Complex microbial inoculant for promoting growth of leguminous plants and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for promoting growth of leguminous plants and application of the complex microbial inoculant, and relates to the technical field of fertilizers and biologication.The complex microbial inoculant is prepared from 25-35 parts of a microbial inoculant, 10-20 parts of a biological active agent, 10-15 parts of a biological activator and 30-55 parts of water. The endophyte 3G1 which is screened and separated from rice seeds and can promote the growth of leguminous roots can be completely and successfully planted in plants by utilizing the effects of preventing and treating plant diseases and / or promoting plant growth of microorganisms, and the growth of the plants is directly influenced; in combination with the functions of low temperature resistance, plant disease resistance and the like of other complex microbial inoculants, the compound microbial inoculants have growth promoting and disease preventing effects on leguminous plants; the biological active agent is lipopeptide, so that the spreading performance of plant leaves can be improved, the absorption of the complex microbial inoculant is promoted, the activity of the microbial inoculant is improved, and the crop yield is promoted to be increased by 3%; the biological activator can provide trace elements in the plant growth process, and is matched with the complex microbial inoculants for use, so that the growth of crop seedlings can be effectively promoted, the stress resistance of the crop seedlings is improved, and the transplanting survival rate of the seedlings reaches up to 98%.

Description

Technical Field

[0001] The invention relates to the fields of fertilizer and biotechnology, and in particular to a composite bacterial agent for promoting the growth of leguminous plants and application thereof. Background Art

[0002] Legumes are an important plant species that are widely grown around the world. They have significant nutritional and economic value. However, in the process of legume cultivation, farmers often face a variety of challenges, 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] In the prior art, although some microbial fertilizers are used to promote the growth of legumes, these fertilizers usually contain only a single type of microorganism and their functions are relatively limited. For example, some fertilizers mainly focus on nitrogen fixation, while ignoring other important soil microbial activities, such as phosphorus dissolution, potassium dissolution and production of plant growth hormones. In addition, single microbial fertilizers are often easily affected by environmental conditions, and their stability and durability are poor, making it difficult to meet the diverse needs of legumes during growth. Summary of the invention

[0004] The present invention aims to overcome the deficiencies of the prior art and provides a composite bacterial agent for promoting the growth of legumes and its application, so as to overcome the deficiencies of the prior art, meet the diversified needs in the legume planting process, and promote the sustainable development of the legume industry.

[0005] The composite bacterial agent for promoting the growth of leguminous plants of the present invention is prepared from 25 to 35 parts of microbial agents, 10 to 20 parts of bioactive agents, 10 to 15 parts of bioactivators and 30 to 55 parts of water, and the above parts are all parts by weight.

[0006] As a further improvement of the present invention, the microbial agent is prepared by mixing the composite bacteria and Rhizobium sp. 3G1 in a ratio of 1-2:1-2; The composite bacteria is prepared from Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens and Microbacterium rubrum in a ratio of 1-2:1-2:1-2:1-2; The effective viable counts of the above-mentioned Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens, Microbacterium rubrum and Rhizobium sp. 3G1 were all greater than 1×10 9 cfu / ml.

[0007] As a further improvement of the present invention, the rhizobium is Rhizobium sp. 3G1, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is October 23, 2024, and its deposit number is CGMCC NO.32319.

[0008] As a further improvement of the present invention, the gene sequence of the rhizobium (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 combination of fucoidan, brown algae oligosaccharide, humic acid, and complex amino acids, which are mixed in a ratio of 1:2:10:3.

[0011] As a further improvement of the present invention, the preparation method of the composite bacterial agent comprises the following steps: S1. Preparation of microbial inoculants: Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens, Microbacterium rubrum, and Rhizobium sp. 3G1 were inoculated into LB culture medium at an inoculation rate of 5%, respectively, and cultured at 37°C and 150 rpm for 48 h; the LB culture medium contained 10 g of peptone, 10 g of sodium chloride, and 5 g of yeast extract powder; after fermentation, a separate bacterial fermentation liquid was obtained, and the number of effective live bacteria in the fermentation liquid was greater than 1×10 9 cfu / ml; Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens and Microbacterium rubrum are mixed in a ratio of 1-2:1-2:1-2:1-2 to prepare a composite bacteria; The composite bacteria and Rhizobium sp. 3G1 are fully mixed at a ratio of 1-2:1-2 to prepare a microbial agent; S2. Preparation of bioactivator: Take fucoidan, brown algae oligosaccharide, humic acid, and complex amino acid combination, mix them evenly in a ratio of 1:2:10:3, and prepare bioactivator; S3. Composite bacterial 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, and stir them thoroughly for 1-2 hours to make the composite bacterial agent.

[0012] The application of the composite bacterial agent for promoting the growth of leguminous plants of the present invention is used in preventing and controlling plant diseases and / or promoting plant growth.

[0013] As a further improvement of the present invention, the plant diseases include Fusarium graminearum ( Fusarium graminearum )、Fusarium( Fusarium spp.), Maize Alternaria Alternaria tenuis Nees), Pyricularia oryzae ( Piricularia oryzae ), Fusarium oxysporum ( Fusarium oxysporum ); the plant is of the Leguminosae family.

[0014] As a further improvement of the present invention, the composite bacterial agent is used by dipping the roots or applying in holes during sowing; drip irrigation or direct spraying is used during plant growth, and the usage amount is 1 to 3 L / mu; The method of dipping the composite bacterial agent in the roots of crops comprises diluting the composite bacterial agent by 10 to 20 times, and dipping the roots of the seedlings after washing away the nutrient soil at the roots into the diluted solution, so that the roots are fully dipped in the bacterial solution before transplanting the seedlings, and immediately taking them out for transplanting after being wetted; The hole-fertilization method is to mix the composite bacterial agent with organic matter when the seedlings are planted, and then apply it into the planting hole, and then cover it with soil; the organic matter includes wheat bran, crushed rice bran, and soybean meal; The mixed application method is to mix the composite bacterial agent with organic matter, spread it on the soil surface or apply it in the soil through furrows, and then cover the soil. It is suitable for large-scale planting, and the usage amount per mu is 1 to 3L; The method of drip irrigation or direct spraying during the plant growth period is to dilute the composite bacterial agent 300-500 times during the plant growth period and before the flowering period, and drip irrigate the roots of the plants or spray them on the leaves, with a usage amount of 1-3L / mu.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The purpose of the present invention is to use microorganisms to prevent and control plant diseases and / or promote plant growth. The endophyte 3G1 that can promote the growth of legume roots screened and separated from rice seeds in this invention was identified as Rhizobium sp., which has the functions of producing IAA2100.88μg / mL and solubilizing 1456.41μg / mL, and can be 100% successfully colonized in plants, directly affecting the growth of plants. Combined with the functions of other composite microbial agents such as resistance to low temperatures and plant diseases, it has the effects of promoting growth and preventing diseases for legumes, thereby improving the quality and efficiency of crops; 2. The bioactive agent of the present invention is lipopeptide, which is a combination of lipopeptide and composite bacterial agent. Lipopeptide can increase the spreading performance of plant leaves, promote the absorption of composite bacterial agent, increase the activity of bacterial agent, and promote the increase of crop yield by 3%; 3. The fucoidan, brown algae polysaccharide, humic acid, composite amino acids, etc. contained in the bioactivator of the present invention can provide trace elements for plant growth. When used in combination with the composite bacterial agent, it can effectively promote the growth of crop seedlings and improve their stress resistance. The transplant survival rate of seedlings is as high as 98%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a qualitative determination of IAA secretion by strain 3G1; Figure 2 This is a schematic diagram of the phosphate solubilization effect of strain 3G1; Figure 3 This is the colony morphology plate image of strain 3G1; Figure 4 This is the growth effect diagram of the strain at different pH; Figure 5 This is the growth effect diagram of the strain under different salt stresses; Figure 6 It is a graphic representation of the inhibitory effect of the composite bacterial suspension on five pathogenic fungi; Figure 7 This is a plate verification diagram of the growth-promoting properties of strain 3G1 on Arabidopsis thaliana, where the left side is the positive control and the right side is strain 3G1; Figure 8 This is a comparison chart of the results of the hydroponic growth promotion test of strain 3G1 on leguminous plants, with the CK control on the left and strain 3G1 on the right. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with specific embodiments and drawings.

[0018] Example 1

[0019] Isolation and screening of Rhizobium 3G1: Take rice seeds, and under sterile conditions, first rinse 10g of rice seeds with sterile water 5 times to wash away surface dust, etc., disinfect the seed surface, soak in 75% ethanol for 3min, soak in 5% sodium hypochlorite for 8min, wash with 75% ethanol for 30s, and finally rinse with sterile water 5-7 times, inoculate it on LB culture medium, and at the same time take the sterilized rice seeds and apply them on TSA plate lightly, culture at 28℃ for 72h, until no colony grows, the isolated bacteria are endophytes. Colonies grow on LB plate, pick colonies with different colors, sizes and shapes, streak purification and separation, and perform IAA production determination on the isolated strains to obtain 3G1, 3G2, and 3G3 strains.

[0020] IAA determination: For qualitative determination of IAA, 3G1 was inoculated into a 14 mL sterile centrifuge tube containing 5 mL LB liquid medium. Each strain was repeated 3 times, and the uninoculated LB medium was used as the control. The culture was shaken at 28°C and 180 r / min for 3 days. Centrifuge for 10 min at 8000 rpm / min, and 50 μL of the bacterial supernatant was drawn onto a white porcelain plate. An equal volume of Spot colorimetric solution was added, in which 50 μL of 3-indoleacetic acid IAA (20 μg / mL) was added to the standard solution. The white porcelain plate was placed in the dark at room temperature, and the color change was observed and recorded within 30 min. Figure 1 As shown, it was found that 3G1 turned pink, which means it can secrete IAA. To prepare the IAA standard curve, accurately weigh 10 mg of IAA, dissolve it with a small amount of ethanol, and then dilute it to 100 mL with distilled water (its concentration is 100 μg / mL) as a stock solution. Then, use the stock solution to prepare a series of standard solutions 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 as working solutions (prepared before use). Add 4 mL of IAA series concentration working solution and 4 mL of colorimetric solution to a 10 mL centrifuge tube in sequence, incubate in the dark for 30 minutes, and compare the color at 530 nm. Draw a standard curve with OD value as the horizontal axis and IAA concentration as the vertical axis.

[0021] For quantitative determination of IAA, strain 3G1 was inoculated in LB culture medium and cultured overnight in a shaker. The OD600 of the bacterial solution was adjusted to 1.0. 2.625 mL of LB liquid culture medium and 20 μL of OD 1.0 starter culture were added to a sterile 15 mL centrifuge tube for independent tryptophan production. Another tube was added with 2 mL of LB culture medium and 625 μL of tryptophan (2 mg / mL) (tryptophan was filtered and sterilized), and 20 μL of OD 1.0 bacterial solution was added to a sterile 15 mL centrifuge tube. Each treatment was repeated 3 times. The centrifuge tube was cultured in a shaker at 28°C, 180 r / min for 48 h. The bacterial solution was pipetted into a 1.5 mL centrifuge tube, centrifuged at 8000 rpm / min for 10 min, 4 mL of supernatant was pipetted, placed in a glass tube, 4 mL of Salkowski's Reagent colorimetric solution was added and shaken, and cultured in the dark at room temperature for 30 min. The production of IAA was measured by measuring the absorbance at 530 nm. According to the values ​​drawn from the standard curve, the mass concentration of IAA produced by 3G1 was measured to be 2100.88 μg / mL.

[0022] The 3G1 strain was isolated and cultured in a shake flask, and the bacterial solution was applied to the phosphate-solubilizing medium. The size of the phosphate-solubilizing circle of the grown colonies was observed and compared with the existing strains with phosphate-solubilizing ability. It was obtained that 3G1 had a phosphate-solubilizing effect, such as Figure 2 shown.

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

[0024] Strain morphology and physiological biochemistry: 3G1 was Gram-negative. It was cultured on LB medium at 28°C for 48 hours and the colony morphology was observed. Figure 3 As shown, the colony morphology is round and convex, the surface is smooth and moist, milky yellow, translucent, and the culture medium is colorless. It can utilize glucose, lactose, fructose, mannitol, liquefy gelatin, decompose organic phosphorus, has nitrogen fixation, can hydrolyze starch, VP test is positive, peroxidase is negative, methyl red test is negative, and does not produce siderophore. The 3G1 strain was identified as rhizobium.

[0025] Growth characteristics of 3G1 at different pH and salt concentrations: The acidity and salt tolerance of the rhizobium were measured, and a growth trend chart was drawn. It was found that its optimal pH was 6.0-8.0. Figure 4 , the salt tolerance reaches 13% NaCl, such as Figure 5 .

[0026] The specific method is as follows: A single colony of Rhizobium 3G1 was picked from the activated solid culture medium and inoculated into 5 ml of LB liquid culture medium. After 12 hours, it was inoculated at a 2% inoculation rate into 250 ml of LB liquid culture medium containing different pH (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) and NaCl (1%, 3%, 5%, 7%, 9%, 11%, 13%). The culture was shaken at 180 rpm and 37°C. The OD600, bacterial count, and pH were measured for 24 hours. The uninoculated culture medium was used as a control. The process was repeated three times.

[0027] Test result 1: In the pH range of 4-10, Rhizobium 3G1 grows best at 6.0-8.0. See Table 1:

[0028] Test result 2: In the range of salt concentration 1-11%, Rhizobium 3G1 can still survive at 13%. See Table 2:

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

[0030] Pipette 0ml, 1ml, 2ml, 3ml, 4ml, 5ml, and 6ml of phosphorus standard solution into a 50ml volumetric flask, dilute to 30ml with distilled water, 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 5ml of molybdenum antimony anti-color developer, shake well, and dilute to volume to obtain standard solutions with phosphorus concentrations of 0.0mg / L, 0.1mg / L, 0.2mg / L, 0.3mg / L, 0.4mg / L, 0.5mg / L, and 0.6mg / L. Let stand at room temperature for 30min, adjust the zero point with 0.0mg / L standard solution as blank control at a wavelength of 700nm, and measure the absorbance. Draw a standard curve with absorbance as the ordinate and phosphorus concentration as the abscissa.

[0031] Centrifuge the Rhizobium 3G1 bacterial solution and the sample at 4000r / min for 20min, draw 5-10ml of the supernatant into a 50ml volumetric flask, dilute to 30ml with water, 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. Then add 5ml of molybdenum antimony anti-coloring agent, shake well, and make up to volume. Let it stand at room temperature for 30min, adjust the zero point with the blank test solution as reference at a wavelength of 700nm, and measure the absorbance.

[0032] Calculation results: Phosphorus content in culture medium (mg / L) = (P×V1×K) / V2 P—Phosphorus concentration obtained from the standard curve (mg / L) K—dilution multiple V1—color developing solution volume (ml) V2—Volume of supernatant aspirated (ml).

[0033] 3G1 growth promotion verification: Seed disinfection: The collected Arabidopsis seeds were dried at room temperature for 1-2 weeks, washed twice with sterile distilled water in a clean bench to remove impurities, soaked in 95% alcohol for 2 min, and washed 5 times with sterile distilled water; Place the sterilized seeds on the sterilized filter paper, pick up the full seeds and spot them on the culture medium, and place them on the tissue culture rack for culturing under light for 8 hours and in the dark for 16 hours; After growing to two leaves, it can be transplanted to MS plates; Dilute the 3G1 bacterial preparation with sterile saline for 10 -2 , 10 -3The CK group was coated with sterile water. Arabidopsis was transplanted to the bacterial liquid plate and cultured on the tissue culture rack for 8 hours of light culture and 16 hours of dark culture. After 15 days of culture, the length of Arabidopsis in each treatment was measured. The growth-promoting effect of the 3G1 group was 15% higher than that of the CK group, as shown in Table 3; the growth-promoting effect diagram is shown in Figure 7 :

[0034] Example 2

[0035] The composite bacterial agent for promoting the growth of leguminous plants of the present invention is prepared from 35 parts of microbial agents, 20 parts of bioactive agents, 12 parts of bioactivators, and 33 parts of water, where the above parts are all by weight. The microbial agent is prepared by mixing composite bacteria and Rhizobium sp. 3G1 in a ratio of 1:2; The composite bacteria is prepared from Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens and Microbacterium rubrum in a ratio of 1:2:1:2; The bioactivator is a combination of fucoidan, brown algae oligosaccharide, humic acid and complex amino acids, which are mixed in a ratio of 1:2:10:3.

[0036] The preparation method of the composite bacterial agent described above is characterized by comprising the following steps: S1. Preparation of microbial inoculants: Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens, Microbacterium rubrum, and Rhizobium sp. 3G1 were inoculated into LB culture medium at an inoculation rate of 5%, and cultured at 37°C and 150 rpm for 48 h; the LB culture medium was composed of 10 g of peptone, 10 g of sodium chloride, and 5 g of yeast extract powder. Distilled water was added to make the volume to 1000 mL, and the mixture was divided into Erlenmeyer bottles after mixing. The mixture was sterilized at 121°C for 30 min. After the fermentation was completed, a separate bacterial fermentation liquid was obtained, and the number of effective live bacteria in the fermentation liquid was greater than 1×10 9 cfu / ml; Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens and Microbacterium rubrum are mixed in a ratio of 1:2:1:2 to prepare a composite bacteria; The composite bacteria and Rhizobium sp. 3G1 were fully mixed at a ratio of 1:2 to prepare a microbial agent; S2. Preparation of bioactivator: Take fucoidan, brown algae oligosaccharide, humic acid, and complex amino acid combination, mix them evenly in a ratio of 1:2:10:3, and prepare bioactivator; S3, composite bacterial agent: weigh 35 parts of the microbial agent prepared in step S1, mix 12 parts of the bioactivator prepared in step S2, 20 parts of the bioactive agent, and 33 parts of water, and shake at 37° C. and 150 rpm for 2 hours to fully mix the composite bacterial agent to prepare the composite bacterial agent.

[0037] Example 3

[0038] The composite bacterial agent for promoting the growth of leguminous plants of the present invention is prepared from 30 parts of microbial agents, 15 parts of bioactive agents, 12 parts of bioactivators, and 43 parts of water, where the above parts are all by weight. The microbial agent is prepared by mixing composite bacteria and Rhizobium sp. 3G1 in a ratio of 1:1; The composite bacteria is prepared from Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens and Microbacterium rubrum in a ratio of 1:2:2:1; The bioactivator is a combination of fucoidan, brown algae oligosaccharide, humic acid and complex amino acids, which are mixed in a ratio of 1:2:10:3.

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

[0040] Example 4

[0041] The composite bacterial agent for promoting the growth of leguminous plants of the present invention is prepared from 35 parts of microbial agents, 15 parts of bioactive agents, 10 parts of bioactivators, and 40 parts of water, where the above parts are all by weight. The microbial agent is prepared by mixing composite bacteria and Rhizobium sp. 3G1 in a ratio of 2:1; The composite bacteria is prepared from Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens and Microbacterium rubrum in a ratio of 2:2:2:1; The bioactivator is a combination of fucoidan, brown algae oligosaccharide, humic acid and complex amino acids, which are mixed in a ratio of 1:2:10:3.

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

[0043] Example 5

[0044] The composite bacterial agent for promoting the growth of leguminous plants of the present invention is prepared from 25 parts of microbial agents, 20 parts of bioactive agents, 12 parts of bioactivators, and 43 parts of water, where the above parts are all by weight. The microbial agent is prepared by mixing composite bacteria and Rhizobium sp. 3G1 in a ratio of 1:2; The composite bacteria is prepared from Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens and Microbacterium rubrum in a ratio of 1:2:2:2; The bioactivator is a combination of fucoidan, brown algae oligosaccharide, humic acid and complex amino acids, which are mixed in a ratio of 1:2:10:3.

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

[0046] The use method of the composite bacterial agent for promoting the growth of leguminous plants of the present invention can be used in preventing and controlling plant diseases and / or promoting plant growth; specifically, it can be used in the following manner: The composite bacterial agent is used by dipping the roots or applying in holes during sowing; drip irrigation or direct spraying is used when the plants are growing, and the usage amount is 1 to 3 L / mu; The method of dipping the composite bacterial agent in the roots of crops comprises diluting the composite bacterial agent by 10 to 20 times, and dipping the roots of the seedlings after washing away the nutrient soil at the roots into the diluted solution, so that the roots are fully dipped in the bacterial solution before transplanting the seedlings, and immediately taking them out for transplanting after being wetted; The hole-fertilization method is to mix the composite bacterial agent with organic matter when the seedlings are planted, and then apply it into the planting hole, and then cover it with soil; the organic matter includes wheat bran, crushed rice bran, and soybean meal; The mixed application method is to mix the composite bacterial agent with organic matter, spread it on the soil surface or apply it in the soil through furrows, and then cover the soil. It is suitable for large-scale planting, and the usage amount per mu is 1 to 3L; The method of drip irrigation or direct spraying during the plant growth period is to dilute the composite bacterial agent 300-500 times during the plant growth period and before the flowering period, and drip irrigate the roots of the plants or spray them on the leaves, with a usage amount of 1-3L / mu.

[0047] The composite bacterial agents prepared in the above-mentioned Examples 2-5 have the function of promoting growth and resisting diseases. When applied to leguminous crops, they can promote the growth of the crops.

[0048] Taking kidney beans as an example, the effects of the prepared composite bacterial agent on the growth promotion of kidney beans are explained in combination with Examples 2 to 5: The kidney bean seeds were germinated on sterilized filter paper, then moved into sterilized seedling bowls for 5 days, and then moved into a 5L hydroponic incubator for further growth for 21 days. The experimental group was inoculated with the composite bacterial agent of Example 2-5 by root irrigation according to the ratio of Example 2-5. Before root irrigation, the soil was moistened with a small amount of clean water, and 15 mL was added, which was equivalent to a 300-fold dilution. There were 16 plants per pot, and 15 mL was inoculated again at intervals of 7 days. The control group was inoculated with an equal amount of LB medium (CK) in the same way (without adding any bacterial agent).

[0049] Culture conditions (24h): 16h light, 28℃, 8h dark, 28℃, 75% humidity. After 21d of culture, plant height, root length, nodule number, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, and root system scan of each treatment were measured and recorded. The comparison parameters are shown in Table 4. The comparison of growth status is shown in Table 4. Figure 8 As shown:

[0050] As can be seen from Table 4, the plant height, root length, number of nodules, aboveground fresh weight, aboveground dry weight, underground fresh weight and underground dry weight of kidney bean were significantly higher than those of the control group, the incidence rate was significantly lower than that of the control group, and the transplant survival rate was greatly improved.

[0051] The plant pathogenic bacteria confrontation using the composite bacterial agent prepared in Example 2: The mycelium growth inhibition method was used to determine the antibacterial rate of the composite bacterial agent against five pathogenic fungi on the confrontation plate, including: Preparation of bacterial suspension: After activating the composite bacterial agents, inoculate them into LB liquid culture medium, place them in a constant temperature shaker at 37°C for 48 hours, centrifuge them at 10000 rpm / min for 10 minutes, collect the bacteria, adjust the bacterial suspension OD600≈1.00 with sterile physiological saline, and set aside; The five preserved crop pathogenic fungi were transferred to PDA medium for activation and cultured at 27°C. After the mycelium grew all over the plate, a plate confrontation method was used. A pathogenic fungus cake with a diameter of 5 mm was inoculated in the center of the PDA plate. The composite bacterial suspension was inoculated 3 cm away from the cake and cultured at 27°C for 6 days. The radius of the inhibition zone between bacteria and different pathogenic fungi was measured, and the inhibition rate was calculated. Inhibition rate = (control radius - inhibition radius) / control radius × 100% Inoculate several pathogenic bacteria cakes with a diameter of 5 mm in the PDA medium, and inoculate different concentration gradient composite bacterial suspension cell filtration sterile solution, broken solution stock solution and dilution solution 3 cm away from the bacterial cakes. Culture at 27 ℃ for 5 to 6 days, and measure the antibacterial radius. On a sterile clean bench, the prepared bacterial suspension was divided into 2 mL EP tubes and placed in a constant temperature water bath at 40, 50, 60, 70, 80, 90 and 100 ℃ for 30 min, and 37 ℃ was used as the control group. After 30 minutes of water bath, take it out and place it on the clean bench for use. Fusarium spp. was used as an indicator bacteria. After activation, several bacterial cakes with a diameter of 5 mm were inoculated in the PDA medium, 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 use. Prepare PDA medium with normal pH, adjust the pH to 4, 5, 6, 7, 8, 9, 10 with the prepared NaOH solution and H2SO4 solution, respectively, and sterilize. Use Fusarium spp. as the indicator bacteria, and use a 5 mm puncher to punch several bacterial cakes after activation. Use an inoculation needle to inoculate PDA medium with different pH conditions, and inoculate 3G1 bacterial suspension for confrontation test. Determine its antibacterial rate after 5-6 days; The composite bacterial suspension has a significant inhibitory effect on the growth of mycelium of 5 kinds of crop pathogenic fungi as shown in Table 5 below. Figure 6 :

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

[0053] The results showed that Examples 2-5 could promote the increase of kidney bean plant height, with an average height of 18.78 cm, while the blank group grew 16.12 cm. Examples 2-5 increased the fresh weight and dry weight of the aboveground part and the fresh weight of the underground part, and promoted the growth of rhizobia in the root system. The incidence rate of Examples 2-5 was only 6%, which was significantly lower than 14% of the blank group. This shows that the composite bacterial agent has good growth-promoting and disease-resistant effects.

[0054] The composite bacterial agent prepared in Examples 2-5 was subjected to an experiment in potted plants using the hole application method to further illustrate the effect of the present invention. The experimental steps and results are as follows: Experimental location: carried out in the greenhouse of Daqing Huali Biotechnology Co., Ltd. Experimental period: May-October 2024; Experimental crop: soybean.

[0055] Experimental methods: 1) Prepare the composite bacterial agent; 2) Dilute the composite bacterial agent of Example 2-5 3 times with sterile water or cold boiled water; 3) Apply 6 ml of compound fungicide per hole according to the 3 kg soil volume of potted plants.

[0056] Index determination: At the end of the growth period, the height of soybean plants, fresh weight above ground, dry weight above ground, fresh weight below ground, dry weight below ground, ammonium nitrogen, available phosphorus and available potassium in the soil were measured. The test data are shown in Table 6 and Table 7:

[0057] The results show that when the composite bacterial agent is applied, the greater the fresh weight / dry weight ratio, the better the growth of the plant. Examples 2-5 are about 60% higher than the CK group on average. The decomposition of nitrogen, phosphorus and potassium in the soil is increased by about 23% on average compared to the CK group. It effectively improves the biomass of leguminous plants, improves the decomposition of nitrogen, phosphorus and potassium in the soil, and improves soil quality.

[0058] The composite microbial agent of the present invention should contain a variety of microorganisms with different biological characteristics, such as self-generated nitrogen-fixing bacteria, phosphate-dissolving bacteria, and plant growth hormone-producing bacteria, so as to achieve comprehensive improvement of the soil environment. Through the interaction and synergistic effect between microorganisms, the composite microbial agent can significantly improve soil fertility, enhance the disease resistance and stress resistance of legumes, and thus improve the yield and quality of legumes.

[0059] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in the field to implement: 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 protection scope required by the present invention.

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

Claims

1. A composite bacterial agent for promoting the growth of leguminous plants, characterized in that The invention is prepared from 25 to 35 parts of microbial agents, 10 to 20 parts of bioactive agents, 10 to 15 parts of bioactivators and 30 to 55 parts of water, and the above parts are all by weight.

2. The composite bacterial agent for promoting the growth of leguminous plants according to claim 1, characterized in that The microbial agent is prepared by mixing composite bacteria and Rhizobium sp. 3G1 in a ratio of 1-2:1-2; The composite bacteria is prepared from Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens and Microbacterium rubrum in a ratio of 1-2:1-2:1-2:1-2; The effective viable counts of the above-mentioned Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens, Microbacterium rubrum and Rhizobium sp. 3G1 were all greater than 1×10 9 cfu / ml.

3. The composite bacterial agent for promoting the growth of leguminous plants according to claim 1, characterized in that The rhizobium is Rhizobium sp. 3G1, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 23, 2024, and its deposit number is CGMCC NO.32319.

4. The composite bacterial agent for promoting the growth of leguminous plants according to claim 3, characterized in that: The gene sequence of the rhizobium (Rhizobium sp.) is shown in SEQ ID NO.

1.

5. The composite bacterial agent for promoting the growth of leguminous plants according to claim 1, characterized in that The bioactive agent is a lipopeptide surfactant.

6. The composite bacterial agent for promoting the growth of leguminous plants according to claim 1, characterized in that The bioactivator is a combination of fucoidan, brown algae oligosaccharide, humic acid and complex amino acids, which are mixed in a ratio of 1:2:10:

3.

7. The composite bacterial agent for promoting the growth of leguminous plants according to claim 1, wherein the preparation method of the composite bacterial agent is characterized in that: The following steps are involved: S1. Preparation of microbial inoculants: Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens, Microbacterium rubrum, and Rhizobium sp. 3G1 were inoculated into LB culture medium at an inoculation rate of 5%, respectively, and cultured at 37°C and 150 rpm for 48 h; the LB culture medium contained 10 g of peptone, 10 g of sodium chloride, and 5 g of yeast extract powder; after fermentation, a separate bacterial fermentation liquid was obtained, and the number of effective live bacteria in the fermentation liquid was greater than 1×10 9 cfu / ml; Bacillus mojavei, Bacillus velez, Bacillus amyloliquefaciens and Microbacterium rubrum are mixed in a ratio of 1-2:1-2:1-2:1-2 to prepare a composite bacteria; The composite bacteria and Rhizobium sp. 3G1 are fully mixed at a ratio of 1-2:1-2 to prepare a microbial agent; S2. Preparation of bioactivator: Take fucoidan, brown algae oligosaccharide, humic acid, and complex amino acid combination, mix them evenly in a ratio of 1:2:10:3, and prepare bioactivator; S3. Composite bacterial 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, and stir them thoroughly for 1-2 hours to make a composite bacterial agent.

8. An application of a composite bacterial agent for promoting the growth of leguminous plants, characterized in that: Used for preventing and controlling plant diseases and / or promoting plant growth.

9. The use of a composite bacterial agent for promoting the growth of leguminous plants according to claim 8, characterized in that: The plant diseases include Fusarium graminearum ( Fusarium graminearum )、Fusarium( Fusarium spp.), Maize Alternaria Alternaria tenuis Nees), Pyricularia oryzae ( Piricularia oryzae ), Fusarium oxysporum ( Fusarium oxysporum ); the plant is of the Leguminosae family.

10. The method for using the composite bacterial agent for promoting the growth of leguminous plants according to claim 8, characterized in that: The composite bacterial agent is used by dipping the roots or applying in holes during sowing; drip irrigation or direct spraying is used when the plants are growing, and the usage amount is 1 to 3 L / mu; The method of dipping the composite bacterial agent in the roots of crops comprises diluting the composite bacterial agent by 10 to 20 times, and dipping the roots of the seedlings after washing away the nutrient soil at the roots into the diluted solution, so that the roots are fully dipped in the bacterial solution before transplanting the seedlings, and immediately taking them out for transplanting after being wetted; The hole-fertilization method is to mix the composite bacterial agent with organic matter when the seedlings are planted, and then apply it into the planting hole, and then cover it with soil; the organic matter includes wheat bran, crushed rice bran, and soybean meal; The mixed application method is to mix the composite bacterial agent with organic matter, spread it on the soil surface or apply it in the soil through furrows, and then cover the soil. It is suitable for large-scale planting, and the usage amount per mu is 1 to 3L; The method of drip irrigation or direct spraying during the plant growth period is to dilute the composite bacterial agent 300-500 times during the plant growth period and before the flowering period, and drip irrigate the roots of the plants or spray them on the leaves, with a usage amount of 1-3L / mu.

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