A strain of Pseudomonas fluorescens LSOJM27 and its application and bacterial agent

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Pseudomonas fluorescens LSOJM27 and its application and bacterial agent. The Pseudomonas fluorescens LSOJM27 was deposited in the China Center for Type Culture Collection on December 29, 2022, with a deposit number of M 20222101. The strain provided by the present invention has a strong phosphate solubilizing ability and is suitable for activating phosphorus in cultivated soil. The phosphate solubilizing bacteria can significantly increase the content of available phosphorus in cultivated soil and reduce the application of phosphate fertilizers.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Pseudomonas fluorescens LSOJM27 and an application and a bacterial agent thereof. Background Art

[0002] Phosphate fertilizer application is one of the important measures to ensure crop yield and quality, but the utilization rate of applied phosphate fertilizer does not exceed 25%. A large amount of phosphorus accumulates in the soil in an ineffective form, which not only leads to soil nutrient imbalance, but also increases the risk of non-point source pollution. More importantly, phosphate rock, the main source of phosphate fertilizer, is a non-renewable resource. Effective soil phosphorus activation technology is urgently needed to efficiently utilize the phosphorus accumulated in the soil and take into account both agricultural production and the sustainable use of phosphorus.

[0003] Phosphate-solubilizing microorganisms can effectively dissolve soil phosphorus that is difficult to use, reduce phosphorus fertilizer fixation, and increase crop phosphorus absorption, thereby improving phosphorus fertilizer utilization and crop yield. They are currently recognized as safe, economical and effective biological measures in the world. However, the types and numbers of efficient phosphate-solubilizing microbial strains that can be isolated are limited, and their adaptability to different environmental soils is poor. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a strain of Pseudomonas fluorescens LSOJM27 and its application and bacterial agent. The strain provided by the present invention has strong phosphate solubilizing ability and is suitable for activating phosphorus in cultivated soil. The phosphate solubilizing bacteria can significantly increase the content of available phosphorus in cultivated soil and reduce the application of phosphate fertilizer.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a strain of Pseudomonas fluorescens (Pseudomonas protegens) LSOJM27, which was deposited in the China Center for Type Culture Collection on December 29, 2022, with a deposit number of M 20222101.

[0007] The present invention also provides the use of the fluorescing Pseudomonas LSOJM27 described in the above technical solution in phosphate solubilization / solution.

[0008] Preferably, the phosphorus in the dissolved / solubilized phosphorus includes inorganic phosphorus and / or organic phosphorus.

[0009] Preferably, the inorganic phosphorus comprises calcium phosphate.

[0010] Preferably, the organic phosphorus comprises calcium phytate.

[0011] The present invention also provides the use of the fluorescing Pseudomonas LSOJM27 described in the above technical solution in the preparation of alkaline phosphatase and acidic phosphatase.

[0012] The present invention also provides the use of the fluorescing Pseudomonas LSOJM27 described in the above technical solution in the preparation of phosphodiesterase.

[0013] The present invention also provides the use of the fluorescing Pseudomonas LSOJM27 described in the above technical solution in the preparation of phytase.

[0014] The present invention also provides a microbial agent containing the Pseudomonas fluorescens LSOJM27 described in the above technical solution.

[0015] Preferably, the bacterial content of Pseudomonas fluorescens LSOJM27 in the microbial agent is 1.0×10 6 -1.0×10 10 cells / ml.

[0016] The beneficial effects of the present invention are:

[0017] The bacterial strain provided by the invention has strong phosphate-solubilizing ability and is suitable for activating phosphorus in cultivated soil. The phosphate-solubilizing bacteria can significantly increase the content of available phosphorus in cultivated soil and reduce the application of phosphate fertilizers.

[0018] The strain provided by the invention has a wide range of substrates for phosphorus solubilization, and can release small molecules such as organic acids to activate inorganic phosphorus that is difficult to utilize in the soil, and can also secrete a variety of phosphatases such as alkaline phosphamonoesterase, acid phosphamonoesterase, phosphodiesterase and phytase to hydrolyze organic phosphorus. The phosphorus solubilizing bacteria can significantly improve the ability of soil phosphorus activation.

[0019] After the efficient phosphate-solubilizing bacteria Pseudomonas protegens LSOJM27 provided by the invention is cultured in a calcium phosphate (inorganic phosphorus) solid culture medium for 5 days, the phosphate-solubilizing index is 2.38, and the soluble phosphorus concentration reaches 402 mg / L.

[0020] The invention provides efficient phosphate-solubilizing bacteria Pseudomonas protegens LSOJM27, which has a phosphate-solubilizing index of 2.86 and a phosphate-solubilizing amount of 228 mg / L after being cultured in a calcium phytate (organophosphorus) solid culture medium for 5 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 This is the fluorescent Pseudomonas protegens LSOJM27 under a scanning electron microscope;

[0023] Figure 2This is the completed map of the whole genome sequencing of Pseudomonas fluorescens (Pseudomonas protegens) LSOJM27;

[0024] Figure 3 This is the effect of applying Pseudomonas protegens LSOJM27 inoculant in Chaoyang and Lishu soil.

[0025] Biological Deposit Description

[0026] Pseudomonas fluorescens LSOJM27, Latin for Pseudomonasprotegens, was deposited in the China Center for Type Culture Collection on December 29, 2022, with the deposit number M 20222101, and the deposit address is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0027] The present invention provides a strain of Pseudomonas fluorescens (Pseudomonas protegens) LSOJM27, which was deposited in the China Center for Type Culture Collection on December 29, 2022, with a deposit number of M 20222101.

[0028] The present invention also provides the use of the fluorescent Pseudomonas LSOJM27 described in the above technical solution in phosphate dissolution. In the present invention, the phosphorus in the phosphate dissolution / solution preferably includes inorganic phosphorus and / or organic phosphorus. In the present invention, the inorganic phosphorus preferably includes calcium phosphate. In the present invention, the organic phosphorus preferably includes calcium phytate.

[0029] The present invention also provides the use of the fluorescing Pseudomonas LSOJM27 described in the above technical solution in the preparation of alkaline phosphatase and acidic phosphatase.

[0030] The present invention also provides the use of the fluorescing Pseudomonas LSOJM27 described in the above technical solution in the preparation of phosphodiesterase.

[0031] The present invention also provides the use of the fluorescing Pseudomonas LSOJM27 described in the above technical solution in the preparation of phytase.

[0032] The present invention also provides a microbial agent, comprising the fluorescing Pseudomonas LSOJM27 described in the above technical solution. In the present invention, the bacterial content of fluorescing Pseudomonas LSOJM27 in the microbial agent is 1.0×10 6 -1.0×10 10 The present invention has no particular limitation on the method for preparing the microbial agent, and the method for preparing the agent using Pseudomonas fluorescens can be used.

[0033] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] 1. Strain domestication and preservation

[0036] (1) Soil from the cultivated layer of farmland in Lishu County, Siping City, Jilin Province was collected and stored in sterile bags at low temperature (dry ice);

[0037] (2) 1.0 g of soil was placed in 100 mL of organic phosphorus screening liquid medium and placed in a constant temperature incubator at 28°C with shaking for 24 hours;

[0038] (3) Take 1 mL of the culture solution and inoculate it into 100 mL of freshly prepared organophosphorus screening liquid medium, and place it in a constant temperature incubator at 28°C for 24 hours with shaking;

[0039] (4) After repeating (3) twice, the culture suspension was diluted 10 4 and 10 5 times, pipette 200 μL onto the organophosphorus screening solid culture medium plate, and place it in a constant temperature incubator at 28°C for 24-48 hours;

[0040] (5) Select the strain with a transparent zone, purify it on an organophosphorus screening plate, and purify the single clone at least 3 times. Mix the fresh bacterial liquid of the purified strain with 50% glycerol in a ratio of 1:1 and store it in a -80°C ultra-low temperature refrigerator.

[0041] 2. Strain Identification

[0042] Collect 1 mL of fresh bacterial solution and extract bacterial genomic DNA using a kit.

[0043] (1) The DNA was used as a template and the 16S rRNA universal primers 27F / 1429R were used for polymerase chain reaction (PCR). The PCR product was sequenced using the Sanger method, and the strain 16S rRNA sequence is shown in SEQ ID No. 1.

[0044] The sequence was compared with the 16S rRNA database of the National Center for Biotechnology Information, and it was determined that LSOJM27 was Pseudomonas protegens.

[0045] (2) Complete whole genome sequencing of the strain using Tell-Seq ( Figure 2), and the similarity between Pseudomonas protegens LSOJM27 and the existing Pseudomonas protegens in the database was 99.19%.

[0046] >Pseudomonas protegens LSOJM27 strain 16S rRNA sequence (SEQ ID No.1)

[0047]

[0048] Example 2

[0049] Determination of phosphorus activation ability of Pseudomonas fluorescens LSOJM27 obtained in Example 1:

[0050] (1) Preparation of phosphate-solubilizing bacteria suspension: Use a clean inoculation loop to pick 1-2 monoclonal colonies on a solid plate, inoculate them into a test tube containing LB medium, and culture them at 28°C with constant temperature and shaking overnight. Transfer the inoculation volume to a flask containing LB medium at a 2% inoculation rate, and culture them at 28°C with constant temperature and shaking until the OD value of the bacterial suspension reaches 0. 600 ≈1.

[0051] (2) Collection of phosphate-solubilizing bacteria: 1 mL of bacterial suspension was taken and placed in a 1.5 mL centrifuge tube, washed three times with sterile water, and resuspended in sterile water.

[0052] (3) 0.2 mL of the washed bacterial suspension was transferred to a flask containing 20 mL of liquid culture medium (NBRIP culture medium for determination of inorganic phosphorus solubility; calcium phytate culture medium for determination of organic phosphorus hydrolysis ability) according to a 1% inoculation volume. The culture was cultured at 28°C with constant temperature shaking for 7 days, and samples were taken every 24 hours. At the same time, 0.2 mL of sterile water was taken to replace the bacterial suspension as a negative control. Three parallels were set for each treatment.

[0053] (4) Let the culture stand at room temperature for 15 min, pour the culture medium into a centrifuge tube, centrifuge at 1500 rpm for 3 min, and collect supernatant A; centrifuge part of supernatant A again at 10000 rpm for 10 min, and collect supernatant B.

[0054] (5) Determine the bacterial concentration: Take 100 μL of supernatant A, add 100 μL of 1 M HCl, and measure the OD using a spectrophotometer. 600 value.

[0055] (6) Determination of available phosphorus content: Take an appropriate amount of supernatant B and determine it using the molybdenum antimony colorimetric method.

[0056] (7) Determine pH value: Use a pH meter to measure the pH value of supernatant B.

[0057] (8) Take 10 μL of the bacterial suspension resuspended in sterile water in (2) and spot it on a solid plate (for determination of inorganic phosphorus solubility: NBRIP medium; for determination of organic phosphorus hydrolysis ability: calcium phytate medium). After incubation at 28°C for 5 days, measure the phosphorus solubility index: phosphorus solubility circle diameter / colony diameter.

[0058] The provided efficient phosphate-solubilizing bacteria Pseudomonas protegens LSOJM27 has the strongest phosphate-solubilizing ability after being cultured in calcium phosphate (inorganic phosphorus) solid culture medium for 5 days, with a phosphate-solubilizing index of 2.38 and a soluble phosphorus concentration of 402 mg / L.

[0059] The highly efficient phosphate-solubilizing bacteria Pseudomonas protegens LSOJM27 was cultured in calcium phytate (organophosphorus) solid culture medium for 5 days, and had the strongest phosphate-solubilizing ability, with a phosphate-solubilizing index of 2.86 and a phosphate-solubilizing amount of 228 mg / L.

[0060] Example 3

[0061] Preparation of inoculants:

[0062] Pick 1-2 monoclonal colonies of Pseudomonas fluorescens LSOJM27 and inoculate them into 5 mL of LB liquid culture medium, and culture them at 28°C and 180 rpm overnight. Transfer them to 100 mL of LB culture medium according to the 1% inoculation amount, and culture them at 28°C and 180 rpm until the logarithmic growth phase. Centrifuge the culture at 8000 rpm for 10 min, discard the supernatant, and collect the bacteria. Resuspend the bacteria in sterile water and centrifuge at 8000 rpm for 10 min. Repeat this step 2-3 times. Finally, resuspend the bacteria in sterile water to OD 600 =0.5 bacterial suspension, 1.0×10 6 cells / ml.

[0063] Example 4

[0064] Strain application:

[0065] The Tell-seq whole genome sequencing data was compared with the Kyoto Encyclopedia of Genes and Genomes (KEGG) genome decryption database and showed that the strain had:

[0066] (1) Genes encoding alkaline / acid phosphomonoesterase, phosphodiesterase and phytase, such as alkalinephospatase D (phoD, EC: 3.1.3.1), acid phosphatase class A (phoN, EC: 3.1.3.2), glycerophosphoryl diester phosphodiesterase (ugpQ, glpQ, EC: 3.1.4.46) and 3-phytase (EC: 3.1.3.8);

[0067] (2) Phosphate transport system functional protein encoding genes (pstS, pstA, pstC, etc.);

[0068] (3) a two-component system of genes encoding phosphorus starvation response transporters (phoR-phoB);

[0069] (4) Genes encoding organic acid synthesis proteins, such as dihydroaeruginoic acid synthetase (pchE), adenosylcobyric acid synthase (cobQ, cbiP, EC: 6.3.5.10), dihydroxy-aciddehydratase (ilvD, EC: 4.2.1.9), and beta-ureidopropionase (pydC, EC: 3.5.1.6), all play an important role in the enzymatic hydrolysis of organic phosphorus and the dissolution of inorganic phosphorus in soil.

[0070] After the strain is prepared into a microbial agent, it can be used as a phosphorus activator, synergist and agricultural microbial agent. The phosphate-solubilizing bacteria can be mixed with fertilizers to increase soil phosphatase activity, activate phosphorus in the soil, increase the effective phosphorus content in the soil, and thus reduce the application of phosphorus fertilizers.

[0071] (1) Determination of alkaline / acid phosphatase activity: Place 1.00 g of fresh soil sample (<2 mm) in a 50 mL conical flask, add 0.2 mL of toluene, 4 ml of buffer (alkaline phosphatase: pH 11.0, acid phosphatase: pH 6.5) and 1 ml of disodium p-nitrophenyl phosphate solution. Shake gently to mix and plug the bottle stopper, incubate at 37°C for 1 hour. After the incubation, add 1 ml of CaCL2 solution and 4 ml of NaOH solution, shake gently to mix, and filter with filter paper. The treatment with substrate solution added after incubation is used as a control. Measure the absorbance of the solution at 400 nm-420 nm;

[0072] (2) Determination of phosphodiesterase activity: Place 1.00 g of fresh soil sample (<2 mm) in a 50 mL conical flask, add 0.2 mL of toluene, 4 mL of pH 8.0 buffer and 1 mL of disodium dinitrophenyl phosphate solution. Shake gently to mix and plug the bottle stopper, incubate at 37°C for 1 hour. After the incubation, add 1 mL of CaCL2 solution and 4 mL of THAM-NaOH solution, shake gently to mix, and filter with filter paper. Use a spectrophotometer to measure the absorbance of the solution at 400 nm-420 nm.

[0073] (3) Determination of active phosphorus content: The Hedley continuous extraction method was used to extract phosphorus from the soil. The bioavailable inorganic phosphorus extracted by cation exchange resin and the unsteady inorganic phosphorus and organic phosphorus adsorbed by the surface soil extracted by 0.5 M NaHCO3 were added together to give the active phosphorus content in the soil.

[0074] High-efficiency phosphate-solubilizing bacteria were added to farmland soils in Shuangta District, Chaoyang City, Liaoning Province (test site 1: available phosphorus 6.0 mg / kg; total phosphorus 769.5 mg / kg; organic phosphorus 378.6 mg / kg) and Lishu County, Siping City, Jilin Province (test site 2: available phosphorus 7.6 mg / kg; total phosphorus 734.7 mg / kg; organic phosphorus 454.3 mg / kg), with an addition rate of 1.0 × 10 6 The soil without phosphate-solubilizing bacteria was used as the control. Samples were taken at 1, 7, 14, 28, 42, and 63 days after incubation, and the phosphatase activity and soil active phosphorus content were determined.

[0075] The experimental results showed that the increase in soil alkaline phosphatase activity in Chaoyang and Lishu was 3.1%-14.1% and 0.8%-19.1% respectively, the increase in soil acid phosphatase activity was 0.5%-18.7% and 5.3%-36.1% respectively, the increase in soil phosphodiesterase activity was 4.0%-15.4% and 13.1%-43.7% respectively, and the increase in soil active phosphorus component content was 16.2%-44.0% and 6.1%-21.7% respectively. The active phosphorus effectiveness of phosphate-solubilizing bacteria can last for more than 2 months ( Figure 3 ).

[0076] Table 1 Effect of high-efficiency phosphate-solubilizing bacteria (mg / kg)

[0077]

[0078] ALP alkaline phosphomonoesterase; ACP acid phosphomonoesterase; PD phosphodiesterase

[0079] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas protegens LSOJM27, which was deposited in the China Center for Type Culture Collection on December 29, 2022, with a deposit number of CCTCC NO: M 20222101.

2. Use of the Pseudomonas LSOJM27 according to claim 1 in phosphate dissolution.

3. The use according to claim 2, characterized in that: The phosphorus in the dissolved / solubilized phosphorus includes inorganic phosphorus and / or organic phosphorus.

4. The use according to claim 3, characterized in that: The inorganic phosphorus includes calcium phosphate.

5. The use according to claim 3, characterized in that: The organic phosphorus includes calcium phytate.

6. Use of the Pseudomonas sp. LSOJM27 according to claim 1 in the preparation of alkaline phosphomonoesterase and acid phosphomonoesterase.

7. Use of the Pseudomonas sp. LSOJM27 according to claim 1 in the preparation of phosphodiesterase.

8. Use of the Pseudomonas LSOJM27 according to claim 1 in the preparation of phytase.

9. A microbial agent, characterized in that: Contains the Pseudomonas LSOJM27 according to claim 1.

10. The microbial agent according to claim 9, characterized in that: The bacterial content of Pseudomonas LSOJM27 in the microbial agent is 1.0×10 6 -1.0×10 10 cells / ml.

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