Phosphate-solubilizing bacteria, their preparation methods and applications

Abstract

This invention belongs to the field of microbial technology, and specifically relates to a highly efficient phosphate-solubilizing bacterium, its preparation method, and its application. The phosphate-solubilizing bacterium is named Pantoea cypripediP11, with the biological preservation number CCTCC NO: M2023159. The highly efficient phosphate-solubilizing bacterium Pantoea cypripediP11 proposed in this invention can convert insoluble phosphorus into soluble phosphorus at a maximum content of 663.59 mg / L, which can increase the available phosphorus content in the soil by 1.46 times. This is stronger than the phosphate-solubilizing ability of the existing phosphate-solubilizing bacterium Burkholderia ubonensis, and it can also produce indoleacetic acid and secrete siderophores to promote plant root growth.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a highly efficient phosphate-solubilizing bacterium, its preparation method, and its application. Background Technology

[0002] Phosphorus is an essential element for plant growth and development, but most phosphorus in the soil exists in the form of insoluble phosphate salts, making it difficult for plants to absorb and utilize. To ensure sufficient phosphorus for plant growth, frequent application of phosphate fertilizers is often necessary. However, heavy metal ions in the soil, such as calcium... 2+ Al 3+ and Fe 3+ Plasma can convert soluble phosphorus in phosphate fertilizers into insoluble phosphorus, resulting in low phosphate fertilizer utilization. In addition, frequent application of phosphate fertilizers leads to a series of soil problems such as soil compaction, acidification, and imbalance of nutrients and microbial communities, which in turn deteriorates the soil ecological environment.

[0003] To address this issue, screening for highly efficient phosphate-solubilizing bacteria to dissolve insoluble phosphorus in the soil will be an important way to reduce the use of phosphate fertilizers and develop green agriculture. Utilizing phosphate-solubilizing bacteria to convert insoluble phosphorus into soluble phosphorus that plants can absorb and utilize increases the content of available phosphorus in the soil, improves the utilization efficiency of phosphate fertilizers, thereby reducing the accumulation of insoluble phosphorus in the soil, alleviating soil compaction and other problems, and also reducing the environmental pollution caused by chemical fertilizers. This is of great significance to the sustainable development of agriculture. Summary of the Invention

[0004] To address the above problems, this invention proposes a highly efficient phosphate-solubilizing bacterium, named as follows: Pantoea cypripedii P11, biological preservation number: CCTCC NO: M 2023159.

[0005] Secondly, the present invention also proposes a method using the aforementioned phosphate-solubilizing bacteria. Pantoea cypripedii Fermentation products obtained from P11 fermentation.

[0006] Thirdly, the present invention provides a microbial agent containing the aforementioned phosphate-solubilizing bacteria. Pantoea cypripedii P11 may contain the aforementioned fermentation products.

[0007] Fourthly, the present invention provides a phosphate-solubilizing bacterial fertilizer, wherein the phosphate-solubilizing bacterial fertilizer contains the aforementioned phosphate-solubilizing bacteria. Pantoea cypripedii P11 or the fermentation product described above.

[0008] Fifthly, the present invention provides a soil ecological environment regulator, wherein the regulator contains the aforementioned phosphate-solubilizing bacteria. Pantoea cypripedii P11 or the fermentation product described above.

[0009] In a sixth aspect, the present invention proposes the inclusion of the aforementioned phosphate-solubilizing bacteria. Pantoea cypripedii The application of P11 or the fermentation product or the microbial agent.

[0010] Furthermore, this includes application in the production of indoleacetic acid and / or siderogenic agents.

[0011] Furthermore, this includes application in promoting plant growth and increasing plant yield; even further, the phosphate-solubilizing bacteria Pantoea cypripedii P11. The concentration of the bacterial suspension in the fermentation product and / or inoculum is 10. 8 cfu / mL.

[0012] Furthermore, it also includes applications in phosphorus dissolution, improving phosphate fertilizer utilization, and / or increasing the soluble phosphorus content in the soil.

[0013] Furthermore, the microbial agent is used to prevent and / or improve soil problems caused by the application of phosphate fertilizers; the soil problems include soil compaction, acidification, nutrient imbalance and microbial community imbalance.

[0014] In a seventh aspect, the present invention provides a highly efficient phosphate-solubilizing bacterium. Pantoea cypripedii The preparation method of P11 includes the following steps:

[0015] Rhizosphere soil samples were collected from 5-10 cm below the surface layer in Jinfeng Village, Zhongming Town, Tongling City.

[0016] Weigh 10g of soil sample into a 250mL Erlenmeyer flask, add 90mL of sterile water, and incubate at 180r·min. -1 Prepare a soil suspension by shaking for 30 minutes, and then prepare 10 times dilutions using the 10-fold dilution method. -2 10 -3 and 10 -4 Soil dilution solution;

[0017] 0.1 mL of soil dilution was spread onto inorganic phosphorus solid culture medium, with each gradient repeated three times. The medium was incubated upside down in an incubator at 28°C.

[0018] After 3-5 days, select colonies with obvious phosphate-solubilizing zones and good growth, transfer them again to inorganic phosphorus solid medium using the streak plating method, subculture three times, and then purify to obtain highly efficient phosphate-solubilizing bacteria. Pantoea cypripedii P11.

[0019] Further, the formula of the inorganic phosphorus solid culture medium is as follows: 18.0g agar, 10.0g glucose, 0.3g NaCl, 0.3g KCl, 0.5g (NH4)2SO4, 0.3g MgSO4·7H2O, 0.036g FeSO4·4H2O, 0.03g MnSO4·4H2O, 5.0g Ca3(PO4)2, 1L distilled water, pH value 7.0-7.2.

[0020] The beneficial effects of this invention are:

[0021] The phosphate-solubilizing bacteria proposed in this invention Pantoea cypripedii P11 can convert insoluble phosphorus into soluble phosphorus up to 663.59 mg / L, increasing the available phosphorus content in the soil by 1.46 times. This is stronger than the phosphorus-solubilizing ability of the existing phosphate-solubilizing bacterium Burkholderia ubonensis (publication number CN111662846B). Furthermore, it can produce indoleacetic acid and secrete siderophores, promoting plant root growth.

[0022] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Phosphate-solubilizing bacteria are shown in the embodiments of the present invention. Pantoea cypripedii Morphological diagram of P11 on LB medium;

[0025] Figure 2 This invention illustrates an embodiment based on phosphate-solubilizing bacteria. Pantoea cypripedii Phylogenetic tree of the l6S rDNA gene sequence of P11;

[0026] Figure 3 Phosphate-solubilizing bacteria are shown in the embodiments of the present invention. Pantoea cypripedii morphological diagram of P11 on inorganic phosphorus solid medium;

[0027] Figure 4 Phosphate-solubilizing bacteria are shown in the embodiments of the present invention. Pantoea cypripedii P11 morphological image of spotting on CAS medium. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The following specific embodiments illustrate the highly efficient phosphate-solubilizing bacteria proposed in this invention. Pantoea cypripedii The process of obtaining P11 and its application effects are explained in detail.

[0030] Example 1 Phosphate-solubilizing bacteria Pantoea cypripedii P11 Filtering

[0031] 1.1 Soil sample collection: Soil samples were collected from 5-10 cm below the surface layer in Jinfeng Village, Zhongming Town, Tongling City;

[0032] 1.2 Culture medium preparation:

[0033] Prepare the inorganic phosphorus liquid culture medium as follows: glucose 10.0g, NaCl 0.3g, KCl 0.3g, (NH4)2SO4 0.5g, MgSO4·7H2O 0.3g, FeSO4·4H2O 0.036g, MnSO4·4H2O 0.03g, Ca3(PO4)2 5.0g, distilled water 1L, pH 7.0–7.2. For the inorganic phosphorus solid culture medium, add 18.0g of agar.

[0034] Prepare LB liquid medium: 10.0g tryptone, 5.0g yeast extract, 10.0g NaCl, 1L distilled water, pH 7.0–7.2. For LB solid medium, add 18.0g agar.

[0035] 1.3 Isolation and purification of phosphate-solubilizing bacteria: Weigh 10g of rhizosphere soil sample into a 250mL Erlenmeyer flask, add 90mL of sterile water, and incubate at 180r·min -1 Prepare a soil suspension by shaking for 30 minutes, and then prepare 10 times dilutions using the 10-fold dilution method. -2 10 -3 and 10 -4 Soil dilution solutions were prepared, with 0.1 mL of each solution spread onto inorganic phosphorus solid medium. Each gradient was replicated three times. The cultures were incubated upside down at 28°C. After 3-5 days, colonies with clear phosphate-solubilizing zones and good growth were picked and transferred back to inorganic phosphorus solid medium using the streak plating method. The cultures were subcultured three times, and finally, a phosphate-solubilizing bacterium with strong phosphate-solubilizing ability was purified. Pantoea cypripedii P11. The culture was preserved using glycerol at -20℃ and -80℃, and deposited on February 20, 2023, at the China Center for Type Culture Collection (Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China). The preservation name is […]. Pantoea cypripedii P11, accession number CCTCC NO: M2023159.

[0036] Example 2 Pantoea cypripedii Identification of P11

[0037] 2.1 Morphological characteristics identification: Gram-negative bacteria, appearing as milky-yellow and opaque colonies on LB liquid medium with a smooth surface, such as... Figure 1 .

[0038] 2.2 16S rDNA Identification: PCR amplification was performed using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The obtained 16S rDNA sequence was uploaded to GenBank for sequence alignment, and the strain was identified as... Pantoea cypripedii Phylogenetic tree such as Figure 2 .

[0039] 16S rDNA sequence:>OP810910.1 Pantoea cypripedii The partial sequence of the P11 16S ribosomal RNA gene is as follows:

[0040]

[0041] 2.3 Physiological and biochemical experiments for identification

[0042] Table 1 Pantoea cypripedii P11 Physiological and Biochemical Characteristics Identification (+: Positive; -: Negative)

[0043]

[0044] The results showed that phosphate-solubilizing bacteria Pantoea cypripedii P11 can utilize xylose and raffinose, but cannot utilize lysine, ornithine, sorbitol, calendula alcohol, or phenylalanine. It has no urease activity, can produce organic acids, can produce gas, can produce tryptophanase to decompose tryptophan into indole, has motility, and cannot decompose sulfur-containing amino acids.

[0045] Example 3: Determination of phosphorus solubility

[0046] 3.1 Pantoea cypripedii Qualitative determination of P11 phosphorus solubility

[0047] Purified phosphate-solubilizing bacteria Pantoea cypripedii P11 was dotted in the center of an inorganic phosphorus solid medium and incubated at 28°C for 7 days. Its growth on the inorganic phosphorus solid medium was as follows: Figure 3 As shown, the diameter of the phosphate-solubilizing zone (D) and the colony diameter (d) were measured in three replicates. D / d is an indicator of the relative phosphate-solubilizing ability of phosphate-solubilizing bacteria. Based on the D / d value, the phosphate-solubilizing ability of the strain can be preliminarily judged; the larger the D / d value, the stronger the phosphate-solubilizing ability. (Phosphate-solubilizing bacteria) Pantoea cypripedii The D / d value of P11 is greater than 3, which proves that it has strong phosphorus solubility. The specific results are shown in Table 2.

[0048] Table 2 Phosphate-solubilizing bacteria Pantoea cypripedii P11 phosphorus solubility

[0049]

[0050] 3.2 Pantoea cypripedii Quantitative determination of P11 phosphorus solubility

[0051] Phosphate-solubilizing bacteria Pantoea cypripedii P11 was inoculated into inorganic phosphorus liquid medium, with uninoculated inorganic phosphorus liquid medium as the control group. The mixture was cultured in a shaker at 28 ℃ for 7 days. The amount of dissolved phosphorus in the supernatant was detected every 24 hours using the molybdenum antimony colorimetric method. The results are shown in Table 3.

[0052] Table 3 Phosphate-solubilizing bacteria Pantoea cypripedii P11 solubility of tricalcium phosphate

[0053]

[0054] It can be seen that phosphate-solubilizing bacteria Pantoea cypripedii After P11 was inoculated into inorganic phosphorus liquid culture medium, the soluble phosphorus content in the solution gradually increased. After 4 days of culture, the soluble phosphorus content reached 630.35 mg / L, and then tended to stabilize. On the 6th day, the soluble phosphorus content reached a maximum of 663.59 mg / L, and on the 7th day of culture, the soluble phosphorus content was 650.99 mg / L.

[0055] Example 4 Pantoea cypripedii P11 Indoleacetic Acid (IAA) Production Capacity Determination

[0056] Salkowski colorimetric solution: 15 mL 0.5 mol / L FeCl3, 300 mL concentrated sulfuric acid (specific gravity 1.84), and 500 mL distilled water. Mix before use and store protected from light. Add 4 mL of the solution to 1 mL of sample. Then, add the isolated and purified phosphate-solubilizing bacteria. Pantoea cypripedii P11 was inoculated into LB liquid medium containing L-tryptophan and cultured at 30℃ for 6 days. The bacterial suspension was centrifuged at 5000 r / min for 10 min, and 1 ml of the supernatant was mixed with 4 ml of Salkowski colorimetric solution. After standing in the dark for 30 min, the absorbance at 530 nm was measured. Triple replicates were set up, and measurements were taken every 24 hours. The IAA content in the phosphate-solubilizing bacteria fermentation broth was calculated based on the standard curve, which represents the IAA production capacity of this phosphate-solubilizing bacteria. The results are shown in Table 4.

[0057] Table 4 Phosphate-solubilizing bacteria Pantoea cypripedii P11 IAA content

[0058]

[0059] It can be seen that phosphate-solubilizing bacteria Pantoea cypripedii After P11 was inoculated into LB liquid medium, the IAA content in the solution gradually increased, reaching a maximum of 41.56 μg / ml after 4 days of culture.

[0060] Example 5 Pantoea cypripedii P11 ferritin determination

[0061] Phosphate-solubilizing bacteria Pantoea cypripediiP11 was inoculated onto CAS agar plates, and a yellow halo appeared around the strain. CAS plate culture medium: Solution A: 60.5 mg CAS, 50 mL distilled water, 10 mL ferric chloride solution (containing 1 mM FeCl3·6H2O, 10 mM HCl); Solution B: 72.9 mg HDTMA (hexadecyltrimethylammonium bromide), 40 mL distilled water; Solution C: Solution A added to Solution B, mixed well, sterilized at 121℃ for 15 min; 2 mL 1 mM calcium chloride solution, 2 mL 1 mM magnesium sulfate solution, adjusted pH to 6.8~7.0. Distilled water was brought to a final volume of 1000 mL, 18 g agar was added, sterilized at 121℃ for 15 min, and when the temperature dropped below 60℃, 50 mL of Solution C was added, mixed well, and plates were prepared. The specific method for determining siderophore production is as follows: Phosphate-solubilizing bacteria... Pantoea cypripedii P11 was spotted in the center of CAS agar plates and incubated at 28°C for 48 hours. The color change around the colonies was observed and recorded. The ratio of siderophore circle diameter to phosphate-solubilizing bacteria colony diameter (D / d) was 3.5. Figure 4 As shown.

[0062] Example 6 Pantoea cypripedii Evaluation of phosphorus solubility in soil (P11)

[0063] Take 5.0g of soil, add 25mL of sterile water, sterilize at 121℃ for 30min, let cool, and then inoculate with phosphate-solubilizing bacteria. Pantoea cypripedii P11 5mL, with a parallel control group of 5mL LB liquid medium inoculated simultaneously, three replicates per group. After standing at 25℃ for 7 days, the supernatant was collected, and the soluble available phosphorus content was determined by the molybdenum-antimony colorimetric method.

[0064] The soluble available phosphorus content in the non-inoculated control group was 48.01 mg / kg, while the soluble available phosphorus content in the experimental group was 118.34 mg / kg. This demonstrates the presence of phosphate-solubilizing bacteria. Pantoea cypripedii P11 can effectively increase the soluble phosphorus content in the soil by 70.33 mg / kg, which is 1.46 times higher than the soluble phosphorus content in the soil.

[0065] Example 7 Pantoea cypripedii P11 Growth-promoting experiment

[0066] Take plump barley seeds, rinse them 5 times with sterile water, and place 50 seeds in sterile water (control group) and 10 in sterile water (control group). 8 cfu / mL Pantoea cypripedii The seeds were soaked in P11 bacterial suspension (experimental group) for 1 min, and then planted in flowerpots containing sterilized soil. The groups were divided into experimental group and control group and cultured at 28℃ for 10 days. The results are shown in Table 5.

[0067] Table 5 Phosphate-solubilizing bacteria Pantoea cypripediiP11 promotes growth

[0068]

[0069] As shown in Table 5, phosphate-solubilizing bacteria Pantoea cypripedii P11 has a good growth-promoting effect on barley seeds.

[0070] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A phosphate-solubilizing bacterium, characterized in that, The phosphate-solubilizing bacteria were named as follows: Pantoea cypripedii P11, biological preservation number: CCTCC NO: M 2023159.

2. A microbial agent, characterized in that, Contains the phosphate-solubilizing bacteria of claim 1 Pantoea cypripedii P11.

3. A phosphate-solubilizing bacterial fertilizer, characterized in that, Contains the phosphate-solubilizing bacteria of claim 1 Pantoea cypripedii P11.

4. A soil ecological environment conditioner, characterized in that, Contains the phosphate-solubilizing bacteria of claim 1 Pantoea cypripedii P11.

5. The phosphate-solubilizing bacteria according to claim 1 Pantoea cypripedii The use of the bacterial agent described in claim 11 or claim 2 in the production of indoleacetic acid and / or siderophores.

6. The phosphate-solubilizing bacteria according to claim 1 Pantoea cypripedii The application of the microbial agent described in claim 11 or claim 2 in promoting barley growth and increasing barley yield.

7. The application according to claim 6, characterized in that, Phosphate-solubilizing bacteria Pantoea cypripedii The bacterial suspension concentration in P11 and / or the bacterial agent is 10. 8 cfu / ml.

8. The phosphate-solubilizing bacteria according to claim 1 Pantoea cypripedii Application of the microbial agent described in P11 or claim 2 in the dissolution of phosphorus, improvement of phosphate fertilizer utilization and / or increase of soluble phosphorus content in soil.

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