Saline-alkali-tolerant growth-promoting pseudomonas sp. DHS-HJ-IAA-2 and application thereof

Abstract

The invention discloses a salt-alkali-tolerant growth-promoting ectopseudomonas DHS-HJ-IAA-2 strain and an application thereof, and belongs to the technical field of microorganisms. According to the invention, a strain of salt-resistant, alkali-resistant and drought-resistant pseudomonas sp. DHS-HJ-IAA-2 is screened, and the pseudomonas sp. DHS-HJ-IAA-2 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.36164. Experimental results show that the ectopseudomonas DHS-HJ-IAA-2 has the functions of producing IAA, fixing nitrogen, dissolving potassium, degrading cellulose and lignin, inhibiting phytopathogen and the like, and can remarkably improve the bacteria abundance of saline-alkali soil, improve soil fertility and promote plant growth in a saline-alkali environment. The invention provides a new strain resource and a technical means for improving saline-alkali soil and planting crops on the saline-alkali soil, and realizes improvement of the saline-alkali soil from two aspects of soil-crop-microorganism comprehensive action and microorganism-plant synergistic action.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a salt-tolerant, alkali-resistant, growth-promoting strain of *Exophytosporum* DHS-HJ-IAA-2 and its applications. Background Technology

[0002] Soil salinization is a prevalent ecological problem and has become a key focus and hot topic in soil ecological restoration. Soil salinization not only directly harms crop growth and reduces land productivity, but also leads to environmental problems such as soil erosion and desertification, hindering healthy economic development. Traditional physical and chemical remediation methods have been applied to some extent in the improvement of saline-alkali land, but they suffer from drawbacks such as water waste, high investment costs, complex engineering, and the potential for secondary environmental pollution. In contrast, microbial remediation utilizes beneficial microorganisms to improve the soil microbial community, reduce soil salinity, increase nutrient utilization, promote the synthesis of soil organic matter, increase available nutrients, regulate plant growth, and enhance plant resistance. It offers advantages such as high cost-effectiveness, no residue in crops, energy and freshwater savings, and wide applicability. Furthermore, it can prevent environmental pollution and soil degradation, demonstrating significant application potential in low-fertility saline-alkali land.

[0003] However, microbial remediation technology still faces two major bottlenecks: ① there are few suitable strains for high salinity and alkalinity; ② the microbial agents have low activity and are difficult to colonize in high salinity and alkalinity environments, making it difficult to exert synergistic effects. Current research in this field often focuses on the growth-promoting effects of microorganisms on plants, lacking consideration of the integrated effects of soil-plant-microorganism interactions and analysis of the soil-improvement effects of synergistic interactions between microorganisms and plants.

[0004] Rhizosphere growth-promoting bacteria, as an important component of soil, are closely related to plant growth and the soil environment in saline-alkali soils. Some plant growth-promoting bacteria can not only dissolve nutrient ions in the soil, secrete plant growth factors, promote plant growth, and increase soil microbial diversity, but also improve soil salinity characteristics and enhance plant salt tolerance. Therefore, utilizing the salt-alkali tolerance and growth-promoting functions of microorganisms can improve the soil environment for plant growth and promote plant development, representing a safe, efficient, and environmentally friendly measure for the remediation of saline-alkali soils. There is an urgent need to isolate and identify rhizosphere growth-promoting bacteria with salt-alkali tolerance and various growth-promoting properties, thereby providing important functional microbial resources and a technical foundation for the preparation of microbial agents for improving saline-alkali land. Summary of the Invention

[0005] The purpose of this invention is to provide a salt-tolerant and growth-promoting strain of *Pseudomonas exogenae* DHS-HJ-IAA-2 and its applications to solve the problems existing in the prior art. This invention provides new microbial resources and technical means for the improvement of saline-alkali soil and the planting of crops on saline-alkali soil, and achieves the improvement of saline-alkali soil from two aspects: the integrated effect of soil-crop-microorganism and the synergistic effect of microorganism-plant.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides an exopseudomonas ( Ectopseudomonas The *Exopseudomonas* sp. DHS-HJ-IAA-2 was deposited on October 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36164, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides the application of the above-mentioned Pseudomonas exogenans DHS-HJ-IAA-2 in the preparation of a growth-promoting bacterial agent that promotes plant growth.

[0008] The present invention also provides a growth-promoting agent, with the above-mentioned Pseudomonas exogenans DHS-HJ-IAA-2 as the active ingredient.

[0009] Furthermore, in the growth-promoting bacterial agent, the viable concentration of *Pseudomonas exogenae* DHS-HJ-IAA-2 is not less than 2 × 10⁻⁶. 8 CFU / mL.

[0010] The present invention also provides the use of the above-mentioned *Exophytosporum exosum* DHS-HJ-IAA-2 or the above-mentioned growth-promoting agent in any of the following: (1) Nitrogen fixation; (2)Production of IAA; (3) Potassium dissolution; (4) Decomposition of cellulose: (5) Decomposes lignin; (6) Inhibit pathogenic fungi.

[0011] Furthermore, the pathogenic fungus is either *Fusarium oxysporum* or *Fusarium oxysporum*.

[0012] The present invention also provides the application of the above-mentioned Pseudomonas exogenans DHS-HJ-IAA-2 or the above-mentioned growth-promoting bacteria in promoting plant growth and / or improving the plant's tolerance to salt and alkali stress.

[0013] The present invention also provides a method for promoting plant growth and / or improving the plant's tolerance to salt and alkali stress, comprising the step of applying the above-mentioned Pseudomonas exogenans DHS-HJ-IAA-2 or the above-mentioned growth-promoting bacterial agent to the plant growth environment.

[0014] Furthermore, the plant growth environment is a saline and / or alkaline environment.

[0015] The present invention also provides the application of the above-mentioned Pseudomonas exogenans DHS-HJ-IAA-2 or the above-mentioned growth-promoting agent in improving saline-alkali soil.

[0016] The present invention also provides a method for improving saline-alkali soil, comprising the step of applying the above-mentioned Pseudomonas exogenae DHS-HJ-IAA-2 or the above-mentioned growth-promoting agent to the saline-alkali soil.

[0017] The present invention discloses the following technical effects: This invention screened a salt-tolerant, alkali-tolerant, and drought-tolerant strain of *Pseudomonas exogenae*, DHS-HJ-IAA-2, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36164. Experimental results show that *Pseudomonas exogenae* DHS-HJ-IAA-2 possesses functions such as high-efficiency IAA production, decomposition of insoluble potassium, nitrogen fixation, degradation of cellulose and lignin, and inhibition of pathogenic fungi in crops. *Pseudomonas exogenae* DHS-HJ-IAA-2 can improve soil physicochemical properties through its potassium solubilization and nitrogen fixation capabilities, providing better conditions for microbial proliferation and crop growth. Simultaneously, the secreted IAA can promote crop root growth and enhance plant adaptability to saline-alkali environments. Plant growth provides a carbon source for root microorganisms, promoting their proliferation. Therefore, *Pseudomonas exogenae* DHS-HJ-IAA-2 can significantly improve soil fertility, increase bacterial abundance in saline-alkali soils, and promote plant growth in saline-alkali environments. Pot and field trials have shown that the application of *Pseudomonas exogenae* DHS-HJ-IAA-2 significantly improves the soil environment and promotes maize growth. This invention provides new microbial resources and technical means for the improvement of saline-alkali soils and the cultivation of crops on saline-alkali soils, achieving soil improvement through both integrated soil-crop-microorganism interactions and synergistic microorganism-plant interactions. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1This is a colony morphology diagram of *Exopseudomonas* DHS-HJ-IAA-2 on LB solid medium. Figure 2 Figure showing the qualitative analysis results of IAA production by *Exopsymonas DHS-HJ-IAA-2*. Figure 3 The image shows the qualitative analysis results of potassium solubilization of Pseudomonas exogenes DHS-HJ-IAA-2 on potassium-solubilizing medium. Figure 4 The image shows the qualitative analysis results of *Exopseudomonas* DHS-HJ-IAA-2 on Ashby nitrogen-free medium. Figure 5 The image shows the clear zone observation results of Pseudomonas exogenans DHS-HJ-IAA-2 on Congo red medium. Figure 6 Figure showing the results of the decolorization of *Pseudomonas exogenae* DHS-HJ-IAA-2 on aniline blue medium; Figure 7 The image shows the results of the salt tolerance test for Pseudomonas exogenae DHS-HJ-IAA-2. Figure 8 The image shows the results of acid and alkali resistance testing for Pseudomonas exogenans DHS-HJ-IAA-2. Figure 9 Figure showing the temperature tolerance test results of Pseudomonas exogenans DHS-HJ-IAA-2; Figure 10 Figure showing the results of drought tolerance testing for Exopseudomonas DHS-HJ-IAA-2; Figure 11 Image showing the morphological observation of Pseudomonas exogenes DHS-HJ-IAA-2 under oil immersion Gram staining experiment; Figure 12 The image shows the cell morphology of *Exopseudomonas* DHS-HJ-IAA-2 under a transmission electron microscope. Figure 13 To construct a phylogenetic tree for the exopsips DHS-HJ-IAA-2 based on the 16S rDNA gene sequence; Figure 14 Figure 1 shows the results of an experiment on the promotion of maize growth in saline-alkali soil by *Pseudomonas exogenans* DHS-HJ-IAA-2. Figure 15 This image shows the effect of Pseudomonas exogenes DHS-HJ-IAA-2 on promoting maize growth in saline-alkali land under field experimental conditions. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] The culture medium formulation of this invention is as follows: LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1000 mL deionized water, pH 7.0-7.2. Add 1.5%-2% agar powder to the solid medium.

[0026] Ashby's nitrogen-free medium: mannitol 10 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, K2SO4 0.3 g / L, CaCO3 5 g / L, with 2% agar powder added to the solid medium.

[0027] Potassium-solubilizing medium: sucrose 10.0 g / L, MgSO4·7H2O 0.5 g / L, CaCO3 1.0 g / L, (NH4)2SO4 1.0 g / L, NaCl 0.1 g / L, yeast extract 0.5 g / L, K2HPO4 2.0 g / L, pH 7.0-7.4. 2% agar powder was added to the solid medium.

[0028] Cellulase fermentation liquid culture medium: CMC-Na 20.0 g / L, (NH4)2SO4 5.0 g / L, KH2PO4 2.0 g / L, FeSO4·7H2O 0.01 g / L, CaCl2 0.5 g / L, NaCl 5.0 g / L, distilled water 1000 mL.

[0029] Congo red solid medium: sodium carboxymethyl cellulose 20 g / L, Na2HPO4 2.5 g / L, KH2PO4 1.5 g / L, peptone 2.5 g / L, agar 20 g / L, Congo red 0.2 g / L.

[0030] LB-Aniline Blue Medium: Peptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, Agar 20 g / L, Aniline Blue 0.1 g / L.

[0031] Liquid enzyme-producing medium: alkali lignin 2 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.1 g / L, CaCl2 0.08 g / L, FeSO4·7H2O 0.05 g / L, MnCl2 0.02 g / L, KH2PO4 1 g / L, peptone 2 g / L.

[0032] Example 1: Isolation of growth-promoting bacteria in the rhizosphere of saline-alkali soil The test soil samples were collected from the Shandong Coastal Soil Salinization Prevention and Saline-Alkali Land Improvement Demonstration Base in Changyi City, Weifang City, Shandong Province (119.498067°E, 37.076731°N). A five-point sampling method was used. After removing the topsoil, soil samples were collected at a depth of 5-15 cm, mixed thoroughly, placed in sterile self-sealing bags, and brought back to the laboratory for storage at 4°C for later use.

[0033] Strain isolation and purification: A 250 mL Erlenmeyer flask containing 90 mL of distilled water and 6 glass beads was sterilized. 10 g of soil sample was weighed and added to the sterilized Erlenmeyer flask. The mixture was shaken at 160 r / min for 60 min, allowed to stand for 10 min, and the supernatant was used for 10-fold serial dilutions. 100 μL of each dilution was taken. -3 -10 -5 The bacterial suspension was spread onto LB agar plates and incubated upside down at 30°C for 24 h. Based on the morphology, size, and color of the colonies on the plates, different single colonies were selected and streaked 3-5 times on LB agar plates for purification to obtain purified strains. These purified strains were then inoculated into slant agar plates, collected, and stored at 4°C for later use, and the strains were numbered.

[0034] A total of 12 different strains were isolated.

[0035] Example 2 Screening of DHS-HJ-IAA-2, a rhizosphere growth-promoting bacterium in saline-alkali soil. Different isolated strains were inoculated onto different functional culture media, and a strain DHS-HJ-IAA-2 was screened out, which simultaneously possesses the functions of IAA production, nitrogen fixation, decomposition of insoluble potassium, degradation of cellulose, degradation of lignin, and inhibition of crop pathogenic fungi. Its colony morphology is as follows: Figure 1 As shown, the specific steps are as follows.

[0036] 1. IAA production capacity of the strain Qualitative tests: The Salkowski colorimetric method was used to screen the IAA production capacity of the purified strains. Bacteria from activated single-colony slants were transferred to LB liquid medium and cultured with shaking at 30°C and 160 r / min. [The text then abruptly shifts to a different topic:] ...at 2% (... v / v The inoculum should be taken from the above bacterial suspension in the logarithmic growth phase (1.0 × 10⁻⁶). 8 CFU / mL -1.0×10 9 Inoculate the bacterial suspension (CFU / mL) into LB liquid medium containing L-tryptophan (100 mg / L) and incubate with shaking at 30 ℃ and 160 r / min for 72 h. Drop 50 μL of the bacterial suspension into a 96-well clear plate, add an equal volume of Salkowski chromogenic reagent, and use IAA standard solution as a positive control. Incubate the 96-well clear plate at room temperature in the dark for 30 min; if the color turns red, the strain has the ability to produce IAA.

[0037] Quantitative test: Centrifuge the bacterial suspension containing L-tryptophan in LB liquid medium, add an equal amount of Salkowski colorimetric reagent to the supernatant, let it stand in the dark for 30 min, and measure the absorbance at 530 nm using the UV method to determine the IAA yield of the strain.

[0038] like Figure 2 As shown, the results indicated that strain DHS-HJ-IAA-2 turned pink after being cultured in LB medium containing tryptophan and reacting with Salkowski colorimetric solution. Further measurement of the absorbance of the bacterial suspension at 530 nm revealed that the IAA content in the fermentation broth was 18.16 mg / L.

[0039] 2. Potassium solubilization function Qualitative test: Take 2 μL of purified strain LB culture and inoculate it onto a potassium-solubilizing solid medium plate containing insoluble potassium feldspar powder. Incubate at 30℃ for 5 days and observe whether transparent oil droplet-like circles appear. The appearance of potassium-solubilizing circle colonies can prove that the strain has potassium-solubilizing ability.

[0040] Quantitative experiment: The prepared bacterial suspension was diluted at 2% ( v / v)The inoculum was transferred to 50 mL of potassium-solubilizing liquid medium and cultured at 30℃ and 180 r / min for 7 days. After centrifugation at 8000 r / min for 10 min, the supernatant was collected and the potassium content in the supernatant was determined by atomic absorption spectrometry.

[0041] like Figure 3 As shown, strain DHS-HJ-IAA-2 produced obvious potassium-solubilizing zones on potassium-solubilizing culture plates, with a potassium-solubilizing zone diameter (D) / colony diameter (d) ratio of 8.5. Quantitative experiments showed that the strain's conversion of potassium feldspar powder reached 16.92 mg / L.

[0042] 3. Nitrogen fixation capacity Qualitative test: The nitrogen fixation capacity of the strain was determined using Ashby nitrogen-free medium. The purified strain was inoculated on Ashby nitrogen-free medium and cultured at 28°C for 5 days. The fact that the strain could grow normally on nitrogen-free Ashby medium indicated that the strain had nitrogen fixation capacity, and the degree of nitrogen fixation capacity was judged based on the colony diameter.

[0043] Quantitative experiments: The nitrogenase activity of the strain was determined using an ELISA nitrogenase kit. The prepared bacterial suspension was mixed at 2% ( v / v) The inoculum was transferred to Ashby liquid medium and cultured on a shaker at 30°C and 180 r / min for 96 h. After centrifuging the fermentation broth at 8000 r / min for 10 min, the supernatant was obtained and nitrogenase activity was measured using an ELISA kit.

[0044] The results of the nitrogen fixation experiment are as follows Figure 4 As shown, strain DHS-HJ-IAA-2 grew well on Ashby nitrogen-free medium, indicating that this strain has nitrogen-fixing function. Quantitative experiments revealed that the nitrogenase activity of the strain reached 303.37 U / L.

[0045] 4. Ability to degrade cellulose Qualitative experiment: The strain DHS-HJ-IAA-2 was transferred to Congo red solid medium and incubated upside down at 30°C for 2 days. The presence of a clear zone was observed.

[0046] Quantitative experiment: strain DHS-HJ-IAA-2 was used at 2% ( v / v) The inoculum was transferred to cellulase-producing fermentation broth and cultured at 30°C and 160 r / min for 2 days. One mL of fermentation broth was centrifuged at 12000 r / min for 5 min at 4°C. 200 μL of crude enzyme solution was thoroughly mixed with 200 μL of substrate and incubated at 50°C for 60 min. Then, 400 μL of dinitrosalicylic acid reagent was added, and the mixture was incubated at 100°C for 10 min. After cooling, the volume was adjusted to 10 mL, and the absorbance at 540 nm was measured.

[0047] like Figure 5 As shown, Congo red staining revealed a distinct clear zone in strain DHS-HJ-IAA-2, with a clear zone diameter / colony diameter ratio of 2.54, indicating that DHS-HJ-IAA-2 is a cellulase-producing cellulose-degrading bacterium. Cellulase activity assays showed that the enzyme activity reached 3.12 U / mL after 48 h.

[0048] 5. Lignin degradation ability Qualitative test: The strain DHS-HJ-IAA-2 was transferred to LB-aniline blue medium and cultured for 3 days. The diameter of the fading zone and the diameter of the colony were measured and their ratio was calculated.

[0049] Quantitative experiment: using strain DHS-HJ-IAA-2 at 2% ( v / v) The inoculum was added to liquid enzyme-producing medium and cultured at 30°C and 180 r / min in a shaker. After 72 h, samples were taken to extract the crude enzyme solution, and the enzyme activity of the strain was determined. The activities of manganese peroxidase (MnP), peroxidase (LiP), and laccase (Lac) were determined using the ABTS method, the 2,6-dimethylphenol method, and the resveratrol method, respectively.

[0050] The ratio of the fading zone diameter to the colony diameter reflects, to some extent, the lignin degradation ability of the strain. Figure 6 As shown, the experimental results indicated that DHS-HJ-IAA-2 produced a distinct fading zone on LB-aniline blue medium, with the ratio of the fading zone diameter to the strain diameter being 6.25. After 3 days of culture, the Mnp, Lip, and Lac enzyme activities were 0.95 U / L, 5.42 U / L, and 7.27 U / L, respectively.

[0051] 6. Antagonistic ability against pathogens The antagonistic ability of DHS-HJ-IAA-2 against pathogenic fungi was determined using the plate confrontation method. Two pathogenic strains (Fusarium wilt of cucumber and Fusarium oxysporum) were activated and inoculated onto one side of a PDA agar plate, 2 cm from the center. 5 μL of the fermentation broth of strain DHS-HJ-IAA-2 was then spotted onto the opposite side of the inoculation point, and the plates were incubated at 30 ℃ for 7 days. The inhibition radius was measured, and the inhibition rate was calculated.

[0052] Inhibition rate % = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100; As shown in Table 1, the experimental results showed that strain DHS-HJ-IAA-2 had significant antagonistic effects on two pathogenic strains (Fusarium wilt of cucumber and Fusarium oxysporum of watermelon), with inhibition rates of 21.7% and 32.2%, respectively.

[0053] Table 1. Antagonistic effect of DHS-HJ-IAA-2 against pathogenic fungi Example 3: Tolerance of salt-alkali rhizosphere growth-promoting bacteria DHS-HJ-IAA-2 to salinity, pH, temperature, and drought. 1. Salt tolerance test Add NaCl to LB liquid medium to achieve a mass concentration of ( ). w / v Six gradients were set up, with concentrations of 2%, 5%, 8%, 10%, 12%, and 15% (g / mL) for the DHS-HJ-IAA-2 bacterial suspension, respectively. v / v The inoculum was sequentially transferred to LB liquid medium with different NaCl concentration gradients, and cultured at 30℃ and 160 r / min for 36 h with shaking. The OD of the culture medium was then measured. 600 value.

[0054] like Figure 7 As shown, the salinity tolerance results indicate that strain DHS-HJ-IAA-2 can grow normally in LB medium with NaCl concentration ≤10%, indicating that strain DHS-HJ-IAA-2 has a certain halophilic function; when the salinity of the medium continues to increase to 12%, the growth of strain DHS-HJ-IAA-2 is inhibited.

[0055] 2. Acid and alkali resistance test The pH of LB medium was adjusted using 1 M NaOH and HCl solutions, respectively. The pH was then increased in a gradient from 4 to 11, resulting in eight gradients. The DHS-HJ-IAA-2 bacterial suspension was added at 2% (…). v / v Inoculum was sequentially transferred to LB liquid medium at different pH values ​​and incubated at 30 °C with shaking at 160 r / min for 36 h. The OD of the culture medium was then measured. 600 .

[0056] like Figure 8 As shown, the experimental results indicate that strain DHS-HJ-IAA-2 can grow normally in culture media with pH values ​​of 7-10, belonging to alkali-tolerant microorganisms. The strain even maintains a certain growth rate at pH 10, with an OD value of [missing information]. 600 It reached 0.356.

[0057] 3. Temperature tolerance test The bacterial suspension of strain DHS-HJ-IAA-2 was prepared at 2% ( v / v The inoculum was sequentially transferred to LB liquid medium and incubated at 160 r / min for 36 h at 15℃, 20℃, 25℃, 30℃, 35℃ and 40℃, respectively. The OD of the culture medium was then measured. 600 value.

[0058] like Figure 9As shown, strain DHS-HJ-IAA-2 can grow within a culture temperature range of 15℃-40℃, with the optimal growth temperature being 25℃-35℃. When the temperature is further increased to 40℃, the biomass of strain DHS-HJ-IAA-2 decreases.

[0059] 4. Drought tolerance test The bacterial suspension of strain DHS-HJ-IAA-2 was prepared at 2% ( v / v The inoculum was sequentially transferred to different concentrations of polyethylene glycol (PEG). 6000 In a drought-resistant medium, the culture was incubated with shaking at 160 r / min for 36 h, and the OD of the culture solution was measured. 600 value.

[0060] like Figure 10 As shown in the figure, the experimental results indicate that strain DHS-HJ-IAA-2 exhibits good drought resistance characteristics and can withstand drought conditions (PEG). 6000 It is grown under conditions where the content is 30%.

[0061] Example 4: Morphological and physiological-biochemical identification of DHS-HJ-IAA-2, a rhizosphere growth-promoting bacterium in saline-alkali soil. 1. Morphological identification Under aseptic conditions, strain DHS-HJ-IAA-2 was transferred to LB agar plates and incubated at 30°C for 24 h. The colony morphology on the plates was then observed.

[0062] On the agar plate, colonies are round, raised, pale yellow, relatively large, translucent, and have a moist, smooth surface. Single colonies are picked and Gram-stained; the results are as follows... Figure 11 As shown, under a microscope, the bacteria appear as short rods, stain red with Gram stain, and are Gram-negative. Transmission electron microscopy observations yielded the following results: Figure 12 As shown, the bacteria are rod-shaped, without flagella or spores, and have a size of 1.6 μm × 0.4 μm.

[0063] 2. Physiological and biochemical identification of bacterial strains Physiological and biochemical identification of the strains was performed with reference to the "Common Bacterial Identification Manual" and the "Bergey's Manual of Bacterial Identification", including 21 indicators detected by API 20NE, 19 enzyme activities detected by API ZYM, API 50CH detection, catalase, oxidase and Biolog GNIII carbon source utilization experiments.

[0064] The results of the API 20NE test for 21 indicators of strain DHS-HJ-IAA-2 are shown in Table 2. The positive results were for NO3 reduction to NO2, glucosidase, glucose, maltose, gluconate, decanoic acid, malic acid, and sodium citrate. The weakly positive results were for mannitol and adipic acid. All other test items were negative.

[0065] Table 2. Results of 21 indicators detected by API 20NE for strain DHS-HJ-IAA-2 Note: "+" indicates positive, "-" indicates negative, and "W" indicates weak positive.

[0066] The results of 19 enzyme activities of strain DHS-HJ-IAA-2 are shown in Table 3. β-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosidase, α-mannosidase, and β-fucosidase were negative.

[0067] Table 3. Results of API ZYM assay for 19 enzyme activities in strain DHS-HJ-IAA-2 Note: "+" indicates positive, "-" indicates negative, and "W" indicates weak positive.

[0068] The API 50CH test results of strain DHS-HJ-IAA-2 are shown in Table 4. Except for D-glucose, D-cellobiose, D-mercaptobiose and starch, which were weakly positive, all other test items were negative.

[0069] Table 4. API 50CH detection results of strain DHS-HJ-IAA-2 Note: "+" indicates positive, "-" indicates negative, and "W" indicates weak positive.

[0070] The Biolog GNIII carbon source utilization results of strain DHS-HJ-IAA-1 are shown in Table 5. The following assays showed negative results: D-trehalose, D-cellobiose, gentiobiose, stachyose, metriose, α-D-lactose, β-formyl-D-glucosidase, D-salicylic acid, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, N-acetyl-D-galactosamine, N-acetylneuraminic acid, D-mannose, L-rhamnose, inosine, D-sorbitol, D-mannitol, D-arabinol, inositol, D-glucose-6-phosphate, D-aspartic acid, and D-serine. All other assays were positive or weakly positive.

[0071] Table 5. Results of Biolog GNIII carbon source utilization assay for strain DHS-HJ-IAA-2 Note: "+" indicates positive, "-" indicates negative, and "W" indicates weak positive.

[0072] As shown in Table 6, strain DHS-HJ-IAA-2 showed positive results for both catalase and oxidase.

[0073] Table 6. Results of catalase and oxidase detection in strain DHS-HJ-IAA-2 Note: "+" indicates positive, "-" indicates negative, and "W" indicates weak positive.

[0074] 3. Molecular biological identification The 16S rDNA sequence of the strain was amplified using existing universal primers 27F and 1492R. The single-band PCR product was purified by ExoSAP-IT and sequenced by Shanghai Genewiz Technology Service Co., Ltd. The nucleotide sequence of the 16S rDNA of strain DHS-HJ-IAA-2 obtained by sequencing is shown in SEQ ID NO.1.

[0075] SEQ ID NO.1:

[0076] The 16S rDNA sequence of strain DHS-HJ-IAA-2 was BLAST-aligned in the GenBank database. Sequences with high homology were selected and sequence analysis was performed using MEGA 7.0 to construct a phylogenetic tree. The phylogenetic tree is shown below. Figure 13 As shown, the results indicate that strain DHS-HJ-IAA-2 is related to *Exopsysporium*. Ectopseudomonas chengduensis The strain L02 (MG719530.1) showed 94% sequence homology. Further analysis, combined with morphological characteristics and physiological and biochemical properties, confirmed that strain DHS-HJ-IAA-2 belongs to the genus *Exopseudomonas*. Ectopseudomona) The strain was named *Exopseudomonas*. Ectopseudomonas sp.)DHS-HJ-IAA-2.

[0077] 4. Strain preservation Exopsymonas ( Ectopseudomonas sp.)DHS-HJ-IAA-2 was deposited on October 13, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36164. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0078] Example 5: Exopseudomonas aeruginosa DHS-HJ-IAA-2 promotes maize growth in saline-alkali soil The saline-alkali soil used in the experiment was taken from Binhai New Area, Tianjin. Straw with a length of about 0.5 cm was added at a mass ratio of 3:1. The total soluble salt content of the soil was 2.3‰ and the pH was 8.5.

[0079] Corn variety: Zhengdan 958.

[0080] *Pseudomonas exogenae* DHS-HJ-IAA-2 was transferred to LB broth for activation and cultured at 30°C and 160 r / min with shaking for 48 h. The fermentation broth was then centrifuged at 4°C and 6000 r / min for 10 min to collect the cells and prepare a solution with a concentration of 2×10⁻⁶. 8 A bacterial suspension of CFU / mL is prepared for use.

[0081] The experiment consisted of two groups. The experimental group was treated with a suspension of *Pseudomonas exogenae* DHS-HJ-IAA-2, and 50 mL of the suspension was applied to the soil every 5 days. The control group received no bacterial treatment, only tap water. Each group had 6 pots (15 cm × 15 cm), with 2 corn seeds sown in each pot. Corn growth was continuously observed after emergence. After 20 days, plant height, fresh weight, dry weight, and other growth indicators were measured. Rhizosphere soil samples were collected from the potted corn plants, and the following indicators were measured in the saline-alkali soil: total soluble salts, organic matter, total nitrogen, available nitrogen, total potassium, available potassium, total phosphorus, and total rhizosphere bacteria count.

[0082] like Figure 14 As shown in Table 8, the experimental results indicate that *Pseudomonas exogenae* DHS-HJ-IAA-2 can significantly promote maize growth. Table 8 shows that the average plant height of maize seedlings treated with the bacterial solution was 38.2 cm, stem diameter was 3.5 mm, above-ground fresh weight was 2.09 g, and dry weight was 0.32 g, all significantly higher than the control group, indicating that the application of the bacterial agent can significantly promote maize seedling growth.

[0083] Table 8. Effects of *Exopseudomonas* DHS-HJ-IAA-2 on potted maize. As shown in Table 9, after applying Pseudomonas exogenans DHS-HJ-IAA-2 bacterial solution to the rhizosphere saline-alkali soil of maize, the total salt content and pH of the soil decreased compared with the control group, while the total nitrogen, available phosphorus, and organic matter content all increased significantly.

[0084] Table 9. Effects of Exopseudomonas DHS-HJ-IAA-2 on saline-alkali soils As shown in Table 10, after inoculation with Pseudomonas exogenes DHS-HJ-IAA-2 bacterial solution, the number of bacteria, fungi and actinomycetes in saline-alkali soil was significantly increased.

[0085] Table 10. Effects of *Exopseudomonas* DHS-HJ-IAA-2 on the abundance of microorganisms in maize rhizosphere soil. Example 6: Exopseudomonas aeruginosa DHS-HJ-IAA-2 promotes maize growth in saline-alkali soil under field experimental conditions. A field experiment was conducted in the saline-alkali land of Binhai New Area, Tianjin. This area is reclaimed land with a total soluble salt content of 2.2‰ and a pH of 8.6, making crops susceptible to salt damage and hindering their growth. The experiment included two treatments: a control group and an experimental group.

[0086] Experimental Procedure: First, straw was spread on the surface of the plot to be treated at a rate of 80 kg / mu. The soil was then tilled four times using a tiller in a cross-tillage pattern to a depth of 20-30 cm, and leveled. The experimental group was irrigated with a solution of *Pseudomonas exogenae* DHS-HJ-IAA-2, spraying the agent to thoroughly wet the top 5 cm of soil every 3-5 days. The control group was irrigated only with tap water. Corn of the Zhengdan 958 variety was planted 20 days later. A real-world image of the corn 60 days after planting is shown below. Figure 15 As shown in Table 11, the chlorophyll and biomass test results for the two treatment groups are presented in Table 12. The soil test results for the two treatment groups after 120 days are shown in Table 12.

[0087] Table 11 Effects of *Exoplasmodium* DHS-HJ-IAA-2 on maize plant growth in a field trial. Table 12. Effects of *Exopseudomonas* DHS-HJ-IAA-2 on saline-alkali soils in field trials. Depend on Figure 15 As shown in Table 11, compared with the control group, irrigation with *Pseudomonas exogenae* DHS-HJ-IAA-2 significantly promoted the growth of maize in saline-alkali soil, specifically by increasing chlorophyll content by 27.2%, aboveground biomass by 25.9%, and underground biomass by 35.9%. Soil index measurements after 120 days showed that the soil pH decreased, total water-soluble salts decreased significantly, and organic matter content increased in the experimental group.

[0088] In summary, the *Pseudomonas exogenae* DHS-HJ-IAA-2 provided in this embodiment of the invention exhibits good salt, alkali, and drought tolerance, and can grow under conditions of salinity ≤10%, pH 6.0~10.0, and 15℃-40℃. *Pseudomonas exogenae* DHS-HJ-IAA-2 possesses functions such as efficient IAA production, nitrogen fixation, potassium solubilization, cellulose decomposition, lignin decomposition, and inhibition of crop pathogens. It can significantly promote crop growth in saline-alkali environments.

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An exopseudomonas bacterium ( Ectopseudomonas sp.)DHS-HJ-IAA-2, characterized in that, The aforementioned *Exopseudomonas* DHS-HJ-IAA-2 was deposited on October 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36164, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The use of the *Pseudomonas exogenae* DHS-HJ-IAA-2 as described in claim 1 in the preparation of a plant growth-promoting bacterial agent.

3. A growth-promoting agent, characterized in that, The active ingredient is the *Exopseudomonas* DHS-HJ-IAA-2 as described in claim 1.

4. The growth-promoting bacterial agent as described in claim 3, characterized in that, In the growth-promoting bacterial agent, the viable bacterial concentration of *Pseudomonas aeruginosa* DHS-HJ-IAA-2 is not less than 2 × 10⁻⁶. 8 CFU / mL.

5. The use of the *Exopseudomonas* DHS-HJ-IAA-2 according to claim 1 or the growth-promoting agent according to claim 3 or 4 in any of the following: (1) Nitrogen fixation; (2)Production of IAA; (3) Potassium dissolution; (4) Decomposition of cellulose: (5) Decomposes lignin; (6) Inhibit pathogenic fungi.

6. The application of the *Exopseudomonas* DHS-HJ-IAA-2 as described in claim 1 or the growth-promoting agent as described in claim 3 or 4 in promoting plant growth and / or improving plant tolerance to salt and alkali stress.

7. A method for promoting plant growth and / or improving plant tolerance to salt and alkali stress, characterized in that, The method includes the step of applying the *Exopseudomonas* DHS-HJ-IAA-2 of claim 1 or the growth-promoting agent of claim 3 or 4 to the plant growth environment.

8. The method as described in claim 7, characterized in that, The plant grows in a saline and / or alkaline environment.

9. The application of the *Exopseudomonas* DHS-HJ-IAA-2 as described in claim 1 or the growth-promoting agent as described in claim 3 or 4 in improving saline-alkali soil.

10. A method for improving saline-alkali soil, characterized in that, The method includes the step of applying the *Exopseudomonas* DHS-HJ-IAA-2 of claim 1 or the growth-promoting agent of claim 3 or 4 to the saline-alkali soil.

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