Microbacterium algeriense and application thereof in improving salt tolerance of crops

Abstract

The application belongs to the technical field of microbial engineering, and particularly relates to a strain of algidbacter algidus and application thereof in improving salt tolerance of crops. Microbacterium algeriense The algidbacter algidus is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20242839. The strain has the dual promoting growth characteristics of high production of iron carrier and secretion of plant growth hormone, and can significantly relieve the inhibition of salt stress on seed germination, seedling emergence and growth of crops. The algidbacter algidus and the microbial inoculant provided by the application have wide application prospects in promoting seed germination, seedling emergence and growth of crops in saline-alkali land.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering technology, specifically relating to a strain of Algerian microbacterium and its application in improving crop salt tolerance. Background Technology

[0002] Planting salt-tolerant crops in saline-alkali land is an important way to develop and utilize saline-alkali land. Soybeans and rapeseed are field crops that are highly advantageous for utilizing saline-alkali land and are also crops that are encouraged to be planted in saline-alkali land. However, soil salinization remains one of the major challenges facing agricultural production. Salt stress can cause damage to cell membrane function, accumulation of toxic substances, inhibition of photosynthesis, and weakened nutrient acquisition, ultimately leading to cell and even the death of the entire plant. Generally, when the soil salinity exceeds 0.1%, the growth of ordinary crops begins to be significantly affected; when the soil salinity exceeds 0.3%, the yield of most crops decreases significantly. Seed germination and seedling stages are critical periods for plant growth and are also the stages most sensitive to salt stress. High concentrations of base ions can severely inhibit seed germination, emergence, plant growth and development, leading to problems such as difficulty in seed emergence after sowing, seedling death and stunted growth after emergence, weak growth, and gaps in the rows.

[0003] Rhizosphere microorganisms, often referred to as the plant's second genome, play a crucial and irreplaceable role in assisting plants to resist adverse conditions such as salt stress. During their growth and metabolism, plant growth-promoting bacteria in the rhizosphere can directly promote plant growth under salt stress through processes such as biological nitrogen fixation, auxin (IAA) production, and activation of phosphorus and potassium nutrients in the soil. They can also alleviate the damage caused by salt stress and improve the plant's adaptability to salt-stressed environments by producing siderophores, secreting aminocyclopropane deaminase, and inducing systemic resistance. Therefore, microorganisms or microbial inoculants have shown great potential in promoting salt-tolerant plant growth.

[0004] To address the challenges faced by soybeans and rapeseed in saline-alkali soil production, such as difficulties in emergence, weak seedling growth, seedling death or stunted growth, and gaps in rows, it is necessary to screen for functional microorganisms with various life-promoting properties that can significantly promote crop seed germination, emergence, and seedling growth under salt stress conditions. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides an Algerian microbacterium strain and its application in improving crop salt tolerance. This Algerian microbacterium strain possesses dual growth-promoting properties, including high iron carrier production and secretion of plant growth hormones, which can significantly alleviate the inhibitory effects of salt stress on crop germination, emergence, and growth.

[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0007] The first aspect of this invention provides a strain of Algerian microbacterium (Microbacterium algeriense The Algerian microbacterium is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242839.

[0008] The Algerian microbacterium strain is a high-side-producing bacterium, with a 72-hour siderophore secretion capacity rating of high (++++). This strain can produce auxin (IAA, indoleacetic acid), and under L-tryptophan (500 mg / L) induction, the average amount of indoleacetic acid secreted after 48 hours is 45.3 μg / mL.

[0009] A second aspect of the present invention provides a microbial agent, wherein the effective component of the microbial agent contains the aforementioned Algerian microbacterium.

[0010] Furthermore, the viable count of *Microbacterium algeriae* in the microbial agent is ≥10. 9 CFU / mL.

[0011] Furthermore, the microbial agent is a germination promoter or a growth promoter.

[0012] A third aspect of this invention provides a method for preparing the aforementioned microbial inoculant, comprising the following steps: inoculating the *Microbacterium algeriae* into LB liquid medium and culturing at 28°C–30°C for 16–18 hours, collecting the bacterial suspension; centrifuging the bacterial suspension to collect the bacterial cells; adding sterile water to the bacterial cells and resuspending the bacterial cells to obtain a bacterial suspension; and adjusting the viable count of *Microbacterium algeriae* in the bacterial suspension to ≥10-1. 9 The microbial agent is obtained by obtaining CFU / mL.

[0013] The fourth aspect of the present invention provides the application of the above-described Algerian microbacterium or microbial agent in improving the salt tolerance of plants.

[0014] Furthermore, the improvement of plant salt tolerance means that under salt stress, it can promote plant seed germination, plant seed emergence, plant seedling growth, or increase plant biomass.

[0015] Furthermore, the plant seeds were soaked in the microbial agent for 2 to 4 minutes, and then the microbial agent was applied to the soil to ensure that the viable count of *Microbacterium alpha-10* per gram of soil was ≥10. 9 CFU promotes the germination of plant seeds.

[0016] Furthermore, the plant is irrigated with the aforementioned microbial agent to ensure that the viable count of *Microbacterium alpha-10* per gram of soil is ≥10. 9 CFU can promote the growth of plant seedlings or increase plant biomass.

[0017] Furthermore, the plant in question is a dryland crop.

[0018] Furthermore, the dryland crop is soybean or rapeseed.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention discloses for the first time a strain of Algerian microbacterium with growth-promoting function ( Microbacterium algeriense M266-2. This strain possesses dual growth-promoting properties, including high siderophore production and secretion of the plant growth hormone IAA, which can significantly alleviate the inhibitory effects of salt stress on crop germination, emergence, and growth. Microbial agents prepared using this *Microbacterium algeriae* M266-2 can significantly improve soybean emergence rate, increase functional leaf area, and promote seedling growth under salt stress conditions, alleviating the inhibitory effects of salt stress on soybean emergence and seedling growth, thereby enhancing soybean's tolerance to salt stress. It can also enhance rapeseed seed germination vitality, specifically by promoting the growth of the radicle and shoots under salt stress.

[0021] As a new microbial resource, *Microbacterium algeriae* (Algeria microbes) Microbacterium algeriense M266-2 and its prepared microbial agents have broad application prospects in promoting seed germination, emergence and growth of dryland crops such as soybeans and rapeseed in saline-alkali land. This provides resource and technical support for the development of microbial agents or microbial fertilizers suitable for agricultural applications in saline-alkali land, and provides material and technical support for generating good ecological and economic benefits through saline-alkali agriculture.

[0022] Currently, regarding Algeria microbacterium ( Microbacterium algeriense Research on this strain has only progressed to the taxonomic identification stage, and its application in saline-alkali land agriculture has not yet been reported. Therefore, this invention not only provides an Algerian microbacterium strain with clear growth-promoting and salt-tolerant functions, but also opens up a completely new application direction for this strain in the field of saline-alkali land agriculture.

[0023] The Algerian microbacterium M266-2 in this invention has the Latin name […]. Microbacterium algeriense The category is named as follows: Microbacterium algeriense M266-2 was deposited on December 18, 2024, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China, with accession number CCTCC NO: M 20242839. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This image shows the colony morphology of Algeria microbacterium M266-2 on LB solid medium.

[0026] Figure 2 A phylogenetic tree constructed based on the 16S rRNA gene sequence of Algeria microbacterium M266-2.

[0027] Figure 3 The growth of Algerian microbacterium M266-2 on CAS detection plates.

[0028] Figure 4 The effect of Algeria microbacterium M266-2 on promoting soybean emergence under 1.5 w / v‰ NaCl stress is shown in the figure. Figure 4 Figure A shows a photograph of a duplicate sample from each treatment group on day 5 of the treatment. Figure 4 Figure B shows photographs of all samples in each treatment group on day 9 of treatment. The four samples in the left column are replicates of the original soil control group (CK). The four samples in the middle column are replicates of the untreated group (NaCl) under 1.5 w / v‰ NaCl stress. The four samples in the right column are replicates of the experimental group (NaCl+M266-2) treated with microbial agent containing strain M266-2 under 1.5 w / v‰ NaCl stress.

[0029] Figure 5 Statistical graphs of soybean emergence rates under the following treatments: original soil (CK), original soil with 1.5 w / v‰ NaCl (NaCl), and original soil with 1.5 w / v‰ NaCl and application of microbial inoculant containing strain M266-2 (NaCl+M266-2). Lowercase letters a and b indicate significant differences between groups.

[0030] Figure 6 The image shows the effect of Algeria microbacterium M266-2 on promoting soybean seedling growth under 1.5 w / v% NaCl stress. The four samples in the left column are replicates of the original soil control group (CK), the four samples in the middle column are replicates of the untreated group (NaCl) under 1.5 w / v‰ NaCl stress, and the four samples in the right column are replicates of the experimental group (NaCl+M266-2) treated with microbial agent containing strain M266-2 under 1.5 w / v‰ NaCl stress.

[0031] Figure 7 Statistical charts of soybean plant height under the following conditions: original soil (CK), original soil with 1.5 w / v‰ NaCl (NaCl), and original soil with 1.5 w / v‰ NaCl and application of microbial inoculant containing strain M266-2 (NaCl+M266-2); Figure 7 Figure A shows the statistical results on day 11 after processing. Figure 7 Figure B shows the statistical results on day 25 after treatment; lowercase letters a, b, and c indicate significant differences between groups.

[0032] Figure 8 Statistical graphs of soybean aboveground fresh weight, root fresh weight and top three leaf area under the following treatments: original soil (CK), original soil with 1.5 w / v‰ NaCl (NaCl), and original soil with 1.5 w / v‰ NaCl and application of microbial agent containing strain M266-2 (NaCl+M266-2). Figure 8 Figure A shows the statistics of fresh weight of soybean aboveground parts. Figure 8 Figure B is a statistical chart of the fresh weight of soybean roots. Figure 8 Figure C is a statistical chart of the area of ​​the third leaf from the bottom of soybean; lowercase letters a, b, and c indicate that there are significant differences between groups.

[0033] Figure 9 A plate image of Algerian microbacterium M266-2 on LB solid medium; Figure 9 Figure A shows the results of streak plate culture of the M266-2 monoclonal strain; Figure 9 Image B shows M266-2 cultured for 72 hours with 10 [units of something] coated on top. -4 Image showing bacterial growth on LB solid medium containing rhizosphere soil suspension.

[0034] Figure 10 The image shows the effect of M266-2 on promoting rapeseed seed germination under salt stress. The seeds on the three plates in the same column are all duplicate samples. Detailed Implementation

[0035] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0036] LB medium: Prepare LB liquid medium by adding 10g tryptone, 5g yeast extract, 10g sodium chloride (NaCl), and 1000mL distilled water, pH 7.0; add 15g agar powder to the LB liquid medium to obtain LB solid medium, and sterilize at 121℃ for 20min.

[0037] CAS solid medium: 60.5 mg of Chromium Azurite S (CAS) was dissolved in 50 mL of deionized water and mixed with 10 mL of 1 mM FeCl3 (prepared with 10 mmol / L HCl) to obtain component A. 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 sterilized LB medium. The mixture was then poured into CAS plates for later use.

[0038] CAS detection solution: Dissolve 60.5 mg of Chromium Azurite S (CAS) in 50 mL of deionized water, and mix with 10 mL of 1 mM FeCl3 (prepared with 10 mmol / L HCl) 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, sterilize at 115 °C for 30 min, and set aside for later use.

[0039] Salkowski colorimetric solution: Mix 1 part of 0.5 mol / L FeCl3 and 49 parts of 35 v / v% HClO4 and store away from light.

[0040] Soil salinization is one of the major challenges facing agricultural production. Salt stress can damage cell membrane function, accumulate toxic substances, inhibit photosynthesis, and weaken nutrient acquisition, ultimately leading to cell and even the death of the entire plant. Generally, when the soil salinity exceeds 0.1%, the growth of ordinary crops begins to be significantly affected; when the soil salinity exceeds 0.3%, the yield of most crops decreases significantly. Seed germination and seedling stages are critical periods for plant growth and are also the most sensitive to salt stress. High concentrations of base ions can severely inhibit seed germination, emergence, plant growth, and development, leading to problems such as difficulty in seed emergence, seedling death and stunted growth, weak growth, and gaps in rows. Rhizosphere microorganisms, known as the plant's second genome, play a crucial and irreplaceable role in assisting plants to resist adversity such as salt stress. Screening for functional strains with various life-promoting properties that can improve crop salt tolerance is of great significance for alleviating the inhibitory effects of salt stress on crop germination, emergence, and growth.

[0041] This invention provides an Algerian microbacterium strain that exhibits significant growth-promoting effects on plants under salt stress. Experiments demonstrate that this strain possesses dual growth-promoting properties, including high siderophore production and secretion of plant growth hormones, and can significantly alleviate the inhibitory effects of salt stress on crop germination, emergence, and growth. Specifically, it promotes soybean emergence after sowing, soybean seedling growth, rapeseed germination, and alleviates the inhibitory effects of salt stress on the growth of rapeseed radicles and shoots.

[0042] Example 1: Isolation, identification and preservation of Microbacterium algeriae M266-2

[0043] Rhizosphere soil from *Suaeda salsa* was collected in June 2022 from a severely saline-alkali land in Binhai, Jiangsu Province, with three replicate samples collected. Soil suspensions of 10⁻⁴, 10⁻⁵, and 10⁻⁶ were obtained using a serial dilution method. 100 µL of each dilution was spread onto LB agar plates containing 2 w / v‰ NaCl and incubated at 28°C for 3 days. Colonies with different morphologies and colors were picked, purified, numbered, and cultured.

[0044] The purified strain was subjected to tests to determine its ability to produce the plant growth hormone indoleacetic acid (IAA) and its siderophore production. Finally, a bacterium that could secrete both IAA and siderophores was screened out.

[0045] like Figure 1 As shown, after culturing this strain on LB solid medium at 28°C for 72 hours, the colonies on LB plates were milky white to pale yellow, deepening in color with prolonged incubation. The colonies were smooth, moist, and glossy, round with neat edges, no wrinkles, easily picked up, and homogeneous in texture (non-mucous). This strain is Gram-positive, aerobic, and weakly anaerobic. It has no spore-forming ability.

[0046] The 16S rRNA gene of this strain was amplified by colony PCR using universal bacterial primers 27F and 1492R. The PCR product was sequenced by a sequencing company, and the 16S rRNA gene sequence is shown in SEQ ID NO.1.

[0047] SEQ ID NO.1:

[0048]

[0049] The nucleotide sequence of primer 27F is shown in SEQ ID NO.2, and the nucleotide sequence of primer 1492R is shown in SEQ ID NO.3;

[0050] SEQ ID NO.2: 5'-AGAGTTTGATCMTGGCTCAG-3';

[0051] SEQ ID NO. 3: 5'-TACGGYTACCTTGTTACGACTT-3'.

[0052] The 16S rRNA gene sequence of the strain was uploaded to EzBioCloud (https: / / www.ezbiocloud.net / ) for homology comparison. The results showed that the 16S rRNA gene sequence of this strain was similar to that of *Microbacterium algeriae* (…). Microbacterium algeriense The sequence similarity of G1(Type) was 99.57%. Further phylogenetic tree construction was performed (e.g.,...). Figure 2 As shown), this strain is also more closely related to *Microbacterium algeriae* in evolution, and the strain was identified as *Microbacterium algeriae*. Microbacterium algeriense In this invention, it is named M266-2.

[0053] Example 2: Determination of the growth-promoting properties of Microbacterium algeriae M266-2

[0054] Preparation of microbial inoculum: Strain M266-2 was inoculated into LB liquid medium and cultured at 28℃ and 180 rpm for 17 h with shaking. After centrifugation, the bacterial cells were resuspended in sterile water, and the OD600 value of the bacterial suspension was adjusted to 1 to obtain a microbial inoculum. The viable count of *Microbacterium algeriae* in this inoculum was 10-1. 9 CFU / mL.

[0055] IAA production capacity assay: 250 µL of microbial inoculum was added to 5 mL of LB liquid medium containing 500 mg / L L-tryptophan, and cultured at 28 °C with shaking at 180 rpm for 48 hours. After centrifugation, 100 µL of the supernatant was added to a 96-well plate containing 100 µL of LSalkowski colorimetric solution, with uninoculated medium as a blank control. After standing in the dark for 30 min, the absorbance was measured at 530 nm. The IAA content in the bacterial suspension was calculated using the IAA standard curve. The results are shown in Table 1. After 48 h of culture, strain M266-2 secreted as much as 45.3 μg / mL ± 10.9 μg / mL of IAA.

[0056] Qualitative detection of siderophores secreted by the strain: Apply 2 μL of microbial inoculum to the center of a CAS plate, repeating three times. Place the plates in a 28℃ incubator and incubate for 4 days. Observe the size of the yellow halo around the colonies; the stronger the siderophore secretion ability, the larger the halo. Results are as follows: Figure 3 As shown, M266-2 has a strong ability to secrete siderophores, and its yellow halo diameter D / colony diameter d value is 4.20.

[0057] Quantitative determination of siderophores secreted by the strain: 300 μL of microbial agent was added to 3 mL of LB medium and cultured at 28℃ and 180 rpm for 72 h. After centrifugation, the supernatant was mixed with an equal volume of CAS detection solution and left to stand for 1 h. The absorbance (As) at 680 nm of the sample was measured. The absorbance at 680 nm after mixing blank LB medium with CAS detection solution was measured as Ar.

[0058] References Manjanatha MG, Loynachan TE, Atherly AG, Tn5 mutagenesis ofChinese Rhizobiumfredii for siderophore overproduction. Soil Biology and Biochemistry, 1992, 24(2): 151-155. Classification of bacterial siderophore production capacity: The As / Ar value is between 1.0 and 0, with an interval of 0.2. Each decrease of 0.2 adds one +. The As / Ar value of strain M266-2 is 0.362, which is in the range of 0.2 to 0.4. The siderophore secretion capacity rating is ++++ (Table 1), which reaches the high production level. Therefore, M266-2 belongs to high siderophore production bacteria.

[0059] Table 1. IAA and siderophore secretion capacity of strain M266-2

[0060]

[0061] Example 3: Algerian microbacterium M266-2 can promote soybean emergence under salt stress.

[0062] Soil collection: Soil samples were collected from the topsoil layer (0cm-20cm) of farmland at Xinyang Agricultural Experiment Station, Yancheng City, Jiangsu Province. The soluble salt content of this soil was 0.5w / v‰.

[0063] Soybean seed surface disinfection: Soybean seeds were disinfected with 75% alcohol for 1 minute, rinsed once with sterile water, then disinfected with 2.5v / v% sodium hypochlorite for 2 minutes, rinsed three times with sterile water, and the water after the last elution was used for plate spreading. After 4 days of incubation, no sterile growth was observed on the plate, indicating that the disinfection was thorough.

[0064] Experimental treatment: Weigh 150g (dry soil) of soil into a culture cup. Three treatments were set up: treatment with 1.5w / v‰ NaCl (NaCl), treatment with 1.5w / v‰ NaCl and application of microbial inoculant (NaCl+M266-2), and control treatment without NaCl and without inoculation (CK).

[0065] In the NaCl+M266-2 group: microbial inoculants were applied to the soil to achieve an inoculum concentration of 10 M266-2 per gram of soil. 9 CFU. Surface-sterilized soybean seeds were immersed in the microbial inoculant prepared in Example 2 and stirred to ensure that the Algerian microbacterium M266-2 strain was fully attached to the seed surface.

[0066] Soybean sowing and cultivation: Soybean seeds of different treatments were sown at a rate of 5 seeds / cup in culture cups at a sowing depth of 1 cm. The soybeans were placed in a smart greenhouse for cultivation, with daytime temperatures controlled at 25℃~30℃ and nighttime temperatures controlled at 18℃~24℃. Water was added daily using a weighing method to maintain soil moisture content at 60% SWHC. After the soybeans developed single leaves, the germination rate was recorded. Thinning was then performed, retaining 2 seedlings of uniform growth per cup. Subsequently, the seedlings were sown at a rate of 10... 9 The inoculum concentration (CFU / g soil) was increased by applying the microbial agent via root drenching to enhance the colonization effect of the strain. Soybean emergence was observed daily during the cultivation period. Soybean emergence was assessed after 5 and 9 days of cultivation as follows: Figure 4 As shown, the soybean emergence rate is as follows: Figure 5 As shown.

[0067] from Figure 4 and Figure 5 The results show that NaCl treatment significantly inhibited soybean emergence, but the application of microbial inoculants completely relieved the inhibitory effect of NaCl stress on soybean emergence. Specifically, NaCl reduced the soybean emergence rate from 85% in the control treatment to 45%, while the emergence rate reached 90% after the application of microbial inoculants, which was comparable to the control treatment without NaCl stress.

[0068] Example 4: Salt stress promotes soybean seedling growth with strain M266-2

[0069] Soil collection, soybean seed surface disinfection, experimental treatment, soybean sowing, and cultivation conditions were the same as in Example 3. After 25 days of cultivation, the soybean growth was as follows: Figure 6 As shown, under NaCl stress, soybean growth was significantly better after applying microbial inoculants than in the non-inoculated treatment. The aboveground plant height of soybeans in each treatment group was statistically analyzed. Figure 7 The aboveground biomass, root biomass, and area of ​​the top three leaves of soybean were measured to obtain... Figure 8 .

[0070] NaCl treatment significantly reduced soybean plant height, aboveground fresh weight, root fresh weight, and area of ​​the top three leaves. P The decrease in <0.05 indicates that soybean growth was significantly inhibited under NaCl stress. Application of microbial inoculants can effectively alleviate the inhibitory effect of NaCl stress on soybean growth, and in some indicators, the treatment with microbial inoculants was even superior to the unstressed control. Specifically, after 11 days of cultivation, the plant height recovered to 9.07 cm after application of microbial inoculants under NaCl stress, which was comparable to the control level. Figure 7 (See Figure A). After 25 days of cultivation, the soybean plant height was 23.5 cm without NaCl stress; the soybean plant height decreased to 20.1 cm under NaCl treatment; after application of microbial inoculant, the soybean plant height was 28.6 cm, exceeding the NaCl-free control group by 22.0%. Figure 7 (Figure B). After 25 days of cultivation, under NaCl stress, the application of microbial inoculants restored the aboveground fresh weight and the area of ​​the top three leaves of the plant to levels comparable to the control treatment without NaCl stress. Figure 8 Figure A Figure 8 (Figure C) , which restored the root fresh weight to 61.1% of the salt-free control treatment ( Figure 8 (Figure B).

[0071] Therefore, microbial agents containing strain M266-2 can significantly alleviate the inhibitory effect of NaCl stress on soybean seedling growth. This is manifested in the fact that after 25 days of cultivation, the aboveground fresh weight and the area of ​​the top three leaves recovered to a level comparable to that of the salt-free stress treatment, and even the plant height exceeded that of the salt-free stress control treatment. It also significantly alleviated the inhibitory effect of NaCl stress on root growth.

[0072] Example 5: Stable colonization of Algeria microbacterium M266-2 in soybean rhizosphere under salt stress

[0073] Soil collection, soybean seed surface disinfection, experimental treatment, soybean sowing, and cultivation conditions were the same as in Example 3. After 25 days of soybean cultivation, soybean rhizosphere soil was collected. For the rhizosphere soil of the group treated with NaCl and microbial inoculant (NaCl+M266-2), 10 μL of original rhizosphere soil was prepared using a gradient dilution method. -4 10 -5 10 -6 Soil suspensions were prepared at multiples of 100 μL. For each dilution, 100 μL of the suspension was placed on an LB agar plate and spread evenly with a spreader. After incubation at 28°C for 3 days, the growth of strain M266-2 was observed.

[0074] Based on the colony morphology characteristics of M266-2 ( Figure 9 Figure A shows the bacterial growth on LB solid medium coated with soil suspension after 3 days of culture. Figure 9(See Figure B). Single colonies with the same morphological characteristics as strain M266-2 were collected from LB solid medium coated with soil suspension using toothpicks. These colonies were named NaCl+M266-2_1RS, NaCl+M266-2_2RS, NaCl+M266-2_3RS, and NaCl+M266-2_5RS, respectively. These single colonies were placed in a PCR reaction system, and the 16S rRNA gene of the colonies was amplified by PCR using universal bacterial primers 27F and 1492R. After verifying the integrity and fragment length of the PCR products using polyester gel electrophoresis, the PCR products were sent to a sequencing company for sequencing. The sequenced sequences were then uploaded to the EzBioCloud database (https: / / www.ezbiocloud.net / ) for homology comparison.

[0075] The results showed that the 16S rRNA gene sequence of the colonies on the plate was consistent with that of the type strain. Microbacterium algeriense The sequence similarity of G1 was 99.07%–99.36% (Table 2), and its taxonomic position was consistent with that of strain M266-2, confirming that the single colonies on the detection plate also belonged to this group. Microbacterium algeriense (Algerian microbacterium).

[0076] Based on the colony morphology characteristics of M266-2 and the 16S rRNA gene identification results of the colonies, single colonies with the same morphology as M266-2 were counted on LB solid medium coated with soil suspension.

[0077] The results showed that strain M266-2 could still colonize the soybean rhizosphere soil at soybean harvest time, with a colonization rate of 0.68 × 10⁻⁶ per gram of soil. 7 CFU ~1.70×10 7 CFU.

[0078] Table 2. Colonization of soybean M266-2 in rhizosphere soil after 25 days of salt stress.

[0079]

[0080] Example 6: Algerian Microbacterium M266-2 promotes rapeseed seed germination under salt stress

[0081] Petri dish preparation: Line two layers of sterile filter paper in a 9cm diameter petri dish, and prepare a total of 15 petri dishes.

[0082] The preparation method of the microbial inoculant is the same as in Example 2.

[0083] Disinfecting rapeseed seeds: Select seeds with intact seed coats and uniform size, immerse the seeds in 75v / v% ethanol for 30 seconds, and wash them once with sterile water; then immerse the seeds in 15w / v% H2O2 for 30 seconds, and wash them three times with sterile water.

[0084] Seed sowing: The salt concentration was set at 150 mM NaCl (treatment labeled 150NaCl); the inoculum was applied at three levels: 1 v / v%, 10 v / v%, and 50 v / v% of the 150 mM NaCl solution volume; sterile water was used as a control (treatment labeled H2O). Each treatment was repeated in triplicate. 10 mL of each treatment solution was added to a petri dish lined with two layers of filter paper, and 15 sterilized seeds were sown in each dish. The petri dishes were incubated at 25°C, and the radicle length and shoot length were measured after 5 days.

[0085] Table 3. Rapeseed radicle length and shoot length under different treatments

[0086]

[0087] Note: Lowercase letters a, b, and c in the table indicate significant differences between groups.

[0088] As shown in Table 3 and Figure 10 As shown, 150 mM NaCl significantly reduced the length of the radicle and shoot compared to the control treatment with sterile water, indicating that NaCl stress significantly inhibited the growth of the radicle and shoot. Inoculation with 10 v / v% microbial agent significantly ( P <0.05) alleviated the inhibitory effect of NaCl on the growth of radicles and shoots. Specifically, NaCl stress significantly reduced the length of radicles from 342.9 mm in the control treatment to 8.1 mm, a decrease of 97.6%. Inoculation with 10 v / v% microbial inoculant increased the radicle length to 21.2 mm, a 1.61-fold increase compared to the NaCl treatment. NaCl stress significantly reduced the length of shoots from 164.3 mm in the control treatment to 21.3 mm, a decrease of 87.0%. Inoculation with 10 v / v% microbial inoculant increased the shoot length to 44.9 mm, a 1.10-fold increase compared to the NaCl treatment. Although inoculation with 1 v / v% and 50 v / v% microbial inoculant also resulted in radicle and shoot lengths exceeding those of the NaCl treatment, these increases were not statistically significant. P >0.05). Therefore, inoculation with 10 v / v% microbial inoculant can significantly alleviate the inhibitory effect of NaCl on the growth of rapeseed embryonic roots and shoots.

[0089] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0090] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of Algerian microbacterium ( Microbacterium algeriense ), characterized in that, The Algerian microbacterium is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242839.

2. A microbial inoculant, characterized in that, The active ingredient of the microbial agent contains the Algerian microbacterium as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The viable count of *Microbacterium algeriae* in the microbial agent is ≥10. 9 CFU / mL.

4. The microbial agent according to claim 2, characterized in that, The microbial agent is a growth promoter or a germination promoter.

5. A method for preparing the microbial inoculant according to claim 2, characterized in that, The procedure includes the following steps: Inoculating the *Microbacterium algeriae* into LB liquid medium and culturing at 28°C–30°C for 16–18 hours, then collecting the bacterial suspension; centrifuging the bacterial suspension to collect the bacterial cells; adding sterile water to the bacterial cells and resuspending them to obtain a bacterial suspension; adjusting the viable count of *Microbacterium algeriae* in the bacterial suspension to ≥102. 9 The microbial agent is obtained by obtaining CFU / mL.

6. The application of *Microbacterium algerianum* as described in claim 1 or the microbial agent as described in claim 2 in improving plant salt tolerance, characterized in that... The improvement of plant salt tolerance refers to promoting soybean emergence, soybean seedling growth, or rapeseed seed germination under salt stress.

7. The application according to claim 6, characterized in that, Soybean seeds were soaked in the aforementioned microbial agent, and the agent was then applied to the soil, ensuring that the viable count of *Algeria ulmoides* per gram of soil was ≥10. 9 CFU promotes soybean emergence.

8. The application according to claim 6, characterized in that, The soybean plants were drenched with the aforementioned microbial agent to ensure that the viable count of *Algeria lysate* per gram of soil was ≥10. 9 CFU promotes the growth of soybean seedlings.

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