Defense pseudomonas L4BXN with phosphate-solubilizing capacity and application of defense pseudomonas L4BXN

Abstract

The invention discloses defensive pseudomonas L4BXN with phosphate solubilizing capacity and application thereof, and belongs to the technical field of phosphate solubilizing bacteria. The pseudomonas with the phosphate-solubilizing capacity is a new strain, namely gram-negative bacteria, the optimum growth temperature is 25-30 DEG C, inorganic phosphorus can be decomposed, and the phosphate-solubilizing amount in 7 days is 169 mg.L <-1 >; when the pseudomonas with the phosphate solubilizing capacity is applied to soil, accumulation of biomass of the overground part and the underground part of arabidopsis thaliana can be remarkably promoted, and particularly increase of root length, root area, root volume, branch number, dry weight and fresh weight is promoted; the provided pseudomonas with the phosphate solubilizing capacity has huge application prospects in plant growth promotion, soil improvement and remediation, and has important significance in promotion of agricultural health and sustainability and improvement of the effective utilization rate of phosphorus in soil.

Description

Technical Field

[0001] The invention relates to the technical field of phosphate-solubilizing bacteria, in particular to a Pseudomonas defensis L4BXN strain with phosphate-solubilizing ability and application thereof. Background Art

[0002] Phosphorus is an important material basis for plant growth and development. It is an important component of nucleic acids, various enzymes, coenzymes, ATP, etc. in plants. These phosphorus-containing substances are vital to cells and plants, and contribute to cell division and flowering and fruiting of plants. They can also promote the growth of lateral roots and fine roots. The amount of phosphorus absorbed by plants is significantly positively correlated with their biomass and yield. However, more than 95% of phosphorus in the soil is in the form of organic or inorganic phosphides, which cannot be directly absorbed and utilized by plants.

[0003] Phosphate-solubilizing bacteria refer to microorganisms, mainly bacteria, that can convert phosphorus in the soil that is difficult for plants to absorb and utilize into an absorbable and utilizeable form. They decompose phosphorus-containing compounds, causing transparent circles to appear around the colonies, thereby increasing the phosphorus content in the soil and promoting the absorption and utilization of phosphorus by plants.

[0004] Phosphate-solubilizing bacteria have important ecological and economic value. By inoculating them, the content of available phosphorus in the soil can be significantly increased, plant growth can be promoted, and crop yield and quality can be improved. In addition, phosphate-solubilizing bacteria can also be used for environmental remediation and soil improvement, helping to solve the problem of soil phosphorus deficiency.

[0005] Therefore, actively screening highly efficient phosphate-solubilizing bacteria and developing phosphate-solubilizing bacteria biofertilizers are of great practical significance to the healthy and sustainable development of agriculture and the efficient use of soil. Summary of the invention

[0006] The purpose of the present invention is to provide a Pseudomonas defense bacteria L4BXN with phosphate solubilizing ability and its application, so as to provide a new strain with phosphate solubilizing effect, so as to contribute to the healthy and sustainable development of agriculture and the efficient utilization of soil.

[0007] To achieve the above-mentioned purpose, the present invention provides a defensive Pseudomonas L4BXN with phosphate-solubilizing ability, classified as Pseudomonas protegens, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on August 6, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 31625.

[0008] Preferably, the 16S rRNA sequence of Pseudomonas defense bacteria L4BXN is shown as SEQ ID NO.1.

[0009] Preferably, the optimum growth temperature of Pseudomonas defensis L4BXN is 25-30°C, and the optimum culture medium is LB culture medium, the components of which are 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0010] The application of the above-mentioned Pseudomonas defensinus L4BXN with phosphate solubilization ability in soil improvement and remediation, which refers to converting organic or inorganic phosphorus in the soil that is not directly usable by plants into phosphorus that can be directly absorbed and utilized by plants.

[0011] A use of the above-mentioned Pseudomonas Defensus L4BXN with phosphate solubilization ability in promoting plant growth, wherein promoting plant growth is promoting the increase of plant root length, root area, root volume, branch number, dry weight or fresh weight.

[0012] A microbial agent, which uses the above-mentioned Pseudomonas defensis L4BXN with phosphate solubilization ability as a main ingredient or an effective ingredient.

[0013] An application of the microbial agent as described above in promoting plant growth, improving and repairing soil.

[0014] Therefore, the present invention provides a strain of Pseudomonas defensinus L4BXN with phosphate solubilization ability and its application, and its specific technical effects are as follows:

[0015] (1) The Pseudomonas with phosphate solubilization ability provided by the present invention is a new strain, a Gram-negative bacterium, with an optimal growth temperature of 25-30°C, capable of decomposing inorganic phosphorus, and a phosphate solubilization capacity of 169 mg·L in 7 days. -1 ;

[0016] (2) Applying the Pseudomonas with phosphate solubilization ability provided by the present invention to the soil can significantly promote the accumulation of aboveground and underground biomass of Arabidopsis thaliana, specifically promoting the increase of root length, root area, root volume, number of branches, dry weight and fresh weight;

[0017] (3) The Pseudomonas with phosphate solubilizing ability provided by the present invention has great application prospects in promoting plant growth, soil improvement and restoration, and is of great significance for promoting healthy and sustainable agriculture and improving the effective utilization rate of phosphorus in the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0019] Figure 1 This is a photo of the LB plate cultured for 3 days in Example 2 of the present invention;

[0020] Figure 2 is the phosphorus standard curve obtained in Example 2 of the present invention;

[0021] Figure 3 is the statistical result of the amount of phosphorus solution in Example 2 of the present invention;

[0022] Figure 4 is the homology comparison result of 16S rRNA of strain L4BXN in Example 3 of the present invention;

[0023] Figure 5 This is a bacterial colony photo in Example 3 of the present invention;

[0024] Figure 6 is the Gram staining result in Example 3 of the present invention;

[0025] Figure 7 These are photos of Arabidopsis cultured for 35 days in Example 4 of the present invention, wherein A is a vertical photo of the Arabidopsis negative control group with sterile water added; B is a plane photo of the Arabidopsis negative control group with sterile water added; C is a vertical photo of the Arabidopsis positive control group with LB culture medium added; D is a plane photo of the Arabidopsis positive control group with LB culture medium added; E is a vertical photo of the Arabidopsis with culture solution added; and F is a plane photo of the Arabidopsis with culture solution added. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0027] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0028] The instruments, equipment, reagents and materials used in the examples were all obtained from commercial sources, the inorganic phosphorus source was calcium phosphate, and the reagents were analytical grade.

[0029] Embodiment 1

[0030] The specific steps for collecting bacteria are as follows:

[0031] In the oak germplasm resource garden of Shenyang Agricultural University, 80 cm away from Quercus liaotungensis, the surface humus layer and soil layer were dug out, and the fine roots with a thickness of 2 mm to 5 mm were found according to the exposed main root system of Quercus liaotungensis. The fine roots were dug out and put into a ziplock bag. The roots were shaken to remove the loose soil at the roots, and then the soil adhering to the root surface was removed with a sterile brush. 10 g of fine roots were placed in sterile PBS buffer, and the volume of the solution was sufficient to cover the sample.

[0032] After ultrasonic treatment, the obtained mixed solution was streaked on a solid LB medium plate. After incubation at 37°C overnight, a single colony at the streaked area on the medium was picked out and placed on a new LB medium plate. This process was repeated until pure colonies were obtained.

[0033] Use a sterile inoculation loop to spot the bacteria on the inorganic phosphorus solid medium, and culture it in a 28℃ incubator for 3 days. Pick the colonies with phosphate solubilization rings and streak them on the LB solid plate, and culture them in a 28℃ incubator. When the colonies grow up, spot the bacteria on the inorganic phosphorus solid medium again, and culture them at 28℃ for 7 days. Streak the colonies with phosphate solubilization rings on the LB solid plate again, and repeat this three times. Finally, select the bacteria that still have phosphate solubilization rings, streak them on the LB plate, and store them in a 4℃ refrigerator.

[0034] Inorganic phosphorus solid culture medium: glucose 10.0 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, potassium chloride 0.3 g, magnesium sulfate 0.3 g, ferrous sulfate 0.03 g, manganese sulfate 0.03 g, calcium phosphate 5.0 g, agar 15.0 g, pH 7.0-7.5 (stored at 25°C).

[0035] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar was added to the solid medium, and no agar was added to the liquid medium.

[0036] Embodiment 2

[0037] The descaling characteristics of the strain obtained in Example 1 were investigated, and the specific steps were as follows:

[0038] (1) Using the phosphate solubility circle method. Prepare an inorganic phosphorus culture medium plate using the method in Example 1, and inoculate the bacteria grown on the LB plate stored in a 4°C refrigerator in Example 1 on the center of the inorganic phosphorus culture medium plate, and culture in a 28°C incubator for 3 days. Observe whether the strain grows and forms a phosphate solubility circle, take photos, measure and record the diameter of the phosphate solubility circle (unit D / cm), the colony diameter (unit d / cm), and calculate the D / d ratio to determine the phosphate solubility ability and efficiency of the strain for different insoluble phosphorus.

[0039] Table 1

[0040] serial number D / d value P1 1.37±0.12b P2 1.53±0.14a P3 1.71±0.06b

[0041] Three strains with D / d greater than 1 were screened and numbered P1, P2, and P3 respectively. The calculation results of the D / d ratios are shown in Table 1. Figure 1 This is a photo of the P3 strain with the largest D / d ratio cultured on an LB plate for 3 days.

[0042] (2) Determine the liquid phosphate solubilizing ability of the strain.

[0043] The three strains obtained in (1) were transferred to inorganic phosphorus liquid culture medium (the components are the same as those of the inorganic phosphorus solid culture medium in Example 1, except that the agar was subtracted) for culture. After mixing evenly, they were placed in a shaking incubator at 28°C and 180 r / min for 7 days. The fermentation liquid was aspirated and three replicates were set up to take the average value. 0.00, 0.50, 1.00, 3.00, 5.00, 10.00, and 15.00 mL of 2 mg / L phosphorus standard solution were respectively aspirated and added to a 50 mL colorimetric tube, and distilled water was added to the 50 mL mark. Then 1 mL of ascorbic acid was added to each tube. After 30 seconds, 2 mL of molybdate solution was added. After shaking and standing for 20 minutes, the absorbance was measured at 700 nm. The results are as follows. Figure 2 As shown, the phosphorus standard curve is y=0.3808x+0.0287, where x is the absorbance at 700nm, y is the effective phosphorus content, and R 2 =0.9928.

[0044] The net release of available phosphorus in the fermentation broth of the strain was calculated according to the following calculation formula to determine the phosphate solubilizing ability of the strain. The uninoculated culture medium was used as a blank control, and the available phosphorus content in the fermentation broth of the phosphate-solubilizing bacteria was determined by the molybdenum blue colorimetric method, with each strain being repeated 3 times.

[0045] The calculation formula is as follows:

[0046] P = K × (V1 ÷ V2);

[0047] Among them, P is the effective phosphorus content; K is the phosphorus content (mg / L) obtained from the standard curve; V1 is the volume of the solution fixed to volume during color development (mL); V2 is the volume of the supernatant absorbed during color development (mL).

[0048] The results are as follows Figure 3 As shown in Figure 2, the amount of phosphorus solubilized by strain P3 was 169 mg·L after 7 days. -1 , named strain L4BXN.

[0049] Embodiment 3

[0050] The phosphate-solubilizing strain L4BXN obtained in Example 2 was identified, and the specific steps were as follows:

[0051] (1) Molecular identification. PCR amplification was performed using the bacterial 16S rRNA universal primers 27F / 1492R. The PCR amplification conditions were: 94°C pre-denaturation for 10 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, for a total of 35 cycles; the reaction was terminated after 72°C extension for 10 min. The PCR amplification system was 25 μL, including 12.5 μL of PCR buffer mix (containing taq enzyme), 1 μL of upstream and downstream primers, 2 μL of DNA template, and 8.5 μL of ddH2O.

[0052] The PCR product was sent to Shanghai Biotech Co., Ltd. for sequencing, and the sequencing result is shown in SEQ ID NO.1. The obtained sequencing sequence was compared on NCBI, and the results showed that the strain L4BXN had the highest homology with the identified Pseudomonas strain (P.protegens), with a homology of 99.82%. The obtained strain L4BXN was a Pseudomonas protegens of the genus Pseudomonas, and its classification name was Pseudomonas protegens. The homology comparison results are shown in Figure 4 shown.

[0053] SEQ ID NO.1:

[0054]

[0055] (2) Morphological observation of Pseudomonas strain L4BXN. The strain L4BXN obtained in Example 2 was inoculated into LB medium and cultured at 28°C until colonies grew. Figure 5 As shown, it can be seen that the colonies are round and yellow, with neat and convex edges and a smooth and moist surface. Figure 6 As shown, the obtained Pseudomonas with phosphate solubilization ability is a Gram-negative bacterium.

[0056] The Pseudomonas strain L4BXN was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on August 6, 2024, with the classification name Pseudomonas protegens. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31625.

[0057] Embodiment 4

[0058] The effect of the bacterial solution of Pseudomonas strain L4BXN obtained in Example 2 on plant growth was investigated as follows:

[0059] (1) This experiment had three treatments: a negative control treatment (CK) with the addition of Pseudomonas strain L4BXN, a positive control treatment (LB medium) with the addition of an equal amount of sterile water without the addition of an inoculum, and 12 replicates for each treatment.

[0060] (2) Seedling cultivation.

[0061] Disinfection of Arabidopsis seeds: Place Arabidopsis seeds in 75% ethanol solution, soak and shake for 5 minutes, then rinse with sterile water 3 to 5 times, take out the sterilized seeds and place them on dry sterile filter paper to remove moisture, and use an inoculation needle to pick the seeds onto 1 / 2MS culture medium, with about 5 seeds per culture dish. Place the inoculated culture medium in a 4°C refrigerator for vernalization, take it out after 3 days, and place it in a constant temperature and light incubator for cultivation. The light cycle of the incubator is 16h light / 8h dark per day, the temperature is 25°C, and the humidity is 60-70%. On the 5th day after seed germination, select seedlings with consistent growth and transplant them into sterilized potting soil (soil ratio of peat soil, perlite and vermiculite is 3:1:1).

[0062] (3) Preparation and addition of bacterial solution. Three bottles were inoculated simultaneously in LB medium, and 100 mL of bacterial solution of Pseudomonas strain L4BXN was cultured in shake flasks. After culturing for 24 h, the OD values ​​of the three bottles of fermentation solution were measured. When the OD value reached 1.0, the bacterial solution was centrifuged, and the precipitate was washed three times with 0.9% saline. Finally, the bacterial body was suspended in sterile water to prepare a bacterial agent, and then the bacterial agent was added to the roots of Arabidopsis plants (within 2 cm of the roots) using a pipette. The bacterial solution was added once on the 3rd and 15th days after transplanting the Arabidopsis seedlings, with 100 μL per gram of soil, and 5 mL of bacterial solution was added to each pot (the bacterial solution concentration was 1×10 9 CFU / mL).

[0063] (4) After 35 days of pot experiment, Arabidopsis plants were harvested and the aboveground and underground biomass, root length, and root surface area of ​​Arabidopsis plants in each treatment were measured. The results are shown in Table 2.

[0064] Table 2

[0065]

[0066]

[0067] The root length, root surface area and number of branches of Arabidopsis thaliana in the L4BXN treatment group were increased by 42%, 48% and 60% respectively compared with those in the CK group; the plant height, fresh weight and dry weight were increased by 23%, 84% and 147% respectively; the growth of Arabidopsis thaliana in the LB medium group was slightly worse than that in the CK group. The dry weight and fresh weight of Arabidopsis thaliana in the phosphate-solubilizing strain L4BXN group were 0.0114g and 0.0562g respectively, while the dry weight of Arabidopsis thaliana in the CK group and LB medium group were 0.0046g and 0.0034g respectively, and the fresh weight was 0.0305g and 0.031g respectively. The root volume of Arabidopsis thaliana in the L4BXN treatment group was 0.007cm 3 The root volumes of Arabidopsis thaliana in the LB medium treatment group and the CK treatment group were 0.003 cm 3 and 0.004cm 3 It can be seen that Arabidopsis thaliana with the addition of the phosphate-solubilizing strain L4BXN has significantly better growth and a more developed root system.

[0068] Therefore, the Pseudomonas with phosphate solubilization ability provided by the present invention is a new strain, a Gram-negative bacterium, with an optimal growth temperature of 25-30°C, capable of decomposing inorganic phosphorus, and a phosphate solubilization amount of 169 mg·L in 7 days. -1; Applying the Pseudomonas with phosphate solubilizing ability provided by the present invention to the soil can significantly promote the accumulation of above-ground and underground biomass of Arabidopsis thaliana, specifically promoting the increase of root length, root area, root volume, number of branches, dry weight and fresh weight; the provided Pseudomonas with phosphate solubilizing ability has great application prospects in promoting plant growth, soil improvement and restoration, and is of great significance to promoting healthy and sustainable agriculture and improving the effective utilization rate of phosphorus in the soil.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of Pseudomonas protegens with phosphate solubilization ability, L4BXN, was deposited in the General Microbiology Center of China National Microbiological Culture Collection on August 6, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31625.

2. The Pseudomonas defensinus L4BXN strain having phosphate solubilizing ability according to claim 1, characterized in that: The 16S rRNA sequence of Pseudomonas defense bacteria L4BXN is shown in SEQ ID NO.

1.

3. The Pseudomonas defensinus L4BXN having phosphate solubilizing ability according to claim 1, characterized in that: The optimal growth temperature of Pseudomonas defensis L4BXN is 25-30°C, and the optimal culture medium is LB medium, the components of which are 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

4. Use of the Pseudomonas defensinus L4BXN having phosphate solubilizing ability according to any one of claims 1 to 3 in soil improvement and restoration, characterized in that: Soil improvement and remediation refers to the conversion of organic or inorganic phosphorus in the soil that cannot be directly used by plants into phosphorus that can be directly absorbed and utilized by plants.

5. Use of the Pseudomonas defensinus L4BXN having phosphate solubilizing ability as claimed in any one of claims 1 to 3 in promoting plant growth, characterized in that: Promoting plant growth means increasing root length, root area, root volume, number of branches, dry weight or fresh weight of plants.

6. A microbial agent, characterized in that: The microbial agent contains the Pseudomonas defensis L4BXN strain having phosphate solubilizing ability as claimed in any one of claims 1 to 3 as a main component or an active component.

7. Use of the microbial agent as claimed in claim 6 in promoting plant growth, improving and repairing soil.

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