (mL·h) were significantly lower than those in Example 4. This indicates that the synergistic treatment of gradient acclimatization and ultraviolet mutagenesis is the key to improving the acid and alkali resistance and growth promotion ability of the strain, and is not a simple replacement for single acclimatization or mutagenesis in the existing technology.

[0046] Comparative Example 2: The culture medium used a single carbon source of 5% glucose and a single nitrogen source of 2% peptone, without adding a complex buffer system, only adding 0.5% potassium dihydrogen phosphate. Other conditions were consistent with Example 4. The formulation was prepared and its performance was measured. The results showed that the viable counts of the strain at pH 2.0 and pH 12.0 were 5.2 × 10⁻⁶. 8 CFU / mL and 4.8×10 8 CFU / mL, viable bacterial count after 12 months of storage was 5.3 × 10⁻⁶. 8 The CFU / g, indoleacetic acid content 22.3 mg / L, phosphorus solubility 19.6 mg / L, and nitrogenase activity 11.2 nmol / (mL·h) are significantly lower than those in Example 4. This indicates that the composite carbon and nitrogen source and composite buffer system used in this invention can synergistically improve the acid and alkali resistance and growth promotion effect of the strain, and is not a simple combination of a single carbon and nitrogen source and a single buffer in the prior art.

[0047] Comparative Example 3: Using the optimized culture medium from Example 2, without fed-batch culture or stress induction, the strain was directly cultured at pH 7.0 for 72 hours under constant temperature. Other conditions were the same as in Example 4. The formulation was prepared and its performance was measured. The results showed that the viable counts of the strain at pH 2.0 and pH 12.0 were 8.6 × 10⁶. 8 CFU / mL and 8.1×10 8 CFU / mL, viable bacterial count after 12 months of storage: 7.5 × 10⁻⁶. 8 The CFU / g, indoleacetic acid content 27.8 mg / L, phosphorus solubility 22.4 mg / L, and nitrogenase activity 14.5 nmol / (mL·h) are lower than those in Example 4. This indicates that the synergistic process of fed-batch culture and stress induction can effectively improve the strain's stress resistance and promote the secretion of metabolites, and is not a simple improvement on conventional culture processes in the prior art.

[0048] Comparative Example 4: No protective agent was added during formulation preparation. Other raw material ratios and preparation processes were consistent with Example 4. The storage stability and field application effect of the formulation were determined. Results showed that after 6 months of storage, the viable bacterial count decreased to 3.2 × 10⁻⁶. 8 After 12 months of storage, the number of viable bacteria was only 1.8 × 10⁻⁶ CFU / g. 8 When applied in the field, the colonization rates of the strain in acidic and alkaline soils were 35% and 32%, respectively, significantly lower than the 68% and 65% in Example 4. This indicates that the protective agent added in this invention can effectively improve the storage stability of the formulation and the soil colonization ability of the strain, solving the technical problems of easy inactivation during storage and difficulty in field colonization of existing formulations.

[0049] Comparative Example 5: A commercially available Bacillus subtilis preparation, a common rhizosphere growth promoter, was used. Its acid and alkali resistance and growth-promoting effect were determined under the same conditions as in Example 4. The results showed that the preparation only functioned normally within a pH range of 5.0 to 8.0. Below pH 4.0 and above pH 9.0, the activity of the strain decreased significantly, with the viable count not exceeding 3 × 10⁻⁶. 8 The CFU / g, indoleacetic acid content of 15.7 mg / L, phosphorus solubility of 14.3 mg / L, and nitrogenase activity of 7.9 nmol / (mL·h) are all significantly lower than those of the formulation of this invention in Example 4. This indicates that the *Macrobrachium cladosporum* of this invention, after synergistic improvement, exhibits significantly superior acid and alkali tolerance and growth-promoting effects compared to existing conventional rhizosphere growth-promoting bacteria, demonstrating outstanding technical advantages.

[0050] Comparative Example 6: The formulation was prepared using a common combination of gradient acclimatization, single carbon source, and no supplemental feeding scheme found in existing technologies. Specifically, the original strain was treated with the gradient acclimatization method of Example 1, the single carbon source medium of Comparative Example 2 was used, and the no-supplemental feeding process of Comparative Example 3 was employed. Other conditions were consistent with Example 4. The results showed that the viable cell counts of the strain at pH 2.0 and pH 12.0 were 7.8 × 10⁻⁶. 8 The concentrations were 7.3 × 10⁸ CFU / mL and 6.5 × 10⁸ CFU / mL, respectively. After 12 months of storage, the viable count of the formulation was 6.5 × 10⁸ CFU / mL. 8 The CFU / g, indoleacetic acid content of 25.1 mg / L, phosphorus solubility of 20.3 mg / L, and nitrogenase activity of 13.1 nmol / (mL·h) are still significantly lower than those in Example 4. Based on prior art search results, this combination scheme is not disclosed in any prior art, and its technical effect is far inferior to that of this invention. This further illustrates that the technical solution of this invention is not a simple combination of prior art, but rather a complete technical system formed through synergistic optimization of each step, producing technical effects that cannot be achieved by combinations of prior art.

[0051] To make the technical solution of this invention clearer and more complete, and to enable those skilled in the art to fully implement this invention, the following supplementary explanations are made regarding the undisclosed key raw material ratios, reagent compositions, strain gene sequences, and metrological standards involved in this invention: (1) Formulation ratio of raw materials: The carrier is a mixture of diatomaceous earth and corn straw powder in a mass ratio of 3:1, the protective agent is a mixture of sodium alginate and chitosan in a mass ratio of 2:1, and the growth promoter is a mixture of humic acid and potassium dihydrogen phosphate in a mass ratio of 5:1. The above ratio is the optimal ratio to achieve the synergistic effect of strain adsorption, encapsulation protection and growth promotion. It can also be adjusted within the range of ±20% to achieve the basic technical effect of the present invention.

[0052] (2) Strain screening reagent: The 0.1% rare earth solution used in Example 1 is a mixed rare earth solution of lanthanum and cerium nitrate, wherein La 3+ With Ce 3+ The molar ratio is 1:1, the solvent is deionized water, and 0.1% is the mass-volume percentage (g / 100mL). This rare earth solution, as a selector, can specifically promote the growth of the target macrosporum falciparum and improve the screening efficiency.

[0053] (3) Measurement standard description: In this invention, all raw material addition ratios expressed in "%" are based on the total mass of the preparation raw materials, the total mass of the culture medium raw materials, and the total mass of the carbon / nitrogen source of the corresponding culture medium before feeding. All volume units are milliliters (mL), mass units are grams (g), temperature units are degrees Celsius (°C), and rotation speed units are revolutions per minute (r / min).

[0054] (4) Raw material specifications: The diatomaceous earth used in this invention is food grade with a mesh size of 200 mesh; the corn straw powder is agricultural grade and passes through an 80-mesh sieve; sodium alginate and chitosan are both food grade with a degree of deacetylation ≥90%; humic acid is agricultural grade with a fulvic acid content ≥50%; the remaining chemical reagents are all analytical grade, and the raw materials used for microbial culture are all biological reagent grade and can be obtained through conventional commercial channels.

[0055] Performance testing and results analysis: Comprehensive performance tests were conducted on the bacterial cultures and formulations of Examples 1 to 5 and Comparative Examples 1 to 6. The test items included acid and alkali resistance, proliferation capacity, secretion of growth-promoting metabolites, storage stability of the formulation, soil colonization capacity, and field growth-promoting effect. All test methods were conventional microbiological and agricultural test methods.

[0056] Test materials and instruments: The test materials were bacterial suspensions of the strains from Examples 1 to 5, preparations from Examples 4 to 5, and bacterial suspensions or preparations of Comparative Examples 1 to 6; acidic soil pH 4.5, alkaline soil pH 9.5, and neutral soil pH 7.0; and wheat seed variety Jimai 44. The testing instruments included a constant temperature incubator, a shaking incubator, a centrifuge, a high-performance liquid chromatograph, a UV spectrophotometer, a root scanning analysis system, and a laser confocal microscope. All instruments were standard laboratory equipment, readily available, and the test materials had clearly defined specifications, ensuring repeatability of the tests.

[0057] Test items and methods: (1) Acid and alkali resistance test: The bacterial cultures of each example and comparative example were inoculated into liquid culture media at pH 2.0, pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, pH 10.0, pH 11.0, and pH 12.0, respectively. The cultures were incubated at 25°C with shaking at 150 rpm for 72 hours. The viable cell count was determined using the plate count method, with a minimum viable cell count of 1.0 × 10⁻⁶. 9 CFU / mL is the acceptable standard. Record the acid and alkali tolerance range of the strain and the number of viable bacteria under different pH conditions.

[0058] (2) Proliferation capacity test: Using the culture conditions of Example 3, strains from each example and comparative example were inoculated, and samples were taken at 0, 24, 48, 72, and 96 hours of culture to determine the OD600 value and viable cell count of the bacterial solution, plot growth curves, calculate the maximum specific growth rate and generation time of the strains, and evaluate the proliferative capacity of the strains.

[0059] (3) Test of secretion of probiotic metabolites: After cultivation, bacterial cultures from each example and comparative example were taken, centrifuged to remove bacterial cells, and the indoleacetic acid content of the supernatant was determined by high performance liquid chromatography, the amount of phosphorus dissolved was determined by molybdenum antimony colorimetric method, and the nitrogenase activity was determined by acetylene reduction method. Each sample was measured in parallel three times, and the average value was taken.

[0060] (4) Formulation storage stability test: The formulations of each example and comparative example were placed in environments of 0°C, 25°C, and 35°C, and samples were taken after 1 month, 3 months, 6 months, and 12 months of storage, respectively. The number of viable bacteria in the formulations was determined by plate counting method, and the changes in the number of viable bacteria were recorded to evaluate the storage stability of the formulations.

[0061] (5) Soil colonization capacity test: The formulations of each example and comparative example were applied to acidic soil, alkaline soil, and neutral soil, respectively, at an application rate of 20 kg per acre. Soil samples were collected 1, 7, 15, 30, and 60 days after application. The number of target strains in the soil was determined by the dilution spread method. The colonization of the strains in the plant rhizosphere was observed by fluorescent labeling technology and laser confocal microscopy to evaluate the soil colonization ability of the strains.

[0062] (6) Field test of growth-promoting effect: Three experimental fields were selected, one with acidic soil, one with alkaline soil, and one with neutral soil. Wheat was planted in all fields. Four control groups (no inoculant applied), one treatment group (Example 4), one treatment group (Example 5), one treatment group (Comparative Example 5), and one treatment group (Comparative Example 6) were also included, with three replicates per group. Each plot was 10 square meters in size and planted according to conventional field management methods. After the wheat matured, plant height, ear length, thousand-grain weight, and yield were measured for each group. Simultaneously, total root length, root surface area, root volume, and the content of available nitrogen, available phosphorus, and available potassium in the soil were measured to evaluate the field growth-promoting and soil-improving effects of the inoculant.

[0063] Test Results and Analysis: The results of the acid and alkali resistance test are shown in Table 1 below: Table 1:

[0064] The acid and alkali resistance test results showed that the strains in Examples 1 to 5 could grow normally within the pH range of 2.0 to 12.0, and the viable cell counts under extreme conditions of pH 2.0 and pH 12.0 met the qualification standards. Among them, the strains in Examples 3 to 5 maintained a high viable cell count even under extreme conditions, demonstrating the effect of optimized process on improving the acid and alkali resistance stability of the strains. Comparative Example 1, without gradient acclimatization and ultraviolet mutagenesis treatment, could only grow within the pH range of 4.0 to 9.0 and could not adapt to extreme acid and alkali environments. Comparative Examples 2 to 4 and 6, although covering the pH range of 2.0 to 12.0, did not meet the qualification standards for viable cell counts under extreme conditions. Comparative Example 5, as a conventional strain preparation, had an acid and alkali resistance range of only pH 5.0 to 8.0, and its activity decreased significantly under extreme conditions. In summary, this invention, through the synergistic treatment of gradient acclimatization and ultraviolet mutagenesis, combined with optimized culture medium and culture process, significantly broadened the acid and alkali resistance range of the strains and improved their growth stability under extreme conditions. The technical effect is significantly better than the prior art and combinations of prior art.

[0065] The results of the proliferation capacity test are shown in Table 2 below: Table 2:

[0066] The proliferation capacity test results showed that the strains of Examples 3 to 5 exhibited the best proliferation performance, with a lag phase shorter than 6 hours, a maximum specific growth rate of 0.38 to 0.40 per hour, and a generation time shortened to 1.73 to 1.83 hours, demonstrating significant advantages compared to other examples and comparative examples. The strains of Examples 1 to 2 showed slightly inferior proliferation performance, but were still superior to all comparative examples. Comparative Example 1, which had not undergone acclimatization and mutagenesis treatment, had the longest lag phase and the slowest proliferation rate. Comparative Example 5, as a conventional strain, showed the worst proliferation performance. Comparative Example 6, using a combination of existing technologies, showed better proliferation performance than Comparative Examples 1 and 5, but still lower than the examples of this invention. These results fully demonstrate that the staged feeding and stress-induced synergistic process employed in this invention can effectively shorten the lag phase of strains and increase the proliferation rate, providing technical support for large-scale strain cultivation and industrial production of formulations, reflecting the practicality and advancement of the technical solution of this invention.

[0067] The results of the test for the secretion of proliferative metabolites are shown in Table 3 below: Table 3:

[0068] The results of the test on the secretion of growth-promoting metabolites showed that the strains in Examples 1 to 5 had significantly better secretion capabilities than the comparative examples. Among them, the strains in Examples 3 and 4 performed best, with indoleacetic acid content reaching 37.9 mg / L, phosphorus solubility reaching 29.9 mg / L, and nitrogenase activity reaching 18.9 nmol / (mL·h). Compared with the strain in the basic culture of Example 1, these three indicators were improved by 49.8%, 45.9%, and 43.2%, respectively, fully demonstrating the synergistic effect of culture medium optimization, staged feeding, and stress induction process. Example 2, by optimizing the composite carbon and nitrogen source and composite buffer system, significantly increased the content of growth-promoting metabolites compared with Example 1, indicating that a suitable nutrient supply and a stable culture environment are important foundations for promoting the secretion of growth-promoting substances by strains. Example 5, using different raw material ratios and process parameters, still maintained a high secretion of growth-promoting metabolites, further verifying the wide applicability of the technical solution of the present invention.

[0069] The secretion levels of growth-promoting metabolites in each comparative example were significantly lower than those in the embodiments of the present invention. Comparative Example 1, which did not undergo gradient acclimatization and UV mutagenesis, had the lowest content of growth-promoting metabolites, indicating that genetic improvement of the strain is a core prerequisite for enhancing its growth-promoting ability. Comparative Example 2, using a single carbon and nitrogen source and a single buffer, could not provide sufficient and comprehensive nutrition for the strain's metabolism, resulting in insufficient secretion of growth-promoting substances. Comparative Example 3, without supplemental feeding and stress induction processes, lacked targeted nutrient supply and stress resistance induction, resulting in limited secretion of metabolites. Comparative Example 4, without the addition of a protective agent, although it did not directly affect the secretion of growth-promoting metabolites... However, combined with the previous storage stability test results, the activity of the strain decreased after storage, indirectly leading to poor growth-promoting effect during field application; Comparative Example 5, as an existing conventional Bacillus subtilis preparation, had a growth-promoting ability far lower than the improved Bacillus coccidioidomyces macrosporum of this invention, highlighting the superiority of the strain selection and improvement of this invention; Comparative Example 6 adopted the existing technology combination scheme, which improved the performance of the comparative example with a single defect, but still did not achieve the effect of the embodiment of this invention, indicating that the technical solution of this invention is not a simple accumulation of existing technologies, but rather a breakthrough improvement in the growth-promoting ability of the strain through the synergistic optimization of each link.

[0070] The results of the formulation storage stability test are shown in Table 4 below: The test index is the number of viable bacteria after storage at different temperatures for 12 months. Examples 1-3 are bacterial suspensions, measured in CFU / mL; Examples 4-5 and all comparative examples are formulations, measured in CFU / g; the pass standard is ≥1.0×10⁻⁶ at 0℃. 9 25℃ ≥8.0×10 8 35℃ ≥ 5.0 × 10 8 25℃ is the standard storage temperature and is a core performance indicator.

[0071]

[0072] Note: Examples 1-3 are bacterial suspensions, while Examples 4-5 and Comparative Examples 1-6 are formulations. The testing units for the two are different, but they are all assessed according to the same qualification standard.

[0073] After being stored for 12 months at three temperatures (0℃, 25℃, and 35℃), the bacterial cultures and preparations of Examples 1-5 all met the preset qualification standards for viable cell counts. Examples 3-4 showed the best performance, maintaining a viable cell count of 1.0 × 10⁻⁶ under conventional storage conditions at 25℃. 9 The grade meets the actual needs of long-distance transportation of agricultural bacterial liquids and room temperature storage of formulations. It reflects the synergistic effects of gradient domestication and ultraviolet mutagenesis synergistic treatment, composite carbon and nitrogen source and buffer system optimization, staged feeding and stress induction process, and protective agent and carrier composite system, effectively reducing the inactivation rate of strains during storage.

[0074] Comparative Example 1, lacking gradient acclimatization and UV mutagenesis, exhibited poor strain resistance, resulting in a significant decrease in viable cell count after storage. Comparative Example 2 used a single carbon and nitrogen source and a single buffer; Comparative Example 3 did not employ fed-batch and stress-induced processes; and Comparative Example 6 used a combination of existing technologies. All three examples showed insufficient strain activity, leading to decreased storage stability. Comparative Example 4, without added preservatives and lacking encapsulation protection, showed only 1.8 × 10⁻⁶ viable cells after 12 months of storage at 25°C. 8 The results were far below the acceptable standard, directly demonstrating the non-obviousness of the protective agent formulation of this invention; the commercially available Bacillus subtilis preparation in Comparative Example 5 lacked acid and alkali resistance design, and its storage stability was significantly lower than that of the strain and preparation of this invention.

[0075] The results of the soil colonization capacity test are shown in Table 5 below: The test indicators were the number of bacterial strains colonizing the soil and the rhizosphere colonization rate of plants 60 days after application; Examples 1-3 were bacterial suspensions, and Examples 4-5 and all comparative examples were formulations, with an application rate of 20 kg per acre; the test soils were acidic soil (pH 4.5) and alkaline soil (pH 9.5); the pass standard was a colony count ≥ 5.0 × 10⁻⁶. 6 CFU / g soil and rhizosphere colonization rate ≥60%, with rhizosphere colonization rate being the core assessment indicator.

[0076] Table 5: ;

[0077] The bacterial strains and preparations of this invention exhibit excellent colonization ability in extremely acidic and alkaline soils: after 60 days of application in acidic and alkaline soils, the colony counts of the bacterial solutions and preparations in Examples 1-5 were all ≥6.0 × 10⁻⁶. 6 With a CFU / g soil concentration and a rhizosphere colonization rate ≥60%, this invention meets the practical application value required by patent law. This effect stems from the synergistic effect of the carrier protectant and growth promoter of this invention. Diatomaceous earth and corn straw powder adsorb the bacterial strains into the plant rhizosphere; sodium alginate and chitosan protect the strains from damage caused by extreme soil acidity and alkalinity; and humic acid and potassium dihydrogen phosphate improve the rhizosphere microenvironment. These three elements form a colonization, protection, and improvement technical system that cannot be easily obtained by those skilled in the art through simple combinations of existing technologies.

[0078] Comparative Example 1 strain, without acid and alkali tolerance modification, was completely inactivated in extremely acidic and alkaline soils and could not colonize; Comparative Example 2 used a single carbon and nitrogen source and a single buffer; Comparative Example 3 did not use feeding and stress induction processes; Comparative Example 6 used a combination of existing technologies. All three strains had insufficient stress resistance, and the rhizosphere colonization rate was less than 50%; Comparative Example 4 did not add a protectant, and the strain was easily destroyed by extreme soil environments, with a rhizosphere colonization rate of only 3532; Comparative Example 5, a commercially available Bacillus subtilis preparation, could not adapt to acidic and alkaline soils, and had the lowest colonization number and colonization rate. This demonstrates the technical advantages of the strain selection and modification of the present invention compared to existing conventional rhizosphere growth-promoting bacteria.

[0079] The results of the field growth-promoting effect test are shown in Table 6 below: The test subjects were the blank control group and all Examples 1-5 and Comparative Examples 1-6; the test crop was wheat Jimai 44; the test soils were a mixture of acidic soil (pH 4.5), alkaline soil (pH 9.5), and neutral soil (pH 7.0); all data are the average of three replicates, and yield per mu (unit of land area) is the core indicator.

[0080]

[0081]

[0082] Compared to the blank control group, the strains and formulations used in Examples 1-5 increased wheat yield by 22.6 to 39.8 per mu, thousand-grain weight by 14.5 to 23.7 per mu, and total root length by 49.5 to 74.8 per mu. Simultaneously, the available nitrogen and phosphorus content in the soil significantly increased. These results demonstrate that the formulations of this invention not only directly promote crop growth through the nitrogen-fixing, phosphorus-solubilizing, and indoleacetic acid-producing functions of the strains, but also achieve a dual effect of promoting growth and improving soil nutrient conditions. This solves the technical problem of poor efficacy of rhizosphere growth-promoting bacteria in extremely acidic and alkaline soils in existing technologies, meets the practicality requirements of patent law, and can be directly applied to agricultural production in low-yield soils such as saline-alkali land and acidic red soil.

[0083] Compared to the commercially available Bacillus subtilis preparation in Comparative Example 5, the yield per acre in Example 4 increased by 23.9%. This is because existing conventional strains lack acid and alkali tolerance design, resulting in a significant decrease in activity in non-neutral soils, thus failing to exert a growth-promoting effect. Compared to the existing technical combination scheme in Comparative Example 6, the yield per acre in Example 4 increased by 12.7. This demonstrates that the gradient domestication ultraviolet mutagenesis, composite carbon and nitrogen source buffer system, staged feeding stress induction, and protective agent carrier composite system of the present invention are not simply a combination of existing technologies, but rather a synergistic optimization of various technical links to form a complete technical system.

[0084] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A rhizosphere-promoting macrosporum preparation resistant to acid and alkali, characterized in that, Based on the total mass of the formulation, it consists of the following raw materials in the following percentages by mass: 50% *Sordaria macrospora* bacterial solution, 40% carrier, 6% protectant, and 4% growth promoter; the strain of *Sordaria macrospora* bacterial solution is *Sordaria macrospora*, strain preservation number CGMCCNO.29081, which can grow normally in an environment with pH 2.0 to 12.0 and a viable count of not less than 1.0 × 10⁻⁶. 9 The strain, with a concentration of CFU / mL, possesses rhizosphere growth-promoting functions including nitrogen fixation, phosphorus solubilization, and indoleacetic acid secretion. The carrier is a mixture of diatomaceous earth and corn straw powder at a mass ratio of 3:1; the protectant is a mixture of sodium alginate and chitosan at a mass ratio of 2:1; and the growth-promoting adjuvant is a mixture of humic acid and potassium dihydrogen phosphate at a mass ratio of 5:

1. After 12 months of storage, the viable bacterial count of this preparation is no less than 1 × 10⁻⁶. 9 CFU / g.

2. The acid- and alkali-resistant rhizosphere growth-promoting macrosporum preparation according to claim 1, characterized in that, The *Fasciola heliotropium* bacterial solution was obtained through gradient acclimatization, ultraviolet mutagenesis, culture in a compound carbon and nitrogen source medium, and staged feeding and stress induction. The indoleacetic acid content of the bacterial solution is not less than 37.9 mg / L, the phosphorus solubility is not less than 29.9 mg / L, and the nitrogenase activity is not less than 18.9 nmol / (mL·h).

3. The acid- and alkali-resistant rhizosphere growth-promoting macrosporum preparation according to claim 1, characterized in that, The formulation is in granular form with a particle size of 1.2 mm and a moisture content of 12%.

4. A method for preparing an acid- and alkali-resistant rhizosphere-promoting macrosporum preparation as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Concentration of bacterial culture: The bacterial culture of *Fasciola heliotropium* was concentrated by centrifugation at a speed of 7000 r / min for 15 minutes until the viable count was not less than 5 × 10⁻⁶. 9 CFU / mL was used to obtain a concentrated bacterial solution. (2) Mixing and stirring: Add the concentrated bacterial solution and the protectant to a sterile mixing tank and stir evenly. Let it stand for 30 minutes, then add the carrier and growth promoter and continue stirring to form a uniform mixture; (3) Molding and drying: After granulation, the mixture is placed in a constant temperature drying oven at 35℃ and dried until the moisture content of the mixture is 12% to obtain granular preparation; (4) Packaging and storage: After aseptic packaging, the granular preparations are stored in a cool and dry place.

5. The preparation method according to claim 4, characterized in that, In step (2), the stirring conditions for the concentrated bacterial solution and the protectant are 25°C and 100r / min; the stirring conditions after adding the carrier and growth promoter are 28°C and 120r / min, and the stirring time is 30 minutes.

6. The preparation method according to claim 4, characterized in that, In step (3), the particle size of the granulated mixture is 1.2 mm.

7. The preparation method according to claim 4, characterized in that, The preparation of the *Macrobrachium cladocarbazin* bacterial solution includes the following steps: (1) Strain screening: wheat rhizosphere soil from the boundary area between saline-alkali land and acidic red soil was collected to prepare the inoculum. After being serially diluted and spread on screening medium with pH 2.0 to 12.0, the inoculum was purified and screened to obtain Sordaria macrospora, which meets the requirements for growth and viable number of bacteria. (2) Synergistic improvement: The selected strains were subjected to gradient domestication and ultraviolet mutagenesis synergistic treatment to obtain target strains with improved acid and alkali resistance and growth promotion ability; (3) Optimized culture: The target strain was inoculated into an optimized culture medium containing a compound carbon and nitrogen source and a compound buffer system. The culture was carried out by a combination of staged feeding culture and stress induction to obtain the culture liquid of *Cytomyces coccidioides*.

8. The preparation method according to claim 7, characterized in that, In step (2), the initial culture medium pH for gradient acclimatization was 7.

0. The pH was decreased by 1.0 for acidic conditions and increased by 1.0 for alkaline conditions, gradually adjusted to pH 2.0 and pH 12.

0. Each pH gradient was cultured for 3 generations, with each generation cultured for 72 hours. The culture conditions were 25℃ and 150r / min shaking culture. For ultraviolet mutagenesis, the bacterial solution of the acclimatized strain was adjusted to an OD600 value of 0.6 and mutagenized for 45 seconds with a 20W ultraviolet lamp at a distance of 30 cm. After mutagenesis, the strain was kept in the dark for 1 hour. The target strain was obtained by primary screening and secondary screening for growth promotion ability.

9. The preparation method according to claim 7, characterized in that, In step (3), the carbon source of the optimized culture medium is a composite carbon source of 3% corn flour, 3% wheat bran and 2% glucose, the nitrogen source is a composite nitrogen source of 2% soybean meal, 1% yeast extract and 0.5% urea, the buffer is a composite buffer system of 1.5% potassium dihydrogen phosphate-dipotassium hydrogen phosphate and 0.5% sodium bicarbonate, and 0.01% magnesium sulfate and 0.005% ferrous sulfate are added at the same time; all percentages are based on the total mass of the optimized culture medium.

10. The preparation method according to claim 7, characterized in that, In step (3), the staged fed culture is as follows: during the 0-48 hour proliferation period, a carbon source mixture of corn flour and glucose in a mass ratio of 1:1 is added at 24 hours, with the amount added being 30% of the total mass of the initial culture medium carbon source; during the 48-96 hour metabolite secretion period, a nitrogen source mixture of soybean meal and yeast extract in a mass ratio of 2:1 is added at 60 hours, with the amount added being 25% of the total mass of the initial culture medium nitrogen source, and 0.2% proline is added at the same time; during the stress induction period, the pH is 7.0 from 0 to 24 hours, and from 24 to 48 hours, the pH is adjusted by 1.0 pH unit every 6 hours to pH 2.0 or pH 12.0, and from 48 to 96 hours, the pH is maintained at pH 2.0 or pH 12.0; the percentage of proline is based on the total mass of the culture medium after feeding.

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