Rhizobium for improving soybean feeding performance, bacterial agent and application thereof

Abstract

The application belongs to the field of agricultural microorganism technology, and discloses rhizobium for improving the feeding performance of soybean, a bacterial agent and application thereof. The rhizobium is Sinorhizobium fredii QYS03, the preservation number of which is CCTCC NO: M 20252632, and is isolated from the rhizobium of a feeding type soybean in a northwest loess upland area. The rhizobium has the abilities of dissolving inorganic phosphorus, organic phosphorus and secreting indole acetic acid, and has a broad spectrum of nodulation characteristics. The application further provides a rhizobium bacterial agent containing the strain and a preparation method thereof, and the number of viable bacteria in the bacterial agent is not less than 2*10 9 cfu / mL. The rhizobium and the bacterial agent can be applied to soybean planting by seed dressing, can significantly promote the growth of soybean stems and leaves and root systems, improve the nodulation and nitrogen fixation capacity, increase the biomass accumulation of soybean, improve the soil fertility, and have a broad application prospect in the field of soybean forage cultivation.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a rhizobium, inoculant, and its application for improving the feed performance of soybeans. Background Technology

[0002] Soybean (Glycine max (L.) Merr.) has core advantages as a forage crop, including high protein content, eco-friendliness, and flexible planting. During the flowering period, the crude protein content of its stems and leaves can reach 18%–22%, and it can achieve "soil nourishment through biological nitrogen fixation," making it suitable for planting on marginal lands and a high-quality forage resource. Forage soybeans primarily grow vegetatively, and their yield and forage quality are closely related to nitrogen and phosphorus nutrition. Soybeans have a high nitrogen requirement at all growth stages, but during the seedling stage, root nodulation is low and symbiotic nitrogen fixation is insufficient, making soil nitrogen the main nitrogen source during the seedling stage. Therefore, rationally regulating nitrogen and phosphorus supply is key to improving the yield and quality of forage soybeans.

[0003] Rhizosphere-promoting bacteria can transform nutrients in the soil that are difficult for plants to utilize. The symbiotic nitrogen fixation through root nodule formation by rhizobia and soybean is an important pathway for soybeans to obtain nitrogen. Inoculation with superior rhizobia has become an important measure to reduce nitrogen fertilizer use and improve the quality and efficiency of forage soybeans. However, in practical applications, the effectiveness of rhizobia is limited by various factors such as symbiotic compatibility, competition from native rhizobia, stress resistance, and soil physicochemical factors. Currently, existing rhizobia exhibit poor adaptability and ineffective nodule-promoting effects in the arid soil environment of the Loess Plateau region in Northwest China. Summary of the Invention

[0004] The purpose of this invention is to provide a rhizobium, inoculant, and its application to improve the feed performance of soybeans, aiming to solve the problems of poor adaptability and ineffective nodulation promotion of existing rhizobia. This invention isolates and screens a strain of *Rhizobium fischeri* QYS03 from feed-grade soybean root nodules in the Loess Plateau region of Northwest China. This strain possesses phosphorus-solubilizing, plant hormone-secreting, and broad-spectrum nodulation characteristics, significantly promoting soybean growth and nitrogen fixation through nodulation, thus improving the feed quality of soybeans. It is well-suited to the soil and climate conditions of the Loess Plateau region and has good application value.

[0005] The technical solution provided by this invention is as follows:

[0006] The first aspect of this invention provides a rhizobium that enhances the feed performance of soybeans. The rhizobium is *Sinorhizobium fredii* QYS03, which was isolated from the root nodules of feed-type soybeans in the Loess Plateau region of Northwest China. It was identified as *Sinorhizobium fredii* by 16S rDNA sequencing and was deposited on November 20, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252632, located at CCTCC, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] Furthermore, the rhizobium has the ability to dissolve inorganic phosphorus and organic phosphorus and secrete indoleacetic acid (plant hormone); the colonies of the rhizobium on YMA Congo red medium are round or nearly round, viscous, smooth, slightly raised, moist, milky white, and 2-4 mm in diameter.

[0008] A second aspect of the present invention provides a rhizobium agent comprising the above-mentioned rhizobium, wherein the rhizobium agent contains fermentation broth or bacterial suspension of *Rhizobium fischeri* QYS03, and the viable count of the strain is not less than 2 × 10⁻⁶. 9 cfu / mL.

[0009] Furthermore, the fermentation broth or bacterial suspension is prepared from YMA medium containing calcium carbonate. The YMA medium containing calcium carbonate is composed of: 10.0 g mannitol, 1.0 g yeast extract, 0.5 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 0.1 g sodium chloride, 3.0 g calcium carbonate, and 1000 mL distilled water, with a pH of 7 ± 0.2. It is used after sterilization at 121°C for 26 min.

[0010] A third aspect of the present invention provides a method for preparing the above-mentioned rhizobium agent, comprising the following steps:

[0011] Preparation of strain culture medium: Pick a single colony of Rhizobium fischeri QYS03 and inoculate it into an Erlenmeyer flask containing YMA liquid medium. Incubate at 28℃ and 180 r / min on a shaker for 48-72 h to obtain strain culture medium.

[0012] Preparation of inoculum: The culture broth of the above-mentioned strain was inoculated into YMA medium containing calcium carbonate at an inoculum volume of 3%~8%, and cultured at 28℃ and 180 r / min for 48-72 h to obtain the rhizobium inoculum. The viable count of Rhizobium Fischeriensis QYS03 in the inoculum was not less than 2×10⁻⁶. 9 cfu / mL.

[0013] The fourth aspect of this invention provides the application of the above-mentioned rhizobium or rhizobium agent in soybean cultivation, specifically:

[0014] Application of soybean seed treatment: Mix soybean seeds with rhizobium inoculant, air dry them, and sow them within 12 hours. The amount of inoculant used is 20-50 mL / kg of soybean seeds.

[0015] Applications to enhance soybean feed performance: This rhizobium or rhizobium agent can promote the growth of soybean stems, leaves and roots, increase the number and fresh weight of soybean root nodules, increase the crude protein content of soybean, reduce the content of neutral / acid detergent fiber, improve the feed quality of soybeans throughout the plant, and also enhance the drought resistance of soybeans.

[0016] Application scenarios: This rhizobium or rhizobium agent can be used alone or in combination with chemical fertilizers or organic fertilizers. It is widely used in soybean forage cultivation, and is especially suitable for the soil and climate conditions of the Loess Plateau region in Northwest China.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] 1. The *Rhizobium fischeri* strain QYS03 screened in this invention is a native strain that is adapted to the soil and climate conditions of the Loess Plateau in Northwest China. It has strong stress resistance and solves the problem of poor adaptability and poor effect of foreign strains. Moreover, this strain has broad-spectrum nodulation characteristics and can form effective symbiosis with multiple soybean varieties.

[0019] 2. This strain has the ability to dissolve inorganic phosphorus and organic phosphorus and secrete indoleacetic acid, which can effectively transform the poor phosphorus source in the soil. At the same time, it secretes plant hormones to promote soybean growth, achieving the dual effect of "promoting growth + supplying fertilizer" and significantly improving the nitrogen fixation ability of soybean nodulation.

[0020] 3. The preparation method of the rhizobium agent of the present invention is simple, the culture conditions are mild, it is easy to produce on a large scale, the number of viable bacteria in the agent is high, the stability is good, the seed dressing operation is simple, and it is suitable for field promotion.

[0021] 4. This strain and inoculant can significantly increase soybean biomass accumulation, improve soybean crude protein content, reduce detergent fiber content, and enhance feed quality. At the same time, when applied in combination with fertilizer, it can improve soil physical and chemical properties, increase soil fertility, and achieve the dual benefits of increasing soybean forage yield and quality and soil fertility, while reducing the amount of chemical fertilizer used, thus possessing both economic and ecological value. Attached Figure Description

[0022] Figure 1 This is a colony morphology diagram of strain QYS03 provided in this embodiment of the invention on YMA Congo red medium;

[0023] Figure 2 The phylogenetic tree of strain QYS03 provided in the embodiments of the present invention;

[0024] Figure 3 This is a comparison of soybean root growth and nodulation after inoculation with the strain QYS03 provided in this embodiment of the invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of this application, and not all embodiments.

[0026] The culture medium formulation used in this invention is as follows:

[0027] YMA liquid culture medium: mannitol 10.0 g, yeast extract 1.0 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.2 g, sodium chloride 0.1 g, distilled water 1000 mL, pH=7±0.2, sterilized at 121℃ for 26 min;

[0028] YMA medium containing calcium carbonate: Add 3.0 g of calcium carbonate to YMA liquid medium, and keep the other components and sterilization conditions the same;

[0029] YMA solid medium: Add 15-20g of agar to the YMA liquid medium. The remaining components, pH and sterilization conditions are the same as those of the YMA liquid medium (mannitol 10.0g, yeast extract 1.0g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate heptahydrate 0.2g, sodium chloride 0.1g, agar 15-20g, distilled water 1000mL, pH=7±0.2, sterilize at 121℃ for 26min).

[0030] YMA Congo Red Medium: Add 0.025g of Congo Red reagent (final concentration 0.0025%) to YMA solid medium, and keep the other components and pH consistent; sterilize the YMA solid medium components first, cool to 50~60℃, add Congo Red reagent, shake well and pour into plates for later use.

[0031] NBRIP liquid medium: 10.0 g glucose, 0.1 g ammonium sulfate, 5 g magnesium chloride hexahydrate, 0.25 g magnesium sulfate heptahydrate, 0.2 g potassium chloride, 5.0 g phosphorus source, 1000 mL distilled water, pH=7.0, sterilized at 121℃ for 26 min;

[0032] King'B liquid medium: 20.0 g peptone, 10.0 mL glycerol, 1.0 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 1000 mL distilled water, pH=7.2, sterilized at 121℃ for 26 min.

[0033] Example 1

[0034] This embodiment provides the isolation and purification process of the new strain, as detailed below:

[0035] Sample collection: In the forage soybean planting area of ​​the Loess Plateau in Northwest China, select healthy forage soybean plants, collect fresh root nodules from the roots, remove impurities from the surface of the root nodules, put them into sterile centrifuge tubes, and store them at 4℃ for later use.

[0036] Surface disinfection: Soak the collected root nodules in 75% ethanol solution for 30 seconds, rinse with sterile water 3 times, then soak in 0.1% mercuric chloride solution for 5 minutes, rinse with sterile water 5 times to remove residual disinfectant on the surface.

[0037] Nodule grinding: Place the sterilized nodules in a sterile mortar, add a small amount of sterile water, and grind thoroughly into a homogenate. Dilute the homogenate to 10% using sterile water in a gradient. -3 10 -4 10 -5 Concentration gradient;

[0038] Spread culture: Take 0.1 mL of each concentration gradient of the dilution and spread it evenly on YMA solid medium plates. Incubate in a constant temperature incubator at 28℃ for 3-5 days and observe the colony growth.

[0039] Initial screening and purification: Select single colonies on the plate whose morphology matches the characteristics of rhizobia, and purify them repeatedly on YMA solid medium using the streak plate method 3-4 times until a pure strain with uniform colony morphology is obtained. Inoculate the pure strain into YMA slant medium, incubate at 28°C and then store at 4°C.

[0040] The isolated and purified strain was examined and observed under a microscope. Based on the microscopic examination results, it was preliminarily identified as a Rhizobium strain.

[0041] Example 2

[0042] This embodiment provides the properties of the strain isolated and purified in Example 1, as follows:

[0043] Colony morphology observation: The strain purified in Example 1 was inoculated onto YMA solid medium and YMA Congo red medium and cultured at 28°C for 4 days. Colony morphology was then observed. Figure 1 As shown, the results indicate that the colonies of this strain are round or nearly round, viscous, smooth, slightly raised, moist, milky white, and 2-4 mm in diameter. On YMA Congo Red medium, the colonies are still milky white and there is no red staining, which is consistent with the characteristics of rhizobium colonies.

[0044] Culture characteristics determination: The strain was inoculated into YMA liquid medium and cultured at temperatures of 20℃, 24℃, 28℃, and 32℃, and shaking speeds of 150 r / min, 180 r / min, and 210 r / min, respectively. The growth rate of the strain was measured. The results showed that the optimal culture temperature for this strain was 28℃ and the optimal shaking speed was 180 r / min. Under these conditions, the strain grew rapidly and entered the stationary phase after 48 h.

[0045] Molecular biological identification: Genomic DNA was extracted from this strain, and PCR amplification was performed using universal primers for Rhizobium 16S rDNA. The amplified products were sequenced, and homology comparisons were performed with the 16S rDNA sequences of known strains in the GenBank database. A phylogenetic tree was then constructed. Figure 2 As shown, the results indicate that this strain shares 99.8% homology with *Sinorhizobium fredii*. Based on phylogenetic analysis, the strain was identified as *Sinorhizobium fredii*, named *Sinorhizobium fredii* QYS03, and deposited with the accession number CCTCC M 20252632.

[0046] Example 3

[0047] This embodiment measures the inorganic phosphorus solubility, organic phosphorus solubility, and indoleacetic acid secretion capacity of the *Rhizobium fischeri* strain QYS03 to verify its growth-promoting properties.

[0048] Phosphorus solubility test

[0049] NBRIP liquid culture media were prepared separately, with tricalcium phosphate as the inorganic phosphorus source and calcium phytate as the organic phosphorus source.

[0050] The Rhizobium fischeri QYS03 was inoculated into 50 mL of the two NBRIP liquid media mentioned above and cultured at 28℃ and 180 r / min for 10 days, with the blank medium without inoculation as the control.

[0051] After the culture was completed, the culture medium was centrifuged at 10000×g to obtain the supernatant. The available phosphorus content in the supernatant was determined by the molybdenum antimony colorimetric method, and the increase in available phosphorus was calculated.

[0052] The results showed that strain QYS03 had an inorganic phosphorus solubility of 81.96 μg / mL and an organic phosphorus solubility of 266.46 μg / mL, indicating a strong phosphorus solubility.

[0053] Indoleacetic acid secretion capacity assay

[0054] Rhizobium fischeri QYS03 was inoculated into 50 mL King'B liquid medium and cultured at 28℃ and 180 r / min for 12 days, with blank medium without inoculation as a control.

[0055] After the culture was completed, the culture medium was centrifuged at 10000×g to obtain the supernatant. An equal volume of Spot colorimetric solution was added, and the mixture was allowed to stand in the dark for 30 min. The indoleacetic acid content was then calculated by colorimetry at a wavelength of 530 nm using a spectrophotometer.

[0056] The results showed that strain QYS03 secreted 26.02 μg / mL of indoleacetic acid, demonstrating a significant plant hormone secretion capacity.

[0057] Preparation of Spot colorimetric solution: Dissolve 1 mL of 0.5 M ferric chloride solution in 50 mL of deionized water, slowly add 30 mL of concentrated sulfuric acid, cool and then make up to 100 mL. Store in a brown reagent bottle for later use.

[0058] Example 4

[0059] This embodiment uses a pot experiment to determine the nodulation ability and growth-promoting effect of the *Rhizobium fischeri* strain QYS03 on soybeans, as detailed below:

[0060] Preparation of potting substrate: Mix vermiculite and perlite in a volume ratio of 3:1, autoclave at 121℃ for 1.5 h, cool and then fill into sterile flower pots for later use;

[0061] Soybean seed treatment: Select plump soybean seeds, disinfect with 75% ethanol for 30 seconds, rinse 3 times with sterile water, place in sterile petri dishes for germination, and select seeds with uniform germination for later use.

[0062] Inoculation treatment: Germinated soybean seeds were sown in potting substrate, with 3 seedlings per pot. After the first pair of true leaves of the seedlings unfolded, the roots of the experimental group were irrigated with a suspension of Rhizobium Fischeriensis QYS03 (live bacteria count 1×10⁻⁶). 9 (cfu / mL), the control group was watered with an equal volume of sterile water, and each treatment was repeated 3 times;

[0063] Cultivation and Measurement: Potted plants were conventionally cultivated in a greenhouse, with sterile water added regularly. Soybeans were harvested when they reached the initial flowering stage, and plant height, dry weight, root system indicators (root length, root surface area, etc.) and root nodule indicators (number of root nodules, total fresh weight of root nodules, etc.) were measured.

[0064] The results are shown in Table 1. Compared with the control group, the soybean plant height and dry weight of the experimental group inoculated with strain QYS03 increased significantly, the root length increased by 145.1%, the root surface area increased by 48.1%, the number of root nodules increased from 5.67 / plant to 165.67 / plant, and the total fresh weight of root nodules increased by 98.3%. This indicates that strain QYS03 can significantly promote soybean root growth and greatly improve soybean nodulation ability. Figure 3 This is a comparative diagram of soybean root growth and nodulation after inoculation with Rhizobium fischeri QYS03. Compared with the control group, the soybean root system in the inoculated group was more developed and the number of root nodules was significantly increased.

[0065] Table 1. Effects of Rhizobium fischeri QYS03 on soybean growth and nodulation.

[0066] Example 5

[0067] This embodiment uses a field trial to determine the effect of the bacterial strain on improving the feed quality of soybeans. The trial was conducted at the National Field Scientific Observation and Research Station for Grassland Agro-ecosystems in Qingyang, Gansu Province, Lanzhou University, as detailed below:

[0068] Rhizobium inoculant preparation

[0069] A single colony of activated Rhizobium fischeri QYS03 was picked and inoculated into 150 mL LB liquid medium and cultured at 28℃ and 180 r / min for 48 h to obtain seed culture.

[0070] The seed culture was inoculated at a rate of 5% into YMA medium containing calcium carbonate and cultured at 28°C and 180 r / min for 72 h to obtain a liquid inoculum. Plate counting showed that the viable cell count in the inoculum was 8.5 × 10⁻⁶. 9 cfu / mL;

[0071] Field trial design: The "LS-4" soybean variety for feed was selected, and two inoculation treatments (no inoculation T0 and inoculation T1) and three nitrogen application levels (0 kg N·hm) were set up. -2 N0, 40 kg N·hm -2 N1, 80 kg N·hm -2 N2), a total of 6 treatments, each treatment was replicated 3 times, and the plot area was 20 m². 2 (4 m × 5 m);

[0072] Sowing treatment: Before sowing, treat soybean seeds with the above-mentioned microbial agent at a rate of 30 mL / kg of soybean seeds. Sow the seeds in holes within 12 hours after air-drying. No topdressing or irrigation is required during the growing period. Conventional field management is followed.

[0073] Index determination: Whole plant samples were collected during the full flowering period of soybeans to determine plant height and dry matter yield. Crude protein content, neutral detergent fiber content, and acid detergent fiber content were determined using conventional feed analysis methods.

[0074] The test results are shown in Table 2. Compared with the uninoculated treatment, the inoculum of strain QYS03 significantly improved soybean growth indicators and feed quality at all nitrogen application levels.

[0075] Under nitrogen-free (N0) conditions, soybean plant height, dry matter yield, and crude protein content increased by 1.84%, 1.07%, and 2.22%, respectively, while neutral / acid detergent fiber content decreased by 3.10% and 1.25%, respectively.

[0076] Apply 40 kg N·hm -2 Under (N1) conditions, the above indicators increased by 7.80%, 10.21%, and 1.73%, respectively, while the detergent fiber content decreased by 1.71% and 2.99%, respectively.

[0077] Apply 80 kg N·hm -2 Under (N2) conditions, the above indicators increased by 7.41%, 9.51%, and 6.14%, respectively, while the detergent fiber content decreased by 1.59% and 5.88%, respectively.

[0078] The results show that the QYS03 inoculant of Rhizobium Fischeriensis can significantly increase the dry matter accumulation and crude protein content of soybeans, reduce the washing fiber content, effectively improve the feed quality of soybeans, and the effect is even better when applied in combination with nitrogen fertilizer.

[0079] Table 2. Effects of Rhizobium Fischeriensis QYS03 inoculum on soybean growth and feed quality.

[0080] In summary, the *Rhizobium fischeri* QYS03 of this invention has excellent growth-promoting and nodulation-forming properties. Its preparation method is simple and easy to scale up. It is also convenient to use as a seed dressing agent. It can significantly improve the feed performance and yield of soybeans and is suitable for the soil environment of the Loess Plateau in Northwest China. It can be widely used in soybean forage cultivation, while reducing the amount of chemical fertilizer used and improving soil fertility. It has significant industrial application value, economic benefits, and ecological benefits.

[0081] The above description is merely the preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A rhizobium that enhances the feed performance of soybeans, characterized in that, The rhizobium is *Sinorhizobium fredii* QYS03, with accession number CCTCC NO: M 20252632, accession date November 20, 2025, and accession address China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The rhizobium according to claim 1, characterized in that, The rhizobium strain has the ability to dissolve inorganic phosphorus, dissolve organic phosphorus, and secrete indoleacetic acid.

3. A rhizobium agent, characterized in that, Fermentation broth or bacterial suspension containing *Sinorhizobium fredii* QYS03 as described in claim 1, wherein the viable count of *Sinorhizobium fredii* QYS03 is not less than 2 × 10⁻⁶. 9 cfu / mL.

4. The rhizobium agent according to claim 3, characterized in that, The fermentation broth or bacterial suspension is prepared from YMA medium containing calcium carbonate.

5. The rhizobium agent according to claim 3, characterized in that, The YMA medium containing calcium carbonate is composed of: 10.0 g mannitol, 1.0 g yeast extract, 0.5 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 0.1 g sodium chloride, 3.0 g calcium carbonate, 1000 mL distilled water, and pH = 7 ± 0.

2.

6. A method for preparing a rhizobium agent as described in any one of claims 3-5, characterized in that, Includes the following steps: S1. Preparation of strain culture medium: Select a single colony of Sinohizobium fredii QYS03 and inoculate it into YMA liquid medium. Incubate at 28℃ and 180 r / min for 48-72 h to obtain strain culture medium. S2. Preparation of bacterial agent: The bacterial culture solution obtained in step S1 is inoculated into YMA medium containing calcium carbonate at an inoculation rate of 3% to 8%, and cultured at 28℃ and 180 r / min for 48-72 h to obtain the rhizobium agent.

7. The application of a rhizobium as described in any one of claims 1-2 or a rhizobium agent as described in any one of claims 3-5 in soybean seed treatment, characterized in that, The application method is as follows: mix the rhizobium agent with soybean seeds, air dry them, and sow them within 12 hours. The amount of the rhizobium agent used is 20-50 mL / kg of soybean seeds.

8. The application according to claim 1, characterized in that, The application aims to achieve at least one of the following effects: (1) promoting the growth of soybean stems, leaves and roots; (2) increasing the number of soybean root nodules and the fresh weight of root nodules; (3) increasing the crude protein content of whole soybean plants, reducing the washing fiber content, and improving feed quality; and (4) enhancing the drought resistance of soybeans.

9. The application according to claim 7, characterized in that, The rhizobium or the rhizobium agent can be applied in combination with chemical fertilizers or organic fertilizers for soybean forage cultivation in the Loess Plateau region of Northwest China.

Images