A nitrogen-fixing bacterial agent and its use in sugarcane planting

Abstract

The application discloses a kind of nitrogen-fixing bacteria agent and its purposes in sugarcane planting, belong to microbial inoculant technical field, the nitrogen-fixing bacteria agent of the present application includes mixed inoculant of the dry powder of bacteria functional group and the dry powder of fungus functional group fixed by sodium alginate gel embedding;Bacterial functional group dry powder contains jelly-like bacillus, bacillus megaterium, saccharomyces cerevisiae and lactobacillus plantarum;Fungus functional group dry powder contains trichoderma viride;The application mode of the present application is that nitrogen-fixing bacteria agent is mixed with base fertilizer according to mass ratio 1: (10-20) after ditching or hole application, or nitrogen-fixing bacteria agent is added in water according to mass ratio 1: (5-8) to obtain inoculant suspension for sugarcane seedling treatment;The present application significantly improves the survival and colonization ability of inoculant in sugarcane high sugar acidic environment, effectively solves the antagonism problem between complex microbial flora, realizes the synergistic effect of microbial flora, realizes the multifunctional synergy of nitrogen fixation, growth promotion, disease resistance and the like through functional substance loading and strain compounding.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and in particular to a nitrogen-fixing inoculant and its use in sugarcane cultivation. Background Technology

[0002] As an important sugar and energy crop, the healthy development of the sugarcane industry is crucial to sugar security and energy strategy. In intensive sugarcane cultivation, excessive application of nitrogen fertilizer has become a common phenomenon, which not only increases production costs but also leads to environmental problems such as soil acidification and compaction, and eutrophication of water bodies. Utilizing nitrogen-fixing microbial agents to partially replace chemical nitrogen fertilizers is an important direction for achieving green and sustainable sugarcane development.

[0003] However, the unique physiology and growing environment of sugarcane place extremely demanding requirements on nitrogen-fixing bacteria inoculants. First, sugarcane plants have extremely high sucrose concentrations, exceeding 20%, while their root exudates and continuously cropped soils are often acidic, resulting in a pH frequently below 5.5. This high-sugar, high-acid endophytic and rhizosphere environment makes it difficult for ordinary nitrogen-fixing bacteria to colonize and survive. Second, commercially available nitrogen-fixing bacteria inoculants are mostly single-strain or simple mixtures, and their strains are often not specifically selected for sugarcane, resulting in poor environmental adaptability, weak competitiveness in the sugarcane rhizosphere, inability to achieve effective endophytic colonization, and unstable field application effects. More importantly, simply mixing strains with different functions often leads to antagonistic interactions between strains, inhibiting each other and causing the inoculant to become ineffective.

[0004] While some existing research has attempted to develop sugarcane-specific microbial agents, significant limitations remain. For example, conventional carriers such as peat moss and vermiculite cannot provide effective protection for microbial strains in acidic soils; simple strain combinations fail to address inter-strain antagonism and cannot achieve time-controlled release of functional strains; furthermore, most microbial agent products have limited functionality, lacking the ability to synergistically integrate multiple functions such as nitrogen fixation, growth promotion, disease resistance, and organic matter decomposition. Some encapsulation techniques primarily focus on protecting single strains, failing to design for the structural and functional synergy of complex microbial communities.

[0005] Therefore, there is an urgent need for a highly efficient compound nitrogen-fixing bacterial agent that can overcome the high sugar and acid stress of sugarcane, effectively solve the antagonism of microbial communities, and synergistically exert multiple fertilizer effects, so as to meet the urgent needs of sugarcane planting for fertilizer reduction and efficiency improvement, soil improvement and green production. Summary of the Invention

[0006] The purpose of this invention is to provide a nitrogen-fixing bacterial agent and its use in sugarcane cultivation, in order to solve the technical bottlenecks of existing nitrogen-fixing bacterial agents, such as difficulty in adapting to the high sugar and acidic environment of sugarcane, antagonistic effects between bacterial species, single function, and unstable field application effects. Through the design of compound microbial communities and microencapsulation technology, the efficient colonization, functional synergy, and time-controlled release of the bacterial agent are achieved, forming a microbial agent that can reduce the amount of chemical nitrogen fertilizer used, promote sugarcane growth, and improve soil health.

[0007] To achieve the above objectives, the present invention provides the following solution: A nitrogen-fixing bacterial agent, the key feature of which is that the nitrogen-fixing bacterial agent comprises a mixed bacterial agent consisting of bacterial functional group dry powder and fungal functional group dry powder immobilized by sodium alginate gel; the bacterial functional group dry powder contains Bacillus jellyoidis, Bacillus megaterium, Saccharomyces cerevisiae and Lactobacillus plantarum; the fungal functional group dry powder contains Trichoderma viride.

[0008] Furthermore, the aforementioned Saccharomyces cerevisiae and Lactobacillus plantarum were pre-adsorbed onto biodegradable starch microspheres, and then encapsulated together with Bacillus jellyoidus and Bacillus megaterium in sodium alginate gel.

[0009] Furthermore, the method for preparing the above-mentioned starch microspheres is to prepare a soluble starch solution with a mass concentration of 10% to 12%, heat it to gelatinize it, and then cool it to 42°C to 48°C; control the inlet air temperature to 150°C to 160°C, the outlet air temperature to 80°C to 85°C, and the feed flow rate to 5mL / min to 10mL / min, and then prepare the starch microspheres by spray drying.

[0010] Specifically, the preparation method of the above-mentioned bacterial functional group dry powder is as follows: Bacillus spp., Bacillus megaterium, Saccharomyces cerevisiae, and Lactobacillus plantarum are prepared into bacterial suspensions respectively; the bacterial suspensions of Saccharomyces cerevisiae and Lactobacillus plantarum are adsorbed onto blank starch microspheres to obtain yeast-starch microspheres and lactic acid bacteria-starch microspheres respectively; sodium alginate solution is mixed with molasses powder and sodium molybdate, and then the Bacillus spp., Bacillus megaterium, yeast-starch microspheres and lactic acid bacteria-starch microspheres are added and mixed evenly to obtain a bacterial functional group mixture; the bacterial functional group mixture is dropped into calcium chloride solution for cross-linking and solidification, the gel particles are collected, and dried to obtain bacterial functional group dry powder.

[0011] More specifically, in every 100 mL of sodium alginate solution, the amount of the above-mentioned molasses powder added is 2 g to 4 g, and the amount of the above-mentioned sodium molybdate added is 0.05 g to 0.15 g; the total amount of the above-mentioned gelatinous Bacillus suspension and Bacillus megaterium suspension added is in a volume ratio of 1:(4 to 5) to the sodium alginate solution; the amount of the above-mentioned yeast-starch microspheres and lactic acid bacteria-starch microspheres added is 2 g to 3 g per 100 mL of sodium alginate solution.

[0012] Specifically, the preparation method of the above-mentioned fungal functional group dry powder is as follows: prepare the green Trichoderma agent into a spore suspension; mix sodium alginate solution with chitin powder, wheat bran powder and zinc sulfate evenly, then add Trichoderma spore suspension and mix evenly to obtain fungal functional group mixture; drip the fungal functional group mixture into calcium chloride solution for cross-linking and solidification, collect the gel particles, and dry to obtain fungal functional group dry powder.

[0013] More specifically, in every 100 mL of sodium alginate solution, the amount of chitin powder added is 3 g to 5 g, the amount of wheat bran powder added is 1.5 g to 2.5 g, and the amount of zinc sulfate added is 0.02 g to 0.04 g; the volume ratio of the Trichoderma spore suspension to the sodium alginate solution is 1:(5 to 6).

[0014] Preferably, the mass concentration of the sodium alginate solution is 2% to 4%; the calcium chloride solution is a calcium chloride solution containing 0.1% to 0.2% calcium carbonate with a mass concentration of 1.0% to 2.0%; and the cross-linking curing conditions are cross-linking curing for 30 to 40 minutes at a rotation speed of 180 r / min to 220 r / min and a temperature of 20℃ to 25℃.

[0015] The optimal mass mixing ratio of the above-mentioned bacterial functional group dry powder and fungal functional group dry powder is (4-6):3.

[0016] The key to the application of the above-mentioned nitrogen-fixing bacteria agent in sugarcane planting is that the above-mentioned application method is to mix the above-mentioned nitrogen-fixing bacteria agent with the base fertilizer at a mass ratio of 1: (10-20) and then apply it in furrows or holes, or to add the nitrogen-fixing bacteria agent to water at a mass ratio of 1: (5-8) to obtain a bacterial suspension for dipping sugarcane seed stalks.

[0017] The present invention discloses the following technical effects: This invention solves three core problems faced by nitrogen-fixing microbial agents for sugarcane through microbial agent design and a special encapsulation process, specifically including the following aspects: This invention significantly improves the survival and colonization ability of the microbial agent in the high-sugar, acidic environment of sugarcane. In a simulated high-acid, high-sugar environment of sugarcane rhizosphere, the samples of this invention maintained a total bacterial survival rate of over 76.8% and a Trichoderma spore survival rate of over 72.0% after 7 days of cultivation, significantly superior to all comparative examples. This demonstrates that the sodium alginate microcapsule encapsulation system used in this invention, combined with internally loaded pH buffers and energy substances, constructs an effective physical and chemical protective barrier for the functional microbial community, enabling it to effectively resist acid stress and hyperosmotic pressure, laying the foundation for successful rhizosphere colonization and efficacy. In contrast, the control 4, using a traditional peat moss carrier, had an extremely low survival rate, highlighting the unique advantages of the encapsulation technology of this invention.

[0018] This invention effectively solves the antagonistic problem among complex microbial communities, achieving synergistic effects. The invention creatively employs a compartmentalized encapsulation strategy, physically isolating bacterial and fungal functional groups, and using starch microspheres for pre-adsorption and isolation of susceptible strains. Experimental results show that the survival rate of strains, especially Trichoderma spores, in the control group (using simple mixed encapsulation) was significantly lower than that in the sample group, confirming the existence and harmfulness of antagonistic effects. The compartmentalized design of this invention effectively avoids this problem, ensuring the stability of the complex microbial community during storage and initial application, creating conditions for synergistic effects.

[0019] This invention achieves multifunctional synergy, including nitrogen fixation, growth promotion, and disease resistance, through the loading of functional substances and the combination of strains. This invention not only loads key cofactors for nitrogenase, such as molybdenum and zinc, as well as carbon and nitrogen sources, into the embedding matrix, but also scientifically combines strains with different functions such as nitrogen fixation, growth promotion, and disease resistance. Pot cultivation experiments using this invention show that sugarcane treated with the sample exhibits significantly better growth indicators than the control, particularly with a more than 57% increase in aboveground fresh weight compared to the blank control, demonstrating its powerful comprehensive fertilizer effect. The control sample 3 showed significantly poorer results, illustrating the crucial role of functional substances in activating and maintaining the activity of the microbial agent. This multifunctional integration allows the microbial agent of this invention to transcend the limitations of single-function microbial agents.

[0020] In summary, the nitrogen-fixing bacterial agent and its application method provided by this invention effectively overcome the bottlenecks of existing technologies and have outstanding advantages such as strong environmental adaptability, high bacterial community stability, multifunctional synergy, and significant application effects. They are of great value in promoting fertilizer reduction and efficiency improvement and sustainable development in sugarcane planting. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The microbial agents used in this invention are all commercially available microbial agents. Among them, Bacillus jellyoides comes from Jinan Jinyuyuan Biotechnology Co., Ltd.; Bacillus megaterium comes from Jinan Jinyuyuan Biotechnology Co., Ltd.; Trichoderma viride comes from Guangzhou Weiyuan Biotechnology Co., Ltd.; Saccharomyces cerevisiae comes from Jinan Jinyuyuan Biotechnology Co., Ltd.; and Lactobacillus plantarum comes from Shaanxi Wensheng Biotechnology Co., Ltd.

[0027] LB liquid medium is Luria-Bertani medium, which consists of 10.0 g peptone, 5.0 g yeast extract, and 10.0 g sodium chloride per liter of deionized water, with the pH adjusted to 7.0.

[0028] PDA liquid medium is the liquid form of potato dextrose agar medium. The composition is 4.0 g potato extract powder and 20.0 g glucose per liter of deionized water, with natural pH.

[0029] YPD liquid medium is a standard nutrient-rich medium for culturing Saccharomyces cerevisiae and other yeasts. It consists of 10.0 g yeast extract, 20.0 g peptone, and 20.0 g glucose per liter of deionized water, at the natural pH value.

[0030] Example 1 This embodiment provides a nitrogen-fixing bacterial agent, the preparation method of which specifically includes the following steps: S1. Preparation of bacterial suspension or spore suspension: S11, Preparation of Bacillus group bacterial suspension: Weigh out Bacillus gelatinosa and Bacillus megaterium, and inoculate them into LB liquid medium at a mass ratio of 2.5%, respectively. Incubate them in a shaker at 36℃ and 180 r / min for 60 h. After the culture was completed, the bacterial pellet was collected by centrifugation at 8000 r / min for 12 min at 4℃. The bacterial precipitate was washed with sterile physiological saline and centrifuged at 8000 r / min for 12 min to remove culture medium residue. Resuspend the bacteria in sterile saline and adjust the bacterial concentration. The concentration of Bacillus spp. in the gelatinous form was 1.3 × 10⁻⁶. 9 CFU / mL, Bacillus megaterium cell concentration 1.5 × 10⁻⁶ 9 CFU / mL, respectively, yielded gelatinous Bacillus suspension and Bacillus megaterium suspension.

[0031] S12, Preparation of Trichoderma spore suspension: Weigh out the *Trichoderma viride* inoculum and inoculate it into PDA liquid medium at a mass ratio of 5.5%. Incubate at 28℃ and 150 r / min for 6 days with shaking. The culture will show a thick green spore mass. Add sterile physiological saline containing Tween 80 to the culture and vortex for 5 minutes to release spores; the volume ratio of sterile physiological saline containing Tween 80 to culture medium is 2:1, wherein the mass concentration of Tween 80 is 0.05%.

[0032] Filter the suspension to remove mycelial fragments and obtain spore filtrate; Centrifuge the spore filtrate at 4℃ and 5000r / min for 12min and collect the spore precipitate; The spore pellet was resuspended in sterile physiological saline with a Tween 80 concentration of 0.05% to adjust the spore concentration to 1.6 × 10⁻⁶. 8 Trichoderma spore suspension was prepared by spores / mL.

[0033] S13. Preparation of Saccharomyces cerevisiae suspension: Saccharomyces cerevisiae was inoculated into YPD liquid medium at a mass ratio of 2.5%; and cultured with shaking at 30℃ and 150 r / min for 28 h. Centrifuge the culture medium at 4℃ and 5000r / min for 12min and collect the bacterial precipitate; The bacterial cell precipitate was washed with sterile physiological saline and centrifuged at 4℃ and 6000r / min for 12min to remove culture medium residue. Resuspend in sterile saline and adjust the final bacterial concentration to 5.2 × 10⁻⁶. 9 A Saccharomyces cerevisiae suspension was prepared by using CFU / mL.

[0034] S14. Preparation of Lactobacillus plantarum suspension: Lactobacillus plantarum was inoculated into MRS liquid medium at a mass ratio of 2.5%; and cultured at 37℃ and 150 r / min for 28 h with shaking. Centrifuge the culture medium at 4℃ and 6000r / min for 12min and collect the bacterial precipitate; The bacterial cell precipitate was washed with sterile physiological saline and centrifuged at 4℃ and 6000r / min for 12min to remove culture medium residue. Resuspend in sterile saline and adjust the final bacterial concentration to 5.4 × 10⁻⁶. 9 A suspension of *Lactobacillus plantarum* was prepared using CFU / mL.

[0035] S2, Grouping and Loading: S21. Preparation of blank starch microspheres: Prepare a soluble starch solution with a mass concentration of 11%, heat to gelatinize, and then cool to 45°C; Starch microspheres were prepared by using a spray dryer with the inlet air temperature controlled at 155℃, the outlet air temperature at 82℃, and the feed flow rate at 8mL / min.

[0036] S22, Bacterial Adsorption: Saccharomyces cerevisiae suspension and Lactobacillus plantarum suspension were mixed with starch microspheres respectively. The mixing ratio was 1g starch microspheres added to 10mL of Saccharomyces cerevisiae suspension and 10mL of Lactobacillus plantarum suspension respectively. Adsorption was carried out at 50 r / min and 4℃ for 2 h, respectively; Starch microspheres with adsorbed bacterial cells were collected by centrifugation at 3000 r / min for 5 min. Unadsorbed bacterial cells were removed by rinsing with sterile physiological saline to obtain yeast-starch microspheres and lactic acid bacteria-starch microspheres, respectively.

[0037] S23. Preparation of bacterial functional group microcapsules: Prepare a 3% sodium alginate solution; add molasses powder and sodium molybdate, and stir to dissolve; wherein, 3g of molasses powder and 0.1g of sodium molybdate are added to every 100mL of sodium alginate solution. At a rotation speed of 100 r / min, a suspension of Bacillus jelly-like material, a suspension of Bacillus megaterium, yeast-starch microspheres, and lactic acid bacteria-starch microspheres were added; wherein the volume ratio of the suspension of Bacillus jelly-like material, the suspension of Bacillus megaterium, and the sodium alginate solution was 1:1:4.5; 2.5 g of yeast-starch microspheres and 2.5 g of lactic acid bacteria-starch microspheres were added per 100 mL of sodium alginate solution; Continue stirring at 100 rpm for 12 minutes to ensure that all components are evenly distributed in the sodium alginate gel precursor, thus obtaining the bacterial functional group mixture.

[0038] The bacterial functional group mixture was dropped into a calcium chloride solution containing 0.15% calcium carbonate and a mass concentration of 1.8%, and cross-linked and cured for 35 minutes at a speed of 200 r / min and a temperature of 22℃; wherein the volume ratio of the bacterial functional group mixture to the calcium chloride solution was 2:10. The bacterial functional group particles were collected by filtration, rinsed with sterile water, and then placed in a drying oven and dried at 32°C for 24 hours to obtain the bacterial functional group dry powder.

[0039] S24. Preparation of fungal functional group microcapsules: Prepare a 3% sodium alginate solution; add chitin powder, wheat bran powder, and zinc sulfate, and stir to dissolve; wherein, for every 100mL of sodium alginate solution, add 4g of chitin powder, 2g of wheat bran powder, and 0.03g of zinc sulfate. Stir at 300 rpm for 35 minutes to ensure uniform mixing; Continue adding Trichoderma spore suspension at 300 r / min, adjust the speed to 100 r / min, and continue stirring for 15 min to obtain fungal functional group mixture; the volume ratio of Trichoderma spore suspension to sodium alginate solution is 1:5.5; The fungal functional group mixture was dropped into a calcium chloride solution containing 0.15% calcium carbonate and a mass concentration of 1.8%, and cross-linked and cured for 35 minutes at a speed of 200 r / min and a temperature of 22℃; the volume ratio of the fungal functional group mixture to the calcium chloride solution was 2:10. The fungal functional group particles were collected by filtration, rinsed with sterile water, and then placed in a drying oven and dried at 32°C for 24 hours to obtain the fungal functional group dry powder.

[0040] S3, Mixing of nitrogen-fixing bacterial products: The bacterial functional group dry powder and the fungal functional group dry powder were mixed at a mass ratio of 5:3 and mixed at a speed of 15 r / min for 35 min to obtain a nitrogen-fixing bacterial agent, which was recorded as sample 1.

[0041] Example 2 This embodiment provides a nitrogen-fixing bacterial agent, the preparation method of which specifically includes the following steps: S1. Preparation of bacterial suspension or spore suspension: S11, Preparation of Bacillus group bacterial suspension: Weigh out Bacillus gelatinosa and Bacillus megaterium, and inoculate them into LB liquid medium at a mass ratio of 2%, respectively. Incubate them in a shaker at 35℃ and 200 r / min for 72 h. After the culture was completed, the bacterial pellet was collected by centrifugation at 9000 r / min for 10 min at 2℃. The bacterial precipitate was washed with sterile physiological saline and centrifuged at 9000 r / min for 10 min to remove culture medium residue. Resuspended in sterile physiological saline, the concentration of Bacillus cells in the gelatinous form was 1.4 × 10⁻⁶. 9 CFU / mL, Bacillus megaterium cell concentration 1.3 × 10⁻⁶ 9 CFU / mL, respectively, yielded gelatinous Bacillus suspension and Bacillus megaterium suspension.

[0042] S12, Preparation of Trichoderma spore suspension: Weigh out the *Trichoderma viride* inoculum and inoculate it into PDA liquid medium at a mass ratio of 5%. Incubate at 27°C and 160 r / min for 7 days with shaking. The culture will show a thick green spore mass. Add sterile physiological saline containing Tween 80 to the culture and vortex for 5 minutes to release spores; the volume ratio of sterile physiological saline containing Tween 80 to culture medium is 2:1, wherein the mass concentration of Tween 80 is 0.05%.

[0043] Filter the suspension to remove mycelial fragments and obtain spore filtrate; Centrifuge the spore filtrate at 2℃ and 5500r / min for 10min and collect the spore precipitate. The spore pellet was resuspended in sterile physiological saline with a Tween 80 concentration of 0.05% to adjust the spore concentration to 1.4 × 10⁻⁶. 8 Trichoderma spore suspension was prepared by spores / mL.

[0044] S13. Preparation of Saccharomyces cerevisiae suspension: Saccharomyces cerevisiae was inoculated into YPD liquid medium at a mass ratio of 2%; and cultured at 28℃ and 160 r / min for 32 h with shaking. Centrifuge the culture medium at 2℃ and 6500r / min for 10min and collect the bacterial precipitate; The bacterial cell precipitate was washed with sterile physiological saline and centrifuged at 2℃ and 6500r / min for 10min to remove culture medium residue. Resuspend in sterile saline and adjust the final bacterial concentration to 5.3 × 10⁻⁶. 9 A Saccharomyces cerevisiae suspension was prepared by using CFU / mL.

[0045] S14. Preparation of Lactobacillus plantarum suspension: Lactobacillus plantarum was inoculated into MRS liquid medium at a mass ratio of 2%; and cultured at 36℃ and 160 r / min for 32 h with shaking. Centrifuge the culture medium at 2℃ and 6500r / min for 10min and collect the bacterial precipitate; The bacterial cell precipitate was washed with sterile physiological saline and centrifuged at 2℃ and 6500r / min for 10min to remove culture medium residue. Resuspend in sterile saline and adjust the final bacterial concentration to 5.3 × 10⁻⁶. 9 A suspension of *Lactobacillus plantarum* was prepared using CFU / mL.

[0046] S2, Grouping and Loading: S21. Preparation of blank starch microspheres: Prepare a 12% (w / w) soluble starch solution, heat to gelatinize, and then cool to 42°C; Starch microspheres were prepared by using a spray dryer with the inlet air temperature controlled at 150℃, the outlet air temperature at 80℃, and the feed flow rate at 5mL / min.

[0047] S22, Bacterial Adsorption: Saccharomyces cerevisiae suspension and Lactobacillus plantarum suspension were mixed with starch microspheres respectively. The mixing ratio was 1g starch microspheres added to 12mL of Saccharomyces cerevisiae suspension and 8mL of Lactobacillus plantarum suspension. Adsorption was carried out at a rotation speed of 60 r / min and a temperature of 2℃ for 2.5 h. Starch microspheres with adsorbed bacterial cells were collected by centrifugation at 3500 r / min for 4 min. Unadsorbed bacterial cells were removed by rinsing with sterile physiological saline to obtain yeast-starch microspheres and lactic acid bacteria-starch microspheres, respectively.

[0048] S23. Preparation of bacterial functional group microcapsules: Prepare a 2% sodium alginate solution; add molasses powder and sodium molybdate, and stir to dissolve; wherein, 4g of molasses powder and 0.05g of sodium molybdate are added to every 100mL of sodium alginate solution. At a rotation speed of 120 r / min, a suspension of Bacillus jelly-like material, a suspension of Bacillus megaterium, yeast-starch microspheres, and lactic acid bacteria-starch microspheres were added; wherein the volume ratio of the suspension of Bacillus jelly-like material, the suspension of Bacillus megaterium, and the sodium alginate solution was 1:1:5; 2 g of yeast-starch microspheres and 3 g of lactic acid bacteria-starch microspheres were added per 100 mL of sodium alginate solution; Continue stirring at 120 rpm for 10 minutes to ensure that all components are evenly distributed in the sodium alginate gel precursor, thus obtaining the bacterial functional group mixture.

[0049] The bacterial functional group mixture was dropped into a calcium chloride solution containing 0.2% calcium carbonate and a mass concentration of 1.0%, and cross-linked and cured for 40 minutes at a speed of 220 r / min and a temperature of 20℃; wherein the volume ratio of the bacterial functional group mixture to the calcium chloride solution was 1:10. The bacterial functional group particles were collected by filtration, rinsed with sterile water, and then placed in a drying oven and dried at 30°C for 26 hours to obtain the bacterial functional group dry powder.

[0050] S24. Preparation of fungal functional group microcapsules: Prepare a 2% sodium alginate solution; add chitin powder, wheat bran powder, and zinc sulfate, and stir to dissolve; wherein, for every 100mL of sodium alginate solution, add 3g of chitin powder, 2.5g of wheat bran powder, and 0.02g of zinc sulfate. Stir at 320 rpm for 30 min to ensure uniform mixing; Continue adding Trichoderma spore suspension at 320 r / min, adjust the speed to 120 r / min, and continue stirring for 10 min to obtain fungal functional group mixture; the volume ratio of Trichoderma spore suspension to sodium alginate solution is 1:5; The fungal functional group mixture was dropped into a calcium chloride solution containing 0.2% calcium carbonate and a mass concentration of 1.0%, and cross-linked and cured for 40 minutes at a speed of 220 r / min and a temperature of 20℃; the volume ratio of the fungal functional group mixture to the calcium chloride solution was 3:10. The fungal functional group particles were collected by filtration, rinsed with sterile water, and then placed in a drying oven and dried at 30°C for 26 hours to obtain the fungal functional group dry powder.

[0051] S3, Mixing of nitrogen-fixing bacterial products: The bacterial functional group dry powder and the fungal functional group dry powder were mixed at a mass ratio of 6:3 and mixed at a speed of 18 r / min for 30 min to obtain a nitrogen-fixing bacterial agent, which was recorded as sample 2.

[0052] Example 3 This embodiment provides a nitrogen-fixing bacterial agent, the preparation method of which specifically includes the following steps: S1. Preparation of bacterial suspension or spore suspension: S11, Preparation of Bacillus group bacterial suspension: Weigh out Bacillus gelatinosa and Bacillus megaterium, and inoculate them into LB liquid medium at a mass ratio of 3%, respectively. Incubate them in a shaker at 37℃ and 160 r / min for 48 h. After the culture was completed, the bacterial pellet was collected by centrifugation at 7000 r / min for 15 min at 5℃. The bacterial precipitate was washed with sterile physiological saline and centrifuged at 7000 r / min for 15 min to remove culture medium residue. Resuspended in sterile physiological saline, the concentration of Bacillus cells in the gelatinous form was 1.2 × 10⁻⁶. 9 CFU / mL, Bacillus megaterium cell concentration 1.1×10⁻⁶ 9 CFU / mL, respectively, yielded gelatinous Bacillus suspension and Bacillus megaterium suspension.

[0053] S12, Preparation of Trichoderma spore suspension: Weigh out the *Trichoderma viride* inoculum and inoculate it into PDA liquid medium at a mass ratio of 6%. Incubate at 29℃ and 140 r / min for 5 days with shaking. The culture will show a thick green spore mass. Add sterile physiological saline containing Tween 80 to the culture and vortex for 5 minutes to release spores; the volume ratio of sterile physiological saline containing Tween 80 to culture medium is 2:1, wherein the mass concentration of Tween 80 is 0.05%.

[0054] Filter the suspension to remove mycelial fragments and obtain spore filtrate; Centrifuge the spore filtrate at 5℃ and 4500r / min for 15min and collect the spore precipitate. The spore pellet was resuspended in sterile physiological saline with a Tween 80 concentration of 0.05% to adjust the spore concentration to 1.2 × 10⁻⁶. 8 Trichoderma spore suspension was prepared by spores / mL.

[0055] S13. Preparation of Saccharomyces cerevisiae suspension: Saccharomyces cerevisiae was inoculated into YPD liquid medium at a mass ratio of 3%; and cultured with shaking at 32℃ and 140 r / min for 24 h. Centrifuge the culture medium at 5℃ and 5500r / min for 15min and collect the bacterial precipitate; The bacterial cell precipitate was washed with sterile physiological saline and centrifuged at 5℃ and 5500r / min for 15min to remove culture medium residue. Resuspend in sterile saline and adjust the final bacterial concentration to 5.4 × 10⁻⁶. 9 A Saccharomyces cerevisiae suspension was prepared by using CFU / mL.

[0056] S14. Preparation of Lactobacillus plantarum suspension: Lactobacillus plantarum was inoculated into MRS liquid medium at a mass ratio of 3%; and cultured at 38℃ and 140 r / min for 24 h with shaking. Centrifuge the culture medium at 5℃ and 5500r / min for 15min and collect the bacterial precipitate; The bacterial cell precipitate was washed with sterile physiological saline and centrifuged at 5℃ and 5500r / min for 15min to remove culture medium residue. Resuspend in sterile saline and adjust the final bacterial concentration to 5.1 × 10⁻⁶. 9 A suspension of *Lactobacillus plantarum* was prepared using CFU / mL.

[0057] S2, Grouping and Loading: S21. Preparation of blank starch microspheres: Prepare a 10% (w / w) soluble starch solution, heat to gelatinize, and then cool to 48°C; Starch microspheres were prepared by using a spray dryer with the inlet air temperature controlled at 160℃, the outlet air temperature at 85℃, and the feed flow rate at 10mL / min.

[0058] S22, Bacterial Adsorption: Saccharomyces cerevisiae suspension and Lactobacillus plantarum suspension were mixed with starch microspheres respectively. The mixing ratio was 1g starch microspheres added to 8mL of Saccharomyces cerevisiae suspension and 12mL of Lactobacillus plantarum suspension. Adsorption was carried out at a rotation speed of 40 r / min and a temperature of 5 °C for 1.5 h. Starch microspheres with adsorbed bacterial cells were collected by centrifugation at 2500 r / min for 6 min. Unadsorbed bacterial cells were removed by rinsing with sterile physiological saline to obtain yeast-starch microspheres and lactic acid bacteria-starch microspheres, respectively.

[0059] S23. Preparation of bacterial functional group microcapsules: Prepare a 4% sodium alginate solution; add molasses powder and sodium molybdate, and stir to dissolve; wherein, 2g of molasses powder and 0.15g of sodium molybdate are added to every 100mL of sodium alginate solution. At a rotation speed of 80 r / min, a suspension of Bacillus jelly-like material, a suspension of Bacillus megaterium, yeast-starch microspheres, and lactic acid bacteria-starch microspheres were added; wherein the volume ratio of the suspension of Bacillus jelly-like material, the suspension of Bacillus megaterium, and the sodium alginate solution was 1:1:4; 3 g of yeast-starch microspheres and 2 g of lactic acid bacteria-starch microspheres were added per 100 mL of sodium alginate solution; Continue stirring at 80 rpm for 15 minutes to ensure that all components are evenly distributed in the sodium alginate gel precursor, thus obtaining the bacterial functional group mixture.

[0060] The bacterial functional group mixture was dropped into a calcium chloride solution containing 0.1% calcium carbonate and a mass concentration of 2.0%, and cross-linked and cured for 30 minutes at a speed of 180 r / min and a temperature of 25℃; wherein the volume ratio of the bacterial functional group mixture to the calcium chloride solution was 3:10. The bacterial functional group particles were collected by filtration, rinsed with sterile water, and then placed in a drying oven and dried at 35°C for 22 hours to obtain the bacterial functional group dry powder.

[0061] S24. Preparation of fungal functional group microcapsules: Prepare a 4% sodium alginate solution; add chitin powder, wheat bran powder, and zinc sulfate, and stir to dissolve; wherein, for every 100mL of sodium alginate solution, add 5g of chitin powder, 1.5g of wheat bran powder, and 0.04g of zinc sulfate. Stir at 280 rpm for 40 minutes to ensure uniform mixing; Continue adding Trichoderma spore suspension at 280 r / min, adjust the speed to 80 r / min, and continue stirring for 20 min to obtain fungal functional group mixture; the volume ratio of Trichoderma spore suspension to sodium alginate solution is 1:6; The fungal functional group mixture was dropped into a calcium chloride solution containing 0.1% calcium carbonate and a mass concentration of 2.0%, and cross-linked and cured for 30 minutes at a speed of 180 r / min and a temperature of 25℃; the volume ratio of the fungal functional group mixture to the calcium chloride solution was 1:10. The fungal functional group particles were collected by filtration, rinsed with sterile water, and then placed in a drying oven and dried at 35°C for 22 hours to obtain the fungal functional group dry powder.

[0062] S3, Mixing of nitrogen-fixing bacterial products: The bacterial functional group dry powder and the fungal functional group dry powder were mixed at a mass ratio of 4:3 and mixed at a speed of 12 r / min for 40 min to obtain a nitrogen-fixing bacterial agent, which was recorded as sample 3.

[0063] Comparative Example 1 This comparative example provides a nitrogen-fixing bacterial agent, the main difference from Example 1 being that it does not perform group encapsulation, but instead simply mixes all bacterial strains and encapsulates them all at once. The specific steps include: S1. Preparation of bacterial suspension or spore suspension: Same as step S1 in Example 1.

[0064] S2, Embedding and Loading: To prepare a 3% sodium alginate solution, add 3g of molasses powder, 0.1g of sodium molybdate, 4g of chitin powder, 2g of wheat bran powder, and 0.03g of zinc sulfate per 100mL. At a rotation speed of 100 r / min, suspensions of five bacteria were added to obtain a bacterial mixture, wherein the volume ratio of Bacillus subtilis suspension, Bacillus megaterium suspension, Saccharomyces cerevisiae suspension, Lactobacillus plantarum suspension, Trichoderma spore suspension and sodium alginate solution was 0.5:0.5:0.5:0.5:1:4.5. The bacterial culture mixture was dropped into a calcium chloride solution containing 0.15% calcium carbonate and a mass concentration of 1.8%, and cross-linked and cured for 35 minutes at a speed of 200 r / min and a temperature of 22℃; wherein the volume ratio of the bacterial culture mixture to the calcium chloride solution was 2:10. The solidified particles were collected by filtration, rinsed with sterile water, and placed in a drying oven at 32°C for 24 hours to obtain nitrogen-fixing bacterial agent dry powder, which was designated as control 1.

[0065] Comparative Example 2 This comparative example provides a nitrogen-fixing bacterial agent, the main difference from Example 1 being that starch microspheres are not used for adsorption and isolation in the bacterial functional group. Specific steps include: S1. Preparation of bacterial suspension or spore suspension: Same as step S1 in Example 1.

[0066] S2, Grouping and Loading: S21. Preparation of bacterial functional group microcapsules: Prepare a 3% sodium alginate solution; add 3g of molasses powder and 0.1g of sodium molybdate per 100mL; add a suspension of Bacillus jelly-like bacteria, Bacillus megaterium, Saccharomyces cerevisiae, and Lactobacillus plantarum at a speed of 100r / min; wherein the volume ratio of the suspension of Bacillus jelly-like bacteria, Bacillus megaterium, Saccharomyces cerevisiae, and Lactobacillus plantarum to the sodium alginate solution is 0.5:0.5:0.5:0.5:4.5; the subsequent steps are the same as in Example 1, S23, to obtain the bacterial functional group dry powder.

[0067] S22. Preparation of fungal functional group microcapsules: Same as step S24 in Example 1, to obtain fungal functional group dry powder.

[0068] S3. Mixing of nitrogen-fixing bacterial agent products: Same as step S3 in Example 1, to obtain nitrogen-fixing bacterial agent, which is designated as control product 2.

[0069] Comparative Example 3 This comparative example provides a nitrogen-fixing bacterial agent, the main difference from Example 1 being that no functional substances are loaded during the encapsulation process. Specific steps include: S1. Preparation of bacterial suspension or spore suspension: Same as step S1 in Example 1.

[0070] S2, Grouping and Loading: S21. Preparation of bacterial functional group microcapsules: Prepare a 3% sodium alginate solution; without adding any functional substances; add gelatinous Bacillus suspension, Bacillus megaterium suspension, yeast-starch microspheres and lactic acid bacteria-starch microspheres at a speed of 100 r / min, in the same proportion as in Example 1 S23; the subsequent steps are the same as in Example 1 S23, to obtain the bacterial functional group dry powder.

[0071] S22. Preparation of fungal functional group microcapsules: Prepare a 3% sodium alginate solution; do not add any functional substances; add Trichoderma spore suspension in the same proportion as in Example 1, S24; follow the same steps as in Example 1, S24 to obtain fungal functional group dry powder.

[0072] S3. Mixing of nitrogen-fixing bacterial agent products: Same as step S3 in Example 1, to obtain nitrogen-fixing bacterial agent, which is designated as control product 3.

[0073] Comparative Example 4 This comparative example provides a nitrogen-fixing bacterial agent, the main difference from Example 1 being the use of peat moss carrier instead of sodium alginate microcapsule encapsulation. Specific steps include: S1. Preparation of bacterial suspension or spore suspension: Same as step S1 in Example 1.

[0074] S2. Preparation of nitrogen-fixing bacterial agents: S21. Preparation of blank starch microspheres: Same as step S21 in Example 1.

[0075] S22, Bacterial Adsorption: Same as step S22 in Example 1.

[0076] S23, peat moss carrier microcapsule encapsulation: Weigh out the dried peat moss carrier as the adsorption matrix; The gelatinous Bacillus suspension, Bacillus megaterium suspension, Saccharomyces cerevisiae suspension, Lactobacillus plantarum suspension prepared in step S1, and the yeast-starch microspheres and lactic acid bacteria-starch microspheres prepared in step S22 were all mixed and added to the peat moss carrier; wherein, 25 mL of gelatinous Bacillus suspension, 25 mL of Bacillus megaterium suspension, 25 mL of Saccharomyces cerevisiae suspension, 2.5 g of yeast-starch microspheres and 2.5 g of lactic acid bacteria-starch microspheres were added to every 100 g of peat moss carrier. Stir for 10 minutes until well mixed to ensure that the bacteria are fully adsorbed onto the carrier; The adsorbed wet bacterial agent was placed in a drying oven and dried at 40°C for 28 hours. The dried block bacterial agent was then crushed and sieved to obtain a solid powdered bacterial agent, which was designated as control 4.

[0077] Analysis and Testing The following experiments were conducted using samples 1-3 and controls 1-4 to verify the colonization ability, growth-promoting effect, and product quality of the nitrogen-fixing bacteria agent of the present invention under simulated special sugarcane conditions.

[0078] I. Tests on the survival and colonization ability of bacterial strains under acidic, high-sucrose conditions Test method: To simulate the high sucrose and acidic environment of sugarcane rhizosphere, a liquid culture medium with pH=5.0 and a sucrose concentration of 20% was prepared. 1.0 g of each test sample or control was weighed and added to 100 mL of the above culture medium under aseptic conditions. The medium was then incubated at 30℃ and 150 r / min with shaking. Samples were taken on day 0 and day 7 of incubation. The total viable number of bacteria representing the bacterial population (using nutrient agar NA medium) and the number of *Trichoderma viride* spores representing the fungal population (using PDA medium supplemented with streptomycin and chloramphenicol to inhibit bacterial growth) in the samples were determined using the dilution plating method. The survival rate of each strain on day 7 was calculated. The results are shown in Table 1.

[0079] Survival rate (%) = (Number of live bacteria on day 7 / Number of live bacteria on day 0) × 100%.

[0080] Table 1: Results of bacterial survival rate determination under acidic high sucrose environment As can be seen from the results in Table 1, the core functional strains of samples 1-3 of the present invention maintained a very high survival rate in a simulated sugarcane rhizosphere environment, indicating that the microcapsule encapsulation and internal loading of functional substances strategy of the present invention effectively protected the bacterial cells and laid the foundation for their successful colonization.

[0081] Control sample 1 underwent only simple mixed embedding, and its survival rate was the lowest. In particular, the survival rate of Trichoderma was significantly reduced, which proved that the antagonistic effect between fungal species was aggravated in the simulated sugarcane rhizosphere environment. The compartmentalized embedding of the present invention is crucial for maintaining the stability of the compound microbial agent.

[0082] The control sample 2, which did not use starch microspheres for isolation, had a lower total bacterial survival rate than the sample group, indicating that starch microspheres played a positive role in the physical isolation and protection of sensitive bacteria.

[0083] The control sample 3, which was not loaded with functional substances, had a significantly lower survival rate than the sample group, demonstrating that functional substances such as molasses and sodium molybdate provided crucial energy and nutritional support for the survival and activity of the strains under adverse conditions.

[0084] Control sample 4 used a peat moss carrier, which had an extremely low survival rate, demonstrating that traditional carriers cannot provide the protection needed to withstand acidic, high-sugar environments.

[0085] II. Sugarcane Potted Plant Growth Promotion Experiment Test method: A pot experiment was conducted using typical acidic sugarcane garden soil (pH 5.2). Each pot contained 5.0 kg of soil, and 50 g of each test inoculant was applied quantitatively. All test inoculant samples or controls were mixed with 500 g of potassium sulfate compound fertilizer (N-P2O5-K2O = 15-15-15) and applied as basal fertilizer in furrows. The blank control group received no inoculant or fertilizer. Each treatment was replicated in 10 pots. Sugarcane tissue culture seedlings with uniform growth were transplanted and managed routinely in a greenhouse. No topdressing was applied during the experiment. After 90 days of growth, plant height, stem diameter, and number of tillers were measured, and the above-ground fresh weight was measured and the mean values ​​were recorded. The results are shown in Table 2.

[0086] Table 2: Results of Sugarcane Potted Plant Growth Promotion Experiment As shown in Table 2, under conditions of no chemical nitrogen fertilizer application, the sugarcane plants treated with samples 1-3 of this invention showed better performance than all control groups and the blank control group in all growth indicators, proving that the microbial agent of this invention has efficient nitrogen fixation and comprehensive growth promotion capabilities, and can partially or completely replace chemical nitrogen fertilizer.

[0087] Among them, control 1 had the worst effect, confirming that the antagonism between the strains led to a significant decrease in the actual field effect; control 2 and control 4 had effects worse than the sample group, indicating that the fine encapsulation process is crucial to maximizing the potential of the microbial agent; control 3 had poor effect, proving that the loaded functional substances have an irreplaceable role in stimulating and maintaining the nitrogen fixation and life-promoting properties of the microbial agent.

[0088] III. Simulation Application Testing A greenhouse pot experiment was conducted. The soil was typical acidic sugarcane garden soil (pH 5.2), with 5.0 kg of soil per pot. Sample 1 was selected for the following treatment: In this experiment, potassium sulfate compound fertilizer (N-P2O5-K2O = 15-15-15) was selected as the base fertilizer. Treatment A was the basal fertilizer mixed application group, and treatment B was the seed dipping treatment group. For specific treatment methods, please refer to Table 3.

[0089] Table 3: Summary of Simulated Application Test Processing Methods Sixty days after planting: plant height, stem diameter, and chlorophyll content (SPAD value) were measured; at harvest: plant height, stem diameter, number of tillers, aboveground fresh weight, and total nitrogen content of leaves were measured. Results are shown in Tables 4 and 5.

[0090] Table 4: Summary of Simulated Application Testing Methods (60 days of test data) Table 5: Summary of Simulated Application Test Processing Methods (Harvest Period Test Data) As shown in Table 4, 60 days after planting, the growth indicators of all treatment groups treated with microbial agents were significantly better than those of the corresponding control groups. In treatment group A, the higher the ratio of microbial agent to basal fertilizer, the better the early growth-promoting effect, indicating that high concentrations of microbial agents help to quickly establish rhizosphere dominance in the early growth stage. In treatment group B, the treatment group with a microbial agent to water ratio of 1:6.5 performed best, indicating that an appropriate inoculation concentration is crucial for the early effectiveness of the microbial agent.

[0091] As shown in Table 5, by harvest time, the overall effect of the mixed application of microbial agent and basal fertilizer (Group A) was better than that of the seed dipping treatment (Group B). Notably, treatment A2 performed best in most indicators, with the highest aboveground fresh weight and leaf total nitrogen content. This indicates that the 1:15 mixing ratio achieved the most durable and efficient symbiosis between the microbial community and the crop, avoiding potential later-stage competition from excessively high concentrations, making it the optimal application ratio for overall benefits. In Group B, treatment B2 remained the best ratio among the seed dipping treatments. All treatments applying the microbial agent showed significantly better results than the control, demonstrating the application value of the microbial agent of this invention.

[0092] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A nitrogen-fixing bacterial agent, characterized in that, The nitrogen-fixing bacterial agent comprises a mixture of bacterial functional group dry powder and fungal functional group dry powder immobilized by sodium alginate gel; the bacterial functional group dry powder contains Bacillus jellyoidus, Bacillus megaterium, Saccharomyces cerevisiae, and Lactobacillus plantarum; the fungal functional group dry powder contains Trichoderma viride.

2. The nitrogen-fixing bacterial agent according to claim 1, characterized in that, The Saccharomyces cerevisiae and Lactobacillus plantarum were pre-adsorbed onto biodegradable starch microspheres, and then encapsulated together with Bacillus jellyoidus and Bacillus megaterium in sodium alginate gel.

3. The nitrogen-fixing bacterial agent according to claim 2, characterized in that, The starch microspheres are prepared by preparing a soluble starch solution with a mass concentration of 10% to 12%, heating and gelatinizing it, and then cooling it to 42°C to 48°C; controlling the inlet air temperature to 150°C to 160°C, the outlet air temperature to 80°C to 85°C, and the feed flow rate to 5 mL / min to 10 mL / min, and then preparing the starch microspheres by spray drying.

4. The nitrogen-fixing bacterial agent according to claim 1, characterized in that, The preparation method of the bacterial functional group dry powder is as follows: Bacillus spp., Bacillus megaterium, Saccharomyces cerevisiae, and Lactobacillus plantarum are prepared into bacterial suspensions respectively; the bacterial suspensions of Saccharomyces cerevisiae and Lactobacillus plantarum are adsorbed onto blank starch microspheres to obtain yeast-starch microspheres and lactic acid bacteria-starch microspheres respectively; sodium alginate solution is mixed with molasses powder and sodium molybdate, and then the Bacillus spp., Bacillus megaterium, yeast-starch microspheres and lactic acid bacteria-starch microspheres are added and mixed evenly to obtain a bacterial functional group mixture; the bacterial functional group mixture is dropped into calcium chloride solution for cross-linking and solidification, the gel particles are collected, and dried to obtain the bacterial functional group dry powder.

5. The nitrogen-fixing bacterial agent according to claim 4, characterized in that, In every 100 mL of sodium alginate solution, the amount of molasses powder added is 2 g to 4 g, and the amount of sodium molybdate added is 0.05 g to 0.15 g; the total amount of gelatinous Bacillus suspension and Bacillus megaterium suspension added is in a volume ratio of 1:(4 to 5) to the sodium alginate solution; the amount of yeast-starch microspheres and lactic acid bacteria-starch microspheres added is 2 g to 3 g per 100 mL of sodium alginate solution.

6. The nitrogen-fixing bacterial agent according to claim 1, characterized in that, The preparation method of the fungal functional group dry powder is as follows: a green Trichoderma agent is prepared into a spore suspension; sodium alginate solution is mixed evenly with chitin powder, wheat bran powder and zinc sulfate, and then Trichoderma spore suspension is added and mixed evenly to obtain a fungal functional group mixture; the fungal functional group mixture is dripped into calcium chloride solution for cross-linking and solidification, the gel particles are collected, and dried to obtain fungal functional group dry powder.

7. The nitrogen-fixing bacterial agent according to claim 6, characterized in that, In every 100 mL of sodium alginate solution, the amount of chitin powder added is 3 g to 5 g, the amount of wheat bran powder added is 1.5 g to 2.5 g, and the amount of zinc sulfate added is 0.02 g to 0.04 g; the volume ratio of Trichoderma spore suspension to sodium alginate solution is 1:(5 to 6).

8. The nitrogen-fixing bacterial agent according to any one of claims 5 or 6, characterized in that, The sodium alginate solution has a mass concentration of 2% to 4%; the calcium chloride solution is a calcium chloride solution containing 0.1% to 0.2% calcium carbonate with a mass concentration of 1.0% to 2.0%. The cross-linking curing conditions are cross-linking and curing for 30 to 40 minutes at a rotation speed of 180 r / min to 220 r / min and a temperature of 20℃ to 25℃.

9. The nitrogen-fixing bacterial agent according to claim 1, characterized in that, The mass mixing ratio of the bacterial functional group dry powder and the fungal functional group dry powder is (4-6):

3.

10. The application of the nitrogen-fixing bacterial agent according to any one of claims 1 to 9 in sugarcane cultivation, characterized in that, The application method is to mix the nitrogen-fixing bacteria agent with the base fertilizer at a mass ratio of 1:(10-20) and then apply it in furrows or holes, or to add the nitrogen-fixing bacteria agent to water at a mass ratio of 1:(5-8) to obtain a bacterial suspension for dipping sugarcane seed stalks.

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