Method for preparing rhizobium agent for drought resistance and seedling protection

Abstract

The application discloses a kind of rhizobium preparation methods for drought resistance and seedling preservation in the field of biological bacterial agent fertilizer, and the method steps include: taking rhizobium fermentation culture to prepare bacterial suspension;Take organic carrier, incorporate modified sea moss cellulose nanocrystal dispersion and CaCl2 solution, then absorb bacterial suspension on organic carrier to obtain bacterial material;After pretreatment, part of the abandoned traditional Chinese medicine residue is oxygen-limited pyrolysis to obtain carbonaceous substance and pyrolysis condensate, then mix carbonaceous substance and pyrolysis condensate with the remaining traditional Chinese medicine residue, and carry out hydrothermal synthesis reaction to obtain traditional Chinese medicine residue derivative carrier;Take liquid fertilizer and adsorb it on traditional Chinese medicine residue derivative carrier to obtain fertilizer;Take bacterial material and fertilizer and mix evenly, and it is available.The rhizobium preparation compared with traditional liquid bacterial suspension can help to avoid the problem of penetration loss after inoculation, prolong the action time of bacterial strain, improve the symbiotic effect with root system, and also simplify the inoculation operation;Liquid fertilizer can also help to improve the drought resistance of alfalfa seedlings and promote their growth and development.

Description

Technical Field

[0001] This invention relates to the field of biological fertilizer technology, specifically to a method for preparing a drought-resistant and seedling-protecting rhizobium agent. Background Technology

[0002] Alfalfa, known as the "king of forage," is not only a core feed resource for sustainable livestock development but also plays a crucial role in ecological restoration, soil carbon sequestration, and crop rotation systems. However, its seedlings have weak drought resistance, resulting in low nodule formation and low seedling survival rates under drought conditions. The symbiotic nitrogen-fixing system between rhizobia and alfalfa is an economical and effective way to utilize natural nitrogen in agricultural production, replacing some chemical fertilizers. Simultaneously, it enhances host stress resistance through mechanisms such as regulating plant ion homeostasis and strengthening antioxidant capacity, making it an important technological direction for reducing fertilizer use and increasing efficiency. However, existing compound microbial agents still need breakthroughs in drought resistance mechanism compatibility and strain synergistic stability. The main problems are, on the one hand, short-term and limited action; and on the other hand, reliance solely on rhizobia, resulting in a single function and generally limited overall drought resistance and seedling protection effects. Developing a rhizobium inoculant that combines efficient nitrogen fixation with drought resistance and seedling protection through strain screening, carrier optimization, and process innovation, thereby simultaneously improving the survival rate and growth vigor of alfalfa seedlings under drought stress, is a key breakthrough for solving alfalfa planting problems in arid regions and promoting the sustainable development of the forage industry. Based on this, this study specifically developed a method for producing a drought-resistant and seedling-protecting rhizobium inoculant, providing technical support for high-quality, high-yield alfalfa production and ecological restoration in arid regions. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing a drought-resistant and seedling-protecting rhizobium agent, which solves the problems of short duration of action and single function of existing rhizobium agents during inoculation and application, thereby improving the overall drought-resistant and seedling-protecting effect of the agent.

[0004] The present invention achieves the above objectives through the following technical solutions:

[0005] A method for preparing a drought-resistant and seedling-protecting rhizobium agent, the method comprising the following steps:

[0006] S1. Take rhizobium for fermentation culture, and then use sterile water to make a bacterial suspension from the cultured bacteria.

[0007] S2. Take an organic carrier, uniformly mix it with modified sea squirt cellulose nanocrystal dispersion and CaCl2 solution, then mix the bacterial suspension with the organic carrier evenly so that the bacterial suspension is adsorbed onto the organic carrier, and air dry to obtain the bacterial material for later use.

[0008] S3. After pretreatment of fresh Chinese medicinal residue, 30-40% of the residue is subjected to oxygen-limited pyrolysis. The carbonaceous material and pyrolysis condensate formed by pyrolysis are collected. The carbonaceous material, pyrolysis condensate and the remaining 60-70% of the residue are then mixed. A structural agent accounting for 1-3% of the total mass of the fresh Chinese medicinal residue before pretreatment is added. A hydrothermal synthesis reaction is carried out. The reaction product is dried to obtain a Chinese medicinal residue derivative carrier.

[0009] S4. Take liquid fertilizer and mix it with the Chinese herbal medicine residue derivative carrier to allow the liquid fertilizer to be adsorbed onto the Chinese herbal medicine residue derivative carrier, and obtain fertilizer for later use.

[0010] S5. Mix the bacterial substrate and the fertilizer evenly to obtain the rhizobium agent.

[0011] A further improvement is that the specific operation of step S1 is as follows: the rhizobium strain is activated into YMA solid medium by streak plating, and cultured in a constant temperature incubator at 28℃ for 2-3 days until single colonies appear. Then, single colonies are selected and transferred to YMA liquid medium, and cultured in a shaker at 28℃ for 12-18 hours. The bacterial suspension is then centrifuged for 8-12 minutes, the supernatant is discarded, and the precipitate is mixed with sterile water at a mass ratio of 1:1 to obtain a bacterial suspension, which is then stored in glycerol at -80℃ for later use.

[0012] A further improvement is that, in step S2, the organic carrier is one of peat moss, well-rotted organic fertilizer, or well-rotted straw powder.

[0013] A further improvement is made in step S2, where the preparation steps of the modified sea squirt cellulose nanocrystal dispersion are as follows: Dry and pulverized sea squirt capsules are added to a NaOH solution and stirred in a water bath at 78-85℃ for 3-5 hours. The reaction product is centrifuged and washed, then redispersed in an acetate-sodium acetate buffer solution. NaClO2 is added, and the mixture is stirred in a water bath at 72-78℃ for 1.5-2.5 hours. The reaction product is washed and dried to obtain sea squirt cellulose. The sea squirt cellulose is then dispersed in a phosphate buffer solution, and 2,2,6,6-tetramethylpiperidine-1-oxide and NaBr are added. After pre-cooling in an ice bath, NaClO solution is added dropwise, maintaining the pH at 10-10.5. The reaction is continued for 2-3 hours, and then ethanol is added to terminate the reaction. The reactants are centrifuged and washed, and then dispersed with deionized water to obtain a modified sea squirt cellulose nanocrystal dispersion with a mass concentration of 0.5-1.5%.

[0014] A further improvement is that, in step S3, the pretreatment refers to first drying the Chinese herbal medicine residue until the moisture content is less than 15%, and then crushing it into uniform particles with a particle size of 2-5 mm.

[0015] A further improvement is that, in step S3, the specific operation of the oxygen-limited pyrolysis is as follows: the Chinese herbal medicine residue is loaded into the pyrolysis furnace, nitrogen gas is first introduced into the furnace at a flow rate of 400-600 mL / min for 20-30 min, and then the temperature is raised to 330-360℃ at a rate of 10-20℃ / min, and the pyrolysis is maintained at this temperature for 60-90 min. During the pyrolysis process, nitrogen gas is continuously introduced at a flow rate of 100-200 mL / min.

[0016] A further improvement is that, in step S3, the structuring agent is montmorillonite or kaolinite.

[0017] A further improvement is that, in step S3, the specific operation of the hydrothermal synthesis reaction is as follows: first, the water content of the reaction system is adjusted to 70-80% with deionized water, then the mixture is stirred thoroughly and reacted at a temperature of 170-190℃ for 4-6 hours.

[0018] A further improvement is that, in step S4, the liquid fertilizer is either calcium nitrate or potassium nitrate, and the concentration of calcium ions or potassium ions in the liquid fertilizer is 4-6 mM.

[0019] A further improvement is that the mass ratio of the bacterial suspension, modified sea squirt cellulose nanocrystal dispersion, CaCl2 solution and the organic carrier is 1:0.3-0.5:0.08-0.12:2-3, the mass ratio of the liquid fertilizer and the traditional Chinese medicine residue derivative carrier is 1:2-4, and the mass ratio of the bacterial substrate and fertilizer is 1.5-2.5:1.

[0020] The beneficial effects of this invention are as follows:

[0021] (1) The rhizobium agent of the present invention is loaded with an organic carrier and combined with liquid fertilizer loaded with Chinese medicine residue derivatives. Compared with traditional liquid bacterial suspension, it helps to avoid the problem of osmosis and loss after inoculation, prolongs the effect of bacterial action, improves the symbiotic effect with root system, and simplifies the inoculation operation without repeated watering. At the same time, liquid fertilizer can also help improve the drought resistance of alfalfa seedlings and promote their growth and development. In addition, the separate loading and concentration control can avoid the influence of fertilizer on bacterial cells.

[0022] (2) The rhizobium agent of the present invention has dual carrier water retention. The organic carrier and the Chinese medicine residue derivative carrier have porous structure and hydrophilic groups, which can adsorb and retain water, improve the microstructure of the rhizosphere soil, increase permeability and water retention, and provide effective protection for the strain and seedlings. In addition, the Chinese medicine residue derivative carrier contains a variety of organic matter, trace elements, etc., which can improve the plant's stress resistance, thereby achieving the dual effect of protecting seedlings and promoting growth.

[0023] (3) The present invention uses a Chinese medicine residue derivative carrier, which has a composite structure with carbonaceous material as the skeleton and newly formed humic acid as the active substance. Compared with the use of naturally decomposed Chinese medicine residue, it has higher production efficiency, better pore structure stability, and can also avoid the influence of miscellaneous bacteria on rhizobia. At the same time, compared with the direct addition of humic acid, it is beneficial to maintain porosity, and the newly formed humic acid molecules are combined with carbonaceous material through chemical bonds (hydrogen bonds, π-π interaction) and are not easily lost.

[0024] (4) By using modified sea squirt cellulose nanocrystal dispersion, the present invention can enhance the loading effect and loading rate of rhizobium suspension on organic carrier, and the oxidation modification treatment can avoid the problem of aggregation and promote the migration of rhizobium to the root system. Attached Figure Description

[0025] Figure 1 This is a graph showing the results of alfalfa plant height measurements in each group during the experiment of this invention;

[0026] Figure 2 This is a graph showing the results of alfalfa aboveground biomass measurement in each group during the experiment of this invention;

[0027] Figure 3 This is a graph showing the results of the determination of the relative water content of alfalfa leaves in each group during the experiment of this invention;

[0028] Figure 4 The graph shows the results of the determination of alfalfa chlorophyll (a+b) content in each group during the experiment of this invention. Detailed Implementation

[0029] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0030] I. Main Raw Materials

[0031] Chinese medicine residue: conventional compound medicinal residue, the components of which include hawthorn, angelica, astragalus, coptis, phellodendron, licorice and atractylodes, the main components of which include 13.21% crude protein and 9.22% crude fiber.

[0032] Rhizobium: *Sinorhizobium meliloti*, provided by Gansu Agricultural University.

[0033] YMA solid medium: yeast extract 1g / L, mannitol 10g / L, K2HPO4 0.5g / L, MgSO4·7H2O 0.2g / L, NaCl 0.1g / L, agar 15g / L.

[0034] YMA liquid culture medium: yeast extract 1g / L, mannitol 10g / L, K2HPO4 0.5g / L, MgSO4·7H2O 0.2g / L, NaCl 0.1g / L.

[0035] Alfalfa seeds: Beijing Zhengdao "WL319" alfalfa seeds.

[0036] Nitrogen-free Hoagland nutrient solution: The formula is shown in Table 1 below:

[0037] Table 1: Nitrogen-Free Hoagland Nutrient Solution Formula

[0038]

[0039] Nitrogen-containing Hogland nutrient solution: 1.26 g / L of the above nitrogen-free Hogland nutrient solution, plus 0.945 g / L of calcium nitrate.

[0040] II. Conducting the Experiment

[0041] Example 1

[0042] A method for preparing a drought-resistant and seedling-protecting rhizobium agent, the method comprising the following steps:

[0043] S1. The Rhizobium strain was activated into YMA solid medium by streak plating and cultured in a constant temperature incubator at 28℃ for 2 days until single colonies appeared. Single colonies were then selected and transferred to YMA liquid medium and cultured in a shaker at 28℃ for 12 hours. The bacterial suspension was then centrifuged for 12 minutes, the supernatant was discarded, and the precipitate was mixed with sterile water at a mass ratio of 1:1 to obtain a bacterial suspension, which was then stored in glycerol at -80℃ for later use.

[0044] S2. Take peat moss as an organic carrier, and uniformly mix it with modified sea squirt cellulose nanocrystal dispersion and CaCl2 solution (0.5 mol / L, the same below). Then mix the bacterial suspension with the organic carrier evenly so that the bacterial suspension is adsorbed onto the organic carrier. Air dry to obtain the bacterial material for later use. The mass ratio of bacterial suspension, modified sea squirt cellulose nanocrystal dispersion, CaCl2 solution and organic carrier is 1:0.3:0.08:2.

[0045] The preparation steps of the modified sea squirt cellulose nanocrystal dispersion are as follows: Take 100g of dried and pulverized sea squirt capsules and add them to 1L of water. The reaction mixture was stirred in a 4% (w / v) NaOH solution at 78°C for 4 hours. The reaction product was centrifuged, washed, and redispersed in 1 L of acetate-sodium acetate buffer solution with pH 4.5. 20 g of NaClO2 was added, and the mixture was stirred in a 75°C water bath for 2 hours. The reaction product was washed and dried to obtain succubus cellulose. 1 g of succubus cellulose was dispersed in 100 mL of phosphate buffer solution with pH 10. 0.016 g of 2,2,6,6-tetramethylpiperidine-1-oxide and 0.1 g of NaBr were added. After pre-cooling in an ice bath, 5 mL of 13% NaClO solution was added dropwise, and the pH was maintained at 10 by adding NaOH dropwise. After 2 hours of reaction, ethanol was added to terminate the reaction. The reaction mixture was centrifuged, washed, and then dispersed in deionized water to obtain a 0.5% modified succubus cellulose nanocrystal dispersion.

[0046] S3. Take fresh Chinese herbal medicine residue and dry it until the moisture content is less than 15%. Then crush it into uniform particles with a particle size of 2±0.2mm. Put 30% of the residue into a pyrolysis furnace. First, pass nitrogen gas through the furnace at a flow rate of 400mL / min for 30min. Then, raise the temperature to 330℃ at a rate of 10℃ / min and keep it at that temperature for 90min. During the pyrolysis process, continuously pass nitrogen gas through the furnace at a flow rate of 100mL / min. Collect the carbonaceous material and pyrolysis condensate formed by pyrolysis. Then, mix the carbonaceous material, pyrolysis condensate and the remaining 70% of the residue, and add montmorillonite, which accounts for 1% of the total mass of the fresh Chinese herbal medicine residue before pretreatment. First, adjust the moisture content of the reaction system to 70% with deionized water, then stir thoroughly and react at 170℃ for 6h. The reaction product is dried to obtain the Chinese herbal medicine residue derivative carrier.

[0047] S4. Take calcium nitrate with an ion concentration of 4mM as liquid fertilizer, mix the liquid fertilizer with the traditional Chinese medicine residue derivative carrier at a mass ratio of 1:2, so that the liquid fertilizer is adsorbed onto the traditional Chinese medicine residue derivative carrier, and obtain fertilizer for later use.

[0048] S5. Take the inoculum and the fertilizer and mix them evenly at a mass ratio of 1.5:1 to obtain the rhizobium agent.

[0049] Example 2

[0050] A method for preparing a drought-resistant and seedling-protecting rhizobium agent, the method comprising the following steps:

[0051] S1. The Rhizobium strain was activated into YMA solid medium by streak plating and cultured in a constant temperature incubator at 28℃ for 3 days until single colonies appeared. Single colonies were then selected and transferred to YMA liquid medium and cultured in a shaker at 28℃ for 15 hours. The bacterial suspension was then centrifuged for 10 minutes, the supernatant was discarded, and the precipitate was mixed with sterile water at a mass ratio of 1:1 to obtain a bacterial suspension, which was then stored in glycerol at -80℃ for later use.

[0052] S2. Take peat moss as an organic carrier, and uniformly mix it with modified sea squirt cellulose nanocrystal dispersion and CaCl2 solution. Then mix the bacterial suspension with the organic carrier evenly so that the bacterial suspension is adsorbed onto the organic carrier. Air dry to obtain the bacterial material for later use. The mass ratio of bacterial suspension, modified sea squirt cellulose nanocrystal dispersion, CaCl2 solution and organic carrier is 1:0.4:0.1:2.5.

[0053] The preparation steps of the modified sea squirt cellulose nanocrystal dispersion are as follows: Take 100g of dried and pulverized sea squirt capsules and add them to 1L of water. In a 5% (w / v) NaOH solution, the mixture was stirred in an 80℃ water bath for 4.5 h. The reaction product was centrifuged, washed, and redispersed in 1 L of acetate-sodium acetate buffer solution with a pH of 4.5. 20 g of NaClO2 was added, and the mixture was stirred in a 75℃ water bath for 2.5 h. The reaction product was washed and dried to obtain succubus cellulose. 1 g of succubus cellulose was dispersed in 100 mL of phosphate buffer solution with a pH of 10. 0.016 g of 2,2,6,6-tetramethylpiperidine-1-oxide and 0.1 g of NaBr were added. After pre-cooling in an ice bath, 8 mL of 13% NaClO solution was added dropwise, and the pH was maintained at 10 by adding NaOH dropwise. After reacting for 2.5 h, ethanol was added to terminate the reaction. The reaction product was centrifuged, washed, and then dispersed with deionized water to obtain a 1% modified succubus cellulose nanocrystal dispersion.

[0054] S3. Take fresh Chinese herbal medicine residue and dry it until the moisture content is less than 15%. Then crush it into uniform particles with a particle size of 4±0.2mm. Put 35% of the residue into a pyrolysis furnace. First, pass nitrogen gas through the furnace at a flow rate of 500mL / min for 25min. Then, raise the temperature to 350℃ at a rate of 15℃ / min and keep it at that temperature for 75min. During the pyrolysis process, continuously pass nitrogen gas through the furnace at a flow rate of 150mL / min. Collect the carbonaceous material and pyrolysis condensate formed by pyrolysis. Then mix the carbonaceous material, pyrolysis condensate and the remaining 65% of the residue. Add 2% of the total mass of fresh Chinese herbal medicine residue before pretreatment with kaolin. First, adjust the moisture content of the reaction system to 75% with deionized water. Then, stir thoroughly and react at 180℃ for 5h. The reaction product is dried to obtain the Chinese herbal medicine residue derivative carrier.

[0055] S4. Take calcium nitrate with an ion concentration of 5mM as liquid fertilizer, mix the liquid fertilizer with the traditional Chinese medicine residue derivative carrier at a mass ratio of 1:3, so that the liquid fertilizer is adsorbed onto the traditional Chinese medicine residue derivative carrier, and obtain fertilizer for later use.

[0056] S5. Take the inoculum and the fertilizer and mix them evenly at a mass ratio of 2:1 to obtain the rhizobium agent.

[0057] Example 3

[0058] A method for preparing a drought-resistant and seedling-protecting rhizobium agent, the method comprising the following steps:

[0059] S1. The Rhizobium strain was activated into YMA solid medium by streak plating and cultured in a constant temperature incubator at 28℃ for 3 days until single colonies appeared. Single colonies were then selected and transferred to YMA liquid medium and cultured in a shaker at 28℃ for 18 hours. The bacterial suspension was then centrifuged for 8 minutes, the supernatant was discarded, and the precipitate was mixed with sterile water at a mass ratio of 1:1 to obtain a bacterial suspension, which was then stored in glycerol at -80℃ for later use.

[0060] S2. Take decomposed straw powder as an organic carrier, and uniformly mix it with modified sea squirt cellulose nanocrystal dispersion and CaCl2 solution. Then mix the bacterial suspension with the organic carrier evenly so that the bacterial suspension is adsorbed onto the organic carrier. Air dry to obtain the bacterial material for later use. The mass ratio of bacterial suspension, modified sea squirt cellulose nanocrystal dispersion, CaCl2 solution and organic carrier is 1:0.5:0.12:3.

[0061] The preparation steps of the modified sea squirt cellulose nanocrystal dispersion are as follows: Take 100g of dried and pulverized sea squirt capsules and add them to 1L of water. In a 6% (w / v) NaOH solution, the mixture was stirred in an 85℃ water bath for 3.5 h. The reaction product was centrifuged, washed, and redispersed in 1 L of acetate-sodium acetate buffer solution with a pH of 4.5. 20 g of NaClO2 was added, and the mixture was stirred in a 78℃ water bath for 1.5 h. The reaction product was washed and dried to obtain succubus cellulose. 1 g of succubus cellulose was dispersed in 100 mL of phosphate buffer solution with a pH of 10. 0.016 g of 2,2,6,6-tetramethylpiperidine-1-oxide and 0.1 g of NaBr were added. After pre-cooling in an ice bath, 10 mL of 13% NaClO solution was added dropwise, and the pH was maintained at 10.5 by adding NaOH dropwise. After reacting for 3 h, ethanol was added to terminate the reaction. The reaction product was centrifuged, washed, and then dispersed in deionized water to obtain a 1.5% modified succubus cellulose nanocrystal dispersion.

[0062] S3. Take fresh Chinese herbal medicine residue and dry it until the moisture content is less than 15%. Then crush it into uniform particles with a particle size of 5±0.2mm. Put 40% of the residue into a pyrolysis furnace. First, pass nitrogen gas through the furnace at a flow rate of 600mL / min for 20min. Then, raise the temperature to 360℃ at a rate of 20℃ / min and keep it at that temperature for 60min. During the pyrolysis process, continuously pass nitrogen gas through the furnace at a flow rate of 200mL / min. Collect the carbonaceous material and pyrolysis condensate formed by pyrolysis. Then mix the carbonaceous material, pyrolysis condensate and the remaining 60% of the residue. Add montmorillonite, which accounts for 3% of the total mass of the fresh Chinese herbal medicine residue before pretreatment. First, adjust the moisture content of the reaction system to 80% with deionized water. Then, stir thoroughly and react at 190℃ for 4h. The reaction product is dried to obtain the Chinese herbal medicine residue derivative carrier.

[0063] S4. Take potassium nitrate with an ion concentration of 6mM as liquid fertilizer, mix the liquid fertilizer with the Chinese herbal medicine residue derivative carrier at a mass ratio of 1:4, so that the liquid fertilizer is adsorbed onto the Chinese herbal medicine residue derivative carrier, and obtain fertilizer for later use.

[0064] S5. Take the inoculum and the fertilizer and mix them evenly at a mass ratio of 2.5:1 to obtain the rhizobium agent.

[0065] Comparative Example 1

[0066] A method for preparing a drought-resistant and seedling-protecting rhizobium agent, the method comprising the following steps:

[0067] S1, same as Example 2.

[0068] S2. Take peat moss as an organic carrier, uniformly mix it with CaCl2 solution, and then mix the bacterial suspension with the organic carrier evenly so that the bacterial suspension is adsorbed onto the organic carrier. Air dry to obtain the bacterial material for later use. The mass ratio of bacterial suspension, CaCl2 solution and organic carrier is 1:0.1:2.5.

[0069] S3, same as Example 2.

[0070] S4, Same as Example 2.

[0071] S5, same as Example 2.

[0072] Comparative Example 2

[0073] A method for preparing a drought-resistant and seedling-protecting rhizobium agent, the method comprising the following steps:

[0074] S1, same as Example 2.

[0075] S2. Take peat moss as an organic carrier, and uniformly mix it with sea squirt cellulose nanocrystal dispersion and CaCl2 solution. Then mix the bacterial suspension with the organic carrier evenly so that the bacterial suspension is adsorbed onto the organic carrier. Air dry to obtain the bacterial material for later use. The mass ratio of bacterial suspension, sea squirt cellulose nanocrystal dispersion, CaCl2 solution and organic carrier is 1:0.4:0.1:2.5.

[0076] The preparation steps of the sea squirt cellulose nanocrystal dispersion are as follows: 100g of dried and pulverized sea squirt capsules are added to 1L of 5% (w / v) NaOH solution and stirred in an 80℃ water bath for 4.5h. The reaction product is centrifuged and washed, and redispersed in 1L of acetate-sodium acetate buffer solution with pH 4.5. 20g of NaClO2 is added, and the mixture is stirred in a 75℃ water bath for 2.5h. The reaction product is washed and dried to obtain sea squirt cellulose. 1g of sea squirt cellulose is then dispersed in 100mL of deionized water. The liquid is homogenized using a high-pressure homogenizer at a pressure of 1500bar until it becomes a homogeneous colloidal state. The liquid is then centrifuged and washed, and dispersed again with deionized water to obtain a sea squirt cellulose nanocrystal dispersion with a mass concentration of 1%.

[0077] S3, same as Example 2.

[0078] S4, Same as Example 2.

[0079] S5, same as Example 2.

[0080] Comparative Example 3

[0081] A method for preparing a drought-resistant and seedling-protecting rhizobium agent, the method comprising the following steps:

[0082] S1, same as Example 2.

[0083] S2, same as Example 2.

[0084] S3. Take fresh Chinese medicine residue, dry it until the moisture content is less than 15%, then crush it into uniform particles with a particle size of 4±0.2mm, and then carry out conventional composting and sterilization treatment (compost base width 1.5m, height 1m, aerobic fermentation and composting for 15 days, followed by pasteurization) to obtain Chinese medicine residue carrier.

[0085] S4. Take calcium nitrate with an ion concentration of 5mM as liquid fertilizer, mix the liquid fertilizer with the Chinese herbal medicine residue carrier at a mass ratio of 1:3, so that the liquid fertilizer is adsorbed onto the Chinese herbal medicine residue carrier, and obtain fertilizer for later use.

[0086] S5, same as Example 2.

[0087] Comparative Example 4

[0088] A method for preparing a drought-resistant and seedling-protecting rhizobium agent, the method comprising the following steps:

[0089] S1, same as Example 2.

[0090] S2, same as Example 2.

[0091] S3. Take fresh Chinese herbal medicine residue and dry it until the moisture content is less than 15%. Then crush it into uniform particles with a particle size of 4±0.2mm. Put all the Chinese herbal medicine residue into a pyrolysis furnace. First, pass nitrogen gas through the furnace at a flow rate of 500mL / min for 25min. Then, raise the temperature to 350℃ at a rate of 15℃ / min and keep it at that temperature for 75min. During the pyrolysis process, continuously pass nitrogen gas through the furnace at a flow rate of 150mL / min. Collect the carbonaceous material formed by pyrolysis. Then, add an equal amount of humic acid (obtained by sampling and testing the product of Example 2) to the carbonaceous material. After mixing evenly, dry the mixture to obtain the mixture carrier.

[0092] S4. Take calcium nitrate with an ion concentration of 5mM as liquid fertilizer, mix the liquid fertilizer with the mixture carrier at a mass ratio of 1:3, so that the liquid fertilizer is adsorbed onto the mixture carrier, and obtain fertilizer for later use.

[0093] S5, same as Example 2.

[0094] III. Performance Testing

[0095] (1) Seed germination

[0096] Select alfalfa seeds that are uniform in size and plump. Disinfect them in 75% alcohol for 8 minutes and rinse them 4-5 times with distilled water. Place the disinfected alfalfa seeds in a petri dish lined with filter paper and vernalize them upside down in a refrigerator at 4℃ for 48 hours. Then, place them in an artificial climate chamber and incubate them upside down (12h / 12h, 25℃ / 20℃) to allow them to germinate. During germination, add distilled water as needed based on the seed's water absorption. Germination should last for 5 days.

[0097] (2) Seedling transplanting

[0098] After germination, alfalfa seedlings that have grown to 2-3 cm are transplanted into plastic pots. 100-mesh quartz sand sterilized with 5% sodium hypochlorite is used as the substrate. One alfalfa seedling is planted in each pot. After transplanting, the alfalfa seedlings are placed in a greenhouse environment (temperature 25±2℃, relative humidity 50±5%). On the 7th day, each pot is watered with 500 mL of nitrogen-containing Hoagland nutrient solution.

[0099] (3) Rhizobium inoculation

[0100] Experimental group: When the first true leaf of the alfalfa plant has fully unfolded (day 14), dig a shallow circular trench 5-8 cm deep on one side of the alfalfa plant (about 3-5 cm from the base of the stem). The trench should be close to the roots, but without damaging them. Sprinkle the amount of rhizobium inoculant used per plant (6 g, i.e., adding the rhizobium inoculants prepared in Examples 1-3 and Comparative Examples 1-4) evenly into the trench, and immediately cover it with soil to completely cover the rhizobium inoculant. Water it thoroughly once. On day 10 after inoculation, irrigate with 500 mL of nitrogen-free Hoagland nutrient solution.

[0101] Traditional inoculum solution group: When the first true leaf of the alfalfa plant has fully unfolded (day 14), inoculate directly with a suspension of rhizobium containing the same bacterial content by direct watering, once every 3 days, for a total of 3 waterings. Similarly, on day 10 after the first inoculation, water with 500 mL of nitrogen-free Hoagland's solution.

[0102] (4) Drought stress treatment

[0103] Using nitrogen-free Hoagland nutrient solution as the substrate, a stress solution with a water potential of -0.6 MPa was prepared by PEG-6000 to simulate drought stress environment. After 16 days of inoculation, the stress solution was changed to irrigation, with irrigation every 2 days, 300 mL each time, for 10 days.

[0104] (5) Sampling

[0105] Alfalfa plants from the experimental group and the conventional bacterial culture group were collected, sealed with tin foil, and quickly frozen in liquid nitrogen and stored at -80°C for the determination of growth indicators.

[0106] (6) Measurement of growth indicators

[0107] Alfalfa plants were selected and the following growth indicators were measured (eight alfalfa plants were randomly selected from each group for each measurement, and the average value of the results was taken):

[0108] ① Plant height: The height of the plant is measured using a centimeter ruler, with the leaf scar of the first cotyledon as the standard for dividing the above-ground and underground parts.

[0109] ② Aboveground biomass: The aboveground and underground parts were divided by the leaf scar of the first cotyledon. The aboveground parts of the alfalfa plant were cut off, weighed and recorded using an electronic balance.

[0110] ③ Relative water content of leaves: determined by saturated weighing method, five leaves were randomly selected from each plant and their fresh weight M was weighed. 鲜 Soak in distilled water for 24 hours, and weigh its saturated fresh weight M. 饱 Then dry it at 110℃ to constant weight, and weigh it as dry weight M. 干 The relative water content of the leaves can be calculated using the following formula:

[0111] Relative water content of leaves = (M 鲜 -M 干 ) / (M 饱 -M 干 ) × 100%

[0112] ④ Chlorophyll (a+b) content: The direct extraction method was used for determination. 0.1g of fresh alfalfa leaf sample was taken and extracted with 5mL of 95% ethanol and 5mL of 80% acetone for 24h. The absorbance was measured at wavelengths of 665nm and 649nm.

[0113] IV. Results Analysis

[0114] Among the various indicators, plant height and aboveground biomass, as growth indicators, can directly reflect the growth status of alfalfa and thus its drought resistance and seedling protection effect. Relative water content is a key indicator for measuring the water status of plants and can effectively reflect the plant's ability to retain water under drought stress. The higher the relative water content of leaves, the stronger the alfalfa's water retention capacity and the stronger its drought resistance. Chlorophyll is a key pigment for photosynthesis, and its content directly affects the plant's photosynthetic capacity. Drought stress will reduce the content of photosynthetic pigments, weaken the plant's photosynthetic capacity, and thus affect the plant's growth and development.

[0115] like Figure 1-4 As shown, the alfalfa plants inoculated with the rhizobium inoculants prepared in Examples 1-3 of this invention all exhibited excellent growth, demonstrating significantly better overall drought resistance and seedling protection effects compared to the traditional inoculant group. Taking Example 2 as an example: its plant height reached 30.6 cm, and its aboveground biomass reached 18.4 g / plant, indicating that inoculation with the rhizobium inoculant prepared in this invention can alleviate drought stress, increase alfalfa plant height, and promote the accumulation of substances in both aboveground and belowground parts; its leaf relative water content reached 75.1%, indicating that inoculation with the rhizobium inoculant prepared in this invention can enhance alfalfa's water retention capacity; its chlorophyll (a+b) content reached 2.66 mg / g, indicating that inoculation with the rhizobium inoculant prepared in this invention can effectively alleviate chlorophyll degradation and reduce the inhibitory effect of drought stress on alfalfa photosynthesis.

[0116] In addition, Comparative Examples 1-4 were all based on the steps of Example 2 with adjustments. Specifically, Comparative Example 1 did not use modified sea squirt cellulose nanocrystal dispersion during the bacterial suspension loading process, Comparative Example 2 used ordinary sea squirt cellulose nanocrystal dispersion during the bacterial suspension loading process, Comparative Example 3 used decomposed and sterilized Chinese herbal medicine residue during the liquid fertilizer loading process, and Comparative Example 4 used a direct mixture of pyrolysis carbonaceous matter and humic acid from Chinese herbal medicine residue during the liquid fertilizer loading process. All of these changes resulted in varying degrees of decrease in the drought resistance and seedling protection effect of the rhizobium agent on alfalfa, further verifying the importance of each measure in this invention.

[0117] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a drought-resistant and seedling-protecting rhizobium agent, characterized in that, The method steps include: S1. Take rhizobium for fermentation culture, and then use sterile water to make a bacterial suspension from the cultured bacteria. S2. Take an organic carrier, uniformly mix it with modified sea squirt cellulose nanocrystal dispersion and CaCl2 solution, then mix the bacterial suspension with the organic carrier evenly so that the bacterial suspension is adsorbed onto the organic carrier, and air dry to obtain the bacterial material for later use. The organic carrier is one of peat, well-rotted organic fertilizer, or well-rotted straw powder; The preparation steps of the modified sea squirt cellulose nanocrystal dispersion are as follows: take dried and pulverized sea squirt capsules, add them to NaOH solution, stir and react in a water bath at 78-85℃ for 3-5h, take the reaction product, centrifuge and wash, redisperse in acetate-sodium acetate buffer, add NaClO2, and stir and react in a water bath at 72-78℃ for 1.5-2.5h. After washing and drying the reaction product, sea squirt cellulose is obtained. Then, the sea squirt cellulose is dispersed in phosphate buffer, 2,2,6,6-tetramethylpiperidine-1-oxide and NaBr are added, pre-cooled in an ice bath, and NaClO solution is added dropwise while maintaining the pH at 10-10.

5. After reacting for 2-3h, ethanol is added to terminate the reaction. Take the reaction product, centrifuge and wash, and then disperse with deionized water to obtain a modified sea squirt cellulose nanocrystal dispersion with a mass concentration of 0.5-1.5%. S3. After pretreatment of fresh Chinese medicinal residue, 30-40% of the residue is subjected to oxygen-limited pyrolysis. The carbonaceous material and pyrolysis condensate formed by pyrolysis are collected. The carbonaceous material, pyrolysis condensate and the remaining 60-70% of the residue are then mixed. A structural agent accounting for 1-3% of the total mass of the fresh Chinese medicinal residue before pretreatment is added. A hydrothermal synthesis reaction is carried out. The reaction product is dried to obtain a Chinese medicinal residue derivative carrier. The structural agent is montmorillonite or kaolinite; The pretreatment refers to first drying the Chinese herbal medicine residue until the moisture content is less than 15%, and then crushing it into uniform particles with a particle size of 2-5mm. The specific operation of the oxygen-limited pyrolysis is as follows: the Chinese herbal medicine residue is loaded into the pyrolysis furnace, nitrogen gas is first passed through the furnace at a flow rate of 400-600 mL / min for 20-30 min, and then the temperature is raised to 330-360℃ at a rate of 10-20℃ / min, and the pyrolysis is held at the temperature for 60-90 min. During the pyrolysis process, nitrogen gas is continuously passed through the furnace at a flow rate of 100-200 mL / min. The specific operation of the hydrothermal synthesis reaction is as follows: first, adjust the water content of the reaction system to 70-80% with deionized water, then stir thoroughly, and react at 170-190℃ for 4-6 hours; S4. Take liquid fertilizer and mix it with the Chinese herbal medicine residue derivative carrier to allow the liquid fertilizer to be adsorbed onto the Chinese herbal medicine residue derivative carrier, and obtain fertilizer for later use. S5. Mix the bacterial substrate and the fertilizer evenly to obtain the rhizobium agent; The mass ratio of the bacterial suspension, modified sea squirt cellulose nanocrystal dispersion, CaCl2 solution, and organic carrier is 1:0.3-0.5:0.08-0.12:2-3; the mass ratio of the liquid fertilizer to the traditional Chinese medicine residue derivative carrier is 1:2-4; and the mass ratio of the bacterial substrate to the fertilizer is 1.5-2.5:

1.

2. The method for preparing a drought-resistant and seedling-protecting rhizobium agent according to claim 1, characterized in that, The specific operation of step S1 is as follows: the rhizobium strain is activated into YMA solid medium by streak plating and cultured in a constant temperature incubator at 28℃ for 2-3 days until single colonies appear. Then, single colonies are selected and transferred to YMA liquid medium and cultured in a shaker at 28℃ for 12-18 hours. The bacterial suspension is then centrifuged for 8-12 minutes, the supernatant is discarded, and the precipitate is mixed with sterile water at a mass ratio of 1:1 to obtain a bacterial suspension, which is then stored in glycerol at -80℃ for later use.

3. The method for preparing a drought-resistant and seedling-protecting rhizobium agent according to claim 1, characterized in that, In step S4, the liquid fertilizer is either calcium nitrate or potassium nitrate, and the concentration of calcium ions or potassium ions in the liquid fertilizer is 4-6 mM.

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