Phosphorus solubilizing bacteria with aluminum tolerance and application thereof

Abstract

The present application relates to a kind of phosphorus solubilizing bacteria with aluminum tolerance and its application, the phosphorus solubilizing bacteria is preserved in China Microbial Culture Collection Center on March 15, 2024, and the preservation number is CGMCC NO.30036.The phosphorus solubilizing bacteria of the present application is obtained by using single-cell raman coupling culture technology (scRACS-Culture), directly from environmental sample, for "in situ" metabolic function, single-cell precision, culture-free, fluorescence-labeled-free mining.The phosphorus solubilizing bacteria of the present application has high solubilizing efficiency for inorganic phosphorus and organic phosphorus in insoluble form, and can tolerate high aluminum ion concentration, and certain aluminum ion concentration even has promoting effect on the solubilizing capacity of the phosphorus solubilizing bacteria of the present application.Therefore, the phosphorus solubilizing bacteria of the present application can be used for preparing soil conditioner or fertilizer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microalgae application, and more particularly, relates to a phosphorus solubilizing bacterium with aluminum tolerance and application thereof. BACKGROUND

[0002] Phosphorus is one of the essential macroelements for plant growth and development, and is one of the main factors limiting the productivity of crop production in agricultural production. More than 80% of phosphorus in soil exists in a fixed form, which is difficult for plants to directly absorb and utilize. Especially in acid soil, phosphorus deficiency is often the biggest limiting factor for crop growth.

[0003] Phosphorus solubilizing bacteria are a class of microorganisms with phosphorus solubilizing effect, which participate in a series of rhizosphere processes of soil phosphorus transformation, cause the dissolution and mineralization of insoluble phosphorus, and thus improve the phosphorus utilization capacity of plants.

[0004] Currently, less attention is paid to phosphorus solubilizing bacteria in acid soil. In acid soil, low pH value and high aluminum environment seriously affect the metabolism and function of microorganisms. Therefore, a phosphorus solubilizing bacterium with aluminum tolerance is needed. SUMMARY

[0005] In the research process, the team screened a plurality of strains with phosphorus solubilizing capacity, one of which has high phosphorus solubilizing capacity for inorganic phosphorus and organic phosphorus.

[0006] Based on the above findings, the present application provides a phosphorus solubilizing bacterium with aluminum tolerance, which was preserved in the China General Microbiological Culture Collection Center on March 15, 2024, with a preservation number of CGMCC NO. 30036.

[0007] The present application also provides the application of the phosphorus solubilizing bacterium in dissociating insoluble phosphorus elements into soluble phosphorus elements.

[0008] In one specific embodiment, the insoluble phosphorus elements exist in the form of calcium phosphate or phytate.

[0009] The present application also provides the application of the above-mentioned phosphorus solubilizing bacterium in preparing a soil conditioner.

[0010] The present application also provides a method for dissociating insoluble phosphorus elements into soluble phosphorus elements, comprising the step of applying the above-mentioned phosphorus solubilizing bacterium to an environment where the insoluble phosphorus elements exist.

[0011] In one specific embodiment, the concentration of aluminum ions in the environment where the insoluble phosphorus elements exist is controlled at 0-4 mM.

[0012] In one specific embodiment, the insoluble phosphorus elements exist in the form of calcium phosphate or phytate.

[0013] The phosphorus solubilizing bacteria of the present application has high phosphorus solubilizing efficiency on inorganic phosphorus and organic phosphorus in insoluble form, and can tolerate high aluminum ion concentration, and even shows a promoting effect on the phosphorus solubilizing ability of the phosphorus solubilizing bacteria of the present application under a certain aluminum ion concentration. Therefore, the phosphorus solubilizing bacteria of the present application can be used for preparing soil improver or fertilizer.

[0014] Microorganism preservation

[0015] The algal strain described in the present application is isolated from red soil paddy soil in Jiangxi Province, China, and is identified by 16S rRNA gene sequencing and morphology. The algal strain belongs to Kosakonia oryzendophytica. On March 15, 2024, it was preserved in the China General Microbiological Culture Collection Center of the Institute of Microbiology of the Chinese Academy of Sciences at No. 1 Beichen West Road, Chaoyang District, Beijing, with the preservation number of CGMCC NO. 30036, named Kosakonia oryzendophytica MKA4, and the Latin name of Kosakonia oryzendophytica. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A is the phosphorus solubilizing ring photos of strain MKA4 on solid culture medium, the left photo is the culture medium with calcium phosphate as the only phosphorus source, and the right photo is the culture medium with phytate as the only phosphorus source; B is the columnar statistical chart of the phosphorus solubilizing ability measured by molybdenum blue colorimetric method, the left column is the phosphorus solubilizing ability to calcium phosphate, and the right column is the phosphorus solubilizing ability to phytate.

[0017] Figure 2 A is the phosphorus solubilizing ring photos of strain MKA4 on solid culture medium, the left photo is the culture medium with calcium phosphate as the only phosphorus source, and the right photo is the culture medium with phytate as the only phosphorus source; B is the columnar statistical chart of the phosphorus solubilizing ability measured by molybdenum blue colorimetric method, the left column is the phosphorus solubilizing ability to calcium phosphate, and the right column is the phosphorus solubilizing ability to phytate.

[0018] Figure 3 A is the phosphorus solubilizing ring photos of strain MKA4 on solid culture medium, the left photo is the culture medium with calcium phosphate as the only phosphorus source, and the right photo is the culture medium with phytate as the only phosphorus source; B is the columnar statistical chart of the phosphorus solubilizing ability measured by molybdenum blue colorimetric method, the left column is the phosphorus solubilizing ability to calcium phosphate, and the right column is the phosphorus solubilizing ability to phytate.

[0019] Figure 4 A is the phosphorus solubilizing ring photos of strain MKA4 on solid culture medium, the left photo is the culture medium with calcium phosphate as the only phosphorus source, and the right photo is the culture medium with phytate as the only phosphorus source; B is the columnar statistical chart of the phosphorus solubilizing ability measured by molybdenum blue colorimetric method, the left column is the phosphorus solubilizing ability to calcium phosphate, and the right column is the phosphorus solubilizing ability to phytate. DETAILED DESCRIPTION

[0020] The principles and characteristics of the present application are described below in conjunction with examples, which are used to explain the present application and are not used to limit the scope of the present application.

[0021] 1. Obtaining of phosphorus solubilizing bacteria

[0022] The soil sample was taken from Yingtan, Jiangxi Province, and the rhizosphere soil sample was randomly taken, and the large particles of gravel, soil blocks, etc. were removed by passing through a 0.6 mm screen, and was stored at 4℃.

[0023] Microorganisms were extracted from soil samples using density gradient centrifugation as follows: 1) Weigh 1g of soil into a centrifuge tube, add 5ml of PBS and 25μl of Tween-20, and mix well to prepare a soil suspension; 2) Slowly add the soil suspension to the upper layer of 80% iohexol solution without disturbing the lower layer; 3) Centrifuge at 4℃ and 14000g for 90min to obtain the layered liquid; 4) Take the cell layer (intermediate layer), transfer it to a new centrifuge tube, wash it, and obtain the soil microorganisms.

[0024] MM medium was prepared using calcium phosphate as the sole phosphorus source, with 50% D2O added. Soil microorganisms were cultured in this medium for 24 hours to identify in-situ metabolically functional cells. Subsequently, using a liquid-phase single-cell Raman sorting coupled culture chip (RAGE-Cell Array) that can highly maintain cell viability, these target cells were efficiently sorted based on Raman spectroscopy. Parallel single-cell culture, with one cell per well (One-Cell-One-Tube) corresponding to the metabolomics information, was then performed to obtain live functional bacteria based on in-situ metabolic function. In this invention, cells with a CDR > 0.15 (CDR value is 2040-2300 cm⁻¹ in the Raman spectrum) were screened. -1 The CD peak area divided by 2800-3100cm -1 The single-cell culture with highly efficient phosphate-solubilizing activity (derived from the sum of the CH area and CD peak area) was placed on the surface of S-LB solid medium in a 24-well plate and incubated at 30℃ for 3-5 days. Obvious single colonies were observed. Single colonies were picked and activated in S-LB liquid medium for species identification. Inorganic phosphorus (calcium phosphate) MM liquid medium: 10.0g glucose, 0.5g (NH4)2SO4, 0.3g NaCl, 0.3g KCl, 0.15g MgSO4, 0.03g FeSO4·7H2O, 0.03g MnSO4·H2O, 2.5g Ca3(PO4)2, 1000mL ultrapure water, pH 7.2.

[0025] 2. Screening and identification of strains' phosphate-solubilizing ability

[0026] After sorting and culturing, several phosphate-solubilizing bacteria were obtained. Preliminary screening of their phosphate-solubilizing ability was performed by culturing the strains in solid media with calcium phosphate / phytate as the sole phosphorus source. All strains exhibited a clear phosphate-solubilizing zone around their colonies, confirming their ability to solubilize both inorganic and organic phosphorus. Simultaneously, the molybdenum blue colorimetric method was used to dynamically monitor the soluble phosphorus content during the strains' growth. The soluble phosphorus content in the culture supernatant after 48 hours was used to further assess and compare the phosphate-solubilizing ability of the strains. Results are as follows: Figure 1As shown, strain MKA4 has good phosphorus solubilization ability for both inorganic phosphorus and organic phosphorus.

[0027] 3. Species identification of strain MKA4

[0028] After the strain was activated, it could form colonies with a diameter of 0.5-2.0 mm on the plates prepared from solid nutrient broth medium, and grow under aerobic conditions at 30°C for 24 h, forming light yellow, round, smooth, slightly convex, opaque, and regular edge colonies. Figure 2 Further studies showed that the strain was an aerobic bacterium, did not produce spores, and was gram-positive. The individual bacterial cells were rod-shaped and non-flagellated, and the cell size was (0.7-1.0) x (1.0-1.5) μm.

[0029] The 16S rDNA of the bacteria was amplified using 16S rDNA universal primers, and the sequence is shown as SEQ ID NO: 1. The 16S rDNA was compared in NCBI, and the constructed phylogenetic tree is shown as Figure 3 As shown, it shows the closest genetic relationship with Kosakonia oryzendophytica.

[0030] The strain was preserved in the China General Microbiological Culture Collection Center on March 15, 2024, with the preservation number CGMCC NO.30036, named Kosakonia oryzendophytica MKA4, and the Latin name Kosakonia oryzendophytica.

[0031] 4. Verification of acid aluminum tolerance function of the strain

[0032] With organic phosphorus (phytate) as the only phosphorus source, the change in the phosphorus solubilization ability of the strain under Al 3+ stress was explored.

[0033] 1) Preparation of Al 3+ concentration gradient medium

[0034] Organic phosphorus (phytate) liquid medium: 10.0 g glucose, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g KCl, 0.15 g MgSO4, 0.03 g FeSO4.7H2O, 0.03 g MnSO4.H2O, 10 g phytate, 1000 mL ultrapure water, pH 5.0. 0, 0.3, 0.8, 1, 2, 4, 5, 10, and 50 mM of Al 3+ were added to the organic phosphorus (phytate) liquid medium, respectively, to obtain Al 3+ concentration gradient organic phosphorus (phytate) liquid medium.

[0035] S-LB liquid medium: 100 g of non-acid soil sample sterilized by 121 °C, 15 min high temperature and high pressure was added to 1 L of ddH2O, shaken for 30 min, then the upper layer was placed in a 50 ml centrifuge tube and centrifuged at 10,000 g for 10 min, the supernatant was passed through a 0.45 μm filter, and the collected liquid was the soil extract. Protein peptone 0.5 g, yeast extract 0.25 g, NaCl 10 g, dissolved in neutral soil filtrate 1 L, pH 7.2.

[0036] 2) The isolated phosphorus solubilizing bacteria Kosakonia oryzendophytica MKA4 was inoculated into S-LB liquid medium, and cultured at 30 °C and 180 r / min for 12 h with an OD value of 0.8-1.0. The strain was centrifuged, and the surface culture medium was discarded, then inoculated into Al 3+ The concentration gradient organic phosphorus (phytate) liquid medium was cultured at 30 °C and 180 r / min, and samples were taken every 24 h. The supernatant was diluted by different times, and the soluble phosphorus content in the supernatant was determined by molybdenum blue colorimetry. The uninfected treatment was used as a control group to explore the effect of acid aluminum stress on the phosphorus solubilizing ability of the strain.

[0037] The results are shown in Figure 4 When the Al 3+ concentration was less than 2 mM, the strain state was not affected, and even the phosphorus solubilizing ability was improved due to the stress of Al 3+ The strain had strong acid aluminum tolerance. When the Al 3+ concentration was higher than 4 mM, the phosphorus solubilizing ability of the strain was inhibited, but the strain still showed high phosphorus solubilizing ability under the aluminum ion concentration of 4 mM.

[0038] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A phosphate-solubilizing bacterium with aluminum tolerance Kosakonia oryzendophytica MKA4, characterized in that, The phosphate-solubilizing bacteria were deposited on March 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.30036.

2. The application of the phosphate-solubilizing bacteria according to claim 1 in dissociating insoluble phosphorus into soluble phosphorus, wherein the insoluble phosphorus exists in the form of calcium phosphate or phytate.

3. The application of the phosphate-solubilizing bacteria according to claim 1 in the preparation of soil conditioners or fertilizers.

4. A method for dissociating sparingly soluble phosphorus into soluble phosphorus, characterized in that, The method includes the step of applying the phosphate-solubilizing bacteria of claim 1 to an environment in which the insoluble phosphorus element is present, wherein the insoluble phosphorus element is present in the form of calcium phosphate or phytate.

5. The method according to claim 4, characterized in that, The concentration of aluminum ions in the environment where the sparingly soluble phosphorus element is present is controlled at 0-4 mM.

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