Streptomycete and application thereof in improving salt resistance of pasture

Abstract

The invention relates to the technical field of microorganisms, in particular to a streptomyces strain and application thereof in improving salt resistance of pasture. The invention provides Streptomyces lamedown Qhu-M14, the Streptomyces lamedown Qhu-M14 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the Streptomyces lamedown Qhu-M14 is CGMCC No.4.8022. The Streptomyces lamedown Qhu-M14 is named as Qhu-M14. The Streptomyces lamedown Qhu-M14 is named as Qhu-M14. The strain belongs to a new species of streptomyces, and after the strain is applied to plants, the growth performance of plant biomass, root development, plant height and the like under the salt stress condition can be remarkably improved, and the salt tolerance of the plants is improved; meanwhile, the plant height growth performance of plants under normal conditions can be improved, plant growth is effectively promoted, strain resources are provided for development of growth-promoting microbial agents and salt-tolerant microbial agents, an efficient and low-cost solution is provided for saline-alkali soil plant planting, and good application prospects and actual values are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a strain of Streptomyces and application thereof in improving the salt resistance of forage. Background Art

[0002] With the continued growth of the global population and the accelerating rate of environmental degradation, the area of ​​salinized land has exceeded 800 million hectares, and this trend is expected to continue to expand, posing a serious threat to global agricultural productivity. Salinized soils typically have high salt contents, which have significant adverse effects on plant growth, manifesting as ion toxicity, membrane damage, osmotic stress, oxidative stress, impaired intracellular pH stability, and a significant decrease in root activity and photosynthetic efficiency. Faced with this challenge, improving agricultural productivity in salinized soils has become an urgent global issue that needs to be addressed.

[0003] Sainfoin and oats are suitable forage grasses for cultivation in the arid and semi-arid regions of Northwest China. They have moderate salt tolerance and are rich in protein, amino acids, and various minerals, making them highly palatable. The cultivation of these forage grasses not only helps to improve soil quality and agricultural productivity, but also effectively alleviates the competition between feed crops and food crops. However, the growth inhibition of salinized soils on plants significantly limits the production of sainfoin and oats. Although the resistance of these crops to salinization stress can be improved through genetic improvement and molecular breeding, these methods are usually time-consuming and costly. Therefore, there is an urgent need to develop innovative strategies to effectively improve their salt tolerance and stress resistance.

[0004] Plant growth-promoting rhizobacteria (PGPB), emerging eco-friendly microorganisms, have been shown to promote plant growth through bioactive substances such as auxin synthesis, phosphate solubilization, nitrogen fixation, and siderophore production. PGPB can also enhance plant stress tolerance by regulating plant redox status, hormone levels, and gene expression. As a cost-effective biotechnology tool, PGPB has shown great potential for enhancing plant adaptability to adverse environmental conditions such as salinity.

[0005] Streptomyces, one of the largest genera in the Actinobacteria phylum, produces a variety of bioactive secondary metabolites, including antimicrobials, phytohormones, and siderophores. These properties enable plants to withstand environmental stress. To date, there have been few reports on the use of Streptomyces in improving the growth and salt tolerance of oats and salinity. Therefore, developing novel plant growth-promoting Streptomyces strains and applying them to forage cultivation in saline soils is an important approach to improving soil quality and increasing agricultural production efficiency in saline-alkali lands. Summary of the Invention

[0006] The invention provides a Streptomyces strain and application thereof in improving the salt resistance of forage.

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

[0008] In a first aspect, the present invention provides Streptomyces eulerae ( Streptomyces olameadowicum ) Qhu-M14, which was deposited on March 3, 2025, at the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named Streptomyces olameadowicum , the deposit number is CGMCC No.4.8022.

[0009] The present invention provides Streptomyces eulerae ( Streptomyces olameadowicum ) Qhu-M14 is a new species of Streptomyces isolated from the soil of the Qinghai meadow habitat. It can improve the ability of plants to tolerate salt stress and has a good growth-promoting effect on plants. It can be used to develop plant growth-promoting or salt-tolerant bacterial agents.

[0010] In a second aspect, the present invention provides a microbial preparation comprising the above-mentioned Streptomyces eulerae ( Streptomyces olameadowicum )Qhu-M14.

[0011] Among the microbial preparations described above, Streptomyces eulerae ( Streptomyces olameadowicum ) Qhu-M14 is preferably present in the form of live bacteria.

[0012] The microbial preparation described above can be a liquid preparation or a solid preparation. The active ingredient of the microbial preparation comprises Streptomyces eulerae ( Streptomyces olameadowicum ) Qhu-M14; it may also contain excipients or carriers permitted in the field of microbial preparations. These excipients or carriers include, but are not limited to, lyoprotectants, biochar, diatomaceous earth, and natural organic materials (e.g., rice bran, soybean flour, soybean meal).

[0013] In a third aspect, the present invention provides a method for preparing the microbial preparation, comprising: Streptomyces olameadowicum ) Steps for culturing Qhu-M14 and obtaining a culture.

[0014] Preferably, the culture temperature is 25-35°C.

[0015] Preferably, the pH of the culture is 6.5-7.5.

[0016] Preferably, the culture medium used in the culture may be ISP2 culture medium or TSB culture medium.

[0017] In a fourth aspect, the present invention provides the Euler meadow Streptomyces ( Streptomyces olameadowicum ) Use of Qhu-M14 or the microbial preparation in improving plant salt tolerance or preparing a product for improving plant salt tolerance.

[0018] The above-mentioned improvement of plant salt tolerance includes improving any one or more of the following indicators of plants under salt stress conditions: biomass, plant height, root development, root length, water content, chlorophyll content, and photosynthesis efficiency.

[0019] In a fifth aspect, the present invention provides the Euler meadow Streptomyces ( Streptomyces olameadowicum ) Use of Qhu-M14 or the microbial preparation in promoting plant growth or preparing a product for promoting plant growth.

[0020] The aforementioned promotion of plant growth includes improving any one or more of the following plant indicators: biomass, plant height, root development, root length, water content, chlorophyll content, and photosynthesis efficiency.

[0021] Preferably, the promoting plant growth is promoting the growth of plants under normal conditions.

[0022] In the present invention, the plants include forage grasses, including but not limited to grasses of the grass family (such as oats, sheepgrass, ryegrass, and Sudan grass), leguminous grasses (such as sainfoin, alfalfa, clover, and sweet clover), and Asteraceae grasses (such as comfrey and sunflower).

[0023] In the present invention, the plants include grasses or legumes. The grasses include grass forages or crops (e.g., oats, wheat, barley, corn, rice, etc.); and the legumes include legume forages or crops (e.g., red beans, soybeans, mung beans, peanuts, chickpeas, broad beans, peas, lentils, etc.).

[0024] In a sixth aspect, the present invention provides a product comprising the Euler meadow Streptomyces ( Streptomyces olameadowicum ) Qhu-M14 or the microbial preparation.

[0025] Preferably, the product has any one or more of the following effects: (1) Improve plant salt tolerance; (2) Promote plant growth.

[0026] Wherein, improving the salt tolerance of plants includes improving any one or more of the following indicators of plants under salt stress conditions: biomass, plant height, root development, root length, water content, chlorophyll content, and photosynthesis efficiency.

[0027] The promoting of plant growth includes improving any one or more of the following indicators of the plant: biomass, plant height, root development, root length, water content, chlorophyll content, and photosynthesis efficiency.

[0028] In the present invention, the products include but are not limited to microbial agents, microbial fertilizers, soil conditioners, etc.

[0029] In a seventh aspect, the present invention provides a method for improving plant salt tolerance and / or promoting plant growth, the method comprising: applying the Streptomyces eulerae ( Streptomyces olameadowicum ) Qhu-M14 or the microbial preparation or the product.

[0030] Preferably, the application method includes watering around the rhizosphere of the plant.

[0031] The beneficial effects of the present invention include at least: the Streptomyces eulerae provided by the present invention ( Streptomyces olameadowicum ) Qhu-M14 is a new species of Streptomyces. After application to plants, it can significantly improve plant biomass, root development, plant height and other growth performance under salt stress conditions, and improve plant salt tolerance; at the same time, it can also improve plant height and other growth performance under normal conditions, effectively promote plant growth, provide beneficial bacterial resources for the development of growth-promoting and salt-tolerant agents, and propose an efficient and low-cost solution for saline-alkali land plant cultivation, with good application prospects and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 This is a phylogenetic tree of the 16S rRNA gene sequence of strain Qhu-M14 constructed based on the neighbor-joining method in Example 1 of the present invention.

[0034] Figure 2 The morphological characteristics of the strain Qhu-M14 in Example 1 of the present invention on different culture media; AG: ISP1~ISP7 culture media; H: Gao's medium No. 1; I: NA medium; J: Czapek medium.

[0035] Figure 3 The microstructure of the strain Qhu-M14 in Example 1 of the present invention; A: scanning electron microscopy (10000×); B: microscopic observation after phenol staining (100×).

[0036] Figure 4 This is the analysis of phospholipid components of strain Qhu-M14 in Example 1 of the present invention; A: ninhydrin staining; B: anisaldehyde staining; C: molybdophosphoric acid staining.

[0037] Figure 5 This is the genome map of strain Qhu-M14 in Example 1 of the present invention.

[0038] Figure 6 Effects of inoculation with Qhu-M14 on the growth phenotypes and physiological parameters of oat seedlings in Example 2 of the present invention; A: Growth phenotypes of oat seedlings inoculated with and without Qhu-M14 under normal and salt stress conditions; BD: Total biomass, aboveground biomass, and belowground biomass of oat seedlings inoculated with and without Qhu-M14, respectively; E, F: Plant height and root length of oat seedlings, respectively; GI: Relative water content, chlorophyll content (SPAD), and maximum photochemical quantum yield of PSⅡ in oat seedlings. NC: Normal control; NaCl: 200 mM NaCl treatment group; NC+M14: Plant seedlings inoculated with Qhu-M14 under normal conditions; NaCl+M14: Plant seedlings inoculated with Qhu-M14 under salt stress. Different lowercase letters indicate significant differences. P <0.05.

[0039] Figure 7 Effects of inoculation with Qhu-M14 on the growth phenotypes and physiological parameters of Sainfoin seedlings in Example 2 of the present invention; A, B: Growth phenotypes of Sainfoin seedlings inoculated with and without Qhu-M14 under normal and salt stress conditions; CE: Total biomass, aboveground biomass, and belowground biomass of Sainfoin seedlings inoculated with and without Qhu-M14, respectively; F, G: Plant height and root length of Sainfoin seedlings, respectively; HJ: Relative water content, chlorophyll content (SPAD), and maximum photochemical quantum yield of PSⅡ of Sainfoin seedlings. NC: Normal control; NaCl: 200 mM NaCl treatment group; NC+M14: Plant seedlings inoculated with Qhu-M14 under normal conditions; NaCl+M14: Plant seedlings inoculated with Qhu-M14 under salt stress. Different lowercase letters indicate significant differences. P <0.05. DETAILED DESCRIPTION

[0040] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0041] Similar model strains used in the following examples Streptomyces avidinii NBRC 13429 T and Streptomyces cirratus NRRL B-3250 T Purchased from China General Microbiological Culture Collection Center.

[0042] Example 1 Isolation and polyphasic taxonomic identification of strain Qhu-M14 The present invention isolated a Streptomyces strain from the soil of a Qinghai meadow habitat (N35°13.4752', E101°28.7727'), and named it Qhu-M14.

[0043] The strain Qhu-M14 was subjected to polyphasic taxonomic identification. The specific experimental methods and results are described below.

[0044] 1. Experimental methods (1) Observation of strain morphological characteristics Test culture medium: ISP1 medium: 10.0 g tryptone, 5.0 g yeast extract powder, 20.0 g agar, dissolved in 1 L ddH2O, pH = 7.2; ISP2 medium: 4.0 g yeast extract powder, 4.0 g glucose, 10.0 g malt extract powder, 20.0 g agar, dissolved in 1 L ddH2O, pH = 7.2; ISP3 medium: 20.0 g oatmeal, 1.0 g NaCl, 0.001 g FeSO₄·7H₂O, 0.001 g MnCl₂·4H₂O, 0.001 g ZnSO₄·7H₂O, 20.0 g agar, dissolved in 1 L ddH₂O, pH = 7.2; ISP4 medium: soluble starch 10.0 g, K2HPO4 1.0 g, NaCl 1.0 g, (NH4)2SO4 2.0 g, CaCO3 2.0 g, MgSO4·7H2O 1.0 g, FeSO4·7H2O 0.001 g, MnCl2·4H2O 0.001 g, ZnSO4·7H2O 0.001 g, agar 20.0 g, dissolved in 1 L ddH2O, pH = 7.2; ISP5 medium: 10.0 g glycerol, 1.0 g L-asparagine, 1.0 g K2HPO4, 0.001 g FeSO4·7H2O, 0.001 g MnCl2·4H2O, 0.001 g ZnSO4·7H2O, 20.0 g agar, dissolved in 1 L ddH2O, pH = 7.2; ISP6 medium: 36.0 g peptone yeast extract iron agar, 1.0 g yeast extract, 20.0 g agar, dissolved in 1 L ddH2O, pH = 7.2; ISP7 medium: 15.0 g glycerol, 1.0 g L-asparagine, 0.5 g L-tyrosine, 0.5 g NaCl, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.001 g FeSO4·7H2O, 0.001 g MnCl2·4H2O, 0.001 g ZnSO4·7H2O, 20.0 g agar, dissolved in 1 L ddH2O, pH = 7.2; Gao's medium No. 1: soluble starch 20.0g, NaCl 0.5g, KNO3 1.0g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, agar 15.0g, dissolved in 1L ddH2O, pH = 7.2 NA medium: 3.0 g beef extract, 5.0 g peptone, 2.5 g glucose, 18.0 g agar, dissolved in 1 L ddH2O, pH = 7.2; Czapek medium: sucrose 30.0 g, NaNO3 3.0 g, KCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, K2HPO4 1.0 g, agar 15.0 g, dissolved in 1 L ddH2O, pH = 7.2.

[0045] The strains were streaked onto ISP1–ISP7, Gao's No. 1, NA, and Czapek medium and cultured at 28°C for 7 days for morphological analysis and observation. Ultrastructure of the strains was observed under a scanning electron microscope using the insert method on ISP2 medium at 28°C for 7 days.

[0046] (2) Analysis of physiological and biochemical characteristics of strain Qhu-M14 The Qhu-M14 strain seed liquid was inoculated into TSB medium at 5% volume and cultured at 5-40°C with shaking at 180 rpm for 7 days. The strain Qhu-M14 was also cultured at 28°C and 180 rpm for 7 days under conditions of pH 3.0-12.0 and sodium chloride concentration 0-10% to evaluate its tolerance to temperature, pH, and salinity. To demonstrate the carbon and nitrogen source utilization abilities of the strain, Qhu-M14 was inoculated into ISP9 carbon utilization medium (1% carbon source, (NH₄)₂SO₄ 2.64 g, KH₂PO₄ 2.38 g, K₂HPO₄ 4.31 g, MgSO₄ 0.49 g, FeSO₄·7H₂O 0.001 g, MnCl₂·4H₂O 0.001 g, ZnSO₄·7H₂O 0.001 g, and agar 15.0 g, dissolved in 1 L ddH₂O, pH 7.2) and nitrogen utilization medium (1% nitrogen source, glucose 1.0 g, K₂HPO₄ 1.0 g, MgSO₄·7H₂O 0.5 g, NaCl 0.5 g, FeSO₄·7H₂O 0.01 g, and agar 20.0 g, dissolved in 1 L ddH₂O, pH 7.4) and cultured at 28°C for 7 d. Catalase, urease, cellulose hydrolysis, H2S production, and starch hydrolysis were tested according to the "Rapid Identification and Systematic Classification of Actinomycetes." Furthermore, the physicochemical properties of alkaline phosphatase, esterase (C4), lipase (C8), trypsin, chymotrypsin, and α-galactosidase were further tested according to the API ZYM kit instructions.

[0047] (3) Analysis of cytochemical classification characteristics of strain Qhu-M14 A 5% volume of Qhu-M14 seed solution was inoculated into TSB medium and cultured at 28°C and 180 rpm for 7 days. The cells were harvested by centrifugation at 4°C and 6000 rpm for 10 min and freeze-dried. Fifty mg of freeze-dried cells were weighed and hydrolyzed at 121°C with 100 μL of 0.5 mol / L HCl and 100 μL of 6 mol / L HCl, respectively. The cells were then separated and chromatographed on microcrystalline cellulose plates using a developing system of ethyl acetate:pyridine:glacial acetic acid:water (8:5:1:1.5, volume ratio) and methanol:pyridine:glacial acetic acid:water (5:0.5:0.125:2.5, volume ratio). The hydrolyzed sugar and amino acid compositions of the whole cells were analyzed. Weigh 100 mg of freeze-dried cells, grind them, add 15 mL of methanol and boil in water for 10 min. After cooling, add 10 mL of chloroform and an appropriate amount of 2% sodium chloride solution until the layers are separated. The organic phase is concentrated at 37 °C and dissolved in chloroform:methanol = 2:1 solution. 254Two-phase thin-layer chromatography (DLC) on silica gel plates (developing system: chloroform:methanol:water = 65:25:4; chloroform:acetic acid:methanol:water = 80:15:12:4, volume ratio) was performed to analyze the phospholipid composition. 100 mg of lyophilized cells were weighed and ground, then added to 40 mL of a 2:1 solution of chloroform:methanol, and shaken at 180 rpm overnight. The supernatant was concentrated to dryness under reduced pressure at 40°C, dissolved in 0.5 mL of acetone, and then added to GF. 254 The silica gel plate was developed with n-hexane: ether = 85:15 (volume ratio) and the relative mobility was measured by scraping under 254 nm UV light. R f =0.8 dark brown band, which was then dissolved in 0.5 mL of acetone and filtered through a 0.22 μm microporous filter before LC-MS / MS analysis. The polar lipid composition of the lyophilized cells was determined using the MIDI Sherlock Microbial Identification System (version 6.0B).

[0048] (4) Whole genome sequencing and bioinformatics analysis of strain Qhu-M14 Strain Qhu-M14 was streaked onto ISP2 medium and cultured at 28°C for 3 days. Single colonies were then picked and transferred to TSB medium. After 3 days of culture at 28°C and 180 rpm, the cells were harvested by centrifugation at 6000 rpm for 10 minutes and washed three times with sterile water. The Qhu-M14 genome was extracted using the CTAB method and used as a template for 16S rRNA gene amplification. The bacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′) were used as 16S rRNA gene amplification primers. The PCR reaction system consisted of 27F and 1492R primers (10 μmol L -1), 0.5 μL each of PCR products, 12.5 μL of TaKaRa Taq™ HotStart High-Fidelity DNA Polymerase (0.05 U / μL), 2 μL of template DNA (80 ng / μL), and 9.5 μL of ddH2O, for a total volume of 25 μL. The reaction protocol was as follows: denaturation at 95°C for 3 minutes, 34 cycles of denaturation at 95°C for 45 seconds, annealing at 56°C for 45 seconds, and extension at 72°C for 90 seconds, followed by extension at 72°C for 10 minutes. After PCR product purification and sequencing, the sequences were aligned in the EzBioCloud database, and sequences of highly similar model strains were downloaded. A phylogenetic tree was constructed using the neighbor-joining method using MEGA 11.0 software, with a bootstrap value of 1,000. Qhu-M14 whole-genome sequencing was performed by Beijing Biomike Biotechnology Co., Ltd. on the PacBio sequencing platform. After sequencing, low-quality and short reads were filtered and assembled using Canu v1.5 software. Gene prediction for the strain was then performed using Prodigal v2.6.3. The assembled and predicted genome information was then used to visualize the genome using Circos v0.66. The dDDH values ​​for Qhu-M14 and its top four most similar strains were calculated using the GGDC website (https: / / ggdc.dsmz.de / ). The average nucleotide identity (ANI) was calculated using pairwise BLAST analysis using JSpecies. The genomic similarity between Qhu-M14 and similar bacterial species was further analyzed to determine whether it was a new species.

[0049] 2. Experimental results (1) Molecular identification of strain Qhu-M14 based on 16S rRNA gene sequence The gene sequence obtained by 16S rRNA gene amplification and sequencing was 1526 bp in size. The 16S rRNA gene sequence was compared with the model strain sequence in the EzBioCloud database and found to be similar to Streptomyces Streptomyces avidinii NBRC 13429 T 、 S. cirratus NRRL B-3250 T and S. vinaceus NBRC 13425 TThe highest similarity is 98.51%, 98.37% and 98.37% respectively, all lower than 98.65% (Mincheol Kim, Hyun-Seok Oh, Sang-Cheol Park and Jongsik Chun. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology, 2014, 64, 346-351.), which may be a potential new species of Streptomyces. In addition, the phylogenetic tree constructed using the neighbor-joining method shows ( Figure 1 ), Qhu-M14 and S. pratensis ch24 T The distance is the closest and they are clustered into one branch, indicating that Qhu-M14 belongs to Streptomyces.

[0050] (2) Morphological analysis of strain Qhu-M14 The strain Qhu-M14 grew well on ISP1~7 medium, Gao's No. 1, NA, and Cha's medium ( Figure 2 After 7 days of culture on ISP2 medium, the colonies of strain Qhu-M14 were beige, dry, wrinkled, and had neat edges. White spores were produced on ISP3, ISP4, and Czapek medium. Scanning electron microscopy revealed that the mycelium of strain Qhu-M14 was a filamentous cylinder ( Figure 3 A). After carbolic acid staining, it was found that the spores of the strain were straight and the spores were cylindrical ( Figure 3 B).

[0051] (3) Physiological and biochemical characteristics of strain Qhu-M14 The results of tolerance analysis showed that Qhu-M14 has an optimal growth temperature of 30°C, a maximum NaCl tolerance of 5%, an optimal growth pH of 7.0, and a similar growth temperature to the most similar strains (Table 1). S. avidinii NBRC 13429 T and S. cirratus NRRLB-3250 TIt also exhibits similar growth characteristics. Carbon source utilization experiments revealed that strain Qhu-M14 can utilize D-mannitol, rhamnose, D-galactose, inositol, fructose, D-arabinose, xylose, ribose, sorbitol, ulose, mannose, and glucose. Regarding nitrogen source utilization, the strain can utilize threonine, arginine, tyrosine, alanine, proline, aspartic acid, serine, glutamic acid, and glycine to maintain growth. Furthermore, Qhu-M14 exhibits lipase activity but lacks the ability to produce H2S or hydrolyze starch. API ZYM test further revealed that the bacteria had activities of alkaline phosphatase, lipase (C4), lipase-like enzyme (C8), leucine arylaminease, valine arylaminease, trypsin, cystine arylaminease, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and α-glucosidase, while the activities of lipase-like enzyme (C14), α-galactosidase, β-galactosidase, β-uronidase, β-glucosidase, N-acetyl-glucosaminidase, α-mannosidase and α-fucosidase were negative.

[0052] By comparing the physiological and biochemical characteristics of the most similar model strain, it was found that the Qhu-M14 strain had certain differences in carbon source utilization and starch hydrolysis. In terms of carbon source utilization, the Qhu-M14 strain was able to utilize D-mannitol and inositol, while S. avidinii NBRC 13429 T and S. cirratus NRRL B-3250 T Cannot be used, and compared to Qhu-M14 and S. cirratus NRRL B-3250 T , S. avidinii NBRC 13429 T Cannot utilize D-arabinose and fructose; in terms of starch hydrolysis, Qhu-M14 S. avidinii NBRC 13429 T There is no corresponding activity, and S. cirratus NRRL B-3250 T Able to hydrolyze starch. In terms of other enzyme activities, Qhu-M14 does not have the activities of lipase (C14), β-galactosidase, N-acetyl-glucosaminidase and α-mannosidase, while similar strains S. avidinii NBRC 13429 T and ​ NRRL B-3250 T Have corresponding activity.

[0053] Table 1. Comparison of physiological and biochemical characteristics between strain Qhu-M14 and the most similar model strain

[0054] Note: +: positive; −: negative.

[0055] (4) Chemical classification characteristics of strain Qhu-M14 TLC analysis of the whole cell hydrolysate of strain Qhu-M14 revealed that the hydrolyzed sugars were glucose and galactose, and the amino acid was LL-2,6-diaminopimelate [(2S,6S)-2,6-Diaminoheptanedioic acid, LL-DAP]. Based on LC-MS analysis of the isopentenylquinone species of strain Qhu-M14, the molecular ion peak [M+H] + The isopentenylquinone is MK-9 (H8). The phospholipids ( ​ ), mainly including phosphatidylethanolamine (PE), which is red when stained with ninhydrin and blue when stained with molybdophosphate; phosphatidylinositol mannosides (PIM), which is green when stained with anisaldehyde; and phosphatidyl glycerol (DPG), which is blue when stained with anisaldehyde and blue when stained with molybdophosphate. The polar lipid components of the strain are mainly Antieiso-C 15:0 (36.32 %)、iso-C 15:0 (16.02 %), C 16:0 (13.64 %)、iso-C 16:0 (8.26 %)、Antiieiso-C 17:0 (6.36 %)、iso-C 17:0 (3.93%), iso-C 14:0 (3.02 %) and C 14:0 (1.49 %), etc.

[0056] (5) Whole genome sequencing analysis of strain Qhu-M14 Whole-genome sequencing on the PacBio sequencing platform revealed a Qhu-M14 genome size of 7.56 Mb with a GC content of 72.58%, consisting of two scaffolds. Scaffod 1 is a bacterial chromosomal gene with a length of 7,468,233 bp, while Scaffod 2 is a bacterial plasmid genome with a size of 96,149 bp. Prodigal v2.6.3 software predicted a total of 6,812 coding genes with a total length of 6,550,323 bp, accounting for 86.59% of the genome ( ​ ).

[0057] Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analysis revealed that strain Qhu-M14 was most similar to the strain ​ NBRC 13429 T The dDDH value and ANI value of Qhu-M14 were the highest, at 40.20% and 85.12%, respectively (Table 2), but both were lower than the threshold values ​​of 70% and 95% for distinguishing new bacteria by dDDH and ANI (TIEDJE JM. DNA-DNA hybridization values ​​and their relationship to whole-genome sequences similarities. International Journal of Systematic and EvolutionaryMicrobiology, 2007, 57(Pt 1):81-91;JAIN C, RODRIGUEZ-R LM, PHILLIPPY AM,KONSTANTINIDIS KT, ALURU S. High throughput ANI analysis of 90K prokaryoticgenomes reveals clear species boundaries. Nature Communications, 2018, 9:51-14), which further indicated that the strain Qhu-M14 belonged to a new species of Streptomyces. Based on the above polyphasic taxonomic data, the strain was named Streptomyces eulerae ( ​ ), the model strain is Qhu-M14.

[0058] Table 2. Comparison of genomic similarity between strain Qhu-M14 and the most similar model strain

[0059] Streptomyces eulerae ( ​ ) Qhu-M14 was deposited on March 3, 2025, at the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified as ​ ​ , the deposit number is CGMCC No.4.8022.

[0060] Example 2 Evaluation of plant growth promotion and salt resistance of strain Qhu-M14 1. Experimental methods Qhu-M14 was streaked onto ISP2 medium and cultured at 28°C for 3 days. A single colony was picked and cultured in TSB medium at 28°C and 180 rpm for 5 days. The cells were collected by centrifugation at 5000 rpm for 10 min and then resuspended in sterile deionized water. The bacterial suspension was adjusted to 1.0×10 8 cfu / mL was used for inoculation experiments.

[0061] Full-grained oats and sainfoin seeds were selected and sterilized with 2% sodium hypochlorite for 5 minutes and 75% ethanol for 45 seconds. After rinsing five times with sterile water, they were sown in round pots (soil matrix consisting of a 1:1:1 mixture of peat, vermiculite, and sand). Five sainfoin seeds and seven oats seeds were sown per pot. Water was applied every three days under a 16-hour light (8000 lux) / 8-hour dark condition at a temperature of 22-25°C. Four potted plant groups were used: a normal culture group (NC), a normal culture seedling inoculated with Qhu-M14 (NC+M14), a salt stress group (NaCl), and a salt-stressed plant seedling inoculated with Qhu-M14 (NaCl+M14). After seed germination, oats and sainfoin were grown for 7 and 24 days, respectively. 30 mL of bacterial suspension was applied to the rhizosphere of the NC+M14 and NaCl+M14 treatments, respectively. An equal volume of sterile deionized water was also applied simultaneously to the NC and NaCl treatments. The rhizosphere was inoculated once every other day. After these two inoculations, 50 mL of a 200 mM NaCl solution was applied to the NaCl and NaCl+M14 treatments, respectively, every other day. Plants were photographed and observed for 10 and 14 days after salt stress, respectively. Plant tissues were collected and analyzed for relevant physiological parameters. Plant height and root length were measured using a tape measure (n = 10) for each treatment group. Total plant biomass, aboveground biomass, and root weight were weighed using a 1 / 10,000 scale. Three seedlings were randomly selected as a replicate, with three replicates for each parameter. 0.2 g of expanded leaf samples of the same plant height were oven-dried at 105°C for 15 minutes and then dried at 80°C to constant weight. The samples were weighed and relative water content was calculated as follows: relative water content (%) = (FW-DW) / (TW-DW) × 100, where FW, TW, and DW represent fresh weight, saturated fresh weight, and dry weight, respectively. The maximum photochemical efficiency of PS II (Fv / Fm) in leaves of the same tissue site in the different treatment groups was measured using a chlorophyll fluorometer (FluorPen FP110). Chlorophyll content was measured using a chlorophyll content meter (SPAD 502 Plus). Three biological replicates were used for each experiment.

[0062] 2. Experimental results Further evaluation of new Streptomyces species through potting and inoculation experiments ​Qhu-M14 improves salt tolerance and promotes growth of oats and legumes. For oats, salt stress severely inhibits seedling growth ( ​ Compared with the normal group (NC), the total biomass, aboveground biomass, underground biomass and plant height were significantly reduced by 26.0%, 23.0%, 40.4% and 27.9% ( ​ BE), while the total biomass, aboveground biomass and plant height of oat seedlings inoculated with Qhu-M14 (NC+M14) increased by 24.8%, 29.7% and 8.8%, respectively, while the underground biomass did not change significantly ( ​ Compared with the salt stress NaCl group, the total biomass, aboveground biomass, root weight and plant height of oat seedlings in the inoculated group (NaCl+M14) increased by 40.0%, 33.2%, 80.8% and 29.2%, respectively, and returned to the level of the normal culture group ( ​ BE). Under salt stress, the root development of oat seedlings was significantly inhibited, and the root length decreased by 31.5%. However, the inoculation of bacterial agent significantly increased the root length of oat seedlings under salt stress by 64.5% ( ​ In addition, the relative water content and chlorophyll content of oat seedlings in the salt stress treatment group decreased significantly by 13.6% and 22.9%, respectively. Compared with the salt stress group, the relative water content and chlorophyll content of oat seedlings in the inoculated microbial agent group were lower than those in the normal culture group, but significantly increased by 7.2% and 20.9%, respectively. The maximum photochemical efficiency of PS II, Fv / Fm, also returned to the level of the normal group ( ​ GI), indicating that Qhu-M14 promoted photosynthesis in oat seedlings under salt stress.

[0063] In summary, the new species of Streptomyces ​ Qhu-M14 not only promotes the growth of oats, but also improves the tolerance of oats to salt stress by improving their physiological indicators under salt stress.

[0064] For evaluation ​ Whether Qhu-M14 also has the effect of promoting growth and resisting salt on legume forage, the effects of Qhu-M14 on the growth phenotype and physiological characteristics of red clover under salt stress were observed. ​ The results showed that under salt stress, the growth of Sainfoin seedlings was significantly inhibited, with leaves wilting and yellowing, and the plants becoming short. However, after inoculation with Qhu-M14, the phenotype of Sainfoin seedlings recovered ( ​A, B). When not inoculated with the fungus, salt stress reduced the biomass of Sainfoin seedlings by 12.7% compared to the normal culture group. However, after inoculation with Qhu-M14, the total biomass and aboveground biomass of Sainfoin seedlings in the normal (NC) and salt stress (NaCl) groups were similar to those of uninoculated Sainfoin seedlings under normal conditions, with no significant differences. However, the underground biomass of the salt stress group inoculated with the fungus decreased ( ​ Compared with the NaCl stress group, the total biomass and aboveground biomass of the seedlings in the salt stress inoculation group increased by 26.5% and 57.2% ( ​ C, D). Under normal culture conditions, the plant height of the inoculated group (NC+M14) of Sainfoin seedlings was significantly higher than that of the uninoculated normal group, increasing by 19.8%. However, under salt stress, the seedling height of the NaCl+M14 group inoculated with the fungus did not differ significantly from that of the normal group. However, compared with the normal culture group, the plant height under NaCl stress was significantly reduced by 17.6%, while Qhu-M14 increased the plant height under salt stress by 27.2% ( ​ In addition, compared with the NC group, salt stress inhibited the root development of Sainfoin seedlings, while the root length of plants inoculated with Qhu-M14 was reversed to the level of the normal group, and the root length of seedlings under salt stress increased by 7.8% ( ​ In terms of relative water content and photosynthesis, the relative water content, chlorophyll content, and maximum photochemical efficiency of PS II (Fv / Fm) of Sainfoin seedlings in the NaCl stress group decreased significantly by 35.7%, 29.3%, and 9.7% compared with the NC group. However, after inoculation with Qhu-M14, these physiological indicators were significantly improved. Compared with the NaCl treatment group, Qhu-M14 increased the relative water content, chlorophyll content, and chlorophyll fluorescence (Fv / Fm) of Sainfoin seedlings by 31.6%, 12.6%, and 8.1% ( ​ These changes in phenotypic and physiological parameters indicate that Qhu-M14 can also alleviate salt stress and promote the growth and development of Sainfoin seedlings, showing a certain degree of universality.

[0065] In summary, the present invention used a polyphasic taxonomic method to identify a strain of Streptomyces isolated from alpine meadow soil and determined that the strain was a new species of Streptomyces, which was named Streptomyces eulerae ( ​ ​ ) Qhu-M14. Furthermore, plant growth promotion and salt tolerance evaluations revealed that Qhu-M14 significantly promoted growth and enhanced salt stress tolerance in both the grass family oat and the legume sainfoin. This suggests that this inoculant has a certain degree of universality in promoting growth and salt tolerance across forage grasses, and is of great significance for the development of new salt-tolerant inoculants and the efficient cultivation of forage grasses in saline-alkali soils.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Streptomyces eulerae ( Streptomyces olameadowicum ) Qhu-M14, characterized by, It is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 4.8022.

2. A microbial preparation, characterized in that: The microbial preparation comprises the Streptomyces eulerae described in claim 1 ( Streptomyces olameadowicum )Qhu-M14.

3. The method for preparing the microbial preparation according to claim 2, characterized in that: The method comprises the steps of: Streptomyces olameadowicum ) Steps for culturing Qhu-M14 and obtaining a culture.

4. Streptomyces eulerae according to claim 1 ( Streptomyces olameadowicum ) Use of Qhu-M14 or the microbial preparation according to claim 2 in improving plant salt tolerance or preparing a product for improving plant salt tolerance.

5. The use according to claim 4, characterized in that Improving the salt tolerance of plants includes improving any one or more of the following indicators of plants under salt stress conditions: biomass, plant height, root development, root length, water content, chlorophyll content, and photosynthesis efficiency.

6. Streptomyces eulerae according to claim 1 ( Streptomyces olameadowicum ) Use of Qhu-M14 or the microbial preparation according to claim 2 in promoting plant growth or preparing a product for promoting plant growth.

7. The use according to claim 6, characterized in that The promoting of plant growth includes improving any one or more of the following indicators of the plant: biomass, plant height, root development, root length, water content, chlorophyll content, and photosynthesis efficiency.

8. The use according to any one of claims 4 to 7, characterized in that The plants include forage grasses; And / or, the plant comprises a grass plant or a leguminous plant.

9. A product, characterized in that The product contains the Euler meadow Streptomyces according to claim 1 ( Streptomyces olameadowicum ) Qhu-M14 or the microbial preparation of claim 2; Preferably, the product has any one or more of the following effects: (1) Improve plant salt tolerance; (2) Promote plant growth.

10. A method for improving plant salt tolerance and / or promoting plant growth, characterized in that: The method comprises: applying the Euler meadow Streptomyces ( Streptomyces olameadowicum ) Qhu-M14 or the microbial preparation according to claim 2 or the product according to claim 9.

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