Synthetic bacterial flora-containing complex microbial agent, and preparation method and application thereof

Abstract

The application provides a complex microbial agent containing a synthetic flora, a preparation method and application thereof, and relates to the technical field of microorganisms. The synthetic flora is composed of Bacillus subtilis YX1 and Pseudomonas marginalis. The Bacillus subtilis YX1 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 31921; the Pseudomonas marginalis is also preserved in the same institution, and the preservation number is CGMCC 1.2844. The synthetic flora has good environmental adaptability and rhizosphere survival ability after being applied to the ginseng rhizosphere. Through the combination of the above specific functional strains, long-term colonization of functional microorganisms in an aluminum stress environment is realized, and an effective solution is provided for the problem of unstable colonization of microbial agents in the ginseng planting process.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a composite microbial agent containing a synthetic microbial community, its preparation method, and its application. Background Technology

[0002] Ginseng is a perennial and precious traditional Chinese medicine with a long cultivation cycle. Cultivated ginseng typically requires 4-6 years, while forest-grown ginseng can grow for over 15 years. During long-term continuous cropping, the soil ecological environment deteriorates, with soil acidification being particularly severe. When the soil pH is below 5, active trivalent aluminum ions (Al2+)... 3+ Large amounts of leaching can cause toxicity to plant roots, manifesting as root tip browning, inhibited elongation, and impaired nutrient absorption, which in turn seriously affects the growth, development, biomass accumulation, and quality formation of ginseng.

[0003] Rhizosphere microorganisms, as important regulators of plant growth, are often referred to as the plant's "second genome." Studies have shown that beneficial microorganisms can reduce environmental Al levels by secreting organic acids and chelating metal ions. 3+ The bioavailability of these strains alleviates aluminum stress; simultaneously, some strains also possess functions such as phosphorus solubilization, nitrogen fixation, and production of plant growth hormones (e.g., indoleacetic acid, IAA), which can promote root development and enhance plant stress resistance. Bacillus subtilis ( Bacillus subtilis ) and marginal pseudomonas ( Pseudomonas marginalis Plant growth promoters (PMPs) are common plant growth-promoting bacteria widely found in soil, and studies have reported that they possess certain aluminum tolerance and promote plant growth. However, in practical applications, the function of a single strain is relatively limited, and due to poor environmental adaptability, weak colonization ability, and susceptibility to competition and exclusion from native microorganisms, their application effects are often unstable, making it difficult to meet the needs of perennial crops like ginseng for continuous microecological regulation throughout their entire growth period.

[0004] In recent years, with the development of synthetic biology and microbial ecology, synthetic microbial communities have gradually become a research hotspot in the field of agricultural microbiology. Compared with single-strain preparations, synthetic microbial communities can achieve more stable rhizosphere colonization and stronger comprehensive growth-promoting effects through functional complementarity and metabolic cooperation among different strains, exhibiting a synergistic effect of "1+1>2". An ideal synthetic microbial community not only possesses characteristics such as controllable structure, well-defined functions, and strong environmental adaptability, but can also simulate the key functions of the natural microbiome through a few core strains, exerting the aforementioned multiple growth-promoting and stress-resistance functions to form a stable and mutually beneficial micro-ecosystem. This results in significant advantages in rhizosphere colonization, functional stability, and niche construction, providing a more comprehensive, efficient, and durable solution for plants to cope with aluminum toxicity stress and other adverse conditions.

[0005] Currently, some compound microbial preparations are still at the stage of simple mixing of multiple strains, lacking scientific basis for formulation, and may even exhibit antagonism within the microbial community, leading to weakened or ineffective function. Furthermore, undesigned microbial community combinations struggle to maintain a stable structure in complex soil environments, limiting their widespread application in agricultural production. Therefore, how to rationally construct a synthetic microbial community that can stably coexist and exert synergistic growth-promoting effects in acidic soil with aluminum toxicity, based on functional characteristics and interaction mechanisms, has become a key technical challenge for improving the cultivation quality of medicinal plants.

[0006] To address the aforementioned issues, it is imperative and urgent to develop a functional synthetic microbial community suitable for ginseng cultivation systems that can effectively alleviate aluminum stress and continuously promote root growth and nutrient absorption.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The primary objective of this invention is to provide a synthetic microbial community that enhances the adaptability of the microbial community to harsh environments through metabolic complementarity and mutual benefit between two microorganisms. This community can maintain high activity under aluminum toxicity stress in acidified soil, thereby achieving long-term regulation of the microenvironment of ginseng roots.

[0009] The second objective of this invention is to provide a compound microbial agent.

[0010] The third objective of this invention is to provide a method for cultivating ginseng under aluminum stress.

[0011] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: This invention provides a synthetic microbial community, wherein the synthetic microbial community includes Bacillus subtilis (… Bacillus subtilis YX1 and Pseudomonas marginalis ( Pseudomonas marginalis ),in: The Bacillus subtilis YX1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 31921; The aforementioned *Pseudomonas marginalis* is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 1.2844. When the synthetic microbial community was applied to the rhizosphere of ginseng and colonized for 90 days in acidified soil with pH ≤ 5, the relative colonization rate was ≥ 55% of the initial inoculum.

[0012] Furthermore, the effective viable count of Bacillus subtilis YX1 and Pseudomonas marginalis is 1:1.

[0013] Furthermore, the synthetic microbial community exhibits synergistic growth characteristics. After culturing in TSB liquid medium containing 100 mM Al2(SO4)3 for 36 hours, the OD600 value of the synthetic microbial community is 12.40~12.63% higher than that of any single control group.

[0014] The present invention provides a compound microbial agent, which includes the above-mentioned synthetic microbial community and an agriculturally acceptable carrier.

[0015] Furthermore, the formulation of the compound microbial agent includes one of the following: liquid microbial solution, powder, or granules.

[0016] Furthermore, the compound bacterial agent is a liquid bacterial solution; Preferably, the liquid bacterial solution is a bacterial strain suspension, and the concentration of the synthetic bacterial community in the bacterial strain suspension is 1×10⁻⁶. 8 ~6×10 8 CFU / mL.

[0017] The above-mentioned synthetic microbial flora or compound microbial agent provided by the present invention is used in the preparation of products for relieving aluminum stress in ginseng and promoting its growth.

[0018] Furthermore, the relief of aluminum stress includes: Enhance the vitality of ginseng roots; Increase the amount of dry weight accumulated in the underground part; Promotes biomass accumulation in both the above-ground and underground parts of ginseng; Extend the colonization time of functional bacteria in the rhizosphere.

[0019] The present invention provides a method for cultivating ginseng under aluminum stress, the method comprising: dipping the roots of ginseng seedlings before transplanting, or drenching the roots during the growth period, wherein the treatment solution contains the synthetic microbial community described above, so that the synthetic microbial community continuously colonizes and plays a role in the rhizosphere of ginseng, thereby continuously improving the growth of ginseng in aluminum-stressed soil.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a synthetic microbial community that, after being applied to the rhizosphere of ginseng, can stably survive for a long period in acidified soil with a pH ≤ 5.0. Experimental results show that this synthetic microbial community consists of *Bacillus subtilis* YX1 (CGMCC No. 31921) and *Pseudomonas limbi* (CGMCC No. 1.2844). Through metabolic complementarity and mutual benefit between the two strains, the synthetic microbial community enhances its adaptability to harsh environments and maintains high activity under aluminum toxicity stress in acidified soil, thereby achieving long-term regulation of the ginseng root microenvironment. Experimental verification shows that when continuously colonized in an aluminum-stressed rhizosphere environment for 90 days, the relative colonization rate is ≥ 55% of the initial inoculum, significantly better than the survival time when a single strain is applied alone.

[0021] The present invention provides a compound microbial agent, which improves the stability and survival rate of the microorganisms during storage and application by combining synthetic microbial communities with synergistic growth characteristics and good environmental adaptability with a suitable carrier, thereby facilitating the effective colonization of the microbial community under field conditions.

[0022] This invention provides a method for cultivating ginseng under aluminum stress. The method involves dipping ginseng seedlings in water before transplanting or drenching them during the growing season to apply the aforementioned synthetic microbial community to the plant roots. This allows *Bacillus subtilis* YX1 and *Pseudomonas marginalis* to directly colonize the rhizosphere. This method helps increase the initial inoculum size and survival rate of the functional microbial community in acidified soil, ensuring its stable bioregulatory function under aluminum stress.

[0023] Biological Preservation Instructions: 1. Bacillus subtilis in this application ( Bacillus subtilis YX1, deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 10, 2024, with accession number CGMCC No. 31921. This strain is a previously disclosed strain; for details, please refer to patent documents such as CN 119162037A.

[0024] 2. *Pseudomonas marginalis* in this application ( Pseudomonas marginalis The *Pseudomonas marginalis* strain with accession number CGMCC 1.2844 is deposited at the China General Microbiological Culture Collection Center (CGMCC). This application's *Pseudomonas marginalis* strain with accession number CGMCC 1.2844 was purchased externally; the purchase link is as follows: https: / / www.cgmcc.net / resources / details?uuid=78d56290-8ef2-11f0-94c6-78ac44479d74.

[0025] Therefore, the *Bacillus subtilis* YX1 (accession number CGMCC No. 31921) and *Pseudomonas marginalis* (accession number CGMCC 1.2844) strains mentioned above in this application are both publicly disclosed and available to the public. This application does not involve the biopreservation of the aforementioned *Bacillus subtilis* YX1 (accession number CGMCC No. 31921) and *Pseudomonas marginalis* (accession number CGMCC 1.2844) strains. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is a growth rate diagram of the synthetic bacterial community provided in Embodiment 2 of the present invention; Figure 2 The synthetic bacterial community provided in Example 3 of this invention was prepared in environments containing different concentrations of Al. 3+ Aluminum resistance diagram of TSB liquid culture medium; Figure 3 This is a graph showing the effect of each treatment group on ginseng growth indicators provided in Embodiment 4 of the present invention; Figure 4 This is a diagram showing the effect of each treatment group on the growth phenotype of ginseng, provided in Example 4 of the present invention. Figure 5 This is a comparison chart of the colonization rates of each treatment group in the rhizosphere of ginseng provided in Example 5 of the present invention; Figure 6 The metabolic exchange heatmap of the synthetic microbial community is shown in the simulation results provided in Embodiment 6 of the present invention. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] According to one aspect of the present invention, a synthetic microbial community, said synthetic microbial community comprising Bacillus subtilis (… Bacillus subtilis YX1 and Pseudomonas marginalis ( Pseudomonas marginalisThe Bacillus subtilis YX1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31921; the Pseudomonas marginalis is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 1.2844. When the synthetic microbial community was applied to the rhizosphere of ginseng and colonized for 90 days in acidified soil with pH ≤ 5, the relative colonization rate was ≥ 55% of the initial inoculum.

[0030] This invention provides a synthetic microbial community that, when applied to the rhizosphere of ginseng, can survive stably for a long period in acidified soil with a pH ≤ 5.0. Experimental results show that this synthetic microbial community consists of *Bacillus subtilis* YX1 (CGMCC No. 31921) and *Pseudomonas limbi* (CGMCC No. 1.2844). Through metabolic complementarity and mutual benefit between the two strains, the synthetic microbial community enhances its adaptability to harsh environments, maintaining high activity in acidification-aluminum toxicity combined stress conditions, thereby achieving long-term regulation of the ginseng root microenvironment. Experimental verification shows that when continuously colonized in an aluminum-stressed rhizosphere environment for 90 days, the relative colonization rate is ≥ 55% of the initial inoculum, significantly better than the survival time when a single strain is applied alone.

[0031] In addition, due to the good environmental adaptability and stable coexistence of the synthetic microbial community, the toxic effects of aluminum ions on ginseng roots are effectively alleviated, nutrient absorption and root development are promoted, and ultimately the biomass accumulation and overall growth status of ginseng are significantly improved.

[0032] In a preferred embodiment of this application, the effective viable count of Bacillus subtilis YX1 and Pseudomonas marginalis is 1:1.

[0033] In a preferred embodiment, the synthesized microbial community consists of Bacillus subtilis YX1 and Pseudomonas marginalis in a 1:1 ratio of viable cells. This ratio has been experimentally verified to help maintain the growth advantage of both strains under co-culture conditions. After 36 hours of shaking culture in TSB liquid medium containing 100 mM Al2(SO4)3, the OD600 value of the synthesized microbial community was significantly higher than that of any single-strain control group, exhibiting superior growth performance, indicating that this ratio is conducive to achieving good interspecific interactions. Furthermore, in pot experiments, after inoculation with this 1:1 ratio, the plant height, root length, aboveground fresh weight, and underground dry weight of ginseng under aluminum stress conditions were all superior to those of the single-strain treatment group, demonstrating that the synthesized microbial community at this ratio has a stronger comprehensive effect in alleviating aluminum stress and promoting ginseng growth.

[0034] In a preferred embodiment of this application, the synthetic bacterial community has synergistic growth characteristics. After being cultured in TSB liquid medium containing 100 mM Al2(SO4)3 for 36 hours, the OD600 value of the synthetic bacterial community is 12.4-16.63% higher than that of any single control group.

[0035] In a preferred embodiment, the synthesized microbial community exhibits synergistic growth characteristics. After culturing in TSB liquid medium containing 100 mM Al2(SO4)3 at 28°C and 180 rpm for 36 hours with shaking, its OD600 value was at least 25% higher than that of the Bacillus subtilis YX1 single-strain control group and the Pseudomonas limbi single-strain control group. This result indicates that the two strains can form an interaction beneficial to each other's growth under co-culture conditions, overcoming the inhibitory effect of high aluminum environment on individual growth. The existence of the above-mentioned synergistic growth characteristics also provides a basis for the synthesized microbial community to maintain high activity under aluminum stress in acidified soil, making it easier for it to colonize in the rhizosphere of ginseng and exert a sustained effect compared to single strains, thereby enhancing its growth-promoting ability and stress resistance regulation effect on plants.

[0036] According to one aspect of the present invention, a compound microbial agent comprises the above-mentioned synthetic microbial community and an agriculturally acceptable carrier.

[0037] The compound microbial agent provided by this invention combines synthetic microbial communities with synergistic growth characteristics and good environmental adaptability with a suitable carrier, thereby improving the stability and survival rate of the microbial community during storage and application, which is conducive to the effective colonization of the microbial community under field conditions.

[0038] In a preferred embodiment of this application, the dosage form of the compound microbial agent includes one of the following: liquid microbial solution, powder, or granules.

[0039] In the preferred embodiment described above, the compound bacterial agent is a liquid bacterial solution; Preferably, the liquid bacterial solution is a bacterial strain suspension, and the concentration of the synthetic bacterial community in the bacterial strain suspension is 1×10⁻⁶. 8 ~6×10 8 CFU / mL.

[0040] According to one aspect of the present invention, the above-mentioned synthetic microbial community or the above-mentioned compound microbial agent provided by the present invention can be widely used in the product preparation process to alleviate aluminum stress in ginseng and promote its growth.

[0041] In a preferred embodiment of this application, the relief of aluminum stress includes: improving the vitality of ginseng roots; increasing the accumulation of dry weight in the underground parts; promoting the accumulation of biomass in the aboveground and underground parts of ginseng; and prolonging the colonization time of functional bacteria in the rhizosphere.

[0042] It should be noted that the ratio of viable Bacillus subtilis YX1 to Pseudomonas marginalis (CGMCC No. 1.2844) in the synthesized bacterial community is 1:1; when the synthesized bacterial community is at a ratio of 1×10 7 After CFU / mL concentration was applied via root irrigation to the rhizosphere soil of ginseng at pH 4.8 with an exchangeable aluminum content of 62 mg / kg, the relative colonization of the synthesized microbial community at day 90 was ≥ 55% of the initial inoculum, and this relative colonization was significantly higher than that of Bacillus subtilis YX1 or Pseudomonas limbi (CGMCC No. 1.2844) under the same conditions. P <0.01).

[0043] According to one aspect of the present invention, a method for cultivating ginseng resistant to aluminum stress includes: dipping ginseng seedlings in water before transplanting, or drenching them during the growing season, wherein the treatment solution contains the synthetic microbial community described above in this application.

[0044] This invention provides a method for cultivating ginseng under aluminum stress. The method involves dipping ginseng seedlings in water before transplanting or drenching them during the growing season to apply the aforementioned synthetic microbial community to the plant roots. This allows *Bacillus subtilis* YX1 and *Pseudomonas marginalis* to directly colonize the rhizosphere. This method helps increase the initial inoculum size and survival rate of the functional microbial community in acidified soil, ensuring its stable bioregulatory function under aluminum stress.

[0045] In pot experiments, this invention demonstrated that ginseng exhibited significantly better plant height, root length, and biomass accumulation in both above-ground and below-ground parts compared to the control group over a 90-day growing period. This indicates that applying the synthetic microbial community via root dipping or drenching can effectively mitigate the toxic effects of aluminum ions on the root system and enhance ginseng's adaptability and growth vigor in low-pH soils. This planting method is simple to operate and suitable for targeted regulation of the rhizosphere microecology in green ginseng cultivation.

[0046] The technical solution of the present invention will be further described below with reference to the embodiments.

[0047] Example 1: Strain Information Sources of the strain:

[0048] Example 2: Construction of synthetic microbial communities and verification of their synergistic growth characteristics Bacillus subtilis YX1 (CGMCC No. 31921) and Pseudomonas marginalis (CGMCC No. 1. 2844) from Example 1 were inoculated into TSB liquid medium and cultured with shaking at 28°C and 180 rpm for 24 hours to allow the bacteria to enter the logarithmic growth phase.

[0049] Subsequently, the cells were centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was discarded, the cells were washed twice with sterile water, resuspended, and the concentration was adjusted to 1×10⁻⁶. 7 CFU / mL was used to obtain a single-strain bacterial suspension. Then, the two bacterial suspensions were mixed at a 1:1 volume ratio to prepare the synthetic bacterial community (SynCom) at a concentration of 1×10⁻⁶. 7 CFU / mL.

[0050] SynCom microbial community was obtained at a concentration of 1×10⁻⁶. 7 CFU / mL.

[0051] The following control group was also established: Control group A (Bs): A bacterial suspension containing only Bacillus subtilis, at a concentration of 1×10⁻⁶. 7 CFU / mL; Control group B (Pm): A bacterial suspension containing only Pseudomonas marginalis, at a concentration of 1×10⁻⁶. 7 CFU / mL.

[0052] Each treatment group was inoculated into TSB liquid medium containing 100 mM Al2(SO4)3 and cultured in a shaker at 28°C and 180 rpm with continuous shaking. Samples were taken every 12 hours, and the absorbance (OD600) of the bacterial solution at 600 nm was measured using a spectrophotometer. The zeroing was performed using blank medium without inoculated strains, and the values ​​were recorded. The monitoring was conducted for a total of 36 hours.

[0053] Figure 1 This is a growth rate diagram of the synthetic bacterial community provided in this embodiment.

[0054] The results are as follows Figure 1 As shown, after 12 hours of culture, the OD600 value of the SynCom group reached 0.733, which was higher than that of control group A (0.474) and control group B (0.296); after 36 hours of culture, the OD600 value of the SynCom group reached 0.961, which was about 12.4% higher than that of control group A (0.855) and about 16.63% higher than that of control group B (0.824).

[0055] The above results indicate that the 1:1 synthetic bacterial community composed of Bacillus subtilis YX1 and Pseudomonas marginalis exhibits a significantly better growth rate than a single strain under aluminum stress, demonstrating a clear synergistic effect.

[0056] Example 3: Verification of the aluminum tolerance of the synthetic bacterial community: The synthetic bacterial community (SynCom) prepared in Example 2 was inoculated into bacteria containing different concentrations of Al. 3+Three biological replicates were set up for each group in TSB liquid medium (added in the form of Al2(SO4)3 at concentrations of 0, 50, 100, 150, and 200 mM).

[0057] After 36 hours of shaking culture at 28℃ and 180 rpm, the OD600 values ​​of each treatment group were measured, and the growth rate relative to the aluminum-free control group was calculated. Specific results are as follows: Figure 2 As shown.

[0058] Figure 2 The synthetic bacterial community provided in this embodiment was prepared in environments containing different concentrations of Al. 3+ Aluminum resistance diagram of TSB liquid culture medium.

[0059] The results are as follows Figure 2 As shown, 50, 100, 150, and 200 mM Al at 36 h 3+ Compared with the aluminum-free control group, the growth rates of the treated cells decreased by 32.26%, 27.4%, 34.44%, and 35.28% at each concentration, respectively. Among these, the 100 mM Al treatment resulted in the largest decrease. 3+ The growth rate decreased the least at different aluminum concentrations. Under different aluminum concentrations, the OD600 value of the bacterial community continuously increased with prolonged culture time, and the growth trend of each concentration group did not show inhibition or decline within 36 hours. These results indicate that the synthetic bacterial community could grow well under all aluminum concentration treatments.

[0060] Example 4: Effects of synthetic microbial communities on the growth and quality of ginseng under aluminum stress Healthy ginseng seedlings of uniform size were selected and transplanted into pots filled with acidified soil (pH 4.8, exchangeable aluminum content 62 mg / kg). The experiment consisted of five treatment groups, each with at least three biological replicates. CK group: Normal soil, without aluminum application or inoculation with microbial agents; Al treatment group: 100 mM aluminum sulfate (Al2(SO4)3) was added, and no inoculum was used; Al+Bs group: Al treatment group + inoculated with Bacillus subtilis single-strain suspension, concentration 1×10⁻⁶ 7 CFU / mL; Al+Pm group: Al treatment group + single bacterial suspension of Pseudomonas aeruginosa inoculated at the edge, concentration 1×10⁻⁶ 7 CFU / mL; Al+SynCom group: Al-treated group + inoculated synthetic microbial community (1:1 mixture, final concentration 1×10⁻⁶) 7 (CFU / mL).

[0061] The above-mentioned experimental groups were treated with fungal inoculant by root irrigation, with 10 mL per plant applied each time, once every 15 days, for a total of 3 applications. The pot experiment lasted for 90 days. After 90 days, the growth indicators of the above-ground and underground parts of ginseng were measured, and the results are shown in the table below. Figure 3 As shown.

[0062] Figure 3 This is a graph showing the effect of each treatment group on ginseng growth indicators in this embodiment.

[0063] Table: Ginseng growth indicators for each treatment group:

[0064] The results showed that aluminum stress significantly inhibited the growth of ginseng. Compared with the Al treatment, the plant height and root length of the Al+Bs treatment group and the Al+Pm treatment group were significantly increased by 42.2% and 39.3%, 52.7% and 55.5%, respectively. The aboveground fresh weight, aboveground dry weight, underground fresh weight, and underground dry weight were significantly increased by 73.3% and 55.5%, 58.3% and 75%, 61.5% and 92.3%, and 22.2% and 60%, respectively, indicating that different single-strain treatments have the ability to alleviate aluminum stress. The ginseng plant height and aboveground fresh weight in the Al+SynCom treatment group were significantly higher than those in the aluminum stress group (59.2% and 104.4% respectively), while the aboveground dry weight, underground fresh weight, underground dry weight, and root length were significantly better, increasing by 62%, 83.3%, 107.6%, and 66.7% respectively. The ginseng biomass accumulation (aboveground fresh weight and underground dry weight) was significantly improved under the synthetic microbial community treatment. The two strains produced a synergistic effect under symbiotic conditions, enhancing the plant's stress resistance system and optimizing nutrient absorption through metabolic complementarity. Therefore, ginseng inoculated with synthetic microbial communities has a good ability to promote growth and alleviate aluminum stress.

[0065] Figure 4 This diagram illustrates the effects of each treatment group on the ginseng growth phenotype in this embodiment.

[0066] Depend on Figure 4 It was found that the overall growth of seedlings treated with Al was significantly inhibited, manifested as reduced aboveground biomass, fewer leaves, and yellowing and curling. Seedlings in the Al+Bs and Al+Pm treatment groups showed significantly improved growth compared to the Al treatment, with increased leaf expansion and aboveground biomass. The Al+SynCom treatment group showed the most significant improvement in overall ginseng seedling growth, with intact plant structure, normal leaf color, and significantly higher aboveground biomass and leaf number than the Al treatment group, and superior to the single-strain treatment group. The results indicate that the Al+SynCom treatment can more effectively alleviate the inhibitory effect of aluminum stress on ginseng seedling growth and has a better effect on promoting normal ginseng seedling growth.

[0067] Example 5: Monitoring of the colonization dynamics of synthetic microbial communities in the rhizosphere of ginseng To assess the survival and colonization persistence of the synthetic microbial community in complex soil environments, rhizosphere soil samples were collected from each treatment group in Example 4 after application, and quantitative analysis was performed on days 15, 45, and 90.

[0068] Rhizosphere soil samples were collected from each treatment group, and total DNA was extracted from the soil. Quantitative detection methods were used to amplify specific gene fragments of the target bacteria, and the copy number of the target bacteria per gram of dry soil was calculated using a standard curve to reflect the colonization changes of microorganisms in the rhizosphere soil under different treatment conditions. Specific results are shown in the table below.

[0069]

[0070] Note: No letter or the same letter in the same column indicates that the difference is not significant. P >0.01); different letters indicate significant differences ( P <0.01).

[0071] Figure 5 This is a comparison chart of the colonization rates of ginseng rhizosphere in each treatment group provided in this embodiment.

[0072] Depend on Figure 5 It was found that the target bacteria were detectable in all inoculation groups on day 15; by day 45, the bacterial counts in the AlBs and AlPm groups had decreased to 63.94% and 56.73% of their initial values, respectively, while the AlSynCom group maintained 78.15% of its initial inoculation on day 45; even on day 90, a stable bacterial community structure was still detectable, with the AlBs and AlPm groups at 44.33% and 40.26%, respectively, and the AlSynCom group at 56.61%; statistical analysis showed that the bacterial community decline rate of the synthetic bacterial group was significantly lower than that of the two single bacterial groups ( P <0.01).

[0073] As can be seen from the above, the synthetic microbial community obtained by constructing a specific dual-bacterial combination with a 1:1 live bacteria ratio in this application not only achieves functional colonization for ≥90 days under aluminum stress, but also exhibits a superior interspecific synergistic stabilization effect compared to any single bacterium in complex soil microecology, fundamentally solving the industrialization bottleneck of "application-induced attenuation" in the cultivation of perennial medicinal plants by microbial preparations.

[0074] Example 6: Analysis of Synthetic Microbial Community Interaction Mechanism Based on Metabolic Network Simulation To further reveal the underlying mechanism of the synergistic effect of this synthetic microbial community, this embodiment conducted an interaction simulation study based on a genome-scale metabolic model (GEM).

[0075] First, the complete genome sequences of Bacillus subtilis YX1 and Pseudomonas limbi were obtained, and their respective metabolic network models were reconstructed using automatic annotation tools (such as RAST and ModelSEED). Then, the metabolic exchange behavior of the two bacteria under co-culture conditions was simulated using the MICOM platform (v0.3.0). The culture medium was set to TSB basic components, including common nutrients such as amino acids, carbon sources, and inorganic ions.

[0076] Figure 6 The simulation results provided in this embodiment show a metabolic heatmap of the synthetic microbial community.

[0077] Depend on Figure 6 It can be seen that there is a significant metabolic complementarity signal between the two bacteria, mainly manifested as follows: Bacillus subtilis provides glycine and Fe³⁺ to Pseudomonas limbi + Key resources such as Pi; feedback release of CO2 and H2 by Pseudomonas marginalis. + It also contains some organic acids, which help regulate the local microenvironment; positive exchange fluxes were also observed at the level of energy-related molecules (such as ATP precursors, dGTP, and dCTP); MICOM predicts that the community growth rate of this assemblage is 0.87 h. - ¹, higher than the maximum rate of any single bacterium cultured alone (Bs: 0.72 h). - ¹;Pm: 0.69 h - ¹).

[0078] The above results indicate that the synergistic growth of this synthetic microbial community is not accidental, but rather based on a stable metabolic mutualistic relationship, providing a mechanistic explanation for its stability in complex environments.

[0079] In summary, this application addresses the industry challenge of unstable colonization of functional microorganisms under the combined stress of acidification and aluminum stress caused by continuous ginseng cropping. This invention is not a simple mixture of strains, but rather based on research findings that *Bacillus subtilis* YX1 (CGMCC No. 31921) and *Pseudomonas limbi* (CGMCC 1.2844) can form a specific "glycine-organic acid-energy" metabolic symbiotic closed loop in aluminum stress environments: the former efficiently provides glycine and bioavailable iron (Fe³⁺). + The latter secretes organic acids to chelate Al 3+ It also releases CO2 to enhance the respiration efficiency of the former. This reciprocal relationship only exists at pH ≈ 4.7~5.0 and Al. 3+The bacteria were significantly activated at concentrations ≥100 mM, and their kinetic equilibrium strictly depended on a 1:1 ratio of viable cells between the two strains. Based on this, the rationally constructed synthetic microbial community broke through the functional boundaries of a single strain, achieving a qualitative leap from "passive tolerance" to "active regulation of the rhizosphere microecology".

[0080] The technical effects achieved by this application include: (1) In the rhizosphere soil of ginseng with pH 4.8 and exchangeable aluminum of 62 mg / kg, the functional bacterial community has been colonized for ≥90 days, and the decline rate of the synthetic bacterial community is significantly lower than that of the two single bacterial groups (P<0.01); (2) In TSB medium containing 100 mM Al2(SO4)3, the OD of 36 hours is significantly lower than that of the two single bacterial groups. 600 The value was 12.4% higher than that of the Bacillus subtilis YX1 single strain group and 16.63% higher than that of the Pseudomonas marginalis single strain group, confirming the synergistic growth effect; (3) After being applied to ginseng potted plants, the dry weight of the underground part increased by 60.0% compared with the aluminum stress control group, the fresh weight of the aboveground part increased by 104.4%, and the physiological indicators such as root vitality and chlorophyll content were significantly improved simultaneously.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A synthetic microbial community, characterized in that, The synthetic microbial community consists of Bacillus subtilis ( Bacillus subtilis YX1 and Pseudomonas marginalis ( Pseudomonas marginalis Composed of, wherein: The Bacillus subtilis ( Bacillus subtilis YX1, deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 31921; The marginal pseudomonas ( Pseudomonas marginalis (), deposited at the China General Microbiological Culture Collection Center, accession number CGMCC 1.2844; When the synthetic microbial community was applied to the rhizosphere of ginseng and colonized for 90 days in acidified soil with pH ≤ 5, the relative colonization rate was ≥ 55% of the initial inoculum.

2. The synthetic microbial community according to claim 1, characterized in that, The effective viable count of Bacillus subtilis YX1 and Pseudomonas marginalis was 1:

1.

3. The synthetic microbial community according to claim 1, characterized in that, The synthesized bacterial community exhibits synergistic growth characteristics. After culturing in TSB liquid medium containing 100 mM Al2(SO4)3 for 36 hours, the OD600 value of the synthesized bacterial community is 12.40~16.63% higher than that of any single control group.

4. A compound microbial agent, characterized in that, The compound microbial agent comprises the synthetic microbial community as described in any one of claims 1 to 3 and an agriculturally acceptable carrier.

5. The compound microbial agent according to claim 4, characterized in that, The formulation of the compound microbial agent includes one of the following: liquid microbial solution, powder, or granules.

6. The compound microbial agent according to claim 5, characterized in that, The compound bacterial agent is a liquid bacterial solution.

7. The compound microbial agent according to claim 6, characterized in that, The liquid bacterial solution is a bacterial strain suspension, and the concentration of the synthetic bacterial community in the bacterial strain suspension is 1×10⁻⁶. 8 ~6×10 8 CFU / mL.

8. The use of the synthetic microbial community according to any one of claims 1 to 3 or the compound microbial agent according to any one of claims 4 to 7 in the preparation of a product for relieving aluminum stress in ginseng and promoting its growth.

9. The application according to claim 8, characterized in that, Relieving aluminum stress in ginseng includes: improving the vitality of ginseng roots; Promote the accumulation of ginseng biomass, wherein the ginseng biomass refers to the fresh weight of the aboveground parts and the dry weight of the underground parts; Extend the colonization time of functional bacteria in the rhizosphere.

10. A method for cultivating ginseng to resist aluminum stress, characterized in that, The method includes: dipping the roots of ginseng seedlings before transplanting, or drenching the roots during the growth period, wherein the treatment solution contains the synthetic microbial community as described in any one of claims 1 to 3.

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