A compound microbial agent suitable for alfalfa-corn rotation mode in meiwa black soil and a preparation method thereof

Abstract

This invention discloses a compound microbial agent and its preparation method suitable for alfalfa-maize rotation patterns in sandy loam black soil. The liquid compound microbial agent is prepared by mixing *Cavalleria zedoaria* JMB-20, *Streptomyces* JX-1, and *Bacillus hygroscopicus* JMB-131 bacterial solutions at a wet weight ratio of 1:1.2:1.5. The solid compound microbial agent is prepared by fermenting a mixture of the liquid compound microbial agent with soybean flour and alfalfa straw powder at a mass ratio of 1:10:200. This invention can promote the formation of soil aggregates through the extracellular polysaccharides secreted by *Bacillus hygroscopicus* JMB-131, and simultaneously promote the formation of a bioporous network through maize roots. After field application, it reduces soil bulk density, increases soil porosity and the proportion of water-stable large aggregates, and significantly improves soil physical properties.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and in particular to a compound inoculant suitable for alfalfa-maize rotation in sandy black soil and its preparation method. Background Technology

[0002] Sandy black soil is one of the soil types in my country, and corn is widely planted in its distribution areas. However, due to its high clay content and rich expansive clay minerals, sandy black soil has undesirable physical properties such as swelling when wet and shrinking when dry, hardening and compaction, high soil bulk density, and low porosity. The problem of soil compaction and hardening severely restricts the penetration and growth of corn roots, thereby affecting the improvement of land productivity.

[0003] Crop diversification is an effective way to improve sandy loam black soil. Among them, the winter alfalfa-summer maize rotation model can initially improve soil structure through the biological porosity effect of alfalfa roots, while increasing soil organic matter content. Microbial inoculants, as environmentally friendly agricultural inputs, can promote plant root development and improve soil aggregate structure through the metabolic activities of functional strains, becoming an important technical means for improving sandy loam black soil.

[0004] Our research team previously screened Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3 in a peanut-maize intercropping system in red soil, and developed a compound microbial agent based on these. This agent can effectively address the chemical barriers of red soil acidification and increase crop yield. However, the soil barriers in red soil are fundamentally different from those in sandy loam black soil. Sandy loam black soil is characterized by physical structural barriers, and existing red soil microbial agents cannot adapt to the high clay content and high bulk density environment of sandy loam black soil, nor can they specifically address the problem of compacted soil. Therefore, targeted screening of functional compound microbial agents that are suitable for alfalfa-maize rotation patterns and can synergistically improve sandy loam black soil compaction and promote maize root growth has significant production application value. Summary of the Invention

[0005] This invention addresses the physical structural obstacles of compacted sandy black soil and the planting characteristics of alfalfa-maize rotation by providing a compound microbial agent with complementary functions and strong environmental adaptability, as well as its preparation method. This achieves integrated regulation of sandy black soil structure improvement and maize root growth promotion, thereby enhancing land productivity under this planting model.

[0006] The present invention adopts the following technical solution:

[0007] A liquid compound microbial agent suitable for alfalfa-maize rotation pattern in sandy loam black soil, wherein the liquid compound microbial agent is composed of Caballeronia zhejiangensis JMB-20 with biological nitrogen fixation function, Streptomyces monticola JX-1 with IAA production and growth promotion function, and Bacillus altitudinis JMB-131 with extracellular polysaccharide secretion to improve soil aggregate structure, in a microbial cell wet weight ratio of 1:1.2:1.5, wherein:

[0008] The accession number of Caballeronia zhejiangensis JMB-20 is CGMCCNo.36620, the accession date is November 13, 2025, and the depositary institution is the China General Microbiological Culture Collection Center.

[0009] The accession number of Bacillus altitudinis JMB-131 is CGMCC No. 36387, the deposit date is October 27, 2025, and the depositary institution is the China General Microbiological Culture Collection Center.

[0010] The accession number of Streptacidiphilus monticola JX-1 is CGMCC No.29822, the accession date is January 31, 2024, and the depositary institution is the China General Microbiological Culture Collection Center.

[0011] All three strains were isolated from the alfalfa-maize rotation system in sandy ginger black soil. They have complementary functions. Zhejiang Cavalleria JMB-20 is responsible for biological nitrogen fixation, providing nitrogen nutrition for maize growth and reducing dependence on chemical nitrogen fertilizer; Highland Bacillus JMB-131 is responsible for secreting extracellular polysaccharides, improving soil aggregate structure, and alleviating soil compaction; Streptomyces JX-1 is responsible for producing IAA, promoting growth, inhibiting soil-borne diseases, and improving crop stress resistance.

[0012] The three bacterial cultures were mixed at a wet weight ratio of 1:1.2:1.5. This ratio was optimized based on the functional characteristics of the strains: Bacillus hygroscopicus JMB-131 had the highest proportion to ensure the core effect of soil improvement; Streptomyces JX-1 was the second highest to balance growth promotion and competition with other strains; and Cavalleria zeylans JMB-20 was the base ratio to meet nitrogen fixation requirements.

[0013] Preferably, the effective viable bacteria concentration of the liquid compound microbial agent is 1×10⁻⁶. 8 -5×10 8CFU / mL was prepared by resuspending in 0.85% sterile physiological saline.

[0014] 1×10 8 -5×10 8 The CFU / mL viable bacterial concentration is the optimal activity concentration for field application, which can ensure that the strains can quickly colonize in sandy ginger black soil, while avoiding soil microecological imbalance caused by excessive strain proliferation.

[0015] 0.85% sterile physiological saline removes fermentation residues and impurities. During resuspension, it neither induces premature metabolism and energy depletion in the bacterial strains nor provides growth substrates for other microorganisms. Because 0.85% sterile physiological saline is an isotonic medium, it effectively protects the cell membrane integrity of the bacterial strains, reducing mortality during resuspension. This ensures the strains remain active and stable after resuspension and before soil application, preventing premature inactivation due to drastic changes in osmotic pressure. The ionic strength of the physiological saline reduces the surface tension of the bacterial solution, improving dispersibility and allowing the bacterial agent to quickly disperse into soil pores after application (especially in heavy clay soil environments), preventing bacterial aggregation. The bacterial solution resuspended in 0.85% sterile physiological saline maintains a stable suspension state during storage and application, preventing rapid sedimentation of the bacterial strains and ensuring a consistent concentration of viable bacteria per unit volume, resulting in uniform application effects. A uniform bacterial solution ensures a consistent bacterial intake per corn plant, guaranteeing consistent field application effects and avoiding uneven growth-promoting and soil-improving effects caused by localized concentration differences.

[0016] This invention also discloses a method for preparing a liquid compound microbial agent, comprising the following steps:

[0017] (1) Cavalleria zeolite JMB-20, Streptomyces JX-1 and Bacillus glacialis JMB-131 were inoculated into solid culture medium to obtain activated Cavalleria zeolite JMB-20, Streptomyces JX-1 and Bacillus glacialis JMB-131;

[0018] (2) The three activated strains were inoculated into liquid culture medium to obtain single strain propagation liquids of Zhejiang Cavalleriae JMB-20, Streptomyces JX-1 and Bacillus hygroscopicus JMB-131;

[0019] (3) The OD600 values ​​of the propagation solutions of each single strain were uniformly adjusted to 0.03-0.05 to obtain the bacterial solutions of *Cavalleria zeolites* JMB-20, *Streptomyces* JX-1, and *Bacillus hygroscopicus* JMB-131.

[0020] (4) Centrifuge each bacterial solution obtained in step (3), discard the supernatant and collect the bacterial precipitate, weigh the wet weight of each strain, take the bacterial precipitate according to the wet weight ratio of Zhejiang Cavalleria JMB-20: Streptomyces JX-1: Bacillus hygroscopicus JMB-131=1:1.2:1.5, add sterile water to resuspend and mix to obtain liquid compound bacterial agent.

[0021] First, the OD600 value of the propagation bacterial solution is uniformly adjusted to 0.03-0.05 to standardize the bacterial concentration, laying the foundation for precise wet weight mixing and avoiding mixing deviations caused by differences in initial bacterial concentrations. After centrifugation to remove the supernatant and fermentation residues, the bacterial cells dispersed in the fermentation broth are quickly collected for compounding, significantly reducing the interference of culture medium impurities on the synergistic effect of the bacterial cells. The functional activity of the compound bacterial agent is higher than that of direct compounding without centrifugation. The resuspension process disperses bacterial aggregates. Combined with the ionic environment of physiological saline, resuspension with a quantitative amount of 0.85% sterile physiological saline allows for precise adjustment of bacterial concentration, resulting in a uniform and stable suspension. After application to the soil, it can quickly diffuse and evenly contact crop roots and soil particles, avoiding localized excessively high or low concentrations and ensuring that the nitrogen fixation range and soil improvement effect cover the entire cultivated layer.

[0022] Preferably, the solid culture medium in step (1) is LB solid culture medium, and the culture conditions are constant temperature static culture at 28℃ for 12h; the liquid culture medium in step (2) is LB liquid culture medium, and the culture conditions are constant temperature shaking culture at 28℃ and 180r / min for 24h.

[0023] LB solid / liquid medium is the optimal medium for the three strains, providing sufficient carbon, nitrogen, and inorganic salt nutrients for their growth. Constant temperature incubation at 28℃ matches the optimal growth temperature for the three strains, increasing their proliferation efficiency by more than 30% compared to other media. During the activation phase, static incubation at 28℃ for 12 hours ensures the strains quickly recover from dormant to active states, resulting in plump colonies with uniform growth. During the propagation phase, shaking incubation at 180 rpm for 24 hours ensures sufficient dissolved oxygen, resulting in a uniform suspension of the strains without cell precipitation. Fixed culture parameters allow for standardized replication of the preparation process, with minimal deviation in viable cell counts between different batches, ensuring batch stability of the subsequent compound bacterial agent.

[0024] Preferably, in step (4), the centrifugation conditions are 4℃, 8000r / min for 12min, the bacterial precipitate is weighed using an electronic analytical balance, and after resuspending, it is gently shaken at 28℃, 100r / min for 10min to ensure that the bacterial strains are fully mixed.

[0025] Centrifugation at 4℃ can reduce the metabolic rate of the strains. 8000r / min×12min is a mild and efficient centrifugation parameter, resulting in high bacterial sedimentation collection rate and high strain survival rate. The weighing accuracy of the electronic analytical balance ensures small weighing error of the wet weight of the bacteria, guaranteeing the accuracy of the ratio of the three strains and avoiding a decrease in functional synergy due to ratio deviation. Gentle shaking at 28℃ and 100r / min for 10min allows the three strains to be completely and uniformly dispersed in the bacterial solution, forming a homogeneous compound bacterial agent system. When applied in the field, the bacterial composition of the bacterial solution is consistent, ensuring the uniformity of functional expression.

[0026] Furthermore, the method of applying the liquid compound microbial agent in the alfalfa-maize rotation system in sandy ginger black soil is to apply it by root irrigation at a rate of 2 mL per plant.

[0027] Rhizosphere irrigation is the optimal application method for liquid microbial agents, as it can directly deliver the bacterial strains to the surface of corn roots, avoiding the adsorption and fixation of the strains by soil clay particles. The colonization rate of the strains in the rhizosphere is higher than that of foliar spraying. The application rate of 2 mL / plant is a precise dosage for corn seedlings, which is suitable for the root growth characteristics of corn seedlings. Applying the microbial agent to the rhizosphere allows the strains to quickly colonize around the corn roots, forming a rhizosphere microecological advantage and rapidly exerting its functions of promoting growth and improving soil.

[0028] This invention also discloses a solid compound microbial agent, which is prepared by fermentation of a liquid compound microbial agent mixed with soybean powder and alfalfa straw powder at a mass ratio of 1:10:200. The effective bacterial concentration in the solid compound microbial agent is ≥10. 9 With CFU / g, it exhibits good storage stability and is suitable for mechanized field application.

[0029] In the solid compound microbial agent described in this application, the mass ratio of the liquid compound microbial agent to soybean flour and alfalfa straw powder is 1:10:200, specifically defined as: total mass of liquid compound microbial agent : dry weight of soybean flour : dry weight of alfalfa straw powder; wherein, the dry weight of soybean flour and alfalfa straw powder is determined by drying soybean flour and alfalfa straw powder in an oven at 105℃ until constant weight, cooling to room temperature, and then weighing the resulting mass using an electronic analytical balance; the total mass of the liquid compound microbial agent is the mass value of the homogeneous microbial solution obtained by direct weighing.

[0030] Before mixed fermentation culture, the liquid compound microbial agent should be thoroughly mixed with soybean powder and alfalfa straw powder to ensure that the liquid microbial agent evenly wets all solid powders, avoids clumping, and ensures colonization and uniform fermentation.

[0031] Solid compound microbial agents overcome the limitations of liquid microbial agents in terms of transportation, storage, and application. They can be applied manually or mechanically, making them suitable for large-scale field planting. Soybean powder provides nitrogen and nutrients for the fermentation strains, while alfalfa straw powder provides carbon and is compatible with the straw return pattern of alfalfa-corn rotation. The two work synergistically to provide sufficient nutrients for the fermentation strains, resulting in a viable cell count of ≥1×10⁻⁶ after fermentation. 9 CFU / g; the mass ratio is clearly defined as "total mass of liquid inoculum: dry weight of soybean powder: dry weight of alfalfa straw powder" to eliminate the interference of raw material moisture fluctuations on the ratio, ensuring minimal deviation in viable cell count between different batches of fermented solid inoculum and batch stability; ≥1×10 9 The CFU / g live bacteria concentration is the optimal concentration for field application, ensuring that even if there is some loss after application, there are still enough live bacteria to colonize and function. A single plant application of 30g is sufficient to ensure that the strains can quickly colonize in sandy ginger black soil and perform soil improvement and growth promotion functions.

[0032] Preferably, the soybean powder is made from mold-free soybeans that have been pulverized, passed through a 40-mesh sieve, dried at 105°C to constant weight, and sterilized by 60Co-γ irradiation, with a moisture content ≤5%; the alfalfa straw powder is made from mold-free alfalfa straw that has been pulverized, passed through a 20-mesh sieve, sterilized by high-pressure steam at 121°C and 0.1MPa, and air-dried, with a moisture content ≤10%.

[0033] Soybean flour is sieved through a 40-mesh sieve, and alfalfa straw flour through a 20-mesh sieve, forming a carrier structure with a mix of coarse and fine particles. This ensures both the aeration of the carrier and the uniform saturation of the liquid inoculant, improving the colonization rate of the bacterial strains within the carrier. Drying at 105℃ to constant weight ensures that the moisture content of the soybean flour is ≤5%, and the moisture content of the air-dried alfalfa straw flour is ≤10%, preventing anaerobic spoilage during fermentation due to excessive moisture in the raw materials and ensuring the smooth progress of solid-state aerobic fermentation. Irradiation sterilization of the soybean flour and high-pressure steam sterilization of the alfalfa straw flour achieve aseptic treatment of the raw materials, preventing competition for nutrients between contaminating microorganisms and the bacterial strains, and ensuring the dominance of the bacterial strains in fermentation.

[0034] The fermentation conditions for the preparation of the solid compound microbial agent are as follows: temperature 25-30℃, time 5-7 days, humidity 60%-70%, natural ventilation, stirring 1-3 times a day during fermentation, stirring for 10 minutes each time, stirring speed 60r / min.

[0035] The optimal fermentation temperature for the three strains is 25-30℃. At this temperature, the spore formation rate and metabolic activity of the strains are the highest. Exponential proliferation of the strains can be achieved in 5-7 days of fermentation, with a viable cell count of 1×10⁻⁶. 9Above CFU / g; a fermentation humidity of 60%-70% is the optimal moisture condition for solid-state aerobic fermentation, ensuring both the moisture requirements for bacterial growth and good aeration of the fermentation substrate to avoid anaerobic fermentation; natural aeration combined with stirring 1-3 times daily further ensures sufficient dissolved oxygen in the fermentation substrate, improving bacterial survival rate; a stirring parameter of 60r / min×10min is gentle stirring, ensuring thorough mixing of the bacterial strain and fermentation carrier while avoiding cell breakage caused by excessive stirring speed, and preventing clumping of the fermentation substrate, ensuring uniform fermentation effect; standardized fermentation parameters allow for large-scale production of solid inoculants, resulting in smaller deviations in the number of viable bacteria in batches of fermentation tanks, meeting the requirements of industrial production.

[0036] Furthermore, the solid compound microbial agent is applied in the alfalfa-maize rotation system in sandy ginger black soil by rhizosphere application at the maize seedling stage, with an application rate of 30g / plant.

[0037] Rhizosphere application of solid microbial agents is the optimal method for applying solid microbial agents during the corn seedling stage. During this stage, corn roots grow vigorously, allowing the microbial strains to quickly colonize the rhizosphere and promote growth. Simultaneously, the extracellular polysaccharides secreted by *Bacillus pyrifolia* JMB-131 can improve rhizosphere soil structure early on, promoting root penetration into compacted sandy loam soil. An application rate of 30g / plant is a precise field dosage, ensuring that the fermentation carrier (soybean powder + alfalfa straw powder) provides continuous nutrition to the microbial strains, allowing them to stably colonize the sandy loam soil for over 30 days, while avoiding cost waste due to excessive dosage. The solid microbial agent applied rhizosphere decomposes slowly, providing nutrition to the microbial strains and increasing soil organic matter content, synergistically improving the structure of the sandy loam soil. After field application, it increases corn root biomass, yield, and porosity, while reducing soil bulk density. The application method requires no specialized equipment; it can be operated manually or with a small fertilizer spreader, making it suitable for large-scale alfalfa-corn rotation fields and highly applicable.

[0038] Beneficial effects:

[0039] 1. Targeted improvement of soil compaction in sandy loam black soil: The compound microbial agent of this invention can promote the formation of soil aggregate structure through the extracellular polysaccharides secreted by Bacillus subtilis JMB-131, and at the same time promote the formation of biological pore network through maize roots. After field application, the soil bulk density decreased from 1.65 g / cm³ to 1.45 g / cm³ (a decrease of 12.2%), the soil porosity increased from 45.0% to 51.4% (an increase of 14.2%), and the proportion of water-stable large aggregates increased by 18.1%, significantly improving the physical properties of the soil.

[0040] 2. Highly promotes maize root growth: The synergistic effect of the three strains of bacteria can significantly improve the biomass and elongation capacity of maize roots. In the pot experiment, maize root biomass increased by 18.7% and root length increased by 15.8%. In the field, maize root biomass increased by 11.4%, enhancing the root system's ability to penetrate compacted soil and laying the foundation for crop growth.

[0041] 3. Achieve a win-win situation of soil improvement and crop yield increase: After field application, corn yield increased by 9.2%, while nitrogen fertilizer utilization rate increased by 15.6%, reducing secondary damage to the soil caused by chemical fertilizer application, and is suitable for the ecological planting model of alfalfa-corn rotation.

[0042] 4. Strong environmental adaptability and good stability: All strains were isolated / adapted to the alfalfa-maize rotation system in sandy black soil. The colonization rate in sandy black soil was still over 82.5% after 30 days, which is much higher than that of exogenous strains. The solid inoculant was stored at room temperature in a sealed and dark place for 12 months, and the decline rate of viable bacteria was still low, which meets the storage and application requirements for field production.

[0043] 5. Simple preparation method and scalable production: The preparation process of the microbial agent of this invention does not require special equipment, the fermentation conditions are mild, the liquid microbial agent can be applied by liquid spraying, and the solid microbial agent can be applied by mechanical spreading, which can meet the production needs of different planting scales. Attached Figure Description

[0044] Figure 1 The strain isolated in Example 1;

[0045] Figure 2 This invention demonstrates the cross-antagonistic effect of three strains of bacteria: Zhejiang Cavalleriae JMB-20, Streptomyces JX-1, and Bacillus hygroscopicus JMB-131.

[0046] Figure 3 The colony morphology of the three strains of *Cavalleriae zearalensis* JMB-20, *Streptomyces* JX-1, and *Bacillus hygroscopicus* JMB-131 of this invention is shown.

[0047] Figure 4 The effect of the liquid compound microbial agent in Example 4 on promoting maize root biomass was shown.

[0048] Figure 5 The effect of the liquid compound microbial agent on promoting corn root growth in Example 4 is shown.

[0049] Figure 6 The effect of the solid compound microbial agent in Example 5 on promoting maize root biomass is shown.

[0050] Figure 7 The effect of the solid compound microbial agent in Example 5 on promoting the root growth of maize is shown.

[0051] Figure 8The effect of the solid compound microbial agent in Example 5 on promoting corn yield in farmland is shown;

[0052] Figure 9 The effect of the solid compound microbial agent in Example 5 on the root biomass promotion of maize in farmland is shown.

[0053] Figure 10 The effect of the solid composite microbial agent in Example 5 on improving the bulk density of farmland soil is shown;

[0054] Figure 11 The effect of the solid compound microbial agent in Example 5 on the porosity of farmland soil is shown.

[0055] Figure 12 The effect of the solid composite microbial agent in Example 5 on the particle size of water-stable aggregates in farmland soil is shown. Detailed Implementation

[0056] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial purchase channels.

[0057] Example 1: Isolation, Screening and Identification of Strains

[0058] 1. Separation

[0059] The strain of this invention was isolated and screened from soil samples from a field experiment. The field experiment was conducted at Longkang Farm, Huaiyuan County, Anhui Province, which belongs to the warm temperate semi-humid monsoon climate zone, with an average annual temperature of 14.8°C and an average annual rainfall of approximately 900 mm. The soil type in this area is sandy black soil developed from lacustrine calcareous sediments, with a sand (>0.05 mm), silt (0.05-0.002 mm), and clay (<0.002 mm) content of 80 g·kg⁻¹. -1 541 g·kg -1 379 g·kg -1 The planting pattern for this experiment was a rotation of winter cover crops (November 2023 to May 2024) and summer maize (June 2024 to October 2024). The cover crop was alfalfa, with a growing season from November 2023 to May 2024 and a growing season rainfall of 355 mm. Specifically:

[0060] Field planting method: Use a small hand-held rotary tiller to shallowly till the land to a depth of 5-10 cm; then, sow alfalfa manually. No fertilization is applied throughout the alfalfa planting process. Harvest manually in May of the following year, removing all above-ground parts. After the cover crop is harvested, plant corn. Each year, first shallowly till the land to a depth of 5 cm using a small hand-held rotary tiller, then sow corn manually with a plant spacing of 30 cm and a row spacing of 60 cm.

[0061] Field fertilization method: The fertilizer application rate during the corn growing season is N 100 kg·hm² -2 P2O5 60 kg·hm -2 K2O 90kg·hm -2 Urea is applied as a nitrogen fertilizer (containing 464 g·kg N). -1 ), superphosphate is used as a phosphate fertilizer (containing 120 g·kg P2O5). -1 Potassium chloride is used as a potassium fertilizer (containing 600 g·kg K2O). -1 Additionally, apply 80 kg·hm² of nitrogen as top dressing during the corn jointing stage. -2 During harvest, the corn cobs are harvested one by one by hand, and then all the stalks are cut and removed.

[0062] Rhizosphere soil from maize in an alfalfa-maize rotation system was collected and diluted with PBS buffer under aseptic conditions. The diluted solution was then spread onto LB agar plates. After multiple purification processes, 50 pure bacterial strains were obtained, numbered JMB-1 to JMB-50. Molecular identification was performed on all strains, such as... Figure 1 As shown.

[0063] 2. Initial screening: 50 strains were screened for growth promotion in maize pots. The root length and root biomass of maize were measured with the control group not inoculated. Five strains with potential growth promotion effects were obtained. The growth promotion effects are shown in Table 1.

[0064] Table 1. Strains with potential growth-promoting effects

[0065] CK JMB-10 JMB-131 JMB-20 JMB-17 JMB-31 JX-1 Compound microbial agent Root length 8.54±1.91 9.44±2.01 12.67±2.23 10.92±2.17 9.66±2.81 10.03±1.61 11.38±2.46 13.22±2.53 Root biomass 0.43±0.15 0.49±0.09 0.55±0.13 0.59±0.07 0.48±0.10 0.55±0.11 0.60±0.18 0.63±0.12

[0066] 3. Secondary Screening: To adapt to the needs of sandy black soil improvement and alfalfa-corn rotation, this invention sets three core functional indicators for secondary screening:

[0067] (1) Biological nitrogen fixation capacity: Nitrogenase activity was determined by acetylene reduction method, and nitrogenases with activity >10 nmolC2H4·h were screened. -1 ·mg -1 The strain containing the protein;

[0068] (2) Extracellular polysaccharide secretion capacity: The phenol-sulfuric acid method was used to screen strains with EPS production >20 mg / L;

[0069] (3) IAA production capacity: The Salkowski colorimetric method was used to screen strains with IAA production >20 mg / L.

[0070] The results of the secondary screening showed that only one of the five strains had a high nitrogen-fixing capacity, namely JMB-20; only one strain had a high extracellular polysaccharide production capacity, namely JMB-131; and no strains had the ability to produce IAA.

[0071] 4. Strain identification and species determination

[0072] Using the DNA of strains JMB-20 and JMB-131 as templates, 16S rDNA was amplified using universal 16S rDNA primers, and its sequence was determined. The 16S rDNA sequencing results of strains JMB-20 and JMB-131 were entered into the NCBI database for BLAST alignment. The results showed:

[0073] JMB-20 is Caballeronia zhejiangensis.

[0074] JMB-131 is Bacillus altitudinis.

[0075] 5. Construction of compound microbial agent combination:

[0076] Given that the indole-3-acetic acid (IAA) synthesis ability of Streptomyces has been confirmed in previous patents, and that Streptomyces has the function of inhibiting soil-borne pathogens, that Bacillus hygroscopicus has the ability to solubilize phosphorus, and that Cavalleria zeylans has the nitrogen-fixing properties, in order to construct a three-in-one compound microbial agent that integrates nitrogen fixation, soil improvement, and growth promotion, and to achieve synergistic effects and ensure the balance between species in the compound microbial agent, this invention selects JMB-20 and JMB-131, and combines them with Streptomyces JX-1, which has been isolated in previous patents, to construct a compound microbial agent, so that it forms a complementary mechanism in terms of phosphorus activation, nitrogen fixation, plant growth hormone synthesis, and antagonism against soil-borne pathogens.

[0077] Single colonies of JMB-20 and JMB-131 were preserved:

[0078] The accession number of *Cavalleria zeolite* JMB-20 from Zhejiang is: CGMCC No. 36620, accession date: November 13, 2025, depositary institution: China General Microbiological Culture Collection Center;

[0079] The accession number of Bacillus hygroscopicus JMB-131 is: CGMCC No.36387, accession date: October 27, 2025, depositary institution: China General Microbiological Culture Collection Center.

[0080] Example 2: Preparation of liquid compound bacterial agent

[0081] 1. After activation, *Cavalleriae zeolites* JMB-20, *Streptomyces* JX-1, and *Bacillus hygroscopicus* JMB-131 were activated. Single colonies were picked and streaked onto LB agar plates and incubated at 28°C for 24 h. The growth status at the colony-to-colony junction of each strain was observed, and the cross-antagonistic characteristics of the three strains were investigated. The results are as follows: Figure 2 As shown.

[0082] After 24 hours of culture on LB medium, the colonies of strain JMB-20 were white, opaque, and moist. Microscopic examination revealed straight or slightly curved bacilli, and Gram staining was negative.

[0083] After 24 hours of culture on LB medium, strain JMB-131 colonies were white and opaque with regular edges, appearing rod-shaped under a microscope, and were Gram-positive. Results showed that all three strains grew normally, with no inhibition zones or growth inhibition observed at the interface, indicating no cross-antagonistic effect and suitable for combined use.

[0084] 2. Under aseptic conditions, *Streptomyces* JX-1 and *Cavalleria zedoaria* JMB-20 and *Bacillus glacialis* JMB-131 isolated from Example 1 were inoculated onto LB solid medium and cultured at 28°C for 12 h until the colonies were plump and growing well, thus obtaining activated *Cavalleria zedoaria* JMB-20, *Streptomyces* JX-1, and *Bacillus glacialis* JMB-131. The morphology of the strains is as follows: Figure 3 As shown.

[0085] 3. Under aseptic conditions, activated *Cavalleria zeolite* JMB-20, *Streptomyces jX-1*, and *Bacillus hygroscopicus* JMB-131 were inoculated into LB liquid medium. The Erlenmeyer flasks were placed in a constant temperature shaking incubator, with a rotation speed of 180 r / min and a temperature of 28℃, and were incubated for 24 h until the bacterial solution became uniformly turbid, thus obtaining the propagation bacterial solutions of single strains of *Cavalleria zeolite* JMB-20, *Streptomyces jX-1*, and *Bacillus hygroscopicus* JMB-131.

[0086] 4. Take the propagation broths of each single strain obtained in step 3, and measure the optical density value (OD600) of the broth at a wavelength of 600 nm using a UV-Vis spectrophotometer, with sterile LB liquid medium as a blank control; add sterile LB liquid medium to each propagation broth, and measure the OD600 value while diluting, and adjust the OD600 value of each strain's propagation broth to a uniform range of 0.03-0.05 to obtain standardized concentrations of *Cavalleria zeolites* JMB-20, *Streptomyces* JX-1, and *Bacillus hygroscopicus* JMB-131 broths for later use.

[0087] 5. Preparation of bacterial strain compounding and liquid compound inoculant

[0088] Collection of bacterial precipitate: The three strains of bacteria with standardized concentration obtained in step 4 were transferred to 500 mL sterile centrifuge bottles and placed in a refrigerated centrifuge. The centrifugation temperature was set to 4℃, the centrifugation speed to 8000 r / min and the centrifugation time to 12 min. After centrifugation, the supernatant was discarded under aseptic conditions, and the bacterial precipitate at the bottom of the bottle was retained.

[0089] Wet weight of bacterial cells: Wipe the outer wall of the sterile centrifuge bottle containing bacterial precipitate with sterile absorbent paper, place it on a 0.01 g electronic analytical balance for accurate weighing, and record the wet weight values ​​of bacterial precipitates of Zhejiang Cavalleriae JMB-20, Streptomyces JX-1, and Bacillus hygroscopicus JMB-131 respectively;

[0090] Prepare the mixture according to the following ratio: According to the wet weight ratio of bacterial cells of Zhejiang Cavalleriae JMB-20: Streptomyces JX-1: Bacillus hygroscopicus JMB-131 = 1:1.2:1.5, use a sterile pipette to take the corresponding mass of bacterial cell precipitate of the three strains and transfer them to the same 1000mL sterile Erlenmeyer flask.

[0091] Resuspension and mixing: Add 0.85% sterile physiological saline to an Erlenmeyer flask containing the mixed bacterial precipitate. During resuspension, gently stir along the flask wall with a sterile glass rod until the bacterial precipitate is completely dispersed and free of clumps, and the resuspended bacterial solution is uniformly turbid. The amount of sterile physiological saline added is such that the effective viable bacteria concentration of the resuspended liquid compound bacterial agent reaches 1×10⁻⁶. 8 -5×10 8 The standard is CFU / mL (the effective viable bacteria concentration is determined by plate count method);

[0092] Product preparation: Place the resuspended bacterial solution in a constant temperature shaker at 28℃ and shake gently at 100r / min for 10min to ensure thorough mixing of the strains. This yields a liquid compound bacterial agent suitable for the alfalfa-corn rotation pattern in sandy ginger and black soil. The bacterial agent should be stored at 4℃.

[0093] Example 3: Preparation of Solid Composite Microbial Agent

[0094] 1. Pretreatment of solid fermentation carrier

[0095] Soybean flour pretreatment: Select soybeans that are free from mold and impurities, grind them and pass them through a 40-mesh sieve to obtain soybean flour; place the soybean flour in a 105℃ oven and dry it to constant weight, with the moisture content controlled below 5%; after cooling to room temperature, sterilize it by 60Co-γ irradiation for later use.

[0096] Alfalfa straw powder pretreatment: Select fresh, mold-free alfalfa straw, air dry it naturally, then crush it and pass it through a 20-mesh sieve to obtain alfalfa straw powder; place the alfalfa straw powder in a 121℃, 0.1MPa high-pressure steam sterilizer for 15 minutes, cool it to room temperature, and then place it in a sterile operating table to air dry until the moisture content is below 10% for later use.

[0097] 2. Solid-liquid mixture

[0098] Under aseptic conditions, the liquid compound microbial agent prepared in Example 2, the soybean powder and alfalfa straw powder pretreated in Step 1 were added to a sterile stainless steel mixer in a ratio of total mass of liquid compound microbial agent: dry weight of soybean powder: dry weight of alfalfa straw powder = 1:10:200. The mixer speed was set to 80 r / min and mixed for 15 min until the liquid compound microbial agent uniformly saturated all solid carriers, with no dry powder clumping or local liquid accumulation, thus obtaining the fermentation substrate. The final moisture content of the fermentation substrate was controlled at 35%-40%, and the moisture content was determined by drying method. If the moisture content was insufficient, sterile water was added to adjust it.

[0099] 3. Solid-state fermentation culture

[0100] The above-mentioned uniformly mixed fermentation base material is aseptically loaded into a sterile stainless steel production tank. The loading amount is 60%-70% of the effective volume of the production tank to ensure sufficient aeration during the fermentation process.

[0101] The production tank was placed in a constant temperature fermentation workshop, and the fermentation temperature was set at 30℃ for solid-state aerobic fermentation. The fermentation cycle was 5 days. During the fermentation period, the stirring device of the production tank was turned on aseptically at 9:00 am every day, with a speed of 60 r / min and stirring for 10 minutes. At the same time, sterile air was introduced at a ventilation rate of 1 vvm to ensure aerobic respiration of the bacteria and promote bacterial reproduction.

[0102] Fermentation process monitoring: On the 3rd day of fermentation, samples were taken and the total number of viable bacteria in the fermentation substrate was determined by the plate count method to ensure that the number of viable bacteria increased exponentially; if local mold was found, sterile liquid compound microbial agent was added immediately (the amount added was 0.5% of the total mass of the fermentation substrate), and the frequency of aeration and stirring was increased.

[0103] 4. Preparation and Testing of Solid Compound Microbial Agents

[0104] After fermentation is complete, the fermentation product is removed from the production tank, placed on a sterile operating table, and air-dried naturally until the moisture content is below 15% to obtain a crude solid compound microbial agent.

[0105] The crude product is passed through a 10-mesh sieve to remove large clumps, resulting in the finished solid compound microbial agent.

[0106] Finished product testing: The total viable count was determined using the plate count method to ensure that the effective total viable count in the solid compound microbial agent was ≥1×10⁻⁶. 9 CFU / g; Simultaneously test for contamination rate, ensuring that the rate of contaminants such as mold and pathogens is less than 0.1%;

[0107] Packaging and storage: The qualified solid compound microbial agent is vacuum-packed in sterile aluminum foil bags and stored in a cool, dry place.

[0108] Example 4: Effect of liquid compound microbial agent on root growth promotion of maize seedlings

[0109] Using potted corn seedlings as the experimental subject, the growth-promoting effect of the liquid compound microbial agent was studied. The specific steps were as follows:

[0110] Cultivation method for corn potted seedlings: Select healthy and plump corn seeds and germinate them for 3 days at 30℃. Select seeds with similar growth and plant them in flower pots filled with sandy ginger black soil. Place them in a light incubator with 12 hours of light and 12 hours of darkness, light intensity of 18000 lx, and constant temperature of 28℃.

[0111] The obtained liquid compound bacterial agent was inoculated into the root system of maize seedlings at a rate of 2 mL, with the treatment of inoculation with 2 mL of sterile water serving as a control. After 21 days of cultivation, the effect of the functional compound microbial propagation solution on the root growth of maize seedlings was statistically analyzed. The results are as follows: Figure 4 , 5 As shown.

[0112] The results showed that inoculating potted maize seedlings with functional compound microbial propagation solution increased maize root biomass and root length by 16.7% and 16.4%, respectively.

[0113] Example 5: Effect of solid compound microbial agent on root growth promotion of potted corn seedlings

[0114] Using potted corn seedlings as the experimental subject, the growth-promoting effect of the solid compound microbial agent was studied. The specific steps were as follows:

[0115] Cultivation method for corn seedlings in pots: Healthy and plump corn seeds were germinated at 30℃ for 3 days. Seeds with similar growth were planted in pots filled with sandy ginger black soil mixed with a solid compound microbial agent prepared according to Example 3. 20g of the agent was applied evenly to each pot. The control group received no microbial agent. The pots were placed in a light incubator with a 12-hour light-dark cycle, a light intensity of 18000 lx, and a constant temperature of 28℃. After 21 days of cultivation, the effect of the solid compound microbial agent on the root development of corn seedlings was statistically analyzed. The results are as follows: Figure 6 , 7 As shown.

[0116] The results showed that inoculating corn pot seedlings with solid compound microbial agents increased corn root biomass and root length by 18.7% and 15.8%, respectively.

[0117] Example 6: The effect of solid compound microbial agents on promoting root growth and optimizing soil structure in maize seedlings in the field.

[0118] The basic measures for field trials, water and fertilizer management, and pest and weed control in this embodiment are consistent with those in Example 1. The experiment was conducted in an alfalfa-maize rotation system field, with experimental plots measuring 20m × 5m. Each treatment was replicated three times. In the maize seedling stage, the solid compound microbial agent prepared in Example 3 was applied around the rhizosphere of the seedlings in the experimental treatment groups at a rate of 30g per plant. The blank control group received no microbial agent, and all other management measures were identical to those in the treatment groups. At harvest time, maize yield, underground biomass, and soil physical properties were measured for each group. Soil bulk density was determined using the ring cutter method, soil porosity was calculated using the formula: Porosity (%) = (Total soil volume - Soil solid volume) / Total soil volume × 100%, and soil aggregates were determined using the wet sieving method.

[0119] The results are as follows Figure 8-12 As shown.

[0120] The results showed that inoculation with the solid compound microbial agent increased maize yield and root biomass by 9.2% and 11.4%, respectively, while reducing soil bulk density by 12.2%, increasing soil porosity by 14.2%, and increasing the proportion of large soil aggregates (particle size >0.25 mm) by 18.1%. This effect primarily stems from the enhanced root growth of maize after inoculation, where the resulting root bioporous network effectively alleviates soil compaction, directly reducing soil bulk density and increasing porosity. Furthermore, the metabolic activities of the functional microorganisms in the compound microbial agent may secrete extracellular polysaccharides and sticky extracellular polymers, promoting the stable formation of soil microaggregates, strengthening aggregate structure, and thus synergistically optimizing soil physical properties.

[0121] Example 7: Determination of the effect of solid compound microbial agents on improving nitrogen fertilizer utilization during the maize planting season

[0122] In this embodiment, the difference method was used to determine the effect of solid compound microbial agent on improving nitrogen fertilizer utilization in the alfalfa-maize rotation system in sandy ginger black soil. The basic experimental conditions were consistent with those in Examples 1 and 6.

[0123] The experiment was conducted in a field of sandy ginger, black soil, alfalfa, and maize rotation at Longkang Farm, Huaiyuan County, Anhui Province. Three treatment groups were established, with three biological replicates per group. The plot size was 20m × 5m.

[0124] Blank group (CK1): No nitrogen fertilizer was applied, and no solid compound microbial agent of the present invention was applied;

[0125] Control group (CK2): Conventional nitrogen fertilizer was applied, but the solid compound microbial agent of this invention was not applied;

[0126] Experimental group (T): The same conventional nitrogen fertilizer was applied, and the solid compound microbial agent of this invention (30g per plant, same as in Example 6) was applied to the rhizosphere of corn seedlings.

[0127] The field water and fertilizer, disease, pest and weed management of each group is the same as in Example 1.

[0128] During the corn harvest period, whole corn plants were collected from each plot. After blanching at 105℃ for 30 minutes, they were dried at 75℃ to constant weight, pulverized and passed through a 60-mesh sieve. The total nitrogen content of the aboveground parts of the plants was determined by the Kjeldahl method, and the total nitrogen uptake per unit area of ​​the aboveground parts of the plants was calculated.

[0129] Nitrogen fertilizer utilization rate calculation formula:

[0130] Nitrogen fertilizer utilization rate (%) = (Total nitrogen uptake by plants in the nitrogen-applied group - Total nitrogen uptake by plants in the control group without nitrogen application) / Actual nitrogen application (pure N) × 100%

[0131] Nitrogen fertilizer utilization improvement value = nitrogen fertilizer utilization rate of experimental group - nitrogen fertilizer utilization rate of control group.

[0132] Because the calculation of nitrogen fertilizer utilization rate in this experiment only selects the aboveground parts of corn (stems, leaves, and ears) as the measurement object, and the underground root system has a low nitrogen content, and the aboveground parts of corn are the core economic output parts in agricultural production, and are the industry's conventional selection parts for nitrogen fertilizer utilization rate measurement, in this experiment, the total nitrogen uptake of the nitrogen-applied group plants is the total nitrogen uptake of the aboveground parts of the corn plants per unit area in the nitrogen-applied group, which is the total nitrogen uptake per unit area of ​​plants in the table below.

[0133] The measurement results are shown in Table 2 below:

[0134] Table 2 Nitrogen fertilizer utilization rate

[0135] Processing group Total nitrogen uptake per unit area of ​​plants (kg / hm²) Nitrogen fertilizer utilization rate (%) CK1 58.2±2.3 - CK2 112.5±3.1 30.17 T 129.8±3.5 39.78

[0136] The Zhejiang Cavalleria JMB-20 in the solid compound microbial agent of this invention has a highly efficient biological nitrogen fixation capacity, which can supplement soil nitrogen; at the same time, the three strains of bacteria work together to promote the growth of maize roots, enhance the root system's ability to absorb soil nitrogen, reduce nitrogen fertilizer leaching and volatilization losses, and ultimately achieve a 9.6% increase in nitrogen fertilizer utilization rate.

[0137] Example 8: Determination of storage stability of solid compound microbial agent

[0138] In this embodiment, the room temperature storage stability of the solid compound microbial agent was determined according to the national standard for agricultural microbial agents, and the decline rate of viable bacteria count after 12 months under sealed and light-proof conditions was clarified to verify the storage performance of the agent.

[0139] Experimental materials: Solid composite microbial agent prepared in Example 3 (initial effective total viable count was 2.5 × 10⁻⁶). 9 The CFU / g count was determined by vacuum sealing in sterile aluminum foil bags and stored at room temperature (25°C, protected from light, and with a relative humidity of 60%-70%).

[0140] Assay method: Samples were taken at 1 month, 3 months, 6 months and 12 months of storage. The total viable count in the solid compound bacterial agent was determined by plate count method (LB solid medium, incubated at 28℃ for 24h). Each group was sampled 3 times and the viable count decline rate was calculated.

[0141] The rate of decline in viable bacteria count (%) = (initial viable bacteria count - viable bacteria count stored for n months) / initial viable bacteria count × 100%.

[0142] The measurement results are shown in Table 3 below:

[0143] Table 3 Storage stability of solid compound microbial agents

[0144] Storage time (months) viable bacteria count (CFU / g) in the room temperature, light-protected group 1 <![CDATA[2.54×10 9 ]]> 3 <![CDATA[2.5×10 9 ]]> 6 <![CDATA[2.4×10 9 ]]> 12 <![CDATA[2.2×10 9 ]]>

[0145] The results show that the solid compound microbial agent of the present invention uses soybean powder and alfalfa straw powder as fermentation carriers, which not only provides nutrient substrates for the strains, but also forms a microenvironment to protect the activity of the strains. At the same time, the fermentation process is optimized to aerobic fermentation at 25-30℃, which enables the strains to form stable spores / dormant bodies. Therefore, after 12 months of storage under sealed and light-proof conditions at room temperature, the effective viable count still remains above 2 billion / g, with good storage stability, meeting the storage and application requirements for field production.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A liquid compound microbial agent suitable for alfalfa-maize rotation pattern in sandy black soil, characterized in that, The liquid compound microbial agent is composed of Caballeronia zhejiangensis JMB-20 (with biological nitrogen fixation function), Streptacidiphilus monticola JX-1 (with IAA-producing growth-promoting function), and Bacillus altitudinis JMB-131 (with extracellular polysaccharide secretion to improve soil aggregate structure) in a wet weight ratio of 1:1.2:1.

5. The accession number of *Cavalleria zeolite* JMB-20 from Zhejiang is: CGMCC No. 36620, accession date: November 13, 2025, depositary institution: China General Microbiological Culture Collection Center; The accession number of Bacillus hygroscopicus JMB-131 is: CGMCC No.36387, accession date: October 27, 2025, depositary institution: China General Microbiological Culture Collection Center; The accession number of Streptomyces JX-1 is CGMCC No.29822, the accession date is January 31, 2024, and the depositary institution is the China General Microbiological Culture Collection Center.

2. The liquid compound microbial agent according to claim 1, suitable for alfalfa-maize rotation in sandy black soil, is characterized in that, The effective viable bacteria concentration of the liquid compound microbial agent is 1×10⁻⁶. 8 -5×10 8 CFU / mL was prepared by resuspending in 0.85% sterile physiological saline.

3. A method for preparing a liquid compound microbial agent suitable for alfalfa-maize rotation pattern in sandy loam black soil, as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) Zhejiang Cavalleria JMB-20, Streptomyces JX-1 and Bacillus hygroscopicus JMB-131 were inoculated into solid culture medium to obtain activated Zhejiang Cavalleria JMB-20, Streptomyces JX-1 and Bacillus hygroscopicus JMB-131; (2) The three activated strains were inoculated into liquid culture medium to obtain single strain propagation broths of Zhejiang Cavalleria JMB-20, Streptomyces JX-1 and Bacillus hygroscopicus JMB-131. (3) The OD600 values ​​of the propagation solutions of each single strain were uniformly adjusted to 0.03-0.05 to obtain the bacterial solutions of *Cavalleria zeolites* JMB-20, *Streptomyces* JX-1, and *Bacillus hygroscopicus* JMB-131. (4) Centrifuge each bacterial solution obtained in step (3), discard the supernatant and collect the bacterial precipitate, weigh the wet weight of each strain, take the bacterial precipitate according to the bacterial wet weight ratio of Zhejiang Cavalleria JMB-20: Streptomyces JX-1: Bacillus hygroscopicus JMB-131=1:1.2:1.5, add sterile water to resuspend and mix to obtain liquid compound bacterial agent.

4. The method for preparing a liquid compound microbial agent suitable for alfalfa-maize rotation pattern in sandy loam black soil according to claim 3, characterized in that, The solid culture medium mentioned in step (1) is LB solid culture medium, and the culture conditions are constant temperature static culture at 28℃ for 12h; the liquid culture medium mentioned in step (2) is LB liquid culture medium, and the culture conditions are constant temperature shaking culture at 28℃ and 180r / min for 24h.

5. The method for preparing a liquid compound microbial agent suitable for alfalfa-maize rotation pattern in sandy loam black soil according to claim 3, characterized in that, In step (4), the centrifugation conditions are 4℃, 8000r / min for 12min. The bacterial precipitate is weighed using an electronic analytical balance. After resuspending, it is gently shaken at 28℃ and 100r / min for 10min to ensure that the bacterial strains are fully mixed.

6. The method of using the liquid compound microbial agent of claim 1, applicable to the alfalfa-maize rotation pattern in sandy loam black soil, in the alfalfa-maize rotation system of sandy loam black soil, characterized in that, The liquid compound bacterial agent is applied by rhizosphere irrigation at a rate of 2 mL per plant.

7. A solid compound microbial agent suitable for alfalfa-maize rotation pattern in sandy black soil, characterized in that, The solid compound microbial agent is prepared by fermentation of the liquid compound microbial agent described in claim 1 with soybean flour and alfalfa straw powder at a mass ratio of 1:10:200, and the effective bacterial concentration in the solid compound microbial agent is ≥10. 9 CFU / g; The mass ratio is the total mass of the liquid compound microbial agent: the dry weight of soybean powder: the dry weight of alfalfa straw powder.

8. A solid compound microbial agent suitable for alfalfa-maize rotation in sandy black soil according to claim 7, characterized in that, The soybean powder is made from mold-free soybeans that have been ground, passed through a 40-mesh sieve, dried at 105℃ to constant weight, and sterilized by 60Co-γ irradiation, with a moisture content of ≤5%. The alfalfa straw powder is made from mold-free alfalfa straw that has been ground, passed through a 20-mesh sieve, sterilized by high-pressure steam at 121℃ and 0.1MPa, and air-dried, with a moisture content of ≤10%.

9. A solid compound microbial agent suitable for alfalfa-maize rotation in sandy black soil according to claim 7, characterized in that, The fermentation conditions are as follows: temperature 25-30℃, time 5-7 days, humidity 60%-70%, natural ventilation, stirring 1-3 times a day during fermentation, stirring for 10 minutes each time, stirring speed 60r / min.

10. The method of using the solid compound microbial agent of claim 7, applicable to the alfalfa-maize rotation pattern in sandy loam black soil, in the alfalfa-maize rotation system of sandy loam black soil, characterized in that, The solid compound microbial agent is applied by rhizosphere application during the corn seedling stage, at a rate of 30g per plant.

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