Enterobacter fluvialis and application thereof

Abstract

The invention discloses Enterobacter fluvialis and application thereof, and belongs to the field of microorganism application, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 23, 2024, and the preservation number is CGMCC NO.32045. The invention further discloses a preparation method of the Enterobacter fluvialis. The strain has the functions of fixing nitrogen, dissolving phosphorus, dissolving potassium and secreting auxin, can effectively promote the growth characteristics of plant height, stem diameter, leaf number, biomass and the like of plants, and improves the photosynthesis efficiency of the plants.

Description

Technical Field

[0001] The invention belongs to the field of microbial application, and in particular relates to a river enterobacter and an application thereof. Background Art

[0002] The application of microorganisms in plant growth is an important research field, especially in modern agriculture and sustainable development. Microorganisms play a variety of roles in promoting plant growth, increasing crop yields, enhancing plant stress resistance and reducing the use of fertilizers and pesticides.

[0003] For example, nitrogen-fixing microorganisms (such as rhizobia and free-living nitrogen-fixing bacteria) can convert atmospheric nitrogen ( ) is fixed as ammonium ions that can be absorbed and utilized by plants ( ). These microorganisms live in symbiosis with plant roots, especially legumes. Nitrogen fixation reduces dependence on chemical nitrogen fertilizers while improving soil fertility.

[0004] Phosphate-solubilizing microorganisms can convert insoluble phosphorus into soluble forms (such as phosphate) by producing organic acids or phosphatases, thereby promoting plant absorption.

[0005] Plant growth-promoting rhizobacteria are a type of beneficial microorganisms that grow in the rhizosphere of plants and can promote plant growth through a variety of mechanisms. Some of them can directly promote plant growth by producing plant hormones (such as auxin, cytokinin, etc.), and can also indirectly improve plant health and yield by inhibiting the growth of pathogenic microorganisms and enhancing plant resistance (such as disease resistance, drought resistance, etc.).

[0006] Some microorganisms can also act as biological control agents to inhibit the growth of plant pathogens. For example, some fungi and bacteria can inhibit pathogens through competition, production of antibiotics, and secretion of enzymes, thus reducing the occurrence of plant diseases.

[0007] The application of microorganisms in plant growth involves a wide range of mechanisms and a variety of microbial types, including nitrogen fixation, phosphorus solubilization, growth promotion, disease resistance, etc. By applying microbial technology, the use of chemical fertilizers and pesticides can be reduced, soil quality can be improved, and the development of sustainable agriculture can be promoted. Summary of the invention

[0008] The purpose of the present invention is to provide a river enterobacter and application thereof.

[0009] The present invention obtains a multifunctional strain with the ability of nitrogen fixation, phosphorus dissolution, potassium dissolution and IAA (auxin) secretion through separation and screening. The strain is classified and named Enterobacter rivulosa ( Enterobacte ramnigenus), and was deposited in the China General Microbiological Culture Collection Center (CGMCC) on September 23, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.32045.

[0010] The nucleotide sequence of Enterobacter fluvial is shown in SEQ ID NO:1.

[0011] The 16S rDNA of the strain was identified as being related to the genus Enterobacter ( Enterobacter ) Enterobacteramnigenus The homology is 99.86%.

[0012] The morphological characteristics of the river enterobacter are as follows: it is a Gram-negative bacterium, has no capsule, no flagella, and the colonies formed on the nutrient agar medium are round milky white colonies with transparent and sticky edges, smooth surfaces and a foul smell.

[0013] The physiological and biochemical characteristics of the river enterobacter are as follows: methyl red and VP tests are negative, it is an alkaline bacteria species, starch hydrolysis is negative, gelatin test is negative, nitrate reduction is negative, oxidase is negative, and indole secretion is negative.

[0014] The separation medium formula of the river enterobacter is: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar powder, and pH 7.2-7.3.

[0015] The formula of the purification culture medium for Enterobacter brucei is: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, and pH 7.0-7.1.

[0016] The riverine Enterobacter strain provided by the present invention has the ability to fix nitrogen, dissolve phosphorus, dissolve potassium and secrete auxin, among which the ability to secrete IAA is the most prominent. The multifunctional growth-promoting characteristics of the strain are of great value in the research and development of biological fertilizers, especially in improving plant hormones. It can effectively promote the growth characteristics of plants such as plant height, stem thickness, number of leaves and biomass, and improve the photosynthesis efficiency of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The morphological characteristics of the strain under a microscope; Figure 2 This is the result of the methyl red experiment on the strain; Figure 3 The results of VP experiment on the strains are shown in the figure; Figure 4 This is the result of the glucose fermentation experiment of the strain; Figure 5This is the result of the starch hydrolysis experiment conducted on the strain; Figure 6 The results of gelatin hydrolysis experiments on strains are shown in Figure 2; Figure 7 The results of the nitrate reduction test for the strain are shown in the figure; Figure 8 This is a graph showing the results of an oxidase experiment on the strain; Fig. 9 The results of the indole experiment for the strain are shown in the figure; Fig.10 This is a diagram showing the results of the cultivation of the strain on nitrogen-free medium; Fig.11 This is a graph showing the results of the cultivation of the strain on an inorganic phosphorus medium; Fig.12 This is a diagram showing the results of the cultivation of the strain on potassium-dissolving medium; Fig.13 This is a diagram showing the results of the strain being cultured on a tryptophan-containing medium and undergoing a color reaction; DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with specific embodiments.

[0019] Preliminary screening: The strain isolated from Actinidia arguta was named R3.

[0020] Purification: The obtained strain was purified by streaking on a plate to obtain colonies of Enterobacter fluvial R3, which were stored on a slant made from a culture medium and placed in a refrigerator for later use.

[0021] The separation medium formula used was: peptone 10 g / L, beef extract 3 g / L, sodium chloride 5 g / L, agar powder 15 g / L, pH 7.2-7.3.

[0022] The formula of the purification medium used is: tryptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, pH 7.0-7.1.

[0023] The purified R3 bacteria were then subjected to physiological, biochemical and biomolecular identification.

[0024] 1. Morphological observation The purified R3 bacterial colony was inoculated on NA (nutrient agar) medium, cultured to the logarithmic growth phase, and then stained. After staining, it was observed under an electron microscope. The observation results are as follows: Figure 1 shown.

[0025] The R3 strain is a Gram-negative bacterium without capsule and flagella. The colonies it forms are round milky white colonies with transparent and sticky edges, smooth surfaces and a foul odor.

[0026] 2. Physiological and biochemical characteristics detection a. Methyl red test Inoculate strain R3 into an MR biochemical tube, culture at 200 r / min and 28°C for 24 hours, add 5 drops of methyl red solution, mix well and observe the results immediately. Red is positive and yellow is negative.

[0027] The results are as follows Figure 2 As shown, the methyl red test result was negative.

[0028] b. VP experiment Inoculate strain R3 in a VP biochemical tube, culture at 200r / min and 28℃ for 24h. After the culture is completed, add VP reagent (6 drops of A solution, 2 drops of B solution), mix well and keep the VP tube still for 0.5-2h. If it turns red, it is positive, and if it does not change color or is brown-yellow, it is negative.

[0029] The results are as follows Figure 3 As shown, the VP test result was determined to be negative.

[0030] c. Glucose glycolysis experiment Pick a single colony of strain R3 and inoculate it into a glucose glycolysis biochemical tube, and inoculate Escherichia coli into another biochemical tube as a control tube. If the contents in the test tube turn yellow, it is judged as a positive reaction, otherwise it is negative.

[0031] The results are as follows Figure 4 As shown in the figure, CK is the control group test tube, Ecoli is the test tube inoculated with Escherichia coli, and the R3 strain is determined to be positive, that is, the acid-producing type.

[0032] d. Starch hydrolysis experiment Inoculate strain R3 on a starch hydrolysis solid plate medium and culture at 28°C for 3 days. After the culture is completed, add iodine solution to the plate medium and wait for the iodine solution to spread evenly before observing whether the colony turns blue. If it turns blue, it is negative, and if it does not turn blue, it is positive.

[0033] The results are as follows Figure 5 As shown, the starch hydrolysis test result was negative.

[0034] e. Gelatin hydrolysis experiment Strain R3 was inoculated on a gelatin hydrolyzed solid plate medium and cultured at 28°C for 3 days. After the culture, the plate medium was placed in a 4°C refrigerator for 30 minutes. If the contents did not solidify and were liquid, it was a positive reaction. If the contents solidified and did not flow, it was a negative reaction.

[0035] The results are as follows Figure 6 As shown, the gelatin hydrolysis test result was negative.

[0036] f. Nitrate reduction experiment Inoculate strain R3 in a nitrate reduction test biochemical tube, and culture at 200r / min and 28℃ for 24h. After the culture, add 2-3 drops of nitrate reduction reagent A and B respectively, and observe the color change after a few seconds. If it turns red, it is a positive reaction; if it does not change color, continue to add a small amount of zinc particles. If it still does not turn red, it is also a positive reaction. If it turns red, it is a negative reaction.

[0037] The results are as follows Figure 7 As shown, the nitrate reduction test result was positive.

[0038] g. Oxidase assay Moisten the oxidase test paper with a small amount of distilled water, pick up a single colony with a disposable sterile inoculation loop and smear it on the test paper. If it turns blue or blue-purple within 30 seconds, it is a positive reaction. If it does not change color, it is a negative reaction.

[0039] The results are as follows Figure 8 As shown, the oxidase test result was negative.

[0040] h. Indole experiment Inoculate strain R3 in a centrifuge tube containing 1 mL of LB liquid medium and culture at 200 r / min and 28°C for 24 hours. After the culture is completed, add 2-3 drops of Kovacs reagent, mix well and observe the results immediately. If a rose-red ring appears on the culture, it is a positive reaction, and if there is no color change, it is a negative reaction.

[0041] The results are as follows Figure 8 As shown, the indole test result was confirmed to be negative.

[0042] Based on the above detection experiments, it can be known that the R3 strain was negative for methyl red and VP, which means it is acidogenic, negative for starch hydrolysis, negative for gelatin test, positive for nitrate reduction, negative for oxidase, and negative for indole secretion.

[0043] 3. Molecular identification The method of direct bacterial amplification of 16S rDNA was adopted. The total DNA of R3 was used as a template, and the PCR amplification primer was selected as the 16SrDNA bacterial universal primer 27F-1492R.

[0044] The 5' end primer is: 5'-AGAGTTTGATCCTGGCTCAG-3'.

[0045] The 3' end primer is: 5'-TACCTTGTTACGACTT-3'.

[0046] 50μL reaction system: 1μL DNA template, 4μL dNTP (2.5mM), 1μL each primer (1mM), 15μL Buffer (10X), 3μL MgCl2 (25mM), 0.5μL Taq enzyme (5UμL-1), 35.5μL ultrapure water.

[0047] PCR reaction conditions: pre-denaturation at 94°C for 2 min, thermal cycling: denaturation at 94°C for 30 s, annealing at 52°C, extension at 72°C for 1 min, for a total of 30 cycles.

[0048] After amplification, 5 μL of the amplified sample was electrophoresed on a 1% agarose gel at 60 V. The target DNA fragment was recovered and then sent to a sequencing company for sequencing.

[0049] When phylogenetic analysis was performed, the measured sequences were spliced ​​and then compared with BLAST in the NCBI database. Strains with relatively high homology and 16S rDNA sequences of common nitrogen-fixing bacteria were selected as reference strains. The phylogenetic tree was then constructed using the neighbor-joining method of MEGA5.05 software. The number of repeated samplings to test the support rate was 1000.

[0050] After BLAST comparison, the strain was identified as Enterobacter ( Enterobacter ) Enterobacteramnigenus The homology is the most similar, with a similarity of 99.86%, which can be determined to be a strain of Enterobacter spp.

[0051] Its SEQ ID NO.1 is: Example 1: The growth-promoting properties of strain R3 were tested. Strain R3 was first qualitatively tested. Strain R3 was inoculated into nitrogen-free medium, inorganic phosphorus medium, potassium-solubilizing medium and King's medium containing tryptophan, respectively, to observe its ability to fix nitrogen, dissolve organic / inorganic phosphorus, dissolve potassium and secrete IAA.

[0052] Ashby nitrogen-free medium was selected as the nitrogen-free medium. It was confirmed that the R3 strain could be stably propagated on the nitrogen-free medium for more than 5 generations. The cultivation results are as follows Fig.10 As shown, it was proved that the R3 strain was a growth-promoting bacterium with nitrogen fixation ability.

[0053] The formula of inorganic phosphorus culture medium is: glucose 10.0g, ammonium sulfate 0.5g, yeast extract powder 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, tricalcium phosphate 5.0g, agar powder 15g (for solid plate), pH 7.0-7.5, total volume 1L.

[0054] The strain R3 was inoculated into inorganic phosphorus medium for cultivation. Fig.11 As shown, there is an obvious transparent circle, indicating that strain R3 has the ability to dissolve phosphate.

[0055] The formula of potassium-dissolving medium is: 5 g glucose, 0.05 g magnesium sulfate heptahydrate, 0.1 g ferric chloride, 2 g calcium carbonate, 3.0 g potassium feldspar powder, 2 g calcium phosphate, 15 g agar powder, pH 6.8-7.0, total volume 1 L.

[0056] Similarly, strain R3 was inoculated into potassium-dissolving medium for cultivation. The results are as follows: Fig.12 As shown, there is an obvious transparent circle, indicating that strain R3 also has the ability to dissolve potassium.

[0057] The formula of King medium containing tryptophan is: 20g peptone, 1.15g dipotassium hydrogen phosphate, 1.5g magnesium sulfate heptahydrate, 15mL glycerol; 100mg tryptophan, total volume 1L.

[0058] Strain R3 was inoculated into King medium containing tryptophan. After the culture was completed, the Salkowski colorimetric method was used to determine whether the strain had the ability to secrete IAA. If the color of the reaction solution turned pink, it indicated that the strain had the ability to secrete IAA.

[0059] The reaction results are as follows Fig.13 As shown, it can be clearly observed that the reaction solution turns pink, proving that strain R3 has the ability to secrete IAA.

[0060] The above qualitative tests show that strain R3 is a multifunctional strain with the functions of nitrogen fixation, phosphorus solubilization, potassium solubilization and IAA secretion.

[0061] Next, the growth-promoting properties of strain R3 were quantitatively tested as follows: a. Determination of nitrogenase activity and net nitrogen fixation Since strain R3 can be continuously and stably propagated for more than 5 generations on Ashby nitrogen-free medium, it is proved that strain R3 is a growth-promoting bacterium with nitrogen fixation ability. Nitrogen fixation efficiency is mainly reflected in the activity of nitrogenase, which is the potential nitrogen fixation ability of the strain. The net nitrogen fixation amount is the nitrogen content remaining in the culture medium after the strain fixes atmospheric nitrogen to meet its own consumption.

[0062] The nitrogenase activity of nitrogen-fixing strains was determined by acetylene reduction method, and the net nitrogen fixation amount of R3 strain was determined by indophenol blue colorimetry.

[0063] Take 0.5mL of 10 8 The bacterial solution with CFU / mL was added into a 25mL Erlenmeyer flask (20mL A Shby medium), cultured at 30°C and 150r / min for 3 days, centrifuged at 10000r / min for 10min, and the supernatant was taken to measure the net nitrogen fixation amount.

[0064] b. Determination of net dissolved phosphorus The molybdenum antimony colorimetric method was used to measure the phosphate solubilization ability of strain R3. 8 CFU / mL) was added into a 25 mL Erlenmeyer flask (20 mL phosphate-solubilizing medium), cultured at 30°C, 150 r / min for 3 days, centrifuged at 10000 r / min for 10 min, and the supernatant was taken to measure the effective phosphorus content of the medium.

[0065] c. Determination of net potassium release The potassium-dissolving ability of strain R3 was measured by flame photometry. 0.5 mL of the test bacteria (10 8 CFU / mL) was added into a 25 mL Erlenmeyer flask (20 mL phosphate-solubilizing medium), cultured at 30°C, 150 r / min for 3 days, centrifuged at 10000 r / min for 10 min, and the supernatant was taken to measure the effective potassium content of the medium.

[0066] d. Determination of net secreted IAA content The strain was selected and inoculated into a nutrient broth medium containing 1.0 g / L of L-tryptophan. The culture was shaken at 30°C for 12 hours. After centrifugation at 12000 rpm, 100 μL of the supernatant was added to a 96-well plate. 100 μL of Salkowski reagent was added and mixed. The color was developed at room temperature in the dark for 30 minutes. The absorbance was measured with an enzyme reader at a wavelength of 530 nm. The uninoculated culture medium was used as a blank control. The IAA concentration was compared with the OD 530 The corresponding IAA concentration was calculated based on the standard curve.

[0067] Through the above-mentioned various tests on strain R3, the final test results showed that the nitrogenase activity of strain R3 was 7.15nmol / h / mL, the net nitrogen fixation amount was 0.13mg / L, the net phosphorus solubilization amount was 45.39mg / L, the net potassium solubilization amount was 4.14mg / L, and the net IAA secretion amount was 130.28mg / L.

[0068] The test results show that the R3 strain has the ability to fix nitrogen, dissolve phosphorus, dissolve potassium and secrete IAA, among which the ability to secrete IAA is the most outstanding. Although the strain can fix atmospheric nitrogen, it also has a certain demand for nitrogen, so the effective nitrogen content that can be used by plants produced by the strain is low. But overall, the multifunctional growth-promoting characteristics of the strain are of great significance in the research and development of later biological fertilizers, especially in terms of improving plant hormones.

[0069] Example 2: Experiments to test the growth-promoting effect of strain R3.

[0070] This experiment adopted a single-factor completely randomized test. The test plants were selected from corn seedlings with consistent growth conditions, and the test strains were selected from the highly efficient and multifunctional growth-promoting bacteria R3. The inoculation method was adopted, and no inoculation was used as the blank control. Three replicates were set for each treatment, and each replicate had 9 seedlings, that is, a total of 54 seedlings in the treatment group and the control group.

[0071] First, prepare the bacterial suspension, activate the strain, and inoculate it into a flask containing 100 mL of LB liquid culture medium at a 1% inoculum, and place it in a shaker at a constant temperature (30°C, 150r / min) for 12 hours. Use a spectrophotometer to measure OD 600 The concentration of bacterial suspension was adjusted to 0.4-0.5 (about 1×10 7 ~1×10 8 CFU / mL) and set aside.

[0072] After the preparation, the inoculation was carried out by root inoculation with bacterial solution. Specifically, when the corn seedlings had two leaves and one heart, 10 mL of bacterial solution was inoculated along the roots with a syringe. After 7 and 14 days, 10 mL was inoculated again. This was inoculated 3 times in total, with watering as a blank control. During the test period, regular watering, weeding, pest control and other management were performed to ensure the normal growth conditions of the test seedlings.

[0073] On the 43rd day after inoculation with strain R3, the growth and photosynthetic characteristics of corn were measured. The growth is shown in Table 1, and the photosynthetic characteristics are shown in Table 2.

[0074] deal with Plant height (cm) Stem diameter (mm) Number of blades Root length (cm) Root coefficient (number) Fresh weight of aboveground parts (g) Fresh weight of underground parts (g) CK 76.83±6.85 b 9.58±1.10 b 7±0.00 b 11.28±1.85 b 9±1.03 b 50.35±7.54 b 8.75±1.66 b R3 93.50±8.55 a 10.95±0.88 a 8±0.00 a 27.38±3.28 a 12±1.79 a 98.72±17.69 a 11.46±2.60 a Table 2 deal with <![CDATA[Net photosynthetic rate P n (μmol CO2·m -2 ·s -1 )]]> <![CDATA[Transpiration rate T r (μmol H2O·m -2 ·s -1 )]]> <![CDATA[Stomatal conductance G s (mmol H2O·m -2 ·s -1 )]]> <![CDATA[Intercellular carbon dioxide concentration C i (μmol·mol -1 )]]> CK 26.43±1.35 b 3.27±0.24 b 166.44±9.18 ab 20.80±0.54 a R3 28.50±1.08 a 3.58±0.19 a 175.33±7.92 a 21.54±0.87 a Note: Different letters represent significant differences, p<0.05.

[0075] By comparing the growth indicators of corn inoculated with strain R3 and corn not inoculated with bacteria (CK), it can be seen very intuitively that after inoculation with strain R3, the growth characteristics of corn seedlings such as plant height, stem thickness, number of leaves and biomass have been significantly improved, and the photosynthetic characteristics of corn can also be improved.

[0076] Therefore, strain R3 has certain application value in promoting plant growth, especially by utilizing the growth-promoting characteristics of strain R3 and applying it in the research and development of biological fertilizers, which has broad application prospects.

Claims

1. A river-borne Enterobacter, characterized in that It is deposited in China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC NO.32045.

2. A river-borne Enterobacter according to claim 1, characterized in that: The nucleotide sequence of Enterobacter fluvial is shown in SEQ ID NO:

1.

3. A river-borne Enterobacter according to claim 1, characterized in that: The morphological characteristics of the river enterobacter are as follows: it is a Gram-negative bacterium, has no capsule, no flagella, and the colonies formed on the nutrient agar medium are round light milky white colonies with transparent and non-sticky edges, smooth surfaces and a foul smell.

4. A river-borne Enterobacter according to claim 1, characterized in that: The physiological and biochemical characteristics of the river enterobacter are as follows: methyl red and VP tests are negative, it is an acidic bacteria species, starch hydrolysis is negative, gelatin test is negative, nitrate reduction is negative, oxidase is negative, and indole secretion is negative.

5. A river-borne Enterobacter according to claim 1, characterized in that: The separation medium formula of the river enterobacter is: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar powder, and pH 7.2-7.

3.

6. A river-borne Enterobacter according to claim 1, characterized in that: The formula of the purification culture medium for Enterobacter brucei is: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, and pH 7.0-7.

1.

7. The use of a river enterobacterium according to any one of claims 1 to 6, characterized in that: The river enterobacterium can be used for nitrogen fixation, phosphorus dissolution, potassium dissolution and growth hormone secretion.

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