A method for preparing a composite microbial agent for promoting crop growth

Abstract

The present invention belongs to the field of agricultural microorganism production, specifically a method for preparing a composite microbial agent for promoting the growth of crops. The agent of the present invention is a mixture of a fermented bacillus amyloliquefaciens broth and a fermented bacillus megaterium broth in a volume ratio of 1:1; the content of bacillus amyloliquefaciens in the final agent is 100-200 billion CFU / mL, the content of bacillus megaterium is 50-100 billion CFU / mL, and the content of indoleacetic acid is 1.4-2.0 mg / mL. The composite microbial agent containing a relatively high concentration of indoleacetic acid provided by the present invention, wherein tryptophan is added to the fermentation process of bacillus amyloliquefaciens in a specific time period so that it secretes a high concentration of indoleacetic acid during the fermentation process, and indoleacetic acid can effectively promote the rooting and germination of the seeds of crops and the growth of stems and leaves in the later stage, and bacillus amyloliquefaciens also secretes some antibiotic metabolites during its growth and reproduction, and these metabolites have a relatively high activity of inhibiting the growth of pathogens and protozoa.

Description

Technical Field

[0001] The invention belongs to the field of agricultural microorganism production, in particular to a method for preparing a composite microbial agent for promoting the growth of crops. Background Art

[0002] Indoleacetic acid is a plant growth regulator that plays an important role in the division, elongation, differentiation of plant cells, seed germination, root development, and vegetative growth. Indoleacetic acid participates in many physiological and biochemical regulation and control in plants, such as cell elongation, cambium cell division, and vascular tissue differentiation. In addition, indoleacetic acid can reduce the toxicity of heavy metals to plants by absorbing and enriching heavy metals in the soil, thereby protecting plants. The application of indoleacetic acid can promote crop growth and increase crop yields, and is therefore widely used in agricultural production.

[0003] However, the production of indoleacetic acid currently mostly adopts chemical synthesis methods. In the production process of chemical synthesis of indoleacetic acid, not only high temperature and high pressure are required, high energy consumption, but also toxic and harmful substances such as potassium cyanide are used, which seriously pollutes the environment and affects the healthy growth of crops and food safety.

[0004] In addition to plants that can produce a small amount of indoleacetic acid, many microorganisms can also produce indoleacetic acid. For example, bacteria such as Bacillus, Bacillus stagnans, and Rhizobium can secrete indoleacetic acid in the form of secondary metabolites. However, the production cycle of indoleacetic acid produced by plants is long, and the production environment and equipment requirements are high, which greatly increases the production cost of indoleacetic acid. Bacteria have the characteristics of simple cultivation, fast growth, short fermentation cycle, and easy large-scale production. The production of indoleacetic acid by microbial fermentation also has the advantages of mild conditions, low energy consumption, no use of toxic and harmful substances, and little environmental pollution.

[0005] Due to the irrational use of chemical fertilizers in modern agriculture, soil compaction is becoming more and more serious. Excess phosphate combines with trace elements such as calcium and magnesium to form insoluble phosphates, which accumulate in the soil, causing the soil to become compacted and the soil permeability to deteriorate. This not only affects the plants' utilization of trace elements and phosphates, but also causes root rot and slow growth of crops due to the deterioration of soil permeability, which seriously restricts the growth quality and yield of crops. The current solution is to apply organic fertilizers and deep plowing. The use of organic fertilizers can alleviate the problem of soil compaction but cannot fundamentally solve the problem of dissolution of insoluble phosphates. The use of organic fertilizers greatly increases agricultural production costs, and the method of deep plowing is not sustainable. The problem of soil compaction has become a difficult problem to solve at present. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a composite microbial agent for promoting the growth of crops and its application

[0007] To achieve the above purpose, the present invention adopts the following technical solution:

[0008] A composite microbial agent for promoting the growth of crops, wherein the agent is a mixture of fermented bacterial liquid of Bacillus amyloliquefaciens and fermented bacterial liquid of Bacillus megaterium in a volume ratio of 1:1; the content of Bacillus amyloliquefaciens in the final agent is 10-20 billion CFU / mL, the content of Bacillus megaterium is 5-10 billion CFU / mL, and the content of indoleacetic acid is 1.4-2.0 mg / mL.

[0009] The fermentation liquid of Bacillus amyloliquefaciens and the fermentation liquid of Bacillus megaterium are mixed, and the pH value of the mixed liquid is adjusted to be neutral, and then the finished bacterial agent is obtained.

[0010] The Bacillus amyloliquefaciens is deposited in the China Center for Type Culture Collection (Bacillus amyloliquefaciens) with the accession number CCTCCM 2018264 and the deposit time on May 14, 2018, and the deposit address is Wuhan University, Wuhan, China.

[0011] The Bacillus megaterium is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee (Bacillus magterium) CGMCC NO.5225 on September 6, 2011. The fermentation broth of Bacillus amyloliquefaciens is added with tryptophan during the fermentation process, and the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0012] A single colony of Bacillus amyloliquefaciens is inoculated into a seed culture medium to obtain a seed liquid, and the seed liquid is inoculated into a fermentation culture medium at an inoculation rate of 5-10% (volume ratio), and fermentation culture is carried out at 32-35° C., pH is controlled at 7.0-7.5, the rotation speed is 300-500RPM, and the dissolved oxygen is maintained at more than 30%. After culturing for 10-12 hours, tryptophan is added to the fermentation system so that the tryptophan in the fermentation system accounts for 5-10 g / L of the total amount, and the culture is continued for 10 hours, so that the final indoleacetic acid content of the fermentation liquid reaches 2.8-4.0 mg / mL, and the Bacillus amyloliquefaciens fermentation liquid is obtained.

[0013] The seed culture medium is added with 4-8 g / L yeast powder, 5-10 g / L peptone, 5-10 g / L sodium chloride, and the pH is 7.0-7.5. The fermentation culture medium is 5-10 g / L sucrose, 4-8 g / L yeast powder, 5-10 g / L protein, 0.5-1 g / L MgSO4·7H2O, and 1-2 g / L potassium dihydrogen phosphate.

[0014] The single colony of Bacillus amyloliquefaciens is inoculated into a seed culture medium, and cultured at 32-35° C. and 150-180 RPM for 15-20 hours to obtain a fermentation liquid of Bacillus amyloliquefaciens.

[0015] The Bacillus megaterium fermentation liquid is obtained by inoculating a single colony of Bacillus megaterium into a seed culture medium to obtain a seed liquid, and the seed liquid is inoculated into a fermentation culture medium at an inoculation amount of 5-10%, and the fermentation culture is carried out for 18-20 hours at 35-37° C., the pH is controlled at 7.2-7.5, the rotation speed is 300-500RPM, and the dissolved oxygen is maintained at more than 30%, so as to obtain the Bacillus megaterium fermentation liquid.

[0016] The single Bacillus megaterium colony is inoculated into a seed culture medium, and cultured at 35-37° C. and 150-180 RPM for 18-20 hours to obtain a Bacillus megaterium fermentation liquid.

[0017] The Bacillus megaterium seed culture medium is prepared by adding 4-8 g / L yeast powder, 5-10 g / L peptone and 5-10 g / L sodium chloride per liter, and the pH value is adjusted to 7.2-7.5.

[0018] The Bacillus megaterium fermentation medium is added with 5-10 g / L sucrose, 4-8 g / L yeast powder, 5-10 g / L peptone, 0.5-4 g / L magnesium sulfate, 1-2 g / L potassium dihydrogen phosphate, 0.5-1 g / L calcium carbonate and 0.05-0.1 g / L ferrous sulfate per liter.

[0019] A composite application of the composite microbial agent for promoting the growth of crops, wherein the composite agent has a promoting effect on the growth of corn and lettuce.

[0020] The advantages of the present invention are:

[0021] The composite microbial agent containing relatively high concentration of indoleacetic acid provided by the present invention is obtained by compounding fermentation broths of two specific strains of Bacillus amyloliquefaciens and Bacillus megaterium, wherein tryptophan is added to the Bacillus amyloliquefaciens in a specific time period during fermentation so that the Bacillus amyloliquefaciens can secrete high concentration of indoleacetic acid during fermentation, and the indoleacetic acid can effectively promote the rooting and germination of seeds of crops and the growth of stems and leaves in the later stage, and the Bacillus amyloliquefaciens can also secrete some antibiotic metabolites during its growth and reproduction, and these metabolites have relatively high activity in inhibiting the growth of pathogenic bacteria and protozoa, and can effectively prevent the occurrence of diseases and insect pests during plant growth. In addition, Bacillus megaterium has a strong ability to dissolve phosphorus and potassium, and can decompose the chelated organic and inorganic phosphorus in the soil into soluble phosphates, while releasing middle elements such as calcium ions and magnesium ions, effectively increasing the utilization of phosphorus, calcium ions and magnesium ions by plants, and reducing the use of middle elements in fertilizers. At the same time, because the chelated phosphorus element is decomposed and utilized, it can effectively solve the problem of soil compaction and improve the looseness of the soil, thereby promoting plant growth and reducing various physiological diseases caused by the lack of middle elements such as calcium and magnesium in plants. At the same time, because the chelated phosphorus element is decomposed and utilized, it can effectively solve the problem of soil compaction and improve the looseness of the soil, thereby reducing various physiological diseases caused by the lack of middle elements such as calcium and magnesium in plants, promoting plant growth vitality, solving the problem of soil compaction, improving fertilizer utilization, enhancing crop metabolism, and improving the quality and yield of crop growth as a whole. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Of course, the described embodiments are only part of the present invention, and other related implementation cases are also within the protection scope of the present invention.

[0023] Bacillus amyloliquefaciens was deposited in China Center for Type Culture Collection (Bacillus amyloliquefaciens) with the accession number CCTCCM 2018264 and the deposit date was May 14, 2018.

[0024] Bacillus megaterium was deposited in the General Microbiology Center (Bacillusmagterium) of China Microbiological Culture Collection Administration CGMCC NO.5225 on September 6, 2011.

[0025] Example 1 (Determination of Optimum Fermentation Temperature)

[0026] The Bacillus amyloliquefaciens strain stored in the strain tube was taken out from the -80°C refrigerator and inoculated on the solid LB plate medium, and cultured in the incubator at 32°C for 24 hours to obtain a single colony of Bacillus amyloliquefaciens. The cultured single colony of Bacillus amyloliquefaciens was inoculated into a shake flask containing 200 ml of seed culture medium, and cultured in a shaker box at 32°C and 180RPM for 18 hours to obtain a first-level seed solution of Bacillus amyloliquefaciens. The 200 ml seed solution was inoculated into four shake flasks containing 400 ml of culture medium and cultured in the above-mentioned culture method to prepare a second-level seed solution. The prepared second-level seed solution was inoculated into four continuous fermentation tanks containing 4 liters of culture medium, respectively at 30°C, 32°C, 35°C, 37°C, pH 7.0, and tryptophan was added to make the fermentation liquid concentration reach 5g / L after 12 hours of culture, and the fermentation was continued. During the period, the indoleacetic acid concentration was sampled and tested, and the optimal temperature range was determined by the experimental group with the maximum indoleacetic acid concentration. The indoleacetic acid concentration in the fermentation liquid was detected by high-performance liquid chromatography.

[0027] temperature Bacterial density (OD) Fermentation broth indoleacetic acid concentration (g / L) 30℃ 13 2.2 32℃ 15 2.8 35℃ 18 2.5 37℃ 15 2.6

[0028] From the data in the table, it can be seen that with the increase of fermentation temperature, the content of indoleacetic acid produced and the bacterial density are in a positive relationship in the early stage and inversely correlated in the later stage. When the temperature is 32°C, the content of indoleacetic acid in the fermentation liquid is the largest, so it can be determined that the optimal fermentation temperature is 32°C.

[0029] Embodiment 2 (optimum pH fermentation range determination)

[0030] Four bottles of Bacillus amyloliquefaciens seed solution, 400 ml each, were prepared in the manner of Example 1 and inoculated into four continuous fermentation tanks containing 4 liters of culture medium. After culturing at pH 6.8, 7.2, 7.5, and 7.8 and a temperature of 32° C. for 12 hours, tryptophan was added to make the fermentation liquid concentration reach 5 g / L, and fermentation was continued. During the fermentation, samples were taken to detect the indoleacetic acid concentration, and the optimal pH range was determined by the experimental group with the maximum indoleacetic acid concentration.

[0031] pH Bacterial density (OD) Fermentation broth indoleacetic acid concentration (g / L) 6.8 15 2.5 7.2 17 3.3 7.5 17 2.9 7.8 16 2.6

[0032] From the data in the table, it can be seen that with the increase of fermentation pH, the content of indoleacetic acid produced and the bacterial density are in a positive relationship in the early stage and inversely correlated in the later stage. When the pH is 7.2, the content of indoleacetic acid in the fermentation broth is the largest, so it can be determined that the optimal fermentation pH is 7.2.

[0033] Example 3 (Determination of the Optimal Tryptophan Concentration)

[0034] Four bottles of Bacillus amyloliquefaciens seed solution, each containing 400 ml, were prepared in the manner of Example 1 and inoculated into four continuous fermentation tanks containing 4 liters of culture medium. After culturing at 32° C. and pH 7.2 for 12 hours, tryptophan was added to make the fermentation solution concentrations 2, 5, 8, and 12 g / L, respectively. The fermentation was continued, and samples were taken during the fermentation to detect the indoleacetic acid concentration and determine the optimal tryptophan addition concentration.

[0035] Tryptophan concentration g / L Bacterial density (OD) Fermentation broth indoleacetic acid concentration (g / L) 2 16 1.5 5 16 3.1 8 17 3.8 12 16 3.5

[0036] From the data in the table, it can be seen that as the tryptophan content in the fermentation broth increases, the content of indoleacetic acid produced is positively correlated with the bacterial density. When the tryptophan content in the fermentation broth reaches 8g / L, the content of indoleacetic acid in the fermentation broth is the highest, and the bacterial density also reaches the highest. Therefore, it can be determined that the optimal tryptophan fermentation concentration is 8g / L.

[0037] Embodiment 4 (Determination of the optimal time for adding tryptophan).

[0038] Four bottles of 400 ml of Bacillus amyloliquefaciens seed solution were prepared in the manner of Example 1, and inoculated into four continuous fermentation tanks containing 4 liters of culture medium, respectively. After culturing at 32° C. and pH 7.2 for 8, 10, 12, and 14 hours, 8 g / L tryptophan was added and fermentation was continued. During the fermentation, samples were taken to detect the indoleacetic acid concentration. The optimal time for adding tryptophan was determined based on the experimental group with the maximum indoleacetic acid concentration.

[0039] Fermentation time (hours) Bacterial density (OD) Fermentation broth indoleacetic acid concentration (g / L) 8 8 2.2 10 12 4.3 12 15 3.5 14 17 2.5

[0040] From the data in the table, it can be seen that as the addition time increases, the content of indoleacetic acid produced in the fermentation broth has a positive relationship with the bacterial density in the early stage and a negative relationship in the later stage. When the tryptophan is added for 10 hours, the content of indoleacetic acid reaches the highest, so it can be determined that the optimal tryptophan addition time is 10 hours. Example 5: (Preparation method of Bacillus amyloliquefaciens and Bacillus megaterium inoculants)

[0041] The Bacillus amyloliquefaciens strain stored in the strain tube was taken out from a -80°C refrigerator and inoculated on a solid LB plate medium, and cultured in an incubator at 32°C for 24 hours to obtain a single colony of Bacillus amyloliquefaciens.

[0042] The single colony of Bacillus amyloliquefaciens obtained by culture is inoculated into a shaking bottle containing 200 ml of seed culture medium, and cultured in a shaking box at 32°C, 180RPM for 18 hours to obtain Bacillus amyloliquefaciens seed liquid. Accurately weigh the starch fermentation medium, put it in a 4-liter fermentation tank, stir and dissolve it, adjust the pH to 7.2 with sodium hydroxide, and sterilize it at 121°C for 20 minutes. After cooling, three bottles of Bacillus amyloliquefaciens seed liquid are respectively connected to a 4-liter fermentation tank, and the inoculation amount is 5%. The fermentation temperature is 32°C, the pH is controlled at 7.2, and the dissolved oxygen is maintained at more than 30%. After culturing for 10 hours, start adding tryptophan sterile solution to the fermentation tank to make its concentration reach 8 grams / L. When the content of the acetic acid fermentation liquid reaches 2.8-4.0 mg / mL, stop fermentation and collect the fermentation liquid.

[0043] The Bacillus megaterium strain stored at -80°C was inoculated on a solid LB plate medium and cultured in an incubator at 35°C for 24 hours to obtain a single colony of Bacillus megaterium.

[0044] The cultured single colony of Bacillus megaterium was inoculated into a shaking flask containing 200 ml of seed culture medium, and cultured in a shaking box at 35° C. and 180 RPM for 18 hours to obtain a Bacillus megaterium seed solution.

[0045] Put the fermentation medium into the fermentation tank, stir and dissolve it thoroughly, adjust the pH to 7.0 with sodium hydroxide, sterilize it at 121°C for 20 minutes, and cool it to 35°C for use. Inoculate the Bacillus megaterium seed liquid obtained in the previous step into a 4-liter fermentation tank containing the fermentation medium, with an inoculum amount of 5%. The fermentation temperature is 35°C, the pH is controlled at 7.0, and the dissolved oxygen is maintained at 30%. Cultivate for 20 hours, and when the Bacillus megaterium spore conversion rate reaches more than 90%, put the tank to collect the fermentation liquid.

[0046] The fermentation liquid of Bacillus amyloliquefaciens and Bacillus megaterium completed in the last fermentation is combined in a batching tank at a volume ratio of 1:1, mixed evenly, and the pH of the mixed liquid is adjusted to neutral, and the finished composite microbial agent is filled. The final finished product contains 10-20 billion CFU / mL of Bacillus amyloliquefaciens, 5-10 billion CFU / mL of Bacillus megaterium, and 1.4-2.0 mg / mL of indoleacetic acid.

[0047] Comparative Example (Preparation method of Bacillus amyloliquefaciens and Bacillus subtilis inoculants).

[0048] The Bacillus amyloliquefaciens strain stored in the strain tube was taken out from a -80°C refrigerator and inoculated on a solid LB plate medium, and cultured in an incubator at 32°C for 24 hours to obtain a single colony of Bacillus amyloliquefaciens.

[0049] The single colony of Bacillus amyloliquefaciens obtained by culture was inoculated into a shaking bottle containing 200 ml of seed culture medium, and cultured in a shaking box at 32°C and 180RPM for 18 hours to obtain Bacillus amyloliquefaciens seed liquid. Accurately weigh the starch fermentation medium, put it in a 4-liter fermentation tank, stir and dissolve it, adjust the pH to 7.2 with sodium hydroxide, and sterilize it at 121°C for 20 minutes. After cooling, three bottles of Bacillus amyloliquefaciens seed liquid were respectively connected to a 4-liter fermentation tank with an inoculation amount of 5%. The fermentation temperature was 32°C, the pH was controlled at 7.2, and the dissolved oxygen was kept greater than 30%. After 10 hours of culture, tryptophan sterile solution was added to the fermentation tank to make its concentration reach 8 g / L. When the indoleacetic acid fermentation liquid content reached 2.8-4.0 mg / mL, the fermentation was stopped and the fermentation liquid was collected.

[0050] The Bacillus subtilis strain stored at -80°C was inoculated on a solid LB plate medium and cultured in an incubator at 32°C for 24 hours to obtain a single colony of Bacillus subtilis.

[0051] The cultured single colony of Bacillus subtilis was inoculated into a shaking flask containing 200 ml of seed culture medium (same as Bacillus amyloliquefaciens seed culture medium), and cultured in a shaking box at 32° C. and 180 RPM for 18 hours to obtain a Bacillus subtilis seed solution.

[0052] Put the fermentation medium (same as Bacillus amyloliquefaciens medium) into the fermentation tank and fully stir and dissolve it, adjust the pH to 7.0 with sodium hydroxide at 121°C, sterilize it for 20 minutes, and cool it to 32°C for use. The subtilis seed liquid obtained in the previous step is inoculated into a 4-liter fermentation tank containing the fermentation medium, and the inoculum amount is 5% (volume ratio). The fermentation temperature is 32°C, the pH is controlled at 7.0, and the dissolved oxygen is kept at 30%. Cultivate for 20 hours, and when the conversion rate of Bacillus subtilis spores reaches more than 90%, put the tank to collect the fermentation liquid.

[0053] The fermentation liquid of Bacillus amyloliquefaciens and Bacillus subtilis completed in the last fermentation is combined in a batching tank at a volume ratio of 1:1, mixed evenly, and the pH of the mixed liquid is adjusted to neutral, and the finished composite microbial agent is filled. The final finished product contains 10-20 billion CFU / mL of Bacillus amyloliquefaciens, 10-20 billion CFU / mL of Bacillus subtilis, and 1.4-2.0 mg / mL of indoleacetic acid.

[0054] Application Example 1

[0055] Sterile physiological saline was used as control CK, the bacterial agent prepared by Bacillus amyloliquefaciens and Bacillus subtilis was experimental group 1 (Comparative Example 1), and the bacterial agent prepared by Bacillus amyloliquefaciens and Bacillus megaterium was experimental group 2. (Example 5) After diluting with physiological saline at 1:50 times, it was applied to the experimental pot containing corn seeds (the experimental pot was filled with compacted experimental soil), and 20 parallel experiments were performed in each group of experiments. The germination and growth status of corn were investigated in the light greenhouse. During the period, each investigation group was appropriately watered with an equal amount of water in the experimental pot to keep the soil moist. When the corn plants in the experimental group grew to more than 15 cm, 10 experimental pots were randomly taken from each group to clean the soil, and the height, root length, and fresh weight of the corn were investigated. The remaining 10 experimental pots in each group were placed in an oven at 105°C for drying to investigate the hardness (bulk density) of the compacted soil.

[0056] By comparing the experimental data of corn plant height, root length and fresh weight in the table below, the plant height of experimental group 2 increased by 28.2%, the root length increased by 25.4%, and the fresh weight increased by 29.8% compared with CK. The increase data of experimental group 1 compared with CK was not significant. The bulk density of experimental group 2 was reduced by 17 compared with CK1, while the bulk density of experimental group 1 was not much different from that of CK, indicating that the addition of Bacillus megaterium significantly improved the original soil compaction, and promoted the growth of corn together with Bacillus amyloliquefaciens. The composite microbial agent has a significant promoting effect on the growth of corn.

[0057] Treatment Group Plant height(CM) Root length (CM) Fresh weight(g) <![CDATA[Soil bulk density (g / cm 3 )]]> CK 14.5 12.2 18.1 1.62 Experimental Group 1 15.9 13.9 20.2 1.60 Experimental Group 2 18.6 15.3 23.5 1.35

[0058] Application Example 2

[0059] Sterile physiological saline was used as control CK, the bacterial agent prepared by Bacillus amyloliquefaciens and Bacillus subtilis was experimental group 1 (Comparative Example 1), and the bacterial agent prepared by Bacillus amyloliquefaciens and Bacillus megaterium was experimental group 2 (Example 5). After being diluted with physiological saline at 1:50 times, it was applied to the experimental pot containing lettuce seeds (the experimental pot was filled with compacted experimental soil). Each group of experiments was done in 20 parallels, and the germination and growth status of lettuce were investigated in the light greenhouse. During this period, each investigation group was appropriately watered with an equal amount of water in CK and the experimental group to keep the soil moist. When the lettuce of the experimental group grew to more than 10 cm, 10 experimental pots were randomly taken from each group to clean the soil, and the height, root length and fresh weight of the lettuce were investigated. The remaining 10 experimental pots in each group were placed in an oven at 105°C for drying, and the hardness of the soil (bulk density) of the plate was investigated.

[0060] By comparing the experimental data of plant height, root length and fresh weight of lettuce in the table below, the plant height of experimental group 2 increased by 23.1%, the root length increased by 26.6%, and the fresh weight increased by 16.8% compared with CK. The increase data of experimental group 1 compared with CK was not significant. The bulk density of soil was measured (soil bulk density determination method NY / T1121.4-2006). The bulk density of experimental group 2 was 14% lower than that of CK. The bulk density of experimental group 1 was not much different from that of CK, indicating that the addition of Bacillus megaterium significantly improved the original soil compaction and promoted the growth of lettuce together with Bacillus amyloliquefaciens.

[0061] The composite microbial agent also has a promoting effect on lettuce.

[0062] Treatment Group Plant height(CM) Root length (CM) Fresh weight(g) <![CDATA[Soil bulk density (g / cm 3 )]]> CK 10.8 7.5 20.2 1.60 Experimental Group 1 11.6 8.3 21.5 1.61 Experimental Group 2 13.3 9.5 23.6 1.38

[0063] As can be seen from the above, the present invention selects the bacillus amyloliquefaciens strain that efficiently produces indoleacetic acid, by optimizing fermentation conditions, tryptophan addition and time and other methods, promotes bacillus amyloliquefaciens to efficiently secrete indoleacetic acid during fermentation, greatly improves the unit content of indoleacetic acid in fermentation liquid, and its fermentation liquid output reaches 4 mg / mL, which is significantly higher than the output of indoleacetic acid fermented by other bacteria, and greatly reduces the production and use cost of microbial agent. And microbial agent of the present invention is compounded with efficient Bacillus megaterium that dissolves phosphorus and potassium, and its phosphorus dissolving ability can reach 200mg / L, which is much higher than the national standard (70mg / L). The addition of Bacillus megaterium fundamentally solves the problem of soil compaction, effectively promotes the utilization of excessive phosphate and trace elements in crops, directly increases the output of crops, and produces good economic benefits.

[0064] The above description is only a preferred implementation example of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A composite microbial agent for promoting the growth of crops, characterized in that: The bacterial agent is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) fermentation broth and Bacillus megaterium ( Bacillus magterium ) The fermentation broth is mixed in a volume ratio of 1:1; the content of Bacillus amyloliquefaciens in the final bacterial agent is 10-20 billion CFU / mL, the content of Bacillus megaterium is 5-10 billion CFU / mL, and the content of indoleacetic acid is 1.4-2.0 mg / mL; The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M2018264 and the deposit date is May 14, 2018; The Bacillus megaterium was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number of CGMCC NO.5225 and the deposit time of September 6, 2011.

2. The composite microbial agent for promoting crop growth according to claim 1, characterized in that: The fermentation liquid of Bacillus amyloliquefaciens and the fermentation liquid of Bacillus megaterium are mixed, and the pH value of the mixed liquid is adjusted to be neutral, and then the finished bacterial agent is obtained.

3. The composite microbial agent for promoting crop growth according to claim 1, characterized in that: The Bacillus amyloliquefaciens fermentation broth is added with tryptophan during the fermentation process.

4. The composite microbial agent for promoting crop growth according to any one of claims 1 to 3, characterized in that: A single colony of Bacillus amyloliquefaciens is inoculated into a seed culture medium to obtain a seed liquid, and the seed liquid is inoculated into a fermentation medium at an inoculation rate of 5-10%, and fermentation is carried out at 32-35° C., pH is controlled at 7.0-7.5, and the rotation speed is 300-500RPM, and the dissolved oxygen is maintained at more than 30%. After culturing for 10-12 hours, tryptophan is added to the fermentation system so that the tryptophan in the fermentation system accounts for 5-10 g / L of the total amount, and the culture is continued for 10 hours, so that the final indoleacetic acid content of the fermentation liquid reaches 2.8-4.0 mg / mL, and the Bacillus amyloliquefaciens fermentation liquid is obtained.

5. The composite microbial agent for promoting crop growth according to claim 4, characterized in that: The seed culture medium is added with 4-8 g / L yeast powder, 5-10 g / L peptone, and 5-10 g / L sodium chloride, and the pH value is 7.0-7.

5.

6. The composite microbial agent for promoting the growth of crops according to claim 4, characterized in that: The fermentation medium is sucrose 5-10 g / L, yeast powder 4-8 g / L, protein 5-10 g / L, MgSO4•7H2O 0.5-1 g / L, potassium dihydrogen phosphate 1-2 g / L.

7. The composite microbial agent for promoting crop growth according to claim 4, characterized in that: The single colony of Bacillus amyloliquefaciens is inoculated into a seed culture medium, and cultured at 32-35° C. and 150-180 RPM for 15-20 hours to obtain a Bacillus amyloliquefaciens seed liquid.

8. The composite microbial agent for promoting crop growth according to claim 1 or 2, characterized in that: The Bacillus megaterium fermentation liquid is obtained by inoculating a single colony of Bacillus megaterium into a seed culture medium to obtain a seed liquid, and the seed liquid is inoculated into a fermentation culture medium at an inoculation amount of 5-10% by volume, and the fermentation culture is carried out for 18-20 hours at 35-37° C., the pH is controlled at 7.2-7.5, the rotation speed is 300-500RPM, and the dissolved oxygen is maintained at more than 30%, so as to obtain the Bacillus megaterium fermentation liquid.

9. The composite microbial agent for promoting the growth of crops according to claim 8, characterized in that: The single Bacillus megaterium colony is inoculated into a seed culture medium, and cultured at 35-37° C. and 150-180 RPM for 18-20 hours to obtain a Bacillus megaterium seed solution.

10. An application of the composite microbial agent for promoting crop growth according to claim 1, characterized in that: The composite bacterial agent promotes the growth of corn and lettuce.