Rhizobium with salt-tolerant growth-promoting capability and application thereof

Abstract

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a rhizobium with salt-tolerant and growth-promoting capabilities and application of the rhizobium. The salt-tolerant growth-promoting bacterium is rhizobium meliloti, the strain number is SmYrd01, the preservation number is CCTCC NO: M 2025024, and the salt-tolerant growth-promoting bacterium is preserved in China Center for Type Culture Collection, Luojia Mountain, Wuchang District, Wuhan City, Hubei Province on January 6, 2025. The strain obtained by the invention comes from rhizosphere soil of halophyte alfalfa in saline-alkali soil of the Yellow River Delta, and is obtained by screening and culturing the halophyte alfalfa. The obtained strain has high salt tolerance, the germination rate and seedling growth indexes of alfalfa under the salt stress condition can be remarkably improved through the strain liquid of the strain, and the germination rate and seedling growth related indexes of alfalfa under the salt stress environment are remarkably improved through rhizobium strain liquid treatment; the bacterial liquid has potential application value in the aspect of improving the salt tolerance of alfalfa, and a theoretical basis is provided for further research of the bacterial liquid in improvement of saline-alkali soil.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and in particular relates to a rhizobium with salt-resistant growth-promoting ability and application thereof. Background Art

[0002] Soil salinization poses a serious challenge to agricultural production and ecosystems, leading to a decline in soil fertility and interfering with normal plant growth, which in turn affects agricultural productivity and ecological diversity. The improvement of saline-alkali land is of great significance to improving soil fertility, promoting sustainable agricultural development, and ensuring national food security. At present, the breeding of salt-tolerant plant varieties and the application of microbial technology provide new ideas for solving the problem of salt stress. However, the breeding cycle of salt-tolerant plants is long and the results are uncertain, while microbial technology has attracted widespread attention due to its high efficiency and diversity. Microbial technology mainly screens salt-tolerant strains or uses functional microorganisms to make bacterial agents, which are applied to soil or crop planting to construct a plant-microbe synergistic restoration system to alleviate the adverse effects of salt stress.

[0003] Rhizobium ( Rhizobia ) as a typical plant growth-promoting rhizobacteria (PGPR), plays an important role in promoting plant growth. Its unique nitrogen-fixing ability enables it to form a symbiotic relationship with leguminous plants, converting atmospheric nitrogen (N2) into ammonia (NH3) that can be used by plants, significantly improving the nitrogen nutrition level of plants. In addition, rhizobia promote plant growth and health through a variety of mechanisms such as secreting plant hormones (such as auxins and cytokinins), improving nutrient utilization (such as dissolving phosphates), and enhancing plant stress resistance. However, there is still a shortage of bacterial species that can be actually applied to social production. In particular, rhizobia that can not only adapt to high-salinity environments, but also effectively promote the salt-tolerant growth of plants grown in saline-alkali lands and their applications are still rare. Summary of the invention

[0004] Based on the above research background, the present invention provides a rhizobium with salt tolerance and growth-promoting ability screened from the rhizosphere soil of alfalfa, a halophyte in the saline-alkali land of the Yellow River Delta ( Sinorhizobium meliloti. ), the strain number is SmYrd01, and it is identified as a new strain belonging to the genus Sinorhizobium, which has the characteristics of strong salt tolerance. The present invention shows that the SmYrd01 strain has good salt tolerance and can promote plant growth or promote plant growth in saline-alkali soil environment.

[0005] Based on the above research content, the first aspect of the present invention provides a rhizobium SmYrd01, which has been deposited in the China Center for Type Culture Collection, the preservation date is January 6, 2025, the preservation address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M 2025024.

[0006] The rhizobium ( Sinorhizobium meliloti. )The nucleotide sequence of SmYrd01 is shown in SEQ ID NO.1.

[0007] The rhizobium ( Sinorhizobium meliloti. )SmYrd01 has good salt tolerance and can promote plant growth or promote plant growth in saline-alkali soil environments.

[0008] The second aspect of the present invention provides a salt-tolerant growth-promoting agent or a plant growth-promoting agent, wherein the salt-tolerant growth-promoting agent or the plant growth-promoting agent contains rhizobium ( Sinorhizobium meliloti. )SmYrd01 or its bacterial solution.

[0009] Preferably, the bacterial solution concentration is not less than 4×10 9 cfu / mL.

[0010] The third aspect of the present invention provides a rhizobium ( Sinorhizobium meliloti. )Application of SmYrd01 or its bacterial solution in promoting plant growth.

[0011] Furthermore, the plant includes alfalfa.

[0012] The present invention also includes rhizobium ( Sinorhizobium meliloti. ) Application of SmYrd01 or its bacterial solution in the preparation of salt-tolerant growth-promoting bacteria or plant growth-promoting bacteria.

[0013] The present invention also includes rhizobium ( Sinorhizobium meliloti. )Application of SmYrd01 or its bacterial solution in improving the salt tolerance of alfalfa.

[0014] Preferably, seed germination and seedling growth can be significantly promoted at a salt concentration of 6‰.

[0015] A fourth aspect of the present invention provides a method for promoting salt-tolerant growth of plants, using rhizobium (Sinorhizobium meliloti. )SmYrd01 strain was used to treat the plants.

[0016] Preferably, the rhizobium (Sinorhizobium meliloti. ) The concentration of SmYrd01 strain was not less than 4×10 9 cfu / mL, and the inoculation amount is 3%~4%.

[0017] The embodiment of the present invention provides a rhizobium strain with salt-tolerant growth-promoting ability, which is convenient for promoting the salt-tolerant growth of plants in saline-alkali land and provides a theoretical basis for further research in saline-alkali land improvement.

[0018] Rhizobium (Sinorhizobium meliloti.) The biological characteristics of the SmYrd01 strain are as follows: the optimum growth temperature is 28 ℃, and it can be cultured aerobically. On YMA plate medium, it forms round or nearly round, sticky, smooth, protruding, grayish white or milky white colonies.

[0019] The present invention shows that the SmYrd01 strain has good salt tolerance and can promote plant growth or promote plant growth in saline-alkali soil environments. After the SmYrd01 strain is prepared into a bacterial agent, the alfalfa seeds are soaked in the bacterial solution. Compared with the blank control, the root length and shoot length of the alfalfa seeds treated with SmYrd01, as well as the root weight and shoot weight of a single plant are increased. Among them, the salt tolerance and growth-promoting effect is most significant under a salt gradient of 6‰, and the root length, shoot length, root weight and shoot weight of alfalfa are increased by 45.04%, 18.49%, 36.08% and 26.21% respectively. Therefore, the SmYrd01 bacterial agent can promote crop growth and has important economic value and practical significance.

[0020] Beneficial effects of the embodiments of the present invention: 1) A rhizobium with salt-tolerant growth-promoting ability provided by the present invention ( Sinorhizobium meliloti. )SmYrd01 analyzed the biological characteristics of the strain, such as salt tolerance, and found that the strain had strong salt tolerance and could grow at a salt concentration of 0.5% to 0.6%, which has important practical significance and value for improving the ecological environment. 2) Rhizobium ( Sinorhizobium meliloti. )SmYrd01 can be prepared into a salt-tolerant growth promoter or plant growth promoter; 3) Rhizobium ( Sinorhizobium meliloti. )SmYrd01 is pollution-free and harmless during use, and can significantly improve the salt tolerance of alfalfa, alleviate salt damage, increase biomass, and promote the growth of alfalfa. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A phylogenetic tree of the strain SmYrd01 provided in an embodiment of the present invention; Figure 2 A graph showing the salt tolerance test results of the strain SmYrd01 provided in an embodiment of the present invention; Figure 3 A salt tolerance curve diagram of the strain SmYrd01 provided in an embodiment of the present invention; Figure 4 The root length of alfalfa under different treatments provided by the embodiment of the present invention; Figure 5 The sprout length of alfalfa under different treatments provided by the embodiment of the present invention; Figure 6 , Figure 7 The biomass of alfalfa under different treatments provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0022] Embodiments of the present embodiment will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present embodiment are shown in the accompanying drawings, it should be understood that the present embodiment can be implemented in various forms and should not be construed as being limited to the embodiments set forth herein. Instead, these embodiments are provided to provide a more thorough and complete understanding of the present embodiment. It should be understood that the drawings and embodiments of the present embodiment are only for exemplary purposes and are not intended to limit the scope of protection of the present embodiment.

[0023] The salt-tolerant growth-promoting bacteria provided in the embodiment of the present invention are Rhizobium ( Sinorhizobium meliloti. ), strain number is SmYrd01, deposit number is CCTCC NO: M 2025024, and was deposited in the China Type Culture Collection Center, Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province on January 6, 2025.

[0024] Example 1 Isolation and purification of strains Healthy alfalfa plants were selected from Wudi County, Binzhou City, Shandong Province. Pink and plump nodules were collected from the lateral roots of the main roots, placed in sample bags, and transported to the laboratory in an ice pack incubator at 4°C. The impurities on the surface of the nodules were rinsed with clean water, and the water was absorbed with sterile filter paper. In the biosafety cabinet, the nodule samples were immersed in 75% ethanol for 3 minutes, 5% sodium hypochlorite solution for 4-6 minutes, and rinsed with sterile water for more than 6 times. The nodules were cut in half with a sterilized knife to expose the internal structure of the rhizobium. The nodules were clamped with sterilized tweezers and streaked on the YMA medium. The streaked culture medium was placed in a constant temperature incubator at 28°C and cultured for 2 to 5 days. Typical colonies that were round, raised, sticky, with neat edges, and slightly transparent or translucent were selected from the plate and purified by streaking in four areas on the YMA medium. The strains after multiple purifications were inoculated into the YMA medium slant and stored at 4°C for a short period of time. The obtained strain was named SmYrd01.

[0025] The composition of YMA solid culture medium is (g / L): 0.5g potassium dihydrogen phosphate, 0.2g magnesium sulfate heptahydrate, 0.1g sodium chloride, 10g D-mannitol, 1.0g yeast powder, 18g agar, 1000ml distilled water, pH 6.8~7.2.

[0026] Figure 1 This is a phylogenetic tree of the strain SmYrd01 provided in the embodiments of the present invention.

[0027] Example 2 Salt tolerance test of rhizobium SmYrd01 The activated rhizobium SmYrd01 was streaked and inoculated into YMA solid medium with salt contents of 0, 0.5, 1, 2, 4, 6, 8, and 10%, respectively, with a total of 5 replicates, and cultured in a biochemical incubator for 72 hours at 28°C to observe its salt tolerance. Figure 2 As shown, rhizobium SmYrd01 grew well at salt contents of 0, 1, 0.5, 2, and 4%, respectively.

[0028] The activated rhizobium SmYrd01 was inoculated into a 250 mL conical flask containing 100 mL YMA medium, and placed in a temperature-controlled shaker for 48 h. The temperature was set to 28°C and the speed was set to 180 r / min. The seed solution was prepared for use. The seed solution was inoculated into YMA medium with different salt contents at a 4% inoculation rate. A total of 5 replicates were set. The culture was shaken in a temperature-controlled shaker for 48 h. The temperature was set to 28°C and the speed was set to 180 r / min. The OD value was measured to determine the salt tolerance of the strain. Figure 3 The salt tolerance curve shown in FIG. 1 shows that the vertical axis represents the bacterial concentration (OD600) and the horizontal axis represents the salt content. Figure 3 As shown in the figure, the rhizobium SmYrd01 still maintains a high bacterial concentration when the salt content is 6%. Figure 3 The results showed that the rhizobium SmYrd01 had a wide adaptability to NaCl and could maintain a high bacterial concentration when the salt concentration reached 6%.

[0029] Example 3 Application of Rhizobium SmYrd01 to Alfalfa Growth In this embodiment, the salt-tolerant growth-promoting bacteria agent is used as an example to promote the salt-tolerant growth of alfalfa.

[0030] 1. Preparation of Rhizobium SmYrd01 Agent The activated SmYrd01 was inoculated into a 250 mL conical flask with 100 mL YMA medium, and placed in a temperature-controlled shaker for 48 h, with the temperature set at 28°C and the speed set at 180 r / min to prepare the seed solution. The seed solution was inoculated into a 500 mL conical flask with 300 mL YMA medium at a 3% to 4% inoculation rate, and was inoculated in a temperature-controlled shaker for 48 h, with the temperature set at 28°C and the speed set at 180 r / min. The cultured fermentation broth was then placed in a centrifuge for centrifugation, the supernatant was discarded, the precipitated bacteria were collected, the centrifugation temperature was set at 4°C, and the speed was set at 10000 r / min.

[0031] The collected precipitated bacteria were resuspended in sterile water (OD600=2±0.01) to obtain a salt-tolerant growth-promoting agent with an effective viable bacterial count of 4×109 cfu / mL for rhizobium SmYrd01.

[0032] 2. Growth-promoting effect of rhizobium SmYrd01 on alfalfa under salt stress In order to verify the effect of rhizobium SmYrd01 on salt tolerance and growth promotion of alfalfa under different salt contents, four salt gradients were set in this experiment, namely 0‰, 2‰, 4‰ and 6‰; two treatments were set in this experiment, namely, adding 4×10 9 cfu / mL of SmYrd01 inoculant and a control group without the addition of the inoculant, with a total of 5 replicates for each treatment. Alfalfa seeds with full grains, uniform size and no pests were selected for a seven-day germination test in an incubator. The culture conditions were set as a photoperiod of 16 h, a temperature of 28°C, a dark treatment of 8 h, a temperature of 22°C, and a humidity of 50%-80%.

[0033] In this embodiment, the growth status of alfalfa seedlings in the SmYrd01 bacterial agent group and the control group on the seventh day, root length, shoot length and total plant biomass, etc. Figure 4-7 shown. Figure 4 The root length of alfalfa under different treatments provided by the embodiment of the present invention; Figure 5 The sprout length of alfalfa under different treatments provided by the embodiment of the present invention; Figure 6 , Figure 7 The biomass of alfalfa under different treatments provided in the embodiments of the present invention.

[0034] The results showed that the plant height and total biomass of alfalfa increased after the addition of the inoculant. The results were particularly significant under a salt gradient of 6‰, where the root length, shoot length, root weight, and shoot weight of alfalfa were 45.04%, 18.49%, 36.08%, and 26.21% higher than those of alfalfa not inoculated with the inoculant, respectively. The application of the SmYrd01 inoculant can significantly improve the salt tolerance and growth-promoting ability of alfalfa under salt stress, slow down salt damage, increase biomass, and thus promote the growth of alfalfa.

Claims

1. A rhizobium with salt tolerance and growth-promoting ability, characterized in that: The rhizobium ( Sinorhizobium meliloti. ) Sm Yrd01 has been deposited in the China Center for Type Culture Collection on January 6, 2025. The deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M 2025024.

2. The rhizobium according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

3. A salt-tolerant growth-promoting bacterial agent or a plant growth-promoting bacterial agent, characterized in that: The salt-tolerant growth-promoting bacteria agent or plant growth-promoting bacteria agent contains the rhizobium or its bacterial solution as described in claim 1.

4. The salt-tolerant growth-promoting bacteria or plant growth-promoting bacteria according to claim 3, characterized in that: The concentration of bacterial solution should not be less than 4×10 9 cfu / mL.

5. Use of the rhizobium or its bacterial solution as claimed in claim 1 in promoting plant growth.

6. The use according to claim 5, characterized in that: The plants include alfalfa.

7. Use of the rhizobium or its bacterial solution as claimed in claim 1 in the preparation of salt-tolerant growth-promoting bacteria or plant growth-promoting bacteria.

8. Use of the rhizobium or bacterial solution thereof as claimed in claim 1 in improving the salt tolerance of alfalfa.

9. A method for promoting salt-tolerant growth of plants, characterized in that: Plants are treated with the rhizobium strain described in claim 1.

10. A method for promoting salt-tolerant growth of plants as claimed in claim 9, characterized in that: The concentration of the rhizobium strain is not less than 4×10 9 cfu / mL, and the inoculation amount is 3%~4%.

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