A composite microbial agent and application thereof in weight loss and yield increase

Abstract

This invention discloses a compound microbial agent and its application in reducing fertilizer use and increasing yield. The compound microbial agent is composed of *Spiralobacter iridis* (…). Niveispirillum irakense YM-6CGMCC No. 37555, Bacillus tekirae ( Bacillus from Tequila This invention comprises Re2CGMCC No. 36764 and attapulgite soil modified by ammoniation and freezing. The composite microbial agent provided by this invention can effectively promote deep root development and plant growth in crops, exhibiting a stronger ability to promote rhizosphere growth. Multiple field trials show that, under conditions of a 20% reduction in chemical fertilizer use, the yields of corn, rice, and soybeans can still maintain growth or even significantly increase, achieving the goal of "reducing fertilizer use and increasing yield." This invention has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a compound microbial agent and its application in reducing fertilizer use and increasing yield. Background Technology

[0002] Chemical fertilizers play a crucial role in maintaining high crop yields in modern agricultural production systems. However, the extensive fertilization model, which has long focused solely on increasing yields, has led to serious resource waste and environmental crises. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), the global utilization rates of nitrogen and phosphate fertilizers in the current season are only 30-50% and 10-25%, respectively. In my country's major grain-producing areas, the negative effects of excessive base fertilizer input are particularly significant: the continuous input of acidic fertilizers accelerates soil acidification and compaction, destroying soil aggregate structure while inhibiting deep root development; excessive application of nitrogen fertilizers leads to a large amount of residual nitrogen causing eutrophication of water bodies or leaching pollution of groundwater through runoff; and excessive application of phosphate fertilizers is easily fixed by soil minerals to form insoluble phosphates, resulting in the common contradiction of "high total phosphorus but scarce available phosphorus." Therefore, in response to the strategic need for "zero growth in chemical fertilizer use," developing new types of bio-fertilizers that can partially replace chemical fertilizers and restore soil microecology has become the core direction of industry development.

[0003] Plant rhizosphere growth-promoting bacteria, as a core component of biofertilizers, possess significant growth-promoting capabilities, including biological nitrogen fixation, phosphorus and potassium solubilization, and the secretion of plant hormones. However, in complex field environments, single-function strains often fail due to metabolic imbalances or insufficient colonization. Specifically, biological nitrogen fixation is an energy-intensive process that consumes large amounts of ATP, and ATP biosynthesis depends on a sufficient supply of phosphorus. In phosphorus-deficient soil environments, single nitrogen-fixing bacteria experience a rapid decline in nitrogenase activity due to energy scarcity; while single phosphate-solubilizing bacteria can activate soil phosphorus, they cannot independently meet the massive nitrogen demands of crop growth. Therefore, relying solely on single-function inoculants is insufficient to overcome the colonization resistance of indigenous microorganisms and achieve stable yield increases in the field. Summary of the Invention

[0004] The purpose of this invention is to provide a compound microbial agent that can reduce fertilizer use and increase yield in agricultural production.

[0005] This invention first protects a compound microbial agent. The compound microbial agent may include *Syntrophus iridis* and *Bacillus tekirae*. The compound microbial agent may function to increase crop yield or reduce crop fertilizer use while increasing yield.

[0006] The compound microbial agent may specifically consist of *Syntrophus iridis* and *Bacillus tekira*.

[0007] The aforementioned compound microbial agents may also include a carrier.

[0008] The aforementioned compound microbial agent may specifically consist of *Syntrophus iridis*, *Bacillus tekira*, and a carrier.

[0009] In any of the above-mentioned compound microbial agents, the effective live bacteria ratio of *Helicobacter iridis* and *Bacillus tekirae* can be 1:(1-2) (e.g., 1:(1-1.5), 1:(1.5-2), 1:1, 1:1.5, or 1:2).

[0010] The aforementioned *Iraqiella iridis* can specifically be *Iraqiella iridis* (… Niveispirillum irakense YM-6. The Iraqi Snow White Spirulina ( Irak Niveispirillum YM-6, its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 37555.

[0011] The aforementioned *Bacillus tekirae* can specifically be *Bacillus tekirae* (… Bacillus tequilensis Re2. The Bacillus tergentii ( Tequila Bacillus Re2, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 36764.

[0012] The carrier described above can be attapulgite that has undergone ammoniation-freezing modification.

[0013] The preparation method of the above-mentioned ammonia-freeze modified attapulgite can be as follows: (1) the colloidal grade attapulgite used as the adsorption matrix is ​​crushed and dispersed in ammonia water, and then frozen at low temperature to obtain a frozen carrier; (2) the frozen carrier is thawed, then dried and crushed to obtain a modified attapulgite carrier with high adsorption activity, that is, attapulgite modified by ammonia-freeze.

[0014] The particle size of the colloidal attapulgite can be 100-200 mesh.

[0015] In step (1), the low temperature can be -50℃ to -70℃ (e.g., -50℃ to -60℃, -60℃ to -70℃, -50℃, -60℃ or -70℃). The freezing time can be 2-4 h (e.g., 2-3 h, 3-4 h, 2 h, 3 h or 4 h).

[0016] The purpose of step (1) is to utilize the in-situ growth and volume expansion effect of ice crystals to effectively depolymerize the clay crystal bundle structure and significantly increase the porosity and specific surface area of ​​the carrier; at the same time, ammonia treatment can increase the active adsorption sites on the surface of the carrier and enhance its affinity for microorganisms and soil nutrient ions.

[0017] In step (2), the drying temperature can be 30℃-50℃ (e.g., 30℃-40℃, 40℃-50℃, 30℃, 40℃ or 50℃).

[0018] When preparing any of the above-mentioned compound bacterial agents, both *Syntrophus iridis* and *Bacillus tekira* are added in the form of bacterial suspension.

[0019] The bacterial suspension can be obtained by inoculating *Syntrophus iridis* or *Bacillus tekirae* into a liquid culture medium. The liquid culture medium can be LB liquid medium.

[0020] The effective viable cell count concentrations in both the *Iraqiella icterus* bacterial suspension and the *Bacillus tektina* bacterial suspension are ≥2.0 × 10⁻⁶. 9 CFU / mL.

[0021] Preferably, when preparing the composite bacterial agent, the liquid-to-solid ratio of the total volume of *Symplocos iridis* and *Bacillus tekiria* bacterial suspension to the carrier is 1L:(0.8-1.2)Kg (e.g., 1L:(0.8-1.0)Kg, 1L:(1.0-1.2)Kg, 1L:0.8Kg, 1L:1.0Kg, or 1L:1.2Kg).

[0022] This invention also protects the application of any of the above-described compound microbial agents, which may be at least one of A1)-A4):

[0023] A1) Increased crop yield; A2) Promotes crop growth; A3) Promotes biological nitrogen fixation in crops; A4) Enhances the crop's ability to capture soil nutrients.

[0024] This invention also protects the application of any of the above-described compound microbial agents in crop fertilizer reduction and yield increase; the fertilizer reduction and yield increase refers to increasing yield while reducing the amount of chemical fertilizer used.

[0025] In any of the above applications, the crop may be corn, soybean, or rice.

[0026] The compound microbial agent provided by this invention has the following advantages: (1) It overcomes the bottleneck of metabolic imbalance of single agents. This invention scientifically combines *Syntrophus iridis* YM-6 with *Bacillus tekira* Re2 to construct a "nitrogen-phosphorus synergistic" microecological symbiotic system. The soil phosphorus activated by *Bacillus tekira* Re2 provides sufficient ATP substrate for the nitrogen fixation process of *Syntrophus iridis* YM-6, significantly improving the colonization and growth promotion ability of functional strains in complex field environments; (2) The improved carrier improves the colonization rate of the compound agent. Unlike the peat or ordinary adsorbents commonly used in traditional agents, this invention uses attapulgite soil modified by "ammoniation-freezing" as a carrier. With its high porosity and specific surface area, this carrier greatly improves the adsorption capacity and survival rate of functional bacteria, effectively alleviating the technical problems of easy inactivation and weak resistance in field colonization of traditional carrier agents; (3) It has significant effects on crop growth promotion and yield increase. In terms of promoting growth, the compound microbial agent provided by this invention can effectively promote deep root development and plant growth, exhibiting a stronger ability to promote rhizosphere growth. Regarding yield increase, multiple field trials have shown that even with a 20% reduction in chemical fertilizer use, the yields of corn, rice, and soybeans can still maintain growth or even show significant growth, achieving the goal of "reducing fertilizer use and increasing yield." This invention has significant application value.

[0027] Deposit description Strain name: Iraqi Snow Spirulina Latin name: Irak Niveispirillum Classification and nomenclature: *Iraqis white spirochete* Irak Niveispirillum Strain number: YM-6 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: January 28, 2026 Collection Center Registration Number: CGMCC No. 37555 Bacterial strain name: Bacillus tekirae Latin name: Tequila Bacillus Classification and nomenclature: Bacillus tekirae Tequila Bacillus Strain number: Re2 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: November 25, 2025 Collection Center Registration Number: CGMCC No. 36764 Attached Figure Description Figure 1 The nitrogenase activity of *Syntrophus iridis* YM-6 and *Bacillus tekira* Re2 was determined by the acetylene reduction method in Example 3.

[0028] Figure 2 The image shows the finished product appearance of the compound microbial agent "Micro Nitrogen No. 1" prepared in Example 4.

[0029] Figure 3 The effect of compound microbial inoculants on reducing fertilizer use and increasing yield in corn fields.

[0030] Figure 4 The effect of compound microbial inoculants on reducing fertilizer use and increasing yield in rice fields.

[0031] Figure 5 The effect of compound microbial inoculants on reducing fertilizer use and increasing soybean yield in the field. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0035] The solutes and their concentrations in the PBS buffer were 8.0 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L disodium hydrogen phosphate and 0.24 g / L potassium dihydrogen phosphate, with water as the solvent and a pH value (25℃) of 7.4 ± 0.1.

[0036] Example 1: Iraqi Snow Spirulina ( Irak Niveispirillum YM-6 CGMCC No. 37555 and Bacillus tekirae ( Tequila Bacillus Separation, identification and preservation of Re2 CGMCC No. 36764 I. Iraqi Snow Spirulina ( Irak Niveispirillum Isolation, identification and preservation of YM-6 CGMCC No. 37555 (I) Isolation of bacteria YM-6 1. Wash and chop the rice root samples (collected from rice paddies in Shunyi District, Beijing). Weigh 1g and add it to a test tube containing 9mL of sterile PBS buffer. Shake for 10 minutes, let stand for 30 seconds, and the supernatant is the rice root stock solution (the dilution at this point is recorded as 10). -1 Add 1 mL of rice root extract to a test tube containing 9 mL of sterile PBS buffer and mix thoroughly (the dilution is 10⁻⁶). -2 Then, take 1 mL from this test tube and add it to another test tube containing 9 mL of sterile PBS buffer. Mix well, and so on to prepare dilutions of different concentrations.

[0037] 2. Using a pipette, take 100 μL of the diluted solution at different concentrations and spread it evenly on ordinary nutrient agar medium. Incubate at 30°C for 2-3 days.

[0038] 3. After completing step 2, inoculate each colony into ordinary broth medium and incubate at 30°C to obtain the corresponding bacterial solution.

[0039] 4. Take 10 μL of the above bacterial solution, dilute it 100 times with sterile PBS buffer, and then take 100 μL of the bacterial solution to inoculate it into ordinary nutrient agar medium and incubate at 30°C for 24 h.

[0040] 5. After completing step 4, use a sterile inoculation loop to pick up a single colony and inoculate it into ordinary broth medium for culture to obtain the corresponding bacterial solution.

[0041] One of the selected bacteria was named Bacterium YM-6.

[0042] (II) Identification of bacteria YM-6 The method used in the references (Dong Xiuzhu, Cai Miaoying (2001) Handbook of Systematic Identification of Common Bacteria. Beijing: Science Press; RE. Buchanan, N.G. Gibbons (1984) Berger's Manual of Bacterial Identification (8th Edition) Translation Group of Berger's Manual of Bacterial Identification, Institute of Microbiology, Chinese Academy of Sciences, Beijing: Science Press) was used to identify bacteria YM-6.

[0043] (1) Genomic DNA was extracted from bacteria YM-6 and used as a template. PCR amplification was performed using primer pair 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and primer 1492R: 5'-TACGACTTAACCCCAATCGC-3' to obtain the PCR amplification product. The PCR amplification product is 16S rDNA.

[0044] (2) Sequencing the PCR amplification products.

[0045] Sequencing results showed that the nucleotide sequence of the 16S rDNA of bacterial YM-6 is shown in SEQ ID No. 1. BLAST analysis of the nucleotide sequence shown in SEQ ID No. 1 in the NCBI Standard Nucleotide database was performed to ultimately determine the genus and species of bacterial YM-6. The results indicated that bacterial YM-6 is related to *Spiralella iridis* (…). Irak Niveispirillum It showed the highest homology with *Synspira iridis*. Therefore, bacterium YM-6 was identified as *Synspira iridis*. Irak Niveispirillum ).

[0046] (III) Preservation Step (I) isolated bacteria YM-6, which was deposited on January 28, 2026, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 37555. The full name of bacteria YM-6 is *Spiralella iridis* (Iraqis white spirochete). Irak Niveispirillum YM-6CGMCC No.37555, also known as Iraqi Snow White Spirulina YM-6.

[0047] II. Bacillus tekiria ( Tequila Bacillus Separation, identification and preservation of Re2 CGMCC No. 36764 (a) Isolation of bacteria Re2 1. Weigh 1g of soil sample (collected from a rare earth mine in Longyan City, Fujian Province), add it to a test tube containing 9mL of sterile PBS buffer and an appropriate amount of glass beads, shake for 10min, let stand for 30s, and the supernatant is the original soil solution (the dilution at this time is recorded as 10). -1 Pipette 1 mL of the supernatant into a test tube containing 9 mL of sterile PBS buffer and mix thoroughly (the dilution is now 10). -2 Then, take 1 mL from this test tube and add it to another test tube containing 9 mL of sterile PBS buffer. Mix well, and so on to prepare dilutions of different concentrations.

[0048] 2. Using a pipette, take 100 μL of the diluted solution at different concentrations and spread it evenly on ordinary nutrient agar medium. Incubate at 37°C for 2-3 days.

[0049] 3. After completing step 2, inoculate each colony into ordinary broth medium and incubate at 37°C to obtain the corresponding bacterial solution.

[0050] 4. Take 10 μL of the above bacterial solution, dilute it 100 times with sterile PBS buffer, and then take 100 μL of the bacterial solution to inoculate it into ordinary nutrient agar medium and incubate at 37°C for 24 h.

[0051] 5. After completing step 4, use a sterile inoculation loop to pick up a single colony and inoculate it into ordinary broth medium for culture to obtain the corresponding bacterial solution.

[0052] One of the selected bacteria was named bacteria Re2.

[0053] (II) Identification of bacteria Re2 The method for identifying bacteria Re2 was found in the references (Dong Xiuzhu and Cai Miaoying (2001). Handbook of Systematic Identification of Common Bacteria. Beijing: Science Press; RE. Buchanan and N.G. Gibbons (1984). Berger's Manual of Bacterial Identification (8th Edition). Translation Group of Berger's Manual of Bacterial Identification, Institute of Microbiology, Chinese Academy of Sciences. Beijing: Science Press).

[0054] (1) Genomic DNA was extracted from bacteria Re2 and used as a template. PCR amplification was performed using primer pair 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and primer 1492R: 5'-TACGACTTAACCCCAATCGC-3' to obtain the PCR amplification product. The PCR amplification product is 16S rDNA.

[0055] (2) Sequencing the PCR amplification products.

[0056] Sequencing results showed that the nucleotide sequence of the 16S rDNA of bacteria Re2 is shown in SEQ ID No. 2. BLAST analysis of the nucleotide sequence shown in SEQ ID No. 2 using the NCBI Standard Nucleotide database ultimately determined the genus and species of bacteria Re2. The results indicated that bacteria Re2 are related to Bacillus tekirae (…). Tequila Bacillus The bacteria Re2 showed the highest homology with Bacillus tekirae ( ). Therefore, Bacterium Re2 was identified as Bacillus tekirae ( ). Tequila Bacillus ).

[0057] (III) Preservation Step (I) isolated bacteria Re2, which was deposited on November 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 36764. The full name of bacteria Re2 is *Bacillus tekirae* (…). Tequila Bacillus Re2 CGMCCNo.36764, abbreviated as Tekira Bacillus Re2.

[0058] Example 2: Preparation of Compound Microbial Agent I. Preparation of YM-6 inoculum of *Spiralobacter iridis* 1. Inoculate a single colony of *Syntrophus iridis* YM-6 into 50 mL of LB liquid medium and culture at 30°C with shaking at 200 r / min for 24-48 h to obtain *Syntrophus iridis* YM-6 seed culture.

[0059] 2. Inoculate the *Iraqiella icariina* YM-6 seed culture at a rate of 1-5% (v / v) into a fermenter containing 3 L LB liquid medium (5 L capacity). Ferment at 30℃ and 200 r / min to obtain an effective viable count concentration ≥2.0 × 10⁻⁶. 9 Fermentation broth of *Syngonium raffinosum* YM-6 at CFU / mL.

[0060] The fermentation broth of *Syntrophus raffinis* YM-6 is the *Syntrophus raffinis* YM-6 inoculum.

[0061] II. Preparation of Bacillus tekiria Re2 inoculum 1. Inoculate a single colony of Bacillus tekirae Re2 into 50 mL of LB liquid medium and culture at 37℃ and 200 r / min for 12-24 h to obtain Bacillus tekirae Re2 seed culture.

[0062] 2. Inoculate the Bacillus tekiria Re2 seed culture at an inoculation rate of 1-5% (v / v) into a fermenter containing 3 L LB liquid medium (5 L capacity), and ferment at 37℃ and 200 r / min to obtain an effective viable count concentration ≥2.0×10⁻⁶. 9 CFU / mL Bacillus tekirae Re2 fermentation broth.

[0063] The fermentation broth of Bacillus tekirae Re2 is the Bacillus tekirae Re2 inoculum.

[0064] III. Preparation of Compound Microbial Agents A compound bacterial agent was prepared by mixing *Synoptera iridis* YM-6 inoculum and *Bacillus tekirae* Re2 inoculum. The volume ratio of *Synoptera iridis* YM-6 inoculum to *Bacillus tekirae* Re2 inoculum in the compound bacterial agent was 1:1.

[0065] Example 3: Evaluation of growth-promoting indicators of *Spiralobacter iridis* YM-6 and *Bacillus tekirae* Re2 I. Determination of Nitrogenase Activity by Acetylene Reduction Method 1. Pre-culture of strains The test strains (Iraqi white spirochete YM-6 or Bacillus tekirae Re2) were inoculated into 3 mL of nitrogen-free liquid culture medium and cultured at the optimal temperature (30℃ for Iraqi white spirochete YM-6 and 37℃ for Bacillus tekirae Re2) with shaking at 200 r / min for 24-48 h to obtain the pre-inoculated bacterial solution.

[0066] The solutes and their concentrations in the nitrogen-free liquid culture medium were sucrose 10.0 g / L, malic acid 5.0 g / L, dipotassium hydrogen phosphate 0.1 g / L, potassium dihydrogen phosphate 0.4 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.1 g / L, anhydrous calcium chloride 0.02 g / L, ferric chloride 0.01 g / L, and sodium molybdate dihydrate 0.002 g / L. The solvent was water, and the pH value (25℃) was 7.2±0.2.

[0067] 2. Semi-solid microaerobic culture Inoculate 375 μL of pre-inoculated bacterial solution (approximately 0.75% (v / v)) into a 125 mL anaerobic bottle containing 50 mL of nitrogen-free semi-solid culture medium. When inoculating, inject the bacterial solution into the middle of the culture medium, seal the bottle with a cotton plug, and incubate for 48 h until a typical microaerophilic bacterial growth film is observed on the subsurface.

[0068] The solutes and their concentrations in the nitrogen-free semi-solid culture medium were sucrose 10.0 g / L, malic acid 5.0 g / L, dipotassium hydrogen phosphate 0.1 g / L, potassium dihydrogen phosphate 0.4 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.1 g / L, anhydrous calcium chloride 0.02 g / L, ferric chloride 0.01 g / L, sodium molybdate dihydrate 0.002 g / L, and agar 1.75 g / L. The solvent was water, and the pH value (25℃) was 7.2±0.2.

[0069] 3. Construction of the acetylene reduction reaction system Seal the anaerobic bottle opening with a sterile rubber stopper, and replace the gas in the top space of the bottle with an airtight syringe: extract 5 mL of air and inject an equal volume of 10% acetylene gas (v / v) to initiate the acetylene reduction reaction. The time point of the first acetylene injection is recorded as zero point, and an uninoculated blank culture medium is set up as a control group.

[0070] 4. Gas chromatography detection The amount of ethylene produced was detected by gas chromatography. The chromatographic conditions were set as follows: injection port temperature 120℃, detector temperature 60℃, column temperature 150℃; argon was used as the carrier gas and the flow rate was 25 mL / min; 100 μL of headspace sample was injected for analysis at specific time points, and the peak area of ​​ethylene was recorded.

[0071] 5. Result Calculation Total protein content was determined using the Bradford assay kit, and nitrogenase activity was defined as the amount of ethylene generated per unit protein content per unit time (nmol C2H4 / min / mg protein).

[0072] Test results are shown Figure 1 ( Niveispirillum irakenseYM-6 refers to *Synspira iridis* YM-6. Bacillus tequilensis Re2 is *Bacillus tekirae* Re2. The results showed that the nitrogenase activity of *Synspira iridis* YM-6 was 40.54 nmol C2H4 / min / mg protein, indicating that *Synspira iridis* YM-6 has nitrogen-fixing ability; however, no ethylene was detected in the acetylene reduction reaction system inoculated with *Bacillus tekirae* Re2, indicating that *Bacillus tekirae* Re2 does not have nitrogen-fixing ability.

[0073] II. Determination of Phosphate Solubility by Molybdenum-Antimony Colorimetric Method 1. Cultivation and Pretreatment (1) A single colony of *Spiritobacter iridis* YM-6 was inoculated into 50 mL of LB liquid medium and cultured at 30℃ and 200 r / min for 24-48 h with shaking to obtain *Spiritobacter iridis* YM-6 seed culture. A single colony of *Bacillus tekirae* Re2 was inoculated into 50 mL of LB liquid medium and cultured at 37℃ and 200 r / min for 24-48 h with shaking to obtain *Bacillus tekirae* Re2 seed culture.

[0074] (2) The seed culture (Spiritus iridis YM-6 seed culture or Bacillus tekirae Re2 seed culture) was inoculated into Monkina inorganic phosphorus liquid medium at an inoculation rate of 1% (v / v) and cultured at the optimal temperature (the optimal temperature of Spiritus iridis YM-6 is 30℃, and the optimal temperature of Bacillus tekirae Re2 is 37℃) and shaking at 200 r / min for 3 days. After the culture was completed, the bacterial precipitate was collected by centrifugation, washed with sterile water and then subjected to ultrasonic disruption. The cell debris was removed by centrifugation again, and 2.5 mL of the disrupted supernatant was taken as the sample to be tested.

[0075] The solutes and their concentrations in the Monkina inorganic phosphorus liquid culture medium were: glucose 10.0 g / L, calcium phosphate 10.0 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate heptahydrate 0.3 g / L, ferrous sulfate heptahydrate 0.03 g / L, and manganese sulfate tetrahydrate 0.03 g / L. The solvent was water, and the pH value (25℃) was 7.5±0.2.

[0076] 2. Color reaction Add 5 mL of molybdenum antimony colorimetric reagent to the sample to be tested, transfer it to a 50 mL volumetric flask and dilute to volume with distilled water; after standing and color development, measure the absorbance value at a wavelength of 700 nm.

[0077] 3. Result Calculation The soluble phosphorus content in the sample was calculated using a potassium dihydrogen phosphate standard curve to characterize the phosphate-solubilizing activity of the strain.

[0078] The test results are shown in Table 1. The results indicate that no soluble phosphorus was detected in the sample of *Spiralia iridis* YM-6, indicating that it does not have phosphorus-solubilizing activity as a nitrogen-fixing bacterium; the phosphorus-solubilizing capacity of the sample of *Bacillus tekirae* Re2 was 15.27 ± 0.77 mg / L, indicating that *Bacillus tekirae* Re2 has phosphorus-solubilizing ability.

[0079] Table 1. Determination of phosphate solubilization activity and indoleacetic acid (IAA) secretion capacity

[0080] III. Salkowski colorimetric method for determining indoleacetic acid (IAA) secretion capacity (1) The overnight activated test strain (Iraqis white spirochete YM-6 or Bacillus tekirae Re2) was inoculated at a 1% (v / v) inoculation rate into R2A liquid medium containing 100 mg / L L-tryptophan and cultured at the optimal temperature (30℃ for Iraqis white spirochete YM-6 and 37℃ for Bacillus tekirae Re2) with shaking at 200 r / min for 24 h or 48 h.

[0081] The solutes and their concentrations in R2A liquid culture medium were: tryptone 0.5 g / L, yeast extract 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, soluble starch 0.5 g / L, glucose 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.05 g / L, and sodium pyruvate 0.3 g / L. The solvent was water, and the pH (25℃) was 7.2 ± 0.2.

[0082] (2) Centrifuge and collect the supernatant. Take 1 mL of the supernatant and mix it with an equal volume of Salkowski colorimetric solution. After reacting in the dark for 30 min, measure the absorbance value at a wavelength of 530 nm. Calculate the content of indoleacetic acid (IAA) in the sample using the indoleacetic acid standard curve.

[0083] The results are shown in Table 1. The results indicate that *Spiralella iridis* YM-6 has the ability to secrete indoleacetic acid (IAA), with IAA production of 24 h and 22.53 ± 6.03 mg / L after 24 h and 48 h of culture, respectively; no IAA secretion activity was detected in *Bacillus tekirae* Re2.

[0084] Therefore, the compound microbial agent prepared in Example 2 establishes a highly efficient microecological symbiotic system through functional complementarity among the strains. Among them, *Syntrophus iridis* YM-6 possesses the ability to fix nitrogen biologically and secrete indoleacetic acid, while *Bacillus tekirae* Re2 possesses phosphorus-solubilizing ability. *Syntrophus iridis* YM-6 and *Bacillus tekirae* Re2 exhibit functional complementarity in their growth-promoting mechanisms, thereby synergistically enhancing the overall growth-promoting and yield-increasing effect of the compound microbial agent: the phosphorus and energy produced by phosphorus solubilization can assist the energy-intensive biological nitrogen fixation process; simultaneously, the auxin—indoleacetic acid—secreted by *Syntrophus iridis* YM-6 optimizes the crop root architecture, further enhancing the plant's ability to capture soil nutrients.

[0085] Example 4: Preparation of Compound Microbial Agent 1. To improve the bacterial agent carrier's loading capacity for bacterial strains and its dispersibility in soil, colloidal attapulgite was selected as the adsorption matrix. Colloidal attapulgite (particle size 100-200 mesh) was pulverized and dispersed in ammonia water to prepare a suspension. The suspension was then frozen and allowed to stand at a low temperature of -50℃ to -70℃ for 2-4 hours to obtain the frozen carrier.

[0086] The purpose of this step is to effectively depolymerize the clay crystal bundle structure by utilizing the in-situ growth and volume expansion effect of ice crystals, thereby significantly increasing the porosity and specific surface area of ​​the carrier; at the same time, ammonia treatment can increase the active adsorption sites on the carrier surface, thereby enhancing its affinity for microorganisms and soil nutrient ions.

[0087] 2. Thaw the frozen carrier and dry and pulverize it at 30℃-50℃ to obtain a modified attapulgite carrier with high adsorption activity.

[0088] 3. The composite microbial agent prepared in Example 2 is sprayed evenly onto the modified attapulgite carrier with high adsorption activity obtained in step 2 at a liquid-to-solid ratio of 1:1 (1L:1Kg) (the liquid-to-solid ratio is strictly controlled to ensure adsorption saturation). The microbial solution is uniformly adsorbed into the microporous structure of the attapulgite. After low-temperature drying and disc granulation, the granular finished product—the composite microbial agent—is obtained.

[0089] The composite microbial agent prepared by the above process has high particle strength, high strain survival rate, and the carrier itself has excellent soil improvement and fertilizer retention performance.

[0090] The appearance of the finished compound microbial agent prepared by the above process is shown in the figure. Figure 2 The composite microbial agent prepared by the above process was named "Micro Nitrogen No. 1".

[0091] Field application effects of the compound microbial inoculants prepared in Examples 5 and 4 I. The Effect of Compound Microbial Inoculants on Fertilizer Reduction (Reduced Chemical Fertilizer Application) and Yield Increase in Corn Fields 1. Experimental site: West of Anghuo Highway, south of Shengli Village, Yushutun Town, Angangxi Demonstration Zone, Qiqihar City; the experimental plot is low-lying, the soil is alkaline meadow soil with medium fertility; the experimental field area is 80 mu.

[0092] 2. Experimental crop variety: maize variety Tianyu 108.

[0093] 3. Test inoculum: The commercial inoculum was a commercially available high-yield-increasing agricultural microbial inoculum; the test inoculum was Micro Nitrogen No. 1 prepared in Example 4.

[0094] 4. Experimental Design: The experiment includes four treatments. The parameters and specific requirements for each treatment will be implemented according to the experimental plan. Treatment 1: Full fertilization; Treatment 2: Lose 20% of weight; Treatment 3: 20% weight loss + commercial bacterial agent; Treatment 4: 20% weight loss + experimental bacterial agent.

[0095] 5. The yield measurement method shall be carried out in accordance with the Ministry of Agriculture and Rural Affairs' method for yield measurement and acceptance of high-yield maize creation projects. Specifically, a random sampling method shall be used, selecting 3 sampling points for each treatment, with 4 rows (row length 7.7 m, area 20 m²) taken from each sampling point. 2 All ears of grain were harvested and weighed at the sampling point. Twenty standard ears were randomly selected for threshing and weighing. The shelling percentage and grain moisture content of the fresh ears were determined, and the final yield per mu (unit of land area) was calculated based on a standard moisture content of 14.0%. The yield per mu calculation formula is as follows: .

[0096] After field sampling and calculation, the yield measurement results are as follows: Figure 3 The yield of corn under the full fertilizer treatment was 751.81 kg / mu; the yield under the 20% fertilizer reduction treatment was 736.63 kg / mu, a decrease of 2.02% compared to the full fertilizer treatment, indicating that simply reducing fertilizer will cause yield loss; on the basis of reducing fertilizer by 20%, the yield of corn under the application of commercial microbial agents rebounded to 790.19 kg / mu, an increase of 5.10% compared to the full fertilizer treatment; and on the basis of reducing fertilizer by 20%, the yield under the application of the experimental microbial agent - Micro Nitrogen No. 1 reached as high as 871.96 kg / mu, an increase of 15.98% compared to the full fertilizer treatment.

[0097] The above results show that, with a 20% reduction in chemical fertilizer application, the yield per acre of Micro-Nitrogen No. 1 provided by this invention is far higher than that of commercially available high-efficiency yield-increasing microbial agents for agriculture.

[0098] II. The effect of compound microbial agents on reducing fertilizer application (reducing chemical fertilizer use) and increasing yield in rice fields 1. Experimental site: West of Anghuo Highway, south of Shengli Village, Yushutun Town, Angangxi Demonstration Zone, Qiqihar City; the experimental plot is low-lying, the soil is alkaline meadow soil with medium fertility; the experimental field area is 10 mu.

[0099] 2. Experimental crop variety: Rice variety Jinshuiyuan 9. Jinshuiyuan 9 was developed by the Rice Research Institute of the Chinese Academy of Agricultural Sciences and received national variety approval in 2015.

[0100] 3. Test inoculum: The commercial inoculum was a commercially available high-yield-increasing agricultural microbial inoculum; the test inoculum was Micro Nitrogen No. 1 prepared in Example 4.

[0101] 4. Experimental Design: The experiment includes four treatments. The parameters and specific requirements for each treatment will be implemented according to the experimental plan. Treatment 1: Full fertilization; Treatment 2: Lose 20% of weight; Treatment 3: 20% weight loss + commercial bacterial agent; Treatment 4: 20% weight loss + experimental bacterial agent.

[0102] 5. The yield measurement method shall be implemented in accordance with the Ministry of Agriculture and Rural Affairs' methods for high-yield rice creation and acceptance. Specifically, a random sampling method shall be used, with 3 sampling points selected for each treatment, and the sampling area at each point ≥ 3 m². 2 The rice plants in the sample area were harvested, threshed, and cleaned. Fresh grain weight, grain yield, and moisture content were measured, and the final yield was calculated based on a standard moisture content of 14.5%. The formula for calculating yield per mu (unit of land area) is as follows: .

[0103] After field sampling and calculation, the yield measurement results are as follows: Figure 4 The yield of rice under the full fertilizer treatment was 642.68 kg / mu; the yield under the 20% fertilizer reduction treatment was 576.82 kg / mu, a 10.25% decrease compared to the full fertilizer treatment, indicating that the basic soil fertility of this plot could not support the nutrient gap caused by the reduction in fertilizer application. Under the same fertilizer reduction conditions (i.e., a 20% reduction in fertilizer application), the yield of rice under the experimental microbial agent—Micro-Nitrogen No. 1—recovered to 651.32 kg / mu. This result was not only significantly better than the fertilizer reduction treatment, but also showed a 1.34% increase in yield compared to the full fertilizer treatment. Under the same fertilizer reduction conditions (i.e., a 20% reduction in fertilizer application), the yield of rice under the commercial microbial agent was 660.94 kg / mu, not significantly different from the experimental results of Micro-Nitrogen No. 1.

[0104] III. The Effect of Compound Microbial Inoculants on Soybean Field Fertilizer Reduction (Reduced Chemical Fertilizer Application) and Yield Increase 1. Experimental site: West of Anghuo Highway, south of Shengli Village, Yushutun Town, Angangxi Demonstration Zone, Qiqihar City; the experimental plot is low-lying, the soil is alkaline meadow soil with medium fertility; the experimental field area is 10 mu.

[0105] 2. Experimental crop variety: Soybean variety Dongsheng 79.

[0106] 3. Test inoculum: The commercial inoculum was a commercially available high-yield-increasing agricultural microbial inoculum; the test inoculum was Micro Nitrogen No. 1 prepared in Example 4.

[0107] 4. Experimental Design: The experiment includes four treatments. The parameters and specific requirements for each treatment will be implemented according to the experimental plan. Treatment 1: Full fertilization; Treatment 2: Lose 20% of weight; Treatment 3: 20% weight loss + commercial bacterial agent; Treatment 4: 20% weight loss + experimental bacterial agent.

[0108] 5. The yield measurement method shall be implemented in accordance with the Ministry of Agriculture and Rural Affairs' method for high-yield soybean creation and acceptance. Specifically, a five-point sampling method shall be used, with five sampling points selected for each treatment, and the sampling area at each point ≥ 3 m². 2 The soybeans in the sample area were harvested whole, threshed, and impurities removed. Fresh weight, seed yield, and moisture content of the seeds were measured, and the final yield was calculated based on a standard moisture content of 13.0%. The formula for calculating yield per mu (unit of land area) is as follows: .

[0109] After field sampling and calculation, the yield measurement results are as follows: Figure 5 The soybean yield under the full fertilizer treatment was 225.19 kg / mu; the yield under the 20% fertilizer reduction treatment was 192.87 kg / mu, a 14.35% reduction compared to the full fertilizer treatment, indicating that the basic soil fertility of the plot could not support the nutrient gap caused by the reduction in fertilizer application. Under the same fertilizer reduction conditions (i.e., a 20% reduction in fertilizer application), the application of commercial microbial agents alleviated some of the yield reduction trend, but the yield only recovered to 216.03 kg / mu, still a 4.07% reduction compared to the full fertilizer treatment, and failed to completely replace the reduced fertilizer application. However, under the same fertilizer reduction conditions (i.e., a 20% reduction in fertilizer application), the application of the experimental microbial agent—Micro-Nitrogen No. 1—recovered the yield to 241.01 kg / mu. This result was not only significantly better than the fertilizer reduction treatment, but also showed a 7.02% increase in yield compared to the full fertilizer treatment.

[0110] In summary, the compound microbial inoculant—Micro-Nitrogen No. 1—prepared in Example 4 demonstrated its ability to reduce fertilizer use and increase yield in the production of three major crops: corn, rice, and soybean. Applying the compound microbial inoculant prepared in this invention not only effectively compensated for the soil nutrient deficit caused by reduced chemical fertilizer use but also achieved a yield increase compared to the full-fertilization treatment. Therefore, the compound microbial inoculant prepared in Example 4 has good broad-spectrum applicability and significant potential for stable and increased yields, and can effectively replace some chemical fertilizers. It has important promotional value for reducing agricultural non-point source pollution and achieving green and sustainable agricultural development.

[0111] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A compound microbial agent comprising *Helicobacter iridis* and *Bacillus tekirae*.

2. The complex bacterial agent according to claim 1, characterized by: The compound microbial agent is composed of *Iraqiella icariina* and *Bacillus tekira*. 3.The complex bacterial agent according to claim 1, characterized in that: The compound microbial agent also includes a carrier.

4. The complex bacterial agent according to claim 3, characterized in that: The compound microbial agent consists of *Helicobacter iridis*, *Bacillus tekira*, and a carrier.

5. The compound microbial agent according to any one of claims 1 to 4, characterized in that: The Iraqi white spirochete is Iraqi white spirochete ( Niveispirillum irakense YM-6; The *Bacillus tekirae* is *Bacillus tekirae* ( Bacillus tequilensis Re2; The Iraqi Snow White Spirulina ( Niveispirillum irakense YM-6, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 37555; The Bacillus tergenta ( Bacillus tequilensis Re2, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 36764.

6. The compound microbial agent according to claim 3 or 4, characterized in that: The carrier is attapulgite that has undergone ammoniation-freezing modification.

7. The compound microbial agent according to any one of claims 1 to 6, characterized in that: In preparing the compound bacterial agent, both *Syntrophus iridis* and *Bacillus tekira* were added in the form of bacterial suspension. Preferably, the concentration of effective viable bacteria in the bacterial liquid of Microspira iraqiensis and the bacterial liquid of Bacillus tequilensis is both ≥2.0×10 9 CFU / mL. Preferably, when preparing the composite bacterial agent, the liquid-to-solid ratio of the total volume of *Ischemicula rabies* bacterial solution and *Bacillus tekirae* bacterial solution to the carrier is 1L:(0.8-1.2)Kg.

8. The application of the compound microbial agent according to any one of claims 1 to 7 is at least one of A1) to A4): A1) Increased crop yield; A2) Promotes crop growth; A3) Promotes biological nitrogen fixation in crops; A4) Enhances the crop's ability to capture soil nutrients.

9. The application of the compound microbial agent according to any one of claims 1 to 7 in crop fertilizer reduction and yield increase; wherein fertilizer reduction and yield increase means increasing yield while reducing the amount of chemical fertilizer used.

10. The application according to claim 8 or 9, characterized in that: The crop is corn, soybean, or rice.

Images