Function identification and application of strain B53 capable of enhancing salt tolerance and growth promotion capability of plants

Abstract

The invention belongs to the technical field of plant growth-promoting bacteria, and discloses functional identification and application of a strain B53 for enhancing salt-tolerant growth-promoting capability of plants. Based on the study on the 16S rRNA gene sequence of the strain B53, the strain B53 is the Exiguobacterium mexicanum, namely, the strain is named as Exiguobacterium mexicanum in taxonomy, and the strain B53 is a strain of Exiguobacterium mexicanum. According to the method, growth of plants such as wild economic plants apocynum venetum, important crops such as corn and forage ryegrass can be promoted by utilizing the Exiguobacterium mexicanum, particularly, the salt tolerance of the plants can be effectively improved, and limitation of saline-alkali soil on plant growth is reduced, so that the method has comprehensive advantages of ecological, economic and social sustainable development.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant growth promoting bacteria, and particularly relates to functional identification of a strain for enhancing plant salt tolerance and growth promotion and application thereof. TECHNICAL BACKGROUND

[0002] Soil salinization is a global environmental problem that is becoming increasingly serious. It not only destroys soil structure and reduces soil fertility, but also directly hinders the normal growth and development of plants by interfering with their physiological metabolism, leading to a decrease in grain yield and quality and restricting agricultural development. Alleviating or reducing the damage of salinization to plants is one of the key challenges in the restoration of saline-alkali land.

[0003] Traditional methods for improving saline-alkali soil include agricultural measures, water conservancy engineering and chemical soil improvement, but they have the disadvantages of high investment, long cycle and slow effect. Using plant growth promoting rhizobacteria (PGPR) to interact with plants to improve plant salt tolerance, promote plant growth, increase yield and nutrient absorption is an important means for improving saline-alkali soil. At present, the related work of PGPR alleviating plant salt stress mainly focuses on crops, including tomato (Lycopersicon esculentum), rice (Oryza sativa), potato (Solanum tuberosum), soybean (Glycine max) and corn (Zea mays), but there are problems such as unstable and unsustainable salt tolerance and growth promotion effect of microorganisms on plants (crops) and fewer available microbial groups. Therefore, screening of microorganism resources with high salt tolerance and growth promotion ability from the rhizosphere of plants in natural saline-alkali environment and applying them to production not only meet the needs of improving crop salt tolerance and increasing yield, but also meet the needs of enhancing the salt tolerance of wild plants and improving saline-alkali areas, and has broad market potential.

[0004] Apocynum venetum L. is a multi-purpose plant resource with ecological, economic and medicinal values in the northwest desert region of China, which can adapt to various harsh environments (such as saline-alkali land, desert edge and Gobi, etc.), has strong stress resistance, has become the preferred plant for ecological restoration and reconstruction in desertification areas, and has great development and utilization potential. However, there is no report on using PGPR to improve the salt stress adaptation ability of Apocynum venetum L. SUMMARY

[0005] In view of the above problems, the present application provides functional identification of a strain of B53 of Mexican Microbacterium isolated from the rhizosphere of Apocynum venetum L. and its application for enhancing plant salt tolerance and growth promotion.

[0006] The implementation process of the present application. A strain B53 capable of promoting plant growth under salt stress is isolated from the rhizosphere of Apocynum venetum L. The 16S rRNA gene of the strain is amplified by PCR and sequenced, and it is found that the length of the 16S rRNA gene of the strain is 1490 bp; through NCBI sequence comparison, the four 16S rDNA sequences with the highest sequence similarity in the NCBI library are selected as reference objects, and the Mega 11 software is used to construct the phylogenetic tree between the strain B53 and the reference strains by the neighbor-joining method; in the phylogenetic tree, the strain B53 and the model strain Exiguobacterium mexicanum strain 8N form a separate cluster evolution branch, that is, the taxonomic name of the strain is Exiguobacterium mexicanum, and the strain is named Exiguobacterium mexicanum B53 (B53 strain for short hereinafter).

[0007] Further, the physiological determination of the strain shows that the Exiguobacterium mexicanum has the functional characteristics of producing peroxidase, catalase and ACC deaminase.

[0008] Another object of the present application is to provide a functional identification method of the strain B53 for enhancing the salt tolerance and growth promotion ability of plants, which comprises:

[0009] The strain is inoculated into LB liquid medium for overnight culture, and then 4 μl of the bacterial liquid is mixed with 196 μl of LB liquid medium containing different NaCl concentrations, and is transferred into a 96-well plate for co-culture for 48 hr, and the OD 600 of the strain is determined every 4 hr by using a Thermo multifunctional enzyme marker to monitor the growth of the strain; it is found that the B53 can maintain good growth under the salt concentration of 100 mmol / L to 400 mmol / L, and grows best at 200 mmol / L.

[0010] Another object of the present application is to provide a preparation method of a bacterial suspension of the strain B53 (Exiguobacterium mexicanum). The method comprises inoculating the strain into LB liquid medium for culture for 24 hr, centrifugation, removal of the supernatant, resuspension with sterile pure water, and dilution to OD 600 1.0, so as to obtain the bacterial suspension.

[0011] Another object of the present application is to provide the application of the bacterial suspension under salt stress conditions for promoting the growth and salt tolerance of Apocynum venetum L., which comprises the following steps: directly using the bacterial suspension to root irrigation treatment on the roots of Apocynum venetum L., the inoculation amount is 5-10 mL, and the strain concentration is not less than 1×10 8 CFU / mL; the application of the bacterial suspension improves the antioxidant activity of the plant and the ability of the plant to resist external salt stress.

[0012] In combination with the above technical solutions and the technical problems solved, the technical solutions to be protected by the present application have the following advantages and positive effects:

[0013] First, the advantages of the present application. The present application discloses a strain of Exiguobacterium mexicanum B53 with salt-tolerant and growth-promoting functions. The strain has the properties of salt tolerance, peroxidase production, catalase production, and ACC deaminase production, and can maintain good growth under the conditions of 100mmol / L to 400mmol / L salt concentration. It has great potential for saline-alkali soil remediation and agricultural production.

[0014] Meanwhile, the present application also provides a new method for determining the salt tolerance of the strain, i.e. determining the growth of the strain in different NaCl concentrations within 48 hours by a multifunctional enzyme marker. The result is more accurate and the operation is simple.

[0015] In addition, the present application also prepares the above-mentioned strain B53 into a growth promoter or a plant salt-tolerant growth-promoting bacterial agent. By directly drip irrigating the bacterial suspension, the activity of plant antioxidant enzymes is enhanced to alleviate salt stress and promote plant growth. Compared with traditional physical and chemical methods, the use of Exiguobacterium mexicanum B53 bacterial solution has no pollution, has little impact on the environment, and has a quick effect.

[0016] In summary, the present application has the advantage of providing a new plant salt-tolerant growth-promoting strain of Exiguobacterium mexicanum B53 and its application method. By using the B53 strain, not only the salt tolerance of apocynum venetum, corn and ryegrass is improved, but also the growth of plants is promoted, thereby reducing the limitation of saline-alkali soil on plants and crops, and thus having potential advantages for the sustainable development of ecology and industrial economy.

[0017] Second, the positive effects of the present application.

[0018] As a new multi-purpose plant resource with ecological, economic and medicinal values in the northwest desert region of China, apocynum venetum not only can prevent wind and sand, conserve water, and improve ecology, but also can be used for medicine and tea making, and has very high ecological and economic values. Taking Yuli County in Xinjiang as an example, the area of wild and artificially planted apocynum venetum is about 1.6 million mu. Relying on the rich apocynum venetum resources, the county has developed apocynum venetum tea, honey, medicine, health products, clothing and other full-industry-chain products. In addition, there are also large areas of apocynum venetum distribution and planting in Ruoqiang and Altay regions. Although apocynum venetum has a certain salt tolerance, the increase of "high salt value" habitats caused by soil salinization exacerbates the problems of low seed germination, difficult seedling planting, slow growth of adult plants and reduced salt tolerance, which restricts the release of ecological and economic values.

[0019] Maize is one of the four major crops in China, and is a high-energy, multi-purpose "food-feed-energy" crop with important production, economic and social value. However, the overall stress tolerance of maize is poor, especially its moderate salt tolerance, which severely limits the development of the maize industry. It is an urgent task in the maize planting industry to further expand its planting range through breeding and cultivation measures.

[0020] Ryegrass has good soil fixation and water purification functions, and has high dry matter and crude protein content, making it a high-quality and high-yield forage grass. Compared to its high cold and poor tolerance, ryegrass has moderate salt tolerance, which limits its planting range and utilization of marginal land.

[0021] China has 1.5 billion mu of saline-alkali land, which is an extremely important available land and arable land reserve resource. Improving plant salt tolerance is crucial for mitigating soil salinization and achieving economic benefits. The strain B53 isolated from the rhizosphere of Apocynum venetum in a high-salt environment and the technical solutions provided in the present application can be applied not only to economic plants such as Apocynum venetum in saline-alkali areas, achieving a 30%-40% increase in Apocynum venetum biomass and synchronous growth of fiber yield and medicinal ingredients, solving the bottleneck problem of the Apocynum venetum industry, but also to the planting of traditional crops such as maize and important forage resources such as ryegrass, expanding the planting range and improving the utilization efficiency of marginal land. Finally, the patent strain can be packaged and transformed into a microbial inoculant, which can be applied to the planting and production of economic crops in saline-alkali areas, achieving the purpose of improving land use efficiency and improving saline-alkali land. Compared with traditional chemical improvement methods, the microbial inoculant has a four-dimensional synergistic mechanism of "salt reduction-resistance-fertilization-remediation", which can achieve low-cost green development of saline-alkali land. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Phylogenetic tree of Exiguobacterium mexicanum B53;

[0023] Figure 2 Salt tolerance test of Exiguobacterium mexicanum B53;

[0024] Figure 3 Effect diagram of the promotion of Exiguobacterium mexicanum B53 on Apocynum venetum under salt stress;

[0025] Figure 4 Data result statistical graph of the effects of Exiguobacterium mexicanum B53 on the plant height and biomass of Apocynum venetum under salt stress; (a) plant height, (b) fresh weight of aboveground part, and (c) fresh weight of underground part;

[0026] Figure 5Figure of data statistics of antioxidant enzyme activity of B53 on guayule under salt stress; (a) peroxidase (POD) activity in guayule plant leaves, (b) catalase (CAT) activity, (c) superoxide dismutase (SOD) activity.

[0027] Figure 6 Figure of data statistics of plant height and biomass of B53 on corn under salt stress; (a) plant height, (b) plant dry weight;

[0028] Figure 7 Figure of data statistics of plant height and biomass of B53 on ryegrass under salt stress; (a) plant height, (b) plant dry weight; DETAILED DESCRIPTION

[0029] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with examples. It should be noted that the specific examples described herein are only used to explain the present application and not to limit the present application.

[0030] The technology provided by the present application aims at the problems of reduced germination rate, limited root development, early leaf senescence and yield reduction of plants in high salt stress environment in saline-alkali soil agricultural production, and proposes to use B53 with multiple growth-promoting properties to improve the physiological adaptability of plants through rhizosphere microbial intervention. The existing chemical modifier or single gene breeding has limitations such as long cycle, high cost or ecological risk in improving salt tolerance, while the B53 strain can be directly planted in the rhizosphere of plants to quickly exert biological effects, realizing low-input, sustainable soil improvement and crop stable yield and increased yield.

[0031] The B53 strain can tolerate high salt stress and has good growth stability, which lays a foundation for its field application. It can maintain a high specific growth rate under the condition of 100 to 400 mmol / L NaCl concentration, especially under the condition of 200 mmol / L NaCl, which shows that the strain has good osmotic regulation ability and cell membrane stability in high osmotic pressure environment. This stability enables it to survive and function in soil with serious salt accumulation, and has a competitive advantage in the soil micro-ecosystem of saline habitat.

[0032] The B53 strain has growth-promoting function. The B53 strain can secrete peroxidase and catalase to resist oxidative stress (cell damage caused by reactive oxygen species) produced by salt stress, thereby rapidly removing harmful hydrogen peroxide and other peroxides produced by adversity metabolism; the ACC deaminase produced by it can reduce the accumulation of ethylene in plants, and alleviate the early senescence response under salt stress. These effects together build a multi-layer protective barrier for plants under adversity conditions.

[0033] The core mechanism of B53 strain in salt stress alleviation also includes inducing or enhancing plant antioxidant system. The activities of peroxidase, superoxide dismutase and catalase in the root system and leaves of plants treated by B53 are significantly improved, which can efficiently scavenge active oxygen free radicals caused by salt stress, reduce the damage of cell membrane lipid peroxidation, and thus maintain the integrity of cell structure and function. This biological induced stress resistance mode has better ecological and economic feasibility than exogenous application of antioxidants.

[0034] In the application process, the B53 strain is prepared into a standardized bacterial suspension after liquid fermentation culture, and is applied to the roots of plants by root irrigation. The bacterial cells colonize in the rhizosphere to form a stable microecological community, continuously release growth-promoting metabolites, and interact positively with the plant roots. The inoculation process can be combined with water-saving technologies such as drip irrigation to achieve precise, quantitative and efficient application, and is suitable for large-scale farmland and saline-alkali land.

[0035] The industrial value of this technology lies in its strong adaptability, low cost and high sustainability. B53 strain is not only suitable for economic crops such as apocynum venetum, but also can be popularized to food crops such as corn, and even to salt-tolerant planting systems of pasture such as ryegrass, which has important significance for improving the comprehensive utilization rate of saline-alkali land, reducing the use of fertilizers and reducing the ecological footprint of agricultural production. By using microbial resources to enhance plant salt tolerance, a feasible biological approach is provided for saline-alkali land agricultural production.

[0036] The embodiment of the application provides a strain B53 capable of promoting plant growth under salt stress, wherein the length of 16S rRNA gene of the strain B53 is 1490 bp; four 16S rDNA sequences with high homology are selected as reference objects, a phylogenetic tree between the strain B53 and reference strains is constructed by using a Mega 11 software by using a neighboring method; in the phylogenetic tree, the strain B53 and a model strain Exiguobacterium mexicanum strain 8N of Exiguobacterium mexicanum form a separate intra-cluster evolutionary branch, and the taxonomic name of the strain is Exiguobacterium mexicanum.

[0037] The Exiguobacterium mexicanum has the growth-promoting properties of producing peroxidase, catalase and ACC deaminase.

[0038] The application further provides a strain B53 function identification method for enhancing plant salt tolerance and growth promotion capability, comprising the following steps:

[0039] The strain is inoculated into LB liquid medium for overnight culture, then 4 μl of bacterial liquid is mixed with 196 μl of LB liquid medium containing different NaCl concentrations, and is transferred into a 96-well plate for co-culture for 48 hours; OD600 The growth of the strain is observed; the strain can maintain good growth trend under the salt concentration of 100mmol / L-400mmol / L, especially the growth trend is the best under the concentration of 200mmol / L.

[0040] Another purpose of the present application is to provide a preparation method of the bacterial suspension of the B53 strain of the Exiguobacterium mexicanum. The method comprises inoculating the strain into LB liquid medium for 24 hours, centrifuging, removing supernatant, resuspending with sterile pure water, diluting to OD 600 1.0, that is, obtaining the bacterial suspension.

[0041] Another purpose of the present application is to provide a specific application method of the bacterial suspension for promoting the growth and salt tolerance of the guayule under the salt stress condition, which comprises the following steps: directly performing root irrigation treatment on the guayule roots with the bacterial suspension, the inoculation amount is 5-10mL, the strain concentration is not less than 1×10 8 CFU / mL; the plant antioxidant activity is improved after the application of the bacterial suspension, thereby helping the plant to resist the external salt stress.

[0042] Another purpose of the present application is to provide a specific application method of the bacterial suspension for promoting the growth and salt tolerance of the corn under the salt stress condition, which comprises the following steps: directly performing root irrigation treatment on the corn roots with the bacterial suspension, the inoculation amount is 10mL, the strain concentration is not less than 1×10 8 CFU / mL; the plant antioxidant activity is improved after the application of the bacterial suspension, thereby helping the plant to resist the external salt stress.

[0043] Another purpose of the present application is to provide a specific application method of the bacterial suspension for promoting the growth and salt tolerance of the corn under the salt stress condition, which comprises the following steps: directly performing root irrigation treatment on the corn roots with the bacterial suspension, the inoculation amount is 10mL, the strain concentration is not less than 1×10 8 CFU / mL; the plant antioxidant activity is improved after the application of the bacterial suspension, thereby helping the plant to resist the external salt stress.

[0044] In the following examples, the materials, reagents and the like are obtained from commercial channels unless otherwise specified.

[0045] Example 1: Isolation, purification, identification and preservation of the salt-tolerant Exiguobacterium mexicanum B53

[0046] 1.1 Preparation of LB solid medium

[0047] The present embodiment first carries out the configuration of LB solid medium. The required raw materials include yeast extract powder 5.0 g, tryptone 10.0 g, NaCl 10.0 g, distilled water 1,000 mL, adjust pH to 7.2, add 15.0-20.0 g agar, stir until completely dissolved. Subsequently, the prepared LB liquid medium is divided into conical flasks, sterilized in a pressure sterilizer at 121°C for 20 min; after sterilization is completed, cool to 50°C, and then divided into disposable petri dishes in a clean bench to prepare LB solid medium, which is placed at 4°C for standby.

[0048] 1.2 Isolation and purification of salt-tolerant rhizosphere strains

[0049] First, take 1 g of the rhizosphere soil of apocynum venetum under salt stress, add it into a conical flask containing 99 mL of sterile water, and shake at a speed of 150 rpm for 30 min. Dilute the soil suspension according to the gradient dilution method, take 200 μL of the soil suspension with dilution degrees of 10 -3 , 10 -4 and 10 -5 , and spread them on LB medium plates, with 3 repeats for each concentration. Incubate at 28°C for 48-72 hr, and observe the growth conditions of the strains. Pick single colonies and transfer them to the same solid medium plates for subculturing by streaking, to obtain pure culture strains.

[0050] 1.3 Identification of the growth-promoting function of salt-tolerant rhizosphere strains

[0051] Determine the abilities of all the strains purified in 1.2 to produce peroxidase, catalase and ACC deaminase, according to the following methods:

[0052] Peroxidase production: add 1-2 drops of Kovacs reagent to the agar slant culture or the blood agar plate colony, and the positive sample shows pink to deep purple color, while the negative sample shows no color change. If the color changes, it indicates that the strain has the function of producing peroxidase.

[0053] Catalase production: take a clean glass slide, add 1 drop of 3-10% H2O2, pick 1 ring of bacterial mat cultured for 24 hr, and smear it in the hydrogen peroxide solution. If bubbles (oxygen) appear, it indicates that the strain is catalase positive, while no bubbles indicate that it is catalase negative.

[0054] ACC deaminase production: take 1 mL of bacterial liquid into 50 mL of sterile DF liquid medium, and incubate at 28°C and 200 rpm for 1 d. Take 1 mL of the cultured bacterial liquid into 50 mL of ADF liquid medium, and incubate at 28°C and 200 rpm for 2 d. If it becomes turbid, it indicates that the strain has the ability to secrete ACC deaminase.

[0055] 1.4 Strain identification

[0056] The screened salt-tolerant growth-promoting bacterial colonies were orange, translucent, smooth-surfaced, easy to pick, and with neat edges. The TIANamp Bacteria DNA Kit was used to extract DNA from the strain, and the universal primers 27F and 1492R were used to amplify the 16S rDNA fragment by PCR. The amplification product was sent to Beijing Genesee Biotechnology Co., Ltd. for bidirectional sequencing, and the spliced results were obtained. The phylogenetic tree was constructed on the MEGA X software, and it was found that the 16S rRNA sequence homology with the Exiguobacterium mexicanum strain 8N strain with the GenBank accession number SAMN29987059 reached 99% ( Figure 1 ), and it was considered to be the same kind of bacteria, named Exiguobacterium mexicanum B53, with the NCBI accession number PV211479.

[0057] 1.5 Strain preservation

[0058] The Exiguobacterium mexicanum of the present embodiment was preserved in the Guangdong Microbial Culture Collection Center on November 18, 2025, with the collection number GDMCC 67289 and the preservation address being No. 59 Building, 5th Floor, Guangzhou Martyrs' Courtyard, 100 Middle Lieth Road. This is convenient for future research and application.

[0059] Example 2: Salt tolerance test of Exiguobacterium mexicanum B53

[0060] 2.1 Preparation of LB liquid medium

[0061] The LB liquid medium was prepared, and the raw materials included yeast extract powder 5.0 g, tryptone 10.0 g, NaCl 10.0 g, distilled water 1,000 mL, and the mixture was stirred until completely dissolved. The pH was adjusted to 7.2, and then the mixture was divided into conical flasks. The flasks were sterilized in a high-pressure sterilization pot at 121℃ for 20 min. After sterilization, the flasks were cooled to room temperature and stored at 4℃ for standby.

[0062] 2.2 Salt tolerance experiment of Exiguobacterium mexicanum B53

[0063] Strain B53 was inoculated into the LB liquid medium and cultured overnight. Then, 4 μl of the bacterial solution was mixed with 196 μl of LB liquid medium containing different concentrations of NaCl (0 mmol / L, 100 mmol / L, 200 mmol / L, and 400 mmol / L), and the mixture was transferred into a 96-well plate for co-culture for 48 hr. The OD 600 was measured every 4 hr using a Thermo multifunctional enzyme marker, and the growth of the strain was observed. The LB liquid medium without bacteria was used as a negative control.

[0064] 2.3 Data Analysis

[0065] The area under the growth curve (AUC) was calculated using Graph Pad Prism (10.1.2) software. Specific TI values ​​were calculated based on the normalized AUC values ​​for all tested concentrations for the strain. Based on the TI value distribution ranging from 0 (completely sensitive, no growth at the lowest tested concentration) to 1 (completely tolerant, no growth inhibition at the highest tested concentration), the strain was categorized into three types: tolerant (TI ≥ 0.75), intermediate (0.75 > TI ≥ 0.50), and sensitive (TI < 0.50). For each concentration, normalized bacterial growth data were statistically compared with the control group using a one-sample t-test or non-parametric test, with statistical significance set at a threshold of P < 0.05.

[0066] 2.4 Results Analysis

[0067] like Figure 2 As shown, *Microbacterium muscarinii* strain B53 can grow in salt solutions of different concentrations, and its growth is inhibited to varying degrees with increasing NaCl concentration, with the best growth observed at a salt concentration of 200 mmol / L. Therefore, 200 mmol / L NaCl was used as the stress concentration for subsequent pot experiments. The AUC and TI values ​​of strain B53 at different salt concentrations were then calculated using Graph Pad Prism (10.1.2) software. The results showed that the TI values ​​were all above 0.75 under all three salt concentration treatments, indicating that this strain is salt-tolerant (Table 1).

[0068] Table 1. Area of ​​growth (AUC) and tolerance index (TI) of strain B53 at different salt concentrations.

[0069]

[0070] Example 3: Application of Microbacterium Mexicanum strain B53 in promoting the growth of Apocynum venetum.

[0071] 3.1 Seedling raising of Apocynum venetum

[0072] Seeds of *Apocynum venetum* collected from Toksun County, Xinjiang, were surface-sterilized by soaking in 70% ethanol for 1 minute and in 3% NaClO solution for 10 minutes. After rinsing 5-6 times with sterile distilled water, the seeds were placed in sterile petri dishes lined with two layers of moist sterile filter paper and cultured in the dark at 28°C. Once the seeds showed signs of sprouting, seedlings with uniform growth were selected and transplanted into seedling trays. The culture medium was prepared by mixing soil and vermiculite in a ratio of 1:1 (v / v).

[0073] 3.2 Preparation of bacterial suspension

[0074] Strain B53 was cultured overnight in LB liquid medium and centrifuged at 5,000 × g for 10 min at 4°C. The supernatant was discarded, and the suspension was resuspended in sterile water. The OD value of the bacterial suspension was adjusted to 1.0 to obtain the bacterial suspension.

[0075] 3.3 Salt stress treatment

[0076] To verify the growth-promoting effect of *Microbacterium gracilistylus* B53 strain on *Apocynum venetum* under different salt stress conditions, two treatments were set up: one with no NaCl (-NaCl) and the other with 200 mmol / L NaCl (+NaCl). Specifically, after *Apocynum venetum* seedlings were transplanted into seedling trays and had 3-4 pairs of true leaves (approximately 15 days), the soil around the roots of the *Apocynum venetum* seedlings was irrigated with 5 mL of a prepared bacterial suspension. The control group was irrigated with sterile water. Irrigation was performed every 3 days for a total of 3 times. On day 30 after transplanting, a salt solution treatment was applied, irrigating with 5 mL of 200 mmol / L NaCl solution (with sterile water as the control) every 2 days for a total of 7 treatments. Each treatment was repeated 6 times, with 6 *Apocynum venetum* plants in each replicate. Samples were collected 15 days after the salt solution treatment ended, and the plant growth and physiological indicators of *Apocynum venetum* were measured.

[0077] 3.4 Index Measurement

[0078] Determination of growth indicators of Apocynum venetum: The plant height of Apocynum venetum was measured with a ruler, and the fresh weight of the plant roots and stems was measured with an electronic balance, with 10 replicates.

[0079] The plant antioxidant enzyme activities were determined according to the methods in "Experimental Techniques in Plant Physiology". Among them, POD activity was determined using the guaiacol method, CAT activity was determined using ultraviolet spectrophotometry, and SOD activity was determined using the nitroblue tetrazolium (NBT) photoreduction method.

[0080] 3.5 Data Processing

[0081] The experimental data were analyzed and plotted using Excel, SPSS (version 19), and Graph Pad Prism (10.1.2) software. The data in the charts are the mean ± standard deviation.

[0082] 3.6 Results show

[0083] The results showed that, regardless of salt treatment, *Microbacterium gracilistylus* B53 promoted plant growth and improved the salt tolerance of plants under salt stress. Figure 3 Compared with the control group, inoculation with strain B53 significantly increased the plant height of *Apocynum venetum* (P<0.05). Under saline solution treatment, the aboveground and belowground biomass of plants treated with strain B53 increased significantly by 46.77% and 64.24%, respectively. Figure 4 ).

[0084] To evaluate the effect of the strains on the salt stress resistance of apocynum plants, the CAT, POD and SOD activities of the plants were determined. The results are shown in Table 2. Figure 5 As shown in Table 2, under salt stress, the POD and CAT enzyme activities in the leaves of the apocynum plants inoculated with the strains were significantly higher than those of the control group (P < 0.05), and the SOD did not change significantly. Under no salt stress, the POD in the leaves of the apocynum plants inoculated with the strains was significantly higher than that of the control group (P < 0.05), the CAT was significantly lower (P < 0.05), and the SOD did not change significantly. Figure 5

[0085] Example 4 Application of Exiguobacterium mexicanum strain B53 to corn growth promotion

[0086] 4.1 Corn seedling culture

[0087] The seeds of Xianyu 1818 purchased from the Gansu Academy of Agricultural Sciences were soaked in a 5% NaClO solution and shaken on a shaker at 150 rpm for 10 min for surface sterilization, and then rinsed with sterile distilled water for 5 times and the surface water was absorbed. The seeds were placed in a sterile culture dish lined with two layers of moist sterile filter paper, and germinated at 25℃ / 20℃ in the dark for 6 days. After the seeds were white, the seedlings with uniform growth were selected and transplanted in flowerpots, and the culture medium was mixed according to the ratio of soil: vermiculite = 1:1 (v / v). Then the plants were grown at 25℃ / 20℃, relative humidity 60%, light and dark cycle 16hr / 8hr.

[0088] 4.2 Preparation of bacterial suspension

[0089] The strain B53 was cultured in LB liquid medium overnight, and centrifuged at 5,000 x g at 4℃ for 10 min. The supernatant was discarded, and the bacterial suspension was obtained by resuspending with sterile water and adjusting the OD value of the bacterial solution to 1.0.

[0090] 4.3 Salt stress treatment

[0091] NaCl (-NaCl) and 100 mmol / L, 200 mmol / L NaCl (+NaCl) treatments were set up to verify the growth promotion effect of Exiguobacterium mexicanum B53 strain on corn under different salt stress conditions. Specifically, 10 mL of bacterial suspension or an equal volume of sterile water was applied to the roots of the seedlings as the inoculation group (+B53) and the non-inoculation group (-B53). Then, 50 mL of solution containing 100 mmol / L, 200 mM NaCl was used to irrigate the soil, and the control was irrigated with an equal amount of sterile water. The water or NaCl solution was replenished twice a week, and the treatment lasted for 4 weeks. There were 6 seedlings in each treatment, and the experiment was repeated three times. The growth indicators were determined 28 days after the start of the treatment.

[0092] 4.4 Determination of indicators ​

[0093] Plant height was measured with a ruler and plant dry weight was measured with an electronic balance, with 10 replicates.

[0094] 4.5 Data processing

[0095] Data were analyzed and plotted using Excel and SPSS (version 19), and the data in the figures are mean ± standard deviation.

[0096] 4.6 Results

[0097] Regardless of the presence of salt stress, B53 promoted the growth of corn, and the effect was not obvious in the absence of salt stress; in the presence of salt stress, the effect was significant (P < 0.05), i.e., inoculation with strain B53 significantly increased the plant height and dry weight of corn under salt stress (P < 0.05). Figure 6 The plant height of the inoculated corn increased by 28% under 100 mmol / L treatment and by 62% under 200 mmol / L treatment; the dry weight of the inoculated corn increased by 29% under 100 mmol / L treatment and by 85% under 200 mmol / L treatment; at the same time, the root system of the inoculated corn was more developed. This proved that strain B53 can effectively promote the growth and biomass accumulation of crops under salt stress.

[0098] Example 5 Application of B53 to ryegrass

[0099] 5.1 Ryegrass seedling culture

[0100] Full and uniform ryegrass (Lolium perenne) seeds were selected, soaked in 15% H2O2 for 10 min, then rinsed with distilled water for 4-5 times, placed in a culture dish lined with two layers of sterile filter paper, and germinated in a 25°C light incubator. After the cotyledon emerged, it was moved to a plastic flowerpot (92 mm in diameter, 62 mm in bottom diameter, and 120 mm in height) containing quartz sand. After transplanting, it was placed in a culture room with a temperature of 22.5°C and a light cycle of 14 hr / 10 hr. When the highest seedling was about 25 cm long, it was treated.

[0101] 5.2 Preparation of bacterial suspension

[0102] Strain B53 was cultured in LB liquid medium overnight, centrifuged at 5,000 x g at 4°C for 10 min. The supernatant was discarded, and the bacterial suspension was resuspended with sterile water to adjust the OD value to 1.0.

[0103] 5.3 Salt stress treatment

[0104] NaCl ( -NaCl) and 100 mmol / L, 200 mmol / L NaCl (+NaCl) treatments were set to verify the growth-promoting effect of M. mesophilicum B53 strain on ryegrass under different salt stress conditions. Specifically, 10 mL of bacterial suspension or an equal volume of sterile water was applied to the roots of seedlings as the inoculation group (+B53) and the non-inoculation group (-B53). Then, 50 mL of a solution containing 100 mmol / L, 200 mM NaCl was used to irrigate the soil, and the control was irrigated with an equal amount of sterile water. The water or NaCl solution was replenished twice a week, and the treatment lasted for 4 weeks. There were 6 seedlings per treatment, and the experiment was repeated three times. The growth indicators were measured 28 days after the start of the treatment.

[0105] 5.4 Indicator measurement

[0106] The height of ryegrass was measured with a ruler, and the dry weight of the plant was measured with an electronic balance, with 10 replicates.

[0107] 5.5 Data processing

[0108] Excel and SPSS (version 19) were used to analyze the data and generate graphs, and the data in the graphs are the mean ± standard deviation.

[0109] 5.6 Result display

[0110] M. mesophilicum B53 generally promotes the growth of ryegrass. Without salt stress, the promotion effect on plant height and dry weight is not obvious; in the presence of salt, it has a significant promoting effect (P<0.05), i.e., inoculation with strain B53 significantly increases the plant height and dry weight of ryegrass under salt stress (P<0.05) Figure 7 ). The height of the inoculated ryegrass increased by 18.8% under 100 mmol / L treatment and by 37.8% under 200 mmol / L treatment; the dry weight of the inoculated ryegrass increased by 53.1% under 100 mmol / L treatment and by 44.8% under 200 mmol / L treatment; at the same time, the root system of the inoculated ryegrass was more developed; and the plant growth and root activity were significantly enhanced, proving that strain B53 has broad application potential in salt-tolerant forage grasses.

[0111] Specific application fields or related products of the present application.

[0112] 1. Specific application fields

[0113] The present application can be particularly applied to the cultivation of apocynum venetum in saline-alkali environments, and its application is described in detail in Example 3. The present application can also be applied to other crops and forage plants grown in saline-alkali environments, as described in detail in Example 4 for corn and in Example 5 for ryegrass.

[0114] Since the strain can be recruited as rhizosphere bacteria by specific plants, it has the potential to promote the salt tolerance and growth ability of plants, and belongs to but is not limited to the technical field of plant growth-promoting bacteria, and particularly relates to application of a strain capable of promoting plant growth under salt stress.

[0115] 2. Related products

[0116] Firstly, a strain with growth promotion and salt tolerance enhancement for plants is isolated and purified from the rhizosphere of a plant grown in a saline-alkali environment, and is identified as Exiguobacterium mexicanum B53 with a NCBI accession number of PV211479, and is preserved in the Guangdong Microbial Culture Collection Center on November 18, 2025 with a collection number of GDMCC 67289.

[0117] Further, the application provides a growth promotion function identification method and a salt tolerance test method of Exiguobacterium mexicanum B53.

[0118] Further, the application provides an implementation method of the salt tolerance and growth promotion function of the strain B53 and a preparation method of a bacterial suspension of the strain B53.

[0119] The application also provides an implementation method of the salt tolerance and growth promotion function of the strain B53 in corn planting, and verifies that the Exiguobacterium mexicanum B53 in the application has the functions of enhancing the salt tolerance and growth promotion of crops such as corn.

[0120] The application also provides an implementation method of the salt tolerance and growth promotion function of the strain B53 in rye grass planting, and verifies that the Exiguobacterium mexicanum B53 in the application has the functions of enhancing the salt tolerance and growth promotion of forage grasses such as rye grass.

[0121] Example 1: System evolution identification of the strain B53

[0122] The strain B53 isolated from the rhizosphere of Apocynum venetum growing in saline-alkali environment was subjected to 16S rRNA gene amplification sequencing, and a sequence of 1490 bp in length was obtained. After BLAST comparison and a series of analysis using NCBI database, it was confirmed that the strain was Exiguobacterium mexicanum, named Exiguobacterium mexicanum B53, with NCBI accession number PV211479, and preserved in Guangdong Microbial Culture Collection Center on November 18, 2025, with collection number GDMCC 67289 and address at No. 59 Building, 5th Floor, Guangzhou Institute, 100 Middle Lieth Road, Guangzhou. This is convenient for future research and application.

[0123] Example 2: Detection of plant growth promoting enzymatic properties of strain B53

[0124] Strain B53 was inoculated into LB solid medium and subjected to peroxidase, catalase and ACC deaminase activity determination. The results showed that strain B53 exhibited strong antioxidant enzyme activity, indicating that it had the potential to enhance plant stress tolerance and promote plant growth by regulating active oxygen metabolism and ethylene synthesis pathway.

[0125] Example 3: Determination of growth curve of Exiguobacterium mexicanum B53 strain under salt stress

[0126] Strain B53 was cultured overnight in LB liquid medium, and 4 μL of bacterial solution was added to 196 μL of LB medium containing different NaCl concentrations (0, 100, 200, 400 mmol / L) in a 96-well plate, and incubated at 28°C. The OD 600 was detected every 4 hours using a Thermo multifunctional enzyme marker, and the growth curve was plotted. The results showed that strain B53 could still grow in 100-400 mmol / L NaCl environment, and could maintain the best growth in 200 mmol / L NaCl, indicating that it had significant salt tolerance and the ability to survive stably under salt stress.

[0127] Example 4: Application of enzyme marker detection method

[0128] The Thermo multifunctional enzyme marker was used to detect the growth curve of strain B53 under different salt concentrations, which had the advantages of automatic data acquisition and high-throughput detection, ensuring the accuracy, repeatability and efficiency in subsequent large-scale application.

[0129] Example 5: Preparation and concentration determination of bacterial suspension for plant growth promoting experiment

[0130] Strain B53 was inoculated into LB liquid medium, and after 24 hr cultivation at 28°C, the supernatant was removed by centrifugation, and the bacterial cells were resuspended in sterile pure water. The concentration was adjusted to OD 600 = 1.0, and a bacterial suspension was obtained. The bacterial concentration was determined by plate dilution coating method. The results showed that when the OD 600 was adjusted to 1.0, the actual concentration of strain B53 was stabilized between 1 x 10 8 and 2 x 10 8 CFU / mL, indicating that this preparation method can ensure the quality and consistency of the bacterial suspension and meet the inoculation standards required for subsequent plant treatment.

[0131] Example 6: Application of strain B53 to promote the growth and salt tolerance of Apocynum venetum

[0132] Apocynum venetum seedlings with consistent growth were selected, and the treatment group was inoculated with bacterial suspension by root irrigation, with an inoculation amount of 5-10 mL and a bacterial concentration of ≥ 1 x 10 8 CFU / mL. The control group was added with the same volume of sterile water. The plants were grown under salt stress conditions containing 200 mmol / L NaCl. The activities of peroxidase, superoxide dismutase, and catalase in the leaves of Apocynum venetum treated for 30 d were all increased by more than 30% compared with the non-inoculated control group, and the plant biomass was significantly higher than that of the control group, proving that strain B53 improved the antioxidant capacity and salt tolerance of Apocynum venetum and promoted the growth of Apocynum venetum.

[0133] Example 7: Application of strain B53 to promote the growth and salt tolerance of maize

[0134] The bacterial suspension of strain B53 (concentration ≥ 1 x 10 8 CFU / mL) was used for 10 mL root irrigation treatment of maize (Xianyu 1818) seedlings, and the plants were cultured in soil containing 100 mmol / L NaCl and 200 mmol / L NaCl. The control group was added with the same volume of sterile water. The results showed that the growth of maize under salt stress was significantly promoted by B53 (P < 0.05): the plant height was increased by 28%-62% compared with the non-inoculated maize, and by 29%-85% compared with the non-inoculated control. It was proved that strain B53 could effectively promote the growth and biomass accumulation of crops under salt stress.

[0135] Example 8: Application of strain B53 to promote the growth and salt tolerance of ryegrass

[0136] Ryegrass was selected as the representative of forage grass, and the same bacterial suspension inoculation treatment and salt treatment method as in Example 7 was used to observe the effect of strain B53 on the growth and salt tolerance of ryegrass under 100 mmol / L and 200 mmol / L NaCl environment. The results showed that the plant height of the inoculated ryegrass increased by more than 18.8%, and the biomass increased by more than 44.8% compared with the control, proving that strain B53 has good application potential in salt-tolerant forage grass production.

[0137] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement within the technical range disclosed by the present application and within the spirit and principle of the present application shall be covered within the protection scope of the present application.

Claims

1. A bacterial strain B53 for enhancing salt tolerance and growth promoting ability in plants, characterized in that, The 16S rRNA gene length of the strain is 1490 bp, a phylogenetic tree is constructed by using Mega 11 software by using the adjacent method, in the phylogenetic tree, the strain and the model strain Exiguobacterium mexicanum strain 8N of Exiguobacterium mexicanum in Mexico form a separate cluster evolution branch, and the strain B53 is Exiguobacterium mexicanum.

2. The strain B53 according to claim 1, characterized in that, It has the stress-tolerant growth-promoting properties of producing peroxidase, catalase and 1-aminocyclopropane-1-carboxylic acid deaminase.

3. A method for functional identification of strain B53, which enhances the salt tolerance and growth promotion ability of plants as described in any one of claims 1-2; characterized in that, Strain B53 was inoculated into LB liquid medium for overnight culture, 4 μL of the bacterial solution was mixed with 196 μL of LB liquid medium containing different NaCl concentrations, and was incubated in a 96-well plate for 48 hr, and OD was measured every 4 hr 600 The growth curve was recorded, in which strain B53 grew well at a NaCl concentration of 100 mmol / L to 400 mmol / L, and the growth trend was best at a NaCl concentration of 200 mmol / L.

4. The method of claim 3, wherein, where OD 600 The assay was performed using a Thermo Multiskan Ascent plate reader.

5. A method for preparing a bacterial suspension of Exiguobacterium mexicanum, characterized in that, Comprising: Strain B53 was inoculated into LB liquid medium and cultured for 24 hr, the supernatant was removed by centrifugation, resuspended with sterile pure water and diluted to OD 600 1.0, to obtain a bacterial suspension, characterized in that the concentration of bacterial bodies in the bacterial suspension is not less than 1 x 10 8 CFU / mL.

6. The use of a bacterial suspension to enhance salt tolerance and promote growth of Apocynum venetum under salt stress conditions, characterized in that, Comprising: The root of apocynum venetum is treated by root irrigation, inoculation amount is 5mL to 10mL, strain concentration is not less than 1×10 8 CFU / mL;the treatment improves the oxidation resistance of the plant, thereby enhancing the tolerance of the plant to salt stress and promoting the growth of the plant;the plant oxidation enzyme includes peroxidase, superoxide dismutase and catalase.

7. Use of a bacterial suspension for enhancing salt tolerance and promoting growth of corn under salt stress conditions, characterized in that, Comprising: The corn roots are subjected to root irrigation treatment, the inoculation amount is 10 mL, the strain concentration is not less than 1 x 10 8 CFU / mL, and the treatment enhances the tolerance of the plant to salt stress and promotes the growth of the plant.

8. Use of a bacterial suspension for enhancing salt tolerance and promoting growth of ryegrass under salt stress conditions, characterized in that, Comprising: Comprising: The rye roots are subjected to root irrigation treatment, the inoculation amount is 10 mL, the strain concentration is not less than 1 x 10 8 CFU / mL, the treatment enhances the tolerance of the plant to salt stress and promotes the growth of the plant.

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