Sinorhizobium sp. BJ, application and preparation method of bacterial agent of Sinorhizobium sp. BJ

Abstract

The invention relates to Sinorhizobium sp. BJ, application and a preparation method of a bacterial agent of Sinorhizobium sp. BJ, the strain is preserved in China Center for Type Culture Collection, the preservation time is November 14, 2025, and the preservation number is CCTCC M 20252477; the sinorhizobium sp. BJ strain has the characteristics of high growth speed, salt and alkali resistance and the like, has growth promoting functions of producing indoleacetic acid, producing siderophores, dissolving phosphorus, producing ammonia, promoting nodulation and the like, and can be used for efficiently synthesizing BJ exopolysaccharides in a simple culture medium to form a viscous liquid microbial inoculum which can be stably preserved. The Sinorhizobium sp. Inoculant disclosed by the invention has the characteristic of stable activity, the BJ exopolysaccharide in the inoculant has the functions of promoting germination, growth and root nodulation of soybeans, and the Sinorhizobium sp. Inoculant can effectively relieve abiotic stress suffered by plants, such as salt and alkali, drought and the like. According to the technical achievement, a novel efficient and economical microbial resource solution is provided for improving the quality and efficiency of leguminous plants in saline-alkali soil and arid regions, and the application prospect is wide.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a species of Rhizobium sinense BJ (… Sinorhizobium sp. BJ), its application and preparation method of its inoculant. Background Technology

[0002] Leguminous plants (such as soybeans, peanuts, peas, and alfalfa) are important global food, oilseed, feed, and cash crops, also possessing nitrogen-fixing and fertilization functions. Rhizobium inoculants are among the earliest developed bio-fertilizer products and are recognized worldwide as the most stable microbial fertilizers, widely used globally. Especially in newly planted areas and newly improved soils, the number of native rhizobia is extremely low, often failing to induce effective nodulation in leguminous plants; therefore, artificial inoculation with root nodules is essential.

[0003] Due to the highly specific symbiotic relationship between rhizobia and legumes, related research has attracted much attention. Although rhizobia are widely distributed in soil, most of them are ineffective or low-efficiency strains, with only a small portion capable of forming nodules with soybeans, and these are mostly concentrated in the genera *Rhizobium* and *Sinopterygium* [Acta Microbiologica Sinica, 2025, 65(4): 1667-1683, Precise Evaluation of Soybean Rhizobia by China Agricultural Microbial Culture Collection Center]. Sinorhizobium ) belongs to fast-growing rhizobia, compared to medium- and slow-growing rhizobia ( Mesorhizobium ) and slow-growing rhizobia ( Bradyrhizobium It has a shorter cell generation time and a faster growth rate. Related strains, for example... Sinorhizobium meliloti 1021 (ATCC51244) Sinorhizobium meliloti SU47 (USDA 1002) Sinorhizobium fredii Most strains, such as USDA 205 (ATCC 35423), require 3–5 days of cultivation on YMA plates to form 2–4 mm colonies, which is a long process.

[0004] Extracellular polysaccharides produced by soybean rhizobia play an important role in symbiotic nitrogen fixation. These extracellular polysaccharides are signaling molecules during the interaction between rhizobia and the host plant. With the assistance of nodulation factors, they enter root hairs, induce cell membrane growth to form infection lines, and then differentiate into root nodules within the plant cortex.

[0005] The commercial production of rhizobium inoculants typically depends on the following factors: the yield-increasing effect of the strain, production cost, the number of viable bacteria after preservation, the formulation, and the application method. Currently, commonly used rhizobium inoculants both domestically and internationally fall into five categories: agar, solid, granules, liquid, and freeze-dried. Among these, solid rhizobium inoculants are the main commercial inoculants, using peat moss, vermiculite, and perlite as carriers. They are widely used due to their advantages such as ease of use, long shelf life, simple production process, and low cost. However, once the functional strains of the core components in these products are inactivated, their application effect will significantly decrease. Summary of the Invention

[0006] To address the aforementioned deficiencies and shortcomings in the prior art, this invention provides a *Rhizobium sinense* BJ (… Sinorhizobium sp. BJ), its application and preparation method of its inoculant.

[0007] To solve the above technical problems: The first objective of this invention is to provide a *Rhizobium sinense* BJ (… Sinorhizobium sp. The *Rhizobium sinense* BJ, deposited at the China Center for Type Culture Collection on November 14, 2025, with accession number CCTCCM 20252477.

[0008] Furthermore, the extracellular polysaccharide produced by the fermentation of Rhizobium sinense BJ consists of basic monosaccharides, neutral monosaccharides, and acidic monosaccharides, accounting for more than 95% of the total mass, with a glucose to galactose molar ratio of 9-10:1.

[0009] The basic monosaccharide is glucosamine; the neutral monosaccharide is glucose, galactose, mannose, fucose, rhamnose, ribose, xylose, and arabinose; and the acidic monosaccharide is galacturonic acid, glucuronic acid, guluronic acid, and mannuronic acid. The molar percentages of the acidic monosaccharides are: guluronic acid (0.12%), mannuronic acid (0.04%), glucuronic acid (0.70%), and galacturonic acid (1.15%), meaning the total molar percentage of acidic monosaccharides is approximately 2%.

[0010] The second objective of this invention is to provide a method for preparing *Rhizobium sinense* inoculant, comprising the following steps: adding 50 mL of seed culture medium to a 250 mL Erlenmeyer flask, adding 100 μL of *Rhizobium sinense* BJ inoculum, and culturing the Erlenmeyer flask at 30°C and 200 rpm on a shaker for 24 h to obtain a primary seed culture; inoculating the primary seed culture at a 6% (v / v) inoculation rate into a 1000 mL Erlenmeyer flask containing 300 mL of fermentation culture medium, and culturing it at 30°C and 200 rpm on a shaker for 12 h to obtain a secondary seed culture; inoculating the secondary seed culture at a 10% (v / v) inoculation rate into a 5 L stirred fermenter containing 3 L of fermentation culture medium; fermenting and culturing at 30°C, pH 7.5-8.0, aeration rate of 0.5-1.0 vvm, and stirring speed of 200-1100 rpm for 64 h to obtain *Rhizobium sinense* BJ inoculant; its viable count is 2.78 × 10⁻⁶. 9 CFU / mL; The viscous fermentation broth formed by the high polysaccharide production of strain BJ can be directly used as a liquid inoculant, with a stability period of up to 180 days, meeting the requirements of the national standard GB 20287-2006 for agricultural microbial inoculants.

[0011] The third objective of this invention is to provide the above-mentioned Rhizobium sinense BJ ( Sinorhizobium Application of *Sp. BJ* or its extracellular polysaccharides, or bacterial agents prepared by the above methods, in improving the growth of leguminous plants and strengthening root nodulation.

[0012] The fourth objective of this invention is to provide the above-mentioned Rhizobium sinense BJ ( Sinorhizobium Application of *Sp. BJ* or its extracellular polysaccharides, or bacterial agents prepared by the above methods, in improving drought and salt stress in leguminous plants.

[0013] The fifth objective of this invention is to provide the above-mentioned Rhizobium sinense BJ ( Sinorhizobium Application of *Sp. BJ* or its extracellular polysaccharides, or microbial agents prepared by the above methods, in increasing the yield of leguminous plants.

[0014] The beneficial technical effects achieved by this invention are as follows: (1) Taking advantage of the ability of Rhizobium sinense strain BJ to efficiently synthesize extracellular polysaccharides in conventional fermentation medium, it was developed into a viscous liquid bacterial agent. It has the advantages of low cost, simple operation, short production cycle and no need to add other stabilizers or synergists, showing the potential for large-scale production. (2) In the liquid bacterial agent, the viable count of strain BJ can reach 2.78 × 10⁻⁶. 9 The cell count was CFU / mL, the BJ polysaccharide content reached 20 g / L, the viable cell count was stable, and after 180 days of storage at room temperature, the viable cell count still reached 6.15 × 10⁻⁶. 8 CFU / mL, meeting the requirements of the national standard GB 20287-2006 for agricultural microbial agents; (3) BJ polysaccharide itself has biological effects such as promoting nodulation, growth, and stress resistance. Liquid bacterial agents rich in BJ polysaccharide can significantly enhance the germination of soybean seeds and the ability of soybean seedlings to resist salt and drought stress. Attached Figure Description

[0015] Figure 1 The present invention is a strain of Rhizobium sinense. Sinorhizobium sp. BJ colony morphology; Figure 2 This invention relates to a strain constructed based on the 16S rDNA gene sequence. Sinorhizobium sp. BJ phylogenetic tree; Figure 3 This invention provides a comprehensive identification of the functional characteristics of Rhizobium chinense BJ in the embodiments of the present invention. Figure 4 The fermentation curve of Rhizobium chinense BJ in a 5L stirred reactor is shown in this embodiment of the invention. Figure 5 The liquid inoculum form of Rhizobium tumefaciens BJ in this embodiment of the invention; Figure 6 This is a high-performance liquid chromatogram of the BJ standard monosaccharide component of Rhizobium chinense in an embodiment of the present invention; Figure 7 This is a high-performance liquid chromatogram of extracellular polysaccharide produced by Rhizobium chinense BJ in an embodiment of the present invention; Figure 8 This is a gel permeation chromatogram of extracellular polysaccharides produced by Rhizobium chinense BJ in an embodiment of the present invention; Figure 9 Images showing the germination status of soybean seeds under salt and drought stress according to embodiments of the present invention; Figure 10 This is a picture showing the growth status of soybean seedlings under non-stress conditions on day 42 in an embodiment of the present invention. Figure 11 Images showing the growth status of soybean seedlings under salt stress on day 42 in this embodiment of the invention. Figure 12 This is a picture showing the growth status of soybean seedlings under drought stress on day 30 in an embodiment of the present invention.

[0016] The preservation information of the rhizobium in this application is as follows: Rhizobium sinense: Classified and named Sinorhizobium sp. BJ, deposited at the China Center for Type Culture Collection (Wuhan University) on November 14, 2025, accession number CCTCC M 20252477; Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0019] Example 1: Isolation and Identification of Rhizobium sinense BJ

[0020] 1. Isolation and extraction of *Rhizobium sinense* BJ: Soil from a vegetable garden in the suburbs of Nanjing was collected. Plant debris was removed, and approximately 1 g of soil was weighed and added to a 250 mL Erlenmeyer flask containing 100 mL of sterile water and glass beads. The flask was shaken at 100 rpm for 60 min to allow the soil particles to fully swell and release the microorganisms. The flask was then allowed to stand for 60 min to allow solids such as sand to settle completely. The supernatant was then serially diluted with sterile water to a concentration of 10. -6 100 μL of the diluted solution was transferred to YMA plates and evenly spread using a triangular spreader. Ten plates were then incubated at 30°C for 24-72 hours. Strains with rapid growth, large colonies, and smooth, moist surfaces were selected. By day 2, colonies had grown to 2-4 mm and exhibited a milky-white, smooth, and viscous surface. These were selected as the target strain and named strain BJ. Under the same culture conditions, fast-growing rhizobia... Sinorhizobium meliloti It takes 3 days for 1021 (ATCC 51244) to grow to 2-4mm. Sinorhizobium meliloti It takes 4 days for SU47 (USDA 1002) to grow to 2-4mm. Sinorhizobium fredii USDA 205 (ATCC 35423) takes 3 days to grow to 2-4 mm. Therefore, strain BJ has a more significant growth rate advantage compared to other fast-growing rhizobia, and has greater potential in the development and production of commercial inoculants. YMA medium formulation: mannitol 10 g / L, yeast extract 1 g / L, CaCO3 1 g / L, NaCl 0.1 g / L, K2HPO4 2 g / L, MgSO4 0.2 g / L, agar powder 15 g / L, pH 7.0.

[0021] 2. Identification of the extracellular polysaccharide production ability of strain BJ: BJ was transferred to a sugar-containing medium, the components of which included 20 g / L glucose, 3 g / L peptone, 2 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar. After incubation at 30℃ for 72 h, the colony morphology was as follows... Figure 1 As shown: the colonies are milky white, smooth, raised, and moist with mucus, indicating that strain BJ has the ability to produce extracellular polysaccharides.

[0022] 3. Identification of BJ strain: 3.1 Extraction of total DNA: Single colonies from step 1 were picked and transferred to LB medium, incubated at 30°C and 200 rpm for 24 h on a shaker, and then rediluted to a concentration of 10⁻⁶. -8 Spread the bacterial culture onto YMA plates, and select fast-growing, large-scale single colonies for incubation on LB agar. Repeat this process three times. Take an appropriate amount of bacterial culture and extract total DNA according to the instructions of the genomic DNA extraction kit (Tiangen Biotech Co., Ltd., DP-302).

[0023] 3.2 PCR amplification of bacterial 16S rDNA: The amplification products were detected by 1% agarose gel electrophoresis using universal bacterial primers 27F and 1492R, and sent to a sequencing company (General Biotechnology Co., Ltd.) for sequencing identification. The gene sequence is shown in SEQ ID NO.1:

[0024] The PCR amplification system includes: 20 μL ddH2O, 25 μL high-fidelity DNA polymerase and mixed substrate, 2 μL each of primers, and 1 μL template; Reaction conditions: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 30 s / 1 kb, 30 cycles followed by 72℃ extension for 5 min.

[0025] The sequencing results were compared using NCBI BLAST analysis software. The results showed that the strain sequence obtained in step 1 was consistent with that of *Rhizobium sinense* (Sinobacterium genus). Sinorhizobium The 16S rDNA gene sequence of the strain showed over 95% homology. Therefore, the strain obtained in this application is *Rhizobium sinense*. Sinorhizobium sp., named Sinorhizobium sp. BJ, its phylogenetic tree is as follows Figure 2 As shown.

[0026] Example 2: Beneficial Functions of Rhizobium sinense BJ Strain on Plants

[0027] In the soil microecological environment, plant growth-promoting probiotics (PGPB) enhance crop growth and development and strengthen their resistance to abiotic stresses through diverse mechanisms of action. These mechanisms are mainly manifested in PGPB directly or indirectly synthesizing various plant hormones, accelerating the mineralization and decomposition of organic matter, and activating the plant's antioxidant system by promoting the absorption and utilization of mineral nutrients such as iron and phosphorus. Through these pathways, PGPB not only significantly promotes plant growth but also effectively enhances the plant's resistance to stress environments. The plant growth-promoting characteristics of the strains were evaluated by measuring their ability to secrete indoleacetic acid (IAA), solubilize phosphorus, produce ammonia, and produce siderophores.

[0028] Quantitative analysis method for indoleacetic acid (IAA): *Rhizobium sinense* BJ seed culture was transferred at a 2% inoculum to LB medium containing 100 mg / L L-tryptophan and cultured at 30℃ and 200 rpm for 5 days. Each day, 1 mL of fermentation broth was sampled, centrifuged at 10000×g for 10 min, and 0.1 mL of the supernatant was mixed with 0.4 mL of colorimetric reagent (5 mL 35% HClO4, 0.1 mL 0.5 M FeCl3) and incubated at room temperature in the dark for 45 min. A standard curve was prepared using IAA standards, and the IAA content in the supernatant of the culture medium was determined.

[0029] Siderophore production capacity determination: The siderophore production capacity of the strain was qualitatively determined using CAS medium. The strain was spotted onto CAS medium and incubated at 30℃ for 5-7 days. The presence of an orange-yellow halo around the colony was observed. The presence of a halo indicates that the strain can synthesize a certain amount of siderophores and secrete them onto the medium, chelating iron ions in the CAS medium and altering the pH of the medium. Therefore, the color of the CAS medium changes from blue to orange-yellow.

[0030] Phosphorus solubility test: A single colony of *Rhizobium sinense* strain BJ was placed in liquid LB medium and shaken for 48 h. 10 μL of the bacterial suspension was then sputtered onto inorganic phosphorus selection medium (sorbitol 10.0 g, (NH4)2SO4 5 g, NaCl 0.3 g, MgSO4 0.3 g, MnSO4 0.3 g, K2SO4 0.3 g, FeSO4 0.03 g, Ca3(PO4)2 5.0 g, agar powder 15.0 g, yeast extract 0.5 g, and tap water to a final volume of 1 L). The medium was incubated at 30℃, and the presence or absence of a clear zone was observed. *Rhizobium sinense* strain BJ was then inoculated into an inorganic phosphorus liquid medium and cultured in shake flasks. The available phosphorus content in the supernatant was then determined. Weigh 0.4390 g of pure potassium dihydrogen phosphate dried at 105℃ and dissolve it in pure water; add 5 mL of concentrated sulfuric acid and bring the volume to 1000 mL to obtain a 100 μg / mL phosphorus standard stock solution; dilute 10 times before use to obtain a 10 μg / mL phosphorus standard working solution. Take six 50 mL volumetric flasks and add 0, 0.5, 1.0, 2.0, 3.0, and 4.0 mL of phosphorus standard working solution, respectively; add distilled water to each flask to 20 mL, add 5 mL of molybdenum antimony colorimetric reagent, shake well, and develop the color at room temperature for 30 min; use a spectrophotometer at a wavelength of 700 nm, with a blank solution (0 mL standard solution) as a reference, to measure the absorbance; plot a standard curve with phosphorus content (μg) on ​​the x-axis and absorbance on the y-axis to obtain a linear regression equation. Take 5 mL of fermentation broth, centrifuge at 8000 r / min for 10 min, take the supernatant and dilute with pure water to a phosphorus concentration in the range of 0.1-1.0 μg / mL, take 5 mL of the diluted sample supernatant into a 50 mL volumetric flask, add colorimetric reagent under the same conditions as the standard curve, make up to volume, develop color, and measure absorbance; calculate the phosphorus content in the sample solution by substituting the absorbance into the regression equation.

[0031] Salt tolerance test: 50 μL of *Rhizobium sinense* BJ seed culture was inoculated into 5 mL of LB medium. NaCl concentrations of 10, 20, 40, 60, 80, and 120 g / L were prepared for each group, with three replicates. The cultures were incubated at 30℃ and 200 rpm for 24 h, using uninoculated culture medium as a blank. The absorbance was recorded at 600 nm.

[0032] Ammonia production capacity test: Select a small number of BJ colonies of Chinese rhizobium and inoculate them into LB medium, repeat 3 times, and incubate at 30℃ and 120r / min for 3 days. Add 0.5mL of Nessler's reagent to each test tube. If a yellow precipitate appears, it indicates that the strain has ammonia production activity.

[0033] The ability of Rhizobium sinense strain BJ to produce indoleacetic acid (IAA) is as follows: Figure 3 As shown in a, strain BJ produced the highest IAA level of 58.2 mg / L on day 3. The siderophore production capacity of *Rhizobium sinense* strain BJ is as follows: Figure 3 As shown in b, a distinct orange halo appeared around the BJ colony on the CAS plate after 24 hours of culture. The *Rhizobium sinense* BJ strain possesses phosphate-solubilizing ability, as shown in Figure b. Figure 3 As shown in c, strain BJ forms a phosphate-solubilizing transparent zone on a solid plate containing calcium phosphate. *Rhizobium sinense* strain BJ exhibits excellent salt tolerance, as shown in... Figure 3 As shown in d, it can survive at a salt concentration of 120 g / L, exhibiting extremely strong salt tolerance. In a liquid medium containing calcium phosphate, strain BJ can produce soluble phosphate in the supernatant by lowering the pH value, such as... Figure 3 As shown in e, the available phosphorus in the supernatant reached its peak of 180 μg / mL on day 5. *Rhizobium sinense* strain BJ exhibits ammonia-producing capabilities, such as... Figure 3 As shown in f, after adding Nessler's reagent and letting it stand for 5 minutes, a clear precipitate can be observed at the bottom of the centrifuge tube.

[0034] Example 3: Preparation method of liquid inoculum of Rhizobium sinense strain BJ

[0035] The seed culture medium consists of: 20 g / L sucrose, 2.3 g / L (NH4)2HPO4, 1 g / L yeast extract, 0.1 g / L MgSO4, 0.05 g / L FeSO4·7H2O, and 0.02 g / L MnSO4. The fermentation medium consisted of: 40 g / L glucose, 2.3 g / L (NH4)2HPO4, 1 g / L yeast extract, 0.1 g / L MgSO4, 0.05 g / L FeSO47H2O, 0.02 g / L MnSO4, 1 g / L antifoaming agent, pH 7.5-8.0, and was sterilized at 121℃ for 20 min.

[0036] (1) Seed culture preparation: Add 50 mL of seed culture medium and 100 μL of BJ bacterial culture to a 250 mL Erlenmeyer flask. Incubate the Erlenmeyer flask at 30 °C and 200 rpm for 24 h to obtain the primary seed culture. Inoculate the primary seed culture at an inoculation rate of 6% (v / v) into a 1000 mL Erlenmeyer flask containing 300 mL of fermentation culture medium. Incubate the flask at 30 °C and 200 rpm for 12 h to obtain the secondary seed culture.

[0037] (2) Fermentation culture: Inoculate the secondary seed culture into a 5L stirred fermenter at an inoculation rate of 10% (v / v). The fermenter contains 3L of fermentation culture medium. Fermentation can be completed after about 64 hours (when the glucose concentration is below 5g / L) under the following conditions: temperature 30℃, pH 7.5-8.0, aeration rate 0.5-1.0vvm, and stirring speed 200-1100rpm.

[0038] Survival period detection of liquid inoculum of Rhizobium sinense strain BJ: The fermentation process of the liquid inoculum of Rhizobium sinense strain BJ is shown in Figure 4, and the morphology of the inoculum is as follows: Figure 5 As shown, it is a pale yellow, viscous liquid with a viscosity of 3650 mPa·s, containing 2.78 × 10⁻⁶ viable bacteria. 9 CFU / mL. According to the national standard GB 20287-2006 for agricultural microbial agents, the effective viable count should be ≥ 200 million / g (mL) (≥ 100 million / g for granular formulations). The effective viable count in the Rhizobium sinense BJ liquid inoculant is 14 times that of the national standard.

[0039] The viscous liquid bacterial agent was dispensed into several sealed plastic bottles and stored at room temperature. Samples were taken every month for serial dilution and plated onto LB agar plates for viable cell count and pH testing. The results are shown in Table 1. The viscous liquid bacterial agent prepared using *Rhizobium sinense* strain BJ retained approximately 85% of its viable cell count after 60 days of storage at room temperature, which is 10 times the national standard; after 180 days of storage, the effective viable cell count was >2.0 × 10⁻⁶. 8 The CFU / mL level still meets the national standard requirements.

[0040] Table 1. Viable bacterial count and pH value of viscous BJ bacterial agent under room temperature storage conditions.

[0041] Comparative example: Rhizobium sinense strain BJ was inoculated into YMA liquid medium without agar and cultured in shake flasks at 30°C for 24 hours. After the culture, without adding any excipients, the liquid inoculum was dispensed into several sealed plastic bottles and stored at room temperature. Every month, samples were mixed and serially diluted, then spread on LB plates to detect the effective viable count and pH value of the inoculum. The results are shown in Table 2. The viable count after 30 days of storage did not meet the national standard requirements.

[0042] Table 2. Viable bacterial count and pH value of non-viscous BJ bacterial agent under room temperature storage conditions.

[0043] Detection of extracellular polysaccharides in viscous liquid inoculum of Rhizobium sinense strain BJ: The viscous inoculum at the end of fermentation was used as the test sample. The polysaccharide yield of the liquid inoculum of strain BJ was found to be as high as 20.95 g / L by alcohol precipitation. The liquid inoculum was used to extract crude polysaccharide, pure polysaccharide and component analysis.

[0044] Extraction method of crude Rhizobium sinense strain BJ extracellular polysaccharide: The liquid bacterial agent was heat-treated at 80℃ for 2h to inactivate microorganisms. After cooling, an equal volume of distilled water was added, and the mixture was placed in an 80℃ water bath for 30min. After centrifugation at 8000r / min for 30min, the supernatant was collected, and 3 times the volume of 95% ethanol was added and mixed. The mixture was then allowed to stand overnight at room temperature. After centrifugation at 8000r / min for 10min, the precipitate was collected and dried in a 60℃ oven to constant weight to obtain crude BJ polysaccharide.

[0045] Extraction method of pure BJ strain extracellular polysaccharide from Rhizobium sinense: Dissolve the crude BJ polysaccharide extract in 100 mL of distilled water by heating and stirring. Add 20 mL of a 4:1 mixture of chloroform and n-butanol (volume ratio), shake for 30 min, centrifuge at 8000 r / min for 30 min, collect the supernatant, and repeat several times until no oily substance appears in the organic phase. Place the deproteinized polysaccharide sample in a dialysis bag with a molecular weight of 10000 Da, concentrate with PEG10000, dialyze for 3 days, and freeze-dry the dialyzed solution to obtain a white, loose, pure BJ polysaccharide.

[0046] Component analysis of extracellular polysaccharides from *Rhizobium sinense* strain BJ: 5 mg of pure BJ polysaccharide extract was placed in an ampoule, 1 mL of 72% sulfuric acid was added, and the mixture was incubated in a 30°C water bath for 1 hour. Water was added to bring the volume to 10 mL, and the mixture was hydrolyzed in a nitrogen-filled, 110°C oven for 2 hours. After cooling to room temperature, 0.5 mL of the extract was placed in a 4 mL centrifuge tube, the pH was adjusted to neutral, and water was added to bring the volume to 1 mL. Then, 0.2 mL of 0.3 M NaOH and 0.4 mL of PMP methanol solution were added, and the mixture was incubated in a nitrogen-filled, 70°C water bath for 60 minutes. After cooling to room temperature, 0.2 mL of 0.3 M HCl was added, and water was added to bring the volume to 2 mL. 1.5 mL of chloroform was added, the mixture was shaken well, and allowed to separate into layers. The lower chloroform layer was discarded, and the aqueous layer was filtered through a 0.45 μm filter membrane and analyzed by high-performance liquid chromatography (HPLC).

[0047] The compositional results of the extracellular polysaccharide of Rhizobium sinense strain BJ are as follows: Figure 6 , Figure 7 As shown. Figure 6The high-performance liquid chromatograms of 15 standard monosaccharide components are shown below: 1 GulA (19.795 min); 2 ManA (20.935 min); 3 Man (22.702 min); 4 G1cN (25.475 min); 5 Rib (26.208 min); 6 Rha (27.090 min); 7 G1cA (28.125 min); 8 GalA (29.618 min); 9 GalN (31.433 min); 10 Glc (32.407 min); 11 GalNAc (33.200 min); 12 Gal (33.933 min); 13 Xy1 (34.767 min); 14 Ara (35.238 min); 15 Fuc (37.210 min). Figure 7 The image shows the high-performance liquid chromatogram (HPLC) of the hydrolyzed BJ polysaccharide. The peaks of the monosaccharide components are as follows: 1. GulA (19.878 min); 2. ManA (20.942 min); 3. Man (22.728 min); 4. G1cN (25.515 min); 5. Rib (26.260 min); 6. Rha (27.123 min); 7. G1cA (28.215 min); 8. GalA (29.37 min). 3 min); 9 was Glc (32.348 min); 10 was Gal (33.967 min); 11 was Xy1 (34.837 min); 12 was Ara (35.272 min); 13 was Fuc (37.503 min); GalN and GalNAc were not detected. Based on peak area calculations, galactose and glucose are the main structural components of monosaccharides, accounting for >95%, and the molar ratio of glucose to galactose is 9~10:1. The total molar percentage of uronic acid is about 2%, of which GulA accounts for 0.117%, ManA 0.042%, G1cA 0.695%, and GalA 1.152%.

[0048] Molecular weight determination of extracellular polysaccharides from *Rhizobium sinense* strain BJ: Pure extracellular polysaccharides from *Rhizobium sinense* strain BJ were analyzed using a 1260 Infinity II MDS gel permeation chromatograph (equipped with a differential refractive index detector and a dual-angle laser scattering detector). The chromatographic column used was a Plaquagel-OH Mixed-H, 8 μm (7.5 × 300 mm) (Agilent, USA). The detection conditions were 45℃, 1.0 mL / min; injection volume 50 μl; mobile phase 0.1 M sodium nitrate (0.01% sodium azide). The gel permeation chromatogram of BJ polysaccharides is shown below. Figure 8 As shown: The weight-average molecular weight (Mw) of BJ polysaccharide is 3.6 × 10⁻⁶. 6Da, the number-average molecular weight (Mn) is 3.4 × 10⁻⁶. 6 Da, Polydispersity Index (PDI) (Mw / Mn) = 1.06. BJ polysaccharides are characterized by high molecular weight polymerization degree and good uniformity. High molecular weight polymerization degree, consistent film-forming and moisturizing properties, can form a uniform protective layer, helping plants retain moisture and reduce salt stress.

[0049] To better illustrate the effects of the BJ inoculum of Rhizobium sinense or the extracellular polysaccharide of the present invention on the drought or salt tolerance of leguminous plants, the following examples use extracellular polysaccharides or inoculum for specific illustration, and soybean seeds and seedlings are selected as the plants.

[0050] Example 4: Effects of extracellular polysaccharides and inoculant of Rhizobium sinense strain BJ on soybean seed germination The pure extracellular polysaccharide of Rhizobium sinense BJ strain was obtained from Example 3. The Rhizobium sinense BJ inoculum was obtained from Example 3. The BJ inoculum was inactivated by heat treatment at 80°C for 2 hours to reduce the number of viable bacteria in the BJ inoculum to 0.

[0051] Select plump, uniformly sized, and intact soybean seeds from the Chinese Academy of Agricultural Sciences ("Xiaoyoudou" variety). Submerge the seeds in 75% alcohol and shake for 15 seconds. Rinse 3-4 times with distilled water to remove the alcohol. Then, submerge the seeds in 50% NaClO solution, shake for 1 minute, let stand for 1 minute, shake again for 1 minute, and rinse 6-7 times with water to remove the NaClO. Finally, blot dry with absorbent paper. Use the paper germination method. Place double-layered filter paper at the bottom of a 90mm diameter circular petri dish. Add the solutions for each group to fully wet the filter paper. Use tweezers to select seeds with intact, wrinkled seed coats. Place 10 seeds in each petri dish, with 5 replicates. Wrap the petri dishes in aluminum foil to protect them from light, then place them in an artificial incubator at 20±1℃ for germination. Add water at the same time every day and change the filter paper every other day.

[0052] A 100 mM sodium chloride solution was used to simulate salt stress, while drought stress was achieved by not adding water during the experiment. The concentrations and groupings of the prepared solutions are shown in Table 3.

[0053] Table 3

[0054] Using "white sprouting" as the germination standard, the number of sprouts was counted daily, and the germination process lasted for 7 days. Germination potential, germination rate, relative germination potential, relative germination rate, and relative salt damage rate of the salt stress group were calculated using the formulas below. Experimental data were processed using Excel 2021 software; multiple comparisons of differences were performed using the LSD method (P < 0.05). Germination potential = (Number of germinated seeds on day 3 / Total number of seeds) × 100%; Germination rate = (Number of germinated seeds on day 7 / Total number of seeds) × 100%; Relative germination potential = (treatment germination potential / control germination potential) × 100%; Relative germination rate = (treatment germination rate / control germination rate) × 100%; Note: Lowercase letters in the same column indicate significant differences between different groups at the same concentration. Different letters indicate significant differences at the 0.05 level, and the same applies below.

[0055] The results are shown in Tables 4-6.

[0056] Table 4

[0057] Table 5

[0058] Table 6

[0059] Soybean seed germination status after 7 days of cultivation is as follows Figure 9 As shown in Tables 4-6 and Figure 9 It can be seen that, using CK as the baseline, BJ polysaccharide and BJ inoculant can significantly promote soybean germination under non-stress conditions. Using salt-stress group S and drought-stress group D as baselines, the addition of BJ polysaccharide and BJ inoculant effectively alleviated the damage caused by salt and drought stress, enhanced the stress resistance of soybeans, and significantly improved the germination rate. This series of data indicates that BJ polysaccharide and inoculant can significantly improve the germination rate of soybeans under stress conditions.

[0060] Example 5: Effects of Rhizobium sinense BJ polysaccharide and inoculant on soybean seedling growth and root nodulation. The pure BJ polysaccharide was obtained from Example 3, the BJ inoculum of Rhizobium sinense was obtained from Example 3, and the inactivated BJ inoculum was obtained by heat treatment at 80°C for 2 hours to reduce the number of effective live bacteria in the BJ inoculum to 0.

[0061] Select plump, uniformly sized, and intact "Xiaoyoudou" soybean seeds from the Chinese Academy of Agricultural Sciences. Submerge the seeds in 75% alcohol and shake for 15 seconds. Rinse 3-4 times with distilled water to remove the alcohol. Then, submerge the seeds in 50% NaClO solution, shake for 1 minute, let stand for 1 minute, shake again for 1 minute, and rinse 6-7 times with water to remove the NaClO. Finally, blot dry with absorbent paper. Divide the sterilized seeds evenly and soak them separately in deionized water, BJ polysaccharide solution, inactivated BJ inoculant, and BJ inoculant for 12 hours. The volume of the soaking solution should be twice the volume of the seeds. The working concentration of polysaccharide is set at 100 mg / L. Wrap the seeds in moist gauze in the dark for 12 hours to promote germination. Rinse the gauze with water for 1 minute after 12 hours, and then continue germination in the dark for another 12 hours. After germination, soybean seeds were grouped and sown in tissue culture bottles, covered with 1 cm of nutrient soil. Each bottle contained 400 g of nutrient soil, and 200 mL of deionized water was added to reach 50% of the field maximum water holding capacity. Water was applied daily with 5 mL of water to maintain a weight of approximately 700 g. The bottles were cultured at 21℃ with 75% ± 5% humidity for 7 days until the soybeans reached the single-leaf stage. Seedlings of uniform size were selected, and 100 mL of each group's solution was added to the tissue culture bottles to bring the nutrient soil to 75% of its maximum water holding capacity. Ten samples were prepared from each group. The concentration of pure BJ polysaccharide and BJ inoculant in the soil was set at 100 mg / kg polysaccharide. The concentrations of the prepared solutions and the groupings are as follows: CK: Deionized water E: 0.4 g pure BJ polysaccharide + deionized water to a final volume of 1 L F: 20 mL of inactivated BJ bacteria agent + deionized water to a final volume of 1 L SF: 20 mL BJ bacterial agent + deionized water to a final volume of 1 L After 42 days of cultivation in each group, the growth of soybean seedlings was as follows: Figure 10 As shown, from the perspective of plant growth appearance, the application of BJ polysaccharide and bacterial agent significantly promoted the growth of soybean seedlings. Data were collected and processed using Excel 2021 software; multiple comparisons were performed using the LSD method (P < 0.05); the results are shown in Table 7 and... Figure 10 As shown.

[0062] Table 7

[0063] From Table 7 and Figure 10As shown in the figure. Compared with the control (CK), the plant height of groups E, F, and SF increased by 17.5%, 23.9%, and 18.2%, respectively; root length increased by 22.9%, 16.9%, and 15.8%; aboveground fresh weight increased by 11.1%, 16.7%, and 8.3%; aboveground dry weight increased by 32.2%, 35.6%, and 27.1%; root fresh weight increased by 100.0%, 90.0%, and 70.0%; and root dry weight increased by 28.6%, 28.6%, and 14.3%. The number of root nodules in the experimental groups also increased significantly, with the group containing live BJ inoculant showing the most outstanding effect on promoting nodulation. This series of data indicates that BJ polysaccharide itself has nodulation-promoting ability, and the BJ inoculant formed by BJ polysaccharide + live BJ bacteria has a more significant nodulation-promoting ability.

[0064] Example 6: Effects of Rhizobium sinense BJ polysaccharide and inoculant on soybean seedling growth and root nodulation under stress. The pure BJ polysaccharide was obtained from Example 3, the BJ inoculum of Rhizobium sinense was obtained from Example 3, and the inactivated BJ inoculum was obtained by heat treatment at 80°C for 2 hours to reduce the number of effective live bacteria in the BJ inoculum to 0.

[0065] Select plump, uniformly sized, and intact "Xiaoyoudou" soybean seeds from the Chinese Academy of Agricultural Sciences. Submerge the seeds in 75% alcohol and shake for 15 seconds. Rinse 3-4 times with distilled water to remove the alcohol. Then, submerge the seeds in 50% NaClO solution, shake for 1 minute, let stand for 1 minute, shake again for 1 minute, and rinse 6-7 times with water to remove the NaClO. Finally, blot dry with absorbent paper. Divide the sterilized seeds evenly and soak them separately in deionized water, BJ polysaccharide reconstituted solution, inactivated BJ inoculant, and BJ inoculant for 12 hours. The volume of the soaking solution should be twice the volume of the seeds. The working concentration of polysaccharide is set at 100 mg / L. Wrap the seeds in moist gauze in the dark for 12 hours to promote germination. Rinse the gauze with water for 1 minute after 12 hours, and then continue germination in the dark for another 12 hours. After germination, soybean seeds were grouped and sown in tissue culture bottles, covered with 1 cm of nutrient soil. Each bottle contained 400 g of nutrient soil, and 200 mL of deionized water was added to achieve 50% of the field maximum water holding capacity. 5 mL of water was added daily to maintain a weight of approximately 700 g. The seeds were cultured at 21℃ with 75% ± 5% humidity for 7 days until they reached the single-leaf stage. Seedlings of uniform size were selected, and 100 mL of the solution from each group was added to the tissue culture bottles to achieve 75% of the field maximum water holding capacity. Ten samples were prepared for each group. The concentration of pure BJ polysaccharide and BJ inoculant in the soil was set at 100 mg / kg polysaccharide. Group S was treated with sodium chloride to simulate salt stress, and Group D was treated without subsequent watering to simulate drought stress. "*" indicates the number of days when the extreme drought level was reached (25% of the field maximum water holding capacity); "#" indicates the number of days when half of the soybeans in the group died. The solution concentrations and groupings are as follows: CK: Deionized water S: 23.4 g NaCl + deionized water, bring to a final volume of 1 L. S+E: 0.4 g pure BJ polysaccharide + 23.4 g NaCl + deionized water to a final volume of 1 L S+F: 20 mL of inactivated BJ bacterial agent + 23.4 g NaCl + deionized water to a final volume of 1 L. S+SF: 20 mL BJ bacterial agent + 23.4 g NaCl + deionized water to a final volume of 1 L. D: Deionized water + no water replenishment during the process D+E: 0.4 g pure BJ polysaccharide + deionized water to a final volume of 1 L + no additional water added during the process. D+F: 20 mL of inactivated BJ bacteria agent + deionized water to a final volume of 1 L + no additional water added during the process. D+SF: 20 mL BJ bacterial agent + deionized water to a final volume of 1 L + do not add water during the process. After 42 days of cultivation in each group, the growth of soybean seedlings was as follows: Figure 11 , 12 As shown, where Figure 12 Group D was photographed after 30 days of cultivation. From the plant growth appearance, the application of BJ polysaccharide and inoculant effectively alleviated salt stress and enhanced the survival ability of soybean seedlings in drought conditions. Data were collected and processed using Excel 2021 software; multiple comparisons were performed using the LSD method (P < 0.05); the results are shown in Tables 8-9.

[0066] Table 8

[0067] from Figures 11-12 As shown in Table 8, the application of BJ polysaccharide and BJ inoculant effectively improved the effects of salt stress on soybean seedlings. Compared with group S, the group with added BJ polysaccharide showed a 26.2% increase in plant height, a 52.3% increase in root length, a 43.7% increase in aboveground fresh weight, a 245.5% increase in root fresh weight, a 144.4% increase in aboveground dry weight, and a 150.0% increase in root dry weight; the group with added inactivated BJ inoculant showed a 25.7% increase in plant height, a 48.6% increase in root length, a 43.7% increase in aboveground fresh weight, a 250.0% increase in root fresh weight, a 127.8% increase in aboveground dry weight, and a 200.0% increase in root dry weight; the group with added BJ inoculant showed a 22.7% increase in plant height, a 47.7% increase in root length, a 31.3% increase in aboveground fresh weight, a 231.8% increase in root fresh weight, a 127.8% increase in aboveground dry weight, and a 150.0% increase in root dry weight. In addition, no nodulation was found in the roots of the S group plants due to salt invasion, while the experimental group plants with added BJ polysaccharide and bacterial agent were still able to form nodules.

[0068] Table 9

[0069] Table 9 shows the growth of soybean seedlings on day 30 of the drought stress group as follows: Figure 12 As shown, the plants in the groups treated with BJ polysaccharide and microbial agents exhibited better growth than those in group D. After 42 days of cultivation, all soybean seedlings in group D died, while a certain number of seedlings survived in the experimental groups treated with BJ polysaccharide and microbial agents. In conclusion, it can be seen that under drought conditions, the application of BJ polysaccharide and microbial agents can effectively alleviate the problem of soybean seedlings entering drought-induced decline and death due to water loss, and significantly prolong the survival time of soybean seedlings in drought environments.

[0070] Example 7: Effects of BJ polysaccharide and BJ inoculant on soybean growth and nodulation in field soybeans. The crude BJ polysaccharide was obtained from Example 3, and the BJ inoculum of Rhizobium sinense was obtained from Example 3. The inactivated BJ inoculum was obtained by heat treatment at 80°C for 2 hours, so that the number of effective live bacteria in the BJ inoculum was 0.

[0071] A field experiment was conducted on soybeans in Xuecheng District, Zaozhuang City, Shandong Province to investigate the actual growth-promoting and yield-increasing effects of BJ polysaccharide and inoculants. "Qihuang 34" soybean seeds were sown in the field. Four experimental groups were set up: a blank control group (CK group), group F (treated with inactivated BJ inoculant), group SF (treated with BJ inoculant), and group E (treated with BJ polysaccharide solution). Each group had three replicates, with a total experimental area of ​​approximately 1 mu (0.067 hectares) per group. The application schemes for each treatment group were as follows: Groups F and SF were treated with inactivated BJ inoculant or BJ inoculant at a rate of 5 kg / mu; Group E had crude BJ polysaccharide redissolved in water to prepare a polysaccharide concentration of 20.5 ± 0.5 g / L, also applied at 5 kg / mu. All solutions were mixed and irrigated using an intelligent water and fertilizer integrated machine, and managed using an automatic field seedling monitoring system. Detailed application formulas are as follows: CK: 40 T water E: 104.75 g BJ polysaccharide crude product + 40 T water F: 5 kg of inactivated BJ bacteria agent + 40 T of water SF: 5 kg BJ bacterial agent + 40 T water Table 10

[0072] After 42 days of cultivation, samples were collected and analyzed from each group. The results are shown in Table 10. The application of BJ polysaccharide and BJ inoculant in soybeans in the field experiment achieved significant growth-promoting and nodulation-promoting effects. Compared with the control group (CK), the root length of groups E, F, and SF increased by 32.3%, 30.8%, and 22.7%, respectively; the stem length increased by 32.8%, 33.6%, and 31.0%, respectively; and the fresh weight increased by 12.6%, 10.3%, and 29.1%, respectively. Both BJ polysaccharide and inactivated BJ inoculant effectively promoted the number of nodules on the roots. The SF group, which received live BJ inoculant, had the highest number of nodules. This indicates that both BJ polysaccharide and BJ inoculant from *Rhizobium sinense* have significant nodulation-promoting abilities, which were also effectively verified under actual field production conditions.

[0073] The crude BJ polysaccharide was obtained from Example 3, and the BJ inoculum of Rhizobium sinense was obtained from Example 3. The inactivated BJ inoculum was obtained by heat treatment at 80°C for 2 hours, so that the number of effective live bacteria in the BJ inoculum was 0.

[0074] A field experiment was conducted on peanuts in Xuecheng District, Zaozhuang City, Shandong Province to investigate the practical effects of BJ polysaccharide and BJ inoculant. Puhua 20 peanut variety was selected and sown in the field. Four experimental groups were set up: a blank control group (CK group), group F (treated with inactivated BJ inoculant), group SF (treated with live BJ inoculant), and group E (treated with BJ polysaccharide reconstituted solution). Each group had three replicates, with a total experimental area of ​​approximately 1 mu (0.067 hectares) per group. The application schemes for each treatment group were as follows: Group F (inactivated BJ inoculant) and group SF (live BJ inoculant) were applied at a rate of 5 kg / mu; Group E (BJ polysaccharide) was prepared by reconstituted crude BJ polysaccharide with water to a concentration of 20.5 ± 0.5 g / L, and also applied at a rate of 5 kg / mu. All solutions were mixed and irrigated using an intelligent water and fertilizer integrated machine, and managed using an automatic field seedling monitoring system. Detailed application formulas are as follows: CK: 40 T water E: 104.75 g BJ polysaccharide crude product + 40 T water F: 5 kg of inactivated BJ bacteria agent + 40 T of water SF: 5 kg BJ bacterial agent + 40 T water Table 11

[0075] After 114 days of cultivation, each group was harvested, and the results are shown in Table 11. The application of BJ polysaccharide and BJ inoculant in the field peanut experiment achieved significant growth-promoting and yield-increasing effects. Compared with the control group (CK), the plant height of groups E, F, and SF increased by 22.9%, 23.9%, and 32.6%, respectively; root length increased by 34.7%, 26.6%, and 30.1%, respectively; fresh weight increased by 35.5%, 30.3%, and 34.5%, respectively; and the yield per 20 plants increased by 18.8%, 25.4%, and 21.2%, respectively. The proportion of large pods in groups CK, E, F, and SF were 53.8%, 62.9%, 62.7%, and 67.8%, respectively. These data indicate that BJ polysaccharide and BJ inoculant have significant yield-increasing and quality-improving effects on peanuts.

[0076] The present invention has been disclosed above with reference to preferred embodiments, but it is not intended to limit the present invention. All technical solutions obtained by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.

Claims

1. A type of Rhizobium sinense BJ ( Sinorhizobium sp. BJ), characterized by: The *Rhizobium sinense* BJ strain is deposited at the China Center for Type Culture Collection (CCTCC) on November 14, 2025, with accession number CCTCC M 20252477.

2. The *Rhizobium sinense* BJ strain according to claim 1 Sinorhizobiumsp. BJ), characterized by: The extracellular polysaccharide produced by the fermentation of Rhizobium sinense BJ consists of basic monosaccharides, neutral monosaccharides, and acidic monosaccharides.

3. The *Rhizobium sinense* BJ strain according to claim 2. Sinorhizobiumsp. BJ), characterized by: The basic monosaccharide is glucosamine; the neutral monosaccharide is glucose, galactose, mannose, fucose, rhamnose, ribose, xylose, and arabinose; the acidic monosaccharide is galacturonic acid, glucuronic acid, guluronic acid, and mannuronic acid.

4. A method for preparing a *Rhizobium sinense* inoculant, characterized in that, Includes the following steps: Add 50 mL of seed culture medium to a 250 mL Erlenmeyer flask, then add 100 μL of *Rhizobium sinense* BJ bacterial culture. Incubate the flask at 30℃ and 200 rpm on a shaker for 24 h to obtain primary seed culture. Inoculate the primary seed culture at 6% (v / v) into a 1000 mL Erlenmeyer flask containing 300 mL of fermentation medium and incubate at 30℃ and 200 rpm on a shaker for 12 h to obtain secondary seed culture. Inoculate the secondary seed culture at 10% (v / v) into a 5 L stirred fermenter containing 3 L of fermentation medium. Ferment and culture at 30℃, pH 7.5-8.0, aeration rate of 0.5-1.0 vvm, and stirring speed of 200-1100 rpm for 64 h to obtain *Rhizobium sinense* BJ bacterial agent; its viable count is 2.78 × 10⁻⁶. 9 CFU / mL; the number of viable bacteria after 180 days of storage was 6.15 × 10⁻⁶. 8 CFU / mL.

5. The *Rhizobium sinense* BJ strain according to claim 1 ( Sinorhizobium The application of sp. BJ) or its extracellular polysaccharide, or the microbial agent prepared according to the method of claim 4, in improving the growth of leguminous plants and strengthening root nodulation.

6. The *Rhizobium sinense* BJ strain according to claim 1 ( Sinorhizobium The application of sp. BJ) or its extracellular polysaccharide, or the microbial agent prepared according to the method described in claim 4, in improving drought and salt stress in leguminous plants.

7. The *Rhizobium sinense* BJ (as described in claim 1) Sinorhizobium The application of sp. BJ) or its extracellular polysaccharides, or the microbial agent prepared according to the method of claim 4, in promoting the growth and increasing the yield of leguminous plants.

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