A modulator for promoting the symbiosis of alfalfa and rhizobium and a preparation method thereof

Abstract

The present application belongs to the technical field of soil microorganism regulation, and particularly relates to a regulator for promoting the symbiosis of alfalfa and rhizobium and a preparation method thereof. The regulator comprises the following raw materials in parts by weight: eugenol 0.58-1.16 parts, lactobacillus peptide 0.1-0.4 parts, emulsification aid 0.03-0.05 parts, and slow-release agent 0.07 parts. The slow-release agent is used to solve the problems of easy degradation of the antibacterial peptide lactobacillus peptide and easy volatilization of eugenol through a slow-release and emulsification process. The regulator can not only ensure the safe germination of alfalfa seeds, but also selectively inhibit the competing bacteria in the soil, significantly improve the niche competition advantage and symbiotic nitrogen fixation efficiency of the exogenous rhizobium, and has the advantages of high efficiency, environmental protection and long field persistence.

Description

Technical Field

[0001] This invention belongs to the field of soil microbial regulation technology, specifically relating to a regulator that promotes the symbiosis between alfalfa and rhizobia and its preparation method. Background Technology

[0002] In modern agricultural production, the long-term excessive application of chemical fertilizers has not only reduced nitrogen use efficiency but also led to serious soil degradation and environmental pollution. Therefore, using alfalfa and rhizobium symbiotic nitrogen-fixing systems to replace part of the nitrogen fertilizer has become an important way to develop green and ecological agriculture. However, in actual field planting, exogenous rhizobia often face strong competition from native dominant microorganisms (such as Bacillus and Streptomyces) for nutrients and physical space, resulting in low colonization efficiency and a significant reduction in their nodulation and nitrogen-fixing capacity. To overcome this colonization barrier, traditional solutions often rely on the application of antibiotic-based chemical bacteriostatic agents to suppress competing bacteria. However, this not only easily leads to an overall imbalance in the soil microbial community and the accumulation of antibiotic-resistant genes but also carries high environmental and ecological risks, completely deviating from the development direction of green agriculture.

[0003] Therefore, there is an urgent need to develop a highly efficient, safe, and environmentally friendly green antibacterial agent that can selectively regulate the structure of soil microbial communities and enhance the field competition and colonization ability of rhizobia. Summary of the Invention

[0004] To address the above issues, this invention provides a regulator that promotes the symbiotic relationship between alfalfa and rhizobia and its preparation method. It combines plant-derived eugenol, which has broad-spectrum antibacterial properties, with nisin, a bacteriocinol that specifically targets Gram-positive bacteria, and constructs a stable water-based microemulsion slow-release system using adjuvants such as sodium alginate. The aim is to overcome the technical bottlenecks of single green antibacterial substances being volatile, poorly water-soluble, and easily enzymatically degraded in soil. While ensuring the safe germination of alfalfa seeds, it significantly enhances the niche competitive advantage and symbiotic nitrogen fixation efficiency of exogenous rhizobia, thus providing a novel symbiotic regulator that is highly efficient, environmentally friendly, and has strong stability in field applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a regulator that promotes the symbiosis between alfalfa and rhizobia. The regulator comprises the following raw materials in parts by weight: 0.58-1.16 parts of eugenol, 0.1-0.4 parts of nisin, 0.03-0.05 parts of emulsifying agent, and 0.07 parts of slow-release agent.

[0006] Furthermore, the emulsifying agent is selected from any one of Tween 80, alkyl glycosides, and soybean lecithin.

[0007] Furthermore, the sustained-release agent is selected from any one of sodium alginate, xanthan gum, and sodium carboxymethyl cellulose.

[0008] Furthermore, the regulator is used to promote symbiosis between alfalfa and rhizobia.

[0009] Furthermore, the alfalfa is selected from the Gannong No. 9 variety seeds.

[0010] Furthermore, the rhizobium is *Sinonovacula LL2*.

[0011] Furthermore, the rhizobium is activated and amplified to obtain a bacterial solution.

[0012] Furthermore, the activation process of the rhizobium uses an activation medium prepared from 5 g / L tryptone, 5 g / L yeast extract, 0.1 g / L CaCl2·6H2O, and 15 g / L agar.

[0013] Furthermore, the amplification process of the rhizobium uses an amplification medium, which is prepared by K2HPO4·3H2O 0.5 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.1 g / L, mannitol 10 g / L, yeast extract 1 g / L, agar 15 g / L, CaCO3·0.01 g / L, and distilled water 1000 mL.

[0014] This invention also provides a method for preparing a regulator that promotes the symbiotic relationship between alfalfa and rhizobia, the specific steps of which are as follows: Step 1: Dissolve nisin in milk, add a slow-release agent and stir continuously until the slow-release agent is completely dissolved and the solution is homogeneous and transparent, thus obtaining a colloidal aqueous phase; Step 2: Mix eugenol with emulsifier to obtain oil phase. Slowly add oil phase to colloidal aqueous phase under high-speed shear emulsification. After addition is complete, continue emulsification for 15 min to ensure sufficient emulsification and dispersion, and obtain emulsion. Step 3: Place the emulsion in a high-pressure homogenizer for homogenization. After homogenization, bring the volume to a final level with sterile water to obtain a regulator that promotes the symbiosis between alfalfa and rhizobia.

[0015] The beneficial effects achieved by this invention are as follows: The regulator for promoting symbiosis between alfalfa and rhizobia provided by this invention combines broad-spectrum antibacterial plant-derived eugenol with nisin, which specifically targets Gram-positive bacteria. This combination selectively suppresses competitive bacteria in the soil (such as Bacillus and Streptomyces) that compete with rhizobia for nutrients and space, without harming the Gram-negative rhizobia. This synergistic effect of "suppressing competition and promoting symbiosis" significantly alleviates colonization resistance, promotes the accumulation of exogenous rhizobia in the rhizosphere, and creates a superior microecological advantage for successful colonization. Addressing the common shortcomings of eugenol's high volatility and poor water solubility, as well as the ease with which polypeptide-based nitroglycerin is degraded by proteases in soil, this invention utilizes emulsifying agents and polymeric slow-release carriers to construct a stable water-based microemulsion slow-release system. This microemulsion form can firmly encapsulate the effective active ingredients, effectively resisting rapid loss and degradation caused by the soil environment, achieving long-lasting and uniform release in the crop rhizosphere, effectively solving the problem of short field retention of single green antibacterial agents. Attached Figure Description

[0016] Figure 1 The results of an investigation into the effects of different concentrations of eugenol on the germination of alfalfa seeds; Figure 2 The results of an investigation into the effects of different concentrations of nifedipine on the germination of alfalfa seeds; Figure 3 The inhibitory effects of different concentrations of eugenol and nisin on Rhizobium sinense LL2 were investigated. Figure 4 The phylum-level community structure of soil bacteria and fungi after treatment with eugenol and nisin; Figure 5 The results of the investigation of nodulation index of alfalfa after inoculation with the regulators prepared in Examples 5-7; Figure 6 The regulators prepared in Examples 5-7 were used to detect enzyme activity in alfalfa after inoculation. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0019] Unless otherwise specified, all methods used in the following embodiments are conventional. Unless otherwise specified, all materials used in the following embodiments are new materials purchased from the market. Specifically, the nisin used in the following embodiments and comparative examples was purchased from Chongqing Tianrun Biological Products Co., Ltd.; the eugenol used was purchased from Jiangxi Xuesong Natural Medicinal Oil Co., Ltd.; the alfalfa used was Gannong No. 9 alfalfa seeds; the rhizobium used was a commercially available strain, *Syngonium sinense* strain LL2, isolated from the root nodules of *Alfalfa sinensis* from Longzhong; the eugenol used was in liquid form and was actually added in volume units. All parts mentioned in this application are parts by weight, calculated at 1.16 g / cm³ of eugenol. 3 Density is converted to volumetric mass.

[0020] In the following examples and comparative examples, the activation culture medium was prepared by mixing and sterilizing 5 g / L tryptone, 5 g / L yeast extract, 0.1 g / L CaCl2·6H2O, and 15 g / L agar, with a pH of 7.0, and in solid or liquid form; The amplification medium is prepared from 0.5 g / L K2HPO4·3H2O, 0.2 g / L MgSO4·7H2O, 0.1 g / L NaCl, 10 g / L mannitol, 1 g / L yeast extract, 15 g / L agar, 0.01 g / L CaCO3, and 1000 mL distilled water, and can be in solid or liquid form.

[0021] Example 1: Investigation of the germination ability of eugenol on alfalfa seeds: Germination tests were conducted according to the "Inspection Procedures for Forage Seeds". Eugenol was used in six concentration gradients: 0, 500, 1000, 2000, 5000, and 10000 μL / L, with 100 mL of each solution. Plump and uniform alfalfa seeds were immersed in each concentration gradient of eugenol solution for 10 h. After soaking, the alfalfa seeds were removed, and the surface moisture was blotted dry with filter paper. 100 seeds from each treatment were evenly placed in 15 cm diameter petri dishes lined with double layers of filter paper. Distilled water was added at a rate of 30 mL / dish for germination testing. Each dish was weighed, and the water consumed in the dish was replenished daily with distilled water (weighing method). Each treatment was replicated in triplicate. From seed germination to day 15, the number of germinated seeds was counted daily, and the germination rate and germination potential of each treatment were calculated. Germination rate = (Total number of germinated seeds within 15 days / Number of tested seeds) × 100%; Germination potential = (Number of germinated seeds on day 4 / Number of tested seeds) × 100%. Results are shown in [link to results]. Figure 1 .

[0022] Example 2: Investigation of the germination ability of alfalfa seeds by nisin: Nisin was used in six concentration gradients (0, 100, 200, 300, 400, and 500 mg / L), with 100 mL of each solution. The germination test method was the same as in Example 1. Germination rate and germination potential were calculated, and the results are shown in [Figure 1]. Figure 2 .

[0023] Example 3: Tolerance study of rhizobium (Rhizobium sinense LL2) to eugenol and nisin Strain activation: The commercially available Chinese rhizobium strain LL2, which was preserved in the Key Laboratory of Grassland Ecosystem of the Ministry of Education at Gansu Agricultural University, was inoculated into activation medium (solid) and incubated in a constant temperature incubator at 28℃ for 18 h. Preparation of bacterial culture: Pick a single colony into an Erlenmeyer flask containing activation medium (liquid), and culture with shaking at 28℃ and 180 rpm for 18 h, and adjust the bacterial concentration to 106 CFU / mL; Agar plate preparation: Use a pipette to draw 2 mL of bacterial solution and spread it evenly on the surface of the amplification medium (solid) using a spreader; Sample addition: The Oxford cup method was used for determination. A sterile Oxford cup was placed in the center of the plate after spreading, and it was gently pressed to make it in close contact with the agar surface. 200 μL of eugenol solution or nisin solution of different gradient concentrations in Example 1 and Example 2 were added to the Oxford cup respectively. The control group was treated with sterile water. Incubation and observation: The plates were incubated at 28℃ for 18 h. The presence of inhibition zones around the Oxford cups was observed, and the diameter of the inhibition zones (mm) was measured using calipers. Results are shown below. Figure 3 .

[0024] Example 4: Effects of eugenol and nisin on soil microbial communities: Soil pretreatment: Soil samples were collected from five different locations at the Lanzhou Pasture Experimental Station of Gansu Agricultural University, at a depth of 3–5 cm below the surface. After thorough mixing, 1 L of each sample was placed in a perforated plastic container. 10 mL of 1000 μL / L eugenol solution and 10 mL of 400 mg / L nisin solution were added to the soil, with sterile water used as the control group. The samples were then thoroughly mixed. The samples were then placed in a light incubator under simulated natural light conditions (60% humidity, 25 ± 2℃) for 7 days. Genome sequencing: High-throughput sequencing technology was used to analyze soil samples treated with eugenol or nisin. Genomic DNA was extracted and its quality was assessed. Primers were then established using stable regions of the sequence, and sample-specific barcode sequences were added. PCR amplification was performed, and the results, including PCR amplification, library establishment and validation, and sequencing on the Illumina Mi Seq platform, are presented below. Figure 4 Tables 1 and 2.

[0025] Example 5: This example provides a regulator that promotes the symbiosis between alfalfa and rhizobia. The regulator comprises the following raw materials in parts by weight: 1.16 parts of eugenol, 0.3 parts of nisin, 0.05 parts of Tween 80, and 0.07 parts of sodium alginate; This embodiment also provides a method for preparing a regulator that promotes the symbiosis between alfalfa and rhizobia, the specific steps of which are as follows: Step 1: Dissolve 0.3 parts of nisin in 100 mL of sterile water, then add 0.07 parts of sodium alginate in a water bath at 40℃ and 400 rpm and continue stirring until the sodium alginate is completely dissolved to obtain a colloidal aqueous phase. Step 2: Vortex mix 1.16 parts of eugenol with 0.05 parts of Tween 80 for 5 min to obtain an oil phase. Slowly add the oil phase to the colloidal aqueous phase under high-speed shear emulsification conditions. The shear speed is 6000 rpm and the dropping rate is 3 mL / min. After the addition is completed, continue emulsification for 15 min to obtain an emulsion. Step 3: Place the emulsion in a high-pressure homogenizer for homogenization at a pressure of 25 MPa, and homogenize continuously for 3 cycles. After homogenization, bring the volume to 1 L with sterile water to obtain a regulator that promotes the symbiosis between alfalfa and rhizobia.

[0026] This embodiment provides a method for verifying inoculation of indoor potted plants, as detailed below: Seed sterilization: Healthy, plump, and uniformly sized Gannong No. 9 alfalfa seeds were sterilized using the DS treatment method. The seeds were placed in a sterilized 50 mL Erlenmeyer flask in a sterile operating table, then soaked in povidone-iodine for 5 minutes, rinsed with sterile water, and finally dried with sterile filter paper and set aside for later use. Seedling cultivation: The experiment used soil culture, which was carried out in a culture room (14 hours of light, 25℃; 10 hours of darkness, 20℃; light intensity 350 mol·m⁻¹). -2 ·s -1(Relative humidity 60%), plump and uniform alfalfa seeds were selected and sown into culture cups with a diameter of 9 cm and a height of 12 cm containing soil after disinfection. 20 seeds were sown in each cup, and 15 seedlings were maintained. A total of 11 treatments were conducted, and each treatment was repeated 6 times. During the plant growth process, nitrogen-free nutrient solution and distilled water were used alternately for irrigation to maintain sufficient other elements and water for the plants. 200 mL was added each time. After 40 days of cultivation, samples were taken for the determination of relevant indicators. Preparation and inoculation of rhizobium culture: Rhizobium stored at -80℃ was streaked onto activation medium (solid) for activation and cultured in a biochemical incubator at 28℃ for 24 h. A single colony was picked up using a sterile inoculation needle and transferred to a sterilized Erlenmeyer flask containing 400 mL of amplification medium (liquid). The flask was then incubated at 28℃ with shaking at 180 rpm for 18 h, until the bacterial count reached 1×10⁻⁶. 9 The concentration of CFU / mL was centrifuged at 4°C and 10,000 rpm for 10 min, the supernatant was discarded, and the solution was resuspended in an equal volume of the regulator prepared in this example. The control group was treated with an equal volume of sterile water. When the seedlings developed to the appearance of the first true leaf, they were inoculated with rhizobium at a rate of 1 mL per plant.

[0027] Example 6: This example provides a regulator that promotes the symbiosis between alfalfa and rhizobia. The regulator comprises the following raw materials in parts by weight: 0.58 parts of eugenol, 0.4 parts of nisin, 0.03 parts of alkyl glycoside, and 0.07 parts of xanthan gum. This embodiment also provides a method for preparing a regulator that promotes the symbiosis between alfalfa and rhizobia, the specific steps of which are as follows: Step 1: Dissolve 0.4 parts of nisin in 100 mL of sterile water, then add 0.07 parts of xanthan gum in a water bath at 40℃ and 400 rpm and continue stirring until the xanthan gum is completely dissolved to obtain a colloidal aqueous phase; Step 2: Vortex mix 0.58 parts of eugenol and 0.03 parts of alkyl glycoside for 5 min to obtain an oil phase. Slowly add the oil phase to the colloidal aqueous phase under high-speed shear emulsification conditions. The shear speed is 6000 rpm and the dropping rate is 3 mL / min. After the addition is complete, continue emulsification for 15 min to obtain an emulsion. Step 3: Place the emulsion in a high-pressure homogenizer for homogenization at a pressure of 25 MPa, and homogenize continuously for 3 cycles. After homogenization, bring the volume to 1 L with sterile water to obtain a regulator that promotes the symbiosis between alfalfa and rhizobia.

[0028] This embodiment provides a method for verifying inoculation of indoor potted plants. The specific steps are the same as in Embodiment 5. The regulator prepared in this embodiment is used in the preparation of rhizobium solution and inoculation steps.

[0029] Example 7: This example provides a regulator that promotes the symbiosis between alfalfa and rhizobia. The regulator comprises the following raw materials in parts by weight: 0.928 parts of eugenol, 0.1 parts of nisin, 0.04 parts of soybean lecithin, and 0.07 parts of sodium carboxymethyl cellulose. This embodiment also provides a method for preparing a regulator that promotes the symbiosis between alfalfa and rhizobia, the specific steps of which are as follows: Step 1: Dissolve 0.1 parts of nisin in 100 mL of sterile water, then add 0.07 parts of sodium carboxymethyl cellulose in a water bath at 40°C and 400 rpm and continue stirring until the sodium carboxymethyl cellulose is completely dissolved to obtain a colloidal aqueous phase. Step 2: Vortex mix 0.928 parts of eugenol and 0.04 parts of soybean lecithin for 5 min to obtain the oil phase. Slowly add the oil phase to the colloidal aqueous phase under high-speed shear emulsification conditions. The shear speed is 6000 rpm and the dropping rate is 3 mL / min. After the addition is completed, continue emulsification for 15 min to obtain the emulsion. Step 3: Place the emulsion in a high-pressure homogenizer for homogenization at a pressure of 25 MPa, and homogenize continuously for 3 cycles. After homogenization, bring the volume to 1 L with sterile water to obtain a regulator that promotes the symbiosis between alfalfa and rhizobia.

[0030] This embodiment provides a method for verifying inoculation of indoor potted plants. The specific steps are the same as in Embodiment 5. The regulator prepared in this embodiment is used in the preparation of rhizobium solution and inoculation steps.

[0031] Nodulation index assessment: The number of effective root nodules and the weight of a single effective root nodule were measured after alfalfa inoculation with the regulators prepared in Examples 5-7. The results are shown in […]. Figure 5 .

[0032] Nitrogen metabolism enzyme activity assay: The nitrogen metabolism enzyme activities of alfalfa treated in Examples 5-7 were measured using a kit for nitrate reductase (NR), glutamine synthase (GS), nitrite reductase (NiR), and glutamate synthase (GOGAT). The results are shown in the table below. Figure 6 .

[0033] Table 1. Detection of abundance and diversity indices of soil bacteria and fungi after treatment with nisin and eugenol.

[0034] Table 2. Dominant bacterial species with soil abundance >1% after treatment with nisin and eugenol.

[0035] Figure 1The results showed that after soaking in different concentrations of eugenol solution, the germination ability and probability of the seeds exhibited a similar developmental pattern: they gradually increased with increasing eugenol concentration and then decreased. Within the concentration range of 0-1000 μL / L, the germination rate was slightly higher than the germination potential, and their curves largely overlapped. This indicates that seed growth was relatively stable and consistent within this range, with most seeds successfully germinating within the first four days. Therefore, a treatment concentration of 1000 μL·L⁻¹ is a critical point, at which the effect of eugenol soaking on the seeds shifts from stimulation to inhibition.

[0036] Figure 2 The results showed that different concentrations of nisin had no significant effect on the germination rate, hard seed rate, and imbibition rate of alfalfa. At concentrations of 100-400 mg·L⁻¹, the germination potential was not significantly different from the control (CK). Starting from 500 mg·L⁻¹, the germination potential was significantly lower than other concentrations, resulting in delayed germination.

[0037] Figure 3 The results showed that eugenol did not produce inhibition zones at concentrations of 500-5000 μL·L⁻¹, except for a 9 mm diameter inhibition zone at 10000 μL / L. According to the reference standard for inhibition zone results, an inhibition zone diameter <10 mm indicates weak or no inhibitory effect. No inhibition zones were produced at any concentration of nisin, indicating that nisin has no significant inhibitory effect on the highly nitrogen-fixing rhizobium LL2. Studies have shown that nisin is a bacteriocin produced by lactic acid streptococci, which mainly has a significant inhibitory effect on Gram-positive bacteria (such as Listeria and Staphylococcus), through its mechanism of disrupting the cell membrane by interfering with cell wall synthesis and pore formation. However, rhizobium is a Gram-negative bacterium, and its outer membrane structure (containing lipopolysaccharides) has natural resistance to nisin; therefore, nisin usually does not have a significant inhibitory effect on it.

[0038] Table 1 shows that the Ace and Chao indices of the eugenol and nisin-treated samples were significantly lower than those of the control, indicating that the species richness of bacteria and fungi in the treated samples was lower than that of the control, and the difference was significant. Furthermore, the Shannon index of the eugenol and nisin-treated samples decreased while the Simpson index increased, indicating that the community evenness decreased and the number of dominant species increased.

[0039] Figure 4The results showed that the dominant bacterial phyla at the phylum level were Proteobacteria, Bacteroidetes, Chlorophylloides, Actinobacteria, Acidobacteria, and Firmicutes in all treatments. The relative abundance of dominant phyla in soil bacterial communities differed significantly after treatment with nisin and eugenol. The relative abundance of Proteobacteria increased by 31.06% and 24.89%, respectively, while that of Bacteroidetes decreased by 9.29% and 11.78%, respectively. The relative abundance of Actinobacteria decreased by 57.98% and 50.62%, respectively, and that of Firmicutes decreased by 84.09% and 65.85%, respectively. The phylum-level structure of soil fungal communities was similar across treatments, with Ascomycota, Basidiomycota, and Moniliformes being the dominant phyla. However, after treatment with nisin and eugenol, the relative abundance of all three phyla decreased significantly. Specifically, Ascomycota decreased by 6.44% and 34.68%, respectively; Basidiomycota decreased by 8.54% and 64.87%, respectively; and Moniliformes decreased by 0.08% and 65.72%, respectively.

[0040] Table 2 shows that *Bacillus* was the dominant genus with the highest relative abundance in the control group. After treatment with eugenol and nisin, the relative abundance of *Bacillus* fell below 1%, and its dominant species was replaced by *Rhizobium*. The abundance of *Rhizobium* in the control group was 1.01%, which was at a low level. After treatment with nisin, the abundance increased to 4.47%, an increase of 342.6%, and after treatment with eugenol, the abundance increased to 3.98%, an increase of 294.1%.

[0041] Figure 5 The results showed that the number of effective root nodules per alfalfa plant and the weight of a single effective root nodule were significantly increased after inoculation with the regulator prepared in Example 5. This indicates that the prepared regulator may have effectively enhanced the field colonization advantage of Rhizobium sinense LL2 by selectively inhibiting competitive soil microorganisms and reshaping the colony structure, thereby significantly promoting the nodulation ability of alfalfa.

[0042] Figure 6 The results showed that the regulators prepared in Examples 5-7 significantly enhanced the activity of key enzymes in leaf diazo metabolism in alfalfa, demonstrating the regulators' ability to fix nitrogen in root nodules, improve the nitrogen supply level in the host plant, and promote nitrogen assimilation and metabolic efficiency in the plant.

[0043] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0044] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A modulator for promoting the symbiosis of Medicago sativa with Rhizobium, characterized in that, The energy-saving agent comprises the following raw materials in parts by weight: 0.58-1.16 parts of eugenol, 0.1-0.4 parts of nisin, 0.03-0.05 parts of emulsifying agent, and 0.07 parts of sustained-release agent; The emulsifying agent is selected from any one of Tween 80, alkyl glycosides and soybean lecithin; The sustained-release agent is selected from any one of sodium alginate, xanthan gum, and sodium carboxymethyl cellulose.

2. The modulator for promoting the symbiosis of Medicago sativa and rhizobium according to claim 1, characterized in that, The alfalfa used was selected from the Gannong No. 9 variety.

3. The modulator for promoting the symbiosis of Medicago sativa and rhizobium according to claim 2, characterized in that, The rhizobium is Chinese rhizobium strain LL2.

4. The modulator for promoting the symbiosis of Medicago sativa and rhizobium according to claim 3, characterized in that, The rhizobium was activated and amplified to obtain a bacterial solution; The activation process of the rhizobia uses an activation culture medium; The amplification process of the rhizobia uses an amplification medium.

5. The modulator for promoting the symbiosis of Medicago sativa and rhizobium according to claim 4, characterized in that, The activation medium was prepared from 5 g / L tryptone, 5 g / L yeast extract, 0.1 g / L CaCl2·6H2O, and 15 g / L agar. The amplification medium was prepared from 0.5 g / L K2HPO4·3H2O, 0.2 g / L MgSO4·7H2O, 0.1 g / L NaCl, 10 g / L mannitol, 1 g / L yeast extract, 15 g / L agar, 0.01 g / L CaCO3, and 1000 mL distilled water.

6. A method for preparing the modulator for promoting the symbiosis of Medicago sativa and Rhizobium of any one of claims 1-5, characterized in that, The specific steps are as follows: Step 1: Dissolve nisin in milk, add a slow-release agent and stir to dissolve, thus obtaining a colloidal aqueous phase; Step 2: Mix eugenol with emulsifier to obtain an oil phase, add the oil phase to the colloidal aqueous phase and emulsify to obtain an emulsion; Step 3: Homogenize and adjust the volume of the emulsion to obtain a regulator that promotes the symbiosis between alfalfa and rhizobia.

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