A strain capable of decomposing multiple insoluble phosphorus sources and its application

Abstract

The present invention discloses a strain having the ability to decompose multiple insoluble phosphorus sources and its application. The present invention relates to the field of microbial technology, and in particular to a strain having the ability to decompose multiple insoluble phosphorus sources and its application. The superpressure Lelliottia provided by the present invention is superpressure Lelliottia (Lelliottia nimipressuralis), and its registration number in the General Microbiological Center of the China Microbiological Culture Collection Administration is CGMCC No.25752. The superpressure Lelliottia BP2 provided by the present invention has the ability to decompose insoluble phosphorus sources, secrete auxin and secrete siderophore, and can promote plant germination and increase plant biomass. The above indicators show that the BP2 bacterial agent has the potential of growth-promoting bacteria and has important application value in the field of grass industry.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a bacterial strain capable of decomposing multiple insoluble phosphorus sources and application thereof. Background Art

[0002] Phosphorus is one of the essential nutrients for the normal growth of plants. It participates in the structural composition and physiological metabolism of plants and is of great significance to the growth and development of plants. However, most of the phosphorus in the soil is fixed by metal ions such as iron, aluminum and calcium, and exists as insoluble phosphates, which are difficult to be used by plants. Therefore, phosphorus limitation is common in terrestrial ecosystems. Grassland is the largest terrestrial ecosystem in my country, accounting for 40% of my country's land area. In recent years, phosphorus resources in grasslands in northern my country have been severely limited, affecting the growth of forage grass.

[0003] The total phosphorus content of grassland soil is very high, but the effective phosphorus content is low. Using biological activation to decompose the insoluble phosphorus in grassland soil is an effective way to increase the available phosphorus in grassland soil. The application of phosphate-solubilizing microorganisms can effectively activate the insoluble phosphorus in grassland soil, reduce costs, and reduce pollution to the environment. Phosphate-solubilizing bacteria have become the most promising option for reducing fertilizer input because of their eco-friendliness, low production costs, and ability to effectively restore soil fertility. Phosphate-solubilizing bacteria can decompose insoluble inorganic phosphorus and organic phosphorus in the soil, increase the content of effective phosphorus in the soil, thereby promoting the absorption and utilization of phosphorus by plants and helping plant growth.

[0004] Hyperbaric Leucorrhea ( Lelliottia nimipressuralis ) is often separated from plants or the air and can produce organic acids such as acetic acid, citric acid and gluconic acid, but there are few reports on its phosphorus solubilization and application. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to improve the growth of plants under low-phosphorus conditions, increase the yield and biomass of plants, and improve the stress resistance of plants.

[0006] In order to solve the above technical problems, the present invention first provides a strain of hyperpressure Leuconostoc.

[0007] The superpressure Leuconostoc provided by the present invention is superpressure Leuconostoc ( Lelliottia nimipressuralis ) CGMCC No.25752, and its registration number at the General Microbiology Center of China Culture Collection Administration is CGMCC No.25752.

[0008] The invention also provides a bacterial agent.

[0009] The bacterial agent provided by the present invention contains the superpressure Leuconostae described above and / or the metabolites or cultures of the superpressure Leuconostae.

[0010] The culture of superpressured Leuconostoc above is a substance obtained by culturing the superpressured Leuconostoc mentioned above in a bacterial culture medium.

[0011] In the above-mentioned microbial agent, in addition to the active ingredient, the microbial agent also contains a carrier. The carrier may be a carrier commonly used in the field of pesticides and biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, soybean flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane.

[0012] The above-mentioned bacterial agents may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0013] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0014] In a specific embodiment of the present invention, the bacterial agent is 10 8 CFU / mL of the overpressured Leucophylla ( Lelliottia nimipressuralis ) BP2 fermentation liquid. Further, the fermentation liquid is prepared according to a method comprising the following steps: the superpressure Leliottia ( Lelliottia nimipressuralis ) BP2 was inoculated into bacterial culture medium and cultured to obtain a concentration of 10 8 The bacterial solution with CFU / mL is the bacterial agent.

[0015] The term "metabolite" refers to the primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process in which microorganisms absorb various nutrients from the outside world and generate substances and energy to maintain life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids and other monomers, as well as various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process in which microorganisms use primary metabolites as precursors to synthesize some substances that have no clear function in the life activities of microorganisms during a certain growth period. The products of secondary metabolism are secondary metabolites, most of which are compounds with relatively complex molecular structures. According to their functions, they can be divided into types such as antibiotics, hormones, alkaloids, toxins, etc.

[0016] In the above, the metabolite can be obtained from the fermentation broth of the overpressure Lelliotetra. The metabolite can be a sterile metabolite of the overpressure Lelliotetra or a bacteria-containing metabolite of the overpressure Lelliotetra. The sterile metabolite of the overpressure Lelliotetra (sterile fermentation filtrate) can be prepared specifically according to the following method: the overpressure Lelliotetra is cultured in a liquid culture medium, and the overpressure Lelliotetra in the liquid culture (fermentation broth) is filtered to remove the sterile metabolite of the overpressure Lelliotetra. The bacteria-containing metabolite of the overpressure Lelliotetra can be prepared specifically according to the following method: the overpressure Lelliotetra is cultured in a liquid fermentation medium, and the fermentation broth is collected, and the fermentation broth is the bacteria-containing metabolite of the overpressure Lelliotetra.

[0017] Furthermore, the bacterial culture medium is LB liquid culture medium.

[0018] The term "culture" refers to a general term for liquid or solid products (all materials in the culture container) that have a microbial community after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0019] The present invention also provides the use of the above-mentioned superpressure Leliottia, the above-mentioned bacterial agent or the above-mentioned culture in at least one of the following:

[0020] (a1) Phosphate dissolution;

[0021] (a2) preparing phosphate-dissolved products;

[0022] (a3) promoting plant growth;

[0023] (a4) preparing products for promoting plant growth;

[0024] (a5) Producing IAA;

[0025] (a6) preparing an IAA-producing product;

[0026] (a7) siderophore production;

[0027] (a8) Preparation of siderophore-producing products

[0028] (a9) Promote plant germination.

[0029] In the above application, the plant growth promotion may be all or part of the following:

[0030] (b1) Promote the increase of plant biomass dry weight under low phosphorus conditions;

[0031] (b2) Promote the increase of plant aboveground biomass dry weight under low phosphorus conditions;

[0032] (b3) Promote the increase of plant underground biomass dry weight under low phosphorus conditions.

[0033] The present invention also provides a method for preparing the above-mentioned bacterial agent, comprising the following steps: using the above-mentioned superpressure Leuconostoc as an active ingredient to obtain the bacterial agent.

[0034] The present invention also provides a bio-organic fertilizer, which contains the super-pressure Lelicia or the bacterial agent or the culture mentioned above.

[0035] The present invention also provides a method for promoting plant growth, comprising the following steps: under low-phosphorus conditions, applying the above-mentioned super-stressed Lelicia or the bacterial agent or the culture to the test plant, thereby promoting plant growth.

[0036] In the above application or method, the plant is a dicotyledonous plant.

[0037] In the above application or method, the dicotyledonous plant is a leguminous plant.

[0038] In the above application or method, the legume plant may be alfalfa or Medicago truncatula.

[0039] Compared with the prior art, the present invention has the following advantages: ① It can decompose a variety of insoluble phosphorus, including: calcium phosphate, calcium phosphate containing 100mM NaCl, hydroxyapatite, and calcium phytate; ② When preparing inorganic mixed phosphorus sources present in grassland soil, overpressured Leuconostoc BP2 can effectively decompose the mixed phosphorus source, and the phosphorus solubilization rate is 43.72%; ③ Overpressured Leuconostoc BP2 can promote the germination of grassland plant Medicago truncatula; ④ Overpressured Leuconostoc BP2 can improve the growth conditions of grassland plants Medicago truncatula and alfalfa, and promote the growth of grassland plants.

[0040] Collection Instructions

[0041] Species name: Leucoderma superbarica

[0042] Latin name: Lelliottia nimipressuralis

[0043] Strain ID: BP2

[0044] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0045] Abbreviation of depository institution: CGMCC

[0046] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0047] Date of deposit: September 19, 2022

[0048] CGMCC Registration Number: CGMCC No.25752 BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the phosphate solubilization circle diagram of strain BP2 on phosphate solubilizing medium.

[0050] Figure 2 This is the phylogenetic tree construction diagram of the 16S rDNA sequence of strain BP2.

[0051] Figure 3 It is strain BP2 ikB Diagram of construction of functional gene phylogenetic tree.

[0052] Figure 4 This is the phosphate solubilization curve of strain BP2 in liquid NBRIP phosphate solubilization medium within 7 days.

[0053] Figure 5 This is the phosphate solubilization of strain BP2 to different insoluble phosphorus.

[0054] Figure 6 This is a picture of the siderophore production of strain BP2 on iron carrier medium.

[0055] Figure 7 This is a comparison chart of the germination rate of Medicago truncatula promoted by strain BP2 from 0 to 10 days.

[0056] Figure 8 This is a comparison chart of the germination of Medicago truncatula treated with strain BP2.

[0057] Fig. 9 This is a diagram showing the effect of strain BP2 in promoting the growth of alfalfa and Medicago truncatula.

[0058] Fig.10 This is a graph showing the biomass changes of alfalfa, Medicago truncatula and Cleistogenes serrata after inoculation with strain BP2. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0060] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0061] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0062] Example 1. Screening, Isolation, Identification and Preservation of Strain BP2

[0063] 1. Isolation of strain BP2

[0064] In July 2020, the roots of the grassland plant Poa annua were dug from the Ulagai Management Area of ​​Xilin Gol League, Inner Mongolia Autonomous Region (118°52′36" E, 45°42′50"N), placed in a sterile centrifuge tube, added with sterile distilled water, centrifuged, and rhizosphere soil was collected. The rhizosphere soil samples were diluted by the ten-fold gradient method, and the dilution concentration was selected as 10 -5 -10 -7 , and then spread on NBRIP medium. Culture at 28℃ for 3 days. Select the strain that can produce obvious and large transparent zone, which is phosphate-solubilizing bacteria and named as strain BP2.

[0065] NBRIP medium: glucose 10.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, pH 7.0-7.5, Ca3(PO4) 25.0 g, agar 18 g-20 g, distilled water 1000 mL. Sterilize with high pressure steam at 115°C for 30 min.

[0066] 2. Identification of Physiological and Biochemical Characteristics of Strain BP2

[0067] The strain BP2 was subjected to 94 phenotypic tests using the Biolog GENE Ⅲ identification plate (Beijing Zhijie Fangyuan Technology Co., Ltd., Cat. No. 1030), including 71 carbon source utilization tests and 23 chemical sensitivity tests. The BP2 bacteria were picked up with a sterile inoculation needle and inoculated into the IF-A inoculation solution (Beijing Zhijie Fangyuan Technology Co., Ltd., Cat. No. 72401) to make a bacterial suspension. The bacterial suspension was aspirated into the pipette tip using an 8-channel pipette, and the bacterial suspension was added to all wells of the microplate in sequence at a volume of 100 µl per well. The culture was cultured in the dark at 30°C for 48 hours, and the reading was performed using the BIOLOG identification system.

[0068] The results showed that strain BP2 could utilize α-D-glucose, L-fructose, α-D-lactose, etc., but could not utilize sucrose, D-fructose, pectin, gelatin, etc. It could withstand acidic environment pH=5 and 1-4% NaCl, and was positive for D-serine, tetradecyl sodium sulfate, and sodium lactate, and negative for nalidixic acid and sodium bromate. The detailed results are shown in Table 1.

[0069] Table 1 Physiological and biochemical properties of strain BP2

[0070]

[0071] 3. Molecular Biological Identification of Strain BP2

[0072] DNA of strain BP2 was extracted and used as a template to amplify the 16S rDNA gene using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACT-3'). gyr The B gene is amplified.

[0073] The amplification system was a 25 μL system: 1 μL DNA template, 1 μL forward primer, 1 μL reverse primer, 12.5 μL 2×Mix, 9.5 μL ddH2O, and the amplification program was: 95°C for 3 min; 95°C for 30 s, 52°C for 30 s, 72°C for 2 min, 35 cycles; 72°C for 10 min. The samples were sent to Beijing Nosai Genome Research Center Co., Ltd. for sequencing. The 16S rDNA gene sequence of strain BP2 is shown in SEQ ID No. 1. ikB The gene sequence is shown in SEQ ID No. 2. After comparison at NCBI, it was found that the 16SrDNA sequence of strain BP2 was similar to Lelliottia nimipressuralis The highest homology, sequence similarity reached 99.7%. Based on 16SrDNA and gyr Phylogenetic tree constructed from B gene sequences Figure 2 , Figure 3 shown.

[0074] Based on the above morphological, physiological and biochemical characteristics and 16S rDNA sequence homology analysis results, the strain BP2 isolated and purified in step 1 was identified as superpressure Leliottia ( Lelliottia nimipressuralis ).

[0075] Hyperbaric Leucorrhea ( Lelliottia nimipressuralis) BP2 was deposited in the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​September 19, 2022, with the deposit number CGMCC 25752. Hereinafter referred to as superpressured Leuconostoc CGMCC No. 25752 or superpressured Leuconostoc BP2 or strain BP2.

[0076] Example 2: Phosphate solubilization characteristics of superpressure Leuconostoc BP2

[0077] In order to determine the phosphate solubilization ability of super-pressured Lelliotetra BP2 on calcium phosphate, calcium phosphate containing 100mM NaCl, aluminum phosphate, iron phosphate, calcium phytate, octacalcium phosphate, calcium hydrogen phosphate, hydroxyapatite and five kinds of mixed inorganic phosphorus, super-pressured Lelliotetra BP2 bacterial solution was added to 9 kinds of phosphate solubilization culture media, and the phosphorus solubilization was determined by molybdenum antimony colorimetric method after cultivation. The specific operation is as follows:

[0078] 1. Prepare phosphate-dissolving medium with calcium phosphate, hydroxyapatite, aluminum phosphate, iron phosphate, calcium hydrogen phosphate, octacalcium phosphate, calcium phytate, calcium phosphate containing 100 mM NaCl, and five mixed inorganic phosphorus as phosphorus sources. The experiment was divided into 9 groups, as follows:

[0079] 1) Calcium phosphate solubilizing medium: glucose 10.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, calcium phosphate 5 g, pH 7.0-7.5, agar 18 g-20 g, distilled water 1000 mL.

[0080] 2) Calcium phosphate-lysed medium containing 100 mM NaCl: glucose 10.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, sodium chloride 5.85 g, calcium phosphate 5 g, pH 7.0-7.5, agar 18 g-20 g, distilled water 1000 mL.

[0081] 3) Hydroxyapatite phosphate-solubilizing medium: glucose 10.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, hydroxyapatite 0.16 g, pH 7.0-7.5, agar 18 g-20 g, distilled water 1000 mL.

[0082] 4) Aluminum phosphate solubilizing medium: glucose 10.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, aluminum phosphate 19.68 mg, pH 7.0-7.5, agar 18 g-20 g, distilled water 1000 mL.

[0083] 5) Ferric phosphate solubilizing medium: glucose 10.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, ferric phosphate 48.71 mg, pH 7.0-7.5, agar 18 g-20 g, distilled water 1000 mL.

[0084] 6) Calcium hydrogen phosphate solubilizing medium: glucose 10.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, calcium hydrogen phosphate 13.16 mg, pH 7.0-7.5, agar 18 g-20 g, distilled water 1000 mL.

[0085] 7) Octacalcium phosphate-dissolving medium: glucose 10.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, octacalcium phosphate 29.35 mg, pH 7.0-7.5, agar 18 g-20 g, distilled water 1000 mL.

[0086] 8) Calcium phytate phosphate-dissolving medium: glucose 10.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, calcium phytate 5 g, pH 7.0-7.5, agar 18 g-20 g, distilled water 1000 mL.

[0087] 9) Mixed phosphate-lysing medium: glucose 10.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl0.2 g, (NH4)2SO40.1 g, aluminum phosphate 10 mg, hydroxyapatite 60 mg, iron phosphate 5 mg, calcium hydrogen phosphate 3 mg, octacalcium phosphate 6 mg, pH 7.0-7.5, agar 18 g-20 g, distilled water 1000 mL.

[0088] 2. Preparation of Super-pressured Lelicia BP2 Inoculum

[0089] The super-pressured Leleuteria BP2 was activated in liquid LB medium. The super-pressured Leleuteria BP2 inoculum was obtained by shaking at 120 r / min and 28°C for 48 hours, and the bacterial count reached 10 9 CFU / mL, as an inoculum of super-pressured Leuconostoc BP2.

[0090] 3. Analysis of phosphate solubilization characteristics of superpressured Leuconostoc BP2

[0091] Centrifuge the super-pressured Lelicia BP2 inoculum at 5000 r / min for 5 min, remove the supernatant, and resuspend the bacterial precipitate in sterile distilled water to adjust the bacterial concentration to 10. 8 CFU / mL, inoculated into 100 mL of 9 phosphate solubilizing media, with an inoculation volume of 1%. The phosphate solubilizing media without inoculation of the agent was used as the control group, and each treatment was repeated three times. The shaker was cultured at 120 r / min for 7 days. The molybdenum antimony colorimetric method was used to test the available phosphorus increment in the culture medium.

[0092] 1) Drawing of phosphorus standard curve: Pipette 0 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of 5 μg / mL phosphorus standard solution into a 50 mL volumetric flask, then add 5 mL of molybdenum antimony colorimetric solution, add deionized water to the scale, and mix well. After standing for 30 minutes, use a spectrophotometer to measure the absorbance at 880 nm. Finally, the phosphorus concentrations in the solution are 0 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, and 0.5 μg / mL, respectively. Then draw a phosphorus standard curve with phosphorus concentration as the horizontal axis and absorbance as the vertical axis. The obtained phosphorus standard curve is: y=0.64914x+0.0011 (x is the concentration of available phosphorus, y is the absorbance value), R 2 =0.99991.

[0093] 2) Determination of soluble phosphorus content in fermentation broth

[0094] Take 1 mL of the fermentation broth with shaking culture and centrifuge at 8000rpm for 10 min, and take the supernatant. After the filtrate is appropriately diluted, take 5 mL of the filtrate and add it to a 50 mL volumetric flask, then add 5 mL of molybdenum antimony colorimetric solution, and add deionized water to the scale. After standing for 30 minutes, use a spectrophotometer to measure the absorbance at 880 nm, and then calculate the soluble phosphorus content based on the phosphorus standard curve. Phosphorus solubility rate = (soluble phosphorus in the BP2 inoculated culture medium - soluble phosphorus in the control group) / total insoluble phosphorus content added.

[0095] The experimental results are shown in Table 2. Figure 4 and Figure 5 . Figure 4The curve of the change of available phosphorus content in calcium phosphate medium within 7 days is shown in Table 2 and Figure 5 It can be seen that the superpressured L. BP2 can significantly solubilize calcium phytate, calcium phosphate, hydroxyapatite and calcium phosphate containing 100mM NaCl, and the phosphate solubilization rates can reach 31.75%, 17.79%, 48.74% and 14.26% respectively. The phosphate solubilization rate of 5 inorganic mixed phosphorus is 43.72%. There is no significant solubilization of iron phosphate, aluminum phosphate, calcium hydrogen phosphate and octacalcium phosphate.

[0096] Table 2 Phosphate solubilization effect of superpressured Leuconostoc BP2

[0097]

[0098] Example 3: Other functions of superpressured Leuconostoc BP2

[0099] The preparation of the superpressure Leuconostoc BP2 bacterial agent is the same as step 2 in Example 2.

[0100] 1. IAA production capacity of hyper-stressed Leleutera BP2

[0101] The IAA yield was determined by colorimetry. Preparation of IAA standard curve: Prepare IAA standard solutions with concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L. Add equal amounts of Salkowski reagent to each concentration of IAA standard solution, mix well, and place in the dark for 30 min, then immediately measure OD using a spectrophotometer. 530 Value, 3 replicates for each concentration gradient. Standard curve: y=0.01008x-0.0026, R 2 =0.99909. The super-pressure Leuconostoc BP2 inoculum (bacterial concentration reached 10 9 CFU / mL) was inoculated into 50 mL LB liquid medium at a 1% inoculation rate, cultured in the dark at 28 °C for 3 days, centrifuged at 10,000 × g for 10 min, and 2 mL of the supernatant was mixed with 2 mL of Salkowski reagent (50 mL, 35% perchloric acid and 1 mL, 0.5 mol / L FeCl3). The sample was placed in the dark at room temperature for 30 min, and the absorbance at 530 nm (OD 530 ), and the IAA production was estimated. The IAA production was 26.42±5.05 mg / L.

[0102] 2. Ability of Superpressured Leuconostoc BP2 to Produce Siderophores

[0103] Preparation of iron carrier solid detection medium: weigh 121 g CAS medium base, 18 g agar, add deionized water to 800 mL, and dissolve by magnetic stirring. Sterilize at 115 °C for 20 min, cool to 60 °C, add 100 mL 10× buffer and 100 mL 10× CAS detection solution preheated at 60 °C to the medium, pour into a culture dish, and inoculate after solidification (the required reagents CAS medium base, 10× buffer and 10× CAS detection solution were purchased from Coolaber, catalog number PM0820-1L).

[0104] The superpressure Leleuteria BP2 was inoculated into the iron carrier solid detection medium with an inoculation needle. After culturing for 7 days, a dissolution zone was found on the iron carrier detection medium. The siderophore dissolution zone was as follows: Figure 6 As shown, it has the ability to produce siderophore.

[0105] 3. Safety testing of hyper-pressured Lelicia BP2

[0106] The overpressured Lelliotetra BP2 was inoculated on my wife's blood agar plate (Beijing Wokai Biotechnology Co., Ltd., Catalog No.: F11359) using an inoculation needle and cultured for 7 days. No hemolysis zone was produced, indicating that the overpressured Lelliotetra BP2 passed the blood agar safety test.

[0107] Example 4: Test on the germination rate promotion of overpressured Leuconostoc BP2

[0108] Seed treatment: Wild Medicago truncatula seeds were collected from grasslands in Inner Mongolia Autonomous Region, soaked in 70% ethanol for 5 min, and rinsed with sterile distilled water 5 times. The seeds were soaked in 5 mL of LB liquid medium cultured with super-pressured Lelicia BP2 inoculum (containing 10 8 CFU / mL) for 1h. The control was 5mL blank LB liquid medium. A disposable sterilized 90 mm round petri dish was selected, three layers of filter paper were laid in the petri dish, soaked with sterile distilled water, and the seeds were inoculated on the petri dish. 100 seeds were inoculated in each petri dish, and 3 replicates were performed for each treatment. The germination of seeds was recorded within 10 days, and the germination rate (%) was calculated as (total number of germinated seeds / total number of seeds) × 100%.

[0109] The experimental results are as follows Figure 7 and Figure 8 As shown: The germination rate of Medicago truncatula was significantly increased by soaking seeds with superpressure Leliconia BP2 inoculant, and the germination rate was 41.33%, while the germination rate of the control group was only 29.33%.

[0110] Example 5: Potted plant growth promotion test

[0111] Preparation of super-pressured Leleuteria BP2 inoculum: Super-pressured Leleuteria BP2 was cultured in LB liquid medium with a shaker at 120 r / min for 24 h, centrifuged at 5000 r / min for 5 min, the supernatant was removed, and the culture was rinsed three times with sterile distilled water. The bacterial solution was resuspended in sterile distilled water to obtain a concentration of 10 8 CFU / mL super-pressured Leuconostoc BP2 inoculum.

[0112] Seeds of Medicago truncatula, Alfalfa flavescentis, and Cleistogenes scabra collected from the wild in Ulagai Grassland, Inner Mongolia Autonomous Region were soaked in 70% ethanol for 5 min and rinsed with sterile distilled water 5 times. A disposable sterilized 90 mm round culture dish was selected, and three layers of filter paper were laid in the culture dish, soaked with sterile distilled water, and the seeds of the above plants were inoculated on the culture dish. After 7 days, the seedlings were transplanted into potted plants. Each treatment was repeated in 8 pots, and one plant was planted in each pot. Seven days after transplanting, BP2 bacterial agent was inoculated with root irrigation at a dose of 1%. The same volume of sterile distilled water was inoculated as a control. The culture medium is sterilized natural grassland soil in Inner Mongolia Autonomous Region. The effect of the potted growth promotion experiment is as follows Fig. 9 The results of the dry weight of the whole plant biomass and the aboveground and underground biomass are shown in Fig.10 The results showed that the super-pressured L. leucoderma BP2 could significantly increase the whole plant biomass dry weight of grassland plants Medicago truncatula and Medicago flavescentis, with an increase rate of 223.83% and 94.01%, respectively. Among them, the BP2 inoculant treatment could significantly increase the aboveground and underground biomass dry weight of Medicago truncatula and the underground biomass dry weight of Medicago flavescentis, with an increase rate of 246.23%, 178.82% and 146.58%, respectively. In the pot experiment, the super-pressured L. leucoderma BP2 did not significantly increase the biomass dry weight of Cleistogenes serrata.

[0113] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Hyperbaric Leucorrhea, characterized in that: The superpressure Leuconostoc is superpressure Leuconostoc ( Lelliottia nimipressuralis )BP2, whose registration number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.25752.

2. A bacterial agent, characterized in that: The bacterial agent contains the superpressure Leuconostoc as described in claim 1.

3. The culture of the superpressure Leucoderma according to claim 1 is a substance obtained by culturing the superpressure Leucoderma according to claim 1 in a bacterial culture medium.

4. Use of the superpressure Leuconostoc as claimed in claim 1, the bacterial agent as claimed in claim 2, or the culture as claimed in claim 3 in at least one of the following: (a1) Phosphate dissolution; (a2) preparing phosphate-dissolved products; (a3) promoting plant growth; (a4) preparing products for promoting plant growth; (a5) Producing IAA; (a6) preparing an IAA-producing product; (a7) siderophore production; (a8) Preparation of siderophore-producing products (a9) promoting plant germination; The plants are Medicago truncatula and / or Medicago fruticosa.

5. A method for preparing the bacterial agent according to claim 2, comprising the following steps: using the superpressure Leuconostoc as described in claim 1 as an active ingredient to obtain the bacterial agent.

6. Bio-organic fertilizer, characterized by: The biological organic fertilizer contains the superpressure Leucoderma described in claim 1 or the bacterial agent described in claim 2 or the culture described in claim 3.

7. A method for promoting plant growth, comprising the following steps: applying the super-pressured Lelicia according to claim 1 or the bacterial agent according to claim 2 or the culture according to claim 3 to the test plant, thereby promoting plant growth; the plant is Medicago truncatula and / or Medicago fruticosa.

Images