A composite microbial agent tapping into indigenous microbial sources and its preparation method and application

Abstract

The present invention discloses a composite microbial agent that taps the source of indigenous microorganisms, and a preparation method and application thereof, and belongs to the field of microorganisms. The present invention taps indigenous microorganisms and isolates a strain of Bacillus amyloliquefaciens, the preservation number of which is CGMCC No. 25581. The above-mentioned Bacillus amyloliquefaciens, Paenibacillus pilorei and Streptomyces freundii are compositely prepared into a composite microbial agent. According to tests, the number of live bacteria of the composite agent is high, and the prepared microbial agent with growth-promoting effect can not only adapt to the climate environment and stabilize growth metabolism, but also has a good growth-promoting effect on the growth of crops such as seed corn and plateau summer vegetables, and can also have a good biological control effect on common diseases of plateau summer vegetables.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a composite microbial agent tapping into indigenous microorganism sources, and a preparation method and application thereof. Background Art

[0002] Under the strategic requirements of national agricultural modernization and sustainable development, the development of new microbial fertilizers to replace part or all of chemical fertilizers will help alleviate ecological security and food safety issues and promote the green ecological development of agriculture. Beneficial microorganisms in microbial fertilizers can secrete plant hormones, stimulate the division of cell formation layers, and promote plant growth and development; they can increase the germination rate of crops, increase biomass, root length and root surface area, promote crop growth, increase leaf area and chlorophyll content, and increase crop yields; they can reduce the nitrate content in fruits and vegetables, increase vitamin C and sugar content, and improve the quality of crops; microbial fertilizers can maintain moisture in the soil, increase the number of microorganisms in the soil, improve the thermal insulation performance of the soil, and improve the physical and chemical properties and fertilizer efficiency of the soil; the beneficial microorganisms in microbial fertilizers exist in large quantities, forming dominant populations in the root system of crops and producing hormones such as antibiotics, which have an inhibitory effect on a variety of crop pathogens; beneficial bacteria can degrade pesticide residues and heavy metal pollution in the soil, thereby improving the quality and quality of crops.

[0003] There has been some research on growth-promoting microbial agents both at home and abroad, but the existing microbial agents have a single performance and require the use of multiple agricultural microbial agents to achieve the desired effect. Different types of microbial agents may have antagonistic effects, making operation very inconvenient. In addition, the effects of existing microbial agents are unstable, and the microbial strains cannot adapt to the climate environment and thus cannot grow and reproduce normally. Summary of the invention

[0004] The purpose of the present invention is to provide a composite microbial agent extracted from indigenous microorganisms and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The microbial agent prepared by extracting indigenous microorganisms in the present invention can not only adapt to the climatic environment and have stable growth metabolism, but also has a good growth-promoting effect on the growth of crops such as seed corn and plateau summer vegetables, and can also have a good biological control effect on common diseases of plateau summer vegetables.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a Bacillus amyloliquefaciens wsy-09, the preservation number of the bacterium is CGMCC No.25581, the preservation time is August 22, 2022, the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides the use of the Bacillus amyloliquefaciens or its fermentation liquid in any of the following:

[0008] (1) Application in the preparation of IAA, iron carrier and ACC deaminase promoter;

[0009] (2) Application in the preparation of antibacterial agents;

[0010] (3) Application in the preparation of growth-promoting agents for promoting crop growth;

[0011] (4) Application in improving crop quality and yield.

[0012] Preferably, the antibacterial agent is a bacterial agent that inhibits the growth of Fusarium oxysporum, Fusarium solani or Alternaria tenuissima.

[0013] Preferably, the crops include plateau summer vegetables and corn.

[0014] The present invention also provides a composite microbial agent of indigenous microbial origin, comprising any of the following:

[0015] (1) The composite microbial agent comprises the Bacillus amyloliquefaciens, Paenibacillus peoviae and Streptomyces fradiae as described in claim 1;

[0016] (2) The composite microbial agent comprises the Bacillus amyloliquefaciens, Paenibacillus pierrei, Streptomyces freundii and auxiliary materials as described in claim 1.

[0017] Preferably, the deposit number of the Paenibacillus pierrei is CGMCC 1.12148, and the deposit number of the Streptomyces freundii is CGMCC 4.7387;

[0018] The mass ratio of the Bacillus amyloliquefaciens, the Paenibacillus pierrei and the Streptomyces freundii is (1-2): (1-2): (1-2).

[0019] Preferably, the auxiliary materials include defatted bran powder, glycerol, zeolite powder and mineral potassium humate.

[0020] The present invention also provides a method for preparing the composite microbial agent, comprising any one of the following methods:

[0021] (1) activating the Bacillus amyloliquefaciens, the Paenibacillus pierreiu and the Streptomyces freundii respectively, and obtaining the Bacillus amyloliquefaciens agent, the Paenibacillus pierreiu agent and the Streptomyces freundii agent through expanded culture;

[0022] (2) The Bacillus amyloliquefaciens, the Paenibacillus pierrei, and the Streptomyces freundii are activated respectively, and after expanded culture, a fermentation broth of Bacillus amyloliquefaciens, a fermentation broth of Paenibacillus pierrei, and a fermentation broth of Streptomyces freundii are obtained respectively, and auxiliary materials are added to each of them to prepare powders, thereby obtaining a Bacillus amyloliquefaciens inoculum, a Paenibacillus pierrei inoculum, and a Streptomyces freundii inoculum.

[0023] Preferably, the auxiliary materials are defatted bran powder, glycerol, zeolite powder and mineral-derived potassium humate; based on the total mass of the Bacillus amyloliquefaciens fermentation broth, the Paenibacillus pierrei fermentation broth or the Streptomyces freundii fermentation broth, the mass percentages of the defatted bran powder, glycerol, zeolite powder and mineral-derived potassium humate added are 50%, 2%, 25% and 1% respectively.

[0024] The present invention also provides an application of the composite microbial agent in any of the following items:

[0025] (1) Application in the preparation of growth-promoting agents for promoting crop growth;

[0026] (2) Application in improving crop quality and yield;

[0027] (3) Application in the prevention and treatment of pathogens or diseases caused by pathogens;

[0028] The crops include highland summer vegetables and corn;

[0029] The pathogenic bacteria include Fusarium oxysporum, Fusarium solani or Alternaria alternata.

[0030] The present invention discloses the following technical effects:

[0031] Different plant rhizospheres have microbial species that are adapted to them. The present invention obtains indigenous microbial strains that are adapted to the plant rhizosphere, which can quickly adapt to the environment, grow, reproduce and metabolize well in the environment, and effectively compete with microorganisms from other communities, ultimately allowing the prepared plant probiotic agent to exert its efficacy.

[0032] The invention adopts a low-temperature spray drying method to prepare a powdered growth-promoting microbial agent, the preparation method is simple and easy to operate, the number of effective live bacteria is high, and the shelf life is long.

[0033] The use of the composite microbial agent prepared by the present invention improves soil quality, increases crop quality and yield, increases the competitiveness and visibility of agricultural products in the industry, is beneficial to increasing farmers' income, reduces the use of chemical agents, is beneficial to protecting people's health, and generates huge social, ecological and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 Phylogenetic tree of Bacillus amyloliquefaciens WSY09 constructed;

[0036] Figure 2 The results of the determination of IAA, siderophore and ACC deaminase production by Bacillus amyloliquefaciens WSY09 strain;

[0037] Figure 3 The results of the antagonistic experiment of Bacillus amyloliquefaciens WSY09 against different pathogens: A: Fusarium oxysporum, B: Fusarium solani, C: Alternaria tenuissima;

[0038] Figure 4 The effects of different bacterial agents on plant height, stem diameter and leaf number of pink tomatoes at different growth stages;

[0039] Figure 5 This is the effect of composite microbial agents on plant height and leaf number of corn at different growth stages. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Sources of the strains involved in the following examples: Paenibacillus pierrei and Streptomyces freundii were purchased from the General Microbiological Center of China Microbiological Culture Collection Administration, with strain collection numbers of CGMCC 1.12148 and CGMCC4.7387, respectively.

[0046] Example 1 Isolation and identification of indigenous microbial strains

[0047] 1. Strain Isolation

[0048] Soil samples were collected from the rhizosphere of healthy vegetables at the plateau summer vegetable planting base in Xinying Township, Yuzhong County, Lanzhou City, Gansu Province, and placed in sampling bags and taken back to the laboratory for use. Weigh 10 g of soil sample and add it to a sterilized triangular flask with glass beads and 90 mL of pure water in advance. Incubate it on a shaker at 30 °C at 200 rpm for 30 min. After standing for 5 min, take 1 mL of the supernatant and add it to a test tube with 9 mL of sterile water in advance. Then perform gradient dilution to 10 -2 -10 -6 g / mL soil suspension for later use. Take 100 μL of the diluted soil bacterial suspension (10-2 -10 -6 g / mL) was evenly spread on LB solid plates (0.5% yeast extract, 1% peptone, 1% sodium chloride, 2% agar, and the rest was water) with a spreading rod, 3 plates were spread for each concentration, the plates were inverted and placed in a 30°C constant temperature incubator for culture, the growth of the colonies on the plates was observed, strains of different morphologies were picked and streaked on the LB plates for purification, and finally the indigenous microbial strain WSY09 was isolated and obtained.

[0049] 2. Strain identification

[0050] The DNA of strain wsy09 was extracted using a kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the 16S rDNA gene sequence of the strain was amplified using the bacterial 16SrRNA specific primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO: 1) and 1492R (5′-GGTTACCTTGTTACGACTT-3′, SEQ ID NO: 2). The amplified product was sent to Shanghai Biotech for sequencing. The sequenced 16S rRNA sequence was submitted to the NCBI website for comparison analysis. The Neighbor Joining phylogenetic tree based on the 16S rRNA sequence was constructed using MEGA11 software, and the calculation model was Kimura2-paremeter. Sequence alignment found that the similarity of Bacillus amyloliquefaciens WSY09 with the published Bacillus amyloliquefaciens strain VITKA1, Bacillus amyloliquefaciens strain Ba13 and Bacillus amyloliquefaciens strain YP6 was 99.95%. The NJ phylogenetic tree constructed based on 16S rRNA gene sequences showed that strain wsy09 had the closest relationship with Bacillus amyloliquefaciens strain VITKA1, Bacillus amyloliquefaciens strain Ba13 and Bacillus amyloliquefaciens strain YP6, see Figure 1 In summary, based on the 16S rDNA sequence comparison results, strain WSY09 was identified as Bacillus amyloliquefaciens.

[0051] 16S rDNA sequence (SEQ ID NO: 3):

[0052]

[0053] The above-mentioned Bacillus amyloliquefaciens WSY09 was deposited in the General Microbiology Center of China Culture Collection Administration on August 22, 2022. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25581.

[0054] Example 2 Analysis of the growth-promoting function of Bacillus amyloliquefaciens WSY09

[0055] 1. Determination of IAA production by strains

[0056] Drawing of IAA standard curve: Prepare 100μg / mL IAA stock solution, and dilute it with distilled water to prepare 5μg / mL, 10μg / mL, 15μg / mL, 20μg / mL and 25μg / mL, and set up 3 groups of repeated experiments for each group. Use Salkowski method for determination, take 1mL of the test solution, add 1mL of Salkowski colorimetric solution, and color development for 30min under light-proof conditions, then quickly use spectrophotometer for colorimetry (530nm), and draw standard curve with statistical data.

[0057] Quantitative determination of IAA secretion by strains: Inoculate Bacillus amyloliquefaciens WSY09 into LB culture medium containing L-tryptophan, culture at 30°C and 180r / min for 72h, centrifuge at 10000r / min for 10min, aspirate 1mL of the centrifugal supernatant, add 4mL Salkowski colorimetric solution, color develop for 30min under light-proof conditions, and then quickly perform colorimetry (530nm) with a spectrophotometer. Measure three sets of data, take the average of the results, and find the corresponding amount of IAA produced on the standard curve.

[0058] 2. Determination of iron carrier production by strains

[0059] Bacillus amyloliquefaciens WSY09 was inoculated in MKB medium and cultured at 30°C and 150r / min for 72h. The bacterial solution was then centrifuged and the supernatant was mixed with CAS detection solution in a 1:1 ratio. After standing for 1h, the absorbance value As was measured at 630nm. Another uninoculated MKB medium was mixed with CAS as a control to measure the absorbance value Ar. Ar-As / Ar represents the relative content of iron carriers in the sample. The lower the value, the higher the iron carrier content.

[0060] MKB medium: Accurately measure 7.5 mL of glycerol, weigh 1.25 g of potassium dihydrogen phosphate, dissolve in deionized water, adjust pH to 7.2, and then make up to 450 mL. Sterilize at 115°C for 30 min. After sterilization, add 5 mL of sterile 5% magnesium sulfate solution and 10% acid hydrolyzed casein solution to every 90 mL of MKB solution, mix well and set aside.

[0061] 3. Determination of ACC deaminase activity produced by strains

[0062] Drawing of standard curve: 100mmol / L α-ketobutyric acid storage solution was prepared with 0.1mol / L Tris-HCl (pH 8.5) buffer, and α-ketobutyric acid solutions with final concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0μmol / mL were prepared by gradient dilution. 200μL of each solution was added to 300μL of 0.2% 2,4-dinitroaniline, incubated at 30℃ for 30min, and 2mL of 2mol / LNaOH solution was added to color aniline, and the absorbance was measured at 540nm. The standard curve was drawn with the concentration of α-ketobutyric acid (mmol / L) as the horizontal axis and the absorbance (A540) as the vertical axis.

[0063] Enzyme activity determination: The screened strains were inoculated into 30 mL DF liquid culture medium and cultured in the dark at 30 ° C and 200 rpm for 72 hours. The bacterial suspension was centrifuged at 4 ° C and 8000 rpm for 10 minutes, and the bacterial precipitate was collected. The bacterial suspension was resuspended in 30 mL DF liquid culture medium, and then cultured at 30 ° C and 200 rpm for 24 hours to induce the ACC deaminase activity of the bacteria. The above bacterial suspension was centrifuged at 4 ° C and 8000 rpm for 10 minutes, the bacterial precipitate was collected, and resuspended in 5 mL 0.1 mol / L Tris-HCL buffer (pH 7.6), and the precipitate was collected after centrifugation at 4 ° C and 8000 rpm for 10 minutes. Add 1mL, 0.1moL / L Tris-HCL buffer (pH7.6) to the bacterial precipitate to suspend the bacteria, then transfer to a 1.5mL centrifuge tube and centrifuge at 11000rpm for 10min; resuspend the bacteria in 600μL, 0.1moL / L Tris-HCL buffer (pH 8.5), add 30μL toluene and vortex for 30s to break the bacterial cells. Pipette 200μL of the broken cell suspension, add 20μL, 0.5moL / L AC solution, mix well and water bath at 30℃ for 15min. Then add 1mL, 0.56moL / L HCl solution to mix well and centrifuge at 11000rpm for 10min. Take 1 mL of the above centrifuged supernatant, add 800 μL of 0.56 mol / L HCl solution, mix well, then add 300 μL of 2 g / L 2,4-dinitrophenylhydrazine solution, and place in a water bath at 30°C for 30 min. Add 2 mL of 2 mol / L NaOH solution for color development and detect OD 540 The OD of the sample was 0.04477 W / m2 and the OD of the sample was 0.0337 W / m2. 540 The values ​​were substituted into the standard curve to calculate the corresponding α-ketobutyric acid content, and the amount of 1 μmol α-ketobutyric acid formed per minute was defined as 1 enzyme activity unit.

[0064] The DF medium is as follows: Component 1 (100 mL): 10 mg molybdenum trioxide, 10 mg boric acid, 11.19 mg manganese sulfate, 78.22 mg copper sulfate pentahydrate, 124.6 mg zinc sulfate heptahydrate; Component 2 (10 mL): 100 mg ferrous sulfate heptahydrate;

[0065] Dissolve component 1 in 100 mL of sterile distilled water, dissolve component 2 in 10 mL of sterile distilled water, and store at low temperature for later use. Take 0.5 mL of component 1, 0.2 mL of component 2, 0.2 g of magnesium sulfate heptahydrate, 2.0 g of citric acid, 2.0 g of ammonium sulfate, 2.0 g of gluconic acid, 2.0 g of glucose, 4.0 g of potassium dihydrogen sulfate, and 6.0 g of disodium hydrogen phosphate, and adjust the volume to 1000 mL, pH 7.0.

[0066] like Figure 2 As shown, the test results show that Bacillus amyloliquefaciens WSY09 can secrete IAA, siderophore and ACC deaminase. The content of IAA gradually increases with the increase of strain culture time, and the IAA content reaches a maximum of 63.53 mg / L at 36h; the relative content of siderophore reaches a maximum of -0.331 at 48h; and the activity of ACC deaminase reaches a maximum of 0.153 U / mg at 36h.

[0067] Example 3 Analysis of the control effect of Bacillus amyloliquefaciens WSY09 on pathogenic microorganisms

[0068] The antagonistic experiment was conducted by plate confrontation culture. The pathogenic microorganisms used were common Fusarium oxysporum, Fusarium solani, and Alternaria tenuissima, which are easy to cause plateau summer vegetable diseases. The pathogenic microorganisms were inoculated into PDA solid culture medium (200g potato, 20g glucose, 15g agar powder, H 2 O 1000mL, sterilize at 121℃ for 15min), and culture in a constant temperature incubator at 28℃ for 3d for activation.

[0069] Punch holes at the edge of the pathogen colony with a sterile puncher with a diameter of 5 mm to make a bacterial cake. Use an inoculation loop to inoculate the bacterial cake to the middle of the diameter of the PDA culture medium. Punch holes with a sterile puncher at a position 1 cm away from the edges of both ends. Take 50 μL of activated Bacillus amyloliquefaciens bacterial solution and inject it into the holes at both ends. After the bacterial solution is completely absorbed, seal the culture dish with a sterile sealing film. Place the culture dish upright in a constant temperature incubator at 28°C and culture for 5 to 7 days. Each probiotic is repeated 3 times for the same plant pathogenic microorganism, and the LB liquid culture medium replaces the bacterial solution in the control group.

[0070] like Figure 3 As shown, the results showed that Bacillus amyloliquefaciens WSY09 had a uniformly good control effect on common pathogenic microorganisms Fusarium oxysporum, Fusarium solani, and Alternaria tenuissima.

[0071] Example 4 Preparation of Growth-Promoting Bacteria

[0072] (1) Preparation of bacterial agent

[0073] Bacillus amyloliquefaciens WSY09 and Paenibacillus pierreiro (CGMCC 1.12148) were activated in LB plate medium, single colonies were picked and transferred to liquid expansion medium for primary culture, and the primary cultures were transferred to liquid expansion medium for secondary culture, so as to obtain Bacillus amyloliquefaciens and Paenibacillus pierreiro inoculants respectively; Streptomyces freundii was activated in Gao's No. 1 plate medium, single colonies were picked and transferred to liquid expansion medium for primary culture, and the primary culture was transferred to liquid expansion medium for secondary culture, so as to obtain Streptomyces freundii inoculants. The composition of the liquid expansion medium is: 5% monohydrate glucose, 5% dextrin, 5% malt powder, 1% sodium chloride and 0.5% magnesium sulfate, pH 7.0; the composition of Gao's medium No. 1 is: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.5g ammonium chloride, 0.01g ferrous sulfate heptahydrate and 20g agar, the pH value is adjusted to 7.4-7.6, and sterilized at 121°C for 20min.

[0074] 50% of 60-mesh defatted bran powder, 2% of glycerol, 25% of zeolite powder and 1% of mineral potassium humate were added to the amyloliquefaciens inoculant according to mass percentage, and the inoculant was prepared into powder by low-temperature spray drying. When the feed rate of low-temperature spray drying was set to 7.5 mL / min and the inlet temperature was 150 °C, the effective viable count of the solid amyloliquefaciens inoculant was 1.0 × 10 9 cfu / mL;

[0075] 50% of 60-mesh defatted bran powder, 2% of glycerol, 25% of zeolite powder and 1% of mineral potassium humate were added to the Paenibacillus piloreus inoculant according to mass percentage, and the inoculant was prepared into powder by low-temperature spray drying. When the feed rate of low-temperature spray drying was set to 7.5 mL / min and the inlet temperature was 140 °C, the effective viable count of the solid Paenibacillus piloreus inoculant was 0.8 × 10 9 cfu / mL;

[0076] 50% of 60-mesh defatted bran powder, 2% of glycerol, 25% of zeolite powder and 1% of mineral potassium humate were added to the Streptomyces freundii inoculum according to mass percentage, and the inoculum was prepared into powder by low-temperature spray drying. When the feed rate of low-temperature spray drying was set to 7.0 mL / min and the inlet temperature was 130 °C, the effective viable count of the solid Streptomyces freundii inoculum was 1.0 × 10 8 cfu / mL.

[0077] (2) Potted plant experiment

[0078] The pot experiment was conducted to determine the growth-promoting effects of different ratios of the three strains on crop seedlings, and then the optimal ratio of Bacillus amyloliquefaciens, Paenibacillus pilorei and Streptomyces freundii was obtained. The experiment set up 4 treatment groups. The fermentation broth of the three strains was first freeze-dried into powder according to the conventional method, and then prepared according to the mass ratio shown below:

[0079] Treatment 1: Bacillus amyloliquefaciens, Paenibacillus pierrei, and Streptomyces freundii = 1:1:1;

[0080] Treatment 2: Bacillus amyloliquefaciens, Paenibacillus pierrei, and Streptomyces freundii = 2:1:1;

[0081] Treatment 3: Bacillus amyloliquefaciens, Paenibacillus pierrei, and Streptomyces freundii = 1:2:1;

[0082] Treatment 4: Bacillus amyloliquefaciens, Paenibacillus pierrei, and Streptomyces freundii = 1:1:2;

[0083] Control group CK: distilled water.

[0084] Each treatment group was fully mixed with distilled water at a mass-to-volume ratio of 1:50, and the inoculants of different treatment groups were directly irrigated into the roots of pakchoy at the 3-leaf stage. Each sample was repeated 3 times. After 30 days of cultivation, samples were taken to measure the agronomic traits (plant height, root length, number of leaves), biomass (fresh weight) and chlorophyll content of pakchoy (Top Yunnong TYS-B handheld chlorophyll meter).

[0085] The results are shown in Table 1 below. Compared with root irrigation with distilled water, the microbial agent group for root irrigation of pakchoi seedlings significantly improved the agronomic traits, fresh weight of the whole plant and chlorophyll content of pakchoi. Compared with the microbial agent treatment groups, the agronomic traits, fresh weight of the whole plant and chlorophyll content of pakchoi in treatments 1 and 2 were significantly higher than those in treatments 3 and 4, indicating that the compound ratio of the three microbial strains has a significant effect on the growth of pakchoi. The experimental effects of treatments 1 and 2 are better than those of treatments 3 and 4, indicating that the content of Bacillus amyloliquefaciens inoculant cannot be less than that of Paenibacillus pierreu and Streptomyces freundii. Considering the growth data of pakchoi in the table below and the cost saving perspective, treatment 1 was finally selected, that is, the compound ratio of Bacillus amyloliquefaciens, Paenibacillus pierreu and Streptomyces freundii = 1:1:1 was the best.

[0086] Table 1 Effects of different bacterial agent ratios on the growth of Chinese cabbage

[0087] Treatment Group Plant height / cm Root length / cm Number of blades Whole plant fresh weight / g Chlorophyll / SPAD Process 1 16.76±0.45a 16.89±0.62a 12.0±0.36a 17.53±0.33a 103.58±1.79a Process 2 16.82±0.38a 16.74±0.27a 13.0±0.52a 17.84±0.46a 105.21±2.06a Process 3 15.03±0.52b 14.68±0.36b 10.0±0.64b 15.27±0.28b 86.79±2.63b Process 4 14.84±0.48b 14.25±0.25b 9.0±0.46b 14.88±0.63b 77.28±3.17bc CK 13.76±0.75c 13.63±0.33c 9.0±0.72b 11.64±0.53c 64.69±1.69c

[0088] Example 5 Verification of the growth-promoting effects of different bacterial agents on plateau summer vegetables

[0089] The test site is located in Heping Town, Yuzhong County, Lanzhou City, and the planting test area of ​​the pilot base of the Institute of Biology, Gansu Academy of Sciences. The tomato variety is "Hezuo 918" pink tomato, which was purchased from the market; there are 5 treatments in the test, namely: treatment 1 (Bacillus amyloliquefaciens), treatment 2 (Bacillus piloreus), treatment 3 (Streptomyces freundii), treatment 4 (Bacillus amyloliquefaciens, Bacillus piloreus and Streptomyces freundii = 1:1:1), and treatment 5 (CK without adding bacteria). The bacteria in each treatment group were fully mixed with water at a mass volume ratio of 1:100, and then directly irrigated to the roots of tomato plants in the seedling stage. Each treatment is a plot, and the experiment adopts a completely randomized block design with a total of three replicates. The growth and yield of tomatoes were measured at the maturity stage.

[0090] The results are as follows Figure 4 As shown in the figure, the effects of the five different treatments on tomato growth are as follows: in each growth stage, the tomato plant height of treatment 1 and treatment 4 was significantly higher than that of treatment 2, treatment 3 and CK, and treatment 2 and treatment 3 were significantly higher than CK; among the treatments at different growth stages, the stem diameter of tomato in treatment 4 was significantly greater than that of other treatments, the stem diameter of treatment 1 was significantly higher than that of other treatments at maturity, there was no significant difference between treatment 1 and other treatments at flowering and fruiting stages, and the stem diameter of the treatment group with added microbial agents was significantly greater than that of CK; the number of leaves in the treatment group with added microbial agents was significantly greater than that of CK at each growth stage, and there was no significant difference in the number of leaves between the treatments; the chlorophyll content of tomatoes in each growth stage was significantly higher than that of CK in the treatment group with added microbial agents, and there was no significant difference between the treatment groups. The above results show that the application of microbial agents has a growth-promoting effect on tomatoes. The growth-promoting effect of applying Bacillus amyloliquefaciens alone is higher than that of applying Paenibacillus pierreu and Streptomyces freundii alone, but its growth-promoting effect is lower than that of applying a composite microbial agent. The growth-promoting effect of applying a composite microbial agent (Bacillus amyloliquefaciens, Paenibacillus pierreu and Streptomyces freundii = 1:1:1) on tomatoes is the best. This shows that Bacillus amyloliquefaciens has a growth-promoting effect on tomatoes. If Bacillus amyloliquefaciens is mixed with Paenibacillus pierreu and Streptomyces freundii in a ratio of 1:1:1, the growth-promoting effect on tomatoes is the best.

[0091] Table 2 Effects of different bacterial agents on tomato yield

[0092] deal with Number of fruits per plant Single fruit weight g Yield Kg / mu T1 7.87±0.17a 287.16±3.78a 4175.41±6.64b T2 7.57±0.21b 264.34±5.72b 3926.45±6.37c T3 7.51±0.31b 266.42±4.34b 3985.51±5.16c T4 8.03±0.24a 288.64±5.83a 4302.16±6.29a CK 7.23±0.27c 217.25±4.14c 3832.43±6.82d

[0093] Table 2 shows the results of tomato yield measurement. The results show that compared with CK, the addition of microbial agents has a significant promoting effect on the number of fruits per plant, fruit weight and yield of tomatoes. The tomato yield of treatment 4 is significantly higher than that of other treatment groups, and treatment 1 is significantly higher than that of treatments 2 and 3. The number of fruits per plant and the weight of fruits per plant of tomatoes in treatments 1 and 4 are significantly higher than those in treatments 2 and 3. The results show that treatment 4 has the highest tomato yield, that is, the application of composite microbial agents can effectively increase tomato yield.

[0094] Table 3 Effects of different bacterial agents on tomato quality

[0095]

[0096] Table 3 shows the tomato quality test results. The results show that the addition of different microbial agents made the Vc, soluble sugar and lycopene contents of tomatoes significantly higher than those of the control group CK, while the titratable acid and nitrate contents were significantly lower than those of CK, indicating that different agents can effectively improve the flavor and quality of tomato fruits, among which treatment 4, i.e., the composite microbial agent, had the best effect on improving tomato quality.

[0097] Rhizosphere soil samples of the tomato ripening treatment group T4 and the control group CK were collected, and the metabolites of the tomato roots under different treatments were detected and analyzed, with 3 replicates for each treatment. The non-targeted metabolome was detected by Biomarker Biotechnology Co., Ltd. The pathways of various differential metabolites, and the annotation results of significantly differential metabolites were classified according to the pathway types in KEGG. The results showed that there were three differential metabolites after comparison between the treatment group and the control group with the addition of growth-promoting microbial agents: tyrosine can generate plastoquinone, ubiquinone and other substances through a series of reactions, and plastoquinone plays a role in electron transport in plant photosynthesis, which is essential for plant growth and development; 12-oxyphytodienoic acid, jasmonic acid belongs to the precursor of pentane compounds, and is also a metabolite of the jasmonic acid synthesis pathway, which is related to the stress response and growth and development of plants; indole-3-acrylic acid is a metabolite of tryptophan, among which tryptophan is an important precursor for auxin biosynthesis in plants. Its structure is similar to indole-3-acetic acid (IAA), which promotes plant growth and development to a certain extent.

[0098] Table 4 Detection and analysis results of tomato root metabolites under different bacterial agent treatments

[0099]

[0100]

[0101] Note: log 2 FC represents the logarithm of the difference in the relative content of the metabolite in the two treated samples. The larger the absolute value, the greater the difference in the relative content of the substance in the two treated samples.

[0102] Example 6 Application of composite microbial agent in seed corn planting

[0103] The test site is located in x050, Dangzhai Town, Ganzhou District. The corn variety is Guoyu 717, and the test area is 1 mu. Three treatments were designed with the use of composite microbial agents (Bacillus amyloliquefaciens, Paenibacillus piloreus and Streptomyces freundii = 1:1:1) as the gradient, namely: 1kg / mu (treatment 1), 2kg / mu (treatment 2), 3kg / mu (treatment 3) and no addition of agents (CK treatment 4). Three replicates were set for each treatment, and the experimental plots were randomly arranged. After the composite microbial agents of each treatment group were fully mixed with water at a mass volume ratio of 1:100, they were added to the drip irrigation belt when the corn grew to the seedling stage. The growth of corn was observed at the seedling stage, jointing stage, large trumpet stage, heading stage, filling stage and harvest stage, and the physiological indicators of corn plant height, leaf number and other physiological indicators under different treatments were measured. After the corn matured, its related agronomic indicators were measured.

[0104] like Figure 5 As shown in the figure, the results showed that there was no significant difference in the height of corn plants under different treatments from the seedling stage to the trumpet stage; the height of corn plants in treatment 2 was the highest in both the heading and filling stages. From the seedling stage to the heading stage, the number of leaves of corn treated with growth-promoting agents was more than that of the control, and in the filling stage, treatment 1, treatment 2 and the control group were more than treatment 3; there was no significant difference in the number of leaves treated with growth-promoting agents in the seedling stage, and the number of leaves from the jointing stage to the filling stage was the highest in treatment 2. Therefore, the addition of 2kg / mu of the composite microbial agent has a significant growth-promoting effect on corn.

[0105] Table 5 Yield traits and yield of corn

[0106]

[0107] As shown in Table 5, the results of agronomic traits test of corn showed that the yield traits of seed corn treated with composite microbial agents were significantly better than those of the control. Treatment 2 was the best in terms of ear weight, ear length, ear diameter, number of kernels per ear, and thousand-kernel weight; the application of composite microbial agents effectively improved the shape and yield of corn yield, but both lower and higher application rates were not conducive to the improvement of the shape and yield of corn yield, and the optimal application rate was 2 kg / mu.

[0108] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composite microbial agent of indigenous microbial origin, characterized in that: Includes any of the following: (1) The composite microbial agent comprises Bacillus amyloliquefaciens, Paenibacillus speoviae and Streptomyces fradiae; (2) The composite microbial agent comprises Bacillus amyloliquefaciens, Paenibacillus pierrei, Streptomyces freundii and auxiliary materials; The deposit number of the Bacillus amyloliquefaciens is CGMCC No. 25581, the deposit number of the Paenibacillus pierrei is CGMCC 1.12148, and the deposit number of the Streptomyces freundii is CGMCC 4.7387.

2. The composite microbial agent according to claim 1, characterized in that: The mass ratio of the Bacillus amyloliquefaciens, the Paenibacillus pierrei and the Streptomyces freundii is (1-2): (1-2): (1-2).

3. The composite microbial agent according to claim 1, characterized in that The auxiliary materials include defatted bran powder, glycerol, zeolite powder and mineral potassium humate.

4. A method for preparing the composite microbial agent according to any one of claims 1 to 3, characterized in that: This includes any of the following methods: (1) activating the Bacillus amyloliquefaciens, the Paenibacillus pierreiu and the Streptomyces freundii respectively, and obtaining the Bacillus amyloliquefaciens agent, the Paenibacillus pierreiu agent and the Streptomyces freundii agent through expanded culture; (2) The Bacillus amyloliquefaciens, the Paenibacillus pierrei, and the Streptomyces freundii are activated respectively, and after expanded culture, a fermentation broth of Bacillus amyloliquefaciens, a fermentation broth of Paenibacillus pierrei, and a fermentation broth of Streptomyces freundii are obtained respectively, and auxiliary materials are added to each of them to prepare powders, thereby obtaining a Bacillus amyloliquefaciens inoculum, a Paenibacillus pierrei inoculum, and a Streptomyces freundii inoculum.

5. The preparation method according to claim 4, characterized in that: The auxiliary materials are defatted bran powder, glycerol, zeolite powder and mineral-derived potassium humate; based on the total mass of Bacillus amyloliquefaciens fermentation broth, Paenibacillus pierrei fermentation broth or the Streptomyces freundii fermentation broth, the mass percentages of the defatted bran powder, glycerol, zeolite powder and mineral-derived potassium humate added are 50%, 2%, 25% and 1% respectively.

6. Use of the composite microbial agent according to any one of claims 1 to 3 in any of the following: (1) Application in the preparation of growth-promoting agents for promoting crop growth; (2) Application in increasing crop yields; (3) Application in the prevention and treatment of pathogens or diseases caused by pathogens; The crops include highland summer vegetables and corn; The pathogenic bacteria are Fusarium oxysporum, Fusarium solani or Alternaria tenuissima.

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