A Burkholderia strain with high phosphate-solubilizing efficiency and its application

Abstract

The present invention discloses a Burkholderia strain with high phosphate-solubilizing efficiency. The strain is Burkholderia arboris strain DP01, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 15, 2022, with the deposit number CGMCC NO. 25536. The phosphate-solubilizing efficiency of this strain can reach 22%. This strain can grow in an acidic culture medium and has a certain acid tolerance. Its phosphate-solubilizing efficiency is significantly higher than that of the inorganic phosphate-solubilizing strains screened from the rhizosphere of Camellia oleifera in previous studies, and it can well alleviate the shortage of phosphorus element supply in the soil for Camellia oleifera cultivation.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a Burkholderia strain with high phosphate-solubilizing efficiency and its application. Background Art

[0002] Camellia oleifera is a unique woody oil crop in China and is of great significance for ensuring China's edible oil security. However, the current yield of Camellia oleifera forests in China is generally low, and the soil phosphorus supply status is one of the key factors restricting the yield of Camellia oleifera. Phosphate-solubilizing bacteria refer to microorganisms in the soil that have the ability to convert phosphorus that is difficult for plants to absorb and utilize into an absorbable form. The soil phosphorus cycle is centered around microbial activities, and microbial activities have a great impact on the transformation and availability of soil phosphorus. Therefore, screening for highly efficient phosphate-solubilizing bacteria in the rhizosphere of Camellia oleifera is of great significance for improving the utilization efficiency of soil phosphorus in Camellia oleifera forests and increasing the yield of Camellia oleifera. However, in the research on inorganic phosphate-solubilizing bacteria in the rhizosphere of Camellia oleifera, it was found that the phosphate-solubilizing efficiency of the screened inorganic phosphate-solubilizing strains is generally about 10%, and the phosphate-solubilizing efficiency is extremely low. For example, among the inorganic phosphate-solubilizing strains screened by Wang Shu et al. in the rhizosphere of Camellia oleifera, the strain with the highest phosphate-solubilizing efficiency is P. auricularis in Pseudomonas, and its phosphate-solubilizing rate is about 11%. At the same time, these strains have poor acid tolerance, and the suitable growth environment has a pH of about neutral. Camellia oleifera is a typical acid-loving plant, and the most suitable growth soil pH value is 5-6. The rhizosphere environment shows a highly specialized acidic environment (pH about 4.5) due to the acid secretion of the roots. As the hot zone of the interaction between plants and soil, the rhizosphere is the gateway for plant nutrient absorption. The availability of phosphorus in this strongly acidic environment is extremely low. Therefore, it is necessary to screen for acid-tolerant phosphate-solubilizing bacteria to participate in the phosphorus cycle in the rhizosphere of Camellia oleifera and promote the growth of Camellia oleifera. The acid tolerance of bacteria can enable them to adapt to some acidic environments and grow in acidic environments. Most of the existing phosphate-solubilizing bacteria strains are suitable for a neutral pH, and few have acid tolerance. Therefore, their adaptability in acidic soils (Camellia oleifera is mainly planted in acidic soils) is poor, and it is difficult to survive or reproduce after being applied to Camellia oleifera soils, seriously restricting the exertion of the phosphate-solubilizing ability of phosphate-solubilizing bacteria. Acid-tolerant strains have good adaptability in Camellia oleifera planting soils, colonize quickly in the rhizosphere of Camellia oleifera, have high phosphate-solubilizing activity, and can significantly promote the growth of Camellia oleifera. Therefore, screening for acid-tolerant phosphate-solubilizing strains in the rhizosphere of Camellia oleifera has become the research focus in the field of efficient nutrient utilization of Camellia oleifera. Summary of the Invention

[0003] In view of the above deficiencies existing currently, the present invention provides a Burkholderia strain with high phosphate-solubilizing efficiency. This strain grows well in an acidic medium, and its growth amount in LB medium at pH = 4.5, pH = 5.0, and pH = 5.5 is significantly higher than that of the control strain (Escherichia coli, Figure 2In the medium under the above three pH conditions, after inoculation and culturing for 12 h, the OD600 of the strain of the present application is as high as 1.2 - 1.8, while the OD600 of the control group is lower than 0.3. This indicates that this strain has a certain acid tolerance, and its phosphorus solubilization efficiency is significantly higher than that of the inorganic phosphorus-solubilizing strains screened by others, and it can well alleviate the shortage of phosphorus in red soil.

[0004] To achieve the above object, the present invention provides a Burkholderia strain with high phosphorus solubilization efficiency. The strain is a Burkholderia arboris strain DP01, which was deposited on August 15, 2022, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a postal code of 100101 and a deposit number of CGMCC NO. 25536.

[0005] The above-mentioned Burkholderia arboris strain DP01 was obtained by the inventor by collecting rhizosphere soil samples of Camellia oleifera in the main production area of Camellia oleifera in Changde, Hunan, which had not been fertilized for more than 10 years, and isolating and purifying a strain of Burkholderia arboris, named DP01.

[0006] The biological characteristics of this strain are as follows:

[0007] After culturing in an inorganic phosphorus medium for 3 - 5 days, its colony shape is regular round, the colony color is milky white, transparent, and the colony edge is wrinkled. As Figure 1 shown, observed under an optical microscope, the morphology of DP01 is rod-shaped and its surface is very smooth.

[0008] Based on the same inventive concept, the present invention also provides a microbial inoculant containing the above-mentioned Burkholderia arboris strain DP01.

[0009] Based on the same inventive concept, the present invention also provides the application of the above-mentioned Burkholderia arboris strain DP01.

[0010] According to one aspect of the present invention, the Burkholderia arboris strain DP01 can decompose inorganic phosphorus.

[0011] According to one aspect of the present invention, the Burkholderia arboris strain DP01 is acid-tolerant.

[0012] Advantages of the present invention: The present invention collected rhizosphere soil samples of Camellia oleifera that had not been applied with any fertilizers for more than 10 years in Changde, the main production area of Camellia oleifera in Hunan. One strain of Burkholderia arboris was isolated and purified therefrom, named DP01, and was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC) on August 15, 2022. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the postal code 100101 and the deposit number CGMCC NO.25536. The phosphate-solubilizing rate of this strain is 22%, and it has acid tolerance; compared with the inorganic phosphate-solubilizing bacteria in the rhizosphere of Camellia oleifera (the general phosphate-solubilizing rate is about 10%), the strain of the present application is significantly higher than the strains identified in the research on inorganic phosphate-solubilizing bacteria in the rhizosphere of Camellia oleifera; it can well alleviate the phenomenon of phosphorus deficiency in red soil. Brief Description of the Drawings

[0013] Figure 1 It is an optical microscope image of the Burkholderia arboris strain DP01 in Example 1 of the present application;

[0014] Figure 2 It is the standard working curve for the determination of phosphate-solubilizing rate described in Example 2 of the present application;

[0015] Figure 3 It is the acid tolerance curve graph of the Burkholderia arboris strain DP01 in Example 3 of the present application under the conditions of pH 4.5, 5.0 and 5.5. Detailed Embodiments

[0016] To make the present invention easier to understand, the present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. Unless otherwise defined, the professional terms used hereinafter have the same meaning as understood by those of ordinary skill in the art; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by well-known methods.

[0017] Example 1

[0018] Screening and Identification of the Burkholderia arboris Strain DP01

[0019] (1)Collect the rhizosphere soil samples of Camellia oleifera that have not been fertilized for more than 10 years in the main production area of Camellia oleifera in Changde, Hunan (abbreviation: soil samples). First, conduct a preliminary screening of phosphate-solubilizing bacteria in the collected soil samples. Specifically: Add 45 mL of prepared normal saline (concentration: 0.9%) to a conical flask, weigh 5 g of soil samples and add them to the conical flask. Then, place the conical flask in a constant-temperature shaker, keep the temperature constant at 28 °C and shake for about 20 min to fully mix the soil samples and normal saline, and take out the soil homogenate; Dilute the soil homogenate into soil suspensions with dilution factors of 10 -4 、10 -5 、10 -6 Use a pipette to suck 100 μL of soil suspensions of each concentration and evenly coat them on the cooled and solidified inorganic phosphorus screening medium. After the added soil suspension penetrates into the medium, seal it with a sealing film, and then leave it stationary in a constant-temperature incubator with the temperature adjusted to 28 °C and culture for 3 - 5 d; After the culture is completed, observe the growth status of phosphate-solubilizing microorganisms cultured on the inorganic phosphorus medium and record; Among them, the inorganic phosphorus medium is 10.0 g of glucose, 0.5 g of ammonium sulfate, 5.0 g of calcium phosphate, 0.2 g of sodium chloride, 0.2 g of potassium chloride, 0.03 g of ferrous sulfate heptahydrate, 0.03 g of manganese sulfate tetrahydrate, 0.5 g of yeast powder, 0.1 g of magnesium sulfate heptahydrate, 1000 mL of distilled water, and adjust the pH to 6.8 - 7.2;

[0020] (2)Enrich the phosphate-solubilizing bacteria that have undergone preliminary screening. Specifically: Select the colonies with relatively obvious phosphate-solubilizing circles on the preliminary screening medium (inorganic phosphorus medium) and inoculate them into LB liquid medium respectively. Place the liquid medium on a shaker with the rotation speed set at 180 r / min and the temperature fixed at 28 °C, and culture for 3 - 5 d; Among them, the inorganic phosphorus medium is 10.0 g of glucose, 0.5 g of ammonium sulfate, 5.0 g of calcium phosphate, 0.2 g of sodium chloride, 0.2 g of potassium chloride, 0.03 g of ferrous sulfate heptahydrate, 0.03 g of manganese sulfate tetrahydrate, 0.5 g of yeast powder, 0.1 g of magnesium sulfate heptahydrate, 1000 mL of distilled water, and adjust the pH to 6.8 - 7.2;

[0021] (3)After the enrichment is completed, dilute the bacterial liquid enriched in the LB liquid medium by the 10-fold gradient dilution method to 10 -3 、10 -4 、10 -5, 100 μL of the diluted bacterial solutions at each concentration gradient were respectively inoculated onto the inorganic phosphorus selection medium. After sealing, they were placed in an incubator and cultured for 3 - 5 days, and colonies with obvious phosphorus dissolution halos were observed. Among them, the inorganic phosphorus medium consisted of 10.0 g of glucose, 0.5 g of ammonium sulfate, 5.0 g of calcium phosphate, 0.2 g of sodium chloride, 0.2 g of potassium chloride, 0.03 g of ferrous sulfate heptahydrate, 0.03 g of manganese sulfate tetrahydrate, 0.5 g of yeast powder, 0.1 g of magnesium sulfate heptahydrate, and 1000 mL of distilled water, and the pH was adjusted to 6.8 - 7.2;

[0022] (4) Six strains with obvious phosphorus dissolution halos were isolated and purified, numbered HP01, HP02, HP03, DP01, DP02, and DP03 respectively. Among them, DP01 had the highest efficiency in dissolving inorganic phosphorus. Its colony shape was regular round, the colony color was milky white, transparent, and the colony edge was wrinkled. Observed under an optical microscope, the morphology of DP01 was rod-shaped and its surface was very smooth, as specifically shown in Figure 1 shown.

[0023] The effective sequence obtained by sequencing DP01 was input into NCBI, and compared and analyzed with the database sequences using Blast software. The results showed that the similarity of the 16S rDNA gene sequence with the genus Burkholderia was 99.79%, indicating that DP01 had the highest homology with the genus Burkholderia. The gene sequence of the strain DP01 of the genus Burkholderia (Burkholderia arboris) is shown in SEQ ID NO.1.

[0024] The gene sequence of the strain DP01 of the genus Burkholderia (Burkholderia arboris) is as follows:

[0025]

[0026] Example 2

[0027] Phosphate-solubilizing characteristics of Burkholderia arboris strain DP01

[0028] Preparation of detection reagents and bacterial solutions:

[0029] (1) Molybdenum-antimony stock solution: Measure 153 mL of sulfuric acid (ρ about 1.84 g / mL) and slowly pour it into about 400 mL of distilled water (stir while adding to prevent splashing). Weigh 10.0 g of ammonium molybdate and dissolve it in 300 mL of water at about 60 °C, then cool. Then slowly add the sulfuric acid solution to the ammonium molybdate solution, and then add 100 mL of 5 g / L potassium antimonyl tartrate solution [K(SbO)C4H4O6·1 / 2H2O], and make up to 1 L with distilled water, and store in a brown bottle.

[0030] (2) Molybdenum-antimony-ascorbic acid color reagent: Dissolve 1.5 g of ascorbic acid (C6H8O8, specific rotation of levorotatory form +21~22 °C) in 100 mL of molybdenum-antimony stock solution (prepare and use immediately).

[0031] (3) 2,4-dinitrophenol [C6H5OH(NO3)2] indicator solution (2.5 g / L): Weigh 0.25 g of 2,6-dinitrophenol and dissolve it in 100 mL of water.

[0032] (4) Preparation of standard phosphorus solution: Weigh potassium dihydrogen phosphate (KH2PO4) and place it in an oven, dry it at 105 °C for 2 h, take it out and weigh 0.439 g, cool and add it to a beaker, pour about 200 mL of distilled water, then add 5 mL of concentrated sulfuric acid solution (ρ about 1.84 g / mL). After the solid particles are completely melted, transfer the solution to a 1000 mL volumetric flask and make up to 1 L with distilled water. Use a pipette to take 5 mL of this solution and add it to a 100 mL volumetric flask, and make up to the standard scale with distilled water to obtain a 5 mg / L standard phosphorus solution. By pipetting the standard phosphoric acid solution, adding molybdenum-antimony-ascorbic acid color reagent, shaking well and making up the volume, a 5.0 mg / L phosphorus standard series solution is obtained. Using the same method, 0, 0.2, 0.5, 1.0, 2.0, 4.0 mg / L phosphorus standard series solutions are obtained, that is, 0, 0.2, 0.5, 1.0, 2.0, 4.0, 5.0 mg / L phosphorus standard series solutions are obtained. After standing at room temperature of 20 °C~25 °C for 4 h, using the 0 mg / L phosphorus standard solution as the reference solution, measure the absorbance value at a wavelength of 720 nm on a spectrophotometer. Taking the absorbance value as the ordinate and the concentration of the standard phosphorus solution as the abscissa, plot the standard working curve of the content of soluble phosphorus. This standard working curve is specifically as Figure 2 shown.

[0033] (5) Preparation of bacterial solution: Prepare an inorganic phosphorus medium (the same as the inorganic phosphorus medium in Example 1). Add 50 mL of the inorganic phosphorus medium to a 300 mL Erlenmeyer flask and sterilize it at 121 °C for 15 min. Under a sterile environment, inoculate the DP01 strain screened in Example 1, and set up a blank control group. Place the Erlenmeyer flask inoculated with the strain and the blank control group on a shaker at 28 °C and 160 r / min, and shake and culture for 3 - 5 d, then take it out to measure the available phosphorus content.

[0034] (6) The obtained culture solution is centrifuged at 4000 r / min for 30 min. Take several milliliters of the supernatant, dilute it with distilled water by an appropriate multiple, add it to a 50 mL volumetric flask, add two drops of the above-mentioned 2,4-dinitrophenol indicator, and adjust the pH to just turn slightly yellow with 1 mol / L sodium hydroxide solution or sulfuric acid solution. Then add 5 mL of the above-mentioned molybdenum antimony anti-color reagent, shake well, and make up the volume to the scale. The blank control is treated in the same way. Color at room temperature (20 - 25 °C) for 4 h, measure the absorbance of the solution with a spectrophotometer at a wavelength of 720 nm, adjust the zero point with the blank control as the reference solution, read the absorbance value, and find out the phosphorus concentration in the test solution on the standard working curve. The content of phosphorus element in the measured bacterial solution is the available phosphorus content produced by the target strain decomposing insoluble phosphorus.

[0035] The diameter of the phosphorus-dissolving circle (D), the diameter of the colony (d), the ratio of the phosphorus-dissolving circle to the colony diameter (D / d), the available phosphorus content in the culture solution before cultivation, the available phosphorus content in the culture solution after cultivation, the phosphorus dissolution amount (P), and the phosphorus dissolution rate (X) of the Burkholderia arboris strain DP01 obtained in Example 1 are shown in Table 1. As shown in Table 1, the ratio of the phosphorus-dissolving circle to the colony diameter D / d is 2.044, and the phosphorus dissolution rate is 22%.

[0036] Table 1:

[0037]

[0038] Among them, X represents the phosphorus dissolution rate (the content of elements in the set blank control group needs to be deducted, unit mg / L, and the phosphorus dissolution rate is the percentage of the insoluble phosphorus that can be dissolved into water-soluble phosphorus per 100 g), P represents the phosphorus dissolution amount (the content of available phosphorus in the bacterial solution), and the phosphorus dissolution rate X is calculated according to the following formula: ; In the formula: P = dilution multiple × α (α is the content of soluble phosphorus measured under the standard curve); V — culture solution volume, mL; W — the content of phosphorus element in the insoluble organic or inorganic phosphorus added to the fermentation broth, g.

[0039] Example 3

[0040] Acid resistance of Burkholderia arboris strain DP01

[0041] The acid resistance experiment of Burkholderia arboris strain DP01 obtained in Example 1 was carried out, and the results are as follows Figure 2 shown, from Figure 2 it can be seen that the growth of Burkholderia arboris strain DP01 obtained in Example 1 was significantly higher than that of the control strain (Escherichia coli, abbreviated as CK in Figure 2 ) under the conditions of pH = 4.5, pH = 5.0 and pH = 5.5. After inoculating into LB medium under the three pH conditions and culturing for 12 h, the OD600 of the strain in this application was as high as 1.2 - 1.8, and the OD600 of the control group was lower than 0.3, indicating that the strain in this application has good acid resistance. The specific process of the acid resistance test is as follows: Prepare liquid LB medium (10.0 g of sodium chloride, 10.0 g of tryptone, 5.0 g of yeast extract), use acetic acid - sodium acetate buffer solution (0.2 mol / L) as the buffer system, adjust the pH value of the medium to 4.5, 5.0 and 5.5 respectively, and make up the volume to 1 L. Inoculate the test strain into the above medium at an inoculation amount of 0.1%, repeat 6 times for each pH medium, and inoculate the same amount of Escherichia coli into the medium solution with the same components (repeat 6 times under each pH condition) as the control. Culture in a constant temperature shaker incubator at 30 °C and 200 r / min for 48 h. Measure the OD value of the bacterial cells at 600 nm every 4 h with an L5 - ultraviolet visible spectrophotometer, and plot the growth curve by taking the average value of OD600 of 6 repetitions and the measurement time. The measurement results are as follows Figure 3 .

[0042] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims

Claims

1. An efficient phosphate-solubilizing Burkholderia strain, characterized in that, The strain is Burkholderia arboris strain DP01, which was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on August 15, 2022, with the deposit number CGMCC NO. 25536.

2. A microbial inoculum containing the strain DP01 described in claim 1.

3. Use of the strain DP01 described in claim 1 in dissolving inorganic phosphorus in the rhizosphere of oil tea.

4. The application according to claim 3, characterized in that The strain DP01 is acid-tolerant.

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