A microbial compound bacterial agent and its application

Abstract

The invention discloses a microbial composite agent and its application, and relates to the field of microbial technology. The microbial composite agent disclosed in the invention is composed of bradyrhizobium WP5 and sphingobacterium WD6. The results show that compared with no inoculation, after inoculation with the microbial composite agent of the present application, the height of soybean plants is significantly increased, the number of nodules is significantly increased, and the root system is more developed and dense; the fresh weight of the aboveground part of soybeans increased by 85.92%; the fresh weight of the underground part of soybeans increased by 59.17%; the fresh weight of soybean nodules increased by 58.83%; the height of soybeans increased by 19.35%; the thickness of soybean stems increased by 17.60%; the total root length increased by 21.58%; the number of root tips increased by 69.88%; the number of branches increased by 21.97%; and the root volume increased by 190.75%. In summary, the co-inoculation of bradyrhizobium WP5 and sphingobacterium WD6 has a significant promoting effect on the growth and development of soybeans.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a microbial compound bacterial agent and application thereof. Background Art

[0002] As the fifth largest food and oil crop in my country, soybean occupies a core position in agriculture. It is not only the main source of protein, but also widely used in animal husbandry feed and food additives. Although in the past soybean planting process, a large amount of chemical fertilizers were used to ensure the increase in yield, excessive fertilization has had a certain impact on soil microbial diversity and fertility, posing a huge challenge to the soil ecosystem. Therefore, it is necessary to relieve soil pressure and promote soybean growth. Therefore, using beneficial microorganisms to enhance soil fertility and improve nutrient utilization, thereby increasing crop yields and increasing plant nutrition is to replace the traditional application of chemical fertilizers to increase soybean yields and reduce the use of chemical fertilizers and pesticides, and promote the green and sustainable development of the soybean industry.

[0003] Soil and root microbial communities are beneficial to plant growth and health. Microorganisms can directly or indirectly affect plant growth and development by secreting secondary metabolites, producing volatile compounds, and regulating the expression of plant functional genes. Using a single strain has problems such as single function and poor adaptability for the complex and metabolically diverse microbial communities in nature. Synthetic bacterial communities composed of different strains can function more efficiently and stably in the soil, systematically regulating various functions related to plant growth, nutritional metabolism, and stress resistance, and "bottom-up" regulating the expression of plant functional genes. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a microbial compound agent. The technical problem to be solved by the present invention is to provide an application of a microbial compound agent for promoting soybean growth and promoting soybean root nodulation.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A microbial compound agent, composed of Bradyrhizobium WP5 and Sphingobacterium WD6;

[0007] The Bradyrhizobium WP5, whose classification name is Bradyrhizobium yuanmingense WP5, has been deposited in Guangdong Microbial Culture Collection Center, with the deposit number: GDMCC NO: 64629, the deposit date: May 17, 2024, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0008] The sphingobacterium thalpophilum WD6, whose classification name is sphingobacterium thalpophilum WD6, has been deposited in the Guangdong Provincial Microbiological Culture Collection Center, with a deposit number of: GDMCC NO: 64628, a deposit date of: May 17, 2024, and a deposit address of: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The preparation method of the microbial compound bacterial agent comprises the following steps: preparing Bradyrhizobium WP5 fermentation liquid and Sphingobacterium WD6 fermentation liquid respectively; mixing the Bradyrhizobium WP5 fermentation liquid and the Sphingobacterium WD6 fermentation liquid in a volume ratio of 1:1 to obtain the microbial compound bacterial agent.

[0010] Application of microbial compound agents in promoting soybean growth.

[0011] The promoting soybean growth is to promote the increase of fresh weight of soybean plants.

[0012] The method of promoting soybean growth is to promote the increase of soybean plant height.

[0013] The method of promoting soybean growth is to promote the increase of stem thickness of soybean plants.

[0014] The method of promoting soybean growth is to increase the total root length, the number of root tips and the root volume of the soybean root system.

[0015] Application of microbial compound agents in promoting soybean root nodulation.

[0016] The method of promoting soybean growth is to promote an increase in the number of soybean root nodules.

[0017] The method of promoting soybean growth is to promote the increase of fresh weight of soybean root nodules.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) The present invention screened and obtained Bradyrhizobium WP5 from soybean nodules, which is classified and named Bradyrhizobium yuanmingense WP5, and has been deposited in Guangdong Microbial Culture Collection Center, with a deposit number of GDMCC NO: 64629, a deposit date of May 17, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0020] 2) The present invention screened and obtained Sphingobacterium thalpophilum WD6 from soybean nodules, which is classified and named Sphingobacterium thalpophilum WD6, and has been deposited in Guangdong Microbial Culture Collection Center, with a deposit number of GDMCC NO: 64628, a deposit date of May 17, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0021] 3) The present invention prepares Bradyrhizobium WP5 fermentation broth and Sphingobacterium WD6 fermentation broth respectively; the Bradyrhizobium WP5 fermentation broth and the Sphingobacterium WD6 fermentation broth are mixed in a volume ratio of 1:1 to obtain a microbial composite bacterial agent. The results showed that compared with CK (no inoculation), after co-inoculation with Bradyrhizobium WP5 and Sphingobacterium WD6, soybean plant height increased significantly, the number of nodules increased significantly, and the root system became more developed and dense; the fresh weight of the aboveground part of soybean was significantly higher than that of the non-inoculation treatment, increasing by 85.92%; the fresh weight of the underground part of soybean was significantly higher than that of the non-inoculation treatment, increasing by 59.17%; the fresh weight of the root nodules of soybean was significantly higher than that of the non-inoculation treatment, increasing by 58.83%; the plant height of soybean was higher than that of the non-inoculation treatment, increasing by 19.35%; the stem diameter of soybean was greater than that of the non-inoculation treatment, increasing by 17.60%; the total root length increased by 21.58%; the number of root tips increased by 69.88%; the number of branches increased by 21.97%; and the root volume increased by 190.75%. In summary, the co-inoculation of Bradyrhizobium WP5 and Sphingobacterium WD6 can increase the length and number of root tips of soybean roots, especially the effect on root volume is more significant, which has a promoting effect on the growth and development of soybean. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the morphological characteristics of Bradyrhizobium WP5;

[0023] Figure 2 is the morphological characteristics of Sphingobacterium WD6;

[0024] Figure 3 This is a plate confrontation effect diagram of Bradyrhizobium WP5 and Sphingobacterium WD6;

[0025] Figure 4 This is a diagram showing the effect of Bradyrhizobium WP5 and Sphingobacterium WD6 co-inoculation on the phenotype of the aboveground part of soybean plants;

[0026] Figure 5 This is a diagram showing the effect of Bradyrhizobium WP5 and Sphingobacterium WD6 co-inoculation on the underground phenotype of soybean plants;

[0027] Figure 6 This is the effect of Bradyrhizobium WP5 and Sphingobacterium WD6 co-inoculation on the fresh weight of the aboveground part of soybean plants;

[0028] Figure 7 This is the effect of Bradyrhizobium WP5 and Sphingobacterium WD6 co-inoculation on the fresh weight of the underground part of soybean plants;

[0029] Figure 8 This is the effect of Bradyrhizobium WP5 and Sphingobacterium WD6 co-inoculation on the fresh weight of soybean nodules;

[0030] Fig. 9 This is the effect of Bradyrhizobium WP5 and Sphingobacterium WD6 co-inoculated on soybean plant height;

[0031] Fig.10 This is the effect of Bradyrhizobium WP5 and Sphingobacterium WD6 co-inoculation on soybean stem diameter;

[0032] Fig.11 This is a graph showing the effect of co-inoculation of Bradyrhizobium WP5 and Sphingobacterium WD6 on soybean root growth. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0034] In the following embodiments:

[0035] The formula of YMA liquid culture medium (1L) is: MgSO4.7H2O 0.2g, K2HPO4 0.8g, KH2PO4 0.2g, CaSO4.2H2O 0.1g, FeCl3 0.01g, Na2aMoO4.2H2O 0.002g, mannitol 20g, yeast extract 0.5g, pH 7.2, high pressure sterilization at 121℃ for 20min.

[0036] The formula of YMA solid culture medium is: MgSO4.7H2O 0.2g, K2HPO4 0.8g, KH2PO4 0.2g, CaSO4.2H2O0.1g, FeCl3 0.01g, Na2aMoO4.2H2O 0.002g, mannitol 20g, yeast extract 0.5g, agar powder 15g, pH 7.2, high pressure sterilization at 121℃ for 20min.

[0037] LB solid medium: peptone 10 g, yeast extract 5 g, sodium chloride 10 g, agar powder 15 g, pH 7.0, high pressure sterilization at 121 °C for 20 min.

[0038] The formula of LB liquid culture medium is: peptone 10g, yeast extract 5g, sodium chloride 10g, pH 7.0, high pressure sterilization at 121°C for 20min.

[0039] Axubei nitrogen-free medium (1L): KH2PO4 0.2g, MgSO 2. 7H2O 0.2g, NaCl 0.2g, CaCO35g, mannitol 10g, CaSO 4. 2H2O 0.1g, make up to volume with ddH2O, adjust pH=6.5.

[0040] The formula of DFa medium (1L) is: Na2HPO4 6g, KH2PO4 4g, MgSO4·7H2O 0.2g, glucose 2g, gluconic acid 2g, citric acid 2g, (NH4)2SO4 2g, FeSO4·7H2O 1mg, H3BO310μg, MnSO4·H2O 11.19μg, ZnSO4·7H2O 124.6μg, CuSO4·5H2O 72.22μg, MnO3 10μg, ACC solution 3.0mmol / L, agar 15-20g, pH7.2.

[0041] The formula of tricalcium phosphate medium (1L) is: glucose 10g, sodium chloride 0.3g, magnesium sulfate 0.3g, manganese sulfate 0.03g, ammonium sulfate 0.5g, potassium chloride 0.3g, iron sulfate 0.03g, calcium phosphate 10g, agar 15g, pH 7.2-7.4.

[0042] The color developing solution was prepared as follows: 5 mL of 95% ethanol, 1 mL of 1% phosphoric acid solution, and 0.2 mL of 10% pure sulfuric acid, mixed well, and then diluted to a 50 mL volumetric flask with sterile water to obtain the Salkowski color developing solution.

[0043] In the following embodiments:

[0044] The soybean samples (nodules) used for strain screening were planted at the Guangxi Zhuang Autonomous Region Maize Research Institute.

[0045] The soybean seeds used in the functional verification experiment were purchased from the Institute of Economic Crops, Guangxi Academy of Agricultural Sciences.

[0046] Example 1

[0047] 1. Isolation and identification of Bradyrhizobium WP5

[0048] Soak fresh nodule samples in 95% ethanol for 3 minutes, 0.5% sodium hypochlorite for 1 minute, and then rinse with sterile water for 5-7 times. Grind the samples thoroughly with a sterile mortar and dilute the tissue homogenate to 10 -2. Pipette 200 μL and spread on YMA plate, set 3 replicates, culture at 28℃ for 5-7 days, observe, when regular, moist, milky white colonies appear on the surface of the plate, promptly purify the colonies with a bacterial loop to a new plate, and inoculate the colonies on YMA liquid medium and culture at 28℃ for about 5-7 days to obtain a bacterial solution with OD=0.5. After molecular biological identification, promptly use 50% glycerol and OD=0.5 bacterial solution at a ratio of 1:1 and store in a -80℃ refrigerator.

[0049] The results are as follows Figure 1 As shown, a bacterial strain was screened from soybean nodules and named WP5 strain. The bacteria were rod-shaped, Gram-negative, and the colonies were round, opaque, white, and had granular structures.

[0050] The 16S sequence of Bradyrhizobium WP5 is shown in SEQ ID NO.1. Based on the above morphological observations and 16S sequence analysis results, it is determined that its taxonomic status is in the same branch as Bradyrhizobium yuanmingense. It is classified and named Bradyrhizobium yuanmingense WP5, and has been deposited in the Guangdong Provincial Microbiological Culture Collection Center with the deposit number: GDMCC NO: 64629, the deposit date: May 17, 2024, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0051] 2. Isolation and identification of Sphingobacterium WD6

[0052] Soak fresh nodule samples in 95% ethanol for 3 minutes, 0.5% sodium hypochlorite for 1 minute, and then rinse with sterile water for 5-7 times. Grind the samples thoroughly with a sterile mortar and dilute the tissue homogenate to 10 -2 . Pipette 200 μL and spread on NA or LB plate, set up 3 replicates, and culture at 28℃ for 24 hours. Observe. When regular, moist, light yellow colonies appear on the surface of the plate, purify the colonies with a bacterial loop to a new plate in time, and inoculate the colonies on NA or LB liquid culture medium and culture at 28℃ for about 24 hours to obtain a bacterial solution with OD=0.5. After molecular biological identification, use 50% glycerol and OD=0.5 bacterial solution in a ratio of 1:1 and store in a -80℃ refrigerator.

[0053] The results are as follows Figure 2 As shown, a bacterial strain was screened from soybean nodules and named as WD6 strain. The bacteria were rod-shaped, Gram-negative, with round and raised colonies, yellow and translucent, smooth and moist surface, and neat edges.

[0054] The 16S sequence of Sphingobacterium WD6 is shown in SEQ ID NO.2. Based on the above morphological observations and 16S sequence analysis results, its taxonomic status is in the same branch as Sphingobacterium thalpophilum. It is classified as Sphingobacterium thalpophilum WD6 and has been deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC NO: 64628, a deposit date of May 17, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0055] 3. Identification of the growth-promoting function of Bradyrhizobium WP5 and Sphingobacterium WD6

[0056] ① Autogenous nitrogen fixation: Use the spotting method to spot 10 μL of bacterial solution with OD=0.5 on the Ashubei nitrogen-free culture medium and culture it at 28°C for 5 days.

[0057] ② IAA production: inoculate the bacterial liquid with OD=0.5 into YMA medium containing L-Try (1 mg / mL) at a ratio of 1%, and culture at 28°C, 180rpm for 5 days; centrifuge the culture solution for 10 minutes, take 200μL of the supernatant and mix it with 200μL of colorimetric solution in equal volumes, and mix 200μL of the supernatant with 200μL of sterile water in equal volumes for the control, and place it in the dark for 0.5h. The IAA production ability is qualitatively judged based on whether color is developed. A pink color after the addition of colorimetric solution indicates that the strain has the ability to produce IAA, while no color is developed in the control.

[0058] ③ Production of ACC deaminase: 10 μL of bacterial solution with OD=0.5 was spotted in DFa medium and cultured at 28°C for 5 days. Bacterial circles appeared after 5 days of culture, indicating that the strain has the ability to utilize ACC nitrogen source.

[0059] ④ Phosphate solubilization ability: 10 μL of bacterial solution with OD=0.5 was spotted in tricalcium phosphate culture medium by the spotting method and cultured at 28°C for 5 days. Complete colonies appeared after 5 days of culture, indicating that the strain has the ability to solubilize phosphate.

[0060] The results are shown in Table 1. Both Bradyrhizobium WP5 and Sphingobacterium WD6 have growth-promoting functions.

[0061] Table 2 Identification of the growth-promoting functions of Bradyrhizobium WP5 and Sphingobacterium WD6

[0062] strain Autogenous nitrogen fixation Produced by IAA ACC deaminase Phosphate solubilization Bradyrhizobium WP5 + + + + Sphingobacterium WD6 + + + +

[0063] 4. Co-culture of Bradyrhizobium WP5 and Sphingobacterium WD6 on plates

[0064] 1) Activation of Bradyrhizobium WP5 and preparation of bacterial solution

[0065] Take out the strain stored in -80℃ refrigerator, place it in the center of YMA medium plate (3cm in diameter), and culture it at 28℃ for 5-7 days until colonies grow on the medium. Inoculate the activated strain into YMA liquid medium, culture it at 28℃ and 180-200rpm for 5-7 days, measure the OD600 of the bacterial solution, and dilute the bacterial solution of Bradyrhizobium WP5 to OD600=0.5.

[0066] 2) Activation of Sphingobacterium WD6 and preparation of bacterial solution

[0067] Take out the strain stored in -80℃ refrigerator, place it in the center of LB medium plate (3cm in diameter), and culture it at 28℃ for 24h, until colonies grow in the medium. Inoculate the activated strain into LB liquid medium, culture it at 28℃ and 180-200rpm for 24h, measure the OD600 of the bacterial solution, and dilute the bacterial solution of Sphingobacterium WD6 to OD600=0.5.

[0068] 2) Co-culture

[0069] Use a pipette tip to inoculate 10 μL of Bradyrhizobium WP5 in the middle of the YMA medium plate, and then inoculate 10 μL of Sphingobacterium WD6 at a distance of 0.6 cm from Bradyrhizobium WP5. Transfer to a 28°C light incubator and culture for 4-5 days to observe the interaction.

[0070] The results are as follows Figure 3 As shown, in the confrontation experiment between Bradyrhizobium WP5 and Sphingobacterium WD6, no inhibitory effect of Sphingobacterium WD6 on Bradyrhizobium WP5 was found; the two bacteria can grow in fusion with each other, and the compatibility of the two bacteria becomes stronger with the longer the co-culture time, and there is no mutual antagonism.

[0071] Example 2

[0072] 1. Preparation of Bradyrhizobium WP5 fermentation broth

[0073] 1) Activate and culture Bradyrhizobium WP5 on YMA solid medium plates at 28°C for 5 to 7 days;

[0074] 2) The activated strain was inoculated into YMA liquid culture medium and cultured at 28°C and 180-200 rpm for 5-7 days to obtain seed solution;

[0075] 3) The seed solution was re-inoculated into a new YMA liquid medium for fermentation. The seed solution inoculation amount was 1% of the volume of the YMA liquid medium. The culture was carried out at 28°C and 180-200 rpm for 5-7 days to obtain a fermentation liquid. The bacterial cell concentration of the obtained fermentation liquid was 0.3×10 9 CFU / mL.

[0076] 2. Preparation of Sphingobacillus WD6 fermentation broth

[0077] 1) Activate and culture Sphingobacterium WD6 on LB solid medium plates at 28°C for 24 hours;

[0078] 2) The activated strain was inoculated into LB liquid culture medium and cultured at 28°C and 180-200 rpm for 24 hours to obtain seed solution;

[0079] 3) The seed solution was inoculated into LB liquid medium, the seed solution inoculation amount was 1% of the volume of the LB liquid medium, and cultured at 28°C and 180-200 rpm for 24 h to obtain a fermentation liquid; the bacterial cell concentration of the obtained fermentation liquid was 0.3×10 9 CFU / mL.

[0080] 3. Preparation of microbial compound agents

[0081] The fermentation broth of Bradyrhizobium WP5 and the fermentation broth of Sphingobacterium WD6 were mixed at a volume ratio of 1:1, and the bacterial concentration was 0.3×10 9 CFU / mL.

[0082] Example 3

[0083] The effects of the mixed bacterial agent of Bradyrhizobium WP5 and Sphingobacterium WD6 on soybean growth are as follows:

[0084] Soybean was used as the test object. The soybean seeds were first germinated, soaked in 75% alcohol for 30s, and then soaked in 1% sodium hypochlorite solution for 10min to sterilize the soybean seeds. After being cleaned with sterile water, they were placed on gauze in a sterilized glass dish, and sterile water was sprayed to keep the gauze moist. Then the glass dish was placed in a clean bench to allow the soybean to germinate. Seedlings with the same germination degree were selected and transferred to a PET bottle with a soil pH of 4.7, about 600g of soil per bottle, and a hole in the bottom of the bottle. The experiment was designed with a 2% inoculation amount (i.e., 2mL of bacterial liquid per 100g of air-dried soil) and a control without inoculation. Each treatment was set up with 4 replicates; one plant per bottle; the moisture content of the experimental soil was controlled at 70%±5% of the field water holding capacity, and the walk-in incubator simulated the normal growth environment of soybeans. The light, temperature, and humidity were suitable. No additional fertilization was performed for all treatments, and the management measures for each treatment were the same. Destructive sampling was performed after 65 days to observe the above-ground and underground plant phenotypes and to measure indicators such as plant height, stem diameter, biomass, and root morphology.

[0085] The results are as follows Figure 4 As shown, the plant height of the soybean plant co-inoculated with Bradyrhizobium WP5 and Sphingobacterium WD6 was significantly higher than that of CK (no inoculation). Inoculation with the combined bacterial agent can effectively and significantly increase the plant height of soybean plants.

[0086] The results are as follows Figure 5 As shown, the roots and nodules co-inoculated with Bradyrhizobium WP5 and Sphingobacterium WD6 were significantly more than those in CK (not inoculated with bacteria). The soybean roots inoculated with the combined bacterial agent were more developed and dense, and the number of nodules was greater.

[0087] The results are as follows Figure 6 As shown, compared with CK (not inoculated with bacteria), the fresh weight of the aboveground part of soybean co-inoculated with Bradyrhizobium WP5 and Sphingobacterium WD6 was significantly higher than that of the non-inoculated treatment, increasing by 85.92%.

[0088] The results are as follows Figure 7 As shown, compared with CK (not inoculated with bacteria), the fresh weight of the underground part of soybean co-inoculated with Bradyrhizobium WP5 and Sphingobacterium WD6 was significantly higher than that of the non-inoculated treatment, increasing by 59.17%.

[0089] The results are as follows Figure 8 As shown, compared with CK (no inoculation), the fresh weight of soybean nodules co-inoculated with Bradyrhizobium WP5 and Sphingobacterium WD6 was significantly higher than that of the no inoculation treatment, increasing by 58.83%.

[0090] The results are as follows Fig. 9 As shown, compared with CK (no inoculation), the soybean plant height co-inoculated with Bradyrhizobium WP5 and Sphingobacterium WD6 was higher than that of the no inoculation treatment, increasing by 19.35%.

[0091] The results are as follows Fig.10 As shown, compared with CK (no inoculation), the stem diameter of soybeans co-inoculated with Bradyrhizobium WP5 and Sphingobacterium WD6 was greater than that of the no inoculation treatment, increasing by 17.60%.

[0092] The results are as follows Fig.11 As shown in the figure, compared with CK (no inoculation), the co-inoculation of Bradyrhizobium WP5 and Sphingobacterium WD6 had a significant effect on the underground growth of soybean. In terms of total root length, the inoculation increased by 21.58% compared with the non-inoculation; in terms of the number of root tips, the inoculation increased by 69.88% compared with the non-inoculation; in terms of the number of branches, the inoculation increased by 21.97% compared with the non-inoculation; in terms of root volume, the inoculation increased by 190.75% compared with the non-inoculation. In summary, the co-inoculation of Bradyrhizobium WP5 and Sphingobacterium WD6 can increase the length and number of root tips of soybean roots, especially the root volume, which has a more significant effect on the growth and development of soybean.

[0093] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A microbial compound agent, characterized in that: It is composed of Bradyrhizobium WP5 and Sphingobacterium WD6; The bradyrhizobium WP5 is classified and named Bradyrhizobium yuanmingense WP5 has been deposited in Guangdong Microbiological Culture Collection Center, with the deposit number: GDMCC NO: 64629, the deposit date: May 17, 2024, and the deposit address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou; The Sphingobacterium WD6 is classified as sphingobacterium thalpophilum WD6 has been deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number: GDMCC NO: 64628, the deposit date: May 17, 2024, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The method for preparing the microbial composite agent according to claim 1, characterized in that: The fermentation liquid of Bradyrhizobium WP5 and the fermentation liquid of Sphingobacterium WD6 are prepared respectively; the fermentation liquid of Bradyrhizobium WP5 and the fermentation liquid of Sphingobacterium WD6 are mixed in a volume ratio of 1:1 to obtain a microbial composite bacterial agent.

3. Use of the microbial composite agent according to claim 1 in promoting soybean growth.

4. The use of the microbial composite agent in promoting soybean growth according to claim 3, characterized in that: The promoting soybean growth is to promote the increase of fresh weight of soybean plants.

5. The use of the microbial composite agent in promoting soybean growth according to claim 3, characterized in that: The method of promoting soybean growth is to promote the increase of soybean plant height.

6. The use of the microbial composite agent in promoting soybean growth according to claim 3, characterized in that: The method of promoting soybean growth is to promote the increase of stem thickness of soybean plants.

7. The use of the microbial composite agent in promoting soybean growth according to claim 3, characterized in that: The method of promoting soybean growth is to increase the total root length, the number of root tips and the root volume of the soybean root system.

8. Use of the microbial compound agent according to claim 1 in promoting soybean root nodulation.

9. The use of the microbial composite agent according to claim 8 in promoting soybean root nodulation, characterized in that: The method of promoting soybean growth is to promote an increase in the number of soybean root nodules.

10. The use of the microbial composite agent according to claim 8 in promoting soybean root nodulation, characterized in that: The method of promoting soybean growth is to promote the increase of fresh weight of soybean root nodules.

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