Soybean rhizobium and application thereof

Abstract

The invention relates to the technical field of microorganisms, and provides a soybean rhizobium and application of the soybean rhizobium, the soybean rhizobium is Chinese rhizobium freudeneri HY47, and the preservation number of the soybean rhizobium is CCTCC (China Center for Type Culture Collection) NO: M 20242695. The Sinorhizobium fredii HY47 has a stable symbiotic nitrogen fixation effect and a high IAA secretion capability, can maintain the growth activity in a soil pH value range of 5-9, can stably grow at 42 DEG C, and is suitable for being used as an excellent strain for increasing the yield of soybeans in Jianghan plain areas.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to soybean rhizobium and application thereof. Background Art

[0002] Soybean is an important food and feed crop in my country, providing high-quality protein and oil resources. However, soybean production in the Jianghan Plain is affected by many factors, among which limited soil fertility and low nitrogen utilization efficiency are more prominent.

[0003] Rhizobia are key microorganisms that can improve the nitrogen absorption efficiency of soybeans through symbiotic nitrogen fixation, which is of great significance for increasing yields. However, the adaptability and activity of existing rhizobia strains in the Jianghan Plain area are not ideal. For example, the rhizobia disclosed in the invention patent with publication number CN103952341A have poor nitrogen fixation ability and poor application effect in the Jianghan Plain area. The present invention aims to screen and cultivate a soybean rhizobium suitable for the Jianghan Plain environment, so that it can promote soybean growth more efficiently. Summary of the invention

[0004] In view of this, the present invention proposes a soybean rhizobium having good nitrogen fixation effect, efficiently promoting soybean growth, and being suitable for the Jianghan Plain environment and application thereof.

[0005] The technical solution of the present invention is implemented as follows: In the first aspect, the present invention provides a soybean rhizobium, wherein the soybean rhizobium is Bradyrhizobium japonicum (Kirchner) Jordan) HY47, and the deposit number is CCTCC NO: M 20242695.

[0006] In a second aspect, the present invention provides a bacterial fertilizer, comprising the above-mentioned Sinorhizobium freundii HY47.

[0007] In a third aspect, the present invention provides the application of bacterial fertilizer in soybean production in the Jianghan Plain area.

[0008] In a fourth aspect, the present invention provides the use of bacterial fertilizer in promoting nitrogen fixation and / or promoting the growth of soybean plants.

[0009] The soybean rhizobium and its application of the present invention have the following beneficial effects compared with the prior art:

[0010] (1) The Sinorhizobium freundii HY47 of the present invention has a stable symbiotic nitrogen fixation effect and a high IAA secretion capacity, can maintain growth activity in the soil pH range of 5-9, and can grow stably under conditions as high as 42°C, and is suitable as an excellent strain for increasing soybean production in the Jianghan Plain area. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 This is a colony morphology diagram of Sinorhizobium freundii HY47 of the present invention. DETAILED DESCRIPTION

[0013] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] The invention provides a soybean rhizobium, which is Bradyrhizobium japonicumHY47, preserved in China Center for Type Culture Collection, with the address at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation date being December 3, 2024, and the preservation number being CCTCC NO: M 20242695.

[0015] The present invention also provides a bacterial fertilizer comprising Sinorhizobium freundii HY47, which can be used for the growth of soybeans in the Jianghan Plain, and can promote soybean nodulation and nitrogen fixation, promote soybean growth, and increase soybean yield.

[0016] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0017] Example 1 Strain selection and characteristics

[0018] 1. Strain source

[0019] Samples were collected from typical soybean-growing soil in the Jianghan Plain area, and the Sinorhizobium freundii strain HY47 with high nitrogen-fixing ability was obtained through isolation and screening.

[0020] 2. Breeding process

[0021] In the high-yield soybean root soil in the Jianghan Plain area, 10 g of rhizosphere soil was weighed, washed three times with sterile water, and placed in a conical flask containing 100 mL of sterile water. It was shaken at 30°C and 180 rpm for 30 minutes and then allowed to stand for 10 minutes to prepare the rhizosphere soil suspension, which was diluted and inoculated into YMA (yeast mannitol) medium.

[0022] The formula of YMA medium is: yeast extract 1g / L, mannitol 10g / L, sodium chloride 0.1g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.2g / L, pH 6.8-7.2. Cultivate on the medium for 24-48h and select colonies with typical rhizobium characteristics.

[0023] The morphological characteristics of the strain HY47 isolated and purified in this example are as follows: the strain HY47 forms smooth, moist milky white colonies on the YMA medium (see Figure 1 ); Microscopic observation showed short rod shape and Gram staining was negative, confirming it to be Sinorhizobium fredii.

[0024] Molecular identification: Using 16S rRNA gene sequence analysis and comparison, it was confirmed that strain HY47 was related to other soybean rhizobium species and had high homology. Its nucleotide sequence is shown in SEQ NO: 1, which is suitable as a special strain for increasing soybean production in the Jianghan Plain area.

[0025] Example 2 Highly efficient nitrogen fixation capability

[0026] Verify the advantage of strain HY47 in nitrogen fixation ability to support its feasibility of promotion and application in the Jianghan Plain area. By comparing its nitrogenase activity and nitrogen fixation effect in actual field application with that of the control strain, determine whether the HY47 strain has a significant nitrogen supply capacity.

[0027] 2.1 Low nitrogen culture medium test

[0028] Low-nitrogen manganese chloride medium was used to inoculate the HY47 strain on the medium, and a blank group was set. The culture conditions were set at 30°C and 120rpm shaking culture for 7 days. The nitrogenase activity was determined by the acetylene reduction method, and the ethylene production (C2H4) was used as an indicator of nitrogenase activity. Principle of acetylene reduction method: Rhizobium nitrogenase reduces acetylene to ethylene, so the ethylene production can be used as an indicator of nitrogenase activity. Take 3mL of the strain culture solution each time and inject it into a vial filled with acetylene gas. After 30 minutes, the ethylene concentration was determined by gas chromatography. The results are shown in Table 1.

[0029] Low nitrogen manganese chloride medium: 10g / L mannitol (as a carbon source), 0.5g / L potassium chloride, 0.5g / L potassium dihydrogen phosphate (provides a phosphorus source and buffering effect), 0.2g / L magnesium sulfate (provides magnesium ions to promote enzyme activity), 0.01g / L manganese chloride (provides trace elements to support nitrogenase activity), and 0.1g / L calcium sulfate (adjusts ion balance). Adjust the pH value of the medium to 7.0-7.2 with sodium hydroxide or hydrochloric acid, divide the medium into portions, sterilize it under high pressure at 121°C for 15-20 minutes, and use it after cooling.

[0030] 2.2 Field nitrogen fixation effect test

[0031] In the typical soybean planting area of ​​Jianghan Plain, an experimental field (20m 2 ), set up three replicate test plots for the treatment group (inoculated with HY47 strain) and the control group (not inoculated with strain). Both groups planted the same variety of soybeans and standardized field management.

[0032] The strain inoculation of the treatment group was carried out before sowing, using the seed coating method. The specific method is as follows:

[0033] S1. Prepare bacterial suspension of HY17 strain with a concentration of 1×10 8 CFU / mL.

[0034] S2. Spray the bacterial solution evenly on the surface of the soybean seeds that have been surface-sterilized, ensuring that each seed is fully covered with the bacterial solution.

[0035] S3. After spraying, let the seeds stand to dry to avoid seeds sticking together. The treated seeds can be used directly for sowing.

[0036] Inoculation amount: Use 10 ml of bacterial solution per kilogram of soybean seeds to ensure that at least a sufficient number of strains (about 1×10 6 CFU / particle).

[0037] Test indicators: Samples were collected in the middle growth period of soybean (about 60 days after sowing), and the nitrogen content, plant height, number of nodules and biomass of the plants were measured. The nitrogen fixation ability of strain HY47 was analyzed. The results are shown in Table 1.

[0038] Table 1. Experimental data of nitrogen fixation capacity

[0039]

[0040] From the nitrogenase activity and field test data, it can be seen that the HY47 strain has a significant advantage in nitrogen fixation ability. The nitrogen content, plant height, and number of nodules in the inoculated group were significantly higher (P < 0.5) than those in the control group, proving that it has a stronger nitrogen fixation effect and is suitable for promotion and application in the Jianghan Plain area. Compared with the control group: the nitrogenase activity of the HY47 strain reached 30.2nmoLC2H4·mg -1 ·h -1 In the treatment group inoculated with HY47 strain, the plant height, nodule number and nitrogen content of soybean increased by 30%, 131% and 52%, respectively.

[0041] Example 3 IAA secretion ability test

[0042] In order to evaluate the IAA secretion ability of the HY47 strain, the present invention observed its growth-promoting effect through colorimetry and field experiments to verify its significant effect on promoting soybean root growth.

[0043] 3.1 Colorimetric detection of IAA secretion

[0044] The HY47 strain was inoculated into R2A medium containing L-tryptophan (200 mg / L) at 30° C. and 120 rpm shaking for 48 h.

[0045] The amount of IAA secretion was detected by the Salkowski colorimetric method: 1 mL of bacterial solution was taken, 1 mL of Salkowski colorimetric solution was added, and the color change was observed after 15 minutes, and quantitative analysis was performed using a spectrophotometer at a wavelength of 530 nm. An IAA standard curve was set up, and the IAA concentration in the bacterial solution was calculated based on the absorbance value. The results are shown in Table 2.

[0046] 3.2 Field growth promotion effect test

[0047] Experimental field design: In the soybean planting area of ​​Jianghan Plain, two adjacent experimental fields were selected as the treatment group (inoculated with HY47 strain) and the control group (not inoculated with strain), and the same field management measures were adopted during the growth period.

[0048] The strain inoculation of the treatment group was carried out during the seedling planting period after sowing, and the inoculation was carried out when the soybean seedlings grew 2-3 true leaves (10-15 days after sowing). The inoculation method was root irrigation, and the specific method was as follows:

[0049] 1. Prepare bacterial suspension of HY17 strain with a concentration of 1×10 8 CFU / mL.

[0050] 2. Apply the bacterial solution directly to the soil at the roots of soybean seedlings, and apply it evenly to each seedling.

[0051] 3. When irrigating the roots, ensure that the bacterial solution penetrates into the soil around the rhizosphere so that the bacterial strain can quickly contact the root system and colonize.

[0052] Inoculation amount: 100 ml of bacterial solution is applied to each soybean seedling to ensure that the number of strains reaches about 1×10 per gram of soil around the root system. 5 CFU and above, meeting the needs of strain growth promotion and colonization.

[0053] Monitoring indicators: Soybean root development indicators were measured in the middle growth period (about 60 days after sowing), including root length, root dry weight and number of nodules. The results are shown in Table 2.

[0054] Table 2. IAA secretion ability experimental data

[0055]

[0056] Judging from the IAA secretion and field test results, the IAA secretion capacity of the HY47 strain was significantly higher than that of the control group, which effectively promoted the development of soybean roots, increased root length, root weight and leaf area, and was beneficial for the plant to absorb nutrients and water and improve stress resistance. Compared with the control group: the IAA secretion of the HY47 strain was 18.5μg / mL, and the root length, root dry weight and leaf area of ​​soybeans in the treatment group inoculated with the HY47 strain increased by 22%, 46% and 49% respectively.

[0057] Example 4 Stability test of HY47 strain under different pH and high temperature conditions

[0058] The growth stability of strain HY47 in the pH range of 5-9 and under different high temperature conditions was evaluated to verify its ability to adapt to the variable soil pH and high temperature environment in the Jianghan Plain area.

[0059] 4.1 pH tolerance test

[0060] The HY47 strain was inoculated in YMA (yeast mannitol) liquid medium, and the pH value of the medium was adjusted to 5, 6, 7, 8 and 9, respectively, with 3 parallel samples under each pH value condition. The culture was cultured at 30°C and 120 rpm for 48 h. After the culture was completed, the OD600 value was measured by a spectrophotometer (600 nm wavelength) as an indicator of strain growth.

[0061] The formula of YMA (yeast mannitol) liquid culture medium is: yeast extract (Yeast Extract) 0.5g / L, mannitol (Mannitol): 10g / L, potassium dihydrogen phosphate (KH2PO4): 0.2g / L, magnesium sulfate (MgSO4·7H2O): 0.1g / L, sodium chloride (NaCl): 0.05g / L, agar: 15g / L, distilled water: 1000mL, pH 6.8-7.0 (adjust to the appropriate range using NaOH or HCl).

[0062] 4.2 High temperature tolerance test

[0063] The HY47 strain was inoculated in YMA (yeast mannitol) liquid medium, and the samples were placed in incubators at 30°C (control), 37°C, 42°C, 45°C, and 50°C, with 3 parallel samples for each temperature condition. The culture was shaken at 120 rpm for 48 hours. After the culture was completed, the OD600 value was measured, and the growth of the strain at different temperatures was used as an indicator to analyze its heat resistance.

[0064] Table 3. pH tolerance experimental data

[0065]

[0066] As shown in Table 3, the HY47 strain can maintain stable growth in the pH range of 5-9, especially showing strong growth activity between pH 6-8, which is suitable for the variable soil pH environment of the Jianghan Plain.

[0067] Table 4. High temperature tolerance test data

[0068]

[0069] As shown in Table 4, the HY47 strain can maintain normal growth at 30-42°C, can still adapt and grow to a limited extent at 45°C, and has almost no growth at 50°C. The results show that the HY47 strain has good heat resistance and is adapted to the high temperature conditions in the Jianghan Plain in summer.

[0070] Example 5 Application Experiment

[0071] Evaluate the effect of HY47 strain on promoting soybean growth under actual production conditions and verify its application potential in the Jianghan Plain area.

[0072] 5.1 Test materials and methods

[0073] Experimental substrate: Select natural soybean planting soil with a depth of 0 to 20 cm, pass through a 5 mm sieve and put into a 240×160 mm flower pot. The weight of each pot is about 2 kg. Use tap water to irrigate the soil and leave it for 2 days to approach the field water holding capacity.

[0074] Seed treatment: Soybean seeds were sterilized in 20% volume fraction hydrogen peroxide for 20 minutes, rinsed with sterile water and germinated for 2 days, and seeds with uniform germination were selected for later use.

[0075] Treatment settings: Three treatment groups and a control group were set up as follows:

[0076] Treatment group: Use HY47 bacterial solution with a concentration of 100 million CFU / mL, dilute to 0.5%, 1.0% and 2.0%, and irrigate the roots respectively. The root irrigation period is 10-15 days after sowing. Root irrigation treatment is carried out when the seedlings grow 2-3 true leaves. The root irrigation volume per pot is 100mL.

[0077] Control group: 100 mL of sterile water was used to irrigate the roots of each pot.

[0078] Each group was set up with 20 pots and three replicates.

[0079] Cultivation management: Sow 5 soybean seeds in each pot, and after emergence, thin out the seedlings to 2 plants per pot. Maintain a consistent growth environment and water and fertilizer management to simulate the management conditions of actual production.

[0080] 5.2 Measurement indicators

[0081] After 30 days of bacterial liquid treatment, growth indicators such as plant height, diameter, leaf width and dry weight of soybean were measured. The results are shown in Table 5.

[0082] Table 5 Soybean growth index

[0083]

[0084]

[0085] As shown in Table 5, under actual production conditions, the 0.5% treatment group inoculated with HY47 strain showed the best growth-promoting effect. Compared with the control group, indicators such as plant height, diameter, leaf width and dry weight were significantly improved.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A soybean rhizobium, characterized in that: The soybean rhizobium is Bradyrhizobium japonicum (Kirchner) Jordan) HY47, and its deposit number is CCTCC NO: M 20242695.

2. A bacterial fertilizer, characterized in that: It includes the Sinorhizobium freundii HY47 described in claim 1.

3. Application of the bacterial fertilizer as claimed in claim 2 in soybean production in Jianghan Plain area.

4. Use of the bacterial fertilizer as claimed in claim 2 in promoting nitrogen fixation and / or growth of soybean plants.

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