Phosphorus-solubilizing bacteria W047 with nitrogen-fixing and siderophore-producing characteristics and application of phosphate-solubilizing bacteria W047

Abstract

The invention discloses a phosphate-solubilizing bacterium W047 with resistance to various heavy metals and application of the phosphate-solubilizing bacterium W047, the taxonomic name of the phosphate-solubilizing bacterium W047 is Burkholderia ambifaria, the phosphate-solubilizing bacterium W047 is preserved in the China General Microbiological Culture Collection Center on January 6, 2025, and the preservation number of the phosphate-solubilizing bacterium W047 is CGMCC No.33300. The phosphate-solubilizing bacterium W047 is obtained by directly separating and purifying arsenic-polluted farmland soil by taking a PVK solid culture medium as a basic culture medium, and has the characteristics of dissolving insoluble inorganic phosphorus (calcium phosphate, iron phosphate and aluminum phosphate), fixing nitrogen, producing iron carriers and the like. The phosphorus-solubilizing strain W047 can remarkably promote dissolution of insoluble phosphorus in phosphate ore, and the soluble phosphorus content in supernatant of phosphate ore slices and powder samples under the condition of shake flakes in a laboratory is 92.94 + / -0.94 mg / L and 173.36 + / -21.17 mg / L. The bacterial strain can activate phosphorus in soil, and compared with a contrast, the available phosphorus in the soil added with bacterial liquid of the bacterial strain is increased by 15.48 + / -2.85 mg / kg after the soil is cultured for 20 days. The multiple functions of the strain W047 enable the strain W047 to have strong viability, and the strain W047 has important significance in enriching strain resources, reducing production cost, developing microbial agents and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a phosphate-solubilizing bacterium W047 with nitrogen fixation and siderophore-producing characteristics and its application. Background Art

[0002] Phosphorus (P) is an indispensable component of nucleic acids (RNA and DNA), proteins, phospholipids, and cofactors such as ATP, and is one of the major essential nutrients for plants. The total phosphorus content in most natural soils is not high, and most of it exists in forms that cannot be absorbed by plants. To meet the growth requirements of crops, phosphorus is often supplied to crops by applying phosphate fertilizers. However, the applied phosphate fertilizers are easily combined with cations such as Ca 2+ 、Fe 3+ 、Al 3+ to form insoluble phosphates. Only 10% - 15% of the phosphorus can be absorbed and utilized by crops in the year of application. Therefore, excessive application of phosphate fertilizers will lead to problems such as overcompensation of soil phosphorus, soil compaction, water eutrophication, and increased economic burden. Therefore, finding an economical and environmentally friendly method is of great significance for improving the availability of phosphorus in the soil and the fertilizer efficiency of phosphate fertilizers.

[0003] Phosphate rock powder directly obtained by crushing phosphate rock is a kind of insoluble phosphate fertilizer with wide sources and low cost, which has the characteristics of slow and long-lasting fertilizer efficiency, but also has problems such as insolubility and low grade, resulting in ineffective release of phosphorus. Phosphate-solubilizing microorganisms dissolve or mineralize insoluble inorganic phosphorus and organic phosphorus in phosphate rock or soil by secreting organic acids, phosphatases, etc., and may also have growth-promoting characteristics such as nitrogen fixation, siderophore production, and auxin production. Therefore, microbial phosphate solubilization is considered an environmentally friendly, green, and sustainable way to decompose insoluble phosphorus in soil or phosphate rock into available phosphorus for plants.

[0004] Currently, the reported patented strains for dissolving phosphate rock powder are Pantoea ananatis (CN107338199A) and Bacillus thuringiensis (CN102191205A), etc. There is no report on using Burkholderia to promote the dissolution of phosphate rock. Therefore, screening new microbial strains with multiple functions is of great significance for enriching the germplasm resources, improving the efficiency of insoluble phosphate fertilizers, and the development of microbial inoculants. Summary of the Invention

[0005] The object of the present invention is to provide a phosphate-solubilizing bacterium W047 with nitrogen fixation and siderophore-producing characteristics and its application. The phosphate-solubilizing bacterium W047 of the present invention has functions such as nitrogen fixation, siderophore production, and phosphate solubilization, and can promote the dissolution of phosphorus in phosphate rock and phosphate rock powder, and increase the content of available phosphorus in the soil.

[0006] To achieve the object of the present invention, the technical solution of the present invention is as follows: A phosphate-solubilizing bacterium W047 with nitrogen fixation and siderophore-producing characteristics and its application. The taxonomic name of the phosphate-solubilizing bacterium W047 is Burkholderia ambifaria, which was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 6, 2025, with the deposit number of CGMCC No. 33300.

[0007] The application of the aforementioned phosphate-solubilizing bacterium W047 in dissolving insoluble phosphates.

[0008] The aforementioned insoluble phosphates are calcium phosphate, iron phosphate or aluminum phosphate.

[0009] The application of the aforementioned phosphate-solubilizing bacterium W047 in dissolving phosphate rock and / or phosphate ore powder.

[0010] The application of the aforementioned phosphate-solubilizing bacterium W047 in increasing the content of soluble phosphorus in soil.

[0011] The separation and purification method of the aforementioned phosphate-solubilizing bacterium W047 is carried out according to the following steps:

[0012] (1) Collect soil samples from arsenic-polluted farmland around high-arsenic coal mining areas;

[0013] (2) Weigh 10 g of arsenic-polluted soil and add it to a 250 mL conical flask containing 90 mL of sterile water and glass beads. Oscillate and disperse it at 28 °C and 150 r / min for 30 min to prepare a soil suspension, and then dilute it 10-fold gradient to prepare soil dilutions of 10 -2 ~10 -7 ;

[0014] (3) Pipette 0.1 mL of the soil dilution and spread it on the PVK solid medium. Invert the petri dish and culture it in an incubator at 28 °C for 3 - 5 d;

[0015] (4) Select colonies with obvious phosphate-solubilizing circles and good growth, and repeatedly streak and purify them 5 times on the PVK solid medium to obtain the phosphate-solubilizing bacterium W047.

[0016] The formula of the aforementioned PVK solid medium is: 10 g of glucose, 0.5 g of yeast powder, 0.5 g of (NH4)2SO4, 0.3 g of NaCl, 0.3 g of KCl, 0.3 g of MgSO4·7H2O, 0.03 g of FeSO4·7H2O, 0.03 g of MnSO4·4H2O, 5 g of calcium phosphate, 20 g of agar, 1 L of ultrapure water, pH 7.0 - 7.6.

[0017] A phosphate-solubilizing microbial inoculum, the active ingredient of the phosphate-solubilizing microbial inoculum includes the aforementioned phosphate-solubilizing bacterium W047.

[0018] A phosphorus-solubilizing microbial inoculant, wherein the active ingredient of the phosphorus-solubilizing microbial inoculant is the aforementioned phosphorus-solubilizing bacterium W047.

[0019] The aforementioned bacterium W047 was isolated from the farmland soil around a high-arsenic coal mining area in Xingren City, Qianxinan Prefecture, Guizhou Province. It is a Gram-negative bacterium, rod-shaped, with an average cell length of 1.1 - 1.4 μm and an average width of 0.40 - 0.45 μm. The suitable growth temperature range is 20 - 37 °C, the pH range is 5 - 8, and it can tolerate 2% NaCl. The strain grows well on the PVK solid medium. After culturing for 7 days at 28 °C, the colony diameter is 9 - 10 mm, the diameter of the phosphorus-solubilizing circle is 18 - 20 mm. The colony is round with a neat edge, the surface is rough and not smooth, and the center of the colony is slightly higher than the edge of the colony.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] The phosphorus-solubilizing bacterium W047 of the present invention has functions such as nitrogen fixation, siderophore production, and phosphorus solubilization. It can promote the dissolution of phosphorus in phosphate rock and phosphate powder, and increase the content of available phosphorus in the soil. Experiments have proved that the phosphorus-solubilizing bacterium W047 can still grow after being passaged 5 times on a nitrogen-free medium, indicating its nitrogen fixation ability. This strain has a strong ability to produce siderophores. After culturing on the CAS plate for 7 days, the ratio of the siderophore circle to the colony diameter (D / d) is 4.14 ± 0.30. The strain W047 has a strong phosphorus-solubilizing ability. Under the condition of shaking flasks in the laboratory for 7 days, the maximum dissolution amounts of insoluble phosphates calcium phosphate, iron phosphate, and aluminum phosphate are 107.85 ± 21.49 mg / L, 26.33 ± 5.56 mg / L, and 28.86 ± 1.41 mg / L respectively. This strain can significantly promote the dissolution of insoluble phosphorus in phosphate rock. Under the condition of continuous culturing of phosphate rock thin slices for 28 days and phosphate rock powder for 7 days in shaking flasks in the laboratory, the soluble phosphorus contents in the supernatant of the thin slice and powder samples are 92.94 ± 0.94 mg / L and 173.36 ± 21.17 mg / L respectively. Compared with the control added with sterile water, the available phosphorus in the soil increased by 15.48 ± 2.85 mg / kg after culturing for 20 days with the bacterial solution of this strain added. Description of the Drawings

[0022] Figure 1 It is the phosphorus-solubilizing effect (front) of strain W047 cultured on the PVK solid medium for 7 days;

[0023] Figure 2 It is the scanning electron micrograph of strain W047;

[0024] Figure 3 It is the phylogenetic tree of strain W047;

[0025] Figure 4Morphology of strain W047 after 5 passages on nitrogen-free medium and cultured for 3 days;

[0026] Figure 5 Effect of strain W047 on siderophore production;

[0027] Figure 6 Maximum phosphate solubilization amount of strain W047 for different insoluble phosphates;

[0028] Figure 7 Phosphate solubilization amounts of strain W047 for phosphate rock flakes (A) and powders (B);

[0029] Figure 8 Dissolution electron probe images of phosphate rock flakes by strain W047 after 28 days of culture (A is the control, B is the addition of W047);

[0030] Figure 9 Change in available phosphorus content after strain W047 was added to the soil and cultured for 20 days. Detailed implementation mode

[0031] The present invention will be further described below in conjunction with the drawings and embodiments, but it shall not be used as a basis for limiting the present invention. Reagents, materials, etc. used in the following embodiments can be obtained from commercial channels without special instructions; test methods used are conventional methods without special instructions.

[0032] PVK solid medium: 10 g of glucose, 0.5 g of yeast powder, 0.5 g of (NH4)2SO4, 0.3 g of NaCl, 0.3 g of KCl, 0.3 g of MgSO4·7H2O, 0.03 g of FeSO4·7H2O, 0.03 g of MnSO4·4H2O, 5 g of calcium phosphate, 20 g of agar, 1 L of ultrapure water, pH 7.0 - 7.6.

[0033] PVK liquid medium: 10 g of glucose, 0.5 g of yeast powder, 0.5 g of (NH4)2SO4, 0.3 g of NaCl, 0.3 g of KCl, 0.3 g of MgSO4·7H2O, 0.03 g of FeSO4·7H2O, 0.03 g of MnSO4·4H2O, 5 g of calcium phosphate, 1 L of ultrapure water, pH 7.0 - 7.6.

[0034] LB liquid medium: 5 g of yeast extract, 10 g of tryptone, 10 g of NaCl, 1.0 L of ultrapure water, adjust the pH to 7.0 - 7.2.

[0035] Beef extract peptone solid medium: 5 g of beef extract, 10 g of peptone, 5 g of NaCl, 20 g of agar, 1.0 L of ultrapure water, adjust the pH to 7.2 - 7.4.

[0036] Beef extract peptone liquid medium: 5 g of beef extract, 10 g of peptone, 5 g of NaCl, 1.0 L of ultrapure water, adjust the pH to 7.2 - 7.4.

[0037] RM medium: 10 g of yeast powder, 2 g of KH2PO4, 2% glucose, 1.0 L of ultrapure water, adjust the pH to 7.2 - 7.4.

[0038] Ashby nitrogen - free medium: 10 g of glucose, 0.2 g of KH2PO4, 0.2 g of NaCl, 0.2 g of MgSO4·7H2O, 0.1 g of CaSO4·2H2O, 5 g of CaCO3, 15 - 20 g of agar, 1 L of ultrapure water, pH 7.2 - 7.4.

[0039] CAS detection medium: 60.5 mg of chrome azurol S (CAS), 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA), 2.645 mg of FeCl3·6H2O, 295.25 mg of NaH2PO4·2H2O, 1213.5 mg of Na2HPO4·12H2O, 125 mg of NH4Cl, 37.5 mg of KH2PO4, 62.5 mg of NaCl, 9000 mg of agar, 1 L of ultrapure water, pH 6.8 ± 0.2.

[0040] Example 1: Isolation, purification and preservation of phosphate - solubilizing bacterium W047

[0041] 1 Isolation and purification of phosphate - solubilizing bacterium W047

[0042] Collect fresh soil samples from the 0 - 20 cm surface layer of arsenic - polluted farmland around a high - arsenic coal mine area in Xingren City, Qianxinan Prefecture, Guizhou Province. Put them into a sterile bag and place the bag in a foam box with ice packs, then bring them back to the laboratory and store them in a 4°C refrigerator. Weigh 10 g of arsenic - polluted soil in time and add it to a 250 mL conical flask containing 90 mL of sterile water and several glass beads. Shake and disperse it at 28°C and 150 r / min for 30 min to prepare a soil suspension, and then dilute it by 10 - fold gradient to prepare 10 -2 ~10 -7 soil dilution solutions. Pipette 0.1 mL of the 10 -2 ~10 -7 gradient soil dilution solutions and spread them on the PVK solid medium. Set three replicates for each gradient, and place the petri dishes upside - down in an incubator at 28°C for 3 - 5 d. Select colonies with obvious phosphate - solubilizing circles and good growth, and streak - purify them 5 times on the PVK solid medium to obtain the phosphate - solubilizing bacterium W047.

[0043] 2 Preservation of phosphate - solubilizing bacterium W047

[0044] In a laminar flow hood, the phosphate-solubilizing strain after secondary screening and purification was inoculated into a beef extract peptone liquid medium and cultured with shaking at 28 °C and 150 r / min for 24 h. It was adjusted with fresh sterilized beef extract peptone liquid medium to have an absorbance value of about 0.6 at a wavelength of 600 nm. 0.5 mL of this bacterial solution and 0.5 mL of 80% glycerol were mixed and placed into a sterile cryotube, and stored in a -80 °C refrigerator.

[0045] Example 2: Identification of Phosphate-Solubilizing Bacterium W047

[0046] 1 Strain Morphology

[0047] The strain W047 obtained in Example 1 was inoculated onto a PVK solid medium and incubated upside down in a constant temperature incubator at 28 °C for 7 d. It was observed that the colony diameter was 9 - 10 mm, the phosphate-solubilizing circle diameter was 18 - 20 mm, the colony was circular with a neat edge, the surface was rough and not smooth, and the center of the colony was slightly higher than the edge of the colony ( Figure 1 ). Gram staining showed that the strain was a Gram-negative bacterium. Observed by scanning electron microscopy, the strain was rod-shaped, with an average cell length of 1.1 - 1.4 μm and an average width of 0.40 - 0.45 μm ( Figure 2 ).

[0048] 2 Conventional Physiological and Biochemical Characteristics

[0049] The utilization of xylose, mannose, maltose, β-galactoside, citrate by strain W047, and physiological and biochemical characteristics such as nitrate reduction, oxidative fermentation ability (OF), gelatin liquefaction, and urease were mainly determined using micro biochemical identification tubes. Fresh bacterial colonies activated on a beef extract peptone solid medium for 48 h were picked and diluted with 0.85% sterile normal saline to about 10 8 CFU / mL. 50 μL - 80 μL (about 1 - 2 drops) of the bacterial suspension was pipetted into micro biochemical identification tubes produced by Guangdong Huankai Microbial Science and Technology Co., Ltd. and incubated in an incubator at 35 °C - 37 °C for 12 h - 48 h, and then the results were observed. The bacterial suspension after activation with LB liquid medium was inoculated into conical flasks containing 50 mL of beef extract peptone liquid medium at pH values of 3.0 - 10.0 (gradient of 1 pH unit) at a volume ratio of 1%, and cultured at 150 rpm and 28 °C for 24 h. Then, the absorbance value was measured at a wavelength of 600 nm using a UV-visible spectrophotometer to determine the optimal growth pH range. The activated bacterial suspension was inoculated into conical flasks containing 50 mL of beef extract peptone liquid medium at a volume ratio of 1%, and cultured at 150 rpm for 24 h at 20 °C, 25 °C, 28 °C, 30 °C, 35 °C, 37 °C, and 40 °C respectively, and OD 600Values were used to determine the optimal growth temperature range. 15 mL of RM medium was placed in a 50 mL conical flask, and the salinity of the medium was adjusted with NaCl to be 0%, 1.0% - 6.0% (gradient 1.0%). The activated bacterial suspension was inoculated at a volume ratio of 1%, and cultured in a shaker at 150 rpm and 28 °C for 24 h, and the OD 600 value was measured to determine the salt tolerance of the strain. The results of conventional physiological and biochemical characteristics identification are shown in Table 1.

[0050] Table 1 Identification results of conventional physiological and biochemical indexes of the strain

[0051]

[0052] Note: "+" indicates positive and "-" indicates negative.

[0053] 3 Molecular biology identification

[0054] The purified strain in Example 1 was transferred to LB medium and cultured overnight. The cells were collected by centrifugation at 8000 rpm for 1 min at room temperature, and the total DNA of the cells was extracted according to the method of Ezup column bacterial genomic DNA extraction kit. The genomic DNA of the bacterium was PCR amplified with the universal primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) of bacterial 16S rRNA gene. The amplified PCR product was entrusted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were subjected to Blast sequence alignment on the NCBI website, and the evolutionary tree was constructed with MEGA11 software using the sequences of several similar strains selected from them. The results are as Figure 3 described. The results showed that the 16S rRNA sequence of the strain W047 isolated and purified in the present invention had the highest homology (99%) with Burkholderia ambifaria strain AMMD (NR 074687.1), and was identified as Burkholderia ambifaria, and the strain was named W047.

[0055] The strain W047 contains the nucleotide sequence shown in SEQ ID No.1.

[0056] Example 3: Nitrogen fixation ability detection

[0057] The strain W047 was spotted onto Ashby nitrogen-free medium and cultured in an incubator at 28 °C for 3 d, and then subcultured on Ashby nitrogen-free medium every 3 d. After subculturing 5 times under the same culture conditions. The results showed that the strain could still grow normally on Ashby nitrogen-free medium after subculturing 5 times ( Figure 4) indicates that this strain has nitrogen fixation ability.

[0058] Example 4: Detection of siderophore production ability

[0059] Spot inoculate strain W047 on the CAS detection medium and incubate it upside down at 28 °C for 2 d. Observe the change in color around the colony. The appearance of a yellow circle around the colony indicates that the strain can secrete siderophores. The effect of siderophore production by strain W047 is as Figure 5 shown. The ratio of the siderophore circle (D) to the colony diameter (d) is 4.14 ± 0.30, indicating that this strain has a strong ability to produce siderophores.

[0060] Example 5: Determination of the phosphorus solubilizing ability of phosphorus solubilizing bacterium W047 on different insoluble phosphates

[0061] 1 Qualitative determination

[0062] Spot inoculate strain W047 on the PVK solid medium with 5 g / L calcium phosphate as the sole phosphorus source, and incubate it upside down in a constant temperature incubator at 28 °C for 7 d. Set a scale, take a photo, and then import it into the ImageJ software to measure the colony diameter (d) and the phosphorus solubilizing circle diameter (D). The results show that the phosphorus solubilizing index (D / d) of the strain is 1.94 ± 0.09.

[0063] 2 Quantitative determination

[0064] Transfer strain W047 to LB medium and activate it overnight. Take out 5 mL of the strain culture solution in a laminar flow hood, measure the absorbance value at a wavelength of 600 nm using an ultraviolet-visible spectrophotometer, and dilute it appropriately with sterilized LB medium according to the absorbance value to adjust its OD 600 value to about 0.6. Dilute the activated strain culture solution overnight according to the determined dilution factor, inoculate the strain into 250 mL conical flasks containing 100 mL of PVK liquid medium (using 5 g / L calcium phosphate, iron phosphate, and aluminum phosphate as phosphorus sources respectively) at a ratio of 1% (v / v), and set up a non-inoculated control. Each treatment has three replicates, and place them in a constant temperature shaking incubator at 28 °C and 150 r / min for continuous culture for 7 d. After the culture is completed, centrifuge at 8000 r / min and 4 °C for 10 min, take the supernatant, and determine the soluble phosphate content by the molybdenum antimony anti-spectrophotometric method.

[0065] The results show that this strain has a strong phosphorus solubilizing ability. After culturing for 7 d under the conditions of a laboratory shaking flask, the maximum dissolution amounts of the insoluble phosphates calcium phosphate, iron phosphate, and aluminum phosphate are 107.85 ± 21.49 mg / L, 26.33 ± 5.56 mg / L, and 28.86 ± 1.41 mg / L respectively ( Figure 6), compared with the control, it increased by 105.65±4.79mg / L, 23.47±2.68mg / L and 26.03±1.24mg / L respectively.

[0066] Example 6: Ability of phosphate-solubilizing bacterium W047 to dissolve phosphate rock flakes and powder

[0067] The phosphate-solubilizing ability of phosphate-solubilizing bacterium W047 was further explored. Guizhou Kaiyang phosphate rock was prepared into rock flakes (about 0.5 mm thick) and powder passing through a 100-mesh sieve (the mass fraction of P2O5 in this phosphate rock was 28.853%, and the mass fraction of elemental phosphorus was 12.592%). 100 mL of PVK liquid medium was placed in a 250 mL conical flask, and the calcium phosphate in it was replaced with phosphate rock flakes and powder. One phosphate rock flake and 5 g / L of phosphate rock powder (phosphorus content 630 mg / L) were placed in each conical flask, and sterilized at 121 °C for 20 min. The strain suspension with an OD 600 value adjusted to about 0.6 after culturing the LB medium overnight was inoculated into the medium containing phosphate rock flakes and powder at 1% (v / v), and cultured with shaking at 28 °C and 150 r / min. The medium containing phosphate rock flakes was continuously cultured for 28 d. About 8 mL of the culture solution was taken out on the 2nd, 7th, 14th, 21st and 28th days in the ultra-clean bench and an equal amount of new sterilized culture solution was added. Centrifuge at 8000 r / min and 4 °C for 10 min, and determine the content of soluble phosphate by molybdenum antimony anti-spectrophotometry. After the phosphate rock flakes on the 28th day were rinsed clean with sterile water, they were placed in an oven and dried at low temperature (30 °C - 40 °C), and entrusted to Beijing Beida Zhihui Microstructure Analysis and Testing Center Co., Ltd. to analyze the elemental composition on the surface of the flakes using an electron probe. The medium containing phosphate rock powder was continuously cultured for 7 d. About 8 mL of the culture solution was taken out in the ultra-clean bench every 1 day and an equal amount of new sterilized culture solution was added. Centrifuge at 8000 r / min and 4 °C for 10 min, and determine the content of soluble phosphate by molybdenum antimony anti-spectrophotometry.

[0068] The results showed that this strain could significantly promote the dissolution of insoluble phosphorus in phosphate rock. When the phosphate rock flake was the sole phosphorus source, the maximum phosphate-solubilizing amount reached 92.94±0.94mg / L on the 7th day ( Figure 7 A); the electron probe results showed that compared with the control, after 28 d of culture, most of the insoluble phosphates on the surface of the phosphate rock flakes had been dissolved ( Figure 8 ). When the phosphate rock powder was the sole phosphorus source, the phosphorus content in the supernatant tended to be stable on the 4th day of culture, and reached the maximum value of 173.36±21.17mg / L on the 6th day during the culture period ( Figure 7 B), and the phosphate-solubilizing rate for the phosphate rock powder (after deducting the phosphate-solubilizing amount of the control) was 27.2%.

[0069] Example 7: Activation effect of phosphate-solubilizing bacterium W047 on soil available phosphorus

[0070] Soil samples of 0-20 cm in depth were collected from the arsenic-contaminated farmland around a high-arsenic coal mine area in Xingren City, Qianxinan Prefecture, Guizhou Province, and placed in a cool and dry place to air-dry naturally. After passing through a 5-mm sieve, they were reserved for use. The soil used was yellow soil, with a pH of 4.50, total phosphorus of 1.48 g / kg, total nitrogen of 1.97 g / kg, and total potassium of 7.48 g / kg. The phosphate-solubilizing bacterium W047 was inoculated into LB medium and activated overnight. Referring to Example 5, the strain suspension was adjusted to an OD 600 value of about 0.6 for standby. Weigh 50 g of soil and put it into a 250-mL plastic bottle. Add the strain suspension according to the ratio of dry soil:bacterial suspension of 5:2 (w / v). The control group was added with an equal amount of sterile water and weighed. The soil moisture was maintained at 50%-60% of the maximum water-holding capacity in the field. Incubate at 25°C ± 1°C in a constant temperature incubator for 20 d. Supplement water with sterile water every about 3 d to keep the weight at the initial level. After the incubation was completed, the soil samples were air-dried and passed through a 2-mm sieve. Weigh 5.00 g and put it into a 200-mL plastic bottle. Add 50 mL of ammonium fluoride-hydrochloric acid extractant (0.03 mol / L ammonium fluoride, 0.025 mol / L hydrochloric acid) at 25°C ± 1°C. Shake and incubate at 25°C ± 1°C and 180 r / min for 30 min, and immediately filter dry with phosphorus-free filter paper. The filtrate was determined with reference to "Soil Testing - Part 7: Determination of Soil Available Phosphorus" (NY / T 1121.7-2014).

[0071] The results showed that compared with the control group added with sterile water, the available phosphorus in the soil increased by 15.48 ± 2.85 mg / kg after 20 d of incubation with the strain suspension added, indicating that the strain had a certain activation effect on soil phosphorus.

[0072] W047 (Burkholderia ambifaria)

[0073] CGMCC NO.33300

[0074] DNA sequence:

[0075] >47 (Burkholderia ambifaria)

[0076] AGTTTGATTCCAGGCTCAGATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGA

[0077] ACGGCAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATC

[0078] GGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACG

[0079] ATCTACGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCTATAGGGTTGGCCGATGGC

[0080] TGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCTGGTCTG

[0081] AGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCA

[0082] GCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGTGTG

[0083] AAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCTTGGTTCTAATA

[0084] TAGCCGGGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAG

[0085] CCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCG

[0086] CAGGCGGTTTGCTAAGACCGATGTGAAATCCCCGGGCTCAACCTCGGAACTGCATTG

[0087] GTGACTGGCAGGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAA

[0088] ATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTG

[0089] ACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACG

[0090] CCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAACGTAGCTAACGC

[0091] GTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACG

[0092] GGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTT

[0093] ACCTACCCTTGACATGGTCGGAATCCTGCTGAGAGGTGGGAGTGCTCGAAAGAGAAC

[0094] CGGCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT

[0095] CCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAAGGAGAC

[0096] TGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATG

[0097] GGTAGGGCTTCACACGTCATACAATGGTCGGAACAGAGGGTTGCCAACCCGCGAGGG

[0098] GGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAGTGCA

[0099] TGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGG

[0100] GTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTTACCAGAAGTGGCTAGTC

[0101] TAACCGCAAGGAGGACGGTCACCACGGTAGGATTCATGACTGGGGTGAAGTCGTAAA

[0102] AAAGGGTAAACCA

Claims

1. A phosphate-solubilizing bacterium W047 with nitrogen fixation and siderophore production characteristics and its application, characterized in that: The phosphate-solubilizing bacterium W047 has the taxonomic name Burkholderia ambifaria and was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on January 6, 2025, with the deposit number CGMCC No. 33300.

2. Use of the phosphate-solubilizing bacterium W047 as described in claim 1 in dissolving insoluble phosphates.

3. The application according to claim 2, characterized in that: The insoluble phosphates are calcium phosphate, iron phosphate or aluminum phosphate.

4. Use of the phosphate-solubilizing bacterium W047 as described in claim 1 in dissolving phosphate rock and / or phosphate powder.

5. Use of the phosphate-solubilizing bacterium W047 as described in claim 1 in increasing the content of soluble phosphorus in soil.

6. The method for isolation and purification of phosphate-solubilizing bacterium W047 according to claim 1, characterized in that, The method for isolation and purification of the phosphate-solubilizing bacterium W047 is carried out according to the following steps: (1) Collect soil samples from arsenic-contaminated farmland around high-arsenic coal mining areas; (2) Weigh 10 g of arsenic-contaminated soil and add it to a 250 mL conical flask containing 90 mL of sterile water and glass beads. Oscillate and disperse it for 30 min at 28 °C and 150 r / min to prepare a soil suspension, and then dilute it by 10-fold gradient to prepare soil dilutions of 10 -2 ~10 -7 ; (3) Pipette 0.1 mL of the soil dilution and spread it on the PVK solid medium. Invert the petri dish and culture it in an incubator at 28 °C for 3 - 5 days; (4) Select colonies with obvious phosphate-solubilizing halos and good growth, and streak and purify them 5 times on the PVK solid medium to obtain the phosphate-solubilizing bacterium W047.

7. The method for isolation and purification of phosphate-solubilizing bacterium W047 according to claim 6, characterized in that, The formula of the PVK solid medium is: 10 g of glucose, 0.5 g of yeast powder, 0.5 g of (NH4)2SO4, 0.3 g of NaCl, 0.3 g of KCl, 0.3 g of MgSO4·7H2O, 0.03 g of FeSO4·7H2O, 0.03 g of MnSO4·4H2O, 5 g of calcium phosphate, 20 g of agar, 1 L of ultrapure water, pH 7.0 - 7.

6.

8. A phosphorus-solubilizing microbial inoculant, characterized in that: The active ingredient of the phosphate-solubilizing microbial inoculant includes the phosphate-solubilizing bacterium W047 as described in claim 1.

9. A phosphorus-solubilizing microbial inoculum, characterized in that: The active ingredient of the phosphate-solubilizing microbial inoculant is the phosphate-solubilizing bacterium W047 as described in claim 1.

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