Geotrophic bacteria SG265 and application thereof

Abstract

The invention provides a geotrophic bacterium SG265 and application thereof, and belongs to the technical field of microbial agents. The geotrophic bacteria SG265 disclosed by the invention is preserved in Guangdong Microbial Culture Collection Center on May 7, 2025, the preservation number is GDMCC No: 66266, and the geotrophic bacteria SG265 is named as Geotalea sp. In taxonomy. The terrestrial nutrient bacteria SG265 are anaerobic nitrogen-fixing bacteria, have iron reduction and nitrogen fixation functions at the same time, can reduce nitrogen into nitrogen fertilizer available for crops under the anaerobic condition, can greatly increase the content of available nitrogen in soil, can effectively promote rice growth, can reduce the application amount of the nitrogen fertilizer, can reduce environmental pollution, and can improve the yield of rice. The method has a good application prospect in agricultural production.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a geotroph SG265 and its application. Background Art

[0002] Rice is the largest food crop in China, and nitrogen is the main limiting factor for rice yield. Biological nitrogen fixation and chemical nitrogen fertilizers are the main nitrogen sources for rice growth. However, the large application of chemical nitrogen fertilizers will cause a certain degree of environmental pollution. Biological nitrogen fixation is an important process in paddy fields, which can maintain the nitrogen balance in flooded paddy fields, reduce the application of chemical fertilizers, and reduce environmental pollution.

[0003] Rice originated from an aquatic environment. Wild rice mostly grows in wetland swamps and requires sufficient water to survive. Traditional paddy fields maintain a water layer through irrigation to ensure that the roots absorb water and nutrients. Research shows that the dominant nitrogen-fixing bacteria in flooded paddy fields are members of the Geobacteraceae family with iron-reducing effects ( Geobacteraceae ), such as the genus Geobacter ( Geobacter ), and the genus Anaeromyxobacter ( Anaeromyxobacter ). Moreover, it has been found that the iron reduction reaction is positively correlated with nitrogen fixation. Increasing the iron reduction ability of paddy fields can improve the nitrogen fixation activity of nitrogen-fixing bacteria. Thus, it can be seen that iron-reducing nitrogen-fixing bacteria play an important role in biological nitrogen fixation in flooded paddy fields. However, most of the current nitrogen-fixing bacteria are aerobic bacteria. For example, the nitrogen-fixing bacteria in the invention with the application number "201910620590.0" and the invention name "A nitrogen-fixing bacterium N24 with nitrogen-fixing effect and its application" and the invention with the application number "201911414175.6" and the invention name "Halophilic nitrogen-fixing bacterium and method for treating chromium-polluted soil in high-saline-alkali environment by using the bacterium" are all aerobic bacteria and cannot adapt to the anaerobic environment of paddy fields.

[0004] The genus Geotroph ( Geotalea ), is a newly discovered class of strictly anaerobic, Gram-negative bacteria in the Geobacteraceae family. The genus Geotroph ( Geotalea ), is a new genus further divided from the genus Geobacter ( Geobacter ). Currently, this genus only contains 3 validly published species, and the research reports on related resources are relatively scarce. The globally culturable microorganisms account for about 1%, while the pure cultures of anaerobic microorganisms account for less than 0.1% of the globally culturable microorganisms, and most anaerobic microorganisms are in an uncultured state. Therefore, it is an urgent problem for those skilled in the art to explore new species of Geotroph with iron reduction and nitrogen-fixing functions. Summary of the Invention

[0005] In view of this, the present invention provides a geotroph SG265 and its application. This strain is an anaerobic bacterium, which can adapt to the anaerobic environment in paddy fields and play a nitrogen-fixing role, and can be applied to paddy fields.

[0006] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions: The present invention provides a strain of soil-bred bacteria SG265, which is deposited in the Guangdong Microbial Culture Collection Center, with the deposit number of GDMCC No: 66266, the deposit date of May 7, 2025, and the deposit address of the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou.

[0007] The present invention also provides a method for preparing the bacterial liquid of the soil-bred bacteria SG265, inoculating the soil-bred bacteria SG265 into a fermentation medium for fermentation culture to obtain the bacterial liquid.

[0008] Preferably, the temperature of the fermentation culture is 28-32 °C, and the time of the fermentation culture is 3-5 d.

[0009] Preferably, the effective viable bacteria count in the bacterial liquid is 1-10×10 8 CFU / mL.

[0010] The present invention also provides a biological bacterial agent, and the microbial bacterial agent includes the soil-bred bacteria SG265 described above.

[0011] Preferably, the dosage form of the microbial bacterial agent includes at least one of agar bacterial agent, liquid bacterial agent, freeze-dried bacterial powder, solid peat powder, oil-dried bacterial agent, granular inoculant, and vacuum infiltration inoculant.

[0012] The present invention also provides the application of the soil-bred bacteria SG265 described above in any one of the following: (1) Application in nitrogen fixation fermentation; (2) Application in biological enzyme production; (3) Application in promoting plant growth and / or increasing yield; (4) Application in improving and fertilizing soil.

[0013] Preferably, the nitrogen fixation fermentation includes soil nitrogen fixation and organic matter fermentation.

[0014] Preferably, the enzyme includes at least one of alkaline phosphatase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase.

[0015] Preferably, the plant includes rice.

[0016] By adopting the above technical solutions, the present invention has the following beneficial effects: (1)The soil-borne bacterium SG265 of the present invention is an anaerobic nitrogen-fixing bacterium, which has both iron reduction and nitrogen fixation functions. It can reduce nitrogen gas to nitrogen fertilizer available to crops under anaerobic conditions, greatly increase the content of available nitrogen in the soil, effectively promote the growth of rice, reduce the application amount of nitrogen fertilizer, and reduce environmental pollution, showing good application prospects in agricultural production.

[0017] (2)The soil-borne bacterium SG265 of the present invention has the ability to produce alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, and can be used as an alternative strain for biological enzyme production and for the development and utilization of the biological enzyme production industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a colony morphology diagram of the soil-borne bacterium SG265.

[0019] Figure 2 It is a phylogenetic tree map of the soil-borne bacterium SG265 of the present invention based on the 16S rRNA gene.

[0020] Figure 3 It is the concentration of Fe(II) produced by the soil-borne bacterium SG265 at different times.

[0021] Figure 4 It is to detect the cytochrome absorption peaks of the strain SG265 at wavelengths of 425, 523 and 524 nm.

[0022] Figure 5 It is for PCR amplification using the nitrogen fixation gene nifH primer, and the electrophoresis diagram of the PCR product.

[0023] Figure 6 It is a schematic diagram of the nitrogenase activity of the strain SG265.

[0024] BIOLOGICAL DEPOSIT DESCRIPTION The soil-borne bacterium ( Geotalea sp.) SG265 of the present invention is deposited in the Guangdong Provincial Microbial Culture Collection Center, with the deposit number GDMCC No: 66266, the deposit date being May 7, 2025, and the deposit address being the 5th floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION OF THE INVENTION

[0025] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Experimental materials: MFM medium (L -1): 2.0 g KHCO3, 0.02 g MgSO4·7H2O, 0.3 g KH2PO4, 1.0 g NH4Cl, 0.1 g MgCl2·6H2O, 0.08 g CaCl2·2H2O, 0.6 g NaCl, 9.52 g HEPES, 10 mL mineral solution [(L -1 ): NTA Trisodium Salt (Free acid) 1.50 g, MgSO4 3.00 g, MnSO4·H2O 0.50 g, NaCl 1.00 g, FeSO4·7H2O 0.10 g, CaCl2·2H2O 0.10 g, CoCl2·6H2O 0.10 g, ZnCl2 0.13 g, CuSO4·5H2O 0.01 g, AlK(SO4)2·12H2O 0.01 g, H3BO3 0.01 g, NaMoO4·2H2O 0.09 g], 10 mL vitamin mixture [(L -1 ): biotin 0.002 g, pantothenic acid 0.005 g, vitamin B12 0.0001 g, p-aminobenzoic acid 0.005 g, lipoic acid (α-) 0.005 g, nicotinic acid 0.005 g, thiamine 0.005 g, riboflavin 0.005 g, pyridoxine hydrochloride 0.01 g, folic acid 0.002 g], pH = 6.8.

[0027] Solid R2A medium (g / L): 0.5 g yeast extract, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g potassium dihydrogen phosphate, 0.024 g anhydrous magnesium sulfate, 0.3 g sodium pyruvate, 15.0 g agar, 1000 mL distilled water, pH = 7.0.

[0028] Liquid R2A medium (g / L): 0.5 g yeast extract, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g potassium dihydrogen phosphate, 0.024 g anhydrous magnesium sulfate, 0.3 g sodium pyruvate, 1000 mL distilled water, pH = 7.0.

[0029] The liquid R2A medium was aerated and deoxygenated for 0.5 h with a gas mixture of N2:CO2 (80:20, vol / vol) to form an anaerobic environment. After sealing with an aluminum cap, it was autoclaved at 121 °C for 20 min.

[0030] Example 1. Isolation, purification and physicochemical property identification of the soil bacterium SG265 1. Sample enrichment culture Soil samples were collected from unfertilized paddy soils. 10 g of paddy soil was weighed and added to 90 mL of sterile MFM liquid medium. N2:CO2 (V / V, 80:20) was used to deoxygenate for 0.5 h to form an anaerobic environment. After sealing with an aluminum cap, it was statically enriched and cultured at 30 °C for 2 weeks.

[0031] 2. Isolation and purification of strains 100 μL of freshly gradient-diluted soil suspension and enrichment solution were taken and spread on modified R2A (added with 20 mM sodium fumarate) plates, and strictly anaerobically cultured at 30 °C for 10 d. Red colonies on the plates were picked and purified by the continuous streaking method until single pure cultures were obtained. All purified strains were preserved at -80 °C with 10% DMSO preservation solution. All operations were carried out in an anaerobic glove box workbench to ensure an anaerobic environment. After culturing on the modified R2A plate for 3 days, the colony shape of strain SG265 was round, with a smooth edge and a smooth surface. The colony was red, and the colony diameter was 0.5 - 1 mm, as Figure 1 shown.

[0032] Gram staining: One drop of 3% KOH solution was added to a glass slide. A loopful of the colony to be tested was picked with an inoculation loop and mixed with the 3% KOH solution on the slide. After 30 - 60 s, the suspension of bacteria in the KOH solution became viscous and even formed a jelly-like substance, which was Gram-negative bacteria. If it showed a uniform suspension and no jelly-like substance appeared, it was Gram-positive bacteria. The Gram staining result showed that strain SG265 was a Gram-negative bacterium.

[0033] 3. Physiological and biochemical characteristics Temperature adaptability: Prepare modified R2A medium (0.5 g yeast extract powder, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g potassium dihydrogen phosphate, 0.024 g anhydrous magnesium sulfate, 0.3 g sodium pyruvate, 6.4 g sodium fumarate, 1000 mL distilled water, pH = 7.0.) plates (1.5% agar). Dip the bacterial solution with an inoculation loop and inoculate it on the plate by the continuous streaking method. Place it at 6, 10, 13, 16, 20, 25, 30, 33, 37, 40 and 42 °C, with 3 parallels set for each temperature. After one week, measure the diameter of the colonies.

[0034] pH adaptability: Prepare modified R2A medium + 1.5% agar plates with pH (5.0 - 9.0, gradient of 0.5). Dip the bacterial solution with an inoculation loop and streak it on the plate by the continuous streaking method. Do 3 parallels for each pH. After one week, measure the diameter of the colonies.

[0035] NaCl concentration adaptability: Prepare modified R2A medium + 1.5% agar plates with NaCl concentrations (0 - 5%, [w / v], gradient of 0.5%). Dip a inoculation loop into the bacterial solution and streak it on the plate using the continuous streaking method. Do 3 replicates for each salt concentration. After one week, measure the diameter of the colonies.

[0036] The results showed that the new strain SG265 tolerated temperatures of 20 - 37 °C, with an optimal growth temperature of 30 °C, tolerated pH values of 5.0 - 9.0, with an optimal pH of 7.0 - 8.0, and could grow at NaCl concentrations of 0 - 0.2% (w / v), with an optimal NaCl growth concentration of 0% (w / v).

[0037] Aerobicity: Streak the strain on a modified R2A plate and place the inoculated R2A plate in a 30 °C incubator for aerobic culture for two weeks.

[0038] Observation found that the strain SG265 did not grow and form colonies on the plate, indicating that the strain SG265 could not grow under aerobic conditions.

[0039] Motility: Prepare modified R2A semi-solid medium (0.5% agar). Scrape the colonies on the plate with an inoculation loop and insert it into the semi-solid medium. After three days, observe the growth status and motility of the strain.

[0040] Observation found that in addition to growth on the inoculated puncture line, feathery or cloudy turbidity growth was visible on both sides of the puncture line, indicating that the strain SG265 had certain motility.

[0041] Enzyme production characteristics: Use the API ZYM enzyme activity detection reagent strip to detect the enzyme production characteristics of the new strain SG265. The operating steps are shown in the instruction manual.

[0042] The results showed that the strain SG265 had the ability to produce alkaline phosphatase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, and did not produce lipase (C4), lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminidase, α-mannosidase, and β-fucosidase.

[0043] Electron donor and electron acceptor tests: In the electron acceptor tests, 10 mM sodium acetate was used as the electron donor in all cases; in all electron donor tests, 10 mM ferric citrate was used as the electron acceptor. The strain SG265 was inoculated into anaerobic sterile MFM medium and cultured at 30 °C. The biomass and changes in the color of the medium were measured to determine the electron acceptor and donor spectra.

[0044] The test results showed that ferric citrate was used as the electron acceptor, and phenol, benzene, pyruvate, lactate, benzoate, propionate, ethanol, methanol, glucose, formate, malate, acetate and succinate were used as electron donors. When sodium acetate was used as the electron donor, fumarate, AQDS and ferric citrate could be used as electron acceptors.

[0045] In all the electron acceptor tests, 10 mM sodium acetate was used as the electron donor; in all the electron donor tests, 10 mM ferric citrate was used as the electron acceptor. The strain SG265 was inoculated into an anaerobic sterile MFM medium and cultured at 30 °C, and the biomass and the color change of the medium were measured to determine the electron acceptor and donor spectra.

[0046] The results showed that when ferric citrate was used as the electron acceptor, phenol, benzene, pyruvate, lactate, benzoate, propionate, ethanol, methanol, glucose, formate, malate, acetate and succinate were used as electron donors. When sodium acetate was used as the electron donor, fumarate, AQDS and ferric citrate could be used as electron acceptors.

[0047] Example 2. Gene identification and phylogenetic analysis of Geobacter SG265 16S rRNA gene identification The isolated strain was amplified by the universal primers for bacterial 16S rRNA gene 27F: 5′-GAG TTTGAT CCT GGC TCA G-3′ (SEQ ID NO.1); 1492R: 5′-ACG GCT ACCTTG TTA CGA CTT-3′ (SEQ ID NO.2).

[0048] PCR reaction program: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 60 s, extension at 72 °C for 90 s, for a total of 30 cycles; finally, extension at 72 °C for 10 min.

[0049] After the PCR reaction, 5 μL of the PCR product was taken and loaded onto a 1% agarose gel. Using 100 bp Marker as the standard molecular weight, electrophoresis was carried out at 100 V for 30 min, and the electrophoresis results were observed using a gel imaging system. The PCR products of the strains with detectable bands were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The ContigExpress software was used to proofread the 16S rRNA gene sequences obtained by sequencing, removing the random bases at both ends, and the obtained valid sequences were submitted to EZBioCloud (https: / / www.ezbiocloud.net / ) and NCBI (https: / / www.ncbi.nlm.nih.gov / ) for sequence alignment analysis. If the alignment results showed that the similarity of the 16S rRNA gene was higher than 98.65%, it was preliminarily judged that it was at the same species-level taxonomic status as the closest strain; otherwise, it was judged as a potential new taxonomic unit.

[0050] The results showed that: SG265 and the closest type strain Geotalea daltonii FRC-32 T had a 16S rRNA gene similarity of 98.2%, lower than the prokaryotic species delineation threshold of 98.65%, and was a potential new species of the genus Geotalea.

[0051] Phylogenetic analysis Based on the alignment results of the EZBioCloud and NCBI databases, the 16S rRNA gene sequences of the type strains similar to the isolated strains in the databases were downloaded. Using the MEGA X software, the Kimura 2-parameter method was used to calculate the evolutionary distance and construct a Maximum Likelihood phylogenetic tree, as Figure 2 shown.

[0052] Genomic ANI and dDDH analysis The digital DNA-DNA hybridization (dDDH) was estimated using the online calculation software GGDC (Genome-to-Genome Distance Calculator), and the average nucleotide identity (ANI) between the bacterium and its type strain was calculated using the ANI Calculator.

[0053] The results showed that the genome size of strain SG265 was 4.1 Mbp, the DNA G+C content was 55.4%, and the ANI and dDDH with the closest type strain Geotalea daltonii FRC-32T were 80% and 22.8% respectively, lower than the prokaryotic species delineation thresholds of 95% and 70%. Therefore, SG265 is a new species of the genus Geotalea.

[0054] Genomic function analysis revealed that SG265 has the core genes of nitrogenase, nifHDK, suggesting that this strain has nitrogen fixation function.

[0055] Example 3. Fatty Acid Detection ① Bacterial Obtaining: Pick about 40 mg of bacteria in the logarithmic phase (cultured for 3 d) and put them into a test tube with a screw cap (specification 13 mm × 100 mm).

[0056] ② Saponification: Add 1.0 mL of saponification reagent into the test tube, tighten the cap, shake the test tube on an oscillator for 5 - 10 s, place it in a boiling water bath for 5 min, take it out and continue to shake for 5 - 10 s, tighten the cap again, continue the water bath for 25 min, remove the test tube, and cool it at room temperature.

[0057] ③ Methylation: Add 2.0 mL of methylation reagent into the test tube, tighten the cap, shake it on an oscillator for 5 - 10 s, conduct a water bath at 80 °C for 10 min, remove the test tube and quickly cool it to room temperature by flushing with tap water.

[0058] ④ Extraction: Add 1.25 mL of extraction reagent, tighten the cap, shake it quickly for 10 min, open the tube cap, and suck out the lower aqueous phase in the test tube with a pipette and discard it.

[0059] ⑤ Washing: Add 3.0 mL of washing reagent into the test tube, tighten the cap, shake it quickly for 5 min, suck out about 2 / 3 volume of the upper organic matter into a GC vial with a syringe for testing.

[0060] ⑥ Detection: The analytical instrument uses the Agilent 7890N gas chromatography system of the United States for detection.

[0061] Detection Procedure: Set the temperature of the vaporization chamber at 250 °C, the temperature of the detector at 300 °C, the flow rate of the carrier gas hydrogen at 2 mL·min -1 , the flow rate of the tail gas nitrogen at 30 mL·min -1 , the injection split ratio at 100:1, the column head pressure at 68.95 kPa; the chromatographic column uses a second-order temperature program. Start heating from 170 °C, increase the temperature by 5 °C per minute. When it reaches 260 °C, increase the temperature by 40 °C per minute. When it reaches 310 °C, hold for 90 s; the injection volume is 1 μL.

[0062] Analysis Software: The fully automatic microbial identification system Sherlock MIS4.5 (Microbial Identification System) and LGS4.5 (Library Generation Software) developed by MIDI Inc. of the United States, which is based on the identification of fatty acid components of microbial cells.

[0063] The results showed that the main fatty acids in strain SG265 were C 14:0 , iso-C 15:0 , C 16:0 3OH, C 16:0 , and Summed Feature 3, and the main respiratory quinone component was MK-8.

[0064] Example 4. Functional determination of soil bacterium SG265 1. Determination of iron reduction ability Standard curve: The standard curve y = 2.0465x - 0.0028 was established, with R 2 = 1.

[0065] Under anaerobic conditions, the SG265 seed fermentation broth was inoculated into an anaerobic test tube containing 10 mL of sterilized 20 mM ferrihydrite medium, with 3 parallels set, and the non-inoculated one served as the blank control.

[0066] Determination of Fe (II): Every 48 h, 0.1 mL of the sample was taken and added to a centrifuge tube containing 0.9 mL of 0.5 M HCl, and the absorbance was measured at a wavelength of 562 nm. The results were as Figure 3 shown.

[0067] Figure 3 shown that SG265 had a high iron reduction ability and reduced most of the Fe (III) in ferrihydrite to Fe (II).

[0068] 2. Cytochrome determination The isolated strain was cultured in modified R2A medium for 3 d. After centrifuging 5 mL of the cultured bacterial solution, it was suspended in 9 mL of 20 mM PIPES buffer (pH 7) and 9 mL of 20 mM PIPES buffer (pH 7) containing 2 mM sodium dithionite. After 6 h of reduction, the differential spectrum of the sodium dithionite-reduced negative air oxidation of the cells was obtained by scanning at a wavelength of 400 - 800 nm with a UV-2600 (SHIMADZU, Japan) spectrophotometer. The results were as Figure 4 shown.

[0069] Figure 4 shown that strain SG265 detected cytochrome absorption peaks at wavelengths of 425, 523, and 524 nm.

[0070] 3. Nitrogen fixation ability determination Nitrogen fixation gene nifH Amplification: Using nifH gene primers Pol-F: 5'-TGC GAY CCS AAR GCB GAC TC-3' (SEQ ID NO.3); Pol-R: 5'-ATS GCC ATC ATY TCR CCG GA-3' (SEQ ID NO.4). PCR amplification was performed on the isolated strain to determine whether it has nitrogen fixation function.

[0071] PCR reaction procedure: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s; annealing at 60 °C for 45 s; extension at 72 °C for 450 s; 40 cycles, and finally extension at 72 °C for 45 s. Take 5 μL of PCR product and load it on a 1% agarose gel. Using 100 bp Marker as the standard molecular weight, electrophoresis was carried out at 100 V for 30 min, and the electrophoresis result was observed with a gel imaging system.

[0072] Using the nitrogen fixation gene nifH The primer pair was used to perform PCR amplification on the isolated strain SG265. The PCR amplification result showed that the amplified nifH gene fragment ( Figure 5 ) indicated that the strain SG265 has nitrogen fixation ability.

[0073] ARA determination of nitrogenase activity: The acetylene reduction method (ARA method) was used to measure the nitrogenase activity. When the SG265 strain was cultured in the modified R2A medium to the logarithmic growth phase, it was transferred to a sterile anaerobic centrifuge tube and centrifuged at 6000 rpm for 10 min to remove the supernatant. After rinsing with 30 mL of modified nitrogen-free medium, it was transferred to an anaerobic bottle exchanged with He / C2H2 (90:10 [vol / vol]) (first sterilize the anaerobic bottle and the anaerobic rubber stopper, evacuate after covering the stopper, and then fill with the mixed gas). Similarly, after rinsing with 30 mL of modified nitrogen-free medium, it was transferred to an anaerobic bottle exchanged with pure He as a negative control. After culturing for 3 d, the content of reduced ethylene was detected by gas chromatography, and the results were as Figure 6 shown.

[0074] The results showed that the strain SG265 has a relatively high nitrogenase activity, reaching 2830 ± 20 μmol C2H4 g −1 protein h −1 .

[0075] Example 5. Preparation of bacterial agent Under sterile conditions, the seed liquid of the strain SG265 was inoculated into the deoxygenated sterile modified R2A liquid medium at an inoculation amount of 1%, and cultured statically (anaerobically) at 30 °C for 5 d to prepare the bacterial agent of the nitrogen-fixing soil bacterium SG265; the effective viable count in the seed liquid of the strain SG265 was 4×108 CFU / mL.

[0076] As can be seen from the above examples, the present invention provides a soil-borne bacterium SG265 and its application. The soil-borne bacterium SG265 of the present invention can play a nitrogen fixation role under anaerobic conditions and can be applied to paddy fields.

[0077] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A soil-cultivated bacterium SG265, characterized in that, Deposited with the Guangdong Microbial Culture Collection Center, the deposit number is GDMCC No: 66266, the deposit date is May 7, 2025, and the deposit address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

2. The method for preparing the bacterial liquid of the bacteria-raising SG265 described in claim 1, characterized in that, Inoculate the soil-cultivated bacterium SG265 into a fermentation medium for fermentation culture to obtain the bacterial liquid.

3. The preparation method according to claim 2, wherein, The temperature of the fermentation culture is 28-32 °C, and the time of the fermentation culture is 3-5 d.

4. The preparation method according to claim 2, characterized in that, The effective viable count in the bacterial liquid is 1 to 10×10 8 CFU / mL.

5. A microbial inoculant, characterized in that, The microbial inoculant includes the soil-cultivated bacterium SG265 described in claim 1.

6. The microbial inoculant according to claim 5, characterized in that, The dosage form of the microbial inoculant includes at least one of agar inoculant, liquid inoculant, freeze-dried bacterial powder, solid peat powder, oil-dried inoculant, granular inoculant and vacuum infiltration inoculant.

7. Use of the soil-cultivated bacterium SG265 described in claim 1 in any of the following: (1) Use in nitrogen fixation fermentation; (2) Use in biological enzyme production; (3) Use in promoting plant growth and / or increasing yield; (4) Use in improving and fertilizing soil.

8. The application according to claim 7, wherein The nitrogen fixation fermentation includes soil nitrogen fixation and organic matter fermentation.

9. The application according to claim 7, wherein The enzyme includes at least one of alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase.

10. The application according to claim 7, wherein, The plant includes rice.

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