Compound microbial agent for repairing uranium-contaminated soil and application thereof

Abstract

The invention provides a compound microbial agent for repairing uranium-contaminated soil and application of the compound microbial agent for repairing the uranium-contaminated soil, and the compound microbial agent for repairing the uranium-contaminated soil is prepared by mixing micromycobacterium and bacillus cereus. Through microbial agent treatment, the inhibition effect on seed germination of the sudangrass is remarkably relieved, and the plant biomass, the stress-resistant enzyme activity, the uranium enrichment coefficient, the total biomass of the sudangrass, the total nitrogen content of soil and the rapidly available phosphorus content are also remarkably improved; in the aspect of uranium pollution remediation efficiency, the complex microbial inoculants can reach 20%; the activity of beta-glucosidase is respectively improved by 10.57%-42.47%, and the activity of urease and the activity of catalase are also synchronously improved; the abundance of proteobacteria and cyanobacteria in a microbial community structure is obviously increased. According to the technical scheme provided by the invention, a uranium pollution treatment mode of'functional fungicide-rhizosphere micro-ecological regulation-plant synergistic remediation 'is constructed. A new thought and method are provided for repairing the uranium-polluted soil, and a scientific basis and a feasible repairing scheme are provided for subsequent large-scale application.

Description

Technical Field

[0001] The present invention belongs to the technical field of ecological restoration, specifically to the technical field of uranium-contaminated soil restoration, and more specifically to the technical field of a composite microbial agent for uranium-contaminated soil restoration and its application. Background Art

[0002] The rapid development of the nuclear industry has increased demand for nuclear fuel and accelerated uranium mining and refining. However, the uranium mining and refining processes generate large quantities of uranium tailings and waste. The uranium in these wastes is dispersed into the soil, air, and groundwater. Because it is non-biodegradable, once it enters the environment or is ingested by organisms, its harmful effects are persistent. Not only can it gradually accumulate in organisms through the respiratory system and food chain, causing internal radiation damage, it can also induce mutations in species, posing a significant threat to human health.

[0003] In the biological remediation of uranium contaminated soil, phytoremediation technology has become the preferred option for the long-term remediation of large, medium- and low-level contaminated sites due to its high cost-effectiveness, strong in-situ operability, and low environmental disturbance. However, the limited number of plant species that hyperaccumulate radioactive uranium, low biomass, low enrichment rates, and long remediation cycles significantly limit its application in large-scale radioactively contaminated soils. Integrating plant growth-promoting bacteria into the phytoremediation system for heavy metal-contaminated soils is an effective strategy for addressing heavy metal contamination in soils. Summary of the Invention

[0004] In response to the technical problems in the prior art that Bacillus cereus B6 does not have the ability to degrade organic phosphorus and produce IAA and that Microvirga sp. M2 has a low ability to produce siderophores, the present application aims to provide a composite microbial agent that can exert the synergistic effect of Bacillus cereus B6 and Microvirga sp. M2, as well as its application in uranium-contaminated soil remediation. Under uranium stress conditions, the agent treatment significantly alleviated the inhibitory effect on Sudan grass seed germination, plant biomass, stress-resistant enzyme activity, uranium enrichment coefficient, and total Sudan grass biomass, and significantly improved the total nitrogen and available phosphorus content in the soil; in terms of uranium pollution remediation efficiency, the composite agent can reach 20%; the β-glucosidase activity increased by 10.57%-42.47%, and the urease and catalase activities also increased simultaneously; the abundance of Proteobacteria and Cyanobacteria in the microbial community structure increased significantly. The technical solution presented in this application establishes a uranium contamination treatment approach combining "functional microbial agents, rhizosphere microecological regulation, and plant-based synergistic remediation." This approach provides new ideas and methods for the remediation of uranium-contaminated soils, and also provides a scientific basis and feasible remediation plan for subsequent large-scale application.

[0005] To achieve this technical purpose, the present invention adopts the following technical solutions:

[0006] On the one hand, the present application provides a composite microbial agent for remediation of uranium-contaminated soil, wherein the composite microbial agent comprises: a mixture of Microvirga sp. M2 fermentation broth and Bacillus cereus B6 fermentation broth in a volume ratio of (1-3): (3-1).

[0007] Preferably, in the composite microbial agent, the fermentation broth of Microvirga sp. M2 and the fermentation broth of Bacillus cereus B6 are mixed in a volume ratio of 1:1.

[0008] Preferably, the fermentation broth of Microvirga sp. M2 is prepared by inoculating a single colony of Microvirga sp. M2 into a sterilized NB liquid culture medium at 37°C and 150 r·min -1 The culture was shaken for 24 h and the bacterial concentration was adjusted to OD600 of 1.0.

[0009] Preferably, the Bacillus cereus B6 fermentation broth is obtained by inoculating a single colony of Bacillus cereus B6 into a sterilized NB liquid culture medium at 37°C and 150 r·min -1 The culture was shaken for 24 h and the bacterial concentration was adjusted to OD600 of 1.0.

[0010] Furthermore, the present application provides the use of the composite microbial agent for remediation of uranium-contaminated soil in the remediation of uranium-contaminated soil.

[0011] The application method comprises planting Sudan grass in uranium-contaminated soil and applying a composite microbial agent to treat the uranium-contaminated soil.

[0012] In the application method, the composite microbial agent is centrifuged at 10000g for 10 minutes, the supernatant is discarded, and the bacteria are collected; the bacteria are rinsed with sterile water and centrifuged three times to remove the residual culture medium; and finally the bacteria liquid is resuspended and mixed with sterile water to OD = 1.0, about 10 8 Use per mL.

[0013] In the application method, Sudan grass seeds with full grains are selected, the seeds are immersed in a 1% sodium hypochlorite solution for 10 minutes for disinfection, the seeds are rinsed with sterile water three times, each time for 1 minute, the seeds are taken out and drained, and then immersed in a composite bacterial agent suspension for 1 hour, the bacterial suspension is poured out, the seeds are dried in a sterile environment, and then planted.

[0014] In the application method, the composite microbial agent is diluted at a ratio of 100:1, and then the bacterial solution is evenly poured near the rhizosphere of each Sudan grass plant, and the application amount for each plant is ensured to be no less than 100 mL.

[0015] By implementing the technical solution of the present invention, the following beneficial effects can be achieved:

[0016] (1) The rhizosphere growth-promoting bacteria Microvirga sp. M2 and Bacillus cereus B6 used in this application do not antagonize each other and can be used to prepare a composite microbial agent. A study was conducted using different ratios, and ultimately, while ensuring the growth-promoting properties of each strain, a 1:1 ratio of Microvirga sp. M2 and Bacillus cereus B6 was selected to prepare a composite agent, achieving good technical results, with organic phosphorus 10.6±2.5 mg / L, inorganic phosphorus 20.9±3.3 mg / l, siderophore SU production 45.18%±2.1, and IAA 22.39±3.2 μg / mL.

[0017] (2) The results of seed germination and potted plant experiments of the composite microbial agent technology for uranium contaminated soil remediation provided by this application showed that under uranium stress conditions, the agent treatment significantly alleviated the inhibitory effect on Sudan grass seed germination, among which the composite agent combined treatment group showed the best growth-promoting effect. The potted plant experiment showed that at 20, 50 and 100 mg·kg -1 At high uranium concentrations, inoculation with different inoculants increased plant biomass by 12.27%-59.81%, enhanced the activity of stress-resistant enzymes such as superoxide dismutase and peroxidase, and increased the uranium enrichment coefficient of Sudan grass. This result confirms that inoculants have the potential to synergistically enhance plant remediation of uranium-contaminated soils by mitigating uranium toxicity and activating plant physiological metabolism.

[0018] (3) In the in-situ test of the composite microbial agent technology solution for uranium contaminated soil remediation provided by this application, the agent treatment significantly increased the total biomass of Sudan grass, with an increase of 55.01%-83.04%, as well as the total nitrogen and available phosphorus content in the soil. In terms of uranium pollution remediation efficiency, the uranium removal rate of a single agent was 15%-18%, while that of the composite agent could reach 20%. Soil enzyme activity analysis showed that the activity of β-glucosidase increased by 10.57%-42.47%, and the activities of urease and catalase also increased simultaneously. In terms of microbial community structure, the abundance of Proteobacteria and Cyanobacteria increased significantly, indicating that the agent optimized the microenvironment of rhizosphere nutrient cycling and pollutant transformation by regulating functional microbial groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is the effect of different combinations of bacterial agents on Sudan grass enzyme activity under uranium stress.

[0020] Figure A shows the results of CAT enzyme activity determination; Figure B shows the results of MDA enzyme activity determination; Figure C shows the results of GSH enzyme activity determination; Figure D shows the results of POD enzyme activity determination; Figure E shows the results of SOD enzyme activity determination; Figure F shows the chlorophyll content; Figure G shows the results of PAL determination; and Figure H shows the results of GSH / GSSG determination.

[0021] Figure 2 Shown are the effects of different bacterial agents on Sudangrass biomass in the field under uranium stress.

[0022] Figure A shows the results of the measurement of the effect of plant height and root length on the biomass of Sudan grass; Figure B shows the results of the measurement of the aboveground and underground weights of Sudan grass.

[0023] Figure 3 Shown is a Venn diagram of the distribution of ASVs in the rhizosphere soil bacteria of Sudangrass.

[0024] In the figure, T1 is the M2+B6 group; T2 is the B6 group; T3 is the M2 group; and T4 is the CK group.

[0025] Figure 4 NMDS analysis results are shown in the figure.

[0026] In the figure, T1 is the M2+B6 group; T2 is the B6 group; T3 is the M2 group; and T4 is the CK group.

[0027] Figure 5 Shown is the analysis of the top 10 phyla and genera of the soil bacterial community.

[0028] Figure A shows the phylum analysis of the top 10 soil bacterial communities; Figure B shows the genus analysis of the top 10 soil bacterial communities; in the figure, T1 is the M2+B6 group; T2 is the B6 group; and T3 is the M2 group.

[0029] Figure 6 Shown are the LDA values ​​and cladogram of LEfSe analysis of soil bacterial microorganisms.

[0030] Figure A shows the LEfSe analysis diagram; Figure B shows the evolutionary branch diagram.

[0031] Figure 7 Shown is a redundancy analysis diagram of microbial species (top 30 genera) and environmental factors.

[0032] Among them, A is the M2+B6 group; B is the B6 group; C is the M2 group; and D is the CK group. DETAILED DESCRIPTION

[0033] The technical solutions of the present invention are described clearly and completely below with reference to the embodiments. It is obvious that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0034] In this embodiment, the reagents used were: uranyl nitrate (UO2(NO3)2·6H2O) purchased from Huawei Ruike Chemical Co., Ltd., IAA (C 10 H9NO2) was purchased from Shanghai Xuanya Biotechnology Co., Ltd., concentrated sulfuric acid (H2SO4) was purchased from Chengdu Kelong Chemical Co., Ltd., iron (III) chloride hexahydrate (FeCl3·6H2O) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., L-tryptophan (C 11 H 12 N2O2) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0035] The instruments used in this application are: high-pressure steam autoclave LDZM-60KGS-Ⅲ Shanghai Shen'an; clean bench SW-CJ-1F Zhejiang Fuxia; optical microscope Eclipse Ci-L Japan Nikon; stereo microscope SMZ25 Japan Nikon; automatic colony counter ZX-400 Hangzhou Zexi; constant temperature oscillating box TS-2102C Shanghai Tiancheng; centrifuge CF1524R United States Celoczech; constant temperature incubator bluepard Shanghai Yiheng; pH meter FiveEasy Plus Shanghai Mettler-Toledo; microplate reader Epoch2 United States Berton; analytical electronic balance Adventurer United States Ohaus; inductively coupled plasma mass spectrometer (ICP / MS) 7700X United States Agilent.

[0036] IAA mother solution: Accurately weigh 10 mg of IAA and dissolve it in a small amount of anhydrous ethanol, then dilute to 100 mL with distilled water to prepare 100 μg mL -1 of IAA stock solution.

[0037] Salkowski colorimetric solution: 4.5 g L -1 FeCl3·6H2O, 10.8 mol L -1 H2SO4 (prepare and use immediately).

[0038] UO2(NO3)2 stock solution: Take 1.05g UO2(NO3)2·6H2O and 100mL distilled water to prepare 5g·L -1 of UO2(NO3)2 mother liquor.

[0039] Nutrient broth (NB): peptone 10.0 g, beef extract powder 3.0 g, sodium chloride 5.0 g, distilled water 1000 mL, adjust the pH to 7.0 ± 0.1, and sterilize at 121 °C for 15 min.

[0040] Nutrient agar medium (NA): Add 15.0 g of agar to the NB medium.

[0041] Organophosphorus bacteria culture medium: glucose 10.0 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, magnesium sulfate 0.3 g, manganese sulfate 0.03 g, potassium sulfate 0.3 g, ferrous sulfate 0.03 g, calcium phosphate 5.0 g, lecithin 0.2 g, agar 15.0 g, distilled water 1000 mL.

[0042] Inorganic phosphate-dissolving bacterial culture medium (PKO): 10.0 g glucose, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g magnesium sulfate, 0.03 g manganese sulfate, 0.3 g potassium sulfate, 0.03 g ferrous sulfate, 5.0 g calcium phosphate, 15.0 g agar, and 1000 mL distilled water.

[0043] Siderophore assay medium (CAS): glucose 100.0 g, peptone 20.0 g, magnesium sulfate heptahydrate 0.5 g, calcium chloride 0.5 g, agar 20.0 g, distilled water 1000 mL.

[0044] IAA production medium: Add 200 mg L-1 tryptophan to the basal medium and sterilize by autoclaving at 121°C for 15 min.

[0045] The Microvirga sp. M2 selected in this application is a strain obtained by our laboratory in the early stage of self-screening. The relevant information of this strain has been disclosed in the invention patent with application number 202411279780.8. The strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 20, 2023, with the deposit number GDMCC No: 63775.

[0046] The Bacillus cereus B6 selected in this application is a strain obtained by our laboratory in the early stage of self-screening. The relevant information of this strain has been disclosed in the invention patent application number 202411168679.5. The strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 7, 2024, with the deposit number GDMCC No: 64588.

[0047] The general public can obtain the above strains by contacting the applicant's research group or purchasing them through public channels such as the China General Microbiological Culture Collection Center (CGMCC).

[0048] In the following content of this application, “Microvirga sp. M2” is referred to as “strain M2” or “M2”; “Bacillus cereus B6” is referred to as “strain B6” or “B6”.

[0049] The plant seeds used in this application are commercially available Sudan grass seeds.

[0050] In this application, the original data were collated using Excel 2010 and statistical analysis was performed using SPSS 25. The results were expressed as mean ± standard error. The data in the figures and tables of this application are all mean ± standard error (mean ± SE).

[0051] Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0052] Example 1: A composite microbial agent for remediation of uranium-contaminated soil

[0053] The present application provides a composite microbial agent for remediation of uranium-contaminated soil, wherein the composite microbial agent comprises: a mixture of Microvirga sp. M2 fermentation broth and Bacillus cereus B6 fermentation broth in a volume ratio of (1-3):(3-1).

[0054] Preferably, in the composite microbial agent, the fermentation broth of Microvirga sp. M2 and the fermentation broth of Bacillus cereus B6 are mixed in a volume ratio of 1:1.

[0055] Preferably, the fermentation broth of Microvirga sp. M2 is prepared by inoculating a single colony of Microvirga sp. M2 into a sterilized NB liquid culture medium at 37°C and 150 r·min -1 The culture was shaken for 24 h and the bacterial concentration was adjusted to OD600 of 1.0.

[0056] Preferably, the Bacillus cereus B6 fermentation broth is obtained by inoculating a single colony of Bacillus cereus B6 into a sterilized NB liquid culture medium at 37°C and 150 r·min -1 The culture was shaken for 24 h and the bacterial concentration was adjusted to OD600 of 1.0.

[0057] Example 2: Application of a composite microbial agent for remediation of uranium-contaminated soil

[0058] The present application provides the use of the composite microbial agent for remediation of uranium-contaminated soil in the remediation of uranium-contaminated soil.

[0059] The application method comprises planting Sudan grass in uranium-contaminated soil and applying a composite microbial agent to treat the uranium-contaminated soil.

[0060] In the application method, the composite microbial agent is centrifuged at 10000g for 10 minutes, the supernatant is discarded, and the bacteria are collected; the bacteria are rinsed with sterile water and centrifuged three times to remove the residual culture medium; and finally the bacteria liquid is resuspended and mixed with sterile water to OD = 1.0, about 10 8 Use per mL.

[0061] In the application method, Sudan grass seeds with full grains are selected, the seeds are immersed in a 1% sodium hypochlorite solution for 10 minutes for disinfection, the seeds are rinsed with sterile water three times, each time for 1 minute, the seeds are taken out and drained, and then immersed in a composite bacterial agent suspension for 1 hour, the bacterial suspension is poured out, the seeds are dried in a sterile environment, and then planted.

[0062] In the application method, the composite microbial agent is diluted at a ratio of 100:1, and then the bacterial solution is evenly poured near the rhizosphere of each Sudan grass plant, and the application amount for each plant is ensured to be no less than 100 mL.

[0063] Example 3: Preparation of bacterial agent

[0064] Pick up the bacterial suspension of two strains stored in -80℃ glycerol tubes, activate them by streaking on NA plates, and culture them at 30℃ for 48h. In the clean bench, use sterile bamboo sticks to pick up the strains respectively, inoculate them on NA plates at the intersection, and culture them at 30℃ for 2d. Observe the growth of the two bacteria at the intersection. If both bacteria grow at the intersection, it means that there is no inhibitory effect between the two strains, or they promote each other. Inoculate M2 and B6 into NB liquid culture medium respectively, at 28℃, 150r·min -1 After 48 h of culture, the OD600 was adjusted to 1.0. The two strains were mixed in different proportions as seed solution, and their growth-promoting characteristics were quantitatively measured.

[0065] Quantitative determination of siderophore production: 5 mL of bacterial solution was placed in a centrifuge tube and centrifuged at 10,000 r / min. -1 Centrifuge for 10 minutes, take 1 mL of the supernatant and thoroughly mix with an equal volume of CAS assay solution. Let the mixture stand for 1 hour, then dilute twice with deionized water. Measure the absorbance (As) at 630 nm, using deionized water as a control for zero adjustment. Measure the absorbance of the uninoculated culture medium using the same method as the reference value (Ar). Perform three replicates for each treatment, and calculate the SU value according to the formula.

[0066] SU=[(A r -As ) / A r ]×100% (Formula 1)

[0067] IAA auxin quantitative determination: 100 μL seed solution was inoculated into 5 mL containing 200 mg·L -1 L-tryptophan in NB medium, 30℃, 150r·min -1 Culture at constant temperature and shake for 48 hours, and measure OD600. -1 After centrifugation for 5 minutes, the supernatant was mixed with Salkowski colorimetric solution in a ratio of 1:2, and OD530 was measured after protection from light for 30 minutes. The IAA yield was calculated based on the IAA standard curve.

[0068] Phosphate solubilization capacity: inoculate the seed solution into a 250ml triangular flask containing 100ml NBRIP culture medium, shake at 30℃ and 170r·min -1 After culturing for 7 days, the available phosphorus content and pH changes in the culture medium were determined. The available phosphorus content was determined using the molybdenum antimony colorimetric method. The culture medium was heated at 5000 r / min. -1 Centrifuge for 10 minutes, take the supernatant and dilute it appropriately, take 1 mL and add it to a volumetric flask (25 mL) filled with 15 mL of distilled water, add 100 μL of dinitrophenol indicator, and add an appropriate amount of 0.05 mol·L -1 Add 2.5 mL of dilute sulfuric acid solution to the volumetric flask until the color of the solution in the volumetric flask turns light yellow. Then add 2.5 mL of molybdenum antimony anti-color developing solution to the volumetric flask and dilute to the mark with distilled water. After inversion to mix, place it in a 25°C incubator for 30 minutes and measure the absorbance of the reaction solution at a wavelength of 700 nm.

[0069] Plate streaking revealed that the two bacterial strains could grow at their intersections, indicating that strains M2 and B6 were not antagonistic, allowing subsequent preparation of a composite bacterial inoculant. Quantitative analysis of the growth-promoting abilities of M2 and B6 in varying ratios, as shown in Table 1, revealed no significant differences in their growth-promoting abilities. Therefore, a 1:1 ratio of M2 to B6 was selected, ensuring the growth-promoting effects of each.

[0070] Table 1: Growth-promoting effects of different ratios of compound microbial agents

[0071] Ratio (M2:B6) Organic phosphorus mg / L Inorganic phosphorus mg / l Siderophore-producing SU IAA μg / mL M2 <![CDATA[11.56±2.5 a ]]> <![CDATA[18.9±3.3 a ]]> <![CDATA[40.18%±2.1 b ]]> <![CDATA[24.39±3.2 a ]]> B6 - <![CDATA[15.2±2.1 b ]]> <![CDATA[48.2%±1.9 a ]]> - 1:1 <![CDATA[10.6±2.5 a ]]> <![CDATA[20.9±3.3 a ]]> <![CDATA[45.18%±2.1 a ]]> <![CDATA[22.39±3.2 a ]]> 1:2 <![CDATA[9.3±3.7 a ]]> <![CDATA[19.54±4.5 a ]]> <![CDATA[42.33%±4.8 b ]]> <![CDATA[17.33±4.5 b <!-- 5 -->]]> 2:1 <![CDATA[8.2±5.1 a ]]> <![CDATA[18.6±1.2 a ]]> <![CDATA[46.22%±5.2 a ]]> <![CDATA[22.86±1.5 a ]]> 1:3 <![CDATA[8.2±1.8 a ]]> <![CDATA[20.3±3.0 a ]]> <![CDATA[40.55±2.5 ab ]]> <![CDATA[18.55±3.2 ab ]]> 3:1 <![CDATA[9.0±2.5 a ]]> <![CDATA[17.88±4.1 a ]]> <![CDATA[43.38±6.5 ab ]]> <![CDATA[23.6±4.8 a ]]>

[0072] Note: Different lowercase letters indicate significant differences (P<0.05)

[0073] Example 4: Seed germination test

[0074] The bacterial solution was centrifuged at 10000g for 10 min, the supernatant was discarded, and the cells were collected. The cells were rinsed with sterile water and centrifuged 3 times to remove the residual culture medium. Finally, the bacterial solution was resuspended and mixed with sterile water to OD = 1.0 (about 10 8 / mL). Select seeds with full grains, soak them in 1% sodium hypochlorite solution for 10 minutes for disinfection, and rinse the seeds with sterile water 3 times, 1 minute each time. (Take 100μL of the last rinse water to apply to the plate to check whether the surface disinfection is thorough). After removing and draining, soak in the bacterial solution for 1 hour, pour out the bacterial suspension, and dry the seeds in a sterile environment. Spread 3 layers of culture dishes with 10mL sterile water (20, 50 and 100mg L -1 uranium solution) was soaked in filter paper, and the seeds were spread on it with tweezers. After spreading, another layer of soaked filter paper was covered. Ten seeds were placed on each plate, and three replicates were set for each treatment group. The plates were cultured in a light incubator at 20°C, 12h / 12h light / dark alternation, and 80% humidity.

[0075]

[0076] Where: Gt refers to the number of seeds germinated on day t, and Dt is the corresponding number of days for seeds to germinate.

[0077] Vitality Index (VI) = GI × Lr (Formula 5)

[0078] Where: Lr is the average root length

[0079] At 0, 50 and 100 mg·L -1 The results of the seed germination experiment under uranium concentration treatment are shown in Table 2. As the uranium concentration increases, the germination potential, germination rate, germination index and vitality index of Sudan grass decrease. The root length shows a significant downward trend, indicating that the root system of Sudan grass is highly sensitive to uranium. The uranium concentration is 50 mg·L -1 When the uranium concentration was 100 mg·L -1 When treated with single bacteria and compound bacteria, the root length and shoot length of the groups were significantly higher than those of the CK control group, increasing by 28.92%-43.57% and 8.36%-15.39% respectively. Among them, the M2+B6 combination of bacteria had the best effect, which had a significant promoting effect on the root system of Sudan grass under different uranium stresses and could alleviate the impact of uranium stress on the root system of Sudan grass.

[0080] Table 2: Effects of different treatments on the germination stage of Sudan grass seeds under uranium stress

[0081]

[0082] Example 5: Potted plant test

[0083] 1. Test methods

[0084] Sudan grass seeds were surface-sterilized and germinated in seedling trays (with a substrate of vermiculite: peat = 1:1). When the seedlings had 2-3 true leaves, 10 seedlings per pot were transplanted into uranium-containing soil (2.5 kg soil per pot) and 20 mg kg -1 , 50mg·kg -1 and 100 mg kg -1 Three soil U 6+ Concentration; each uranium concentration was set to four inoculation treatments: CK, M2, B6, and M2+B6, for a total of 12 different treatments, with 5 replicates for each treatment. Plants were grown in a greenhouse with a light intensity of 16 h / darkness of 8 h, 25°C, and a relative humidity of 54%. After one week of seedling acclimatization, 300 mL of the prepared 100-fold diluted bacterial solution (with a bacterial count of approximately 10 6 CFU / mL), while the control group received an equal amount of distilled water. Seven days later, the second application of bacteria was performed, followed by weekly application. Samples were collected 35 days later, and 10 plants were randomly selected from each treatment group to measure indicators such as plant height, root length, aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight.

[0085] Plant stress-resistant enzyme activities: Malondialdehyde (MDA), chlorophyll, soluble protein content, catalase (CAT), phenylalanine ammonia lyase (PAL), peroxidase (POD), and superoxide dismutase (SOD) activities were measured in leaves. Reduced glutathione (GSH) and oxidized glutathione (GSSG) activities were measured using kits provided by Suzhou Grace Biotechnology Co., Ltd.

[0086] Determination of uranium content in plants: Grind and mix the dried plants from each treatment separately, accurately weigh 300 mg of sample into a 50 mL digestion container, add 6-8 mL of aqua regia and 1 mL of hydrogen peroxide, place on a graphite hot plate and digest at 120-200°C for 20-60 minutes until the sample is completely digested, remove the acid, cool, filter, and adjust the volume. Determine by ICP-MS instrument.

[0087] Surface-sterilized Sudangrass seeds were placed in seedling trays for germination and then transplanted into round pots filled with 2.5 kg of simulated uranium-contaminated soil. Ten plants were grown per pot in a greenhouse under conditions of 16 h light / 8 h dark, 25°C, and 54% relative humidity. All potted plants were regularly watered and treated with inoculants according to the treatment settings. Samples were collected after 35 days of growth, and plant physiological parameters were measured. Five replicates were set for each treatment group.

[0088] 2. Experimental Results

[0089] (1) Effects of microbial agents on physiological indicators of Sudan grass

[0090] At 20, 50 and 100 mg kg -1 Table 3 shows the effects of inoculants M2 and B6 on the growth of sudangrass under uranium stress. With the exception of plant height, all other growth indicators of the treated plants were higher than those of the control. Root length, leaf fresh-dry weight, and root fresh-dry weight of sudangrass significantly increased (P < 0.05) compared to the control under different uranium concentrations, increasing by 11.72%-23.53%, 22.38%-35.40%, 19.58%-53.60%, 21.74%-52.17%, 15.89%-21.52%, and 18.78%-53.93%, respectively. The M2+B6 treatment had the greatest combined effect, indicating that both M2 and B6 alone and the M2+B6 combination can alleviate the stress induced by uranium on sudangrass growth and promote its growth.

[0091] Table 3: Effects of different microbial agents on Sudan grass growth under uranium stress

[0092]

[0093] (2) Effects of microbial agents on enzyme activity of Sudangrass under uranium stress

[0094] See attached Figure 1 As shown, without the application of microbial inoculants, as uranium stress concentration increased, the levels of GSH, POD, SOD, and soluble protein generally decreased, while the plant-toxic MDA content continued to increase. Under the same uranium stress, the enzyme activity of sudangrass treated with microbial inoculants was higher in all treatment groups than in the control group. The M2+B6 treatment combination had the best overall effect. CAT, GSH, and GSH / GSSG were significantly higher than those in the control group under different stresses (P < 0.05), increasing by 6.36%-22.17%, 7.69%-78.61%, and 39.05%-60.96%, respectively. MDA content was significantly lower than that in the control group under different stresses (P < 0.05), decreasing by 17.29%-40.23%. Microbial inoculants can positively affect the activities of most enzymes during sudangrass growth, and their positive effects on enzyme activity became more pronounced with increasing uranium stress.

[0095] (3) Effect of microbial agents on the enrichment of Sudan uranium

[0096] As the concentration of soil uranium stress increased, the uranium content in both the aboveground and root parts of Sudan grass showed a significant dose-dependent increase, indicating that the accumulation of uranium in the plant body was positively correlated with the level of external pollution. It is worth noting that the uranium content in the aboveground parts of each treatment group was significantly lower than that in the roots. -1 Under uranium stress conditions, the inoculation of microorganisms increased the uranium transfer coefficient from roots to aboveground parts, indicating that the microorganisms promoted uranium migration. -1 In the treatment group, although the uranium concentration in the roots of the treatment group was not significantly different from that of the CK group, the total uranium enrichment in the treatment group was significantly higher than that in the control group by promoting the growth of root biomass (p<0.05). -1 When the uranium concentration in the roots of the strain treatment group increased by 49.66%-83.46% compared with the CK group, among which the M2+B6 composite bacterial agent treatment showed the strongest enrichment effect (p<0.01). -1 ), the uranium accumulation in the roots of the M2 single bacteria treatment was significantly higher than that of the B6 and composite treatment groups (p<0.05).

[0097] Table 4: Effects of different bacterial agents on uranium enrichment in Sudan grass under uranium stress

[0098]

[0099] Note: Different lowercase letters indicate significant differences between different treatments at the same concentration (p < 0.05); Different uppercase letters indicate significant differences between different concentrations at the same treatment (p < 0.05)

[0100] Example 6: In situ repair experiment

[0101] 1. Test methods

[0102] (1) Field trial design

[0103] A contaminated farmland 10 kilometers away from a uranium tailings mine in Renhua County, Shaoguan City, Guangdong Province is used as a remediation site. Renhua County is located in the northern part of Guangdong Province, at the junction of Guangdong, Hunan and Jiangxi provinces, and at the southern foot of the Nanling Mountains. Its geographical coordinates are 24°57′38″-25°04′36″ north latitude and 113°39′53″-113°46′57″ east longitude. The terrain is mainly mountainous and hilly (accounting for about 90%), with complex landforms and typical Danxia landforms. The region has a mid-subtropical monsoon climate with an average annual temperature of 19.7°C and an annual precipitation of 1858mm. It is cold in winter and spring and hot in summer and autumn. The temperature difference between day and night is significant, and the autumn temperature is higher than the spring temperature.

[0104] Field trials using strains M2 and B6 for soil heavy metal U remediation began in late April 2024. Based on preliminary laboratory work, four treatments were designed: 1) CK treatment (blank control, without any microbial addition); 2) M2 treatment; 3) B6 treatment; and 4) combined treatment. Each treatment consisted of three replicates, resulting in six plots measuring 3 m x 6 m. Sudan grass seeds were sown evenly within the rows, with 20 cm spacing between rows. Treatments were separated by 30 cm-wide drainage ditches or protective rows. The microbial agent was diluted 100:1 and evenly watered near the rhizosphere of each plant, ensuring that at least 100 mL was applied to each plant. A control group received the same amount of water. Microbial agents were applied every two weeks after planting, and plants were harvested on the 120th day. Normal field management was maintained throughout the experiment. Samples were collected from 15 plants per replicate per treatment, for a total of 45 plants collected across the three replicates.

[0105] (2) Sampling and testing

[0106] The plant height, root length, aboveground fresh weight, underground fresh weight, and uranium content in plant tissues of the harvested plant samples were determined using the same methods as above.

[0107] Sampling points were evenly distributed using a five-point method. Soil samples were collected layer by layer at a depth of 20 cm using a stainless steel soil augers. Immediately after collection, impurities such as rocks and roots were removed, and the soil was sealed and labeled in sterile polyethylene bags. Soil uranium content and major soil chemical properties were determined using the same methods as above. Soil enzyme activity was determined: Sucrase activity was determined using the 3,5-dinitrosalicylic acid colorimetric method, urease using the indophenol blue colorimetric method, phosphatase using the disodium phenyl phosphate colorimetric method, and catalase using ultraviolet spectrophotometry, using kits provided by Suzhou Gres Biotechnology Co., Ltd.

[0108] To collect rhizosphere soil from plants, fine roots from healthy plants were first selected. A small hoe was used to obtain a root-soil mixture. Non-rhizosphere soil was gently shaken off, retaining approximately 1 mm of rhizosphere soil adhering to the root surface. The mixture was placed in a sterile sampling bag and stored at low temperatures. Total microbial DNA was extracted and purified from 0.5 g of soil using a soil DNA kit (Omega Bio-Tek, USA). The quality and concentration of the extracted DNA were determined using NanoDrop and agarose gel electrophoresis. PCR amplification was performed using 1 ng / L DNA as a template using primers 341F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') targeting the 16S rRNA V3-V4 region. Sequencing was commissioned by Beijing Novogene Technology Co., Ltd. QIIME (Quantitative Insights into Microbial Ecology version 2) was used to filter, trim, and remove detected chimeric sequences from the raw data. First, representative reads of each amplicon sequence variant (ASV) were selected using the QIIME2 platform. Second, a set of representative reads were annotated and aligned with the Silva database using the default configuration of q2-feature-classifier. Alpha and beta diversity indices were calculated. R language (Version 4.2.2) was used for species relative analysis and significance test plotting at the phylum and genus levels, RDA analysis and plotting, and Bray-Curtis distance algorithm was used for NMDS (non-metric multi-dimensional scaling) analysis and LEfSe analysis. The screening value of LDAScore was 3.

[0109] 2. Test results

[0110] (1) Effects of microbial agents on the growth of Sudan grass

[0111] Except for plant height, all other growth indicators of the plants treated with the fungus agent were significantly higher than those of the control group (P<0.05). Figure 2As shown in the results, the plant height, root length, aboveground weight, and underground weight of the inoculant-treated groups increased by 11.85%-23.59%, 6.22%-10.81%, 43.57%-50.62%, and 18.52%-42.59%, respectively, compared with the control group. The M2+B6 treatment group showed the best effect, with plant height, root length, aboveground weight, and underground weight increased by 23.59%, 10.81%, 50.62%, and 42.59%, respectively, compared with the control group. The aboveground weight and underground weight of the inoculant-treated groups increased by 3.31%-50.41% and 11.11%-36.11%, respectively, compared with the control group. These results indicate that both the M2 and B6 single inoculants and the M2+B6 combined inoculant can promote the growth of Sudangrass under uranium stress in the field.

[0112] (2) Effect of microbial agents on the accumulation efficiency of Sudan uranium

[0113] In the field, uranium concentrations in the aboveground parts of Sudangrass were low, with uranium primarily accumulating in its roots. The effects of different treatments on soil uranium concentrations and plant accumulation in the experimental plots are shown in Table 5. In the control (CK) field, soil uranium concentrations were 21.2 mg / kg before treatment, which decreased to 18.93 mg / kg after treatment, with a removal rate of 10.31%. Uranium concentrations in the aboveground parts of Sudangrass were 0.1 mg / kg, and in the underground parts were 3.68 mg / kg. In the M2 field, soil uranium concentrations were 20 mg / kg before treatment, which decreased to 16.9 mg / kg after treatment, with a removal rate of 15.36%. Uranium concentrations in the underground parts of plants reached 4.21 mg / kg. In the B6 field, soil uranium concentrations were higher, at 24.53 mg / kg before treatment, but decreased to 20.1 mg / kg after treatment, with a removal rate of 17.45%. Uranium concentrations in the underground parts of plants were 3.91 mg / kg. The M2+B6 treatment group showed the most significant effect, reducing soil uranium concentration from 23.65 mg / kg to 18.8 mg / kg, achieving a removal rate of 20.19%. Uranium concentrations in the plant's underground compartments rose to 4.77 mg / kg, while those in the aboveground compartments reached 0.18 mg / kg, both of which were the highest among the treatment groups. These results indicate that different bacterial agents play a positive role in enriching uranium in the soil, enhancing the remediation efficiency of Sudangrass.

[0114] Table 5: Uranium content in plants above and below ground and in soil before and after treatment in different treatment groups

[0115]

[0116] Note: Different lowercase letters indicate significant differences (P<0.05)

[0117] (3) Effects of microbial agents on the physical and chemical properties and enzyme activities of Sudangrass rhizosphere soil

[0118] Soil enzyme activity is involved in various biochemical processes within the soil, such as the decomposition and synthesis of humus; the decomposition of plant and animal debris and microbial residues, and the hydrolysis and transformation of the organic compounds they synthesize; and the oxidation and reduction reactions of certain inorganic compounds. Soil enzyme activity generally reflects the relative intensity of biochemical processes within a given soil ecological context. Measuring the activity of corresponding enzymes provides an indirect understanding of the transformation of specific substances within the soil.

[0119] Table 6 shows the significant effects of different microbial treatments on enzyme activities in the rhizosphere soil of Sudangrass. The combined M2+B6 treatment group exhibited the highest performance in acid phosphatase and catalase activities, increasing by 8.9% and 16.1%, respectively, compared to the control group. The M2 treatment group significantly increased glucosidase and FDA hydrolase activities by 42.5% and 70.3%, respectively, with significant differences compared to the control group (P < 0.05). The B6 treatment group ranked first in sucrase activity, increasing by 22.4% compared to the control group. Furthermore, the urease activities of the single microbial treatments (M2 and B6) were 317.89 μg / d / g and 301.08 μg / d / g, respectively, both higher than the control group's 279.83 μg / d / g, although no statistical differences were observed between treatment groups. Notably, the M2+B6 combination treatment significantly improved the activities of multiple enzymes: glucosidase increased by 42.5%, acid phosphatase by 8.9%, sucrase by 16.1%, catalase by 21.0%, and FDA hydrolase by 63.0%. In contrast, while single-agent treatments (M2 and B6) also improved the activities of certain enzymes to varying degrees, the overall effect was inferior to the M2+B6 combination.

[0120] Table 6: Effects of different microbial agents on soil enzyme activities in Sudan grass root system

[0121]

[0122]

[0123] Note: Different lowercase letters indicate significant differences (P<0.05)

[0124] (4) Effects of microbial agents on the rhizosphere microbial community of Sudan grass

[0125] The ASV list was obtained by extracting total DNA from the rhizosphere soil of Sudan grass and performing high-throughput sequencing. Figure 3As shown in the figure, 1443 bacterial ASVs were shared across treatment groups. The M2+B6 and M2 groups had more unique ASVs, while the B6 and M2 groups shared more ASVs. These results indicate significant overlap and differences in microbial community structure among the M2+B6, B6, M2, and CK treatment groups, reflecting the impact of different treatments on microbial community composition.

[0126] Alpha diversity analysis showed that the sequencing coverage of all treatment groups was higher than 0.99, indicating that the microbial community data were highly representative. There were no significant differences in species richness (chao1, observed_features) and sequencing depth (goods_coverage) between the treatment and control groups (P>0.05). However, the chao1 index (M2 group 2822.1±143.65 vs. CK group 2689.22±93.61) and observed_features (M2 group 2720±147.16 vs. CK group 2593±88.84) of the treatment groups showed an upward trend. In terms of community evenness (pielou_e) and diversity (shannon), the single-bacteria and composite-bacteria treatment groups were significantly higher than the control group (P<0.05). The results of non-metric multidimensional scaling (NMDS) analysis are shown in the appendix. Figure 4 The results showed that the bacterial community structures of the 12 Sudangrass rhizosphere soil samples were significantly different (stress = 0.0771, P = 0.001). The model fit was good, and the differences between the groups were statistically significant. There was no overlap in the ordination plot for all treatment group samples, indicating that the different treatments significantly altered the composition of the rhizosphere microbial community. Among them, sample T2 from the B6 group and sample T3 from the M2 group had the closest planar distance, indicating that although the two groups were significantly different, they may share some functional groups or be regulated by similar environmental factors. The control group (T4) was distributed farther away from each treatment group (T1, T2, and T3), further confirming that the application of microbial agents (M2, B6, and their combined treatments) affected the composition of the Sudangrass rhizosphere soil bacterial community.

[0127] The bacterial community at the phylum level in the rhizosphere soil of Sudan grass was analyzed in the Appendix. Figure 5As shown in A, the results show that Proteobacteria and Actinobacteriota are the core microbial communities in the rhizosphere soil of Sudangrass, and their dynamic changes are closely related to the treatment with the inoculant. Specifically, the relative abundance of Proteobacteria in the single inoculant treatment groups (M2, B6) increased significantly, but in the M2+B6 treatment group, the proportion of Actinobacteria exceeded that of Proteobacteria. In addition, the abundance of Gemmatimonadota in the treatment group was significantly increased compared with the CK group (52.38%-200%), especially in the M2+B6 group, reaching a peak. At the same time, the relative abundance of Acidobacteriota in the M2+B6 group decreased significantly, which may be directly related to the increase in pH caused by the inoculant treatment. In addition, it was found that the abundance of Chloroflexi and Cyanobacteria fluctuated differently with the type of microbial agent. For example, the proportion of Cyanobacteria increased in the B6 group, which may be due to its nitrogen fixation ability and the cooperation of plant root secretions to optimize nitrogen utilization efficiency. Figure 5 As shown in Figure B, the relative distribution ranged from 22.78% to 29.46%. Chujiaibacter and Rhodanobacter from the Proteobacteria phylum, and Sphingomonas from the Bacteroidetes phylum, dominated the control group. However, the proportion of Chujiaibacter decreased significantly across the different treatments, while the proportion of Streptomyces increased by 26.31% to 115.78%.

[0128] LDA value distribution histogram in the attached figure Figure 6 A shows species with LDAscore greater than the set value of 3, i.e., biomarkers with statistical differences between groups. The length of the bar graph represents the effect size of the different species (i.e., LDA score). Figure 6In Figure B, the circles radiating from the inside out represent the taxonomic levels from phylum to genus (or species). Each small circle at a different taxonomic level represents a taxon at that level, and the diameter of the small circle is proportional to the relative abundance. Species with no significant differences are uniformly colored yellow, and the biomarkers of differentially expressed species are colored according to the group. Red nodes indicate microbial groups that play an important role in the red group, and green nodes indicate microbial groups that play an important role in the green group. The M2+B6 group contains a total of 19 microbial species, including the Nitrospiraceae and Solibacteraceae families of the Gemmatimonadaceae phylum, and the Nitrosomonadaceae family of the Acidobacteria phylum. These microorganisms play an important role in nitrogen cycling and soil ecology, for example, participating in ammonia oxidation and nitrification. In addition, the M2+B6 group also includes some microorganisms related to plant rhizosphere ecology, such as Bryobacteraceae and Xanthobacteraceae, which may play a role in promoting plant growth and decomposing soil organic matter. The B6 group has a total of 17 microorganisms, covering multiple phyla such as Proteobacteria and Firmicutes. Among them, microorganisms of the Pseudomonadales and Bacillales orders have excellent environmental adaptability and metabolic diversity, and are able to degrade a variety of organic pollutants and survive in extreme environments. The M2 group has a total of 8 microorganisms, mainly concentrated in the Beijerinckiaceae, Acetobacteraceae, and Pseudomonadaceae families. These microorganisms possess unique advantages in organic acid production and environmental adaptation. For example, members of the Bezierinaceae family can fix nitrogen and grow in poor soils, while members of the Pseudomonadaceae family are known for their broad metabolic capabilities and environmental adaptability, playing an important role in a variety of ecological environments, including soil, water, and plant surfaces. The CK group contained a total of 13 microbial species, and as a control group, it included a variety of microorganisms similar to those in the T1 group, such as the Solibacteraceae and Xanthobacteraceae families. In addition, the CK group also contained some unique microbial species, such as the genera Chujiaibacter and Devosia.

[0129] Redundancy analysis (RDA) was used to analyze the relationship between the top 30 bacterial genera and environmental factors. Figure 7As shown, the main drivers of changes in microbial community structure are TN and organic matter, with changes in TN content having the greatest impact and being the primary factor influencing soil bacterial communities. Eleven bacterial genera were positively correlated with TN, including Bryobacter, Sphingomonas, and Gemmatimona. Seven bacterial genera were positively correlated with organic matter, including Mycobacterium and Streptomyces. These genera were primarily found in the M2+B6 treatment group and the M2 and B6 treatment groups.

[0130] Based on the above description, it can be seen that the present application provides a composite microbial agent capable of exerting a synergistic effect between Bacillus cereus B6 and Microvirga sp. M2, and its application in combination with Sudan grass in the remediation of uranium-contaminated soil. The experimental results described in the above examples show that:

[0131] In terms of the protective effect of uranium on seed germination under uranium stress, the composite bacterial agent can better promote the root and shoot length of seedlings under uranium stress, which is significantly higher than that of the untreated control group, and the comprehensive effect is better than that of the single bacterial treatment. In the absence of uranium, the germination rate, germination potential, and germination index of the composite bacterial agent treatment are the highest (significant), and the vitality index, root length and shoot length index are second only to the B6 treatment group (no significant difference); 50mg L -1 and 100 mg L -1 Under different uranium concentrations, the M2 treatment performed best in germination rate, germination potential, germination index and root length, the B6 treatment was best in promoting bud growth, and the composite bacterial agent treatment achieved better comprehensive indicators.

[0132] Similarly, in the potted plant experiment simulating uranium contaminated soil, compared with the single bacteria treatment group, the composite bacterial agent treatment also achieved better comprehensive plant height, root length, and dry and fresh weight growth indicators, and was significantly higher than the untreated control group. -1 Under the soil uranium concentration, the composite bacterial agent treatment significantly promoted the enrichment of uranium by roots and promoted the transport of uranium; 100mgL -1 Under the soil uranium concentration, B6 promoted transport, M2 promoted enrichment, and the composite bacterial agent showed a comprehensive effect.

[0133] In addition, field trials more realistically reflect the application effects of single bacteria and microbial agents in actual contaminated sites. Not only was the remediation efficiency of the microbial agent-Sudangrass combined remediation system evaluated, but ecological indicators such as soil enzyme activity and microbial communities were also measured. Microbial agents also promoted the ecological restoration process of the soil. In the field, mixed bacteria treatments produced the highest biomass, and plants also had the best uranium enrichment and transport effects. Compared with single bacteria, in complex field environments, composite microbial agents performed the best and were more stable in promoting growth and enhancing uranium removal. Composite microbial agents also showed a good overall effect.

[0134] The above embodiments are only for illustrating the technical concept and features of the present invention in a specific scenario. Its purpose is to enable people who need this technology to understand the content of the present invention and implement it. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A composite microbial agent for remediation of uranium-contaminated soil, characterized in that: The composite microbial agent comprises: Microvirga sp. M2 fermentation broth and Bacillus cereus B6 fermentation broth mixed in a volume ratio of (1-3): (3-1).

2. The composite microbial agent according to claim 1, wherein The composite microbial agent is obtained by mixing Microvirga sp. M2 fermentation broth and Bacillus cereus B6 fermentation broth in a volume ratio of 1:

1.

3. The composite microbial agent according to claim 1, wherein The fermentation broth of Microvirga sp. M2 is prepared by inserting a single colony of Microvirga sp. M2 into a sterilized NB liquid culture medium at 37°C and 150 r·min. -1 The culture was shaken for 24 h and the bacterial concentration was adjusted to OD600 of 1.

0.

4. The composite microbial agent according to claim 1, wherein The Bacillus cereus B6 fermentation broth is prepared by inoculating a single colony of Bacillus cereus B6 into a sterilized NB liquid culture medium at 37°C and 150 r·min. -1 The culture was shaken for 24 h and the bacterial concentration was adjusted to OD600 of 1.

0.

5. Use of the composite microbial agent for remediation of uranium-contaminated soil according to any one of claims 1 to 4 in remediation of uranium-contaminated soil.

6. The use according to claim 5, characterized in that The application method comprises planting Sudan grass in uranium-contaminated soil and applying a composite microbial agent to treat the uranium-contaminated soil.

7. The use according to claim 6, characterized in that In the application method, the composite microbial agent is centrifuged at 10000g for 10 minutes, the supernatant is discarded, and the bacteria are collected; the bacteria are rinsed with sterile water and centrifuged three times to remove the residual culture medium; and finally the bacteria liquid is resuspended and mixed with sterile water to OD = 1.0, about 10 8 Use per mL.

8. The use according to claim 6, wherein: In the application method, Sudan grass seeds with full grains are selected, the seeds are immersed in a 1% sodium hypochlorite solution for 10 minutes for disinfection, the seeds are rinsed with sterile water three times, each time for 1 minute, the seeds are taken out and drained, and then immersed in a composite bacterial agent suspension for 1 hour, the bacterial suspension is poured out, the seeds are dried in a sterile environment, and then planted.

9. The use according to claim 6, characterized in that In the application method, the composite microbial agent is diluted at a ratio of 100:1, and then the bacterial solution is evenly poured near the rhizosphere of each Sudan grass plant, and the application amount for each plant is ensured to be no less than 100 mL.

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