Enterobacter campanulata and application thereof

Abstract

The invention discloses enterobacter rolvestris and application thereof, the classification name of the enterobacter rolvestris is enterobacter rolvestris, the enterobacter rolvestris is preserved in China General Microbiological Culture Collection Center on July 12, 2024, the preservation number is CGMCC No.31282, and the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain is higher in salt tolerance which can reach 8.5%, and has acid resistance, alkali resistance and higher IAA (indoleacetic acid) production capacity.

Description

Technical Field

[0001] The invention relates to Enterobacter logankampii and application thereof, belonging to the field of biotechnology. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Enterobacteriaceae can promote plant growth, improve plant resistance, increase crop yield and improve crop quality.

[0004] The polyamine-producing Enterobacter cloacae DJ has a higher biomass and grows faster in a medium with arginine as a substrate, and its optimal growth condition is neutral to alkaline conditions. The DJ bacteria were cultured in a simulated saline-alkali environment and their growth was observed. The results showed that the DJ bacteria had stronger motility under saline-alkali conditions, indicating that it has better adaptability under saline-alkali conditions and is suitable for growth in saline-alkali environments.

[0005] The GL-2 strain is Enterobacter ludwigii. A pot experiment was used to study the promoting effect of GL-2 on wheat growth. The GL-2 strain significantly increased the fresh weight of wheat roots, the number of tillers and the dry weight of roots.

[0006] The TIA062 strain is Enterobacter cloacae; the optimal culture conditions are liquid volume 100mL (250mL Erlenmeyer flask), initial pH=7.0, culture temperature 37℃, rotation speed 180r / min, and inoculation size 3%; the optimized culture medium components are 1% corn starch, 1.5% peptone, and 0.3% CaCO3. Under this optimal condition, the colony count obtained after TIA062 culture is 1.325×1011cfu / mL.

[0007] Endophytic Enterobacter cloacae TMX-6 has the ability to secrete indole-3-acetic acid (IAA) and siderophores, solubilize phosphate, solubilize potassium and fix nitrogen. The secretion amount of IAA is 54.89±2.34 mg / L, and the contents of dissolved organic phosphorus and available potassium are 120.76±3.69 mg / L and 9.4±2.07 mg / L. It can promote the growth of rice under thiamethoxam stress, significantly enhance the root activity of rice and increase the fresh weight of rice.

[0008] Ludwig Enterobacter FYP1101 was isolated and purified based on the spread-streaking technique using phytic acid phosphate medium, and the bacteria with nitrogen fixation and inorganic phosphorus decomposition ability were initially screened using Ashby medium and inorganic phosphorus medium, and then the purified bacteria were analyzed for their ability to fix nitrogen, decompose phytic acid phosphate, decompose inorganic phosphorus, produce siderophores, produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, and produce indoleacetic acid (IAA). A pot experiment was used to analyze the growth characteristics of salt-stressed wheat under different treatments, and the salt tolerance reached 8%, and it had a strong nitrogen fixation ability [nitrogenase activity was 2.59 nmol C2H4 / (h·mg protein)], phytic acid phosphorus decomposition ability (2.70μg / mL), inorganic phosphorus decomposition ability (4.29μg / mL), iron carrier production ability (D / d is 2.88), ACC deaminase activity [7.32μmol α-ketobutyric acid / (h·mg protein)], IAA production ability (24.93mg / L). It significantly increased root length (increase of about 46%) and promoted the growth of wheat seedlings.

[0009] However, the Enterobacteriaceae in the existing technology are not comprehensive in the ability to produce IAA, produce iron carriers, fix nitrogen, solubilize potassium, dissolve phosphorus, and utilize ACC; their salt-alkali resistance and growth-promoting abilities need to be improved. Summary of the invention

[0010] In view of the shortcomings of the prior art, the present invention screens a strain Enterobacter roggenkampii strain that is resistant to acid, alkali, salt and has the ability to promote growth, and determines the optimal culture conditions, thereby optimizing the growth-promoting ability of the strain, providing a theoretical basis for the subsequent preparation of plant growth-promoting bacterial preparations.

[0011] The technical solution adopted by the present invention is as follows:

[0012] In the first aspect of the present invention, a strain of Enterobacter roggenkampii is provided, which is classified and named Enterobacter roggenkampii strain, deposited in the China General Microbiological Culture Collection Center on July 12, 2024, with a deposit number of CGMCC No.31282, and the address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] The colony characteristics and bacterial morphology of the described Enterobacter logankamp are as follows: the colony diameter is 2-4 mm, round, with irregular edges, opaque, grayish white or slightly yellow, raised in the middle, smooth surface, bright surface, moist, sticky texture, easy to pick up, shiny, and uniform texture; the bacterial body is short rod-shaped and has no spores.

[0014] The physiological and biochemical characteristics of the Enterobacter logenkamp include: an optimal growth temperature of 30°C; a Gram-negative rod-shaped bacterium; it can produce acid and gas by fermenting glucose, and can utilize sucrose, arabinose, glucose, mannitol, lactose, D-sorbitol, and maltose as carbon sources; it cannot hydrolyze gelatin and cannot utilize cellulose.

[0015] Qualitative and quantitative determination of the functions of the Enterobacter logankampii:

[0016] It can utilize ACC deaminase, produce siderophore (D / d is 36.03%), fix nitrogen, and dissolve organic phosphorus (D / d is 55.79%).

[0017] Sdny-150 was inoculated into a culture medium containing tryptophan (glucose 2%; peptone 1%; yeast extract 1%; NaCl 0.5%; pH 7.0-7.2, sterilized at 121°C for 20 min) and cultured in a shaking incubator at 28°C, 200 rpm for 4 days. The bacterial suspension was centrifuged at 10000 rpm for 10 min, and the supernatant was added with an equal volume of Salkowski (50 mL 35% HClO4 + 1 mL 0.5 M FeCl3) colorimetric solution, and allowed to stand in the dark for 30 min. The OD was determined by spectrophotometry. 530 The content of IAA in the fermentation broth per unit volume was calculated, and the content of IAA was calculated by the standard curve of IAA gradient dilution. The IAA content produced by Sdny-150 was 49.39 μg / mL by determination.

[0018] In a second aspect of the present invention, a microbial mixture is provided, characterized in that it contains at least the strain of the present invention. The microbial mixture may contain other plant growth-promoting strains and microorganisms that coexist with the strain of the present invention.

[0019] In the third aspect of the present invention, a product comprising the strain of the present invention or the microbial mixture of the present invention is provided. The product may be a bacterial agent, a bacterial fertilizer or a composition.

[0020] Preferably, the product includes a carrier; the carrier may be a solid carrier or a liquid carrier. The solid carrier or the liquid carrier are conventional carrier materials, wherein the solid carrier may be selected from clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, corn flour, soybean flour, polyvinyl alcohol and / or polyglycol; the liquid carrier may be vegetable oil, mineral oil or water.

[0021] Preferably, the product is in powder form, solution form, suspension form, tablet form, granule form or other forms.

[0022] Further preferably, the powder form may be a freeze-dried powder form; and the tablet form may be a tablet form containing the freeze-dried powder and optional nutrients (such as vitamins, mineral salts, etc.).

[0023] The active ingredient of the product is Enterobacter logankamp, ​​which can be present in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate.

[0024] In a fourth aspect of the present invention, a fermentation liquid, fermentation product or metabolite of Enterobacter logankamp is provided.

[0025] In a fifth aspect of the present invention, a fermentation method of the Enterobacter logenkamp is provided, which comprises inoculating the Enterobacter logenkamp into a fermentation medium for fermentation culture.

[0026] In the sixth aspect of the present invention, there is provided a use of the strain of the present invention, the microbial mixture of the present invention, or the product of the present invention comprising the strain of the present invention or the microbial mixture of the present invention, or the fermentation broth or fermentation product or metabolite of the present invention in promoting plant growth.

[0027] Preferably, the plant growth particularly includes promoting the growth of wheat.

[0028] In a seventh aspect of the present invention, a method for promoting wheat growth is provided, which comprises the step of contacting the surrounding environment of the wheat rhizosphere with the strain of the present invention, Enterobacter logankamp.

[0029] Preferably, the rhizosphere surrounding environment is wheat rhizosphere soil.

[0030] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects:

[0031] The present invention aims to utilize Enterobacter roggenkampii strain or its metabolites to stimulate plant hormones, improve nutrient absorption efficiency, promote root growth, etc., to promote plant growth and increase crop yields. At the same time, Enterobacter roggenkampii strain has good salt and alkali tolerance, which can improve the resistance of plants to adversity. By introducing Enterobacterroggenkampii strain or utilizing its metabolites, it helps to reduce dependence on chemical fertilizers, cope with problems such as land salinization and excessive use of chemical fertilizers, and improve crop yield and quality, thereby achieving sustainable agricultural production.

[0032] The strain of the present invention has a stronger salt tolerance of up to 8.5%, has a relatively superior acid and alkali resistance, and can grow in a pH range of 4-11.0. It has a good IAA production capacity (49.39 μg / ml), produces siderophore D / d of 36.03%, can utilize ACC deaminase, and has the ability to fix nitrogen and dissolve organic phosphorus. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 :The bacterial morphology and microscopic morphology of Sdny-150 strain.

[0035] Figure 2 : Growth curve of Sdny-150 strain.

[0036] Figure 3 :The test results of the iron carrier production capacity of Sdny-150 strain.

[0037] Figure 4 :Test results of nitrogen fixation ability of Sdny-150 strain.

[0038] Figure 5 :The test results of the phosphorus (organic phosphorus) solubilizing ability of Sdny-150 strain.

[0039] Figure 6 : Experimental results of the ability of Sdny-150 strain to dissolve phosphorus (inorganic phosphorus).

[0040] Figure 7 : Graph showing the test results of the ability of the Sdny-150 strain to utilize ACC.

[0041] Figure 8 : Light absorption values ​​of Sdny-150 strain grown at different pH values.

[0042] Fig. 9 : Experimental results of salt tolerance of Sdny-150 strain.

[0043] Fig.10 : Wheat seedling growth chart. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0045] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0046] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0047] Example 1 Screening of Enterobacter logankampii of the present invention

[0048] (1) Screening process

[0049] First, strains with the ability to promote plant growth are screened out from the soil environment and purified.

[0050] Weigh 10g of soil sample and put it into 90mL of sterile water to make soil bacterial solution. Take 5 test tubes containing 9mL of sterile water and press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Under sterile operation conditions, use a 1 mL sterile pipette to draw 1 mL 10 -1 The concentration of bacterial solution was 10 -2 . Dilute to 10 -5 . Take 10 -4 , 10 -5 Spread 0.2mL of each dilution on the PDA medium, and inoculate 2 plates for each dilution. Place the plate inoculated with the soil suspension in a biochemical incubator and culture at 28°C for 5-6 days. Pick different single colonies with good growth from the plate, streak them on the PDA medium, and culture them in a constant temperature incubator at 28°C for 3-6 days. After culture, observe whether it is pure bacteria. If it is not pure, pick colonies with different morphologies and streak them on the PDA medium plate, and repeat the picking and purification steps. Culture the purified strains on a solid slant medium at 28°C for 3-6 days and store them at 4°C.

[0051] (2) Colony characteristics and bacterial morphology

[0052] The colony diameter is 2-4mm, round, with irregular edges, opaque, grayish white or slightly yellow, raised in the middle, smooth surface, bright surface, moist, sticky, easy to pick up, shiny, and uniform texture; the bacteria are short rod-shaped and have no spores.

[0053] like Figure 1 , bacterial morphology and microscopic morphology of Sdny-150 strain.

[0054] (3) Physiological and biochemical characteristics

[0055] The optimum growth temperature is 30°C; it is a Gram-negative rod-shaped bacterium; it can ferment glucose to produce acid and gas; it can utilize sucrose, arabinose, glucose, mannitol, lactose, D-sorbitol, and maltose as carbon sources; it cannot hydrolyze gelatin and cannot utilize cellulose.

[0056] (4) 16S rDNA sequence analysis

[0057] The 16SrDNA of the strain was sequenced using a TIANGEN bacterial extraction kit to determine that it belonged to the genus Enterobacter cloacae. Combining colony morphology, physiological and biochemical characteristics and 16SrDNA sequence analysis, the strain of the present invention was identified as Enterobacter roggenkampii strain.

[0058] The following sequence is the 16S rDNA of Sdny-150, as shown in SEQ ID NO: 1:

[0059] AGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAACCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCC CGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATTGCTGAGGtTATTAACCTCAACACCTTCCTCCCCGCTGAA AGTACTTTACAACCCGAAGGCCTTTTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATTGTCGCCTAGGTGAGCCGTTAC CCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACCCGGGAGCAAGCT

[0060] Example 2

[0061] By changing the culture conditions, such as temperature, pH value, culture medium components, etc., the strain is cultured and proliferated, and a growth curve is drawn to determine the optimal culture conditions and strain growth cycle.

[0062] Four treatments were set at 25°C, 30°C, 37°C, and 45°C, with three replicates for each treatment, and the optimal growth temperature was determined to be 30°C.

[0063] Five treatments were set from pH 5 to 9, with three replicates for each treatment. The pH of the LB liquid medium was adjusted with 1 mmol / L NaOH and 0.5 mmol / L HCl, and the optimal growth pH of the strain was determined to be 7.

[0064] Optimize the culture medium and screen the carbon source: Use LB culture medium as the base culture medium, add different carbon sources (such as sucrose, arabinose, glucose, mannitol, lactose, D-sorbitol, maltose), use bacterial concentration as an indicator, and use no added carbon source as a control to determine that the best carbon source is glucose. Set a mass concentration gradient of 1, 1.5, 2, 2.5, and 3 g / L, and determine that the optimal concentration of glucose is 2 g / L. Nitrogen source screening: Liquid culture medium uses different nitrogen sources to replace the yeast extract powder (peanut cake powder, corn steep liquor powder, beef extract powder, peptone, ammonium nitrate, and urea) in LB culture medium, add 2 g / L of glucose, use bacterial concentration as an indicator, determine that the best nitrogen source is yeast extract powder, set a mass concentration gradient of 1, 1.5, 2, 2.5, and 3 g / L, and determine that the optimal concentration of yeast extract powder is 1 g / L. As above, replace the peptone in LB culture medium, and determine that the best combination and optimal concentration are 1 g / L of yeast extract powder and peptone. Inorganic salt screening: Different inorganic salts were added to the liquid culture medium to replace the sodium chloride in the LB culture medium (potassium dihydrogen phosphate, magnesium sulfate, potassium chloride, calcium chloride, zinc sulfate, copper sulfate, manganese sulfate, and iron sulfate), and 2g / L of glucose, 1g / L of yeast extract powder and peptone were added. The optimal inorganic salt was determined to be sodium chloride, with no inorganic salt added as the control. The mass concentration gradients of 1, 1.5, 2, 2.5, and 3g / L were set respectively, and other conditions remained unchanged. The optimal sodium chloride concentration was screened out to be 0.5g / L.

[0065] Take Sdny-150 seed liquid and inoculate it in 100ml culture medium (glucose 2g; peptone 1g; yeast extract powder 1g; NaCl 0.5g; water 100ml; pH 7.0-7.2, sterilized at 121℃ for 20min) at a 0.1% inoculum, and culture it at 28℃, 200rpm for 48h. Measure it every 2h and draw a growth curve (such as Figure 2 ). The Sdny-150 strain entered the logarithmic phase after 4 hours and the stable phase after 18 hours.

[0066] The purified strains were qualitatively and quantitatively tested for their ability to produce IAA, siderophore, nitrogen fixation, phosphate solubilization and ACC utilization under appropriate conditions.

[0067] IAA production: Sdny-150 was inoculated into a culture medium containing tryptophan (glucose 2%; peptone 1%; yeast extract 1%; NaCl 0.5%; pH 7.0-7.2, sterilized at 121°C for 20 min), and cultured in a shaking incubator at 28°C, 200 rpm for 4 days. The bacterial suspension was centrifuged at 10,000 rpm for 10 min, and the supernatant was added with an equal volume of Salkowski (50 mL 35% HClO4 + 1 mL 0.5 M FeCl3) colorimetric solution, and allowed to stand in the dark for 30 min. The OD was determined by spectrophotometry. 530 The content of IAA in the fermentation broth per unit volume was calculated by the standard curve of IAA gradient dilution. The OD of Sdny-150 after 4 days of culture was 530 The value is 1.7, and substituting it into the standard curve equation y=0.034x-0.0206, the IAA production of Sdny-150 is 49.39μg / ml.

[0068] Iron carrier production: Inoculate the purified strain into CAS solid medium and invert at 28℃ for 3-5 days. If an orange-yellow halo is produced around the colony, it is judged to have the ability to produce iron carriers. Measure the diameter of the orange iron carrier halo (D) and the colony diameter (d), and calculate the iron carrier production efficiency (Dd) / d×100%. Sdny-150 can produce an orange-yellow halo around the colony (such as Figure 3 ), the results of measurement and calculation showed that the siderophore production efficiency of Sdny-150 strain was 36.03%.

[0069] Nitrogen fixation: Inoculate the Sdny-150 strain in Ashby nitrogen-fixing medium, repeat 3 times for each strain. Incubate at 28℃ for 3 days and observe the growth of the strain. If the strain grows normally, it is judged to have the ability to fix nitrogen. After 6 generations of continuous subculture on Ashby nitrogen-fixing medium, the Sdny-150 strain can still grow on nitrogen-free medium, indicating that the strain has the ability to fix nitrogen (such as Figure 4 ).

[0070] Phosphorus solubilization ability: Phosphorus in soil exists in two forms: organic phosphorus and inorganic phosphorus. Therefore, the phosphate solubilization ability of the strain was determined using organic phosphorus solid culture medium and inorganic phosphorus solid culture medium. The strain was inoculated on the surface of the two culture media and cultured at 28℃ for 3 days. The results showed that Sdny-150 had the ability to solubilize organic phosphorus (such as Figure 5 ) and inorganic phosphorus (such as Figure 6) ability. By observing the diameter of the phosphate solubilization circle and the colony diameter formed, measuring the diameter of the phosphate solubilization circle (D) and the colony diameter (d), and calculating the ratio between them, the phosphate solubilization efficiency [(Dd) / d×100%] of the strain was determined. After measurement and calculation, it was found that the efficiency of Sdny-150 strain in dissolving organic phosphorus was 56.25%, and the efficiency of dissolving inorganic phosphorus was 10%. The strain had a stronger ability to dissolve organic phosphorus.

[0071] Ability to utilize ACC: The activated Sdny-150 strain was first streaked and inoculated in DF medium, and then incubated at 37°C for 24 to 36 hours. The strain in DF medium was then transferred to ADF medium with ACC as the sole nitrogen source. If the strain can grow normally, it means that the strain can utilize ACC and has ACC dehydrogenase activity; otherwise, it does not. The Sdny-150 strain was transferred from DF medium to ADF medium and still maintained its activity and could grow normally (e.g. Figure 7 ).

[0072] Under appropriate conditions, the concentration of bacterial suspension at different pH and salt concentration is measured to determine its acid, alkali and salt resistance.

[0073] The following pH gradient was set: 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, the pH value was adjusted with 1mmol / L NaOH and 0.5mmol / L HCl, the inoculum size was 0.1%, and the culture was shaken at 30°C for 48h, and the light absorption value (OD600) was measured. The results showed that the Sdny-150 strain could grow in the pH range of 4.0-11.0 (such as Figure 8 ).

[0074] Salt tolerance evaluation: Inoculate the seed solution in a medium containing 0%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% (w / v) NaCl, with an inoculum of 0.1%, shake culture at 30℃ for 48h, and measure the light absorption value (OD600). The results showed that the strain could grow at a NaCl concentration of 5%-8.5% (w / v). At 9%, the growth of the strain was about half of the maximum growth (such as Fig. 9 ).

[0075] Enterobacter roggenkampii strain was marked with two antibiotics, rifampicin and ampicillin, and its colonization ability was determined.

[0076] Colonization ability determination: First, the Sdny-150 strain was marked with two antibiotics, rifampicin and ampicillin. Rifampicin solution was added to a triangular flask containing 20 mL LB liquid culture medium to a final concentration of 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 120 μg / mL, 160 μg / mL, 200 μg / mL, and 300 μg / mL. 100 μL of activated bacterial solution was added to the culture medium containing 10 μg / mL rifampicin, and cultured at 28°C for 36 to 48 hours. 100 μL of bacterial solution in the bottle was transferred to the culture medium containing 20 μg / mL rifampicin for culture. In this way, mutant strains with rifampicin resistance can be screened out by culturing in sequence. The mutant strain with rifampicin resistance was marked with ampicillin resistance using the same method, and the strain was gradually cultured in a medium containing 10μg / mL, 20μg / mL, 50μg / mL, 100μg / ml, 150μg / mL, and 200μg / mL ampicillin, so that the strain obtained dual resistance marking. The strain's nitrogen fixation, phosphate solubilization, IAA production, and ACC utilization effects were verified to ensure that it maintained its original growth-promoting ability. The above strains were cultured and made into 10 8 CFU / mL of bacterial suspension was used to treat cucumber potted plants with root irrigation. The amount of bacterial suspension added to each pot was 50 mL, and 3 pots were used for each treatment. Rhizosphere soil samples were taken 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, and 10 weeks after root irrigation. The gradient dilution method was used to take 10 -2 , 10 -3 and 10 -4 The diluted soil dilutions were spread on LB medium containing both rifampicin and ampicillin resistance and cultured in a 30°C incubator for 24 to 72 h. The number of strains in the soil was counted and calculated, and the growth-promoting ability of the recovered strains was tested to verify the colonization ability of the Sdny-150 strain in the soil.

[0077] The results showed that the Sdny-150 strain grew rapidly, entered the logarithmic phase 4 hours after inoculation, and reached the stable phase 18 hours after inoculation, and then maintained a stable bacterial count. After double antibody labeling, the strain still maintained the growth-promoting effect, and the growth-promoting ability did not change significantly. The results are shown in Table 1. The number of live bacteria showed a certain downward trend after different time of root irrigation with bacterial solution. After 1 week of treatment, the number of live bacteria was maintained at 1.5×10 per gram of soil. 5 CFU, and the number of viable bacteria was maintained at 3×10 per gram of soil 3 weeks after treatment. 4 CFU, and the number of viable bacteria was maintained at 1×10 per gram of soil 4 weeks after treatment. 3 Above CFU, a certain number of live bacteria ensures the growth-promoting effect of the strain, providing a guarantee for its application as a growth-promoting agent.

[0078] Table 1

[0079]

[0080] Example 3

[0081] In the plant planting area, the prepared Enterobacter agent is mixed with an appropriate amount of water and applied to the soil by root irrigation. The plant growth is observed regularly. Finally, the plant growth, yield and quality are evaluated by comparing the control group with the Enterobacter agent and the non-application control group to verify the effect of Enterobacter in promoting plant growth.

[0082] Select seeds with full and round grains and soak them in a culture dish filled with 70% ethanol for 15 minutes to disinfect and sterilize the wheat seeds. Then rinse them with sterile water 5 times, add sterile water to completely immerse the wheat seeds in water overnight to allow them to absorb enough water. Transfer the soaked seeds to a culture dish covered with absorbent paper, add sterile water to completely soak the absorbent paper, protect from light, and germinate at 28°C for 2 to 3 days. Put the successfully germinated wheat with comparable growth into a hydroponic device with 1% addition of Sdny-150, use the added culture medium as a blank control, expose to light for 14 hours and darkness for 8 hours. After 14 days of culture, observe the growth of the wheat seedlings (such as Fig.10 ), and measured the root length, seedling height, dry weight and fresh weight respectively. The results are shown in Table 2. After applying the Sdny-150 strain, the root length and seedling height were increased, and the fresh weight and dry weight were also increased, which had a significant promoting effect on the growth of wheat.

[0083] Table 2

[0084]

[0085] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Enterobacter logenkamp, ​​characterized in that The bacteria was classified and named Enterobacter roggenkampiistrain, and was deposited in the China General Microbiological Culture Collection on July 12, 2024, with the deposit number: CGMCC No.31282. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial mixture, characterized in that: The characteristic is that it at least contains the strain of the present invention. The microbial mixture may contain other strains that promote plant growth and microorganisms that coexist with the strain of the present invention.

3. A product comprising the Enterobacter logankampii according to claim 1 or the microbial mixture according to claim 2.

4. The product according to claim 3, characterized in that: The product is in the form of powder, solution, suspension, tablet, granule or other forms.

5. A fermentation broth, a fermentation product or a metabolite comprising the Enterobacter logankampii according to claim 1.

6. A fermentation method of Enterobacter logankampii according to claim 1, characterized in that: The Enterobacter logenkampii is inoculated into a fermentation medium for fermentation culture to obtain the product.

7. Use of the Enterobacter logenkampii according to claim 1, the microbial mixture according to claim 2, the product according to claim 3 or 4, or the fermentation liquid or fermentation product or metabolite according to claim 5 in promoting plant growth.

8. The use according to claim 7, characterized in that: The plant growth particularly includes promoting the growth of wheat.

9. A method for promoting wheat growth, characterized in that: The method comprises the step of contacting the surrounding environment of wheat rhizosphere with the strain of the present invention, Enterobacter logankampii.

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