Alkali-resistant rhizosphere growth-promoting bacterium for magnesium-polluted soil and application thereof

Abstract

The invention belongs to the technical field of bioremediation of polluted soil in mining areas, and particularly relates to an alkali-resistant rhizosphere growth-promoting bacterium for magnesium-polluted soil and application of the alkali-resistant rhizosphere growth-promoting bacterium. The magnesium dust polluted soil alkali-resistant growth-promoting rhizobacteria is C-1, the classification name is Klebsiella pasteurii, the magnesium dust polluted soil alkali-resistant growth-promoting rhizobacteria is preserved in China General Microbiological Culture Collection Center on August 15, 2025, the preservation number is CGMCC No.35635, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Klebsiella pasteurii C-1 disclosed by the invention has the characteristic of secreting auxin IAA (Indoleacetic Acid), the concentration of the produced IAA can reach 28.90 mg / L, various agronomic indexes of plants in an alkaline environment are obviously improved, and the growth promoting effect is good.

Description

Technical Field

[0001] This invention belongs to the field of bioremediation technology for contaminated soil in mining areas, specifically relating to an alkali-tolerant rhizosphere growth-promoting bacterium in magnesium-contaminated soil and its application. Background Technology

[0002] Liaoning Province has the most concentrated reserves of magnesite in my country, with proven reserves of approximately 3.05 × 10⁻⁶. 9 This mineral deposit, accounting for 85% of the national total, is mainly distributed in Haicheng, Dashiqiao, and Xiuyan areas, and is characterized by high grade, shallow burial, and ease of mining. However, the exploitation of this advantageous resource and the development of the industry have led to significant local environmental problems: waste ore is piled up everywhere, occupying land and disrupting the ecological balance; extensive mining and stripping methods and dust and smoke emissions from processing plants amount to approximately 3.8 × 10⁻⁶ tons annually. 5 This leads to soil pollution and compaction; plant growth is slow, leaves are scorched, and production levels decline significantly. Especially around the re-firing kilns, large areas of white crust remain on the soil surface; long-term dust deposition causes soil alkalization and calcium... 2+ / Mg 2+ Problems such as imbalanced soil composition, severe release of soil nutrients, decreased microbial activity, and reduced soil biofertility severely hinder plant growth, with only a small number of tall trees such as elm and poplar surviving. Therefore, it is urgent to develop targeted remediation technologies to fundamentally improve soil quality in mining areas, ensure long-term and sustainable remediation effects, and improve the plant growth environment.

[0003] The formation process of sedimentary crusts in magnesite mining areas involves magnesium oxide dust undergoing a series of morphological changes under precipitation and anaerobic conditions to form magnesium oxysulfate hydrates, resulting in a hard, cement-like crust. Herbaceous plants such as Kochia scoparia, Cassia tora, and Suaeda salsa, along with woody plants like jujube, elm, black locust, Amorpha fruticosa, and poplar, serve as rapid remediation plants. Microbial inoculants are an effective means to enhance the remediation performance of enriched plants. The microbial community in the mining area soil can absorb some magnesium, alleviating the toxicity of excessive magnesium to plant roots, activating some poorly absorbed magnesium, promoting magnesium absorption by plants, and increasing the nutrients needed for plant growth through microbial secretions. Soil microorganisms also participate in regulating soil carbon and nitrogen cycles. Rhizosphere growth-promoting bacteria can promote soil nitrogen fixation, improve soil texture, and promote plant growth. Therefore, screening alkali-tolerant rhizosphere growth-promoting bacteria from magnesium dust-contaminated rhizosphere soils and then combining them with pioneer plants to remediate contaminated soils is of significant feasibility. Summary of the Invention

[0004] The purpose of this invention is to provide an alkali-tolerant rhizosphere growth-promoting bacterium for magnesium-contaminated soil and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A rhizosphere growth-promoting bacterium tolerant to magnesium dust-contaminated soil, wherein the alkali-tolerant rhizosphere growth-promoting bacterium is C-1 and is classified as Klebsiella pastoris. Klebsiella pasteurii The specimen was deposited on August 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35635, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The application of a alkali-tolerant rhizosphere growth-promoting bacterium in magnesium dust-contaminated soil, wherein the strain is used to promote plant growth under alkaline soil environmental stress in magnesite mining areas.

[0007] Furthermore, the strain can promote plant growth under alkaline stress, synthesize nitrogen-containing organic matter, and / or convert atmospheric nitrogen into ammonia that is available to plants.

[0008] At the same time, the strain can improve the plant's stress resistance, especially its alkali tolerance.

[0009] The plant in question is Suaeda salsa or other suitable plants.

[0010] An application of an alkali-tolerant rhizosphere growth-promoting bacterium in magnesium dust-contaminated soil, wherein the strain is used to prepare bio-fertilizer.

[0011] The bio-fertilizer can increase the available carbon, nitrogen, and phosphorus content in the soil, improve various agronomic indicators and stress resistance of plants, promote plant growth, and serve as a functional microbial agent for improving barren and saline-alkali land.

[0012] Application of an alkaline-tolerant rhizosphere growth-promoting bacterium in magnesium dust-contaminated soil, wherein the strain is used for soil nitrogen fixation.

[0013] A plant growth promoting agent containing alkali-tolerant rhizosphere growth-promoting bacteria from magnesium dust-contaminated soil.

[0014] Furthermore, it contains alkali-tolerant rhizosphere growth-promoting bacteria, bacterial suspensions, metabolites, or fermentation broths in soil contaminated with magnesium dust.

[0015] A bio-fertilizer containing alkali-tolerant rhizosphere growth-promoting bacteria from magnesium dust-contaminated soil.

[0016] Furthermore, it contains alkali-tolerant rhizosphere growth-promoting bacteria, bacterial suspensions, metabolites, or fermentation broths in soil contaminated with magnesium dust.

[0017] A soil nitrogen-fixing bacterial agent containing the aforementioned alkali-tolerant rhizosphere growth-promoting bacteria in magnesium dust-contaminated soil.

[0018] Furthermore, it contains alkali-tolerant rhizosphere growth-promoting bacteria, bacterial suspensions, metabolites, or fermentation broths in soil contaminated with magnesium dust.

[0019] The beneficial effects of this invention are as follows: (1) This invention collects rhizosphere soil from plants in saline-alkali land polluted by magnesium dust in magnesite mining areas, and isolates Klebsiella pastoris, which has high alkali tolerance and rhizosphere growth-promoting ability, from this soil. Klebsiella pasteurii C-1 can maintain stable and excellent genetic traits even after three generations of purification.

[0020] (2) The Klebsiella Pasteurella of the present invention ( Klebsiella pasteurii C-1 is an indigenous microorganism with rhizosphere growth-promoting ability. It can tolerate alkaline environments with a pH as high as 9. Under alkaline environmental stress, it has a nitrogen-fixing effect and can enhance the nitrogen-fixing effect of the soil.

[0021] (3) The Klebsiella Pasteurella of the present invention ( Klebsiella pasteurii C-1 has the characteristic of secreting auxin IAA, and the concentration of IAA produced can reach 28.90 mg / L. Under alkaline conditions, various agronomic indicators of plants are significantly improved, and the growth-promoting effect is good.

[0022] (4) The Klebsiella Pasteurella of the present invention ( Klebsiella pasteurii C-1 can be applied to microbial inoculant fertilizers. After applying this bacterial solution to alkaline soils contaminated with magnesium dust, plant height increased significantly, with an average increase of 10.89%-13.47% compared to the control group after 90 days of growth. It also significantly affected root density, increasing it by 30.81%-32.49% compared to the control group.

[0023] (5) The Klebsiella Pasteurella of the present invention ( Klebsiella pasteurii C-1 can enhance plant stress resistance, improve the soil environment, and promote plant growth. Using this strain to prepare bio-fertilizer can improve the soil environment and promote plant growth, with broad application prospects and the potential for industrial production. Attached Figure Description

[0024] Figure 1 Growth curves of alkali-tolerant rhizosphere growth-promoting bacteria under different pH treatments; Figure 2 Images of alkaline-tolerant rhizosphere growth-promoting bacteria plates and their H2O2 production are shown below; (a) shows the streaking morphology of the three bacteria on LB medium; (b) shows the catalase production capacity of the three bacteria; (c) shows the nitrogen-fixing zones of the three bacteria; (d) shows the growth status of B bacteria on nitrogen-free medium; and (e) shows the color change of B bacteria producing siderophores. Figure 3 The growth-promoting ability of alkali-tolerant rhizosphere growth-promoting bacteria is shown in (a) for phosphorus content, (b) for IAA content, (c) for SU content, (d) for color change reaction when measuring IAA ability, and (e) for color change reaction when measuring siderophore production ability of strain B. Figure 4 Klebsiella pastoris ( Klebsiella pasteuriiScanning electron microscope of C-1; Figure 5 Klebsiella pastoris ( Klebsiella pasteurii Phylogenetic tree of C-1; Figure 6 The main growth indicators and changes of the plants are shown in (a) plant height; (b) fresh weight / dry weight; (c) chlorophyll and nitrogen content at 60 days; and (d) chlorophyll and nitrogen content at 90 days. Figure 7 A graph showing plant root length, total root weight, and total root density; Figure 8 The content of soluble sugars, proteins, and stress resistance indicators. Detailed Implementation

[0025] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0026] Numerous specific details are set forth in the following description to provide a full understanding of the invention, such as the use of Suaeda salsa in pot experiments. However, the invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0027] Example 1: Isolation and purification of alkali-tolerant rhizosphere microorganisms 1. Materials The experimental materials used in this study included rhizosphere soil samples from *Suaeda salsa*, LB solid medium, Ashby solid medium, total phosphorus medium (TPM), CAS medium, and Salkowski solution. Also included were an electronic balance, autoclave, laminar flow hood, biochemical incubator, drying oven, water bath, shaker, pH meter, 4°C freezer, -80°C freezer, micropipette, and various other instruments, reagents, and consumables used for the culture and identification of common microbial strains.

[0028] 1.1 Collection and preservation of soil samples for testing Soil samples were collected from the rhizosphere soil of *Suaeda salsa* growing in the magnesium dust-contaminated soil area of ​​the magnesite mining area in Bali Town, Haicheng City, Liaoning Province. A five-point sampling method was used to collect the soil. The collected soil samples were brought back to the laboratory under low-temperature conditions and stored in a 4°C refrigerator before microbial screening.

[0029] 1.2 Preparation of culture medium (1) LB solid medium: 10g tryptone, 5g yeast extract, 20g NaCl, 20g agar, pH=8.6±0.1 (25℃), 1L, sterilized at 121℃ for 20min; (2) Ashby solid medium: C6H 14 O6 (mannitol) 10.0g, NaCl 0.2g, KH2PO4 0.2g, CaCO3 5.0g, MgSO4·7H2O 0.2g, CaCO3 0.1g, pH=7.2±0.1 (25 ℃), 1L, sterilized at 121℃ for 30min; (3) Phosphate-soluble medium: glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, KCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g, MnSO4·H2O 0.03g, Ca3(PO4)2 5.0g, agar 20g, pH 7.0-7.5±0.2 (25℃), 1L, sterilized at 121℃ for 30min; 2. Experimental Procedure 2.1 Screening of rhizosphere microorganisms The specific dilution method is as follows: Weigh 1.0g of rhizosphere soil sample, grind it in a mortar and pestle, and add it to an Erlenmeyer flask containing 50ml of sterile physiological saline (0.85% NaCl). Shake on a shaker at 200r / min and constant temperature of 28℃ for 2 hours. After natural sedimentation for 10 minutes, take 100μL of the central supernatant into a centrifuge tube containing 900μL of sterile water to obtain 10 -1 A soil sample solution of 100 μL concentration was prepared, and after thorough mixing, 100 μL of the solution was taken. -1 The soil sample solution was placed in a centrifuge tube containing 900 μL of sterile water to obtain 10... -2 Concentration of soil sample solution. Dilute stepwise using a concentration gradient method, setting the concentration gradient to 10. -1 10 -2 10 -3 10 -4 10 -5 50 μL of each concentration gradient was spread onto LB solid medium. The spread LB solid medium plates were incubated in a biochemical incubator at 28℃ for 48 h, and the colony growth on the plates was observed.

[0030] 2.2 Alkali tolerance of rhizosphere microorganisms - growth curve determination The isolated and purified strains were inoculated into LB liquid medium with pH values ​​of 7, 8, and 9, respectively, and incubated at 28°C and 120 rpm. -1 The bacterial strain was cultured in a constant temperature shaker, and its growth was observed. 5 mL of bacterial solution was taken at 0, 3, 6, 10, 12, 14, 22, 26, 33, 36 and 48 h, respectively. Uninoculated LB liquid medium was used as a blank control, and the absorbance was measured at 600 nm using a UV spectrophotometer.

[0031] 2.3 Methods for preserving alkali-tolerant rhizosphere microorganisms Short-term storage: Inoculate the alkali-resistant strains obtained from the initial screening into LB solid medium, incubate at 28°C for 2 days, and then store in a refrigerator at 4°C. If the storage period exceeds 14 days, the strains need to be reactivated for preservation.

[0032] Long-term preservation: Use the glycerol preservation method. Mix the bacterial solution with sterilized 30% glycerol in a 1:1 ratio and store 3 portions in a -80℃ refrigerator for later use.

[0033] 3. Experimental Results The colonies obtained from the initial screening were purified. The purified strains were streaked onto Ashby nitrogen-free agar plates and TPM inorganic phosphorus agar plates, respectively, and incubated upside down in a 28°C incubator for 2–3 days. Colonies exhibiting vigorous growth and distinct morphological characteristics on both media were selected. Figure 1 It is evident that the three strains (strain A, strain B, and strain C) still grew well in LB liquid medium at pH 9, and even during the decline phase, their OD values ​​remained above 1.5, indicating that all three strains possess good alkali tolerance. Thus, three excellent alkali-tolerant strains with both nitrogen fixation and phosphorus solubilization functions were initially obtained, and their growth-promoting properties will be further determined.

[0034] Example 2: Screening of alkali-tolerant rhizosphere microorganisms in soil 1. Reagent preparation (1) CAS medium: Chromium azurite S 60.5mg, HDTMA 72.9mg, Fe2Cl3·6H2O 2.645mg, Na2HPO4·12H2O 1213.5mg, NH4Cl 125mg, KH2PO4 37.5mg, NaCl 62.5mg, agar 9000mg, pH=6.8±0.1 (25℃), 1L, sterilized at 116℃ for 30min; (2) MKB liquid culture medium: 5g casein amino acids, 15ml glycerol, 2.5g K2HPO4, 2.5g MgSO4·7H2O, pH 7.2±0.2 (25℃), 1L, sterilized at 121℃ for 20min; (3) IAA liquid culture medium: 25g LB liquid culture medium, 0.5g L-tryptophan, 1L distilled water, pH 7.0-7.2; (4) IAA stock solution: Accurately weigh 10 mg of auxin, dissolve it in a small amount of ethanol, and make up to 100 mL with deionized water. Store in the dark. (5) Salkowski solution: 450 mL of 35% HClO, 0.5 mol·L⁻¹ -1 FeCl3 1mL; 2. Experimental Procedure 2.1 Growth-promoting characteristics of alkali-tolerant rhizosphere microorganisms 1) Nitrogen fixation effect The isolated and purified strains were inoculated onto Ashby solid medium plates. The formation of a clear zone indicated nitrogen fixation ability, and the size of the clear zone indicated the strength of nitrogen fixation ability.

[0035] 2) Quantitative determination of phosphorus solubility Under acidic conditions, soluble phosphorus reacts with ammonium molybdate to form a complex. After color development to blue, the absorbance is measured at 700 nm, and quantification is performed using a standard curve.

[0036] 3) Determination of iron production capacity The isolated and purified strain was inoculated onto CAS detection medium plates. The ability of the strain to produce siderophores was initially determined based on the size of the yellow halo around the colony. Then, colonies with obvious halos were picked and inoculated into MKB liquid medium and cultured at 28℃ and 150r / min. The bacterial culture was centrifuged after 3 days and 5 days of culture. The supernatant was mixed with the CAS detection solution at a ratio of 1:1. After standing for 1 hour, the absorbance (As) was measured at 630nm.

[0037] 4) Determination of ability to produce indole-3-acetic acid Qualitative detection of IAA-PGPR: Inoculate 0.5 mL of bacterial suspension into 50 mL of IAA liquid medium and incubate at 30℃ and 170 rpm for 24 h on a shaker. Transfer 100 μL of bacterial suspension to a microplate, with an uninoculated medium as a negative control. Add an equal volume of Salkowski colorimetric reagent and incubate at 25℃ in the dark for 30 min. If the solution turns pink, it indicates the ability to produce IAA; the deeper the color, the stronger the ability.

[0038] IAA-PGPR Quantitative Detection: Prepare a series of standard solutions of 0-100 μg / mL from the IAA stock solution. After adding the colorimetric reagent, react at room temperature in the dark for 30 min. Measure the absorbance at 550 nm and plot a standard curve. Take a certain amount of bacterial culture, centrifuge at 4000 r / min for 10 min, take 3 mL of the supernatant, add an equal volume of colorimetric reagent, mix well, and measure the absorbance at 550 nm after 30 min in the dark. Substitute the absorbance into the standard curve to calculate the IAA yield.

[0039] 4) Determination of catalase production capacity Add 600 μL of 3% hydrogen peroxide solution to a 1.5 mL centrifuge tube, then add 400 μL of bacterial culture to the solution. Observe the generation of bubbles after 30 seconds. The presence of bubbles indicates a positive catalase test, while the absence of bubbles indicates a negative test.

[0040] 2.2 Identification and Morphological Characterization of Alkali-Tolerant Rhizosphere Growth-Promoting Bacteria The extraction and sequencing of 16S rDNA from the bacterial strains were performed by Shanghai Paisennong Biotechnology Co., Ltd. Genomic DNA was extracted using a column chromatography method. Universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-TACGGCTACCTTGTTACGACTT-3) were used for PCR amplification of the 16S rDNA. The purified PCR products from each bacterial strain were sequenced using an ABI 3730-XL sequencer. The sequenced nucleotide sequences were aligned using the BLAST function provided by the NCBI database, and a phylogenetic tree was constructed using MEGA 11 software.

[0041] 3. Experimental Results 3.1 Screening of alkali-tolerant rhizosphere growth-promoting bacteria Three strains (strain A, strain B, and strain C) were simultaneously streaked onto nitrogen-free medium. After 24 hours of incubation, all strains grew well and formed clear colonies. Figure 2 a) Take 200 μL of bacterial suspension and inoculate it onto a blank paper disc. It can be seen that all three bacteria produced a clear transparent zone. Figure 2 c) indicates that all three bacteria have significant nitrogen-fixing capabilities. Measurements with a ruler showed that the clear zone of bacteria A was ±1.1 cm, while that of strains B and C was ±1.4 cm. Therefore, strains B and C have relatively stronger nitrogen-fixing abilities. In addition, experiments were conducted on the three bacteria's abilities to solubilize phosphorus, produce IAA, produce siderophores, and produce hydrogen peroxide. The results showed that all three bacteria produced gas bubbles, indicating a positive catalase response. Figure 2 b), strain B showed the strongest phosphate-solubilizing effect and IAA production capacity at 5 days. Figure 3 a, 3b), and B bacteria will produce a yellow halo on the CAS detection medium plate ( Figure 2 e), quantitative testing revealed that it had the strongest iron-producing capacity ( Figure 3 e). Therefore, after comprehensive comparison and selection (Table 1), strain B was found to have a strong potential plant growth-promoting ability.

[0042] Table 1. Growth-promoting characteristics of alkali-tolerant rhizosphere growth-promoting bacteria

[0043] 3.2 Identification and Characterization of Alkali-Tolerant Rhizosphere Growth-Promoting Bacteria As shown in the figure ( Figure 2 As shown in d), strain B colonies on LB agar are yellow, smooth, slightly raised, and produce a pale yellow pigment covering the entire plate. Under an electron microscope, the bacteria appear as short rods with a surface exhibiting typical dense, cone-shaped / spiny, sharp protrusions resembling frosting. Individual bacteria are approximately 1.358 μm × 598 nm in size. Figure 4The 16S rDNA sequence obtained from sequencing was subjected to BLAST alignment analysis in NCBI, and the results showed that strain B was similar to... Klebsiella pasteurii The sequence similarity of SPARK836C1 (NR 180640.1) reached 99.44%. Phylogenetic tree construction results showed that bacteria B and... Klebsiella pasteurii Clustered in the same branch ( Figure 5 Based on morphological and physiological-biochemical identification, strain B was determined to be Klebsiella pastoris. Klebsiella pasteurii The DNA sequence was deposited on August 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.35635. The specific DNA sequence is as follows:

[0044] Example 3 Klebsiella Pasteurella ( Klebsiella pasteurii C-1's effect on plant growth 1. Experimental Procedure Moderately contaminated soil collected from the magnesite mining area (physicochemical properties of the contaminated soil: pH 8.94, EC 223 μS / cm, moisture content 4.64%, SOC 21.26 g / kg) was placed into 1.5-gallon plastic flowerpots (height 18 cm, mouth diameter 17 cm, bottom diameter 13 cm) with perforations at the bottom, with each pot weighing 2.5 kg. Regarding the treatment of the bacterial suspension: *Klebsiella pastoris* strain (… Klebsiella pasteurii C-1 was cultured in LB medium until the logarithmic growth phase, centrifuged, and resuspended in sterile water to prepare a bacterial suspension. The OD of this bacterial suspension was... 600 The value is 1.1 (at this time, the bacterial concentration is 7×10). 8 (CFU / ml).

[0045] The experimental groups (J1 and J2) were given 5% and 10% of the soil weight per pot, respectively, and mixed thoroughly.

[0046] The control group (CK) was treated with sterile water instead.

[0047] The purchased Suaeda salsa seeds were evenly scattered in each pot in the same amount, and the plants were allowed to grow. Observations and records were kept daily. The height of the Suaeda salsa plants was measured on days 60 and 90. On day 90, the Suaeda salsa plants were harvested and rhizosphere soil samples were collected. A series of agronomic traits, such as the fresh / dry weight of the aboveground / underground parts and root density, were measured.

[0048] 2. Experimental Results like Figure 6 As shown in Figure a, the changes in the main agronomic indicators of Suaeda salsa after 90 days of growth are as follows: The average plant height of the control group (CK) reached 39.03 cm; the average plant height of the group with 5% rhizosphere growth-promoting bacteria (J1) reached 43.28 cm, an increase of 10.89% compared to the average plant height of CK; the average plant height of the group with 10% rhizosphere growth-promoting bacteria (J2) reached 44.28 cm, an increase of 13.47% compared to the average plant height of CK. Therefore, the group with 10% rhizosphere growth-promoting bacteria (J2) was more effective in increasing plant height, but the difference between the J1 and J2 groups was not significant.

[0049] Figure 6Biomass accumulation is a comprehensive reflection of the light energy utilization capacity and environmental adaptability of *Suaeda salsa*. The addition of rhizosphere growth-promoting bacteria effectively increased the fresh / dry weight of both the aboveground and underground parts of *Suaeda salsa*. Compared with the control group (CK), the most significant effect was observed in experimental group J1, where the fresh weight of the aboveground and underground parts increased by 54.07% and 133.12%, respectively, showing a significant difference. Compared with the control group (CK), experimental group J2 showed an increase in the fresh weight of the aboveground and underground parts of 5.63% and 88.62%, respectively.

[0050] like Figure 6 As shown in c and d, compared with the control group CK, the chlorophyll content of experimental groups J1 and J2 increased by 26.71% and 34.36% respectively after 90 days of plant growth.

[0051] Depend on Figure 7 Analysis of root length, total root weight, and total root density showed that, compared with the control group (CK), the root length, total root weight, and total root density of the J1 experimental group increased by 50%, 118.02%, and 30.81%, respectively; while those of the J2 experimental group increased by 20.31%, 84.01%, and 32.49%, respectively. The root length and total root weight of the J1 experimental group increased significantly, but there was no significant difference in total root density between the J1 and J2 groups.

[0052] In conclusion, the addition of Klebsiella pasteurii C-1 significantly promoted plant growth. Comprehensive analysis revealed that the group with 5% rhizosphere growth-promoting bacteria (J1) exhibited better plant growth and higher biomass.

[0053] Example 4: Effects of alkali-tolerant rhizosphere growth-promoting bacteria on plant stress resistance 1. Experimental Procedure Potted Suaeda salsa were planted in moderately polluted soil in a magnesite mining area. Three treatments were set up: the original soil control (CK, watered with sterile water), the soil with 5% rhizosphere growth-promoting bacteria (J1 treatment group), and the soil with 10% rhizosphere growth-promoting bacteria (J2 treatment group), with three replicates. Physiological indicators and enzyme activity indicators of the plants were measured 90 days after planting.

[0054] The values ​​of soluble sugar / protein content, malondialdehyde, proline, plant peroxidase, catalase, and SOD activity were measured in the leaves to evaluate the changes in various physiological indicators of Suaeda salsa after 90 days of growth.

[0055] 2. Experimental Results like Figure 8 As shown, the physiological indicators of Suaeda salsa change over 60 days of growth.

[0056] Leaf soluble sugar content (see Figure 8(a) Compared with the control group, the groups with 5% rhizosphere growth-promoting bacteria (J1) and 10% rhizosphere growth-promoting bacteria (J2) showed increases of 5.47% and 19.71%, respectively; the content of soluble sugars in plants was positively correlated with plant resistance. Specifically, the higher the soluble sugar content, the less likely the plant cells would lose water under stress, and the greater the chance of survival, i.e., the higher the resistance. This experiment shows that rhizosphere growth-promoting bacteria treatment increased the stress resistance of Suaeda salsa.

[0057] Leaf soluble protein content (see) Figure 8 (b) Compared with the CK group, the addition of 5% rhizosphere growth-promoting bacteria (J1) and 10% rhizosphere growth-promoting bacteria (J2) increased by 188.49% and 123.95%, respectively. Soluble protein index reflects the physiological state of plants. Its increase is a sign of vigorous growth, sufficient nutrition and strong stress resistance. Therefore, the treatment of rhizosphere growth-promoting bacteria group promoted plant growth and improved its stress resistance.

[0058] Leaf malondialdehyde (MDA) content (see Figure 8 (c) Compared with the CK group, there was no significant difference in group J1, while group J2 decreased by 15.71%. MDA is a product of lipid peroxidation in plants, and its content can reflect the degree of stress damage suffered by plants. Excessive MDA content can damage plant cells, leading to impaired membrane and cell function. This indicates that plants with 10% rhizosphere growth-promoting bacteria exhibit better stress resistance.

[0059] Proline content (see) Figure 8 (d): Compared with the CK group, the groups with 5% rhizosphere growth-promoting bacteria (J1) and 10% rhizosphere growth-promoting bacteria (J2) showed increases of 8.51% and 79.95%, respectively. The proline content in plants reflects the stress resistance of plants to a certain extent, indicating that rhizosphere growth-promoting bacteria treatment can improve the stress resistance of plants.

[0060] Catalase content (see) Figure 8 (e) Compared with the control group, the groups with 5% rhizosphere growth-promoting bacteria (J1) and 10% rhizosphere growth-promoting bacteria (J2) showed increases of 219.04% and 118.33%, respectively. The catalase content in plants plays a crucial role, primarily functioning to scavenge hydrogen peroxide (H2O2), protect cells from oxidative stress damage, and enhance plant adaptability. Therefore, rhizosphere growth-promoting bacteria treatment significantly increased the catalase content in plant leaves, improving their resistance to oxidative stress.

[0061] Peroxidase content (see) Figure 8(f) Compared with the control group, the groups with 5% rhizosphere growth-promoting bacteria (J1) and 10% rhizosphere growth-promoting bacteria (J2) showed increases of 180.35% and 111.20%, respectively. Plant peroxidase (POD) plays a crucial role in antioxidant detoxification, growth and development regulation, enhanced defense capabilities, and reduced membrane system damage, directly impacting plant stress resistance and survival. This indicates that rhizosphere growth-promoting bacteria treatment significantly increased peroxidase content in plant leaves, thereby improving their stress resistance and survival.

[0062] Plant SOD activity (see) Figure 8 (g) Compared with the CK group, the groups with 5% rhizosphere growth-promoting bacteria (J1) and 10% rhizosphere growth-promoting bacteria (J2) showed increases of 173.11% and 98.12%, respectively. SOD (superoxide dismutase) is a protective enzyme whose main function is to scavenge superoxide anions (O2). - This prevents lipid peroxidation of biofilms, thus protecting them. High SOD activity means that plants can more effectively remove harmful substances produced during metabolism. This experiment demonstrates that adding rhizosphere-promoting bacteria to magnesium dust-contaminated soil helps Suaeda salsa better remove harmful substances from its body.

[0063] The enhancement of plant stress resistance is closely related to the optimization of plant growth characteristics: on the one hand, the accumulation of stress-resistant substances such as soluble sugars and soluble proteins not only strengthens the cell's osmotic regulation capacity to maintain water balance, but also reserves sufficient energy and nutrients for plant growth and development; on the other hand, the reduction in malondialdehyde content (J2 group) and the increase in the activity of antioxidant enzymes (CAT, POD, SOD) effectively mitigate the oxidative damage caused by magnesium dust pollution and maintain the stability of cell structure and metabolic function. These improvements in stress resistance ultimately enhance the plant's adaptability to polluted soil, allowing it to maintain good growth even in adverse environments.

[0064] In summary, this invention collected rhizosphere soil from *Suaeda salsa* in a magnesite mining area, and after multiple initial plate screenings, isolated a strain of *Klebsiella pastoris* with high alkali-tolerant nitrogen-fixing ability from the soil. Klebsiella pasteurii C-1, after three generations of purification, maintained stable and desirable genetic traits. Klebsiella pastoris ( Klebsiella pasteurii C-1 can improve the stress resistance of Suaeda salsa and improve the soil environment, promoting plant growth.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. It is understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and all such changes should be included within the scope of protection of the present invention.

Claims

1. A rhizosphere growth-promoting bacterium tolerant to magnesium-contaminated soil, characterized in that: The alkali-tolerant rhizosphere growth-promoting bacteria in the magnesium dust-contaminated soil is C-1, classified and named Klebsiella pastoris. Klebsiella pasteurii The specimen was deposited on August 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35635, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of the alkali-tolerant rhizosphere growth-promoting bacteria in magnesium-contaminated soil as described in claim 1, characterized in that: The strain was used to promote plant growth under alkaline environmental stress.

3. The application according to claim 2, characterized in that: The plant in question is Suaeda salsa.

4. The application of the alkali-tolerant rhizosphere growth-promoting bacteria in magnesium-contaminated soil as described in claim 1, characterized in that: The strain is used to prepare microbial inoculant fertilizer.

5. The application of the alkali-tolerant rhizosphere growth-promoting bacteria in magnesium-contaminated soil as described in claim 1, characterized in that: The strain is used for soil nitrogen fixation.

6. A plant growth promoting microbial agent, characterized in that: The rhizosphere growth-promoting bacteria for magnesium-contaminated soil as described in claim 1.

7. A microbial inoculant fertilizer, characterized in that: The rhizosphere growth-promoting bacteria for magnesium-contaminated soil as described in claim 1.

8. A soil nitrogen-fixing bacterial agent, characterized in that: The rhizosphere growth-promoting bacteria for magnesium-contaminated soil as described in claim 1.

9. The microbial agent according to any one of claims 6-8, characterized in that: The product contains the alkali-tolerant rhizosphere growth-promoting bacteria, bacterial suspension, metabolites, or fermentation broth of magnesium-contaminated soil as described in claim 1.

Images