Bradyrhizobium strain SBE2501, compound bacteria and application of bradyrhizobium strain SBE2501 and compound bacteria in soil carbon sequestration

Abstract

The invention provides a bradyrhizobium strain SBE2501, compound bacteria and application of the bradyrhizobium strain SBE2501 and the compound bacteria in soil carbon sequestration, and belongs to the technical field of microorganisms. The invention provides a bradyrhizobium strain SBE2501, and the preservation number of the bradyrhizobium strain SBE2501 is CGMCC (China General Microbiological Culture Collection Center) NO. The invention also provides a compound bacterium, which comprises the bradyrhizobium strain SBE2501 and a rhizosporium heterotropoides strain. The strain SBE2501 can effectively promote plant growth, strengthen nitrogen and phosphorus absorption of plants, enhance soil carbon sequestration capability, promote soil carbon component accumulation and improve the content of glomalin in soil and carbon conversion key enzyme activity. The strain SBE2501 and the heterosporium rhizosporium strain show an excellent synergistic effect, and the plant growth promoting effect and the soil fertility can be remarkably enhanced. The invention provides a feasible technical approach for improving crop yield and soil fertility.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a slow-growing rhizobium strain SBE2501, a compound strain, and its application in soil carbon sequestration. Background Technology

[0002] The continued accumulation of greenhouse gas emissions has exacerbated global warming, leading to abnormally high temperatures, altered precipitation patterns, and frequent extreme weather events. This severely disrupts normal crop growth cycles and limits yield potential. Against this backdrop, enhancing the carbon sequestration capacity of agricultural systems and increasing soil carbon storage are not only key pathways to mitigating climate change but also urgent needs for improving soil structure, enhancing crop climate adaptability, and ensuring food security and sustainable agricultural development.

[0003] In modern agricultural production, fertilizer application is a core means of increasing crop yield per unit area. Rational application can increase crop yield per unit area by 55% to 65%. Currently, more than half of grain production depends on fertilizer input. However, traditional fertilizers have a low match between nutrient release and crop needs. A large amount of unabsorbed nitrogen and phosphorus nutrients are easily leached and lost, which not only wastes resources but also causes ecological and environmental problems such as soil compaction and eutrophication of water bodies. Summary of the Invention

[0004] In view of this, the present invention provides a slow-growing rhizobium ( Bradyrhizobium The strain SBE2501 (sp.) can promote plant growth, enhance soil carbon sequestration capacity, and improve soil fertility.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a slow-growing rhizobium ( Bradyrhizobium strain SBE2501 (sp.), with accession number CGMCC NO.36389.

[0006] This invention provides a compound microorganism comprising the slow-growing rhizobium strain SBE2501 and *Rhizocystis heteromorpha* (… Rhizophagus irregularis ).

[0007] The present invention provides a fermentation product, including the fermentation product of the slow-growing rhizobium strain SBE2501 or the fermentation product of the complex bacteria.

[0008] The present invention provides a microbial fertilizer comprising at least one of the following: the slow-growing rhizobium strain SBE2501, the compound bacteria, and the fermentation product.

[0009] This invention provides the application of the slow-growing rhizobium strain SBE2501, the compound bacteria, the fermentation product, or the microbial fertilizer in promoting plant growth and / or enhancing soil carbon sequestration capacity.

[0010] Preferably, promoting plant growth includes increasing plant biomass and / or promoting nutrient absorption by the plant.

[0011] Preferably, the enhancement of soil carbon sequestration capacity includes at least one of the following: promoting the accumulation of soil carbon components, increasing the content of soil globulin, and increasing the activity of key enzymes for soil carbon transformation.

[0012] The present invention provides a method for promoting plant growth and / or enhancing soil carbon sequestration capacity, characterized in that it includes: treating the soil with the slow-growing rhizobium strain SBE2501, the compound bacteria, the fermentation product, or the microbial fertilizer.

[0013] Preferably, the soil is treated to ensure that the spore content of *Rhizoctonia solani* strain is above 3 spores / g, and the content of *S. stomatologica* strain SBE2501 is above 7 × 10⁻⁶. 6 More than one per gram.

[0014] Preferably, the plant includes grasses (Poaceae).

[0015] Compared with the prior art, the present invention has the following advantages: This invention provides a slow-growing rhizobium ( Bradyrhizobium Strain SBE2501 (CGMCC NO.36389) was isolated from maize rhizosphere soil. It effectively promotes plant growth, enhances nitrogen and phosphorus absorption, and significantly increases plant biomass, aboveground and underground fresh weight, plant height, and stem diameter. It also enhances soil carbon sequestration, promotes soil carbon accumulation, and increases soil glomerulimycin content and the activity of catalase, a key enzyme in carbon conversion.

[0016] This invention provides a compound microorganism comprising the slow-growing rhizobium strain SBE2501 and *Rhizocystis heteromorpha* (… Rhizophagus irregularisThe strain, *Rhizobium stenogenum*, a type of intracellular rhizosphere growth-promoting bacterium (PGPR), exhibits excellent synergistic effects when co-inoculated with *Rhizospora heterophylla*. This significantly enhances plant growth promotion and soil fertility. *Rhizobium stenogenum* provides nitrogen to *Rhizospora heterophylla* through biological nitrogen fixation, promoting spore germination and hyphal growth, thereby increasing mycorrhizal infection rate. Simultaneously, it stimulates the host plant roots to secrete flavonoids, inducing successful colonization of *Rhizospora heterophylla*. Conversely, *Rhizospora heterophylla* can enhance the host plant's nodulation ability and nitrogen fixation rate by expanding its extraroot hyphal network to form "hyphae bridges." The mutually beneficial symbiotic relationship established by the two effectively makes up for the shortcomings of single inoculation with Rhizoctonia solani. The resulting compound bacteria can overcome the problems of low infection efficiency, unstable growth promotion effect and poor environmental adaptability of existing single microbial agents in practical applications. After inoculation, it can not only promote plant growth, but also enhance the soil carbon sequestration capacity, achieving multiple goals of increasing crop yield and soil organic carbon content. This provides a new idea for the development of efficient compound biological agents and a feasible technical approach for improving maize yield and soil fertility in dryland farming areas.

[0017] Biological Preservation Instructions Slow-growing rhizobium strain SBE2501, classified as Bradyrhizobium sp. was deposited on October 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.36389.

[0018] Sphingosomalid strain SBE2513, classified as Sphingobium sp. was deposited on October 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.36388. Attached Figure Description

[0019] Figure 1 The images show the colony morphology of the slow-growing rhizobium strain SBE2501 on YMA plates and its fermentation in liquid YMA medium. Figure 2 Phylogenetic tree of Slow-growing rhizobium SBE2501; Figure 3 A diagram showing the relationship between mycorrhizal development and plant growth. Figure 4 The graph shows the effects of microbial agents on maize growth and development. Figure 5 The graph shows the effect of inoculants on the mineral nutrient content of maize plants. Figure 6 The graph shows the effects of microbial agents on soil carbon composition and globulin content. Figure 7 The figure shows the effect of microbial agents on soil enzyme activity. Detailed Implementation

[0020] This invention provides a slow-growing rhizobium ( Bradyrhizobium strain SBE2501 (sp.), with accession number CGMCC NO.36389.

[0021] In this invention, the *S. stomatologica* strain SBE2501 was isolated from maize rhizosphere soil. Colonies formed on YMA medium are round, opaque or translucent, white, raised, with smooth edges and a granular structure. The 16S rDNA sequence of strain SBE2501 is shown in SEQ ID NO: 1. Morphological characteristics and molecular identification results indicate that strain SBE2501 is *S. stomatologica* (…). Bradyrhizobium sp.).

[0022] The slow-growing rhizobium strain SBE2501 described in this invention can promote plant growth, enhance the absorption of nitrogen and phosphorus by plants, and significantly increase plant biomass, aboveground and underground fresh weight, plant height and stem diameter; it can also enhance soil carbon sequestration capacity, promote soil carbon component accumulation, and increase soil globulin content and the activity of key carbon conversion enzymes.

[0023] This invention provides a compound microorganism comprising the slow-growing rhizobium strain SBE2501 and *Rhizocystis heteromorpha* (… Rhizophagus irregularis ).

[0024] This invention does not specifically limit the source of the *Rhizocarpium heterophyllum*, and any conventional source in the art can be used. In this invention, the main utilization is the ability of *Rhizocarpium heterophyllum* to form a symbiotic relationship with the host plant, creating arbuscular mycorrhizae, thereby promoting plant growth. In this embodiment of the invention, the *Rhizocarpium heterophyllum* strain AH01 is used as an example to verify the effect of the composite fungus. *Rhizocarpium heterophyllum* strain AH01 is based on existing technology (Jin Chen, Keqing Lin, Tao Huang, Xiaowan Geng, Zishan Li, Boyan Wang, Qingchen Xiao, Xiaoyu Li. 2024. The active effect of...). Rhizophagus irregularisThe method for isolating and identifying *Inoculants on maize endophyticbacteria community* (iMetaOmicse23.DOI: https: / / doi.org / 10.1002 / imo2.23) is disclosed in existing techniques (Wang Youshan, Wang Xiaoyan, Zhang Shubin, et al. Two new records of endophyticbacteria species in China). Dominikia indica and Rhizophagus irregularis [J]. Journal of Northeast Forestry University, 2022, 50(04):74-77+88. DOI:10.13759 / j.cnki.dlxb.2022.04.011). The slow-growing rhizobium strain SBE2501 and *Rhizospora heterophylla* synergistically promote plant growth, enhance soil carbon sequestration, and improve soil fertility. Specifically, the slow-growing rhizobium strain SBE2501 provides nitrogen to *Rhizospora heterophylla* through biological nitrogen fixation, promoting spore germination and mycelial growth, thereby increasing mycorrhizal infection rate. Simultaneously, the slow-growing rhizobium strain SBE2501 also induces the host plant roots to secrete flavonoids, thereby inducing successful colonization of *Rhizospora heterophylla*. Conversely, *Rhizospora heterophylla* can enhance the host plant's nodulation ability and nitrogen fixation rate by expanding the extraroot hyphal network to form "hyphae bridges."

[0025] The present invention provides a fermentation product, including the fermentation product of the slow-growing rhizobium strain SBE2501 or the fermentation product of the complex bacteria.

[0026] In this invention, the ferment of the slow-growing rhizobium strain SBE2501 is preferably prepared by the following method: the slow-growing rhizobium strain SBE2501 is inoculated onto yeast mannitol agar (YMA) slant for activation culture, followed by expansion culture in YMA liquid medium to obtain the ferment. The temperature of the activation culture and the expansion culture is preferably 23-28℃, more preferably 24-26℃, and most preferably 25℃. The activation culture time is preferably 3-8 days, more preferably 4-6 days, and most preferably 5 days. The expansion culture is preferably cultured to the logarithmic phase. The YMA liquid medium preferably contains the following components: mannitol 10 g / L, yeast extract 1 g / L, calcium carbonate 0.2 g / L, magnesium sulfate 0.2 g / L, sodium chloride 0.1 g / L, dipotassium hydrogen phosphate 0.5 g / L, and the pH value is preferably 7.0±0.2. The yeast mannitol agar medium preferably has 1.5% agar added to the YMA liquid medium. The effective viable count of *S. vegetative rhizobium* strain SBE2501 per milliliter of fermentation product is preferably not less than 10. 7 One, more preferably 10 8 ~1012 10, the optimal number is 10. 9 The fermentation product of the compound bacteria preferably includes the fermentation product of the individually packaged slow-growing rhizobium strain SBE2501 and the Rhizocystis heterophylla agent. In this invention, the main advantage is that the Rhizocystis heterophylla agent contains Rhizocystis heterophylla. The Rhizocystis heterophylla spore content in the Rhizocystis heterophylla agent is preferably 50-70 spores / g. This invention does not impose any special limitations on the preparation method of the Rhizocystis heterophylla agent; conventional Rhizocystis heterophylla culture methods in the art can be used. For example, healthy, active spores are selected and transferred to tap water in a petri dish. The petri dish is placed in a culture dish or covered and stored at 4°C for at least 48 h. After microscopic examination, all abnormal spores are removed. Sterile distilled water is then used, and the spores are stored at 4°C for another 24-48 h to further remove atypical spores. Three to four days before inoculation, clean spores are extracted and separated by wet sieving and placed in petri dishes according to their morphology. After sterilizing 50-cell seedling trays with 10% bleach, each cell was filled with sterilized boiling sand substrate to approximately 2 / 3 of its height. Under a stereomicroscope, plump spores were selected and placed on the substrate, covered with 1-2 cm of sterilized substrate, followed by the sowing of 2-3 sorghum seeds on the surface, then covered with another 0.5 cm of substrate. After watering, the trays were moved to a light chamber for initial cultivation. After cultivation, the seedling-bearing culture was removed, and approximately 1 / 4 of the culture was aseptically cut for wet sieving and microscopic examination to confirm root infection. The remaining 3 / 4 of the infected seedlings were vertically transplanted into the center of a plastic pot filled with 1 / 3 of sterilized substrate, and the surrounding area was filled with substrate. After watering, 25-30 sorghum seeds were evenly sown, covered with 0.5 cm of soil, and the trays were moved back to the light chamber for continued cultivation for 4-5 months. At harvest, the above-ground parts are first cut off, and the culture pots are placed in a stable temperature and humidity environment to dry. Finally, all the culture is collected to obtain the *Arbuscular Mycorrhizalis* agent, which is a cultivation substrate containing roots, mycelium, and spores. In this invention, the *Arbuscular Mycorrhizalis* agent preferably includes an agent containing *Arbuscular Mycorrhizalis* strain AH01 (BGC number). In this embodiment, the *Arbuscular Mycorrhizalis* strain AH01 agent was purchased from the "Arbuscular Mycorrhizal Fungi Germplasm Resource Bank (BGC)" of the Institute of Plant Nutrition and Resource Environment, Beijing Academy of Agricultural and Forestry Sciences. The *Arbuscular Mycorrhizalis* agent is a cultivation substrate containing spores and mycelium, and the spore content in the *Arbuscular Mycorrhizalis* agent is 50-70 spores / g.

[0027] Based on the characteristics of the SBE2501 strain and the compound bacteria in promoting plant growth and enhancing soil fertility, the present invention provides a microbial fertilizer comprising at least one of the SBE2501 slow-growing rhizobium strain, the compound bacteria, and the fermentation product.

[0028] In this invention, the preparation method of the microbial fertilizer is not particularly limited; any microbial fertilizer preparation scheme well known in the art can be used. For example, it can be obtained by mixing the fermentation product of the slow-growing rhizobium strain SBE2501, the heteromorphic rhizocystis agent, and excipients. This invention does not specifically limit the composition of the excipients; any microbial fertilizer excipient components well known in the art can be used, such as macroelements, mesoelements, microelements, and components that promote soil aggregate formation and reduce soil compaction.

[0029] This invention provides the application of the slow-growing rhizobium strain SBE2501, the compound bacteria, the fermentation product, or the microbial fertilizer in promoting plant growth and / or enhancing soil carbon sequestration capacity.

[0030] In this invention, the plant preferably includes grasses, more preferably at least one of maize, sorghum, wheat, and rice. The plant parts preferably include the above-ground parts and / or the underground parts. In this embodiment, the maize variety Zhengdan 958 is used as an example to illustrate its application in promoting plant growth and enhancing soil carbon sequestration capacity. Zhengdan 958 was bred by the Institute of Food Crops, Henan Academy of Agricultural Sciences, and purchased from Henan Shengdao Seed Industry Co., Ltd.

[0031] In this invention, promoting plant growth preferably includes increasing plant biomass and / or promoting nutrient absorption by the plant. Increasing plant biomass preferably includes at least one of the following: increasing plant fresh weight, plant height, and stem diameter. Promoting nutrient absorption preferably includes increasing the nitrogen and / or phosphorus content in the plant.

[0032] This invention compared the effects of a single *S. truncatella* strain SBE2501 (NM-BR), a complex of SBE2501 and *Rhizopus heterophyllus* strain AH01 (AM-BR), and a single *Sphingomonas* strain (NM-SP), and a complex of Sphingomonas and *Rhizopus heterophyllus* strain AH01 (AM-SP) on plant growth. The results showed that *S. truncatella* strain SBE2501 promoted *Rhizopus heterophyllus* infection, with a better effect than the *Sphingomonas* and *Rhizopus heterophyllus* complex (AM-SP). All treatments promoted maize growth, increased nitrogen and phosphorus content in both aboveground and underground parts of the plant, enhanced nitrogen and phosphorus absorption, and significantly increased plant biomass, aboveground and underground fresh weight, plant height, and stem diameter. The AM-BR complex showed the best effect.

[0033] In this invention, the enhancement of soil carbon sequestration capacity preferably includes at least one of the following: promoting the accumulation of soil carbon components, increasing the content of soil globulin, and increasing the activity of key enzymes for soil carbon transformation.

[0034] This invention further compared the effects of different treatments on soil carbon sequestration. The results showed that all treatments promoted the accumulation of soil carbon components, with the AM-BR combined treatment showing the most significant improvement. The synergistic effect of mycorrhizae and bacteria enhanced the sequestration of soil organic carbon, dissolved organic carbon, and total carbon, while optimizing the soil carbon-nitrogen ratio. All treatments increased the content of soil globulin-associated soil proteins (GRSPs). The AM-BR combination had a particularly prominent promoting effect on easily extractable GRSPs and total GRSPs. Furthermore, under the AM-BR combined application conditions, the activity of key enzymes such as catalase and dehydrogenase was significantly enhanced, effectively improving soil carbon cycling efficiency.

[0035] The present invention provides a method for promoting plant growth and / or enhancing soil carbon sequestration capacity, comprising: treating the soil with the slow-growing rhizobium strain SBE2501, the compound bacteria, the fermentation product, or the microbial fertilizer.

[0036] In this invention, the plant is preferably the same as the plant described in the above applications, and the plant growth and soil carbon sequestration capacity enhancement are preferably the same as those described in the above applications, which will not be repeated here.

[0037] In this invention, the soil treatment aims to ensure that the spore content of *Rhizoctonia solani* strains in the soil is preferably 3 spores / g or higher, more preferably 4.2-8.1 spores / g, and most preferably 5 spores / g. The content of the *S. solani* strain SBE2501 in the soil is 7 × 10⁻⁶. 6 More than 100 cells / g, preferably 8×10 6 ~9×10 6 The optimal value is 8.3 × 10⁻⁶ units / g. 6 per g.

[0038] To further illustrate the present invention, the 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.

[0039] Experimental materials YMA liquid medium contains the following components: mannitol 10 g / L, yeast extract 1 g / L, calcium carbonate 0.2 g / L, magnesium sulfate 0.2 g / L, sodium chloride 0.1 g / L, dipotassium hydrogen phosphate 0.5 g / L, and Rh trace elements 0.32 g / L, with a pH of 7.0 ± 0.2. Yeast mannitol agar medium is YMA liquid medium supplemented with 1.5% agar.

[0040] The formula for the 1 / 2 modified Hoagland culture medium is shown in Table 1.

[0041] Table 1.2 Modified Hoagland Culture Medium Formula

[0042] Example 1: Screening method for strains Slow-growing rhizobia ( Bradyrhizobium sp.) SBE2501 and Sphingosomalidobacterium (sp.) SBE2501 and Sphingosomalidobacterium Sphingomonas sp.) SBE2513 was isolated from maize rhizosphere soil, with Sphingomonas as the control strain.

[0043] The method for isolating and obtaining the slow-growing rhizobium SBE2501 is as follows: 1. Constructing the microbial bank: Fresh corn rhizosphere soil was collected from the black soil region of Northeast China, and mixed with water at a ratio of 10... -4 10 -5 10 -6 10 -7 10 -8 The solution was diluted according to the specified dilution ratio, and then placed in a 96-well cell culture plate containing YMA and incubated at room temperature (25°C) for 2 weeks. At the end of the culture, a concentration of 10-1 was selected. -6 The 96-well cell culture plates were used for subsequent bacterial bank construction.

[0044] Take 10 µL of bacterial culture from a 96-well cell culture plate, add 16.6 µL of alkaline lysis buffer (pH 12 containing 25 mM NaOH and 0.2 mM Na2-EDTA) and lyse at 95°C for 30 min. Add 40 mM Tris-HCl buffer (pH = 7.5) and mix well to obtain the bacterial DNA. Store at -20°C.

[0045] The DNA of the preserved bacterial culture was amplified by the first round of PCR: the universal amplification primers for bacteria 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 3) were selected.

[0046] The amplification system consisted of: 3 µL template, 3 µL 10×Buffer, 2.4 µL 2.5 mM dNTPs, 0.3 µL each of 10 µM upstream and downstream primers, and 0.15 µL 5 U / µL HS taq. The amplification program was: 95℃ for 5 min pre-denaturation; 25 cycles of 95℃ for 10 s, 55℃ for 15 s, and 72℃ for 1 min; final incubation at 72℃ for 5 min; and storage at 4℃. PCR amplification reagents were purchased from Thermo Fisher Scientific.

[0047] In the second round of PCR, the template was the product from the first round of PCR, diluted 40-fold. The primers used were 27F and 1492R primers with tags (barcodes) and the adapters required for sequencing. The amplification system and procedure were the same as in the first round of PCR. After both rounds of PCR amplification, the obtained DNA samples were subjected to agarose gel electrophoresis. The gel was then recovered according to the kit instructions. Finally, the concentration of the recovered samples was detected using Nanodrop. Qualified samples were sequenced to preliminarily identify the types of bacteria in the soil.

[0048] 2. Isolation, purification, and identification of *S. stomatologic plexiformis* SBE2501: The bacteria identified by sequencing were isolated and purified. From 96-well cell culture plates, wells containing identified bacteria were selected, and 10 µL of bacterial culture was streaked onto YMA plates for purification, with three replicates per well. The strain was cultured at 28°C for 3 days for further purification, repeated twice until pure colonies were obtained for morphological identification. The pure strain was then selected and cultured in YMA liquid medium at 28°C and 180 rpm for 3 days for 16S sequencing confirmation.

[0049] Morphological identification: According to the *Handbook of Systematic Identification of Common Bacteria*, strain SBE2501 was physiologically and biochemically identified. Strain SBE2501 is rod-shaped (0.5~0.9 × 1.2~3.0 μm), Gram-negative. In YMA medium, colony growth is slow; after 5~7 days, the colony diameter usually does not exceed 1 mm. The colonies are round, opaque or translucent, white, raised, with smooth edges, a granular structure, and a small diameter. The liquid culture is moderately turbid. A morphological image of strain SBE2501 is shown below. Figure 1 As shown.

[0050] Figure 2 As shown. Strain SBE2501 and *Syntrophus stolonifera* (… Bradyrhizobium The strain SBE2501 of this invention showed the highest homology (99%) with *S. sp.*, and combined with identification results based on bacterial morphology and growth conditions, it was determined that the strain is *S. sp.*. Bradyrhizobium sp.).

[0051] Example 2 Preparation method of Slow-growing Rhizobium inoculum: Slow-growing Rhizobium strain SBE2501 was inoculated onto yeast mannitol agar slant for activation. After culturing at 25℃ for 5 days, it was transferred to YMA culture medium for further cultivation. The mixture was shaken at 180 rpm for 48 hours until the logarithmic growth phase was reached, yielding a liquid Slow-growing Rhizobium inoculum. The liquid inoculum contained 1 × 10⁻⁶ bacteria. 9 per mL.

[0052] Preparation method of Sphingomonas sphingosine monophosphate inoculum: Sphingomonas strain SBE2513 was inoculated onto LB slant for activation, then cultured at 25℃ for 5 days, and then transferred to LB broth for further culture. The mixture was shaken at 180 rpm for 48 hours until the logarithmic growth phase was reached, yielding a liquid Sphingomonas sphingosine monophosphate inoculum. The liquid inoculum contained 1 × 10⁻⁶ bacteria. 9 per mL.

[0053] Preparation method of arbuscular mycorrhizal fungal inoculant: The arbuscular mycorrhizal fungal inoculant is *Rhizocystis heterophylla* (… Rhizophagus irregularis The strain AH01 inoculant was purchased from the "Arbuscular Mycorrhizal Fungi Germplasm Resource Bank (BGC)" of the Institute of Plant Nutrition and Resource Environment, Beijing Academy of Agricultural and Forestry Sciences. The inoculant is a cultivation substrate containing spores and mycelia, with a spore content of 50-70 spores / g.

[0054] A method to promote corn plant growth and soil carbon sequestration includes the following steps: a. Seedling selection and cultivation: The seedling substrate, including vermiculite, river sand, and perlite, is subjected to high-temperature and high-pressure sterilization. The sterilized river sand, vermiculite, and perlite are mixed in a mass ratio of 2:1:1, with a total substrate mass of 600g. The high-temperature and high-pressure sterilization parameters are: 121℃, 1×10⁻⁶. 5 kPa, 2h; Disinfection of culture containers: Disinfect the potted container (16 cm in diameter × 12 cm in diameter × 11 cm in height) with 75% alcohol, wipe it repeatedly with sterile water and set aside; Select plump corn seeds, disinfect the surface with 10% hydrogen peroxide for 10 min, rinse repeatedly with distilled water, place the seeds in a tray with a moist cotton layer at the bottom, and place it in a 25℃ incubator to germinate; Select germinated seeds to cultivate in sterilized seedling substrate, and when the corn seedlings grow to two leaves and one heart, divide them into experimental groups, prepare for transplanting and inoculation treatment; b. Experimental Groups: The experiment was conducted in pots and was a two-factor randomized trial. The experimental factors included whether or not arbuscular mycorrhizal fungi (AMF) inoculants were applied (2 levels: no inoculation denoted as NM, inoculation denoted as AM) and different types of bacterial inoculants (3 levels: including application of Sphingomonas denoted as SP, application of Slow-growing Rhizobium denoted as BR, and no application as control CK). There were a total of 6 treatments, each of which was replicated 5 times, for a total of 30 plants. The experimental groups are as follows.

[0055] NM-CK: No inoculation with bacterial agent; NM-SP: Inoculation with Sphingomonas sphingosine mononucleosis; NM-BR: Inoculation with slow-growing rhizobium inoculum; AM-CK: Inoculation with arbuscular mycorrhizal fungi; AM-SP: Inoculation with arbuscular mycorrhizal fungi and Sphingomonas inoculum; AM-BR: Inoculate with arbuscular mycorrhizal fungi and slow-growing rhizobium inoculants.

[0056] b. Transplanting seedlings and inoculating with arbuscular mycorrhizal fungi: Select corn seedlings with uniform growth for transplanting; The NM-CK, NM-SP and NM-BR groups were transplanted directly without inoculation with arbuscular mycorrhizal fungi; For groups AM-CK, AM-SP, and AM-BR, 2 / 3 of the mixed substrate was first spread evenly at the bottom of the pot, and then inoculated with arbuscular mycorrhizal fungi. The "sandwich" inoculation method was used to inoculate the arbuscular mycorrhizal fungi, and the fungicide was evenly applied to the mixed substrate. Then, a 2 cm layer of substrate was placed on top, and finally, the mixed substrate was used to complete the transplanting of the corn seedlings. The application rate of arbuscular mycorrhizal fungi was 50.00 g per plant.

[0057] c. Inoculate with Sphingomonas sphingosine mononitrate or Sphaerozoopterygium spp.: Use a pipette to inoculate each corn seedling with 5 mL of Sphaerozoopterygium sphingosine mononitrate or Sphaerozoopterygium spp., and finally place the transplanted corn seedlings in a greenhouse for cultivation; Greenhouse parameters: temperature (28±2)℃ day / (18±2)℃ night, light intensity 4000 Lx, photoperiod 14h / d.

[0058] d. Fertilizer management: On the day of transplanting, apply only sterilized water to ensure the survival of corn seedlings. Water every 2-3 days to maintain the moisture content of the mixed substrate at 70%. Supplement with 1 / 2 of the modified Hoagland nutrient solution once a week. Harvest the plants after 6 weeks of cultivation in the greenhouse.

[0059] After harvesting, maize plant growth and development indicators were measured, including plant height, stem diameter, aboveground fresh weight, underground fresh weight, and biomass. Mycorrhizal infection rate was determined using the tribenzene blue staining method. Total phosphorus content in maize leaves and roots was determined using the molybdenum-antimony colorimetric method. Total nitrogen content in maize leaves and roots was determined using the Kjeldahl method. Soil dissolved organic carbon (DOC) was extracted with distilled water and quantified using a TOC analyzer. Soil organic carbon (SOC) content was determined using the potassium dichromate oxidation-external heating method. Total soil carbon (TC), total nitrogen (TN), and carbon-to-nitrogen ratio (C:N) were determined using Vario EL. III. Elemental analyzer; Gastromycin-associated soil protein (GRSP) was extracted with citrate buffer, using bovine serum albumin as a standard, and quantitatively analyzed using the Bradford method for easily extractable globulin (EE-GRSP) and total globulin (T-GRSP) content; The activity of extracellular enzymes (catalase, dehydrogenase, α-glucosidase, β-glucosidase, α-galactosidase) related to soil carbon fixation was determined using enzyme-linked immunosorbent assay (ELISA), in which soluble antigens or antibodies were bound to a solid-phase carrier such as polystyrene, and qualitative and quantitative detection was performed by utilizing the specific binding of antigens and antibodies to conduct an immune reaction.

[0060] (1) Plant growth and mycorrhizal development are shown in Table 2. Figure 3 and Figure 4 The results of the plant mineral nutrient content test are shown in Table 3 and Figure 5 .

[0061] Table 2. Effects of different compound biological agents on mycorrhizal infection and plant growth and development in maize.

[0062] Note: Different lowercase letters represent p Significance at the ≤0.05 level.

[0063] like Figure 3 As shown, Figure 3 In the middle (a), the results of mycorrhizal infection observation show that the maize roots have been successfully infected by arbuscular mycorrhizal fungi. The mycorrhizal plants have formed obvious vesicle structures inside the roots, and there are obvious extraroot hyphae. Figure 3 (c) shows the mycorrhizal infection rate. The AM-CK treatment, inoculated with arbuscular mycorrhizal fungi alone, had a mycorrhizal infection rate of 54.49%. The AM-SP combined inoculation treatment increased the rate by 4.57%, and the AM-BR combined inoculation treatment significantly increased it by 10.31%. This indicates that both bacteria can help arbuscular mycorrhizal fungi infect maize roots, and BR showed a better effect in improving the mycorrhizal infection rate. Furthermore, Figure 3(b) shows the growth phenotype of maize plants. The growth phenotypes of maize plants under the combined inoculation of the two bacteria with arbuscular mycorrhizal fungi were compared. Compared with no inoculation, plant growth and development were effectively improved after inoculation alone (NM-SP / NM-BR) or combined inoculation with arbuscular mycorrhizal fungi (AM-SP / AM-BR), and the combined treatment had a more significant effect on promoting plant growth.

[0064] like Figure 4 As shown, both bacteria significantly promoted plant growth, and the combined treatment with arbuscular mycorrhizal fungi exhibited a synergistic effect, particularly in increasing biomass, aboveground and underground fresh weight, plant height, and stem diameter. Among these, the AM-BR combined inoculation showed the most significant growth-promoting effect. Regarding biomass, regardless of whether arbuscular mycorrhizal fungi were inoculated, the biomass of plants inoculated with SP and BR was higher than that of uninoculated plants, with the AM-BR combined treatment showing the most significant improvement, indicating that mycorrhizae and bacteria synergistically promoted plant dry matter accumulation. In terms of aboveground fresh weight, the bacterial inoculation treatment alone was higher than the control, and the aboveground fresh weight of the combined inoculation with arbuscular mycorrhizal fungi was further increased. Meanwhile, the underground fresh weight showed the same trend as the aboveground part; both SP and BR treatments significantly increased root fresh weight, reaching the highest level under the combined treatment of arbuscular mycorrhizal fungi and bacteria, indicating that mycorrhizae and bacteria synergistically enhanced plant growth and root development. Regarding plant height and stem diameter, inoculation with bacteria (especially AM-BR) under AM treatment had a more significant effect on promoting plant height, with AM-BR treatment showing a significantly higher effect than other treatments, indicating that this combination had the most outstanding effect on plant growth and development.

[0065] Table 3. Effects of different compound biological agents on mineral nutrient content in maize plants.

[0066] Note: Different lowercase letters represent p Significance at the ≤0.05 level.

[0067] like Figure 5 As shown, BR significantly promotes nitrogen and phosphorus absorption in plants, and exhibits a strong synergistic effect when co-inoculated with arbuscular mycorrhizal fungi, significantly increasing nitrogen and phosphorus content in both aboveground and belowground parts of the plant. In contrast, SP has a weaker promoting effect on nitrogen and phosphorus absorption. Regarding aboveground nitrogen content, regardless of whether arbuscular mycorrhizal fungi are inoculated, inoculation with BR (13.45~18.66 g kg) significantly increases nitrogen content. -1 The nitrogen content in the aboveground parts of the treated plants was significantly higher than that of the control (CK) (11.46–12.13 g / kg). -1 ) and SP (10.86~11.44 g kg) -1The treatments included mycorrhizae and BR. Among them, the AM-BR combined inoculation treatment showed the most significant increase in aboveground nitrogen content. The trend of underground nitrogen content was basically consistent with that of the aboveground parts. BR treatment also significantly increased underground nitrogen content, and the increase was more pronounced under the AM-BR combined inoculation treatment, indicating that mycorrhizae and BR synergistically promoted nitrogen accumulation in the aboveground parts and the root system's ability to absorb nitrogen. Regarding aboveground phosphorus content, both SP and BR treatments increased its content, with the AM-BR combined treatment showing the highest aboveground phosphorus content. Underground phosphorus content also showed that the BR treatment was significantly higher than the control, reaching its maximum value under the AM-BR combined treatment, significantly superior to other treatments, indicating that mycorrhizae and BR have a synergistic effect in promoting phosphorus absorption in plants.

[0068] (2) The results of soil carbon composition and globulin-related soil protein content detection are shown in Table 4 and Figure 6 The results of the detection of extracellular enzyme activities related to soil carbon fixation are shown in Table 5 and Figure 7 .

[0069] Table 4. Effects of different compound biological agents on carbon composition and globulin content in maize rhizosphere soil.

[0070] Note: Different lowercase letters represent p Significance at the ≤0.05 level.

[0071] like Figure 6 As shown, both bacteria promoted the accumulation of soil carbon components and exhibited a synergistic effect when co-inoculated with arbuscular mycorrhizal fungi. Regardless of whether arbuscular mycorrhizal fungi were inoculated, SP (2.04–2.29 mg / kg) -1 ) and BR (2.16~2.42 mg kg) -1 The dissolved organic carbon content of the treated groups was higher than that of the control group (1.95~2.28 mg kg). -1 Among the various treatments, the AM-BR combined treatment showed the most significant improvement, and the trend of soil organic carbon content change was basically consistent with that of dissolved organic carbon. Both SP and BR treatments increased soil organic carbon content, and the AM-bacterial combined treatment further increased it, demonstrating the synergistic effect of mycorrhizae and bacteria in enhancing soil organic carbon sequestration. Furthermore, both SP and BR treatments were significantly higher than the control, with the AM-BR treatment showing the highest total soil carbon content, indicating that mycorrhizae and bacteria synergistically enhanced the soil's total carbon accumulation capacity. Regarding the carbon-nitrogen ratio, all bacterial treatments increased the soil carbon-nitrogen ratio, with the AM-bacterial combined treatment (such as AM-BR) showing the largest increase, indicating that the combined application of mycorrhizae and bacteria helps optimize the soil carbon-nitrogen balance and enhance carbon pool stability.

[0072] Furthermore, both bacteria effectively increased GRSP content and exhibited a synergistic enhancement effect when co-inoculated with arbuscular mycorrhizal fungi, particularly in regulating the contribution of GRSP to soil organic carbon. The AM-BR combination showed the most significant promoting effect on GRSP. Regardless of whether arbuscular mycorrhizal fungi were inoculated or not, the EE-GRSP content in the SP and BR treatments was higher than that in the CK treatment, and both co-inoculation treatments were superior to single inoculation treatments. The trend of total GRSP changes was basically consistent with that of easily extractable GRSP; both SP and BR treatments increased the total GRSP content, with a more significant increase under co-inoculation conditions of arbuscular mycorrhizal fungi and bacteria, indicating that the synergistic effect of mycorrhizae and bacteria is beneficial to the accumulation and retention of GRSP.

[0073] Table 5 Effects of different compound bio-agents on enzyme activity in maize rhizosphere soil

[0074] Note: Different lowercase letters represent p Significance at the ≤0.05 level.

[0075] like Figure 7 The results show the effects of three treatments (CK, SP, and BR) on the activities of key enzymes in soil carbon transformation under both non-inoculation (NM) and AM (arbuscular mycorrhizal fungi) inoculation conditions. The results indicate that AM inoculation significantly increased the activities of catalase, dehydrogenase, β-glucosidase, and β-galactosidase. These enzymes participate in soil carbon transformation by protecting microbial function, promoting the oxidation and decomposition of organic matter, and catalyzing carbohydrate hydrolysis, respectively. Increased activity signifies improved carbon cycle efficiency. Only α-glucosidase showed the highest activity under NM conditions with the SP treatment. Under AM conditions, there was no significant difference between BR and SP treatments, but both were higher than CK. In summary, BR treatment is the optimal exogenous regulation method for enhancing the activities of soil carbon transformation-related enzymes, and its effect is even more pronounced when combined with AM inoculation, providing data support for strengthening soil carbon cycling through regulatory measures.

[0076] 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. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A slow-growing rhizobium ( Bradyrhizobium strain SBE2501 (sp.) is characterized by, The accession number is CGMCC NO.36389.

2. A compound bacteria, characterized in that, Including the slow-growing rhizobium strain SBE2501 as described in claim 1 and Rhizocystis heteromorpha ( Rhizophagus irregularis ).

3. A fermentation product, characterized in that, This includes the fermentation product of the slow-growing rhizobium strain SBE2501 as described in claim 1 or the fermentation product of the complex bacteria as described in claim 2.

4. A microbial fertilizer, characterized in that, It includes at least one of the following: the slow-growing rhizobium strain SBE2501 of claim 1, the complex bacteria of claim 2, and the ferment of claim 3.

5. The application of the slow-growing rhizobium strain SBE2501 of claim 1, the compound bacteria of claim 2, the fermentation product of claim 3, or the microbial fertilizer of claim 4 in promoting plant growth and / or enhancing soil carbon sequestration capacity.

6. The application according to claim 5, characterized in that, Promoting plant growth includes increasing plant biomass and / or promoting nutrient absorption by the plant.

7. The application according to claim 5, characterized in that, The enhancement of soil carbon sequestration capacity includes at least one of the following: promoting the accumulation of soil carbon components, increasing the content of soil globulin, and increasing the activity of key enzymes for soil carbon transformation.

8. A method for promoting plant growth and / or enhancing soil carbon sequestration capacity, characterized in that, include: Soil is treated with the slow-growing rhizobium strain SBE2501 as described in claim 1, the compound bacteria as described in claim 2, the fermentation product as described in claim 3, or the microbial fertilizer as described in claim 4.

9. The method according to claim 8, characterized in that, The soil treatment aims to ensure that the spore content of *Rhizoctonia solani* strain in the soil is above 3 spores / g, and the content of *S. stomatologica* strain SBE2501 is above 7 × 10⁻⁶. 6 More than one per gram.

10. The method according to claim 8 or 9, characterized in that, The plants mentioned include grasses (Poaceae).

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