Adhesion of the strain of Myxococcus M. Wang-R7 and its application

Abstract

The present application relates to the field of microbial technology, and particularly to a strain of Xanthomonas campestris M.Wang-R7 and its application. Ensifer adhaerens The present application provides Xanthomonas campestris (Xanthomonas campestris) M.Wang-R7, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 39391. The strain can significantly promote the seed germination of rice under salt stress conditions, improve the growth ability of rice seedlings, significantly improve the antioxidant capacity and osmotic regulation capacity of rice, regulate the ion homeostasis of plants, thereby slowing down the salt damage phenomenon of rice seedlings, and can be applied to improve the growth of rice in saline-alkali soil, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to an adhesive sword fungus M. Wang-R7 and its applications. Background Technology

[0002] Soil salinization has numerous negative impacts on soil properties, plant growth, and agricultural production. In arid and semi-arid regions, salinization is particularly severe, and in some semi-humid coastal areas, it also affects crop growth, reducing land productivity and biodiversity. In recent years, soil salinization has intensified due to climate change factors such as rising sea levels increasing saline groundwater levels and irrigation with saline or high-pH water during periods of insufficient rainfall, as well as anthropogenic factors. Reports indicate that in saline-alkali soils with a pH of 9.8, the yields of tolerant, moderately tolerant, and sensitive rice varieties decreased by 25%, 37%, and 68%, respectively. Soil salinization has become a significant constraint on the further stable development of regional agriculture.

[0003] Salt stress can significantly inhibit the performance of plants throughout the entire developmental stage. For example, salt stress can lead to a decrease in rice seed germination rate, weakened early seedling vigor, reduced biomass accumulation, inhibited taproot elongation, and inhibited tillering, ultimately resulting in reduced panicle quality.

[0004] Salt stress mainly originates from Na + and Cl - Excessive accumulation of sodium leads to ion toxicity, osmotic stress, and sodium ions. + / K + The disruption of homeostasis directly impairs membrane transport processes and cellular metabolism. Soil water potential decreases with increasing salt concentration, making water absorption difficult for roots and even leading to dehydration. Salt ions enter the plant through the roots, disrupting the ion balance within the plant, resulting in stunted growth and leaf wilting and yellowing. Salt ions inhibit root tissue elongation and division, as well as lateral root development. Furthermore, significant changes in physiological and biochemical characteristics can be detected in seedlings under salt stress, including disordered mineral nutrient absorption, inhibited functional protein synthesis, and impaired reactive oxygen species metabolism leading to membrane integrity damage.

[0005] With the continuous advancement of the green transformation and upgrading of modern agriculture, the role of plant growth-promoting bacteria in stimulating plant growth and improving their stress resistance through direct or indirect mechanisms has attracted increasing attention. Studies have shown that growth-promoting bacteria have been used as a major component of biopesticides, biofertilizers, and bioinhibitors. Currently, growth-promoting bacteria have garnered widespread attention in agriculture and have been successfully used as field soil inoculants to improve plant growth and yield, demonstrating broad application prospects. However, it remains necessary to develop new strains to enhance the plant growth-promoting effects under salt stress. Summary of the Invention

[0006] This invention provides a strain of *M. Wang-R7* and its applications.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides *Achillea millefolium* (… Ensifer adhaerens M. Wang-R7, this strain was deposited on June 24, 2026, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as *M. Wang-R7*. Ensifer adhaerens Latin name Ensifer adhaerens The accession number is CGMCC No. 39391.

[0009] The *M. Wang-R7* strain provided by this invention was isolated from the root system of rice in saline-alkali land in Lianyungang and is an endophytic fungus of rice roots. This strain can grow on YMA plates with exogenous salt concentrations of 0-4% and pH 7-11, and exhibits good salt tolerance and growth-promoting properties in rice. Under salt stress conditions, it can effectively promote rapid seed germination, aboveground and root growth during the seedling stage, increase chlorophyll content, enhance antioxidant activity in rice under salt stress, and reduce sodium content. + It increases the sodium and potassium ion content and improves the sodium ion to potassium ion ratio, effectively alleviating the sodium ion concentration problem in rice growing in saline-alkali soil. + Salt stress caused by accumulation, etc.

[0010] In a second aspect, the present invention provides a microbial agent comprising the *M. Wang-R7* strain described in the first aspect above.

[0011] Preferably, the *M. Wang-R7* bacteria in the bacterial agent are present in live form.

[0012] Preferably, the active ingredient of the bacterial agent comprises or is composed of *M. Wang-R7*.

[0013] In addition to the active ingredient, the microbial agent may also contain excipients permitted in the field of microbial preparations. The microbial agent may be a liquid or solid dosage form. The excipients may be selected according to different formulation types and requirements, including but not limited to fillers, carriers, and protective agents.

[0014] Thirdly, the present invention provides a rice salt-tolerant growth promoter, wherein the rice salt-tolerant growth promoter comprises the *M. Wang-R7* or the bacterial agent.

[0015] Preferably, the active ingredient of the rice salt-tolerant growth promoter includes the *M. Wang-R7* or the fungal agent, or is composed of the *M. Wang-R7* or the fungal agent.

[0016] Fourthly, the present invention provides a method for preparing the rice salt-tolerant growth promoter described in the third aspect above, the method comprising: inoculating the *M. Wang-R7* or the bacterial agent into a liquid culture medium, culturing with shaking at 28-30°C to obtain a bacterial solution, centrifuging the bacterial solution to obtain a bacterial precipitate, and resuspending the bacterial precipitate.

[0017] Preferably, the liquid culture medium is YMB liquid culture medium.

[0018] Preferably, the culture temperature is 28-30℃.

[0019] Preferably, the culture is shaken until the bacterial count reaches OD200. 600 The result is 1, and the bacterial solution is obtained.

[0020] Preferably, the centrifugation is performed at 5000-6000 rpm for 5 minutes.

[0021] Preferably, the resuspension is performed using water or a buffer solution.

[0022] Preferably, after resuspension, a washing step may be included. The washing may be as follows: after resuspension with an equal volume of ultrapure water by shaking, centrifugation at 5000-6000 rpm for 5 min, discarding the supernatant, and repeating 1-2 times before resuspension.

[0023] Fifthly, the present invention provides the application of the *M. Wang-R7*, the bacterial agent, or the rice salt-tolerant growth promoter in improving the growth performance of rice seedlings under salt stress.

[0024] In this invention, the salt tolerance promotion includes enhancing the salt stress tolerance of rice seedlings and promoting the growth and development of rice seedlings under salt stress conditions.

[0025] Sixthly, the present invention provides any one or more of the following applications of the *M. Wang-R7*, the fungal agent, or the rice salt-tolerant growth promoter: (1) Improve the germination rate of rice seeds under salt stress; (2) Promotes root growth in rice seedlings under salt stress; (3) Increase the plant height, fresh weight and / or dry weight of rice seedlings under salt stress.

[0026] Specifically, improving the germination rate of rice seeds under salt stress means promoting rapid germination of rice seeds under salt stress.

[0027] The promotion of rice seedling root growth under salt stress is manifested in increasing total root length, root volume and / or root surface area.

[0028] In a seventh aspect, the present invention provides the application of the *M. Wang-R7*, the bacterial agent, or the rice salt-tolerant growth promoter in increasing the chlorophyll content of rice seedlings under salt stress.

[0029] The preferred chlorophyll content is the leaf chlorophyll content.

[0030] Eighthly, the present invention provides one or more of the following applications of the *M. Wang-R7*, the fungal agent, or the rice salt-tolerant growth promoter: (1) Reduce Na+ in rice seedling leaves during salt stress + content; (2) Increase root K in rice seedlings under salt stress + content; (3) Reduce Na+ in rice seedling leaves during salt stress + and K + Content ratio; (4) Reduce root Na+ in rice seedlings under salt stress + and K + Content ratio.

[0031] In a ninth aspect, the present invention provides one or more of the following applications of the *M. Wang-R7*, the fungal agent, or the rice salt-tolerant growth promoter: (1) Increase the enzyme activity of SOD, CAT and / or POD in the leaves of rice seedlings under salt stress; (2) Reduce malondialdehyde content in rice leaves during the seedling stage under salt stress; (3) Increase the L-proline content in rice leaves during the seedling stage under salt stress.

[0032] In a tenth aspect, the present invention provides a method for improving the growth performance of rice seedlings under salt stress tolerance, the method comprising: treating the roots of rice seeds or seedlings with the *M. Wang-R7*, the fungal agent, or the rice salt-tolerant growth promoter before sowing.

[0033] Preferably, the treatment of rice seeds includes: soaking the seeds in the aforementioned *M. Wang-R7*, the bacterial agent, or the rice salt-tolerant growth promoter.

[0034] Preferably, the treatment of the seedling roots includes root irrigation.

[0035] The beneficial effects of this invention include at least the following: This invention provides a salt-tolerant growth-promoting bacterium for rice seedlings—*M. Wang-R7*, which significantly improves the salt tolerance and growth-promoting ability of rice seedlings under salt stress, significantly promotes seed germination and seedling growth under salt stress, and significantly alleviates the salt stress suffered by rice. It also significantly promotes root length, root surface area, and root volume in rice, significantly improving the antioxidant capacity and osmotic regulation of rice. This helps to improve the salt tolerance of rice from multiple aspects, including ion homeostasis, osmotic regulation, and mitigation of oxidative damage, thereby promoting the growth of rice seedlings and alleviating the growth problems of rice in saline-alkali land. This salt-tolerant growth-promoting bacterium has broad application prospects, providing more and more efficient salt-tolerant growth-promoting bacterium options for the utilization of saline-alkali land, and providing a theoretical basis and technical support for using plant growth-promoting bacteria to alleviate crop salt damage. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 This is a diagram showing the results of streaking culture of *M. Wang-R7* on YMA medium in Example 1 of this invention.

[0038] Figure 2 The image shows the growth of *M. Wang-R7* on YMA medium with different concentrations of NaCl (top image) and different pH values ​​(bottom image) in Example 1 of this invention.

[0039] Figure 3 This is a scanner image of the root system of rice seeds germinating under different microbial environments under salt stress in Example 2 of the present invention.

[0040] Figure 4 This is a diagram showing the germination rate of rice seeds under salt stress in different microbial environments in Example 2 of the present invention.

[0041] Figure 5 The figure shows the germination and growth of rice seeds under different microbial environments under salt stress in Example 2 of the present invention. Here, af represents fresh weight, plant height, total root length, root volume, root surface area, and average root diameter, respectively.

[0042] Figure 6 These are photos of potted rice seedlings grown under salt stress in different microbial environments, as shown in Example 3 of this invention.

[0043] Figure 7The plant height of potted rice seedlings under salt stress in different microbial environments is shown in Example 3 of this invention.

[0044] Figure 8 The figures represent the fresh weight and dry weight of potted rice seedlings under salt stress in different microbial environments in Example 3 of this invention, where af represents the fresh weight of leaves, the dry weight of leaves, the fresh weight of stems, the dry weight of stems, the fresh weight of roots, and the dry weight of roots, in that order.

[0045] Figure 9 The figures represent the chlorophyll content (SPAD), L-proline content, antioxidant enzyme activity, and malondialdehyde content of potted rice seedlings under salt stress in different microbial environments in Example 3 of this invention.

[0046] Figure 10 The Nai of potted rice seedlings under salt stress in different microbial environments in Example 3 of this invention is an example of this invention. + and K + Content, where af is the leaf Na + Content, Na + Content, leaf K + Content, root K + Content, leaf Na + / K + , root Na + / K.

[0047] Figures 1-10 In this context, R7 represents strain M. Wang-R7; P < 0.05 P < 0.01 P < 0.001 P < 0.0001. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples can be obtained commercially or prepared according to conventional methods in the art. In the following examples, the rice material used for testing is Huajing 5. All strains used were obtained from Nanjing Agricultural University. Among them, strains R1, R2, R3, R4, M.Wang-R7 (accession number CGMCC No. 39391), R9, and R13 are strains with salt tolerance and growth-promoting potential isolated by the applicant from rice roots. R12 is a reported nitrogen-fixing bacterium (…) preserved in the laboratory. Rhizobium japonicum USDA110) (Win, KT, S. Wasai-Hara, F. Tanaka, AZ Oo, K.Minamisawa, Y. Shimoda and H. Imaizumi-Anraku (2023). Synergistic N2-fixation and salt stress mitigation in soybean through dual inoculation of ACCdeaminase-producing Pseudomonas and Bradyrhizobium. Scientific Reports.).

[0050] The YMA medium formulation used in the following examples is as follows: Yeast Extract 0.4 g / L + Mannitol 10 g / L + K₂HPO₄ 0.5 g / L + NaCl 0.1 g / L + MgSO₄·7H₂O 0.2 g / L + CaCl₂·2H₂O 0.1 g / L + Agar 15 g / L, pH 7.0, Ultrapure Water: 1 L. The YMB medium formulation is based on the YMA medium formulation with the agar removed.

[0051] Example 1 Salt-tolerant growth-promoting bacteria in rice seedlings (Adhesive capsulatum) Ensifer adhaerens Isolation, screening and identification of M. Wang-R7 1. Isolation and screening of rice salt-tolerant growth-promoting bacteria strain M. Wang-R7 Weigh approximately 2.0-5.0g of rice roots from saline-alkali land in Lianyungang and place them in a sterile, enzyme-free centrifuge tube. Add sodium hypochlorite for surface sterilization, wash 3-4 times with sterile water, then grind in a sterile mortar with 1-2mL of double-distilled water. Serially dilute to 10 in sterile 10mL centrifuge tubes. -3 and 10 -4A mixed suspension of varying concentrations was prepared. 100 µL of the suspension was diluted and spread onto YMA medium. After three streak plate tests, single colonies were obtained for purification. The purified *M. Wang-R7* strain could be transferred to YMB liquid medium using a disposable inoculating loop. The loop was shaken 4-5 times and incubated at 28°C for 48-72 hours. The bacterial suspension was then mixed with 40% glycerol at a 1:1 volume ratio in a 2 mL cryovial and stored at -80°C. For the purified salt-tolerant growth-promoting *M. Wang-R7* strain obtained through streak plate testing, well-growing single colonies could be cultured using the same method for later use.

[0052] 2. Identification of rice salt-tolerant growth-promoting bacterium M. Wang-R7 strain (1) Biological identification: When this strain was cultured on YMA medium at 28℃ for 48h, the colonies were nearly round, moist without raised wrinkles, milky white, and opaque. In the early stage of growth, single colonies were 1-2 mm in size, with white gelatinous material at the edge. Figure 1 YMA plates with different salt and alkali concentrations were prepared using NaCl and NaOH, respectively. The growth of the strains on YMA plates with pH 7–11 and exogenous NaCl additions of 1%–5% is shown below. Figure 2 As shown, strains M.Wang-R7, R2, and R9 can all grow on YMA plates with exogenous addition of 1%–4% NaCl and on YMA plates with pH 7–11, indicating that they can survive in saline-alkali soils.

[0053] (2) Systematic classification and identification: PCR amplification was performed using the universal bacterial primers 27F (SEQ ID No.2: 5'-AGAGTTTGATCMTGGCTCAG-3') / 1492R (SEQ ID No.3: 5'-CGGYTACCTTGTTACGACTT-3'), and the 16S rDNA sequence is shown in SEQ ID No.1.

[0054] The sequence shown in SEQ ID No. 1 is as follows:

[0055] The 16S rRNA gene sequence of the strain was aligned with the nucleotide sequence of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). The results showed that the sequence was consistent with seq NR_113893.1. Ensifer adhaerens The homology with strain NBRC 100388 was 100%. Based on colony characteristics, strain M. Wang-R7 was identified as *Strombus amygdalinus*. Ensifer adhaerens ).

[0056] *M. Wang-R7* was deposited on June 24, 2026, at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as *M. Wang-R7*. Ensifer adhaerens The accession number is CGMCC No. 39391.

[0057] Example 2: Test on the salt tolerance promoting effect of salt-tolerant growth-promoting bacterium M. Wang-R7 on rice seed germination Preparation method of rice salt-tolerant growth promoter: The strain was inoculated into YMB liquid medium and cultured at 180 rpm and 30℃ until OD. 600 =1 (i.e., 10) 8 The bacterial suspension was obtained by centrifuging at 5500 rpm for 5 min, discarding the supernatant, and then adding an equal volume of ultrapure water. After resuspending by shaking, the suspension was diluted 100 times to obtain the rice salt tolerance growth promoter (concentration of 10 CFU / mL). 6CFU / mL). Select Huajing No. 5 rice seeds, disinfect with NaClO solution (2.5% v / v) for 30 min, rinse repeatedly with ultrapure water, soak for 30 min, and then soak in the prepared rice salt-tolerant growth promoter for 2 h. Sterilize a 90 mm glass plate with two layers of qualitative filter paper at 115℃ for 30 min, then dry it in a 65℃ oven with sterilized 5 mL pipette tips and tweezers. Place 20 sterilized and soaked yellow, plump rice seeds (remove any seeds floating in the pure water) on the plate, immediately add 5 mL of pure water, and after all seeds are completely added, immediately and evenly add 5 mL of the treatment liquid (100 mM sodium chloride solution / ultrapure water / the prepared rice salt-tolerant growth promoter dissolved in 100 mM sodium chloride solution) around the plate and mix thoroughly. Protect from light for the first 3 days, and then cultivate in light after germination. The entire cultivation process followed a 16h:8h day-night cycle, with the petri dish lids kept closed. The daytime illuminance was 9000 Lux, the temperature was 25±5℃ during the day and 20±5℃ at night, and the relative humidity was 50%±10% during the day and 60%±6% at night. The number of seeds germinating was recorded. On the fourth day, 1 mL of sterile water was added to maintain a salt stress concentration of 100 mM. Finally, the growth of the rice was statistically analyzed after 4 and 8 days of treatment.

[0058] Multiple treatment groups were set up, with 3 replicates in each group, including: (1) only exogenous addition of sterilized ultrapure water (control group, CK); (2) exogenous addition of 100mM NaCl without exogenous addition of rice salt tolerance promoters (salt stress control group, Salt); (3) exogenous addition of 100mM NaCl and exogenous addition of a single rice salt tolerance promoter (R1, R2, R3, R4, M.Wang-R7, R9, R12, R13) or synthetic microbial community Syncom; wherein, the synthetic microbial community Syncom only contained rice root isolates R1, R2, R3, R4, M.Wang-R7 (preservation number CGMCC No.). The ratio of viable counts of R1, R2, R3, R4, M.Wang-R7, R9, R12 and R13 was 1:1:1:1:1:1:1:1.

[0059] Rice seed germination and root growth under salt stress inoculated with different salt-tolerant growth promoters are as follows: Figure 3 As shown.

[0060] Rice seed germination rate, plant height, fresh weight, and total root length were measured: 4 and 8 days after seed germination treatment, the number of rice seed buds (bud length greater than half the seed length) and the number of surviving seeds were recorded, and the rice seed germination rate was calculated; 8 days later, the plant height (distance between the base of the plant root and the top) of the treated and control plants was measured using a ruler; immediately after collecting seedlings, the surface moisture was absorbed with absorbent paper before measuring the fresh weight, and the total root length was measured and recorded using a root scanner.

[0061] See the results below. Figure 4 , Figure 5 The results showed that under 100 mM salt stress, the germination rate of rice treated with the salt-tolerant growth promoter M. Wang-R7 was significantly higher on day four compared to the salt-stressed control, but there was no significant difference on day eight, indicating that strain M. Wang-R7 is beneficial for the rapid germination and growth of rice seeds under salt stress. Furthermore, under salt stress, rice inoculated with M. Wang-R7 and its synthetic community Syncom (R1+R2+R3+R4+M. Wang-R7+R9+R12+R13) had higher plant height and fresh weight than rice without the growth promoter. Compared to the treatment without the growth promoter, plant height at the germination stage increased by 20.08% and 16.62%, respectively, and fresh weight increased by 10.39% and 12.16%, respectively. Compared with rice without growth promoters, rice inoculated with M. Wang-R7 and the synthetic microbial community Syncom under salt stress showed improved root growth. Total root length increased by 31.36% and 30.20%, respectively; root volume increased by 63.48% and 90.84%, respectively; root surface area increased by 45.59% and 56.39%, respectively; and average root diameter increased by 10.18% and 19.18%, respectively. These results indicate that strain M. Wang-R7 is beneficial for rice germination under salt stress and has the potential to construct highly efficient synthetic communities.

[0062] Example 3: Salt-tolerant growth-promoting test of salt-tolerant bacterium M. Wang-R7 on rice seedlings. Preparation method of rice salt-tolerant growth promoter: The strain was inoculated into YMB liquid medium and cultured at 180 rpm and 30℃ until OD. 600 =1 (i.e. 10) 8 The bacterial suspension was obtained by centrifuging at 5500 rpm for 5 min, discarding the supernatant, and then resuspending in an equal volume of ultrapure water. The suspension was then diluted 10-fold to obtain a rice salt-tolerant growth promoter (concentration of 10 CFU / mL). 7 (CFU / mL) is used for potted plant irrigation.

[0063] Salt stress treatment during seedling stage: The test materials were rice seedling strains with salt tolerance and growth-promoting potential screened at the seedling stage according to the method described in Example 1 (R1, R2, R3, R4, M. Wang-R7 (CGMCC No. 39391), R9, R12, R13), R9, R12, R13, and the commercially available salt-tolerant growth-promoting control strain Bacillus belyssus (…). Bacillus velezensisSQR9, isolated and identified by the Organic Fertilizer Team of the Department of Plant Nutrition, College of Resources and Environment, Nanjing Agricultural University (i.e., Jiangsu Provincial Key Laboratory of High Technology Research on Resource Utilization of Solid Waste), were prepared as rice salt-tolerant growth promoters according to the above methods. Among them, strain R3 was cultured and resuspended in LB liquid medium (LB medium formula: trypton 10 g / L; yeast extract 5 g / L; NaCl 10 g / L; ultrapure water: 1L) under the same culture conditions. Seeds of the Huajing No. 5 rice variety were selected, disinfected with NaClO solution (2.5% v / v concentration) for 30 minutes, rinsed repeatedly with ultrapure water, and soaked for 30 minutes. Seedlings were then raised in pure water and cultured in a 30cm x 20cm x 10cm height container until they reached the size of three leaves. Plants with uniform growth were then transplanted into the prepared "Xingxing Xiangnong" seedling substrate (Jiangsu Xingnong Machinery Technology Co., Ltd., Invention Patent No. ZL202510448249.7). Immediately after transplanting, 100mL of pure water was added to the pots to submerge the substrate. The same amount of water was added daily. After 4-5 days of cultivation, 50mL of the aforementioned prepared rice salt-tolerant growth promoter (concentration 10) was applied as a root irrigation inoculation. 7 Rice seedlings were cultured in pots under CFU / mL and salt stress treatment (using plastic boxes with dimensions of 9cm long × 9cm wide × 8.5cm high, with about 250g of substrate in each pot and 2 rice seedlings transplanted into each pot). Each treatment was replicated 10 times, and samples were collected after 14 days of culture. There were 3 treatment groups: (1) only distilled water was added exogenously (control group, CK); (2) 100mM NaCl was added exogenously without the addition of rice salt tolerance promoter (salt stress control group, Salt); (3) 100mM NaCl was added exogenously with the addition of a single rice salt tolerance promoter or synthetic bacteria. The entire culture process was 16h:8h day and night. The daytime illuminance of the dysprosium lamp was 10000Lux, the temperature was 25±5℃ during the day and 20±5℃ at night, and the relative humidity was 50%±10% during the day and 60%±6% at night. After 14 days of treatment, the experimental results of rice seedling pot cultivation in different treatment groups are shown in the figure. Figure 6 .

[0064] The detection methods and results for various indicators of rice under salt stress are as follows: 1. Measurement of rice seedling height, fresh weight, and dry weight Fourteen days after salt stress treatment, the plant height (distance from the base of the stem to the top of the plant) of the treatment and control groups was measured using a ruler. Healthy and uniformly growing rice plants were selected, and the roots were thoroughly washed with pure water. The water attached to the surface of the seedlings was absorbed with absorbent paper. The fresh weight of the above-ground and underground parts (the weight measured immediately after collecting fresh plants) was measured. After measurement, the plants were placed in an oven at 105℃ for 30 minutes to kill the green, and then dried at 65℃ for about 2-3 days until constant weight was achieved. The dry weight of the plants was then measured.

[0065] See the results below. Figure 7 , Figure 8 The results show that under 100mM salt treatment, rice plants inoculated with salt-tolerant growth promoters such as M.Wang-R7 generally had higher plant height and fresh weight than rice plants without growth promoters. Compared with the treatment without growth promoters, the treatment inoculated with M.Wang-R7 increased plant height by 8.81%, and the fresh weight of roots, stems, and leaves increased by 101.20%, 111.79%, and 138.42%, respectively, while the dry weight increased by 49.52%, 62.19%, and 69.82%, respectively. This indicates that applying salt-tolerant growth promoter M.Wang-R7 under salt stress can significantly alleviate salt damage and promote rice growth.

[0066] 2. Chlorophyll content determination On day 14 after salt stress treatment, the SPAD values ​​of fully expanded new leaves of rice in pot experiments were measured during the day using a handheld SPAD chlorophyll meter to represent chlorophyll content. Five replicates were performed for each treatment. Results are as follows: Figure 9 As shown, under the condition of adding 100mM salt, the chlorophyll content of rice with added salt-tolerant growth promoter was significantly higher than that of the group without growth promoter, and the chlorophyll content of rice inoculated with M.Wang-R7 was the highest, which was 21.26% higher than that of the salt stress treatment without growth promoter.

[0067] 3. Determination of antioxidant enzyme activity For specific solution preparation methods, refer to "Experimental Guide to Plant Stress Physiology" edited by Shi Haitao. The activity of superoxide dismutase (SOD) was determined using the nitroblue tetrazolium (NBT) solution method, the activity of guaiacol peroxidase (POD) was determined using the guaiacol method, and the activity of catalase (CAT) was determined using an ultraviolet microplate.

[0068] The results are as follows Figure 9 As shown, under 100 mM salt treatment, compared with the treatment without growth promoter, the activity of antioxidant enzymes in the leaves of rice seedlings treated with growth promoter was significantly increased. Inoculation with M. Wang-R7 strain significantly increased the enzyme activities of SOD and CAT, by 792.24% and 1311.54% respectively compared with the treatment without growth promoter, which is beneficial for the gradual reduction of superoxide anions to hydrogen peroxide, and finally to oxygen, thus alleviating oxidative stress under salt stress.

[0069] 4. Determination of malondialdehyde content Because salt and alkali stress causes membrane oxidation in plants, leading to the production of large amounts of malondialdehyde (MDA), the level of MDA can be used to determine the degree of cell membrane damage and the ability of tested salt-tolerant and growth-promoting strains such as M. Wang-R7 to alleviate oxidative stress. The MDA content was determined using fresh root and leaf samples from a potted plant experiment subjected to salt stress. Following the same experimental instructions, 0.3g of fresh rice leaves were taken, rinsed with deionized water, blotted dry with filter paper, and ground in a mortar with liquid nitrogen. Approximately 0.1g of leaf sample was accurately weighed into a 10mL centrifuge tube using a 0.01% balance. 5mL of 5% TCA solution was pipetted in and thoroughly mixed. The mixture was centrifuged at 3000 r / min at 4℃ for 10 min. 2mL of the supernatant was collected, and 2mL of 0.25% TBA was added. The mixture was incubated in a 100℃ water bath for 30 min, then rapidly cooled in an ice bath before collecting the supernatant. The absorbance values ​​(A) were measured at 450 nm, 532 nm, and 600 nm. The malondialdehyde concentration was calculated using the following formula: C (μmol / L) = 6.45 (A) 532 -A 600 -0.56×A 450 .

[0070] Then, the MDA content per unit fresh tissue sample (μmol / g) was converted and analyzed.

[0071] The results are as follows Figure 9 As shown, under 100 mM salt treatment, compared with the treatment without growth promoter, the MDA content in rice leaves during the seedling stage was generally reduced in the treatment group with added growth promoter. Under non-stress conditions, the MDA content in plant leaves was extremely low. Under salt stress, the MDA content in rice leaves decreased after exogenous addition of rice salt-tolerant growth promoter compared with the uninoculated control. Compared with the treatment without growth promoter, the MDA content in the leaves of Huajing 5 rice decreased by 73.91% after inoculation with M. Wang-R7 strain. Combined with the above enzyme activity results, it further indicates that the application of salt-tolerant growth promoter M. Wang-R7 under salt stress is beneficial to alleviate oxidative damage under salt stress and promote rice growth.

[0072] 5. Determination of L-proline (hereinafter referred to as proline, Pro) content in leaves Approximately 0.1 g (record sample weight) of leaf material ground in liquid nitrogen was accurately weighed using a 0.01 g balance and mixed with 1 mL of sulfosalicylic acid solution (3%, w / v). The mixture was centrifuged at 10,000 rpm for 10 min. 1 mL of the supernatant was mixed with an equal volume of glacial acetic acid-ninhydrin reagent (5 g ninhydrin dissolved in 120 mL glacial acetic acid and 80 mL 2 mol / L phosphoric acid, heated and stored in the dark). The mixture was then reacted in a 95°C water bath for 30 min, followed by rapid ice bath cooling to terminate the reaction. After cooling, 2 mL of toluene was added and mixed by inversion. After standing, the phases separated. The absorbance of the upper red solution was measured at 520 nm using toluene as a blank control. A proline standard curve was plotted to calculate the proline concentration in the sample. The proline content is expressed in mg / mL. Calculation method: Proline content (mg / g) = Proline concentration (mg / mL) × Sample volume (mL) / Sample weight (g).

[0073] See results Figure 9 As shown in the figure, the Proline content in rice leaves significantly increased under the 100 mM salt treatment. Furthermore, the Proline content in leaves treated with the salt-tolerant growth promoter was generally higher than that in the salt-treated group without the promoter. The leaves inoculated with *M. Wang-R7* and the synthetic bacterial community *Syncom* showed even higher Proline content, increasing by 51.81% and 55.23% respectively compared to the treatment without the promoter. This indicates that applying the salt-tolerant growth promoter *M. Wang-R7* under salt stress can alleviate osmotic stress and promote rice growth by increasing proline content.

[0074] 6. Determination of the relative ratio of sodium ion to potassium ion content Weigh the leaves and roots of the rice used in the further salt tolerance test, crush and sieve 0.3g of the sample, and weigh 0.045-0.055g of dried plant sample (the plants were blanched at 105℃ for 30min, then dried at 65℃ to constant weight) (record the weight of each sample tube). Place the sample into a digestion tube, accurately record the weight, and add 5 mL of sulfuric acid (H2SO4) to each digestion tube. Use a digestion tube with only 5 mL of H2SO4 as a blank control to eliminate background interference. Let stand overnight for at least 12 hours. Then, digest in a digestion oven (280℃) for 1 hour, cool for 10min, add 3ml of hydrogen peroxide (H2O2), and digest in the digestion oven for another 30min until colorless. Finally, make up to 100mL with deionized water, filter into a 10mL centrifuge tube, and determine the Na+ content using a flame photometer. + and K + The ion content and the statistical ratio of sodium ion to potassium ion content (Na) + / K + ).

[0075] See results Figure 10Under the condition of adding 100mM salt, the Na+ content in rice seedling leaves decreased. + / K + The content was generally higher; compared with the treatment without the addition of growth promoters, the Na content in the leaves and roots of rice seedlings treated with salt-tolerant growth promoters was higher. + / K + The content of most strains was significantly reduced. Under salt stress, inoculation with M. Wang-R7 strain and the synthetic bacterial group Syncom significantly reduced Na content in rice leaves compared to no inoculation. + The content of K in the root system was increased. + The concentration of sodium and potassium in the root system and leaves was adjusted, thereby mitigating the ion toxicity of salt stress on rice growth. Similar to the effect of the commercial growth-promoting bacterium Bacillus vesiculosus SQR9, inoculation with strain M. Wang-R7 alone can effectively regulate the ion homeostasis of rice.

[0076] In conclusion, rice seeds supplemented with salt-tolerant growth-promoting bacteria M.Wang-R7 can germinate and grow rapidly under salt stress, with significant improvements in plant height, dry and fresh weight, chlorophyll content, and root growth of rice seedlings. It can effectively regulate the ion content in the plant and improve the salt tolerance of rice by regulating antioxidant enzyme pathways and increasing proline content.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Adhesive sword fungus ( Ensifer adhaerens M.Wang-R7, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39391.

2. A microbial agent, characterized in that, It contains the *M. Wang-R7* strain as described in claim 1.

3. A rice salt-tolerant growth promoter, characterized in that, The rice salt-tolerant growth promoter comprises the *M. Wang-R7* strain of claim 1 or the fungal agent of claim 2.

4. The preparation method of the rice salt-tolerant growth promoter according to claim 3, characterized in that, The method includes: inoculating the *M. Wang-R7* strain of claim 1 or the bacterial agent of claim 2 into a liquid culture medium, culturing with shaking at 28-30°C to obtain a bacterial solution, centrifuging the bacterial solution to obtain a bacterial precipitate, and resuspending the bacterial precipitate.

5. Any one or more of the following applications of the *M. Wang-R7* as described in claim 1, the fungal agent as described in claim 2, or the rice salt-tolerant growth promoter as described in claim 3: (1) Improve the germination rate of rice seeds under salt stress; (2) Promotes root growth in rice seedlings under salt stress; (3) Increase the plant height, fresh weight and / or dry weight of rice seedlings under salt stress.

6. The application of the *M. Wang-R7* as described in claim 1, the fungal agent as described in claim 2, or the rice salt-tolerant growth promoter as described in claim 3 in increasing the chlorophyll content of rice seedlings under salt stress.

7. Any one or more of the following applications of the *M. Wang-R7* as described in claim 1, the fungal agent as described in claim 2, or the rice salt-tolerant growth promoter as described in claim 3: (1) Reduce Na+ in rice seedling leaves during salt stress + content; (2) Increase root K in rice seedlings under salt stress + content; (3) Reduce Na+ in rice seedling leaves during salt stress + and K + Content ratio; (4) Reduce root Na+ in rice seedlings under salt stress + and K + Content ratio.

8. Any one or more of the following applications of the *M. Wang-R7* as described in claim 1, the fungal agent as described in claim 2, or the rice salt-tolerant growth promoter as described in claim 3: (1) Increase the enzyme activity of SOD, CAT and / or POD in the leaves of rice seedlings under salt stress; (2) Reduce malondialdehyde content in rice leaves during the seedling stage under salt stress; (3) Increase the L-proline content in rice leaves during the seedling stage under salt stress.

9. A method for improving the salt stress tolerance and growth performance of rice seedlings, characterized in that, The method includes treating the roots of rice seeds or seedlings with the *M. Wang-R7* of claim 1, the fungal agent of claim 2, or the rice salt-tolerant growth promoter of claim 3 before sowing. The improvement of rice seedlings' tolerance to salt stress growth performance includes any one or more of the following: (1) Improve the germination rate of rice seeds under salt stress; (2) Promotes root growth in rice seedlings under salt stress; (3) Increase the plant height, fresh weight and / or dry weight of rice seedlings under salt stress.

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