Rahnella and application thereof

Abstract

The invention discloses rahnella and application thereof, and belongs to the technical field of microbiology and biology, the rahnella S5-11 strain provided by the invention is a new species of rahnella, and the preservation number of the rahnella is GDMCC No: 63732. The strain S5-11 has relatively strong tolerance to selenate and selenite, the selenite and the selenite can be reduced into nano elemental selenium, a method for preparing a large amount of nano selenium by utilizing a bioreactor is established, and the strain S5-11 can be used for production of nano selenium and bioremediation of selenium-polluted environment and has a wide application prospect. Besides, the strain has good plant growth promoting ability, can promote crop growth in low-selenium soil, and can relieve stress of selenium on plants in high-selenium soil.

Description

Technical Field

[0001] The invention relates to the fields of microbiology and biotechnology, in particular to Rahn's bacterium and application thereof. Background Art

[0002] Selenium (Se) is an essential trace element for humans and animals, and plays an irreplaceable role in maintaining life activities and health. However, the range between the bioavailability and toxicity of selenium is narrow, and excessive intake may lead to selenium poisoning. The biological activity of selenium is closely related to its chemical form. Among the different chemical forms of selenium, selenate [Se(VI)] and selenite [Se(IV)] show high toxicity, while selenomethionine (SeMet) and selenomethylselenocysteine ​​(MeSeCys) are relatively safe forms of selenium. In recent years, a new form of selenium, selenium nanoparticles (SeNPs), has become a hot topic of research due to its excellent biological activity and relatively low biological toxicity. SeNPs not only show great application potential in the biomedical field, but also show their unique advantages in the fields of food additives and feed additives.

[0003] Microbial cell factories play an important role in the production of nano-selenium. Studies have shown that a large number of microorganisms have the ability to reduce selenite and selenate to synthesize nano-selenium, and this process significantly reduces the biological toxicity of selenium oxides. Nano-selenium-synthesizing bacteria are widely distributed in multiple phyla in nature, including Proteobacteria, Acidobacteria, and Firmicutes, including both Gram-positive and Gram-negative bacteria, such as Bacillus, Enterobacter, Pseudomonas, Citrobacter and other microorganisms, which play a key role in the biogeochemical cycle of selenium.

[0004] Rahnella belongs to the family Yersiniaceae and the order Enterobacterales. It is a facultative anaerobic Gram-negative bacterium widely distributed in soil, water and plant rhizosphere. There are currently 13 officially published species in this genus. It is reported that multiple strains of Rahnella aquatilis have plant growth-promoting and biocontrol effects and are typical plant growth-promoting rhizosphere bacteria (PGPR). For example, the aquatic Rahn's bacteria HX2 strain can dissolve inorganic phosphorus, produce growth-promoting factors such as indoleacetic acid (IAA), siderophore, pyrroloquinoline quinone (PQQ), and synthesize functional substances such as antimicrobial proteins, low-temperature lactase, and extracellular polysaccharides, and has multiple functions such as promoting seed germination, seedling growth, nutrient absorption, and inhibiting bacteria and diseases; it can tolerate 95mM Se(IV) and at least 600mM Se(VI), and reduce Se(VI) and Se(IV) to synthesize spherical nano-selenium particles with a particle size of 100-200nm. However, research on selenium metabolism, nano-selenium synthesis, and plant growth-promoting ability of other types of Rahn's bacteria is relatively lacking. Summary of the invention

[0005] The object of the present invention is to provide a Rahn's bacterium to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In the first aspect, the present invention provides a Rahnella S5-11 strain isolated from high-selenium soil, which is named Rahnella sp. S5-11, preserved in Guangdong Microbial Culture Collection Center, with a preservation number of GDMCC No: 63732, a preservation date of August 11, 2023, and a preservation address of the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] In a second aspect, the present invention provides an application of the above-mentioned Rahnella S5-11 in synthesizing nano-selenium. Rahnella S5-11 is inoculated into a culture medium supplemented with sodium selenite and / or sodium selenate, and after culturing for a period of time, the nano-selenium is separated and purified from the culture solution.

[0009] Furthermore, in the culture medium added with sodium selenite and / or sodium selenate, the initial sodium selenate concentration is 1-400 mM, the initial sodium selenite concentration is 1-80 mM, and preferably the initial selenium concentration is 2, 5, 10 and 20 mM.

[0010] In a third aspect, the present invention provides a use of Rahnella S5-11 in promoting plant growth in common soil. Specifically, Rahnella is used to promote plant growth in low-selenium soil.

[0011] In a fourth aspect, the present invention provides a use of Rahnella S5-11 in alleviating the stress of selenium on plants in high-selenium soil. Specifically, Rahnella alleviates the stress of selenium on plants in high-selenium soil.

[0012] Furthermore, the plants include at least corn.

[0013] In a fifth aspect, the present invention provides an application of Rahnella S5-11 in the remediation of a selenium-contaminated environment.

[0014] The Rahnella sp. S5-11 provided by the present invention has strong tolerance to selenate and selenite, can reduce selenite and selenate to nano-elemental selenium (nano-selenium), can be used for the production of nano-selenium and the bioremediation of selenium-contaminated environments, and has broad application prospects. In addition, the strain has good plant growth-promoting ability, can promote crop growth in ordinary (low-selenium) soil, and can alleviate the stress of selenium on plants in high-selenium soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The colony and bacterial morphological characteristics of the Raenella S5-11 provided by the present invention are shown in FIG. 1 , wherein a is the colony characteristics of the Raenella S5-11 on TSA culture medium, and b is the bacterial morphological characteristics of S5-11 observed by TEM.

[0016] Figure 2 The present invention provides a 16S rRNA gene phylogenetic tree of Rahnella S5-11.

[0017] Figure 3 The present invention provides the complete genome and core gene phylogenetic tree of Rahnella S5-11.

[0018] Figure 4 This is a graph showing the test results of the tolerance and reducing ability of Rahnella S5-11 to Se(VI) and Se(IV) in a preferred embodiment of the present invention.

[0019] Figure 5 This is a graph showing the yield of elemental selenium synthesized by reducing Se(VI) and Se(IV) by Laenella S5-11 in a preferred embodiment of the present invention.

[0020] Figure 6 The physicochemical characteristics of nano-selenium synthesized by Laenella S5-11 in a preferred embodiment of the present invention; wherein, a is a TEM photo of nano-selenium synthesized by reducing 5mM Se(IV) by Laenella S5-11, b is the EDX energy spectrum of the nano-selenium particles indicated by the arrow in the TEM photo, and c is the Raman spectrum of nano-selenium.

[0021] Figure 7 This is a graph showing the effect of applying Rahnella S5-11 on the biomass and root development of corn in normal and high-selenium soils in a preferred embodiment of the present invention.

[0022] Figure 8 This is a graph showing the effect of applying Rahnella S5-11 on selenium absorption and enrichment of corn in ordinary and high-selenium soils in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0024] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0025] The embodiment of the present invention aims to develop a method for synthesizing nano-selenium by utilizing the biotransformation ability of a new strain of Rahn's bacteria. Through multiphase classification identification and establishment of a fermentation process for nano-selenium, it is expected to conduct systematic strain identification and mass-produce safe and efficient nano-selenium supplements to meet the needs of food, feed and biomedical fields. At the same time, the strain has good plant growth-promoting ability, can promote crop growth in low-selenium soil, and can alleviate selenium stress on plants in high-selenium soil.

[0026] The following examples are examples of practical applications of the technical solutions of the present invention, but are not limited thereto. The reagents and experimental equipment involved are all commercially available products. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0027] Example 1: This example provides a method for isolating and identifying the Rahn's bacterium S5-11 strain, as follows:

[0028] S1. Isolation and screening of Rahn's bacterium S5-11: The rhizosphere soil of the selenium hyperaccumulator Cardamine amphibians was collected from the high-selenium area of ​​Yutangba, Enshi, Hubei Province. 5 g of soil was added with 95 mL of sterile saline, and the mixture was extracted at 25°C and 150 rpm for 30 min. After dilution in a 10-fold gradient, 100 μL of each concentration was spread on a NA plate (Oxoid) supplemented with selenate and selenite, and cultured at 28°C for 2 days. Selenate and selenite reducing bacteria were screened based on whether the colonies were red (synthesized elemental selenium), and the bacteria were streaked and purified to obtain a strain S5-11 that can synthesize elemental selenium.

[0029] S2. Polyphasic classification and identification of Rahnella S5-11: Based on morphological, genomic, and physiological and biochemical characteristics, Rahnella S5-11 was identified as a new species of the genus Rahnella. The details are as follows:

[0030] (1) Analysis of colony and bacterial morphology: After culturing in TSA medium (BD BACTO) at 28°C for 2 days, the colony morphology of Rahnella S5-11 was Figure 1 The colonies are round, with a diameter of 1.5-3.0 mm, neat edges, moist surface, and no pigment. Figure 1 b, S5-11 bacteria are short rod-shaped with flagella, 1.5–2.0 μm long and 0.8–1.2 μm wide. At the same time, S5-11 produces swimming circles on semi-solid culture medium, indicating that it has motility.

[0031] T 、Rouxiella silvae 213 T ,Rouxiella chamberiensis130333 T and Rahnella in usitata DSM 30078 T ,Rahnella contaminans Lac-M11 T The highest similarity was found, with sequence similarity ranging from 98.8–99.5%, all exceeding 98.7%.

[0032] The 16S rRNA gene phylogenetic tree constructed using the neighbor-joining method using MEGA software is shown in Figure 2 The figure shows the Bootstrap value greater than 50%, the brackets are the GenBank accession numbers of the strain 16S rRNA gene sequences, and the ruler represents the 1% nucleotide substitution rate. T 、Rouxiella silvae 213 T ,Rouxiella chamberiensis130333 T ,Rahnellacontaminans Lac-M11 T and Rahnella inusitata DSM 30078 T However, the 16S rRNA gene alone cannot effectively distinguish between Rahnella and Rouxiella species, and further genomic analysis is needed.

[0033] (3) Genome sequencing and genome similarity analysis: The whole genome of Raenella S5-11 was sequenced using the Illumina NovaSeq platform, and the genome size was measured to be 5.45 Mb and the GC content was 52.4 mol% (as shown in Table 1). Genome-based species identification was carried out using the Model (Strain) Genome Server (TYGS, https: / / tygs.dsmz.de), and the results showed that S5-11 was a potential new species.

[0034] Table 1 Sequence information used for genome analysis of Rahnella S5-11 and ANI and dDDH values ​​of closely related bacteria

[0035]

[0036] The average nucleotide similarity (ANI) between the genomes of Rahnella S5-11 and its closely related bacteria was further calculated using JSpeciesWS (https: / / jspecies.ribohost.com / jspeciesws / #analyse), and the digital DNA-DNA hybridization (dDDH) was calculated using GGDC Calculator 3.0 (http: / / ggdc.dsmz.de). The results showed that strain S5-11 had a similar genome to Rahnella SAP-17. T and Rahnella contaminans Lac-M11 T The genome similarity of Raenella S5-11 was the highest, with ANI values ​​of 94.3% and 92.8%, and dDDH values ​​of 60.5% and 52.3%, respectively. Both ANI and dDDH were below the species threshold (ANI = 95%–96%, dDDH = 70%), indicating that Raenella S5-11 is a new species of the genus Raenella (as shown in Table 1).

[0037] (4) Genome phylogenetic analysis: The whole genome phylogenetic tree constructed using TYGS, such as Figure 3 As shown, Figure 3 a is a phylogenetic tree based on the whole genome constructed using the TYGS tool. The branch length is calculated according to the GBDP evolutionary distance formula d5. The branch values ​​shown are from 100 GBDP pseudo-bootstrap values. The average branch support rate is 96.5%, and the ruler represents 2% nucleotide difference. Figure 3 b is a phylogenetic tree based on 92 single-copy core genes constructed using the UBCG tool. The data at the nodes represent the gene support index (GSI), and the scale represents 10% nucleotide difference. Figure 3 This indicates that S5-11 and Rahnella laticis SAP-17 are clustered in the same large branch. T and Rahnella contaminansLac-M11 T Clustered in a small branch, indicating that it belongs to the genus Rahn's Figure 3 The UBCG tool (www.ezbiocloud.net / tools / ubcg) was used to extract 92 common single-copy core genes in the genome and draw a phylogenetic tree of concatenated gene sequences, which also showed that Rahnella S5-11 belonged to the genus Rahnella and was similar to Rahnella laticis SAP-17. Tand Rahnella contaminans Lac-M11 T The closest relative ( Figure 3 b).

[0038] (5) Analysis of physiological and biochemical characteristics: Through physiological and biochemical characteristics detection, S5-11 is a facultative anaerobic bacterium with a growth temperature range of 4–37°C and a growth pH range of 4–11, and can tolerate 7% NaCl. It is oxidase-negative and catalase-positive, and can reduce nitrate. The API 20E identification results show that S5-11 produces ornithine decarboxylase, but does not produce β-galactosidase, arginine dihydrolase, lysine decarboxylase, urease or tryptophan deaminase; does not produce indole or hydrogen sulfide, and does not decompose gelatin; can use citric acid to ferment glucose, mannitol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose, but does not ferment inositol. API The results of 50CH identification showed that S5-11 could utilize the following 23 carbon sources to produce acid: glycerol, L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, dulcitol, D-mannitol, D-sorbitol, N-acetylglucosamine, arbutin, esculin, salicin, D-cellobiose, D-lactose, D-melibiose, sucrose, D-trehalose, D-raffinose, and gentiobiose.

[0039] and the closely related species R. laticis SAP-17 T and R.contaminans Lac-M11 TIn comparison, the differential physiological and biochemical characteristics of Rahnella S5-11 are β-galactosidase activity and acid production using the following carbon sources: D-adonitol, D-sorbitol, methyl-α-D-mannopyranoside, D-maltose, D-arabitol, gluconate and 5-ketogluconate (as shown in Table 2).

[0040] Table 2 Differential physiological and biochemical characteristics of Rahnella S5-11 and its closely related species

[0041]

[0042] It should be noted that the physiological and biochemical results of the above-mentioned closely related bacteria are from the literature Jeon D, Kim IS, Lee SD. Rahnella laticis sp.nov.and Rahnella contaminans sp.nov., and emended description of the genus Rahnella[J].International Journal of Systematic and Evolutionary Microbiology, 2021, 71(7).

[0043] Based on the above-mentioned 16S rRNA gene and genome phylogenetic analysis as well as morphological characteristics and physiological and biochemical properties, strain S5-11 was identified as a new species of the genus Rahnella. It has been deposited in the Guangdong Microbial Culture Collection Center with the deposit number GDMCC No: 63732. The deposit date is August 11, 2023. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0044] Example 2: This example verifies the tolerance and reduction ability of Rahnella S5-11 to selenate and selenite in solid culture medium, as follows:

[0045] Prepare TSA solid medium (BD Bacto), wait for it to cool to about 60°C, add filter-sterilized sodium selenate and sodium selenite solutions (3M) respectively, shake gently to mix, and pour on the plate to prepare selenium-containing plates containing 0-400mM sodium selenate and 0-100mM sodium selenite. Pick a single colony of Raenella S5-11 and inoculate it into a TSB (BD Bacto) test tube, shake and incubate at 28°C and 150rpm for 12h for activation, adjust the bacterial solution OD600 to 0.8, use it as a seed solution, and dilute it 10 times with sterile saline (marked as 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 ), take 2.5 μL of each gradient and drop it onto the selenium-containing plate, blow it dry with sterile air, and then culture it at 28°C for 2 days to observe the growth of the strain.

[0046] Results Figure 4 , Laenella S5-11 can tolerate at least 400mM sodium selenate and 80mM sodium selenite, and the colonies on the selenium-containing plates are bright red, indicating that S5-11 can reduce sodium selenate and sodium selenite to synthesize red elemental selenium.

[0047] Example 3: This example verifies the efficiency of reducing Se(VI) and Se(IV) to synthesize nano-selenium by Laenella S5-11 in liquid culture medium, as follows:

[0048] A single colony of S5-11 was selected and inoculated into an LB tube (5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, pH = 7.0–7.2, sterilized at 121°C for 20 min), and cultured at 28°C and 150 rpm for 12 h for activation. The bacterial solution OD was adjusted. 600 The inoculum concentration was 0.8, which was used as the seed solution. The LB shake flask was inoculated with 1% inoculum, and the corresponding volume of sodium selenate or sodium selenite mother solution (filter sterilized) was added to make the initial sodium selenate concentration 1-400mM and the initial sodium selenite concentration 1-80mM, and then cultured at 28°C and 150rpm for 2 days.

[0049] The yield of elemental selenium was determined by sodium sulfide spectrophotometry. A 1M Na2S solution was prepared with distilled water (prepared and used immediately), 500μL of the fermentation broth to be tested was centrifuged at 12000rpm for 5min, the supernatant was discarded, and the broth was washed three times with sterile saline. The supernatant was discarded, 1mL of Na2S solution (1M) was added, and the mixture was mixed thoroughly and reacted for 1h. The broth was centrifuged at 12000rpm for 5min, and the absorbance at a wavelength of 500nm was measured by taking the reaction supernatant. The yield of nano-selenium in the fermentation broth was calculated based on the nano-selenium standard curve. The results showed that Rahnella S5-11 was able to reduce sodium selenate and sodium selenite to synthesize nano-selenium. The nano-selenium yield results under the conditions of initial selenium concentrations of 2, 5, 10 and 20mM are shown in Table 1. Figure 5 The yield was in the range of 0.7–1.9 mM. Among them, the yield of nano-selenium was the highest at 10 mM Se(IV) and 2 mM Se(VI) concentrations, reaching 1.9 mM and 0.5 mM respectively, while the conversion rate of nano-selenium was the highest at 2 mM Se(VI) and Se(IV) concentrations, reaching 43.4% and 23.4% respectively.

[0050] Example 4: This embodiment provides a method for producing nano-selenium by fermenting Rahnella S5-11 in a fermenter, which specifically includes the following steps:

[0051] S1. Activation of strains: Streak out the Rahn's bacterium S5-11 on an LB plate for activation culture and culture at 28°C for 48 h.

[0052] S2. Seed culture: Select activated Rahn's bacterium S5-11 and inoculate it into LB liquid culture medium at 28°C and 150 rpm for shaking culture for 12-16 hours as seed solution.

[0053] S3, Nano-selenium fermentation: Fermentation medium (glucose 20g / L, yeast extract 10g / L, soy peptone 10g / L, sodium chloride 5g / L, ammonium sulfate 2g / L, ammonium chloride 1g / L, calcium chloride 0.3g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.3g / L, pH 7.0–7.2) was prepared in a fermenter according to 70% liquid volume, and sterilized at 121°C for 20min. After the culture medium is cooled, the prepared selenite solution (1M, sterilized by filtration or high temperature and high pressure sterilization) is added to control the final concentration of selenite in the culture medium to 10mM, and then the S5-11 seed solution is inoculated at 2% inoculation amount, the fermentation temperature is controlled at 28±0.5°C, the rotation speed is 180rpm, the ventilation volume is 0.5vvm, and the fermentation culture is cultured for 24h. The nano-selenium content of the fermentation broth is determined by sodium sulfide spectrophotometry, and the yield of nano-selenium in the fermentation broth is calculated according to the nano-selenium standard curve.

[0054] The results showed that after 24 hours of fermentation in the fermenter, the yield of nano-selenium synthesized by reducing 10mM sodium selenite by Laenella S5-11 reached 4.8mM, which was 150% higher than the shake flask yield and the fermentation time was shortened by half.

[0055] Example 5: This example verifies the physicochemical characteristics of the synthesis of nano-selenium by reducing Se(VI) and Se(IV) by Rahnella S5-11, as follows:

[0056] Pick a single colony of S5-11 and inoculate it into an LB test tube. Cultivate it at 28°C and 150 rpm for 12 hours to activate it. Adjust the OD value of the bacterial solution. 600 The inoculum was transferred to an LB shake flask at 1% inoculation, and a corresponding volume of sodium selenite mother solution (filtered and sterilized) was added to make the initial selenium concentration 10 mM. The flask was placed at 28°C and 150 rpm for 48 hours of shaking culture. The shape, particle size, element type and other physical and chemical characteristics of the elemental selenium synthesized by Rahnella S5-11 were analyzed by transmission electron microscopy (TEM) and X-ray energy dispersive spectrometer (EDS). The crystal form of the elemental selenium was analyzed by Raman spectroscopy. The results are as follows: Figure 6 shown.

[0057] The results showed that the elemental selenium synthesized by reducing Se(IV) by Laenella S5-11 was nano-sized. The nano-selenium was nearly spherical and distributed in the particle size range of 50–700 nm. It was mainly distributed in the bacterial cell membrane, and a small amount was distributed inside and outside the cell ( Figure 6 a). Through EDS spectrum analysis of the nanoparticles shown in the figure, characteristic peaks of selenium at 1.37keV, 11.22keV and 12.50keV can be seen, indicating that the nanoparticles synthesized by the strain are nano-selenium ( Figure 6 b). Raman spectrum visible at 253cm -1 The characteristic Raman shift peak at shows that the crystal form of nano-selenium is amorphous ( Figure 6 c).

[0058] Example 6: This example verifies that Rahnella S5-11 can synthesize protease, siderophore and IAA, and has plant growth promotion potential, as follows:

[0059] Prepare the CAS medium for siderophore detection (Haibo Biotechnology) and the protease detection medium (tryptone 5.0g, yeast extract 3.0g, glucose 1.0g, agar 15.0g, distilled water 1000mL, pH7.0, autoclave at 121℃ for 30min, add 10% sterile skim milk when cooled to about 50℃, mix well and pour on the plate). After activating Rahn's bacteria S5-11 on LB medium, inoculate it on the siderophore and protease detection medium, culture at 28℃ for 5 days, and observe whether a transparent circle is formed around the colony. DF (5.0 g of peptone, 1.5 g of yeast extract, 1.5 g of beef extract, 5.0 g of NaCl, 1000 mL of distilled water, pH 7.0, sterilized at 121° C. for 30 min) and DF+Try culture medium (5.0 g of peptone, 1.5 g of yeast extract, 1.5 g of beef extract, 5.0 g of NaCl, 0.5 g of tryptophan, 1000 mL of distilled water, pH 7.0, sterilized at 121° C. for 30 min) were prepared for indoleacetic acid (IAA) detection. After activating Rahn's bacterium S5-11 on LB medium, it was inoculated into DF and DF+Try culture medium at an inoculum size of 1%, and cultured at 28° C. and 150 rpm for 7 days. The bacterial solution was centrifuged at 12000 rpm for 5 min, and the IAA content in the bacterial solution was determined by the Salkowkin colorimetric method.

[0060] The results showed that Rahn's bacterium S5-11 was able to produce proteases and siderophores, and synthesized 2.9 mg / L and 14.1 mg / L of IAA in DF and DF+ medium, respectively, indicating that this strain has the potential to promote plant growth.

[0061] Example 7: This example verifies the effect of Rahnella S5-11 on corn growth and selenium absorption, as follows:

[0062] The test soil was collected from the surface soil (0–20 cm) in Pinggu District, Beijing, with an organic matter content of 7.8 g / kg, available phosphorus of 34.0 g / kg, available potassium of 51.4 g / kg, and total selenium content of 49.6 μg / kg (low selenium soil). The sampling area is a wasteland abandoned all year round, and no fertilizer has been applied in recent years. The soil was collected and transported back to the laboratory for natural air drying, and then sieved through a 2 mm sieve for use. The test corn variety was Zhengdan 958. Before sowing, the surface was disinfected with 10% H2O2 for 10 min, cleaned with deionized water and placed on moist filter paper, germinated in a constant temperature incubator at 25 ° C, and transplanted when the buds were about 1 cm long. A total of 4 treatments were set up in the experiment: ①CK (normal soil blank control), ②S5-11 (normal soil inoculated with S5-11), ③Se-CK (normal soil added with 1 mg / kg sodium selenate), and ④S11+Se (normal soil added with 1 mg / kg sodium selenate and inoculated with S5-11 at the same time).

[0063] The culture of S5-11 was prepared as follows: a single colony of S5-11 was inoculated into an LB tube, incubated at 28°C and 150 rpm for 24 h, transferred to an LB shake flask at 1% inoculum, and incubated at 28°C and 150 rpm until the culture OD reached 0. 600 The cells were collected by centrifugation at 7000 rpm for 8 min, washed three times with sterile saline and resuspended to a bacterial solution OD 600 The bacterial suspension was 1.0 and stored in a refrigerator at 4°C for later use.

[0064] The potted experiment used a plastic flower pot with a diameter of 17.9 cm and a height of 18.8 cm. A layer of nylon mesh was laid at the bottom of the pot to prevent soil leakage and plant root growth. Each pot was filled with 3.0 kg of sieved air-dried soil. 3.75 mL of 10 mM Na2SeO4 solution was added to each pot in the selenium treatment group and mixed with the soil before use, so that the soil selenium content was 1 mg / kg. The same amount of distilled water was added to each pot in the treatment group without selenium. 300 mL of bacterial suspension was added to each pot in the S5-11 bacterial agent treatment group, mixed with the soil before use. The same amount of physiological saline was added to each pot without inoculation of bacterial species. After the soil was prepared, one corn was sown in each pot, weighed and supplemented with distilled water every day to keep the soil moisture content at about 60% of the field water holding capacity. The temperature during cultivation was 10–28 °C.

[0065] Maize aerial parts, root and soil samples were collected 50 days after maize sowing. The fresh weight of aerial parts and root system was measured, and the root system samples were cleaned and spread on a plexiglass tray for root scanning. The root length, root diameter, root surface area and root volume were calculated using WinRHIZO software (ReagentInstrument Inc., Quebec, Canada). In addition, the dry weight of the samples was measured after drying, and they were crushed and mixed evenly with a ball mill (mm400, Retsch, Germany). 0.20 g of the sample was weighed and placed in a digestion tube. After adding 5 mL of HNO3 and standing overnight, it was digested in a digestion furnace at 180 ° C for 3 h, and the volume was adjusted to 50 mL with ultrapure water. 4 mL of the digestion solution was added with 1 mL of 6 M hydrochloric acid (super pure), and reduced in a water bath at 95 ° C for 3 h. The selenium content of the plants was determined using a hydride generation-atomic fluorescence spectrometer (HG-AFS) (Jitian Instrument Co., Ltd., Beijing).

[0066] Results Figure 7 and Figure 8 After inoculation of Rahnella S5-11 in ordinary soil, the aboveground and root biomass of corn increased significantly, with the aboveground fresh weight increasing by 108%, the dry matter increasing by 133%, and the root fresh weight and dry weight increasing by 52% and 48%, respectively ( Figure 7a and b). Adding 1 mg / kg of selenate to the soil significantly inhibited the growth of corn plants, while inoculation of Rahnella S5-11 effectively alleviated the biological toxicity caused by selenium stress, increasing the fresh weight of the aboveground part and roots of corn in high-selenium soil by 134% and 101%, respectively, and the dry weight by 150% and 42%, respectively ( Figure 7 a and b). Root scanning results showed that the total root length, root surface area and root volume of maize roots increased significantly in normal soil after inoculation with S5-11, while the average diameter remained unchanged ( Figure 7 c and d); High selenium in the soil caused a significant decrease in the average root diameter, total root length, root surface area, and root volume of maize, while inoculation with S5-11 alleviated the biological toxicity caused by selenium, and the total root length, root surface area, and root volume of maize were significantly increased ( Figure 7 c and d).

[0067] In addition, in normal soil, the inoculation of Rahnella S5-11 increased the selenium content of corn roots by 175%, while the selenium content of the aboveground parts decreased by 42%, inhibiting the transport of selenium from the roots to the aboveground parts, resulting in a decrease in the selenium transport coefficient (TF) ( Figure 8 a). After selenate was applied to the soil, the selenium enrichment in the plant body increased significantly, and the selenium content in the aboveground part and root system of corn increased to 76.5 mg / kg and 80.5 mg / kg, indicating that the applied selenium can be effectively absorbed by corn ( Figure 8 b). In high-selenium soil, inoculation of Rahnella S5-11 had no significant effect on the selenium content of the aboveground part of corn, but it caused the selenium content of the corn root system to decrease by 35%, which may be because Rahnella S5-11 reduced Se(VI) to elemental selenium, thereby reducing the absorption and enrichment of selenium by the root system ( Figure 8 b).

[0068] The above results show that inoculation with Rahn's bacteria S5-11 can significantly promote the growth and root development of corn plants and alleviate the stress of high-selenium soil on plants. In low-selenium soil, inoculation with S5-11 inhibited the transport of selenium from the roots to the aboveground parts, while in high-selenium soil, inoculation with S5-11 inhibited the absorption and enrichment of selenium by the roots.

[0069] Based on the above-mentioned ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above-mentioned description. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. Rahn's bacterium, characterized in that The strain was named Rahnella sp. S5-11 and deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 63732.

2. Use of the Rahnella as claimed in claim 1 in synthesizing nano-selenium.

3. The use according to claim 2, characterized in that: The method comprises the following steps: inoculating the Rahn's bacteria into a culture medium added with sodium selenite and / or sodium selenate, culturing for a period of time, and separating and purifying nano-selenium from the culture solution.

4. The use according to claim 3, characterized in that: In the culture medium added with sodium selenite and / or sodium selenate, the initial sodium selenate concentration is 1-400 mM, and the initial sodium selenite concentration is 1-80 mM.

5. Use of the Rahnella as claimed in claim 1 in promoting plant growth in low-selenium soil.

6. The use according to claim 5, characterized in that: The plants include at least corn.

7. Use of the Rahnella as claimed in claim 1 in alleviating the stress of selenium on plants in high-selenium soil.

8. The use according to claim 7, characterized in that: The plants include at least corn.

9. Use of the Rahnella as claimed in claim 1 in repairing environmental selenium pollution.

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