CN106167771B - A kind of Bacillus megaterium D122 and its inoculum and preparation method of inoculum - Google Patents

Abstract

The invention relates to the technical field of microbial inoculants, in particular to a Bacillus megaterium D122, an inoculum and a preparation method for the inoculum. The preparation method of the bacterial agent includes the following steps: (1) separation and screening; (2) purification and preservation; (3) culture medium; (4) preparation of the D122 bacterial agent to obtain the Bacillus megaterium D122 bacterial agent. The Bacillus megaterium D122 has nitrogenase activity and the function of secreting auxin indoleacetic acid.

Description

A kind of Bacillus megaterium D122 and its inoculum and preparation method of inoculum

technical field

The invention relates to the technical field of microbial inoculants, in particular to a Bacillus megaterium D122, inoculum and a preparation method thereof.

Background technique

Agricultural production has long relied on chemical fertilizers, which not only wastes a lot of non-renewable resources, but also leads to problems such as poor soil quality, degradation of agricultural product quality and environmental pollution, which greatly affects the healthy survival of human beings.

Bacillus megaterium ( Bacillus megaterium ) is a kind of bacteria that dissolves phosphorus and promotes potassium and nitrogen fixation. It can well degrade phosphorus and potassium in the soil that cannot be used by plants, improve soil fertility, and at the same time fix nitrogen in the air to promote crop yield. However, the products produced by my country's microbial fertilizer industry show many problems, such as low number of effective viable bacteria, high rate of miscellaneous bacteria, and short validity period, which seriously affect farmers' enthusiasm for use and market development. Domestic research on Bacillus megaterium on microbial fertilizers mainly focuses on strain selection, phosphorus solubilizing effect, optimization of fermentation conditions and interaction with potassium solubilizing bacteria, etc., while the research on Bacillus megaterium inoculant protective agent is rare. .

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of Bacillus megaterium D122, which has nitrogenase activity and the function of secreting auxin indoleacetic acid in view of the above-mentioned deficiencies in the prior art.

Another object of the present invention is to provide a Bacillus megaterium D122 bacterial agent for the above-mentioned deficiencies in the prior art, which has high nitrogenase activity and can secrete auxin, has a wide range of agricultural applications and high economic benefits.

Another object of the present invention is to provide a method for preparing a Bacillus megaterium D122 inoculum in view of the above-mentioned deficiencies in the prior art, which is simple and mature in technology and can be produced on a large scale.

The object of the present invention is achieved through the following technical solutions.

A Bacillus megaterium D122, the Bacillus megaterium D122 is preserved in the China Center for Type Collection (China Center for Type Collection), the address is Wuhan University, China, and the classification name is Bacillus megaterium D122, preserved The date is December 15, 2015, and the deposit number is CCTCC NO: M2015751.

The Bacillus megaterium D122 has the DNA sequence of Sequence Listing No. 1.

The nitrogenase activity of the Bacillus megaterium D122 is 500-600 nmol/(mL·h), it can produce indoleacetic acid in the process of growth and metabolism, and the secretion amount of indoleacetic acid is 100-200 mg/L.

nitrogenase activity assay

1. Test method

The nitrogenase activity of each strain was determined by acetylene reduction method. The preserved strains were activated with VM-Ethanol solid medium, and one loop of bacterial cells was picked with an inoculation loop into a 1.5 mL sterile centrifuge tube, diluted with sterile water, and inserted into a 5 mL half-filled tube according to the same inoculum amount. In a 10 mL test tube of solid medium, seal it with a reverse rubber stopper. After culturing at 37°C for 24 hours, inject 1/10 volume of 10% acetylene gas, continue to culture for 24 hours, extract 0.5 mL of gas from the test tube and inject it into a gas chromatograph (Beijing Tianpu Analytical Instrument Factory SP-2100) to measure acetylene, Ethylene content. Calculate nitrogenase activity according to the following formula (Beijing Agricultural University Microbiology Specialty, 1986):

C =( h _x × c × V )/(24.9 × h _s × t ) (2.1)

Wherein, h _x is the sample peak area value; h _s is the standard C ₂ H ₄ peak area value; c is the standard C ₂ H ₄ concentration (nmol/mL);

V is the culture vessel volume (mL); t is the sample incubation time (h); C is the generated C ₂ H ₄ concentration [nmol/(mL·h)].

2. Test results

Combining formula (2.1) and nitrogenase activity determination in Figure 4, we know that nitrogenase activity is 500-600 nmol/(mL·h). It can be shown that Bacillus megaterium D122 can produce specific nitrogenase in the metabolic process, which can autogenously fix nitrogen in the atmosphere.

Auxin qualitative assay

(1) Pick the strains and inoculate them respectively ( OD ₆₀₀ =1.0, 0.5mL bacterial suspension) into 250mL conical flasks containing 50mL King liquid medium, 3 replicates per group.

(2) After the inoculation is completed, place the triangular flask on a shaker, 28°C, 125rpm, and cultivate for 3 days, to be tested.

(3) Put 50 µL of the above bacterial suspension grown on King liquid medium for 3 days into a transparent centrifuge tube, and add 50 µL of colorimetric solution at the same time.

(4) Set a positive control and a negative control, add 50 µL of plant growth hormone (IAA) at a concentration of 10 mg/L to the positive control, and add 50 µL of colorimetric solution at the same time. Add 50µL of King liquid medium and 50µL of colorimetric solution to the negative control.

(5) Place the positive control solution, negative control solution and assay solution on a white ceramic plate and place it at room temperature for 15 minutes, observe the color change, if the color turns red, it is positive, indicating that IAA can be secreted, and the darker the color, the more secreted The stronger the ability of IAA; if it does not change color, it is negative, indicating that the strain cannot secrete IAA, which is used as a judgment basis for judgment analysis.

The medium and reagent formulations are: King medium (1 L): peptone 20 g, K ₂ HPO ₄ 1.725 g, MgSO ₄ ·7H ₂ O 1.5 g, glycerol 15 mL, tryptophan 0.1 g, and distilled water 1000 mL.

Quantitative determination of growth hormone

Referring to the method of Riberio et al. with slight modifications. Strain D122 was inoculated into LB liquid medium containing 1 g/L tryptophan at 30 °C, 180 r/min, and shaken for 48 h. Centrifuge the culture medium at 10,000 r/min for 5 min, add 100 mL of supernatant to a 96-well plate, and mix with 100 mL of Salkowski's reagent (1 mL of 0.5 mol/L FeCl ₃ and 49 mL of 35% perchloric acid) at room temperature. After standing for 30 min, the absorbance was measured with a microplate reader at a wavelength of 530 nm. A standard curve was prepared with different concentrations of IAA standard substances, and the determination results are shown in Table 1.

Table 1 Determination results of strain D122 IAA strain D141 D52 D87 D122 IAA concentration (mg/L) 485.68 364.20 294.16 158.36

It can be seen from Table 1 and Figure 5 that the auxin concentration of Bacillus megaterium D122 is 158.36 mg/L. Therefore, it can be judged that Bacillus megaterium D122 can produce IAA in the process of growth and metabolism, and has the function of promoting crop growth.

A method for preparing a bacterial agent of Bacillus megaterium D122, comprising the following steps:

(1) Separation and screening

The soil sample containing the nitrogen-fixing bacteria colony of Bacillus megaterium D122 was selected, and the nitrogen-fixing bacteria colony was obtained by separating and screening the culture medium;

(2) Purification and preservation

The nitrogen-fixing bacteria colonies obtained by separation and screening were streaked and purified on the purification and preservation medium, and a single colony of Bacillus megaterium D122 was obtained by culturing and separating, and the single colony was stored for use; the single colony of Bacillus megaterium D122 was shown in Figure 1.

Specifically, streak and purify the colonies obtained by separation and screening on the purification and preservation medium, culture at a constant temperature of 30° C. for 36-48 hours to isolate a single colony of Bacillus megaterium D122, and store the single colony that appears on the plate in a test tube, Incubate at a constant temperature of 30°C for 36-48 hours, and store in a refrigerator at 2-5°C, preferably in a refrigerator at 4°C.

(3) Culture medium

The activated Bacillus megaterium D122 slant is inoculated in a sterilized liquid medium, and shake cultured to obtain a microbial liquid fermentation broth;

(4) Preparation of D122 inoculum

The mixed solution of sodium chloride, sodium acetate and water is used as a liquid protective agent, added to the cultured liquid fermentation broth and mixed, and placed for 24h to determine the initial bacterial count of 200-400 million/mL to obtain Bacillus megaterium D122 Liquid bacteria.

Screening experiment of liquid protective agent

On the basis of experiments, sucrose, sodium chloride, molasses and sodium acetate were selected as the screening objects of liquid protective agents. Add it to the cultured liquid fermentation broth in a certain proportion, mix well, adjust the water, and place it for 24h to measure the initial bacterial count. It was placed in a cool place for 30 days, and the number of viable bacteria was determined again, and the treatment without protective agent was used as the control (CK). Seven treatments were set, and each treatment was repeated three times, see Table 2.

Table 2 Screening of protective agents (unit: volume ratio of fermentation broth/%) sucrose Sodium chloride molasses Sodium acetate Process 1 10 7 Process 2 10 3 Process 3 10 1.5 Process 4 7 3 Process 5 7 1.5 Process 6 3 1.5 CK 0 0 0 0

The effect of adding protective agent on the effective viable count and shelf life of microbial inoculum: add the selected protective agent to the prepared inoculum D122 in a certain proportion, and store it in a sealed room at room temperature. Place for 24h to measure the initial bacterial count, measure the viable bacterial count every 1-2 months, and take the treatment without protective agent as the control (CK).

Screening results of inoculum protective agent: According to the effect of adding different protective agents on the effective viable count and shelf life of microbial inoculum, the optimal formula of inoculant protective agent was determined to be 7% sodium chloride + 1.5% sodium acetate.

In the step (4), the mass concentration of sodium chloride is 6-8%, and the mass concentration of sodium acetate is 1-2%.

The specific method for separation and screening in the step (1) is as follows: selecting soil samples, adding sterile water containing Tween 80, shaking, standing, taking the supernatant and centrifuging, discarding the supernatant, retaining the sediment, adding The sterile water of Tween 80 is suspended, centrifuged, and the precipitation is removed, the supernatant is centrifuged, the supernatant is discarded, the precipitate is retained, and the precipitate is suspended with a phosphate buffer to obtain a sample solution; take the above-mentioned sample solution and phosphate buffer Suspended and mixed to obtain a diluted bacterial suspension; the diluted bacterial suspension was heated in a water bath, and after natural cooling, it was sucked and coated on a nitrogen-free medium, and cultured to obtain nitrogen-fixing bacteria colonies.

More specifically, the methods of separation and screening are:

Take a 500 g soil sample from Huangnitang Farm, Changping Town, Dongguan City, Guangdong Province, add 3 L of sterile water containing 0.01% Tween 80, shake it for 10 min, let it stand for half an hour, take the supernatant and centrifuge it at 4820 rpm for 20 min at 20°C . Discard the supernatant, keep the precipitate, add 30-50 mL of sterile water containing 0.01% Tween 80 to suspend, and centrifuge the liquid at 5000 rpm for 5s at 20°C to remove the precipitate, and pour the supernatant into a dry sterile centrifuge. Centrifuge the tube at 6000 rpm for 10 min at 20 °C, discard the supernatant, keep the precipitate, and suspend the precipitate with 10 mL of pH 7.0 phosphate buffer to obtain a sample solution; take 1 mL of the above sample solution and 9 mL of phosphate buffer Suspended and mixed, that is, 10-fold diluted bacterial suspension. The diluted bacterial suspension was placed in a 75°C water bath and heated for 15 min. After natural cooling, 100 μL was drawn and spread on a nitrogen-free medium, and cultured at 30°C for 36-48 hours to obtain nitrogen-fixing bacteria colonies containing Bacillus megaterium D122.

In the step (1), the separation and screening medium is made from the following raw materials: CaCO ₃ 1.0-1.4 g, MgSO ₄ ·7H ₂ O 0.6-1.2 g, K ₂ HPO ₄ 1.0-2.0 g, NaCl 0.1- 0.4g, _{FeSO4 · 7H2O} 0.001-0.005g, _NaMO4 ·2H2O 0.05-0.1g, sucrose 5-10g, agar 18-20g, distilled water 1000ml, the pH of the separation and screening medium is 7.1- 7.4. The separation and screening medium of the present invention belongs to an improved nitrogen-free medium.

The specific method for purification and preservation is as follows: streaking and purifying the colonies obtained by separation and screening on a purification and preservation medium, and culturing at a constant temperature of 30° C. for 36-48 hours to isolate a single colony of Bacillus subtilis D122. The single colonies that appeared on the plate were stored in test tubes, incubated at a constant temperature of 30°C for 36-48 hours, and stored in a refrigerator at 4°C. Bacillus megaterium D122 was deposited in the China Center for Type Culture Collection with the deposit number: CCTCCM 2015751.

(2) The formula of the purified preservation medium is: beef extract 3.0 g, peptone 10.0 g, sodium chloride 5.0 g, agar 18.0 g, distilled water 1000 ml, and the pH of the purified preservation medium is 7.0-7.4.

In the step (3) medium cultivation, the medium is made of the following raw materials: CaCO ₃ 1.0-1.4 g, MgSO ₄ ·7H ₂ O 0.6-1.2 g, K ₂ HPO ₄ 1.0-2.0 g, NaCl 0.1 -0.4g, FeSO ₄ ·7H ₂ O 0.001-0.005g, NaMO ₄ ·2H ₂ O 0.05-0.1g, sucrose 5-10g, agar 18-20g, distilled water 1000ml, the pH of the medium is 7.1-7.4.

The following steps are also included between the steps (2) and (3):

(S1) Gram staining: Gram-stain the purified single colony, and screen to obtain positive bacteria;

(S2) Spore staining: the positive bacteria are stained with spores, and single colonies of Gram-positive bacteria containing spores are obtained by screening.

Spore staining and its Gram stain

Gram staining and spore staining are two routine methods for bacterial identification, and staining can narrow the identification. Unstained bacteria have very little difference in refractive index with the surrounding environment and are extremely difficult to observe under a microscope. After Gram staining, the bacteria are in sharp contrast with the environment, and the morphology, arrangement and some species of bacteria can be clearly observed as Gram-positive (G ⁺ ) or Gram-negative bacteria (G ^- ) for classification identification. Gram-negative bacteria generally have safety hazards, and structural characteristics are discarded after direct high-temperature sterilization in the application process of agricultural microorganisms. Spore staining dyes the spores in the bacteria, which can visually observe the size, position, shape and other characteristics of the spores, and further narrow the identification range. Bacillus has the characteristics of long shelf life and easy storage, and has a wide range of applications in agricultural microbial products.

1. Gram stain

(1) Smear: In a sterile operating table, take a glass slide and bake it slightly above a flame lamp to remove impurities on the glass slide. Drop a drop of sterile water in the center of the slide, pick a single colony into the water drop, and spread it evenly with a cauterized inoculation loop. Pass the sample slide 3 times back and forth over the fire light to fix the cells.

(2) Initial dyeing: Add 2-5 drops of ammonium oxalate crystal violet dye solution dropwise, dye for 1 min, pour off the dye solution, rinse with running water until no purple color is present.

(3) Mordant dyeing: Rinse off the residual water with newly prepared iodine solution (1.0g iodine, 2.0g potassium iodide, 300.0mL distilled water), then cover the coated surface with iodine solution for 1min, and then wash with water.

(4) Decolorization: After removing the residual water, add 95% alcohol dropwise to decolorize for about 15-20 seconds, then rinse with running water immediately.

(5) Counterstaining: add 1 drop of safranin dyeing solution dropwise, dye for 3-5min, wash with water and blot dry with absorbent paper.

(6) Microscopic examination: place the slides under an optical microscope to observe the staining results.

2. Spore staining

In a sterile bench, take a slide and bake briefly over a flame lamp to remove impurities from the slide. Drop a drop of sterile water in the center of the slide, pick a single colony droplet, and spread it evenly with a cauterized inoculation loop. Pass the sample slide 3 times back and forth over the fire light to fix the cells. Add 1-2 drops of phenolic alkaline fuchsin staining solution dropwise to the area where the cells are coated, and stain for 3 min. The stain solution was rinsed with distilled water, and after air-drying, the slides were observed under a light microscope.

As shown in Figures 2 and 3, it can be seen from the results of Gram staining and spore staining that Bacillus megaterium D122 is a Gram-positive bacterium, rod-shaped, and contains spores.

A microbial inoculum of Bacillus megaterium D122 is prepared by the method for preparing a microbial inoculum of Bacillus megaterium D122.

Beneficial effects of the present invention:

(1) In the present invention, Bacillus megaterium D122 is used as the starting strain, and its nitrogenase activity and auxin secretion ability are measured, and it is found that strain D122 has efficient nitrogenase activity and IAA secretion ability. The nitrogenase activity of the Bacillus megaterium D122 is 500-600 nmol/(mL·h), it can produce indoleacetic acid in the process of growth and metabolism, and the secretion amount of indoleacetic acid is 100-200 mg/L.

(2) In order to improve the spore survival rate of Bacillus megaterium D122 bacterial agent, the present invention adds a specific protective agent to reduce the mortality rate of spores in the process of processing and storage, effectively reduce the cost of the product, and improve the economic benefit, which is beneficial for future microorganisms. Provide guidance on the production of fertilizers.

(3) A certain proportion of protective agent is added to the preparation method of Bacillus megaterium D122 bacterial agent involved in the present invention, so that the survival rate of the bacteria can be maintained at a high level, and the storage stability of the microbial fertilizer product and the shelf life of the product can be effectively prolonged. life.

Description of drawings

The invention will be further described by using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the present invention. For those of ordinary skill in the art, under the premise of no creative work, other Attached.

Figure 1 is a colony diagram of the isolated Bacillus megaterium D122 single colony magnified 10×100 times under a microscope.

Figure 2 is a graph showing the results of Gram staining of Bacillus megaterium D122.

Figure 3 is a graph showing the staining results of Bacillus megaterium D122 spores.

Figure 4 is a graph showing the results of determination of nitrogenase activity of Bacillus megaterium D122.

Figure 5 is a colorimetric effect diagram of Bacillus megaterium D122 IAA.

Detailed ways

The present invention will be further described with reference to the following examples.

Example 1

One kind of Bacillus megaterium D122 in this example, the Bacillus megaterium D122 is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCCM 2015751. The Bacillus megaterium D122 has the DNA sequence of Sequence Listing No. 1.

The nitrogenase activity of the Bacillus megaterium D122 is 500 nmol/(mL·h), it can produce indoleacetic acid in the process of growth and metabolism, and the secretion amount of indoleacetic acid is 100 mg/L.

A method for preparing a bacterial agent of Bacillus megaterium D122, comprising the following steps:

(1) Separation and screening

The soil sample containing the nitrogen-fixing bacteria colony of Bacillus megaterium D122 was selected, and the nitrogen-fixing bacteria colony was obtained by separating and screening the culture medium;

(2) Purification and preservation

The nitrogen-fixing bacteria colonies obtained by separation and screening are purified on the purification preservation medium, and the single colonies of Bacillus megaterium D122 are obtained by culturing and separating, and the single colonies are stored for future use;

(3) Culture medium

The activated Bacillus megaterium D122 slant is inoculated in a sterilized liquid medium, and shake cultured to obtain a microbial liquid fermentation broth;

(4) Preparation of D122 inoculum

The mixed solution of sodium chloride, sodium acetate and water is used as a liquid protective agent, and is added to the cultured liquid fermentation broth according to a certain proportion, mixed, placed, and the initial bacterial count is determined to be 200 million/mL to obtain Bacillus megaterium D122 Bacterial agent.

In the step (4), the mass concentration of sodium chloride is 6%, and the mass concentration of sodium acetate is 1%.

The specific method for separation and screening in the step (1) is as follows: selecting soil samples, adding sterile water containing Tween 80, shaking, standing, taking the supernatant and centrifuging, discarding the supernatant, retaining the sediment, adding The sterile water of Tween 80 is suspended, centrifuged, and the precipitation is removed, the supernatant is centrifuged, the supernatant is discarded, the precipitate is retained, and the precipitate is suspended with a phosphate buffer to obtain a sample solution; take the above-mentioned sample solution and phosphate buffer Suspended and mixed to obtain a diluted bacterial suspension; the diluted bacterial suspension was heated in a water bath, and after natural cooling, it was sucked and coated on a nitrogen-free medium, and cultured to obtain nitrogen-fixing bacteria colonies.

In the step (1), the separation and screening medium is made from the following raw materials: CaCO ₃ 1.0g, MgSO ₄ ·7H ₂ O 0.6g, K ₂ HPO ₄ 1.0g, NaCl 0.1g, FeSO ₄ ·7H ₂ O 0.001g, NaMO ₄ ·2H ₂ O 0.05g, sucrose 5g, agar 18g, distilled water 1000ml, the pH of the separation and screening medium was 7.1.

A microbial inoculum of Bacillus megaterium D122 is prepared by the method for preparing a microbial inoculum of Bacillus megaterium D122.

Example 2

The difference between this embodiment and Embodiment 1 is that the following steps are further included between the steps (2) and (3) of this embodiment:

(S1) Gram staining: Gram-stain the purified single colony, and screen to obtain positive bacteria;

(S2) Spore staining: the positive bacteria are stained with spores, and single colonies of Gram-positive bacteria containing spores are obtained by screening.

Specifically, the Gram staining method is:

(1) Smear: In a sterile operating table, take a glass slide and bake it slightly above a flame lamp to remove impurities on the glass slide. Drop a drop of sterile water in the center of the slide, pick a single colony into the water drop, and spread it evenly with a cauterized inoculation loop. Pass the sample slide 3 times back and forth over the fire light to fix the cells.

(2) Initial dyeing: Add 2-5 drops of ammonium oxalate crystal violet dye solution dropwise, dye for 1 min, pour off the dye solution, rinse with running water until no purple color is present.

(3) Mordant dyeing: Rinse off the residual water with newly prepared iodine solution (1.0g iodine, 2.0g potassium iodide, 300.0mL distilled water), then cover the coated surface with iodine solution for 1min, and then wash with water.

(4) Decolorization: After removing the residual water, add 95% alcohol dropwise to decolorize for about 15-20 seconds, then rinse with running water immediately.

(5) Counterstaining: add 1 drop of safranin dyeing solution dropwise, dye for 3-5min, wash with water and blot dry with absorbent paper.

(6) Microscopic examination: place the slides under an optical microscope to observe the staining results.

Specifically, the spore staining method is:

In a sterile bench, take a slide and bake briefly over a flame lamp to remove impurities from the slide. Drop a drop of sterile water in the center of the slide, pick a single colony droplet, and spread it evenly with a cauterized inoculation loop. Pass the sample slide 3 times back and forth over the fire light to fix the cells. Add 1-2 drops of phenolic alkaline fuchsin staining solution dropwise to the area where the cells are coated, and stain for 3 min.

The rest of the content of this embodiment is the same as that of Embodiment 1, and will not be repeated here.

Example 3

The difference between this example and Example 1 or 2 is that the nitrogenase activity of the Bacillus megaterium D122 in this example is 520 nmol/(mL·h), which can produce indoleacetic acid in the process of growth and metabolism , the secretion of indoleacetic acid was 120 mg/L.

For the Bacillus megaterium D122 bacterial preparation, the initial bacterial count was determined to be 250 million/mL.

In the step (4), the mass concentration of sodium chloride is 6.5%, and the mass concentration of sodium acetate is 1.5%.

In the step (1), the separation and screening medium is made from the following raw materials: CaCO ₃ 1.2g, MgSO ₄ ·7H ₂ O 0.8g, K ₂ HPO ₄ 1.5g, NaCl 0.2g, FeSO ₄ ·7H ₂ O 0.002g, NaMO ₄ ·2H ₂ O 0.06g, sucrose 6g, agar 18g, distilled water 1000ml, the pH of the separation and screening medium was 7.2.

The rest of the content of this embodiment is the same as that of Embodiment 1 or 2, and will not be repeated here.

Example 4

The difference between this example and Example 1 or 2 is that the nitrogenase activity of the Bacillus megaterium D122 in this example is 550 nmol/(mL·h), which can produce indoleacetic acid in the process of growth and metabolism , the secretion of indoleacetic acid was 150 mg/L.

For the Bacillus megaterium D122 bacterial preparation, the initial bacterial count was determined to be 300 million/mL.

In the step (4), the mass concentration of sodium chloride is 7%, and the mass concentration of sodium acetate is 1.5%.

In the step (1), the separation and screening medium is made from the following raw materials: CaCO ₃ 1.3 g, MgSO ₄ ·7H ₂ O 1.0 g, K ₂ HPO ₄ 1.8 g, NaCl 0.3 g, FeSO ₄ ·7H ₂ O 0.004g, NaMO ₄ ·2H ₂ O 0.09g, sucrose 8g, agar 19g, distilled water 1000ml, the pH of the separation and screening medium was 7.3.

The rest of the content of this embodiment is the same as that of Embodiment 1 or 2, and will not be repeated here.

Example 5

The difference between this example and Example 1 or 2 is that the nitrogenase activity of the Bacillus megaterium D122 in this example is 580 nmol/(mL·h), which can produce indoleacetic acid in the process of growth and metabolism , the secretion of indoleacetic acid was 180 mg/L.

For the Bacillus megaterium D122 bacterial preparation, the initial bacterial count was determined to be 400 million/mL.

In the step (4), the mass concentration of sodium chloride is 7.5%, and the mass concentration of sodium acetate is 1.5%.

In the step (1), the separation and screening medium is made of the following raw materials: CaCO ₃ 1.4g, MgSO ₄ ·7H ₂ O 1.2g, K ₂ HPO ₄ 2.0g, NaCl 0.4g, FeSO ₄ ·7H ₂ O 0.005g, NaMO ₄ ·2H ₂ O 0.1g, sucrose 10g, agar 20g, distilled water 1000ml, and the pH of the separation and screening medium was 7.4.

The rest of the content of this embodiment is the same as that of Embodiment 1 or 2, and will not be repeated here.

Species identification of the 16S rDNA sequence of Bacillus megaterium D122 of the present invention

Bacteria are small in size and simple in shape. Traditional methods of identifying bacteria are often based on their different physiological and biochemical responses as the main basis for classification and identification. Since the late 1970s, the internationally accepted "official" or "official" classification of bacteria has been based on the Bergey's Manual of Identification Bacteriology. In physiological and biochemical identification, one or several physiological indicators usually do not conform to the unique properties of the strain, and it is difficult to identify the strain clearly. At present, the method of bacterial identification usually combines the physiological and biochemical indicators of the strain with the molecular biological characteristics, and draws relatively reliable conclusions. Among them, the 16S rRNA gene evolution and development system based on DNA sequence analysis has become a commonly used technical means for the identification of bacterial heterogeneity in the world (Kim et al, 2004; Prap et al, 1997).

The ribosomal 16S rDNA gene sequence is about 1550 bp in length and consists of alternating conserved and variable regions. All bacterial 16S rDNA fragments can be amplified using universal primers designed in conserved regions. The basic principle of 16S rDNA sequence analysis technology is to extract 16S rDNA fragments from microbial samples, obtain the sequence information of 16S rDNA through cloning, sequencing or enzyme digestion, and probe hybridization, and then compare with the sequence data of 16S rDNA database or other data, Determine its position in the evolutionary tree to identify possible microbial species present in the sample. Universal primers designed using the conserved regions of 16S rDNA fragments will not complement non-bacterial DNA, while differences in bacterial 16S rDNA variable regions can be used to distinguish different bacteria. Therefore, it is generally accepted that the final identification certificate is obtained by sequencing the 16S rDNA of a certain strain.

1. Method:

(1) PCR reaction system (25 μL):

10×PCR Buffer 2.5μL

dNTP (2.5mM) m 2.0μL

Primer 27F (10μM) 0.5μL

Primer 1492R (10μM) 0.5μL

DNA template 100ng

Taq enzyme (5U/μL) 0.5μL

ddH ₂ O 19 μL

(2) PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 80 s, 35 cycles, and extension at 72°C for 10 min. DNA sequencing was performed using an ABI 3730 xl DNA Analyzer (Applied Biosystems).

2. Sequencing results

tcgagcgaac tgattagaag cttgcttcta tgacgttagc ggcggacggg tgagtaacac

gtgggcaacc tgcctgtaag actgggataa cttcgggaaa ccgaagctaa taccggatag

gatcttctcc ttcatgggag atgattgaaa gatggtttcg gctatcactt acagatgggc

ccgcggtgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat gcatagccga

cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca

gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg

aaggctttcg ggtcgtaaaa ctctgttgtt agggaagaac aagtacgaga gtaactgctc

gtaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa

tacgtaggtg gcaagcgtta tccggaatta ttgggcgtaa agcgcgcgca ggcggtttct

taagtctgat gtgaaagccc acggctcaac cgtggagggt cattggaaac tggggaactt

gagtgcagaa gagaaaagcg gaattccacg tgtagcggtg aaatgcgtag agatgtggag

gaacaccagt ggcgaaggcg gctttttggt ctgtaactga cgctgaggcg cgaaagcgtg

gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt

tagagggttt ccgcccttta gtgctgcagc taacgcatta agcactccgc ctggggagta

cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt

ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct gacaactcta

gagatagagc gttccccttc gggggacaga gtgacaggtg gtgcatggtt gtcgtcagct

cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca

gcatttagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga

cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa

agggctgcaa gaccgcgagg tcaagccaat cccataaaac cattctcagt tcggattgta

ggctgcaact cgcctacatg aagctggaat cgctagtaat cgcggatcag catgccgcgg

tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc

gaagtcggtg gagtaaccgt aag

3. Homology analysis

The bacteria were identified as Bacillus megaterium , Bacillus megaterium.

The invention is researched and developed by the project of an innovative and entrepreneurial team introduced by Guangdong Province, and the prepared Bacillus megaterium D122 and its inoculum have broad market prospects and high economic benefits.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that , the technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

<0001>

SEQUENCE LISTING

<110> Dongguan Baode Biological Engineering Co., Ltd.

<120> A kind of Bacillus megaterium D122 and its inoculum and preparation method of inoculum

<130>0

<160>1

<170>PatentIn version 3.3

<210>1

<211>1403

<212> DNA

<213> 16s rDNA gene sequence of Bacillus megaterium D122

<400>1

tcgagcgaac tgattagaag cttgcttcta tgacgttagc ggcggacggg tgagtaacac 60

gtgggcaacc tgcctgtaag actgggataa cttcgggaaa ccgaagctaa taccggatag 120

gatcttctcc ttcatgggag atgattgaaa gatggtttcg gctatcactt acagatgggc 180

ccgcggtgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat gcatagccga 240

cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300

gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360

aaggctttcg ggtcgtaaaa ctctgttgtt agggaagaac aagtacgaga gtaactgctc 420

gtaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa 480

tacgtaggtg gcaagcgtta tccggaatta ttgggcgtaa agcgcgcgca ggcggtttct 540

taagtctgat gtgaaagccc acggctcaac cgtggagggt cattggaaac tggggaactt 600

gagtgcagaa gagaaaagcg gaattccacg tgtagcggtg aaatgcgtag agatgtggag 660

gaacaccagt ggcgaaggcg gctttttggt ctgtaactga cgctgaggcg cgaaagcgtg 720

gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt 780

tagagggttt ccgcccttta gtgctgcagc taacgcatta agcactccgc ctggggagta 840

cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt 900

ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct gacaactcta 960

gagatagagc gttccccttc gggggacaga gtgacaggtg gtgcatggtt gtcgtcagct 1020

cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca 1080

gcatttagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1140

cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa 1200

agggctgcaa gaccgcgagg tcaagccaat cccataaaac cattctcagt tcggattgta 1260

ggctgcaact cgcctacatg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1320

tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1380

gaagtcggtg gagtaaccgt aag 1403

Claims (3)

1. A Bacillus megaterium D122, wherein: the bacillus megaterium D122 is preserved in China center for type culture Collection with the preservation number of CCTCCM 2015751.

2. The bacillus megaterium D122 according to claim 1, wherein: the bacillus megaterium D122 has a DNA sequence of a sequence table NO. 1.

3. The bacillus megaterium D122 according to claim 1, wherein: the azotase activity of the bacillus megaterium D122 is 500-600 nmol/(mL-h), the indole acetic acid can be produced in the growth and metabolism process, and the secretion amount of the indole acetic acid is 100-200 mg/L.

Images