Loess rhizobium and duplex bag rhizobium inoculant adopting loess rhizobium

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to loess rhizobium and a duplex bag rhizobium inoculant adopting the loess rhizobium. The rhizobium can provide nitrogen nutrition guarantee for good growth of Astragalus mongholicus plants as a Zhongmongolian medicinal material, solves the important problem of long-term lack of nitrogen nutrition in soil, and is extremely urgent to screen and develop the rhizobium with high nitrogen fixation efficiency and high competitive nodulation efficiency. The loess rhizobium duplex bag rhizobium inoculant provided by the invention has a growth promoting effect on plants and can be inoculated at any time in the whole growth period of the plants. The preparation method and the application method are simple and easy to implement, the problem that liquid dosage forms are prepared from radix astragali rhizobium is solved, rhizobium can be inoculated at any time, the nodulation rate and the nitrogen fixation rate are increased, and therefore the yield and the quality of radix astragali are improved; the rhizobium inoculant is prepared by adopting a duplex bag method, so that the problem of low nodulation rate of the rhizobium inoculant under the condition of nutrition deficiency is solved, and on the other hand, the problem that perennial astragalus mongholicus is inoculated with the inoculant for multiple times is solved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a loess rhizobium and a double-bag rhizobium agent using it. Background Technology

[0002] Astragalus is one of the most widely used traditional Chinese medicinal herbs. Astragalus polysaccharides in astragalus have effects such as enhancing the immunity of humans and livestock, and strengthening the body's defenses. With the increasing awareness of health and wellness, the production of traditional Chinese medicinal materials has ushered in a period of development.

[0003] Currently, commercially available Astragalus membranaceus is mainly produced through artificial cultivation. However, the cultivation process lacks scientific fertilization guidance, with indiscriminate application of chemical fertilizers in pursuit of higher yields. This leads to nitrogen loss, soil acidification, decreased organic matter content, and frequent pest and disease outbreaks, significantly reducing both the yield and quality of Astragalus membranaceus. As a legume, Astragalus membranaceus can interact with rhizosphere microorganisms to form nodules and fix nitrogen. Rhizobia play a highly efficient symbiotic nitrogen-fixing role, providing nitrogen nutrition for the healthy growth of Astragalus membranaceus, a traditional Chinese and Mongolian medicinal herb. This addresses the long-standing problem of nitrogen deficiency in the soil, continuously increasing the accumulation of effective components in Astragalus membranaceus and improving its quality. Therefore, screening and developing rhizobia with high nitrogen-fixing efficiency and high competing nodulation efficiency is extremely urgent. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned problems and provide a rhizobium in loess soil and a rhizobium agent in double-bag bags using the same. The rhizobium and the agent have a growth-promoting effect on plants and can be inoculated at any time during the entire growth cycle of plants.

[0005] The above objective is achieved by the following technical solution: A rhizobium of loess soil, characterized by: This rhizobium is deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 4, 2024, with accession number CGMCC NO.31858.

[0006] The method for culturing rhizobium in loess soil is characterized by: The loess rhizobium was inoculated into a fermentation medium and cultured. This culture medium comprises the following components: Potassium dihydrogen phosphate 0.25 g / L, dipotassium hydrogen phosphate 0.25 g / L, MgSO4•7H2O 0.2 g / L, NaCl 0.1 g / L, yeast extract 3 g / L, mannitol 10 g / L, pH 6.6~7.2.

[0007] The method for culturing rhizobium in loess soil is characterized by: The cultivation method and conditions were as follows: shaking culture, cultivation temperature 27-29℃, cultivation time 2-3 days, and rotation speed 180 rpm.

[0008] A dual-bag rhizobium agent using the aforementioned loess rhizobium, characterized in that: This rhizobium agent contains loess rhizobium with accession number CGMCC NO.31858; This rhizobium agent contains trace elements that promote nitrogen fixation.

[0009] The trace elements include the following components: H3BO32.86 g / L, MnSO41.81 g / L, ZnSO40.22 g / L, CuS04·5H2O 0.8 g / L, H2MoO40.02 g / L.

[0010] The dual-bag rhizobium agent is suitable for Astragalus membranaceus, and it can improve the nodulation rate and nitrogen fixation capacity of Astragalus membranaceus. The addition of trace elements while applying the rhizobium agent ensures the smooth progress of the nodulation and nitrogen fixation process in barren areas.

[0011] The method of using the dual-bag rhizobium agent is characterized by: This application method involves applying the rhizobium agent by one or more of the following methods: seed dressing (after drying the seeds in the shade before planting), spraying, or drip irrigation. The viable count of rhizobia in the rhizobium agent is not less than 1×10⁻⁶. 8 CFU / mL.

[0012] The beneficial effects of this invention are: (1) The liquid dosage form preparation method and application method of the present invention are simple and easy to implement, which solves the problem of preparing liquid dosage form of Astragalus rhizobium. Rhizobium can be inoculated at any time to improve the nodulation rate and nitrogen fixation rate, thereby improving the yield and quality of Astragalus. (2) Currently, the Astragalus rhizobium agent on the market is prepared by only one type of rhizobium and protectant. In comparison, this application uses a double-bag method to prepare the rhizobium agent, which solves the problem of low nodulation rate under nutrient deficiency conditions. On the other hand, it solves the problem of multiple inoculation of perennial Mongolian Astragalus with fungal agent. Attached Figure Description

[0013] The attached image is as follows: Figure 1 This is a colony diagram of rhizobium in loess soil involved in this invention; Figure 2This is a comparison chart of experimental results in the double-layer pot inoculation experiment of Embodiment 5 of the present invention. In the chart, data such as plant height, fresh weight of plants, number of nodules, root length, fresh weight of roots and diameter of Astragalus membranaceus in the control group and the inoculation group were collected and statistically analyzed.

[0014] Preservation of biological materials: The rhizobium in Inner Mongolia rhizobium involved in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is September 4, 2024, the accession number is CGMCC NO.31858, and the name is *Rhizobium inner Mongolia* subsp. *loessense*. Rhizobium mongolense subsp.loessense ). Detailed Implementation

[0015] The Astragalus rhizobium provided by this invention was isolated and differentiated from samples of Astragalus rhizobium grown in agriculture in Naiman Banner, Tongliao City, Inner Mongolia. The colonies are characterized as round, colorless, smooth, raised, and moist. After processing the strain... recA Gene sequence analysis identified it as *Astragalus rhizobium*. This strain is also deposited in the Agricultural Product Quality Inspection Laboratory of Inner Mongolia University for Nationalities. The *Astragalus rhizobium* provided by this invention exhibits high nodulation rate and strong nitrogen fixation capacity, which can reduce the amount of chemical nitrogen fertilizer used in *Astragalus* cultivation, reduce nitrogen fertilizer pollution to the environment, promote the green cultivation of *Astragalus* in Inner Mongolia, and better protect the local ecological environment.

[0016] Example 1: Preparation method of loess rhizobium inoculant.

[0017] The refrigerated loess rhizobium was transferred to YMA solid medium and cultured at 28°C for three days.

[0018] The rhizobium of loess was inoculated into YMA liquid medium and cultured at 28°C for three days to obtain seed culture. The seed culture was then transferred to YMA liquid medium and cultured at 28°C for three days.

[0019] The YMA culture medium comprises the following components: Potassium dihydrogen phosphate 0.25 g / L, dipotassium hydrogen phosphate 0.25 g / L, MgSO4•7H2O 0.2 g / L, NaCl 0.1 g / L, yeast extract 3 g / L, mannitol 10 g / L, pH 6.6~7.2 (adjust the pH of the culture medium to the range of 6.6~7.2).

[0020] The cultivation method and conditions were as follows: shaking culture, cultivation temperature 27–29℃ (preferably 28℃), cultivation time 2–3 days (preferably 3 days), and rotation speed 180 rpm. After cultivation, the viable count of *Rhizobium loessense* was 1 × 10⁻⁶. 8 CFU / mL.

[0021] After the bacterial solution is mixed, it is aseptically filled. Separately, aseptic trace elements are also filled in. The two components together form the loess rhizobium double-bag rhizobium agent.

[0022] The trace elements include the following components: H3BO32.86 g / L, MnSO41.81 g / L, ZnSO40.22 g / L, CuS04·5H2O 0.8 g / L, H2MoO40.02 g / L.

[0023] Adding trace elements while applying rhizobium inoculants ensures the smooth progress of nitrogen fixation and nodulation in barren areas.

[0024] Example 2: Application method of rhizobium inoculants.

[0025] This embodiment provides a method for improving the nodulation rate and nitrogen fixation capacity of Astragalus membranaceus. The method includes the following steps: mixing the two components in a double-layered bag, treating the seeds with a rhizobium agent, air-drying them, and then sowing them. This method can effectively improve the survival rate of Astragalus membranaceus in the early stages of its growth.

[0026] Seeds can be treated with rhizobium inoculation in double-bag bags and sown after air-drying. Alternatively, they can be inoculated into the roots of Astragalus membranaceus throughout its growth cycle via the irrigation system.

[0027] Since Astragalus is a perennial plant, the rhizobium inoculants currently on the market are only inoculated once at the time of sowing by seed dressing, and cannot be inoculated with rhizobium in the following years, which seriously affects the nitrogen fixation effect of Astragalus. The liquid rhizobium inoculant of the present invention can be applied to the roots of Astragalus through the irrigation system, so that the rhizobium can reach the roots to form nodules and fix nitrogen.

[0028] Example 3: Isolation and identification of Astragalus rhizobium.

[0029] The rhizobium of this invention was isolated and differentiated from samples of Astragalus membranaceus rhizobia grown in Naiman Banner, Tongliao City, Inner Mongolia, and is deposited at Inner Mongolia University for Nationalities, strain number IMUNJ 23057. The colonies of this invention are characterized by being round, colorless, smooth, raised, and moist, with no color. See attached figure for details. Figure 1 .

[0030] Gene identification: Total DNA was extracted from rhizobia using the GUTC method and amplified by PCR. recAThe gene sequences were compared with those of the model strain for phylogenetic analysis.

[0031] Example 4: Screening for high-efficiency strains under vermiculite conditions.

[0032] First, the symbiotic nitrogen-fixing ability of Astragalus membranaceus grown in vermiculite was verified by inoculating the first batch of rhizobium populations isolated from Naiman, Inner Mongolia. The screening indicators included: leaf chlorophyll content, plant height, aboveground dry weight, underground dry weight, number of root nodules, and root nodule dry weight.

[0033] Competitive nodulation under vermiculite conditions: Rhizobium was inoculated into Astragalus membranaceus, and the growth indicators of Astragalus membranaceus were measured.

[0034] Verification of nitrogen fixation capacity of soil mixed with vermiculite: Soil from Naiman, Inner Mongolia, was mixed with vermiculite and inoculated separately under different nitrogen conditions, while mixed strains were also inoculated to verify the nitrogen fixation and growth promotion capacity of the high-efficiency strains in the soil.

[0035] Example 5: Double-layer pot inoculation experiment.

[0036] 1. Seed germination.

[0037] Select plump, undamaged Astragalus membranaceus seeds, ensuring their integrity and health. Pour the seeds into a sterile Erlenmeyer flask and add enough 95% ethanol to completely cover the seed surface. Gently shake the flask for 30 seconds. The ethanol removes oils and impurities from the seed surface and reduces surface tension, preparing the seeds for subsequent sterilization. After shaking for 30 seconds, discard the ethanol.

[0038] Next, add a 2.4% sodium hypochlorite solution, ensuring complete coverage of the seeds. Gently shake the Erlenmeyer flask to ensure even contact of the sodium hypochlorite solution with the seeds, and sterilize for 5 minutes. After sterilization, discard the sodium hypochlorite solution and rinse the seeds with sterile, cooled, and sterile water. Each time you wash, pour in sterile water and gently shake the flask to thoroughly remove any sodium hypochlorite residue. Repeat the rinsing process 5-7 times to ensure complete removal of any sodium hypochlorite residue to avoid affecting subsequent experiments. After rinsing, evenly distribute the sterile seeds on a 0.8% water agar plate, ensuring sufficient space between seeds to facilitate uniform germination.

[0039] Finally, place the plates in a 28°C constant temperature incubator and incubate in the dark for 36-48 hours to promote seed germination.

[0040] 2. Prepare the inoculation solution.

[0041] 2.1 Rhizobium activation and culture.

[0042] First, obtain the Rhizobium strain to be inoculated from the strain bank. Using a sterile inoculation loop, pick an appropriate amount of cells from the inoculum tube and inoculate it onto the surface of a pre-prepared solid culture medium (yeast mannitol agar, YMA).

[0043] After inoculation, the culture medium is placed in a constant temperature incubator at 28°C for 2-3 days until obvious and uniform single colony growth is observed, ensuring the health and consistency of the colonies.

[0044] 2.2 Transfer to liquid culture.

[0045] Once a single colony has grown well on the solid culture medium, select a healthy single colony and pick up an appropriate amount of bacteria using a sterile inoculation loop.

[0046] The bacterial cells were then inoculated into a pre-prepared liquid culture medium (YMA) and placed on a shaker at 28°C for shake culture at 150-200 rpm.

[0047] Rhizobium is usually cultured in liquid medium until the logarithmic growth phase (approximately 24 hours), at which point the cell concentration reaches the required OD600 value.

[0048] 2.3 Bacterial washing.

[0049] When the OD600 value of the bacterial culture reaches 1.0-2.0, stop the culture and remove the culture flask from the shaker to prepare for bacterial cell collection. Pour the bacterial culture into a sterile centrifuge tube and centrifuge at low speed (4000-5000 rpm) for 10 minutes to precipitate the bacterial cells.

[0050] After centrifugation, carefully discard the supernatant, retaining the precipitated bacterial cells. Add 0.8% sterile saline to the centrifuge tube, gently resuspend the bacterial cells, and ensure thorough mixing by gentle agitation. This step aims to wash the bacterial cells and remove residual culture medium components. Repeat this washing step 1-2 times to ensure the purity of the bacterial suspension.

[0051] 2.4 Adjust the OD600 value.

[0052] The washed bacterial cells were resuspended in 0.8% sterile physiological saline, and the OD600 value was measured using a spectrophotometer. If necessary, the bacterial suspension was diluted with sterile physiological saline to adjust the OD600 to 0.2. After adjustment, the bacterial suspension was stored under sterile conditions for use in subsequent experiments.

[0053] 3. Preparation of a double-layer bottle structure.

[0054] 3.1 Preparation and sterilization of vermiculite.

[0055] Use sterile instruments or your hands to thoroughly mix the vermiculite with a low-nitrogen nutrient solution diluted 20 times. During mixing, ensure that each portion of vermiculite is evenly moistened, but not over-moistened.

[0056] The standard for judging the moisture content of a mixture is: when vermiculite is squeezed in the hand, it should form a loose, uncompacted clump structure without any liquid seepage.

[0057] The mixed vermiculite is placed into a heat-resistant sterile bag or sterilization container, ready for autoclaving. The vermiculite is steam-sterilized in an autoclave at 121°C for 90 minutes. After sterilization, the vermiculite is allowed to cool naturally to room temperature before use.

[0058] 3.2 Preparation of canning jars and plastic cups.

[0059] Add an appropriate amount of deionized water to the canning jar and seal it with sealing film. Select a plastic cup of suitable size, ensuring its height is suitable for placing in the canning jar while leaving some air space. Cut an appropriate amount of sterile gauze strips and thread them through the small holes at the bottom of the plastic cup, allowing one end of the gauze strip to extend outside the cup. Both the canning jar and the plastic cup with the gauze strip need to be sterilized. Place them separately in a heat-resistant sterilization bag or container and autoclave them at 121°C for 30 minutes. After sterilization, remove the plastic cup and place it on a sterile operating table to dry for later use.

[0060] 4. Assemble the double-layered bowl in the clean bench.

[0061] Place all sterilized materials on a laminar flow hood, ensuring the entire assembly process is performed in a sterile environment to avoid external contamination. Take a sterilized glass jar and place a sterilized plastic cup inside, ensuring the gauze strip at the bottom of the cup is in contact with the water in the jar. Ensure the plastic cup is securely placed in the jar, leaving adequate space between the cup opening and the jar to allow for ventilation.

[0062] Evenly fill the sterilized and cooled vermiculite into the plastic cup, gently pressing it down but not too tightly to ensure sufficient air circulation for root growth. The vermiculite should be at a moderate height to guarantee optimal root growth conditions.

[0063] 5. Seedlings and cultivation.

[0064] In the previous step, sterilized and germinated Astragalus mongholicus seeds were obtained. Select healthy and successfully germinated seeds, ensuring that each seed has a distinct radicle (root tip).

[0065] On the clean bench, remove the assembled double-layered pot. Gently place each healthy germinating seed into the double-layered pot, ensuring the root end is facing down and in contact with the vermiculite. The previously adjusted OD600 to 0.2 bacterial suspension is now at the standard inoculation concentration. Using a sterile pipette, draw 1 mL of the rhizobium suspension and evenly drop it around the seed roots, ensuring the rhizobia quickly contact and adhere to the root tip.

[0066] After sealing, the double-layered pots were gently transferred to an artificial light cultivation room. The cultivation room was set to a constant temperature of 28°C to simulate the natural growth temperature of plants, and the light cycle was set to 16 hours of light / 8 hours of darkness.

[0067] On the third day of seed culture, use sterile scissors or a scalpel to cut a small cross-shaped notch in the double-layer pot sealing film. The cross-shaped notch should be located at the seedling growth position and be of appropriate size.

[0068] During the 45-day cultivation period, the humidity of the double-layered pots needs to be checked regularly. The vermiculite absorbs moisture from the deionized water in the jars through the gauze strips at the bottom, maintaining a moderate level of moisture. The deionized water in the jars should be replenished periodically as needed to prevent the vermiculite from drying out excessively and affecting plant root growth. The plants go through stages such as germination, growth, and root nodule formation during cultivation. Plant growth should be observed regularly, and the development of leaves, stems, roots, and root nodule formation should be recorded.

[0069] The experimental results, after statistical analysis, are shown in the attached figure. Figure 2 As shown.

[0070] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A rhizobium of loess soil, characterized in that: This rhizobium is deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 4, 2024, with accession number CGMCC NO.31858.

2. The method for culturing rhizobia in loess soil according to claim 1, characterized in that: The loess rhizobium was inoculated into a fermentation medium and cultured. This culture medium comprises the following components: Potassium dihydrogen phosphate 0.25 g / L, dipotassium hydrogen phosphate 0.25 g / L, MgSO4•7H2O 0.2 g / L, NaCl 0.1 g / L, yeast extract 3 g / L, mannitol 10 g / L, pH 6.6~7.

2.

3. The method for culturing rhizobium in loess soil according to claim 2, characterized in that: The cultivation method and conditions were as follows: shaking culture, cultivation temperature 27-29℃, cultivation time 2-3 days, and rotation speed 180 rpm.

4. A dual-bag rhizobium agent using the loess rhizobium as described in claim 1, characterized in that: This rhizobium agent contains loess rhizobium with accession number CGMCC NO.31858; This rhizobium agent contains trace elements that promote nitrogen fixation.

5. The double-bag rhizobium agent according to claim 4, characterized in that: The trace elements include the following components: H3BO3 2.86 g / L, MnSO4 1.81 g / L, ZnSO4 0.22 g / L, CuS04 •5H2O 0.8 g / L, H2MoO40.02 g / L.

6. The double-bag rhizobium agent according to claim 4 or 5, characterized in that: The aforementioned double-bag rhizobium agent is suitable for Astragalus membranaceus, and it can improve the nodulation rate and nitrogen fixation capacity of Astragalus membranaceus. By adding trace elements while applying the rhizobium agent, the process of nodulation and nitrogen fixation can be ensured to proceed smoothly in barren areas.

7. The double-bag rhizobium agent according to claim 6, characterized in that: The method of using the dual-bag rhizobium agent is as follows: Apply the rhizobium agent using one or more of the following methods: seed dressing, spraying, or drip irrigation system; The viable count of rhizobia in the rhizobium agent is not less than 1×10⁻⁶. 8 CFU / mL.

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