Compound preparation containing nitrogen-fixing bacteria and application of compound preparation in yield increase of sugarcane

Abstract

The invention discloses a compound preparation containing nitrogen-fixing bacteria and application of the compound preparation in sugarcane yield increase, and belongs to the technical field of microorganisms. Comprising a complex microbial inoculant and biochar loaded with the complex microbial inoculant, and the complex microbial inoculant is composed of the following strains: Azospirillum brasilense ACCC 04138, Bacillus mucilaginosus ACCC 19749 and Pseudomonas fluorescens CICC 23919. Azospirillum brasilense, bacillus mucilaginosus and pseudomonas fluorescens are compounded to prepare a complex microbial inoculant, and the complex microbial inoculant is specifically combined with biochar prepared from bagasse and mushroom residues, so that the acid soil structure can be remarkably improved, and the sugarcane yield and sugar content can be increased. The invention provides a novel complex microbial inoculant for sugarcane planting, and provides a scientific basis for improvement of sugarcane yield and quality and improvement of acid soil.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a composite preparation containing nitrogen-fixing bacteria and its application in increasing the yield of sugarcane. BACKGROUND

[0002] Sugarcane, as an important sugar and energy crop, has a long growth cycle and a large nitrogen requirement. In traditional cultivation, the use of chemical nitrogen fertilizer (such as urea) not only leads to high production costs, but also causes a series of problems: low nutrient utilization rate: the pH value of red soil in southern China is low (4.5-5.5), and the cation exchange capacity is low. Chemical nitrogen fertilizer is easily lost through leaching and volatilization, causing soil nitrogen surplus and the risk of water eutrophication; soil quality degradation: long-term application of chemical nitrogen fertilizer leads to imbalance of soil microbial community, soil compaction, and decline of sugarcane root activity; decline of sugarcane quality and stress resistance: excessive chemical nitrogen fertilizer leads to reduced sugar content in sugarcane and easy lodging under drought, typhoon and other stress conditions.

[0003] To solve the above problems, existing technologies have attempted to apply nitrogen-fixing bacteria to sugarcane cultivation. Some use a single nitrogen-fixing bacteria as a microbial inoculant, but this has the following defects: low nitrogen fixation efficiency: the survival rate of a single nitrogen-fixing bacteria in a red soil acidic environment is very low, and it cannot form a stable symbiotic relationship with the sugarcane root system, so the cumulative nitrogen fixation amount during the growth period cannot meet the nitrogen requirement of sugarcane; single function: only nitrogen supply is considered, the demand for phosphorus and potassium by sugarcane is not addressed, and there is no improvement in soil acidification; unstable application effect: the nutrient requirements of sugarcane at different growth stages (seedling stage, tillering stage, and elongation stage) are not considered, and the single application method leads to fertilizer loss in the later period, resulting in low yield improvement and far from meeting production needs. Some use a mixture of nitrogen-fixing bacteria and other bacteria, such as the invention patent CN114467668A "Method for promoting the growth of sugarcane", which discloses a mixture of nitrogen-fixing bacteria, soil conditioner, and organic fertilizer. However, this patent requires the use of multiple raw materials to make the soil conditioner (mixed slurry made from metakaolin and lime water, sugarcane leaf powder, humic acid, and filter mud), four types of bacteria to make the composite inoculant (Sporidesmium scoliospermum, S. hoohinai, Bacillus subtilis, and Lactobacillus reuteri), organic fertilizer (composed of sugarcane residue, wood ash, spirulina farmyard manure, wheat bran, and chicken manure), and compound fertilizer (composed of urea, humic acid, monoammonium phosphate, magnesium sulfate, zinc sulfate, and polyaspartic acid), and a slow-release agent (composed of phenylpropyl emulsion, guar gum, and chitosan) to coat the compound fertilizer. As can be seen, this technology involves the simultaneous use of multiple raw materials, which is complex and cumbersome to operate. Moreover, the technology does not verify the use of the composite inoculant in improving the soil, and only provides experimental data on yield, lacking more detailed evidence of the effects of the raw materials.

[0004] Therefore, developing a compound preparation which can adapt to the acidic environment of red soil, give consideration to the functions of "nitrogen fixation-phosphorus release-soil improvement-stress resistance", and adapt to the nutrient demand of sugarcane growth period, becomes the key to solve the problem of sugarcane production. SUMMARY

[0005] The application aims to provide a compound preparation containing nitrogen-fixing bacteria and its application in increasing the yield of sugarcane, so as to solve the problems in the prior art. The compound preparation is prepared by compounding Azospirillum brasilense, Bacillus mucilaginosus and Pseudomonas fluorescens, and then combined with the biochar prepared from sugarcane residue and mushroom residue, so that the structure of acidic soil can be significantly improved and the yield and sugar content of sugarcane can be increased.

[0006] To achieve the above object, the application provides the following scheme.

[0007] The application provides a compound preparation containing nitrogen-fixing bacteria, which comprises a compound microbial agent and biochar loaded with the compound microbial agent, and the compound microbial agent is composed of the following strains: Azospirillum brasilense ACCC04138, Bacillus mucilaginosus Krassilnikov ACCC 19749 and Pseudomonas fluorescens CICC 23919.

[0008] Preferably, in the compound microbial agent, the mass ratio of the Azospirillum brasilense, the Bacillus mucilaginosus and the Pseudomonas fluorescens is (1-2):(0.5-2):(1-2), and the mass ratio of the Azospirillum brasilense and the biochar is 1:(5-8).

[0009] Preferably, the biochar comprises sugarcane residue and mushroom residue, and the mass ratio of the sugarcane residue and the mushroom residue is (1-2):(1-2).

[0010] The application further provides a preparation method of the compound microbial agent, which comprises the following steps.

[0011] Azospirillum brasilense bacterial liquid, Bacillus mucilaginosus bacterial liquid and Pseudomonas fluorescens bacterial liquid are prepared through fermentation culture respectively.

[0012] Sugarcane residue and mushroom residue are mixed and pyrolyzed at high temperature to prepare biochar.

[0013] The Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid are mixed, then sprayed on the biochar, and intermittently stirred and mixed for 1-2 hours, then the Pseudomonas fluorescens bacterial liquid is added and stirred for 1-2 hours to obtain the compound microbial agent.

[0014] Preferably, the fermentation medium for preparing the Azospirillum brasilense bacterial liquid and the Pseudomonas fluorescens bacterial liquid is a nutrient broth medium, and the fermentation medium for preparing the nutrient broth medium is a silicate bacterial culture medium.

[0015] Preferably, the high-temperature pyrolysis temperature is 500 DEG C, and the time is 3 hours.

[0016] Preferably, the intermittent stirring condition is that after spraying the Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid on the biochar, stirring is started at 50 rpm for 5 minutes, and then stirring is performed once every 20 minutes under the same condition.

[0017] Preferably, the optimal mass ratio of the Azospirillum brasilense bacterial liquid, the Bacillus mucilaginosus bacterial liquid and the Pseudomonas fluorescens bacterial liquid is 1.8:1:1.8.

[0018] The optimal mass ratio of the sugarcane residue and the mushroom residue is 1.5:1.

[0019] The optimal mass ratio of the Azospirillum brasilense bacterial liquid and the biochar is 1:8.

[0020] The application further provides application of the composite preparation in improving soil structure of a sugarcane field.

[0021] The application further provides application of the composite preparation in improving yield and sugar content of sugarcane.

[0022] The application discloses the following technical effects:

[0023] The application provides a new composite bacterial agent for sugarcane planting, and provides a scientific basis for improving yield and quality of sugarcane and improving acid soil. DETAILED DESCRIPTION

[0024] The detailed description should not be considered as limiting the application, but rather as a description of certain embodiments of the application.

[0025] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an individual value from the range can be expressly disclosed herein to mean that each and every intermediate value of the range is also expressly disclosed. All individual values and subranges from the disclosed ranges are specifically included in the scope of the present application. When a range is disclosed of the form, "from X to Y," or "from about X to Y," it is meant "X, Y, and any intermediate range and value thereof that self-evidently falls within the ambit of the disclosure."

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0027] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from consideration of the description and practice of the application. The description and examples are illustrative only.

[0028] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0029] The following examples involve the following strains: Azospirillum brasilense ACCC04138, purchased from China General Microbiological Culture Collection Center (CGMCC); Bacillus mucilaginosus Krassilnikov ACCC 19749, purchased from China General Microbiological Culture Collection Center (CGMCC); Pseudomonas fluorescens CICC 23919, purchased from China Industrial Culture Collection Center (CICC).

[0030] Culture medium formula:

[0031] LB solid medium: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, agar 20 g, water 1000 mL, pH 7.0. Sterilized at 121 °C for 15 min.

[0032] Nutrient Broth Medium: Proteose peptone 5 g, beef extract powder 3 g, sodium chloride 5 g, agar 15 g, distilled water 1000 mL, pH 7.0. Sterilized at 121℃ for 15 min.

[0033] Silicate Bacteria Medium: Yeast extract 0.3 g, sucrose 10 g, ammonium sulfate 0.5 g, calcium carbonate 0.5 g, magnesium sulfate heptahydrate 0.5 g, potassium phosphate dibasic 1 g, distilled water 1000 mL, pH 7.2. Sterilized at 121℃ for 15 min.

[0034] Example 1 A composite preparation containing nitrogen-fixing bacteria

[0035] (1) Strain culture

[0036] The Azospirillum brasiliense (ACCC 04138) was first activated on LB solid medium, then inoculated into nutrient broth medium, and cultured at 30℃ with 180 rpm shaking for 48 h to obtain Azospirillum brasiliense bacterial liquid; the bacterial concentration was above 10 8 CFU / mL.

[0037] The Bacillus mucilaginosus (ACCC 19749) was first activated on LB solid medium, then inoculated into silicate bacteria medium, and cultured at 30℃ with 180 rpm shaking for 48 h to obtain Bacillus mucilaginosus bacterial liquid; the bacterial concentration was above 10 8 CFU / mL.

[0038] The Pseudomonas fluorescens (CICC 23919) was first activated on LB solid medium, then inoculated into nutrient broth medium, and cultured at 30℃ with 180 rpm shaking for 48 h to obtain Pseudomonas fluorescens bacterial liquid; the bacterial concentration was above 10 8 CFU / mL.

[0039] (2) Carrier preparation

[0040] The bagasse and mushroom residue were respectively screened, impurities were removed, and dried, then mixed according to the mass ratio of bagasse to mushroom residue of 1:1, wrapped with double-layer aluminum foil paper, and placed in a muffle furnace for pyrolysis at 550℃ for 3h to prepare biochar;

[0041] (3) Composite preparation preparation

[0042] The Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid prepared in step (1) are mixed in a mass ratio of 1:1, sprayed on the biochar, and intermittently stirred (stirring at a speed of 50 rpm for 5 min after adding the bacterial strain, and then stirring every 20 min) for 1 h, then the Pseudomonas fluorescens bacterial liquid (the mass ratio of the Azospirillum brasilense bacterial liquid to the Pseudomonas fluorescens bacterial liquid is 1:0.5) is added and stirred for 1 h to obtain a composite preparation; the mass ratio of the Azospirillum brasilense bacterial liquid to the biochar is 1:5.

[0043] Example 2: A composite preparation containing nitrogen-fixing bacteria

[0044] (1) Strain culture

[0045] The same as step (1) of Example 1.

[0046] (2) Carrier preparation

[0047] The bagasse and the mushroom residue are respectively screened, impurities are removed, and then dried, and then mixed according to a mass ratio of 1:2, wrapped with double-layer aluminum foil paper, and placed in a muffle furnace for pyrolysis at a high temperature of 550°C for 3 h to prepare biochar.

[0048] (3) Composite preparation preparation

[0049] The Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid prepared in step (1) are mixed in a mass ratio of 1:1, sprayed on the biochar, and intermittently stirred (stirring at a speed of 50 rpm for 5 min after adding the bacterial strain, and then stirring every 20 min) for 1 h, then the Pseudomonas fluorescens bacterial liquid (the mass ratio of the Azospirillum brasilense bacterial liquid to the Pseudomonas fluorescens bacterial liquid is 1:0.5) is added and stirred for 1 h to obtain a composite preparation; the mass ratio of the Azospirillum brasilense bacterial liquid to the biochar is 1:5.

[0050] Example 3: A composite preparation containing nitrogen-fixing bacteria

[0051] (1) Strain culture

[0052] The same as step (1) of Example 1.

[0053] (2) Carrier preparation

[0054] The bagasse and the mushroom residue are respectively screened, impurities are removed, and then dried, and then mixed according to a mass ratio of 1:2, wrapped with double-layer aluminum foil paper, and placed in a muffle furnace for pyrolysis at a high temperature of 550°C for 3 h to prepare biochar.

[0055] (3) Composite preparation preparation

[0056] The Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid prepared in step (1) are mixed according to a mass ratio of 1.8:1, and then sprayed on the biochar, and intermittently stirred and mixed for 1 h, and then the Pseudomonas fluorescens bacterial liquid (the mass ratio of the Azospirillum brasilense bacterial liquid to the Pseudomonas fluorescens bacterial liquid is 1:1) is added and stirred for 1 h, to obtain a composite preparation; the mass ratio of the Azospirillum brasilense bacterial liquid to the biochar is 1:8.

[0057] Example 4: A composite preparation containing nitrogen-fixing bacteria

[0058] (1) Strain culture

[0059] The same as step (1) of Example 1.

[0060] (2) Carrier preparation

[0061] The bagasse and the mushroom residue are respectively screened, impurities are removed, and then dried, and then mixed according to a mass ratio of 2:1, wrapped with double-layer aluminum foil paper, and placed in a muffle furnace for pyrolysis at a high temperature of 550 ℃ for 3 h, to prepare biochar;

[0062] (3) Composite preparation preparation

[0063] The Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid prepared in step (1) are mixed according to a mass ratio of 2:1, and then sprayed on the biochar, and intermittently stirred and mixed for 1 h, and then the Pseudomonas fluorescens bacterial liquid (the mass ratio of the Azospirillum brasilense bacterial liquid to the Pseudomonas fluorescens bacterial liquid is 1:2) is added and stirred for 1 h, to obtain a composite preparation; the mass ratio of the Azospirillum brasilense bacterial liquid to the biochar is 1:6.

[0064] Example 5: A composite preparation containing nitrogen-fixing bacteria

[0065] (1) Strain culture

[0066] The same as step (1) of Example 1.

[0067] (2) Carrier preparation

[0068] The bagasse and the mushroom residue are respectively screened, impurities are removed, and then dried, and then mixed according to a mass ratio of 1.5:1, wrapped with double-layer aluminum foil paper, and placed in a muffle furnace for pyrolysis at a high temperature of 550 ℃ for 3 h, to prepare biochar;

[0069] (3) Composite preparation preparation

[0070] The Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid prepared in step (1) are mixed according to a mass ratio of 1:2, and then sprayed on the biochar, and intermittently stirred and mixed for 1 h, and then the Pseudomonas fluorescens bacterial liquid (the mass ratio of the Azospirillum brasilense bacterial liquid to the Pseudomonas fluorescens bacterial liquid is 1:1) is added and stirred for 1 h, to obtain a composite preparation; the mass ratio of the Azospirillum brasilense bacterial liquid to the biochar is 1:6.

[0071] Example 6: A composite preparation containing nitrogen-fixing bacteria

[0072] (1) Strain culture

[0073] The same as step (1) of Example 1.

[0074] (2) Carrier preparation

[0075] The sugarcane residue and the mushroom residue are respectively screened, impurities are removed, and then dried, and then mixed according to a mass ratio of 1.5:1, wrapped with double-layer aluminum foil paper, and placed in a muffle furnace for pyrolysis at a high temperature of 550°C for 3 h, to prepare biochar.

[0076] (3) Composite preparation preparation

[0077] The Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid prepared in step (1) are mixed according to a mass ratio of 3:1, and then sprayed on the biochar, and intermittently stirred and mixed for 1 h, and then the Pseudomonas fluorescens bacterial liquid (the mass ratio of the Azospirillum brasilense bacterial liquid to the Pseudomonas fluorescens bacterial liquid is 2:1) is added and stirred for 1 h, to obtain a composite preparation; the mass ratio of the Azospirillum brasilense bacterial liquid to the biochar is 1:8.

[0078] Comparative Example 1

[0079] Compared with Example 3, the difference lies in that the composite preparation is not prepared using a carrier. The preparation steps are as follows:

[0080] (1) Strain culture

[0081] The same as step (1) of Example 1.

[0082] (2) The Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid prepared in step (1) are mixed according to a mass ratio of 1.8:1, and then intermittently stirred and mixed for 1 h, and then the Pseudomonas fluorescens bacterial liquid (the mass ratio of the Azospirillum brasilense bacterial liquid to the Pseudomonas fluorescens bacterial liquid is 1:1) is added and stirred for 1 h, to obtain a composite preparation.

[0083] Comparative Example 2

[0084] Compared with Example 3, the difference lies in that the three bacterial liquids are simultaneously mixed and added into the carrier. The preparation steps are as follows:

[0085] (1) Strain culture

[0086] The step (1) of Example 1.

[0087] (2) Carrier preparation

[0088] The bagasse and mushroom residue were respectively screened, impurities were removed and dried, then mixed according to the mass ratio of 1.5:1, wrapped with double aluminum foil paper and placed in a muffle furnace, and pyrolysis was carried out at a high temperature of 550 DEG C to prepare biochar;

[0089] (3) Preparation of composite preparation

[0090] The Brazil nitrogen-fixing spirillum liquid, bacillus mucilaginosus liquid and pseudomonas fluorescens liquid prepared in step (1) were mixed and sprayed on the biochar, and intermittent stirring was carried out for 2h to obtain a composite preparation; wherein the mass ratio of Brazil nitrogen-fixing spirillum liquid and bacillus mucilaginosus liquid was 1.8:1, and the mass ratio of Brazil nitrogen-fixing spirillum liquid and pseudomonas fluorescens liquid was 1:1; the mass ratio of Brazil nitrogen-fixing spirillum liquid and biochar was 1:8.

[0091] Comparative example 3

[0092] Compared with Example 3, the difference lies in that only Brazil nitrogen-fixing spirillum liquid and bacillus mucilaginosus liquid are added. The preparation steps are as follows:

[0093] (1) Strain culture

[0094] The preparation method of Brazil nitrogen-fixing spirillum liquid and bacillus mucilaginosus liquid in step (1) of Example 1.

[0095] (2) Carrier preparation

[0096] The bagasse and mushroom residue were respectively screened, impurities were removed and dried, then mixed according to the mass ratio of 1.5:1, wrapped with double aluminum foil paper and placed in a muffle furnace, and pyrolysis was carried out at a high temperature of 550 DEG C to prepare biochar;

[0097] (3) Preparation of composite preparation

[0098] The Brazil nitrogen-fixing spirillum liquid and bacillus mucilaginosus liquid prepared in step (1) were mixed according to the mass ratio of 1.8:1, sprayed on the biochar, and intermittent stirring was carried out for 1h to obtain a composite preparation; the mass ratio of Brazil nitrogen-fixing spirillum liquid and biochar was 1:8.

[0099] Comparative example 4

[0100] Compared with Example 3, the difference lies in that only Brazil nitrogen-fixing spirillum liquid and pseudomonas fluorescens liquid are added. The preparation steps are as follows:

[0101] (1) Strain culture

[0102] The preparation method of the Azospirillum brasilense bacterial liquid and the Pseudomonas fluorescens bacterial liquid in step (1) of Example 1.

[0103] (2) Carrier preparation

[0104] The bagasse and the mushroom residue were respectively screened, impurities were removed, and drying was performed, and then the bagasse and the mushroom residue were mixed according to a mass ratio of 1.5:1, wrapped with double-layer aluminum foil paper, and placed in a muffle furnace for pyrolysis at a high temperature of 550 DEG C to prepare biochar;

[0105] (3) Preparation of composite preparation

[0106] The Azospirillum brasilense bacterial liquid and the Pseudomonas fluorescens bacterial liquid prepared in step (1) were mixed according to a mass ratio of 1:1, sprayed on the biochar, and intermittently stirred and mixed for 1h to obtain a composite preparation; the mass ratio of the Azospirillum brasilense bacterial liquid to the biochar was 1:8.

[0107] Comparative Example 5

[0108] Compared with Example 3, the difference lies in that only the Bacillus mucilaginosus bacterial liquid and the Pseudomonas fluorescens bacterial liquid are added. The preparation steps are as follows:

[0109] (1) Strain culture

[0110] The preparation method of the Bacillus mucilaginosus bacterial liquid and the Pseudomonas fluorescens bacterial liquid in step (1) of Example 1.

[0111] (2) Carrier preparation

[0112] The bagasse and the mushroom residue were respectively screened, impurities were removed, and drying was performed, and then the bagasse and the mushroom residue were mixed according to a mass ratio of 1.5:1, wrapped with double-layer aluminum foil paper, and placed in a muffle furnace for pyrolysis at a high temperature of 550 DEG C to prepare biochar;

[0113] (3) Preparation of composite preparation

[0114] The Bacillus mucilaginosus bacterial liquid and the Pseudomonas fluorescens bacterial liquid prepared in step (1) were mixed according to a mass ratio of 1:1.8, sprayed on the biochar, and intermittently stirred and mixed for 1h to obtain a composite preparation; the mass ratio of the Pseudomonas fluorescens bacterial liquid to the biochar was 1:8.

[0115] Comparative Example 6

[0116] Compared with Example 3, the difference lies in that only the Azospirillum brasilense bacterial liquid is added. The preparation steps are as follows:

[0117] (1) Strain culture

[0118] The preparation method of the Azospirillum brasilense bacterial solution in step (1) of Example 1.

[0119] (2) Carrier preparation

[0120] The bagasse and mushroom residue were respectively screened, impurities were removed, and dried, and then mixed according to a mass ratio of 1.5:1, wrapped with double-layer aluminum foil paper, and placed in a muffle furnace for pyrolysis at a high temperature of 550 DEG C to prepare biochar;

[0121] (3) Preparation of composite preparation

[0122] The Azospirillum brasilense bacterial solution prepared in step (1) was sprayed on the biochar, and intermittent stirring and mixing were performed for 1 h to obtain a composite preparation; the mass ratio of the Azospirillum brasilense bacterial solution to the biochar was 1:8.

[0123] Comparative Example 7

[0124] Compared with Example 3, the difference lies in that intermittent stirring is not performed. The preparation steps are as follows:

[0125] (1) Strain culture

[0126] The same as step (1) of Example 1.

[0127] (2) Carrier preparation

[0128] The bagasse and mushroom residue were respectively screened, impurities were removed, and dried, and then mixed according to a mass ratio of 1.5:1, wrapped with double-layer aluminum foil paper, and placed in a muffle furnace for pyrolysis at a high temperature of 550 DEG C to prepare biochar;

[0129] (3) Preparation of composite preparation

[0130] The Azospirillum brasilense bacterial solution prepared in step (1) was sprayed on the biochar, and intermittent stirring and mixing were performed for 1 h to obtain a composite preparation; the mass ratio of the Azospirillum brasilense bacterial solution to the biochar was 1:8.

[0131] Comparative Example 8

[0132] Compared with Example 3, the difference lies in that only bagasse is used as the raw material of the biochar. The preparation steps are as follows:

[0133] (1) Strain culture

[0134] The same as step (1) of Example 1.

[0135] (2) Carrier preparation

[0136] The bagasse was screened, impurities were removed and dried, wrapped with double-layer aluminum foil paper and placed in a muffle furnace for pyrolysis at a high temperature of 550°C to prepare the biochar;

[0137] (3) Preparation of the composite preparation

[0138] The Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid prepared in step (1) were mixed according to a mass ratio of 1.8:1, then sprayed on the biochar, and intermittently stirred and mixed for 1 h, then the Pseudomonas fluorescens bacterial liquid (the mass ratio of the Azospirillum brasilense bacterial liquid to the Pseudomonas fluorescens bacterial liquid was 1:1) was added and stirred for another 1 h to obtain the composite preparation; the mass ratio of the Azospirillum brasilense bacterial liquid to the biochar was 1:8.

[0139] Comparative Example 9

[0140] Compared with Example 3, the difference lies in that only mushroom residue is used as the raw material of the biochar. The preparation steps are as follows:

[0141] (1) Strain culture

[0142] The same as step (1) of Example 1.

[0143] (2) Carrier preparation

[0144] The mushroom residue was screened, impurities were removed and dried, wrapped with double-layer aluminum foil paper and placed in a muffle furnace for pyrolysis at a high temperature of 550°C to prepare the biochar;

[0145] (3) Preparation of the composite preparation

[0146] The Azospirillum brasilense bacterial liquid and the Bacillus mucilaginosus bacterial liquid prepared in step (1) were mixed according to a mass ratio of 1.8:1, then sprayed on the biochar, and intermittently stirred and mixed for 1 h, then the Pseudomonas fluorescens bacterial liquid (the mass ratio of the Azospirillum brasilense bacterial liquid to the Pseudomonas fluorescens bacterial liquid was 1:1) was added and stirred for another 1 h to obtain the composite preparation; the mass ratio of the Azospirillum brasilense bacterial liquid to the biochar was 1:8.

[0147] The viable cell count of the composite preparation products prepared in Examples 1-6 and Comparative Examples 1-9 above was determined by plate counting method. The results are shown in Table 1.

[0148] Table 1: Viable cell count statistical results

[0149]

[0150] From the experimental data in Table 1, it can be seen that for the production of composite preparation, the type and amount of bacteria used, the addition method of bacteria, the treatment method during the preparation of bacteria, and the selection of carrier raw materials all have a direct impact on the viable count of bacteria in the composite bacteria agent. The selection and amount of bacteria affect the coexistence state and the speed of dominant bacteria formation. The addition method of bacteria and the treatment method during the preparation of bacteria (adding Pseudomonas fluorescens and the other two bacteria separately, and performing intermittent stirring after adding each bacteria) can ensure that bacteria with consistent nutritional and environmental needs avoid competition, first meeting the needs of one bacteria, and then because the Bradyrhizobium brazilense and Bacillus mucilaginosus are mixed with the carrier, the porous environment and nutritional components of biochar promote the coexistence of the two bacteria as dominant bacteria, and some secondary metabolites are also produced. When Pseudomonas fluorescens is added later, there are more nutrients, and the growth of each bacteria is not affected by the competition for nutrients with Bradyrhizobium brazilense. In order to avoid the better nutritional competition of Bradyrhizobium brazilense as the dominant bacteria, the amount of Pseudomonas fluorescens added needs to be controlled to ensure the coexistence of the three bacteria. After adding the bacteria, intermittent stirring is needed, on the one hand to meet the needs of oxygen, and on the other hand to avoid the rapid reproduction of any one of the bacteria in a large amount of biochar at one time, affecting the coexistence of the bacteria. According to years of research experience in bacteria and agriculture, the inventors have found that sugarcane residue and mushroom residue contain dietary fiber, various amino acids, vitamins, calcium, zinc, and other trace elements, which can provide nutrition, and after high-temperature treatment, the density difference between the two can form a porous structure with different size pores. The presence of mycelium in mushroom residue not only provides a carrier, but also is rich in organic matter and enzymes, which is of great significance for improving soil structure and increasing crop yield. Preparing biochar from sugarcane residue alone can only meet the needs of the carrier, and the nutrients are not enough to support the stable coexistence of the three bacteria. Mixing it with mushroom residue at a specific ratio to prepare biochar can meet the needs of both the carrier and the nutrients, allowing the three bacteria to develop and coexist in balance.

[0151] Application Example

[0152] Experimental site: a certain planting household in Jiangzhou District, Chongzuo City, Guangxi.

[0153] Experimental time: March-December 2024.

[0154] Experimental crop: sugarcane (Guimei No. 42).

[0155] Experimental grouping: 10 mu of sugarcane of the grower were randomly divided into equal-area plots, and the composite preparation prepared by Examples 1-6 and Comparative Examples 1-9 was used for fertilization experiments, and the blank control group was not used in the composite preparation prepared by any of the above groups. Three parallel repeated experiments were set up using the same composite inoculant.

[0156] Method of use: 1.5 kg / mu of the composite inoculant was applied once 20 days before planting; 1.0 kg / mu was applied once at the tillering stage and 100 days after planting, respectively. Other fertilization and management were carried out according to the conventional sugarcane planting and management methods.

[0157] The soil pH value, organic matter content, and nitrogen-fixing bacteria count were determined before planting, and the soil pH value, organic matter content, and nitrogen-fixing bacteria count were determined 6 months after planting (determined according to the conventional dilution and plating method, and the culture medium was a nitrogen-free culture medium). The results are shown in Table 2.

[0158] Table 2 Experimental results

[0159]

[0160] The organic matter content growth rate (%) = (soil organic matter content before planting-soil organic matter content 6 months after planting) / soil organic matter content before planting x 100.

[0161] The nitrogen-fixing bacteria count growth rate (%) = (soil nitrogen-fixing bacteria count before planting-soil nitrogen-fixing bacteria count 6 months after planting) / soil nitrogen-fixing bacteria count before planting x 100.

[0162] From the data in Table 2 above, it can be seen that the composite preparation of Examples 1-6 of the present application can improve the organic matter content and nitrogen-fixing bacteria count in the soil, and the pH value does not change significantly, and significantly improves the soil structure. In the blank control group, the pH value, organic matter content, and nitrogen-fixing bacteria count hardly change, and there is no obvious improvement in the change of soil structure; the comparative example group is beneficial to the increase of organic matter content and nitrogen-fixing bacteria count to a certain extent, but the effect is not better than that of Examples 1-6, and the effect of Example 3 is the most outstanding.

[0163] In order to further prove the function of the composite preparation of the present application, at the same time as the above experiment, considering that the amount of nitrogen fertilizer used in the conventional planting of sugarcane in China is excessive, a treatment of reducing the amount of nitrogen fertilizer was set up as a comparison, Treatment I: the total amount of nitrogen fertilizer during the entire growth period was halved, and the others were the same as the blank control; Treatment II: the total amount of conventional fertilization during the entire growth period was halved + the composite preparation prepared by Example 3, and the others were the same as Example 3. The yield and sugar content of the harvested sugarcane were determined after different treatments, and the results are shown in Table 3.

[0164] Table 3 Yield and sugar content determination results

[0165]

[0166] As can be seen from the data in Table 3, after the application of the composite preparation of Examples 1-6 of the present application, the yield and sugar content of sugarcane are significantly improved compared with the blank control group and the comparison example group, and the effect of Example 3 is the best, which is consistent with the data in Tables 1 and 2. After the nitrogen fertilizer is reduced by half and mixed with the composite preparation, the yield and sugar content of sugarcane can still be significantly improved, and the effect is equivalent to that of Example 3, which shows that the composite preparation prepared by the present application can significantly reduce the use amount of nitrogen fertilizer, and will not affect the yield and sugar content of sugarcane, which has important significance for alleviating the negative effects caused by long-term overuse of nitrogen fertilizer.

[0167] The above-described examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A compound preparation containing nitrogen-fixing bacteria, characterized in that, The invention includes a compound microbial agent and biochar loaded with the compound microbial agent, the compound microbial agent being composed of the following strains: Azospirillum brasilense ACCC04138, Bacillus mucilaginosus Krassilnikov ACCC 19749, and Pseudomonas fluorescens CICC 23919.

2. The compound formulation as described in claim 1, characterized in that, In the compound microbial agent, the mass ratio of *Azotobacter brasiliensis*, *Bacillus mucilaginosus* and *Pseudomonas fluorescens* is (1-2):(0.5-2):(1-2), and the mass ratio of *Azotobacter brasiliensis* to the biochar is 1:(5-8).

3. The compound formulation as described in claim 1, characterized in that, The biochar includes bagasse and mushroom residue, and the mass ratio of bagasse to mushroom residue is (1-2):(1-2).

4. A method for preparing the compound microbial agent according to any one of claims 1-3, characterized in that, Includes the following steps: The cultures of *Azotobacter brasiliensis*, *Bacillus mucilaginosus*, and *Pseudomonas fluorescens* were prepared by fermentation and culture, respectively. Sugarcane bagasse and mushroom residue are mixed and pyrolyzed at high temperature to produce biochar; After mixing the bacterial solutions of *Azotobacter brasiliensis* and *Bacillus mucilaginosus*, the mixture was sprayed onto the biochar and stirred intermittently for 1-2 hours. Then, *Pseudomonas fluorescens* bacterial solution was added and stirring was continued for 1-2 hours to obtain a compound bacterial agent.

5. The preparation method according to claim 4, characterized in that, The fermentation medium used to prepare *Azotobacter brasiliensis* and *Pseudomonas fluorescens* bacterial cultures was nutrient broth medium, while the fermentation medium used to prepare the nutrient broth medium was silicate bacteria medium.

6. The preparation method according to claim 4, characterized in that, The high-temperature pyrolysis temperature is 500℃ and the time is 3 hours.

7. The preparation method according to claim 4, characterized in that, The intermittent stirring conditions are as follows: after spraying the biochar with *Braziliana zoiformis* and *Bacillus mucilaginosus* solutions, stir at 50 rpm for 5 minutes, and then stir under the same conditions once every 20 minutes.

8. The preparation method according to claim 4, characterized in that, The optimal mass ratio of the *Azotobacter brasiliensis* bacterial suspension, *Bacillus mucilaginosus* bacterial suspension, and *Pseudomonas fluorescens* bacterial suspension is 1.8:1:1.

8. The optimal mass ratio of sugarcane bagasse to mushroom residue is 1.5:1; The optimal mass ratio of the *Braziliana nigrostriatus* bacterial solution to biochar is 1:

8.

9. The application of the compound formulation as described in any one of claims 1-3 in improving the soil structure of sugarcane fields.

10. The application of the compound formulation according to any one of claims 1-3 in increasing sugarcane yield and sugar content.

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