Pantoea dispersa YWZ-3 and application thereof in acid-resistant and aluminum-toxicity-resistant

Abstract

This invention relates to the fields of agriculture and microbiology, and discloses a strain of *Umbrella dispersalis* YWZ-3 and its application in acid resistance and aluminum toxicity mitigation. The strain is *Umbrella dispersalis* (… Dispersed pantoea YWZ-3 was deposited on April 2, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, with accession number CCTCC M 2026581. This invention relates to the dispersible pantothenic acid (PVA) Pant disperse YWZ-3 possesses the functions of alkali production, aluminum detoxification, phosphorus solubilization, potassium solubilization, nitrogen fixation, EPS production, and plant growth hormone production, making it suitable for use in acidic environments with high aluminum concentrations. Through studies of the strain's biochemical characteristics and the physiological functions of inoculation with YWZ-3, it was revealed that this strain has the ability to remove aluminum toxicity, significantly increase environmental pH, and produce plant hormones under aluminum stress, thus enhancing its potential in agricultural production and sustainable development.

Description

Technical Field

[0001] This invention relates to the fields of agricultural cultivation and microbiology, specifically to a strain of dispersible pantothecin YWZ-3, and its applications, including acid resistance and aluminum detoxification. Background Technology

[0002] Aluminum is the most abundant metallic element in the Earth's crust, accounting for approximately 8.3%. Under normal conditions, it exists as insoluble silicates or aluminum oxide, which are non-toxic to plants. However, under acidic conditions (pH < 5), some aluminum will exist as exchangeable aluminum (Al₂O₃). 3+ Aluminum exists in the form of aluminum (Al), which is highly toxic to plants. Acidic soils are the primary environment for aluminum toxicity. Soil acidification activates aluminum in the soil, inhibiting root elongation and affecting the absorption and utilization of water and nutrients by plant roots, thus impacting plant growth and development. Most plants experience growth inhibition under extremely low concentrations (micromolar levels) of aluminum stress. Al in cultivated crops... 3+ Aluminum can also accumulate in the human body through the food chain, harming human health. Aluminum levels exceeding a certain threshold can cause irreversible damage. Aluminum toxicity is considered one of the most significant limiting factors for crop yields in acidic soils. Therefore, research on aluminum removal from acidic soils is of great importance.

[0003] Currently, the main method for treating aluminum toxicity is to apply agricultural lime (calcium carbonate) to raise the soil pH value, thereby reducing the concentration of free aluminum and mitigating its toxicity to crops. However, these measures have drawbacks such as insufficient deep soil improvement, easy soil compaction, and easy soil acidification. Therefore, developing a new environmentally friendly and efficient solution to aluminum toxicity is imperative.

[0004] Biological methods offer low-pollution and high-efficiency aluminum removal capabilities. This method is an emerging soil improvement technology that alleviates the toxicity of aluminum to plants by introducing beneficial microorganisms (such as rhizosphere growth-promoting bacteria and mycorrhizal fungi). Compared with traditional methods such as applying lime, its main advantages lie in the fact that microorganisms can directly reduce aluminum toxicity by secreting organic acids to chelate active aluminum ions in the soil, and indirectly reduce aluminum toxicity in the soil by alkalizing the local pH of the rhizosphere soil through their own metabolic activities. Beneficial microorganisms can live in symbiosis with plants, improving the soil microenvironment by secreting plant regulatory hormones and enhancing the plant's antioxidant defense capabilities, thereby increasing the crop's tolerance to aluminum stress and promoting crop growth.

[0005] Although biological methods have many advantages, existing technologies still have some problems and limitations. For example, commonly used microorganisms such as Bacillus cereus, Burkholderia, and Pseudomonas only remove 20%–30% of aluminum under laboratory shake-flask conditions. Burkholderia isolated from sugarcane planting soil showed a removal rate of only 22.42% after 48 h of shake-flask culture in LB medium (pH 4.5) containing 1 mmol / L (27 mg / L) of free aluminum, indicating a low aluminum removal capacity. This significantly limits the application of biological methods for aluminum detoxification. Therefore, exploring new, highly efficient aluminum-removing microorganisms and their applications has become an urgent issue in the research of biological methods for aluminum detoxification. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a dispersible pantothenic bacterium, YWZ-3, with functions including aluminum solubilization, phosphorus solubilization, nitrogen fixation, potassium solubilization, pH increase, and secretion of plant hormones and extracellular polysaccharides. This invention isolates a novel aluminum-solubilizing bacterium from aluminum-rich soil. It can grow under acidic, high-aluminum-toxicity conditions (e.g., pH 3-6, Al content 20-60 mg / L), reducing free aluminum concentration and producing plant hormones such as IAA and ACC deaminase. It has the potential to become a special fertilizer strain. This research aims to provide excellent strain resources for the development of microbial fertilizers.

[0007] A strain of acid-resistant dispersible pantothecin YWZ-3 was deposited on April 2, 2026, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, with accession number CCTCC NO: M 2026581, and classified as follows: Pantoea dispersa YWZ-3.

[0008] The dispersible pantothecin YWZ-3 has the following morphological and physiological and biochemical characteristics: after being cultured on LB solid medium at 30℃ for 1-3 days, the colonies are round, slightly yellow, with neat, smooth and moist edges, and raised in the middle, with a diameter of 1-3 mm; it can grow with glucose as a carbon source, is urease negative, and has positive nitrogen fixation ability.

[0009] The dispersible pantothenic acid YWZ-3 has the potential to be used as an agricultural microbial agent, applied directly to the root soil of plants suffering from aluminum toxicity.

[0010] The dispersed pantothenic bacteria can achieve a biomass (OD600) of over 4.0 when cultured for 96 h in an acidic, aluminum-rich environment, such as pH 3-6 and active aluminum concentration of 20-60 mg / L, with almost no biomass reduction compared to neutral conditions.

[0011] The dispersed pantothecin has a high capacity for phosphorus solubilization, potassium solubilization, production of plant hormones and extracellular polysaccharides, and also has a certain nitrogen-fixing capacity.

[0012] Based on this, the dispersed pantothenic bacteria have the ability to raise the pH value of acidic environments.

[0013] An alumina-dissolving bacterium, said alumina-dissolving bacterium comprising the aforementioned dispersible pantothecin YWZ-3.

[0014] A microbial inoculant comprising the aforementioned dispersible pantothecin YWZ-3.

[0015] A culture obtained by fermentation of the dispersible pantothenic acid YWZ-3.

[0016] The application of the aforementioned dispersible pantothenic acid YWZ-3, the aforementioned microbial agent, or the aforementioned culture in reducing the concentration of free aluminum. Specifically, in an environment with a pH of 3-6 and an aluminum concentration of 20-60 mg / L, the environment can be a soil environment or a water environment, wherein the water environment and soil environment can also be a coexistence of both.

[0017] To simulate the above-mentioned environment, in some embodiments, a culture medium was used as the environment for aluminum reduction experiments. In some preferred cases, after culturing for 96 h in a culture medium with pH 4.5 and aluminum concentrations of 20, 40, and 60 mg / L, the free aluminum concentration can be reduced by more than 55%, more preferably by more than 60%, and even more preferably by more than 80%.

[0018] The application of the aforementioned dispersible pantothecin YWZ-3, the aforementioned microbial agent, or the aforementioned culture in improving the pH value of aquatic or soil environments, with a pH of 3-6.

[0019] To simulate the above environment, in some embodiments, liquid culture medium is used as the environmental object. For example, after culturing in liquid culture medium with pH values ​​of 3, 4, 5, 6 or 7 for 96 h, the pH can be raised to about 8.5.

[0020] The application of the aforementioned dispersible pantothecin YWZ-3, the aforementioned microbial agent, or the aforementioned culture in soil or aquatic environments to dissolve insoluble phosphorus or potassium. (The phosphorus source for the insoluble phosphorus is one or more of tricalcium phosphate or lecithin, and the potassium source for the insoluble potassium is potassium feldspar).

[0021] To simulate the aforementioned soil or water environment, in some embodiments, a solid culture medium is used as the environmental object, which also contains insoluble phosphorus or potassium, and the experiment is conducted in an environment in which insoluble phosphorus or potassium is dissolved.

[0022] The application of the aforementioned dispersible pantothenic acid YWZ-3, the aforementioned microbial inoculant, or the aforementioned culture in nitrogen fixation in soil or aquatic environments. (The nitrogen fixation refers to the ability to fix nitrogen from the air; after three subcultures in Assumption medium without a nitrogen source, 0.18 mg / L of ammonia nitrogen can be fixed in 5 days).

[0023] To simulate the aforementioned soil or aquatic environments, in some embodiments, nitrogen fixation experiments were conducted from the air using Ashube nitrogen-free culture medium.

[0024] The application of the dispersible pantothecin YWZ-3, the microbial agent, or the culture in shake flask culture for the production of plant hormones, wherein the plant hormone is one or more of 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) and indole-3-acetic acid (IAA).

[0025] The aforementioned dispersible pantothecin YWZ-3, the aforementioned microbial agent, or the aforementioned culture can produce extracellular polysaccharides in an acidic aluminum-rich medium (pH 4.5, active aluminum ion content within 60 mg / L), with a content between 328 and 453 mg / L.

[0026] The application is to be carried out in acidic environments (pH 3-6) and / or environments under aluminum stress (20-60 mg / L). These environments refer to aquatic environments, soil environments, or a combination of both.

[0027] In the application of the dispersible pantothenic acid YWZ-3, the microbial agent, or the culture in promoting plant growth performance, preferably, the specific method of application is as follows: after preparing the dispersible pantothenic acid YWZ-3 into a bacterial solution, the bacterial solution is injected into the root soil of the plant.

[0028] Compared with existing technologies, the advantages of this invention are: The aluminum-dissolving bacteria of the present invention Pantoea dispersa YWZ-3 exhibits beneficial effects such as high acid resistance, high aluminum resistance, phosphorus solubility, potassium solubility, nitrogen fixation, IAA production, ACC deaminase production, extracellular polysaccharide production, and pH enhancement in the culture environment. This bacterium has the potential to remediate red acidic soils in southern China and farmland contaminated with free aluminum. Specifically, it can reduce the concentration of free aluminum ions in the environment by increasing the environmental pH and through the chelating effect of extracellular polysaccharides. Furthermore, when applied to farmland as an agricultural microorganism, it can increase the environmental pH, promote plant growth, alleviate soil compaction and acidification, and enhance soil fertility. It can reduce the use of chemical products such as lime in traditional methods of aluminum toxicity remediation, and has broad application prospects. Attached Figure Description

[0029] Figure 1 Colony diagram of dispersed pantothenic acid YWZ-3 on LB medium.

[0030] Figure 2 Phylogenetic tree of dispersible pantothenic acid YWZ-3.

[0031] Figure 3 The strain characteristics of the dispersing pantothenic bacteria YWZ-3 are shown in the diagram (A decomposes inorganic phosphorus, B decomposes organic phosphorus, C decomposes potassium, D fixes nitrogen).

[0032] Figure 4 A graph showing the characteristics of dispersing pantothenic acid YWZ-3 cultured at different pH values ​​without stress (A: growth rate; B: pH value).

[0033] Figure 5 The growth curves of dispersible pantothenic acid YWZ-3 under different aluminum concentration stresses (pH 4.5) were obtained.

[0034] Figure 6 The graph shows the remaining Al content of dispersed pantothenic acid YWZ-3 under different aluminum concentration stresses (pH 4.5).

[0035] Figure 7 A graph showing pH changes of different aluminum concentrations (pH 4.5) to disperse pantothenic acid YWZ-3.

[0036] Figure 8 Figure showing the production of extracellular polysaccharides (PS) of dispersed pantothenic acid YWZ-3 under different aluminum concentration stresses (pH 4.5).

[0037] Figure 9 Figure 1 shows the plant hormone production of dispersible pantothenic acid YWZ-3 under different aluminum concentration stresses (pH 4.5) (A: IAA, B: ACC deaminase). Detailed Implementation

[0038] The present invention will be further described in detail below with reference to specific examples. The embodiments given are only for explaining the present invention and do not limit the scope of application of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0039] The screening medium used was LB solid medium (g / L): tryptone 10, yeast extract 5 and sodium chloride 10, agar powder 15, pH 7.2~7.5, sterilized at 121℃ for 20 min.

[0040] The fermentation medium was a modified LB liquid medium (g / L): glucose 1, yeast extract 1, sodium chloride 10, soybean peptone 10, pH 4.5, sterilized at 115℃ for 30 min.

[0041] Organophosphorus hydrolysis identification medium using Monkina solid medium (g / L): glucose 10.0, (NH4+) 4）2SO4 0.5, Mg MgSO4·7H2O 0.3, NaCl 0.3, KCl 0.3, FeSO4·7H2O 0.03, MnSO4·H2O 0.03, CaCO3 5.0, lecithin 0.2, agar powder 15, pH 7.0~7.5, sterilize at 115℃ for 30 min.

[0042] Organophosphate liquid medium using Monkina liquid medium (g / L): glucose 10.0, (NH4+) 4）2 SO4 0.5, MgSO4·7H2O 0.3, NaCl 0.3, KCl 0.3, FeSO4·7H2O 0.03, MnSO4·H2O 0.03, CaCO3 5.0, lecithin 0.2, pH 7.0~7.5, sterilize at 115℃ for 30 min.

[0043] Inorganic phosphorus hydrolysis identification medium using tricalcium phosphate solid medium (g / L): glucose 10.0, (NH4)2SO4 0.5, yeast extract 0.5, NaCl 0.3, KCl 0.3, FeSO4·7H2O 0.03, MnSO4 4· H2O 0.03, Ca3(PO4)2 5.0, agar powder 15.0, pH 7.0~7.5, sterilize at 115℃ for 30 min.

[0044] Inorganic phosphorus liquid culture medium using tricalcium phosphate liquid medium (g / L): glucose 10.0, (NH4+) 4）2 SO4 0.5, yeast extract 0.5, NaCl 0.3, KCl 0.3, FeSO4·7H2O 0.03, MnSO4·H2O 0.03, Ca3(PO4)2 5.0, pH 7.0~7.5, sterilized at 115℃ for 30 min.

[0045] Potassium-solubilizing medium using potassium feldspar solid medium (g / L): sucrose 5.0, glucose 5.0, (NH4+) 4）2 SO4 0.5, yeast powder 0.5, Mg SO4·7H2O 0.3, Na2HPO4 2.0, FeSO4·7H2O 0.03, MnSO4·H2O 0.03, potassium feldspar 2.0, agar powder 15.0, pH 7.0~7.5, sterilize at 115℃ for 30 min.

[0046] Urease identification medium (g / L): peptone 1.0, glucose 1.0, NaCl 5.0, Na₂HPO₄ 1.2, KH₂PO₄ 0.8, phenol red 0.012, agar powder 15.0, pH 6.6-7.0. Add 950 mL of distilled water, heat to boiling, stir until completely dissolved, dispense into Erlenmeyer flasks (95 mL each), and autoclave at 121℃ for 15 minutes. Cool to 50-55℃, add 5 mL of sterile 40% urea solution to each 95 mL of medium, mix well, dispense into test tubes, and form slant agar for later use.

[0047] The nitrogen-fixing medium was Ashube nitrogen-free medium (g / L): sucrose 10.0, K2HPO4 0.5, MgSO4·7H2O 0.2, NaCl 0.2, CaCO3 1.0, agar powder 15, pH 7.0, sterilized at 115℃ for 30 min.

[0048] Salkowski's colorimetric solution: purchased from Feijing Biotechnology Co., Ltd.

[0049] Aluminum reagent: Dissolve 0.25 g of rose red ammonium tricarboxylate and 5.0 g of gum arabic in 250 mL of water, warm until dissolved, then add 87.0 g of ammonium acetate. After the ammonium acetate dissolves, add 145 mL of 15% hydrochloric acid solution and dilute to 500 mL. Filter if necessary. This solution is valid for one month.

[0050] Nessler's reagent: purchased from the Bickman Biotech Taobao flagship store.

[0051] Example 1 Alumina-dissolving bacteria ( Pantoea dispersa Separation and screening of YWZ-3 Preliminary screening of strains Soil samples were collected from alumina-rich acidic soil near Yichang City, Hubei Province. They were placed in sterile bags and brought back to the laboratory, where they were stored at 4℃. 10 g of fresh soil sample was weighed and added to 90 mL of sterile physiological saline, shaken at 100 rpm for 15 min, and then incubated at 28℃ for 24 h. After enrichment, the supernatant was diluted to 10⁻⁶. -3 10 -4 10 -5 Three gradients were used, with 350 µL of each dilution spread onto the screening solid culture medium (containing 25 mg / L Al). 3+ On the plate, 3 replicates per group. Incubate upside down in a 30℃ incubator for 4-5 days and observe bacterial growth. Select vigorous colonies and repeatedly streak them for purification. After purification, select single colonies and transfer them to beef extract peptone slant for storage at 4℃ for later use.

[0052] YWZ-3, a highly acid- and aluminum-resistant alumina-solving bacterium, was obtained through preservation at the China Center for Type Culture Collection. This strain is classified as *Panthera dispersans*. Pantoea dispersa (This is a partial encapsulation of a genera and its contents, deposited at the China Center for Type Culture Collection, accession number CCTCCNO: M 2026581, and classified as follows:) Pantoea dispersa YWZ-3.

[0053] Example 2 Alumina-dissolving bacteria Pantoea dispersa Identification of YWZ-3 1. Morphological characteristics The bacterial strain YWZ-3 was prepared into a bacterial suspension. A small amount of the bacterial suspension was inoculated onto an LB solid plate using the streak plate method and cultured at 30 °C for 3 days.

[0054] Figure 1 The image shows the colony morphology of YWZ-3. The results indicate that the colonies are round, pale yellow and translucent, with a smooth and slightly moist surface, regular edges, no halo, and a slightly raised center, with a diameter of 1-2 mm. Preliminary identification based on Bergey's Manual of Bacteriology is Pantotheca dispersalis.

[0055] 2.16S rDNA gene sequence analysis Bacterial DNA was extracted using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0. Using the bacterial DNA as a template, PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTT ACGACTT-3') for the bacterial 16S rRNA gene.

[0056] Amplification reaction system: template 7 µL, dNTP 4 µL, 5 × Ps Buffer 10 µL, primer 1 (27F) 2 µL, primer 2 (1492R) 2 µL, PrimerSTAR HS enzyme 0.5 µL, dd H2O 24.5 µL.

[0057] Reaction conditions: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 56 °C annealing for 30 s, 72 °C extension for 1.5 min, 30 cycles; 72 °C extension for 10 min; and finally, hold at 4 °C.

[0058] The amplification products were detected by agarose gel electrophoresis. Amplification samples with single bands and appropriate fragment sizes were selected and sent to Wuhan Sangon Biotech Co., Ltd. for sequencing. Sequence alignment was performed using NCBI. The 16S rDNA sequence of strain YWZ-3 is shown in SEQ ID NO:1: The results showed that the screened strain YWZ-3 was related to *Panthera dispersans*. Pantoea dispersa Similarity 99.59%, phylogenetic tree as follows Figure 2 As shown, the confidence level is 98%. Therefore, YWZ-3 is identified as... Pantoea dispersa.

[0059] The aluminum-dissolving bacteria Pantoea dispersa Preservation information for YWZ-3: Preservation date: April 2, 2026; Preservation institution: China Center for Type Culture Collection (CCTCC); Accession number: CCTCC M 2026581; Preservation address: Wuhan University, Wuhan, China.

[0060] Example 3 Determination of phosphorus-solubilizing capacity of inorganic phosphorus (tricalcium phosphate) Solid inorganic phosphorus screening medium was prepared using 5 g / L Ca3(PO4)2 as the phosphorus source. 10 µL of YWZ-3 culture broth activated in LB medium for 16 h was spotted onto the inorganic phosphorus screening medium plate and incubated statically at 30 ℃. After 4 days, the size of the colony zones and phosphate-solubilizing zones were observed and measured. The phosphate-solubilizing index was calculated as: phosphate-solubilizing zone diameter / colony zone diameter.

[0061] Take 20 mL of YWZ-3 culture medium activated in LB medium for 16 h, centrifuge at 8000 rpm for 5 min, discard the supernatant, wash the bacterial cells three times with 20 mL of sterile physiological saline (0.9% NaCl), and resuspend in physiological saline, adjusting OD600 to 1.0. Take 100 µL of the resuspended bacterial solution into tricalcium phosphate liquid medium, incubate at 30 ℃ on a shaker at 200 rpm for 4 days, centrifuge, collect the supernatant, and filter through a 0.22 μm filter membrane. Determine the soluble phosphorus content in the supernatant using spectrophotometry.

[0062] Determination of soluble phosphorus content in supernatant by phosphomolybdic blue spectrophotometry: Dilute the sample solution with pure water to obtain a diluted solution. Transfer 10.00 mL of the diluted solution to a 100 mL Erlenmeyer flask, add 1.00 mL of (1+35) sulfuric acid to make the pH value less than 1; then add 5.00 mL of potassium persulfate solution (40 g / L), simmer over low heat for nearly 30 minutes, adding water as needed during boiling to keep the volume between 25 and 30 mL, and remove from heat and cool to room temperature.

[0063] Adjust the pH to 3-10 using sodium hydroxide solution, transfer the solution to a 50 mL volumetric flask, add 2.00 mL of ammonium molybdate solution (26 g / L) and 1.00 mL of ascorbic acid solution (100 g / L), dilute to the mark with water, shake well, and let stand at room temperature for 10 min. Measure the absorbance at 710 nm using a 1 cm cuvette with a blank, and read the result directly. Substitute the result into the standard curve to obtain the phosphorus content of the diluted solution, and multiply this by the dilution factor to obtain the phosphorus content in the sample.

[0064] Table 1. Solubility effect of dispersible pantothenic acid YWZ-3 on poorly soluble inorganic phosphorus.

[0065] Figure 3 Figure (A) shows the phosphate solubility circle of inorganic phosphorus. Table 1 shows that the phosphorus solubility index of YWZ-3 bacteria for inorganic phosphorus is 2.41, and the solubility of tricalcium phosphate is 64.6 mg / L after 4 days.

[0066] Example 4 Determination of the ability to dissolve organic phosphorus (lecithin) Take 10 µL of YWZ-3 culture medium that has been activated in LB medium for 16 h, spot it onto a Monkina agar plate and incubate statically. After 4 days, observe and measure the size of the colony zones and phosphate-solubilizing zones, and calculate the phosphate-solubilizing index.

[0067] Take 20 mL of YWZ-3 culture medium activated in LB medium for 16 h, centrifuge at 8000 rpm for 5 min, discard the supernatant, wash the bacterial cells three times with 20 mL of sterile physiological saline (0.9% NaCl), and resuspend in physiological saline, adjusting OD600 to 1.0. Take 100 µL of the resuspended bacterial solution into Monkina liquid medium, incubate at 30 ℃ on a shaker at 200 rpm for 4 days, centrifuge, collect the supernatant, and filter through a 0.22 μm filter membrane. Use the phosphomolybdic blue spectrophotometric method described in Example 3 to determine and calculate the soluble phosphorus content in the supernatant.

[0068] Figure 3 Figure (B) in the table shows the organophosphate solubilization circle diagram. Table 2 shows that the phosphorus solubilization index of YWZ-3 bacteria for lecithin is 2.73, and the solubilization capacity of lecithin after 4 days is 252.7 mg / L.

[0069] Table 2. Solubility effect of dispersible pantothecin YWZ-3 on poorly soluble organophosphates.

[0070] Example 5 Potassium solubility test (potassium feldspar) A solid potassium-solubilizing screening medium was prepared using potassium feldspar as the potassium source. 10 µL of YWZ-3 culture broth activated in LB medium for 16 h was spotted onto the medium plate and incubated statically at 30 ℃. After 4 days, the presence of transparent oil droplet-like colonies was observed and determined.

[0071] Figure 3 Figure (C) in the diagram is a potassium-solubility plate diagram, from which... Figure 3 Figure (C) shows that YWZ-3 bacteria can form transparent oil droplet-shaped colonies on potassium-solubilizing plates, indicating that the YWZ-3 strain has a significant ability to dissolve potassium feldspar.

[0072] Example 6 Nitrogen fixation capacity determination Using Assumption nitrogen-free medium as the nitrogen-fixing medium, 10 µL of YWZ-3 culture solution activated in LB medium for 16 h was spotted onto the medium plate and incubated statically in a 30 ℃ incubator for 3 days. The resulting single colonies were then streaked again on Assumption nitrogen-free medium. After repeating the operation 3 times, it was observed whether the colonies could still grow on Assumption nitrogen-free medium.

[0073] Take 20 mL of YWZ-3 culture medium activated in LB medium for 16 h, centrifuge at 8000 rpm for 5 min, discard the supernatant, wash the bacterial cells three times with 20 mL of sterile physiological saline (0.9% NaCl), and resuspend in physiological saline, adjusting OD600 to 1.0. Take 100 µL of the resuspended bacterial solution into Assab nitrogen-free liquid medium, incubate at 30 ℃ on a shaker at 200 rpm for 5 days, centrifuge, collect the supernatant, and filter through a 0.22 μm filter membrane. Determine the nitrogen-fixing capacity of the supernatant using spectrophotometry.

[0074] Figure 3 Figure (D) in the diagram is a nitrogen fixation plate plot, from... Figure 3 Figure (D) shows that the YWZ-3 strain can still produce obvious colonies after multiple streaks (3 generations) on nitrogen-fixing plates, and after 5 days of shaking culture in Assumption medium without nitrogen source, the ammonia nitrogen content was measured to be 0.18 mg / L, indicating that the YWZ-3 strain has nitrogen-fixing ability.

[0075] Example 7: Characteristics of bacterial strains under different pH values ​​without aluminum stress LB medium at different pH levels was prepared using 30% hydrochloric acid and 10% sodium hydroxide. Four pH gradients were used, with three replicates for each concentration. After sterilization, activated aluminothermic bacteria YWZ-3 were inoculated with an initial biomass OD600 of 0.1. The medium was cultured at 30 °C for 96 h, and OD600 and pH were measured every 24 h.

[0076] Figure 4Figure (A) shows the growth changes (OD600) at different pH levels without stress. Figure 4 As can be seen in (A), aluminum-solubilizing bacteria Pantoea dispersa YWZ-3 can grow at pH 4-7, and its biomass is not less than 4.5 after 96 hours. This indicates that YWZ-3 has acid tolerance.

[0077] Figure 4 Figure (B) shows the pH changes at different initial pH values ​​under no-stress conditions. Figure 4 As can be seen in (B), aluminum-solubilizing bacteria Pantoea dispersa The pH of YWZ-3 showed an upward trend after 4 days of cultivation, reaching pH 8-9, indicating that YWZ-3 can effectively increase the environmental pH value through its own physiological activities.

[0078] Example 8: Aluminolysis characteristics under different aluminum stresses at (pH 4.5) acidity 1. Changes in biomass, pH, and free aluminum content LB medium with a pH of 4.5 was prepared using 30% hydrochloric acid and 10% sodium hydroxide, and dispensed into 12 Erlenmeyer flasks, 50 mL per flask. The flasks were sterilized at 115°C for 30 min. A 5.4 g / L aluminum sulfate solution was prepared, filtered, sterilized, and added to the medium to create four gradients of available aluminum concentrations: 0, 20, 40, and 60 mg / L, with three replicates per group. Then, 1 mL of activated YWZ-3 was inoculated into each flask, and the medium was incubated at 30°C for 96 h. OD600, pH, and free aluminum content were sampled at appropriate time points.

[0079] Figure 5 The graph shows the growth variation of YWZ-3 under different aluminum stresses. The results indicate that YWZ-3 under 0, 20, 40, and 60 mg / L Al... 3+ All of them can grow, and the growth rate (OD600) can reach 5~6 in 96 h, indicating that YWZ-3 has a strong aluminum tolerance.

[0080] Figure 6 The graphs showing the aluminum removal residue of the bacterial strain YWZ-3 at different aluminum concentrations reveal that after culturing YWZ-3 under aluminum stress of 0, 20, 40, and 60 mg / L for 96 hours, the aluminum content decreased to 3.41 mg / L in the 20 mg / L group, 17.7 mg / L in the 40 mg / L group, and 24.6 mg / L in the 60 mg / L group. The low-dose group achieved 80% aluminum removal, and the high-dose group also achieved 55%–60%, indicating that YWZ-3 possesses a strong aluminum removal capacity.

[0081] Figure 7The graph shows the pH changes of the bacterial strain under different aluminum concentrations. The pH values ​​of all groups inoculated with YWZ-3 reached 8-9 after 96 h of culture, indicating that YWZ-3 can produce alkaline substances under acidic aluminum stress, thereby changing the culture environment. Furthermore, the increase in pH is also an important way for YWZ-3 to detoxify aluminum.

[0082] 2. Changes in extracellular polysaccharides The fermentation broth cultured for 96 h in Example 1 was collected, centrifuged at 4 °C and 10,000 rpm for 10 min, and the supernatant was collected. Extracellular substances were extracted from the fermentation broth using a low-temperature alcohol precipitation method. The mixture was placed in a boiling water bath for 10 min to denature the proteins. After natural cooling, it was centrifuged at 4 °C and 10,000 rpm for 15 min to remove the proteins, retaining the supernatant. 2.0 mL of the supernatant was taken, and 6 mL of pre-cooled anhydrous ethanol was added. The mixture was then subjected to low-temperature extraction at 4 °C for 24 h. After centrifugation at 4 °C and 10,000 rpm for 15 min, the supernatant was removed, and the extracellular substances were retained. The mixture was then dried in an oven at 50 °C. The polysaccharide (PS) content in the extracellular material was determined using the phenol-sulfuric acid method.

[0083] Figure 8 The extracellular polysaccharide (PS) yield was determined under different aluminum ion concentrations. The results showed that higher aluminum ion concentrations resulted in higher PS content, reaching a maximum of 453 mg / L (60 mg / L Al). 3+ Extracellular polysaccharides not only provide protection for the strain under aluminum stress, but also chelate free aluminum, which may be another important pathway for YWZ-3 to detoxify aluminum.

[0084] Example 9: Growth-promoting ability of bacterial strains under different aluminum stresses (pH 4.5) LB medium with a pH of 4.5 was prepared using 30% hydrochloric acid and 10% sodium hydroxide, and dispensed into 12 Erlenmeyer flasks (50 mL each). The flasks were sterilized at 115°C for 30 min. A 5.4 g / L aluminum sulfate solution was prepared, filtered, sterilized, and added to the medium to create four gradients of available aluminum concentrations: 0, 20, 40, and 60 mg / L. A 10 g / L L-tryptophan stock solution was sterilized separately and added to the medium to achieve a concentration of 300 mg / L. The medium was incubated at 30°C and 200 rpm for 96 h. At 96 h, samples were taken and centrifuged at 10,000 rpm at 4°C for 10 min. The supernatant and bacterial cells were collected separately. The IAA content in the supernatant and the ACC deaminase activity in the bacterial cells were determined colorimetrically.

[0085] The Salkowski colorimetric method was used for measurement. Salkowski colorimetric solution A and solution B were mixed. The culture medium that had been cultured for 96 h was centrifuged at 10000 r / min and 4℃ for 10 min. 1 mL of the supernatant was taken and mixed with 1 mL of the mixed Salkowski colorimetric solution. After standing in the dark for 30 min, the absorbance at 530 nm was quickly measured using a spectrophotometer. The IAA content was calculated using a standard curve.

[0086] ACC deaminase activity is expressed as the amount of α-butanone produced per µmol per hour by which each milligram of bacterial protease catalyzes the deamination of ACC. The unit of enzyme activity is α-butanone µmol / (mg·h). The specific measurement method is as follows: 96-h bacterial cells were washed with 0.1 mol / L Tris-HCl buffer (pH 7.6), then resuspended in 0.1 mol / L Tris-HCl buffer (pH 7.6). 30 μL of toluene was added, and the cells were rapidly shaken for 30 s to lyse the cells. 100 µL of the crude enzyme solution was stored at 4 °C for protein concentration determination. Separately, 200 μL of the crude enzyme solution was mixed with 20 μL of 0.5 mol / L ACC, incubated at 30 °C for 15 min, and then 1 mL of 0.56 mol / L HCl was added to terminate the reaction. A blank control without ACC was used. The mixture was centrifuged at 12000 rpm for 5 min. Take 1 mL of the supernatant and add 800 μL of 0.56 mol / L HCl and 300 μL of 0.2% 2,4-dinitrophenylhydrazine solution to dissolve it completely. Maintain the temperature at 30 ℃ for 30 min, then add 2 mL of 2 mol / L NaOH. Measure the yield of α-butanone at 540 nm. Protein concentration was detected using the BCA micro-reaction kit.

[0087] 1. IAA production Figure 9 (A) shows the changes in IAA under different aluminum stresses, indicating that aluminum-dissolving bacteria... Pantoea dispersa YWZ-3 can still produce IAA under aluminum stress of 20-60 mg / L. Its concentration range is 4.5-18.2 mg / L.

[0088] 2. ACC deaminase activity Figure 9 (B) shows the ACC deaminase activity under different aluminum stresses. The results indicate that YWZ-3 can produce ACC deaminase under aluminum stress ranging from 20 to 60 mg / L. Aluminum stress promotes the production of ACC deaminase. Without aluminum stress, the ACC deaminase activity is only 11.2 U, while under 60 mg / L aluminum stress, the ACC deaminase activity can reach 57.2 U.

[0089] As can be seen from the above results, the above embodiments are merely preferred technical solutions of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art can make different modifications and variations to the descriptions and technical solutions in the above embodiments. Any modifications, improvements, or equivalent substitutions made without departing from the spirit and principles of the present invention should be protected within the scope of the rights of the present invention.

Claims

1. A strain of *Panthera dispersa* YWZ-3 was classified and named *Panthera dispersa*. (Pantoea dispersa) It was deposited on April 2, 2026, at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC NO: M 2026581, and classified as follows: Pantoea dispersa YWZ-3.

2. An aluminum-lysis bacterium, characterized by, The aluminolytic bacteria comprise the dispersible pantothenic bacteria YWZ-3 as described in claim 1.

3. A culture or microbial inoculum, characterized in that, The cultured microbial agent is obtained by fermentation culture of the dispersible pantothenic acid YWZ-3 as described in claim 1.

4. The application of the dispersible pantothecin YWZ-3 as described in claim 1, the aluminum-dissolving bacteria as described in claim 2, or the culture or microbial agent as described in claim 3 in alleviating or controlling aluminum toxicity.

5. The application of the dispersible pantothecin YWZ-3 of claim 1, the aluminum-dissolving bacteria of claim 2, or the culture or microbial agent of claim 3 in improving the pH value of the environment.

6. The use of the dispersible pantothenic acid YWZ-3 of claim 1, the aluminum-dissolving bacteria of claim 2, or the culture or microbial agent of claim 3 in dissolving insoluble phosphorus or potassium.

7. The application of the dispersible pantothecin YWZ-3 of claim 1, the aluminothermic bacteria of claim 2, or the culture or microbial agent of claim 3 in nitrogen fixation.

8. The use of the dispersible pantothecin YWZ-3 of claim 1, the aluminothermic bacteria of claim 2, or the culture or microbial agent of claim 3 in the production of plant hormones or extracellular polysaccharides.

9. Use according to claim 8, characterized in that, The plant hormone is indole-3-acetic acid and / or 1-aminocyclopropane-1-carboxylic acid deaminase.

10. Use according to any one of claims 4 to 9, characterized in that, The application is performed in acidic environments and / or aluminum-stressed environments, which are soil or aquatic environments.

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