Bradyrhizobium sp. and application thereof

Abstract

The application discloses a Bradyrhizobium sp. and application thereof, and belongs to the technical field of nitrogen-fixing symbiotic microorganisms. The Bradyrhizobium sp. is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 27064. The strain has good growth-promoting characteristics, and has strong nitrogen tolerance and broad spectrum. In the case of reducing the nitrogen fertilizer application amount, the strain can significantly increase the number of rhizobia, the number of lateral branches, the length of main stems and the number of fruit needles of peanuts in the flowering stage, and improve the yield of peanuts in the maturing stage, thereby providing a strain source for obtaining rhizobium agents suitable for peanut planting of various varieties, providing a technical basis for reducing nitrogen fertilizer application and saving planting cost, providing an efficient and stable growth-promoting microbial germplasm resource, expanding oil crop symbiotic germplasm resources in China, and providing a basis for research and development of efficient and stable microbial fertilizers and application technologies.

Description

Technical Field

[0001] This invention belongs to the field of nitrogen-fixing symbiotic microbial technology, specifically relating to a slow-growing rhizobium and its applications. Background Technology

[0002] Slow-growing rhizobia are a type of Gram-negative bacteria widely distributed in soil. Peanuts can form root nodules through symbiosis with slow-growing rhizobia, fixing atmospheric nitrogen into ammonia, which the plant can absorb and utilize. Statistics show that root nodules can provide peanuts with 60-65% of their nitrogen, partially replacing chemical nitrogen fertilizers to meet the nitrogen requirements for peanut growth, demonstrating significant nitrogen-saving potential while also reducing environmental pollution from chemical fertilizers. Based on a planting area of ​​4.9 million hectares, this could save 710,000 tons of urea, 1.42 billion yuan in expenses, and 1.065 million tons of coal per growing season. Therefore, the symbiosis between slow-growing rhizobia and peanuts has enormous potential in reducing nitrogen fertilizer application, protecting the environment, and saving energy.

[0003] Soil nitrogen content significantly affects nitrogen fixation efficiency in root nodules. Excessive nitrogen concentration significantly inhibits nitrogen fixation in root nodules, and the degree of inhibition is positively correlated with fertilizer application rate and timing. Excessive nitrogen in the soil reduces root nodule formation by inhibiting the attachment and infection of rhizobia to plant roots and by activating nitrogen feedback regulation, hormone feedback regulation, and the plant's automatic nodule-forming regulation system.

[0004] When symbiotic relationships are difficult to establish, plant growth and yield become more dependent on nitrogen fertilizer, leading to further environmental damage from nitrogen fertilizer and hindering further improvements in crop yield and quality. Nitrogen-tolerant rhizobia can break this cycle. These rhizobia can form nodules and form symbiotic relationships with leguminous crops in high-nitrogen soils, providing nitrogen sources for plants through nitrogen fixation, thereby reducing the crop's dependence on and demand for nitrogen fertilizer. This ensures high crop yields and improves crop quality without or with reduced nitrogen fertilizer application.

[0005] Although peanut rhizobium inoculants offer numerous advantages in agricultural production, their effectiveness is highly inconsistent due to the fact that each peanut rhizobium species has its own preferred peanut variety. Therefore, promoting and expanding the application of peanut rhizobium inoculants currently faces significant challenges. The key issue at present is how to screen for peanut rhizobium strains that are nitrogen-tolerant, have a wide adaptability, conserve nitrogen fertilizer, and promote high peanut yields, in order to further develop rhizobium inoculants suitable for peanut cultivation. Summary of the Invention

[0006] This invention provides a slow-growing rhizobium (Bradyrhizobium sp.), which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 27064.

[0007] The aforementioned slow-growing rhizobia exhibit good nitrogen tolerance, broad-spectrum symbiotic activity, growth-promoting function, and nitrogen-saving and high-yield-promoting capabilities. Based on this, the present invention provides the application of the aforementioned slow-growing rhizobia in promoting plant growth and / or saving nitrogen and promoting high yield.

[0008] This invention provides a rhizobium inoculant containing the aforementioned slow-growing rhizobium. Furthermore, the inoculant may also contain other conventional excipients.

[0009] The formulation of the above-mentioned rhizobium inoculant can be selected from liquid inoculant, powder or granule.

[0010] In one specific embodiment, the inoculant is a liquid inoculant, which is obtained by culturing the aforementioned slow-growing rhizobia using TY liquid culture medium; the concentration of slow-growing rhizobia in the liquid inoculant is 1×10⁻⁶. 10 ~ 1×10 11 cfu / mL.

[0011] This invention provides the application of the above-mentioned rhizobium inoculant in promoting plant growth and / or saving nitrogen and promoting high yield.

[0012] The aforementioned slow-growing rhizobia or rhizobium inoculants can replace part of the nitrogen fertilizer and, together with phosphate and potassium fertilizers, form microbial fertilizers. Based on this, the present invention provides the application of the aforementioned slow-growing rhizobia or rhizobium inoculants in the preparation of microbial fertilizers.

[0013] This invention provides a microbial fertilizer containing the aforementioned slow-growing rhizobium or rhizobium inoculum. The microbial fertilizer may also contain phosphate and potassium fertilizers.

[0014] This invention provides a rhizobium seed coating agent, which is composed of the above-mentioned slow-growing rhizobium inoculum, trace element mother liquor, and sodium carboxymethyl cellulose solution.

[0015] The above-mentioned slow-growing rhizobium culture solution was obtained by culturing the slow-growing rhizobium in TY liquid medium; the concentration of slow-growing rhizobium in the culture solution was 1×10⁻⁶. 10 ~ 1×10 11 cfu / mL.

[0016] The composition of the above-mentioned trace element stock solution is as follows: H3BO3 2.86g / L, MnSO4 1.81g / L, CuSO4·5H2O 0.80g / L, ZnSO4 0.22g / L, H2MoO4 0.02g / L, with the balance being water; wherein, the function of the trace elements is to provide nutrients for plants.

[0017] The above-mentioned sodium carboxymethyl cellulose solution was selected from an aqueous solution containing sodium carboxymethyl cellulose at a final concentration of 10 g / L, i.e., a solution concentration of 1%.

[0018] This invention provides a seed coating method, the steps of which are as follows:

[0019] Add trace element stock solution to the above slow-growing rhizobium inoculum solution, mix well to form a mixed inoculum solution; then spray the mixed inoculum solution evenly on the seed surface and air dry; use sodium carboxymethyl cellulose solution to coat the air-dried seeds again, and air dry.

[0020] The volume ratio of the trace element stock solution to the slow-growing rhizobium culture solution is selected as 1:1000. That is, the amount of trace element stock solution added to the culture solution can be selected as: 1 mL of the trace element stock solution per 1000 mL of the culture solution.

[0021] The dosage of the above mixed bacterial solution is 150 mL / 30 kg of plant seeds.

[0022] The above-mentioned sodium carboxymethyl cellulose solution is used at a rate of 150 mL / 30 kg of plant seeds to maintain the adhesion and humidity of rhizobia.

[0023] In this invention, the plant is selected from legumes; preferably peanuts.

[0024] The beneficial effects of this invention are as follows:

[0025] The slow-growing rhizobium provided by this invention possesses good growth-promoting properties, strong nitrogen tolerance, and broad-spectrum activity, making it a biosafe strain. Under conditions of reduced nitrogen fertilizer application, this strain can significantly increase the number of root nodules, lateral branches, main stem length, and pegs in peanuts during the full-blown stage, thereby increasing peanut yield at maturity. This provides a source of strains for obtaining rhizobium agents suitable for various peanut varieties, offers a technical basis for reducing nitrogen fertilizer application and saving planting costs, provides a highly efficient and stable growth-promoting microbial germplasm resource, expands the symbiotic microbial germplasm resources of oil crops in my country, and lays the foundation for the development of highly efficient and stable microbial fertilizers and their application technologies. Attached Figure Description

[0026] Figure 1 The colony morphology of SBJ24 on TY medium.

[0027] Figure 2Gram staining image of SBJ24 cells.

[0028] Figure 3 The nodulation of peanuts inoculated with SBJ24 under different concentrations of KNO3 treatment. Detailed Implementation

[0029] 1. Test materials

[0030] The culture media, reagents, and other materials used in this invention are as follows:

[0031] TY medium: Dissolve 5g tryptone, 3g yeast extract and 0.6g CaCl2 in an appropriate amount of deionized water, then bring the volume to 1000mL with deionized water, adjust the pH to 6.8~7.2, add 15~20g agar (solid medium), and sterilize at 121℃ for 30min.

[0032] YMA medium: mannitol 10.0g, K2HPO4 0.25g, KH2PO4 0.25g, MgSO4 0.1g, NaCl 0.1g, yeast extract 3.0g, deionized water 1L, pH 6.8~7.0, agar 15~20g (solid medium), sterilized at 121℃ for 20min.

[0033] Blood agar medium: 18g peptone, 1g yeast extract, 5g NaCl, 15-20g agar, 1000mL deionized water, pH 6.8-7.2. After sterilizing at 121℃ for 20min, add 5% (5mL / 100mL) defibrinated sheep blood to the medium after it cools to 50℃, mix well and pour into plates.

[0034] 20× Low-nitrogen nutrient solution for plants: Dissolve 0.075g of ferric citrate, 0.03g of Ca(NO3)2, 0.136g of K2HPO4, 0.46g of CaSO4, 0.075g of KCl, 0.06g of MgSO4·7H2O, and 20mL of trace element stock solution in an appropriate amount of deionized water, and then make up to 1000mL with deionized water.

[0035] Trace element stock solution: Dissolve 2.86g of H3BO3, 0.02g of H2MoO4, 0.22g of ZnSO4, 0.8g of CuSO4·5H2O, and 1.81g of MnSO4 in an appropriate amount of deionized water, and then make up to 1000mL with deionized water.

[0036] Sterile saline: Dissolve 0.8g NaCl in 100mL deionized water and sterilize at 121℃ for 30min.

[0037] Water agar culture: Add 0.6g of agar powder to 100mL of deionized water and sterilize at 121℃ for 30min.

[0038] 5mM KNO3 solution: Dissolve 50.5g KNO3 in 100mL of deionized water and sterilize at 121℃ for 30min.

[0039] Sodium carboxymethyl cellulose solution (seed surface protectant): Dissolve 1g of sodium carboxymethyl cellulose (800~1200mPa·s) in 100mL of deionized water at 60℃, and stir until a transparent paste-like solution is obtained.

[0040] The nitrogen, phosphorus, and potassium fertilizers applied in the field are conventional urea, calcium magnesium phosphate, and potassium sulfate.

[0041] Primer information is shown in Table 1:

[0042] Table 1

[0043]

[0044] The genome extraction kit, Taq Mix for PCR amplification, and ddH2O were all from Kangrun Biotechnology Co., Ltd.

[0045] 2. Isolation and Identification of Microbial Strains

[0046] On June 7, 2021, peanut root samples were collected at the Shandong Peanut Research Institute base in Laixi City, Shandong Province. The samples were placed in resealable bags and stored at 4°C. Root nodules were removed from the peanut roots and placed in 10mL sterile centrifuge tubes. 5mL of 75% ethanol solution was added, and the tubes were shaken for 1 minute for sterilization. The ethanol solution was then discarded. 5mL of sodium hypochlorite solution (sodium hypochlorite:water = 1:5) was added to the centrifuge tubes, and the tubes were shaken for 7 minutes for sterilization. The sodium hypochlorite solution was then discarded. Sterile water was added to the centrifuge tubes, and the root nodules were washed 8 times. The residual liquid was discarded to obtain sterile root nodules. The sterile root nodules were placed in another sterile PCR tube, and the nodules were crushed using a sterile pipette tip. The obtained root nodule juice was streaked onto YMA solid medium. The culture was incubated in the dark at 28°C for 14 days. Single colonies were picked, streaked three times for purification, and then stored. This bacterial strain was designated SBJ24.

[0047] Following the methods described in Bergey's Manual of Bacterial Identification (8th Edition), strain SBJ24 was identified by morphological and physiological-biochemical characteristics. The specific results are as follows:

[0048] (1) Morphological characteristics

[0049] Strain strain SBJ24 was inoculated onto TY solid medium and incubated at 28°C. Its colony morphology included: small, white, round colonies with a smooth, opaque surface. Figure 1 As shown.

[0050] (2) Biological characteristics

[0051] like Figure 2 As shown, the strain is a red bacillus after Gram staining, indicating that the strain is a Gram-negative bacterium.

[0052] (3) Genetic characteristics

[0053] 16S rDNA sequence sequencing:

[0054] Genomic DNA was extracted from strain SBJ24 and amplified by PCR using universal primers for the 16S rDNA gene.

[0055] The amplification system was (50 μL):

[0056] 25 μL Taq Mix, 22 μL ddH2O, 1 μL forward primer 16S rDNA P1 (concentration 10 μmol / L), 1 μL reverse primer 16S rDNA P6 (concentration 10 μmol / L), and 1 μL DNA template.

[0057] The amplification reaction conditions are:

[0058] The PCR was performed in 30 cycles: 95℃ for 5 min; 94℃ for 1 min; 60℃ for 30 s; 72℃ for 90 s; and a final extension at 72℃ for 10 min. PCR primer synthesis and sequencing were performed by Qingke Sequencing Company.

[0059] Sequencing results:

[0060] The paired-end sequencing sequences were assembled using DNAMAN software, yielding a 1357 bp 16S rDNA gene sequence, the nucleotide sequence of which is shown in SEQ ID No:1.

[0061] 16S rDNA gene (SEQ ID No:1):

[0062]

[0063] BLAST comparison on the NCBI website showed that the 16S rDNA gene of strain SBJ24 had the highest similarity to the 16S rDNA gene of strain Bradyrhizobium guangzhouense CCBAU 51670, with 100% homology. Based on its morphological and biological characteristics, this strain was identified as Bradyrhizobium sp. This bacterium was deposited on April 12, 2023, at the 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, 100101, China), with accession number CGMCC No. 27064.

[0064] 3. Safety testing

[0065] Strains of strain SBJ24 were inoculated into blood agar medium and incubated at 28°C for 14 days. The presence or absence of hemolysis zones was observed. The appearance of hemolysis zones indicates that the strain has hemolytic activity and poses a potential threat to humans and animals; therefore, the strain should not be used in microbial fertilizers. The absence of hemolysis zones indicates that the strain has no hemolytic activity and is a safe strain that can be used in microbial fertilizers. The results showed that after 14 days of incubation on blood agar plates, no hemolysis zones appeared, indicating negative hemolytic activity. This demonstrates that the strain is a safe strain and can be safely used in microbial fertilizers.

[0066] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0067] Example 1

[0068] Nitrogen resistance test:

[0069] Prepare a low-nitrogen nutrient solution for plants by mixing 100 mL of the solution with 1 kg of vermiculite, adding an appropriate amount of deionized water to keep the vermiculite moist, and sterilizing at 121℃ for 90 min. Select seeds of the uniform variety *Huayu 917* for disinfection using the following procedure: first, disinfect with 75% alcohol solution for 1 min; second, disinfect with sodium hypochlorite solution (sodium hypochlorite to water ratio 1:4) for 10 min; third, rinse 8 times with sterile water; finally, place the seeds on water agar medium and germinate in the dark for 4 days. Compare the results of strain SBJ24 and the control strain *Bradyrhizobium zhanjiangense* CCBAU 51778.T (Li, YH, et al. *Bradyrhizobium nanningense* sp. nov., *Bradyrhizobium guangzhouense* sp. nov. and *Bradyrhizobium zhanjiangense* sp. nov., isolated from effective nodules of peanut in Southeast China. *Systematic and Applied Microbiology*, 2019, 42: 126002.) The cultures were inoculated into TY liquid medium and cultured at 28°C with shaking at 180 rpm for 5 days. The OD of the bacterial culture was measured using sterile physiological saline. 600nm The concentration was adjusted to 0.2. Peanuts were grown using a double-layer pot method (Li, YH, et al. Bradyrhizobium guangdongense sp. nov. and Bradyrhizobium guangxiense sp. nov., isolated from effective nodules of peanut. International Journal of Systematic and Evolutionary Microbiology, 2015, 65: 4655-4661.). The upper layer of the double-layer pot contained sterile vermiculite from the above steps, and the lower layer contained sterile water. The upper layer of vermiculite absorbed the sterile water from the lower layer through a sterile gauze. Germinated peanut seeds were planted in vermiculite and inoculated with 1 mL of SBJ24 bacterial suspension or control rhizobium suspension, serving as the SBJ24 treatment group and the control rhizobium treatment group, respectively. Each treatment group included three treatment methods: adding 1 mL of sterile physiological saline, 1 mL of 5 mM KNO3 solution, and 2 mL of 5 mM KNO3 solution, respectively, to form the 0 mM KNO3 treatment, 5 mM KNO3 treatment, and 10 mM KNO3 treatment. The peanuts were cultured in a greenhouse at 28℃ with 12 hours of artificial light followed by 12 hours of darkness. The number of root nodules was measured on day 40 post-inoculation.

[0070] The experimental results are shown below ( Figure 3 ):

[0071] In the SBJ24 treatment group, the number of peanut root nodules in the 0 mM KNO3 treatment, 5 mM KNO3 treatment (1 mL / bottle), and 10 mM KNO3 treatment (2 mL / bottle) were 122, 25, and 8, respectively. In the control rhizobium treatment group, the number of peanut root nodules in the 0 mM KNO3 treatment, 5 mM KNO3 treatment, and 10 mM KNO3 treatment were 67, 6, and 5, respectively.

[0072] The results show that, compared with the control rhizobium, SBJ24 was still able to form more root nodules under 5 mM KNO3 treatment. This indicates that SBJ24 can form root nodules even when nitrogen inhibits peanut nodulation and nitrogen fixation, meaning that the SBJ24 strain has good nitrogen tolerance.

[0073] Example 2

[0074] Peanut symbiosis spectrum detection:

[0075] The field soil was divided into 5L flowerpots, each with the same soil weight, and watered thoroughly. Strains SBJ24 were inoculated into TY liquid medium and cultured at 28°C with shaking at 180 rpm for 5 days. The OD of the bacterial culture was measured using sterile physiological saline. 600nm Adjust to 0.2. Select peanut seeds of consistent quality, including varieties such as Luhua 11, Yuhua 9326, Rihua 1, Huayu 36, Huayu 22, Zhonghua 24, Huayu 917, and Baisha 1016. Spray the seed surface evenly with rhizobium inoculum solution and air-dry in the dark. Coat the air-dried seeds evenly with sodium carboxymethyl cellulose solution and air-dry in the dark. Plant the seeds in pots, with each peanut variety including 3 pots treated with SBJ24 and 3 pots inoculated with Bradyrhizobium zhanjiangense CCBAU 51778. T Three pots were used for a positive treatment and three pots were used for a negative treatment without root nodule inoculation, with three seeds planted in each pot. The peanuts were placed in a greenhouse, watered every two days, and cultivated for 40 days. The above-ground dry weight and root nodule number of the peanut plants were measured.

[0076] The experimental results are shown below:

[0077] Compared with the negative control without rhizobium inoculation, SBJ24 increased the aboveground dry weight of Yuhua 9326, Rihua 1, Huayu 36, Huayu 22, Zhonghua 24, Huayu 917, and Baisha 1016 by 30.61%, 28.18%, 10.49%, 6.43%, 14.11%, 16.36%, and 46.13%, respectively; and increased the number of root nodules of Luhua 11, Yuhua 9326, Rihua 1, Huayu 22, Zhonghua 24, Huayu 917, and Baisha 1016 by 168.78%, 1660.00%, 874.19%, 307.50%, 17.35%, 230.61%, and 237.50%, respectively.

[0078] Compared with the negative control without rhizobium inoculation, Bradyrhizobium zhanjiangense CCBAU51778 T The aboveground dry weight of Yuhua 9326, Rihua 1, Huayu 36, Huayu 22, Zhonghua 24, Huayu 917, and Baisha 1016 was increased by 68.18%, 41.74%, -17.74%, 1.55%, 2.30%, -9.92%, and 39.98%, respectively; the number of root nodules of Luhua 11, Yuhua 9326, Rihua 1, Huayu 22, Zhonghua 24, Huayu 917, and Baisha 1016 was increased by 584.12%, 3822.22%, 1148.39%, -28.57%, -55.39%, -17.35%, and 329.17%, respectively.

[0079] In summary, SBJ24 increased the aboveground dry weight and root nodule number of seven peanut varieties, compared to the control rhizobium Bradyrhizobium zhanjiangense CCBAU 51778. T It was able to increase the aboveground dry weight of 5 peanut varieties and the number of root nodules in 4 peanut varieties. This result indicates that SBJ24 has a broader spectrum of growth-promoting and nodulation-promoting effects than the control strain.

[0080] I. Field Application

[0081] Before applying it in the field, the following preparations should be made:

[0082] Strain activation and scale-up culture:

[0083] The SBJ24 strain, stored at -80℃, was activated on YMA solid medium using a three-zone streak method and incubated at 28℃ for 14 days. Single colonies were then inoculated into TY liquid medium for expansion and cultured at 28℃ and 180 rpm on a shaker until the bacterial concentration reached 1×10⁻⁶. 10~11 CFU / mL. Before use, add trace element stock solution (1 mL / 1000 mL) to the bacterial agent and mix well.

[0084] Peanut seed coating:

[0085] Spray the mixed inoculant evenly onto the seed surface until it is moist, then place it in a cool, shady place to dry. After drying, treat the seeds with a 1% sodium carboxymethyl cellulose solution (150 mL / 30 kg of seeds) to maintain the inoculant's adhesion and moisture, then continue drying in the shade.

[0086] Watering before sowing:

[0087] Sowing can be done manually or by machine. Water the soil 3 days before sowing to ensure the necessary moisture, which is conducive to the survival of the strain and the germination of the seeds.

[0088] Peanut field cultivation, as shown below:

[0089] The experiment was conducted at the Shandong Peanut Research Institute base in Laixi City, Shandong Province. The peanut variety used was the locally common high-oleic acid variety (Huayu 917). The sowing date was April 30, 2022. Since the conventional pure nitrogen application rate is 120 kg / ha, the pure nitrogen application rate was 60 kg / ha when nitrogen was reduced by 50% in the experimental field. Based on this, the urea application rate was deduced to be 135 kg / ha when nitrogen was reduced by 50%. The conventional application rates of phosphate fertilizer (calcium magnesium phosphate) and potassium fertilizer (potassium sulfate) were 750 kg / ha and 240 kg / ha, respectively. The conventional fertilization levels are found in the literature (Wang Chunxiao et al. Effects of different nitrogen fertilizer application rates and organic fertilizer application on peanut senescence and nodulation. Peanut Journal, 2023, 52(2): 1-7.). All fertilizers were mixed and applied to the soil as base fertilizer in one application.

[0090] The land was prepared and ridged using conventional methods, with ridges 0.8m wide and 0.4m apart. One acre was divided into 6 equal plots. Two treatments were included: a control without inoculation and a treatment inoculated with a biological agent. Each treatment had 3 plots, and all plots were randomly distributed. The biological agent used was *Bacillus stolonifera* SBJ24.

[0091] Peanuts were planted in two rows per ridge, with two seeds per hole and a hole spacing of 0.02m. Subsequent peanut cultivation followed standard procedures. Peanut yield was measured in each plot on September 27, 2022, specifically by measuring a 2m section of peanut plants at their uniform growth (measuring the dry weight of the shelled peanuts). The yield per hectare was calculated using the following formula:

[0092] Peanut yield per mu = (Dry weight of peanuts / (0.85 × 2) × 666.7 × 15)

[0093] The experimental results are shown in Table 2:

[0094] Table 2

[0095]

[0096] Table 2 shows that, under the premise of reducing nitrogen fertilizer application by 50%, the slow-growing rhizobium SBJ24 can promote peanut growth during the flowering period and peanut yield during the harvest period. This indicates that this strain has significant potential for field application in reducing nitrogen and promoting high yield, and can be used as a resource for developing stable and efficient microbial fertilizer strains.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A slow-growing rhizobium ( Bradyrhizobium sp. ), characterized in that, The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 27064.

2. The application of the slow-growing rhizobium as described in claim 1 in promoting peanut growth and / or saving nitrogen in peanuts and / or increasing peanut yield.

3. A rhizobium inoculant, characterized in that, Contains the slow-growing rhizobium as described in claim 1.

4. The rhizobium inoculant according to claim 3, characterized in that, The formulation of the microbial agent is selected from liquid, powder, or granules.

5. The rhizobium inoculant according to claim 4, characterized in that, The concentration of slow-growing rhizobia in the liquid bacterial agent is 1×10⁻⁶. 10 ~ 1×10 11 cfu / mL.

6. The use of the slow-growing rhizobium of claim 1 or the rhizobium inoculant of claim 3 in the preparation of microbial fertilizers.

7. A microbial fertilizer, characterized in that, Contains the slow-growing rhizobium of claim 1 or the rhizobium agent of claim 3.

8. A rhizobium seed coating agent, characterized in that, It consists of slow-growing rhizobium inoculum, trace element mother liquor, and sodium carboxymethyl cellulose solution; The slow-growing rhizobium culture solution was obtained by culturing the slow-growing rhizobium of claim 1 using TY liquid medium; the concentration of slow-growing rhizobium in the culture solution was 1×10⁻⁶. 10 ~ 1×10 11 cfu / mL; The composition of the trace element mother liquor is as follows: H3BO3 2.86 g / L, MnSO4 1.81 g / L, CuSO4·5H2O 0.80 g / L, ZnSO4 0.22 g / L, H2MoO4 0.02 g / L, with the balance being water; The sodium carboxymethyl cellulose solution is selected from a 1% sodium carboxymethyl cellulose aqueous solution, and its preparation method is as follows: dissolve 10g of sodium carboxymethyl cellulose in 1L of deionized water at 60℃ and stir until a transparent paste-like solution is obtained.

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