Yorkykia rajinensis and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a Yorkinia lejininckii strain and application thereof. The specific technical scheme is as follows: the strain of Yokenella regensburgei PSI-02 has the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO: 34762, and the strain of Yokenella regensburgei PSI-02 has the preservation number of CGMCC NO: 34762. The invention provides a new strain of Yorkinia lejingui, which can tolerate high-concentration sodium selenite and can be used for reducing the sodium selenite into nano-selenium. Meanwhile, the strain can secrete IAA and siderophores and has the capacity of dissolving inorganic phosphorus and fixing nitrogen. The invention provides a novel growth-promoting strain which is efficient, sustainable and wide in application prospect for selenium nutrient enrichment and ecological restoration of a polluted area.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Yorkella reginsburgensis and application thereof. Background Art

[0002] Certain microorganisms can transform selenium in various ways, including reduction, oxidation, methylation, and demethylation, which play an important role in regulating the form of selenium in the environment. Biogenic nanoselenium synthesized by microorganisms has the advantages of high solubility, strong stability, high bioavailability, and low toxicity, and has shown great potential in promoting crop growth, preventing and controlling pests and diseases, and reducing environmental pollution.

[0003] Studies have demonstrated that some microorganisms can reduce selenate or selenite to nanoselenium particles, thereby enhancing plant selenium uptake and improving soil productivity. However, few known microbial resources possess this ability. Therefore, developing plant growth-promoting bacteria that can tolerate high concentrations of selenite and convert it to nanoselenium is crucial for promoting agricultural selenium biofortification and the application of nanomaterials. Summary of the Invention

[0004] The present invention aims to provide a strain of Yorkella reginsburgii and application thereof.

[0005] To achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a strain of Yorkella regensburgei PSI-02, deposited in the China General Microbiological Culture Collection Center (CGMCC) on June 5, 2026, with the deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO: 34762.

[0006] Correspondingly, a bacterial preparation prepared using the Yorkshire spp. PSI-02; or a bacterial preparation containing the Yorkshire spp. PSI-02.

[0007] The application of the Yorkshire spp. PSI-02 or the bacterial preparation in the preparation of nano-selenium, or in the preparation of indoleacetic acid, or in the preparation of iron carriers, or in the dissolution of inorganic phosphorus, or in nitrogen fixation, or in promoting seed germination, promoting plant growth, preventing and controlling plant diseases, and improving plant stress resistance.

[0008] The present invention has the following beneficial effects: It provides a new strain of Yorkella regensburgei (PSI-02). PSI-02 is able to tolerate high concentrations of sodium selenite and can reduce sodium selenite to nano-selenium. Furthermore, PSI-02 secretes IAA and siderophores, dissolves inorganic phosphorus, and has nitrogen-fixing capabilities, making it an ideal biofertilizer additive. This invention provides a new, highly efficient, sustainable, and promising growth-promoting strain for selenium nutrient enrichment and ecological restoration of contaminated areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is the standard curve for detecting nano-selenium content;

[0010] Figure 2 is the phylogenetic tree of strain PSI-2;

[0011] Figure 3 Figure 1 is the colony morphology of strain PSI-2 on LB solid medium containing 20 mM sodium selenite;

[0012] Figure 4 This is the morphological picture of strain PSI-2 observed under a 1000x microscope after Gram staining;

[0013] Figure 5 This is a schematic diagram of the tolerance of strain PSI-02 to sodium selenite;

[0014] Figure 6 Schematic diagram of the reduction of 0.5, 1, 2, 5, and 10 mM sodium selenite by strain PSI-02 within 72 h;

[0015] Figure 7 Schematic diagram of the growth of strain PSI-02 in nitrogen-free medium;

[0016] Figure 8 Schematic diagram of IAA production by strain PSI-02;

[0017] Figure 9 This is the standard curve for IAA concentration detection;

[0018] Figure 10 Schematic diagram of the siderophore production of strain PSI-02;

[0019] Figure 11 This is a schematic diagram of the solubility of inorganic phosphorus by strain PSI-02;

[0020] Figure 12 The standard curve for soluble phosphorus concentration detection was used. DETAILED DESCRIPTION

[0021] The present invention provides a new strain of Yorkella regensburgei PSI-02, which was deposited in the China General Microbiological Culture Collection Center (CGMCC) on June 5, 2025, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO: 34762.

[0022] The Yorkia reginsburgi bacterium is capable of converting sodium selenite into nano-selenium.

[0023] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The data obtained are the average values ​​obtained after at least three repetitions, and the data obtained in each repetition are valid data.

[0024] Example 1: Screening and identification of microorganisms

[0025] 1. Screening

[0026] Soil from a tea garden in Bajiao Township, Enshi City, Hubei Province was collected. 10 g of soil sample was weighed and placed in a sterilized conical flask containing 90 mL of deionized water. The sample was shaken at 30°C and 180 rpm for 30 minutes and then allowed to stand for 10 minutes. 2 mL of the soil suspension supernatant was added to LB liquid medium containing 100 mM selenium for enrichment culture. After shaking culture at 30°C and 200 rpm for 2 days, the enriched bacterial suspension was screened by dilution and spreading (10 -2 , 10 -3 , 10 -4 , 10 -5 ) Isolate on LB solid medium containing 5 mM sodium selenite. Red single colonies are the target colonies. Observe the growth and morphology of the microorganisms. Select individual colonies with distinct morphological characteristics and good growth. Repeat the plate streak method to isolate and purify until multiple single colonies appear on the LB solid medium. Inoculate the purified strain onto LB solid medium slants in test tubes and store in a refrigerator at 4°C.

[0027] Finally, the selenium reducing ability of the five isolated selenium-reducing bacteria (Se1, Se2, Se3, Se4, and Se5) was determined using the following method:

[0028] Use an inoculating loop to select bacteria from the LB slant and inoculate them into 100 mL of LB liquid medium containing 5 mM sodium selenite. After 24 hours, collect 1 mL of the bacterial sample, centrifuge at 5000 g for 10 minutes, and discard the supernatant. Wash the precipitate three times with 1 mL of saline. Disperse the collected bacteria and red precipitate thoroughly with 1 mL of 1M Na2S solution. After 1 hour of reaction, centrifuge at 10,000 rpm for 30 minutes. The supernatant is collected and the absorbance at 500 nm is measured on a spectrophotometer. Substitute the absorbance value into the standard curve to calculate the corresponding sodium selenite concentration, the amount of converted SeNPs, and the conversion rate.

[0029] Prepare the standard curve for SeNPs detection: pipette 0.1M sodium selenite solution into test tubes to prepare 1mL of 1-10μmol / mL selenite gradient solution. Add 25μmol / mL hydroxylamine hydrochloride solution to each test tube containing selenite to ensure that SeO3 2- Quantitative reduction to Se 0 After the contents of the test tube were dried under nitrogen flow, 1 mL of 1 M Na2S solution was added. The test tube was gently mixed (avoid vortexing). After 1 hour, the absorbance of the reddish-brown selenium solution was measured using a spectrophotometer at a wavelength of 500 nm. The standard curve prepared was as follows: Figure 1 As shown, R 2 =0.9965, showing a good linear relationship.

[0030] Within 24 hours, Se1, Se2, Se3, Se4 and Se5 converted 2.03, 1.44, 1.56, 1.35 and 1.55 mM nano-selenium respectively in LB liquid culture medium containing 5 mM sodium selenite, with conversion rates reaching 40.6%, 28.8%, 31.2%, 27.0% and 31.0% respectively.

[0031] Among them, Se-1 bacteria had the strongest selenium reduction ability and was named PSI-02.

[0032] 2. Identification and preservation of strain PSI-02

[0033] The bacterial liquid DNA was extracted, and 16S rDNA was amplified by PCR and sequenced. The obtained 16S rDNA sequence is shown in SEQ ID NO: 1.

[0034] Phylogenetic tree Figure 2 As shown, the strain had a similarity of up to 99% with Yorkella regensburgei NBRC 102600, and was preliminarily identified as Yorkella regensburgei.

[0035] The colony morphology of strain PSI-2 on LB solid medium containing 20mM sodium selenite is shown in the figure below. Figure 3 As shown. Drop a drop of sterile water on a clean glass slide, pick up a trace of strain PSI-02 with an inoculation loop to make a bacterial film on the slide and let it dry. Stain with crystal violet for 1 minute, stain with iodine solution for 1 minute, decolorize for 30 seconds, counterstain with Tsar stain for 2 minutes, and then dry. Cover with a coverslip and observe the staining under a microscope. The bacterial morphology is as follows Figure 4 As shown, the bacteria are red short rods, Gram-negative bacteria, and have no spores.

[0036] Based on the comprehensive sequencing molecular identification results and physiological and biochemical characteristics, strain PSI-02 was identified as Yorkella regensburgei and deposited in the China General Microbiological Culture Collection (CGMCC) on June 5, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO: 34762.

[0037] Example 2: Demonstration of the ability of strain PSI-02 to tolerate sodium selenite

[0038] The strain PSI-02 was inoculated on LB solid slant medium and cultured at 28°C for activation. The activated strain on the slant medium was inoculated into 100 mL LB liquid medium using an inoculating loop and cultured in a shaker at 28°C and 180 rpm until the OD value of the bacterial solution reached 0. 600 =1.0, obtain seed solution.

[0039] 1 mL of the seed solution was inoculated into 10 mL of LB liquid culture medium containing 0, 100, 200, 300, 400, and 500 mM sodium selenite, respectively. After culturing at 28°C and 180 rpm for 3 days, the tolerance of the culture medium to sodium selenite was observed based on the color of the culture medium.

[0040] The results are as follows Figure 5 The results showed that the strain PSI-02 could still synthesize red SeNPs at a concentration of 300 mM sodium selenite, indicating that the strain PSI-02 could tolerate high concentrations of sodium selenite.

[0041] Example 3: Demonstration of the ability of strain PSI-02 to synthesize nano-selenium

[0042] The seed solution of strain PSI-02 was prepared according to the method of Example 2.

[0043] 1 mL of the seed solution was inoculated into 100 mL of LB liquid medium containing 0, 0.5, 1.0, 2.0, 5.0, or 10 mM sodium selenite. The cultures were shaken at 28°C and 180 rpm. The culture medium from the 0.5 mM concentration group was sampled every 12 hours, while the remaining groups were sampled every 24 hours for determination of nano-selenium and sodium selenite content. Three replicates were performed for each group, and the results were averaged.

[0044] The method for determining the content of SeNPs (nanoselenium nanoparticles) is the same as that in Example 1.

[0045] Determination of nano-selenium content in samples: Collect 1 mL of sampled bacterial liquid, centrifuge at 5000 g for 10 minutes, and remove the supernatant. Wash the precipitate three times with 1 mL of physiological saline, centrifuge at 5000 g for 10 minutes, and collect the supernatant and bacteria. The collected bacteria and red precipitate are used to determine the nano-selenium content, and the determination method is the same as Example 1. The collected supernatant is centrifuged at 8000 r / min for 20 minutes at 4°C, then filtered through a 0.22 μm filter membrane, and the residual sodium selenite content is determined by inductively coupled plasma mass spectrometry according to the HJ 700-2014 water quality 65 element detection method.

[0046] The results are as follows Figure 6 The results showed that strain PSI-02 could completely convert 0.5 mM sodium selenite into nano-selenium within 12 h, and achieved 100%, 95%, 55% and 30% conversion rates for 1 mM, 2 mM, 5 mM and 10 mM sodium selenite, respectively, within 72 h.

[0047] Example 4: Demonstration of other abilities of strain PSI-02

[0048] 1. Nitrogen fixation ability

[0049] Prepare Ashby medium (nitrogen-free medium): mannitol (Mannitol) 10 g / L, dipotassium hydrogen phosphate (K2HPO4) 0.2 g / L, magnesium sulfate (MgSO4·7H2O): 0.2 g / L, sodium chloride (NaCl) 0.2 g / L, calcium carbonate (CaCO3) 5 g / L, agar (Agar) 15 g / L.

[0050] 10 μL OD 600 = 1.0) was applied to the surface of Ashby culture medium. The inoculated culture dish was inverted and incubated in a 30°C incubator for 3 days. After 3 days of incubation, the surface of the culture medium was observed for the formation of colonies.

[0051] The results are as follows Figure 7 The results showed that colonies were formed, indicating that the strain PSI-02 had the ability to fix nitrogen and could use nitrogen in the atmosphere as a nitrogen source.

[0052] 2. IAA production capacity test

[0053] Prepare Salkowski reagent: Mix 15 mL of 0.5 mol / L FeCl3 solution with 300 mL of concentrated H2SO4 and 500 mL of deionized water. Store in a brown bottle at 4°C in the dark.

[0054] Prepare tryptophan stock solution: Weigh 0.1 g of L-tryptophan and dissolve it in 10 mL of distilled water to prepare a 10 mg / mL tryptophan stock solution.

[0055] 1 mL of strain PSI-02, which has reached the logarithmic growth phase in LB liquid medium, was inoculated into LB liquid medium containing 100 mg / L L-tryptophan. The culture was shaken at 180 rpm at 28°C in the dark for 48 hours. The bacterial culture was then centrifuged at 4000 g for 20 minutes. 1 mL of the supernatant was then mixed with 2 mL of Salkowski reagent (dark). The culture was allowed to stand in the dark for 30 minutes and the color change was observed. The results were as follows. Figure 8 As shown, the culture medium turned pink (YR), indicating the ability to produce IAA.

[0056] Use a spectrophotometer to measure the absorbance of the culture medium at 535 nm. Preparation of IAA standard curve: weigh 10 mg IAA (Sigma), dissolve it in 10 mL distilled water, and prepare 1 mg / mL IAA stock solution. When using, dilute with distilled water to the required concentration (0, 10, 20, 30, 40, 50, 60 μg / mL), take 1 mL and add 2 mL Salkowski reagent respectively, mix and shake well, incubate in the dark for 30 minutes at room temperature, and measure OD 535 The vertical axis is IAA concentration, OD 535 As the horizontal axis, draw a standard curve, such as Figure 9 As shown. According to the OD of the sample 535 The IAA concentration was calculated from the standard curve and the result was 45.56 ± 1.35 μg / mL.

[0057] This indicates that strain PSI-02 has the ability to secrete IAA, and therefore has the potential to promote seed germination and plant growth and development, and can be used as a plant growth promoter.

[0058] 3. Quantitative detection of siderophore production capacity

[0059] Prepare low-iron culture medium (for inducing siderophore production): glucose 4 g / L, asparagine 2 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, and NaCl 0.1 g / L.

[0060] Prepare CAS detection solution (for siderophore detection): weigh 60.5 mg of Chrome Azurol S (CAS) and dissolve it in 50 mL of distilled water.

[0061] Prepare the iron solution: weigh 10 mg of FeCl3·6H2O and dissolve it in 10 mL of 10 mM HCl.

[0062] Prepare HDTMA solution: weigh 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA) and dissolve it in 40 mL of distilled water.

[0063] Prepare buffer: Weigh 30.24 g of PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) and dissolve it in 900 mL of distilled water. Adjust the pH to 6.8 with NaOH and make up to 1 L.

[0064] Prepare CAS assay solution: Slowly add 1 mL of iron solution to 50 mL of CAS solution and mix thoroughly. Add 40 mL of HDTMA solution to the mixture and mix thoroughly. Add 50 mL of PIPES buffer and mix thoroughly. Make up to 100 mL and store in a brown bottle at 4°C protected from light.

[0065] Inoculate 1 mL of strain PSI-02, which has reached the logarithmic growth phase in LB liquid medium, into low-iron medium and culture in a shaker at 28°C and 180 rpm for 48 hours. Take 1 mL of culture medium and centrifuge at 4000g for 20 minutes. Take 500 μL of supernatant and add 500 μL of CAS detection solution. Mix well. Incubate in the dark for 1 hour and observe the color change. The results are as follows: Figure 10 As shown, the solution turned orange (yellow), indicating that strain PSI-02 was able to produce siderophores.

[0066] The absorbance was measured at 630 nm using a spectrophotometer and the siderophore activity was calculated as:

[0067] Siderophore activity (%) = [(OD 630 Blank-OD 630 Sample) / OD 630 blank] × 100%.

[0068] The blank was a mixture of low iron culture medium without bacteria inoculation and CAS detection solution. The results showed that the relative siderophore production of strain PSI-02 was 63.91±2.36%.

[0069] Strain PSI-02 has the ability to secrete siderophores, and therefore has the potential to promote iron absorption and utilization by plants, alleviate plant iron deficiency symptoms, and promote plant nutrient accumulation.

[0070] 4. Inorganic phosphorus dissolving ability test

[0071] Prepare phosphate solubilization medium: glucose 10 g / L, calcium phosphate 5 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.2 g / L, potassium chloride 0.2 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.002 g / L, FeSO4·7H2O 0.002 g / L, yeast extract 0.5 g / L.

[0072] Prepare molybdenum blue reagent (for determining soluble phosphorus): Prepare Reagent A: 5% ammonium molybdate solution. Weigh 5g of ammonium molybdate and dissolve it in 100mL of distilled water. Prepare Reagent B: 0.25% ascorbic acid solution. Weigh 0.25g of ascorbic acid and dissolve it in 100mL of distilled water (prepare immediately before use). Prepare Reagent C: 2.5M sulfuric acid solution. Slowly add 13.9mL of concentrated 98% sulfuric acid to 86.1mL of distilled water and mix thoroughly. Mix Reagent A, Reagent B, and Reagent C in a 1:1:2 volume ratio to obtain the molybdenum blue working solution. Prepare immediately before use.

[0073] Inoculate 20 μL of strain PSI-02 that has reached the logarithmic growth phase in LB liquid medium into 100 mL of phosphate-soluble medium and culture in a shaker at 28°C and 180 rpm for 3 days. Take 1 mL of culture medium, centrifuge at 4000g for 20 minutes, and collect the supernatant. Take 1 mL of supernatant, add 2 mL of molybdenum blue working solution, mix well, and let it stand for 20 minutes. Observe the color change of the culture medium. The results are as follows: Figure 11 As shown: the culture medium turned blue (YR), indicating that strain PSI-02 has the ability to solubilize inorganic phosphorus.

[0074] Use a spectrophotometer to measure the absorbance at 880nm. Preparation of standard curve: Prepare 0, 5, 10, 20, 50, 60, 70, 80, 90, 100μg / mL KH2PO4 standard solutions. Take 1mL of standard solution, add 2mL of molybdenum blue working solution, mix well, and let it stand for 20min. Use a spectrophotometer to measure the absorbance at 880nm. Use KH2PO4 concentration as the horizontal axis, OD 880 As the vertical axis, draw the standard curve, such as Figure 12 The concentration of soluble phosphorus in the sample was calculated based on the standard curve.

[0075] Phosphate solubilization capacity (μg / mL) = soluble phosphorus concentration in sample - soluble phosphorus concentration in blank control. The blank control was liquid phosphate solubilization medium without bacteria inoculation. The results showed that the phosphate solubilization capacity of strain PSI-02 was 103.23 ± 9.90 μg / mL.

[0076] Strain PSI-02 has the ability to fix nitrogen and dissolve inorganic phosphorus, so it can provide nitrogen sources for plants, promote plant growth, increase the effectiveness of phosphorus, improve the utilization rate of phosphorus fertilizer by plants, improve the soil environment, and enhance the stress resistance of plants.

[0077] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A strain of Yorkella regensburgei PSI-02, characterized by: It was deposited in the China General Microorganism Culture Collection Center on June 5, 2025, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO: 34762.

2. A bacterial preparation prepared using the Yorkia reginsburg PSI-02 according to claim 1.

3. A bacterial preparation comprising the Yorkshire spp. PSI-02 according to claim 1.

4. Use of the Yorkella reginsburgii PSI-02 described in claim 1 or the bacterial preparation described in claim 2 or 3 in the preparation of nano-selenium.

5. Use of the Yorkshire spp. PSI-02 of claim 1 or the bacterial preparation of claim 2 or 3 in the preparation of indoleacetic acid.

6. Use of the Yorkshire spp. PSI-02 of claim 1 or the bacterial preparation of claim 2 or 3 in the preparation of siderophores.

7. Use of the Yorkia reginsburgii PSI-02 according to claim 1 or the bacterial preparation according to claim 2 or 3 in dissolving inorganic phosphorus.

8. Use of the Yorkella reginsburgii PSI-02 according to claim 1 or the bacterial preparation according to claim 2 or 3 in nitrogen fixation.

9. Use of the Yorkella reginsburgii PSI-02 according to claim 1 or the bacterial preparation according to claim 2 or 3 in promoting seed germination and plant growth.

10. Use of the Yorkella reginsburgii PSI-02 according to claim 1 or the bacterial preparation according to claim 2 or 3 in preventing and controlling plant diseases and improving plant stress resistance.

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