Wheat rhizosphere growth promoting composite microbial inoculant, and preparation method and application thereof

Abstract

The application discloses a wheat rhizosphere growth promoting composite microbial agent and a preparation method and application thereof, and belongs to the technical field of agricultural microorganisms.The wheat rhizosphere growth promoting composite microbial agent has the functions of nitrogen fixation, phosphorus dissolution and IAA production, and is beneficial to nutrient utilization of wheat; after wheat seedlings are connected with the wheat rhizosphere growth promoting composite microbial agent, the nitrogen absorption efficiency can be significantly improved, and the growth of the wheat is promoted, so that the purpose of reducing fertilizer and increasing efficiency is achieved; moreover, the wheat rhizosphere growth promoting composite microbial agent has an antagonistic effect on multiple pathogenic fungi such as false smut fungus, fusarium graminearum, fusarium solani and the like, and can be used for biological control of wheat.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a wheat rhizosphere growth-promoting compound microbial agent, its preparation method, and its application. Background Technology

[0002] Wheat is the second most widely cultivated crop globally. Nitrogen is a key nutrient for plant growth and development, playing a crucial role in chlorophyll formation, photosynthesis, and protein synthesis, and is a major factor limiting crop yield and quality. Moderate application of nitrogen fertilizer can promote crop growth and increase yield. However, in current agricultural production, farmers often over-apply nitrogen fertilizer to boost wheat yields, while wheat itself has limited nitrogen absorption and utilization capacity. Excessive fertilization not only fails to increase yield but may also lead to serious environmental pollution.

[0003] Currently, common measures to improve nitrogen absorption and utilization in wheat include developing high-nitrogen-utilization-rate wheat varieties, improving field management and cultivation techniques, and using microbial agents to replace some chemical fertilizers. Fertilization plays a crucial role in agricultural production, and seeking scientific and rational fertilization methods is one of the effective management strategies for increasing wheat yields. To increase wheat production, especially in developing countries, many countries have over-relyed on chemical fertilizers to ensure stable and efficient grain production, leading to a sharp increase in grain output. However, long-term reliance on chemical fertilizers to increase wheat yields not only increases economic costs but also causes irreversible damage to the environment. Therefore, China urgently needs to develop scientific and green fertilization methods to increase wheat yields while minimizing environmental pollution such as eutrophication and ensuring the healthy development of the ecosystem. Currently, the use of microbial fertilizers in my country is relatively low, especially in wheat cultivation. Improving the nitrogen absorption and utilization rate in wheat and reducing the input of inorganic nitrogen fertilizers have become major demands for green wheat production and sustainable development in my country.

[0004] Therefore, for soils with nitrogen-efficient wheat varieties, screening and constructing a rhizosphere growth-promoting compound microbial agent that can stably colonize in the soil and significantly promote wheat nitrogen absorption and growth, and developing a highly efficient and stable multifunctional microbial fertilizer, are effective measures to ensure high-quality, high-efficiency, and green wheat production. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a wheat rhizosphere growth-promoting compound microbial agent, its preparation method and application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a wheat rhizosphere growth-promoting compound microbial agent comprising: Bacillus velezensis 9H23-2 and Bacillus aryabhattai 66H147-3.

[0008] The Bacillus velezensis 9H23-2 was deposited on January 22, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number CGMCC NO.33489.

[0009] Bacillus aryabhattai 66H147-3 was deposited on January 22, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number CGMCC NO.33490.

[0010] Preferably, in the wheat rhizosphere growth-promoting compound microbial agent, Bacillus velezensis 9H23-2 and Bacillus aryabhattai 66H147-3 exist in the form of cultured live bacteria, bacterial suspension or fermentation broth.

[0011] Preferably, the wheat rhizosphere growth-promoting compound microbial agent is prepared by mixing a bacterial suspension of Bacillus velezensis 9H23-2 and a bacterial suspension of Bacillus aryabhattai 66H147-3 in a volume ratio of 1:1.

[0012] Furthermore, the bacterial suspension of Bacillus velezensis 9H23-2 was prepared by the following method:

[0013] Bacillus velezensis 9H23-2 was inoculated into LB liquid medium and cultured in a constant temperature shaking incubator at 28℃ and 180rpm for 24h. After centrifugation, the bacterial precipitate was collected and resuspended in sterile distilled water to prepare a bacterial suspension.

[0014] The bacterial suspension of Bacillus aryabhattai 66H147-3 was prepared by the following method:

[0015] Bacillus aryabhattai 66H147-3 was inoculated into LB liquid medium and cultured in a constant temperature shaking incubator at 28℃ and 180rpm for 24h. After centrifugation, the bacterial precipitate was collected and resuspended in sterile distilled water to prepare a bacterial suspension.

[0016] More preferably, the OD values ​​of the bacterial suspensions of Bacillus velezensis 9H23-2 and Bacillus aryabhattai 66H147-3 are... 600 The values ​​are all 0.80.

[0017] In a second aspect, the present invention provides the application of the above-mentioned wheat rhizosphere growth-promoting compound microbial agent in improving the nitrogen absorption efficiency of wheat.

[0018] In soybeans, nitrogen absorption is primarily achieved through symbiotic nitrogen fixation with rhizobia; however, in wheat, non-symbiotic nitrogen fixation is dominant. In the wheat rhizosphere growth-promoting compound microbial agent of this invention, *Bacillus velezensis* 9H23-2 and *Bacillus aryabhattai* 66H147-3 are the main nitrogen fixation contributors in wheat, which can improve nitrogen absorption efficiency and increase nitrogen accumulation in the aboveground parts.

[0019] In a third aspect, the present invention provides the application of the above-mentioned wheat rhizosphere growth-promoting compound microbial agent in the following (1) or (2):

[0020] (1) Prepare products that inhibit the growth of pathogens;

[0021] (2) Prevention and control of wheat diseases caused by pathogens.

[0022] In the above applications, the pathogens are *Fusarium graminearum*, *Fusarium graminearum*, and / or *Fusarium solani*.

[0023] In the above applications, the wheat diseases caused by pathogens include: wheat stem base rot, wheat scab, and wheat root rot.

[0024] The beneficial effects of this invention are:

[0025] 1. The compound microbial agent S3 provided by the present invention is composed of Bacillus velezensis 9H23-2 and Bacillus aryabhattai 66H147-3, which can significantly improve the absorption and utilization of nitrogen in wheat and promote plant growth.

[0026] 2. Bacillus velezensis 9H23-2 in this invention has the ability to fix nitrogen and produce indoleacetic acid; Bacillus aryabhattai 66H147-3 can achieve the functions of nitrogen fixation, phosphorus solubilization and indoleacetic acid production.

[0027] 3. The compound microbial agent S3 provided by this invention has antagonistic effects on a variety of pathogens such as Fusarium graminearum, Fusarium graminearum, and Fusarium solani, and has a wide range of applications in promoting plant health.

[0028] 4. The bacterial strain described in this invention will not cause any harm to wheat seedlings and can coexist well with wheat. Furthermore, this strain originates from the rhizosphere soil of nitrogen-efficient wheat varieties and is not pathogenic to humans or animals. In addition, the bacterial strains in the bacterial suspension described in this invention have strong adaptability, the compound bacterial agent has stable performance, and the preparation method is simple and low-cost, thus showing broad application prospects. Attached Figure Description

[0029] Figure 1 This is a colony diagram of Bacillus bellis 9H23-2 of the present invention on LB solid medium.

[0030] Figure 2 This is a colony diagram of Bacillus 66H147-3 of the present invention on LB solid medium.

[0031] Figure 3 This is a Gram staining image of Bacillus berleis 9H23-2 of the present invention.

[0032] Figure 4 This is a Gram staining image of Bacillus 66H147-3 of the present invention.

[0033] Figure 5 This is a colony diagram of Bacillus belyss 9H23-2 cultured on Assumption nitrogen-free medium for 7 days.

[0034] Figure 6 This is a colony diagram of Bacillus 66H147-3 cultured on Assumption nitrogen-free medium for 7 days.

[0035] Figure 7 The color development of Bacillus belyss 9H23-2 and Bacillus argentis 66H147-3 under the Salksowski staining method.

[0036] Figure 8 The figure shows the results of a pot experiment on the compound microbial agent S3 under normal nitrogen fertilizer levels.

[0037] Figure 9The growth index of potted wheat under normal nitrogen fertilizer levels using the compound microbial agent S3 is shown in the figure.

[0038] Figure 10 The figure shows the plate antagonistic experiment of Bacillus belyss 9H23-2, Bacillus argentis 66H147-3, and compound inoculum S3 against Fusarium oxysporum.

[0039] Figure 11 The figure shows the plate antagonistic effect of Bacillus belyss 9H23-2, Bacillus argentea 66H147-3, and compound bacterial agent S3 on Fusarium graminearum.

[0040] Figure 12 The figure shows the plate antagonistic effect of Bacillus belyss 9H23-2, Bacillus argentea 66H147-3, and compound inoculant S3 on Fusarium solani. Detailed implementation method:

[0041] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0042] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be described in detail below with reference to specific embodiments. Unless otherwise specified, experimental conditions not detailed in the embodiments are generally based on conventional conditions or conditions recommended by the reagent company; reagents, consumables, etc., used in the following embodiments can be obtained commercially unless otherwise specified.

[0043] Example 1: Isolation and Identification of Strains

[0044] 1. Isolation of bacterial strains:

[0045] 1g of rhizosphere soil from the experimental field (36°10′N, 117°9′E) of the Shandong Agricultural University Resource and Environment Experimental Station (36°10′N, 117°9′E) of a high-nitrogen-utilization wheat variety was added to 10mL of sterile water and mixed thoroughly to prepare 10 -1 Diluent, take 1 mL of 10 -1 Add the diluent to 9 mL of sterile water and mix well to prepare 10 -2 Diluent, and so on, prepare 10 of each. -3 10 -4 10 -5 10 -6 Diluent, take 10 -3 10 -4 10 -5 10 -6 The diluted solution was spread onto LB solid medium and incubated at 28°C.

[0046] Single colonies with different morphological characteristics were purified, and the growth-promoting properties of the screened strains were determined. Two strains with the best growth-promoting effect and the ability to significantly increase nitrogen accumulation in wheat seedlings were selected. One strain was named 9H23-2, and the other was named 66H147-3. Both strains were stored in glycerol tubes.

[0047] 2. Identification of the strain:

[0048] (1) Morphological and physiological-biochemical characteristics analysis of the strain:

[0049] The colony morphology of strain 9H23-2 on LB agar plates is as follows: Figure 1 As shown, the colonies are round, light yellow in the center, with irregular edges, opaque, and wrinkled / raised on the surface. The colony morphology of strain 66H147-3 on LB agar plates is as follows. Figure 2 As shown, the colonies are round, white, opaque, dry, and have relatively regular edges.

[0050] Gram staining was performed on strains in the stable phase. The Gram staining result of strain 9H23-2 was purple. Figure 3 This is a Gram-positive bacterium, appearing as a rod-shaped organism under a light microscope. Its growth temperature is 18–37℃, pH 7.28, with an optimum growth temperature of 28℃. The Gram staining result of strain 66H147-3 is purple. Figure 4 It is a Gram-positive bacterium that appears as a rod-shaped organism under a light microscope. Its growth temperature ranges from 18 to 37°C, with a pH of 5.43, and the optimum growth temperature is 28°C.

[0051] (2) Molecular biological identification:

[0052] The genomes of strains 9H23-2 and 66H147-3 were extracted using a genome extraction kit. Using these genomes as templates, the 16S rDNA sequences were amplified using the universal 16S rDNA primers 27F (5'-GAG AGT TTG ATC CTG GCT CAG-3') and 1492R (5'-ACG GAT ACCTTG TTA CGA CTT-3').

[0053] The PCR reaction system (25 μL) consisted of: 2.5 μL 10× buffer, 2 μL dNTPs, 0.5 μL upstream primer 27F, 0.5 μL downstream primer 1492R, 0.15 μL Taq enzyme, 1 μL DNA template, and 18.35 μL ultrapure water. The PCR program was: 94℃ pre-denaturation for 10 min, 94℃ denaturation for 40 s, 53℃ annealing for 40 s, 72℃ extension for 90 s, 35 cycles, followed by a 10 min extension at 72℃ and incubation at 10℃. 1% agarose gel electrophoresis showed that the PCR product bands were single and approximately 1.5 kb in size. The PCR products from both strains were sent to Qingdao Ruiboxingke Sequencing Co., Ltd. for sequencing. The 16S rDNA sequence of strain 9H23-2 is shown in SEQ ID No. 1; the 16S rDNA sequence of strain 66H147-3 is shown in SEQ ID No. 2.

[0054] Sequence alignment of the sequencing results on EZBioCloud (https: / / www.ezbiocloud.net / identify) revealed that 9H23-2 showed 99.92% sequence similarity to *Bacillus belyescens*, and 66H147-3 showed 100% similarity to *Bacillus argentea*. Therefore, 9H23-2 was identified as *Bacillus belyescens*, and 66H147-3 as *Bacillus argentea*.

[0055] Based on the combined morphological and molecular biological identification results, strain 9H23-2 was identified as *Bacillus velezensis*, and strain 66H147-3 was identified as *Bacillus aryabhattai*. The screened and isolated strains 9H23-2 and 66H147-3 were bio-preserved. Depository institution: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Deposit date: January 22, 2025; Accession number for *Bacillus velezensis* 9H23-2: CGMCC NO. 33489; Accession number for *Bacillus aryabhattai* 66H147-3: CGMCC NO. 33490.

[0056] Example 2: Determination of growth-promoting properties of Bacillus velezensis 9H23-2 and Bacillus aryabhattai 66H147-3

[0057] Purified single colonies of 9H23-2 and 66H147-3 were inoculated into LB liquid medium and activated by shaking culture at 28℃ and 180 rpm for 24-48 h. Their growth-promoting abilities were then measured. The main growth-promoting indicators measured included nitrogen fixation ability, phosphorus solubility, and IAA production ability.

[0058] (1) Nitrogen fixation capacity determination:

[0059] 9H23-2 and 66H147-3 were inoculated onto Assumption nitrogen-free medium and incubated at 28°C. Bacterial colonies were observed after 3 days of incubation. Normal growth indicated nitrogen-fixing ability of the strain. The nitrogen-fixing ability of strain 9H23-2 was determined as follows: Figure 5 As shown in the figure, the nitrogen fixation capacity test results of strain 66H147-3 are as follows: Figure 6 As shown. Both strains 9H23-2 and 66H147-3 described in this invention exhibit good nitrogen fixation effects. Nitrogen standard curves were plotted: 0.0 mL, 0.2 mL, 0.5 mL, 1.0 mL, 3.0 mL, and 7.0 mL of potassium nitrate standard solution were added to 25 mL colorimetric tubes using a pipette. The tubes were then diluted to 10 mL with pure water, and 5 mL of potassium persulfate solution was added. The tubes were wrapped with gauze. The digestion was carried out at 121℃ for 30 min. After cooling, the tubes were shaken thoroughly. 1 mL of hydrochloric acid solution was added, and the volume was diluted to 25 mL with sterile water. Using pure water as a reference, the absorbance of each gradient of potassium nitrate solution was measured at 220 nm and 275 nm using a quartz cuvette. The corrected absorbance at zero concentration was denoted as Ab, and the corrected absorbance of other gradient standard solutions was denoted as AS. The difference between AS and Ab was denoted as Ar. A nitrogen standard curve was plotted with Ar values ​​corresponding to each gradient standard solution as the ordinate and total nitrogen content (ug) as the abscissa. Nitrogen fixation activity was determined: the strain was activated in LB liquid medium, and 1% of the bacterial solution was added to 100 mL of sterile Assumption liquid medium. The culture was incubated at 28℃ and 200 rpm / min for 5 days. The bacterial solution was then centrifuged at 10000 rpm / min for 15 min. 10 mL of the supernatant was then added to a 25 mL colorimetric tube, along with 1 mL of hydrochloric acid solution. The volume was adjusted to 25 mL with sterile water. Using pure water as a reference, the absorbance of each treated bacterial solution was measured at 220 nm and 275 nm. Ar values ​​were calculated using the above method, and the total nitrogen content in the bacterial solution was calculated using the following formula. The results showed that the nitrogen contents of 9H23-2 and 66H147-3 reached 1073.75 mg / L and 974.58 mg / L, respectively.

[0060] (2) Phosphorus solubility determination:

[0061] The culture medium used to determine the phosphorus solubility of the strain was PKO (Monginna Inorganic Phosphorus Medium), with the following ingredients: glucose 10 g / L, (NH4)2SO4 0.5 g / L, NaCl 0.3 g / L, MgSO4·7H2O 0.3 g / L, MnSO4·H2O 0.03 g / L, KCl 0.3 g / L, FeSO4·7H2O 0.03 g / L, Ca3(PO4)2 5 g / L, and agar 15 g / L.

[0062] The strain 66H147-3 described in this invention has a good phosphorus-solubilizing effect, and the ratio of the diameter (D) of the phosphorus-solubilizing zone to the diameter (d) of the colony is 1.60.

[0063] (3) IAA production capacity determination:

[0064] The Salksowski colorimetric method was used to qualitatively determine the IAA production capacity of strains 66H147-3 and 9H23-2. R2A liquid medium containing 200 mg / L L-tryptophan (uninoculated) was used as a blank control, and IAA was used as a positive control. The results are as follows: Figure 7 As shown, both bacterial solutions were pink, indicating that both strains had the ability to produce IAA. A quantitative determination of IAA production capacity was then performed, and the amounts of IAA secreted by strain 66H147-3 and strain 9H23-2 were measured to be 4.87 and 6.36 mg / L, respectively.

[0065] Example 3: Preparation of a compound microbial agent for promoting wheat rhizosphere growth

[0066] (1) Preparation of Bacillus velezensis 9H23-2 bacterial suspension:

[0067] Strain strain 9H23-2 was inoculated into LB liquid medium at a rate of 1% (v / v). After incubation at 28°C and 180 rpm for 24 h in a constant temperature shaking incubator, the fermentation broth was transferred to sterile centrifuge tubes and centrifuged at 5000 rcf for 10 min at room temperature. The supernatant was then carefully discarded, and the bacterial cells were resuspended in sterile distilled water. The OD was then measured. 600 The absorbance value at 600 nm was adjusted to 0.80 to prepare a suspension of Bacillus velezensis 9H23-2.

[0068] (2) Preparation of Bacillus aryabhattai 66H147-3 bacterial suspension:

[0069] Strain strain 66H147-3 was inoculated into LB liquid medium at a rate of 1% (v / v). After incubation at 28°C and 180 rpm for 24 h in a constant temperature shaking incubator, the fermentation broth was transferred to sterile centrifuge tubes and centrifuged at 5000 rcf for 10 min at room temperature. The supernatant was then carefully discarded, and the bacterial cells were resuspended in sterile distilled water. The OD was then measured. 600 The absorbance value at 600 nm was adjusted to 0.80 to prepare a suspension of Bacillus aryabhattai 66H147-3.

[0070] (3) Preparation of wheat rhizosphere growth-promoting compound microbial agent:

[0071] A wheat rhizosphere growth-promoting compound microbial agent was prepared by mixing Bacillus velezensis 9H23-2 bacterial suspension and Bacillus aryabhattai 66H147-3 bacterial suspension at a volume ratio of 1:1, and named compound microbial agent S3.

[0072] Experiment Example 1: Wheat Seedling Growth Promotion Experiment

[0073] 1. Test method:

[0074] Soil and vermiculite were mixed in a 1:1 mass ratio to form the soil for planting wheat. Nitrogen, phosphorus, and potassium fertilizers used were prepared urea solution, sodium dihydrogen phosphate solution, and potassium sulfate solution, respectively, under normal nitrogen fertilizer treatment conditions (N: 0.32 g / kg, P: 0.15 g / kg, K: 0.15 g / kg). After disinfection, wheat seeds were germinated at 28℃ for 3 days, then the soil was lightly watered before planting in seedling trays about 1-2 cm below the soil surface. The seedlings were then placed in a greenhouse for cultivation. A growth promotion experiment was conducted after 3 days of seedling growth, with four treatment groups: control (CK), single-graft 9H23-2 treatment (9H23-2), single-graft 66H147-3 treatment (66H147-3), and compound microbial agent S3 treatment (S3).

[0075] 9H23-2 treatment group: The Bacillus velezensis 9H23-2 bacterial suspension prepared in Example 3 was applied to the seedling pots, 10 mL per pot.

[0076] 66H147-3 treatment group: The Bacillus aryabhattai 66H147-3 bacterial suspension prepared in Example 3 was applied to the seedling pots, 10 mL per pot.

[0077] S3 treatment group: The compound microbial agent S3 prepared in Example 3 was applied to the seedling pots, 10 mL per pot.

[0078] CK group: Apply an equal volume of sterile distilled water.

[0079] Other culture conditions remained consistent across all treatment groups. On day 21 after the bacterial suspension interacted with wheat plants, samples were taken to measure SPAD, plant height, fresh weight, dry weight, and aboveground nitrogen accumulation, using the following methods:

[0080] SPAD measurement: Insert the leaf into the SPAD instrument clamp, ensuring that the leaf completely covers the sensing area. Measure 5 areas on each leaf and take the average value to obtain the SPAD value.

[0081] Plant height measurement: Wheat seedlings of each treatment were taken out, their above-ground parts were straightened, and the vertical height from the seed to the highest growth point of the above-ground part was measured. Each treatment was repeated 24 times to obtain the plant height value.

[0082] Fresh weight and dry weight determination: After cutting off the roots, weigh the above-ground part of the wheat seedling to obtain the fresh weight value; then dry it in an oven and measure the dry weight value.

[0083] Determination of nitrogen accumulation in aboveground parts: Crush the aboveground leaves and add them to a test tube. Add concentrated sulfuric acid and then digest the mixture using a digester. Add hydrogen peroxide every 20 minutes until the liquid becomes clear and transparent. Transfer the liquid to a centrifuge tube and use a flow analyzer to determine the nitrogen accumulation in the aboveground parts of each treatment.

[0084] 2. Test Results:

[0085] like Figure 8 and Figure 9 As shown in the figure, under normal nitrogen fertilizer treatment conditions (N: 0.32 g / kg, P: 0.15 g / kg, K: 0.15 g / kg), wheat seedlings inoculated with bacterial suspensions of 9H23-2, 66H147-3, and compound microbial agent S3 showed improved biomass and other growth indicators compared to uninoculated wheat seedlings. Compound microbial agent S3, compared to the two single strains, maximized the accumulation of nitrogen nutrients in wheat, promoting wheat growth. Specifically, inoculation with 9H23-2, 66H147-3, and S3 increased fresh weight by 8.2%, 20.72%, and 77.78% respectively compared to the control (CK) treatment; and increased dry weight by 4.05%, 17.56%, and 62.2% respectively compared to the CK treatment. Inoculation with 9H23-2, 66H147-3, and S3 increased nitrogen accumulation in the aboveground parts by 13.89%, 15.62%, and 56.55%, respectively, compared to the control (CK) treatment.

[0086] Experimental Example 2: Antagonistic Test against Pathogens

[0087] The *F. pseudograminearum* used in the antagonism test is described in the journal article "Research progress on the occurrence, damage and prevention of *Fusarium crownrot* caused by *Fusarium pseudograminearum*"; the pathogen *F. graminearum* is described in the journal article "Functional analysis of the glutathione S-transferases from *Thinopyrum* and its derivatives on wheat *Fusarium head blight resistance*".

[0088] 1. Antagonism test against the pathogen of wheat stem rot (F. pseudograminearum):

[0089] The antagonistic experiment used PDA medium. The mycelial cakes of Fusarium graminearum pathogens were placed in the middle of new PDA medium, and 2 μL of Bacillus velezensis 9H23-2 bacterial suspension, Bacillus aryabhattai 66H147-3 bacterial suspension and compound bacterial agent S3 prepared in Example 3 were dotted around them respectively. The medium was placed in an inverted incubator at 28℃ and the antagonistic effect was observed after 7 days of incubation.

[0090] like Figure 10 As shown in the figure. The results indicated that *Fusarium graminearum*, which causes wheat stem base disease, was clearly separated from 9H23-2 and S3 at their growth sites, indicating that 9H23-2 and S3 had significant antagonistic effects on the pathogen *Fusarium graminearum*, while 66H147-3 had no antagonistic ability against *Fusarium graminearum*. The inhibition rate of 9H23-2 against *Fusarium graminearum* was 63.5%, while that of S3 was 67%. This demonstrates that under the same inoculum concentration, strains 9H23-2 and 66H147-3 in the compound inoculant S3 can synergistically enhance the antibacterial activity against *Fusarium graminearum*.

[0091] 2. Antagonism test against Fusarium graminearum, the pathogen of wheat scab.

[0092] The antagonistic experiment used PDA medium. The mycelial cakes of Fusarium graminearum pathogens were placed in the middle of a new PDA medium. 2 μL of Bacillus velezensis 9H23-2 suspension, Bacillus aryabhattai 66H147-3 suspension, and compound bacterial agent S3 prepared in Example 3 were dotted around the medium. The medium was then placed in an inverted incubator at 28℃ and cultured for 7 days to observe the antagonistic effect.

[0093] like Figure 11 As shown in the figure. The results indicated that *Fusarium graminearum*, the causal agent of wheat scab, was clearly separated from 9H23-2 and S3 at their growth sites, indicating that 9H23-2 and S3 had significant antagonistic effects against *Fusarium graminearum*, while 66H147-3 had no antagonistic effect. The inhibition rate of 9H23-2 against *Fusarium graminearum* was 60.6%, while that of S3 was 67.2%. This demonstrates that under the same inoculum concentration, strains 9H23-2 and 66H147-3 in the compound inoculant S3 can synergistically enhance the antibacterial activity against *Fusarium graminearum*.

[0094] 3. Antagonism test against wheat root rot pathogen (Fusarium solani):

[0095] The antagonistic experiment used PDA medium. The mycelial cakes of Fusarium solani pathogens were placed in the middle of new PDA medium, and 2 μL of Bacillus velezensis 9H23-2 suspension, Bacillus aryabhattai 66H147-3 suspension and compound bacterial agent S3 prepared in Example 3 were dotted around them respectively. The medium was placed in an inverted incubator at 28℃ and the antagonistic effect was observed after 7 days of incubation.

[0096] like Figure 12 As shown in the figure. The results indicated that *Fusarium solani*, which causes wheat root rot, was clearly separated from 9H23-2 and S3 at its growth sites, indicating that 9H23-2 and S3 had significant antagonistic effects on *Fusarium solani*, while 66H147-3 had no antagonistic effect. Specifically, 9H23-2 showed an inhibition rate of 55.5% against *Fusarium solani*, while S3 showed an inhibition rate of 57.6%.

[0097] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A compound microbial agent for promoting rhizosphere growth in wheat, characterized in that, The wheat rhizosphere growth-promoting compound microbial agent is composed of Bacillus belysinus (B. belysinus). Bacillus velezensis )9H23-2 bacterial suspension and Bacillus argentis ( Bacillus aryabhattai The bacterial suspension of 66H147-3 was prepared by mixing the two bacteria at a volume ratio of 1:

1. The Bacillus belesi ( Bacillus velezensis The accession number for 9H23-2 is CGMCC NO. 33489, and the described Bacillus aureus ( Bacillus aryabhattai The accession number for 66H147-3 is CGMCC NO. 33490; The Bacillus belesi ( Bacillus velezensis The bacterial suspension of 9H23-2 was prepared by the following method: Bacillus berberis ( Bacillus velezensis )9H23-2 was inoculated into LB liquid medium and cultured in a constant temperature shaking incubator at 28℃ and 180 rpm for 24 h. After centrifugation, the bacterial precipitate was collected and resuspended in sterile distilled water to prepare a bacterial suspension. The Bacillus argentis ( Bacillus aryabhattai The bacterial suspension of 66H147-3 was prepared by the following method: Bacillus argentis ( Bacillus aryabhattai 66H147-3 was inoculated into LB liquid medium and cultured in a constant temperature shaking incubator at 28℃ and 180 rpm for 24 h. After centrifugation, the bacterial precipitate was collected and resuspended in sterile distilled water to prepare a bacterial suspension.

2. The wheat rhizosphere growth-promoting compound microbial agent according to claim 1, characterized in that, The Bacillus belesi ( Bacillus velezensis )9H23-2 bacterial suspension and Bacillus argentis ( Bacillus aryabhattai OD of bacterial suspension of 66H147-3 600 The values ​​are all 0.

80.

3. The application of the wheat rhizosphere growth-promoting compound microbial agent according to claim 1 or 2 in improving the nitrogen absorption efficiency of wheat.

4. The application according to claim 3, characterized in that, Specifically, improving the nitrogen absorption efficiency of wheat means increasing the nitrogen accumulation in the aboveground parts of wheat.

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