Efficient phosphate-solubilizing bacteria and application thereof

Abstract

The embodiment of the invention discloses an efficient phosphate-solubilizing bacterium and application thereof. The phosphate-solubilizing bacterium is acinetobacter calcoaceticus AP1, is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms on September 29, 2024, and has the preservation number of CGMCC No.32104. The phosphate-solubilizing bacterium can be used for preparing the phosphate-solubilizing bacterium. The phosphate-solubilizing bacterium provided by the invention is high in phosphate-solubilizing speed, can dissolve insoluble phosphorus in a short time, provides a novel phosphate-solubilizing strain for soil with low phosphorus utilization rate, can improve the phosphorus utilization rate of the soil and reduce the application of phosphate fertilizer, and has a good application prospect in the aspect of microbial fertilizer production.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of microbial technology, and in particular to a highly efficient phosphate-dissolving bacteria and an application thereof. Background Art

[0002] Phosphorus is one of the important elements necessary for plant growth and one of the three essential nutrients for plants. It is involved in many aspects such as plant energy transfer, the synthesis of genetic material and the construction of cell structure, and plays a vital role in the growth and development of plants. However, phosphorus deficiency exists in large areas of agricultural soil in my country, and phosphorus in the soil is not as easily absorbed by plants as nitrogen and potassium. It often exists in the form of insoluble phosphates, which is difficult for plant roots to directly absorb and utilize. In order to solve the current situation of soil phosphorus deficiency, in the process of agricultural planting in my country, phosphorus is generally supplemented in the form of increased application of phosphorus fertilizers. However, the long-term application of phosphorus fertilizers not only increases the cost of agricultural production, but may also lead to soil structure degradation, groundwater pollution, eutrophication of water bodies and other environmental problems.

[0003] In recent years, the technology of using microorganisms to activate insoluble phosphorus in the soil has gradually attracted attention. For example, phosphorus-dissolving bacteria and phosphate-dissolving bacteria can convert insoluble calcium phosphate, aluminum phosphate, etc. in the soil into soluble phosphorus that can be absorbed and utilized by plants by secreting organic acids or enzymes. This method of activating phosphorus through microorganisms can not only reduce dependence on chemical phosphorus fertilizers, but also improve the utilization efficiency of phosphorus in the soil, improve soil structure, and reduce the risk of environmental pollution, thereby achieving the goal of green agriculture.

[0004] However, the existing phosphate-dissolving bacteria generally have the problem of insignificant field application effects. The main reason may be that environmental conditions have a great influence on microbial activity, phosphate-dissolving bacteria are difficult to survive and colonize in the soil for a long time, and different strains have great differences in adaptability to soil phosphorus sources. In view of this situation, screening out a highly efficient phosphate-dissolving bacteria strain to improve the utilization rate of crops to phosphorus in the soil, thereby reducing the application of phosphate fertilizer, is of great strategic significance for the development of my country's crop industry and the reduction of environmental pollution and energy waste. Summary of the invention

[0005] To this end, an embodiment of the present invention provides a highly efficient phosphate-dissolving bacteria and application thereof.

[0006] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to the first aspect of an embodiment of the present invention, the present invention provides a phosphate-solubilizing bacterium, wherein the phosphate-solubilizing bacterium is Acinetobacter calcoaceticus AP1, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 29, 2024, with a deposit number of CGMCC No.32104.

[0008] According to a second aspect of an embodiment of the present invention, the present invention provides a bacterial agent containing the phosphate-dissolving bacteria as described above.

[0009] According to a third aspect of an embodiment of the present invention, the present invention provides a phosphate-dissolving bacterial fertilizer containing the phosphate-dissolving bacteria as described above.

[0010] According to a fourth aspect of an embodiment of the present invention, the present invention provides the use of the phosphate-dissolving bacteria, the bacterial agent, and the phosphate-dissolving bacterial fertilizer as described above in dissolving phosphate, improving the utilization rate of phosphate fertilizer, or increasing the soluble phosphorus content in soil.

[0011] Furthermore, the soil contains sparingly soluble phosphates.

[0012] Furthermore, the poorly soluble phosphate is calcium phosphate.

[0013] The embodiments of the present invention have the following advantages:

[0014] The phosphate-dissolving bacteria provided by the invention has a fast phosphate-dissolving speed and can dissolve poorly soluble phosphorus in a relatively short period of time, thus providing a new type of phosphate-dissolving strain for soil with low phosphorus utilization rate, thereby improving soil phosphorus utilization rate and reducing the application of phosphate fertilizers, and has good application prospects in the production of microbial fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0016] Figure 1 A colony characteristic diagram of the phosphate-dissolving bacteria provided by the present invention;

[0017] Figure 2 A phylogenetic tree of 16S rRNA gene sequences of the phosphate-solubilizing bacteria and related species provided by the present invention;

[0018] Figure 3 The results of the determination of the phosphate-dissolving effect of the solid culture medium of phosphate-dissolving bacteria provided by the present invention;

[0019] Figure 4The present invention provides the change of water-soluble phosphorus content over time. DETAILED DESCRIPTION

[0020] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1

[0022] Isolation and identification of phosphate-dissolving bacteria (Acinetobacter calcoaceticus)

[0023] 1. Isolation of Acinetobacter calcoaceticus

[0024] (1) Soil sample collection: Soil samples were collected from corn soil 0-20 cm below the surface in Eryuan County, Dali City, Dali Bai Autonomous Prefecture, Yunnan Province.

[0025] (2) Culture medium preparation:

[0026] Prepare inorganic phosphorus liquid culture medium: 10.0g glucose, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g magnesium sulfate, 0.03g manganese sulfate, 0.3g potassium sulfate, 0.03g ferrous sulfate, 5.0g calcium phosphate, 1L distilled water, pH 7.0-7.5, and add 15.0g agar to the inorganic phosphorus solid culture medium.

[0027] Prepare LB liquid medium: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of NaCl, 1 L of distilled water, pH 7.0-7.2, and LB solid medium plus 18.0 g of agar.

[0028] (3) Isolation and purification of phosphate-solubilizing bacteria: Weigh 10 g of soil sample into a 250 ml conical flask, add 90 mL of sterile water and shake at 180 r / min for 30 min to prepare a soil suspension. Then use the 10-fold dilution method to prepare 10 -1 , 10 -2 and 10 -30.1 mL of soil dilution was taken and spread on inorganic phosphorus solid culture medium. Each gradient was repeated three times and cultured upside down in a 28°C incubator. After 0.5 days, colonies with obvious phosphate-dissolving rings and good growth were picked out and transferred to inorganic phosphorus solid culture medium again by streaking method. After three subcultures, a phosphate-dissolving bacterium Acinetobacter calcoaceticus with strong phosphate-dissolving ability was finally purified and preserved with glycerol at -80°C.

[0029] 2. Identification of Acinetobacter calcoaceticus

[0030] (1) Morphological identification: Gram-negative bacteria appear as white, opaque colonies with smooth surfaces on inorganic phosphate media, such as Figure 1 .

[0031] (2) 16S rRNA gene identification:

[0032] PCR amplification was performed using bacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′), and the resulting 16S rRNA gene sequence was uploaded to Genbank for sequence comparison. The strain was identified as Acinetobacter calcoaceticus, and the phylogenetic tree is shown in Figure 2 .

[0033] 16S rRNA gene sequence: >0P810910.1 Pantoea cypripedii P1116 S ribosomal RNA gene, partial sequence see SEQ ID NO: 1.

[0034] The strain was deposited, the deposit name is Acinetobacter calcoaceticus AP1, the deposit unit is General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is September 29, 2024, and the deposit number is CGMCC No.32104.

[0035] Example 2

[0036] Phosphate solubilization ability and effect detection of Acinetobacter calcoaceticus

[0037] 1. Determination of the phosphate solubilizing effect of Acinetobacter calcoaceticus solid culture medium

[0038] The purified phosphate-dissolving strain was streaked onto LB solid medium (composition: 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g NaCl, 1 L distilled water, pH 7.0-7.2, 18.0 g agar), and the activated strain was cultured at 28°C. A single colony was picked and inoculated into LB liquid medium (composition: 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g NaCl, 1 L distilled water, pH 7.0-7.2), and cultured at 28°C and 180 rpm until the bacterial density OD 600 =0.5-0.9 to obtain bacterial suspension.

[0039] Take 200 μL of bacterial suspension (OD 600 About 0.8) was dropped in the center of the inorganic phosphorus solid culture medium (composition: 10.0g glucose, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g magnesium sulfate, 0.03g manganese sulfate, 0.3g potassium sulfate, 0.03g ferrous sulfate, 5.0g calcium phosphate, 1L distilled water, pH 7.0-7.5, agar 15.0g), placed in a 28°C constant temperature incubator and cultured for 5 days. The ratio (D / d) of the diameter of the phosphate solubilization circle (D) to the diameter of the colony (d) was measured to preliminarily determine the phosphate solubilization ability of the strain (repeated 3 times under the same treatment). The results showed that the ratio (D / d) of the diameter of the phosphate solubilization circle (D) to the diameter of the colony (d) were 1.62, 1.59, and 1.66, respectively. For specific effects, see Figure 3 .

[0040] 2. Determination of the phosphate solubilizing effect of Acinetobacter calcoaceticus in liquid culture medium

[0041] In a 500 mL Erlenmeyer flask, 50 mL of sterilized inorganic phosphorus liquid medium (composition: glucose 10.0 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, magnesium sulfate 0.3 g, manganese sulfate 0.03 g, potassium sulfate 0.3 g, ferrous sulfate 0.03 g, calcium phosphate 5.0 g, distilled water 1 L, pH 7.0-7.5) was placed and inoculated with 200 μL of bacterial suspension (OD 600 The culture was carried out at 25°C and 200 r / min for 3 days, and the dissolved phosphorus content within 60 hours was measured at a wavelength of 700 nm by molybdenum antimony spectrophotometry. Figure 4 The amount of dissolved phosphorus in the inorganic phosphorus liquid medium showed a trend of first increasing and then stabilizing. After the strain was inoculated, the amount of dissolved phosphorus increased rapidly, reaching a maximum value at 12h, and then the growth tended to be slow. The strain released a large amount of soluble phosphorus in the inorganic phosphorus liquid medium, with the highest amount reaching 124mg / L, and the phosphorus dissolution rate was very fast, reaching 45mg / L·h at the fastest.

[0042] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

[0043] Sequence Listing

[0044] <110> China Agricultural University

[0045] <120> A kind of high-efficiency phosphate-dissolving bacteria and its application

[0046] <130> GG241396922A

[0047] <160> 1

[0048] <170> PatentIn version 3.5

[0049] <210> 1

[0050] <211> 1938

[0051] <212> DNA

[0052] <213> Artificial sequence

[0053] <400> 1

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Claims

1. A phosphate-dissolving bacterium, characterized in that: The phosphate-soluble bacteria is Acinetobacter calcoaceticus AP1, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 29, 2024, with a deposit number of CGMCC No.32104.

2. A bacterial agent, characterized in that Contains the phosphate-dissolving bacteria as described in claim 1.

3. A phosphate-dissolving bacterial fertilizer, characterized in that: Contains the phosphate-dissolving bacteria as described in claim 1.

4. Use of the phosphate-dissolving bacteria according to claim 1, the bacterial agent according to claim 2, and the phosphate-dissolving bacterial fertilizer according to claim 3 in dissolving phosphate, improving the utilization rate of phosphate fertilizer, or increasing the soluble phosphorus content in soil.

5. The use according to claim 4, characterized in that: The soil contains poorly soluble phosphates.

6. The use according to claim 5, characterized in that: The poorly soluble phosphate is calcium phosphate.

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