Bacillus thuringiensis as well as application and preparation thereof

Abstract

The invention provides bacillus thuringiensis MPEB0014827, which is preserved in the Guangdong Microbial Culture Collection Center (GDMCC), the preservation address is the fifth floor of the building 59, No. 100 courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 65170. The invention further provides a preparation. The preparation comprises the bacillus thuringiensis. The invention also provides an application of the bacillus thuringiensis in preparation of a fertilizer. The bacillus thuringiensis MPEB0014827 provided by the invention can be used for preparing a fertilizer.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and more specifically, to Bacillus thuringiensis and its applications and preparations. Background Art

[0002] Plant growth-promoting rhizobacteria (PGPR) are a class of beneficial bacteria colonizing the rhizosphere of plants, which mainly promote plant growth by biological nitrogen fixation, dissolving mineral elements, producing growth hormones, producing siderophores, antagonizing pathogenic bacteria causing plant diseases, etc. Nitrogen, phosphorus, and potassium are the three elements with the largest demand for plant growth, and their roles in plant photosynthesis and energy metabolism synthesis are indispensable. Nitrogen source is a limiting nutrient for plant growth. Plants need a large amount of nitrogen fertilizer for growth and development, while there is a large amount of nitrogen resources in the atmosphere that are difficult to utilize. Autotrophic nitrogen-fixing bacteria in the plant rhizosphere can provide nitrogen source for plants.

[0003] Microbial fertilizer refers to a microbial product containing beneficial microorganisms, and the crop obtains fertilizer effects through the life activities of specific microorganisms. Microbial fertilizer can optimize the soil microbial community structure and make the soil change towards a direction more beneficial to plant growth. The biological fertilizer developed by using excellent growth-promoting bacteria isolated and screened from different environments and the rhizosphere of different plant communities not only has fertilizer effects, but also can reduce environmental and food pollution and non-renewable energy consumption caused by the use of pesticides and fertilizers in agricultural production. With the continuous increase of environmental pressure, the application of PGPR agents will become an important research direction for sustainable agricultural development. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides a Bacillus thuringiensis MPEB0014827, which has strong nitrogen fixation and siderophore production capabilities, has the capabilities of phosphorus solubilization, potassium solubilization and IAA production, and has great application potential in promoting plant growth.

[0005] The present invention provides a Bacillus thuringiensis MPEB0014827, which is deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC), the deposit address: 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the deposit number is: GDMCC No: 65170.

[0006] The present invention also provides a preparation, including the above-mentioned Bacillus thuringiensis.

[0007] The present invention also provides the application of the above-mentioned Bacillus thuringiensis in the preparation of fertilizers.

[0008] The Bacillus thuringiensis MPEB0014827 provided by the present invention is a plant growth-promoting rhizobacterium that can fix nitrogen, dissolve phosphorus, dissolve potassium, secrete IAA, and produce siderophores, providing the nutrients and growth-promoting substances required by plants for a long time, thereby reducing the harm of chemical fertilizers to the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Figure showing the growth state of Bacillus thuringiensis MPEB0014827 of the present application in an MHA solid plate.

[0010] Figure 2 Figure showing the Gram staining of Bacillus thuringiensis MPEB0014827 of the present application.

[0011] Figure 3 It is the standard working curve of ammonium nitrogen.

[0012] Figure 4 Figure showing the results of the color reaction of Bacillus thuringiensis MPEB0014827 of the present application.

[0013] Figure 5 Figure showing the amount of ammonium nitrogen secreted by Bacillus thuringiensis MPEB0014827 of the present application.

[0014] Figure 6 Figure showing the phosphorus-dissolving ability of Bacillus thuringiensis MPEB0014827 of the present application.

[0015] Figure 7 It is the standard working curve of the phosphorus standard solution.

[0016] Figure 8 Figure showing the potassium-dissolving ability of Bacillus thuringiensis MPEB0014827 of the present application.

[0017] Figure 9 It is the standard working curve of the IAA standard solution.

[0018] Figure 10 Figure showing the siderophore-producing ability of Bacillus thuringiensis MPEB0014827 of the present application.

[0019] The Bacillus thuringiensis MPEB0014827 provided by the present invention has the taxonomic name of Bacillus thuringiensis and was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on September 20, 2024, with the deposit number: GDMCC No: 65170; the deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION OF THE INVENTION

[0020] The following embodiments can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0021] The Bacillus thuringiensis of the present application is from the surface soil of Lianhuage, Motuo Town, Motuo County, Tibet. Isolation and purification steps: Collect soil samples, make a series of diluted bacterial solutions with sterile water, coat the bacterial solutions of each gradient on MHA solid medium (beef powder 6 g / L, soluble starch 1.5 g / L, acid hydrolyzed casein 17.5 g / L, agar 17 g / L, pH 7.3, sterilized at 121 °C for 30 min), and place them in an incubator at 28 °C for cultivation; repeatedly pick single colonies on different dilution concentrations for streak plate cultivation, and subculture them 4 - 5 generations respectively to obtain pure and morphologically consistent single colonies. After isolation and purification, a strain named MPEB0014827 was obtained.

[0022] Phenotypic identification: Cultivate the strain MPEB0014827 on an MHA solid plate at 30 °C for 24 h. The strain grows well on the MHA medium, and the colony is round and light yellow ( Figure 1 ), perform Gram staining on the strain, the strain is rod-shaped and shows as a Gram-positive bacterium ( Figure 2 ).

[0023] Molecular biology identification: Perform molecular biology - 16S rDNA sequencing on the strain MPEB0014827. The sequencing results are compared according to the sequence homology of Gene Bank and phylogenetic analysis is carried out. The strain H38 is in the same branch as Bacillus thuringiensis, and its homology is over 99%. The strain MPEB0014827 was identified as Bacillus thuringiensis, and this strain was deposited for biological preservation.

[0024] Determination of the nitrogen fixation ability of the strain

[0025] Qualitative determination: After activating the strain, inoculate it into Ashby nitrogen-free medium (C6H 14 O6 10 g, K2HPO4 0.2 g, NaCl 0.2 g, MgSO4·7H2O 0.2 g, CaCO3 5 g, CaSO4 0.1 g, agar 15 g, distilled water 1 L, pH 7.0), and cultivate it at 30 °C for 5 days. If the strain can grow on the above medium, it has nitrogen fixation ability.

[0026] Quantitative determination: The common indophenol blue - spectrophotometry is used for the quantitative determination of nitrogen fixation ability.

[0027] Preparation of ammonium nitrogen standard solution: Accurately weigh 0.2358 g of (NH4)2SO4 and dissolve it in 100 ml of water to obtain an ammonium nitrogen stock solution with an ammonium nitrogen content of 500 μg / ml. Then dilute it 10 times to obtain an ammonium nitrogen standard solution with a content of 50 μg / ml.

[0028] 1.25% sodium nitroferricyanide solution: Accurately weigh 0.3622 g of Na2Fe(CN)5NO·2H2O, add water to make up the volume to 25 ml, and store it at 4°C. The reagents used are domestic analytical pure.

[0029] Solution A: Take 400 μL of sodium nitroferricyanide solution and add it to 20 mL of 5% phenol, mix and make up the volume to 100 mL. Store it in the dark at 4°C to avoid turning red-brown when exposed to light.

[0030] Solution B: Accurately weigh 2 g of C6H5Na3O7, 2.50 g of NaOH, and 3.5 ml of NaClO, mix and dissolve them in 500 mL of water, and store it in the dark at 4°C.

[0031] Preparation of ammonium nitrogen standard curve: Take 6 test tubes, number them in sequence, add the corresponding reagents according to Table 1, vortex and mix evenly, place them in a constant temperature water bath at 37°C for color reaction for 20 min, take them out and cool to room temperature, measure their OD values at a wavelength of 637 nm, observe and record the data, and draw the ammonium nitrogen standard curve (see Figure 3 ).

[0032] Table 1 Preparation of ammonium nitrogen standard curve

[0033]

[0034] Inoculate the strain into Ashby liquid medium at an inoculation amount of 1% (v / v). At the same time, set up a control group without inoculation, with three replicates in each group. After culturing in a shaker at 28°C and 180 r / min for 24 hours, take 100 μL of the supernatant, collect it in a sterile EP tube, add 5 mL of Solution A, then add 5 mL of Solution B, vortex and mix evenly, place it in a constant temperature water bath at 37°C for color reaction for 20 min. The darker the color, the stronger the nitrogen fixation ability ( Figure 4 ), take it out and cool to room temperature, then measure its OD637 value. Substitute the value into the ammonium nitrogen standard curve to obtain the ammonium nitrogen content secreted by the strain as 48.40 μg / mL ( Figure 5 ).

[0035] Determination of phosphate-solubilizing ability of the strain

[0036] Inorganic phosphorus medium formula: Glucose 10.0 g, magnesium chloride hexahydrate 5.0 g, magnesium sulfate heptahydrate 0.25 g, potassium chloride 0.2 g, ammonium sulfate 0.1 g, tricalcium phosphate 5.0 g, agar 15 g, distilled water 1 L, pH 6.8 - 7.0.

[0037] Qualitative determination: Take 10 μL of the strain and spot inoculate it on the inorganic phosphorus solid medium. Incubate at 28 °C for 7 d, with 3 replicates set. If a transparent circle is formed by the strain on the inorganic phosphorus medium, it indicates that the strain has the ability to dissolve phosphorus ( Figure 6 showing the formation of a transparent circle).

[0038] Drawing of the phosphorus standard working curve: Take 8 25-mL stoppered graduated tubes and add 0.0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 12.0 mL of phosphate standard solution respectively. Add ultrapure water to 12.5 mL and then add 1.5 mL of molybdenum antimony anti-color reagent to obtain phosphorus standard series solutions with concentrations of 0, 0.08, 0.16, 0.32, 0.64, 0.96, 1.28, 1.92 mg / L. Using water as the reference, perform colorimetry at 700 nm with a spectrophotometer and draw the corresponding working curve of phosphorus content ( Figure 7 ).

[0039] Quantitative detection: The molybdenum antimony anti-colorimetric method commonly used was adopted to quantitatively detect the phosphorus-dissolving ability of the strain. Inoculate the strain into MH(B) liquid medium and culture it on a shaker at 37 °C and 170 rpm until its OD600 = 0.7. Inoculate it into NBRIP liquid medium at an inoculation amount of 1%, and at the same time inoculate an equal amount of MH(B) liquid medium as a blank control. Set three replicates for each treatment and culture it on a shaker at 30 °C and 170 rpm for 7 days. Take 6 mL of samples on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days of culture. At the same time, measure the blank control group, and use the molybdenum antimony anti-colorimetric method to measure the soluble phosphorus content. After deducting the absorbance of the blank control group, substitute it into the phosphorus standard working curve, and the maximum phosphorus-dissolving amount of Bacillus thuringiensis MPEB0014827 in NBRIP medium is 173.81 mg·L -1 .

[0040] Determination of the potassium-dissolving ability of the strain

[0041] Formula of the potassium-dissolving medium: Sucrose 10.0 g, disodium hydrogen phosphate 1.0 g, ammonium sulfate 0.5 g, magnesium sulfate 1.0 g, yeast extract 0.2 g, ferrous sulfate 0.03 g, calcium carbonate 2.5 g, potassium feldspar powder 10.0 g, agar powder 15.0 g. Add distilled water to make up to 1000 mL, with pH 7.0 - 7.5.

[0042] Take 10 μL of the strain and spot inoculate it on the potassium-dissolving medium. Incubate at 28 °C for 5 d, with 3 replicates set. The experimental results are as Figure 8 shown, and a transparent circle is formed on the potassium-dissolving medium, indicating that the strain has the potassium-dissolving ability.

[0043] Determination of the IAA-producing ability of the strain

[0044] The IAA standard curve was prepared by gradient dilution method: IAA standard solutions with concentrations of 0, 5, 10, 20, 30, 40, and 50 mg / L were prepared, and the absorbance values were measured at 530 nm and the IAA standard curve was plotted( Figure 9 ).

[0045] The ability of the strain to produce IAA was determined by Salkowski colorimetric method. 1% (v / v) of the strain was added to MHB medium (containing 100 μg·mL -1 L-tryptophan, 17.5 g / L of casein hydrolysate, 5.0 g / L of beef extract powder, 1.5 g / L of starch, pH 7.0 - 7.2, sterilized at 121 °C for 30 min) and cultured (at 28 °C, 180 r·min -1 , for 24 h). The bacterial liquid was centrifuged at 10000 r·min -1 for 10 min at room temperature. 2 mL of the supernatant was taken and placed in a test tube, and an equal volume of Salkowski colorimetric solution was added. Each treatment was repeated 3 times. After standing in the dark at room temperature for 30 min, after color development, the absorbance value was measured at 530 nm. According to the standard curve, the IAA production of the strain was calculated to be 2.3309 mg / L.

[0046] Determination of the ability of the strain to produce siderophores

[0047] KMB medium: 15 mL of glycerol, 5 g of iron-free casein amino acids, 2.5 g of KH2PO4, 2.5 g of MgSO4·7H2O, made up to 1 L with deionized water, pH = 7.4, 18 g / L of agar powder.

[0048] CAS detection medium: 60.5 mg of chrome azurol S, 72.9 mg of cetyltrimethylammonium bromide, 10 mL of 1 mmol / L FeCl3·6H2O, 50 mL of 0.1 mol / L phosphate buffer, 940 mL of distilled water, 0.9% of agar.

[0049] 10 μL of the strain was inoculated onto the KMB iron-free medium plate and cultured for 1 day. After 1 day, obvious single colonies grew on the KMB iron-free medium plate. When the sterilized CAS detection medium cooled to about 40 °C, 10 mL of it was poured onto each KMB iron-free plate. After standing for 1 h, the color change of each plate was observed. The control group was the KMB plate without inoculation. The experimental results are as Figure 10 shown. A large orange halo appeared on the CAS detection medium, indicating that the strain could produce siderophores.

[0050] Therefore, the Bacillus thuringiensis provided in this application has strong nitrogen fixation and siderophore production abilities, has the abilities of phosphorus solubilization, potassium solubilization and IAA production, and has great application potential in promoting plant growth.

[0051] Those skilled in the art should understand that the above embodiments are only exemplary embodiments, and various changes, substitutions, and alterations can be made without departing from the spirit and scope of the present invention.

Claims

1. A Bacillus thuringiensis MPEB0014827, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit number: GDMCC No: 65170.

2. A preparation comprising the Bacillus thuringiensis according to claim 1.

3. Use of the Bacillus thuringiensis according to claim 1 or the preparation according to claim 2 in the preparation of fertilizer.

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