A functional composite synthetic bacterial community as well as a preparation method and application thereof

Abstract

The application provides a functional composite synthetic bacterial flora and a preparation method and application thereof, and belongs to the technical field of microorganisms. The synthetic bacterial flora comprises Bacillus velezensis, Bacillus sphaericus, Paenibacillus frigidicola and Sporosarcina koreensis. The four bacteria in the synthetic bacterial flora can realize symbiotic interaction through metabolic function differentiation and cross-nutrition feeding, and form a strong metabolic dependence relationship relying on a carbon-amino acid-purine three-dimensional nutrition supply network. The synthetic bacterial flora not only improves the common tolerance of the bacterial flora to rhizosphere penetration stress, and guarantees stable colonization of the bacterial flora, but also produces a superimposed growth promoting effect through multi-metabolic pathway synergistic complementation. The results of the examples show that the synthetic bacterial flora provided by the application can significantly promote the growth of soybeans as a microbial inoculant, and can also significantly improve the prevention and treatment effect of soybean root rot. The synthetic bacterial flora provided by the application provides a theoretical basis and practical reference for promoting the growth of soybeans and preventing and treating soybean root rot.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a functional complex synthetic microbial community, its preparation method, and its application. Background Technology

[0002] Soybeans are an important oilseed crop worldwide and are widely cultivated globally. Soybean diseases mainly include fungal diseases such as root rot, downy mildew, gray spot, brown spot, and rust. Among these, root rot is the most widespread and damaging, becoming a key bottleneck restricting the sustainable development of the soybean industry. Currently, chemical control remains the primary method for controlling this disease. However, the long-term and extensive use of chemical pesticides not only causes environmental pollution and pesticide residues but also easily leads to pesticide resistance in pathogens. Compared with traditional chemical pesticides, microbial inoculants have advantages such as being environmentally friendly, residue-free, and less prone to developing resistance, and have become an important component of green pest control technologies.

[0003] Rhizosphere microorganisms are closely related to plant health, and the structure and function of the microbial community directly determine the plant's growth status and disease resistance. Based on the principles of microbial community ecology, artificially constructed functional microbial communities play an increasingly important role in resisting harmful organism invasion, controlling soil diseases, and increasing plant biomass. However, the rhizosphere microbiome is a highly dynamic system, and single functional strains often face problems such as difficulty in colonization and unstable activity, making it difficult to sustain their growth-promoting or disease-preventing effects. Therefore, the research focus of biocontrol using agricultural microorganisms has shifted from single strains to the community level of the microbiome. By constructing functional composite microbial communities with good biocompatibility, complementary biocontrol mechanisms, and synergistic effects, an interactive network can be formed to jointly regulate plant health. Synthetic communities (SynComs), by mimicking the assembly rules and functional characteristics of natural microbial communities, can form a stable ecological niche occupancy effect in the rhizosphere, effectively inhibiting pathogen invasion. This not only overcomes the shortcomings of single biocontrol strains in the field environment but is also an effective way to improve the biocontrol effect of soil-borne diseases.

[0004] In recent years, the design of synthetic microbial communities has shifted from simple "functional superposition" to precise assembly based on metabolic interactions. Cross-feeding networks among strains are a core mechanism for community functional stability. Metabolic interactions not only encompass the cross-strain exchange and complementary utilization of basic metabolites such as amino acids, organic acids, and vitamins, but also involve the synergistic secretion and dynamic regulation of signaling molecules and secondary metabolites; these multi-level metabolic interaction networks collectively maintain the structural stability and functional efficiency of the microbial community. Currently, research on synthetic microbial communities targeting soybean root rot is still limited, especially the lack of systematic reports on simplified microbial communities precisely designed based on metabolic interaction mechanisms. Furthermore, existing studies mostly focus on evaluating the disease control efficacy of the microbial community, and the analysis of the metabolic interaction networks among community members is still insufficient, limiting the rational design and optimization of synthetic microbial communities. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a synthetic microbial community that can be used synergistically to prevent and control soybean root rot and significantly promote soybean growth.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides a synthetic microbial community, comprising: *Bacillus belye*, *Bacillus spheroidosa*, *Bacillus repens*, and *Dystrophus koreanum*; the viable count ratio of *Bacillus belye*, *Bacillus spheroidosa*, *Bacillus repens*, and *Dystrophus koreanum* in the synthetic microbial community is 1:1:1:1; the total viable count of the synthetic microbial community is ≥2.0 × 10⁻⁶. 7 CFU / mL.

[0007] Preferably, the *Bacillus belyssus* includes *Bacillus belyssus* YH1 with accession number CGMCC No. 39711; the *Bacillus spheroidae* includes *Bacillus spheroidae* YH2 with accession number CGMCC No. 39712; the *Bacillus thuringiensis* includes *Bacillus thuringiensis* YH5 with accession number CGMCC No. 39713; and the *Bacillus koreana* includes *Bacillus koreana* YH17 with accession number CGMCC No. 39714.

[0008] This invention provides a method for preparing the synthetic microbial community described in the above technical solution, comprising the following steps: Each bacterium was inoculated into a liquid culture medium for cultivation. The bacterial resuspension was collected and adjusted to obtain a bacterial solution with an equal number of viable bacteria to obtain the seed solution of each bacterium. Equal volumes of seed cultures of each bacterium were mixed to obtain a synthetic bacterial community.

[0009] Preferably, the liquid culture medium includes LB medium; the culture temperature is 25~30℃; and the culture time is 16~24h.

[0010] This invention provides a bacterium, including any one of Bacillus sporeans YH17, Bacillus repens YH5, Bacillus belyssus YH1, and Bacillus spheroidosa YH2; the preservation number of Bacillus sporeans YH17 is CGMCC No. 39714; the preservation number of Bacillus repens YH5 is CGMCC No. 39713; the preservation number of Bacillus belyssus YH1 is CGMCC No. 39711; and the preservation number of Bacillus spheroidosa YH2 is CGMCC No. 39712.

[0011] This invention provides the application of the synthetic microbial community described in the above-described technical solution, the synthetic microbial community prepared by the preparation method described in the above-described technical solution, or the bacteria described in the above-described technical solution in the preparation of pathogen-inhibiting agents; the pathogen includes Fusarium solanum.

[0012] This invention provides the application of the synthetic microbial community described in the above-described technical solution, the synthetic microbial community prepared by the preparation method described in the above-described technical solution, or the microorganism described in the above-described technical solution in promoting plant growth and / or preventing plant diseases; the plant includes soybean.

[0013] Preferably, the plant disease includes root rot.

[0014] This invention provides a method for promoting soybean growth and / or preventing soybean diseases, comprising: The synthetic microbial community described in the above technical solution, the synthetic microbial community prepared by the preparation method described in the above technical solution, or the microorganism described in the above technical solution are applied during the soybean growth process.

[0015] Preferably, the application period of the synthetic microbial community includes the soybean two-true-leaf stage and / or three-leaf stage; the application amount of the synthetic microbial community or the bacteria is 50~300 mL / plant.

[0016] The beneficial effects of this invention are: This invention provides a synthetic microbial community comprising: *Bacillus belye*, *Bacillus spheroidosa*, *Bacillus thymophilus*, and *Bacillus koreana*. The four strains in this synthetic community achieve symbiotic interaction through metabolic functional differentiation and cross-feeding of nutrients: different strains divide the supply of carbon sources, various amino acids, purine nucleic acid precursors, vitamin precursors, and osmotic protective substances; strains mutually absorb metabolites secreted by each other to compensate for their own synthetic deficiencies; and simultaneously reduce overall energy consumption through purine salvage synthesis and methyl metabolism coupling, forming a strong metabolic dependency relationship based on a three-dimensional carbon-amino acid-purine nutrient supply network. This not only enhances the community's collective tolerance to rhizosphere osmotic stress and ensures stable colonization, but also produces a synergistic growth-promoting effect and disease control effect through the synergistic complementarity of multiple metabolic pathways. The results of the embodiments show that the synthetic microbial community provided by this invention, when applied as a microbial agent in soybean growth, can significantly promote soybean growth and significantly improve the control effect of soybean root rot. The synthetic microbial community provided by this invention provides a theoretical basis and practical reference for promoting soybean growth and controlling soybean root rot.

[0017] Biological Preservation Instructions Bacillus belye YH1, Latin scientific name Bacillus velezensis It was deposited at the China General Microbiological Culture Collection Center on July 15, 2026, with accession number CGMCC No. 39711.

[0018] Bacillus spheroidae YH2, Latin scientific name Bacillus stercoris It was deposited at the China General Microbiological Culture Collection Center on July 15, 2026, with accession number CGMCC No. 39712.

[0019] Cold-resistant Bacillus repens YH5, Latin scientific name Peribacillus frigoritolerans It was deposited at the China General Microbiological Culture Collection Center on July 15, 2026, with accession number CGMCC No. 39713.

[0020] Korean spore-forming ... Sporosarcina koreensis It was deposited at the China General Microbiological Culture Collection Center on July 15, 2026, with accession number CGMCC No. 39714. Attached Figure Description

[0021] 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.

[0022] Figure 1This experiment involved a potted plant inoculation to verify the pathogen of Fusarium solani; A shows the growth traits of soybeans inoculated with Fusarium solani; B shows the growth traits of soybeans treated with sterile water. Figure 2 To assess the control effect of synthetic microbial flora on soybean root rot and its impact on soybean growth indicators; a) plant height, b) root weight, c) number of soybean pods, d) aboveground dry weight, e) underground dry weight, f) leaf nitrogen content, g) soybean growth phenotype of the ck treatment group, h) soybean growth phenotype of the synthetic microbial flora treatment group, and i) the effect of synthetic microbial flora on the control effect of soybean root rot on soybean root rot and soybean growth indicators. F. solani Soybean growth phenotype, j represents the application rate. F. solani Soybean growth phenotype, k is the root rot disease incidence index, and l is the rhizosphere. F. solani Density determination, m represents the relative efficacy of the synthetic microbial community against soybean root rot; in the figure Indicates significant difference (Duncan's multiple comparisons, n=6-8, P<0.05); Figure 3 A circular heatmap showing the interactions among the core strains of the SynCom microbial community; Figure 4 This is a matrix diagram of interactions among the core strains of the SynCom microbial community. Figure 5 To simplify the effect of synthetic microbial communities (SynComs) on soybean growth; A~E represent the phenotypic characteristics of soybean plants in different treatment groups, F represents plant height, G represents fresh weight, H represents aboveground dry weight, I represents aboveground dry weight, and J represents nitrogen content; in the figure, the same letter indicates no significant difference (P>0.05), and different letters indicate significant differences (P≤0.05); Figure 6 A heatmap of differential metabolite clustering among SBC5 strains; A represents... B. velezensis YH1 and B. stercoris YH2 differential metabolite clustering analysis heatmap, B is B. velezensis YH1 and P. frigoritolerans YH5 differential metabolite clustering analysis heatmap, C is B. velezensis YH1 and S. koreensis YH17 differential metabolite clustering analysis heatmap, D is... B. stercoris YH2 and P. frigoritolerans YH5 differential metabolite clustering analysis heatmap, E is... B. stercoris YH2 and S. koreensis YH17 differential metabolite clustering analysis heatmap, F is... P. frigoritolerans YH5 and S. koreensisYH17 differential metabolite clustering analysis heatmap. The horizontal axis represents different strains, and the vertical axis represents the differential metabolites compared in the group. Red indicates high expression of the substance, and blue indicates low expression of the substance (P<0.05, n=3). Figure 7 The effects of applying YH1, YH2, YH5 and YH17 individually on the growth promotion of soybeans are shown in the graph. Detailed Implementation

[0023] This invention provides a synthetic microbial community, comprising: *Bacillus belye*, *Bacillus spheroidosa*, *Bacillus repens*, and *Dystrophus koreanum*. As an optional embodiment of this invention, the viable count ratio of *Bacillus belye*, *Bacillus spheroidosa*, *Bacillus repens*, and *Dystrophus koreanum* in the synthetic microbial community is 1:1:1:1; the viable count of the synthetic microbial community is ≥2.0 × 10⁻⁶. 7 CFU / mL can be ≥1×10⁻⁶ 8 CFU / mL can be 1.0 × 10⁻⁶. 8 ~1.0 × 10 10 CFU / mL, or 1.0 × 10⁻⁶. 9 CFU / mL. As an optional embodiment of the present invention, the *Bacillus belyssus* includes *Bacillus belyssus* YH1 with accession number CGMCC No. 39711; the *Bacillus spheroidae* includes *Bacillus spheroidae* YH2 with accession number CGMCC No. 39712; the *Bacillus cytosolicus* includes *Bacillus cytosolicus* YH5 with accession number CGMCC No. 39713; and the *Bacillus koreana* includes *Bacillus koreana* YH17 with accession number CGMCC No. 39714.

[0024] In this invention, *Bacillus belyssus* YH1 in the synthetic bacterial community serves as the core amino acid and basic carbon source donor. It can specifically secrete multiple essential amino acids such as arginine, leucine, isoleucine, and phenylalanine, while simultaneously releasing pyruvate and uracil. Pyruvate, as a primary degradation product of central carbon metabolism, provides a universal carbon source and energy for YH5 and YH17, making it the main supplier of amino acids and basic energy to the bacterial community. *Bacillus spheroidae* YH2 in the synthetic bacterial community provides osmotic protection, amino acid and special organic acid supply. It can efficiently secrete amino acids such as lysine, methionine, and glutamic acid, as well as guanine and methylmalonic acid, supplementing the community's carbon and nitrogen nutrition and purine metabolic substrates. It can secrete betaine to alleviate rhizosphere osmotic stress, protect all symbiotic strains, and provide methyl groups to assist YH17 in completing purine methylation modification, achieving deep metabolic coupling. It can also produce histamine and aromatic special organic acids (2-(4-methylphenyl)-ethyl-6-hydroxybenzoic acid), supplying the community with organic acid precursors and perfecting osmotic regulation metabolism. The synthetic bacterial community includes the cold-resistant *Bacillus subtilis* YH5, which predominantly secretes purines such as adenine, hypoxanthine, and allopurinol. YH1 and YH17 can directly utilize these substances to synthesize nucleotides via the purine salvage pathway, eliminating the energy-intensive de novo purine synthesis, reducing the overall metabolic energy consumption of the bacterial community, and providing raw materials for nucleic acid synthesis. In this synthetic bacterial community, *Bacillus koreana* YH17 forms a synergistic amino acid secretion pair, jointly secreting valine, proline, β-alanine, creatine, and pyroglutamic acid. β-alanine is a precursor for pantothenic acid (vitamin) synthesis, compensating for the deficiency in vitamin synthesis within the bacterial community and ensuring adequate micronutrients for overall growth. In this invention, the synthetic bacterial community exhibits significant metabolic functional differentiation: YH1 and YH2 are responsible for amino acid and carbon source supply, YH5 provides purine bases, and YH17 supplements essential amino acids and vitamin precursors (β-alanine is a precursor for pantothenic acid synthesis). This three-dimensional cross-feeding architecture of "carbon source-amino acid-purine base" is a key foundation for the stable function of the synthetic microbial community.

[0025] The synthetic microbial community provided by this invention achieves symbiotic interaction through metabolic functional differentiation and cross-feeding of nutrients, and forms a strong metabolic dependence relationship by relying on the three-dimensional nutrient supply network of carbon-amino acid-purine. It not only enhances the community's common tolerance to rhizosphere osmotic stress and ensures stable colonization, but also produces a superimposed growth-promoting effect through the synergistic complementarity of multiple metabolic pathways.

[0026] This invention provides a method for preparing the synthetic microbial community described in the above technical solution, comprising the following steps: Each bacterium was inoculated into a liquid culture medium for cultivation. The bacterial resuspension was collected and adjusted to obtain a bacterial solution with an equal number of viable bacteria to obtain the seed solution of each bacterium. Equal volumes of seed cultures of each bacterium were mixed to obtain a synthetic bacterial community.

[0027] As an optional embodiment of the present invention, the liquid culture medium includes LB medium; the culture temperature can be 25-30℃, or 25, 26, 27, 28, 29, or 30℃; the culture time can be 16-24h, or 16, 17, 18, 19, 20, 21, 22, 23, or 24h; the culture process can be accompanied by shaking; the shaking speed can be 180 r / min. After the culture is completed, the present invention obtains the culture solutions of each bacterium. After obtaining the culture solutions of each bacterium, the present invention preferably collects the bacterial cells in the culture solutions of each bacterium. The method of collecting the bacterial cells in the present invention can be centrifugation; the centrifugation speed can be 8000 r / min; the centrifugation time can be 10 min. After collecting the bacterial cells, the present invention preferably washes the bacterial cells. In the present invention, the washing can be performed using sterile physiological saline; the washing can be performed twice. After washing, the present invention preferably resuspends the bacterial cells using sterile physiological saline to obtain the seed culture of each bacterium. After obtaining the seed liquids of each bacterium, the present invention mixes the seed liquids of each bacterium in equal volumes to obtain a synthetic bacterial community.

[0028] This invention provides a bacterium, comprising any one of *Bacillus koreana* YH17, *Bacillus repens* YH5, *Bacillus belyssus* YH1, and *Bacillus spheroidosa* YH2; the preservation number of *Bacillus koreana* YH17 is CGMCC No. 39714; the preservation number of *Bacillus repens* YH5 is CGMCC No. 39713; the preservation number of *Bacillus belyssus* YH1 is CGMCC No. 39711; and the preservation number of *Bacillus spheroidosa* YH2 is CGMCC No. 39711. In this invention, the bacterium can promote soybean growth, increasing soybean plant height, stem diameter, fresh weight, and dry weight. The bacterium also has a certain control effect on soybean root rot disease.

[0029] This invention provides the application of the synthetic microbial community described in the above-described technical solution, the synthetic microbial community prepared by the preparation method described in the above-described technical solution, or the bacteria described in the above-described technical solution in the preparation of pathogen-inhibiting agents; the pathogen includes Fusarium solanum.

[0030] This invention provides the application of the synthetic microbial community described in the above-described technical solutions, the synthetic microbial community prepared by the preparation method described in the above-described technical solutions, or the microorganisms described in the above-described technical solutions in promoting plant growth or controlling plant diseases; the plant includes soybean. As an optional embodiment of this invention, the plant disease includes root rot.

[0031] This invention provides a method for promoting soybean growth or preventing soybean diseases, comprising: The synthetic microbial community described in the above-mentioned technical solution, the synthetic microbial community prepared by the preparation method described in the above-mentioned technical solution, or the microorganism described in the above-mentioned technical solution are applied during the soybean growth process. As an optional embodiment of the present invention, the application period of the synthetic microbial community includes: the soybean two-true-leaf stage and / or the three-leaf stage; the application amount of the synthetic microbial community can be 50~300 mL / plant, 50~200 mL / plant, or 60 mL / plant.

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] The pathogen used in the following embodiments of the present invention is Fusarium solani (…). Fusarium solani For more information on the Fusarium moniliforme that causes solanaceous diseases, please refer to [Chen Xijun, Chen Ying, Shi Tong, Jiang Dongyang, Ling Huangui, Chen Chen. Symptoms, pathogen types and physiological differentiation of common soybean diseases. Soybean Science and Technology. 2023.(2):27-37].

[0034] Example 1 I. Experimental Methods 1. Collection of soil samples and infected plants The sampling site was located in the soybean experimental field of the Heihe Branch of the Heilongjiang Academy of Agricultural Sciences, Heihe City, Heilongjiang Province, China (50°14′58″N, 127°26′54″E), and the sampling time was September 10, 2024. This area has a cold-temperate continental monsoon climate, and the soil type is black soil. Approximately 500 g of mixed sample was collected and placed in a sterile sampling bag.

[0035] 2. Isolation and identification of culturable bacteria Weigh 2.0 g of soil sample and place it in a 500 mL Erlenmeyer flask containing 200 mL of sterile water for shaking incubation (30℃, 180 r / min, 4–6 h). Dilute the soil suspension 10-fold serially to 100 g. -4 and 10 -5The concentrations were determined, and the cultures were plated on tryptone soybean broth (TSB) (Becton Dickinson, Franklin Lakes, NJ, USA), beef extract nutrient broth (NB) (Qingdao Haibo Biotechnology Co., Ltd., Qingdao, China), and LB (Luria-Bertani) medium (Qingdao Haibo Biotechnology Co., Ltd., Qingdao, China) and incubated (30℃, 2–5 days). Individual colonies with distinct morphologies were selected based on their characteristics for purification. DNA was extracted from each strain, and the full-length 16S rRNA gene sequence was amplified using universal primers. The PCR products were sent to Guangdong Megagene Technology Co., Ltd. for sequencing and identification.

[0036] 3. Verification experiment on reinoculation of soybean root rot pathogen Select healthy soybean seeds (Heihe 43 variety), and disinfect them by soaking in 1% NaClO solution for 2 minutes, then in 75% alcohol for 1 minute. Rinse with sterile water 5-6 times, and place them in a tray containing sterilized vermiculite to moisten and germinate for 1 day before use. Fusarium wilt of Solanum (… Fusarium solani The pathogen inoculation was performed according to the method described by Wang et al. (2024). Sterile water was used as the control (CK), and the incidence of disease was investigated during period V1 (approximately 15 days).

[0037] References [Wang MY, Du YQ, Yan JL, Wang D, Zhang YY, Zhu JQ (2024)Identification and biological characteristics analysis of a pathogen of strawberry root rot. Chin Agric Sci Bull 40:113~120].

[0038] 4. Screening of strains antagonistic to Fusarium solani and determination of inhibition rate The plate confrontation method was used to screen for bacteria that antagonize Fusarium solani.

[0039] First, Fusarium oxysporum was inoculated into PDA solid medium and cultured until colonies with a diameter of 1.5-2 cm grew (24℃, 2d). Then, the bacteria to be tested were cultured in liquid for 16 h (30℃, 180 r·min). -1 The antibacterial ability of the strains was observed by streaking the bacterial colony 1 cm away from the colony and culturing at 30℃. Strains with antibacterial activity against Fusarium solani were preliminarily screened.

[0040] The inhibition rate was determined using the cross-hatching method. Fusarium solani was activated and cultured on solid PDA medium (24℃, 3 days). After colonies covered the entire plate, 7 mm diameter mycelial blocks were punched out from the outer edge of the colonies using a sterile punch. These mycelial blocks were placed in the center of the PDA medium and cultured until they reached 1.5–2 mm in diameter (24℃, 2 days). Oxford cups (6 mm in diameter) were placed at equal intervals at the four corners, 20 mm from the mycelial block. 50 μL of a uniform concentration (OD) was added to each Oxford cup. 600 =0.1 INFINITE 200 PRO (TECAN, Australia) bacterial culture medium, with an uninoculated antagonistic bacterial culture medium as the control group. Each treatment was repeated in triplicate, and the cultures were incubated at 28°C for 7 days before the inhibition rate was measured. Each group had three replicates. The inhibition rate formula is as follows: .

[0041] 5. Study on the effects of synthetic microbial communities on soybean growth and root rot prevention Preparation of pathogen spore suspension: *Fusarium solani* var. *solani* was inoculated onto PDA plates and cultured at 28°C for 7 days. After sporulation, the spores were washed with sterile water, filtered through four layers of sterile gauze, counted using a hemocytometer, and the spore concentration was adjusted to 3.6 × 10⁻⁶. 5 Units / mL are available for use.

[0042] Preparation of synthetic bacterial suspension: Each SBC strain (those exhibiting an inhibition rate of over 60% against *Fusarium solani* in step 4) was inoculated into LB liquid medium and cultured at 30°C and 180 r / min with shaking for 24 h at 4°C. The cultured bacterial suspension was centrifuged at 8000 r / min for 10 min to collect the bacterial cells. After washing twice with sterile physiological saline, the suspension was resuspended, and the concentration of each strain was adjusted to 1.0 × 10⁻⁶. 8 Synthetic bacterial culture (SBC) suspension was prepared by mixing equal volumes of CFU / mL.

[0043] Preparation of experimental materials: The soybean variety used was Heihe 43. Seeds were surface-sterilized (immersed in 75% ethanol for 30 seconds, 2% sodium hypochlorite solution for 10 minutes, and rinsed 5 times with sterile water), and then germinated in a 28℃ constant temperature incubator for 24 hours. Seeds with uniform germination were selected for pot experiments. The soil was autoclaved at 121℃ for 2 hours (sterilized 3 times at 24-hour intervals), dried, and then sieved through a 2mm sieve for later use. The flowerpots were 15 cm × 20 cm, and each pot contained 500–600 g of sterilized soil.

[0044] The inhibitory effect of synthetic microbial flora on soybean root rot was determined using an indoor pot experiment. Germinated soybean seeds were sown in sterile pots, 3 seeds per pot, covered with 2 cm of soil, and placed in a light incubator for routine cultivation (temperature 25℃, photoperiod 14 h / 10 h, light intensity 8000 lx, relative humidity above 70%). After emergence, seedlings were thinned, retaining one healthy plant per pot with uniform growth. When the soybeans reached the three-leaf stage, inoculation treatments were administered, with four treatment groups: Treatment 1: Control group (CK), each pot was watered with 50 mL of sterile water; Treatment 2: Synthetic microbial flora treatment (SBC), each pot was watered with 50 mL of synthetic microbial flora suspension; Treatment 3: Pathogen group (FUS), each pot was watered with 50 mL of Fusarium solani spore suspension. Treatment 4: Synthetic microbial community + pathogen group (SBC + FUS): First, inoculate with 50 mL of synthetic microbial community suspension, then 24 h later, inoculate with 50 mL of pathogen spore suspension by irrigation. The final pathogen concentration was the same as in the FUS group. Each treatment had 8-10 replicates, arranged completely randomly. After inoculation, the plants were kept in a light incubator and watered regularly and quantitatively to maintain soil moisture content at 60%-70% of field capacity.

[0045] Disease investigation and index determination: Disease investigation was conducted 14 days after inoculation with the pathogen. Disease severity was determined by slightly modifying the method reported by Ye et al. (2020), using a 5-point grading system: 0, no disease symptoms, normal plant growth. 1, slight wilting of lower leaves, less than 1 / 4 of the plant is wilted, no obvious root lesions. 2, wilting of 1 / 4 to 1 / 2 of the plant, brown lesions appear on the roots, and lateral roots are reduced. 3, more than half of the plant is wilted, most of the roots are necrotic, and the taproot turns brown and rots. 4, the entire plant dies or dies, and the roots are completely rotten.

[0046] References [Ye WW, Zeng DD, Xu M, Li pathogens. J Integr Agric 19:2056~2063].

[0047] The disease index formula is as follows, where n1-n4 are the number of soybean plants in the specified category, and Nt is the total number of soybean plants in all four categories. The control effect is calculated as a percentage, as shown in the following formula, where the control group refers to the FUS group, and the treatment group refers to the SBC+FUS group.

[0048] ; ; Growth index measurement: The following growth indexes were measured simultaneously during the disease survey: plant height, root weight, aboveground fresh weight, underground fresh weight, number of pods, aboveground dry weight, underground dry weight, chlorophyll content, and total nitrogen content of the plant (TYS-4N plant nutrient analyzer, Beijing Jinkelida Electronic Technology Co., Ltd.).

[0049] 6. Interactions among core microorganisms: determination of supernatant. The number of strains in the synthetic microbial community was simplified by using in vitro co-culture of strains (Spent Medium, SM).

[0050] Each core strain was inoculated into 20 mL of LB medium and cultured with shaking until the growth plateau phase (37℃, 170 r / min for 48 h). The culture was then centrifuged at 8000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm sterile filter membrane (Millipore SLGV033RS Merck USA) to obtain the culture medium residue (SM) for each strain, which was stored at -20℃ for later use.

[0051] The test strain was recultured in LB medium and adjusted to the same concentration (OD). 600 =0.1), and inoculated into each of the above SMs at a volume ratio of 1%. A control group inoculated into fresh LB medium was included. All treatments were incubated at 37℃ and 170 r / min with shaking for 48 h, and the OD of each culture medium was measured. 600 value.

[0052] Interstrain interaction strength as expressed by OD 600 Waste liquid / OD 600 Fresh culture medium ratios are used. A ratio >1 indicates that the SM has a promoting effect on the tested strain, a ratio <1 indicates an inhibitory effect, and a ratio ≈1 indicates no significant effect. Each treatment has three biological replicates.

[0053] 7. Determination of microbial interactions By measuring OD 600 Waste liquid / OD 600Fresh culture media were used to determine microbial interactions (Hamid et al. 2017). Specifically, to classify interactions between strains, a t-test was used to compare experimental OD values. 600 Waste liquid / OD 600 Is the fresh culture medium significantly greater or less than 1? If OD600 waste / OD 600 If the values ​​of fresh culture medium were significantly greater than or less than 1 (P<0.05), the interaction between the two strains was classified as positive (+) or negative (-). For the remaining samples (P>0.05), the interaction was classified as neutral (0). The interaction was represented by a relationship matrix: "- / -": competition; "0 / -" or "- / 0": amensalism; "0 / 0": no interaction between strains; "+ / 0" or "0 / +": commensalism; "- / +" or "+ / -": parasitism; "+ / +": mutualism.

[0054] 8. Metabolic function analysis of the core strain The core strains were cultured in LB medium (37℃, 170 r / min, 48 h) until the stable growth phase. The culture medium was collected and centrifuged at 8000 r / min for 10 min to remove bacterial cells. The supernatant was filtered through a 0.22 μm sterile filter to obtain the culture medium residue (Spent Medium, SM) containing extracellular metabolites. Fresh LB medium was used as a control. Chromatographic separation was performed using a Vanquish ultra-high performance liquid chromatograph (Thermo Fisher Scientific, Waltham, MA, USA). The chromatographic column was a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm). Mobile phase A was an aqueous solution containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and mobile phase B was acetonitrile. The sample pan temperature was 4℃, the injection volume was 2 μL, and gradient elution was used. Primary mass spectrometry (MS) was performed using an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific). 1The data were acquired by mass spectrometry and secondary mass spectrometry (MS / MS), and the instrument control software was Xcalibur (version 4.4, Thermo). The main mass spectrometry parameters were as follows: sheath gas flow rate 50 Arb; auxiliary gas flow rate 15 Arb; capillary temperature 320 ℃; primary mass spectrometry resolution 60,000; secondary mass spectrometry resolution 15,000; collision energy gradient SNCE 20 / 30 / 40; spray voltage 3.8 kV for positive ion mode and -3.4 kV for negative ion mode. The raw data were converted to mzXML format by ProteoWizard software (version 3.0.24054) and then metabolite identification was performed using R package [7]. The database used was inhouse_database (version 4.0). The metabolites were qualitatively analyzed by comparing the retention time, accurate molecular weight, secondary mass spectrometry fragments and other information of the metabolites in the sample with those in the database; relative quantitative analysis was performed based on the peak area.

[0055] 9. Effects of SynComs (Simplified Microbial Community) on Soybean Growth A comprehensive comparison of 15 core strains was conducted, and strains with cross-feeding interaction functions and high interaction indices were selected to construct the following simplified synthetic bacterial communities: SBC3 (3 strains), SBC4 (5 strains), and SBC8 (8 strains). Simultaneously, the initial 15 whole bacterial combinations (SBC, OD) were used as the basis for further analysis. 600 = 0.1) and sterile water (CK) were used as controls. Among them, SBC8 (YH1, YH2, YH5, YH17, YH21, YH22, YH33 and DJ1), SBC4 (YH1, YH2, YH5, YH17), and SBC3 (YH17, YH22, YH33) were used.

[0056] An indoor pot experiment was used to evaluate the growth-promoting effect of simplified synthetic microbial communities on soybean growth. All treatments were not inoculated with pathogens to eliminate disease interference and to examine the growth-promoting function of the microbial communities independently. Each treatment was randomly assigned with 8–10 replicates. Culture conditions and measurement indicators were the same as above. Growth indicators were measured 6 weeks after inoculation. Soil samples from each treatment were subjected to high-throughput sequencing for community composition analysis.

[0057] Preparation of synthetic bacterial suspension: Each SBC strain was inoculated onto LB liquid medium and cultured at 30℃ with shaking at 180 r / min for 24 h. The cultured bacterial suspension was centrifuged at 8000 r / min for 10 min (4℃) to collect the bacterial cells. The cells were washed twice with sterile physiological saline and resuspended. The concentration of each strain was adjusted to 1.0 × 10⁻⁶. 8 Synthetic bacterial culture (SBC) suspension was prepared by mixing equal volumes of CFU / mL.

[0058] Preparation of experimental materials: The soybean variety used was Heihe 43. Seeds were surface-sterilized (immersed in 75% ethanol for 30 seconds, 2% sodium hypochlorite solution for 10 minutes, and rinsed 5 times with sterile water), and then germinated in a 28℃ constant temperature incubator for 24 hours. Seeds with uniform germination were selected for pot experiments. The soil was autoclaved at 121℃ for 2 hours (sterilized 3 times at 24-hour intervals), dried, and then sieved through a 2mm sieve for later use. The flowerpots were 15 cm × 20 cm, and each pot contained 500–600 g of sterilized soil.

[0059] The inhibitory effect of synthetic microbial flora on soybean root rot was determined using an indoor pot experiment. Germinated soybean seeds were sown in sterile pots, 3 seeds per pot, covered with 2 cm of soil, and placed in a conventional incubator (temperature 25℃, photoperiod 14 h / 10 h, light intensity 8000 lx, relative humidity above 70%). After emergence, seedlings were thinned, retaining one healthy plant per pot. Inoculation was performed when the soybeans reached the three-leaf stage, with four treatment groups: Treatment 1: Control group (CK) was watered with 50 mL of sterile water per pot.

[0060] Treatment 2: Synthetic microbial community SBC3: Water each pot with 50 mL of synthetic microbial community SBC3 bacterial suspension.

[0061] Treatment 3: Synthetic microbial community SBC4: Water each pot with 50 mL of synthetic microbial community SBC4 bacterial suspension.

[0062] Treatment 4: Synthetic microbial community SBC8: Water each pot with 50 mL of synthetic microbial community SBC8 bacterial suspension.

[0063] Treatment 5: Synthetic microbial community (SBC): Water each pot with 50 mL of SBC bacterial suspension.

[0064] Each treatment was replicated 8-10 times, in a completely randomized manner. After inoculation, the plants were kept in a light incubator and watered regularly and quantitatively to maintain soil moisture content at 60%-70% of field capacity.

[0065] Relevant indicators were measured 6 weeks after vaccination.

[0066] 10. Data Analysis Data were compiled using Excel and statistically analyzed using SPSS 26.0 software. Differences between groups were assessed using one-way ANOVA combined with Duncan's multiple comparison test, with P < 0.05 considered statistically significant. Graph plots were generated using Graph Padprism 10 and R.

[0067] II. Experimental Results 1. Isolation and identification of soil-cultured bacteria A total of 118 bacterial strains were isolated from soil samples from soybean planting areas in Heihe. After identification by 16S rRNA gene sequence, these strains belonged to 39 genera and species, with Bacilli being the most abundant group.

[0068] Subsequent screening revealed that 12 strains exhibited an inhibition rate of over 60% against Fusarium solani.

[0069] The nucleotide sequences of the 16S rRNA of the 12 strains are shown in SEQ ID NO.1~12.

[0070] The nucleotide sequence of the 16S rRNA of strain YH1 is shown in SEQ ID NO.1, specifically:

[0071] The nucleotide sequence of the 16S rRNA of strain YH2 is shown in SEQ ID NO.2, specifically:

[0072] The nucleotide sequence of the 16S rRNA of strain YH4 is shown in SEQ ID NO.3, specifically:

[0073] The nucleotide sequence of the 16S rRNA of strain YH5 is shown in SEQ ID NO.4, specifically:

[0074] The nucleotide sequence of the 16S rRNA of strain YH17 is shown in SEQ ID NO.5, specifically:

[0075] The nucleotide sequence of the 16S rRNA of strain YH21 is shown in SEQ ID NO.6, specifically:

[0076] The nucleotide sequence of the 16S rRNA of strain YH24 is shown in SEQ ID NO.7, specifically:

[0077] The nucleotide sequence of the 16S rRNA of strain YH26 is shown in SEQ ID NO.8, specifically:

[0078] The nucleotide sequence of the 16S rRNA of strain YH22 is shown in SEQ ID NO.9, specifically:

[0079] The nucleotide sequence of the 16S rRNA of strain YH31 is shown in SEQ ID NO.10, specifically:

[0080] The nucleotide sequence of the 16S rRNA of strain YH33 is shown in SEQ ID NO.11, specifically:

[0081] The nucleotide sequence of the 16S rRNA of strain YH37 is shown in SEQ ID NO.12, specifically:

[0082] Based on morphological, physiological and biochemical characteristics and 16S rRNA identification, the taxonomic position of the 12 strains was finally determined to be Bacillus belesiensis (B. belesiensis). Bacillus velezensis YH1, Bacillus spheroidae ( Bacillus stercoris YH2, *Priscilla argentea* ( Priestia aryabhattai YH4, cold-resistant Bacillus subtilis ( Peribacillus frigoritolerans YH5, Korean Bacillus subtilis ( Sporosarcina koreensis YH17, Serratia procumbens ( Serratia plymuthica YH21, Microbacterium natoriense YH22, Hydroxybacterium oxidans ( Microbacterium hydrocarbonoxydans YH24, Simple Bacillus ( Peribacillus simplex YH26, Bacillus pumilus ( Bacillus pumilus YH31, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens YH33, *Pseudomonas arsenic-oxidizing* ( Pseudomonas arsenicoxydans YH37. Three other strains were used for further research: Bacillus amyloliquefaciens (…). Bacillus amyloliquefaciens TF28, Bacillus belysinus ( Bacillus velezensis DJ1, Bacillus subtilis ( Bacillus subtilis GB519.

[0083] The strains used subsequently include: Bacillus amyloliquefaciens TF28, which has been disclosed in patent CN 110655925 A. Bacillus bereaves DJ1 has been disclosed in the literature [Ma Junxiu, Wu Haoqiong, Jiang Wei, et al. Screening, identification and efficacy study of broad-spectrum antagonistic bacterial strains against soft rot pathogens of vegetables [J]. Biotechnology Bulletin, 2023, 39(7):228-240.DOI:10.13560 / j.cnki.biotech.bull.1985.2022-1349.]. Bacillus subtilis GB519 has been disclosed in the literature [Zhang Xueming, Zhu Feng, Chen Yubo, et al. Isolation and identification of gray mold pathogens of strawberries and the disease prevention and growth promotion effects of Bacillus subtilis GB519 [J]. Jiangsu Agricultural Sciences, 2025, 53(17): 154-159].

[0084] 2. Verification experiment on reinoculation of soybean root rot pathogen The results of the verification experiment on reinoculation with Fusarium oxysporum, the disease of solanum, showed that inoculation 15 days after planting... F. solani The soybean plants in the treatment group showed a 100% susceptibility rate to root rot, exhibiting typical symptoms such as yellowing and brown spots on seedling leaves, stunted growth, and weak development. Figure 1In the control group (CK), the main root turns brown or black, the number of fibrous roots is significantly reduced and they turn brown and rot. The plant can continue to grow through the infection point, but the entire root system is severely rotten, and a large number of lateral roots and root hairs die. In the control group (CK), soybean plants are vigorous, with dark green leaves, well-developed root systems, and no symptoms of disease. Figure 1 (B)

[0085] 3. Screening of strains with inhibitory effects against Fusarium solani and determination of inhibition rate. The 118 bacteria selected through the plate confrontation method were targeted... F. solani Antibacterial activity was determined. The results showed that 36 strains exhibited varying degrees of antagonistic activity against *F. solani*. The inhibition rate of these 36 strains was then measured, and 15 strains (YH1, YH2, YH4, YH5, YH17, YH21, YH22, YH24, YH26, YH31, YH33, YH37, TF28, DJ1, and N2) showed inhibition rates above 60% (see Table 1 for details). Strains TF28, DJ1, and N2 were not selected in this experiment and were used as control strains.

[0086] Table 1. Inhibition rate of screened strains against F. solani

[0087] 4. The effect of constructing synthetic microbial communities on pot control of soybean root rot, such as... Figure 2 As shown.

[0088] Fifteen bacterial strains with inhibition rates above 60% were used to construct a synthetic microbial community (SynCom, or SBC). Four treatments were used to evaluate their control efficacy against soybean root rot in pot experiments. Results showed no disease incidence in the ck and SBC treatment groups, while the SBC+FUS treatment resulted in a disease incidence of 15-20%. The FUS treatment group exhibited the highest disease incidence, around 80%. Figure 2 (k). Disease index results showed that SBC inoculation reduced the incidence of soybean root rot by approximately 75%. In the SBC+FUS and FUS treatment groups... F. solani The density is significantly increased ( Figure 2 In the middle group (l), the disease index of SBC group decreased significantly, and the relative efficacy reached more than 70%. Figure 2 (m). Plant growth traits showed that the CK treatment group grew normally ( Figure 2 In the SBC treatment group (g), the plants were robust, with dark green leaves and more branches, showing a significant growth-promoting effect. Figure 2 (h). The SBC+FUS treatment group showed no obvious disease symptoms (h). Figure 2 (i). The FUS treatment group exhibited typical disease symptoms, including stunted growth, yellowing and wilting of leaves, partial lodging, and stem base rot, and was the most severely affected group. Figure 2The results of six growth indicators—plant height, root weight, aboveground fresh weight, underground fresh weight, pod number, and nitrogen content—showed that the CK group plants grew normally, but were relatively shorter in height compared to the SBC and SBC+FUS treatment groups. Figure 2 (a) FUS treatment alone significantly reduced root weight, but SBC+FUS treatment completely reversed the inhibitory effect of the pathogen on the root system, with root weight significantly higher than the CK group, showing a clear overcompensation effect. Figure 2 (b) SBC treatment significantly increased soybean pod number, and the SBC+FUS treatment maintained a high level of reproductive growth even under pathogen stress, while FUS treatment alone led to a significant decrease in pod number, indicating that SBC plays an important protective role in soybean yield formation. Figure 2 (c). Furthermore, FUS vaccination alone significantly reduced both aboveground and belowground dry weight ( Figure 2 The SBC treatment had a protective effect on plant biomass accumulation; the FUS treatment alone also significantly reduced plant nitrogen content and inhibited nitrogen absorption or metabolism. The nitrogen content of both the SBC and SBC+FUS treatments was higher than that of the FUS treatment, but the difference from the CK group was not significant. Figure 2 (f)

[0089] 5. Simplified Synthetic Microbiota - In Vitro Interactions Among Core Microorganisms, such as Figures 3-4 As shown.

[0090] Figure 3 OD values ​​based on cultures of 15 core strains in fresh culture medium and strain waste liquid 600 Average values ​​are used to construct a circular heatmap to illustrate the interactions. Each radial sector corresponds to the residual culture medium (SM) produced by one active strain, and each concentric ring corresponds to one test strain (i.e., the strain inoculated into the SM). Color scheme: Red (OD). 600 >1) Represents growth promotion; White / light color (OD) 600 ≈1) represents no significant effect; blue (OD) 600 <1) represents inhibited growth. Figure 4 Based on OD 600 Waste liquid / OD 600 The ratio of fresh culture medium was analyzed for significance using a t-test, generating a simplified interaction matrix. In the figure, "+" indicates a t-test result significantly greater than 1 (promoting effect); "-" indicates a t-test result significantly less than 1 (inhibiting effect); "0" indicates no significant difference between the t-test result and 1 (neutral, no overlap); (P<0.05, n = 3).

[0091] We used a supernatant co-culture method to simplify the number of strains in the synthetic microbial community. Based on OD 600The values ​​are visually represented in the form of a ring heatmap, which shows the in vitro interaction network among the 15 tested strains. Figures 3-4 The results showed that the interactions among strains exhibited obvious modular clustering characteristics. Based on OD... 600 Waste liquid / OD 600 The interaction symbol matrix constructed by the T-test significance analysis of the fresh culture medium ratio clearly showed the directional pattern of interactions among the 15 core strains. The results showed that reciprocal (+ / +) relationships were densely distributed in the lower left region of the matrix. YH2 and YH5, YH17, YH21, YH22, and YH33 all showed strong bidirectional promotion, indicating broad metabolic compatibility between Bacillus and members of the Actinobacteria and Proteobacteria phyla, forming a stable core framework for the community. In contrast, the rows / columns of YH4, YH26, YH31, YH37, and TF28 were predominantly blue (- / -) and light blue (0 / -), exhibiting broad-spectrum inhibition or partial harm effects. The pivotal roles of YH2 and YH33 (5 groups + / +), YH5 and YH17 (4 groups + / +), and YH21 and YH22 (3 groups + / +) were clarified for several strains.

[0092] Based on this, a simplified microbial community was further established. First, members YH17, YH21, YH22, YH33, and YH5 from the + / + mutualistic dense zone were retained, while symbiotic bridging strains YH1, YH2, and DJ1 were integrated to maintain network connectivity. Simple superposition of 0 / 0 neutral relationships was avoided, ensuring that each new member formed a positive interaction with the existing network. The resulting simplified combinations SBC8 (YH1, YH2, YH5, YH17, YH21, YH22, YH33, and DJ1), SBC4 (YH1, YH2, YH5, and YH17), and SBC3 (YH17, YH22, and YH33) ensured both functional integrity and maximized ecological stability.

[0093] 6. Simplify the impact of SynComs on soybean growth Based on the results of in vitro interactions among core microorganisms, a simplified synthetic microbial community was used in a soybean pot experiment. Several physiological indicators, including plant height, fresh weight, aboveground dry weight, and nitrogen content, were measured. Figure 5 The results showed that several synthetic bacterial groups all had a growth-promoting effect on soybeans, and their growth traits were better than those of the CK control. Figure 5 The soybean plant height measurements for the three groups (SBC 4, SBC 8 ... Figure 5The fresh weight measurements were as follows: 8.920±0.6657 (SBC 8), 6.702±0.5789 (CK), 9.422±0.8522 (SBC 3), 9.688±1.382 (SBC 4), and 10.41±0.6760 (SBC 5). Figure 5 The aboveground dry weight measurements were as follows: 2.013±0.2163 (SBC), 2.001±0.1962 (SBC 4), 1.858±0.1378 (SBC 8), 1.610±0.1811 (SBC 3), and 1.431±0.1403 (CK). Figure 5 The nitrogen content determination results showed: 12.90±0.3495 (SBC 4), 12.86±0.2283 (SBC), 12.63±0.2202 (SBC 8), 12.63±0.2007 (SBC 3), 12.21±0.3340 (CK) ( ). Figure 5 (J). Synthetic communities constructed from different strains were superior to those constructed from ck, with SBC4 forming a multifunctional community. Bacillus velezensis YH 1、 Bacillus stercoris YH 2、 Peribacillus frigoritolerans YH 5、 Sporosarcina koreensis YH17 showed better results than other combinations. The strains included *Bacillus belyssae* YH1, *Bacillus spheroidae* YH2, *Bacillus cytotoxicus* YH5, and *Bacillus koreana* YH17. This study used an in vitro interaction matrix to screen for positive / negative interactions among the core strains. The constructed simplified synthetic microbial community exhibited significant growth-promoting and disease-suppressing effects in pot experiments. The four-strain combination (SBC 4) showed the best results, while the 15-strain whole-cell combination (SBC) did not show synergistic effects, suggesting possible competition or antagonism between the strains.

[0094] This invention has deposited *Bacillus belyssus* YH1, *Bacillus spheroidae* YH2, *Bacillus cytotoxicus* YH5, and *Bacillus koreana* YH17 at the China General Microbiological Culture Collection Center, respectively. *Bacillus belyssus* ( Bacillus velezensis The preservation number of YH 1 is CGMCC No. 39711; Bacillus spheroidae ( Bacillus stercoris The preservation number of YH 2 is CGMCC No. 39712; *Bacillus subtilis* (a cold-resistant strain) Peribacillus frigoritolerans YH 5 has the accession number CGMCC No. 39713; Bacillus thuringiensis (Korean Bacillus) Sporosarcina koreensis YH17 has the accession number CGMCC No. 39714.

[0095] 7. Study on the interaction mechanism among SBC4 strains based on metabolomics To elucidate the functional complementarity mechanism among the four strains in the SBC4 synthetic bacterial community, non-targeted metabolomics analysis was performed on the culture supernatant of each strain. The results are as follows: Figure 6 As shown. Six sets of cross-culture heatmaps revealed the differences in metabolic secretion profiles and cross-feeding patterns among the strains. Four strains formed a complex cross-secretion network of metabolites, involving multiple metabolic categories such as amino acids, vitamins, purine / pyrimidine bases, and organic acids. YH1 specifically secreted multiple amino acids, including arginine, isoleucine, leucine, and phenylalanine, as well as pyruvate and uracil, and was a core amino acid donor in the bacterial community. Figure 6 (A~C). YH2 specifically secretes high levels of lysine, methionine, and glutamate, while also secreting guanine and methylmalonate. Figure 6 (A, D, E). It also secretes high levels of betaine. Furthermore, histamine and 2-(4-methylphenyl)-ethyl-6-hydroxybenzoic acid, among other special metabolites, were detected in the YH2 secretion profile, serving as osmotic protectants and organic acid suppliers. YH5 ( P. frigoritolerans () Figure 6 It metabolizes purines (B, D, F) in the middle purine group, and has high levels of hypoxanthine, adenine, and allopurinol secretion, providing nucleic acid synthesis precursors for other strains. YH17 ( S. koreensis () Figure 6 C, E, and F form a synergistic amino acid secretion pair, jointly secreting valine, proline, β-alanine, creatine, and pyroglutamic acid. Heatmaps reveal significant metabolic functional differentiation within the bacterial community: YH1 and YH2 are responsible for amino acid and carbon source supply, YH5 provides purine bases, and YH17 supplements essential amino acids and vitamin precursors (β-alanine is a precursor for pantothenic acid synthesis). This three-dimensional cross-feeding structure of "carbon source-amino acid-purine base" is a key foundation for the simplified and stable function of the SBC4 community. Untargeted metabolomics revealed a complex cross-secretion network of metabolites among the four cross-genera strains, thus promoting growth in plant height, fresh weight, and aboveground dry weight. B. velezensis YH 1 and B. stercoris YH2 secretes central carbon metabolites such as pyruvate and glutamate, which play the role of "primary degradation products," providing... P. frigoritolerans YH 5、 S. koreensisYH 17 provides a universal carbon and energy source, conforming to the theoretical framework of a tertiary trophic structure. In SBC 4, the betaine secretion of YH 2 may not only protect itself and symbiotic strains from rhizosphere osmotic stress, but may also support the purine methylation modification of YH 17 by providing methyl groups, forming a deep coupling of metabolic functions. The adenine and hypoxanthine secreted by YH5 can be directly used for nucleotide synthesis by strains with purine salvage synthesis pathways, such as YH1 and YH17, avoiding the high energy consumption of de novo synthesis. The four strains in SBC 4 each secrete unique combinations of metabolites, while relying on other strains to provide different essential nutrients, forming a highly intertwined metabolic dependency network. This is the structural basis for the stable colonization and continuous growth-promoting function of this bacterial community in the complex rhizosphere environment.

[0096] In summary, the synthetic bacterial community SBC 4 (SBC 4) provided by this invention... Bacillus velezensis YH1, Bacillus dung YH2, Peribacillus frigoritolerans YH5 Sporosarcina koreensis YH17 exhibits disease resistance and growth-promoting effects on soybeans. Metabolomics analysis confirmed a clear metabolic division of labor and functional complementarity among the SBC4 strains. Through cross-supply of carbon sources, amino acids, and purine-pyrimidine metabolites, a stable three-dimensional cooperative network of "carbon source-amino acid-purine base" was constructed, effectively reducing the metabolic burden on the microbial community and maintaining its structural and functional stability. In summary, the screening and acquisition of the structurally stable and highly effective synthetic microbial community SBC4 can provide a theoretical basis and strain resources for the green control of soybean root rot and the improvement of rhizosphere microecology.

[0097] Example 2 1. Preparation of pathogen spore suspension: *Fusarium solani* var. *solani* was inoculated onto PDA plates and cultured at 28°C for 7 days. After sporulation, the spores were washed with sterile water, filtered through four layers of sterile gauze, counted using a hemocytometer, and the spore concentration was adjusted to 3.6 × 10⁻⁶. 5 Units / mL are available for use.

[0098] 2. Preparation of bacterial suspension: Strains YH1, YH2, YH5, and YH17 were inoculated into LB liquid medium and cultured at 30℃ and 180 rpm with shaking for 24 h at 4℃. The cultured bacterial suspensions were collected by centrifugation at 8000 rpm for 10 min, washed twice with sterile physiological saline, and resuspended. The concentration of each strain was adjusted to 1.0 × 10⁻⁶. 8 CFU / mL was used for subsequent experiments.

[0099] Preparation of synthetic bacterial suspension: YH1, YH2, YH5, and YH17 strains were inoculated into LB liquid medium and cultured at 30℃ and 180 r / min with shaking for 24 h. The cultured bacterial suspension was centrifuged at 8000 r / min for 10 min (4℃) to collect the bacterial cells. The cells were washed twice with sterile physiological saline and resuspended. The concentration of each strain was adjusted to 1.0 × 10⁻⁶. 8 A synthetic bacterial suspension (SBC4 strain) was prepared by mixing equal volumes of CFU / mL.

[0100] 3. Preparation of experimental materials: The soybean variety used was Heihe 43. Seeds were surface-sterilized (immersed in 75% ethanol for 30 seconds, 2% sodium hypochlorite solution for 10 minutes, and rinsed 5 times with sterile water), and then germinated in a 28℃ constant temperature incubator for 24 hours. Seeds with consistent germination were selected for pot experiments. The soil was autoclaved at 121℃ for 2 hours (sterilized 3 times consecutively at 24-hour intervals), dried, and then sieved through a 2 mm sieve for later use. The flowerpots were 15 cm × 20 cm, and each pot contained 500–600 g of sterilized soil.

[0101] 4. An indoor pot experiment was conducted to determine the control effects of various fungi on soybean root rot and their growth-promoting effects on soybeans. Germinated soybean seeds were sown in sterile soil pots, 3 seeds per pot, covered with 2 cm of soil, and placed in a light incubator for routine cultivation (temperature 25℃, photoperiod 14 h / 10 h, light intensity 8000 lx, relative humidity above 70%). After emergence, seedlings were thinned, retaining one healthy plant per pot with uniform growth.

[0102] 5. When soybeans reached the three-leaf stage, inoculation treatments were carried out. The growth-promoting experiment included five treatment groups: Treatment 1: Control group (CK), each pot was watered with 50 mL of sterile water. Treatment 2: YH1 treatment group: each pot was watered with 50 mL of YH1 bacterial suspension. Treatment 3: YH2 treatment group: each pot was watered with 50 mL of YH2 bacterial suspension. Treatment 4: YH5 treatment group: each pot was watered with 50 mL of YH5 bacterial suspension. Treatment 5: YH17 treatment group: each pot was watered with 50 mL of YH17 bacterial suspension. Each treatment had 8-10 replicates, completely randomized. After inoculation, the soybeans were kept in a light incubator and watered regularly and quantitatively to maintain soil moisture content at 60%-70% of field capacity. Fifteen days after irrigation with the bacterial suspension, the growth indicators of soybeans in each group were recorded.

[0103] 6. When soybeans reach the three-leaf stage, inoculation treatments were conducted. The experiment verifying the control effect on soybean root rot included six treatment groups: Treatment 1: Each pot was watered with 50 mL of sterile water, and after 24 h, inoculated with 50 mL of pathogen spore suspension. Treatment 2: Each pot was watered with 50 mL of YH1 bacterial suspension, and after 24 h, inoculated with 50 mL of pathogen spore suspension. Treatment 3: Each pot was watered with 50 mL of YH2 bacterial suspension, and after 24 h, inoculated with 50 mL of pathogen spore suspension. Treatment 4: Each pot was watered with 50 mL of YH5 bacterial suspension, and after 24 h, inoculated with 50 mL of pathogen spore suspension. Treatment 5: Each pot was watered with 50 mL of YH17 bacterial suspension, and after 24 h, inoculated with 50 mL of pathogen spore suspension. Treatment 6: Each pot was watered with 50 mL of (SBC4 strain) bacterial suspension, and after 24 h, inoculated with 50 mL of pathogen spore suspension.

[0104] Each treatment was replicated 8-10 times, in a completely randomized manner. After inoculation, the plants were kept in a light incubator and watered regularly and quantitatively to maintain soil moisture content at 60%-70% of field capacity.

[0105] Disease survey and index determination: Disease survey was conducted 15 days after inoculation with pathogen. The criteria for evaluating disease severity were the same as in Example 1.

[0106] 7. Results (1) Growth indicators of each treatment group in the growth-promoting experiment are as follows: Figure 7 As shown in the figure. The results indicate that different strains of bacteria have varying effects on promoting soybean growth. YH1 significantly increased soybean plant height, fresh weight, plant dry weight, and root length. The YH17 treatment showed the best performance in plant height, fresh weight, and root length. YH2 and YH5 had weaker growth-promoting effects, with some indicators showing no significant advantage over the control. Overall, YH1 and YH17 have outstanding growth-promoting potential and can effectively promote the growth of soybean aboveground parts and roots.

[0107] (2) The growth of soybeans in each treatment group and the disease control effect in the verification experiment of soybean root rot control are shown in Table 2.

[0108] Table 2. Control efficacy of each treatment group against soybean root rot

[0109] Table 2 shows that the disease index of the CK control group was 6.25. The disease index of YH1 was 3.57, with a relative control effect of 42.88%, indicating disease prevention efficacy. Under the combined conditions of SBC (4 strains of bacteria), the control effect of soybean root rot was significantly improved, and the four strains achieved a synergistic effect in controlling soybean root rot.

[0110] 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, characterized in that, include: Bacillus belesiensis ( Bacillus velezensis ), Bacillus spheroidae ( Bacillus stercoris ), cold-resistant Bacillus subtilis ( Peribacillus frigoritolerans ) and Korean Bacillus ( Sporosarcina koreensis The ratio of viable bacteria of *Bacillus belyssioides*, *Bacillus spheroidae*, *Bacillus frostridae*, and *Bacillus koreana* in the synthetic bacterial community is 1:1:1:1; the total viable count of the synthetic bacterial community is ≥2.0 × 10⁻⁶. 7 CFU / mL.

2. The synthetic microbial community according to claim 1, characterized in that, The *Bacillus belyssus* includes *Bacillus belyssus* YH1 with accession number CGMCC No. 39711; the *Bacillus spheroidae* includes *Bacillus spheroidae* YH2 with accession number CGMCC No. 39712; the *Bacillus cytosus* includes *Bacillus cytosus* YH5 with accession number CGMCC No. 39713; and the *Bacillus koreana* includes *Bacillus koreana* YH17 with accession number CGMCC No. 39714.

3. The method for preparing the synthetic microbial community according to claim 1 or 2, characterized in that, Includes the following steps: Each bacterium was inoculated into a liquid culture medium for cultivation. The bacterial resuspension was collected and adjusted to obtain a bacterial solution with an equal number of viable bacteria to obtain the seed solution of each bacterium. Equal volumes of seed cultures of each bacterium were mixed to obtain a synthetic bacterial community.

4. The preparation method according to claim 3, characterized in that, The liquid culture medium includes LB medium; the culture temperature is 25~30℃; and the culture time is 16~24h.

5. A bacterium, characterized in that, This includes any one of Bacillus sporeans YH17, Bacillus repens YH5, Bacillus belyssus YH1, and Bacillus spheroidosa YH2; the preservation number of Bacillus sporeans YH17 is CGMCC No. 39714; the preservation number of Bacillus repens YH5 is CGMCC No. 39713; the preservation number of Bacillus belyssus YH1 is CGMCC No. 39711; and the preservation number of Bacillus spheroidosa YH2 is CGMCC No. 39712.

6. The use of the synthetic microbial community of claim 1 or 2, the synthetic microbial community prepared by the preparation method of claim 3 or 4, or the bacteria of claim 5 in the preparation of agents to inhibit pathogens; wherein the pathogen includes Fusarium solani.

7. The application of the synthetic microbial community of claim 1 or 2, the synthetic microbial community prepared by the preparation method of claim 3 or 4, or the microorganism of claim 5 in promoting plant growth and / or controlling plant diseases; wherein the plant includes soybean.

8. The application according to claim 7, characterized in that, The plant diseases mentioned include root rot.

9. A method for promoting soybean growth and / or preventing soybean diseases, characterized in that, include: The synthetic microbial community described in claim 1 or 2, the synthetic microbial community prepared by the preparation method described in claim 3 or 4, or the microbial community described in claim 5 are applied during the soybean growth process.

10. The method according to claim 9, characterized in that, The application period of the synthetic microbial community or the microorganism includes the soybean 2-true-leaf stage and / or 3-leaf stage; the application amount of the synthetic microbial community is 50~300 mL / plant.

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