A Haloxylon ammonium glutamate bacterium that enhances crop salt tolerance and its application

Abstract

This invention belongs to the field of microbial application technology, specifically relating to a halophyte bacterium that improves crop salt tolerance and its application. The halophyte bacterium is *Glutamicibacter halophytocola* G37-2, with accession number CCTCC NO: M 20242581, deposited at the China Center for Type Culture Collection on November 18, 2024. The halophyte bacterium *Glutamicibacter halophytocola* G37-2 described in this invention possesses growth-promoting properties such as producing auxin and forming biofilms. It can also significantly promote soybean emergence and seedling growth under salt stress, and promote rapeseed seed germination, radicle, and shoot growth. Furthermore, the halophyte bacterium can significantly increase soybean emergence rate, increase soybean functional leaf area, promote seedling growth, alleviate the inhibitory effect of salt stress on soybean emergence and seedling growth, thereby enhancing soybean's adaptability to salt stress; it can also promote rapeseed seed germination and alleviate the inhibitory effect of salt stress on rapeseed radicle and shoot growth.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, specifically relating to a Haloxylon ammonium glutamate bacterium that improves crop salt tolerance and its application. Background Technology

[0002] China ranks third in the world in terms of saline-alkali land area, totaling approximately 1.5 billion mu (100 million hectares), of which 500 million mu (20 million hectares) have development and utilization potential, accounting for more than 10% of China's total arable land. Effective development of saline-alkali land is of great significance for alleviating the imbalance between land supply and demand and ensuring food security. In recent years, planting salt-tolerant crops on saline-alkali land has become an important means of rationally utilizing this land.

[0003] Soybeans and rapeseed are field crops with significant advantages in utilizing saline-alkali land. Soybeans are not only an important source of high-quality protein in the diet, but also a moderately salt-tolerant crop, capable of growing in slightly to moderately saline-alkali land, thus expanding their planting range. As a legume, soybeans can fix nitrogen through symbiotic relationships with rhizobia, improving soil fertility, enhancing nutrient conditions in saline-alkali land, and reducing reliance on chemical fertilizers. Rapeseed is an important oilseed crop and a major overwintering crop in many provinces. It also possesses a certain degree of salt and alkali tolerance, absorbing water and nutrients from deeper soil layers through its deep root system, further enhancing its adaptability to saline-alkali environments. Furthermore, rapeseed flowers have ornamental value. Therefore, planting soybeans and rapeseed in saline-alkali land has unique advantages in utilizing and improving saline-alkali soil, and is expected to generate both economic and ecological benefits, showing broad prospects.

[0004] However, high concentrations of basic ions in the soil can cause osmotic stress, oxidative stress, and ion toxicity in plants, severely impacting their growth. Current research has shown that soybeans grown in saline-alkali soils exhibit significant declines in key traits, including a 25% decrease in plant height, a 26.6% decrease in pod number, a 33% decrease in seed yield, and a 13% decrease in 100-seed weight. NaCl-induced salt stress can reduce soybean yield by as much as 40%, or even result in complete crop failure. Seed germination and seedling stages are critical periods for plant growth and are also the most sensitive to salt-alkali stress. Saline-alkali soils commonly suffer from problems such as difficulty in seed emergence after sowing, seedling death and stunted growth after emergence, and weak growth, all of which severely hinder yield formation. 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

[0005] The purpose of this invention is to provide a halotrophic glutamate bacterium that improves crop salt tolerance, and can be used to alleviate the impact of salt stress on crop growth.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a halophilic glutamate bacterium that improves crop salt tolerance. The halophilic glutamate bacterium is Glutamicibacterhalophytocola G37-2, with accession number CCTCC NO: M 20242581, and was deposited at the China Center for Type Culture Collection on November 18, 2024.

[0008] A second aspect of the present invention provides a bacterial suspension, which is obtained by culturing the aforementioned *Haloxyglutamic acid bacillus*. The culturing process is as follows:

[0009] Inoculate *Bacillus halophilus* into LB medium and incubate at 28℃–30℃ and 180 rpm for 16–18 hours. Collect the cells and adjust the viable count of *Bacillus halophilus* to 10⁻⁶ cells / mL with sterile water. 9 The bacterial suspension is obtained by measuring CFU / mL.

[0010] Preferably, the cultivation process is as follows:

[0011] Bacillus halophilus 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 Bacillus halophilus was adjusted to 10⁻⁶ cells / mL with sterile water. 9 The bacterial suspension is obtained by measuring CFU / mL.

[0012] A third aspect of the present invention provides the application of the aforementioned *Haloxyglutamate* or the aforementioned bacterial suspension, characterized in that the application refers to any one of the following:

[0013] 1) Promotes crop emergence;

[0014] 2) Promotes crop seedling growth;

[0015] 3) Promotes crop seed germination;

[0016] 4) Improve crop salt tolerance.

[0017] Preferably, the crop includes either soybean or rapeseed.

[0018] A fourth aspect of the present invention provides a microbial growth promoter comprising the aforementioned *Haloxyglutamate*, wherein the viable count of *Haloxyglutamate* in the microbial growth promoter is 10-1. 9 CFU / mL.

[0019] A fifth aspect of the present invention provides a microbial germination promoter, characterized in that the microbial germination promoter comprises the aforementioned *Haloxyglutamate*, wherein the viable count of *Haloxyglutamate* in the microbial germination promoter is 10-1. 9 CFU / mL.

[0020] Preferably, the microbial growth promoter or microbial germination promoter further includes microbiologically acceptable excipients.

[0021] Preferably, the microbiologically acceptable excipients include at least one of fillers, binders, disintegrants, lubricants, and antacids.

[0022] Preferably, the filler includes any one of sodium alginate, polyacrylamide, and zeolite.

[0023] Preferably, the adhesive comprises any one of starch paste, hydroxypropyl methylcellulose, and povidone.

[0024] Preferably, the disintegrant includes any one of starch, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose.

[0025] Preferably, the lubricant includes any one of microalgae-based biolubricant, mucin, and tannic acid.

[0026] Preferably, the antacid comprises any one of phosphate buffer, bicarbonate buffer, and Tris buffer.

[0027] Preferably, the application method of the microbial growth promoter or microbial germination promoter includes either root irrigation or seed soaking.

[0028] The preservation information for biological material samples involved in this invention is as follows:

[0029] *Glutamicibacter halophytocola* G37-2 was deposited at the China Center for Type Culture Collection (CCTCC) on November 18, 2024. The proposed taxonomic name is *Glutamicibacter halophytocola* G37-2, the accession number is CCTCC NO: M 20242581, and the deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. For ease of description, *Glutamicibacter halophytocola* G37-2 will be abbreviated as G37-2 in this invention.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] This invention provides a *Glutamicibacter halophytocola* strain that enhances crop salt tolerance. The strain is *Glutamicibacter halophytocola* G37-2, with accession number CCTCC NO: M20242581, and was deposited at the China Center for Type Culture Collection on November 18, 2024. This invention marks the first isolation of a salt-tolerant and growth-promoting *Glutamicibacter halophytocola* G37-2 from the roots of *Salvia splendens* in coastal saline-alkali land of Jiangsu Province. This bacterium exhibits IAA production and biofilm-producing characteristics, significantly promoting soybean emergence and seedling growth under salt stress, and also promoting rapeseed seed germination, radicle, and shoot growth. Microbial germination promoters and growth promoters further prepared using *Glutamicibacter halophytocola* G37-2 can significantly improve soybean emergence rate, increase soybean functional leaf area, promote seedling growth, alleviate the inhibitory effect of salt on soybean seed germination and root growth, and thus enhance soybean's adaptability to salt stress. They can also promote rapeseed seed germination and alleviate the inhibitory effect of salt stress on rapeseed embryo, root, and shoot growth. Therefore, *Glutamicibacter halophytocola* G37-2 and microbial germination promoters and growth promoters containing G37-2 show broad application prospects in promoting seed germination, emergence, and growth of crops such as soybean and rapeseed in saline-alkali land. This provides important resource and technical support for developing microbial agents or microbial fertilizers suitable for saline-alkali land, and also lays the material and technical foundation for creating significant ecological and economic benefits.

[0032] This invention also discloses the application of the aforementioned Haloxylon ammonium glutamate in promoting soybean emergence under salt stress. Soil pot experiments showed that, under salt stress with a NaCl concentration of 1.5‰, inoculation with strain G37-2 doubled the emergence rate compared to the uninoculated control.

[0033] Furthermore, this invention also discloses the application of the aforementioned Haloxylon ammonium glutamate bacteria in promoting soybean growth under salt stress. Soil pot experiments showed that when 1.5‰ NaCl was added to the soil to simulate salt stress, immersing soybean seeds in OD... 600 A 1.0 g / L G37-2 bacterial suspension was used for seed inoculation. The G37-2 bacterial suspension was inoculated into the soil before soybean sowing. Seven days after soybean sowing, OD2 was applied again to the soybean roots via root drenching. 600 A suspension of G37-2 bacteria with a concentration of 1.0 was cultured for 25 days. The G37-2 strain significantly alleviated the inhibitory effect of NaCl stress on soybean growth, restoring indicators such as plant height, aboveground fresh weight, area of ​​the top three leaves, and root fresh weight to levels close to or exceeding those of the control without NaCl.

[0034] This invention also discloses the application of the aforementioned Haloxylon ammonium glutamate bacterium in promoting rapeseed germination under salt stress. Inoculation with strain G37-2 completely relieved the inhibitory effect of NaCl stress on rapeseed germination rate. Under 150 mM NaCl stress, compared with the uninoculated control, inoculation with 10% G37-2 bacterial solution increased the radicle length and shoot length by 2.19 times and 1.03 times, respectively. Attached Figure Description

[0035] Figure 1 Photographs of the colony morphology of Bacillus halophilus G37-2 on LB solid medium.

[0036] Figure 2 Phylogenetic tree of the 16S rRNA gene of Haematococcus glutamate G37-2.

[0037] Figure 3 The images show the effect of 1.5‰ NaCl stress on promoting soybean emergence using Haloxylon ammodendron G37-2. A: Growth status 5 days after sowing; B: Growth status 9 days after sowing.

[0038] Figure 4 Soybean emergence rate under pristine soil, with 1.5‰ NaCl stress and inoculated with G37-2 treatment.

[0039] Figure 5 The diagram shows the effect of 1.5‰ NaCl stress on the growth of soybean seedlings by Haloxylon ammonium glutamate G37-2. A: 4 parallel samples of the CK group; B: 4 parallel samples of the NaCl group; C: 4 parallel samples of the NaCl + G37-2 group.

[0040] Figure 6 The figures show the soybean plant heights under the following conditions: original soil, 1.5‰ NaCl stress, and 1.5‰ NaCl stress with inoculation with G37-2. A: Soybean plant height after 11 days of cultivation; B: Soybean plant height after 25 days of cultivation.

[0041] Figure 7 The growth indicators are: original soil, 1.5‰ NaCl stress, and 1.5‰ NaCl stress with G37-2 inoculation. A: Soybean aboveground fresh weight; B: Root fresh weight; C: Area of ​​the top three leaves.

[0042] Figure 8 The diagram shows the effect of G37-2 on promoting rapeseed seed germination under salt stress. A to E represent the results of H2O, 150NaCl, 150NaCl+1%G37-2, 150NaCl+10%G37-2, and 150NaCl+50%G37-2, respectively. The three rows represent three parallel experiments for the corresponding groups. Detailed Implementation

[0043] 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.

[0044] The inventive concept of this invention is as follows:

[0045] Plants and microorganisms have undergone hundreds of millions of years of co-evolution, forming a close symbiotic functional group. Root-associated microorganisms have evolved various mechanisms to enhance the host's salt tolerance, playing a vital and irreplaceable role in mitigating the damage caused by salt stress to plants. Some plant growth-promoting bacteria can promote plant growth by producing auxins or reduce the level of harmful ethylene in plants by secreting ACC deaminase, thereby alleviating the negative effects of salt stress. In addition, they can also reduce the damage caused by salt stress by producing siderophores and secreting extracellular polysaccharides. Currently, the most widely used salt-tolerant plant growth-promoting bacteria are mainly concentrated in the genera *Bacillus* and *Pseudomonas*. Regarding *Glutamicibacter halophytocola*, Chinese patent CN202210024727.8 discloses the application of *Glutamicibacter halophytocola* G2 in organic waste composting, biogas slurry, eutrophic water treatment, and black and odorous water treatment; Chinese patent CN202411073717.9 discloses the application of *Glutamicibacter halophytocola* DL1 in carbon fixation. However, there are currently no reports on the growth-promoting effects of *Glutamicibacter halophytocola* on crops such as soybeans and rapeseed under salt stress, or on its ability to improve their salt stress adaptability.

[0046] The purpose of this invention is to provide a Haloxylon ammonium glutamate strain that can promote the growth of soybeans and rapeseed under salt stress, thereby improving the emergence rate of soybeans, promoting the growth of soybean seedlings, and alleviating the inhibition of rapeseed germination and root growth by salt, thus enhancing the tolerance of the two crops to salt stress.

[0047] 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.

[0048] The reagent preparation method used in this invention is as follows:

[0049] 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.

[0050] Salkowski colorimetric solution: Mix 1 part of 0.5 mol / L FeCl3 and 49 parts of HClO4 (35% by volume) in the specified volume ratio and store away from light.

[0051] Crystal violet staining solution with a mass percentage of 0.1%: Weigh 0.1g of crystal violet, add it to 10mL of anhydrous ethanol, stir thoroughly until completely dissolved, add water to make up to 100mL, mix well, and set aside.

[0052] PBS buffer: Weigh 8g sodium chloride, 0.2g potassium chloride, 3.63g disodium hydrogen phosphate, and 0.24g potassium dihydrogen phosphate, dissolve them in 900mL distilled water, add water to make up to 1000mL, and store at room temperature for later use.

[0053] Example 1

[0054] A type of halophilic glutamate bacterium that enhances crop salt tolerance, as detailed below:

[0055] 1. Isolation, identification and growth-promoting characteristics determination of strain G37-2.

[0056] Strain G37-2 was isolated and screened from the root system of *Sargassum fusiforme* in a saline-alkali land in Binhai, Jiangsu Province. On June 15, 2022, three healthy *Sargassum fusiforme* plants were collected from a severely saline-alkali land in Binhai, Jiangsu Province, and brought back to the laboratory in sterile bags. The plant roots were carefully cut off with scissors, washed with tap water to remove surface soil, and then rinsed three times with sterile water. The roots were then disinfected with 75% ethanol for 5 minutes, rinsed once with sterile water, and then disinfected with 2.5% sodium hypochlorite for 5 minutes, followed by three rinses with sterile water to disinfect the root surface. 100 μL of the final elution water was spread onto LB agar plates. After 5 days of incubation, no colonies grew on the plates, indicating thorough disinfection. Approximately 1.5 g of root tissue was cut using sterile scissors, placed in a sterile mortar, and 1.5 mL of sterile water was added. The mixture was then ground until a homogenate was formed. 100 μL of homogenate was taken from each sample and spread evenly on an LB agar plate, then incubated at 28°C for 4 days. After incubation, colonies with different morphologies and colors were picked, purified, numbered, and stored.

[0057] The purified strain was subjected to tests to determine its characteristics, such as production of indole-3-acetic acid (IAA) and biofilm formation.

[0058] The method for IAA determination is as follows: The purified bacterial strain was inoculated into LB liquid medium containing 100 mg / L L-tryptophan and cultured with shaking at 28℃ and 180 rpm for 48 h. After culturing, 100 μL of bacterial culture was mixed with 100 μL of Salkowski colorimetric solution and added to a 96-well plate. A blank control was also set up, i.e., 100 μL of uninoculated culture medium was mixed with 100 μL of Salkowski colorimetric solution. The 96-well plate was placed in the dark for 30 min, and then the color change of the mixture was observed in a bright environment. If the strain produces IAA, the mixture will turn pink, and the deeper the pink, the stronger the strain's ability to produce IAA.

[0059] The method for determining the biofilm-forming ability of the bacterial strain is as follows: The strain was inoculated into LB liquid medium and cultured at 28℃ and 180 rpm for 17 h. The bacterial culture was then diluted with LB medium at a volume ratio of 1:100. At this point, the OD... 600 ≈0.1. Add 200 μL of diluted bacterial solution to a 96-well plate and incubate at 28°C for 24 h. Wash away unattached cells with PBS buffer. Stain adherent cells with 100 μL of 0.1% crystal violet solution for 30 min. Rinse with PBS buffer to remove excess stain, air dry, and add 200 μL of 95% ethanol. After the crystal violet is completely dissolved, measure the absorbance at 595 nm, denoted as As; use LB medium as a control, denoted as Ac. Assessment of bacterial biofilm formation ability: As / Ac < 1 indicates no biofilm production; As / Ac > 1 indicates biofilm production.

[0060] Based on the strain's IAA production and biofilm formation capabilities, a strain was screened. After 48 hours of culture, this strain produced 13.8 μg / mL of IAA, and after 24 hours of culture, its biofilm formation ratio (As / Ac) was 1.77. This strain was named G37-2, and its colony morphology on LB agar is shown below. Figure 1 As shown.

[0061] The G37-2 strain was cultured on LB broth. A small amount of fresh G37-2 cells was taken with a sterile toothpick and added to the PCR reaction system. The 16S rRNA gene was amplified by PCR using universal primers 27F and 1492R. The PCR product was then sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. The sequencing results of the PCR product are shown in SEQ ID NO.3. The obtained 16S rRNA gene sequence was uploaded to EzBioCloud for homology comparison. Sequences of bacterial species with high similarity to the G37-2 sequence were downloaded, and a phylogenetic tree was constructed using MEGA 5.0. The results are as follows: Figure 2As shown, the 16S rRNA gene sequence of this bacterium clusters on the same branch as Glutamicibacter halophytocola KLBMP 5180 (Type), and the sequence similarity of the strains reaches 100%. This strain was identified as Glutamicibacter halophytocola.

[0062] The primer sequences for 27F and 1492R are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0063] 27F, SEQ ID NO. 1: 5'-AGAGTTTGATCMTGGCTCAG-3'.

[0064] 1492R, SEQ ID NO. 2: 5'-TACGGYTACCTTGTTACGACTT-3'.

[0065] The website address for EzBioCloud is https: / / www.ezbiocloud.net / .

[0066] >G37-2, SEQ ID NO.3:

[0067]

[0068] Example 2

[0069] An application of a halophilic glutamate bacterium to improve crop salt tolerance is detailed below:

[0070] 1. Under salt stress, strain G37-2 promotes soybean emergence.

[0071] Soil tested: Soil from the topsoil layer (0cm to 20cm) of farmland at the Xinyang Experimental Station of Jiangsu Coastal Agricultural Research Institute, Jiangsu Academy of Agricultural Sciences, Yancheng City, was collected. The soluble salt content of this soil was 0.5‰.

[0072] Potting setup: Weigh 150g of test soil (dry soil) into culture cups. Three treatments were set up: ① NaCl treatment, i.e., adding 1.5‰ NaCl to the test soil; ② NaCl + G37-2 treatment, i.e., adding 1.5‰ NaCl to the test soil and inoculating with strain G37-2; ③ Control treatment CK, i.e., no NaCl added to the test soil and no inoculation. The NaCl + G37-2 treatment was specifically as follows: strain G37-2 was inoculated into LB liquid medium, cultured at 28℃ with shaking at 180rpm for 17h, the cells were collected by centrifugation, and the cell count was adjusted 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.

[0073] Soybean sowing and cultivation in the three treatments: Five soybean seeds were sown in each culture cup. The culture cups were placed in a glass greenhouse with a daytime temperature of 28℃ and a nighttime temperature of 22℃. Water was added regularly to maintain the soil moisture content at 60% of the soil's maximum water holding capacity. After 7 days of cultivation, once the single leaves had unfolded, the germination rate was recorded. Thinning was performed, retaining two seedlings of uniform growth per cup, and seedlings were transplanted using the root irrigation method at 10... 9 The strain was re-inoculated at an inoculum concentration of CFU / g soil to enhance colonization. Results were as follows: Figure 3 and Figure 4As shown, the addition of 1.5‰ NaCl significantly inhibited soybean emergence, and strain G37-2 completely relieved the inhibitory effect of NaCl stress on soybean emergence. Specifically, NaCl treatment reduced the soybean emergence rate from 85% in the control treatment to 45%. After inoculation with strain G37-2, the emergence rate of the NaCl + G37-2 treatment returned to 90%, comparable to the control treatment, indicating that strain G37-2 completely relieved the inhibitory effect of NaCl stress on soybean emergence.

[0074] Figure 4 In the bar chart, different lowercase letters indicate significant differences (P < 0.05).

[0075] 2. The effect of strain G37-2 on promoting soybean seedling growth under salt stress.

[0076] The methods for preparing the test soil, potting setup, bacterial suspension preparation and inoculation, soybean sowing and cultivation were the same as described above. Twenty-five days after soybean sowing, plant height, aboveground fresh weight, root fresh weight, and functional leaf area were measured. The functional leaf was the third leaf from the top.

[0077] The results are as follows Figure 5 , Figure 6 and Figure 7 As shown, compared with the control group without NaCl, the addition of NaCl significantly inhibited soybean growth, manifested as a significant decrease in plant height, aboveground fresh weight, area of ​​the top three leaves, and root fresh weight. Inoculation with strain G37-2 effectively relieved the inhibitory effect of NaCl on soybean growth, and even exceeded the control treatment in some indicators. Specifically, 11 days after sowing, compared with the control treatment, NaCl treatment reduced soybean plant height by 54.9%, while inoculation with G37-2 restored plant height to a level comparable to the control. 25 days after sowing, the plant height of soybeans under NaCl treatment was 12.8% lower than the control, while inoculation with G37-2 not only relieved the inhibitory effect of NaCl on plant height but even resulted in plant height exceeding that of the control group without NaCl. 25 days after sowing, compared with the control group, NaCl treatment reduced soybean aboveground fresh weight and area of ​​the top three leaves by 27.8% and 31.4%, respectively; inoculation with G37-2 restored aboveground fresh weight and area of ​​the top three leaves to a level comparable to the control. NaCl treatment reduced root fresh weight to 46.2% of the control treatment, while inoculation with G37-2 restored root fresh weight to 73.7% of the control treatment. These studies indicate that strain G37-2 can significantly alleviate the inhibitory effect of NaCl stress on soybean growth, restoring plant height, aboveground fresh weight, area of ​​the top three leaves, and root fresh weight to levels close to or exceeding the control.

[0078] Figure 6 and Figure 7 In the bar chart, different lowercase letters indicate significant differences (P < 0.05).

[0079] 3. Under salt stress, strain G37-2 stably colonized the soybean rhizosphere.

[0080] The test soil, pot setup, bacterial suspension preparation and inoculation, soybean sowing and culture conditions were the same as described above. After 25 days of soybean culture, rhizosphere soil from the three treatments was collected using the root-shaking method. For the treatment with added NaCl and inoculated with G37-2, 1g of soil was weighed into a test tube containing 9mL of sterile water and labeled as 10. -1 Dilution: Shake at 28℃ and 180 rpm for 30 min. Using a sterile pipette, transfer 100 μL of the suspension into a centrifuge tube containing 900 μL of sterile water, mix well, and label as 10. -2 Dilution. Repeat the above steps to prepare 10 [units / units] sequentially. -3 10 -4 10 -5 10 -6 Diluted suspensions. Using a sterile pipette, pipette 100 μL of a 10⁻⁶ dilution. -4 10 -5 and 10 -6 Soil suspension was dropped onto the surface of LB solid medium and spread evenly. The plates were inverted and incubated at 28°C for 72 hours. Plates with an appropriate colony count, typically 30–300 colonies per plate, were selected. Based on the colony morphology of G37-2, the colonies were counted to determine the colony density of the inoculated strain G37-2 in the rhizosphere soil. Single colonies with the same morphology as the inoculated strain G37-2 were picked up with a toothpick and placed in a 50 μL PCR reaction system. The 16S rRNA gene was amplified by PCR using universal bacterial primers 27F and 1492R. The PCR product was sent to Beijing Liuhe Huada Genomics Co., Ltd. for sequencing. The obtained 16S rRNA gene sequence was uploaded to EzBioCloud for homology comparison. The results showed that the 16S rDNA sequence of the colonies on the detection plate had 99.78%–99.93% sequence similarity to that of *Glutamicibacter halophytocola* KLBMP 5180(Type) (Table 1), indicating that the colonies belonged to *Glutamicibacter halophytocola*, consistent with the biological taxonomic position of the inoculated strain G37-2. Counting of single colonies with the same morphology as those inoculated with G37-2 showed that G37-2 could still colonize in the soybean rhizosphere soil at soybean harvest time, i.e., 25 days after sowing, with a colony size of 1.38 ± 0.16 × 10⁻⁶. 7 CFU / g soil.

[0081] Table 1. Colonization of soybean variety G37-2 in rhizosphere soil 25 days after sowing under salt stress.

[0082]

[0083] 4. The effect of strain G37-2 on promoting rapeseed seed germination under salt stress.

[0084] Preparation of bacterial culture: Strain G37-2 was inoculated into LB liquid medium and cultured at 28℃ and 180 rpm for 17 h with shaking. The bacterial cells were collected by centrifugation, resuspended in 150 mM NaCl solution, and the OD of the bacterial culture was adjusted. 600 The value is 1.0, and the viable count is 10. 9 CFU / mL, for later use.

[0085] Petri dish preparation: Line two layers of sterile filter paper into a 9cm diameter petri dish, and prepare a total of 15 petri dishes.

[0086] Seed disinfection: Soak rapeseed seeds in 75% ethanol for 30 seconds and rinse once with sterile water; then soak the seeds in 15% H2O2 for 30 seconds and rinse three times with sterile water to ensure that the seed surface is sterile.

[0087] The salt concentration settings are shown in Table 2.

[0088] Table 2. Salt concentration treatment of rapeseed seeds

[0089]

[0090]

[0091] In Table 2, each group is processed with 3 replicates.

[0092] Seed sowing: Add 10 mL of the solution corresponding to the group in Table 2 to each petri dish. Sow 15 sterilized rapeseed seeds per dish in the petri dish. Place the petri dishes in an incubator at 25℃ and incubate for 5 days. Measure the radicle length and shoot length afterward.

[0093] Table 3 shows that under 150 mM NaCl stress, the germination rate, radicle, and shoot growth of rapeseed were significantly inhibited. Compared with the sterile water control, 150 mM NaCl reduced the germination rate by 11.1%, shoot length by 87.0%, and radicle length by 97.6%. Inoculation with 1%, 10%, and 50% G37-2 bacterial solutions all restored the germination rate to a level comparable to the control. Inoculation with a certain concentration of G37-2 bacterial solution significantly alleviated the inhibitory effect of NaCl stress on root and shoot growth. Specifically, under NaCl stress, the radicle length of rapeseed was only 8.1 mm, and the shoot length was only 21.3 mm. Inoculation with 10% G37-2 bacterial solution significantly increased the radicle length and shoot length to 25.9 mm and 43.3 mm, respectively, representing increases of 2.19 times and 1.03 times compared to the NaCl treatment. Furthermore, inoculation with 50% G37-2 bacterial solution also significantly increased the radicle length of rapeseed, by 51.8% compared to the NaCl treatment. In conclusion, inoculation with G37-2 bacterial solution can significantly alleviate the inhibitory effect of NaCl stress on rapeseed germination, and inoculation with 10% G37-2 bacterial solution is particularly effective in alleviating the inhibitory effect of NaCl on radicle and shoot growth. The effect of strain G37-2 on promoting rapeseed seed germination under NaCl stress is shown in the figure. Figure 8 .

[0094] Table 3. Rapeseed radicle length and shoot length under different treatments

[0095] <![CDATA[H2O]]> 90.0±4.7a 342.9±10.8a 164.3±21.1a 150NaCl 80.0±6.7b 8.1±0.7d 21.3±4.2c 150NaCl + 1%G37-2 91.1±3.8a 15.8±2.7cd 37.3±3.7bc 150NaCl + 10% G37-2 91.1±3.8a 25.9±4.1b 43.3±2.9b 150NaCl + 50% G37-2 91.1±3.8a 19.3±1.4bc 32.4±5.3bc

[0096] Note: In Table 3, different lowercase letters in the same column of germination rate, radicle length and shoot length indicate significant differences (P<0.05).

[0097] 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.

[0098] 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 halophilic glutamate bacterium that enhances crop salt tolerance, characterized in that, The Haloxyglutamate bacillus is Glutamicibacter halophytocola G37-2, with accession number CCTCC NO: M 20242581, 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 the *Haloxyglutamate* bacillus described in claim 1, and the culturing process is as follows: Inoculate *Bacillus halophilus* into LB medium and incubate at 28°C–30°C and 160–200 rpm for 16–18 hours. Collect the cells and adjust the viable count of *Bacillus halophilus* 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: Bacillus halophilus 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 Bacillus halophilus was adjusted to 10⁻⁶ cells / mL with sterile water. 9 The bacterial suspension is obtained by measuring CFU / mL.

4. The application of the *Haloxyglutamate* bacillus 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 crop emergence; 2) Promotes crop seedling growth; 3) Promotes crop seed germination; 4) Improve crop salt tolerance; The crop includes either soybean or rapeseed.

5. A microbial growth promoter, characterized in that, The microbial growth promoter comprises the *Haloxyglutamate* bacillus according to claim 1, wherein the viable count of *Haloxyglutamate* bacillus in the microbial growth promoter is 10-1. 9 CFU / mL.

6. A microbial germination promoter, characterized in that, The microbial germination promoter comprises the *Haloxyglutamate* bacillus as described in claim 1, wherein the viable count of *Haloxyglutamate* bacillus 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, The 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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