A strain of Bacillus nicotinoic acid BD-5-3 and its application

Abstract

The present invention provides a strain of Bacillus nicotinoic acid BD-5-3 and an application thereof, belonging to the field of biotechnology, and the preservation number of Bacillus nicotinoic acid is: CCTCC NO: M 20232178. The Bacillus nicotinoic acid BD-5-3 provided by the present invention can be used for phosphate solubilization.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a strain of Bacillus nicotinoic acid BD-5-3 and an application thereof. Background Art

[0002] Phosphorus is a component of many important compounds in plants. It can not only promote better photosynthesis, root development and flower bud formation in plants, thereby increasing yields, but also improve plant stress resistance and adaptability. However, due to the unique interaction characteristics between phosphorus and other elements and compounds, and its poor fluidity in the soil, its utilization efficiency is much lower than that of nitrogen and potassium fertilizers. The utilization rate of phosphorus fertilizers in my country is about 10%-25%, and the average utilization rate is 14%. At present, the total phosphorus content in the soil of most tea gardens in my country is high, but the effective phosphorus content available for tea trees to absorb and utilize is very low, and most of it exists in the form of insoluble phosphorus. Organic phosphorus has the effects of promoting root growth, enhancing reproductive organ development, and improving tea quality on tea trees. Therefore, for a long time, many tea gardens have relied on the application of large amounts of organic phosphorus fertilizers to alleviate the problem of soil phosphorus deficiency, but it may affect the absorption of other elements by tea trees and lead to soil quality degradation, acidification, and deterioration of soil physical and chemical properties.

[0003] Phosphorus-solubilizing microorganisms play an important role in activating soil phosphorus and improving the biological effectiveness of phosphorus. The organic phosphorus in tea garden soil mainly comes from the dead branches and leaves of plants in the tea garden, root residues, animal feces and corpses, etc., which are rich in organic phosphorus compounds, but the effective phosphorus content that can be directly absorbed and utilized by tea trees is very low. Therefore, phosphorus-solubilizing microorganisms are needed to participate in degradation. With the metabolism of microorganisms, the phosphorus is released to improve the absorption and utilization rate of organic phosphorus by tea trees, thereby improving the yield and quality of tea. Therefore, the application of phosphorus-solubilizing microorganisms is an effective way to alleviate the problem of organic phosphorus utilization in tea garden soil. Summary of the invention

[0004] In order to overcome the problems existing in the prior art, the present invention provides a strain of Bacillus nicotinoic acid BD-5-3 and application thereof.

[0005] To achieve the above-mentioned object, the first aspect of the present invention provides a strain of Bacillus nicotinate BD-5-3, whose deposit number is: CCTCC M 20232178.

[0006] A second aspect of the present invention provides a bacterial agent, wherein the active ingredient of the bacterial agent is the Bacillus nicotinoic acid BD-5-3 according to claim 1.

[0007] The third aspect of the present invention provides the use of the above-mentioned Bacillus nicotinoic acid BD-5-3 and bacterial agent in phosphate solubilization.

[0008] Through the above technical solution, the present invention can at least achieve the following beneficial effects:

[0009] First, the Bacillus nicotinoic acid BD-5-3 of the present invention is isolated from the soil and is easier to colonize in the soil than the engineered bacteria.

[0010] Secondly, the Bacillus nicotinoic acid BD-5-3 of the present invention has good organic phosphorus and inorganic phosphorus solubilizing ability, and can effectively increase the effective phosphorus content in the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a physical picture of the experiment for determining the ability of phosphate-solubilizing bacteria BD-5-3 to decompose organic phosphorus in the present invention;

[0012] Figure 2 It is the phylogenetic tree of the phosphate-solubilizing bacteria BD-5-3 in the present invention.

[0013] Biological Deposit

[0014] The present invention provides Bacillus nicotinic acid, which has the classification number Bacillus niacini, It was deposited in the China Center for Type Culture Collection on November 10, 2023, with the deposit number: CCTCC M 20232178, and the deposit address: Wuhan University, Wuhan City, Hubei Province, China. DETAILED DESCRIPTION

[0015] Unless otherwise specified, the materials and reagents used in the present invention are commercially available.

[0016] The culture medium involved in this embodiment is as follows:

[0017] Solid medium for organophosphorus bacteria: 0.2 g lecithin, 10.0 g glucose, calcium carbonate (CaCO 3 )1.0 g, yeast extract powder 0.5 g, sodium chloride (NaCl) 0.3 g, ammonium sulfate ((NH4)2 SO4) 0.5 g, potassium chloride (KCl) 0.3 g, magnesium sulfate (MgSO 4 )0.3 g, ferrous sulfate (FeSO 4 )0.03 g, manganese sulfate (MnSO 4 )0.03 g, agar powder 20.0 g, distilled water 1000 mL, pH 7.0-7.5, sterilize in an autoclave at 121 ℃ for 20 min.

[0018] Liquid medium for organophosphorus bacteria①: 0.2 g lecithin, 10.0 g glucose, calcium carbonate (CaCO 3 )1.0 g, yeast extract powder 0.5 g, sodium chloride (NaCl) 0.3 g, ammonium sulfate ((NH 4 ) 2 SO4 )0.5 g, potassium chloride (KCl)0.3 g, magnesium sulfate (MgSO 4 )0.3 g, ferrous sulfate (FeSO 4 )0.03 g, manganese sulfate (MnSO 4 )0.03 g, purified water 1000 mL, pH 7.0-7.5, sterilized in a high pressure sterilizer at 121 °C for 20 min.

[0019] Liquid medium for organophosphorus bacteria②: 2.0 g calcium phytate, 10.0 g glucose, 0.2 g sodium chloride (NaCl), 0.2 g ammonium sulfate (NH 4 ) 2 SO 4 )0.1 g, potassium chloride (KCl)0.2 g, magnesium sulfate (MgSO 4 )0.25 g, purified water 1000 mL, pH 7.0-7.5, sterilized in a high pressure sterilizer at 121 °C for 20 min.

[0020] Liquid medium for inorganic phosphorus-dissolving bacteria①: Calcium phosphate (Ca 3 (PO 4 ))2.0 g, glucose 10.0 g, sodium chloride (NaCl) 0.3 g, ammonium sulfate ((NH 4 ) 2 SO 4 )0.5 g, potassium chloride (KCl)0.3 g, magnesium sulfate (MgSO 4 ) 0.3 g, ferrous sulfate (FeSO4) 0.03 g, manganese sulfate (MnSO4) 0.03 g, purified water 1000 mL, pH 7.2~7.4, sterilize in a high pressure sterilizer at 121 ℃ for 20 min.

[0021] Liquid medium for inorganic phosphorus-dissolving bacteria②: iron phosphate (FePO 4 )4.86 g, glucose 10.0 g, sodium chloride (NaCl) 0.3 g, ammonium sulfate ((NH 4 ) 2 SO 4 )0.5 g, potassium chloride (KCl)0.3 g, magnesium sulfate (MgSO 4 )0.3 g, ferrous sulfate (FeSO 4 )0.03 g, manganese sulfate (MnSO 4 )0.03 g, purified water 1000 mL, pH 7.0, sterilized in an autoclave at 121 °C for 20 min.

[0022] Liquid culture medium for inorganic phosphorus-dissolving bacteria ③: aluminum phosphate (AlPO4 )3.93 g, glucose 10.0 g, sodium chloride (NaCl) 0.3 g, ammonium sulfate ((NH 4 ) 2 SO 4 )0.5 g, potassium chloride (KCl)0.3 g, magnesium sulfate (MgSO 4 )0.3 g, ferrous sulfate (FeSO 4 )0.03 g, manganese sulfate (MnSO 4 )0.03 g, purified water 1000 mL, pH 7.0, sterilized in an autoclave at 121 °C for 20 min.

[0023] Beef extract peptone medium: NaCl 5 g, peptone 10 g, beef extract 3 g, distilled water 1000 mL, pH 7.4~7.6, sterilized in a high pressure sterilizer at 121 ℃ for 20 min.

[0024] 1. Phosphate-solubilizing bacteria BD-5-3 ( Bacillus niacini ) separation and screening

[0025] Preliminary screening of strains

[0026] Soil samples were collected from the ancient tea garden of Bingdao Laozhai in Bingdao Village, Mengku Town, Shuangjiang County, Lincang City, Yunnan Province, and were placed in sterile bags and brought back to the laboratory for storage at 4°C. Weigh 2g of soil and add it to 200ml of sterile water containing glass beads, then place it in a constant temperature air bath shaking incubator for shaking culture for 4 hours (30°C, 180r / min). After enrichment, take the supernatant to prepare a soil dilution with a concentration of 10-3~10-5 for use. Use a pipette to draw 200 microliters of dilutions of different concentrations and place them on the organic phosphorus solid culture medium. After evenly spreading, invert and culture in a 30°C biochemical incubator for 3~4 days, and repeat 3 times for each concentration. After the colonies are cultured, pick the strains with different morphologies and transparent circles in the culture dish for repeated streaking and purification. After purification, pick a single colony and transfer it to the slant of beef extract peptone culture medium at 4°C for storage.

[0027] 2. Determination of the ability of phosphate-dissolving bacteria to degrade organic phosphorus

[0028] The phosphate-solubilizing strains screened initially were spotted on four grids of lecithin solid medium and cultured in a 30°C incubator for 48 h. Figure 1 The diameter of the phosphorus dissolving zone (D) and the diameter of the colony (d) were observed and measured, and the phosphorus dissolving index was calculated as (phosphorus dissolving zone diameter / colony diameter).

[0029] Prepare organic phosphorus liquid screening medium ①② with lecithin and calcium phytate as the only phosphorus source, respectively, and set up 3 replicates for each phosphorus source. The preserved phosphate-solubilizing bacteria BD-5-3 was inoculated into beef extract peptone liquid medium, cultured in a shaking incubator at 30℃ and 180r / min for 24h, and 500 μL of seed liquid was inoculated into organic phosphorus liquid medium ①②, and fermented in a shaking incubator at 30℃ and 180r / min for 5d. Take 10mL of fermentation liquid every day and centrifuge at 4000r / min for 10min. Use the molybdenum antimony colorimetric method to take the supernatant after centrifugation of the uninoculated fermentation liquid as the blank control, measure the absorbance at a wavelength of 700nm, and calculate the soluble phosphorus increment according to the following formula:

[0030] Soluble phosphorus increment = (Aa) * V 0 / (b*V 1 ).

[0031] Note: A-absorbance value of fermentation broth supernatant; a-intercept of phosphorus standard curve, which is 0.0719 in this test; b-slope of phosphorus standard curve, which is 0.537 in this test; V0-volume of colorimetric tube, 25mL; V1-volume of fermentation broth supernatant, 2.5mL.

[0032] The results are shown in Table 1 and Table 2:

[0033] Table 1 Phosphate solubilization of phosphate-solubilizing bacteria BD-5-3 on lecithin plates

[0034]

[0035] Table 2 Decomposition ability of phosphate-solubilizing bacteria BD-5-3 on two different organic phosphorus sources

[0036]

[0037] The test results showed that phosphate-solubilizing bacteria BD-5-3 ( Bacillus niacini ) has a phospholipid solubility index of 1.80. Figure 1 The figure shows the phosphate solubilization circle formed by BD-5-3 on the solid medium containing phosphatidylcholine. The maximum decomposition capacity of phosphatidylcholine within 5 days is 2.44 mg / L, and the maximum decomposition capacity of calcium phytate is 23.08 mg / L.

[0038] 3. Determination of the ability to dissolve inorganic phosphorus (tricalcium phosphate, iron phosphate and aluminum phosphate are inorganic phosphorus)

[0039] The preserved phosphate-solubilizing bacteria BD-5-3 were inoculated into beef extract peptone liquid medium, cultured in a shaking incubator at 30°C and 180r / min for 24h, and 500 μL of seed solution was inoculated into inorganic phosphorus liquid medium ①②③, and cultured in a shaking incubator at 30°C and 180r / min for 5d. Every day, 10mL of fermentation liquid was centrifuged at 4000r / min for 10min, and the supernatant after centrifugation of the uninoculated fermentation liquid was used as a blank control by the molybdenum antimony colorimetric method, and the soluble phosphorus content was determined at a wavelength of 700nm. The results are shown in Table 3:

[0040] Table 3 Decomposition ability of phosphate-solubilizing bacteria BD-5-3 on three different inorganic phosphorus sources

[0041]

[0042] The results showed that phosphate-solubilizing bacteria BD-5-3 ( Bacillus niacini ) The maximum decomposition capacity for tricalcium phosphate is 25.66 mg / L, the maximum decomposition capacity for iron phosphate is 27.34 mg / L, and the maximum decomposition capacity for aluminum phosphate is 7.87 mg / L.

[0043] 4. Phosphate-solubilizing bacteria BD-5-3 ( Bacillus niacini ) identification - morphological characteristics

[0044] (1) Morphology

[0045] After the strain was cultured on a beef extract peptone medium plate at 30°C for 3 days, its morphology was observed. The results showed that the colonies were round, white, wavy, opaque, shiny, and waxy in texture; under an electron microscope, the bacteria were Gram-positive.

[0046] (2) Physiological and biochemical characteristics

[0047] The physiological and biochemical characteristics of the strain were determined with reference to the bacterial identification manual. The results showed that the strain was positive for gelatin reaction and nitrate reduction reaction, but negative for starch hydrolysis reaction, citric acid reaction, sucrose and glucose fermentation reaction, methyl red reaction and urease reaction.

[0048] (3) 16S rDNA gene sequence analysis

[0049] After culturing the phosphate-solubilizing bacteria BD-5-3 in the slant medium, it was sent to Qingke Biological Company to help identify the strain. Sequencing used the TSINGKE plant DNA extraction kit, and the bacterial DNA was extracted according to its instruction manual. The extracted DNA was amplified using a PCR instrument. The primers were 16SrDNA bacterial amplification universal primers 36F-1492R, and the sequences were 36F: 5-TCTGGCTCAGGATGAACGC-3, 1492R: 5-GGTTACCTTGTTACGACTT-3. The PCR reaction program was as follows: pre-denaturation 98℃2min; cycle stage 98℃10s, 56℃10s, 72℃10s / kb, 35 cycles; extension stage 72℃5min. PCR reaction system: 1×TSE101 gold mix 45μL, upstream primer 2μL, downstream primer 2μL. DNA template 1μL. The amplified PCR product was subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue) to detect whether the PCR product was qualified. The amplified product was sequenced, and the sequencing result is shown in SEQ ID NO.1. The sequencing result was Blast aligned at NCBI (www.ncbi.nlm.nih.gov / BLAST / ), and the phylogenetic tree was constructed using MEGA 6.0 software.

[0050] Identification results: The strain was identified as Bacillus nicotinicus, and ( Bacillus niacini ) with a similarity of 99.93%, and the phylogenetic tree is as follows Figure 2 shown.

[0051] 5. Determination of soil phosphorus solubilizing capacity

[0052] The conventional soil (CK) without inoculation (basic physical and chemical properties of soil, available phosphorus 7.53 mg / kg) and the conventional soil (P-BD-5-3) with inoculation were set up, and 3 replicates were set up for each treatment. 150 g of the conventional soil was weighed into a 250 mL beaker, and the beaker was shielded with tin foil. A concentration of 1×10 8 15mL of BD-5-3 bacterial suspension prepared with sterile water at CFU / mL was mixed thoroughly, and the same amount of sterile water was used as a control. Cultured at 30℃, and samples were taken after 10 days of culture to determine the available phosphorus content in the soil using the sodium bicarbonate method. The results showed that the available phosphorus content in the soil without inoculation was 8.82mg / kg, and the available phosphorus content in the soil with inoculation was 8.93mg / kg. Compared with CK, the phosphate-solubilizing bacteria BD-5-3 ( Bacillus niacini ) increased the available phosphorus content in the soil by 1.25%.

[0053] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus nicotinate ( Bacillus niacini )BD-5-3, its deposit number is: CCTCC NO:M20232178.

2. A bacterial agent, characterized in that: The active ingredient of the bacterial agent is the Bacillus nicotinoic acid BD-5-3 described in claim 1.

3. Use of the Bacillus nicotinic acid BD-5-3 according to claim 1 or the bacterial agent according to claim 2 in phosphate solubilization, wherein the phosphorus is: lecithin, calcium phytate, calcium phosphate, iron phosphate and / or aluminum phosphate.

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