Synthetic flora with effect of promoting plant growth and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a synthetic flora with an effect of promoting plant growth and application of the synthetic flora. In the synthetic flora provided by the invention, both the chaetococcus RB13 (the preservation number is CGMCC No.37455) and the pseudomonas 5asv2 (the preservation number is CGMCC No.37454) are indigenous microorganisms, are safe and pollution-free to the environment, do not generate any harmful residue in the application process, and do not damage the balance of the original soil microbial community. Meanwhile, due to good adaptability and high growth promoting efficiency to low-temperature stress, the high-temperature-resistant plant growth promoting agent can be widely applied to agricultural production in high and cold areas, and the problems that crops in the plateau cold areas grow slowly and the yield is low are solved. A new technical approach is provided for agricultural sustainable development of the Qinghai-Tibet Plateau and even other high-cold and high-altitude areas, and the method has important significance for guaranteeing plateau grain safety and ecological environment.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a synthetic microbial community that promotes plant growth and its applications. Background Technology

[0002] Wetlands, as one of the three major ecosystems, are vital to human survival and development, providing essential biodiversity and ecosystem services, and playing a key role in hydrological regulation, biogeochemical cycles, and biodiversity conservation. The Qinghai-Tibet Plateau, known as the Earth's "Third Pole" and "Asia's Water Tower," is highly sensitive to global climate change, serving as a magnifying glass for global climate change. Due to its unique extreme environment of low temperatures, strong radiation, drought, and high salinity, the alpine wetlands of the Qinghai-Tibet Plateau have nurtured a diverse and unique array of microbial resources, considered a "repository" of ancient extreme-environment microorganisms. These microorganisms possess unique metabolic characteristics and extremely high scientific research value.

[0003] Currently, the exploration and utilization of soil microbial resources in typical wetlands of the Qinghai-Tibet Plateau remain relatively limited. Decoding the functional genes of these microbial strains, revealing the genetic characteristics of the microbial communities, and establishing a systematic germplasm resource bank of soil microorganisms in Qinghai-Tibet Plateau wetlands will help protect microbial diversity in climate-sensitive areas and provide a theoretical basis for exploring the special characteristics, adaptation mechanisms, and evolutionary history of life under extreme environmental conditions. Highland barley (barley from the plateau) is one of the main food crops in the Qinghai-Tibet Plateau region, growing year-round in a high-altitude, oxygen-deficient, and infertile environment. For a long time, local agricultural production has faced problems such as slow crop growth due to high temperatures and low soil nutrient availability. Although some growth-promoting microbial agents are currently used to increase crop yields, most are derived from microorganisms in plains areas with normal temperatures, and their activity is often insufficient in the extreme environment of the low temperature and high altitude of the Qinghai-Tibet Plateau, resulting in unsatisfactory effects. Therefore, it is urgent to screen native microorganisms from the special environment of the Qinghai-Tibet Plateau to construct functional microbial communities adapted to the high-altitude environment and with significant growth-promoting effects, to improve the growth and yield of crops such as highland barley. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a synthetic microbial community that promotes plant growth and its application. The synthetic microbial community provided by this invention can stably colonize the rhizosphere of barley under low-temperature conditions (10℃–20℃), enhancing the nutrient turnover and supply capacity of the rhizosphere, thereby promoting root development and driving plant growth.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a synthetic microbial community that promotes plant growth, including *Trichophyton mentagrophytes* (…). Trichococcus sp.) RB13 and Pseudomonas (sp.) RB13 and Pseudomonas ( Pseudomonassp.) 5asv2; the preservation number of the *Trichophyton spp.* RB13 is CGMCC No. 37455, and the preservation number of the *Pseudomonas 5asv2* is CGMCC No. 37454.

[0006] Preferably, the ratio of viable bacteria of Trichophyton rubrum RB13 to Pseudomonas 5asv2 in the synthetic bacterial community is 0.5 to 2:1.

[0007] This invention provides a compound microbial agent, the effective components of which include the synthetic microbial community described in the above technical solution.

[0008] Preferably, the dosage form of the compound microbial agent includes a liquid formulation or a solid formulation; when the dosage form of the compound microbial agent is a liquid formulation, the viable count of the compound microbial agent is ≥1×10⁻⁶. 8 CFU / ml; when the compound microbial agent is in solid dosage form, the viable count of the compound microbial agent is ≥1×10⁻⁶. 8 CFU / g.

[0009] This invention provides a method for preparing the compound microbial agent described in the above technical solution, comprising: After activating Trichophyton rubrum RB13 and Pseudomonas 5asv2, they were inoculated into a culture medium and cultured to the logarithmic growth phase; the culture medium included tryptic soy peptone liquid medium.

[0010] Preferably, the culture temperature is 25-30℃.

[0011] This invention provides the application of the synthetic microbial community or the compound microbial agent described in the above-mentioned technical solutions in one or more of 1)-3): 1) Promotes plant growth; the plants include barley; 2) Improve soil fertility; 3) Enhance the activity of rhizosphere soil enzymes; the rhizosphere soil enzymes include one or more of the following: urease, alkaline phosphatase, alkaline protease, β-glucosidase, α-glucosidase and β-galactosidase.

[0012] Preferably, promoting plant growth includes promoting plant growth in a low-temperature environment; the temperature range of the low-temperature environment is 10℃–20℃.

[0013] The present invention provides a method for promoting plant growth, comprising: applying the synthetic microbial community or the compound microbial agent described in the above technical solution to the rhizosphere region of the plant.

[0014] Preferably, the viable count of the synthetic microbial community in the plant rhizosphere after application is ≥1×10⁻⁶. 8 CFU / g soil.

[0015] Compared with the prior art, the present invention has the following advantages: 1) Strong adaptability to high altitude and cold, with significant growth-promoting effect: The synthetic microbial flora provided by this invention, including *Trichophyton spp.* RB13 and *Pseudomonas spp.* 5asv2, both originate from high-altitude wetlands above 4400 meters and are tolerant of harsh environments such as low temperatures. Even at low temperatures (e.g., around 10℃), the synthetic microbial flora they form maintains high activity, effectively promoting barley growth. Greenhouse pot experiments showed that, compared to the uninoculated control (CK), inoculation with the synthetic microbial flora of this invention (RB13+5asv2) significantly promoted the root growth and development of barley: the total root length increased from 917.19 cm to 1694.90 cm, an increase of approximately 84.79%; the root surface area increased from 302.06 mm² / cm² to 1694.90 cm. 2 Increased to 444.78 mm 2 The root system volume increased by approximately 47.25%; the total root volume increased from 8.76 cm³. 3 Increased to 11.02 cm 3 The average root diameter increased by approximately 25.80% from 0.86 mm to 1.04 mm, an increase of approximately 20.93%; and the root weight increased by approximately 16.63% from 9.98 g to 11.64 g. These improvements were significantly greater than those achieved by single-strain inoculation in terms of total root length, surface area, volume, and root biomass (the root length increase from RB13 or 5asv2 alone was approximately 34%–52%), demonstrating a synergistic effect between the two strains (the root length of the synthetic community was further increased by approximately 21% compared to the optimal single strain).

[0016] 2) Improved soil nutrient availability and health: After applying the compound microbial agent prepared from the synthetic microbial community of this invention, the activities of various enzymes in the rhizosphere soil of barley were significantly enhanced, which is beneficial to soil nutrient transformation and supply. Specifically, soil urease activity was significantly increased (approximately 567% higher than the control), indicating accelerated nitrogen mineralization and release in the soil; alkaline phosphatase activity increased by approximately 46.4%, promoting the mineralization of organic phosphorus; protease activity increased by approximately 18%, β-glucosidase by an average of approximately 25.4%, and β-galactosidase by approximately 8%, indicating that soil carbon and nitrogen cycle-related processes were enhanced. In summary, the microbial community of this invention not only promotes plant growth but also enhances the activity of key mineralizing enzymes and nutrient transformation functions in the rhizosphere soil, providing sustainable ecological benefits.

[0017] 3) Environmentally Friendly and with Broad Application Prospects: The synthetic microbial community provided by this invention includes *Trichophyton rubrum* RB13 and *Pseudomonas 5asv2*, both of which are indigenous microorganisms. They are environmentally safe and pollution-free, producing no harmful residues during application and not disrupting the original soil microbial community balance. Furthermore, due to their excellent adaptability to low-temperature stress and high growth-promoting efficiency, they can be widely applied to agricultural production in high-altitude and cold regions, solving the problems of slow growth and low yields of crops in these areas. This invention provides a new technological approach for the sustainable agricultural development of the Qinghai-Tibet Plateau and other high-altitude and cold regions, and is of great significance for ensuring food security and the ecological environment of the plateau.

[0018] Biological Preservation Instructions Trichophyton RB13, classified as Trichococcus sp. was deposited on January 19, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 37455.

[0019] Pseudomonas 5asv2, classified as Pseudomonas sp. was deposited on January 19, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 37454. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 The images show the colony morphology and SEM images of the strains screened in Example 1 of the present invention (from top to bottom, strains 5asv2 and RB13). Figure 2 This is Example 2 of the present invention, which illustrates the effect of single-strain / complex microbial community laboratory conditions on the root morphology indicators of highland barley. Figure 3 This is Example 2 of the present invention, which examines the effects of single-strain / complex microbial community laboratory conditions on the content of physicochemical factors in the rhizosphere soil of highland barley.

[0022] Figure 4 This is Example 2 of the present invention, which examines the effect of single-strain / complex microbial community laboratory conditions on rhizosphere soil enzyme activity in highland barley rhizosphere soil. Detailed Implementation

[0023] This invention provides a synthetic microbial community that promotes plant growth, including *Trichophyton mentagrophytes* (…). Trichococcus sp.) RB13 and Pseudomonas (sp.) RB13 and Pseudomonas ( Pseudomonas sp.) 5asv2; the preservation number of *Trichophyton spp. RB13 is CGMCC No. 37455, and the preservation number of *Pseudomonas 5asv2* is CGMCC No. 37454. In one embodiment, the viable count ratio of *Trichophyton spp. RB13* to *Pseudomonas 5asv2* in the synthesized bacterial community is 0.5~2:1. In another embodiment, the viable count ratio of *Trichophyton spp. RB13* to *Pseudomonas 5asv2* in the synthesized bacterial community is 1:1.

[0024] The synthetic microbial community provided by this invention, comprising *Trichophyton rubrum* RB13 and *Pseudomonas 5asv2*, was isolated from soil samples of Yamdrok Lake, a high-altitude wetland on the Qinghai-Tibet Plateau. Both are indigenous microorganisms, environmentally safe and pollution-free, producing no harmful residues during application and not disrupting the original soil microbial community balance. Furthermore, due to their excellent adaptability to low-temperature stress and high growth-promoting efficiency, they can stably colonize the rhizosphere of barley under low-temperature conditions, enhancing rhizosphere nutrient turnover and supply capacity, thereby promoting barley root development and driving plant growth. This can be widely applied to agricultural production in high-altitude and cold regions, solving the problems of slow growth and low yield of crops in these areas. This invention provides a new technical approach for the sustainable agricultural development of the Qinghai-Tibet Plateau and other high-altitude and cold regions, and is of great significance for ensuring food security and the ecological environment on the plateau.

[0025] Based on the above advantages, this invention provides a compound microbial agent, the effective component of which includes the synthetic microbial flora described in the above technical solution. As one embodiment, the dosage form of the compound microbial agent includes a liquid formulation or a solid formulation; when the dosage form of the compound microbial agent is a liquid formulation, the viable count of the compound microbial agent is ≥1×10⁻⁶. 8 CFU / ml; when the compound microbial agent is in solid dosage form, the viable count of the compound microbial agent is ≥1×10⁻⁶. 8 CFU / g.

[0026] Based on the above advantages, the present invention provides a method for preparing the compound microbial agent described in the above technical solution, comprising: activating Trichophyton mentagrophytes RB13 and Pseudomonas 5asv2 respectively, and then inoculating them into a culture medium and culturing them to the logarithmic growth phase; wherein the culture medium comprises tryptic soy peptone liquid medium.

[0027] As one implementation method, the culture temperature is 25-30°C.

[0028] Based on the above advantages, the present invention provides the application of the synthetic microbial community or the compound microbial agent described in the above technical solutions in one or more of 1)-3): 1) Promotes plant growth; the plants include barley; 2) Improve soil fertility; 3) Enhance rhizosphere soil enzyme activity; the rhizosphere soil enzymes include one or more of urease, alkaline phosphatase, alkaline protease, β-glucosidase, α-glucosidase, and β-galactosidase. The synthetic microbial community provided by this invention can promote rhizosphere nutrient turnover and supply capacity, and improve rhizosphere soil ecological function.

[0029] As one implementation, promoting plant growth includes promoting plant growth under low-temperature conditions; the temperature range of the low-temperature environment is 10℃–20℃, within which the synthetic microbial community provided by the present invention can still maintain high activity and play a growth-promoting role.

[0030] Based on the above advantages, this invention provides a method for promoting plant growth, comprising: applying the synthetic microbial community or the compound microbial agent described in the above-described technical solution to the rhizosphere region of a plant. As one embodiment, the plant includes barley. During the plant's growing season, by applying the synthetic microbial community provided by this invention, the activity of key enzymes related to carbon, nitrogen, and phosphorus transformation in the rhizosphere soil can be enhanced, improving the efficiency of rhizosphere nutrient transformation and the supply of available nutrients, thereby promoting root growth and development and enhancing plant vigor.

[0031] As one implementation method, the application method includes one or more of root irrigation, trench application, and soil mixing application.

[0032] As one implementation method, the viable count of the synthetic microbial community in the plant rhizosphere after application is ≥1×10⁻⁶. 8 CFU / g soil, this application rate is the amount that can achieve the effect of promoting growth and establishing stable colonization.

[0033] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a synthetic microbial community that promotes plant growth and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0034] In the embodiments of the present invention, unless otherwise stated, all culture media used are conventional culture media. Specifically, the basic culture medium used for strain isolation and culture in the present invention is TSB medium, the formulation of which is as follows (per 1 L of medium): 17.0 g tryptone, 3.0 g soy peptone, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, and 2.5 g glucose; the initial pH of the medium is adjusted to about 7.0 with NaOH, and then autoclaved at 121°C for 20 minutes before use. If solid culture is required, 1.5% agar can be added to the above TSB medium.

[0035] Example 1: Screening and identification of growth-promoting bacterial strains in alpine wetland soils of the Qinghai-Tibet Plateau (1) Sample collection: Soil samples from the wetland near Yamdrok Lake (approximately 4441 m above sea level), a typical alpine wetland on the Qinghai-Tibet Plateau, were selected as the sample source for strain screening. Sampling was conducted in August 2023, and five representative soil samples were randomly collected from the surface layer (0–10 cm) at the edge of the wetland. The collected soil samples were placed in sterile self-sealing bags, transported to the laboratory at low temperature, and stored at 4°C for later use.

[0036] (2) Screening method for strains: To obtain native microorganisms with growth-promoting potential, strains were isolated from soil samples using dilution plating and high-throughput pure culture. 10 g of each soil sample was added to an Erlenmeyer flask containing 90 mL of sterile water, shaken at room temperature for 30 min, and allowed to stand for 5 min. The supernatant was then serially diluted 10-fold to 10⁻⁶. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 .

[0037] The dilution and plating method was as follows: 100 μL of each dilution gradient was plated onto TSB solid medium plates (repeated in parallel), and incubated at 28°C for 5 days. During the incubation period, individual colonies with different morphologies were picked and subcultured for purification to obtain a series of pure bacterial strains.

[0038] The method for high-throughput pure culture is as follows: Take 10 -3 10 -4 10 -5 10 -6 10 -7 The diluted solution was placed in 96-well cell culture plates containing TSB and incubated for 2 weeks at room temperature in the dark (25°C). At the end of the culture, a concentration of 10... -5 10 -6 10 -7 The 96-well cell culture plates were used for subsequent bacterial bank construction.

[0039] Take 10 µL of bacterial culture from a 96-well cell culture plate, add 16.6 µL of alkaline lysis buffer (pH 12 containing 25 mM NaOH and 0.2 mM Na2-EDTA) and lyse at 95°C for 30 min. Add 40 mM Tris-HCl buffer (pH = 7.5) and mix well to obtain the bacterial DNA. Store at -20°C.

[0040] The DNA from the preserved bacterial culture was amplified by the first round of PCR: the universal amplification primers for bacteria 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2) were selected.

[0041] The amplification system consisted of: 3 µL template, 3 µL 10×Buffer, 2.4 µL 2.5 mM dNTPs, 0.3 µL each of 10 µM upstream and downstream primers, and 0.15 µL 5 U / µL HS taq. The amplification program was: 95℃ for 5 min pre-denaturation; 95℃ for 10 s, 55℃ for 15 s, 72℃ for 1 min, for 25 cycles; final incubation at 72℃ for 5 min; storage at 4℃. PCR amplification reagents were purchased from Thermo Fisher Scientific.

[0042] In the second round of PCR, the template was the product from the first round of PCR, diluted 40-fold. The primers used were 27F and 1492R primers with tags (barcodes) and the adapters required for sequencing. The amplification system and procedure were the same as in the first round of PCR. After both rounds of PCR amplification, the obtained DNA samples were subjected to agarose gel electrophoresis. The gel was then recovered according to the kit instructions. Finally, the concentration of the recovered samples was detected using Nanodrop. Qualified samples were sequenced to preliminarily identify the types of bacteria in the soil.

[0043] (3) Preliminary screening of strains with growth potential: The isolated strains were preliminarily screened using functional culture media. This included: ① Phosphate solubilization capacity determination: The bacteria were inoculated onto organic and inorganic phosphorus media (NBRIP solid medium) and cultured at 30°C for 7 days. If a clear zone appeared around the colony after 7 days, it indicated that the strain had phosphate solubilization capacity. The larger the diameter of the clear zone, the stronger the phosphate solubilization capacity of the strain. Strains with high phosphate solubilization capacity were screened out. ② Siderophore secretion capacity determination: The test strains were inoculated onto CAS solid detection medium and cultured at 30°C for 24-48 h. The presence of a pale yellow halo around the bacteria was observed to determine its siderophore secretion capacity. ③ Nitrogen fixation capacity determination: Ashby's nitrogen-free medium was used for growth observation to screen out strains that could grow well. ④ Bacterial sulfur reduction capacity determination: Fresh (18-24 h) cultured bacteria were selected and grown on SIM (sulfide indole kinetic) medium. The SIM medium was inserted halfway into the tube. Incubate the test tubes aerobically at 35±2℃ (with loose caps) for approximately 24 hours. Observe the change in black color on the medium and screen out strains with high sulfur reduction capacity; ⑤ Carbon fixation capacity determination: Inoculate the test strains in a carbon-free liquid culture medium and observe their growth to screen out strains that can grow well. Carbon-free culture medium: ammonium sulfate 2.6g, potassium dihydrogen phosphate 2.4g, dipotassium hydrogen phosphate 5.7g, magnesium sulfate heptahydrate 1.0g, copper sulfate pentahydrate 0.0064g, ferrous sulfate heptahydrate 0.0011g, manganese chloride tetrahydrate 0.0079g, zinc sulfate heptahydrate 0.0015g, distilled water 1L, pH 7.0.

[0044] (4) Strain identification: The strains 5asv2 and RB13, which possessed all functions, were screened and identified. Figure 1 Taxonomic identification was performed using a combination of alkaline lysis buffer and buffer to extract DNA from the strain. Universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO.3) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.4) were used to select the V1-V9 regions of the 16S rRNA gene for detection. After sequencing the PCR products, the sequences were submitted to the NCBI GenBank database for BLAST alignment analysis.

[0045]

[0046] The obtained sequences were uploaded to NCBI. Homology comparisons were performed with sequences in gene banks using BLAST analysis. Strain 5asv2 was compared with *Pseudomonas* (…). Pseudomonas The strain 5asv2 of this invention showed the highest homology (99%) with Pseudomonas aeruginosa (sp.). Combined with identification results based on bacterial morphology and growth conditions, it was determined that this strain is Pseudomonas aeruginosa (sp.). Pseudomonas sp.).

[0047]

[0048] The obtained sequence was uploaded to NCBI and compared with *Trichophyton* spp. ( Trichococcus Based on the homology of over 99% with certain known strains of *Trichophyton mentagrophytes*, and combined with identification results such as its culture characteristics and growth conditions, the strain RB13 of this invention is determined to be *Trichophyton mentagrophytes*. Trichococcus Both of the above-mentioned functional bacteria are deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession numbers as described above. In this invention, strains RB13 and 5asv2 are referred to as "RB13 strain" and "5asv2 strain," respectively.

[0049] (5) Antagonistic test before constructing the synthetic microbial community: To assess the feasibility of the mixed interaction of the two strains, the antagonistic effect between RB13 and 5asv2 was tested. The streak confrontation method was used: RB13 strain was inoculated at one end of a TSB agar plate, and 5asv2 strain was inoculated at the opposite end. The plates were incubated at 28°C for 5 days, and the growth boundary between the two strains was observed to see if an inhibition zone was formed. The results showed that the colonies of RB13 strain and 5asv2 strain could coexist in contact, and no obvious antagonistic inhibition was observed.

[0050] Example 2: Pot experiment on the effects of synthetic microbial community RB13+5asv2 on the growth of highland barley and improvement of rhizosphere soil function (1) Preparation of synthetic microbial agents: After activating and culturing strains RB13 and 5asv2, bacterial suspensions were prepared. The specific method was as follows: strains RB13 and 5asv2 were inoculated into tryptic soy peptone liquid medium (TSB) and cultured at 28°C with shaking for 48 h to obtain the logarithmic growth phase bacterial suspension.

[0051] (2) Pot experiment design: A pot experiment of barley was conducted under greenhouse conditions to evaluate the effect of the synthetic microbial community of the present invention on the growth of barley. The test soil was taken from the topsoil (0–20 cm) of Yamdrok Lake wetland in Tibet, air-dried and sieved (5 mm). Its basic physicochemical properties were measured before use: pH 7.75, organic matter about 28.27 g / kg, total phosphorus 0.08 g / kg, total nitrogen 1.45 g / kg, total potassium 1.17 g / kg, total sulfur 4.08 g / kg, alkaline nitrogen 75 mg / kg, available phosphorus 12 mg / kg, available potassium 201 mg / kg, and available sulfur 68.31 mg / kg.

[0052] Four treatments were set up for the experiment: (a) CK control (no inoculation with bacterial agent, only an equal volume of sterile water); (b) RB13 single-strain inoculation; (c) 5asv2 single-strain inoculation; (d) RB13 + 5asv2 synthetic bacterial co-inoculation. Each treatment had 3 replicates, for a total of 12 pots. Before sowing, barley seeds were disinfected by soaking in 2% hypochlorous acid and rinsed with sterile water. Seedlings were raised at 28℃. When the barley reached the two-leaf-one-heart stage, they were transplanted into flower pots, each filled with 2.5 kg of air-dried soil. Immediately afterwards, the bacterial agent was inoculated at the base of the plants (10 ml of bacterial solution per pot, containing approximately 1 × 10⁻⁶ live bacteria). 8 (CFU / ml). The control group was inoculated with 10 ml of sterile water. After sowing, the surface of the potting soil was gently mixed to ensure thorough contact between the inoculant and the soil. During the experiment, the greenhouse daytime temperature was 22–25℃ and the nighttime temperature was 16–18℃, close to the diurnal temperature range conditions in spring on the Qinghai-Tibet Plateau. An equal amount of sterile water was regularly added to maintain the soil moisture content at approximately 60% field capacity. No other chemical fertilizers or inoculants were applied.

[0053] (3) Growth index determination: Thirty days after inoculation of barley, when the plants entered the jointing stage, the experiment was stopped for harvest analysis. First, the plants were carefully removed from the pots, and the plant height and fresh weight were measured. Then, the soil around the roots was gently washed with clean water, and the roots of three plants with similar growth were scanned on a root scanner (WinRHIZO root analysis system) to obtain root morphological index data, including: total root length (cm), root surface area (cm²). 2 ), average root diameter (mm), total root volume (cm³) 3 The average value of three plants per pot was taken for each treatment, and the results are shown in Table 1. The results showed that, compared with the control group, the growth of barley plants inoculated with growth-promoting bacteria was improved to varying degrees. Specifically, both RB13 and 5asv2 single-strain treatments improved barley root growth: for example, the average total root length of barley treated with RB13 was 1397.47 cm, an increase of 52.36% compared to the control (917.19 cm); the average root length of barley treated with 5asv2 was 1229.27 cm, an increase of 34.03% compared to the control. The RB13 + 5asv2 combined microbial community treatment showed the most significant effect, with a total root length reaching 1694.90 cm, an increase of 84.79% compared to the control. Furthermore, the root surface area of ​​the combined microbial community treatment increased from 302.06 mm². 2 Increased to 444.78 mm 2 (Increased by 47.25%), total root volume increased from 8.76 cm. 3 Increased to 11.02 cm 3 (Increased by 25.80%), the average root diameter increased from 0.86 mm to 1.04 mm (thickened by 20.93%). Figure 2The figure visually compares various indicators of barley root systems under different treatments. It shows that both RB13 and 5asv2 strains, applied alone, promote barley root development, but the combined application of the two strains has a more pronounced growth-promoting effect, achieving a synergistic effect.

[0054] Table 1. Effects of single-strain / complex microbial community laboratory conditions on root morphology in highland barley.

[0055] Note: Different lowercase letters represent p Significance at the ≤0.05 level, and the same applies to the table below.

[0056] (4) Analysis of soil physicochemical and enzymatic properties: The rhizosphere soil remaining after removing the plants from the pots of each treatment was collected, mixed, and the nutrient content and enzyme activity of the soil were measured to evaluate the impact of the inoculant on the soil ecological function. Soil organic carbon and organic matter content were determined by potassium dichromate oxidation method. Total carbon and total nitrogen content were determined by elemental analysis using a tin boat embedding method. Total phosphorus and total potassium were determined by molybdenum antimony colorimetric method and flame photometry, respectively. Soil total sulfur content was determined by nitric acid-perchloric acid digestion and ICP-OES. Alkali-available nitrogen, available phosphorus, and available potassium were determined by alkaline diffusion method, molybdenum antimony colorimetric method, and flame photometry, respectively. Available sulfur was determined by Ca(H2PO4)2 -HOAc or CaCl2 extraction, followed by ICP-OES determination. Soil enzyme activity indicators include: β-glucosidase (using p-nitrophenyl-β-D-galactoside as a substrate, hydrolyzing to p-nitrophenol, the product is yellow with a characteristic absorption peak at 400 nm, unit μg / (g·h)); β-galactosidase (determined using p-nitrophenyl-β-D-galactoside as a substrate, unit μg / (g·h)); alkaline phosphatase (using disodium phenyl phosphate as a substrate, under alkaline conditions, soil alkaline phosphatase catalyzes the hydrolysis of the substrate to phenol and disodium hydrogen phosphate. Phenol reacts with 4-aminoantipyrine, and after oxidation by potassium ferricyanide, it generates a quinone derivative. The activity of alkaline phosphatase can be calculated based on the intensity of the red color, unit mg / ( (g·24h)); Protease (using casein as a substrate, under neutral conditions, soil alkaline protease catalyzes the hydrolysis of the substrate to tyrosine, and tyrosine reduces phosphomolybdic acid compounds to produce tungsten blue, which has a characteristic absorption peak at 680nm. The activity of soil alkaline protease can be determined by the color intensity, in μmol / (g·24h)); Urease (using urea as a substrate, soil urease enzymatically hydrolyzes the substrate to produce ammonia, and ammonia reacts with phenol-sodium hypochlorite under room temperature conditions to produce blue indophenol. The color intensity is directly proportional to the amount of ammonia produced. The amount of ammonia is measured colorimetrically to express urease activity, in mg / (g·24h)); Dehydrogenase (hydrogen acceptors 2, 3, 5) - Triphenyltetrazolium chloride (TTC) accepts hydrogen during cellular respiration and is reduced to triphenylformazan (TPF). TPF is red and has a maximum absorption peak at 485 nm. The absorbance at 485 nm is used to determine the soil dehydrogenase activity, expressed in μg / (g·h). All measurements were performed using three replicate soil samples, and the average values ​​were taken. Some results are summarized in Table 2. Figure 3 The comparison of soil physicochemical factors in barley root system under different treatments is presented in a visual way.

[0057] The results showed that single-strain and synthetic microbial treatments had little impact on soil organic carbon, organic matter, and basic fertility indicators such as total nitrogen, total phosphorus, and total potassium. The overall levels of each treatment were similar (organic carbon approximately 16.17–16.97 g / kg, organic matter approximately 27.87–29.23 g / kg; total nitrogen approximately 1.40–1.45 g / kg, total phosphorus approximately 0.081–0.094 g / kg, and total potassium approximately 1.03–1.17 g / kg), with no significant differences, indicating that inoculation treatments did not disrupt the basic stability of soil physicochemical properties. Regarding changes in nutrient availability and key elements, the synthetic microbial treatment showed a more prominent comprehensive advantage. Firstly, in terms of sulfur, the total sulfur content in the synthetic microbial treatment reached 4.64 g / kg, significantly higher than the control (4.08 g / kg) and also higher than the two single-strain treatments, suggesting that the synthetic microbial community is more conducive to the accumulation or transformation of sulfur in the rhizosphere. Secondly, the alkaline nitrogen in the synthetic microbial community treatment increased to 81.3 mg / kg, showing an upward trend compared to the control (75±4.36 mg / kg) and the single-strain treatment (73–74 mg / kg), indicating its greater potential in promoting nitrogen mineralization and enhancing soil nitrogen supply capacity. Furthermore, regarding available phosphorus and potassium, the available phosphorus in the soil was slightly lower than the control in both the single-strain and synthetic microbial community treatments, while the available potassium was lower than the control. This may be because the inoculated bacteria promoted nutrient absorption by plants, leading to a relative decrease in the residual available phosphorus and potassium content in the soil.

[0058] Table 2. Effects of single-strain / complex microbial community laboratory conditions on the physicochemical factors of barley rhizosphere soil.

[0059] However, the significant increase in soil enzyme activity is even more noteworthy. As shown in Table 3, compared to the control soil, the soil treated with the RB13+5asv2 synthetic microbial community exhibited a substantial increase in the activity of all major enzymes. Specifically, urease activity increased from 0.09 mg / g·24h in the control to 0.60 mg / g·24h, an increase of 566.7%, indicating a significant acceleration in urea decomposition and nitrogen cycling in the soil; alkaline phosphatase activity increased by approximately 46.4% (from 0.69 to 1.01 mg / g·24h), which is beneficial for the mineralization and release of organophosphorus compounds; protease activity increased by approximately 18% (from 2.66 to 3.14 μmol / g·24h), β-glucosidase activity increased by approximately 25.4% (from 4.25 to 5.33 μg / g·h), and β-galactosidase activity increased by approximately 8% (from 5.88 to 6.35 μg / g·h). In single-strain treatment, RB13 had a certain effect on increasing the activity of phosphatase and protease, while 5asv2 made a slight contribution to increasing β-glucosidase and β-galactosidase, but the enzyme-promoting effects of both were not as good as the combined effect of the synthetic bacterial community. Figure 4The study showcased a comparison of key enzyme activities (urease, phosphatase, glucosidase, etc.) between treatments, providing a more intuitive view of the comprehensive improvement in the activities of various enzymes in soil treated with synthetic microbial communities.

[0060] Table 3. Effects of single-strain / complex microbial community laboratory conditions on enzyme activity in the rhizosphere soil of highland barley.

[0061] In summary, the pot experiment results of Example 2 fully demonstrate that the RB13+5asv2 synthetic microbial community provided by this invention can effectively colonize the rhizosphere of highland barley under the simulated environment of the Qinghai-Tibet Plateau, significantly promoting the growth and development of highland barley plants, especially root growth. Simultaneously, this microbial community can also improve the biochemical properties of the rhizosphere soil and enhance soil nutrient supply capacity. This dual function of promoting crop growth and improving soil ecology makes the microbial agent of this invention have significant application value and promotion potential in actual agricultural production.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A synthetic microbial community with plant growth-promoting effects, characterized in that, Including Trichophyton ( Trichococcus sp.) RB13 and Pseudomonas (sp.) RB13 and Pseudomonas ( Pseudomonas sp.) 5asv2; the preservation number of the *Trichophyton spp.* RB13 is CGMCC No. 37455, and the preservation number of the *Pseudomonas 5asv2* is CGMCC No. 37454.

2. The synthetic microbial community according to claim 1, characterized in that, The ratio of viable bacteria of Trichophyton rubrum RB13 and Pseudomonas 5asv2 in the synthetic bacterial community is 0.5~2:

1.

3. A compound microbial agent, characterized in that, The active ingredient includes the synthetic microbial community as described in claim 1 or 2.

4. The compound microbial agent according to claim 3, characterized in that, The dosage form of the compound microbial agent includes liquid or solid formulations; when the dosage form of the compound microbial agent is a liquid formulation, the viable count of the compound microbial agent is ≥1×10⁻⁶. 8 CFU / ml; when the compound microbial agent is in solid dosage form, the viable count of the compound microbial agent is ≥1×10⁻⁶. 8 CFU / g.

5. The method for preparing the compound microbial agent according to claim 3 or 4, characterized in that, include: After activating Trichophyton RB13 and Pseudomonas 5asv2 respectively, they were mixed and inoculated into a culture medium and cultured until the logarithmic growth phase; the culture medium included tryptic soy peptone liquid medium.

6. The preparation method according to claim 5, characterized in that, The culture temperature is 25-30℃.

7. The use of the synthetic microbial community according to claim 1 or 2, or the compound microbial agent according to claim 3 or 4, or the compound microbial agent prepared by the preparation method according to claim 5 or 6, in one or more of 1)-3): 1) Promotes plant growth; the plants include barley; 2) Improve soil fertility; 3) Enhance rhizosphere soil enzyme activity; the rhizosphere soil enzymes include: One or more of urease, alkaline phosphatase, alkaline protease, β-glucosidase, α-glucosidase, and β-galactosidase.

8. The application according to claim 7, characterized in that, The promotion of plant growth includes promoting plant growth in a low-temperature environment; the temperature range of the low-temperature environment is 10℃–20℃.

9. A method for promoting plant growth, characterized in that, include: The synthetic microbial community as described in claim 1 or 2, or the compound microbial agent as described in claim 3 or 4, or the compound microbial agent prepared by the preparation method described in claim 5 or 6, is applied to the rhizosphere region of plants.

10. The method according to claim 9, characterized in that, After application, the viable count of the synthetic microbial community in the plant rhizosphere is ≥1×10⁻⁶. 8 CFU / g soil.

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