Method for preparing super-stable methylobacterium nodulans pre-inoculation inoculant and application method thereof

Abstract

The application discloses a preparation method of super-stable Astragalus sinicus rhizobium pre-inoculation inoculant. The method comprises the following steps: step one, optimizing culture medium; the optimized Astragalus sinicus rhizobium special culture medium GSG is as follows: glycerol 5-20 g / L, sodium succinate 0.1-0.5 g / L, sodium glutamate 0.1-0.5 g / L, potassium dihydrogen phosphate 0.1-0.8 g / L, disodium hydrogen phosphate 0.1-1 g / L, magnesium sulfate heptahydrate 0.1-0.5 g / L, yeast powder 1-2 g / L and proteose peptone 0.1-1 g / L; step two, preparing a freeze-drying protective agent; step three, preparing Astragalus sinicus rhizobium freeze-dried agent; and step four, determining the viable cell count of Astragalus sinicus rhizobium freeze-dried bacterial powder and storing in a warehouse. The application has the advantages that the survival period of rhizobium on seeds is prolonged to 3-6 months, and the nitrogen fixation efficiency is improved. The application further discloses an application method of the super-stable Astragalus sinicus rhizobium pre-inoculation inoculant.

Description

Technical Field

[0001] This invention relates to the field of rhizobium application technology, specifically to a method for preparing an ultra-stable *Astragalus clover* rhizobium pre-inoculation agent, and more specifically, to a method for preparing a lyophilized formulation of the ultra-stable *Astragalus clover* rhizobium pre-inoculation agent. This invention also relates to an application method for the lyophilized formulation of the ultra-stable *Astragalus clover* rhizobium pre-inoculation agent. Background Technology

[0002] Milk vetch, a legume green manure crop unique to China, is widely cultivated in paddy fields in the south. It is typically sown in autumn and plowed back into the soil the following spring, providing abundant organic matter and nutrients to the paddy fields. It significantly increases the organic matter and nitrogen, phosphorus, and potassium content of the paddy fields. In the era before chemical fertilizers, milk vetch was a major source of fertilizer for paddy fields. The widespread use of chemical fertilizers has drastically reduced the planting area of ​​milk vetch, and the overuse of fertilizers has led to a series of environmental and health consequences. Therefore, the country has proposed a zero-growth strategy for chemical fertilizers and is gradually reducing their usage. Rice is my country's largest grain crop, with an annual planting area of ​​450 million mu (approximately 30 million hectares). Rice cultivation consumes a large amount of chemical fertilizers. To achieve the strategic goal of reducing chemical fertilizer application, it is essential to reduce fertilizer use in rice cultivation. Against this backdrop, planting milk vetch has become an important measure for reducing chemical fertilizer application.

[0003] Milk vetch is a legume that can form nodules with rhizobia and fix nitrogen, providing additional nitrogen fertilizer for paddy fields. Inoculation with a high-quality milk vetch rhizobia inoculant can improve nodulation and symbiotic nitrogen fixation efficiency, as well as fresh grass yield. Each acre of milk vetch green manure yields 2000–4000 kg of fresh grass, and each 1000 kg can fix approximately 3.75 kg of nitrogen, equivalent to 15–30 kg of urea per acre. Emission reduction benefit estimate: For every ton of nitrogen fertilizer produced and applied in China, 13.5 tons of CO2 are emitted (New technologies reduce greenhouse gasemissions from nitrogenous fertilizer in China Wei-feng Zhang, Zheng-xia Dou, Pan He, Xiao-Tang Ju, David Powlson, Dave Chadwick, David Norse, Yue-Lai Lu, Ying Zhang, Liang Wu, Xin-Ping Chen, Kenneth G. Cassman, and Fu-Suo Zhang, PNAS. 2013, vol. 110(21) 8375–8380).

[0004] Outlook: The rice-growing area in the south is approximately 300 million mu (20 million hectares), while the dryland area in the north suitable for green manure cultivation reaches 500 million mu (33 million hectares), with a vast area fallow in winter, most of which is suitable for milkvetch cultivation. Currently, green manure cultivation only accounts for about 10% (30 million mu) of rice paddy area. If the cultivation area of ​​rice-green manure milkvetch and dryland green manure is further increased, the economic and ecological benefits will be enormous.

[0005] To achieve efficient nitrogen fixation, inoculation with rhizobia is necessary to form root nodules on the roots of milkvetch. Generally, inoculating with superior rhizobia, and ensuring their survival on the milkvetch roots, can increase nitrogen fixation efficiency by approximately 10-30%. Currently, the most commonly used milkvetch rhizobia inoculant is in liquid form. Milkvetch rhizobia is a Gram-negative bacterium that does not produce desiccation-resistant spores. Liquid milkvetch rhizobia inoculants dry out within minutes of seed treatment, resulting in a very short survival period for the rhizobia on seeds. Over 90% of the rhizobia on milkvetch seeds die within 24 hours. Therefore, liquid rhizobia inoculants can only be used for seed treatment at sowing. Farmers typically pour milkvetch seeds from bags into large plastic basins or other containers, open the rhizobia packaging, and mix the rhizobia solution with the milkvetch seeds at a ratio of 3-5 ml / kg of seeds. This complex field treatment process is what makes farmers reluctant to use rhizobia. Furthermore, the short survival time of liquid rhizobia on seeds affects nitrogen fixation efficiency (currently, only by treating milkvetch seeds with milkvetch rhizobia at the time of sowing and immediately sowing can the effect of increasing nitrogen fixation efficiency by 10-30% be achieved. If the seeds are treated in advance, the milkvetch rhizobia die quickly, and the improvement in nitrogen fixation efficiency by milkvetch rhizobia cannot reach 10-30%, or even the improvement effect is zero). To overcome this constraint, a new technology and product are needed to greatly extend the survival period of rhizobia on seeds. Rhizobia would be mixed with seeds during seed processing, so farmers would receive seeds already inoculated with rhizobia, eliminating the need for further treatment at sowing. This would extend the survival period of rhizobia on seeds from 1-2 days to 3-6 months. This technology will break through the bottleneck in the application of milkvetch rhizobia and greatly improve the application rate of rhizobia in my country.

[0006] Because *Rhizobium clavatum* adapts to the high-humidity ecological environment of paddy fields and thrives in humid conditions, it has a long survival period in water but struggles to survive in dry environments. Enabling *Rhizobium clavatum* to survive under dry conditions presents a significant technical challenge, which is the main reason why there is currently no *Rhizobium clavatum* dry powder formulation available. There are currently no precedents for the production technology and application of *Rhizobium clavatum* pre-inoculation agents, either domestically or globally.

[0007] Therefore, it is necessary to develop a method for preparing and applying Rhizobium tumefaciens dry powder to prolong its survival period. Summary of the Invention

[0008] The primary objective of this invention is to provide a method for preparing an ultra-stable pre-inoculated agent of *Astragalus membranaceus* rhizobium, which significantly extends the survival period of *Astragalus membranaceus* on seeds. During seed processing, the freeze-dried *Astragalus membranaceus* rhizobium powder is mixed with the seeds. Since the seeds purchased by farmers are already inoculated with rhizobium, there is no need to mix the seeds with rhizobium again before sowing. This extends the survival period of rhizobium on seeds from 1-2 days to 3-6 months, greatly increasing the application rate of rhizobium in my country.

[0009] The second objective of this invention is to provide a method for applying an ultra-stable pre-inoculation agent of *Rhizobium arvense*, which extends the survival period of *Rhizobium arvense* on seeds from 1-2 days to 3-6 months, greatly improving the application rate of *Rhizobium arvense* in my country and increasing nitrogen fixation efficiency.

[0010] To achieve the first objective of this invention, the technical solution is as follows: a method for preparing an ultra-stable *Astragalus membranaceus* rhizobium pre-inoculation agent, characterized by comprising the following steps.

[0011] Step 1: Optimize the culture medium;

[0012] The optimized GSG culture medium for Rhizobium oxyclover is as follows:

[0013] Glycerol 5–20 g / L, sodium succinate 0.1–0.5 g / L, monosodium glutamate 0.1–0.5 g / L, potassium dihydrogen phosphate 0.1–0.8 g / L, disodium hydrogen phosphate 0.1–1 g / L, magnesium sulfate heptahydrate 0.1–0.5 g / L, yeast extract 1–2 g / L. Peptone 0.1–1 g / L;

[0014] Step 2: Prepare the freeze-drying protectant;

[0015] Step 3: Prepare the freeze-drying agent of *Astragalus membranaceus* rhizobium;

[0016] (1) Culture of Rhizobium from Astragalus membranaceus:

[0017] 2 tons of fermentation tank-grade culture medium and 1.4 tons of GSG astragalus rhizobium-specific culture medium.

[0018] The fermentation conditions were: temperature 28℃, pH 6.8-7.2, dissolved oxygen controlled between 50% and 85%, stirring speed 150 rpm, aeration rate 0.3-1.0 vvm, and fermentation time 68 hours.

[0019] (2) The cultured Astragalus membranaceus inoculum was concentrated using a continuous centrifuge at a concentration ratio of 80:1, that is, 1.4 tons of Astragalus membranaceus inoculum was concentrated into 17.5 liters of Astragalus membranaceus inoculum concentrated inoculum.

[0020] (3) Add a certain amount of freeze-drying protectant to the concentrated solution of Astragalus membranaceus root nodules;

[0021] (4) Pre-freeze the concentrated mixed bacterial solution of milkvetch root nodules at -50℃ for 4-10 hours;

[0022] (5) Place the pre-frozen concentrated mixed bacterial solution of purple clover root nodules into a freeze dryer;

[0023] The freeze-drying conditions were: pressure 0.02 mBar, cold trap temperature -50℃;

[0024] After freeze-drying for 48 hours, the freeze-dried mycelium powder of Rhizobium cuspidatum was extracted.

[0025] Step 4: Determine the viable count of the freeze-dried Rhizobium tumefaciens powder and store it in a warehouse.

[0026] In the above technical solution, glycerol is 9-11 g / L, sodium succinate is 0.1-0.3 g / L, monosodium glutamate is 0.2-0.4 g / L, potassium dihydrogen phosphate is 0.4-0.6 g / L, disodium hydrogen phosphate is 0.9-1 g / L, magnesium sulfate heptahydrate is 0.1-0.3 g / L, and yeast extract is 1.1-1.3 g / L. Peptone 0.7–0.9 g / L.

[0027] In the above technical solution, in step one, the optimized GSG culture medium for *Astragalus membranaceus* rhizobia is:

[0028] Glycerol 10 g / L, Sodium succinate 0.2 g / L, Monosodium glutamate 0.3 g / L, Potassium dihydrogen phosphate 0.5 g / L, Disodium hydrogen phosphate 0.95 g / L, Magnesium sulfate heptahydrate 0.2 g / L, Yeast extract 1.2 g / L Peptone 0.8 g / L.

[0029] In the above technical solution, in step two, the amount of freeze-drying protectant added is 40%-60% (V / W) of the concentrated clover rhizobium solution, and the mixture is stirred for 10-20 minutes to fully dissolve the freeze-drying protectant, thereby obtaining the concentrated mixed clover rhizobium solution.

[0030] In the above technical solution, in step two, the components of the freeze-drying protectant are selected from at least a variety of emulsifiers, humectants, preservatives, binders, skim milk powder, thickeners, surfactants, amino acids, antioxidants, and buffers.

[0031] The method for preparing the freeze-drying protectant is as follows: simply mix the components of the freeze-drying protectant in a certain proportion until homogeneous;

[0032] The emulsifier used is lecithin powder;

[0033] The moisturizers selected are trehalose and sorbitol;

[0034] Sodium alginate was chosen as the preservative.

[0035] The adhesive used is polyvinylpyrrolidone;

[0036] The thickener is selected from one or more of xanthan gum, dextrin, and guar gum;

[0037] The surfactant is selected from betaine and mercaptoethanol.

[0038] The amino acid selected is one of monosodium glutamate, proline, or glycine.

[0039] Ascorbic acid was chosen as the antioxidant.

[0040] K2HPO4 was selected as the buffer. 4、 KH2PO4.

[0041] In the above technical solution, the formulation of the freeze-drying protectant in step two is as follows:

[0042] Lecithin powder 20%; Trehalose 15%; Sorbitol 5%; Sodium alginate 20%; Polyvinylpyrrolidone 10%; Skim milk powder 20%; Xanthan gum 1%; Betaine 2%; Monosodium glutamate 7%.

[0043] In the above technical solution, in step two, the formulation of the freeze-drying protectant is as follows: 20% lecithin powder; 15% trehalose; 5% sorbitol; 20% sodium alginate; 10% polyvinylpyrrolidone; 20% skim milk powder; 0.1% mercaptoethanol; 5% proline; and 4% dextrin.

[0044] In the above technical solution, in step two, the formulation of the freeze-drying protectant is as follows: 20% lecithin powder; 15% trehalose; 5% sorbitol; 20% sodium alginate; 10% polyvinylpyrrolidone; 20% skim milk powder; 1% ascorbic acid; 1% guar gum; 5% glycine; 1.5% K2HPO4; 1.5% KH2PO4.

[0045] To achieve the second objective of this invention, the technical solution is as follows: a method for applying an ultra-stable *Astragalus membranaceus* rhizobium pre-inoculation agent, characterized by comprising the following steps.

[0046] Step 1: Dilute the freeze-dried powder of Rhizobium oxyclover;

[0047] The bacterial count of the freeze-dried Rhizobium tumefaciens powder was diluted to 100-200 billion / gram using a suitable powder carrier; the powder carrier was selected from at least one of corn starch, kaolin, and phosphate rock powder.

[0048] Step 2: Prepare the antioxidant solution;

[0049] The prepared combined antioxidant is: propyl gallate : β-(4-hydroxyphenyl-3,5-di-tert-butyl)propionate n-octadecyl alcohol ester = 5 : 1. The formulated amounts of propyl gallate and β-(4-hydroxyphenyl-3,5-di-tert-butyl)propionate n-octadecyl alcohol ester are dissolved in propylene glycol to a concentration of 1% (w / v). This is the antioxidant solution.

[0050] Step 3: Before seed dressing, mix the antioxidant solution with the freeze-dried powder of Rhizobium clavatum to obtain Rhizobium clavatum powder; Antioxidant solution: freeze-dried powder of Rhizobium clavatum = 0.05:100;

[0051] Step 4: Seed dressing;

[0052] Mix the astragalus root nodule powder with clean astragalus seeds, the ratio of astragalus root nodule powder to clean astragalus seeds is 5:1000;

[0053] The powder of Astragalus membranaceus rhizobia was electrostatically adsorbed onto the surface of Astragalus membranaceus seeds, and the Astragalus membranaceus seeds with the powder adsorbed were stored in a warehouse.

[0054] Step 5: Periodically measure the number of viable bacteria on the milkvetch seeds after seed dressing in Step 4. After seed dressing, milkvetch seeds do not need to be treated with rhizobia again before sowing; they can be sown directly.

[0055] In the above technical solution, the method for determining the number of viable bacteria on the treated milkvetch seeds in step five is as follows:

[0056] Accurately weigh 100 seeds of the treated milk vetch and place them in a 50 ml sterile container. Add 10 ml of sterile buffer solution and 20% skim milk powder solution. Shake thoroughly to wash off the milk vetch rhizobia from the treated milk vetch seeds. This is the first tube. Dilute the milk vetch rhizobia solution in the first tube by 10 times to make two more tubes, which are the second and third tubes, respectively.

[0057] Take 0.1 ml of *Astragalus membranaceus* rhizobium solution from tubes 1, 2, and 3 respectively and spread it on YEM plates; after incubation at 28°C for 4 days, check the number of *Astragalus membranaceus* rhizobium colonies.

[0058] Calculation formula:

[0059] The number of viable milk vetch rhizobia bacteria per milk vetch seed = (N x 10) n+1 )÷100

[0060] Where: N represents the number of colonies on the plate; n represents the corresponding dilution tube.

[0061] The "ultra-stable" nature of the *Acer clover* rhizobium pre-inoculation agent refers to its ability to survive on seeds for more than 3 months (after storage at 15°C for 6 months, the viable number of rhizobia on each *Acer clover* seed is approximately 1347 CFU, greater than 1000 CFU, meeting the requirements of practical production). In contrast, existing *Acer clover* rhizobia generally survive on seeds for only 3-5 days (i.e., existing liquid agents only survive on seeds for 1-5 days; after storage at 15°C for 1-5 days, the viable number of rhizobia on each *Acer clover* seed is less than 1000 CFU, failing to meet the requirements of practical production).

[0062] The present invention has the following advantages:

[0063] This method significantly extends the survival period of rhizobium on seeds. When processing seeds, freeze-dried rhizobium powder is mixed with the seeds. Farmers can then purchase seeds that are already inoculated with rhizobium, eliminating the need for seed dressing before sowing. This extends the survival period of rhizobium on seeds from 1-2 days to 3-6 months, greatly increasing the application rate of rhizobium in my country and improving nitrogen fixation efficiency. Detailed Implementation

[0064] The following detailed description of embodiments of the present invention does not constitute a limitation thereof, but is merely illustrative. The description aims to make the advantages of the present invention clearer and easier to understand.

[0065] This invention is a pre-inoculation agent of *Rhizobium clavatum* with freeze-dried mycelial powder as the core, and a special protectant is developed to enable *Rhizobium clavatum* to survive on the surface of dried seeds for 3-6 months, resulting in an ultra-stable pre-inoculation agent.

[0066] A method for preparing an ultra-stable pre-inoculated agent of *Astragalus membranaceus* rhizobium includes the following steps:

[0067] Step 1: Optimize the culture medium;

[0068] The optimized GSG culture medium for Rhizobium oxyclover is as follows:

[0069] Glycerol 5–20 g / L, sodium succinate 0.1–0.5 g / L, monosodium glutamate 0.1–0.5 g / L, potassium dihydrogen phosphate 0.1–0.8 g / L, disodium hydrogen phosphate 0.1–1 g / L, magnesium sulfate heptahydrate 0.1–0.5 g / L, yeast extract 1–2 g / L. Peptone 0.1–1 g / L;

[0070] Step 2: Prepare the freeze-drying protectant;

[0071] Step 3: Prepare the freeze-drying agent of *Astragalus membranaceus* rhizobium;

[0072] (1) Culture of Rhizobium from Astragalus membranaceus:

[0073] 2 tons of fermentation tank-grade culture medium and 1.4 tons of GSG astragalus rhizobium-specific culture medium.

[0074] The fermentation conditions were: temperature 28℃, pH 6.8-7.2, dissolved oxygen controlled between 50% and 85%, stirring speed 150 rpm, aeration rate 0.3-1.0 vvm, and fermentation time 68 hours.

[0075] (2) The cultured Astragalus membranaceus inoculum was concentrated using a continuous centrifuge at a concentration ratio of 80:1, that is, 1.4 tons of Astragalus membranaceus inoculum was concentrated into 17.5 liters of Astragalus membranaceus inoculum concentrated inoculum.

[0076] (3) Add a certain amount of freeze-drying protectant to the concentrated solution of Astragalus membranaceus root nodules;

[0077] (4) Pre-freeze the concentrated mixed bacterial solution of milkvetch root nodules at -50℃ for 4-10 hours;

[0078] (5) Place the pre-frozen concentrated mixed bacterial solution of purple clover root nodules into a freeze dryer;

[0079] The freeze-drying conditions were: pressure 0.02 mBar, cold trap temperature -50℃;

[0080] After freeze-drying for 48 hours, the freeze-dried mycelium powder of Rhizobium cuspidatum was extracted.

[0081] Step 4: Determine the viable count of the freeze-dried Rhizobium tumefaciens powder and store it in a warehouse.

[0082] Further, the concentrations are as follows: glycerol 9–11 g / L, sodium succinate 0.1–0.3 g / L, monosodium glutamate 0.2–0.4 g / L, potassium dihydrogen phosphate 0.4–0.6 g / L, disodium hydrogen phosphate 0.9–1 g / L, magnesium sulfate heptahydrate 0.1–0.3 g / L, and yeast extract 1.1–1.3 g / L. Peptone 0.7–0.9 g / L.

[0083] Furthermore, in step one, the optimized GSG culture medium for *Astragalus membranaceus* rhizobia is as follows:

[0084] Glycerol 10 g / L, Sodium succinate 0.2 g / L, Monosodium glutamate 0.3 g / L, Potassium dihydrogen phosphate 0.5 g / L, Disodium hydrogen phosphate 0.95 g / L, Magnesium sulfate heptahydrate 0.2 g / L, Yeast extract 1.2 g / L Peptone 0.8 g / L.

[0085] Further, in step two, the amount of freeze-drying protectant added is 40%-60% (V / W) of the concentrated clover rhizobium solution, and the mixture is stirred for 10-20 minutes to fully dissolve the freeze-drying protectant, thus obtaining the concentrated mixed clover rhizobium solution.

[0086] Furthermore, in step two, the components of the freeze-drying protectant are selected from at least a variety of emulsifiers, humectants, preservatives, binders, skim milk powder, thickeners, surfactants, amino acids, antioxidants, and buffers;

[0087] The method for preparing the freeze-drying protectant is as follows: simply mix the components of the freeze-drying protectant in a certain proportion until homogeneous;

[0088] The emulsifier used is lecithin powder;

[0089] The moisturizers selected are trehalose and sorbitol;

[0090] Sodium alginate was chosen as the preservative.

[0091] The adhesive used is polyvinylpyrrolidone;

[0092] The thickener is selected from one or more of xanthan gum, dextrin, and guar gum;

[0093] The surfactant is selected from betaine and mercaptoethanol.

[0094] The amino acid selected is one of monosodium glutamate, proline, or glycine.

[0095] Ascorbic acid was chosen as the antioxidant.

[0096] K2HPO4 was selected as the buffer. 4、 KH2PO4.

[0097] Furthermore, in step two, the formulation of the lyophilization protectant is as follows:

[0098] Lecithin powder 20%; Trehalose 15%; Sorbitol 5%; Sodium alginate 20%; Polyvinylpyrrolidone 10%; Skim milk powder 20%; Xanthan gum 1%; Betaine 2%; Monosodium glutamate 7%.

[0099] Furthermore, in step two, the formulation of the freeze-drying protectant is as follows: 20% lecithin powder; 15% trehalose; 5% sorbitol; 20% sodium alginate; 10% polyvinylpyrrolidone; 20% skim milk powder; 0.1% mercaptoethanol; 5% proline; and 4% dextrin.

[0100] Furthermore, in step two, the formulation of the freeze-drying protectant is as follows: 20% lecithin powder; 15% trehalose; 5% sorbitol; 20% sodium alginate; 10% polyvinylpyrrolidone; 20% skim milk powder; 1% ascorbic acid; 1% guar gum; 5% glycine; 1.5% K2HPO4; and 1.5% KH2PO4.

[0101] The application method of the aforementioned ultra-stable Astragalus membranaceus rhizobium pre-inoculation agent includes the following steps:

[0102] Step 1: Dilute the freeze-dried powder of Rhizobium oxyclover;

[0103] The bacterial count of the freeze-dried Rhizobium tumefaciens powder was diluted to 100-200 billion / gram using a suitable powder carrier; the powder carrier was selected from at least one of corn starch, kaolin, and phosphate rock powder.

[0104] Step 2: Prepare the antioxidant solution;

[0105] The prepared combined antioxidant has a strong protective effect and consists of: propyl gallate : β-(4-hydroxyphenyl-3,5-di-tert-butyl)propionate n-octadecyl alcohol ester = 5 : 1. The prescribed amounts of propyl gallate and β-(4-hydroxyphenyl-3,5-di-tert-butyl)propionate n-octadecyl alcohol ester are dissolved in propylene glycol to a concentration of 1% (w / v), which is the antioxidant solution.

[0106] Step 3: Before seed dressing, mix 5 ml of antioxidant solution with 100 g of freeze-dried Astragalus membranaceus powder to obtain Astragalus membranaceus powder;

[0107] Step 4: Seed dressing;

[0108] Mix 5 grams of Astragalus membranaceus rhizobium powder with 1000 grams of clean Astragalus membranaceus seeds. The Astragalus membranaceus rhizobium powder is electrostatically adsorbed onto the surface of the Astragalus membranaceus seeds. Store the Astragalus membranaceus seeds with the adsorbed Astragalus membranaceus rhizobium powder in a warehouse at a constant temperature of 15℃.

[0109] Step 5: Periodically measure the number of viable bacteria on the milkvetch seeds after seed dressing in Step 4. After seed dressing, milkvetch seeds do not need to be treated with rhizobia again before sowing; they can be sown directly.

[0110] Furthermore, in step five, the method for determining the number of viable bacteria on the treated milkvetch seeds is as follows:

[0111] Accurately weigh 100 seeds of the treated milk vetch and place them in a 50 ml sterile container. Add 10 ml of sterile buffer solution and 20% skim milk powder solution. Shake thoroughly to wash off the milk vetch rhizobia from the treated milk vetch seeds. This is the first tube. Dilute the milk vetch rhizobia solution in the first tube by 10 times to make two more tubes, namely the second and third tubes (i.e., the second tube is 10 times the dilution of the first tube; the third tube is 10 times the dilution of the second tube).

[0112] Take 0.1 ml of *Astragalus membranaceus* rhizobium solution from tubes 1, 2, and 3 respectively and spread it on YEM plates; after incubation at 28°C for 4 days, check the number of *Astragalus membranaceus* rhizobium colonies.

[0113] Calculation formula:

[0114] The number of viable milk vetch rhizobia bacteria per milk vetch seed = (N x 10) n+1 )÷100

[0115] Where: N represents the number of colonies on the plate coated in the third tube;

[0116] n represents the corresponding dilution tube.

[0117] For example: Suppose there are 35 colonies on the plate coated in the third tube.

[0118] The number of viable bacteria per seed = (35 x 10) 3+1 )÷100

[0119] = 3500 CFU / seed.

[0120] Wherein: cfu (colony forming unit) is the number of viable bacteria in biological terms.

[0121] Example 1

[0122] A highly stable pre-inoculated agent of *Rhizobium oxyclover*, the preparation method of its lyophilized formulation includes the following steps.

[0123] Step 1: Optimize the culture medium;

[0124] The culture conditions of bacteria have a significant impact on bacterial survival, and optimizing the culture medium is a necessary condition for the development of ultra-stable Rhizobium oxyclover inoculants.

[0125] The optimized GSG culture medium for *Astragalus membranaceus* rhizobia in this embodiment is:

[0126] Glycerol 10 g / L, Sodium succinate 0.2 g / L, Monosodium glutamate 0.3 g / L, Potassium dihydrogen phosphate 0.5 g / L, Disodium hydrogen phosphate 0.95 g / L, Magnesium sulfate heptahydrate 0.2 g / L, Yeast extract 1.2 g / L Peptone 0.8 g / L;

[0127] Step 2: Prepare the freeze-drying protectant;

[0128] Freeze-drying involves two steps: deep freezing and dehydration. Deep freezing causes intracellular freezing, damaging cell structure and leading to cell death. Vacuum drying causes cells to lose almost all water, resulting in drastic changes in cell structure, particularly cell membrane damage, which also leads to cell death. Therefore, developing cryoprotectants is a crucial technical measure.

[0129] Rhizobium tumefaciens thrives in humid environments and is particularly intolerant of dryness. It is extremely sensitive to dry conditions, making it very difficult to prepare stable freeze-dried bacterial powder. Low-temperature storage is beneficial for the preservation of Rhizobium tumefaciens. Freeze-dried bacterial powder has a very small volume and occupies relatively little refrigeration space, which is perfectly acceptable in production practice. Therefore, there is an urgent need to develop freeze-drying protectants for Rhizobium tumefaciens.

[0130] In step two, the amount of freeze-drying protectant added is 50% (V / W) of the concentrated alfalfa rhizobium solution. Stir for 10-20 minutes to fully dissolve the freeze-drying protectant and obtain the concentrated alfalfa rhizobium mixed solution.

[0131] In step two, the formulation of the lyophilization protectant is as follows:

[0132] Lecithin powder 20%; Trehalose 15%; Sorbitol 5%; Sodium alginate 20%; Polyvinylpyrrolidone 10%; Skim milk powder 20%; Xanthan gum 1%; Betaine 2%; Monosodium glutamate 7%;

[0133] Step 3: Prepare the freeze-drying agent of *Astragalus membranaceus* rhizobium;

[0134] (1) Culture of Rhizobium from Astragalus membranaceus:

[0135] 2 tons of fermentation tank-grade culture medium and 1.4 tons of GSG astragalus rhizobium-specific culture medium.

[0136] The fermentation conditions were: temperature 28℃, pH 6.8-7.2, dissolved oxygen controlled between 50% and 85%, stirring speed 150 rpm, aeration rate 0.3-1.0 vvm, and fermentation time 68 hours.

[0137] (2) The cultured Astragalus membranaceus inoculum was concentrated using a continuous centrifuge at a concentration ratio of 80:1, that is, 1.4 tons of Astragalus membranaceus inoculum was concentrated into 17.5 liters of Astragalus membranaceus inoculum concentrated inoculum.

[0138] (3) Add a certain amount of freeze-drying protectant to the concentrated solution of Astragalus membranaceus root nodules;

[0139] (4) Pre-freeze the concentrated mixed bacterial solution of milkvetch root nodules at -50℃ for 4-10 hours;

[0140] (5) Place the pre-frozen concentrated mixed bacterial solution of purple clover root nodules into a freeze dryer;

[0141] The freeze-drying conditions were: pressure 0.02 mBar, cold trap temperature -50℃;

[0142] After freeze-drying for 48 hours, the freeze-dried mycelium powder of Rhizobium cuspidatum was extracted.

[0143] Step 4: Determine the viable count of the freeze-dried Rhizobium tumefaciens powder and store it in a warehouse.

[0144] In this embodiment, the bacterial count of the fermentation broth was determined by plate counting, and the bacterial count was 4.8 billion / mL.

[0145] Centrifugal concentration: 1.4 tons of rhizobium fermentation broth was concentrated to 17.5 kg using a continuous centrifuge manufactured by Jiangsu Yixing Huading Company. The number of Rhizobium alginate after concentration was determined by plate count method (380 billion / mL), and the centrifugal recovery rate was 83%.

[0146] Freeze-drying: Take 500 ml of concentrated bacterial solution and mix it separately with freeze-drying protectant. The ratio of protectant added is 50%.

[0147] The freeze-dried bacterial powder was packaged in brown glass bottles, sealed, and stored at 4°C and 20°C respectively. Bacterial counts were determined periodically using the plate method.

[0148] To reduce the damage to the bacteria caused by drastic changes in osmotic pressure, the freeze-dried bacterial powder sample was suspended and diluted with sterilized 20% skim milk powder each time the viable bacteria count was determined.

[0149] Example 2

[0150] A super-stable pre-inoculation agent for Rhizobium oxycloverae, the preparation method of its lyophilized formulation is the same as in Example 1, except that: in step two, the formulation of the lyophilization protectant is as follows: 20% lecithin powder; 15% trehalose; 5% sorbitol; 20% sodium alginate; 10% polyvinylpyrrolidone; 20% skim milk powder; 0.1% mercaptoethanol; 5% proline; and 4% dextrin.

[0151] Example 3

[0152] A super-stable pre-inoculation agent for *Rhizobium oxyclover* is prepared by the same method as in Example 1, except that in step two, the freeze-drying protectant is formulated as follows: 20% lecithin powder; 15% trehalose; 5% sorbitol; 20% sodium alginate; 10% polyvinylpyrrolidone; 20% skim milk powder; 1% ascorbic acid; 1% guar gum; 5% glycine; 1.5% K₂HPO₄; and 1.5% KH₂PO₄.

[0153] Example 4

[0154] A highly stable pre-inoculation agent for *Astragalus membranaceus* rhizobia, prepared by freeze-drying in the same manner as in Example 1, except that the optimized *Astragalus membranaceus* rhizobia-specific culture medium GSG is as follows:

[0155] Glycerol 5 g / L, sodium succinate 0.1 g / L, monosodium glutamate 0.1 g / L, potassium dihydrogen phosphate 0.8 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, yeast extract 2 g / L. Peptone 1 g / L.

[0156] Example 5

[0157] A highly stable pre-inoculation agent for *Astragalus membranaceus* rhizobia, prepared by freeze-drying in the same manner as in Example 1, except that the optimized *Astragalus membranaceus* rhizobia-specific culture medium GSG is as follows:

[0158] Glycerol 20 g / L, Sodium succinate 0.5 g / L, Monosodium glutamate 0.5 g / L, Potassium dihydrogen phosphate 0.1 g / L, Disodium hydrogen phosphate 0.1 g / L, Magnesium sulfate heptahydrate 0.1 g / L, Yeast extract 1 g / L Peptone 0.1 g / L.

[0159] Example 6

[0160] A highly stable pre-inoculation agent for *Astragalus membranaceus* rhizobia, prepared by freeze-drying in the same manner as in Example 1, except that the optimized *Astragalus membranaceus* rhizobia-specific culture medium GSG is as follows:

[0161] Glycerol 11 g / L, Sodium succinate 0.1 g / L, Monosodium glutamate 0.2 g / L, Potassium dihydrogen phosphate 0.4 g / L, Disodium hydrogen phosphate 0.9 g / L, Magnesium sulfate heptahydrate 0.3 g / L, Yeast extract 1.3 g / L Peptone 0.7 g / L.

[0162] Example 7

[0163] A highly stable pre-inoculation agent for *Astragalus membranaceus* rhizobia, prepared by freeze-drying in the same manner as in Example 1, except that the optimized *Astragalus membranaceus* rhizobia-specific culture medium GSG is as follows:

[0164] Glycerol 9 g / L, sodium succinate 0.3 g / L, monosodium glutamate 0.4 g / L, potassium dihydrogen phosphate 0.6 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.1 g / L, yeast extract 1.1 g / L Peptone 0.9 g / L.

[0165] Comparative Example 1

[0166] A pre-inoculation agent for *Rhizobium clavatum*, the method for preparing its lyophilized formulation includes the following steps:

[0167] Step 1: Prepare the culture medium;

[0168] Comparative Example 1 uses YMA, a commonly used culture medium for Rhizobium clavatum, namely yeast extract mannitol medium:

[0169] Mannitol 10 g / L, yeast powder 0.3 g / L, K2HPO4 0.65 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.13 g / L.

[0170] Step 2: Preparation of *Astragalus membranaceus* Rhizobium lyophilization agent;

[0171] (1) Culture of Rhizobium from Astragalus membranaceus:

[0172] 2 tons of fermentation tank-filled culture medium and 1.4 tons of YMA Astragalus membranaceus rhizobium culture medium,

[0173] The fermentation conditions were: temperature 28℃, pH 6.8-7.2, dissolved oxygen controlled between 50% and 85%, stirring speed 150 rpm, aeration rate 0.3-1.0 vvm, and fermentation time 68 hours.

[0174] (2) The cultured Astragalus membranaceus inoculum was concentrated using a continuous centrifuge at a concentration ratio of 80:1, that is, 1.4 tons of Astragalus membranaceus inoculum was concentrated into 17.5 liters of Astragalus membranaceus inoculum concentrated inoculum.

[0175] (4) Pre-freeze the concentrated bacterial solution of *Astragalus membranaceus* root nodules at -50°C for 4-10 hours;

[0176] (5) Place the pre-frozen concentrated mixed bacterial solution of purple clover root nodules into a freeze dryer;

[0177] The freeze-drying conditions were: pressure 0.02 mBar, cold trap temperature -50℃;

[0178] After freeze-drying for 48 hours, the freeze-dried mycelium powder of Rhizobium cuspidatum was extracted.

[0179] Step 4: Determine the viable count of the freeze-dried Rhizobium tumefaciens powder and store it in a warehouse.

[0180] Comparative Example 2

[0181] A super-stable pre-inoculation agent of Rhizobium clavatum is prepared by the same method as in Example 1, except that: no freeze-drying protectant was prepared in Comparative Example 2, and no freeze-drying protectant was added to the concentrated Rhizobium clavatum solution during the preparation of the freeze-dried agent.

[0182] Comparative Example 3

[0183] A highly stable pre-inoculated agent of *Rhizobium affine* is prepared by the same method as in Example 1, except that the protectant used for the freeze-dried *Rhizobium affine* strain in Comparative Example 3 is: 1% glycerol + 10% sucrose + 0.5% KI. Five parts of *Rhizobium affine* inoculum solution are mixed with one part of the protectant and then freeze-dried.

[0184] The test results show that the survival rate of *Rhizobium clavatum* in the freeze-dried powder of the ultra-stable *Rhizobium clavatum* pre-inoculation agent prepared in Comparative Example 3 is about 5%, which cannot meet the requirements of production practice.

[0185] Example 8

[0186] An ultra-stable pre-inoculation agent for *Astragalus membranaceus* rhizobia, the application method of which includes the following steps.

[0187] Step 1: Dilute the freeze-dried powder of Rhizobium oxyclover;

[0188] The freeze-dried mycelium powder of *Rhizobium clavatum* prepared in Example 3 was used for seed survival tests. After freeze-drying, the bacterial count of the freeze-dried mycelium powder of *Rhizobium clavatum* was first determined, and the bacterial count of the freeze-dried mycelium powder of *Rhizobium clavatum* prepared in Example 3 was diluted to 100-200 billion / gram with an appropriate powdered carrier, corn starch.

[0189] Step 2: Prepare the antioxidant solution;

[0190] The prepared combined antioxidant has a strong protective effect and consists of: propyl gallate : β-(4-hydroxyphenyl-3,5-di-tert-butyl)propionate n-octadecyl alcohol ester = 5 : 1. The prescribed amounts of propyl gallate and β-(4-hydroxyphenyl-3,5-di-tert-butyl)propionate n-octadecyl alcohol ester are dissolved in propylene glycol to a concentration of 1% (w / v), which is the antioxidant solution.

[0191] Step 3: Before seed dressing, mix 5 ml of antioxidant solution with 100 g of freeze-dried Astragalus membranaceus powder to obtain Astragalus membranaceus powder;

[0192] Step 4: Seed dressing;

[0193] Mix 5 grams of Astragalus membranaceus powder with 1000 grams of clean Astragalus membranaceus seeds in a resealable plastic bag. The Astragalus membranaceus powder will be electrostatically adsorbed onto the surface of the Astragalus membranaceus seeds. Place the Astragalus membranaceus seeds with the powder adsorbed into a 15°C constant temperature incubator. To reduce interference from other bacteria on the seed surface in the Astragalus membranaceus detection, the Astragalus membranaceus seeds can be washed twice with tap water and once with sterile water. After drying, they are clean Astragalus membranaceus seeds.

[0194] Step 5: Periodically measure the number of viable bacteria on the milkvetch seeds after seed treatment in Step 4. After seed treatment, milkvetch seeds do not need to be treated with rhizobia again before sowing; they can be sown directly.

[0195] In step five, the method for determining the number of viable bacteria on the treated milkvetch seeds is as follows:

[0196] Taking 100 seeds each time by counting is time-consuming and prone to errors. Touching each seed may also cause the rhizobia to fall off. Therefore, the average weight of 100 seeds of the tested milkvetch varieties was measured beforehand.

[0197] In this embodiment, 100 astragalus seeds were weighed, and the average weight of 100 astragalus seeds was obtained by weighing them ten times. The weights of the 100 astragalus seeds in this embodiment were as follows:

[0198] 0.378g, 0.366g, 0.373g, 0.364g, 0.357g, 0.363g, 0.366g, 0.365g, 0.366g, 0.371g;

[0199] (0.378+0.366+0.373+0.364+0.357+0.363+0.366+0.365+0.366+0.371)÷10=0.367 (grams)

[0200] Accurately weigh 100 seeds of treated milk vetch (0.367 g), place them in a 50 ml sterile container, add 10 ml of sterile buffer solution and 20% skim milk powder solution, shake thoroughly to wash off the milk vetch rhizobia from the treated milk vetch seeds, this is the first tube; continue to dilute the milk vetch rhizobia solution in the first tube with a 10-fold dilution to 2 more tubes, which are the second and third tubes respectively;

[0201] Take 0.1 ml of *Astragalus membranaceus* rhizobium solution from tubes 1, 2, and 3 respectively and spread it on YEM plates; after incubation at 28°C for 4 days, check the number of *Astragalus membranaceus* rhizobium colonies.

[0202] Calculation formula:

[0203] The number of viable milk vetch rhizobia bacteria per milk vetch seed = (N x 10) n+1 )÷100

[0204] Where: N represents the number of colonies on the plate;

[0205] n represents the corresponding dilution tube.

[0206] In this embodiment: the plate coated in the third tube had 35 colonies.

[0207] The number of viable bacteria per seed = (35 x 10) 3+1 )÷100

[0208] = 3500 CFU / seed.

[0209] CFU (colony forming unit) refers to the number of viable bacteria in a organism.

[0210] Example 9

[0211] An ultra-stable pre-inoculation agent for Rhizobium oxyclover is used in the same way as in Example 8, except that an antioxidant solution is not prepared.

[0212] Example 10

[0213] A super-stable pre-inoculation agent for Rhizobium clavatum, the application method of which is the same as in Example 8, except that the prepared combined antioxidant is: propyl gallate : β-(4-hydroxyphenyl-3,5-di-tert-butyl)propionate n-octadecyl alcohol ester = 4 : 1.

[0214] Example 11

[0215] A super-stable pre-inoculation agent for Rhizobium clavatum, the application method of which is the same as in Example 8, except that the prepared combined antioxidant is: propyl gallate : β-(4-hydroxyphenyl-3,5-di-tert-butyl)propionate n-octadecyl alcohol ester = 6 : 1.

[0216] Comparative Example 4

[0217] A highly stable pre-inoculation agent of *Astragalus membranaceus* rhizobium was developed. Its application method is the same as in Example 8, except that: in step one, the freeze-dried *Astragalus membranaceus* rhizobium powder prepared in Comparative Example 1 was diluted; an antioxidant solution was not prepared, and *Astragalus membranaceus* rhizobium powder was directly used to coat clean *Astragalus membranaceus* seeds. Analysis of experimental results:

[0218] (1) To compare the effects of the two culture media on cell stability, the viable cell count and total cell count of the *Rhizobium clavatum* pre-inoculated inoculum prepared in Comparative Examples 2 and 1 were measured before and after freeze-drying. The culture media of Comparative Examples 2 and 1 were freeze-dried without any protectant (10 mL of bacterial solution was directly freeze-dried). The results are shown in Table 1 below:

[0219] Table 1. Results of total bacterial count determination before and after freeze-drying of culture media without cryoprotectant.

[0220] Comparative Example 1 (YMA medium) <![CDATA[1.74x10 9 CFU / mL]]> <![CDATA[17.4x10 9 ]]> <![CDATA[1.41x10 8 ]]> 0.81％ Comparative Example 2 (GSG medium) <![CDATA[4.82x10 9 CFU / mL]]> <![CDATA[48.2x10 9 ]]> <![CDATA[1.09x10 9 ]]> 2.27％

[0221] As shown in Table 1 above, under the same culture volume, the number of *Rhizobium clavatum* produced by Comparative Example 2 (GSG medium) was 2.77 times that of Comparative Example 1 (YMA medium). More importantly, the number of *Rhizobium clavatum* surviving after freeze-drying of the mycelial powder prepared by Comparative Example 2 (GSG medium) was 7.73 times that of the mycelial powder prepared by Comparative Example 1 (YMA medium). This demonstrates the important role of the GSG medium of the present invention in the pre-inoculation of *Rhizobium clavatum* (under the same conditions, it can greatly increase the number of *Rhizobium clavatum* and greatly improve the survival rate of *Rhizobium clavatum* in the freeze-dried *Rhizobium clavatum* powder).

[0222] (2) To compare the protective effects of different freeze-drying protectants of the present invention on the freeze-drying process of Rhizobium clavatum, the viable counts of the Rhizobium clavatum pre-inoculated inoculum prepared in Examples 1, 2, and 3 above were measured before and after freeze-drying. 10 ml of bacterial solution was directly freeze-dried. The results are shown in Table 3 below:

[0223] Table 3 shows the results of viable bacterial count determination of the *Astragalus membranaceus* rhizobium pre-inoculation agents prepared in Examples 1, 2, and 3 of this invention before and after freeze-drying.

[0224] Example 1 (GSG medium) 571 grams 250 billion / gram 8％ Example 2 (GSG medium) 582 grams 238.3 billion / gram 8％ Example 3 (GSG medium) 603 grams 255.2 billion / gram 8％

[0225] (3) To compare the protective effects of different freeze-drying protectants of the present invention on the freeze-drying process of Rhizobium clavatum, 10 ml of bacterial solution was directly freeze-dried and stored for the same period of time under the same environmental conditions (i.e., under the same conditions). The viable count of Rhizobium clavatum pre-inoculated inoculum prepared in Examples 1, 2, and 3 above was measured after storage. The results are shown in Tables 4 and 5 below:

[0226] Table 4. Results of viable bacterial count determination of *Astragalus membranaceus* pre-inoculation agents prepared in Examples 1, 2, and 3 after storage.

[0227]

[0228] Table 5. Results of viable cell count determination of *Astragalus membranaceus* pre-inoculation agents prepared in Examples 1, 2, and 3 after storage.

[0229]

[0230]

[0231] As shown in Tables 4 and 5 above, the freeze-dried *Astragalus membranaceus* powder prepared in Examples 1, 2, and 3 of this invention, when stored at 4°C for one year, exhibited a maximum survival rate of 74.9%, meeting the requirements of practical production. The freeze-dried *Astragalus membranaceus* powder prepared in Examples 1, 2, and 3 of this invention also showed a maximum survival rate of 38.0% at room temperature (20°C), meeting the requirements of practical production. The results indicate that low-temperature storage is beneficial for the preservation of *Astragalus membranaceus* rhizobia, and the freeze-dried powder has a very small volume, occupying relatively little refrigeration space, which is perfectly acceptable in practical production.

[0232] (4) To compare the protective effect of the oxidizing agent of the present invention on *Rhizobium clavatum* after inoculation onto *Rhizobium clavatum* seeds, the viable number of *Rhizobium clavatum* on *Rhizobium clavatum* seeds prepared in Examples 8, 9, 10, 11 and Comparative Example 4 were measured after seed dressing with the *Rhizobium clavatum* pre-inoculation agents and stored under the same environmental conditions for the same period of time (i.e., under the same conditions). The results are shown in Table 6 below:

[0233] Table 6. Results of the determination of viable bacteria count of *Astragalus membranaceus* rhizobia on *Astragalus membranaceus* seeds.

[0234]

[0235] Once inoculated onto seeds, rhizobia are in a dry state with a moisture content of around 8%. The survival rate of liquid rhizobia drops drastically when transferred from a liquid to a dry environment. Traditional peat powder has a moisture content of approximately 45%, and when the moisture content drops below 30%, the rhizobia die rapidly. In an extremely dry environment of 8%, *Acer truncatum* rhizobia survives on *Acer truncatum* seeds for only 1-5 days (as shown in Comparative Example 4 in Table 6). Ensuring the survival of rhizobia in such an extremely dry environment (approximately 8% moisture content) remains a global challenge.

[0236] The freeze-dried bacterial agent has a moisture content of 4-8%, allowing the rhizobia to adapt to a dry environment. However, oxidative stress is the main factor causing the death of rhizobia on seeds. As shown in Table 6 above, under the same conditions, the survival rate of *Rhizobia clover* rhizobia in seeds treated with antioxidants (as in Example 8) during storage was much higher than that without antioxidants (as in Example 9). Furthermore, under the same conditions, the survival rate of *Rhizobia clover* rhizobia in seeds treated with antioxidants of specific components and ratios (as in Example 8) during storage was much higher than that without antioxidants of other component ratios (as in Examples 10 and 11). This demonstrates that the antioxidant in Example 8 of this invention has a strong antioxidant protective effect on *Rhizobia clover* rhizobia on seeds.

[0237] Experiments show that a viable count of rhizobium per seed (above 1000 CFU) ensures normal nodulation and nitrogen fixation activity. Conversely, a significantly lower count below 1000 CFU per seed reduces nodulation and nitrogen fixation. Therefore, 1000 CFU / seed is generally used as an indicator of the effectiveness of rhizobium inoculation in small-seeded legumes like milkvetch. Previously, milkvetch rhizobium peat powder or liquid formulations were difficult to maintain long-term survival on seeds, with viable counts dropping below 1000 CFU within 1-3 days after inoculation. This meant seeds had to be treated with fungicide before sowing, and this limitation discouraged farmers from inoculating with rhizobium, hindering the realization of its significant economic and ecological benefits in my country. The preparation and application of the ultra-stable *Rhizobium clavatum* of this invention optimizes three key technical aspects: optimizing the fermentation medium to obtain more cells, optimizing the freeze-drying protectant to improve cell survival during freeze-drying, and optimizing the antioxidant to protect the survival of rhizobium on the seed surface. This achieves a technological breakthrough, extending the survival period of *Rhizobium clavatum* on seeds from a few days to more than six months. This new technology will change the current status of rhizobium application and make a significant contribution to reducing fertilizer use and CO2 emissions.

[0238] The alfalfa rhizobium inoculant prepared according to this invention has been successfully tested in southern China. Currently, the alfalfa planting area in southern China is approximately 30 million mu (about 2 million hectares). After inoculation with the alfalfa rhizobium inoculant prepared according to this invention, it can replace approximately 450,000 to 900,000 tons of urea (2,600 yuan / ton), saving approximately 1.17 to 2.34 billion yuan. Saving 450,000 to 900,000 tons of urea is equivalent to reducing carbon dioxide emissions by 6.07 to 12.15 million tons. In addition, alfalfa green manure can increase rice yield by about 5%. Seed treatment with the alfalfa rhizobium inoculant prepared according to this invention, followed by sowing within 3 to 6 months, can increase the nitrogen fixation efficiency of alfalfa by 10-30%.

[0239] Emission reduction benefit estimate: The production of 1 ton of nitrogen fertilizer emits approximately 4.524 tons of CO2. Rhizobium tumefaciens can replace approximately 450,000 to 900,000 tons of urea, thus reducing CO2 emissions by 1,017,900 to 2,035,800 tons. Similarly, after inoculation with the Rhizobium tumefaciens agent prepared according to this invention, 13 million mu of dryland green manure in my country can reduce CO2 emissions by approximately 530,000 to 700,000 tons in the nitrogen fertilizer production process, achieving significant economic benefits.

[0240] All other unspecified parts belong to the prior art.

Claims

1. A method for preparing a pre-inoculation agent of ultra-stable *Astragalus membranaceus* rhizobium, characterized in that: Includes the following steps, Step 1: Optimize the culture medium; The optimized GSG culture medium for Rhizobium oxyclover is as follows: Glycerol 5–20 g / L, sodium succinate 0.1–0.5 g / L, monosodium glutamate 0.1–0.5 g / L, potassium dihydrogen phosphate 0.1–0.8 g / L, disodium hydrogen phosphate 0.1–1 g / L, magnesium sulfate heptahydrate 0.1–0.5 g / L, yeast extract 1–2 g / L, peptone 0.1–1 g / L; Step 2: Prepare the freeze-drying protectant; In step two, the freeze-drying protectant is formulated with emulsifier, humectant, preservative, binder, skim milk powder, thickener and amino acid, and also contains a combination of surfactant or antioxidant and buffer. The method for preparing the freeze-drying protectant is as follows: simply mix the components of the freeze-drying protectant in a certain proportion until homogeneous; The emulsifier is lecithin powder; the amount of lecithin powder used is 20%; The humectants are trehalose and sorbitol; the amount of trehalose is 15%, and the amount of sorbitol is 5%. The preservative is sodium alginate; the amount of sodium alginate used is 20%; The adhesive is polyvinylpyrrolidone; the amount of polyvinylpyrrolidone used is 10%; The amount of skim milk powder used is 20%; The thickener is one or more of xanthan gum, dextrin, and guar gum; the amount of xanthan gum used is 1%; the amount of dextrin used is 4%; and the amount of guar gum used is 1%. The amino acid is one of monosodium glutamate, proline, and glycine; the amount of monosodium glutamate used is 7%; the amount of proline used is 5%; and the amount of glycine used is 5%. The surfactant is one of betaine and mercaptoethanol; the amount of betaine is 2%; the amount of mercaptoethanol is 0.1%; or, The antioxidant is ascorbic acid; the amount of ascorbic acid used is 1%; The buffers selected are K2HPO4 and KH2PO4; the amount of K2HPO4 used is 1.5%; the amount of KH2PO4 used is 1.5%. The amount of freeze-drying protectant added is 40%-60% (w / v) of the concentrated alfalfa rhizobium solution. Step 3: Prepare the freeze-drying agent of *Astragalus membranaceus* rhizobium; (1) Culture of Rhizobium from Astragalus membranaceus: 2 tons of fermentation tank-grade culture medium and 1.4 tons of GSG astragalus rhizobium-specific culture medium. The fermentation conditions were: temperature 28℃, pH 6.8-7.2, dissolved oxygen controlled between 50% and 85%, stirring speed 150 rpm, aeration rate 0.3-1.0 vvm, and fermentation time 68 hours. (2) The cultured Astragalus membranaceus inoculum was concentrated using a continuous centrifuge at a concentration ratio of 80:1, that is, 1.4 tons of Astragalus membranaceus inoculum was concentrated into 17.5 liters of Astragalus membranaceus inoculum concentrated inoculum. (3) Add a certain amount of freeze-drying protectant to the concentrated solution of Astragalus membranaceus root nodules; (4) Pre-freeze the concentrated mixed bacterial solution of milkvetch root nodules at -50℃ for 4-10 hours; (5) Place the pre-frozen concentrated mixed bacterial culture of milkvetch root nodules into a freeze dryer; The freeze-drying conditions were: pressure 0.02 mBar, cold trap temperature -50℃; After freeze-drying for 48 hours, the freeze-dried mycelium powder of Rhizobium cuspidatum was extracted. Step 4: Determine the viable count of the freeze-dried Rhizobium tumefaciens powder and store it in a warehouse.

2. The method for preparing the ultra-stable *Astragalus membranaceus* rhizobium pre-inoculation agent according to claim 1, characterized in that: Glycerol 9–11 g / L, sodium succinate 0.1–0.3 g / L, monosodium glutamate 0.2–0.4 g / L, potassium dihydrogen phosphate 0.4–0.6 g / L, disodium hydrogen phosphate 0.9–1 g / L, magnesium sulfate heptahydrate 0.1–0.3 g / L, yeast extract 1.1–1.3 g / L, peptone 0.7–0.9 g / L.

3. The method for preparing the ultra-stable *Astragalus membranaceus* rhizobium pre-inoculation agent according to claim 2, characterized in that: In step one, the optimized GSG culture medium for Rhizobium oxyclover is as follows: Glycerol 10 g / L, sodium succinate 0.2 g / L, monosodium glutamate 0.3 g / L, potassium dihydrogen phosphate 0.5 g / L, disodium hydrogen phosphate 0.95 g / L, magnesium sulfate heptahydrate 0.2 g / L, yeast extract 1.2 g / L, peptone 0.8 g / L.

4. The method for preparing the ultra-stable *Astragalus membranaceus* rhizobium pre-inoculation agent according to claim 3, characterized in that: In step two, stir for 10-20 minutes to fully dissolve the freeze-drying protectant and obtain a concentrated mixed bacterial solution of Astragalus membranaceus root nodules.

5. The method of applying the ultra-stable *Astragalus membranaceus* rhizobium pre-inoculation agent according to any one of claims 1 to 4, characterized in that: Includes the following steps, Step 1: Dilute the freeze-dried powder of *Astragalus membranaceus* rhizobium; The bacterial count of the freeze-dried Rhizobium tumefaciens powder was diluted to 100-200 billion / gram using a suitable powder carrier; the powder carrier was selected from at least one of corn starch, kaolin, and phosphate rock powder. Step 2: Prepare the antioxidant solution; The prepared combined antioxidant is: propyl gallate : β-(4-hydroxyphenyl-3,5-di-tert-butyl)propionate n-octadecyl alcohol ester = 5 :

1. The prescribed amounts of propyl gallate and β-(4-hydroxyphenyl-3,5-di-tert-butyl)propionate n-octadecyl alcohol ester are dissolved in propylene glycol to a concentration of 1% (w / v). This is the antioxidant solution. Step 3: Before seed dressing, mix the antioxidant solution with the freeze-dried powder of Rhizobium clavatum to obtain Rhizobium clavatum powder; Antioxidant solution: freeze-dried powder of Rhizobium clavatum = 0.05:100; Step 4: Seed dressing; Mix the astragalus root nodule powder with clean astragalus seeds, the ratio of astragalus root nodule powder to clean astragalus seeds is 5:1000; The powder of Rhizobium tumefaciens adsorbed onto the surface of the Rhizobium tumefaciens seeds by electrostatic adsorption, and the Rhizobium tumefaciens seeds adsorbed with the powder are stored in a warehouse. Step 5: Periodically measure the number of viable bacteria on the milkvetch seeds after seed dressing in Step 4. After seed dressing, milkvetch seeds do not need to be treated with rhizobia again before sowing and can be sown directly.

6. The application method of the ultra-stable *Astragalus membranaceus* rhizobium pre-inoculation agent according to claim 5, characterized in that: In step five, the method for determining the number of viable bacteria on the treated milkvetch seeds is as follows: Accurately weigh 100 seeds of the treated milk vetch and place them in a 50 ml sterile container. Add 10 ml of sterile buffer solution and 20% skim milk powder solution. Shake thoroughly to wash off the milk vetch rhizobia from the treated milk vetch seeds. This is the first tube. Dilute the milk vetch rhizobia solution in the first tube by 10 times to make two more tubes, which are the second and third tubes, respectively. Take 0.1 ml of *Astragalus membranaceus* rhizobium solution from tubes 1, 2, and 3 respectively and spread it on YEM plates; after incubating at 28°C for 4 days, check the number of *Astragalus membranaceus* rhizobium colonies; Calculation formula: The number of viable milk vetch rhizobia bacteria per milk vetch seed = Where: N represents the number of colonies on the plate; n represents the corresponding dilution tube.

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