A compound microbial agent for promoting the growth of leguminous plants and its application

Abstract

This invention relates to the fields of fertilizers and biotechnology, specifically a compound microbial agent for promoting legume growth. The compound microbial agent is composed of 25-35 parts microbial agent, 10-20 parts bioactive agent, 10-15 parts bioactivator, and 30-55 parts water. This invention utilizes the role of microorganisms in preventing plant diseases and / or promoting plant growth. Endophytic bacteria 3G1, isolated from rice seeds, promotes legume root growth and has a 100% successful colonization rate, directly impacting plant growth. Combined with the low-temperature resistance and plant disease resistance functions of other compound microbial agents, it has growth-promoting and disease-preventing effects on legumes. The bioactive agent, a lipopeptide, increases leaf spread, promotes the absorption of the compound microbial agent, increases its activity, and increases crop yield by 3%. The bioactivator provides trace elements necessary for plant growth; when used in conjunction with the compound microbial agent, it effectively promotes seedling growth, improves seedling resistance, and achieves a transplant survival rate as high as 98%.

Description

Technical Field

[0001] This invention relates to the fields of fertilizers and biotechnology, and in particular to a compound microbial agent for promoting the growth of leguminous plants and its application. Background Technology

[0002] Legumes are an important plant family, widely cultivated globally due to their significant nutritional and economic value. However, growers often face various challenges in legume cultivation, including insufficient soil fertility, frequent pests and diseases, and unstable yields. These problems not only affect the yield and quality of legumes but also increase production costs, limiting the sustainable development of the legume industry.

[0003] While some microbial fertilizers are currently used to promote the growth of legumes, these fertilizers typically contain only a single type of microorganism, resulting in relatively limited functionality. For example, some fertilizers primarily focus on nitrogen fixation while neglecting other important soil microbial activities, such as phosphorus solubilization, potassium release, and the production of plant growth hormones. Furthermore, single-microbial fertilizers are often susceptible to environmental conditions, exhibiting poor stability and persistence, making it difficult to meet the diverse needs of legumes during their growth process. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art by providing a compound microbial agent for promoting the growth of legumes and its application, thereby overcoming the deficiencies of the prior art, meeting the diverse needs in the legume planting process, and promoting the sustainable development of the legume industry.

[0005] The present invention provides a compound microbial agent for promoting the growth of legumes, which is made of 25-35 parts of microbial agent, 10-20 parts of bioactive agent, 10-15 parts of bioactivator, and 30-55 parts of water, all of which are parts by weight.

[0006] As a further improvement of the present invention, the microbial agent is prepared by mixing compound bacteria and Rhizobium sp. 3G1 in a ratio of 1 to 2: 1 to 2. The compound bacteria are prepared by Bacillus moghaves, Bacillus belye, Bacillus amyloliquefaciens, and Microbacterium rubrum in a ratio of 1-2:1-2:1-2:1-2; The above-mentioned Bacillus mogarevica, Bacillus belye, Bacillus amyloliquefaciens, Microbacterium rubrum, and Rhizobium sp. 3G1 all had effective viable counts greater than 1×10⁻⁶. 9 cfu / ml.

[0007] As a further improvement of the present invention, the rhizobium is Rhizobium sp. 3G1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on October 23, 2024, with accession number CGMCC NO.32319.

[0008] As a further improvement of the present invention, the gene sequence of the rhizobium sp. is shown in SEQ ID NO. 1.

[0009] As a further improvement of the present invention, the bioactive agent is a lipopeptide surfactant.

[0010] As a further improvement of the present invention, the bioactivator is a mixture of fucoidan, brown algae oligosaccharide, humic acid, and a complex amino acid, in a ratio of 1:2:10:3.

[0011] As a further improvement of the present invention, the preparation method of the compound microbial agent includes the following steps: S1. Preparation of microbial inoculants: *Bacillus moghaves*, *Bacillus belye*, *Bacillus amyloliquefaciens*, *Microbacterium rubrum*, and *Rhizobium sp.* 3G1 were inoculated into LB culture medium at a rate of 5%, and cultured at 37°C and 150 rpm for 48 hours. The LB culture medium consisted of 10 g peptone, 10 g sodium chloride, and 5 g yeast extract. After fermentation, separate bacterial fermentation broths were obtained, each with an effective viable cell count greater than 1 × 10⁻⁶. 9 cfu / ml; A compound bacteria was prepared by mixing Bacillus moghavus, Bacillus belye, Bacillus amyloliquefaciens, and Microbacterium rubrum in a ratio of 1-2:1-2:1-2:1-2; The compound bacteria and Rhizobium sp. 3G1 were thoroughly mixed at a ratio of 1-2:1-2 to prepare a microbial inoculum. S2. Preparation of bioactivator: Take fucoidan, brown algae oligosaccharide, humic acid and compound amino acid combination, mix them evenly in a ratio of 1:2:10:3 to prepare bioactivator; S3. Compound microbial agent: Weigh 25-35 parts of the microbial agent obtained in step S1, 10-15 parts of the bioactivator obtained in step S2, 10-20 parts of the bioactive agent, and 30-55 parts of water, mix them together, stir thoroughly for 1-2 hours until homogeneous, and the compound microbial agent can be prepared.

[0012] The present invention relates to the application of a compound microbial agent for promoting the growth of leguminous plants, used in the prevention and control of plant diseases and / or the promotion of plant growth.

[0013] As a further improvement of the present invention, the plant diseases include Fusarium graminearum (… Fusarium graminearum Fusarium ( ) Fusarium spp., Maize (Alternaria zeylanica) Alternaria tenuis Nees), Inari spores ( Piricularia oryzae Fusarium oxysporum ( Fusarium oxysporum Diseases caused by one or more of the following: The plant is a legume.

[0014] As a further improvement of the present invention, the compound microbial agent is applied by root dipping or hole application during sowing; during plant growth, it is applied by drip irrigation or direct spraying, with a dosage of 1-3 L / mu. The method of dipping the roots of crops with the compound microbial agent involves diluting the compound microbial agent 10 to 20 times, immersing the roots of seedlings (after washing away the nutrient soil) in the diluted solution, ensuring the seedlings are fully coated with the microbial solution before transplanting, and immediately removing them for transplanting after they are wet. The described hole application method involves mixing the compound microbial agent with organic matter and applying it into the planting hole when transplanting seedlings, and then covering it with soil; the organic matter includes wheat bran, crushed rice husks, and soybean meal; The method of mixed application is to mix the compound microbial agent with organic matter, then spread it on the soil surface or apply it in the soil in furrows, and then cover it with soil. It is suitable for large-scale planting, and the dosage is 1-3L per acre. The method of drip irrigation or direct spraying during the plant growth period involves diluting the compound microbial agent 300 to 500 times before flowering and then drip-irrigating the plant roots or spraying it on the leaves, with a dosage of 1 to 3 L / acre.

[0015] Compared with existing technologies, the advantages of this invention are as follows: 1. The purpose of this invention is to utilize the role of microorganisms in preventing and controlling plant diseases and / or promoting plant growth. This invention isolates endophytic bacteria 3G1 from rice seeds, which promotes the root growth of leguminous plants. Identified as a rhizobium sp., it possesses functions such as producing 2100.88 μg / mL of IAA and 1456.41 μg / mL of phosphorus solubilization, and can successfully colonize plants 100% of the time, directly affecting plant growth. Combined with the low-temperature resistance and plant disease resistance functions of other compound microbial agents, it has growth-promoting and disease-preventing effects on leguminous plants, thereby improving crop quality and efficiency. 2. The bioactive agent of this invention is a lipopeptide, which is combined with a compound microbial agent. The lipopeptide can increase the leaf spreading performance of plants, promote the absorption of the compound microbial agent, increase the activity of the microbial agent, and promote a 3% increase in crop yield. 3. The bioactivator of this invention contains fucoidan, brown algae polysaccharide, humic acid, and complex amino acids, which can provide trace elements in the plant growth process. When used in combination with the compound microbial agent, it can effectively promote the growth of crop seedlings, improve their stress resistance, and achieve a seedling transplant survival rate of up to 98%. Attached Figure Description

[0016] Figure 1 This is a graph showing the qualitative determination of IAA secreted by strain 3G1. Figure 2 A schematic diagram illustrating the phosphorus solubilization effect of strain 3G1; Figure 3 This is a plate image showing the colony morphology of strain 3G1. Figure 4 This diagram illustrates the effect of different pH levels on the growth of the strain. Figure 5 This diagram illustrates the effects of different salt stresses on the growth of the strain. Figure 6 The diagram illustrates the inhibitory effect of the compound bacterial suspension on five pathogenic fungi. Figure 7 The image shows a plate validation diagram of the growth-promoting properties of strain 3G1 on Arabidopsis thaliana, with the positive control on the left and strain 3G1 on the right. Figure 8 The image shows a comparison of the hydroponic growth-promoting effects of strain 3G1 on leguminous plants. The left image represents the control (CK), and the right image represents strain 3G1. Detailed Implementation

[0017] The following description, in conjunction with specific embodiments and accompanying drawings, provides further clarification of this specification.

[0018] Example 1

[0019] Isolation and screening of Rhizobium 3G1: Rice seeds were collected and, under aseptic conditions, 10g of rice seeds were rinsed five times with sterile water to remove surface dust and other contaminants. The seeds were then disinfected by soaking in 75% ethanol for 3 minutes, followed by soaking in 5% sodium hypochlorite for 8 minutes, and then rinsing with 75% ethanol for 30 seconds. Finally, the seeds were rinsed 5-7 times with sterile water. The seeds were then inoculated onto LB agar plates. Simultaneously, sterilized rice seeds were lightly pressed onto TSA plates and incubated at 28°C for 72 hours. The isolated bacteria were considered endophytes if no colonies grew. When colonies grew on LB plates, colonies of different colors, sizes, and morphologies were picked, streaked, purified, and isolated. The isolated strains were then subjected to IAA production testing to obtain strains 3G1, 3G2, and 3G3.

[0020] IAA Assay: For qualitative IAA determination, 3G1 strains were inoculated into 14 mL sterile centrifuge tubes containing 5 mL of LB liquid medium. Each strain was inoculated three times, with uninoculated LB medium as a control. The cultures were incubated at 28℃ and 180 rpm for 3 days with shaking. After centrifugation at 8000 rpm for 10 min, 50 μL of the supernatant was transferred to a white porcelain plate. An equal volume of Spot colorimetric solution was added, including 50 μL of 3-indoleacetic acid (IAA) (20 μg / mL) as the standard solution. The plate was placed in the dark at room temperature, and the color change was observed and recorded within 30 min. Figure 1 As shown, 3G1 turned pink, indicating the secretion of IAA. To establish the IAA standard curve, 10 mg of IAA was accurately weighed, dissolved in a small amount of ethanol, and then diluted to 100 mL with distilled water (concentration 100 μg / mL) as a stock solution. Then, a series of standard solutions with concentrations of 0, 100 μg / mL, 75 μg / mL, 50 μg / mL, 25 μg / mL, 20 μg / mL, 15 μg / mL, 10 μg / mL, 5 μg / mL, and 1 μg / mL were prepared using the stock solution as working solutions (prepared fresh each time). 4 mL of the IAA working solution and 4 mL of colorimetric solution were added sequentially to 10 mL centrifuge tubes, incubated in the dark for 30 min, and measured at 530 nm. A standard curve was plotted with OD value on the x-axis and IAA concentration on the y-axis.

[0021] For IAA quantitative determination, strain 3G1 was inoculated into LB broth and cultured overnight on a shaker. The OD600 of the bacterial culture was adjusted to 1.0. 2.625 mL of LB broth and 20 μL of OD1.0 starter culture were added to a sterile 15 mL centrifuge tube for independent tryptophan production. In another centrifuge tube, 2 mL of LB broth and 625 μL of tryptophan (2 mg / mL) (sterilized by filtration) were added, along with 20 μL of OD1.0 bacterial culture in a sterile 15 mL centrifuge tube. Each treatment was repeated three times. The centrifuge tubes were incubated at 28°C and 180 rpm for 48 h on a shaker. The bacterial culture was then transferred to a 1.5 mL centrifuge tube, centrifuged at 8000 rpm for 10 min, and 4 mL of the supernatant was transferred to a glass tube. 4 mL of Salkowski's Reagent colorimetric solution was added, and the mixture was shaken well and incubated in the dark at room temperature for 30 min. The production of IAA was measured by measuring the absorbance at 530 nm. Based on the values ​​plotted from the standard curve, the measured IAA concentration produced by 3G1 reached 2100.88 μg / mL.

[0022] The 3G1 strain was isolated and cultured in shake flasks. The bacterial suspension was spread onto phosphate-solubilizing medium, and the size of the phosphate-solubilizing zone was observed after colonies grew. Comparison with existing phosphate-solubilizing strains confirmed that 3G1 possesses phosphate-solubilizing properties. Figure 2 As shown.

[0023] The phosphorus-solubilizing medium has the following formula: glucose 10g, ammonium sulfate 0.5g, potassium chloride 0.3g, sodium chloride 0.3g, ferrous sulfate 0.03g, magnesium sulfate 0.3g, manganese sulfate 0.03g, calcium phosphate 10g, water 1L, agar 18-20g, and pH adjusted to 7.0-7.5.

[0024] Strain morphology and physiological and biochemical properties: 3G1 strain was Gram-negative and cultured on LB medium at 28°C for 48 hours. Colony morphology was observed as follows. Figure 3 As shown, the colonies are round and raised, with a smooth, moist surface, milky yellow in color, and translucent. The culture medium is colorless. It can utilize glucose, lactose, fructose, mannitol, and gelatin liquefaction; it can decompose organophosphates, has nitrogen-fixing properties, and can hydrolyze starch. It is positive for VP testing, negative for peroxidase, and negative for methyl red testing. It does not produce ferrophosphate. Strain 3G1 was identified as a rhizobium.

[0025] Growth characteristics of 3G1 at different pH and salt concentrations: The acid, alkali and salt tolerance of this rhizobium was determined and a growth trend graph was plotted. It was found that its optimal pH was 6.0-8.0. Figure 4 It can withstand salt concentrations up to 13% NaCl, such as Figure 5 .

[0026] The specific method is as follows: A single colony of Rhizobium 3G1 was picked from activated solid medium and inoculated into 5 ml of LB liquid medium. After 12 h, 2% of the colony was inoculated into 250 ml of LB liquid medium containing different pH values ​​(4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) and NaCl values ​​(1%, 3%, 5%, 7%, 9%, 11%, 13%). The culture was carried out at 180 rpm and 37 °C in a shaker. After 24 h of culture, the OD600, bacterial count, and pH were measured. The culture was repeated three times with a control culture medium without inoculation.

[0027] Result 1: Within the pH range of 4-10, Rhizobium 3G1 showed optimal growth at pH 6.0-8.0. See Table 1:

[0028] Result 2: Within a salt concentration range of 1-11%, Rhizobium 3G1 can still survive at 13%. See Table 2:

[0029] Determination of phosphorus solubility: The phosphorus solubility of this Rhizobium sp. 3G1 was determined to be 1456.41 μg / mL by the molybdenum antimony colorimetric method.

[0030] Pipettes of 0 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, and 6 ml of phosphorus standard solution into 50 ml volumetric flasks, respectively. Dilute with distilled water to 30 ml, add 2 drops of dinitrophenol indicator, and adjust the pH with sodium hydroxide solution or dilute sulfuric acid solution until the solution is just slightly yellow. Add 5 ml of molybdenum antimony colorimetric reagent, shake well, and dilute to volume to obtain standard solutions with phosphorus concentrations of 0.0 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, and 0.6 mg / L. Incubate at room temperature for 30 min, and measure the absorbance at 700 nm, using the 0.0 mg / L standard solution as a blank control as the zero point. Plot a standard curve with absorbance on the ordinate and phosphorus concentration on the abscissa.

[0031] Centrifuge the Rhizobium 3G1 bacterial suspension at 4000 rpm for 20 min. Transfer 5-10 ml of the supernatant to a 50 ml volumetric flask, dilute to 30 ml with water, add 2 drops of dinitrophenol indicator, and adjust the pH to a slightly yellow color with sodium hydroxide or dilute sulfuric acid solution. Then add 5 ml of molybdenum-antimony anti-chromic reagent, shake well, and dilute to volume. Let stand at room temperature for 30 min, and measure the absorbance at 700 nm using a blank solution as a reference for zeroing.

[0032] Result calculation: Phosphorus content in culture medium (mg / L) = (P × V1 × K) / V2 P—The concentration of phosphorus (mg / L) was obtained from the standard curve. K—Dilution factor V1 — Volume of colorimetric solution (ml) V2 — Volume of supernatant (ml) collected.

[0033] 3G1's facilitation and validation: Seed disinfection: Dry the collected Arabidopsis seeds at room temperature for 1-2 weeks, wash them twice with sterile distilled water in a clean bench to remove impurities, soak them in 95% alcohol for 2 minutes, and wash them five times with sterile distilled water. After sterilization, the seeds were placed on sterilized filter paper, and plump seeds were picked up and dotted on the culture medium. The seeds were then placed on a tissue culture rack for culture under light for 8 hours and under darkness for 16 hours. Once the plant has grown to two leaves, it can be transplanted onto an MS plate. The 3G1 bacterial agent was diluted with sterile physiological saline at a concentration of 10 mg / L. -2 10 -3The bacterial culture was spread onto solid culture medium and allowed to dry completely. Simultaneously, the control (CK) group was coated with sterile water. Arabidopsis thaliana were then transplanted onto bacterial culture plates and cultured on tissue culture racks under light for 8 hours followed by 16 hours in the dark. After 15 days of culture, the length of Arabidopsis thaliana in each treatment was measured. The growth-promoting effect of the 3G1 group was 15% higher than that of the CK group (see Table 3); the growth-promoting effect diagram is shown below. Figure 7 :

[0034] Example 2

[0035] The present invention provides a compound microbial agent for promoting the growth of legumes, which is made of 35 parts of microbial agent, 20 parts of bioactive agent, 12 parts of bioactivator, and 33 parts of water, all of which are parts by weight. The microbial agent is prepared by mixing compound bacteria and Rhizobium sp. 3G1 in a 1:2 ratio. The compound bacteria are prepared by Bacillus mogarevae, Bacillus belye, Bacillus amyloliquefaciens, and Microbacterium rubrum in a ratio of 1:2:1:2. The bioactivator is a mixture of fucoidan, brown algae oligosaccharide, humic acid, and a complex amino acid, in a ratio of 1:2:10:3.

[0036] The preparation method of the aforementioned compound microbial agent is characterized by comprising the following steps: S1. Preparation of microbial inoculants: *Bacillus moghaves*, *Bacillus belye*, *Bacillus amyloliquefaciens*, *Microbacterium rubrum*, and *Rhizobium sp.* 3G1 were inoculated into LB culture medium at a rate of 5%, and cultured at 37℃ and 150 rpm for 48 h. The LB culture medium consisted of 10 g peptone, 10 g sodium chloride, and 5 g yeast extract, diluted to 1000 mL with distilled water, mixed thoroughly, dispensed into Erlenmeyer flasks, and sterilized at 121℃ for 30 min. After fermentation, separate bacterial fermentation broths were obtained, each with an effective viable cell count greater than 1 × 10⁻⁶. 9 cfu / ml; A compound bacteria was prepared by mixing Bacillus moghavus, Bacillus belye, Bacillus amyloliquefaciens, and Microbacterium rubrum in a ratio of 1:2:1:2. The compound bacteria and Rhizobium sp. 3G1 were thoroughly mixed at a ratio of 1:2 to prepare a microbial inoculum. S2. Preparation of bioactivator: Take fucoidan, brown algae oligosaccharide, humic acid and compound amino acid combination, mix them evenly in a ratio of 1:2:10:3 to prepare bioactivator; S3, Compound Microbial Agent: Weigh 35 parts of the microbial agent prepared in step S1, and mix 12 parts of the bioactivator, 20 parts of the bioactive agent, and 33 parts of water prepared in step S2. Shake at 37°C and 150 rpm for 2 hours to ensure that the compound microbial agent is fully mixed.

[0037] Example 3

[0038] The present invention provides a compound microbial agent for promoting the growth of legumes, which is made of 30 parts of microbial agent, 15 parts of bioactive agent, 12 parts of bioactivator, and 43 parts of water, all of which are parts by weight. The microbial agent is prepared by mixing compound bacteria and Rhizobium sp. 3G1 in a 1:1 ratio. The compound bacteria are prepared by Bacillus mogarevae, Bacillus belye, Bacillus amyloliquefaciens, and Microbacterium rubrum in a ratio of 1:2:2:1. The bioactivator is a mixture of fucoidan, brown algae oligosaccharide, humic acid, and a complex amino acid, in a ratio of 1:2:10:3.

[0039] The preparation method is the same as in Example 2.

[0040] Example 4

[0041] The present invention provides a compound microbial agent for promoting the growth of legumes, which is made of 35 parts of microbial agent, 15 parts of bioactive agent, 10 parts of bioactivator, and 40 parts of water, all of which are parts by weight. The microbial agent is prepared by mixing compound bacteria and Rhizobium sp. 3G1 in a 2:1 ratio. The compound bacteria are prepared by Bacillus mogarevae, Bacillus belye, Bacillus amyloliquefaciens, and Microbacterium rubrum in a ratio of 2:2:2:1. The bioactivator is a mixture of fucoidan, brown algae oligosaccharide, humic acid, and a complex amino acid, in a ratio of 1:2:10:3.

[0042] The preparation method is the same as in Example 2.

[0043] Example 5

[0044] The present invention provides a compound microbial agent for promoting the growth of legumes, which is made of 25 parts of microbial agent, 20 parts of bioactive agent, 12 parts of bioactivator, and 43 parts of water, all of which are parts by weight. The microbial agent is prepared by mixing compound bacteria and Rhizobium sp. 3G1 in a 1:2 ratio. The compound bacteria are prepared by Bacillus mogarevae, Bacillus belye, Bacillus amyloliquefaciens, and Microbacterium rubrum in a ratio of 1:2:2:2; The bioactivator is a mixture of fucoidan, brown algae oligosaccharide, humic acid, and a complex amino acid, in a ratio of 1:2:10:3.

[0045] The preparation method is the same as in Example 2.

[0046] The present invention discloses a method for using a compound microbial agent to promote the growth of leguminous plants, which can be used in the prevention and control of plant diseases and / or the promotion of plant growth; specifically, it can be used according to the following method: The compound microbial agent is applied by root dipping or hole application during sowing; during plant growth, it is applied by drip irrigation or direct spraying at a rate of 1-3 L / mu. The method of dipping the roots of crops with the compound microbial agent involves diluting the compound microbial agent 10 to 20 times, immersing the roots of seedlings (after washing away the nutrient soil) in the diluted solution, ensuring the seedlings are fully coated with the microbial solution before transplanting, and immediately removing them for transplanting after they are wet. The described hole application method involves mixing the compound microbial agent with organic matter and applying it into the planting hole when transplanting seedlings, and then covering it with soil; the organic matter includes wheat bran, crushed rice husks, and soybean meal; The method of mixed application is to mix the compound microbial agent with organic matter, then spread it on the soil surface or apply it in the soil in furrows, and then cover it with soil. It is suitable for large-scale planting, and the dosage is 1-3L per acre. The method of drip irrigation or direct spraying during the plant growth period involves diluting the compound microbial agent 300 to 500 times before flowering and then drip-irrigating the plant roots or spraying it on the leaves, with a dosage of 1 to 3 L / acre.

[0047] The compound microbial agents prepared in Examples 2-5 above have the properties of promoting growth and resisting disease. When applied to legume crops, they can promote crop growth.

[0048] The following uses kidney beans as an example, combined with Examples 2-5, to illustrate the effect of the prepared compound microbial agent on the growth promotion of kidney beans: Kidney bean seeds were germinated on sterilized filter paper, then transferred to sterilized seedling trays for 5 days, and then transferred to a 5L hydroponic incubator for 21 days of further growth. The experimental group was inoculated with the compound microbial agent prepared according to the ratios in Examples 2-5 using the root irrigation method. Before root irrigation, the soil was moistened with a small amount of water, and 15 mL of the inoculant was added (equivalent to a 300-fold dilution). Each tray contained 16 seedlings, and another 15 mL was added after a 7-day interval. The control group was inoculated with an equal volume of LB medium (CK) (without any added microbial agent) using the same method.

[0049] Culture conditions (24h): 16h light, 28℃; 8h darkness, 28℃; 75% humidity. After 21 days of culture, the plant height, root length, number of root nodules, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, and root system scan of each treatment were measured and recorded. The results of the comparative parameters are shown in Table 4 below, comparing the growth states. Figure 8 As shown:

[0050] As shown in Table 4, the height, root length, number of root nodules, above-ground fresh weight, above-ground dry weight, underground fresh weight, and underground dry weight of kidney beans were significantly higher than those of the control group, while the incidence rate was significantly lower than that of the control group, and the transplant survival rate was greatly improved.

[0051] The plant pathogen confrontation using the compound microbial agent prepared in Example 2: The mycelial growth inhibition method was used to determine the inhibition rate of the compound inoculant against five pathogenic fungi on plate confrontation, including: Preparation of bacterial suspension: After activating the compound bacterial agent separately, inoculate it into LB liquid medium, place it in a constant temperature shaker at 37℃ for 48h, centrifuge at 10000rpm / min for 10min, collect the bacterial cells, adjust the OD600 of the bacterial suspension to ≈1.00 with sterile physiological saline, and set aside for later use; Five types of crop pathogenic fungi were transferred to PDA medium for activation and cultured at 27 ℃. After the mycelium covered the plate, a 5 mm diameter pathogenic fungal disc was inoculated in the center of the PDA plate using the plate confrontation method. A compound bacterial suspension was inoculated 3 cm away from the mycelial disc. The plate was cultured at 27 ℃ for 6 days. The radius of the inhibition zone between the bacteria and the different pathogenic fungi was measured, and the inhibition rate was calculated. Inhibition rate = (Control radius - Inhibition radius) / Control radius × 100% Several pathogenic bacterial discs with a diameter of 5 mm were inoculated into PDA medium. At a distance of 3 cm from the discs, different concentration gradients of compound bacterial suspension, cell filtration sterile solution, lysate stock solution, and dilution solution were inoculated. The culture was incubated at 27 ℃ for 5–6 days, and the inhibition radius was measured. On a sterile laminar flow hood, the prepared bacterial suspension was dispensed into 2 mL EP tubes and placed in constant temperature water baths at 40, 50, 60, 70, 80, 90, and 100 ℃ for 30 min, with 37 ℃ serving as the control group. After 30 min, the tubes were removed and placed in a laminar flow hood for later use. Using Fusarium spp. as an indicator bacterium, several 5 mm diameter bacterial discs were inoculated into PDA medium after activation, and 3G1 fermentation broth treated at different temperatures was inoculated to conduct a confrontation experiment. Prepare 1 mol·L⁻¹ NaOH solution and 1 mol·L⁻¹ H₂SO₄ solution for later use. Prepare PDA medium with normal pH, and adjust the pH to 4, 5, 6, 7, 8, 9, and 10 respectively using the prepared NaOH and H₂SO₄ solutions, then sterilize. Use Fusarium spp. as an indicator bacterium, activate it, and then punch it into several mycelial cakes using a 5 mm punch. Inoculate these cakes into PDA medium at different pH conditions using an inoculation needle, and then inoculate them with 3G1 bacterial suspension for confrontation tests. Measure the inhibition rate after 5–6 days. The compound bacterial suspension showed significant inhibitory effects on the mycelial growth of five crop pathogenic fungi (see Table 5 below). The inhibition effects were observed on plate samples. Figure 6 :

[0052] As shown in Table 5, the compound microbial agent prepared in Example 2 has a good inhibitory effect on Fusarium graminearum, Fusarium spp., Maize Alternaria tenuis Nees, Piricularia oryzae, and Fusarium oxysporum.

[0053] The results showed that Examples 2-5 promoted the increase of kidney bean plant height, with an average growth of 18.78 cm, compared to 16.12 cm in the control group. Examples 2-5 increased the fresh and dry weight of the aboveground parts and the fresh weight of the underground parts, and promoted the growth of rhizobia in the roots. The disease incidence rate in Examples 2-5 was only 6%, significantly lower than the 14% in the control group. This indicates that the compound microbial agent has good growth-promoting and disease-resistant effects.

[0054] The following experiments, conducted using the hole application method in potted plants, further illustrate the effectiveness of the present invention with the compound microbial agents prepared in Examples 2-5. The experimental steps and results are as follows: Experiment location: The experiment was conducted in a greenhouse at Daqing Huali Biotechnology Co., Ltd. Experiment period: May-October 2024; Experimental crop: soybean.

[0055] Experimental methods: 1) Prepare the compound microbial agent; 2) Dilute the compound bacterial agent of Examples 2-5 by 3 times with sterile water or cooled boiled water, etc. 3) Apply 6 ml of compound microbial agent to the hole according to the amount of soil for 3 kg of potted plants.

[0056] Indicator Measurement: At the late growth stage, soybean plant height, above-ground fresh weight, above-ground dry weight, underground fresh weight, underground dry weight, and soil ammonium nitrogen, available phosphorus, and available potassium were measured. Experimental data are shown in Tables 6 and 7.

[0057] The results showed that a higher fresh weight / dry weight ratio after applying the compound microbial agent indicated better plant growth, with examples 2-5 showing an average increase of approximately 60% compared to the control group. The decomposition of nitrogen, phosphorus, and potassium in the soil was improved by an average of approximately 23% compared to the control group. This effectively improved the biomass of leguminous plants and enhanced the decomposition of nitrogen, phosphorus, and potassium in the soil, thus improving soil quality.

[0058] The compound microbial agent of this invention should contain a variety of microorganisms with different biological characteristics, such as free-living nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and plant growth hormone-producing bacteria, to achieve comprehensive improvement of the soil environment. Through the interaction and synergistic effect between microorganisms, the compound microbial agent can significantly improve soil fertility, enhance the disease resistance and stress resistance of legumes, thereby improving the yield and quality of legumes.

[0059] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0060] 16S rRNA sequence of strain 3G1:

Claims

1. A compound microbial agent for promoting the growth of leguminous plants, characterized in that... It is made of 25-35 parts of microbial inoculant, 10-20 parts of bioactive agent, 10-15 parts of bioactivator, and 30-55 parts of water. All of the above parts are by weight. The microbial agent is prepared by mixing compound bacteria and Rhizobium sp. 3G1 in a ratio of 1-2:1-2. The compound bacteria are prepared by Bacillus moghaves, Bacillus belye, Bacillus amyloliquefaciens, and Microbacterium rubrum in a ratio of 1-2:1-2:1-2:1-2; The above-mentioned Bacillus mogarevica, Bacillus belye, Bacillus amyloliquefaciens, Microbacterium rubrum, and Rhizobium sp. 3G1 all had effective viable counts greater than 1×10⁻⁶. 9 cfu / ml; The rhizobium mentioned is Rhizobium sp. 3G1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is October 23, 2024, and its accession number is CGMCC NO. 32319.

2. The compound microbial agent for promoting the growth of leguminous plants according to claim 1, characterized in that, The gene sequence of the rhizobium sp. is shown in SEQ ID NO.

1.

3. The compound microbial agent for promoting the growth of leguminous plants according to claim 1, characterized in that... The bioactive agent is a lipopeptide surfactant.

4. The compound microbial agent for promoting the growth of leguminous plants according to claim 1, characterized in that... The bioactivator is a mixture of fucoidan, brown algae oligosaccharide, humic acid, and a complex amino acid, in a ratio of 1:2:10:

3.

5. The compound microbial agent for promoting the growth of leguminous plants according to claim 1, wherein the preparation method of the compound microbial agent is characterized in that, Includes the following steps: S1. Preparation of microbial inoculants: *Bacillus moghaves*, *Bacillus belye*, *Bacillus amyloliquefaciens*, *Microbacterium rubrum*, and *Rhizobium sp.* 3G1 were inoculated into LB culture medium at a rate of 5%, and cultured at 37°C and 150 rpm for 48 hours. The LB culture medium consisted of 10 g peptone, 10 g sodium chloride, and 5 g yeast extract. After fermentation, separate bacterial fermentation broths were obtained, each with an effective viable cell count greater than 1 × 10⁻⁶. 9 cfu / ml; A compound bacteria was prepared by mixing Bacillus moghavus, Bacillus belye, Bacillus amyloliquefaciens, and Microbacterium rubrum in a ratio of 1-2:1-2:1-2:1-2; The compound bacteria and Rhizobium sp. 3G1 were thoroughly mixed at a ratio of 1-2:1-2 to prepare a microbial inoculum. S2. Preparation of bioactivator: Take fucoidan, brown algae oligosaccharide, humic acid and compound amino acid combination, mix them evenly in a ratio of 1:2:10:3 to prepare bioactivator; S3. Compound microbial agent: Weigh 25-35 parts of the microbial agent prepared in step S1, 10-15 parts of the bioactivator prepared in step S2, 10-20 parts of the bioactive agent, and 30-55 parts of water, mix them together, stir thoroughly for 1-2 hours until homogeneous, and the compound microbial agent can be prepared.

6. The application of the compound microbial agent for promoting the growth of leguminous plants according to claim 1, characterized in that, Used for promoting the growth of legumes.

7. The method of using the compound microbial agent for promoting the growth of leguminous plants according to claim 1, characterized in that: The compound microbial agent is applied by root dipping or hole application during sowing; during plant growth, it is applied by drip irrigation or direct spraying at a rate of 1-3 L / mu. The method of dipping the roots of crops with the compound microbial agent involves diluting the compound microbial agent 10 to 20 times, immersing the roots of seedlings (after washing away the nutrient soil) in the diluted solution, ensuring the seedlings are fully coated with the microbial solution before transplanting, and immediately removing them for transplanting after they are wet. The described hole application method involves mixing the compound microbial agent with organic matter and applying it into the planting hole when transplanting seedlings, and then covering it with soil; the organic matter includes wheat bran, crushed rice husks, and soybean meal; The method of mixed application is to mix the compound microbial agent with organic matter, then spread it on the soil surface or apply it in the soil in furrows, and then cover it with soil. It is suitable for large-scale planting, and the dosage is 1-3L per acre. The method of drip irrigation or direct spraying during the plant growth period involves diluting the compound microbial agent 300 to 500 times before flowering and then drip-irrigating the plant roots or spraying it on the leaves, with a dosage of 1 to 3 L / acre.

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