Application of new mycobacterium spp. Strain and sudangrass in combined remediation of uranium-contaminated soil

Abstract

The invention discloses an application of a novel mycobacterium spp. Strain and sudangrass in combined remediation of uranium-contaminated soil. According to the invention, a new strain Microvirga sp.M2 is separated from a sandy soil sample of a tailing dam in the southwest region, and the bacterial colony of the new strain Microvirga sp.M2 is light pink, round, glossy in surface, convex in middle, smooth and tidy in edge, thin in texture and easy to emulsify on NA; the cells are rod-shaped, the ends of the cells are round, and the cells are independent, paired or short-chain-cluster-shaped, have no flagellum and do not generate spores. The micromycobacterium sp.M2 strain not only has the characteristics of nitrogen fixation, phosphorus solubilization, siderophore synthesis and IAA (Indoleacetic Acid) production, but also has good tolerance to uranium; meanwhile, the strain also has good growth promoting ability, has a better protective effect on relieving the toxicity of uranium to seed germination stress, can relieve the toxicity of uranium to plant growth, and can significantly increase the biomass of plants and the enrichment amount of uranium.

Description

Technical Field

[0001] The invention relates to the technical field of microbial soil restoration, and in particular to the technical field of application of a new strain of Microbacterium truncatum and Sudan grass in the restoration of uranium-contaminated soil. Background Art

[0002] With the urgent global demand for clean energy, nuclear power has developed rapidly as a low-carbon and efficient form of energy. However, the rapid growth of nuclear power has also brought about a sharp increase in demand for natural uranium, which has directly promoted the substantial expansion of the uranium mining and metallurgy industry. A series of operations in uranium mining, smelting and later application in mineral mining will cause soil pollution in the surrounding environment due to the large amount of industrial pollutants such as tailings and tailings water containing radioactive uranium and other heavy metals. Therefore, how to effectively repair and treat these uranium-contaminated soils has become an urgent problem to be solved in the current field of environmental protection.

[0003] Conventional remediation technologies for uranium-contaminated soil mainly use physical and chemical means to transfer and fix uranium and its compounds in the soil, reducing the migration capacity and bioavailability of uranium in the soil. However, the high cost and inevitable secondary contamination make these treatment methods difficult to promote and apply on a large scale.

[0004] Bioremediation is a treatment technology that uses naturally occurring or specially cultivated biological materials to convert toxic pollutants in the soil into non-toxic pollutants under controllable environmental conditions. Among them, phytoremediation technology has been widely accepted worldwide due to its advantages such as low cost, little impact on the environment, and no need to change the soil structure. It has become the most suitable and most promising method for treating uranium-contaminated soil.

[0005] Sudan grass is an annual herbaceous plant of the genus Sorghum in the Poaceae family. It has a well-developed root system and a strong ability to adapt to the environment. Studies have found that Sudan grass has a certain resistance to medium and low concentrations of uranium stress. Researchers have found that the combined restoration of plants and microorganisms can effectively solve the shortcomings of plant restoration, such as small biomass, poor adaptability, and low enrichment efficiency. By building a symbiotic relationship between plants and microorganisms, the advantages of both can be fully utilized to achieve efficient restoration of uranium-contaminated soil. Summary of the invention

[0006] Conventional remediation technology for uranium-contaminated soil has high costs and inevitable secondary pollution, making it difficult to promote and apply its treatment method on a large scale. The present invention aims to provide a new strain of Microvirga sp. and Sudan grass for the combined restoration of uranium-contaminated soil. The present invention isolates a new strain of Microvirga sp. M2 from sandy soil samples of uranium tailings dams in the southwest region. Its colonies are light pink, round, shiny, convex in the middle, smooth and neat edges, thin in texture, and easy to emulsify on NA; the cells are rod-shaped with round ends, single, paired or short-chain clusters, without flagella, and do not produce spores. The new strain of Microvirga sp. M2 not only has the characteristics of nitrogen fixation, phosphate solubilization, iron carrier synthesis and IAA production, but also has good tolerance to uranium. At the same time, the new strain of Microvirga sp.M2 also has good growth-promoting ability, can alleviate the toxicity of uranium to plant growth, can significantly increase the biomass of plants and the amount of uranium enrichment, and has a good protective effect on alleviating the toxicity of uranium to seed germination stress. After inoculation with the new strain of Microvirga sp.M2, the uranium content of the roots increased significantly, and the enrichment coefficient reached 1.11; indicating that the inoculation of the new strain of Microvirga sp.M2 can promote the enrichment of uranium in Sudan grass roots, and has the potential to be used as a microorganism combined with plants to repair soil uranium contamination.

[0007] In order to achieve the above technical effects, the present invention is implemented through the following technical solutions.

[0008] In a first aspect, the present invention provides a new strain Microvirga sp. M2, which is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC No: 63775, and its 16Sr RNA genome sequence is shown in SEQ ID NO: 1.

[0009] The novel strain Microvirga sp., numbered M2, provided by the present invention, is isolated from a sandy soil sample of a tailings dam in the southwest region. The strain is sequenced by 16S rRNA genome sequence and whole genome sequencing, and the sequence length is 1475bp. After sequencing, the sequence is submitted to the GenBank database, and compared and analyzed by NCBI and EzBioCloud databases. The phylogenetic tree analysis based on the 16S rRNA genome sequence shows that the novel strain Microvirga sp., numbered M2, is most closely related to strains of the genus Microvirga: M. calopogonii CCBAU 65841 T Similarity 98.65% with M.makkahensis SV1470 TSimilarity 98.58% with M. pakistanensis NCCP-1258 T The similarity is 98.44%, and the similarity with other species of the genus is 98.15%-94.11%. After a series of polyphasic classification identifications, it was determined that the strain numbered M2 is a new species of the genus Microvirga, with typical characteristics of new strains, and is tentatively named Microvirga sp.M2.

[0010] The Microvirga sp. M2 provided by the present invention is subjected to the above-mentioned well-known and recognized strain system molecular level identification in the art, combined with multiphase classification system identification analysis based on morphological identification, physiological and biochemical characteristics, etc. The Microvirga sp. M2 provided by the present invention is different from the standard model strain with the closest homology to the genus Microvirga in many aspects, and it is confirmed that the strain numbered M2 belongs to a new strain of the genus Microvirga, and it is named Microvirga sp. M2. The strain has been deposited in Guangdong Microbial Culture Collection Center, address: 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, deposit date: September 20, 2023, and deposit number: GDMCC No: 63775.

[0011] In the present invention, the growth temperature range of the Microvirga sp. M2 strain is 20° C.-45° C., the pH is 6-9, and the salinity (v / w) is 0%-1%.

[0012] The Microvirga sp. M2 strain has the characteristics of nitrogen fixation, phosphate solubilization, iron carrier synthesis and IAA production.

[0013] In a second aspect, the present invention provides a method for culturing a new strain Microvirga sp. M2: picking a single colony of the uranium-resistant Microvirga sp. M2 strain, inoculating it into 50 mL of NA liquid culture medium and shaking it at 30° C. for 24 hours as a seed solution, aspirating 5 μL of the bacterial solution and culturing the bacterial solution at a constant temperature of 30° C. in a fermentation medium by a spot plate method.

[0014] The fermentation medium is one or more of silicate bacterial medium, Ashutosh medium, Pikovakaya's medium, organophosphorus medium, CAS detection medium, skimmed milk powder medium, sodium carboxymethyl cellulose medium and bromocresol purple color development medium.

[0015] In a third aspect, the present invention provides an application of a new strain Microvirga sp. M2 in promoting the growth of Sudan grass.

[0016] In a fourth aspect, the present invention provides a method for preparing a new strain of Microvirga sp. M2 inoculant, the preparation method comprising: using NA liquid culture medium, centrifuging a bacterial solution cultured at 30°C, 150r / min constant temperature shaking for 24h, collecting the bacterial cells, washing with sterile water three times to remove the residual culture medium, and then resuspending the bacterial solution with sterile water to mix to 10 8 CFU / mL, that is, the bacterial agent is obtained.

[0017] In a fifth aspect, the present invention provides an application of a Microvirga sp. M2 bacterial agent and Sudan grass in the repair of uranium contaminated soil.

[0018] Through the above technical scheme, the present invention obtains the following technical effects:

[0019] (1) The Microvirga sp. M2 used in the present invention has been identified at the molecular level by a well-known and recognized strain system in the art. Combined with the multi-phase classification system identification analysis based on morphological identification, physiological and biochemical characteristics, the Microvirga sp. M2 provided by the present invention has multiple differences from the standard model strain with the closest homology to the Microvirga genus, confirming that the strain numbered M2 belongs to a new strain of the Microvirga genus, which not only has the characteristics of nitrogen fixation, phosphorus solubilization, iron carrier synthesis and IAA production, but also has good tolerance to uranium. At the same time, Microvirga sp. M2 also has good growth-promoting ability, can alleviate the toxicity of uranium to plant growth, can significantly increase the biomass of plants and the enrichment of uranium, and has a good protective effect on alleviating the toxicity of uranium to seed germination stress.

[0020] (2) After inoculation with Microvirga sp. M2 under 100 mg / kg uranium stress, the root length, leaf length, and dry fresh weight of Sudan grass increased significantly, and the significant differences in leaf length and dry fresh weight compared with those under non-uranium stress disappeared, indicating that Microvirga sp. M2 can alleviate the toxic effects of uranium stress on plant growth. By measuring the uranium content in plant roots and leaves, it can be seen that Sudan grass mainly relies on the root system to enrich uranium, and the root enrichment coefficient for uranium is 0.74. After inoculation with Microvirga sp. M2, the uranium content in the root increased significantly, and the enrichment coefficient reached 1.11.

[0021] (3) Under 100 mg / kg uranium stress, after inoculation with Microvirga sp. M2, the activities of antioxidant enzymes were all improved, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased significantly, the proportion of reduced glutathione increased significantly, and the MDA content decreased significantly. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown is a scanning electron micrograph of strain Microvirga sp. M2.

[0023] Figure 2 Shown is the phylogenetic tree of strain Microvirga sp. M2 constructed based on 16S rRNA sequences.

[0024] Figure 3 Shown is the growth curve of Microvirga sp.M2 under different uranium concentrations.

[0025] Figure 4 Shown is a graph showing the growth-promoting properties of Microvirga sp. M2.

[0026] Figure 5 Shown is a comparison of the growth promotion of Sudan grass by strain Microvirga sp.M2 under uranium stress.

[0027] Figure 6 The graph shows the growth-promoting effect of strain Microvirga sp.M2 on Sudan grass under uranium stress.

[0028] Figure 7 Shown is a comparison of the effects of inoculation with Microvirga sp. M2 on Sudan grass biomass, enzyme activity and uranium content under uranium stress. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. The following embodiments and features in the embodiments can be combined with each other without conflict.

[0030] The present invention uses Excel 2010 to sort the original data, and SPSS25 to perform single factor variance analysis and principal component analysis. The data in the figures and tables of this article are all mean values ​​± standard errors.

[0031] The basic culture medium used in the present invention is: NA culture medium, R2A culture medium, LB culture medium; silicate bacterial culture medium, Ashuber culture medium, Pikovakaya's culture medium, organophosphorus culture medium, CAS detection culture medium, skim milk powder culture medium, sodium carboxymethyl cellulose culture medium and bromocresol purple color development culture medium.

[0032] The uranium-containing culture medium is: add 5.0 g L of filtered sterilized uranium to the NA culture medium. -1 UO2(NO3)2 mother solution to the corresponding concentration. All culture media selected in the present invention are conventional culture media in the art.

[0033] Example 1: Morphological characteristics of Microvirga sp. M2

[0034] The present invention isolates a new strain of Microvirga sp.M2 from a sandy soil sample of a tailings dam in the southwest region, inoculates the new strain of Microvirga sp.M2 onto a NA medium, obtains a single colony by streaking the plate, and cultures the single colony at 30°C for 3 days on the NA medium. The colony morphology is shown in the attached Figure 1 As shown, the colonies are light pink, round, shiny, convex in the middle, with smooth and neat edges, thin texture, and easy to emulsify on NA. The cells are rod-shaped, 0.8-1μm wide, 1.7-2μm long, with rounded ends, and are single, paired, or in short chain clusters. T Different from the new strain Microvirga sp.M2, the cells do not have flagella.

[0035] The new strain Microvirga sp.M2 was centrifuged at 10000×g for 10 min to collect cells that entered the stationary phase, heated at 70°C for 10 min, and then re-inoculated on a fresh NA plate for a spore formation test. The cell culture of strain Microvirga sp.M2 was heat sensitive (70°C, 10 min), and no spores were observed under a microscope, indicating that Microvirga sp.M2 did not produce spores.

[0036] Example 2: Identification of Microvirga sp. M2 strain

[0037] The bacterial genome extraction kit (TIANGEN, DP302) was used to extract the strain DNA. The total DNA was used as a template and PCR was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACCTTGTTACGACTT-3'). The amplification system was 30 μL: template DNA 2 μL, primer 27F 1 μL, primer 1492R 1 μL, PCR Buffer Mix 15 μL, double distilled water 11 μL. Amplification program: 94℃4min; 94℃30s, 65℃40s, 72℃90s, 30cycles; 72℃10min. After the amplification product was qualified by 1% agarose electrophoresis, the sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd. After sequencing, the sequences were submitted to the GenBank database. After comparative analysis between the NCBI and EzBioCloud databases, the 16S rRNA sequences of strains with similar sequences were selected for download, and the phylogenetic tree was constructed using MEGA7.0 software.

[0038] After PCR amplification and sequencing, the 16S rRNA sequence of the new strain Microvirga sp.M2 was 1475bp long (GenBank No.: OR593354), and its closest relationship to the strains of Microvirga genus was: "M.calopogonii" CCBAU 65841 T Similarity 98.65% with M.makkahensis SV1470 T Similarity 98.58% with M. pakistanensis NCCP-1258 T The similarity is 98.44%, and the similarity with other species of the genus is 98.15%-94.11%. See the attached phylogenetic tree constructed based on 16S rRNA sequences. Figure 2 As shown, the new strain Microvirga sp.M2 and M.makkahensis SV1470 T 、M.pakistani NCCP-1258 T 、M.arsenatis3D203 T The strains are closely related but form a separate branch. The new strain numbered M2 is a new species of the genus Microvirga.

[0039] The genomic DNA of the strain was further extracted and sequenced using the Nanopore PromethION and Illumina NovaSeqPE150 platforms to obtain the complete genome of the new strain Microvirga sp.M2 (Genbank sequence number: CP134063-CP134065). Based on the phylogenetic tree constructed based on the genome sequence, it is most closely related to M.makkahensis and M.pakistani, forming a separate branch. According to the data in Table 1, the ANI value of the new strain Microvirga sp.M2 of Microvirga and the six closely related species in the genus Microvirga is 80.88%-84.5%, which is lower than the species threshold of 95%. The dDDH value between the new strain Microvirga sp.M2 and the six closely related species was 24.8%-29.3%, which is also far below the critical value of 70% for the species, proving that the strain numbered M2 is a new species of the genus Microvirga sp. and is named Microvirga sp.M2. The strain has been deposited in the Guangdong Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, deposit date: September 20, 2023, and deposit number: GDMCC No: 63775.

[0040] Table 1: ANI and dDDH values ​​between the genomes of strain Microvirga sp. M2 and related bacteria

[0041]

[0042] Example 3: Physiological and biochemical characteristics of Microvirga sp. M2

[0043] The physiological and biochemical characteristics of this example were determined using API 20NE, API 50CH, API ZYM and Biolog GEN III microplates. The fatty acid composition of cell membranes was determined using the MIDI fatty acid analysis system. The polar ester composition was analyzed using two-way thin layer chromatography (TLC). The cell respiration quinones were analyzed using HPLC.

[0044] Microvirga sp.M2 was positive for oxidase, lipase (C14), leucine arylaminase, acid phosphatase, β-glucuronidase, nitrate reductase, and assimilation of arabinose, while the closely related strains were weakly positive or negative.

[0045] Table 2 gives the detailed phenotypic characteristics and species description of strain Microvirga sp.M2 and its closely related species. T 、M. pakistanensis NCCP-1258 T 、M.subterranea FaiI4 T Physiological and biochemical characteristics of Microvirga sp.M2 strain: The growth temperature range is 20℃-45℃, pH is 6-9, and salinity (v / w) is 0%-1%. Cytochrome oxidase, catalase, and nitrate reduction are positive, and it hydrolyzes esculin and gelatin, does not produce H2S, and is negative for urease. Alkaline phosphatase, acid phosphatase, esterase (C4), lipid esterase (C8), naphthol-AS-BI-phosphohydrolase, leucine arylaminase, and β-Glucuronidase are positive. It can utilize 23 carbon sources: dextrin, glucosamine, mannose, D-galactose, D-trehalose, D-fructose-6-PO4, gelatin, L-alanine, L-aspartic acid, L-glutamic acid, L-glutamine, D-galacturonic acid, L-galacturonan, D-gluconic acid, D-glucuronic acid, glucuronamide, mucic acid, D-glucaric acid, L-malic acid, Tween 40, β-hydroxy-D,Lbutyric acid, pyruvic acid, acetic acid, and can utilize L-arabinose, D-arabinose, ribose, L-xylose, D-xylose, ribitol, galactose, glucose, mannose, rhamnose, esculin, D-lyxose, and D-trehalose to produce acid. The main respiratory quinone is Q10, and the main fatty acid components are, Summed Feature 8, C 19:0 cycloω8c,C 16:0 and C 18:0 It is consistent with several other related bacteria, but there are differences in the content.

[0046] Table 2: Physiological and biochemical characteristics of strain Microvirga sp.M2.

[0047]

[0048]

[0049] In the table: 1. Microvirga sp.M2; 2. M.makkahensis SV1470T ;3.M.pakistanensis NCCP-1258 T ; 4. M.subterranea FaiI4 T .Note: All data were obtained under the same conditions. +, positive; -, negative; w, weakly positive. & Data from API 20E; # Data from ZYM; * Data from API 50CH. The four strains were positive for aescin, gelatin, alkaline phosphatase, esterase (C4), lipid esterase (C8), and Naphthol-AS-BI-phosphohydrolase. The four strains were able to utilize L-arabinose, D-xylose, Esculin, D-glucose, and D-fucose to produce acid. Summed feature 8 by C 18:1 ω7c and / or C 18:1 ω6c composition.

[0050] Example 4: Detection of uranium resistance of strain Microvirga sp. M2

[0051] Preparation of OD 600 The seed culture solution with a concentration of 1.0 was inoculated into uranium-containing medium with uranium concentrations of 0, 100, 200, 400, 600, and 800 mg L -1 The uranium-containing medium was inoculated at 2%. Each treatment was repeated 3 times. The culture was kept at 30°C for 48 hours. The blank medium was used as a control. The absorbance of the bacterial solution at 600 nm was measured every 2 hours. The time was used as the horizontal axis, and the absorbance OD was used as the horizontal axis. 600 The value is the vertical axis, and the strain is plotted at 0-800 mg mL -1 Growth curves at different uranium concentrations.

[0052] Uranium standard curve: Take 300 μL of uninoculated culture medium with different uranium concentrations, add 0.5 mL HCl, 1.0 mL ETA, and 2.0 mL arsenazo in sequence, dilute to 10.0 mL with distilled water, and mix well as the test solution. Take 200 μL of the test solution and measure the OD 652 , with uranium concentration as the horizontal axis, OD 652 As the ordinate, draw a standard curve.

[0053] Determination of residual uranium concentration in bacterial solution: After 48h of shaking culture, the bacterial solution was heated at 10000r / min. -1 Centrifuge for 5 minutes, take 300 μL of supernatant, and measure the absorbance at 652 nm according to the standard curve to determine the remaining uranium concentration in the bacterial solution.

[0054] The obtained data were used to calculate the adsorption rate:

[0055]

[0056] The growth curve of strain Microvirga sp.M2 in medium containing different concentrations was determined. The results are shown in the attached Figure 3 As shown, when the uranium concentration is between 0 and 600 mg L -1 The growth curve of Microvirga sp.M2 was S-shaped, 100L -1 and 200 mg L -1 Uranium has a promoting effect on the growth of the strain; when the uranium concentration of the culture medium is 400 mg L -1 The growth was slightly inhibited, the delay period increased, and the bacterial count decreased after entering the stable period; the uranium concentration in the culture medium exceeded 700 mg L -1 However, by measuring the remaining uranium concentration in the supernatant of the fermentation broth, it was found that the uranium concentration in the culture medium did not decrease, and the strain Microvirga sp.M2 had no adsorption or absorption effect on the uranium in the culture medium.

[0057] Example 5: Determination of the growth-promoting properties of strain Microvirga sp. M2

[0058] A single colony of the uranium-resistant Microvirga sp.M2 strain was picked and inoculated into 50 mL NA liquid medium and shaken at 30°C for 24 hours as a seed solution. About 5 μL of bacterial solution was aspirated and plated to silicate bacterial culture medium, Asubei culture medium, Pikovakaya's culture medium, organophosphorus culture medium, CAS detection culture medium, skim milk powder culture medium, sodium carboxymethyl cellulose culture medium and bromocresol purple color development culture medium, and cultured at 30°C to observe whether a transparent circle is produced around the colony. 100 μL of bacterial suspension was mixed with 200 μL Salkowski colorimetric solution on a white ceramic plate, and the culture solution without bacterial solution was used as a blank control. The white ceramic plate was placed in a dark place at room temperature for 30 minutes to observe whether the color turned red. If the color turned red, it means that the strain has the ability to produce IAA, and the darker the color, the stronger the ability to produce IAA.

[0059] The results are shown in the attached Figure 4The strain Microvirga sp.M2 can grow well on the Axubei medium, indicating that it has the ability to fix nitrogen; it can produce transparent hydrolysis circles on inorganic phosphorus medium (PKO) and organic phosphorus medium plates, indicating that it has the ability to solubilize phosphorus; it produces transparent hydrolysis circles on CAS detection medium, indicating that the strain can synthesize iron carriers. After the bacterial suspension is mixed with the Salkowski colorimetric solution, it presents a light pink color, indicating that it can produce IAA. The strain Microvirga sp.M2 has the characteristics of nitrogen fixation, phosphorus solubilization, iron carrier synthesis and IAA production, and has good potential to promote plant growth.

[0060] Example 6: Uranium-resistant growth-promoting assay of strain Microvirga sp. M2

[0061] The NA liquid medium was used to culture the bacterial solution at 30°C and 150 r / min constant temperature for 24 h, and the bacterial cells were collected by centrifugation. The residual medium was removed by washing with sterile water for 3 times, and the bacterial solution was resuspended and mixed with sterile water until OD = 1.0 (about 10 8 CFU / mL) and used as bacterial solution for later use.

[0062] Select Sudan grass seeds with full grains, disinfect them with 1% (m / V) sodium hypochlorite solution for 10 minutes, rinse the seeds with sterile water 3 times, 1 minute each time, and then soak them in sterile water for 7 hours. After taking them out and draining, soak them in bacterial solution for 2 hours, dry them in a sterile environment and set them aside. The filter paper germination bed method was used for seed germination. Under heavy metal uranium stress, uranyl nitrate was prepared into 3 concentrations of 0, 50, and 100 mg / L. Seeds without bacterial solution treatment were used as controls. Three replicates were set for each treatment. 15 seeds were placed in each culture dish and cultured in a light incubator at 20°C, 12h / 12h light and dark alternation, and 80% humidity for 7 days. After 7 days, the germination rate was counted, the root length and shoot length were measured, and the germination potential, germination index, and vitality index were calculated. The calculation formulas are shown in (1)-(4).

[0063]

[0064] In the formula: Gt refers to the number of seeds germinated on day t, and Dt is the corresponding number of germination days for seeds.

[0065] Vitality index (VI) = GI × S (4)

[0066] Where: S is the fresh weight of seedlings on the specified date.

[0067] Take the seeds with full grains, place them in the seedling tray for germination and seedling raising, and transplant them into a round flower pot with an inner diameter of 20 cm and a height of 14 cm after the seedlings grow the fourth true leaf. The flower pot is pre-filled with 2 kg of 100 mg kg -1After one week of seedling acclimatization, 300 mL of the prepared 100-fold diluted bacterial solution was applied, and the bacterial count was about 10 6 cfu mL -1 , the control group was given an equal amount of distilled water. Apply an equal amount of water every day according to the situation to maintain a certain moisture content in the soil. The second application of bacteria was carried out 7 days later, and the bacterial solution was applied 4 times in total. After 35 days, the plant height, root length, stem thickness, number of leaves, leaf width, leaf length, aboveground fresh weight, and underground fresh weight were measured. The plant samples were placed in an oven, fixed at 105°C for 0.5h, and then dried at 80°C to constant weight, and the aboveground dry weight and underground dry weight were measured.

[0068] Fresh leaves from plants at the same growth stage were taken from each treatment group: the activity of phenylalanine ammonia lyase (PAL) was determined by ammonium molybdate colorimetry; the superoxide dismutase (SOD) was determined by nitroblue tetrazolium (NBT) method; the peroxidase (POD) activity was determined by guaiacol method; the catalase (CAT) activity was determined by ammonium molybdate colorimetry; the polyphenol oxidase (PPO) activity was determined by pyrogallol colorimetry; the malondialdehyde (MDA) content was determined by thiobarbituric acid (TBA) method; the reduced glutathione (GSH) and oxidized glutathione (GSSG) contents were determined by spectrophotometry, and the ratio of reduced to oxidized glutathione (GSH / GSSG) was calculated.

[0069] The dried plant samples were ground and mixed respectively, and 300 mg of the sample was accurately weighed into a 50 mL digestion container, 6-8 mL of aqua regia and 1 mL of hydrogen peroxide were added. The sample was placed on a graphite heating plate and digested at 120-200 ° C for 20-60 min until the sample was completely digested. The acid was removed, and the sample was filtered after cooling and the volume was fixed. The uranium content was determined using an ICP-MS instrument.

[0070] Example 7: Protective and promoting effects of Microvirga sp. M2 on germination of Sudan grass under uranium stress

[0071] As can be seen from Table 3, in the uninoculated CK group, under the stress of 50 mg / kg uranium, the root length, shoot length, germination index and vitality index of Sudan grass seeds after germination were reduced, and the root length and vitality index decreased significantly; under the stress of 100 mg / kg uranium, the root length, shoot length, germination index and vitality index of Sudan grass were significantly reduced; indicating that 50 mg / kg and 100 mg / kg of uranium had a significant toxic effect on the germination of Sudan grass seeds. Despite this, there was no significant difference in germination potential and germination rate under uranium stress. It can be seen that 50-100 mg / kg uranium cannot inhibit the germination of seeds, but has a significant toxic effect on the seedlings after germination.

[0072] After inoculation with Microvirga sp.M2, the germination indexes of Sudan grass were all improved compared with those of the uninoculated CK group under the same uranium concentration; under non-uranium stress and uranium stress, the vitality index of Sudan grass seeds and the root length of seedlings were significantly improved after inoculation with Microvirga sp.M2. From the perspective of germination index, the root and vitality index of new seedlings showed the strongest response to uranium stress. These two sensitive indexes were significantly improved after inoculation with Microvirga sp.M2, indicating that Microvirga sp.M2 has a good protective effect on alleviating the toxicity of uranium to seed germination.

[0073] Table 3: Effects of strain Microvirga sp.M2 on germination of Sudan grass seeds under uranium stress

[0074]

[0075] Example 8: Effect of Microvirga sp. M2 on the growth and uranium enrichment of Sudan grass under uranium stress

[0076] The results are shown in the attached Figure 5 , when inoculated with Microvirga sp.M2 under non-uranium stress, the root length, leaf length and fresh weight of Sudan grass increased significantly, and the dry weight also increased but did not reach a significant level, indicating that Microvirga sp.M2 can promote the growth of Sudan grass under non-uranium stress. Under 100 mg / kg uranium stress, the root length, leaf length, and dry and fresh weight of Sudan grass that was not inoculated with bacterial solution decreased significantly, and its growth was inhibited. After inoculation with Microvirga sp.M2 under 100 mg / kg uranium stress, the root length, leaf length, and dry and fresh weight of Sudan grass increased significantly, and the significant differences in leaf length and dry and fresh weight compared with non-uranium stress disappeared, indicating that Microvirga sp.M2 can alleviate the toxic effect of uranium stress on plant growth. The uranium content in plant roots and leaves was determined, and the results are shown in the attached Figure 6 It can be seen that Sudan grass mainly relies on its root system to enrich uranium, and the root enrichment coefficient of uranium is 0.74. After inoculation with Microvirga sp.M2, the uranium content in the roots increased significantly, and the enrichment coefficient reached 1.11. In contrast, after inoculation with Microvirga sp.M2, the uranium content in Sudan grass leaves also increased, and the uranium transfer coefficient was 0.03. This shows that inoculation with Microvirga sp.M2 can promote the enrichment of uranium in Sudan grass roots.

[0077] Under the stress of heavy metals such as uranium, plants may accumulate a large number of reactive oxygen free radicals in their bodies, leading to oxidative stress. In the oxidative stress response, the antioxidant enzyme system plays a key role, such as superoxide dismutase (SOD) to remove excess superoxide anions in cells, peroxidase (POD) and catalase (CAT) to decompose hydrogen peroxide, and phenylalanine ammonia lyase. PAL, as a key enzyme in the metabolism of phenylpropanoids in plant cells, regulates plant resistance and growth and development. Reduced glutathione (GSH), as an important antioxidant sulfhydryl substance in cells, plays an important role in cell antioxidant and amino acid transmembrane transport, and has a weakening effect on uranium toxicity. The ratio of reduced to oxidized forms (GSH / GSSG) can better reflect the redox state of plant cells. At the same time, glutathione can chelate with uranium (VI) through carboxyl groups to form insoluble substances, significantly reducing uranium toxicity. MDA is caused by membrane lipid peroxidation when plants fail to clear free radicals in their bodies in time, and can be used to measure the severity of stress damage to plants.

[0078] The results are shown in the attached Figure 7 Under uranium stress, the overall activity of the antioxidant enzyme system of Sudan grass decreased, and the activities of phenylalanine ammonia lyase (PAL), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO) decreased significantly; the proportion of oxidized peptide glutathione (GSSG) and the content of malondialdehyde (MDA) increased significantly, showing the toxic effect of uranium on plants. Under non-uranium stress, the antioxidant enzyme activities of Sudan grass did not change significantly when inoculated with Microvirga sp.M2 strain. In comparison, under 100 mg / kg uranium stress, after inoculation with Microvirga sp. M2, the activities of antioxidant enzymes were all improved, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased significantly, the proportion of reduced glutathione increased significantly, and the MDA content decreased significantly; the antioxidant enzyme activity of Sudan grass after inoculation with Microvirga sp. M2 was no longer different from that of Sudan grass growing normally under non-uranium stress, indicating that the inoculation with Microvirga sp. M2 strain eliminated and alleviated the toxicity of uranium to plants by increasing the activity of antioxidant enzymes and reducing the contents of GSSG and MDA.

[0079] The above-described embodiments are part of the embodiments of the present invention, but not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but rather represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A strain of Microvirga sp.M2, characterized in that: The strain is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 63775, and its 16S rRNA genome sequence is shown in SEQ ID NO:

1.

2. A strain of Microvirga sp. M2 according to claim 1, characterized in that: The growth temperature range of the Microvirga sp. M2 strain is 20° C. to 45° C., the pH is 6 to 9, and the salinity (v / w) is 0% to 1%.

3. A strain of Microvirga sp. M2 according to claim 1, characterized in that: The strain has the characteristics of nitrogen fixation, phosphate solubilization, iron carrier synthesis and IAA production.

4. A method for culturing Microvirga sp. M2, characterized in that: A single colony of the uranium-resistant Microvirga sp. M2 strain was picked and inoculated into 50 mL of NA liquid culture medium and shaken at 30°C for 24 h as seed solution. 5 μL of the bacterial solution was aspirated and cultured at a constant temperature of 30°C in the fermentation medium by spot plating.

5. The method for culturing Microvirga sp. M2 according to claim 4, characterized in that: The fermentation medium is one or more of silicate bacterial medium, Ashutosh medium, Pikovakaya's medium, organophosphorus medium, CAS detection medium, skimmed milk powder medium, sodium carboxymethyl cellulose medium and bromocresol purple color development medium.

6. Application of Microvirga sp.M2 in promoting the growth of Sudan grass.

7. A method for preparing a Microvirga sp.M2 bacterial agent, characterized in that: The preparation method is as follows: using NA liquid culture medium, centrifuging the bacterial solution cultured at 30°C, 150r / min constant temperature shaking for 24 hours, collecting the bacterial cells, washing them with sterile water for 3 times to remove the residual culture medium, and then resuspending the bacterial solution with sterile water to mix until 10 8 CFU / mL, that is, the bacterial agent is obtained.

8. An application of the Microvirga sp. M2 bacterial agent as described in claim 7 and Sudan grass in the restoration of uranium contaminated soil.

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