A strain of ground-growing bacteria SG265 and its application

Abstract

The present invention provides a strain of geotrophic bacteria SG265 and its application, belonging to the technical field of microbial agents. The geotrophic bacteria SG265 of the present invention was deposited in Guangdong Province Microbiological Culture Collection Center on May 7, 2025, with the deposit number GDMCC No: 66266, and its taxonomic name is Geolocation sp. The geotrophic bacteria SG265 of the present invention is an anaerobic nitrogen-fixing bacterium with both iron reduction and nitrogen fixation functions. It can reduce nitrogen gas into nitrogen fertilizer that can be used by crops under anaerobic conditions. It can significantly increase the content of available nitrogen in the soil, effectively promote rice growth, reduce the amount of nitrogen fertilizer applied, and reduce environmental pollution. It has good application prospects in agricultural production.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a geotrophic bacterium SG265 and applications thereof. Background Art

[0002] Rice is my country's top grain crop, and nitrogen is the primary limiting factor in rice yield. Biological nitrogen fixation and chemical nitrogen fertilizers are the primary nitrogen sources for rice growth, but the large-scale application of chemical nitrogen fertilizers can cause a certain degree of environmental pollution. Biological nitrogen fixation is a key process in rice fields, maintaining nitrogen balance in flooded paddies, reducing the need for chemical fertilizers, and minimizing environmental pollution.

[0003] Rice originates from aquatic environments. Wild rice mostly grows in wetlands and swamps and requires sufficient water to survive. Traditional paddy fields are irrigated to maintain a water layer to ensure that the roots absorb water and nutrients. Studies have shown that the dominant nitrogen-fixing bacteria in flooded rice fields are the iron-reducing Geobacteraceae ( Geobacteraceae ) members, such as Geobacter ( Geobacter ) and Anaerobic Myxobacterium ( Anaeromyxobacter ), and it was found that the iron reduction reaction was positively correlated with nitrogen fixation. Increasing the iron reduction capacity of paddy fields can increase the nitrogen fixation activity of nitrogen-fixing bacteria. It can be seen that iron-reducing nitrogen-fixing bacteria play an important role in biological nitrogen fixation in flooded rice fields. However, most of the current nitrogen-fixing bacteria are aerobic bacteria. For example, the nitrogen-fixing bacteria in the application number "201910620590.0" and the invention name "A nitrogen-fixing bacterium N24 with nitrogen-fixing effect and its application" and the application number "201911414175.6" and the invention name "Halophous nitrogen-fixing bacteria and the method for treating chromium-contaminated soil in a high-salt-alkali environment" are aerobic bacteria and cannot adapt to the anaerobic environment of paddy fields.

[0004] Geotrophic bacteria ( Geotalea ) is a newly discovered type of strictly anaerobic, Gram-negative bacteria in the family Geobacteraceae. Geotalea ) is from the genus Geobacter ( Geobacter ) is a new genus further delineated from the genus. Currently, this genus contains only three published species, and research reports on related resources are relatively scarce. While approximately 1% of the world's culturable microorganisms are culturable, pure cultures of anaerobic microorganisms account for less than 0.1% of this global total, leaving the majority of anaerobic microorganisms uncultured. Therefore, discovering new species of geotrophic bacteria with iron-reducing and nitrogen-fixing capabilities is a pressing challenge for those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a geotrophic bacterium SG265 and its application. The bacterium is an anaerobic bacterium that can adapt to the anaerobic environment in rice paddies and play a nitrogen-fixing role, and can be used in rice paddies.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a geotrophic bacterium SG265, which is deposited in Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No: 66266, a deposit date of May 7, 2025, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The present invention also provides a method for preparing a bacterial liquid of the geotrophic bacterium SG265, wherein the geotrophic bacterium SG265 is inoculated into a fermentation medium for fermentation culture to obtain the bacterial liquid.

[0009] Preferably, the fermentation temperature is 28-32° C., and the fermentation time is 3-5 days.

[0010] Preferably, the effective viable bacteria count in the bacterial solution is 1~10×10 8 CFU / mL.

[0011] The present invention also provides a biological microbial agent, wherein the microbial agent includes the geotrophic bacteria SG265.

[0012] Preferably, the dosage form of the microbial agent includes at least one of agar agent, liquid agent, freeze-dried powder, solid peat powder, oil-dried agent, granular inoculant and vacuum infiltration inoculant.

[0013] The present invention also provides the use of the geotrophic bacteria SG265 in any of the following:

[0014] (1) Application in nitrogen-fixing fermentation;

[0015] (2) Application in bio-enzyme production;

[0016] (3) Application in promoting plant growth and / or increasing yield;

[0017] (4) Application in improving and fertilizing soil.

[0018] Preferably, the nitrogen fixation fermentation includes soil nitrogen fixation and organic matter fermentation.

[0019] Preferably, the enzyme comprises at least one of alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase.

[0020] Preferably, the plant comprises rice.

[0021] By adopting the above technical solution, the present invention has the following beneficial effects:

[0022] (1) The geotrophic bacteria SG265 of the present invention is an anaerobic nitrogen-fixing bacterium that has both iron reduction and nitrogen fixation functions. It can reduce nitrogen gas into nitrogen fertilizer that can be used by crops under anaerobic conditions, significantly increase the content of available nitrogen in the soil, effectively promote rice growth, reduce the amount of nitrogen fertilizer applied, and reduce environmental pollution. It has good application prospects in agricultural production.

[0023] (2) The geotrophic 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 bio-enzyme production and the development and utilization of the bio-enzyme production industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the colony morphology of the geotrophic bacterium SG265.

[0025] Figure 2 This is the phylogenetic tree of the 16S rRNA gene of the ground bacteria SG265 of the present invention.

[0026] Figure 3 The concentration of Fe(Ⅱ) produced by geotrophic bacteria SG265 at different times.

[0027] Figure 4 The cytochrome absorption peaks were detected at wavelengths of 425, 523, and 524 nm for strain SG265.

[0028] Figure 5 To utilize nitrogen fixation genes nif Primers were used for PCR amplification, and electrophoresis of PCR products was shown.

[0029] Figure 6 Schematic diagram of nitrogenase activity of strain SG265.

[0030] Biological Deposit Description

[0031] The geotrophic bacteria of the present invention ( Geotalea sp.) SG265 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 66266 and the deposit date of May 7, 2025. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0032] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Experimental Materials:

[0034] 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, NaCl1.00 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, niacin 0.005 g, thiamine 0.005 g, riboflavin 0.005 g, pyridoxine hydrochloride 0.01 g, folic acid 0.002 g], pH = 6.8.

[0035] Solid R2A medium (g / L): 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, 15.0 g agar, 1000 mL distilled water, pH = 7.0.

[0036] Liquid R2A medium (g / L): 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, 1000 mL distilled water, pH = 7.0.

[0037] Liquid R2A culture medium was aerated and deoxygenated with a mixed gas of N2:CO2 (80:20, vol / vol) for 0.5 h to create an anaerobic environment, sealed with an aluminum cap, and then sterilized by autoclaving at 121°C for 20 min.

[0038] Example 1. Isolation, purification and physicochemical properties of geotrophic bacteria SG265

[0039] 1. Sample enrichment and culture

[0040] Soil samples were collected from unfertilized paddy soil. 10 g of paddy soil was weighed and added to 90 mL of sterile MFM liquid medium. The medium was deoxygenated with N2:CO2 (V / V, 80:20) for 0.5 h to create an anaerobic environment. The soil was sealed with an aluminum cap and then incubated at 30 °C for 2 weeks.

[0041] 2. Isolation and Purification of Strain

[0042] Take 100 μL of gradient diluted fresh soil suspension and enrichment solution, spread it on the modified R2A (added with 20 mM sodium fumarate) plate, and culture it strictly anaerobically at 30 ℃ for 10 days. Pick the red colonies on the plate and purify them by continuous streaking until a single pure culture is obtained. All purified strains are stored at -80 ℃ in 10% DMSO preservative solution. All operations are carried out in an anaerobic glove box workbench to ensure an anaerobic environment. After 3 days of culture on the modified R2A plate. The colony shape of strain SG265 is round, with smooth edges and a smooth surface. The colonies are red and the colony diameter is 0.5~1 mm. Figure 1 shown.

[0043] Gram staining: Add one drop of 3% KOH solution to a glass slide. Use a loop to pick a loopful of the bacterial colonies to be tested and mix them evenly with the 3% KOH solution on the slide. After 30-60 seconds, if the bacterial suspension in the KOH solution becomes viscous and jelly-like, it is a Gram-negative bacterium. If it is a homogeneous suspension without a jelly-like substance, it is a Gram-positive bacterium. The Gram staining results indicate that strain SG265 is a Gram-negative bacterium.

[0044] 3. Physiological and biochemical characteristics

[0045] 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) on 1.5% agar plates. Use a loop to dip the bacterial solution and inoculate it onto the plates by continuous streak method. Incubate the plates at 6, 10, 13, 16, 20, 25, 30, 33, 37, 40, and 42°C, with three replicates at each temperature. After one week, measure the diameter of the colonies.

[0046] pH adaptability: Prepare pH (5.0-9.0, gradient of 0.5) modified R2A medium + 1.5% agar plates. Use a loop to dip the bacterial solution and streak on the plate using the continuous streak method. Make three parallel streaks for each pH value. After one week, measure the diameter of the colonies.

[0047] Adaptability to NaCl concentration: Prepare NaCl concentrations (0-5%, [w / v], gradient of 0.5%) modified R2A medium + 1.5% agar plates. Use a loop to dip the bacterial solution and streak the plates using the continuous streak method. Make three parallel streaks for each salt concentration. After one week, measure the diameter of the colonies.

[0048] The results showed that the new strain SG265 could tolerate temperatures of 20-37 ℃, with an optimal growth temperature of 30 ℃, a pH range of 5.0-9.0, and an optimal pH of 7.0-8.0. It could grow at NaCl concentrations of 0-0.2% (w / v), with the optimal NaCl growth concentration being 0% (w / v).

[0049] Aerobicity: Streak the strain onto a modified R2A plate and place the inoculated R2A plate in a 30°C constant temperature incubator for aerobic culture for two weeks.

[0050] It was observed that strain SG265 did not grow on the plate to form colonies, indicating that strain SG265 could not grow under aerobic conditions.

[0051] Motility: Prepare a modified R2A semi-solid medium (0.5% agar), scrape the colonies from the plate with an inoculation loop, insert them into the semi-solid medium, and observe the growth and motility of the strain after three days.

[0052] It was observed that in addition to the growth on the inoculated puncture line, feathery or cloudy turbid growth was seen on both sides of the puncture line, indicating that strain SG265 had a certain motility.

[0053] Enzyme production characteristics: Use API ZYM enzyme activity test strips to detect the enzyme production characteristics of the new strain SG265. The operating steps are shown in the instructions.

[0054] The results showed that strain SG265 had the ability to produce alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but did not produce lipid enzymes (C4), lipid esterase (C8), lipase (C14), leucine arylaminease, valine arylaminease, cystine arylaminease, trypsin, chymotrypsin, α-galactosidase, β-galactosidase, β-uronidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminidase, α-mannosidase and β-fucosidase.

[0055] Electron Donor and Acceptor Assays: 10 mM sodium acetate was used as the electron donor in all electron acceptor assays, and 10 mM ferric citrate was used as the electron acceptor in all electron donor assays. Strain SG265 was inoculated into sterile, anaerobic MFM medium and cultured at 30°C. Biomass and medium color changes were measured to determine the electron acceptor and donor profiles.

[0056] The experimental results show that ferric citrate is an electron acceptor, while phenol, benzene, pyruvate, lactate, benzoate, propionate, ethanol, methanol, glucose, formate, malate, acetate, and succinate are electron donors. When sodium acetate is used as an electron donor, fumarate, AQDS, and ferric citrate can act as electron acceptors.

[0057] In all electron acceptor experiments, 10 mM sodium acetate was used as the electron donor; in all electron donor experiments, 10 mM ferric citrate was used as the electron acceptor. Strain SG265 was inoculated into sterile, anaerobic MFM medium and cultured at 30°C. Biomass and medium color changes were measured to determine the electron acceptor and donor profiles.

[0058] 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 could serve as electron donors. When sodium acetate was used as the electron donor, fumarate, AQDS, and ferric citrate could serve as electron acceptors.

[0059] Example 2. Gene identification and phylogenetic analysis of geotrophic bacteria SG265

[0060] 16S rRNA gene identification

[0061] Bacterial 16S rRNA gene universal primers

[0062] 27F: 5′-GAG TTTGAT CCT GGC TCA G-3′ (SEQ ID NO. 1);

[0063] 1492R: 5′-ACG GCT ACCTTG TTA CGA CTT-3′ (SEQ ID NO. 2) was amplified from the isolated strain.

[0064] 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, and extension at 72 °C for 90 s, for a total of 30 cycles; and final extension at 72 °C for 10 min.

[0065] After the PCR reaction, 5 μL of PCR product was spotted on a 1% agarose gel. Electrophoresis was performed at 100 V for 30 min using a 100 bp marker as a standard molecular weight. The results were visualized using a gel imaging system. PCR products from strains with detected bands were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16S rRNA gene sequences obtained were proofread using ContigExpress software, and random bases at the beginning and end were removed. Valid sequences were submitted to EZBioCloud (https: / / www.ezbiocloud.net / ) and NCBI (https: / / www.ncbi.nlm.nih.gov / ) for sequence alignment. If the alignment results showed a 16S rRNA gene similarity greater than 98.65%, the strain was preliminarily classified at the same taxonomic level as the closest strain; otherwise, it was identified as a potential new taxon.

[0066] The results showed that SG265 and the closest model strain Geotalea daltonii FRC-32 T The 16S rRNA gene similarity of the two strains was 98.2%, which was lower than the prokaryotic species definition threshold of 98.65%, making it a potential new species of the genus Geotrophicum.

[0067] Phylogenetic analysis

[0068] Based on the comparison results of EZBioCloud and NCBI databases, the 16S rRNA gene sequences of model strains similar to the isolated strains were downloaded from the database. The evolutionary distance was calculated using the Kimura 2-parameter method and the maximum likelihood phylogenetic tree was constructed using MEGA X software. Figure 2 shown.

[0069] Genomic ANI and ddDH analysis

[0070] Digital DNA-DNA hybridization (dDDH) was estimated using GGDC online calculation software (Genome-to-Genome Distance Calculator), and the average nucleotide identity (ANI) between bacteria and their model strains was calculated using ANI Calculator.

[0071] The results showed that the genome of strain SG265 was 4.1 Mbp and its DNA G+C content was 55.4%. Its ANI and dDDH ratios compared to its closest type strain, Geotalea daltonii FRC-32T, were 80% and 22.8%, respectively, below the prokaryotic species definition thresholds of 95% and 70%, respectively. Therefore, SG265 is a new species of the genus Geotalea.

[0072] Genome functional analysis revealed that SG265 has the nitrogenase core gene nifHDK, suggesting that the strain has nitrogen fixation function.

[0073] Example 3. Fatty acid detection

[0074] ① Obtain bacteria: Pick about 40 mg of bacteria in the logarithmic phase (cultured for 3 days) and place them in a test tube with a screw cap (specifications: 13 mm × 100 mm).

[0075] ② Saponification: Add 1.0 mL of saponification reagent to a test tube, tighten the lid, oscillate the test tube on an oscillator for 5–10 seconds, place in a boiling water bath for 5 minutes, remove the tube and continue oscillating for 5–10 seconds, tighten the lid again, continue in the boiling water bath for 25 minutes, remove the tube, and cool to room temperature.

[0076] ③ Methylation: Add 2.0 mL of methylation reagent to the test tube, tighten the lid, oscillate on a shaker for 5-10 seconds, place in an 80°C water bath for 10 minutes, remove the test tube and quickly rinse with tap water to cool to room temperature.

[0077] ④ Extraction: Add another 1.25 mL of extraction reagent, tighten the lid, shake rapidly for 10 min, open the tube lid, use a pipette to aspirate the lower aqueous phase of the test tube and discard it.

[0078] ⑤ Washing: Add 3.0 mL of washing reagent to the test tube, tighten the lid, shake rapidly for 5 minutes, and use a syringe to draw out about 2 / 3 of the volume of the upper layer of organisms into a GC vial for detection.

[0079] ⑥Detection: The analytical instrument used was the American Agilent 7890N gas chromatography system.

[0080] Detection procedure: Set the vaporization chamber temperature to 250 °C, the detector temperature to 300 °C, and the flow rate of the carrier gas hydrogen to 2 mL min -1 The flow rate of the tail gas nitrogen was 30 mL min -1 The injection split ratio was 100:1 and the column head pressure was 68.95 kPa. The column was heated using a two-step temperature program, starting from 170 °C at a rate of 5 °C per minute to 260 °C, then increasing by 40 °C per minute to 310 °C, where it was maintained for 90 s. The injection volume was 1 μL.

[0081] Analysis software: Sherlock MIS4.5 (Microbial Identification System) and LGS4.5 (Library Generation Software), fully automatic microbial identification systems based on the identification of fatty acid composition of microbial cells, developed by MIDI Company of the United States.

[0082] 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, the main respiratory quinone component was MK-8.

[0083] Example 4. Functional assay of geotrophic bacteria SG265

[0084] 1. Determination of iron reducing ability

[0085] Standard curve: Develop a standard curve y=2.0465x-0.0028, R 2 =1.

[0086] Under anaerobic conditions, the fermentation liquid of SG265 seeds was inoculated into anaerobic test tubes containing 10 mL of sterilized 20 mM ferrihydrite medium. Three parallel tubes were set up, and the uninoculated tube served as a blank control.

[0087] Determination of Fe(II): 0.1 mL of sample was taken every 48 h and added to a centrifuge tube containing 0.9 mL of 0.5 M HCl. The absorbance was measured at a wavelength of 562 nm. The results were as follows: Figure 3 shown.

[0088] Figure 3 It was shown that SG265 had a high iron reducing ability and reduced most of the Fe(III) in ferrihydrite to Fe(II).

[0089] 2. Cytochrome Determination

[0090] The isolated strain was grown in modified R2A medium for 3 days. After centrifugation, 5 mL of the culture was resuspended in 9 mL of 20 mM PIPES buffer (pH 7) and 9 mL of 2 mM sodium dithionite in 20 mM PIPES buffer (pH 7). After 6 hours of reduction, the dithionite reduction-negative air oxidation difference spectrum of the cells was obtained by scanning at 400–800 nm using a UV-2600 (SHIMADZU, Japan) spectrophotometer. The results are shown in Figure 2. Figure 4 shown.

[0091] Figure 4 It was shown that strain SG265 detected cytochrome absorption peaks at wavelengths of 425, 523, and 524 nm.

[0092] 3. Determination of nitrogen fixation capacity

[0093] nitrogen-fixing genes nif Amplification: Using nif Gene primers

[0094] Pol-F: 5′-TGC GAY CCS AAR GCB GAC TC-3′ (SEQ ID NO.3);

[0095] Pol-R: 5′-ATS GCC ATC ATY TCR CCG GA-3′ (SEQ ID NO. 4) was amplified by PCR to determine whether the isolated strain had the nitrogen fixation function.

[0096] The PCR reaction procedure was as follows: 95°C initial denaturation 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, followed by a final extension at 72°C for 45 s. Five μL of PCR product was spotted on a 1% agarose gel and electrophoresed at 100 V for 30 min using a 100 bp marker as the molecular weight standard. The results were visualized using a gel imaging system.

[0097] Using nitrogen-fixing genes nif The primers were used to amplify the isolate SG265 by PCR. The results of PCR amplification showed that nif Gene fragments ( Figure 5 ), indicating that strain SG265 has the ability to fix nitrogen.

[0098] ARA determination of nitrogenase activity: The acetylene reduction method (ARA method) is used to measure nitrogenase activity. When the SG265 strain is cultured in the modified R2A medium to the logarithmic growth phase, it is transferred to a sterile anaerobic centrifuge tube, centrifuged at 6000 rpm for 10 min, and the supernatant is removed. After rinsing with 30 mL of modified nitrogen-free medium, it is transferred to an anaerobic bottle exchanged with He / C2H2 (90:10 [vol / vol]) (first sterilize the anaerobic bottle and anaerobic rubber stopper, cover it with the stopper, evacuate, and then fill it with mixed gas). Similarly, after rinsing with 30 mL of modified nitrogen-free medium, it is transferred to an anaerobic bottle exchanged with pure He as a negative control. After culturing for 3 days, the content of reduced ethylene is detected by gas chromatography, and the results are as follows. Figure 6 shown.

[0099] The results showed that strain SG265 had a high nitrogenase activity of 2830±20 μmol C2H4 g −1 protein h −1 .

[0100] Example 5. Preparation of bacterial agent

[0101] Under sterile conditions, the seed liquid of strain SG265 was inoculated into deoxygenated sterile modified R2A liquid medium at a 1% inoculum rate and cultured statically (anaerobically) at 30°C for 5 days to prepare the inoculum of nitrogen-fixing soil bacteria SG265. The effective viable cell count in the seed liquid of strain SG265 was 4×10 8 CFU / mL.

[0102] As can be seen from the above examples, the present invention provides a geotrophic bacterium SG265 and its application. The geotrophic bacterium SG265 of the present invention can perform nitrogen fixation under anaerobic conditions and can be applied to rice paddies.

[0103] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A strain of ground mushroom ( Geotalea sp .) SG265, characterized in that, It is deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 66266, the deposit date is May 7, 2025, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The method for preparing the bacterial solution of the geotrophic bacterium SG265 according to claim 1, characterized in that: Inoculating the geotrophic bacteria SG265 into a fermentation medium for fermentation culture to obtain the bacterial liquid, wherein the fermentation medium is a modified R2A liquid medium; The fermentation temperature is 28-32°C, and the fermentation time is 3-5 days; The effective viable bacteria count in the bacterial solution is 1~10×10 8 CFU / mL.

3. A microbial agent, characterized in that: The microbial agent includes the geotrophic bacteria SG265 described in claim 1.

4. The microbial agent according to claim 3, characterized in that The dosage form of the microbial inoculant includes at least one of agar inoculant, liquid inoculant, freeze-dried inoculant powder, solid peat powder, oil-dried inoculant, granular inoculant and vacuum infiltration inoculant.

5. Use of the geotrophic bacterium SG265 according to claim 1 in any of the following: (1) Application in nitrogen-fixing fermentation; (2) Application in bio-enzyme production; the enzyme is at least one of alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase.

6. The use according to claim 5, characterized in that The nitrogen fixation fermentation includes soil nitrogen fixation and organic matter fermentation.

Images