Phosphorus solubilizing bacterial strain PSBS-2 and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a phosphorus solubilizing strain PSBS-2 and application thereof. The phosphorus solubilizing strain PSBS-2 is classified as Niallia circulans, and is preserved in the Guangdong Microbial Culture Collection Center on May 14, 2025, with a preservation number of GDMCC No:66329. The phosphorus solubilizing strain is screened to obtain the strain PSBS-2, which is identified as Niallia circulans through morphological observation and molecular biological identification. Through a phosphorus solubilizing capacity test of the strain PSBS-2, it is found that the strain PSBS-2 has strong phosphorus solubilizing capacity and can convert insoluble phosphate into a form that can be absorbed and utilized, which is of great significance to improve soil phosphorus utilization rate, maintain soil health and realize sustainable development of agricultural economy, and can provide strain resource support for development of related microbial agents, biological fertilizers or soil conditioners.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a phosphorus solubilizing strain PSBS-2 and application thereof. BACKGROUND

[0002] Phosphorus (P) is one of the essential nutrients for plant growth and development, and participates in key physiological processes such as energy transfer, cell division and synthesis of genetic material. However, due to the influence of factors such as red soil parent material, high temperature and heavy rainfall in subtropical regions, and frequent exchange of water and heat, the problems of infertility and hardening of red soil in southern China are particularly serious. More than 95% of phosphorus in red soil exists in the form of insoluble phosphates such as calcium phosphate, iron phosphate and aluminum phosphate, which cannot be directly absorbed and utilized by plants. In traditional agricultural production, chemical phosphorus fertilizer is mainly used to supplement available phosphorus, but the utilization rate of phosphorus fertilizer in the current season is less than 20%, and long-term overuse not only increases planting costs, but also causes red soil hardening, leading to a decrease in biodiversity, and also causes environmental problems such as eutrophication of water bodies around farmland. Therefore, it is of great significance to choose a long-term effective and safe method to improve soil phosphorus utilization rate for maintaining soil health and achieving sustainable development of agricultural economy.

[0003] In recent years, soil microbial phosphorus solubilizing technology has attracted widespread attention due to its environmental friendliness and sustainability. Soil phosphate solubilizing microorganisms (PSM), also known as phosphorus solubilizing bacteria, are a group of microorganisms that can convert insoluble phosphates that are difficult for plants to absorb into soluble phosphates, thereby improving the biological availability of soil phosphorus. More than 20 genera of phosphate solubilizing microorganisms have been reported, among which the bacteria with phosphorus solubilizing function mainly include Bacillus, Pseudomonas, Erwinia, Burkholderia, Serratia, Azotobacter, Rhizobium, Salmonella, Chromobacterium, Alcaligenes, Thiobacillus and Escherichia.

[0004] The present inventors have studied the rhizosphere soil microorganisms of the tea plants (tea group plants, Theaceae, Camellia) in Xishan tea group in Guiping City, Guangxi, and screened a native strain PSBS-2 with phosphorus solubilizing function. The phosphorus solubilizing function strain was identified by morphological observation and 16S rRNA sequence sequencing. The native strain PSBS-2 is Niallia circulans, which is of great significance for improving the utilization rate of soil available phosphorus, improving the degraded red soil, and realizing the soil health conservation and quality improvement in subtropical regions and the sustainable development of agricultural economy. Currently, there is no relevant report on Niallia circulans.

[0005] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general context of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is already known in any country in the world. SUMMARY

[0006] The present application aims to provide a phosphorus solubilizing bacterial strain PSBS-2 and its application, which can decompose insoluble phosphate in soil, improve soil phosphorus availability, and help improve soil phosphorus utilization rate.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions.

[0008] The first object of the present application is to provide a phosphorus solubilizing bacterial strain PSBS-2, which is taxonomically named Niallia circulans, and was preserved in Guangdong Microbial Culture Collection Center on May 14, 2025, with the preservation number of GDMCC No:66329.

[0009] The second object of the present application is to provide the application of the phosphorus solubilizing bacterial strain PSBS-2 in decomposing insoluble phosphate in the environment.

[0010] Further, the insoluble phosphate includes calcium phosphate and iron phosphate.

[0011] The third object of the present application is to provide the application of the phosphorus solubilizing bacterial strain PSBS-2 in improving soil phosphorus utilization rate.

[0012] The fourth object of the present application is to provide the application of the phosphorus solubilizing bacterial strain PSBS-2 in preparing microbial inoculants.

[0013] The fifth object of the present application is to provide the application of the phosphorus solubilizing bacterial strain PSBS-2 in preparing biofertilizer.

[0014] The sixth object of the present application is to provide the application of the phosphorus solubilizing bacterial strain PSBS-2 in soil conditioner.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] The present application carries out screening on phosphorus solubilizing strains, and obtains strain PSBS-2. Through morphological observation and molecular biological identification, it is determined that the strain PSBS-2 is Niallia circulans. Through the phosphorus solubilizing capacity test of the strain PSBS-2, it is found that the strain PSBS-2 has strong phosphorus solubilizing capacity, and can convert insoluble phosphate into a form that can be absorbed and utilized, which has important significance for improving soil phosphorus utilization rate, maintaining soil health and realizing sustainable development of agricultural economy, and can provide strain resource support for the development of related microbial agents, biological fertilizers or soil conditioners.

[0017] Preservation information

[0018] The phosphorus solubilizing strain PSBS-2 is taxonomically named Niallia circulans, and was preserved in the Guangdong Microbial Culture Collection Center on May 14, 2025, and the address is: No. 59, Building 5, Institute of Microbiology, Guangdong Academy of Sciences, 100, Martyrs Road, Guangzhou City, Guangdong Province, and its preservation number is GDMCC No: 66329. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a colony morphology diagram of the strain PSBS-2;

[0020] Figure 2 It is an evolutionary tree of the strain PSBS-2;

[0021] Figure 3 It is a standard curve diagram of the soluble phosphorus content;

[0022] Figure 4 It is a molybdenum-antimony anti-color comparison diagram of the strain PSBS-2;

[0023] Figure 5 It is a line graph of the phosphorus solubilizing rate of the strain PSBS-2 under different pH environments;

[0024] Figure 6 It is a line graph of the phosphorus solubilizing rate of the strain PSBS-2 under different culture times;

[0025] Figure 7 It is the phosphorus solubilizing rate of the strain PSBS-2 on different phosphorus sources. DETAILED DESCRIPTION

[0026] The technical solutions of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments.

[0027] 1. Strain screening and identification

[0028] 1.1 Soil sample collection

[0029] During 2023-2025, the soil at the depth of 10-20 cm in the rhizosphere of tea plants in the typical tea plantation of Xishan tea in Guiping City, Guangxi (23.412204 °N, 110.051338 °E) was collected by five-point sampling method, placed in a sterile sealed bag, and stored at -20 °C for screening of phosphate-solubilizing bacterial strains.

[0030] 1.2 Culture medium

[0031] LB solid medium: tryptone 10.0 g / L, yeast extract powder 5.0 g / L, sodium chloride 10.0 g / L, agar 15 g / L.

[0032] LB broth medium: tryptone 10.0 g / L, yeast extract powder 5.0 g / L, sodium chloride 10.0 g / L, pH 7.0±0.1 (30 °C).

[0033] Phosphate-solubilizing solid medium: glucose 10.0 g / L, ammonium sulfate 0.5 g / L, yeast extract powder 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, tricalcium phosphate 5.0 g / L, agar 15 g / L.

[0034] Phosphate-solubilizing medium: glucose 10.0 g / L, ammonium sulfate 0.5 g / L, yeast extract powder 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, tricalcium phosphate 5.0 g / L, pH 7.0-7.5 (30 °C).

[0035] Calcium phosphate / aluminum phosphate / iron phosphate medium: glucose 10.0 g / L, ammonium sulfate 0.5 g / L, yeast extract powder 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, tricalcium phosphate / aluminum phosphate / iron phosphate 5.0 g / L, pH 7.0-7.5 (30 °C).

[0036] 1.3 Isolation and purification of strains

[0037] 5.0 g of fresh soil sample was weighed into a 150 mL conical flask containing about 10 sterile glass beads, 45 mL of sterile water was added, and the flask was shaken at 140 r / min for 30 min at 30 °C. After standing for 15 min, 100 μL of the soil suspension was taken from the flask and added to a 1 mL centrifuge tube containing 900 μL of sterile water to obtain a 10 -1 fold dilution. Then, 10 -1Dilute 100 μL of the solution 10 times in a test tube containing 900 μL of sterile water, and obtain serial dilutions of 10 -6 , 10 -7 , and 10 -8 concentrations, respectively, by sequentially operating. Take 100 μL of the dilution and evenly spread it on the dephosphorizing solid medium, and then invert it in a 30℃ incubator for 3 days. After three times of single colony purification by three-zone streaking on LB solid medium, the strain is stored for standby use.

[0038] 1.4 Screening of dephosphorizing strains

[0039] After purification, the strains are spread on dephosphorizing solid medium at 10 -6 and 10 -7 concentrations of bacterial liquid dilutions, respectively. After the colonies grow, their growth conditions are observed.

[0040] Seven strains with transparent circles around the colonies are screened, and it is judged that they have dephosphorizing functions. One of them is named PSBS-2.

[0041] 1.5 Morphological observation of strain PSBS-2

[0042] The strain PSBS-2 is inoculated on LB solid medium, and then incubated at 30℃. The colony morphology at 3 days is shown in Figure 1 .

[0043] As can be seen from Figure 1 , the colony morphology of the strain PSBS-2 is round, milky white, non-transparent, smooth and moist on the surface, easy to pick out, and the color does not change in the late growth stage.

[0044] 1.6 Molecular biological identification of strain PSBS-2

[0045] The Sanger method is used to sequence the strain PSBS-2, and the universal primer 27F and 1492R are selected as primers to amplify the 16S rRNA coding gene of the target strain. The universal primer sequences are shown in Table 1, and the PCR amplification system is shown in Table 2.

[0046]

[0047]

[0048] The PCR amplification program is as follows: 95℃, 5min; 95℃ 30s, 56℃ 30s, 72℃ 90s, 25 cycles; 72℃, 10min.

[0049] The purified PCR product was sequenced for 16S RNA, and the sequencing results are shown in the sequence table SEQ ID No. 1. The 16S RNA gene sequence was compared with the NCBI database, the species information of the similar sequence was obtained, and the phylogenetic tree was constructed (see Figure 2 ), and the species information was determined by homologous alignment method, and PSBS-2 was determined as Niallia circulans, which was named as Niallia circulans PSBS-2.

[0050] 1.7 Phosphorus solubilization test of strain PSBS-2

[0051] 1.7.1 Preparation of bacterial liquid

[0052] The strain PSBS-2 was inoculated in LB broth medium, and cultured at 28℃ in a 140r / min shaker to the logarithmic growth phase (about 12h), and then centrifuged at 4℃ and 3000r / min for 10min. The precipitate was washed with sterilized deionized water, resuspended and centrifuged for 3 times, and finally resuspended with 0.9% sodium chloride solution to make the OD 600 value to 1.0, and the bacterial liquid of strain PSBS-2 was obtained.

[0053] 1.7.2 Soluble phosphorus standard curve

[0054] 1.7.2.1 Related reagents

[0055] Potassium antimony tartrate solution: weigh 0.3g potassium antimony tartrate and dissolve in water, and dilute to 100mL.

[0056] Molybdenum antimony stock solution: weigh 10.0g ammonium molybdate and dissolve in 300mL water at 60℃, and cool. Then, 181mL of concentrated H2SO4 is slowly injected into 800mL water, stirred and cooled; then the diluted H2SO4 is injected into the ammonium molybdate solution, stirred and cooled, and then 100mL of 3g / L potassium antimony tartrate solution is added, and finally diluted with water to 2L, and poured into a brown bottle for standby.

[0057] Molybdenum antimony anti-color developing agent: weigh 0.5g ascorbic acid (C6H8O6 left-handed, optical rotation +21-22°) and dissolve in 100mL molybdenum antimony stock solution.

[0058] Standard phosphorus stock solution: after drying KH2PO4 (super pure) at 105℃ for 2h, weigh 0.4390g, dissolve in water, add 5mL of concentrated H2SO4, then add water to constant volume to 1000mL, and store in the refrigerator for long-term use.

[0059] Standard phosphorus solution: take 5mL of standard phosphorus stock solution in a 100mL volumetric flask, and constant volume.

[0060] 1.7.2.2 Draw soluble phosphorus standard curve

[0061] Respectively, 0, 1.00, 2.00, 3.00, 4.00, 5.00 mL standard phosphorus solution (5 mg / L) was taken in 25 mL colorimetric tube, 5.00 mL molybdenum antimony anti-color developing agent was added, and distilled water was added to constant volume, and the concentration of phosphorus standard series solution was 0.00, 0.20, 0.40, 0.60, 0.80, 1.00 mg / L. After placing at room temperature 20-25℃ for 30 min, with 0 mg / L phosphorus standard solution as reference solution, the absorbance value was measured at wavelength 880 nm on spectrophotometer. Finally, the standard curve of soluble phosphorus was drawn with absorbance as ordinate and concentration of standard phosphorus solution as abscissa, and the results were shown in Figure 3 .

[0062] From Figure 3 it can be seen that the standard curve equation of soluble phosphorus is: y=0.7366x+0.0047, the regression equation determination coefficient R 2 =0.9997.

[0063] 1.7.3 Quantitative test of strain PSBS-2

[0064] The phosphorus solubilizing medium was prepared with 50 mL, and 1 mL of strain PSBS-2 liquid was added after sterilization. The test was set up with 3 replicates, and the blank control was without bacteria. It was transferred to 30℃, 140 r / min culture, and 1 mL of culture solution was taken after 72 h centrifugation at 12000 r / min for 5 min. Part of the supernatant was diluted and the effective phosphorus content was determined by molybdenum antimony anti-colorimetric method. The results showed that the effective phosphorus content in the culture solution was 322.63 mg / L, and the phosphorus solubilizing amount of strain PSBS-2 was 322.63 mg / L. The results were shown in Figure 4 .

[0065]

[0066] 1.7.3 Effect of different pH environment on phosphorus solubilizing rate of strain PSBS-2

[0067] Prepare 50mL of phosphate solubilization medium, and use 0.1mol / L NaOH solution and 0.1mol / L HCl solution to adjust the pH value of the phosphate solubilization medium to 4.0±0.1, 5.0±0.1, 6.0±0.1, 7.0±0.1, 8.0±0.1, and 9.0±0.1, respectively. After sterilizing the series of phosphate solubilization medium with different pH values, add 1mL of strain PSBS-2 bacterial solution. Set up 3 replicates for each treatment, and set up a blank control. Transfer to 30℃ and 140r / min for culture. After 72h, take 1mL of culture solution and centrifuge at 12000r / min for 5min. Take part of the supernatant for dilution, determine the effective phosphorus content by molybdenum antimony colorimetric method, and calculate the phosphate solubilization rate. Results are shown in Figure 5 .

[0068]

[0069] Depend on Figure 5 It can be seen that when the pH value is 6.0-9.0, the phosphate solubilization rate of strain PSBS-2 in the solution is more obvious, ranging from 1.65% to 20.20%; among them, when the pH value is 7.0, the phosphate solubilization rate of strain PSBS-2 is the highest.

[0070] 1.7.4 Effect of different culture times on the phosphate solubilization rate of PSBS-2 strain

[0071] Prepare 50mL of phosphate solubilization medium, add 1mL of strain PSBS-2 bacterial solution after sterilization, set up 3 replicates, and use the blank control without bacterial solution. Transfer to 30℃ and 140r / min for cultivation. Take 1mL of culture solution after 24h, 48h, 72h, 96h, and 120h of cultivation, centrifuge at 12000r / min for 5min, and take part of the supernatant for dilution. Determine the available phosphorus content by molybdenum antimony colorimetry and calculate the phosphorus solubility rate. Figure 6 .

[0072] Depend on Figure 6 It can be seen that the phosphate solubility rate of strain PSBS-2 in the solution was 18.16-20.10% within 24-120 hours; among them, when the culture time was 120 hours, the phosphate solubility rate of strain PSBS-2 was the highest.

[0073] 1.7.5 Phosphate solubilization rate of strain PSBS-2 on different phosphorus sources

[0074] Prepare 50mL of calcium phosphate, aluminum phosphate, and iron phosphate culture media, respectively. After sterilization, add 1mL of strain PSBS-2 bacterial solution. The experiment was repeated 3 times, and a corresponding blank control was set. Transfer to 30℃ and 140r / min for incubation. After 72h, take 1mL of culture solution and centrifuge at 12000r / min for 5min. Take part of the supernatant for dilution, and determine the available phosphorus content by molybdenum antimony colorimetry, and calculate the phosphorus solubility rate. The results are shown in the table.Figure 7 .

[0075] Depend on Figure 7 It can be seen that when the phosphorus sources are iron phosphate and calcium phosphate, the strain has phosphate solubilization efficiency, and the phosphate solubilization rates are 0.24% and 18.71%, respectively; among them, the strain PSBS-2 has the greatest phosphate solubilization efficiency for calcium phosphate.

[0076] In summary, the strain PSBS-2 of the present invention has a strong phosphate-solubilizing ability at a pH of 6-9 and 30°C, and is thermophilic and acidophilic (30°C, pH 6-7), which is of great significance for improving soil phosphorus utilization, maintaining soil health, and achieving sustainable development of the agricultural economy. It can also provide strain resource support for the development of related microbial agents, biofertilizers, or soil conditioners.

[0077] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A phosphate-solubilizing strain PSBS-2, characterized in that: The phosphate-solubilizing strain PSBS-2 is used to decompose calcium phosphate and iron phosphate; the taxonomic name of the phosphate-solubilizing strain PSBS-2 is Neillella circulans ( Niallia circulans ), deposited in Guangdong Provincial Microbiological Culture Collection on May 14, 2025, with the deposit number GDMCC No: 66329.

2. Use of the phosphate-solubilizing strain PSBS-2 according to claim 1 in improving soil phosphorus utilization.

3. Use of the phosphate-solubilizing strain PSBS-2 according to claim 1 in the preparation of a microbial agent.

4. Use of the phosphate-solubilizing strain PSBS-2 according to claim 1 in preparing biofertilizer.

5. Use of the phosphate-solubilizing strain PSBS-2 according to claim 1 in preparing a soil conditioner.

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