Use of azotobacter paspali dr43-29 in promoting growth of soybean

Abstract

The application provides application of Azospirillum brasilense Dr43-29 in promoting growth of soybeans and belongs to the technical field of microbial function development. The application provides application of Azospirillum brasilense Dr43-29 in promoting growth of soybeans, and it is found that Azospirillum brasilense Dr43-29 can promote increase of fresh weight of whole soybean plants, soybean plant height and soybean root fresh weight, and can promote soybean nodule formation in combination with Bradyrhizobium elkanii. The application further provides a soybean seed coating agent as well as a preparation method and application thereof, and the soybean seed coating agent can be used for increasing soybean yield in soybean planting and has a great application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial function development technology, specifically involving the application of Azotobacter brasiliensis Dr43-29 in promoting soybean growth. Background Technology

[0002] Currently, soybean cultivation still faces numerous bottlenecks: On the one hand, environmental factors such as soil degradation (e.g., compaction, salinization), nutrient imbalances (nitrogen, phosphorus, potassium), and continuous cropping obstacles hinder soybean root development, reduce the number of root nodules, and lower nitrogen fixation efficiency, thus affecting plant growth and yield. On the other hand, excessive application of chemical fertilizers in pursuit of high yields not only increases planting costs but also damages soil microbial communities, causes eutrophication in water bodies, and leads to a decline in soybean quality, which is inconsistent with the trend of green agriculture. Therefore, developing efficient, environmentally friendly growth-promoting technologies adapted to the growth characteristics of soybeans has become a key requirement for overcoming the bottlenecks in soybean production.

[0003] Microbial preparations have become an important direction for the green transformation of agriculture due to their advantages such as promoting growth and improving quality, ameliorhizium anisopliae, and reducing chemical inputs. Among them, *Azotomyces brasiliensis* (… Azospirillum brasilense As a type of growth-promoting microorganism widely distributed in soil, *Azospirillum brasiliensis* has been proven to have significant growth-promoting effects on various crops, showing broad application prospects in agricultural production. However, in current technologies, most research on the application of *Azospirillum brasiliensis* focuses on non-leguminous crops such as corn and wheat, with limited development and application of specific strains for soybeans. The symbiotic nitrogen-fixing system between soybeans and rhizobia is highly specific; common nitrogen-fixing microorganisms often struggle to form synergistic effects with soybeans and may even compete for ecological niches or nutrient resources, inhibiting rhizobium infection and nodulation, resulting in poor growth-promoting effects. Furthermore, reported *Azospirillum brasiliensis* strains suffer from problems such as limited functionality, weak environmental adaptability, and unstable nitrogen-fixing efficiency, making it difficult to adapt to different soil conditions (such as acidic soils and saline soils) and soybean variety characteristics, thus limiting the effectiveness of large-scale application. Therefore, developing new *Azospirillum brasiliensis* strains and related preparations for promoting soybean growth has significant practical significance and application value for promoting green and high-yield soybean cultivation and ensuring sustainable agricultural development. Summary of the Invention

[0004] To address the problems existing in the prior art, the primary objective of this invention is to provide *Azotobacter brasiliensis* (…). Azospirillum brasiliense The application of Dr43-29 in promoting soybean growth, wherein the preservation number of the Brazilian azotospirobacter Dr43-29 is CGMCC No.35622.

[0005] A second objective of this invention is to provide *Azotobacter brasiliensis* (… Azospirillum brasiliense The combined use of Dr43-29 and rhizobium in promoting soybean nodulation.

[0006] A third objective of this invention is to provide a soybean seed coating agent, its preparation method, and its application.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides *Azotobacter brasiliensis* (… Azospirillum brasiliense The application of Dr43-29 in promoting soybean growth, wherein the preservation number of the Brazilian azotospirobacter Dr43-29 is CGMCC No.35622.

[0008] Preferably, the Brazilian azospirobacter Dr43-29 promotes the increase of whole plant fresh weight, soybean plant height and soybean root fresh weight.

[0009] This invention also provides *Azotobacter brasiliensis* (… Azospirillum brasiliense The combined application of Dr43-29 and rhizobium in promoting soybean nodulation, wherein the preservation number of Dr43-29 is CGMCC No. 35622.

[0010] Preferably, the rhizobium includes *Syntrophus esculenta*.

[0011] Preferably, the *Syntrophus esculenta* includes *Syntrophus esculenta* BNCC 232076.

[0012] The present invention also provides a soybean seed coating agent comprising a mixed bacterial solution of *Azotrophus brasiliensis* Dr43-29 and *Syntrophus esculenta* BNCC 232076; wherein the bacterial ratio of *Azotrophus brasiliensis* Dr43-29 and *Syntrophus esculenta* BNCC 232076 is 1:1; and the preservation number of *Azotrophus brasiliensis* Dr43-29 is CGMCC No. 35622.

[0013] Preferably, the soybean seed coating agent further includes a film-forming agent and sterile water; the volume ratio of the film-forming agent, mixed bacterial solution and sterile water is 1:(0.02~0.1):(3.90~3.98).

[0014] This invention also provides a method for preparing the above-mentioned soybean seed coating agent, comprising the following steps: culturing *Azotobacter brasiliensis* Dr43-29 and *Syntrophus elegans* BNCC 232076 respectively to obtain cultures; centrifuging the cultures to collect the bacterial cells; and resuspending the bacterial cells at OD200. 600 =0.6 to obtain bacterial solution; mix the bacterial solution of *Azotobacter brasiliensis* Dr43-29, the bacterial solution of *Syntropha escherichia coli* BNCC 232076, the film-forming agent and sterile water to obtain soybean seed coating agent.

[0015] The present invention also provides a method for coating soybean seeds, using the above-mentioned soybean seed coating agent, and coating soybean seeds at a mass ratio of 1:200 to soybean seed coating agent.

[0016] The present invention also provides the application of the soybean seed coating agent, the preparation method of the soybean seed coating agent, or the soybean seed coating method in soybean cultivation.

[0017] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention provides for the first time *Azotobacter brasiliensis* (… Azospirillum brasiliense The application of Dr43-29 in promoting soybean growth: Dr43-29 of Azotobacter brasiliensis can significantly promote the increase of soybean whole plant fresh weight, soybean plant height and soybean root fresh weight. In addition, when used in combination with Rhizobium esculentum, it can significantly increase the number of nodules and fresh weight of soybeans, and promote the increase of soybean yield.

[0018] The soybean seed coating agent provided by this invention uses *Azotomyces brasiliensis* (… Azospirillum brasiliense Dr43-29 and Slow-growing Rhizobium esculentum are coated together on soybean seeds as a coating, which can effectively promote the growth of soybean plants and significantly increase the fresh weight of soybean plants and the number and weight of nodules. It has great application prospects as a biological inoculant for leguminous crops.

[0019] Biological Preservation Instructions Azotoxinus brasiliensis Dr43-29, classified and named Azotoxinus brasiliensis ( Azospirillum brasilense (), deposited by: China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC No. 35622, deposit date: August 14, 2025. Attached Figure Description

[0020] Figure 1 The classification and identification results of *Azotobacter brasiliensis* Dr43-29; Figure 2 Soybean growth after 30 days; Figure 3 Soybean growth status after 60 days of growth; Figure 4 Statistical analysis of soybean growth parameters after 30 days of growth; Figure 5 Photo of soybean nodules; Figure 6 Statistics on the number of soybean nodules; Figure 7 Statistics on the fresh weight of soybean nodules; Figure 8 Statistical results of soybean yield. Detailed Implementation

[0021] This invention provides *Azotobacter brasiliensis* (… Azospirillum brasiliense The application of Dr43-29 in promoting soybean growth: Dr43-29, with accession number CGMCC No. 35622, was deposited on August 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The *Azotomyces brasiliensis* Dr43-29 described in this invention can promote the increase of whole-plant fresh weight, soybean plant height, and soybean root fresh weight, and promote soybean leaf development. Preferably, *Azotomyces brasiliensis* Dr43-29 is used as a component of soybean seed coating agents to promote soybean growth.

[0022] This invention also provides *Azotobacter brasiliensis* (… Azospirillum brasiliense This invention relates to the combined application of Dr43-29 and rhizobia in promoting soybean nodulation. The *Azotrophus brasiliensis* Dr43-29 has the preservation number CGMCC No. 35622, and the rhizobia preferably includes *Rhizotrophus esculenta*, more preferably *Rhizotrophus esculenta* BNCC 232076. This invention, through the combined action of *Azotrophus brasiliensis* Dr43-29 and *Rhizotrophus esculenta*, can effectively promote soybean plant growth, significantly increase the fresh weight of soybean plants and the number and weight of nodules, and exert a synergistic effect. Preferably, the application of this invention involves using *Azotrophus brasiliensis* Dr43-29 and *Rhizotrophus esculenta* together as components of a soybean seed coating agent to promote soybean growth.

[0023] This invention also provides a soybean seed coating agent comprising a mixed bacterial solution of *Azotobacter brasiliensis* Dr43-29 and *Rhizobium argentea* BNCC 232076; the bacterial ratio of *Azotobacter brasiliensis* Dr43-29 to *Rhizobium argentea* BNCC 232076 is 1:1; the preservation number of *Azotobacter brasiliensis* Dr43-29 is CGMCC No. 35622. Preferably, the soybean seed coating agent of this invention further comprises a film-forming agent and sterile water, wherein the film-forming agent is polyvinylpyrrolidone and polyvinyl alcohol; the mass ratio of polyvinylpyrrolidone to polyvinyl alcohol is 5:1; the polyvinylpyrrolidone is any one of polyvinylpyrrolidone K60, polyvinylpyrrolidone K70, and polyvinylpyrrolidone K80; and the average degree of polymerization of the polyvinyl alcohol is 2000-2500. The volume ratio of the film-forming agent, the mixed bacterial solution, and sterile water is 1:(0.02~0.1):(3.90~3.98), preferably 1:0.05:3.95. The concentration of the mixed bacterial solution in this invention is preferably 5×10⁻⁶. 7 CFU / mL ~5×10 8 CFU / mL, more preferably 1×10⁻⁶ 8 CFU / mL.

[0024] This invention also provides a method for preparing the above-mentioned soybean seed coating agent, comprising the following steps: culturing *Azotobacter brasiliensis* Dr43-29 and *Syntrophus elegans* BNCC 232076 respectively to obtain cultures; centrifuging the cultures to collect the bacterial cells; and resuspending the bacterial cells at OD200. 600 =0.6 to obtain bacterial solution; mix the bacterial solution of *Azotobacter brasiliensis* Dr43-29, the bacterial solution of *Syntropha escherichia coli* BNCC 232076, the film-forming agent and sterile water to obtain soybean seed coating agent.

[0025] As an optional implementation, the present invention cultured *Regenerative Rhizobium elutiliforme* BNCC232076 in beef extract peptone medium and *Azotobacter brasiliensis* Dr43-29 in LB medium, both at 28°C to OD. 600 =1.0. Then, centrifuge at 4000 rpm for 10 min, collect the bacterial cells, and resuspend the bacterial cells in sterile water to OD. 600 =0.6 to obtain the bacterial suspension. The concentration of *Azotobacter brasiliensis* Dr43-29 in the suspension was 1×10⁶. 8 The concentration of *Retiralaria elegans* BNCC 232076 in the bacterial culture was 1×10⁻⁶ CFU / mL. 8 CFU / mL.

[0026] This invention also provides a method for coating soybean seeds, using the aforementioned soybean seed coating agent at a mass ratio of soybean seeds to soybean seed coating agent of 1:200. The coating method of this invention can be configured as follows: selecting seeds that are uniform in size, plump, and undamaged; covering all seeds with the coating agent according to the mass ratio of seeds to seed coating agent; and then spreading them out indoors to air dry.

[0027] The present invention also provides the application of the soybean seed coating agent, the preparation method of the soybean seed coating agent, or the soybean seed coating method in soybean cultivation.

[0028] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0029] In a specific embodiment of the present invention, the slow-growing rhizobium BNCC 232076 was purchased from Beina Biotechnology Co., Ltd.

[0030] Unless otherwise specified, the following embodiments are all conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Example 1 Isolation, purification and identification of Azospirillum brasiliense Dr43-29: 1. Separation: Preparation of soil extract: Weigh 5g of soil sample into a 1000mL Erlenmeyer flask and add 200mL of sterile water to the Erlenmeyer flask; mix the above mixture thoroughly by shaking on a shaker at 37℃ (220rpm, 30min); let the resulting mixture stand for 30min; filter the mixture through a 70μm filter membrane to obtain the soil extract.

[0033] Prepare dilution gradient solutions to determine the optimal dilution concentration. Add 4500µL, 1500µL, 500µL, 167µL, 56µL, and 19µL of soil extract to reagent bottles containing 1L of 10% TSB solution to prepare gradient dilutions of 222×, 666×, 2000×, 6000×, 18000×, and 54000×.

[0034] The diluent was aliquoted into 96-well cell culture plates. In a biosafety cabinet, 160 µL of the diluent was transferred into each well of the 96-well cell culture plate, and each vial of diluent was transferred to three 96-well cell culture plates. A 10% TSB solution without soil extract was transferred to three 96-well cell culture plates as a negative control.

[0035] Incubate the culture plate in the dark for 1 week.

[0036] 2. Purification: The optimal dilution concentration (ODC) was initially determined. After one week, the growth of bacteria in the 96-well cell culture plate was observed. If about 30% of the wells in the 96-well cell culture plate showed visible turbidity, the dilution concentration corresponding to this 96-well cell culture plate was taken as the optimal dilution concentration.

[0037] Prepare sample gradient dilutions. Based on the optimal dilution concentration (ODC) initially determined in the preliminary experiment, prepare 1 / 3×ODC, ODC, and 3×ODC gradient dilutions using 10% TSB solution.

[0038] The diluent was aliquoted into 96-well cell culture plates. In a biosafety cabinet, 160 µL of the diluent was transferred into each well of the 96-well cell culture plate, with each vial being transferred to 30–45 cell culture plates. 10% TSB solution without soil extract was transferred to 3 96-well cell culture plates as a negative control.

[0039] The culture plates were incubated in the dark for 2 weeks.

[0040] Two weeks later, the bacterial growth in the 96-well cell culture plates was observed. Approximately 30% of the wells were retained as visibly turbid 96-well cell culture plates. Using a multipipe, 10 µL of sample was transferred from each well of the retained 96-well cell culture plates into a 96-well PCR plate and stored at -20°C for later bacterial identification.

[0041] 3. Identification: PCR amplification of the hypervariable region V5-V7 of the bacterial 16S rRNA gene was performed using primer pairs 799F and 1193R. The PCR system (20 µL) consisted of: 10 µL 2×PCR Taq Mix (Beijing Bomei Biotechnology Co., Ltd.), 0.4 µL forward primer (5 µM), 0.4 µL reverse primer (5 µM), 0.5 µL DNA template, and 8.7 µL ddH2O. The PCR program was: 95℃ for 30 s pre-denaturation, 55℃ for 30 s annealing, 72℃ for 30 s extension, for 35 cycles. The amplified products were subjected to 1% agarose gel electrophoresis. After confirming the band size to be approximately 400 bp, the PCR products were sequenced. The sequences were compared with known sequences in the GenBank database, and a phylogenetic tree analysis was performed. The results are shown below.Figure 1 As shown. The results confirmed that the purified strain was *Azotobacter brasiliensis* (…). Azospirillum brasiliense It was named *Azotobacter brasiliensis* Dr43-29.

[0042] Azotobacter brasiliensis Dr43-29 was deposited on August 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China. Accession number: CGMCC No. 35622.

[0043] Example 2 Brazilian azospira ( Azospirillum brasiliense Culture of Dr43-29: Take one tube of Azotobacter brasiliensis ( Azospirillum brasiliense Dr43-29 glycerol bacterium was streaked in three zones on LB agar plates and incubated upside down at 37°C for 36 h. Single colonies were then picked and transferred to LB liquid medium, and incubated statically at 37°C for 24 h to obtain a seed culture. This seed culture was then inoculated into LB liquid medium at a 0.1% (v / v) inoculum and incubated statically at 37°C for 24 h to obtain the fermentation broth of *Azotocinobacter brasiliensis* Dr43-29. The viable count in the fermentation broth was 10-1. 8 CFU / mL.

[0044] Example 3 A soybean seed coating agent: the volume ratio of film-forming agent (polyvinylpyrrolidone K70 and polyvinyl alcohol with an average degree of polymerization of 2000 in a mass ratio of 5:1), mixed bacterial solution, and sterile water is 1:0.05:3.95. The mixed bacterial solution is a mixture of *Azotobacter brasiliensis* Dr43-29 and *Staphylococcus aureus* BNCC 232076; the bacterial counts of *Azotobacter brasiliensis* Dr43-29 and *Staphylococcus aureus* BNCC 232076 are 1:1.

[0045] Preparation method: Take Brazilian azospira ( Azospirillum brasiliense Dr43-29 was inoculated into LB liquid medium and incubated statically at 28°C until OD... 600 After reaching a concentration of 1.0, the fermentation broth of *Azotobacter brasiliensis* Dr43-29 was obtained. The fermentation broth was centrifuged at 4000 rpm for 10 min, and the bacterial cells were collected and resuspended in sterile water to an OD value of 1.0. 600 =0.6 for later use (bacterial concentration is 10) 8 (CFU / mL).

[0046] Take the strain of *Syntrophus esculenta* ( Bradyrhizobium elkanii (BNCC.232076), inoculated into beef extract peptone medium, and incubated statically at 28°C until OD. 600=1.0 to obtain the fermentation broth of *Rhizobium esculentum*. The fermentation broth was centrifuged at 4000 rpm for 10 min, the bacterial cells were collected, and the cells were resuspended in sterile water to OD0.05. 600 =0.6 for later use (bacterial concentration is 10) 8 (CFU / mL).

[0047] Equal volumes of *Azotrophus brasiliensis* Dr43-29 bacterial solution and *Syntrophus esculenta* Rhizobium argentiflorum solution were mixed to obtain a mixed bacterial solution. A film-forming agent, the mixed bacterial solution, and sterile water were then mixed in a specific ratio to obtain a seed coating agent.

[0048] Example 4 A method for coating soybean seeds: Select seeds that are uniform in size, plump, and intact. Put two plastic bags of the same size together. According to the mass ratio of seeds to seed coating agent prepared in Example 3, put the seeds and seed coating agent into the inner plastic bag, tie the bag opening, and then quickly rub with both hands until all the seed surfaces are covered with the viscous coating agent. Spread them out indoors to dry, and the coating is complete.

[0049] Experimental Example 1 1. Culture of *Syntrophus esculenta* strain and *Dr43-29* strain The experiment used the strain of *Syntrophus esculenta* (Syntrophus esculenta). Bradyrhizobium elkanii BNCC, number 232076 (referred to as Rhizobium in the experiment) and Dr43-29 strain.

[0050] Rhizobium strains were cultured in beef extract peptone medium, and Dr43-29 strain was cultured in LB medium, both at 28°C until OD. 600 =1.0. Then, both cultures were centrifuged at 4000 rpm for 10 min, the bacterial cells were collected, and the cells were resuspended in sterile water to OD0.0. 600 =0.6 for later use. The bacterial concentration of strain Dr43-29 is 10. 8 CFU / mL, the concentration of rhizobium in the culture was 10. 8 CFU / mL.

[0051] 2. Seed coating Soybean field trials were conducted through seed coating, with individual and / or co-coating of Rhizobium and / or Dr43-29 strains, respectively.

[0052] Rhizobium-coated seed group: The seed coating agent was prepared as follows: 1 mL film-forming agent (polyvinylpyrrolidone K70 and polyvinyl alcohol with an average degree of polymerization of 2000 in a mass ratio of 5:1) + 50 μL of Rhizobium inoculum + 3950 μL of sterile water.

[0053] Dr43-29 strain alone coated group (Dr43-29): The seed coating agent was prepared as follows: 1 mL film-forming agent (polyvinylpyrrolidone K70 and polyvinyl alcohol with an average degree of polymerization of 2000 in a mass ratio of 5:1) + 50 μL Dr43-29 strain culture + 3950 μL sterile water.

[0054] Rhizobium + Dr43-29 co-coating group: The seed coating agent was prepared as follows: 1 mL film-forming agent (polyvinylpyrrolidone K70 and polyvinyl alcohol with an average degree of polymerization of 2000 at a mass ratio of 5:1) + 50 μL mixed bacterial solution (rhizobium and Dr43-29 strain at a bacterial count ratio of 1:1) + 3950 μL sterile water.

[0055] Control group: The seed coating agent was prepared as 1 mL of film-forming agent (polyvinylpyrrolidone K70 and polyvinyl alcohol with an average degree of polymerization of 2000 in a mass ratio of 5:1) + 4000 μL of sterile water.

[0056] Seed coating: Select seeds that are uniform in size, plump, and intact. Put two plastic bags of the same size together. According to the mass ratio of seeds to seed coating agent of 1:200, put the seeds and seed coating agent into the inner plastic bag, tie the bag opening, and then quickly rub with your hands until all the seed surfaces are covered with the sticky coating agent. Spread them out indoors to dry, and the coating is complete.

[0057] 3. Field planting Seeds of each coating treatment were planted in 4 random plots, with a plot area of ​​2m×15m, row spacing of 45cm, and plant spacing of 8cm.

[0058] 4. Sampling and testing (1) After 30 and 60 days of growth, soybean plants of each coating treatment were collected to evaluate their growth (plant height, whole plant fresh weight, root fresh weight) and nodulation (number of nodules and root nodule fresh weight). Four replicates were set up, and three plants were sampled in each replicate.

[0059] Figure 2 Soybean plants after 30 days of growth; Figure 3 Soybean plants were grown for 60 days. Results showed that soybean plants coated solely with Dr43-29 strain exhibited superior aboveground and root growth compared to the control group at both 30 and 60 days. When Dr43-29 was co-coated with rhizobia, the two strains showed a synergistic effect on soybean growth. The soybeans in the "Rhizo+Dr43-29" co-coated group had more developed root systems and larger leaves compared to the control group.

[0060] Figure 4The growth parameters of soybeans after 30 days of growth are statistically analyzed. In the figure, A represents the total plant fresh weight, B represents the plant height, and C represents the root fresh weight. The results showed that soybean plants coated with Dr43-29 strain alone had significantly different plant height, total plant fresh weight, and root fresh weight compared to the control group. At 30 days of growth, the total plant fresh biomass of soybean plants coated with Dr43-29 strain alone and those coated with Dr43-29 strain and rhizobium were 26.80 g and 52.90 g, respectively, while the control group was only 22.95 g, representing significant increases of 16.78% and 130.50%, respectively (p<0.05). The plant heights of Dr43-29 strain alone and co-coated with rhizobia were 47.13 cm and 62.96 cm, respectively, while the control group was only 43.30 cm, representing significant increases of 8.85% and 45.40% (p<0.05). The root biomass of Dr43-29 strain alone and co-coated with rhizobia were 2.67 g and 3.70 g, respectively, while the control group was only 1.43 g, representing significant increases of 86.71% and 158.74% (p<0.05). Therefore, during the soybean seedling stage, Dr43-29 strain significantly promotes soybean plant growth, and Dr43-29 strain and rhizobia exert a synergistic effect.

[0061] Figure 5 Photo of soybean nodules. Figure 6 For the statistics of soybean nodule count, Figure 7 This study statistically analyzed the fresh weight of soybean nodules. The results showed that soybean plants co-coated with Dr43-29 strain and rhizobium had an increased nodule count (from 49.60 to 56.60) and a nodule weight (from 518.50 mg / plant to 877.43 mg / plant) compared to soybean plants coated with rhizobium alone. This indicates a synergistic effect between Dr43-29 strain and rhizobium in soybean nodulation.

[0062] (2) After the soybean plants matured, the yield of soybeans for each coating treatment was determined. Four randomized plots were set up for repeated sampling, with each plot having an area of ​​2m². 2 The result is as follows Figure 8 As shown in the figure. The results showed that the soybean yield of the Dr43-29 strain alone was significantly different from that of the control. The soybean yields of the Dr43-29 strain alone and the soybean yields of the Dr43-29 strain co-coated with rhizobium were 25.16 g and 30.56 g, respectively, while the control group was only 23.93 g, which was significantly higher than the control group by more than 15.29% and 27.72% (p<0.05).

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. *Azospira brasiliensis* ( Azospirillum brasiliense The application of Dr43-29 in promoting soybean growth is characterized by, The preservation number of the Brazilian azospira Dr43-29 is CGMCC No. 35622; the Brazilian azospira Dr43-29 promotes the increase of soybean whole plant fresh weight, soybean plant height and soybean root fresh weight.

2. *Azospira brasiliensis* ( Azospirillum brasiliense The combined application of Dr43-29 and rhizobia in promoting soybean nodulation is characterized by, The preservation number of *Azotrophus brasiliensis* Dr43-29 is CGMCC No. 35622. The rhizobia include *Rhizobium esculentum*, which includes *Rhizobium esculentum* BNCC 232076. The bacterial ratio of *Azotrophus brasiliensis* Dr43-29 to *Rhizobium esculentum* BNCC 232076 is 1:

1.

3. A soybean seed coating agent, characterized in that, The mixture comprises a bacterial culture of *Azotrophus brasiliensis* Dr43-29 and *Staphylococcus aureus* BNCC 232076; the bacterial ratio of *Azotrophus brasiliensis* Dr43-29 to *Staphylococcus aureus* BNCC 232076 is 1:1; the preservation number of *Azotrophus brasiliensis* Dr43-29 is CGMCC No. 35622.

4. The soybean seed coating agent according to claim 3, characterized in that, The soybean seed coating agent further includes a film-forming agent and sterile water; the volume ratio of the film-forming agent, mixed bacterial solution and sterile water is 1:(0.02~0.1):(3.90~3.98).

5. The method for preparing the soybean seed coating agent according to any one of claims 3 to 4, characterized in that, Includes the following steps: *Azotrophus brasiliensis* Dr43-29 and *Syntrophus esculenta* BNCC 232076 were cultured separately to obtain cultures. The cultures were collected by centrifugation, and the bacterial cells were resuspended at OD200. 600 =0.6 to obtain bacterial solution; mix the bacterial solution of *Azotobacter brasiliensis* Dr43-29, the bacterial solution of *Syntropha escherichia coli* BNCC 232076, the film-forming agent and sterile water to obtain soybean seed coating agent.

6. A method for coating soybean seeds, characterized in that, The soybean seed coating agent according to any one of claims 3 to 4 is used for coating at a mass ratio of soybean seed to soybean seed coating agent of 1:

200.

7. The application of the soybean seed coating agent according to any one of claims 3 to 4, the preparation method of the soybean seed coating agent according to claim 5, or the soybean seed coating method according to claim 6 in soybean cultivation.

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