Stutzeri Kunming and application thereof for improving salt tolerance of crops

Abstract

The application belongs to the technical field of microbial application, and particularly relates to Stutzerimonas kunmingensis for improving salt tolerance of crops and application thereof. The Stutzerimonas kunmingensis is Stutzerimonas kunmingensis S246, the preservation number is CCTCC NO: M 20242582, and the Stutzerimonas kunmingensis S246 is preserved in China Center for Type Culture Collection on November 18, 2024. The strain can synthesize indole acetic acid and secrete iron carrier. The strain is used as an active ingredient to be applied to soybeans and oilseed rape, and the strain can significantly improve the emergence rate of dry crop soybeans in salt stress soil, increase the functional leaf area of soybeans, and promote the growth of soybean seedlings. The strain promotes the seed germination of oilseed rape in a salt stress environment, relieves the inhibition of salt stress on the growth of the radicle and bud of oilseed rape, and further enhances the adaptation of dry crop soybeans and oilseed rape to salt stress. Therefore, the Stutzerimonas kunmingensis S246 has a wide application prospect in promoting the seed germination, emergence and growth of crops such as soybeans and oilseed rape in saline-alkali farmland.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, specifically relating to a *Stizemonas kunmingensis* strain that improves crop salt tolerance and its application. Background Technology

[0002] Soil salinization is one of the major abiotic stress factors threatening global agricultural production. High concentrations of basic ions in saline-alkali soils can cause osmotic stress, ion toxicity, and oxidative stress to plants, severely impacting their growth and development, leading to slow growth, wilting, and even death.

[0003] Soybeans and rapeseed are important economic and oilseed crops. Planting soybeans and rapeseed in saline-alkali land holds the promise of simultaneously improving, utilizing, and generating economic benefits from this land. Therefore, soybeans and rapeseed are highly advantageous field oilseed crops for utilizing saline-alkali land and are crops whose cultivation in such land is encouraged. However, seed germination and seedling stages are critical periods for plant growth and are also the most sensitive to salt and alkali stress. Both soybeans and rapeseed are dryland crops, and the primary problems encountered in planting these two dryland crops in saline-alkali land are: difficulty in seed emergence after sowing, weak seedling growth, seedling death or stunted growth, and gaps in the rows, all of which severely impact crop yield. Therefore, there is an urgent need to provide a new strategy that can significantly alleviate the stress of salt on crop growth. Summary of the Invention

[0004] The purpose of this invention is to provide a *Stizemonas kunmingensis* strain that improves crop salt tolerance and can be used to alleviate the effects of salt stress on crop growth.

[0005] The technical solution adopted in this invention is:

[0006] This invention provides a *Stutzerimonas kunmingensis* strain that improves crop salt tolerance. The *Stutzerimonas kunmingensis* strain is S246, with accession number CCTCC NO: M20242582, and was deposited at the China Center for Type Culture Collection on November 18, 2024.

[0007] A second aspect of the present invention provides a bacterial suspension, which is obtained by culturing *Stizemonas kunmingensis*, and the culturing process is as follows:

[0008] *Stizemonas kunmingensis* was inoculated into LB medium and cultured at 28℃–30℃ and 160 rpm–200 rpm for 16–18 hours. The bacterial cells were collected, and the viable count of *Stizemonas kunmingensis* was adjusted to 10⁻⁶ cells / mL with sterile water. 9 The bacterial suspension is obtained by measuring CFU / mL.

[0009] Preferably, the cultivation process is as follows:

[0010] *Stizemonas kunmingensis* was inoculated into LB medium and cultured at 28°C and 180 rpm for 17 h. The bacterial cells were collected, and the viable count of *Stizemonas kunmingensis* was adjusted to 10⁻⁶ cells / mL with sterile water. 9 The bacterial suspension is obtained by measuring CFU / mL.

[0011] A third aspect of the present invention provides the application of the aforementioned *Stizemonas kunmingensis* or the aforementioned bacterial suspension, characterized in that the application refers to any one of the following:

[0012] 1) Promotes crop emergence;

[0013] 2) Promotes crop seedling growth;

[0014] 3) Promotes crop seed germination;

[0015] 4) Improve crop salt tolerance.

[0016] Preferably, the crop includes either soybean or rapeseed.

[0017] A fourth aspect of the present invention provides a microbial growth promoter comprising *St. kunmingii*, wherein the viable count of *St. kunmingii* in the microbial growth promoter is 10-1. 9 CFU / mL.

[0018] The fifth aspect of this invention provides a microbial germination promoter, wherein the microbial germination promoter comprises *Stizemonas kunmingensis*, and the viable count of *Stizemonas kunmingensis* in the microbial germination promoter is 10-1. 9 CFU / mL.

[0019] Preferably, the microbial growth promoter or microbial germination promoter further includes microbiologically acceptable excipients.

[0020] Preferably, the microbiologically acceptable excipients include at least one of fillers, binders, disintegrants, lubricants, and antacids.

[0021] Preferably, the filler includes any one of sodium alginate, polyacrylamide, and zeolite.

[0022] Preferably, the adhesive comprises any one of starch paste, hydroxypropyl methylcellulose, and povidone.

[0023] Preferably, the disintegrant includes any one of starch, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose.

[0024] Preferably, the lubricant includes any one of microalgae-based biolubricant, mucin, and tannic acid.

[0025] Preferably, the antacid comprises any one of phosphate buffer, bicarbonate buffer, and Tris buffer.

[0026] Preferably, the application method of the microbial growth promoter or microbial germination promoter includes any one of spraying, root irrigation, seed dressing, and seed soaking.

[0027] The preservation information for biological material samples involved in this invention is as follows:

[0028] *Stutzerimonas kunmingensis* S246, deposited on November 18, 2024, at the China Center for Type Culture Collection (CCTCC), with the proposed taxonomic name *Stutzerimonas kunmingensis* S246, accession number CCTCC NO: M 20242582, and address at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China. For ease of description, *Stutzerimonas kunmingensis* S246 will be abbreviated as S246 in this invention.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] This invention provides a *Stutzerimonas kunmingensis* strain that enhances crop salt tolerance. The *Stutzerimonas kunmingensis* S246, with accession number CCTCC NO: M20242582, was deposited at the China Center for Type Culture Collection on November 18, 2024. This invention marks the first isolation, identification, and preservation of a *Stutzerimonas kunmingensis* S246 strain with salt-tolerant growth-promoting properties. This strain was isolated from the rhizosphere soil of *Suaeda salsa* in a severely saline-alkali area in coastal Jiangsu Province. This strain not only exhibits growth-promoting properties by secreting indoleacetic acid and producing siderophores, but also promotes the emergence and seedling growth of soybeans and the germination of rapeseed seeds under salt stress. This invention further utilizes this strain as an active ingredient applied to soybeans and rapeseed, which can significantly improve the emergence rate of drought-resistant soybeans in salt-stressed soils, increase the functional leaf area of ​​soybeans, and promote soybean seedling growth; it also promotes rapeseed seed germination in salt-stressed environments, alleviates the inhibitory effect of salt stress on the growth of rapeseed radicles and shoots, and thus enhances the adaptability of dryland crops such as soybeans and rapeseed to salt stress. Therefore, *Stutzerimonas kunmingensis* S246 and microbial germination and growth promoters containing S246 show broad application prospects in promoting seed germination, emergence, and growth of crops such as soybeans and rapeseed in saline-alkali farmland. This provides a novel microbial solution for crop production in saline-alkali land, and also provides important resource and technical support for developing microbial agents or microbial fertilizers suitable for saline-alkali land, providing material and technical support for generating good ecological and social benefits.

[0031] This invention also discloses the application of *Stutzerimonas kunmingensis* S246 in promoting soybean emergence under salt stress. Soil pot experiments showed that under salt stress with a NaCl concentration of 1.5‰ w / w, inoculation with strain S246 significantly increased soybean emergence rate by 1.22 times compared to the uninoculated control.

[0032] Furthermore, this invention also discloses the application of *Stutzerimonas kunmingensis* S246 in alleviating the growth-inhibiting effect of salt stress on soybeans. Soil pot experiments showed that adding 1.5‰ NaCl to improved farmland soil in the coastal area of ​​Jiangsu Province to simulate salt stress, and immersing soybean seeds in OD... 600 A 1.0% S246 bacterial suspension was used for seed inoculation, and OD was applied to the soil before soybean sowing. 600 A bacterial suspension of *Stizemonas kunmingensis* S246 at a concentration of 1.0 was applied to the soybean roots again via root drenching 10 days after soybean sowing.600 S246 bacterial suspension with a concentration of 1.0 significantly promoted soybean seedling growth. Twenty-five days after soybean sowing, under NaCl stress, compared to the uninoculated control, S246 inoculation increased aboveground fresh weight by 28.2%, root fresh weight by 23.3%, soybean plant height by 35.2%, and the area of ​​the top three leaves by 25.9%.

[0033] Meanwhile, this invention also discloses the application of *Stutzerimonas kunmingensis* S246 in promoting rapeseed germination under salt stress. Germination experiments in a petri dish culture system showed that under 150 mM NaCl stress, inoculating with 1% bacterial solution to promote OD... 600 Inoculating a 1.0% S246 bacterial culture solution into a salt solution can significantly alleviate the inhibitory effect of salt stress on the growth of rapeseed radicles and shoots, increasing radicle length by 1.64 times and shoot length by 1 time. Attached Figure Description

[0034] Figure 1 Photographs of colony morphology of *Stizemonas kunmingensis* S246 on LB solid medium.

[0035] Figure 2 Phylogenetic tree of the 16S rRNA gene of *Stizemonas kunmingensis* S246.

[0036] Figure 3 The growth of *Stizemonas kunmingensis* S246 on CAS detection plates.

[0037] Figure 4 The images show the effect of *S. kunmingiensis* S246 on promoting soybean emergence under 1.5‰ NaCl stress. A: Growth status 5 days after sowing; B: Growth status 9 days after sowing.

[0038] Figure 5 Soybean emergence rate under pristine soil, with 1.5‰ NaCl stress and inoculation with S246.

[0039] Figure 6 The image shows the effect of 1.5‰ NaCl stress on the growth of soybean seedlings by *Stizemonas kunmingensis* S246. A: Parallel samples of the 4 CK groups; B: Parallel samples of the 4 NaCl groups; C: Parallel samples of the 4 NaCl+G37-2 groups.

[0040] Figure 7 Soybean plant heights under original soil, 1.5‰ NaCl stress, and 1.5‰ NaCl stress with S246 inoculation: A: Soybean plant height after 11 days of culture; B: Soybean plant height after 25 days of culture.

[0041] Figure 8 The growth indicators are: original soil, 1.5‰ NaCl stress, and 1.5‰ NaCl stress with S246 inoculation. A: Soybean aboveground fresh weight; B: Root fresh weight; C: Area of ​​the top three leaves.

[0042] Figure 9 The colony morphology of strain S246 on LB solid medium is shown in Figure A: Colony morphology of S246; Figure B: Colony morphology of strain S246 after 72 hours of incubation at a dilution of 10. -4 Colony morphology.

[0043] Figure 10 The diagram shows the effect of strain S246 on promoting rapeseed seed germination under NaCl stress. A to E represent the results of H2O, 150 NaCl, 150 NaCl + 1% S246, 150 NaCl + 10% S246, and 150 NaCl + 50% S246, respectively. The three rows represent three parallel experiments for the corresponding groups. Detailed Implementation

[0044] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0045] The inventive concept of this invention is as follows:

[0046] Through hundreds of millions of years of co-evolution, microorganisms and plants have formed a close symbiotic functional relationship. Microorganisms colonizing the rhizosphere of plants have evolved a variety of complex and sophisticated mechanisms to alleviate the damage caused by salt stress. Some plant growth-promoting bacteria can alleviate the inhibitory effect of salt stress on plant growth and help plants improve their salt tolerance by producing growth hormones, secreting 1-aminocyclopropane-1-carboxylate deaminase, producing siderophores, inducing systemic resistance in plants, and regulating gene expression in plants in a tissue-specific manner. Currently, the most widely used plant growth-promoting bacteria are mainly distributed in the genera *Bacillus*, *Pseudomonas*, *Azospirillum*, *Rhizobium*, and *Burkholderia*. *Stizemona* is a relatively new genus of bacteria identified in 2022. Regarding *Stutzerimonas* bacteria, Chinese patent CN202411774383.8 discloses the application of a marine bacterium, *Stutzerimonas balearica* WH-1, in the degradation of triazine herbicides, and Chinese patent CN202411645499.1 discloses the application of a salt-tolerant denitrifying bacterium, *Stutzerimonas degradans* BP, in the biological denitrification of high-salt wastewater. Regarding *Stutzerimonas kunmingensis*, Chinese patent CN202411786319.1 discloses the application of *Stutzerimonas kunmingensis* 9-4 in water denitrification. No research has been reported on the application of *Stutzerimonas kunmingensis* in improving the salt tolerance of dryland crops such as soybeans and rapeseed.

[0047] The purpose of this invention is to provide a strain of *Stizemonas kunmingensis* that can promote the growth of soybeans and rapeseed under salt stress, and to alleviate the inhibitory effects of salt stress on soybean emergence and seedling growth, as well as on rapeseed germination, radicle and shoot growth, thereby enhancing the tolerance of soybeans and rapeseed to salt stress.

[0048] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0049] The reagent preparation method used in this invention is as follows:

[0050] Each liter of LB medium contains: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000mL distilled water, pH 7.0, which is used to prepare the liquid medium; then add 20g agar powder to the liquid medium to make the solid medium, and sterilize at 121℃ for 20min.

[0051] CAS solid medium: 60.5 mg Chromium azurite S dissolved in 50 mL of deionized water, and mixed with 10 mL of 10 mmol / L solution. -1 Component A was prepared by mixing 1 mM FeCl3 with HCl. 72.9 mg of HDTMA was dissolved in 40 mL of deionized water to obtain component B. Component A was slowly added to component B, sterilized at 115°C for 30 min, and then mixed with 900 mL of LB to obtain CAS solid medium. HDTMA, or hexadecyltrimethylammonium bromide, is a type of ammonium bromide.

[0052] CAS detection solution: Dissolve 60.5 mg of Chromium Azurite S in 50 mL of deionized water and mix with 10 mL of 1 mM FeCl3 to obtain component A. Dissolve 72.9 mg of HDTMA in 40 mL of deionized water to obtain component B. Slowly add component A to component B and sterilize at 115 °C for 30 min.

[0053] Salkowski colorimetric solution: Mix 1 part of 0.5 mol / L FeCl3 and 49 parts of 35% HClO4 by volume, and store away from light.

[0054] Example 1

[0055] A strain of *Stizemonas kunmingensis* that enhances crop salt tolerance is described below:

[0056] 1. Isolation, identification and preservation of strain S246.

[0057] On June 15, 2022, rhizosphere soil of *Suaeda salsa* was collected from a severely saline-alkali land in Binhai, Jiangsu Province. 10g of rhizosphere soil was weighed into an Erlenmeyer flask containing 90mL of sterile water and shaken at 180rpm for 30min to obtain 10... -1 Dilute the soil suspension, then take 1 mL of 10 -1 The diluted soil suspension was placed in a test tube containing 9 mL of sterile water, mixed well, and yielded 10. -2 Dilute the soil suspension. Repeat the above steps four times to obtain a dilution of 10. -3 10 -4 10 -5 10 -6 Soil suspension. Take 100 μL of each diluted to 10... -4 10 -5 10 -6 Soil suspension was dripped onto LB solid medium containing 2% NaCl, spread with a spreader, and the plate was incubated at 28°C for 4 days. Colonies of different morphologies were picked, numbered, purified, and cultured on a large scale.

[0058] For the purified strain, its ability to produce IAA and siderophores was determined. The strain was inoculated with a solution containing 100 mg L... -1 L-tryptophan was cultured in LB broth at 28°C with shaking at 180 rpm for 48 h. 100 μL of the bacterial culture was mixed with an equal volume of Salkowski colorimetric solution in a 96-well plate, using uninoculated medium as a control. After reacting in the dark for 30 min, the solution color was observed. A pink solution indicated IAA production, with a deeper pink indicating stronger IAA production. The IAA-producing strain was further tested using the CAS assay to determine its IAA production capacity. This invention screened and obtained a strain that both secretes IAA and produces siderophores. After 48 h of culture, this strain produced 37.15 μg / ml of IAA. -1 After 72 hours, the siderophore production capacity was at a medium to high level, and the siderophore activity unit was 44.6%.

[0059] After culturing the strain on LB solid medium for 3 days, the colony morphology is as follows: Figure 1 As shown, the colonies are milky white to pale yellow, slightly raised or flat, with a smooth, moist, and glossy surface. The colonies are easily picked up and are non-mucous. This strain is a Gram-negative bacterium with flagella, capable of motility, and requires aerobic properties.

[0060] A small amount of fresh bacterial cells was taken with a sterile toothpick and placed into a 50 μL PCR reaction system. The 16S rRNA gene was amplified by PCR using universal bacterial primers 27F and 1492R. The amplified product was sequenced by Beijing Liuhe Huada Genomics Co., Ltd., yielding the 16S rDNA sequence of this strain, as shown in SEQ ID NO.3. The sequence was submitted to the EzBioCloud database for homology comparison analysis. The comparison results are shown below. Figure 2 The results showed that the 16S rDNA sequence of this strain was 99.39% similar to that of the type strain Stutzerianus kunmingensis HL22-2. Therefore, the strain was identified as Stutzerianus kunmingensis and named Stutzerianus kunmingensis S246.

[0061] The primer sequences for 27F and 1492R are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0062] 27F, SEQ ID NO. 1: 5'-AGAGTTTGATCMTGGCTCAG-3'.

[0063] 1492R, SEQ ID NO. 2: 5'-TACGGYTACCTTGTTACGACTT-3'.

[0064] The website address for EzBioCloud is https: / / www.ezbiocloud.net / .

[0065] S246, SEQ ID NO.3:

[0066]

[0067] The quantitative determination of IAA is as follows:

[0068] After culturing strain S246 in LB liquid culture at 28℃ and 180 rpm for 17 h, the bacterial cells were collected by centrifugation and adjusted to OD500 with sterile water. 600 A value of 1 yields the seed culture. Take 250 μL of the seed culture and add it to a 5 mL container containing 500 mg L. -1 In L-tryptophan-containing LB broth, the experiment was repeated three times. After 48 h of incubation at 28°C and 180 rpm with shaking, the supernatant was collected by centrifugation at 8000 rpm for 10 min. 100 μL of the supernatant was mixed with an equal volume of Salkowski colorimetric solution in a 96-well plate, with uninoculated medium as a blank control. The plate was incubated in the dark for 30 min before measuring the absorbance at 530 nm. Calculations based on the IAA standard curve showed that strain S246 produced 37.15 μg / mL of IAA after 48 h of incubation. -1 The results are shown in Table 1.

[0069] Qualitative detection of siderophore production by the strain is as follows:

[0070] Strain S246 was inoculated into LB liquid medium and cultured at 28°C with shaking at 180 rpm for 18 h. The bacterial cells were collected by centrifugation, and sterile water was added to adjust the OD value. 600 The seed culture of S246 was obtained by setting the value to 1. 2 μL of the seed culture was pipetted into the center of a CAS plate using a sterile pipette tip, repeated three times. After incubation at 28°C for 5 days, the size of the yellow halo surrounding the colonies was observed, and the diameter of the yellow halo and the colony diameter were measured. The results are as follows: Figure 3 As shown, the ratio of the yellow halo diameter to the colony diameter of S246 is 2.13, indicating a strong ability to secrete siderophores.

[0071] Quantitative detection of siderophores produced by the strain is as follows:

[0072] Seed culture of strain S246 was prepared according to the aforementioned method for "qualitative detection of siderophore production by strain". 300 μL of seed culture was inoculated into 3 mL of LB liquid medium and cultured at 28℃ with shaking at 180 rpm for 72 h. After centrifugation, the supernatant was mixed with an equal volume of CAS detection solution, and after standing for 1 h, the OD was measured. 680 The value, denoted as As, is calculated using LB medium mixed with CAS detection solution as a blank control. 680The value is denoted as Ar. According to the classification of bacterial siderophore production capacity by Manjanatha MG, Loynachan TE, Atherly AG. Tn5 mutagenesis of Chinese Rhizobium fredii for siderophore overproduction. Soil Biology and Biochemistry, 1992, 24(2): 151-155, the As / Ar ratio ranges from 1 to 0 with increments of 0.2, increasing by one plus sign for each decrease of 0.2. The results show that the As / Ar value of strain S246 is 0.446, indicating a siderophore secretion capacity of +++, which is considered above average.

[0073] Table 1. IAA and siderophore production capacity of strain S246

[0074]

[0075] In summary, the *Stutzerimonas kunmingensis* strain of this invention can produce IAA, and under the induction of 500 mg / mL L-tryptophan, it secretes 37.15 μg / mL of IAA in 48 hours. This strain has high siderophore activity, with a siderophore activity unit of 44.6% after 72 hours, which is at a medium-to-high level.

[0076] Example 2

[0077] An application of *Stizemonas kunmingensis* to improve crop salt tolerance is detailed below:

[0078] 1. Under salt stress, strain S246 promotes soybean emergence.

[0079] Preparation of bacterial suspension: Strain S246 was inoculated into LB liquid medium and cultured at 28°C with shaking at 180 rpm for 17 h. After centrifugation at 8000 rpm for 5 min, the bacterial cells were collected and adjusted to OD500 with sterile water. 600 The count is 1, and the viable count is 10. 9 CFU / mL was used to obtain a bacterial suspension.

[0080] Soybean seed disinfection: Soybean seeds were disinfected with 75% alcohol (by volume) for 1 minute, rinsed once with sterile water, then disinfected with 2.5% NaClO (by volume) for 2 minutes, and rinsed three times with sterile water. The water from the last elution was used for plate spreading. After 5 days of incubation, no sterile growth was observed on the plates, indicating that the disinfection was thorough.

[0081] Soil and potting setup: Soil from the 0cm–20cm topsoil layer of farmland at Xinyang Agricultural Experiment Station, Yancheng City, Jiangsu Province was collected. This soil was derived from long-term improvement of coastal saline-alkali soil and had a soluble salt content of 0.5‰. 150g of soil (dry weight) was weighed into culture cups. Three treatments were set up: ① NaCl treatment (1.5‰ NaCl added to the soil); ② NaCl + S246 treatment (1.5‰ NaCl added to the soil and inoculated with strain S246); ③ Control treatment (CK) (no NaCl added to the soil and no inoculation). The NaCl + S246 treatment involved inoculating strain S246 into LB liquid medium, incubating at 28℃ with shaking at 180rpm for 17 hours, collecting the cells by centrifugation, and adjusting the cell count to OD using sterile water. 600 =1, at which point the number of viable bacteria is approximately 10. 9 CFU / mL was used to obtain a bacterial suspension. This bacterial suspension was then inoculated into the soil to achieve an inoculum concentration of 10. 9 CFU / g soil. Soybean seeds were disinfected with 75% alcohol (v / v) for 1 min, rinsed once with sterile water, then disinfected with 2.5% NaClO for 2 min, and rinsed three times with sterile water. The final wash water was used for plate spreading and incubated for 5 days. Confirmation of sterile growth indicated thorough disinfection. The disinfected soybean seeds were then immersed in OD... 600 In a bacterial suspension of 1:1 for 2 minutes, stir thoroughly to ensure the bacteria adhere evenly to the seed surface. Re-inoculate with the strain after the soybean single leaf has unfolded to enhance colonization.

[0082] Soybean sowing and cultivation in the three treatments: Soybean seeds were sown at a rate of 5 seeds / cup in cultivation cups. The soybeans were then cultivated in a smart greenhouse with a daytime temperature of 28℃ and a nighttime temperature of 22℃. Water was replenished regularly to maintain soil moisture content at 60% of maximum water holding capacity. After the soybeans developed single leaves, the germination rate was recorded. Thinning was then performed, retaining 2 seedlings per cup of uniform growth. The thinning was repeated at a rate of 10 seedlings / cup. 9 The S246 strain was inoculated with an inoculum of CFU / g soil by root drenching to enhance its colonization effect.

[0083] Inoculation with S246 5 and 9 days after sowing promoted soybean emergence as follows: Figure 4 As shown, the germination rate is as follows: Figure 5 As shown, 1.5‰ NaCl stress significantly inhibited soybean emergence, and inoculation with strain S246 completely relieved the inhibitory effect of NaCl on emergence. Specifically, 1.5‰ NaCl stress reduced the soybean emergence rate from 85% in the control treatment to 45%, and inoculation with strain S246 restored the emergence rate to a level comparable to the control, indicating that strain S246 relieved the inhibitory effect of NaCl on emergence.

[0084] Figure 5In the bar chart, different lowercase letters indicate significant differences (P < 0.05).

[0085] 2. Strain S246 promotes seedling growth.

[0086] The preparation of bacterial suspension, S246 inoculation, test soil and pot setup, soybean sowing and culture conditions were the same as in the experiment described above. After 25 days of culture, soybeans were harvested, and the aboveground fresh weight, root fresh weight, plant height, and functional leaf area were measured.

[0087] The results are as follows Figure 6 , Figure 7 and Figure 8 As shown, the addition of NaCl significantly weakened soybean growth, resulting in a marked decrease in plant height, aboveground fresh weight, root fresh weight, and area of ​​the top three leaves. Inoculation with strain S246 effectively alleviated the inhibitory effect of NaCl stress on soybean growth, and in some indicators, the S246 inoculation treatment even surpassed the unstressed control treatment. Specifically, after 11 days of culture, compared with the control treatment, NaCl stress reduced soybean plant height by 54.9%, while inoculation with S246 restored plant height to a level comparable to the control. After 25 days of culture, the plant height of soybeans under NaCl treatment was 12.8% lower than the control without NaCl, and inoculation with S246 not only relieved the inhibitory effect of NaCl stress on plant height but even surpassed the control by 17.9%. Twenty-five days after sowing, NaCl treatment reduced the aboveground fresh weight, root fresh weight, and area of ​​the top three leaves of soybean by 27.8%, 53.8%, and 31.4%, respectively, compared to the control without NaCl. Inoculation with S246 strain restored the aboveground fresh weight and area of ​​the top three leaves to levels comparable to the control without NaCl stress, and restored the root fresh weight to 57.0% of the control without NaCl stress. These studies indicate that strain S246 can significantly alleviate the inhibitory effect of NaCl stress on soybean growth, restoring plant height to levels exceeding the control, and restoring aboveground fresh weight and area of ​​the top three leaves to levels comparable to the control. It also has a significant effect on alleviating the inhibitory effect of NaCl stress on root biomass.

[0088] Figure 7 and Figure 8 In the bar chart, different lowercase letters indicate significant differences (P < 0.05).

[0089] 3. Under salt stress, strain S246 stably colonized the soybean rhizosphere.

[0090] The preparation of the bacterial suspension, S246 inoculation, test soil and pot setup, and soybean sowing and cultivation conditions were the same as in the previous experiment. After 25 days of soybean cultivation, the rhizosphere soil was collected. For the NaCl+S246 treatment, 1g of the collected rhizosphere soil was weighed into a test tube containing 9mL of sterile water, and shaken at 28℃ and 180rpm for 30min. Using a sterile pipette, 100μL of the suspension was added to a centrifuge tube containing 900μL of sterile water, mixed well, and labeled as 10. -2 Dilution. Repeat the above steps to finally prepare 10. -4 10 -5 10 -6 Suspension at dilution. For 10 -4 10 -5 10 -6 Soil suspension: Take 100 μL of each dilution of suspension onto the surface of LB solid medium, spread the suspension with a spreader, and incubate at 28℃ for 72 h. Observe the growth of the target strain.

[0091] Based on the colony morphology of S246 on LB solid medium before inoculation, see Figure 9 Single colonies with morphology identical to S246 were selected from LB solid medium coated with soil suspension after 72 hours of culture. Colony PCR was performed using universal bacterial primers 27F and 1492R to amplify the 16S rRNA gene of the colonies. The PCR product was sequenced by Beijing Liuhe Huada Genomics Co., Ltd. to obtain the 16S rRNA gene sequence, which was then uploaded to EzBioCloud for homology comparison. The results showed that the 16S rRNA gene sequence of the colonies had 99.07%-99.36% sequence similarity to the type strain Stutzerimonas kunmingensis HL22-2 (Table 2), confirming that its taxonomic position was consistent with that of the inoculated strain S246, both being *Stutzerimonas kunmingensis* S246.

[0092] Based on the colony morphology and 16S rRNA gene identification results of S246, single colonies with the same morphology as S246 on LB solid medium coated with soil suspension were counted and analyzed. The results showed that at harvest time 25 days after soybean sowing, the inoculated strain S246 could still colonize in the soybean rhizosphere soil, and the colonization number reached 3.27 ± 0.83 × 10⁻⁶. 6 CFU / g soil.

[0093] Table 2. Colonization of soybean S246 in rhizosphere soil 25 days after soybean sowing under salt stress.

[0094]

[0095] 4. Under salt stress, strain S246 promotes rapeseed seed germination.

[0096] Petri dish preparation: Take petri dishes with a diameter of 9cm, and line each dish with two layers of sterile filter paper. Prepare a total of 15 dishes.

[0097] Preparation of bacterial suspension: S246 strain was inoculated into LB liquid medium and cultured on a shaker at 28°C and 180 rpm for 18 h. After centrifugation to collect the bacterial cells, the cells were resuspended in 150 mM NaCl solution, and the bacterial suspension was adjusted to OD200. 600 The value is 1.0, and the viable count is 10. 9 CFUmL -1 ,spare.

[0098] Rapeseed disinfection: Select rapeseed seeds of uniform size with intact seed coats. Immerse the rapeseed seeds in 75% ethanol (by volume) for 30 seconds, then rinse the seeds once with sterile water. Next, immerse the seeds in 15% H2O2 (by mass) for 30 seconds, and rinse the seeds three times with sterile water.

[0099] The experimental treatment settings and rapeseed seed sowing settings are as follows: three treatments are shown in Table 3.

[0100] Table 3 Experimental Treatment Settings for Rapeseed Seeds

[0101] Group Handling method 150NaCl 150mM NaCl <![CDATA[H2O]]> <![CDATA[H2O]]> 150NaCl + 1%S246 150mM NaCl + 1% (v / v) S246 150NaCl + 10%S246 150mM NaCl + 10% (v / v) S246 150NaCl + 50%S246 150mM NaCl + 50% S246 (by volume)

[0102] Each treatment in Table 3 was performed in triplicate. 10 mL of the corresponding treatment solution was added to each dish, and 15 sterilized seeds were sown in each dish. Radicle length and shoot length were measured after 5 days of incubation at 25°C.

[0103] Under NaCl stress, strain S246 effectively promoted rapeseed germination as follows: Figure 10 As shown in Table 4, the root and shoot lengths of rapeseed are as follows. NaCl stress significantly inhibited rapeseed germination, leading to impaired radicle and shoot growth. Inoculation with a certain amount of S246 bacterial solution significantly alleviated the inhibitory effect of NaCl stress on radicle and shoot growth. As shown in Table 4, NaCl treatment significantly reduced radicle and shoot length, with radicle length decreasing by 87.0% and shoot length decreasing by 97.6%. Inoculation with 1% S246 significantly increased radicle and shoot length compared to the NaCl treatment, increasing radicle length by 1.64 times and shoot length by 1 time compared to the NaCl stress group. Inoculation with 10% S246 also significantly increased radicle length compared to the NaCl stress group. Therefore, when the S246 addition amount is 1%, it can significantly alleviate the inhibitory effect of NaCl stress on rapeseed radicle and shoot growth, and when the S246 addition amount is 10%, it can significantly alleviate the inhibitory effect of NaCl stress on rapeseed radicle growth.

[0104] Table 4. Rapeseed root length and shoot length after 5 days of cultivation.

[0105]

[0106] Note: In Table 4, in the columns for radicle length and shoot length, different lowercase letters in the same column indicate significant differences between treatments (P < 0.05).

[0107] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0108] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A strain of *Stizemonas kunmingensis* that enhances crop salt tolerance, characterized in that... The *Stizemonas kunmingensis* is... Stutzerimonas kunmingensis S246, with accession number CCTCC NO: M 20242582, was deposited at the China Center for Type Culture Collection on November 18, 2024.

2. A bacterial suspension, characterized in that, The bacterial suspension was obtained by culturing *Stizemonas kunmingensis* as described in claim 1. The culturing process is as follows: *Stizemonas kunmingensis* was inoculated into LB medium and cultured at 28°C–30°C and 160–200 rpm for 16–18 hours. The bacterial cells were collected, and the viable count of *Stizemonas kunmingensis* was adjusted to 10⁻⁶ cells / mL with sterile water. 9 The bacterial suspension is obtained by measuring CFU / mL.

3. The bacterial suspension as described in claim 2, characterized in that, The cultivation process is as follows: *Stizemonas kunmingensis* was inoculated into LB medium and cultured at 28°C and 180 rpm for 17 h. The bacterial cells were collected, and the viable count of *Stizemonas kunmingensis* was adjusted to 10⁻⁶ cells / mL with sterile water. 9 The bacterial suspension is obtained by measuring CFU / mL.

4. The application of *Sterospora kunmingensis* as described in claim 1 or the bacterial suspension as described in claim 2, characterized in that, The application refers to any one of the following: 1) Promotes soybean emergence under salt stress; 2) Alleviating the inhibition of soybean growth under salt stress; 3) Promotes the germination of rapeseed seeds under salt stress.

5. A microbial growth promoter, characterized in that, The microbial growth promoter comprises *St. kunmingense* as described in claim 1, wherein the viable count of *St. kunmingense* in the microbial growth promoter is 10-1. 9 CFU / mL.

6. A microbial germination promoter, characterized in that, The microbial germination promoter comprises *St. kunmingiensis* as described in claim 1, wherein the viable count of *St. kunmingiensis* in the microbial germination promoter is 10-1. 9 CFU / mL.

7. The microbial growth promoter as described in claim 5 or the microbial germination promoter as described in claim 6, characterized in that, The microbial growth promoter or microbial germination promoter also includes microbiologically acceptable excipients.

8. The microbial growth promoter or microbial germination promoter as described in claim 7, characterized in that, Microbiologically acceptable excipients include at least one of fillers, binders, disintegrants, lubricants, and antacids.

9. The microbial growth promoter as described in claim 5 or the microbial germination promoter as described in claim 6, characterized in that, The application methods of the microbial growth promoter or microbial germination promoter include any one of spraying, root irrigation, seed dressing, and seed soaking.

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