P.megatherium STPM1 and application thereof

Abstract

The invention provides a P.megatherium STPM1 strain and application thereof, the strain is separated from rhizosphere of a native salt-tolerant plant-suaeda salsa in saline-alkali soil of Hexi Corridor, and is named as P.megatherium STPM1, and the preservation number is CGMCC No. 36250. The invention also provides a method for preparing the P.megatherium STPM1 strain. The strain has the capacities of efficiently dissolving potassium, dissolving phosphorus, fixing nitrogen and secreting auxin, can promote the growth and development of barley and increase the crop yield, and meanwhile, by activating an osmotic regulation and anti-oxidation defense system, induces ABA secretion and regulates related gene expression (up-regulating HvNCED3 and HvABA2 and down-regulating HvHAB1 and HvHAB2), the salt tolerance of barley is improved. The strain STPM1 disclosed by the invention can be applied to a plurality of aspects of preparing saline-alkali soil microbial inoculants, preparing plant growth promoters, improving saline-alkali soil and the like.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of Priestella giantiflora STPM1 and its applications. Background Technology

[0002] Soil salinization is a major abiotic stress factor restricting crop growth and yield, posing a serious threat to sustainable agricultural development and food security. my country has vast areas of saline-alkali land, with the Hexi Corridor in the northwest being a major barley-producing region and also a severely affected area by salinization. High-salt environments severely inhibit crop growth and yield formation through multiple mechanisms, including osmotic stress, ion toxicity, and oxidative stress.

[0003] Utilizing plant growth-promoting rhizobacteria (PGPR) to enhance crop salt tolerance is a green and sustainable strategy. PGPR are beneficial bacteria that live freely in the rhizosphere or specifically colonize the root surface or interior of plants, directly or indirectly promoting plant growth and stress resistance. Direct growth-promoting mechanisms include biological nitrogen fixation, phosphorus solubilization, potassium solubilization, and the secretion of plant hormones such as indole-3-acetic acid (IAA) and abscisic acid (ABA). Indirect mechanisms do not directly provide nutrients or hormones but rely on antagonizing pathogens and alleviating salt stress to create a more favorable growth microenvironment for plants. Studies have shown that various PGPR strains significantly improve the salt tolerance of host crops by regulating plant ion balance, osmotic substance accumulation, antioxidant enzyme activity, and stress-response gene expression.

[0004] However, current PGPR technology faces the following prominent bottlenecks in saline-alkali land agriculture, especially in its application to barley: ① Scarcity of high-efficiency strains: Existing PGPR strain libraries generally have limited salt tolerance (usually <10% NaCl) and single functions, lacking composite strains with both ultra-high salt tolerance and multiple growth-promoting functions; ② Poor ecological adaptability and unstable application: Strains originating from ordinary habitats have poor ecological adaptability, leading to difficulties in colonization, poor competitiveness, and unstable growth-promoting effects in harsh saline-alkali lands; ③ Insufficient crop specificity: Research and products on specific PGPR agents for barley, an important saline-alkali land crop, are almost non-existent; ④ Unclear systemic mechanism of action: Existing research is mostly limited to phenotypic observation, with insufficient understanding of how PGPR systematically regulates crop physiology, biochemistry, and molecular networks, which restricts its targeted development.

[0005] Therefore, developing PGPR that can adapt to saline-alkali environments and improve the salt tolerance of barley has important research and application value. Summary of the Invention

[0006] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a strain of *Priestella giantiflora* STPM1 and its applications. This invention isolates a salt-tolerant strain of *Priestella giantiflora* STPM1 from the rhizosphere of the halophilic crop *Suaeda salsa*. It systematically conducts morphological and molecular identification, salt tolerance, and growth-promoting characteristics analysis, and comprehensively evaluates its effects on barley seed germination, seedling growth, physiological and biochemical characteristics, and salt stress response gene expression under salt stress. This provides a theoretical basis and strain resources for the development and field application of microbial inoculants for saline-alkali land.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a strain of Priestia megaterium, which was isolated from the rhizosphere of Suaeda salsa, a native salt-tolerant plant in the saline-alkali land of the Hexi Corridor, and named Priestia megaterium STPM1, with the accession number CGMCC No. 36250.

[0008] The present invention also provides an application of the STPM1 described above in improving the salt tolerance of barley, wherein it is at least one of (1)-(4): (1) Promotes barley germination under salt stress; (2) Promotes the growth of barley seedlings under salt stress; (3) Improve the osmotic regulation capacity of barley under salt stress; (4) Improve the antioxidant capacity of barley under salt stress.

[0009] The present invention also provides an application of the STPM1 described above in the preparation of microbial inoculants for saline-alkali land.

[0010] The present invention also provides an application of the STPM1 described above in the preparation of plant growth promoters.

[0011] The present invention also provides an application of the STPM1 described above in improving saline-alkali soil.

[0012] The present invention also provides a method for improving the salt tolerance of plants by inoculating the STPM1 strain into plant seeds, seedling roots or the soil in which they grow.

[0013] Preferably, the plant is barley.

[0014] Preferably, the inoculation method is seed soaking or root irrigation.

[0015] This invention has significant technical advantages compared to existing technologies: 1. This invention provides a strain of *Pleurotus ostreatus* STPM1, isolated from the rhizosphere of *Suaeda salsa*, a native salt-tolerant plant found in the saline-alkali soils of the Hexi Corridor. This specific ecological origin determines that STPM1 is a naturally domesticated, highly adapted "native" functional bacterium to high-salt-alkali environments. Compared to strains from ordinary habitats, it is expected to exhibit stronger colonization ability, ecological competitiveness, and survival stability in real saline-alkali soils. This is key to overcoming the industry pain point of unstable field effects of existing PGPR inoculants. Secondly, strain STPM1 is a Gram-positive spore-forming bacillus. Its spore structure endows it with extremely strong resistance to harsh conditions such as drought, high temperature, ultraviolet radiation, and nutrient deficiency. This biological characteristic makes STPM1 easily developed into a standardized solid inoculant with good storage, convenient transportation, and long shelf life, providing unparalleled natural advantages for its commercial production and large-scale promotion.

[0016] 2. The *Priestella giantiflora* STPM1 strain of the present invention has the ability to efficiently solubilize potassium, phosphorus, fix nitrogen, and secrete auxin. On the one hand, STPM1 promotes the growth and development of barley and increases crop yield. On the other hand, it enhances the salt tolerance of barley through various pathways. The application of the strain STPM1 of the present invention produced significant multidimensional and synergistic beneficial effects on barley under salt stress: (1) Agronomic traits: significantly improved seed germination rate, promoted seedling vegetative growth and biomass accumulation, and enhanced photosynthetic efficiency; (2) Physiological defense: systematically activated osmotic regulation (proline) and antioxidant defense system (SOD, CAT, POD), effectively alleviating oxidative damage; (3) Molecular regulation: by activating the ABA core hormone signal and reprogramming the related gene expression network (upregulating HvNCED3 and HvABA2, downregulating HvHAB1 and HvHAB2), the stress response ability of barley was optimized from the root, and a lasting and systematic improvement in salt tolerance was achieved.

[0017] 3. This invention systematically elucidates for the first time the complete action chain of STPM1 through "microbial signaling → plant hormones → gene network → physiological response → phenotypic improvement," particularly the mechanism by which it specifically induces ABA secretion and reprograms the plant's endogenous ABA signaling network under salt stress. This unpredictability lays a solid foundation for precise application. STPM1 has been developed into a dedicated microbial inoculant technology for improving barley salt tolerance, and its comprehensive growth-promoting effects have been rigorously verified through experiments, demonstrating its potential as a functional microbial fertilizer and contributing to the management of saline-alkali land and the promotion of green and sustainable agricultural development.

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1This is an observation of the colony morphology and staining of strain STPM1 in Example 1 of the present invention; A and D are the results of colony morphology, Gram staining, capsule staining and spore staining, respectively.

[0020] Figure 2 This is the 16S rRNA phylogenetic tree of strain STPM1 in Example 1 of this invention; the numbers of the branch nodes are bootstrap values ​​based on 1000 replicates; the numbers in parentheses are the GenBank accession numbers of the reference strain; the scale bar corresponds to 0.02 nucleotide substitution sites.

[0021] Figure 3 This is an analysis of the tolerance of strain STPM1 to NaCl and pH in Example 2 of the present invention; A, C: growth curves of STPM1 under different NaCl concentrations (A) and initial pH (C); B, D: specific growth rates under the corresponding treatments; error bars represent standard deviation (n=3), and different lowercase letters indicate that the differences between treatments reached a significant level (P < 0.05).

[0022] Figure 4 This is the detection of the growth-promoting characteristics of strain STPM1 in Example 2 of the present invention; A: detection of nitrogen fixation (left), phosphorus solubilization (middle) and potassium solubilization (right) activities; B, C: STPM1’s ability to synthesize IAA (B) and ABA (C); error bars represent standard deviation (n=3), and different lowercase letters represent differences between different treatments reaching a significant level (P < 0.05).

[0023] Figure 5 This is the effect of STPM1 inoculation on barley seed germination under salt stress in Example 3 of the present invention; A, B: Germination phenotype (A) and germination rate changes (B) of seeds under different concentrations of NaCl treatment; C, D: Germination phenotype (C) and germination dynamic curve (D) of seeds without inoculation and inoculated with STPM1 under 200 mM NaCl stress; Control-Un: Normal culture, without M1 inoculation; Control-M1: Normal culture with M1 inoculation; NaCl-Un: Salt stress, without M1 inoculation; NaCl-M1: Salt stress with M1 inoculation; Error bars represent standard deviation (n=3), and different lowercase letters represent differences between different treatments reaching a significant level (P < 0.05).

[0024] Figure 6This invention relates to the effect of STPM1 inoculation on barley seedling growth under salt stress in Example 4. A: Growth phenotypes of barley seedlings under different NaCl concentrations; B: Comparison of phenotypes of seedlings (top) and roots (bottom) under 300 mM NaCl stress, with and without STPM1 inoculation; CH: Plant height (C), root length (D), fresh weight (E), leaf width (F), tiller number (G), and chlorophyll content (H) of seedlings in each treatment group; Control-Un: Normal culture, without M1 inoculation; Control-M1: Normal culture with M1 inoculation; NaCl-Un: Salt stress, without M1 inoculation; NaCl-M1: Salt stress with M1 inoculation; Error bars represent standard deviation (n=3), and different lowercase letters indicate significant differences between treatments (P < 0.05).

[0025] Figure 7 This invention relates to the effect of STPM1 inoculation on the physiological indicators of barley under salt stress in Example 4. AD: proline content and CAT, SOD, POD activities; error bars represent standard deviation (n=3), and different lowercase letters represent significant differences between different treatments (P < 0.05).

[0026] Figure 8 This is the effect of STPM1 inoculation on the antioxidant capacity of barley under salt stress in Example 4 of the present invention; A: DAB staining for H2O2 accumulation in leaves; B: relative intensity of DAB staining; C: MDA content; D: H2O2 content; error bars represent standard deviation (n=3), and different lowercase letters represent differences between different treatments reaching a significant level (P < 0.05).

[0027] Figure 9 This refers to the effect of STPM1 inoculation on the ABA signaling pathway in barley in Example 4 of this invention; A: ABA content; BE: relative expression levels of HvHAB1 (B), HvHAB2 (C), HvNCED3 (D), and HvABA2 (E) genes; error bars represent standard deviation (n=3), and different lowercase letters represent significant differences between different treatments (P < 0.05). Detailed Implementation

[0028] The culture medium used in this invention has the following composition: LB medium (g / L): peptone 10.0, yeast extract 5.0, NaCl 10.0; add agar powder 15.0 when preparing solid medium.

[0029] Ashby nitrogen-free medium (g / L): mannitol 10.0, CaCO3 10.0, K2HPO4 0.2, MgSO4·7H2O 0.2, NaCl 0.2, Na2MoO4·H2O 0.005, FeSO4·7H2O 0.001, agar powder 15.0.

[0030] Monkina Inorganic Phosphorus Medium (g / L): Glucose 10.0, Ca3(PO4)2 5.0, (NH4)2SO4 0.5, MgSO4 0.3, K2SO4 0.3, NaCl 0.3, FeSO4 0.03, MnSO4 0.03, Agar Powder 15.0.

[0031] Alexandrov medium (potassium-solubilizing medium) (g / L): sucrose 5.0, Na2HPO4 2.0, MgSO4 0.5, CaCO3 0.1, FeCl3 0.005, soil minerals 1.0, agar powder 18.0.

[0032] Other reagents used were as follows: Gram staining (G1060), capsular staining (G1130), spore staining (G1133), chlorophyll (BC0990), malondialdehyde (BC0020), proline (BC0290), hydrogen peroxide (BC3590), plant tissue reactive oxygen species (G4815), peroxidase (BC0090), catalase (BC0200), and superoxide dismutase (BC0170) detection kits were all purchased from Beijing Solarbio Technology Co., Ltd. Reagents and kits for RNA extraction (9769S), reverse transcription (RR036A), high-fidelity PCR (PrimeSTAR® Max, TCH047), and quantitative real-time PCR (TB Green® PremixEx Taq™ II, RR820A) used in molecular biology experiments were purchased from Takara Engineering (Dalian) Co., Ltd. All routine chemical reagents were of analytical grade.

[0033] Example 1 This example describes the isolation and identification of strain STPM1.

[0034] 1. Strains Isolation (1) Sampling: Samples were taken from a saline-alkali land in the Hexi Corridor of Zhangye City, Gansu Province. The roots of the native salt-tolerant plant Suaeda salsa, which was growing well, were dug up, put into clean self-sealing bags, marked, and brought back to the laboratory in an insulated box.

[0035] (2) Isolation: Place the plant roots into a sterilized conical flask, add an appropriate amount of sterile water, and seal with a sealing film. Place the conical flask in a shaker at 28 ℃ and shake at 200 r / min for 8-12 h. Take the culture medium, dilute it 1000 times, and spread it on the surface of LB solid medium containing 10% NaCl. Incubate at 28 ℃ for 3-5 days. Transfer the grown single colonies to LB liquid medium containing 10% NaCl and incubate at 28 ℃ and 200 r / min for 3-5 days.

[0036] (3) Purification: Pick a single colony from the streaked isolate and inoculate it into LB medium containing 10% NaCl. After purification three times, the target strain is obtained.

[0037] 2. Strain identification (1) Morphological identification Figure 1 The images show the colony morphology and Gram staining of the strain. A: Colony morphology; B: Gram staining; C: Capsule staining; D: Spore staining. The colony forms with a moist, smooth surface, neat edges, and is opaque, appearing as milky-white raised bumps. After Gram staining, the images under an optical microscope show that the strain is Gram-positive, has a capsule structure, and can produce spores.

[0038] (2) 16S rRNA gene sequencing and phylogenetic analysis The purified bacterial strain was subjected to a series of physiological and biochemical experiments, and the 16S rDNA gene of the strain was amplified and sequenced. The primer sequences are as follows: and The PCR product was sent to a company for sequencing (Shanghai Sangon Biotech). The sequencing results were compared with the NCBI database, and a phylogenetic tree was constructed using MEGA 11.0 software. The results showed a 98% sequence similarity to the standard strain of *Priestia megaterium*, CACC119 (accession number: CP140268.1). A phylogenetic tree constructed based on the 16S rRNA gene sequence showed that this strain clustered with *Priestia megaterium* CACC119 in the same high-support evolutionary branch. Figure 2 ).

[0039] Based on comprehensive morphological characteristics and 16S rRNA gene sequence analysis, the strain was identified as *Priestia megaterium*, named STPM1 (Salt-tolerant *Priestia megaterium* 1), and has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36250.

[0040] Example 2 This embodiment is a functional verification of strain STPM1.

[0041] First, a suspension of *Priscilla megaterium* STPM1 was prepared by inoculating a single activated STPM1 colony onto LB liquid medium. Oscillating culture to mid-log (OD) 600 ≈ 1.0); then, centrifuge at 5000 r / min for 5 min to collect the bacterial cells, discarding the supernatant; add sterile water to resuspend the bacterial cells, centrifuge again, repeat this "resuspend-centrifuge" step 3-5 times; finally, resuspend the washed bacterial cells in sterile water and adjust the final concentration of the bacterial suspension to approximately 1.0. .

[0042] 1. Salt tolerance test of strain STPM1 The activated STPM1 strain was prepared into a concentration of The bacterial suspension was inoculated into LB liquid medium (pH 6.8) with different NaCl concentrations (1%, 3%, 6%, 9%, 12%, 13%). The strain was cultured with shaking for 48 h. During the culture period, samples were taken at regular intervals to determine the colony count (CFU / mL) and calculate the specific growth rate to assess the salt tolerance of the strain.

[0043] Growth characteristics of strain STPM1 under different NaCl concentrations, such as Figure 3 As shown in A and B, the strain grew well under 1%–6% NaCl conditions, with the cell concentration continuously increasing with culture time. The optimal growth was observed at 1% NaCl, reaching a maximum concentration of [missing value]. When the NaCl concentration was increased to 9% and 12%, the bacterial growth gradually decreased after 36 h. Under 13% NaCl conditions, the growth of the strain was completely inhibited. (Specific growth rate analysis results...) Figure 3 B) Further analysis showed that NaCl concentrations below 6% had no significant effect on cell growth, but above this concentration, the specific growth rate decreased significantly. These results indicate that STPM1 has strong salt tolerance, with a maximum tolerance concentration of 12%.

[0044] 2. Tests on the acid and alkali resistance of strain STPM1 The activated STPM1 strain was prepared into a concentration of The bacterial suspension was inoculated into LB liquid medium with different initial pH values ​​(4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0). The strain was cultured with shaking for 48 hours. During the culture period, samples were taken regularly to determine the colony count (CFU / mL) and the specific growth rate was calculated to assess the alkali tolerance of the strain.

[0045] The growth of strain STPM1 under different initial pH conditions is as follows: Figure 3 As shown in C and D, this strain cannot grow at pH 4.0 and 10.0, and its growth rate is lower than that of other strains. It can grow well within a pH range of 5.0-9.0, and the bacterial concentration tends to stabilize after 30 h of culture. The highest specific growth rate is observed at pH 8.0 and 9.0, both exceeding [a certain value]. This indicates that the strain is well-suited for growth in a slightly alkaline environment (pH 7.0-9.0).

[0046] 3. Detection of growth-promoting characteristics of strain STPM1 (1) Detection of nitrogen fixation, phosphorus solubility and potassium solubility Freshly activated bacterial strains were inoculated onto Ashby's nitrogen-free medium, Monkina's inorganic phosphorus medium, and Alexandrov's medium (potassium-solubilizing medium), respectively, and cultured at 28 ℃ for 5 days. The nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing abilities of the strains were qualitatively assessed by observing colony growth on nitrogen-free medium, the formation of phosphate-solubilizing zones on inorganic phosphorus medium, and growth on potassium-solubilizing medium. Results are as follows: Figure 4 As shown in Figure A, this strain can grow normally and form relatively clear colonies on both nitrogen-free and potassium-solubilizing media, indicating that it has the potential for nitrogen fixation and potassium solubilization; however, although it can grow in inorganic phosphorus medium, it does not form obvious phosphorus-solubilizing zones, indicating that its phosphorus-solubilizing ability is relatively weak.

[0047] (2) Detection of auxin and abscisic acid secretion capacity The activated STPM1 strain was prepared into a concentration of The bacterial suspension was transferred to LB liquid medium and LB medium containing 1%, 3%, 6%, and 9% NaCl, respectively, and cultured with shaking at 28 ℃ and 200 r / min. After 24 h of culture, samples were collected, centrifuged at 5000 r / min for 5 min, and the supernatant was used to determine the contents of the auxin indole-3-acetic acid (IAA) and abscisic acid (ABA). The IAA content was determined by the Salkowski colorimetric method. The supernatant was mixed with an equal volume of Salkowski reagent, reacted in the dark for 30 min, and the absorbance was measured at 530 nm. The IAA concentration was calculated according to the standard curve. The ABA content was detected using a kit (ml103715) from Shanghai ELISA Biotechnology Co., Ltd. Analysis of plant hormone secretion capacity showed ( Figure 4 After culturing in LB medium for 24 h, STPM1 began to secrete IAA and ABA, and both hormones maintained high yields at different salt concentrations, with IAA reaching a maximum yield of 25.82 mg / L. ABA secretion was specifically promoted with increasing salt concentration, and its production was only inhibited at 9% NaCl.

[0048] The results of this example show that strain STPM1 has good salt tolerance and growth-promoting ability.

[0049] Example 3 This example demonstrates the promoting effect of inoculating strain STPM1 on the germination of barley seeds under salt stress.

[0050] (1) Determine the salt stress concentration for barley seed germination. Using the barley variety "Ganpi 8" as the test material, plump and uniformly sized seeds were selected. The seeds were surface-sterilized with 10% NaClO solution for 8 min and rinsed 3-5 times with sterile water. After surface sterilization, the seeds were placed in petri dishes lined with double-layered filter paper, and 0, 100, 150, 200, and 250 mM NaCl aqueous solutions were added until the filter paper was completely moistened. Each treatment was repeated in triplicate, with 100 seeds per replicate. After 3 days of constant temperature and dark culture, the germination status of the seeds was observed. The germination rate was calculated based on the standard that the radicle breaks through the seed coat by ≥2 mm. The salt concentration that has a significant inhibitory effect on seed germination but does not cause a lethal effect was screened out.

[0051] The results showed that the seed germination rate gradually decreased with increasing NaCl concentration. Figure 5 (A, B). Among them, the germination rate of 200 mM NaCl treatment decreased to below 50% after 3 days, so this concentration was selected as the inoculation salt stress condition for seed germination.

[0052] (2) Effects of inoculation with strain STPM1 on barley seed germination under salt stress Based on the selected salt concentration, stress conditions were set, and germination experiments were conducted using the following four treatments: ① Control-Un (normal culture, uninoculated M1 (STPM1)); ② Control-M1 (normal culture, inoculated M1); ③ NaCl-Un (salt stress, uninoculated M1); ④ NaCl-M1 (salt stress, inoculated M1). After surface sterilization, seeds were placed in petri dishes lined with double-layered filter paper. For the salt stress treatment group, 200 mM NaCl aqueous solution was added until the filter paper was completely wetted. For the normal culture group, sterile water was used for wetting. For the inoculation group, the prepared inoculum was resuspended in sterile water or 200 mM NaCl solution, and the final concentration of the inoculum suspension was adjusted to... Each treatment was replicated in triplicate, with 100 seeds per replicate. The seeds were cultured at 23°C in the dark for 3 days to investigate the effect of strain STPM1 on seed germination under salt stress.

[0053] Figure 5C and D represent the seed germination phenotypes and germination curves under salt stress after inoculation with STPM1, respectively; Control-Un: normal culture, without STPM1 inoculation; Control-M1: normal culture with M1 inoculation; NaCl-Un: salt stress, without M1 inoculation; NaCl-M1: salt stress with M1 inoculation. The results showed that under normal culture conditions, STPM1 inoculation (CK-M1) significantly accelerated seed germination, achieving a germination rate of 63.0% after 24 h, significantly higher than the 40.0% of the uninoculated control group (CK-Un) (P < 0.05). Under 200 mM NaCl stress, seed germination was significantly inhibited, but the germination rate of the inoculated group (NaCl-M1) was consistently significantly higher than that of the uninoculated salt stress group (NaCl-Un), ultimately reaching 61.0%. These results indicate that STPM1 not only effectively promotes barley seed germination under normal conditions but also significantly alleviates the inhibitory effect of salt stress on seed germination.

[0054] Example 4 This example illustrates the promoting effect and mechanism of inoculated strain STPM1 on the growth of barley seedlings under salt stress.

[0055] (1) Determine the salt stress concentration for barley seedling growth. A pot experiment was conducted to evaluate the effects of different salt concentrations on the growth of barley seedlings. Nutrient soil and vermiculite were mixed at a volume ratio of 3:1 and sterilized at high temperature, then dispensed into 10 cm diameter pots. After normal seed germination, seedlings of uniform growth (4 seedlings per pot) were transplanted and placed in a pot with a photoperiod of 16 h / 8 h and a day / night temperature range of [missing information]. Seedlings were cultured under a relative humidity of 65±5%. Salt stress treatment was initiated 5 days after seedling emergence, with irrigation with 150, 200, 250, 300, and 350 mM NaCl solutions every 3 days, using an equal volume of sterile water as a control. After approximately two weeks of continuous treatment, seedling phenotypes were recorded, and the salt concentrations that significantly inhibited seedling growth without causing lethality were ultimately selected.

[0056] Figure 6 A represents the growth phenotypes of barley seedlings under different salt concentrations. As the NaCl concentration increased, seedling growth was gradually inhibited. The 300 mM NaCl treatment significantly inhibited seedling growth, while the 350 mM NaCl treatment essentially halted seedling growth. Therefore, 300 mM NaCl was selected as the stress concentration to investigate the STPM1 mitigation effect.

[0057] (2) Effects of inoculation with strain STPM1 on the growth phenotype of barley seedlings under salt stress Then, based on the determined salt stress concentration at the seedling stage, four treatments were set up, with each treatment repeated three times. After the barley seeds germinated, seedlings with uniform sprout length were selected and transplanted into pots, with four seedlings planted in each pot. The seedlings were cultured under conditions of a 16-h photoperiod / 8-h dark period, a temperature of 25 ℃, and a relative humidity of approximately 65%. After 10 days of seedling growth (three-leaf stage), the following treatment groups were set up: ① Control-Un (normal culture, without M1 inoculation); ② Control-M1 (normal culture, inoculated with M1); ③ NaCl-Un (salt stress, without M1 inoculation); ④ NaCl-M1 (salt stress, inoculated with M1). For the inoculation group, the prepared bacterial agent was resuspended in sterile water or a 300 mM NaCl solution, and the final concentration of the bacterial suspension was adjusted to... Water every 3 days. After 2-3 weeks of cultivation, measure plant height, root length, above-ground fresh weight, leaf width, number of tillers, and chlorophyll content.

[0058] Figure 6 BH represents seedling growth under salt stress after inoculation. B: seedling growth phenotype, C: plant height, D: root length, E: fresh weight, F: leaf width, G: number of tillers, H: chlorophyll content. The results showed that under normal conditions, inoculation with STPM1 (CK-M1) promoted seedling growth, with plant height, leaf width, fresh weight, and chlorophyll concentration increasing by 15.9%, 19.4%, 5.5%, and 61.1% respectively compared to the control group (CK-Un). Under salt stress conditions (NaCl-Un), seedling growth and root development were significantly inhibited, while inoculation with STPM1 (NaCl-M1) effectively alleviated salt damage symptoms. Compared with the NaCl-Un group, the NaCl-M1 group showed increases in plant height, root length, fresh weight, leaf width, tiller number, and chlorophyll content of seedlings by 46.1%, 34.2%, 297.4%, 64.9%, 167.7%, and 353.7%, respectively, with particularly significant increases in biomass accumulation and photosynthetic pigment content. These results indicate that STPM1 can significantly enhance the salt tolerance of barley seedlings by promoting plant growth and maintaining photosynthetic function.

[0059] (3) Effects of inoculation with strain STPM1 on physiological parameters of barley under salt stress Figure 7 This study investigated the effects of STPM1 inoculation on physiological parameters of barley under salt stress. A: proline content; B: catalase (CAT) activity; C: superoxide dismutase (SOD) activity; D: peroxidase (POD) activity. The results showed that STPM1 significantly promoted the accumulation of proline, an osmotic regulator, increasing it by 33.6% and 174.3% under normal and salt stress conditions, respectively, compared to the uninoculated group. Figure 7 A). Regarding antioxidant enzyme systems ( Figure 7In the control-M1 group, the activities of CAT, SOD, and POD increased by 54.4%, 78.5%, and 115.8% respectively compared with the control-Un group. Under salt stress, the activities of the three enzymes in the NaCl-M1 group increased by 54.8%, 42.1%, and 45.1% respectively compared with the NaCl-Un group. This indicates that inoculation with STPM1 can effectively promote the activity of the barley antioxidant enzyme system under normal and salt stress conditions.

[0060] Figure 8 This study investigated the effect of STPM1 inoculation on the antioxidant capacity of barley under salt stress. A: DAB staining of H2O2 accumulation in leaves; B: Relative intensity of DAB staining; C: Malondialdehyde (MDA) content; D: Hydrogen peroxide (H2O2) content. DAB histochemical ROS staining and H2O2 content analysis showed that leaves inoculated with SPM1 had lighter staining and significantly reduced H2O2 accumulation. Furthermore, analysis of MDA content, an indicator of membrane lipid peroxidation, further indicated that inoculation reduced MDA by 27.8% under normal conditions and by 17.7% under salt stress. These results demonstrate that STPM1 systematically improves the salt tolerance of barley seedlings by synergistically promoting proline accumulation, enhancing the activity of antioxidant enzyme systems, and mitigating membrane lipid peroxidation damage.

[0061] (4) Inoculation of strain STPM1 under salt stress activates the ABA signaling pathway in barley. ABA plays an important regulatory role in plant salt stress response. Based on the fact that STPM1 has the ability to secrete ABA and that its secretion is enhanced under salt stress, this invention systematically analyzed the effects of this strain on the endogenous ABA content and the expression of key genes in the ABA signaling pathway in barley plants.

[0062] Figure 9 The study investigated the effect of STPM1 inoculation on ABA signaling in barley. A: ABA content; B: relative expression level of HvHAB1 gene; C: relative expression level of HvHAB2 gene; D: relative expression level of HvNCED3 gene; E: relative expression level of HvABA2 gene. Results showed that under normal conditions, STPM1 inoculation had no significant effect on endogenous ABA levels in plants. However, under salt stress conditions, the ABA accumulation in the inoculated group was significantly higher than that in the uninoculated group. Figure 9 A). Gene expression analysis results ( Figure 9 The study further demonstrated that inoculation under salt stress significantly upregulated the expression levels of key ABA synthesis genes HvNCED3 and HvABA2, while downregulating the transcription levels of negative ABA signaling regulators HvHAB1 and HvHAB2. These results indicate that STPM1 activates the ABA-mediated salt stress response mechanism by promoting ABA biosynthesis and regulating the expression of key genes in the signaling pathway, thereby systematically enhancing the salt tolerance of barley.

[0063] In summary, the plant rhizosphere salt-tolerant growth-promoting bacterium STPM1 of the present invention can tolerate 12% NaCl, produce acidic substances to lower the pH of the culture medium, dissolve calcium phosphate, fix atmospheric nitrogen, dissolve potassium feldspar, and produce plant growth hormones. Therefore, the salt-tolerant growth-promoting bacterium STPM1 has the potential to act as a PGPR, promoting barley growth and mediating its salt tolerance.

[0064] The STPM1 of this invention achieves a comprehensive enhancement of salt tolerance through a complete signal transduction and response chain: "microbial ABA secretion → initiation of endogenous ABA signaling in plants → upregulation of synthetic genes (HvNCED3, HvABA2) → downregulation of negative regulators (HvHAB1, HvHAB2) → activation of physiological defenses (antioxidant / osmotic regulation) → phenotypic improvement (growth promotion)." This is distinct from ordinary growth-promoting bacteria with unclear mechanisms or single functions. Furthermore, HvHAB1 and HvHAB2 are key negative regulators (PP2C protein phosphatases) in the ABA signaling pathway, acting like "brakes" in the pathway. This invention reveals that STPM1 can significantly downregulate the expression of these two genes. Its molecular biological significance lies in relieving the inhibition of the ABA signaling pathway, thus allowing the plant's stress response to be in a "pre-activated" or "continuously activated" preparatory state even without continuous stress. This is crucial for achieving durable, systemic salt tolerance.

[0065] *Priestella* genus has a broad host spectrum, and the core mechanisms of action of STPM1—osmotic regulation (proline), antioxidant defense (SOD / CAT / POD), and ABA signaling pathway regulation—represent conserved salt tolerance strategies in the plant kingdom. Therefore, this strain shows promise for application in gramineous crops such as wheat, maize, and rice, as well as in economically important crops grown in saline-alkali soils such as cotton and sugar beets.

[0066] One of the innovative aspects of STPM1 lies in its ability to specifically enhance ABA secretion under salt stress. Since ABA is a core hormonal signaling agent for plants to cope with various adversities such as drought and low temperature, STPM1 has significant potential application value in improving the overall stress resistance of plants, including drought and cold resistance. This provides technological space for developing a series of biological agents based on the same core strain, which can be used to prepare microbial inoculants for saline-alkali land, improve saline-alkali soil, and prepare plant growth promoters.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A strain of Priestia megaterium, characterized in that, The said Priestia megaterium is isolated from the rhizosphere of Suaeda salsa, a native salt-tolerant plant in the Hexi Corridor saline-alkali land, and named as Priestia megaterium STPM1, with the preservation number of CGMCC No. 36250.

2. Use of STPM1 according to claim 1 for increasing salt tolerance in barley, characterized in that, At least one of (1)-(4): (1) promoting the germination of barley under salt stress; (2) promoting the growth of barley seedlings under salt stress; (3) improving the osmotic regulation ability of barley under salt stress; (4) improving the antioxidant capacity of barley under salt stress.

3. Use of the STPM1 of claim 1 in the preparation of a saline-alkali land microbial inoculant.

4. Use of the STPM1 of claim 1 in the preparation of a plant growth promoter.

5. Use of the STPM1 of claim 1 in the improvement of saline-alkali land soil.

6. A method for increasing salt tolerance in plants, characterized by, The said STPM1 strain is inoculated into plant seeds, seedling roots or soil where the plant grows.

7. The method of claim 6, wherein, The said plant is barley.

8. The method of claim 6, wherein, The said inoculation mode is seed soaking or root irrigation.

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