Roribobacter sp., synthetic flora containing Roribobacter sp. And arbuscular mycorrhizal fungi, complex microbial inoculant and application of complex microbial inoculant in soil nitrogen fixation and plant growth promotion

Abstract

The invention belongs to the technical field of biological bacterial agents and crop cultivation, and particularly relates to a Roribobacter sp. Strain, a synthetic flora containing Roribobacter sp. And arbuscular mycorrhizal fungi, a composite bacterial agent and application of the composite bacterial agent in soil nitrogen fixation and plant growth promotion. According to the invention, a strain of Rosebacter sp. SBE2523 is obtained through separation and is subjected to biological preservation, and the Rosebacter sp. Has the effects of promoting plant growth and improving the nitrogen fixation capability of soil. On the basis, the invention provides a synthetic flora containing the roribacter sp. SBE2523 and the arbuscular mycorrhizal fungi and a complex microbial inoculant containing the synthetic flora, through the synergistic symbiotic effect of the two microorganisms, the effect superior to that of the single bacterium of the roribacter sp. SBE2523 is achieved, the effects of promoting plant growth and improving the nitrogen fixation capacity of soil are more remarkable, and the effect is better than that of the single bacterium of the roribacter sp. SBE2523. The problems that an existing single microbial agent is low in infection efficiency, unstable in growth promoting effect, poor in environmental adaptability and the like in practical application are solved, and therefore the multiple purposes of improving the crop yield and fixing nitrogen in soil are achieved.

Description

Technical Field

[0001] This invention belongs to the field of biological agents and crop cultivation technology, specifically involving a strain of Lorvastatinus, a synthetic microbial community containing Lorvastatinus and arbuscular mycorrhizal fungi, a compound microbial agent, and its application in soil nitrogen fixation and promoting plant growth. Background Technology

[0002] Nitrogen is an essential nutrient element for crop growth and development. In intensive agricultural production, the high nitrogen demand of crops is mainly met by the input of chemical nitrogen fertilizers. This not only involves huge energy consumption and production costs, but also causes a series of serious environmental problems such as soil acidification, water eutrophication, and greenhouse gas emissions. Although certain soil microorganisms can perform biological nitrogen fixation, this process is often accompanied by strong nitrogen absorption by plants and continuous crop harvesting, resulting in widespread nitrogen deficiency in the soil. At the same time, modern farming methods reduce nutrient return, further exacerbating soil nitrogen imbalance and disrupting the natural nitrogen cycle. Both nitrogen deficiency and excess have a significant impact on the health and productivity of global ecosystems. Although humans have invested far more resources in nitrogen management and regulation than in other elements, how to effectively integrate biological nitrogen fixation, optimize soil nitrogen cycling, and maintain nitrogen dynamic balance remains a core scientific challenge and practical bottleneck for achieving sustainable agricultural transformation.

[0003] However, the nitrogen fixation resources and their efficiency in natural soils are far from being effectively utilized. Current traditional strategies aimed at enhancing biological nitrogen fixation all face significant limitations: First, while inoculation with a single microbial agent is simple, some exogenous strains often fail in complex field environments due to insufficient competitiveness and unstable rhizosphere colonization, leading to functional inactivation and fluctuating effects. Second, while optimizing agronomic practices (such as leguminous crop rotation or increased application of organic fertilizer) can improve the soil environment, nitrogen fixation is slow and limited by regional climate, soil, and planting systems, resulting in insufficient universality. Third, while breeding highly efficient symbiotic crops is the fundamental direction, it is constrained by scarce germplasm resources, long breeding cycles, and the inhibitory effect of field water and fertilizer management on nitrogen fixation efficiency. The common shortcoming of these traditional approaches is that they have not fundamentally solved the core dilemma of functional microorganisms in real farmland: "difficulty in survival, poor colonization, and weak function." Therefore, agricultural practice urgently needs a new biological nitrogen fixation technology that can actively adapt to complex environments and stably perform its functions.

[0004] In recent years, the interdisciplinary integration of synthetic biology and microbial ecology has provided new insights into solving these challenges. Synthetic microbial communities, through rational design, combine specific strains with complementary functions and mutually beneficial symbiosis to construct structurally stable and functionally efficient "microbial teams." However, in this field, there is still a lack of single bacteria and synthetic microbial communities that possess both highly efficient nitrogen fixation and multiple growth-promoting functions. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *L. kurtosis*, a synthetic microbial community containing *L. kurtosis* and arbuscular mycorrhizal fungi, a compound microbial agent, and their application in soil nitrogen fixation and promoting plant growth. The *L. kurtosis* SBE2523, the synthetic microbial community containing *L. kurtosis* SBE2523, the microbial agent containing *L. kurtosis* SBE2523, or the compound microbial agent containing the synthetic microbial community all have the effect of promoting plant growth and improving soil nitrogen fixation capacity.

[0006] This invention provides a strain of Helicobacter pylori SBE2523, the preservation number of which is CGMCCNO.36390.

[0007] The present invention also provides a bacterial agent comprising the *Lonicera japonica* SBE2523 described in the above technical solution.

[0008] Preferably, the bacterial agent includes fermentation broth of Helicobacter pylori SBE2523 or bacterial suspension of Helicobacter pylori SBE2523.

[0009] Preferably, the effective viable concentration of Helicobacter pylori SBE2523 in the bacterial agent is ≥1×10⁻⁶. 9 CFU / mL.

[0010] The present invention also provides a synthetic microbial community, including arbuscular mycorrhizal fungi and the *Lyctalobacterium* SBE2523 described in the above technical solution.

[0011] Preferably, the arbuscular mycorrhizal fungus includes Rhizocystis heteromorpha.

[0012] The present invention also provides a compound microbial agent, which includes the synthetic microbial community described in the above technical solution.

[0013] Preferably, the compound microbial agent comprises an arbuscular mycorrhizal fungus agent and a *L. roxburghii* agent, wherein the mass ratio of the arbuscular mycorrhizal fungus agent to the volume ratio of the *L. roxburghii* agent is 10 g: 1 mL; the arbuscular mycorrhizal fungus agent comprises spores, mycelia, infected roots, and a cultivation substrate, wherein the spore content in the arbuscular mycorrhizal fungus agent is 50-70 spores / g; and the effective viable concentration of *L. roxburghii* SBE2523 in the *L. roxburghii* agent is ≥1×10⁻⁶. 9 CFU / mL.

[0014] The present invention also provides the application of the above-described *Lonicera japonica* SBE2523, the above-described microbial agent, the above-described synthetic microbial community, or the above-described compound microbial agent in improving soil nitrogen fixation capacity and / or promoting plant growth.

[0015] The present invention also provides a method for improving soil nitrogen fixation capacity and / or promoting plant growth, comprising the following steps: inoculating the microbial agent or the compound microbial agent described in the above technical solution into the cultivation soil or cultivation substrate.

[0016] Beneficial effects: This invention provides a strain of *L. kurtosis*, a synthetic microbial community containing *L. kurtosis* and arbuscular mycorrhizal fungi, a compound microbial agent, and their applications in soil nitrogen fixation and plant growth promotion. This invention isolates and biopreserves a strain of *L. kurtosis* SBE2523, which possesses the ability to promote plant growth and enhance soil nitrogen fixation capacity. Based on this, this invention provides a synthetic microbial community containing *L. kurtosis* SBE2523 and arbuscular mycorrhizal fungi, and a compound microbial agent containing this synthetic microbial community. Through the synergistic effect of the two microorganisms, it achieves effects superior to that of a single *L. kurtosis* SBE2523 strain, with more significant effects in promoting plant growth and enhancing soil nitrogen fixation capacity. It overcomes the problems of low infection efficiency, unstable growth-promoting effects, and poor environmental adaptability of existing single-microbial agents in practical applications, thereby achieving multiple goals of increasing crop yield and soil nitrogen fixation, and providing effective technical support for the development of green and low-carbon agriculture.

[0017] Biological Preservation Instructions Loropetalum bisporus SBE2523, biologically classified as Rhodanobacter sp. was deposited on October 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.36390, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0018] 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.

[0019] Figure 1 The images show the colony morphology of *Lonicera japonica* SBE2523 cultured on LB solid medium, the fermentation diagram in liquid LB medium, and the phylogenetic tree in Example 1. Figure 2 The effect of the synthetic microbial community in Example 2 on maize mycorrhizal infection rate and growth and development; Figure 3 The effect of the synthetic microbial community in Example 2 on nitrogen absorption, accumulation and translocation in maize plants; Figure 4 This illustrates the effect of the synthetic microbial community in Example 2 on the differential expression of nitrogen metabolism-related genes in maize plant roots. Figure 5The effect of the synthetic microbial community on the nitrogen content of maize rhizosphere soil in Example 2; Figure 6 The effect of the synthetic microbial community in Example 2 on the activity of nitrogen-related extracellular enzymes in maize rhizosphere soil; exist Figures 2-3 and Figures 5-6 Different letters represent p Significance at the ≤0.05 level. Detailed Implementation

[0020] This invention provides a strain of Helicobacter pylori SBE2523, the preservation number of which is CGMCCNO.36390.

[0021] The *L. roxburghii* SBE2523 described in this invention was isolated from the rhizosphere soil of fresh maize in the black soil region of Northeast China. Physiological and biochemical identification confirmed it to be a Gram-negative bacillus, belonging to the facultative anaerobic family. The colonies are pale yellow, with a smooth surface and regular edges. Molecular identification showed that it shared 99% homology with *L. roxburghii*. Combining physiological and biochemical identification with molecular identification, it was confirmed as *L. roxburghii* and named *L. roxburghii* SBE2523.

[0022] This invention also provides a microbial agent comprising *L. coli* SBE2523 as described in the above-described technical solution. As one embodiment, the microbial agent comprises a fermentation broth of *L. coli* SBE2523 or a bacterial suspension of *L. coli* SBE2523. As one embodiment, the effective viable concentration of *L. coli* SBE2523 in the microbial agent is ≥1×10⁻⁶. 9 CFU / mL, further increased to 1×10 9 CFU / mL. As one embodiment, the cultivation method of the bacterial agent includes: inoculating *L. aerobicans* SBE2523 onto LB slant medium for activation; inoculating the activated *L. aerobicans* SBE2523 into LB liquid medium and culturing with shaking until the logarithmic growth phase to obtain the bacterial agent. As one embodiment, the invention further includes solid-liquid separation of the *L. aerobicans* SBE2523 cultured to the logarithmic growth phase, resuspending the bacterial cells to obtain a *L. aerobicans* SBE2523 bacterial suspension, which is the bacterial agent. As one embodiment, the shaking culture temperature is 24-26℃, or 25℃; the shaking culture time is 4-6 days, or 5 days; the shaking culture rotation speed is 180 rpm.

[0023] This invention also provides a synthetic microbial community, comprising arbuscular mycorrhizal fungi and *L. roxburghii* SBE2523 as described in the above-mentioned technical solution. As one embodiment, the arbuscular mycorrhizal fungi include *Rhizocystis heterophylla*. This invention does not specifically limit the source of the arbuscular mycorrhizal fungi; conventionally purchased or self-isolated arbuscular mycorrhizal fungi are acceptable. As one embodiment, the mass-to-volume ratio of arbuscular mycorrhizal fungal spores to *L. roxburghii* SBE2523 in the synthetic microbial community is 10:1.

[0024] This invention also provides a compound microbial agent, which includes the synthetic microbial community described in the above-mentioned technical solution. As one embodiment, the compound microbial agent includes an arbuscular mycorrhizal fungal agent and a *L. kurtosis* agent, with a mass-to-volume ratio of the arbuscular mycorrhizal fungal agent to the *L. kurtosis* agent of 10:1. As one embodiment, the arbuscular mycorrhizal fungal agent includes spores, mycelia, infected roots, and a cultivation substrate; the spore content in the arbuscular mycorrhizal fungal agent is 50-70 spores / g; and the effective viable concentration of *L. kurtosis* SBE2523 in the *L. kurtosis* agent is ≥1×10⁻⁶. 9 CFU / mL. This invention does not specifically limit the preparation method of the arbuscular mycorrhizal fungal inoculant; conventional methods for preparing arbuscular mycorrhizal fungal inoculants (inoculum) in the art can be used.

[0025] The present invention also provides the application of the above-described *Lonicera japonica* SBE2523, the above-described microbial agent, the above-described synthetic microbial community, or the above-described compound microbial agent in improving soil nitrogen fixation capacity and / or promoting plant growth.

[0026] In the compound microbial community or compound microbial agent described in this invention, Lorvastatin SBE2523 can promote the infection of plant roots by arbuscular mycorrhizal fungi, thereby enabling the two to better synergistically promote plant growth and / or improve the nitrogen fixation capacity of the soil.

[0027] In one implementation, promoting plant growth includes at least one of the following: enhancing plant nitrogen absorption and storage, increasing plant nitrogen content, promoting plant nitrogen transfer and accumulation, promoting nitrogen utilization capacity for plant protein synthesis, promoting plant stem development, increasing plant biomass, increasing the fresh weight of plant aboveground parts, and increasing the fresh weight of plant underground parts. In another implementation, promoting plant stem development includes increasing plant height and / or stem diameter. In one implementation, improving soil nitrogen fixation capacity can be achieved by regulating soil nitrogen transformation and utilization, promoting soil nitrogen transformation and microbial metabolism, and enhancing soil nitrogen cycling.

[0028] In one embodiment, the plant includes grasses, and further includes maize.

[0029] This invention also provides a method for improving soil nitrogen fixation capacity and / or promoting plant growth, comprising the following steps: inoculating the microbial agent or the compound microbial agent described in the above-mentioned technical solution into cultivation soil or cultivation substrate. As one embodiment, this invention employs a "sandwich" inoculation method to inoculate the crop cultivation soil or cultivation substrate with an arbuscular mycorrhizal fungal agent, then transplanting plant seedlings into the cultivation soil or cultivation substrate inoculated with the arbuscular mycorrhizal fungal agent, and then inoculating the cultivation soil or cultivation substrate with the plant seedlings with a *L. romaejasminoides* agent before cultivation management. As one embodiment, the mass ratio of the arbuscular mycorrhizal fungal agent to the cultivation soil or cultivation substrate is 1-3:10-20, or it can be 1:10 or 3:20. As one embodiment, the inoculation amount of the *L. romaejasminoides* agent, based on the number of plants, is 5-7 mL / plant, or it can be 5 mL / plant or 7 mL / plant.

[0030] 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.

[0031] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the detection methods involved are all conventional methods unless otherwise specified.

[0032] Example 1 Isolation, purification and identification of Helicobacter pylori SBE2523 Constructing the microbial bank: Fresh corn rhizosphere soil from the black soil region of Northeast China was collected and mixed at a ratio of 10... -4 ~10 -8 After serial dilution, the samples were incubated at 25°C for 2 weeks in 96-well plates containing LB medium. 10 samples were selected. -6For dilutions of the culture, take 10 µL of bacterial culture, add 16.6 µL of alkaline lysis buffer (25 mM NaOH, 0.2 mM Na2-EDTA, pH=12), lyse at 95℃ for 30 min, then add 16.6 µL of neutralization buffer (40 mM Tris-HCl, pH=7.5) and mix well to obtain bacterial DNA, and store at -20℃. Two rounds of PCR amplification were performed: The first round used the universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:2) / 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO:3). The system contained 3 µL template, buffer, dNTPs, primers, and HSTaq enzyme. The program was 95℃ for 5 min; 25 cycles (95℃ for 10 s, 55℃ for 15 s, 72℃ for 1 min; 72℃ for 5 min). The second round used a 40-fold dilution of the first-round product as template, employing the same primer pair with barcodes and sequencing adapters. The system and program were the same as before. The amplified products were purified by agarose gel electrophoresis and gel recovery, and the concentration was determined using Nanodrop. Qualified samples were sent for Illumina sequencing.

[0033] Isolation and Purification: Bacteria identified through bioinformatics analysis were isolated and purified. Identified bacterial colonies were selected from wells of a 96-well cell culture plate, and 10 µL of bacterial suspension was streaked onto LB solid medium for purification, with three replicates per well. The strains were cultured at 28°C for 3 days for further purification, repeated twice until pure colonies were obtained. Pure strains were then cultured on LB liquid medium at 28°C and 180 rpm for 3 days for 16S sequencing confirmation. LB liquid medium consisted of 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 1 L distilled water, pH 7.2 ± 0.2. For LB solid medium, all other components remained the same, but 1.5%–2% agar was added.

[0034] Analysis and identification: Strain SBE2523 was physiologically and biochemically identified as a Gram-negative bacillus (0.5–1.0 × 1.0–2.0 μm), belonging to the facultative anaerobic class. Colonies are pale yellow in color, with a smooth surface and regular edges. Figure 1 As shown in (a). The bacterial culture obtained by inoculating strain SBE2523 into LB liquid medium and shaking at 180 rpm for 48 h is shown in Figure (a). Figure 1

[0035] The 16S rDNA sequence was aligned using NCBI Blast and a phylogenetic tree was constructed using MEGA 5.1, as follows: Figure 1 (c) shows the relationship with Helicobacter pylori ( Rhodanobacter The homology with *L.* was 99%. Based on the combined morphological and molecular results, strain SBE2523 was identified as *L.* sp. Rhodanobacter sp.) Example 2 Nitrogen fixation effect of combined treatment with *Lycium oryzae* and arbuscular mycorrhizal fungi on maize rhizosphere soil This example uses *L. romaejasminoides* and arbuscular mycorrhizal fungi isolated in Example 1 as a composite microbial group, combined at a solid-liquid ratio of 10 g:1 mL, to verify the nitrogen fixation effect in maize rhizosphere soil. The arbuscular mycorrhizal fungi used, *Rhizophagus irregularis*, are disclosed in the following literature: [Wang Youshan, Wang Xiaoyan, Zhang Shubin, et al. Two new records of *Dominikia indica* and *Rhizophagus irregularis* in China [J]. Journal of Northeast Forestry University, 2022, 50(04):74-77+88. DOI:10.13759 / j.cnki.dlxb.2022.04.011].

[0036] Specifically, select plump corn seeds, disinfect the surface with 10% hydrogen peroxide for 10 minutes, rinse repeatedly with distilled water, place the seeds in a tray with a moist cotton layer at the bottom, germinate the corn seeds at 25℃ for 2 days, then select the germinated seeds to sow in a seedling substrate containing 80 g of sterilized seedling substrate, and when the corn seedlings grow to two leaves and one heart, prepare for transplanting and inoculation treatment.

[0037] Four treatments were set up: CK (no fungal agent applied), AM (10g arbuscular mycorrhizal fungal agent applied), RH (1mL Lorobacterium tumefaciens agent applied), and AM+RH (10g arbuscular mycorrhizal fungi and 1mL Lorobacterium tumefaciens agent applied).

[0038] Select corn seedlings with uniform growth for transplanting. First, spread 2 / 3 of the mixed substrate evenly at the bottom of a sterile pot (16 cm diameter at the top × 12 cm diameter at the bottom × 11 cm height; sterilize the pot with 75% alcohol before use, and wipe it repeatedly with sterile water). Inoculate 50 g of arbuscular mycorrhizal fungal inoculant using the "sandwich" inoculation method, applying the inoculant evenly onto the mixed substrate. Then cover the top with a 2 cm layer of substrate, and finally cover with sterilized mixed substrate (121℃, 1×10⁻⁶). 5Transplanting of corn seedlings was completed at kPa, 2 h. The cultured bacterial suspension was transferred to sterile 50 mL centrifuge tubes and centrifuged at 5000 rpm. Sterile water was added to reselect the suspension, and centrifugation was repeated twice to remove the culture medium. Then, sterile water was added to adjust the OD of the bacterial suspension to approximately 0.5. The suspension was then diluted with sterile water at a volume ratio of 1:100 (bacterial suspension: sterile water). The diluted bacterial suspension (concentration 1×10⁻⁶) was then ready. 9 Inoculate each corn seedling with 5 mL of the inoculant (CFU / mL) into the sterilized corn planting substrate (river sand: vermiculite: perlite volume ratio of 2:1:1). Finally, place the transplanted corn seedlings in a greenhouse for cultivation under the following conditions: temperature 28±2℃ day / 18±2℃ night, light intensity 4000 Lx, and photoperiod 14 h / d.

[0039] On the day of transplanting, only sterilized water was applied to ensure the survival of the corn seedlings. Water was applied every 2-3 days to maintain the moisture content of the mixed substrate at 70%. Half of the modified Hoagland nutrient solution (formulation shown in Table 1) was added once a week. The plants were harvested after 6 weeks of cultivation in a greenhouse (25℃, 16 h light, 8 h darkness).

[0040] Mycorrhizal infection rate, plant biomass, root and leaf nitrogen content, plant root transcriptome, soil nitrogen components (total nitrogen, nitrate nitrogen, and ammonium nitrogen), and enzyme activity during soil nitrogen transformation were measured for each treatment. The results are as follows: Figures 3-6 As shown.

[0041] Table 1.2 Modified Hoagland Culture Medium Formula

[0042] The effects of single bacteria and synthetic microorganisms on maize mycorrhizal infection rate and growth and development, such as Figure 2 As shown, the mycorrhizal infection rate of maize roots differed significantly among different treatments. The infection rate of uninoculated AMF-treated roots (CK and RH) was 0%, indicating that pre-inoculation sterilization eliminated interference from native AMF. The mycorrhizal infection rate of AM-inoculated alone reached 54%, while the infection rate of the AM / RH combined treatment significantly increased to 70%. This suggests that *L. roxburghii* can effectively promote AMF infection of maize roots and strengthen the establishment of mycorrhizal symbiosis.

[0043] Furthermore, comparing the growth phenotypes of maize plants under different treatments, compared with no inoculation (CK), plant growth and development were effectively improved after inoculation with AM alone, RH alone, or a combination of AM and RH. The synthetic microbial community treatment had a more significant effect on promoting plant growth. Regarding plant height, the CK treatment had the lowest plant height, the AM alone treatment showed a slight increase, the RH alone treatment further increased plant height, and the AM+RH combination treatment reached the highest, indicating that AM and RH synergistically promoted longitudinal plant growth. As for stem diameter, the CK treatment had a stem diameter of approximately 8.6 mm, both AM and RH alone treatments increased by about 1 mm, and the AM+RH combination treatment significantly increased stem diameter by 10.5 mm, indicating that both *L. oryzae* and the synthetic microbial community promoted stem development, but the synthetic microbial community had the most prominent effect.

[0044] In terms of biomass, regardless of whether AM fungi were inoculated, the biomass of plants inoculated with RH was higher than that of uninoculated plants (CK). The AM+RH combined treatment showed the most significant increase in biomass (5 g), which was much higher than that of CK (2.8 g), indicating that mycorrhizal and bacterial treatments synergistically promoted the accumulation of plant dry matter. In terms of aboveground fresh weight, the aboveground fresh weight of AM and RH treatments alone was higher than that of the control, while the aboveground fresh weight of AM+RH combined treatment was further increased. The performance of underground fresh weight was consistent with that of the aboveground parts. Both AM and RH treatments significantly increased the fresh weight of the root system, and the highest value was achieved under the AM+RH combined treatment, indicating that mycorrhizal and rhizosphere treatments synergistically enhanced plant growth and root development.

[0045] In summary, both *Lycium oryzae* and synthetic microorganisms have a significant promoting effect on plant growth, but the synthetic microorganisms (AM+RH) have the most significant promoting effect on plant growth, especially in increasing biomass, aboveground and underground fresh weight, plant height and stem diameter. This indicates that the synergistic effect of arbuscular mycorrhizal fungi and *Lycium oryzae* treatment can comprehensively enhance the growth and development of maize plants.

[0046] The effects of single bacteria and synthetic microbial communities on nitrogen uptake, accumulation, and translocation in maize plants, such as Figure 3As shown, different biological agent treatments significantly affected the nitrogen absorption, translocation, and accumulation capacity of maize plants, with the combined treatment of arbuscular mycorrhizal fungi and Helicobacter pylori (AM+RH) exhibiting the most prominent effect. Regarding aboveground nitrogen content, the uninoculated (CK) treatment had a content of approximately 11.5 g / kg, the single inoculation treatment showed a slight increase, while the AM+RH combined treatment significantly increased the aboveground nitrogen content (approximately 12.6 g / kg), indicating that AM and RH synergistically promoted nitrogen accumulation in the aboveground parts. The trend in underground nitrogen content was similar; compared to the CK, the content of the single AM ​​inoculation treatment was not significantly different, while both the AM+RH combined treatment and the RH treatment showed significant effects, indicating that the combined treatment enhanced root nitrogen absorption and storage.

[0047] In terms of nitrogen transfer coefficient, the CK treatment was only 1.5, while the transfer coefficient of the AM+RH combined treatment was significantly higher than that of other treatments (1.6), indicating that this combination is more conducive to transporting nitrogen absorbed by the roots to the aboveground parts. In terms of nitrogen accumulation coefficient, the CK treatment was only about 0.37, while the AM or RH single inoculation treatment significantly increased to 0.56, and the AM+RH combined treatment further increased to 0.59, indicating that AM and RH synergistically enhanced the plant's ability to accumulate nitrogen in the environment.

[0048] In summary, the AM and RH combined treatment was most effective in increasing plant nitrogen content and promoting nitrogen transfer and accumulation. AM or RH treatment alone could also improve nitrogen-related indicators to varying degrees, indicating that the synthetic microbial community treatment (AM+RH) has a positive regulatory effect on nitrogen absorption and distribution in maize.

[0049] The effects of single bacteria and synthetic microbial communities on the differential expression of nitrogen metabolism-related genes in maize plant roots, such as Figure 4As shown, differentially expressed gene pathways related to nitrogen were screened using plant transcriptome analysis. Combined with metabolic pathway functions and nitrogen metabolism-related biological processes, differentially expressed gene pathways related to nitrogen were further identified. The results showed that the cyanoamino acid metabolism pathway was significantly upregulated under AM+RH combined treatment, indicating that the combined treatment enhanced the metabolism of cyanoamino acids (nitrogen storage forms). The arginine biosynthesis pathway was significantly upregulated under both AM and AM+RH treatments, indicating that arbuscular mycorrhizal fungi (AM) dominate the biosynthesis of arginine (nitrogen-containing amino acids), reflecting changes in plant nitrogen assimilation capacity after AM inoculation, and potentially enhancing nitrogen uptake by regulating this pathway. The glutathione metabolism pathway was significantly upregulated under AM+RH treatment, and also showed an upregulation trend under AM treatment, indicating that the synthetic microbial community enhanced nitrogen allocation efficiency through glutathione metabolism (a nitrogen-dependent precursor). Nicotinic acid and nicotinamide metabolism pathways were also significantly upregulated under AM+RH treatment. The metabolism pathway was significantly upregulated, indicating that the combined treatment activated this nitrogenous coenzyme (such as NAD). + NADP + The synthesis of aminoacyl-tRNA indirectly supports the activity of nitrogen metabolism-related enzymes; the aminoacyl-tRNA biosynthesis pathway was also significantly upregulated under AM+RH treatment, indicating that the combined treatment promoted the nitrogen utilization process of protein synthesis through this pathway (dependent on nitrogen-containing amino acids).

[0050] The effects of single bacteria and synthetic microbial communities on nitrogen content in maize rhizosphere soil, such as Figure 5 As shown, different biological agents exhibited significant differences in their regulatory effects on different forms of nitrogen in the soil. The combined treatment of arbuscular mycorrhizal fungi and Helicobacter pylori (AM+RH) showed outstanding performance in regulating soil nitrogen forms. Regarding dissolved organic nitrogen, the uninoculated (CK) treatment had the lowest content (0.034 mg / kg), while the AM treatment alone significantly increased it to 0.122 mg / kg, and the RH treatment alone reached 0.062 mg / kg. The AM+RH combined treatment had the highest dissolved organic nitrogen content (0.176 mg / kg), indicating that AM and RH synergistically promoted the dissolution and activation of organic nitrogen in the soil. As for total soil nitrogen, the CK treatment had the lowest content (0.095 g / kg), while the RH treatment alone increased it to 0.106 g / kg, and the AM treatment alone reached 0.135 g / kg. The AM+RH combined treatment had the highest total soil nitrogen content (0.150 g / kg), indicating that AM and RH synergistically increased the total nitrogen reserve in the soil.

[0051] Furthermore, both single and double inoculation treatments significantly reduced soil nitrate nitrogen content and slightly inhibited soil ammonium nitrogen accumulation, indicating that microorganisms may preferentially absorb nitrate and ammonium nitrogen from the soil and convert them into intracellular organic nitrogen compounds. Increased microbial biomass activated the available nutrient content (DON) in the soil, leading to increased direct plant uptake, but total nitrogen content also increased due to the increase in microbial biomass. In summary, the AM+RH combined treatment can activate soil organic nitrogen, increase total nitrogen reserves, and promote plant uptake of inorganic nitrogen (nitrate and ammonium nitrogen). Furthermore, AM or RH treatments alone can regulate soil nitrogen forms to varying degrees, indicating that the synthetic microbial community (AM+RH) has a synergistic regulatory effect on soil nitrogen transformation and utilization.

[0052] Effects of single bacteria and synthetic microbial communities on the activity of nitrogen-related extracellular enzymes in maize rhizosphere soil, such as Figure 6 As shown, different bio-agent treatments significantly regulate the activity of enzymes related to nitrogen cycling and microbial activity in soil. Urease activity in the uninoculated (CK) treatment was approximately 0.320 mol / L / min, increasing to 0.387 mol / L / min in the AM alone treatment, while the AM+RH combined treatment significantly increased urease activity to 0.455 mol / L / min, indicating that AM and RH synergistically enhanced the soil's urea decomposition capacity, facilitating nitrogen mineralization. Dehydrogenase activity in the CK treatment was approximately 26.2 mmol / L / min, increasing to 30.1 mmol / L / min in the AM alone treatment, reaching 28.7 mmol / L / min in the RH alone treatment, and reaching the highest dehydrogenase activity (33.3 mmol / L / min) in the AM+RH combined treatment. This indicates that the combined bio-agent significantly enhanced the metabolic activity of soil microorganisms, indirectly reflecting the activity level of the nitrogen cycle. Compared to the control (CK) treatment, single inoculation with AM and double inoculation with AM+RH significantly increased soil protease activity by 26.6% and 19.3%, respectively, indicating that mycorrhizal fungi play a dominant role in promoting soil protein decomposition, and their secreted glomeruli can provide substrates for nitrogen supply. The catalase activity in the CK treatment was approximately 2.270 mmol / L / min, which increased to 2.799 mmol / L / min in the AM treatment alone, and reached 2.483 mmol / L / min in the RH treatment alone. The catalase activity in the AM+RH combined treatment further increased to 2.997 mmol / L / min, indicating that the synthetic microbial community effectively enhanced the soil's antioxidant stress resistance and ensured the activity of nitrogen cycle-related microorganisms.

[0053] In summary, the synthetic microbial community (AM+RH) significantly enhanced the activities of soil urease, dehydrogenase, protease, and catalase. AM or RH treatment alone also enhanced enzyme activity to varying degrees, indicating that the synergistic effect of mycorrhizal and rhizosphere treatments can further promote nitrogen transformation and microbial metabolism by activating soil enzyme activity, thereby strengthening the soil nitrogen cycle.

[0054] 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 strain of Helicobacter pylori ( Rhodanobacter sp.) SBE2523, the preservation number of the L. sp. is CGMCC NO.36390.

2. A microbial agent, characterized in that, Includes the Lorax bacillus SBE2523 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent includes fermentation broth of Helicobacter pylori SBE2523 or bacterial suspension of Helicobacter pylori SBE2523.

4. The microbial agent according to claim 2 or 3, characterized in that, The effective viable concentration of *Lonicera japonica* SBE2523 in the bacterial agent is ≥1×10⁻⁶. 9 CFU / mL.

5. A synthetic microbial community, characterized in that, Including arbuscular mycorrhizal fungi ( Rhizophagus irregularis (and the Loropetalum oryzae SBE2523 as described in claim 1).

6. The synthetic microbial community according to claim 5, characterized in that, The arbuscular mycorrhizal fungi include Heterostigma heterophyllous.

7. A compound microbial agent, characterized in that, The compound microbial agent includes the synthetic microbial community as described in claim 5 or 6.

8. The compound microbial agent according to claim 7, characterized in that, The compound microbial agent comprises arbuscular mycorrhizal fungi and *L. roxburghii* inoculum, with a mass-to-volume ratio of arbuscular mycorrhizal fungi to *L. roxburghii* inoculum of 10 g:1 mL. The arbuscular mycorrhizal fungi inoculum includes spores, mycelia, infected roots, and cultivation substrate, with a spore content of 50-70 spores / g. The *L. roxburghii* inoculum contains an effective viable concentration of *L. roxburghii* SBE2523 ≥1×10⁻⁶. 9 CFU / mL.

9. The application of the L. bleach SBE2523 of claim 1, or the bacterial agent of any one of claims 2 to 4, or the synthetic bacterial group of claim 5 or 6, or the compound bacterial agent of claim 7 or 8, in improving soil nitrogen fixation capacity and / or promoting plant growth.

10. A method for improving soil nitrogen fixation capacity and / or promoting plant growth, characterized in that, The method includes the following steps: inoculating the microbial agent according to any one of claims 2 to 4 or the compound microbial agent according to claim 7 or 8 into the cultivation soil or cultivation substrate.

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