Saline-alkali tolerant plant growth promoting enterobacter and application thereof

Abstract

The invention relates to a salt and alkali tolerant plant growth promoting enterobacter and application thereof, and belongs to the technical field of microorganism application and agricultural biotechnology, the salt and alkali tolerant plant growth promoting enterobacter is Enterobacter cancerogenic enterobacter PGPR AT1, and the bacterial agent contains the Enterobacter cancerogenic enterobacter PGPR AT1. The growth-promoting enterobacter is applied to plant growth promotion under severe saline-alkali stress conditions or normal growth conditions. The salt-alkali tolerant plant growth promoting enterobacter can directly or indirectly synergistically promote microorganisms for crop growth, and can be prepared into an efficient and stable microbial agent. The microbial agent can achieve a synergistic effect from three aspects of soil, root systems and plant physiology, the physiological stress of crops in the saline-alkali soil is remarkably relieved, the yield, biomass and stress resistance of the crops are fundamentally improved, and long-acting and sustainable improvement on the saline-alkali soil is achieved.

Description

Technical Field

[0001] This invention belongs to the fields of microbial application and agricultural biotechnology, and specifically relates to a salt-tolerant plant growth-promoting Enterobacter and its application in soybeans. Background Technology

[0002] The constraints of saline-alkali stress on agricultural production are complex and severe: it triggers osmotic stress and ion toxicity through high salinity; high pH levels lead to reduced nutrient availability (such as phosphorus, iron, and zinc) and soil structure degradation. The combined effect of these factors significantly reduces crop emergence rates and inhibits root development, ultimately resulting in severe losses in yield and quality. Therefore, combining the potential of saline-alkali land with the challenges of food security is of paramount importance.

[0003] Microbial improvement technology, due to its environmental friendliness and potential long-term effects, has become an important direction in the research of saline-alkali land management and crop stress resistance enhancement. To address the limitations imposed on crop growth by saline-alkali stress, developing environmentally friendly, efficient, and sustainable management and improvement technologies is crucial. Plant growth-promoting rhizobacteria (PGPR) are a class of beneficial bacteria living in or endophytically within plant rhizospheres, demonstrating great potential in enhancing crop resistance to abiotic stresses (including saline-alkali stress). PGPR technology is widely recognized as a green and sustainable biotechnology, and as an environmentally friendly, green, and sustainable biotechnology, it is receiving increasing attention. PGPR not only promotes plant growth but also has biocontrol functions; it is a class of beneficial bacteria that live in the rhizosphere soil or attach to plant roots, promoting plant growth. Through regulating hormone levels, improving nutrient acquisition, enhancing antioxidant and osmotic protection, and forming biofilms and EPS to improve soil structure, PGPR can alleviate ion toxicity and osmotic stress under saline-alkali stress, thereby improving crop adaptability. The core of this technology is to use PGPR to enhance the crop's resistance to salt and alkali stress. Summary of the Invention

[0004] This invention provides a salt-tolerant plant growth-promoting Enterobacter and its application. The Enterobacter is a strain with extremely strong salt and alkali tolerance and significant plant growth-promoting effect. The invention also describes the application of this strain in the preparation of biological agents for dicotyledonous plants to resist severe salt and alkali stress or normal environment, and to achieve yield increase.

[0005] The salt-tolerant plant growth-promoting enterobacter of this invention is Enterobacter cancerogenus PGPRAT1, which is deposited at the China Center for Type Culture Collection (CCTCC) on August 18, 2025, with accession number CCTCC NO: M 20251848, and deposited at Wuhan University, China.

[0006] The salt-tolerant plant growth-promoting Enterobacter bacterium agent of this invention contains Enterobacter cancerogenus PGPR AT1.

[0007] The preparation method of the salt-tolerant plant growth-promoting Enterobacter inoculant of the present invention is as follows: Enterobacter cancerogenus PGPR AT1 described in this invention is inoculated into a bacterial culture medium and cultured to obtain OD. 600 A bacterial solution with a value of 0.5-1.0 is a growth-promoting enterobacterial agent for salt-tolerant plants.

[0008] This invention relates to the application of Enterobacteriaceae in promoting plant growth under severe saline-alkali stress or normal growth conditions.

[0009] Furthermore, the plant is a dicotyledonous plant; the dicotyledonous plant may be a legume or a cruciferous plant.

[0010] Furthermore, the legume is soybean, and the cruciferous plant may be Arabidopsis thaliana.

[0011] Furthermore, after the Enterobacter aerogenes is prepared into an inoculum, it is mixed with a carrier material and dried to prepare microbial fertilizer in granular or powder form.

[0012] Furthermore, the carrier material is peat moss, vermiculite, or humic acid.

[0013] The application of the salt-tolerant plant growth-promoting Enterobacter cancerogenus PGPR AT1, PGPR AT1 inoculum, or PGPR AT1 culture described in this invention in at least one of the following.

[0014] The above applications can specifically be any of the following:

[0015] (a1) Enhance the plant's ability to resist severe salt and alkali stress;

[0016] (a2) To prepare products that enhance the resistance of plants to severe salinity and alkalinity;

[0017] (a3) Promotes plant growth;

[0018] (a4) Prepare products that promote plant growth;

[0019] (a5) Phosphorus-dissolving capacity under severe saline-alkali stress;

[0020] (a6) Prepare phosphorus-soluble products under severe salt and alkali stress conditions;

[0021] (a7) The ability to fix nitrogen under severe saline-alkali stress;

[0022] (a8) Prepare nitrogen-fixing products under severe salt and alkali stress conditions.

[0023] (a9) Production of heparin under severe saline-alkali stress;

[0024] (a10) Prepare a product that produces ferrophiles under severe salt and alkali stress.

[0025] In the above applications, the promotion of plant growth is manifested in all or part of the following:

[0026] (b1) Promotes root elongation in plants under severe saline-alkali stress or normal growth conditions;

[0027] (b2) Promotes increased plant dry weight under severe saline-alkali stress or normal growth conditions;

[0028] (b3) Promotes the increase of plant fresh weight under severe saline-alkali stress or normal growth conditions;

[0029] (b4) Promote plant growth under severe saline-alkali stress or normal growth conditions;

[0030] (b5) Promotes increased leaf length in plants under severe saline-alkali stress or normal growth conditions;

[0031] (b6) Promotes increased leaf width in plants under severe saline-alkali stress or normal growth conditions;

[0032] (b7) Reduce leaf wilting in plants under severe saline-alkali stress or normal growth conditions;

[0033] (b8) Promotes an increase in chlorophyll content in plants under severe saline-alkali stress or normal growth conditions;

[0034] (b9) Promotes a decrease in proline content in plants under severe saline-alkali stress or normal growth conditions;

[0035] (b10) Promotes a reduction in malondialdehyde (MDA) content in plants under severe saline-alkali stress or normal growth conditions;

[0036] (b11) Promotes the increase of plant antioxidant enzyme activity under severe saline-alkali stress or normal growth conditions;

[0037] Furthermore, the present invention also provides a method for preparing the above-mentioned microbial agent, wherein the above-mentioned PGPR AT1 is used as the active ingredient to obtain the microbial agent.

[0038] Furthermore, the present invention utilizes the aforementioned Enterobacter cancerogenus PGPR AT1 to provide a bio-organic fertilizer, wherein the bio-organic fertilizer contains the aforementioned PGPR AT1 or a bacterial agent or the aforementioned culture.

[0039] Furthermore, the present invention provides a method for promoting plant growth, comprising the following steps: applying the above-mentioned PGPR AT1 or bacterial agent or the above-mentioned culture to the test plants under severe salt-alkali stress conditions or normal growth conditions.

[0040] The *Enterobacter cancerogenus* PGPR AT1 described in this invention, or the prepared bacterial agent or culture thereof, can be used to promote plant growth. Application can be done by drip addition.

[0041] The beneficial effects of this invention are:

[0042] The beneficial effects of this invention are that by utilizing the salt-tolerant plant growth-promoting enterobacteria described in this invention, the growth of crops in saline-alkali land can be significantly improved, their yield and biomass can be greatly increased, and their tolerance to adversity can be enhanced. At the same time, it has a long-term and sustainable effect on improving the soil environment.

[0043] Experiments have demonstrated that the salt-tolerant plant growth-promoting Enterobacter cancerogenus PGPR AT1 provided by this invention can improve the resistance of Arabidopsis thaliana and soybean to severe salt-alkali stress. Specifically, this is manifested in a significant increase in dry weight, fresh weight, and root length, an increase in aboveground part height (i.e., increased plant height), an increase in leaf length and width, and changes in physiological and biochemical properties. Biochemical experiments on Enterobacter cancerogenus PGPR AT1 as a growth-promoting bacterium have proven that Enterobacter cancerogenus PGPR AT1 has strong tolerance to severe salt-alkali stress and possesses the ability to solubilize phosphorus, fix nitrogen, and act as a siderophore. These indicators are important evidence that Enterobacter cancerogenus PGPR AT1 has the potential to be a growth-promoting bacterium tolerant to severe salt-alkali stress. The above experiments show that the extremely salt-tolerant plant growth-promoting Enterobacter cancerogenus PGPR AT1 has very important application value in the field of soil remediation in severely saline-alkali areas.

[0044] This invention relates to a salt-tolerant plant growth-promoting enterobacterium that can directly or indirectly synergistically promote crop growth, and it is prepared into a highly efficient and stable microbial agent. This agent can work synergistically at three levels: soil, root system, and plant physiology, significantly alleviating the physiological stress of crops in saline-alkali land, fundamentally improving their yield, biomass, and stress resistance, and achieving long-term, sustainable improvement of saline-alkali land. Attached Figure Description

[0045] Figure 1 Phylogenetic tree of the extremely salt-tolerant plant growth-promoting enterobacter cancerogenus PGPR AT1 constructed in Example 1;

[0046] Figure 2 Effects of PGPR AT1 addition on the growth status of Arabidopsis thaliana in severe saline-alkali conditions;

[0047] Figure 3 Effects of PGPR AT1 addition on salt-alkali tolerance germination ability of soybean seeds;

[0048] Figure 4 Effects of PGPR AT1 addition on the salt-alkali tolerance growth status of soybeans;

[0049] Figure 5 The effect of PGPR AT1 addition on soybean photosynthesis;

[0050] Figure 6 Effects of PGPR AT1 addition on physiological indicators of soybean seedlings;

[0051] Figure 7 PGPR AT1 salt and alkali resistance test;

[0052] Figure 8 Detection of phosphorus solubility of PGPR AT1;

[0053] Figure 9 Detection of nitrogen fixation capacity of PGPR AT1;

[0054] Figure 10 Detection of PGPR AT1 siderophore secretion capacity. Detailed Implementation

[0055] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0057] Example 1: Screening and identification of PGPR AT1, a salt-tolerant plant growth-promoting enterobacterium of the present invention.

[0058] The screening method for obtaining *Enterobacter cancerogenus*, which promotes the growth of extremely salt-tolerant plants, is carried out according to the following steps:

[0059] 1. Add 5 g of soybean rhizosphere soil sample (June 2023, Da'an City, Jilin Province) to 50 mL of sterile distilled water, stir for 20 min, let stand for 15 min, then take 1 mL of the supernatant and add it to a sterile test tube containing 9 mL of sterile water and mix thoroughly (the dilution at this point is recorded as 10). -1 Then, the dilution method described above is used to dilute the solution sequentially, resulting in a concentration of 10. -1 10 -2 10-3, 10 -4 10 -5 10 -6 10 -7 Different dilutions of bacterial suspension. Take 0.1 mL of each dilution and spread it evenly on LB solid medium. Incubate at 30°C for 2-3 days.

[0060] 2. After completing step 1, pick a single colony from the LB solid medium and purify it repeatedly at least 3 times to obtain a single strain. The LB liquid medium consists of 10 g tryptone, 5 g yeast extract, and 30 g sodium chloride, dissolved in distilled water, adjusted to pH 8.0, and brought to a final volume of 1 L. It is then sterilized at 121℃ for 15 min and cooled for later use.

[0061] LB solid medium: Add agar to LB liquid medium to a final concentration of 15 g / L, sterilize at 121°C for 15 min. Cool to approximately 55°C, pour into petri dishes, and cool until solidified before use.

[0062] 3. The morphological characteristics and molecular identification of the single strains obtained through screening are as follows:

[0063] (1) Morphological characteristics:

[0064] The single strains screened in this embodiment exhibit round, milky-white, opaque colonies with smooth edges and a raised center on solid LB medium. They are Gram-negative bacteria with long rod-shaped cells, 3-10 μm in length.

[0065] (2) Molecular identification:

[0066] 1) Genomic DNA extraction

[0067] Single bacterial strains were inoculated into liquid LB medium and incubated at 37°C for 48 hours to obtain a bacterial suspension. The bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was washed with sterile water and centrifuged again, discarding the supernatant to obtain purified bacterial cells. Genomic DNA was then extracted from the bacterial cells according to the instructions of the Promega Genomic DNA Purification Kit.

[0068] 2) 16S rDNA amplification primer sequence

[0069] Table 1. Primer sequences for 16S rDNA amplification

[0070]

[0071] Table 2 PCR reaction system

[0072]

[0073] The reaction conditions were as follows: pre-denaturation at 98℃ for 3 min; 98℃ for 15 sec, 62℃ for 30 sec, 72℃ for 30 sec, with a cycle number of 35; final extension at 72℃ for 10 min, and storage at 4℃.

[0074] 3) PCR product detection and purification

[0075] The PCR product was analyzed by 1.0% agarose gel electrophoresis in 3µL and the band characteristics were observed.

[0076] The PCR product purification was performed according to the standard operating procedure for magnetic bead purification in GB / T 40171-2021 "General Rules for Detection of DNA Extraction and Purification Kits Using Magnetic Beads".

[0077] The obtained sequencing sequences were compared with the nucleic acid data in GenBank using BLAST (National Center for Biotechnology Information [http: / / www.ncbi.nlm / nih.gov]). The results showed that strain PGPR AT1 had 98.55% homology with Enterobacter cancerogenus. A phylogenetic tree was constructed using MOLECULAREVOLUTIONARY GENETIC ANALYSIS software (MEGA 7.0), as follows... Figure 1 As shown, it can form a stable evolutionary branch with the Enterobacter cancerogenu sequence, therefore, it is named Enterobactercancerogenus PGPR AT1. Figure 1Phylogenetic tree of Enterobacter cancerogenus PGPR AT1.

[0078] The obtained single strain was identified as *Enterobacter cancerogenus*, a salt-tolerant plant growth promoter, and named *Enterobacter cancerogenus* PGPR AT1. It was obtained on August 11, 2023, at the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun City, Jilin Province. PGPR AT1 in this embodiment can grow on solid LB medium.

[0079] Example 2 Preparation of the Salt-Tolerant Plant Growth-Promoting Enterobacter Inoculum Agent of the Present Invention

[0080] The specific preparation method is as follows: The *Enterobacter cancerogenus* PGPR AT1 strain, preserved at -80℃ in Example 1, was awakened on LB solid medium. A single colony was picked and inoculated into a 500 mL Erlenmeyer flask containing 100 mL of LB liquid medium. The flask was incubated at 30℃ and 130 r / min for 24-36 h to obtain the OD. 600 Bacterial solution with a value of 1.0 (bacterial solution concentration of 2×10⁻⁶) 8 The resulting bacterial solution (CFU / mL) is the prepared PGPR AT1 bacterial agent.

[0081] Example 3: Application of the highly salt-tolerant plant growth-promoting enterobacterium PGPR AT1 in improving the salt-alkali stress resistance of Arabidopsis thaliana.

[0082] The petri dish used in this example has dimensions of 9 cm × 9 cm, and the specific steps are as follows:

[0083] I. Preparation of Culture Medium

[0084] Mixed alkaline solid culture medium: 1 Adjust the pH of the 2MS solid medium to 8.0, sterilize at 121℃ for 60 min, add NaHCO3:Na2CO3=5:1 to the medium cooled to about 55℃ to a final concentration of 4 mM, then pour the medium into petri dishes (20-25 mL per petri dish), and allow it to cool naturally to obtain a mixed salt-alkali solid medium.

[0085] Mixed salt and alkali + bacterial agent (PGPR AT1) solid culture medium: Take a petri dish containing mixed salt and alkali solid culture medium, stand it upright, and use a coating stick to coat a small amount of the PGPR AT1 bacterial agent prepared in Example 2 above on the surface of the culture medium in the lower quarter of the dish, and let it air dry naturally.

[0086] II. Effects of PGPR AT1 inoculum on Arabidopsis thaliana's resistance to severe salt and alkali stress

[0087] The cultivation conditions were: 22℃, 16 h light / 8 h dark, and light intensity of 8000-12000 Lux.

[0088] 1) Seed Surface Sterilization: Wild-type Arabidopsis thaliana Columbia-0 (Col-0) subtype seeds were used for the experiment. a. Soaked in 2.6% (v / v) sodium hypochlorite solution for 10 min; b. Then rinsed three times with sterile ultrapure water to remove residual sodium hypochlorite; c. Sterilized with 75% (v / v) anhydrous ethanol for 5 min to enhance the sterilization effect; d. Thoroughly rinsed four times with sterile ultrapure water to completely remove ethanol residue, ensuring seed viability and a sterile environment for subsequent culture.

[0089] 2) After completing step 1), Arabidopsis seeds were sown on solid culture media. The control group used a mixed salt-alkali stress solid culture medium (referred to as "mixed salt-alkali medium"). The treatment group used a mixed salt-alkali stress solid culture medium supplemented with a microbial agent (referred to as "mixed salt-alkali + microbial agent medium"), and vernalized at 4℃ for 2 days. Special attention should be paid to this: to investigate the effect of microbial agent secretions on Arabidopsis growth, the seeds in the treatment group were strictly limited to the upper quarter or more of the solid culture medium to avoid direct contact between the seeds or seedling root tips and the PGPR AT1 microbial agent pre-coated at the bottom of the medium during the early stages of growth. The plants were then cultured vertically, and observations and statistics were performed on the Arabidopsis root tips before they came into contact with the microbial agent (this experiment mainly studies the effect of microbial agent secretions on Arabidopsis growth).

[0090] 3) After vernalization, the culture dishes were adjusted to a vertical culture mode and placed under suitable light and temperature conditions (temperature 22℃, 16 h light / 8 h dark, light intensity 8000-12000 Lux). The culture will last for 7 days or longer, and growth phenotype observation and statistical analysis will be performed before the Arabidopsis root tips come into contact with the pre-coated inoculum area to ensure that the experimental results mainly reflect the potential growth-promoting effects of the inoculum's volatiles or soluble secretions.

[0091] Under normal growth conditions (Control pH=5.8) and mixed salt-alkali stress conditions (NaHCO3:Na2CO3=5:1 to a final concentration of 4 mM Na), + Simultaneously, the sample was cultured with PGPR AT1 at pH=8.0 for 7 days, and the growth was recorded by taking pictures and measuring the root length. Figure 2The effect of PGPR AT1 supplementation on the growth status of Arabidopsis thaliana under severe saline-alkali conditions was investigated. After 7 days of vertical culture, the growth phenotypes of Arabidopsis thaliana seedlings showed significant differences among different treatment groups: Taproot elongation: Under saline-alkali stress, compared with the control group (mixed saline-alkali medium), the treatment group coated with PGPR AT1 significantly alleviated the taproot growth capacity of Arabidopsis thaliana seedlings, with a significant increase in average taproot length. This indicates that the secretion of PGPR AT1 effectively alleviated the inhibitory effect of mixed saline-alkali stress on taproot growth. Figure 2 A).

[0092] To further evaluate whether the PGPR AT1 inoculant could improve the salt and alkali tolerance of Arabidopsis thaliana plants from seedling to maturity, 1-week-old Arabidopsis thaliana seedlings were treated with a mixed salt and alkali solution until they reached 4-week-old mature Arabidopsis thaliana (Control: pH=5.8; Alkali:NaHCO3:Na2CO3=5:1 to a final concentration of 150 mM Na). + Simultaneously at pH=8.0), where CK represents the treatment with and without PGPR AT1 inoculant, aboveground growth: Arabidopsis treated with mixed saline-alkali showed almost no growth, exhibiting yellowing, wilting, and even death. Seedlings treated with mixed saline-alkali with PGPR AT1 inoculant showed good aboveground growth advantage, specifically larger leaf area and deeper green leaf color, indicating improved photosynthetic capacity and nutritional status. Under mixed saline-alkali stress, PGPR AT1 inoculant, without direct contact with the root system, significantly enhanced the tolerance and growth vitality of Arabidopsis seedlings through its secretions or volatiles, demonstrating a significant plant growth-promoting effect and improving the plant's salt and alkali tolerance. Figure 2 B).

[0093] 4) Use ImageJ software to measure and count the taproot length of Arabidopsis seedlings from step 3 (n=80).

[0094] The statistical results are shown in Table 3 (Table 3: Statistics on the taproot length of Arabidopsis thaliana). Significance was evaluated using one-way ANOVA. *** (P < 0.001).

[0095] Table 3. Statistics on taproot length of Arabidopsis thaliana

[0096] Types of solid culture media Average taproot length (cm) 1 / 2MS solid culture medium 3.146 <![CDATA[ 1 / 2MS + bacterial agent solid culture medium]]> 3.352 Mixed salt-alkali solid culture medium 0.2313 Mixed salt-alkali + bacterial agent solid culture medium <![CDATA[2.252 *** ]]>

[0097] The results showed that under severe salt-alkali stress, the average taproot length of Arabidopsis seedlings on solid culture medium coated with PGPR AT1 was significantly higher than that on the control solid culture medium (without PGPR AT1) (***, P < 0.0001). This indicates that PGPR AT1 can significantly promote Arabidopsis growth and improve its salt-alkali tolerance.

[0098] Example 4: Application of salt-tolerant plant growth-promoting enterobacter PGPR AT1 in improving the salt-alkali stress resistance of soybean seed germination

[0099] Mixed saline solution (300 mM, Na2CO3: NaHCO3 = 1:5, pH 11.0): Dissolve 0.95391 g Na2CO3 and 6.80481 g NaHCO3 in 300 mL of distilled water for later use.

[0100] Soybean seed germination culture conditions: 25℃; 16 h light / 8 h dark; light intensity of 30-50 µmol / m² / s (approximately 2000-4000 Lux).

[0101] Soybean (Glycine max) Williams 82 seeds were used as experimental material. a. The seeds were first rinsed with tap water for 15 min; b. They were washed with 75% (v / v) ethanol for 1 min, and rinsed with ethanol 5 times consecutively; d. They were washed 5 times consecutively with sterile ultrapure water to completely remove residual ethanol, ensuring the sterility of the experimental environment and the viability of the seeds; e. Screening and sowing: Healthy and plump soybean seeds that had fully absorbed water after the water absorption treatment were selected, and 10 seeds were evenly placed in a sterilized culture medium (90 mm in diameter) lined with two layers of filter paper. Each treatment was repeated 10 times.

[0102] The germination experiment included four treatments: a water control group (Group 1), a water + bacterial agent group (Group 2), a mixed saline-alkali solution control group (Group 3), and a mixed saline-alkali solution + *Enterobacter cancerogenus* PGPR AT1 bacterial agent group (Group 3). Each group was incubated at 25℃ for 3 days. Germination was determined by the radicle breaking through the seed coat. Germination rates were calculated by photographing and recording the number of germinating seeds, comparing the differences between treatments, and thus evaluating the effect of PGPR AT1 on the germination performance of soybean seeds under severe saline-alkali stress.

[0103] Figure 3 The effect of adding PGPR AT1 on the salt-alkali tolerance germination ability of soybean seeds, from Figure 3 It can be seen that the mixed saline-alkali solution control group (group 3) significantly inhibited soybean seed germination, with a germination rate significantly lower than that of the water control (group 1); after adding PGPR AT1 inoculant to the mixed saline-alkali solution (group 4), seed germination significantly recovered, and the germination rate significantly increased, showing a mitigating effect on severe saline-alkali stress; in addition, the water + inoculant group (group 2) also showed a certain germination-promoting trend compared to the water control. Figure 3In summary, PGPR AT1 inoculant demonstrated excellent germination promotion and resistance to severe salt and alkali stress during the soybean seed germination stage.

[0104] Example 5: Application of the highly salt-tolerant plant growth-promoting enterobacterium PGPR AT1 in improving the resistance of soybean to severe salt and alkali stress

[0105] Mixed saline solution (300 mM, Na2CO3: NaHCO3 = 1:5, pH 11.0): Dissolve 9.5391 g Na2CO3 and 68.0481 g NaHCO3 in 3000 mL of distilled water for later use.

[0106] Soybean seedling culture conditions: 25℃; 16 h light / 8 h dark; light intensity of 30-50 µmol / m² / s (approximately 2000-4000 Lux).

[0107] 1) Using sterilized vermiculite:humus = 1:2 as the cultivation substrate, soybeans were planted in culture pots with a diameter of 15 cm, one seed per pot, and each group was repeated 5 times. The soybeans were cultivated in a greenhouse with 16 h light / 8 h darkness and a temperature of 25℃ until the first trifoliate compound leaf (with uniform growth) grew.

[0108] 2) Eighteen pots of soybean seedlings thoroughly irrigated with distilled water were randomly divided into two groups: a control group and a water + microbial agent group, with 18 pots in each group. Eighteen pots of soybean seedlings thoroughly irrigated with mixed alkaline solution were also randomly divided into two groups: a mixed alkaline solution control group and a mixed alkaline solution + microbial agent group, with 9 pots in each group. While the water + microbial agent group and the mixed alkaline solution + microbial agent group were treated with distilled water and mixed alkaline solution respectively for the first time, the prepared PGPR AT1 microbial agent (OD) was evenly dripped onto the roots of each soybean seedling. 600 =1), 2 mL per pot, and then cultured for 15 days; no bacterial agent was applied to the water control group and the saline-alkali solution control group, and they were cultured under the same conditions for 15 days.

[0109] 3) Observation of the growth and development phenotypes of soybean seedlings in each group.

[0110] The full seedling phenotype of soybean seedlings after 15 days of growth is shown in the figure. Figure 4 ( Figure 4The effect of PGPR AT1 addition on the salt-alkali tolerance of soybean was investigated. Group 1 was a water control group, group 2 was a water + inoculant group, group 3 was a mixed salt-alkali solution control group, and group 4 was a mixed salt-alkali solution + inoculant group. Under severe salt-alkali stress, soybean seedling growth was significantly inhibited, manifested as decreased plant height and impaired root development, specifically including significantly slowed taproot elongation, suppressed lateral root development, and decreased overall root extension capacity. In contrast, under the mixed salt-alkali + inoculant group treated with PGPR AT1, seedling growth was significantly improved: aboveground growth recovered, and plant height was higher than the control group treated only with alkali; root growth was significantly promoted, manifested as an increase in total root length, suggesting enhanced root exploration and absorption capacity in response to adversity. Figure 4 The above results indicate that PGPR AT1 can effectively alleviate the inhibitory effect of salt-alkali stress on soybean seedlings, promote root-shoot coordinated growth, and enhance seedling adaptability and growth vigor to unfavorable soil environments.

[0111] 4) Mixed salt-alkali stress (300 mM Na) + The effects of PGPR AT1 inoculant treatment for 15 days on the plant length and fresh weight of soybean seedlings under pH=11 conditions.

[0112] Statistical results showed that the average plant length and fresh weight of soybean seedlings treated with PGPR AT1 inoculant were significantly higher than those of the water control group without PGPR AT1 inoculant treatment. Under mixed saline-alkali treatment conditions, the average fresh weight of soybean seedlings, including whole plant fresh weight, aboveground fresh weight, underground fresh weight, and relative survival rate, decreased significantly compared with the water control group, while the alkali + inoculant group treated with PGPR AT1 inoculant showed a significant increase (Table 4: Statistical analysis of whole plant fresh weight, aboveground fresh weight, underground fresh weight, and relative survival rate of soybean seedlings). This indicates that the application of PGPR AT1 inoculant under normal and heavily saline-alkali treatment conditions can significantly promote the growth of soybean seedlings. Significance was evaluated by one-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0113] Table 4. Statistics on whole plant fresh weight, aboveground fresh weight, underground fresh weight, and relative survival rate of soybean seedlings.

[0114] Average fresh weight (g) Fresh weight of above-ground parts (g) Fresh weight of underground part (g) Relative survival rate Water control group 1 7.77 3.91 3.09 95% Water + bacterial agent group 2 8.34 4.11 3.86 95% Mixed saline solution control group 3 <![CDATA[2.42 *** ]]> <![CDATA[1.16 *** ]]> <![CDATA[1.24 *** ]]> <![CDATA[12% *** ]]> Mixed saline solution + bacterial agent group 4 5.62 3.31 2.69 86%

[0115] 5) Effects of PGPR AT1 inoculant treatment for 15 days on the photosynthetic capacity of soybean seedlings under severe saline-alkali stress.

[0116] Soil salinization is a serious abiotic stress in agricultural ecosystems, posing a significant threat to crop growth and yield. Especially under severe salinity stress, the physiological metabolic activities of plant seedlings are severely disrupted, with photosynthesis being one of the most significantly affected processes. Under severe salinity stress, soybean seedlings exhibit chlorosis, yellowing, edge necrosis, and even wilting of leaves. Figure 5 Adding the effect of PGPR AT1 on soybean photosynthesis, from Figure 5 It can be seen that the leaf damage of soybean seedlings treated with the mixed salt-alkali + inoculant group of PGPR AT1 inoculant was significantly alleviated. Figure 5 A). Chlorophyll is the "vanguard" in photosynthesis, responsible for absorbing, transferring, and converting light energy. There is a strong positive correlation between chlorophyll content and photosynthetic capacity; it is one of the most direct indicators for assessing plant health and photosynthetic efficiency. Under mixed saline-alkali treatment conditions, the total chlorophyll content of soybean seedlings in the mixed saline-alkali solution control group was significantly lower than that in the water control group, severely impacting the photosynthetic capacity of soybean seedlings and affecting soybean growth and development. The total chlorophyll content of soybean seedlings in the mixed saline-alkali solution + inoculant group treated with PGPR AT1 inoculant was significantly higher than that in the mixed saline-alkali solution control group without PGPR AT1 inoculant treatment, improving the photosynthetic capacity of soybeans and enhancing their ability to withstand saline-alkali stress. Figure 5 B). Maintaining high leaf integrity and chlorophyll content ultimately results in a significant enhancement of photosynthetic capacity, laying a solid physiological foundation for the growth and development of soybeans under saline-alkali stress.

[0117] 6) Effects of PGPR AT1 inoculant treatment for 15 days on some physiological indicators of soybean seedlings under severe salt-alkali stress.

[0118] Under abiotic stress (such as alkali stress), reactive oxygen species (ROS) rapidly accumulate in plants. Their strong oxidizing properties can damage cellular macromolecules, leading to membrane lipid peroxidation and the generation of malondialdehyde (MDA). MDA levels can indicate the rate and intensity of lipid peroxidation, indirectly reflecting the degree of oxidative damage to tissues. Proline (Pro) plays an important role in cellular osmotic regulation and redox homeostasis; changes in its content are often used to characterize stress intensity. The body removes excess ROS through antioxidant systems, including both enzymatic (such as superoxide dismutase SOD and catalase CAT) and non-enzymatic systems. The overall level of various large and small molecules and enzymes in both systems determines the overall antioxidant capacity.

[0119] The contents of MDA and Pro, the activities of CAT and SOD, and the levels of superoxide anion and hydrogen peroxide in soybean seedlings were determined separately (see...). Figure 6 , Figure 6The effects of PGPR AT1 addition on physiological indicators of soybean seedlings. Under mixed saline-alkali treatment, the Pro and MDA contents of soybean seedlings in the mixed saline-alkali control group were significantly higher than those in the water control group, indicating that saline-alkali stress significantly aggravated osmotic and oxidative damage. In contrast, after application of PGPR AT1 inoculant (mixed saline-alkali solution + inoculant group), the Pro and MDA contents of seedlings were significantly lower than those in the mixed saline-alkali control group, while the activities of CAT and SOD were significantly increased. Significant differences were determined by one-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0120] Example 6: Analysis of alkaline and neutral salt tolerance of the highly salt-tolerant plant growth-promoting Enterobacter PGPR AT1 strain

[0121] Add NaHCO3 to 100 mL of LB liquid medium 3: Add 1 mL of PGPR AT1 bacterial culture (OD) to Na2CO3 at a ratio of 5:1 to final concentrations of 0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, and 300 mM (pH 11.0). 600 =1) Incubate the cells, and measure the OD using a spectrophotometer every 12 hours. 600 value. Figure 7 To test the salt and alkali tolerance of PGPR AT1, the results showed that the growth of PGPR AT1 strain in a 150 mM alkaline salt mixture did not change significantly. While its proliferation decreased with increasing concentration, it still maintained a certain level of growth and reproduction, and could survive even at 300 mM. Figure 7 A) indicates that the PGPR AT1 strain has a high ability to resist alkaline salt stress.

[0122] Add NaCl to 100 mL of LB liquid medium to final concentrations of 0 M, 0.8 M, 1.2 M, 1.6 M, 2.0 M, 2.2 M, 2.6 M, and 3.0 M. Add 1 mL of PGPR AT1 bacterial suspension (OD200). 600 =1) Incubate the cells, and measure the OD using a spectrophotometer every 12 hours. 600 The results showed that the PGPR AT1 strain could still survive in a 2.2 M NaCl solution, and it could also survive at a high NaCl concentration of 3.0 M. This indicates that the PGPR AT1 strain has a high resistance to neutral salt stress.

[0123] Example 7 Analysis of the growth-promoting properties—phosphorus-solubilizing ability—of the growth-promoting enterobacter PGPR AT1 in extremely salt-tolerant plants

[0124] Phosphorus in soil exists in both organic and inorganic forms. In alkaline soils, inorganic phosphorus mainly exists as insoluble tricalcium phosphate, which is difficult for plants to absorb directly, thus constituting a significant factor limiting plant growth and productivity. Phosphorus-solubilizing microorganisms can lower the extracellular microenvironment pH and react with calcium by secreting organic acids (such as citric acid, oxalic acid, and gluconic acid produced from glucose metabolism) and related metabolites. 2+ The isocations form complexes or chelates, thereby significantly increasing the solubility of insoluble phosphates and converting them into inorganic phosphorus forms (mainly orthophosphate) that can be utilized by plants.

[0125] To evaluate the phosphate-solubilizing ability of strain PGPR AT1, plate screening and phenotypic verification were performed using PKO selective medium containing insoluble calcium phosphate. The PKO medium (per liter) formulation was: glucose 10.0 g, tricalcium phosphate 5.0 g, magnesium chloride 5.0 g, ammonium sulfate 0.1 g, potassium chloride 0.2 g, magnesium sulfate 0.25 g, agar 17.0 g, pH 7.0.

[0126] Figure 8 For the detection of phosphorus solubility of PGPR AT1, combined with Figure 8 The results showed that after 72 h of inoculation of PGPR AT1 onto PKO plates (Control), a clearly visible transparent dissolution zone appeared around the colonies, indicating that PGPR AT1 can effectively dissolve tricalcium phosphate in the culture medium and release soluble inorganic phosphorus. Figure 8 A). To further determine whether PGPR AT1 also possesses phosphate-solubilizing ability under severe saline-alkali stress, PGPR AT1 was inoculated onto PKO plates at pH=11 with a sodium ion concentration of 300 mM (mmol / L). The formation of a clear zone reflected the ability of PGPR AT1 to secrete acidic metabolites and promote tricalcium phosphate dissolution under severe saline-alkali stress. This clear zone formation reflects the strain's ability to secrete acidic metabolites and promote tricalcium phosphate dissolution in a solid substrate.

[0127] Based on this, the phosphorus solubility level of PGPR AT1 was quantitatively determined using the molybdenum antimony colorimetric method. A standard curve was established using orthophosphate standard solution, and the linear regression equation was y = 0.183x + 0.2651 (R² = 0.9907), indicating that the quantitative method has good linear correlation. According to the standard curve, the concentration of soluble inorganic phosphorus generated by PGPR AT1 under culture conditions was 140.34 mg / L, and the concentration of soluble inorganic phosphorus generated under severe salt-alkali stress was 102.84 mg / L. Figure 8 B). Statistical analysis was performed using one-way ANOVA to test for significance, and the results showed that the differences were extremely significant (***, P < 0.001).

[0128] Based on the combined results of plate phospholysis phenotype and quantitative colorimetric analysis, PGPR AT1 exhibited significant and stable phosphorus-solubilizing ability. It also showed a certain phosphorus-solubilizing effect under severe saline-alkali stress, effectively converting insoluble calcium phosphate into a phosphorus source that can be utilized by plants. It has the potential application prospect of being a growth-promoting rhizosphere bacterium (PGPR) to improve phosphorus availability in saline-alkali soils.

[0129] Example 8 Analysis of the growth-promoting characteristics—nitrogen fixation capacity—of the growth-promoting enterobacter PGPR AT1, a highly salt-tolerant plant growth-promoting bacterium.

[0130] Ashby medium (1 L): KH2PO4 0.2 g, NaCl 0.2 g, MgSO4·7H2O 0.2 g, K2SO4·2H2O 0.2 g, CaCO3 5 g, glucose 5 g, mannitol 5 g, agar 10 g, pH 7.0, diluted to volume with deionized water.

[0131] To evaluate the nitrogen-fixing capacity of the strain, growth experiments were conducted using Ashby selective medium without available inorganic or organic nitrogen sources. Bacteria lacking nitrogen-fixing capacity served as a negative control. The control strain and the tested PGPR AT1 strain were inoculated onto Ashby solid medium plates and Ashby solid medium at pH 11 with a sodium ion concentration of 300 mM, respectively. The plates were incubated upside down at 30 °C to minimize interference from condensation on colony formation. Figure 9 To test the nitrogen fixation capacity of PGPR AT1, growth was observed after incubation for a predetermined time. PGPR AT1 grew stably on Ashby nitrogen-free medium, producing viscous colonies with neat edges and smooth surfaces, exhibiting a typical mucoid phenotype. In contrast, the negative control did not grow under the same conditions. To further determine whether PGPR AT1 also possesses nitrogen fixation capacity under severe saline-alkali stress, PGPR AT1 was inoculated into Ashby solid medium at pH 11 with a sodium ion content of 300 mM. Under severe saline-alkali stress, PGPR AT1 also grew normally, indicating that PGPR AT1 can reduce atmospheric nitrogen to usable nitrogen through its own nitrogen fixation system (such as nitrogenase complex), thereby maintaining growth and enhancing its tolerance to severe saline-alkali conditions.

[0132] Example 9: Analysis of the growth-promoting characteristics of *PGPR AT1*, a highly salt-tolerant plant growth-promoting enterobacterium, including its ability to secrete siderophores.

[0133] CAS medium (250 mL): To 200 mL of sterilized medium D, add 0.2 mL of sterilized 1 mM calcium chloride, 4 mL of 1 mM magnesium sulfate, 2 mL of 20% glucose, and 6 mL of 10% acid-hydrolyzed casein, mix well, then add 37.8 mL of sterilized dye solution C and mix well to obtain CAS medium. Solution A: 0.024 g resazurin (CAS) dissolved in 20 mL of distilled water, then thoroughly mixed with 4 mL of 1 mM ferric chloride solution; Solution B: 0.03 g hexadecyltrimethylammonium bromide dissolved in 16 mL of distilled water. Slowly add 24 mL of solution A to 16 mL of solution B and mix thoroughly to obtain dye solution C. Culture medium D (200 mL): 20 mL of 10 × MM9 salt solution, 6.04 g of piperazine diethanolsulfonic acid dissolved in 150 mL of distilled water, the two were mixed and the pH was adjusted to 6.8 with 50% sodium hydroxide solution, and 8 g of agar was added.

[0134] Mixed saline-alkali CAS medium (250 mL): Add 0.2 mL of sterilized 1 mM calcium chloride, 4 mL of 1 mM magnesium sulfate, 2 mL of 20% glucose, and 6 mL of 10% acid-hydrolyzed casein to 200 mL of sterilized medium D, mix well, and then add 37.8 mL of sterilized dye solution C. Mix well to obtain CAS medium. Solution A: 0.024 g resazurin (CAS) dissolved in 20 mL of distilled water, then thoroughly mixed with 4 mL of 1 mM ferric chloride solution; Solution B: 0.03 g hexadecyltrimethylammonium bromide dissolved in 16 mL of distilled water. Slowly add 24 mL of solution A to 16 mL of solution B and mix thoroughly to obtain dye solution C. Culture medium D (200 mL): 20 mL of 10 × MM9 salt solution, dissolved in 150 mL of distilled water with 6.04 g piperazine diethanolsulfonic acid, mixed well, and adjusted to pH 10.5 with 50% sodium hydroxide solution, then 8 g of agar was added. After sterilization, the solution was cooled to 50°C, and then sterilized mixed saline-alkali solution (300 mM, Na2CO3: NaHCO3 = 1:5) was added: 0.95391 g Na2CO3 and 6.80481 g NaHCO3.

[0135] MKB medium (100 mL): 0.5 g tyrosine, 1.5 mL glycerol, 0.25 g potassium dihydrogen phosphate, 0.25 g magnesium sulfate heptahydrate, mix well, and adjust pH to 7.2 with 50% sodium hydroxide solution.

[0136] Take 10 μL of the activated PGPR AT1 strain and inoculate it onto a CAS solid plate or a CAS solid plate mixed with saline-alkali, and incubate it upside down at 30°C. If the PGPR AT1 strain produces a distinct orange-yellow transparent zone on the CAS plate, it indicates that the strain has the ability to secrete siderophores. The size and color of the transparent zone indicate the strain's ability to produce siderophores; the larger the transparent zone and the darker the color, the stronger the strain's ability to produce siderophores. Figure 10 To detect the siderophore secretion capacity of PGPR AT1, from Figure 10 It can be seen that the PGPR AT1 strain can also produce a distinct orange-yellow transparent zone on mixed saline-alkali CAS medium plates, indicating that this strain still has the ability to secrete heptaphilin under saline-alkali stress conditions. Figure 10 A). Quantitative experiment: The strain was inoculated into 5 mL of MKB liquid medium and cultured at 150 r / min and 30℃ for 48 h. The culture was centrifuged at 5000 rpm for 10 min, and 1 mL of the supernatant (reference value, Ar) was collected. 1 mL of uninoculated MKB liquid medium was added, and the mixture was combined with the CAS detection solution at a 1:1 ratio. After reacting at room temperature for 1 h, the OD value (As) was measured at 630 nm, using deionized water as a control.

[0137] The calculation formula is: SU(%) = [(Ar - AS) / Ar]×100%

[0138] A higher ratio indicates a stronger ability of the strain to produce siderophores. Quantitative detection results showed that PGPR AT1 secreted 70.34±1.09% of siderophores under normal growth conditions and 40.73±0.97% under mixed salt-alkali stress conditions. Figure 10 B). Significance was evaluated by one-way ANOVA (***, P < 0.001). The above results indicate that PGPR AT1 has a strong ability to secrete ferrophages under normal growth conditions, and also has a certain ability to secrete ferrophages under saline-alkali stress conditions.

Claims

1. A salt-tolerant plant growth-promoting enterobacterium, characterized in that, The salt-tolerant plant growth-promoting Enterobacter is Enterobacter cancerogenus PGPR AT1, deposited at the China Center for Type Culture Collection (CCTCC) on August 18, 2025, with accession number CCTCC NO: M 20251848.

2. A growth-promoting Enterobacter bacillus agent for salt-tolerant plants, characterized in that, This bacterial agent contains Enterobacter cancerogenus PGPR AT1.

3. The method for preparing the salt-tolerant plant growth-promoting Enterobacter agent according to claim 2, characterized in that, The preparation method is as follows: Enterobacter cancerogenus PGPR AT1 is inoculated into a bacterial culture medium and cultured to obtain OD. 600 A bacterial solution with a value of 0.5-1.0 is a growth-promoting enterobacterial agent for salt-tolerant plants.

4. The application of the growth-promoting Enterobacter as described in claim 1 in promoting plant growth under severe saline-alkali stress or normal growth conditions.

5. The application according to claim 4, characterized in that, The plant is a dicotyledonous plant; the dicotyledonous plant may be a legume or a cruciferous plant.

6. The application according to claim 5, characterized in that, The legume is soybean, and the cruciferous plant may be Arabidopsis thaliana.

7. The application according to claim 4, characterized in that... After being prepared into a microbial agent, Enterobacter for Proliferation is mixed with a carrier material and dried to produce microbial fertilizer in granular or powder form.

8. The application according to claim 7, characterized in that... The carrier material is peat, vermiculite, or humic acid.

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