Preparation method and application of complex microbial agent for promoting alfalfa growth

Abstract

The application provides a preparation method and application of a complex microbial agent for promoting alfalfa growth, and relates to the technical field of agricultural microorganisms. The complex microbial agent is prepared by mixing a bacterial solution and a biological stimulant solution. The bacterial solution is a rhizobium solution, and the biological stimulant is a fucoidan solution. The volume ratio of the rhizobium solution to the fucoidan solution is 1:2-4. The complex microbial agent solves the bottleneck problem that a single rhizobium agent is unstable in actual application and is easily affected by the environment. The alfalfa treated by the complex microbial agent can improve the seedling emergence rate, plant height, biomass and stress resistance of the alfalfa.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural microorganism technology, in particular to a preparation method of a compound microbial agent for promoting alfalfa growth and application thereof. BACKGROUND

[0002] Alfalfa (Medicago sativa L.) is one of the most important high-quality legume forages in the world, widely planted for its high protein content and strong adaptability, and is known as the "king of forage". It plays an indispensable role in the development of animal husbandry, soil improvement and ecological environment construction. However, in actual production, the growth of alfalfa is often restricted by many factors, such as soil infertility, drought stress, etc., resulting in unstable yield and quality, which is difficult to meet the growing demand.

[0003] At present, the main measures to promote the growth of alfalfa include the application of chemical fertilizers and agricultural microbial agents. Although chemical fertilizers can quickly supplement nutrients, long-term overuse will lead to a series of environmental problems such as soil compaction, acidification, and water eutrophication, and does not meet the development concept of green agriculture. In contrast, microbial agents are attracting attention due to their environmental friendliness and sustainability. Among them, the application of rhizobial agents is the most common, which can form a symbiotic nitrogen fixation system with alfalfa root system, converting free nitrogen in the air into plant-absorbable ammonia, thereby reducing the amount of nitrogen fertilizer.

[0004] However, the existing single microbial agent (such as single rhizobial agent) has the bottleneck problems of unstable effect, poor stress resistance, and limited growth promotion effect in actual application. After the microbial agent is applied to the soil, the survival rate of the strain is easily affected by environmental conditions (such as drought, pH, and competition with indigenous microorganisms), resulting in low colonization success rate and inability to fully exert its growth promotion function.

[0005] Therefore, it is of great practical significance to develop a compound microbial agent with reasonable formula, stable effect, and the ability to significantly promote alfalfa growth and enhance its stress resistance, for promoting the green and sustainable development of alfalfa industry. SUMMARY

[0006] To solve the above technical problems, the present application provides a compound microbial agent for promoting alfalfa growth, which is mixed by a microbial solution and a biological stimulant solution, the microbial solution is a rhizobial solution, and the biological stimulant is a brown algal oligosaccharide solution (AOS); the volume ratio of the rhizobial solution to the brown algal oligosaccharide solution is 1:2-4.

[0007] The rhizobium bacterial solution is obtained by culturing Rhizobium sp. MCDC3 strain, the Rhizobium sp. MCDC3 has a preservation number of CCTCC NO: M20231118, a 16S rDNA sequence as shown in SEQ ID NO: 1, and a preservation date of June 28, 2023;

[0008] The rhizobium bacterial solution is obtained by culturing Rhizobium sp. MCDC3 strain, the Rhizobium sp. MCDC3 has a preservation number of CCTCC NO: M20231118, a 16S rDNA sequence as shown in SEQ ID NO: 1, and a preservation date of June 28, 2023;

[0009] Preferably, the volume ratio of the rhizobium bacterial solution to the alginate oligosaccharide solution is 1:2.

[0010] Preferably, the OD 600 value of the rhizobium bacterial solution is 0.6-0.7.

[0011] Preferably, the mass-volume concentration of the alginate oligosaccharide solution is 0.5 g / L-1.5 g / L.

[0012] In another aspect, the present application provides a method for preparing the composite microbial agent, comprising the following steps:

[0013] S1, preparing a rhizobium bacterial solution: inoculating Rhizobium sp. MCDC3 strain into YMA liquid medium, and culturing at 25-28℃ and 150-200 r / min for 2-3 days to obtain a rhizobium bacterial solution with an OD 600 value of 0.6-0.7, and then diluting with pure water at a ratio of 1:3;

[0014] S2, preparing a biological stimulant solution: dissolving alginate oligosaccharide in water to prepare an alginate oligosaccharide solution with a mass-volume concentration of 0.5 g / L-1.5 g / L;

[0015] S3, compounding: mixing the rhizobium bacterial solution obtained in step S1 with the alginate oligosaccharide solution obtained in step S2 at a volume ratio of 1:1-3 to obtain the composite microbial agent.

[0016] Preferably, in step S1, the YMA liquid medium is prepared from the following components: mannitol 10 g, yeast powder 3 g, MgSO4·7H2O 0.2 g, NaCl 0.1 g, K2HPO4 0.25 g, KH2PO4 0.25 g, and the medium is sterilized at 121℃ for 15 min after being adjusted to 1L and pH 7.0.

[0017] In a third aspect, the application provides application of the complex microbial agent in promoting growth of alfalfa, wherein alfalfa seeds are soaked in 300 μM gibberellin solution for 6-7 h, then dried in the shade for 12 h, and the complex microbial agent is applied by irrigation at the time of alfalfa sowing, so as to improve one or more of the seedling emergence rate, plant height, root length and biomass of alfalfa, and to enhance the drought resistance of alfalfa.

[0018] Preferably, the alfalfa is Caoyuan No. 3 mixed clover.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] 1. The complex microbial agent provided by the application is mixed from a bacterial solution and a biological stimulant solution, the bacterial solution is rhizobium solution, and the biological stimulant is brown algal oligosaccharide solution; the volume ratio of the rhizobium solution to the brown algal oligosaccharide solution is 1:2-4 (preferably 1:2), which solves the bottleneck problem that a single rhizobium agent is unstable in effect and is easily affected by the environment; experimental results show that the treatment group using the complex microbial agent has the highest alfalfa seedling emergence rate, which is increased by 22.72%, the plant height and the biomass are significantly increased, for example, the plant height of treatment 5 is 1.43 times that of the control group, the aboveground biomass is increased by 3.5 times, and the stress resistance is enhanced, and the alfalfa performs well under drought stress, which proves the effectiveness of the complex microbial agent in promoting growth of alfalfa and increasing yield.

[0021] 2. The biological stimulant (brown algal oligosaccharide, AOS) can significantly improve the survival and reproduction capacity of rhizobium as an adsorption carrier; experiments show that after the cultured rhizobium is mixed with AOS, the effective viable count is increased from 3.1×10 8 CFU / g to 6.3×10 8 CFU / g, which proves that after the complex microbial agent provided by the application is applied to soil, the colonization success rate and survival rate of rhizobium are higher, so that the growth-promoting effect is more stable and more durable.

[0022] 3. The rhizobium is compounded with the biological stimulant, which solves the bottleneck problem that a single rhizobium agent is unstable in effect and is easily affected by the environment in actual application. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Seedling emergence rates of alfalfa under different treatments;

[0024] Figure 2 Influence of the complex microbial agent on seedling emergence of alfalfa;

[0025] Figure 3 Influence of the complex microbial agent on single alfalfa plant;

[0026] Figure 4For effective viable cell number determination; wherein A is only rhizobium MCDC3 effective viable cell number determination; B is rhizobium MCDC3 and algal oligosaccharide AOS mixed and cultured effective viable cell number determination; C is rhizobium MCDC3 first culture and then mixed into algal oligosaccharide AOS effective viable cell number determination. DETAILED DESCRIPTION

[0027] Example 1

[0028] A composite microbial agent for promoting alfalfa growth is mixed by a bacterial solution and a biological stimulant solution, the bacterial solution is rhizobium bacterial solution, the biological stimulant is algal oligosaccharide solution; the volume ratio of the rhizobium bacterial solution to the algal oligosaccharide solution is 1:2.

[0029] The rhizobium bacterial solution is obtained by culturing Rhizobium sp. MCDC3 strain, the Rhizobium sp. MCDC3 has a preservation number of CCTCC NO: M20231118, and a 16S rDNA sequence as shown in SEQ ID NO: 1.

[0030] Further, the OD 600 value of the rhizobium bacterial solution is 0.6.

[0031] Further, the mass-volume concentration of the algal oligosaccharide solution is 0.5 g / L.

[0032] Example 2

[0033] A composite microbial agent for promoting alfalfa growth is mixed by a bacterial solution and a biological stimulant solution, the bacterial solution is rhizobium bacterial solution, the biological stimulant is algal oligosaccharide solution; the volume ratio of the rhizobium bacterial solution to the algal oligosaccharide solution is 1:4.

[0034] The rhizobium bacterial solution is obtained by culturing Rhizobium sp. MCDC3 strain, the Rhizobium sp. MCDC3 has a preservation number of CCTCC NO: M20231118, and a 16S rDNA sequence as shown in SEQ ID NO: 1.

[0035] Further, the OD 600 value of the rhizobium bacterial solution is 0.7.

[0036] Further, the mass-volume concentration of the algal oligosaccharide solution is 1.5 g / L.

[0037] Example 3

[0038] The compound microbial agent for promoting alfalfa growth is mixed by a bacterial liquid and a biological stimulant solution, the bacterial liquid is rhizobium bacterial liquid, and the biological stimulant is a brown algal oligosaccharide solution; the volume ratio of the rhizobium bacterial liquid to the brown algal oligosaccharide solution is 1:3.

[0039] The rhizobium bacterial liquid is obtained by culturing a nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 strain, the nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 has a preservation number of CCTCC NO:M20231118, and a 16S rDNA sequence is shown as SEQ ID NO:1.

[0040] Further, the OD 600 value of the rhizobium bacterial liquid is 0.6-0.7.

[0041] Further, the mass-volume concentration of the brown algal oligosaccharide solution is 0.5g / L-1.5g / L.

[0042] Example 4

[0043] A method for preparing a compound microbial agent, comprising the following steps:

[0044] S1, preparing a rhizobium bacterial liquid: inoculating a nitrogen-fixing rhizobium (Rhizobium sp.) MCDC3 strain into YMA liquid medium, and culturing at 25°C and 150r / min for 2 days to obtain a rhizobium bacterial liquid with an OD 600 value of 0.6, and then diluting with pure water at a ratio of 1:3;

[0045] S2, preparing a biological stimulant solution: dissolving brown algal oligosaccharide in water to prepare a brown algal oligosaccharide solution with a mass-volume concentration of 0.5g / L-1.5g / L;

[0046] S3, compounding: mixing the rhizobium bacterial liquid obtained in step S1 with the brown algal oligosaccharide solution obtained in step S2 at a volume ratio of 1:1 to obtain the compound microbial agent.

[0047] Further, in step S1, the YMA liquid medium is prepared from the following components: mannitol 10g, yeast powder 3g, MgSO4·7H2O 0.2g, NaCl 0.1g, K2HPO4 0.25g, KH2PO4 0.25g, and the medium is sterilized at 121°C for 15 min after being adjusted to 1L and pH 7.0.

[0048] Example 5

[0049] A method for preparing a compound microbial agent, comprising the following steps:

[0050] S1. Preparation of Rhizobium inoculum: Rhizobium sp. MCDC3 strain was inoculated into YMA liquid medium and cultured with shaking at 28℃ and 200 r / min for 3 days to obtain OD. 600 The Rhizobium inoculum solution with a value of 0.7 was then diluted with purified water at a ratio of 1:3.

[0051] S2. Preparation of biostimulant solution: Dissolve the alginate oligosaccharide in water to prepare an alginate oligosaccharide solution with a mass-volume concentration of 0.5 g / L-1.5 g / L;

[0052] S3. Compound preparation: The rhizobium inoculum solution obtained in step S1 and the brown algae oligosaccharide solution obtained in step S2 are mixed at a volume ratio of 1:3 to obtain the compound inoculum agent.

[0053] Furthermore, the formulation of the YMA liquid culture medium is the same as in Example 4.

[0054] Example 6

[0055] A method for preparing a compound microbial agent includes the following steps:

[0056] S1. Preparation of Rhizobium inoculum: Rhizobium sp. MCDC3 strain was inoculated into YMA liquid medium and cultured with shaking at 26℃ and 150-200 r / min for 2.5 days to obtain OD. 600 The Rhizobium inoculum solution with a value of 0.65 was then diluted with purified water at a ratio of 1:3.

[0057] S2. Preparation of biostimulant solution: Dissolve the brown algae oligosaccharide in water to prepare a brown algae oligosaccharide solution with a mass-volume concentration of 1.0 g / L;

[0058] S3. Compound preparation: The rhizobium inoculum solution obtained in step S1 and the brown algae oligosaccharide solution obtained in step S2 are mixed at a volume ratio of 1:2 to obtain the compound inoculum agent.

[0059] Furthermore, the formulation of the YMA liquid culture medium is the same as in Example 4.

[0060] Experimental Section

[0061] 1. Materials and Methods

[0062] 1.1 Test Materials

[0063] The test seeds used in this invention were alfalfa (Grassland No. 3 hybrid alfalfa), and the microbial inoculant (rhizobium: Rhizobium sp. MCDC3 strain; endomycorrhizal fungi: Rhizocystis strain (purchased from Nanjing Cuijingyuan Biotechnology Co., Ltd.); biostimulants (fucoidan), plant growth regulators (gibberellin), YMA medium.

[0064] 1.2 Experimental Design

[0065] 1.2.1 Preparation of microbial inoculants

[0066] (1) Preparation of Rhizobium inoculum solution

[0067] Rhizobium strains were inoculated into YMA culture medium using an inoculation loop and cultured in a shaker at 25-28℃ and 150-200 r / min for 2-3 days to prepare a rhizobium culture solution with an OD value of 0.6-0.7. The rhizobium culture solution was then diluted with purified water at a ratio of 1:3.

[0068] The YMA culture medium formula is as follows: 10g mannitol, 3g yeast powder, 0.2g MgSO4·7H2O, 0.1g NaCl, 0.25g K2HPO4, 0.25g KH2PO4, bring the volume to 1L, adjust the pH to 7.0, and sterilize the culture medium at 121℃ for 15 min.

[0069] (2) Preparation of endomycorrhizal fungal solution

[0070] Weigh 0.3g of endomycorrhizal fungus agent, dissolve it in purified water, and bring the volume to 500ml.

[0071] 1.2.2 Preparation of Single and Compound Microbial Agents

[0072] (1) Preparation of biostimulant solution

[0073] Weigh 0.5g of brown algae oligosaccharide powder, add it to purified water to dissolve, and make up to 500 ml.

[0074] (2) Preparation of single and compound microbial agents

[0075] The single and compound bacterial agents are obtained by mixing the Rhizobium inoculum solution, the Rhizocystis inoculum solution, and the biostimulant solution in the specified volume ratio (Table 1).

[0076] Table 1 Experimental Design for Different Inoculant Ratios

[0077]

[0078] 1.2.3 Application of different inoculant ratios to alfalfa

[0079] (1) Alfalfa seed treatment

[0080] Weigh 51.9555 mg of gibberellin powder, dissolve it in purified water, and bring the volume to 500 ml to prepare a 300 μM gibberellin solution.

[0081] Soaking alfalfa seeds in 300 μM gibberellin solution for 6-7 h, then air-drying for 12 h, ready for use.

[0082] (2) Alfalfa potting

[0083] Gibberellin soaked alfalfa seeds were used in the potting method, the humidity of the light incubator was 60%, the temperature was 25℃, the light treatment was 16 h; the temperature was 22℃, the dark treatment was 8 h. 9 treatment groups and 1 CK group were set up, each treatment was set up 3 times, 9 seeds were planted in each repeat. Among them, the sowing day was the 0th day, 60 ml of single or compound microbial agent was poured into each pot according to different microbial agent ratio test design on the 0th day, and the control group was poured with 60 ml of pure water. When the plants grew to 22 d, drought stress lasted for 14 d, and after the drought stress was completed, the rehydration test was carried out, and pure water was supplemented every 2 d, for a total of 60 d.

[0084] (3) Phenotype data determination

[0085] The phenotype trait indexes of the 60th day in this experiment mainly include: emergence rate, plant height, root length, biomass (aboveground biomass and underground biomass). In order to ensure the accuracy of the measurement index, the minimum scale of the ruler is 0.5 mm to measure the plant height and root length; when measuring the biomass, first separate the aboveground and underground parts of the plant, then use the electronic balance with the minimum scale of 0.01 g to weigh and record them respectively.

[0086] 1.2.4 Effect of biological stimulant on the number of effective Rhizobium bacteria

[0087] (1) Determination method

[0088] Take 0.01 g of alginate oligosaccharide powder, take 10 ml of YMA culture solution containing Rhizobium strain, mix well, and place in a 25-28℃, 150-200 r / min shaking bed for 2 d of shaking culture. After culture, the Rhizobium bacterial suspension containing alginate oligosaccharide was gradient diluted, 10 −6 0.1 ml of Rhizobium bacterial suspension containing biological stimulant was added to YMA solid culture medium, and gently coated. After the surface of the culture medium solidified, the plate was placed in an incubator at 28℃ for 2 d of culture.

[0089] (2) Counting and calculation

[0090] After culture, observe the growth of colonies on the plate. Choose plates with 30-300 colonies for counting, and record the number of colonies on each plate. According to the dilution factor, calculate the number of effective Rhizobium bacteria per gram (or per milliliter) of sample, the calculation formula is: effective viable count (CFU / mL) = number of colonies on the plate × dilution factor × 10.

[0091] 2 Results and analysis

[0092] 2.1 Effects of single and complex bacterial inoculants on seedling emergence of alfalfa

[0093] The results showed that, as shown in Table 1, all the different treatments could emerge normally, and the emergence rate reached 75% or above. Single and complex bacterial inoculants had different degrees of promoting effect on the emergence of alfalfa. The emergence rate of treatment 5 was 100%, and the emergence rates of treatments 4, 6 and 9 were also relatively high, all around 92%. The emergence rate of treatment 1 was the lowest, only about 77%. The emergence rate of the control group (CK) was about 81%, which was significantly lower than that of treatments 4, 5, 6 and 9, but higher than that of treatments 1 and 8, etc. This indicated that different treatments had different degrees of regulation effect on the emergence rate, and the effect of treatment 5 was the best. Figure 1 2.2 Effects of single and complex bacterial inoculants on alfalfa seedlings

[0094] In order to evaluate the effects of single and complex bacterial inoculants on alfalfa seedlings, the plant height, root length and biomass (aboveground + underground) of alfalfa seedlings were measured, as shown in Table 2.

[0095] Figure 2 Compared with the control group (CK), all the groups of bacterial inoculation treatment could improve the plant height of alfalfa seedlings. Among them, the effects of treatment 5 (10.34 cm) and treatment 1 (9.38 cm) were the most outstanding, which were 2.69 times and 2.44 times of CK (3.84 cm), respectively, and reached a significant difference level with CK.

[0096] In terms of root development, treatment 1 (17.4 cm) and treatment 5 (19.46 cm) also performed best, and the root length increased by 14.4% and 27.9% compared with CK (15.21 cm), respectively. Treatment 1 had significant difference with CK and some other treatments (such as treatments 3, 4 and 8), which showed its specific advantage in promoting root elongation.

[0097] In terms of aboveground biomass, the aboveground biomass of treatment 5 (0.18 g) and treatment 1 (0.15 g) was the highest, which was 4.5 times and 3.75 times of CK (0.04 g), respectively, and the growth was extremely significant.

[0098] In terms of underground biomass, the underground biomass of treatment 5 (0.19 g) was also the highest, which increased by 90% compared with CK (0.10 g), and reached a significant difference (a vs c).

[0099] In terms of underground biomass, the underground biomass of treatment 5 (0.19 g) was also the highest, which increased by 90% compared with CK (0.10 g), and reached a significant difference (a vs c).

[0100] ​The total biomass of treatment 5 (0.38 g) accumulated most significantly, which was 2.71 times of CK (0.14 g). The total biomass of treatment 1 (0.31 g) was the second, which was 2.21 times of CK. Both of them were significantly higher than the control group.

[0101] The experimental results showed that inoculating bacterial agents, especially treatment 5 of compound bacterial agents, could significantly promote the growth of alfalfa seedling height, root extension and biomass accumulation (including aboveground and underground parts), and the comprehensive growth-promoting effect was better than most single bacterial agent treatments.

[0102] Table 2 Effects of single and compound bacterial agents on alfalfa seedlings

[0103]

[0104] 2.3 Effects of single and compound bacterial agents on single alfalfa plant

[0105] Selecting the three groups with the best growth (treatment 1, treatment 5, and treatment 7) and comparing them with the CK group, the growth of single alfalfa plant was observed, and it was found that Figure 3 treatment 1, treatment 5, and treatment 7 were better than the CK group, indicating that single bacterial agents and compound bacterial agents could promote plant growth. The single alfalfa plant of treatment 5 had branches (red circle position), and the average aboveground biomass was increased by 4.5 times compared with the CK group (0.04 g). During the drought stress, it was found that treatment 5 showed good drought resistance in single plant growth, which proved that the combination of rhizobium and biological stimulants in compound bacterial agents not only had obvious growth-promoting effect and increased biomass, but also effectively enhanced the adaptability to adversity.

[0106] 2.4 Effects of biological stimulants on the effective number of rhizobium

[0107] Through pot experiment, it was found that the combination of rhizobium and biological stimulants in treatment 5 was more conducive to plant growth. To verify whether the biological stimulants had an impact on rhizobium, the effective number of rhizobium was determined, and it was found that Figure 4 as shown in the table, the addition of biological stimulants had different degrees of influence on the effective number of rhizobium (Rhizobium sp.) MCDC3, and the effective number of rhizobium after adding biological stimulants showed an increasing trend. Rhizobium Figure 4 For the determination of effective number of rhizobium; in the figure, A is the determination of effective number of rhizobium MCDC3 containing only rhizobium, and the effective number of rhizobium was 3.1×10 8 ​CFU / g; B is the determination of the effective viable cell number of rhizobium MCDC3 mixed with algal oligosaccharide AOS, after 2 days of mixed culture of rhizobium MCDC3 and biological stimulant algal oligosaccharide AOS, the distribution of the effective viable cell number is dispersed, and the number is 2.3 x 10 8 CFU / g; C is the determination of the effective viable cell number of rhizobium MCDC3 mixed with algal oligosaccharide AOS, after 2 days of mixed culture of rhizobium MCDC3 and biological stimulant algal oligosaccharide AOS, the distribution of the effective viable cell number is dispersed, and the number is 2.3 x 10 8 CFU / g, which is 1.03 times the effective viable cell number of rhizobium MCDC3 on the plate; the results show that the biological stimulant as an adsorption carrier is conducive to the survival and reproduction of rhizobium, but rhizobium should not be mixed with biological stimulants for culture in a shaker, which is not conducive to the survival and reproduction of the strain.

[0108] 3 Conclusion

[0109] In this study, the nine different bacterial agent ratio tests all have different effects on the growth of alfalfa plants, and the composite bacterial agent shows stronger stability in promoting growth efficiency and stress resistance than the single bacterial agent. Compared with CK, the treatment of mixing rhizobium and biological stimulants in the composite bacterial agent has a significant effect on the growth, stress resistance and yield of alfalfa plants. The effect of biological stimulants on rhizobium is verified, and it is found that the biological stimulants as an adsorption carrier can increase the number of effective viable cell number of rhizobium after culture, which is more conducive to the survival and reproduction of rhizobium. The composite bacterial agent with a ratio of 1:2 has a more lasting effect on plant growth promotion, and the ratio of 1:1 or 1:4 shows an inhibitory effect. The optimal synergistic ratio between rhizobium and biological stimulants is 1:2, and deviating from this ratio may break the ecological balance of the plant rhizosphere microenvironment, turning from mutualism to competition or inhibition.

[0110] In summary, when the rhizobium bacterial solution and the biological stimulant solution are compounded into a composite bacterial agent at a ratio of 1:2, the data is most balanced, and the effect is most significant.

Claims

1. A complex microbial agent for promoting the growth of alfalfa, characterized by, The bacteria liquid is rhizobium bacteria liquid, and the biological stimulant solution is a brown algal oligosaccharide solution; The volume ratio of the rhizobium bacteria liquid to the brown algal oligosaccharide solution is 1:

2. The rhizobium bacteria liquid is obtained by culturing Rhizobium sp. MCDC3 strain, the Rhizobium sp. MCDC3 has a preservation number of CCTCC NO: M20231118, and the 16S rDNA sequence is shown as SEQ ID NO:

1. The OD600 value of the rhizobium bacteria liquid is 0.6-0.

7. The mass-volume concentration of the brown algal oligosaccharide solution is 1.0 g / L. The preparation method of the rhizobium bacteria liquid is as follows: inoculating Rhizobium sp. MCDC3 strain into YMA liquid medium, and culturing at 25-28 °C and 150-200 r / min for 2-3 days to obtain rhizobium bacteria liquid with an OD600 value of 0.6-0.7, and then diluting with pure water at a ratio of 1:

3.

2. The complex microbial agent for promoting alfalfa growth according to claim 1, characterized by, The preparation method of the complex microbial agent comprises the following steps: S1, preparing rhizobium bacteria liquid; S2, preparing a biological stimulant solution: dissolving brown algal oligosaccharide in water to prepare a brown algal oligosaccharide solution with a mass-volume concentration of 0.5 g / L-1.5 g / L; S3, compounding: mixing the rhizobium bacteria liquid obtained in step S1 with the brown algal oligosaccharide solution obtained in step S2 at a volume ratio of 1:1-3, to obtain the complex microbial agent.

3. The complex microbial agent for promoting alfalfa growth according to claim 1, characterized by, The formula of the YMA liquid medium comprises the following components: mannitol 10 g, yeast powder 3 g, MgSO4·7H2O 0.2 g, NaCl 0.1 g, K2HPO4 0.25 g, KH2PO4 0.25 g, and the medium is adjusted to 1 L, and the pH is adjusted to 7.0, and then the medium is sterilized at 121 °C for 15 min.

4. The complex microbial agent for promoting the growth of alfalfa according to any one of claims 1-3 is applied to promote the growth of alfalfa, alfalfa seeds are soaked in 300 μM gibberellin solution for 6-7 h, and then dried in the shade for 12 h, and the complex microbial agent is applied for irrigation when alfalfa is sown.

5. The use of the composite microbial agent for promoting alfalfa growth according to claim 4, characterized in that, It is used for improving one or more of the seedling emergence rate, plant height, root length and biomass of alfalfa.

6. The use of the composite microbial agent for promoting alfalfa growth according to claim 4, characterized in that, It is used for enhancing the drought resistance of alfalfa.

7. The use of the composite microbial agent for promoting alfalfa growth according to claim 4, characterized in that, The alfalfa variety is grassland No. 3 hybrid alfalfa.

Images