Halotolerant halomonas sp. and application thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses a halophilic halomonas and application thereof. Franzmannia qiaohouensis The halomonas is separated from the millet rhizosphere soil of saline-alkali land, has a strain number STM-122, a preservation number CGMCC No. 36349, is identified and classified by 16S rRNA gene sequence identification and phylogenetic analysis, and has the functions of phosphorus solubilization, EPS production, auxin production and iron carrier production. Through seed treatment by bacterial suspension, the germination energy and germination rate of millet seeds under saline-alkali stress can be significantly improved, and the indexes such as seedling root length and plant height are promoted; meanwhile, a complete preparation method of the fermentation bacterial fertilizer of the strain is provided, and the obtained fermentation bacterial fertilizer has a viable bacterial count ≥1×10 8 CFU / g. The application can enrich the salt-tolerant microbial resources, and provide technical support for the planting of millet and other crops in saline-alkali land.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a salt-tolerant Halomonas bacterium and its applications. Background Technology

[0002] Salinization not only exacerbates osmotic stress and ion toxicity in plants, inhibiting crop absorption of water and nutrients, but also interferes with soil microbial activity and community structure, affecting the material cycling and energy exchange of the soil-plant-microbe system. In recent years, the ecological function of soil microorganisms in regulating saline-alkali stress has gradually attracted attention. As the most active component of the soil ecosystem, microorganisms play an irreplaceable role in organic matter decomposition, nutrient cycling, rhizosphere interactions, and environmental adaptation. Numerous studies have shown that specific rhizosphere microorganisms can significantly enhance plant salt tolerance through multiple mechanisms, such as regulating hormone metabolism, enhancing antioxidant capacity, and improving ion balance. Microbial growth-promoting mechanisms are gradually becoming one of the important strategies for improving crop stress resistance.

[0003] Plant growth-promoting rhizobacteria (PGPRs) are a class of functional microorganisms that can stably colonize the rhizosphere and promote plant growth and stress adaptation through multiple pathways. PGPRs can enhance plant survival and growth under saline-alkali stress by synthesizing plant hormones such as auxin (IAA), gibberellin (GA), and abscisic acid (ABA), inducing antioxidant enzyme activity, maintaining Na⁺ / K⁺ ion homeostasis, and promoting nitrogen fixation, phosphorus solubilization, and siderophore synthesis. Simultaneously, PGPRs can regulate the rhizosphere microecological environment and enhance the synergistic effect of plant-microbe interactions, making them an important biological resource for improving the microecological environment of saline-alkali land and increasing crop quality and yield.

[0004] However, current research on salt-tolerant growth-promoting bacteria mainly focuses on Bacillus, whose screening, functional verification, and inoculant preparation technologies are relatively mature. In contrast, systematic research, mechanism analysis, and application technologies for Haloxylon ammodendron, another important group of salt-tolerant microorganisms, are severely lacking. This has resulted in the underdevelopment of strains with potential salt tolerance advantages, limiting the diversity of salt-tolerant microbial resources and the expansion of application scenarios. Furthermore, existing microbial inoculant technologies primarily focus on mainstream crops such as wheat and corn, with insufficient development of rhizosphere microorganisms for millet, a specialty grain crop in saline-alkali areas. This makes it difficult to meet the needs of diversified agricultural development in saline-alkali lands, necessitating technological breakthroughs to fill this gap. Summary of the Invention

[0005] To address the shortcomings of existing technologies, such as insufficient development of *Haloxylon* strains and limited coverage of microbial agents for specialty grain crops in saline-alkali regions, this invention isolates and screens *Haloxylon* strains with specific functions from the rhizosphere soil of millet in saline-alkali areas. Through systematic identification, its classification and functional characteristics are clarified, and supporting strain application and microbial fertilizer fermentation technologies are developed. This aims to fill the technological gap in the field of salt-tolerant growth-promoting bacteria, enrich salt-tolerant microbial resources, and provide support for the growth of millet and other crops under saline-alkali stress. To achieve this technical objective, this invention provides the following technical solution.

[0006] First, this invention provides a strain of *Haloxylon ammodendron* STM-122, which was isolated from the rhizosphere soil of *Smilax china* in saline-alkali land. Its preservation information is as follows:

[0007] Bacterial species name: Halomonas

[0008] Latin name: Franzmannia qiaohouensis

[0009] Strain number: STM-122

[0010] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0011] Collection institution abbreviation: CGMCC

[0012] Address of depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0013] Deposit date: October 27, 2025

[0014] Accession number: CGMCC No.36349.

[0015] Furthermore, the *Haloxymonas* STM-122 possesses the ability to solubilize phosphorus, produce EPS, auxin, and siderophores.

[0016] Furthermore, the STM-122 colonies of the halometabolite are pale yellow, round, with neat edges, smooth surface and slightly raised, and are Gram-negative bacilli. The 16S rRNA gene sequence of the STM-122 halometabolite is shown in SEQ ID NO.1.

[0017] Secondly, the present invention provides the application of the above-mentioned Halomonas STM-122 in promoting crop growth in saline-alkali environments.

[0018] Furthermore, in the above applications, the crop is millet, wheat, corn, soybean, or foxtail millet.

[0019] Furthermore, the application includes: soaking crop seeds in a bacterial suspension prepared from the *Haloxymonas* STM-122, wherein the concentration of the bacterial suspension is 1 × 10⁻⁶. 7 ~1×10 9 CFU / mL, soaking time is 1~3h.

[0020] Furthermore, the application includes: applying the *Haloxymonas* STM-122 into crop planting soil after processing it into a microbial fertilizer, wherein the viable count of *Haloxymonas* in the microbial fertilizer is ≥1×10⁻⁶. 8 The CFU / g content of the microbial fertilizer is ≤5%, and the pH value of the microbial fertilizer is 6.5~7.5.

[0021] Thirdly, the present invention claims protection for a fermented microbial fertilizer containing the aforementioned Halomonas STM-122, wherein the fermented microbial fertilizer is prepared by fermentation of a mixture of microbial suspension, brown sugar and organic substrate in a mass ratio of 1:0.4~0.6:4~6, and the organic substrate is peanut cake, soybean meal, cottonseed cake or straw powder.

[0022] Furthermore, in this fermented microbial fertilizer, the microbial suspension is prepared by the following method:

[0023] The *Haloxymonas* STM-122 was inoculated into liquid LB medium and cultured with shaking at 28-32°C and 170-190 rpm for 10-14 h to obtain the seed culture.

[0024] After centrifuging the seed culture, discard the supernatant, wash the bacterial cells 2-3 times with PBS buffer, then resuspend the bacterial cells in the same PBS buffer and adjust the bacterial suspension concentration to 1×10⁻⁶. 7 ~1×10 9 The concentration of the PBS buffer is 0.1-0.15 mmol / L and the pH value is 7.0-7.4.

[0025] Furthermore, in this fermented microbial fertilizer, the fermentation conditions are as follows: fermentation temperature 28~30℃, fermentation cycle 14~16 days, aeration for 25~35 minutes per day for the first 4~6 days of fermentation, aeration for 25~35 minutes every 1~3 days for the 6th~16th days of fermentation, and the substrate moisture content is controlled at 50~60% during the fermentation process.

[0026] Compared with the prior art, the present invention, "a salt-tolerant halomonas strain and its application," has the following beneficial effects:

[0027] This invention is the first to isolate, screen, and identify Halomonas STM-122 from the rhizosphere soil of millet in saline-alkali land. It breaks through the limitation of existing research on salt-tolerant growth-promoting bacteria, which is mostly focused on Bacillus genus. It fills the gap in the systematic research and application of Halomonas genus in the field of salt-tolerant growth-promoting bacteria, significantly enriches the salt-tolerant microbial resource library, and provides a new direction for the diversified development and application scenario expansion of salt-tolerant growth-promoting bacteria resources.

[0028] This invention confirms that the *Haloxymonas* STM-122 has been identified as possessing the functions of phosphorus solubilization, production of EPS (extracellular polysaccharides), auxin (IAA), and siderophores. It can act on crops through multiple mechanisms: Phosphorus solubilization involves secreting organic acids and enzymes to decompose organic phosphorus in the soil (such as phytic acid, phospholipids, and nucleic acids), providing the necessary phosphorus to plants through enzymatic hydrolysis; EPS can improve soil physical structure and enhance soil water and fertilizer retention capacity by increasing soil aggregate stability, creating a stable microenvironment for crop roots; simultaneously, the bacteria prevent nutrient imbalance and osmotic stress by secreting EPS, improving bacterial tolerance to salt stress, thus benefiting both microorganisms and plants in the environment; the IAA secreted by the bacteria can antagonize the production of plant ROS, protecting the bacteria from ROS toxicity, while promoting bacterial colonization in plant roots, thereby promoting crop growth and improving nutrient absorption; siderophores alter the iron content in the plant rhizosphere soil through chelation and inhibit the growth of pathogenic microorganisms by competing for iron with them, thereby directly or indirectly promoting plant growth. Germination tests and pot experiments have shown that this strain can significantly improve the germination potential and germination rate of millet under saline-alkali stress, promote seedling root growth and plant height, effectively alleviate the inhibitory effect of saline-alkali environment on crop growth, and provide key technical support for the stable planting of millet, a specialty grain crop in saline-alkali areas.

[0029] Based on the aforementioned characteristics of Halomonas STM-122, this invention provides a practical fermented microbial fertilizer with a viable count ≥1×10⁻⁶. 8 With CFU / g, a miscellaneous bacteria rate of ≤5%, and a pH value of 6.5~7.5, it can also adapt to different production needs by replacing salt-tolerant culture media and fermentation substrates (such as replacing peanut cake with soybean meal or straw powder).

[0030] This invention provides a complete technical process from strain isolation and screening, phylogenetic identification, to bacterial suspension preparation and bacterial fertilizer fermentation, with high reproducibility. At the same time, the halomonas STM-122 can also be applied to mainstream crops such as wheat, corn, and soybeans, providing an efficient and sustainable biological solution for the diversified development of agriculture in saline-alkali land, the development of arable land resources, and the restoration of ecological functions. Attached Figure Description

[0031] Figure 1 This is a streak image of Halomonas STM-122 on an LB agar plate.

[0032] Figure 2 This is a Gram-stained morphological image of Halomonas STM-122.

[0033] Figure 3 The image shows the electrophoretic detection results of 16S rRNA from Halomonas STM-122.

[0034] Figure 4 This is a neighbor-joining (NJ) phylogenetic tree of the 16S rRNA gene of Halomonas STM-122.

[0035] Figure 5 This image shows the qualitative results of the growth-promoting ability assay for Halomonas STM-122. It includes three experimental groups: siderophore production, phosphate solubilization, and growth factor production.

[0036] Figure 6 This is a comparative chart of millet seed growth indicators under salt-alkali stress. CK represents the blank control group, MS represents the salt-alkali stress group, and MS-STM-122 represents the salt-alkali stress + STM-122 treatment group; different letters (a, b, c) represent significant differences between groups.

[0037] Figure 7 This image shows a comparison of millet seed germination on day 7 of cultivation. From left to right, the groups are: blank control group (CK), salt-alkali stress group (MS), and salt-alkali stress + STM-122 treatment group (MS-STM-122).

[0038] Figure 8 This is a comparative chart showing the growth of millet seedlings on day 50 of cultivation. From left to right, the groups are: blank control group (CK), salt-alkali stress group (MS), and salt-alkali stress + STM-122 treatment group (MS-STM-122). Detailed Implementation

[0039] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] This embodiment describes the isolation and screening of Halomonas STM-122.

[0042] I. Experimental Materials

[0043] Soil samples: collected from the rhizosphere soil of millet in saline-alkali land, at a depth of 10-20 cm, where the main distribution layer of millet roots is located. After sampling, the samples were placed in sterile sealed bags, stored at low temperature, and brought back to the laboratory for processing as soon as possible.

[0044] Culture media: 10% LB medium (tryptone 10g / L, yeast extract 5g / L, NaCl 100g / L, agar 16g / L, dissolved in deionized water and adjusted to pH 7.0, autoclaved at 121℃ for 20min); liquid LB medium (same composition as 10% LB medium, without agar, sterilization conditions as above).

[0045] Reagents and instruments: sterile phosphate buffer (pH 7.0), 30% sterile glycerol, 10% sodium hypochlorite solution, 75% ethanol; clean bench, constant temperature incubator (accuracy ±1℃), high-speed centrifuge, -80℃ ultra-low temperature freezer, pipettes (10μL, 100μL, 1000μL), sterile culture dishes (90mm in diameter), sterile conical flasks (250mL).

[0046] II. Experimental Procedure

[0047] Soil suspension preparation: In a clean bench, accurately weigh 5g of the above-mentioned saline-alkali land millet rhizosphere soil sample and place it in a 250mL sterile Erlenmeyer flask containing 45mL of sterile phosphate buffer (pH 7.0). After sealing, place the flask on a shaker and shake at 150rpm / min for 30min to obtain a concentration of 10. -1 Soil suspension. Subsequently, using a serial dilution method, 10 liters were sequentially diluted with sterile phosphate buffer. -1 Soil suspension diluted to 10 -2 10 -3 10 -4 10 -5 10 -6 Concentration gradients were used, and each dilution process was performed under strict aseptic conditions.

[0048] Spread culture: from 10 -2 10 -4 10 -6 200 μL of each of the three concentration gradients of soil dilution was taken and evenly spread onto 10% LB agar plates. Three parallel petri dishes were prepared for each concentration gradient to ensure the repeatability of the experiment. The spread agar plates were inverted and placed in a 30°C incubator for 3-4 days in the dark, with regular observation of colony growth during the period.

[0049] Strain purification: After obvious colony growth appears on the culture medium plates, based on the differences in colony morphology, single colonies of different morphologies are picked using a sterile inoculation loop and inoculated separately onto fresh 10% LB medium plates. Purification is then performed using the four-zone streak method. The streaked plates are then incubated at 30°C for 3-4 days. If contaminating bacteria (with morphology different from the target colonies) appear on the plates, the four-zone streak procedure is repeated until the colonies on the plates have completely identical morphologies, at which point the strain is considered purified.

[0050] Strain preservation: Pick a purified single colony and inoculate it into a sterile Erlenmeyer flask containing 10 mL of liquid LB medium. Incubate at 30 °C and 150 rpm / min for 2 days to obtain bacterial suspension. Take 400 μL of bacterial suspension and mix it thoroughly with 600 μL of sterile 30% glycerol. Transfer it to a sterile cryovial, label it with the strain number, and store it in an ultra-low temperature freezer at -80 °C for long-term storage.

[0051] III. Test Results

[0052] Through the above isolation and screening process, a total of 174 purified bacterial strains were obtained from the rhizosphere soil samples of *Eriobotrya japonica* in saline-alkali land. Among them, strain STM-122 exhibited characteristic morphology on 10% LB agar plates: pale yellow, round colonies with regular edges, smooth and slightly raised surfaces, and a colony diameter of approximately 1-2 mm (see [link to article]). Figure 1 (STM-122 strain streak morphology diagram); Gram staining identification confirmed that this strain is a Gram-negative bacillus (see...) Figure 2 (The image shows the morphology of strain STM-122 after Gram staining). Further identification of this strain as the target halomonas strain STM-122 was subsequently performed.

[0053] Example 2

[0054] This embodiment describes the phylogenetic identification of Halomonas STM-122.

[0055] I. Experimental Materials

[0056] Strain sample: Halomonas STM-122 purified and preserved in Example 1.

[0057] Reagents: 0.05% NaOH solution (filtered and sterilized), PCR reaction mixture (containing Taq DNA polymerase, dNTPs, and buffer), 16S rRNA gene amplification primers (forward primer 27F, reverse primer 1492R), 6× Loading Buffer, nucleic acid dye, 1× TAE buffer, and DNA Marker.

[0058] Instruments: PCR instrument, electrophoresis instrument, gel imaging analyzer, clean bench, vortex mixer, micropipette (10μL, 100μL).

[0059] II. Experimental Procedure

[0060] (a) Extraction of bacterial DNA

[0061] Inside the clean bench, use a sterile inoculation loop to pick up an appropriate amount of single colony of Halomonas STM-122 and place it in a centrifuge tube containing 100 μL of filtered and sterilized 0.05% NaOH solution. Use a vortex mixer to shake thoroughly to ensure that the bacteria and alkaline solution are evenly mixed.

[0062] Place the centrifuge tubes into a PCR instrument and process them according to the DNA extraction procedure shown in Table 1 to break the bacterial cell wall and release DNA.

[0063] Table 1. DNA Extraction Procedure

[0064]

[0065] (II) Bacterial 16S rRNA gene amplification

[0066] Prepare the PCR reaction system according to the ratio shown in Table 2. Add each component to a sterile PCR tube in sequence, mix gently, and then centrifuge briefly (1-2 seconds) to concentrate the liquid at the bottom of the tube.

[0067] Table 2. PCR reaction system

[0068]

[0069] Place the PCR tube containing the PCR reaction system into the PCR instrument and run the reaction according to the 16S rRNA gene amplification program shown in Table 3.

[0070] Table 3.16S rRNA gene amplification program

[0071]

[0072] (III) Electrophoresis detection and sequencing

[0073] After the PCR amplification reaction is completed, take 5 μL of PCR product and mix it with 1 μL of 6×Loading Buffer and 0.5 μL of nucleic acid dye to obtain an electrophoresis sample.

[0074] Prepare a 1% agarose gel (containing nucleic acid dye), add the electrophoresis sample to the gel wells, and add DNA marker as a molecular weight reference. Electrophore in 1×TAE buffer at 120V for 30 min.

[0075] After electrophoresis, the agarose gel is placed in a gel imaging analyzer and observed and recorded under ultraviolet light. If the PCR product bands are single and bright, the amplification is considered successful.

[0076] The amplified PCR products were sent to a sequencing institution (Qingke Biotechnology). First-generation sequencing was performed using forward primer 27F (CGGTGAATACGTTCYCGG) and reverse primer 1492R (AAGGAGGTGATCCRGCCGCA). Poor-quality sequences in the sequencing peaks were removed to obtain the 16S rRNA gene sequence of Halomonas STM-122, which is about 1500 bp in length.

[0077] (iv) Phylogenetic analysis

[0078] The 16S rRNA gene sequence of the obtained Halomonas STM-122 was uploaded to the NCBI database. The Blast tool was used to perform homology comparison in the Nucleotidecollection (nr / nt) database. The taxonomic position of the strain was preliminarily determined based on the sequence similarity (similarity ≥97.00% was judged as the same species).

[0079] Sequences of related strains with high homology to the STM-122 sequence of Halomonas were selected from the database, and phylogenetic analysis was performed using MEGA11 software: First, multiple sequences were aligned using the ClusterW tool, and then the Kimura2-parameter model was used to construct a neighbor-joining (NJ) phylogenetic tree through Bootstrap analysis (1000 replicates).

[0080] III. Test Results

[0081] Electrophoresis results show (see) Figure 3 The PCR product of the 16S rRNA gene of Halomonas STM-122 showed a single bright band at approximately 1500 bp, consistent with the expected fragment size, indicating successful amplification.

[0082] Sequencing revealed that the 16S rRNA gene sequence is shown in SEQ ID NO.1. Blast homology alignment results showed that this sequence had ≥97.00% sequence similarity to strains of the genus *Haloxylon*; combined with the NJ phylogenetic tree (… Figure 4 As can be seen, strain STM-122 clustered with strains of the genus *Haloxylon*. Therefore, strain STM-122 can be identified as a strain of the genus *Haloxylon*, and its classification name is *Haloxylon*. Franzmannia qiaohouensis The halometabolite STM-122 is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36349.

[0083] Example 3

[0084] This embodiment describes the identification of the growth-promoting function of Halomonas STM-122.

[0085] I. Experimental Materials

[0086] Strains sample: The purified and preserved Halomonas STM-122 in Example 1 was identified as a Halomonas strain in Example 2.

[0087] Identification media: Prepare protease production medium, cellulase production medium, amylase production medium, phosphate solubilization medium, potassium solubilization medium, nitrogen fixation medium, IAA (auxin) production detection medium, siderophore production detection medium, and EPS (Extracellular Polymeric Substances) production detection medium according to Table 4. All media are sterilized by autoclaving at 121°C for 20 min before use.

[0088] Table 4. Components of the identification culture medium

[0089]

[0090] Reagents and instruments: Salkowski test solution (for IAA quantification), CAS (chrome azure S) staining solution (for siderophore quantification), molybdenum anti-chromogenic reagent (for phosphorus content determination); flame spectrophotometer (for potassium content determination), gas chromatograph (for acetylene reduction method for nitrogen fixation quantification), clean bench, constant temperature incubator, microplate reader, pH meter.

[0091] II. Experimental Procedure

[0092] (a) Qualitative determination of reproductive function

[0093] Enzyme production capacity assay: Single colonies of *Haloxylon amyloides* STM-122 were picked and inoculated onto protease-producing, cellulase-producing, and amylase-producing agar plates, respectively, and incubated at 30°C for 5–7 days. After incubation, the clear zone was observed by adding trichloroacetic acid solution to the protease-producing medium, Congo red solution to the cellulase-producing medium, and iodine solution to the amylase-producing medium. The presence of a clear zone indicated that the strain possessed the corresponding enzyme production capacity.

[0094] Determination of phosphorus solubilization, phosphorus solubilization, potassium solubilization, and nitrogen fixation capabilities: STM-122 of Halomonas bacillus was inoculated into phosphorus solubilization, phosphorus solubilization, potassium solubilization, and nitrogen fixation media and cultured at 30℃ for 7-10 days. Observe whether a clear zone appears around the colony in the phosphorus solubilization and phosphorus solubilization media (indicating phosphorus solubilization and phosphorus solubilization capabilities), whether there are signs of potassium salt dissolution in the potassium solubilization media, and whether the strain grows normally in the nitrogen fixation media (indicating nitrogen fixation capabilities).

[0095] IAA, siderophore, and EPS production capacity assays: Halomonas STM-122 was inoculated into IAA production detection medium (containing tryptophan), siderophore production detection medium (CAS medium), and EPS production detection medium. The culture was kept at 30℃ for 5-7 days. The color changes of the IAA production medium, whether the blue color in the siderophore production medium turned into orange, and whether there was a mucous substance in the colonies of the EPS production medium (indicating EPS production) were observed.

[0096] (II) Quantitative determination of reproductive function

[0097] EPS production capacity determination: *Haloxymonas hydrophila* strain STM-122 was inoculated into EPS-producing fermentation medium and cultured on a shaker for 48 h. Crude polysaccharide was extracted from the fermentation broth using the ethanol precipitation method. 10 mL of fermentation broth was placed in a 50 mL centrifuge tube, centrifuged at low temperature to remove bacterial cells, and the supernatant was mixed with twice the volume of pre-cooled ethanol. After standing at 4℃ for 12 h, the mixture was centrifuged at 10000 r / min for 10 min. The resulting precipitate was dissolved and diluted to volume with sterile distilled water to obtain the crude polysaccharide solution. Its OD value was determined using the phenol-sulfuric acid method. 490 Absorbance; a glucose standard curve was prepared using glucose standard solutions of different concentrations (0.20, 0.40, 0.60, 0.80, 1.00 mg / mL), yielding Y = 0.0007X + 0.0061 (R²). 2 =0.9991), and the polysaccharide content of the strain was calculated based on the standard curve.

[0098] Phosphate-solubilizing capacity determination: *Haloxylon ammodendron* STM-122 was inoculated into 100 mL of Monkina organic phosphorus liquid medium. The medium was incubated at 28 °C with shaking (180 r / min). A control medium without *Haloxylon ammodendron* STM-122 was used. Each treatment was repeated three times. After 4 days of shaking incubation, the fermentation product was filtered through a 0.22 μm microporous membrane to remove the bacterial cells, yielding a phosphate-solubilizing filtrate sample, which was stored at 4 °C. The phosphorus content was determined using the molybdenum-antimony reagent method. The specific steps are as follows: (1) Preparation of the molybdenum-antimony reagent: Weigh 5g of potassium antimony tartrate and dissolve it in 1L of deionized water to prepare a 5mg / L potassium antimony tartrate solution; weigh 5g of ammonium molybdate and dissolve it in 225mL of deionized water, then slowly add 76.5mL of concentrated sulfuric acid while stirring. After the liquid temperature drops to room temperature, add 100mL of the 5g / L potassium antimony tartrate solution, and dilute to 500ml with deionized water. Shake well and store in a color bottle for later use to prepare an ammonium molybdate-sulfuric acid solution. Before use, add 1.5g of L-ascorbic acid to every 100mL of ammonium molybdate-sulfuric acid solution (molybdenum-antimony reagent). (2) Phosphorus standard curve preparation: Pipette 0, 1.0, 2.0, 3.0, 4.0, and 5.0 ml of 5 mg / L KHPO4 standard solution into 50 mL volumetric flasks, and simultaneously add an equal volume of blank solution to the sample solution used for colorimetric determination. Add 2-3 drops of dinitrophenol indicator, and then adjust the solution to a slightly yellow color with 0.5 mol / L NaHCO3 solution. Finally, accurately add 5 mL of molybdenum antimony reagent, shake well, and dilute to volume with deionized water. Measure the absorbance at a wavelength of 700 nm. Plot the absorbance value on the ordinate (Y) and the phosphorus standard solution concentration on the abscissa (X), obtaining Y = 0.0011X + 0.009(R² / X²) ... 2 =0.9922). (3) Take 1 mL of phosphorus filtrate sample stored at 4℃, add it to a 50 mL volumetric flask, dilute with deionized water to about 3 / 5 of the total volume, add 1~2 drops of dinitrophenol indicator, and adjust the solution to just turn slightly yellow with 0.5 mol / L NaHCO3 solution. Accurately add 5 mL of molybdenum antimony reagent, make up to volume and shake well, and after standing for 30 min, perform colorimetric measurement at a wavelength of 700 nm using an uninoculated Monkina organic phosphorus culture medium as a reference solution for dissolving organic phosphorus. Adjust the absorbance of the reference solution to 0, perform colorimetric measurement, and read the absorbance value at 700 nm. Substitute the absorbance value into the phosphorus standard curve equation to obtain the increased soluble phosphorus content in the liquid culture medium.

[0099] Siderotrophic capacity determination: 5% of *Haloxylon ammonium* STM-122 was inoculated into MKB medium (casein amino acids 5 g / L, glycerol 15 mL / L, K₂HPO₄ 2.5 g / L, MgSO₄·7H₂O 2.5 g / L, pH 7.2) and cultured at 30°C and 180 r / min for 48 h. The fermentation broth was centrifuged at 3500 r / min for 15 min to remove the bacterial cells. 3 mL of the supernatant was mixed with an equal volume of CAS reagent and reacted at room temperature for 1 h. The absorbance (As) of the solution at 630 nm was then measured. 3 mL of blank medium was mixed with an equal volume of CAS reagent, and its absorbance was measured as a reference value (Ar) according to the above method. The relative content of siderotrophs (SU) was calculated as ((Ar-As) / Ar) * 100%.

[0100] IAA production capacity determination: Weigh 10.0 mg of IAA standard, dissolve it in a small amount of anhydrous ethanol, and then dilute to 100 mL with distilled water to prepare a stock solution. Dilute the stock solution to standard solutions with concentrations of 10, 20, 30, 40, and 50 μg / mL. Add 2 mL of Salkowski colorimetric reagent to 2 mL of the standard solution, place in the dark for 30 min to allow for color reaction, and measure the OD. 530 The absorbance at the specified location was measured, and a standard curve was plotted based on the data. *Haloxylon ammodendron* STM-122 was inoculated into LB broth containing L-tryptophan and cultured on a shaker at 30°C and 180 rpm for 3 days. 2 mL of the bacterial suspension was centrifuged for 10 min, and the supernatant was transferred to an equal volume of Salkowski chromogenic solution. The mixture was placed in the dark for 30 min to allow for the color reaction, and the OD was measured and recorded using a spectrophotometer. 530 The absorbance, expressed in terms of OD 530 Plotting a standard curve with the x-axis value and IAA concentration as the y-axis yields Y = 0.0195X + 0.0756 (R0). 2 =0.9928), the concentration of IAA produced by Halomonas STM-122 was calculated using the regression equation of the standard curve.

[0101] III. Test Results

[0102] Qualitative results: *Haloxylon ammodendron* STM-122 showed a white, mucous-like substance in the EPS-producing fermentation medium, indicating its EPS-producing ability; a clear phosphate-solubilizing zone appeared on the Monkina organic phosphorus medium, indicating its phosphate-solubilizing ability; a distinct orange-yellow siderophore chelation zone appeared on the CAS detection plate, indicating its siderophore-producing ability; a red color reaction occurred when the sterile filtrate of *Haloxylon ammodendron* STM-122 was mixed with Salkowski colorimetric solution, indicating its IAA-producing ability; however, no corresponding positive reactions were observed on the protease-producing, amylase-producing, cellulase-producing, phosphate-solubilizing, potassium-solubilizing, and nitrogen-fixing media, indicating that STM-122 possesses the ability to solubilize phosphate, produce EPS, auxin (IAA), and siderophores (see...). Figure 5 (STM-122 growth-promoting capacity qualitative measurement diagram).

[0103] Quantitative determination results: Further quantitative determination of the phosphorus solubilization, EPS production, IAA production, and siderophore production capacity of *Haloxylon ammodendron* STM-122 was performed. The results are as follows: The soluble organic phosphorus content increased by 310 mg / L in *Montagne chinensis* organic phosphorus culture medium was determined by the molybdenum-antimony colorimetric method; the EPS production of *Haloxylon ammodendron* STM-122 was 794.14 mg / L after extraction of crude polysaccharide solution by ethanol precipitation and calculation by phenol-sulfuric acid method; the IAA production of *Haloxylon ammodendron* STM-122 was 21.82 mg / L in LB medium containing tryptophan; after mixing the sterile filtrate of *Haloxylon ammodendron* STM-122 with CAS detection solution and reacting in the dark for 1 hour, the relative production of ferrophile was calculated to be 16.40%. Quantitative analysis showed that STM-122 could solubilize phosphorus, and the production of EPS, IAA, and the relative content of siderophores all reached the effective growth-promoting threshold. Its ability to produce enzymes and synthesize growth-promoting substances can meet the needs of crop growth in saline-alkali environments, further verifying the growth-promoting functional characteristics of this strain.

[0104] Example 4

[0105] This example describes the germination test of Halomonas STM-122.

[0106] I. Experimental Materials

[0107] Strains and seeds: STM-122 of Halomonas strain isolated and purified in Example 1; millet seeds (the variety is Yixuan Dahong Millet, the main variety cultivated in local saline-alkali land, with plump grains and free from diseases and pests).

[0108] Reagents and culture media: 10% sodium hypochlorite solution, 75% ethanol, sterile distilled water; liquid LB medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0, sterilized at 121℃ for 20min); mixed saline solution (formulation: Na2CO3:NaHCO3:NaCl:Na2SO4=1:9:1:9, concentration 80mmol / L, prepared with sterile distilled water).

[0109] Instruments: Clean bench, constant temperature incubator (accuracy ±1℃), shaker, centrifuge, sterile culture dishes (90mm in diameter), sterile filter paper, pipettes (1mL, 5mL).

[0110] II. Experimental Procedure

[0111] (I) Preparation of bacterial suspension

[0112] The STM-122 strain of Halomonas was taken out from the -80℃ ultra-low temperature freezer and streaked onto LB agar plates in a clean bench. The strain was then incubated at 28℃ for 2 days to activate it. This process was repeated twice to ensure the activity of the strain.

[0113] Pick a single activated colony and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of liquid LB medium. Incubate at 30 °C and 180 rpm with shaking for 16 h. Centrifuge at 8000 rpm for 10 min, discard the supernatant, wash the precipitate three times with sterile water, resuspend, and adjust the bacterial suspension concentration to 1 × 10⁻⁶. 8 The STM-122 bacterial suspension was obtained by measuring CFU / mL (verified by plate counting method) and stored at 4℃ for later use.

[0114] (II) Seed disinfection and soaking

[0115] Select uniform and plump millet seeds, soak them in 10% sodium hypochlorite solution for 5 minutes, then soak them in 75% ethanol for 60 seconds, and rinse them three times with sterile distilled water to complete surface disinfection.

[0116] The sterilized seeds were divided into three groups for treatment, and each group underwent the following soaking treatment:

[0117] Control group (CK): Seeds were soaked in sterile distilled water for 2 hours and then air-dried in a clean bench.

[0118] Salt-alkali stress group (MS): Seeds were soaked in sterile distilled water for 2 hours and then air-dried in a clean bench.

[0119] Salt-alkali stress + bacterial suspension treatment group (MS-STM-122): The inoculum was soaked in STM-122 bacterial suspension for 2 hours and then air-dried in a clean bench.

[0120] (III) Germination Test Procedure

[0121] Inside the laminar flow hood, two layers of sterile filter paper were placed on the bottom of a 9mm diameter sterile petri dish. 100 treated seeds were placed in each petri dish, and five replicates were set up.

[0122] Add 5 mL of sterile distilled water to the CK group culture dish, and add 5 mL of 80 mmol / L mixed saline solution to the MS group and MS-STM-122 group culture dishes, ensuring that the filter paper is fully moistened but without excess liquid.

[0123] Place all petri dishes in a 25°C constant temperature incubator and incubate in the dark. During this period, add a small amount of the corresponding liquid daily to keep the filter paper moist.

[0124] (iv) Indicator Measurement

[0125] Germination potential was calculated on day 3 of cultivation: Germination potential = (Number of germinated seeds on day 3 / Total number of seeds tested) × 100%;

[0126] Germination rate was calculated on day 7 of cultivation: Germination rate = (Number of germinated seeds on day 7 / Total number of seeds tested) × 100%.

[0127] III. Test Results

[0128] Table 5. Seed germination indicators

[0129]

[0130] As shown in Table 5, salt and alkali stress significantly inhibited the germination of millet seeds: compared with the CK group, the germination potential, germination rate and germination rate of the MS group decreased significantly, with the germination rate decreasing by about 50%, indicating that the 80 mmol / L mixed salt and alkali solution posed a significant stress on the germination of millet seeds.

[0131] Treatment with STM-122 bacterial suspension significantly alleviated salt-alkali stress: the germination potential of the MS-STM-122 group was 107.98% higher than that of the MS group, the germination rate was 98.05% higher, and the root length, shoot length, root fresh weight, and shoot fresh weight were all significantly increased. Figure 6 This indicates that *Haloxylon ammodendron* STM-122 can effectively promote the germination ability of millet seeds under salt-alkali stress (see the seed germination status of each group on day 7). Figure 7 ).

[0132] Example 5

[0133] This example describes a pot experiment of Halomonas STM-122.

[0134] I. Experimental Materials

[0135] Strains and seeds: Halomonas STM-122 isolated and purified in Example 1; millet seeds (full grains, free from diseases and pests) from the same source as in Example 4.

[0136] Soil and reagents: Nutrient soil, sterilized after passing through a 2mm sieve (121℃, 2h); mixed saline-alkali solution (same as in Example 4, 80mmol / L); STM-122 bacterial suspension (concentration 1×10⁻⁶). 8 CFU / mL, prepared by the same method as in Example 4); sterile distilled water.

[0137] Instruments and apparatus: plastic flower pots (15cm in diameter), constant temperature and light incubator (16h light / 8h darkness, 26℃), measuring tape, electronic balance (0.01g accuracy), vernier calipers.

[0138] II. Test Methods

[0139] Each flowerpot was filled with 400g of sterilized nutrient soil, watered several times with distilled water, and then evaporated to a suitable humidity. Millet seeds that had been sterilized in Example 4 were sown. Each treatment group was repeated in 3 pots. One week after emergence, 50 seedlings were transplanted. When the seedlings reached the three-leaf stage, each group was treated as follows:

[0140] CK group: Water each pot with 50mL of sterile distilled water, and then water with 30mL of distilled water every 3 days in the later stage;

[0141] MS group: Water each pot with 50mL of mixed saline-alkali solution, and then water with 30mL of mixed saline-alkali solution every 3 days in the later stage;

[0142] MS-STM-122 group: 50mL of mixed saline-alkali solution was poured into each pot. In the later stage, 30mL of mixed saline-alkali solution was poured in every 3 days. 30mL of STM-122 bacterial suspension was poured in the next day. A total of 3 times were poured.

[0143] The light exposure was set for 16 hours, the darkness for 8 hours, and the room temperature was 26°C. The placement of different treatment groups was changed every 5 days to eliminate environmental differences, and the culture period was 50 days.

[0144] After cultivation, carefully remove the plant, rinse the soil around the roots, and measure the following indicators:

[0145] Plant height: The vertical height from the base of the stem to the top of the plant;

[0146] Root length: The length of the taproot from the root tip to the base of the root;

[0147] Stem diameter: diameter at the base of the stem (measured with vernier calipers);

[0148] Fresh weight: Fresh weight of the above-ground and underground parts of the plant (weighed by electronic balance).

[0149] III. Test Results

[0150] Table 6. Seedling growth indicators

[0151]

[0152] As shown in Table 6, salt-alkali stress significantly inhibited the growth of millet: compared with the CK group, the plant height, stem diameter, fresh weight and root length of the MS group were all lower, indicating that 80 mmol / L mixed salt-alkali solution significantly inhibited the growth of millet seedlings.

[0153] STM-122 bacterial suspension can effectively alleviate salt and alkali stress: the MS-STM-122 group had higher plant height, stem diameter, fresh weight, and root length than the MS group, and the plant growth was significantly better than that of the MS group. Seedling growth at 50 days was as follows... Figure 8 As shown in the figure. The results confirmed that Halomonas STM-122 can significantly promote the growth and development of millet seedlings under salt-alkali stress.

[0154] Example 6

[0155] This embodiment describes the microbial fertilizer fermentation of Halomonas STM-122.

[0156] I. Experimental Materials

[0157] Strains: STM-122 of Halomonas strain isolated and purified in Example 1.

[0158] Culture medium and fermentation substrate: liquid LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0, sterilized at 121℃ for 20 min); peanut cake (crushed through a 40-mesh sieve), brown sugar; 0.12 mmol / L PBS buffer (pH 7.2, sterilized at 121℃ for 20 min).

[0159] Instruments and equipment: Clean bench, constant temperature shaking incubator, high-speed centrifuge, sterile fermenter (5L), pH meter, colony counter, autoclave.

[0160] II. Experimental Procedure

[0161] (I) Strain activation and bacterial suspension preparation

[0162] The STM-122 strain was taken out from the -80℃ ultra-low temperature freezer and streaked onto a 10% LB medium plate in a clean bench. It was then activated by incubation at 28℃ for 2 days. This process was repeated twice to ensure the activity of the strain.

[0163] Select a single activated colony and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of liquid LB medium. Incubate at 28 °C and 180 rpm for 12 h to obtain the seed culture.

[0164] Centrifuge the seed culture at 8000 rpm for 10 min, discard the supernatant, wash the bacterial cells twice with 0.12 mmol / L PBS buffer (centrifuge at 8000 rpm for 5 min each time), resuspend, and adjust the OD of the bacterial suspension using a spectrophotometer. 600 =0.8, store at 4℃ for later use.

[0165] (II) Fermentation substrate preparation

[0166] Prepare the raw materials according to the mass ratio of bacterial suspension: brown sugar: peanut cake = 1:0.5:5 (on dry weight): Mix the crushed peanut cake with brown sugar evenly, add the above STM-122 bacterial suspension, stir for 15~20 minutes until the substrate is fully moistened, and adjust the substrate moisture content to 50~60%.

[0167] (III) Fermentation process control

[0168] The prepared substrate is loaded into a sterile fermentation tank (filling the tank to 60% of its volume) and placed in a constant temperature fermentation chamber at 28~30℃ to ferment in the dark for 15 days.

[0169] For the first 5 days of fermentation, aerate the substrate for 30 minutes daily to promote aerobic cell growth. From day 6 to day 15, aerate every 2 days to maintain a loose substrate. Monitor the substrate temperature regularly during this period; if it exceeds 35°C, stir and cool it down promptly to ensure a stable fermentation environment.

[0170] (iv) Quality testing of microbial fertilizer

[0171] After fermentation, the following indicators of the microbial fertilizer were tested:

[0172] Viable cell count determination: The dilution plating method was used. 10g of the microbial fertilizer sample was added to 90mL of sterile physiological saline, serially diluted, and then plated onto 10% LB agar plates. The plates were incubated at 30℃ for 48 hours. Colony counts were then performed to calculate the viable cell count (≥1×10⁻⁶). 8 CFU / g).

[0173] Contamination rate determination: Using the same dilution method as described above, spread the sample onto LB medium (to measure total bacterial count) and STM-122 selective medium (to measure target bacterial count). Contamination rate = (total bacterial count - target bacterial count) / total bacterial count × 100% (requirement ≤ 5%).

[0174] Physicochemical index testing: Observe the appearance of the microbial fertilizer (it should be odorless and loose in texture); take 10g of microbial fertilizer, add 90mL of sterile distilled water, shake for 30min and let stand, and measure the pH of the supernatant with a pH meter (it should be 6.5~7.5).

[0175] III. Test Results

[0176] After 15 days of fermentation, the viable bacteria count of the prepared STM-122 microbial fertilizer reached 4.9 × 10⁻⁶.8 With a CFU / g concentration, a contamination rate of 2.6%, a pH of 7.0, and a brown, loose appearance without any putrid odor, this microbial fertilizer meets all the standards for qualified microbial fertilizer. It can be used directly in field trials or packaged and stored at 4℃ (shelf life ≥ 6 months), providing a stable microbial inoculant for crops grown in saline-alkali soils.

[0177] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A salt-tolerant Halomonas strain ( Franzmannia qiaohouensis STM-122, characterized in that, The halometabolite STM-122 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36349.

2. The *Haloxymonas* STM-122 according to claim 1, characterized in that, The halomonas STM-122 strain possesses the ability to solubilize phosphorus, produce EPS, auxin, and siderophores.

3. The application of the *Haloxylon ammodendron* STM-122 according to claim 1 in promoting crop growth under saline-alkali conditions, characterized in that... The crop in question is millet.

4. The application according to claim 3, characterized in that, The application includes: soaking crop seeds in a bacterial suspension prepared from the *Haloxymonas* STM-122, wherein the concentration of the bacterial suspension is 1 × 10⁻⁶. 7 ~1×10 9 CFU / mL, soaking time is 1~3h.

5. The application according to claim 3, characterized in that, The application includes: applying the *Haloxymonas* STM-122 microbial fertilizer to crop planting soil, wherein the viable count of *Haloxymonas* STM-122 in the microbial fertilizer is ≥1×10⁻⁶. 8 The CFU / g content of the microbial fertilizer is ≤5%, and the pH value of the microbial fertilizer is 6.5~7.

5.

6. A fermented microbial fertilizer comprising *Haloxylon ammodendron* STM-122 as described in claim 1, characterized in that, The fermented microbial fertilizer is made by mixing and fermenting microbial suspension, brown sugar and organic substrate in a mass ratio of 1:0.4~0.6:4~6. The organic substrate is peanut cake, soybean meal, cottonseed cake or straw powder.

7. The fermented microbial fertilizer according to claim 6, characterized in that, The bacterial suspension was prepared by the following method: The *Haloxymonas* STM-122 was inoculated into liquid LB medium and cultured with shaking at 28-32°C and 170-190 rpm for 10-14 h to obtain the seed culture. After centrifuging the seed culture, discard the supernatant, wash the bacterial cells 2-3 times with PBS buffer, then resuspend the bacterial cells in the same PBS buffer and adjust the bacterial suspension concentration to 1×10⁻⁶. 7 ~1×10 9 The concentration of the PBS buffer is 0.1-0.15 mmol / L and the pH value is 7.0-7.

4.

8. The fermented microbial fertilizer according to claim 6, characterized in that, Fermentation conditions are as follows: fermentation temperature 28~30℃, fermentation cycle 14~16 days, aeration for 25~35 minutes per day for the first 4~6 days of fermentation, aeration for 25~35 minutes every 1~3 days from the 6th to the 16th day of fermentation, and control of substrate moisture content to 50~60% during fermentation.

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