Application and method of Streptomyces S16-1 in promoting the growth and salt stress tolerance of Leymus chinensis

Abstract

This invention provides the application and method of Streptomyces S16-1 in promoting the growth and salt stress tolerance of Leymus chinensis, belonging to the field of microbial technology. Streptomyces S16-1 was isolated from saline soil, with the preservation number CCTCC M 20251940, and its 16S rDNA sequence is shown in SEQ ID NO:1. The Streptomyces S16-1 has functional activities such as salt and alkali tolerance, phosphorus solubilization, nitrogen fixation, and production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. The microbial fertilizer is obtained by expanding the culture of Streptomyces S16-1. Compared with chemical fertilizers, the microbial fertilizer used in this invention is more environmentally friendly. It can not only improve soil fertility and help plants improve their salt and alkali resistance, but also promote the balance and diversity of soil microbial communities, which helps maintain the health and stability of the soil ecosystem.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically the application and method of Streptomyces S16-1 in promoting the growth and salt stress tolerance of Leymus chinensis. Background Technology

[0002] my country has a wide distribution of saline soils, covering a total area of ​​over 500 million mu (approximately 33 million hectares). Among them, saline arable land with agricultural development potential accounts for more than 10% of the total arable land area in the country.

[0003] Currently, the improvement of saline-alkali soils mainly relies on physical, chemical, and biological methods. Physical methods primarily address the issue by regulating soil water and salt transport, inhibiting water evaporation, and improving the structure of the topsoil. Chemical methods utilize the principle of acid-base neutralization to reduce the content of exchangeable sodium ions in the soil. Biological improvement methods mainly rely on the application of organic fertilizers, the planting of salt-tolerant crops, and the application of microbial fertilizers. Compared to physical and chemical methods, biological improvement methods, especially the screening and utilization of rhizosphere functional microorganisms that can enhance the salt and alkali tolerance of plants and their preparation into microbial fertilizers, have significant advantages such as low cost, environmental friendliness, and strong sustainability. Therefore, they represent a current research hotspot and important direction in the field of saline-alkali soil improvement.

[0004] However, existing microbial fertilizers still face three major bottlenecks in the improvement of saline soils: the strains have single functions and are difficult to coordinately cope with multiple stresses such as soil nutrient imbalance, poor structure and ion toxicity; their survival and colonization ability in high salinity environments is limited, resulting in unstable field effects. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an application and method of Streptomyces S16-1 in promoting the growth and salt stress tolerance of Leymus chinensis, as detailed below: Application of a Streptomyces sp. S16-1 strain for preparing a seed coating agent to improve the growth of Leymus chinensis in high-salinity soil; the Streptomyces sp. S16-1 strain has the accession number CCTCCM 20251940, and its 16S rDNA sequence is shown in SEQ ID NO:1.

[0006] Moreover, the salinity of the high-salinity soil is 1‰-5‰.

[0007] Furthermore, the Streptomyces sp. S16-1 strain was isolated from soil and deposited on September 1, 2025, at the China Center for Type Culture Collection.

[0008] Moreover, the Streptomycete sp. S16-1 is a Gram-positive bacterium that grows under both aerobic and anaerobic conditions.

[0009] Furthermore, the Streptomycete sp. S16-1 possesses functional activities such as salt tolerance, phosphorus solubilization, nitrogen fixation, and production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase.

[0010] On the other hand, the present invention provides a method for the seed coating agent, wherein Streptomycete sp. S16-1 is cultured in an expanded culture medium, centrifuged to obtain bacterial sludge, and resuspended in sterile physiological saline to prepare a seed coating agent.

[0011] Furthermore, the culture medium used for the expanded culture is R2A liquid medium, and the culture conditions are: temperature 28±0.5℃, pH 7.2±0.2.

[0012] Thirdly, the present invention provides a microbial fertilizer obtained by expanding the culture of the Streptomycete sp. S16-1 strain.

[0013] Fourthly, the present invention provides a method for promoting plant growth under salt stress, wherein the microbial fertilizer is applied to the plant or the high-salinity soil in which the plant grows, and the plant is sheepgrass.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The Streptomycete sp. S16-1 strain provided by this invention has multiple functions such as salt tolerance, phosphorus solubilization, nitrogen fixation, and ACC deaminase production. It can not only help Leymus chinensis alleviate osmotic stress and ethylene toxicity caused by salt stress (through ACC deaminase), but also improve the nutrient deficiency and imbalance problems that are common in saline soils through phosphorus solubilization and nitrogen fixation. It effectively overcomes the bottleneck of the single function of existing microbial fertilizers and provides a comprehensive solution for plant growth in saline-alkali land.

[0015] 2. Applying the Streptomycete sp. S16-1 strain provided by this invention as a seed coating agent or microbial fertilizer to Leymus chinensis can significantly improve the seed germination rate, seedling survival rate and biomass accumulation of Leymus chinensis in high salinity soil environment; the strain reduces the level of stress ethylene in the plant through its ACC deaminase activity, alleviates the inhibition of plant growth by salt, and thus effectively enhances the tolerance of Leymus chinensis to salt stress.

[0016] 3. The Streptomycete sp. S16-1 provided by this invention can grow under both aerobic and anaerobic conditions, and can survive and function in different types of soil environments, including arid, barren, or saline-alkali soils, thereby improving the practical application effect of microbial fertilizers.

[0017] 4. Compared with chemical fertilizers, the microbial fertilizer used in this invention is more environmentally friendly. It can not only improve soil fertility, but also promote the balance and diversity of soil microbial communities, which helps to maintain the health and stability of the soil ecosystem.

[0018] 4. The Streptomyces S16-1 and its microbial fertilizer provided by this invention can be obtained from the soil, the materials are simple and widely available, there is no need to use them in combination with other fertilizers, they are convenient to use and have significant effects.

[0019] 5. The Streptomycete sp. S16-1 provided by this invention can significantly improve the germination rate of alfalfa and sheepgrass seeds. Experimental results show that the germination rate of alfalfa increased from 78.67% to 95.33%, and the germination rate of sheepgrass increased from 75.33% to 88.00%, thus effectively ensuring the seedling emergence rate under adverse conditions. At the same time, this strain can significantly promote plant growth in both ordinary soil and saline-alkali / saltified soil, not only increasing plant height, root length and biomass, but also enhancing the salt tolerance of plants under salt stress, effectively alleviating the inhibition of growth by adverse conditions, and promoting their healthy growth under saline-alkali conditions. Attached Figure Description

[0020] Figure 1 Colonies formed from soil suspension on R2A medium; Figure 2 This refers to the colony formed by Streptomyces S16-1 of the present invention on R2A medium; Figure 3 The comparison results of Streptomyces S16-1 in the GenBank database; Figure 4 The presence of a clear zone in Streptomyces S16-1 on phosphate-solubilizing medium; Figure 5 The bacterial sludge was obtained by centrifugation after shaking culture of Streptomyces S16-1. Figure 6 The effect of Streptomyces microbial fertilizer on the growth of alfalfa after planting; Figure 7 The effects of Streptomyces microbial fertilizer on the physicochemical properties of soil after planting Leymus chinensis. Detailed Implementation

[0021] The technical solutions of the present invention will be described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] This invention involves the isolation and cultivation of Streptomyces by the Grassland Research Laboratory of the Chinese Academy of Agricultural Sciences, followed by the preparation of Streptomyces microbial fertilizer and pot experiments. Shanghai Paisenno conducted subsequent genome sequencing and phylogenetic tree construction.

[0023] Example 1 1. Weigh 10 g of soil sample from the salinized plot, place it in a 250 mL Erlenmeyer flask, add 100 mL of sterile distilled water, mix thoroughly, and obtain 10 g of soil sample. -1 Soil suspension of a certain concentration; 2. Take 1 mL of the above soil suspension into a sterile 10 mL centrifuge tube, add 9 mL of sterile distilled water, mix thoroughly, and obtain 10 mL of the suspension. -2 For a soil suspension of a certain concentration, repeat the above steps to dilute the solution to a final concentration of 10. -6 ; 3. On a sterile laminar flow hood, take 10... -3 Up to 10 -6 100 μL of each concentration of soil suspension was spread onto R2A solid culture medium plates. The plates were then placed in a 25°C incubator and inverted for 5-7 days. After incubation, various microbial colonies (such as...) were visible on the plates. Figure 1 (As shown). Based on colony morphology, single colonies suspected to be *Streptomyces* were picked and repeatedly isolated and purified on fresh R2A plates using the streak plating method until a pure culture was obtained. The purified single colony was named S16-1 and transferred to R2A slant medium, then incubated at 28°C to obtain *Streptomyces* S16-1. Figure 2 As shown.

[0024] Whole-genome sequencing was performed by Beijing Housheng Biotech Co., Ltd., and the 16S rRNA gene sequence of Streptomyces S16-1 was analyzed for homology in the GenBank database. Figure 3 As shown, Streptomyces S16-1 has 100% similarity to Streptomyces sp., and the specific 16S rDNA sequence is shown in SEQ ID NO:1.

[0025] Example 2 1. Weigh 15 g of soil sample from the salinized plot, place it in a 250 mL Erlenmeyer flask, add 150 mL of sterile distilled water, mix thoroughly, and obtain 10 g of soil sample. -1 Soil suspension of a certain concentration; 2. Take 1 mL of the above soil suspension into a sterile 10 mL centrifuge tube, add 9 mL of sterile distilled water, mix thoroughly, and obtain 10 mL of the suspension. -2 For a soil suspension of a certain concentration, repeat the above steps to dilute the solution to a final concentration of 10. -6 ; 3. On a sterile, ultra-clean workbench, absorb 10 -3 -10 -6 100 μL of concentrated soil suspension was spread on R2A solid medium to obtain multiple bacterial strains. Streptomyces S16-1 was isolated and purified, and Streptomyces S16-1 was cultured at 28℃ on R2A medium. 4. The purified Streptomyces S16-1 strain was inoculated onto Monkina medium and cultured at 28±0.5℃ in complete darkness for 3-4 days. The strain was identified by its ability to grow on the medium and the presence of a transparent area near the strain, indicating its phosphate-solubilizing ability. Figure 4 As shown; 5. The purified Streptomyces S16-1 was inoculated into Ashby nitrogen-free medium and cultured at 28±0.5℃ in complete darkness for 3-4 days. It was found that it could grow on this medium, which confirmed that it had nitrogen-fixing ability. 6. The purified Streptomyces S16-1 was inoculated into liquid DF medium and liquid ADF medium and cultured on a shaker for 3 days. The absorbance of each culture was measured using an enzyme-linked immunosorbent assay (ELISA) reader (Table 1). The absorbance of the culture in ADF medium was greater than that in DF medium, which proves that the strain can produce ACC deaminase. Table 1. Comparison of absorbance of Streptomyces in ADF and DF media

[0026] 7. The purified Streptomyces S16-1 strain was inoculated onto R2A medium supplemented with different NaCl concentrations: 0.4 mol / L, 0.8 mol / L, 1.2 mol / L, 1.6 mol / L, and 2 mol / L. After incubation at 28°C in complete darkness for 5-7 days, the strain showed growth on all media, confirming its salt tolerance. 8. The purified Streptomyces S16-1 strain was inoculated onto R2A medium, which was prepared by adjusting the pH with NaHCO3 to pH 7, 7.5, 8, 8.5, and 9. After incubation at 28°C in complete darkness for 5-7 days, the strain was found to grow on all media, demonstrating its ability to grow in alkaline environments.

[0027] Example 3 Streptomyces S16-1 was transferred to liquid R2A medium and cultured on a shaker. All components of the culture medium (reagents) used for shaking culture were of analytical grade. Afterwards, the medium components were removed by centrifugation to obtain Streptomyces mycelium sludge. Figure 5 The image shows a Streptomyces microbial sludge. The Streptomyces microbial sludge was dissolved in sterile physiological saline to prepare a Streptomyces microbial solution. Sterilized alfalfa seeds and sheepgrass seeds were soaked in the Streptomyces microbial solution to coat the alfalfa seeds.

[0028] Example 4 Soil samples were collected from the grazing-prohibited area at the Sharqin Experimental Station of the Grassland Research Institute, Chinese Academy of Agricultural Sciences. Pot experiments were conducted in a greenhouse at the Grassland Research Institute, Chinese Academy of Agricultural Sciences. Coated / uncoated alfalfa seeds and coated / uncoated sheepgrass seeds prepared in Example 3 were cultured in sterile petri dishes. 50 seeds were placed in each petri dish to calculate the germination rate. Seven days after germination, seedlings with uniform growth were transplanted for pot experiments. Both groups of seeds were kept under identical environmental conditions. The germination rates of the two groups are shown in Table 2 below. Two variables were set for the pot experiments: inoculant and salinity. N represents no sodium chloride added, S represents 3‰ sodium chloride added, M represents alfalfa, and Y represents sheepgrass. Aboveground biomass, belowground biomass, and plant height were measured after 60 days. The results are shown in Tables 2 and 3. Figure 6 As shown in the figure. The results showed that inoculation with microbial fertilizer could improve the germination rate of alfalfa seeds, aboveground biomass, underground biomass, and plant height.

[0029] Table 2 Comparison of coated and uncoated seeds

[0030] Table 3 Comparison of coated and uncoated seeds

[0031] The results show that Streptomyces S16-1 seed coating agent significantly promoted growth and improved salinity resistance in both alfalfa and Leymus chinensis. However, under saline soil conditions, the germination rate of Leymus chinensis seeds increased by 12.67 percentage points (from 75.33% to 88.00%), significantly higher than that of alfalfa (6.66 percentage points from 78.67% to 85.33%). Furthermore, during normal growth, the germination rate of Leymus chinensis was far lower than that of alfalfa. Meanwhile, Leymus chinensis exhibited stronger adaptability in saline environments; the plant height, root length, and biomass of its coated plants were significantly higher than those of the uncoated control group, and all indicators maintained high growth potential under salt stress. In contrast, although alfalfa also showed significant improvement in saline soil, the absolute values ​​of its overall growth indicators were still significantly lower than those in ordinary soil, indicating that its improvement in salt stress tolerance is still somewhat limited. In particular, in terms of biomass accumulation, the biomass of Leymus chinensis in saline soil with coating treatment was significantly higher than that of the control group, and it still maintained a high biomass yield under salt stress; while alfalfa, although showing a significant increase, had a low baseline biomass value in saline soil, and its actual growth was still significantly inhibited by salt.

[0032] In summary, Streptomyces S16-1 seed coating agent showed significant growth-promoting and salinization-resistant effects on both alfalfa and Leymus chinensis, effectively improving seed germination rate, plant height, root length, and biomass. In comparison, Leymus chinensis showed a more significant effect from the coating treatment, indicating that this strain has superior application potential in improving the salinization adaptability of Leymus chinensis in saline-alkali soils.

[0033] Example 5 Soil samples were collected from a grazing-prohibited area at the Sharqin Experimental Station of the Grassland Research Institute, Chinese Academy of Agricultural Sciences. Pot experiments were conducted in a greenhouse at the Grassland Research Institute, Chinese Academy of Agricultural Sciences. Coated / uncoated alfalfa seeds and coated / uncoated sheepgrass seeds prepared in Example 3 were seedled in petri dishes. Seven days after germination, seedlings with uniform growth were transplanted for pot experiments. Both groups of seeds were ensured to be under the same environmental conditions. Two variables were set in the pot experiments: inoculant and salinity. N represents no sodium chloride added, S represents 3‰ sodium chloride added, M represents alfalfa, and Y represents sheepgrass. After 60 days, the soil contents of total nitrogen, organic carbon, available phosphorus, ammonium nitrogen, and nitrate nitrogen were measured, as follows: Figure 7 As shown in the figure. The results showed that inoculation with microbial fertilizer could increase the content of organic carbon, total nitrogen, ammonium nitrogen, and available phosphorus in the soil where alfalfa was planted.

[0034] Table 4. Effects of adding microbial fertilizer on different soil types for alfalfa cultivation

[0035] Table 5. Effects of adding microbial fertilizer on different soil types for planting Leymus chinensis

[0036] Through Tables 4 and 5 and Figure 7 The experimental results show that the Streptomyces S16-1 inoculant coating treatment significantly improved soil fertility in both alfalfa and sheepgrass plantings. Specifically, for alfalfa, the inoculant coating demonstrated comprehensive soil improvement advantages in saline-alkali soils. Compared with uncoated seeds, the coating treatment significantly increased the content of total nitrogen, organic carbon, and ammonium nitrogen in saline-alkali soils. Available phosphorus, in particular, was significantly increased in both soil types, with a more pronounced increase in saline-alkali soils. However, in ordinary soils, the coating treatment had little or no effect on improving total nitrogen and organic carbon, even showing a slight decrease, indicating that its fertility-enhancing effect may be limited under non-stress conditions.

[0037] For Leymus chinensis (Table 5), inoculant coating showed a better effect on improving soil fertility, especially in saline-alkali soils. The coating treatment caused the organic carbon content in saline-alkali soils to surge to 36.591 g / kg, far exceeding the uncoated treatment and any treatment in ordinary soils. Simultaneously, the available phosphorus content also reached its highest level. Notably, the soil organic carbon and available phosphorus content cultivated by the Leymus chinensis coating treatment in saline-alkali soils even exceeded the optimal values ​​for alfalfa under the same conditions, demonstrating extremely strong potential for improving saline-alkali soils.

[0038] Comprehensive analysis shows that the Streptomyces S16-1 inoculant coating can effectively improve the fertility of rhizosphere soil, especially for saline-alkali soils, where its effect is far superior to that of ordinary soils. Comparing two plants, the inoculant exhibits a better synergistic effect in the Leymus chinensis saline-alkali soil system, with a particularly significant increase in the accumulation of organic carbon and available phosphorus; while for alfalfa, the soil improvement is mainly reflected in the activation of nitrogen and phosphorus nutrients, indicating that the inoculant provided by this invention combined with Leymus chinensis may have greater application value in the rapid fertilization and ecological restoration of saline-alkali soils.

Claims

1. The application of a Streptomyces sp. S16-1 strain, characterized in that, This is used to prepare a seed coating agent to improve the growth of Leymus chinensis in high-salinity soil; the Streptomyces sp. S16-1 strain has the preservation number CCTCC M 20251940, and its 16S rDNA sequence is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The salinity of the high-salinity soil is 1‰-5‰.

3. The application according to claim 1, characterized in that, The Streptomycete sp. S16-1 is a Gram-positive bacterium that grows under both aerobic and anaerobic conditions.

4. The application according to claim 1, characterized in that, The Streptomycete sp. S16-1 possesses functional activities including salt tolerance, phosphorus solubilization, nitrogen fixation, and production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase.

5. A method for preparing the seed coating agent of claim 1, characterized in that, Streptomycete sp. S16-1 was cultured in an expanded culture medium, centrifuged to obtain bacterial sludge, and resuspended in sterile physiological saline to prepare a seed coating agent.

6. The method according to claim 5, characterized in that, The culture medium used for the expanded culture was R2A liquid medium, and the culture conditions were: temperature 28±0.5℃, pH 7.2±0.

2.

7. A microbial fertilizer, characterized in that, It was obtained by expanding the culture of Streptomycete sp. S16-1 as described in claim 1.

8. A method for promoting plant growth under salt stress, characterized in that, The microbial fertilizer of claim 8 is applied to plants or high-salinity soil in which plants grow, wherein the plant is sheepgrass.

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