CN110438036B - Nitrogen-fixing bacterium N24 with nitrogen-fixing effect and application thereof - Google Patents
Nitrogen-fixing bacterium N24 with nitrogen-fixing effect and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of plant rhizosphere growth-promoting bacteria, and particularly discloses a nitrogen-fixing bacterium N24 with a nitrogen-fixing effect and application thereof. The Azotobacter (Azotobacter chroococcum) N24 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, and the preservation number is CCTCC NO: and M2019281. The invention separates a strain of Azotobacter N24 from wheat rhizosphere soil, and identifies the Azotobacter N24 as Azotobacter (Azotobacter chroococcum) by morphological, physiological and biochemical characteristics and genetics. The strain can fix nitrogen in the atmosphere, can greatly increase the content of quick-acting nitrogen in soil, improves the utilization rate of nitrogen elements by plants, further promotes the growth of the plants, reduces the use amount of chemical fertilizers, improves the crop yield, and has important significance in agricultural production.
Description
Technical Field
The invention relates to the technical field of plant rhizosphere growth-promoting bacteria, and particularly relates to a nitrogen-fixing bacterium N24 with a nitrogen-fixing effect and application thereof.
Background
Nitrogen is a mineral nutrient element with the largest demand of plants, and is one of essential nutrient elements for the normal growth and development of the plants. According to statistics, the nitrogen content of soil in China is between 0.2 and 2g/kg of dry soil, and the content of most nitrogen is below 1g/kg of dry soil. The large-area fertilizer application solves the problem of nitrogen deficiency of soil, not only has high cost, but also causes the problem of environmental pollution. Biological nitrogen fixation can provide about 60 percent of globally available reduced nitrogen every year, has great potential in solving the problem of nitrogen sources, and is a long-standing measure for developing the productivity of farming land. In addition, compared with chemical fertilizers, the biological nitrogen fixation technology is new, low in cost and small in pollution, is a hotspot of current research, and has a better application prospect.
The microorganism is the core of soil fertility, the number of microorganisms in the soil is huge, and the types of microorganisms are very many, and many microorganisms play an important role in the conversion and supply of nutrients such as nitrogen, phosphorus, potassium and the like in the soil. The key to develop the biological nitrogen fixation technology is to screen out a bacterial strain which has high nitrogen fixation enzyme activity and can efficiently fix nitrogen. The nitrogen-fixing bacteria are one of very important functional groups in the soil ecosystem and play an irreplaceable role in the soil nitrogen circulation. The self-azotobacter is distributed more in a soil plough layer, the self-azotobacter at the rhizosphere of crops can provide nitrogen nutrition for the crops through the action of azotase, so that the nitrogen nutrition has a certain effect on improving the yield of the crops, and meanwhile, the bacteria can also generate a large amount of plant hormone, thereby influencing the physiological process of the plants to promote the growth of the plants.
For example, Reinhold et al (1993) isolated azovibrio spp from the pioneer plant grass Carrageena (Leptochlo tusca) in Baystein saline-alkali soil demonstrated that the biological yield per year of Carrageen grown in saline-alkali soil without applying any nitrogen fertilizer all the year round can reach 20-40 t/hm2It is closely related to the action of nitrogen-fixing vibrio. The endogenous nitrogen-fixing bacteria existing in some gramineous plants and host plants form a high-efficiency nitrogen-fixing system-an endogenous nitrogen-fixing system in a long-term co-evolution process. Researches prove that the endogenetic nitrogen fixation bacteria avoid the inhibition of compound nitrogen and the competition of indigenous microorganisms, are more favorable for fully exerting the nitrogen fixation efficiency, secrete the nitrogen fixation product to be directly supplied to plants for absorption, and show higher nitrogen fixation efficiency. Meanwhile, the plant growth promoter also has the functions of secreting auxin, dissolving phosphorus, enhancing disease resistance of plants, resisting stress and the like.
Although various azotobacteria exist in the prior art, separating and screening new plant rhizosphere azotobacteria and researching the growth promoting effect of the azotobacteria on plants still have important significance in the aspects of reducing the use of potassium fertilizer, improving the yield of crops and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a nitrogen-fixing bacterium N24 with a nitrogen-fixing effect.
The invention also aims to provide application of the azotobacter N24 in improving the quick-acting nitrogen content of plant rhizosphere soil.
The invention also aims to provide application of the azotobacter N24 in preparation of a preparation for promoting plant growth.
The invention also aims to provide application of the azotobacter N24 in preparation of a preparation for improving plant height.
The invention also aims to provide application of the azotobacter N24 in preparation of a preparation for increasing the dry weight of plants.
The invention also aims to provide application of the azotobacter N24 in preparation of a preparation for improving nitrogen content of plants.
It is another object of the present invention to provide a method for promoting plant growth.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention separates a nitrogen-fixing bacterium N24 from wheat rhizosphere soil, identifies the nitrogen-fixing bacterium in the aspects of morphology, physiological and biochemical characteristics and genetics, and verifies that the nitrogen-fixing bacterium is Azotobacter sp. The result of the determination of the azotobacter activity shows that the azotobacter activity of the strain is higher and is 152.56nmol/gh, nitrogen in the atmosphere can be effectively fixed, the content of quick-acting nitrogen in soil can be greatly increased, the utilization rate of nitrogen elements by plants is improved, the growth of the plants is further promoted, the using amount of a chemical fertilizer is reduced, and the yield of crops is improved.
Therefore, the invention provides a nitrogen-fixing bacterium N24 with nitrogen-fixing function, wherein the nitrogen-fixing bacterium (Azotobacter sp. CCNWSX1904) N24 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, and the preservation number is CCTCC NO: and M2019281. The preservation address is Wuhan university in China.
The Azotobacter sp.CCNWSX1904N 24 has the following morphological, physiological and biochemical characteristics:
after the Azotobacter sp.CCNWSX1904N 24 is cultured on the Ashby culture medium for 24 hours, the colony is circular, the edge is neat, the colony is semitransparent, and the colony appears dark brown when the colony grows to the later stage; under the microscope, gram staining is negative, the cell is oval, capsular, and does not produce spores. The strain can grow by using glucose, citric acid and lactose as carbon sources, the indole experiment, catalase and catalase reaction are positive, and the VP reaction and methyl red experiment are negative.
The 16SrDNA sequence of the Azotobacter sp.CCNWSX1904N 24 is shown as SEQ ID NO: 1.
The invention also claims application of the azotobacter N24 in improving the quick-acting nitrogen content of plant rhizosphere soil.
The invention also claims application of the azotobacter N24 in preparation of a preparation for promoting plant growth.
The invention also claims application of the azotobacter N24 in preparation of a preparation for improving plant height.
The invention also claims application of the azotobacter N24 in preparation of a preparation for increasing the dry weight of plants.
The invention also claims application of the azotobacter N24 in preparation of a preparation for improving the nitrogen content of plants.
Preferably, the plant is wheat.
The invention also claims a method for promoting plant growth, which is to apply 0.5-2 mL of the bacterial liquid of the azotobacter N24 in plant rhizosphere soil, wherein the effective viable count in the bacterial liquid is 108～109one/mL.
Preferably, the application amount of the bacterial liquid is 1mL, and the effective viable count in the bacterial liquid is 108one/mL.
Compared with the prior art, the invention has the following beneficial effects:
the invention separates a nitrogen fixing bacterium N24 from wheat rhizosphere soil, and identifies the nitrogen fixing bacterium N24 as nitrogen fixing bacterium (Azotobacter sp. CCNWSX1904) by morphological, physiological and biochemical characteristics and genetics. The strain can fix nitrogen in the atmosphere, can greatly increase the content of quick-acting nitrogen in soil, improves the utilization rate of nitrogen elements by plants, further promotes the growth of the plants, reduces the use amount of chemical fertilizers, improves the crop yield, and has important significance in agricultural production.
Drawings
FIG. 1 shows the results of the enzyme activities of different azotobacteria.
FIG. 2 is a phylogenetic tree of strain N24.
FIG. 3 shows the variation of plant height, dry weight and nitrogen content of wheat inoculated with the strain N24.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Ashby medium: glucose 10g, K2HPO4 0.2g，NaCl 0.2g，MgSO4·7H2O 0.2g，K2SO40.2g， CaCO35g, 15g of agar and 1000mL of distilled water.
LB solid medium: 5g of yeast extract, 10g of tryptone, 10g of NaCl, 15g of agar and 1000mL of water.
Salkowski developing solution: 0.5M FeCl3 1mL，H2SO430mL and 50mL of distilled water.
EXAMPLE 1 screening of Azotobacter
1. Azotobacteria prescreening
Wheat plants growing for about 7 months are collected from farmlands in Yangling areas of Shaanxi province in 9 months in 2017, and soil loosely combined with wheat roots is shaken off by force. The plant roots are cut down and placed into a 10mL sterilized centrifuge tube, 5mL sterile water is injected into the centrifuge tube, and then ultrasonic treatment is carried out, so that rhizosphere soil tightly combined with the plant roots is dissolved in the water. The collected soil sample is yellow loam, the sample has 24.03mg/kg of quick-acting phosphorus, 126.17mg/kg of quick-acting potassium, 56.28mg/kg of alkaline hydrolysis nitrogen, 21.06g/kg of organic matters and 8.27 of pH.
Diluting the aqueous solution by a series of 10-fold dilutions, and respectively taking 5 μ L of dilution with 10-5、10-6And 10-7The suspension was spread on an Ashby medium and cultured at 28 ℃ for 5 days. Transferring the grown strain to LB solid culture medium for storage.
2. Azotase activity assay
A10 mL bottle with a rubber stopper was filled with Ashby medium and inclined, and the 11 strains grown on the Ashby medium were inoculated on the inclined surfaces, respectively, and cultured at 28 ℃ for 24 hours. 1mL of air was evacuated from the bottle with a syringe, and 1mL of C was injected with the syringe2H2And reacting at 28 ℃ for 12 h. Then, 1mL of the gas was evacuated from the bottle, and C was measured by Shimadzu GC-14C gas chromatograph2H4And C2H2Simultaneously determining the known concentration C2H4And C2H2And making a standard curve, converting the standard curve to obtain the amount of the generated ethylene, and according to a formula: azotase activity ═ C2H4Mol number (nmol)/[ cell weight (g). The reaction time (h)]And calculating the activity of the azotase.
The test result shows that the nitrogen-fixing enzyme activity of the strain N24 (namely CCNWSX1904) is high and is 152.56nmol/gh (shown in figure 1).
3. Production of IAA Standard Curve
Accurately weighing 10mg of IAA, dissolving with a small amount of ethanol, and then diluting to 10mL with deionized water to obtain 1mg/mL IAA standard stock solution. Accurately sucking a certain amount of standard stock solution into a 10mL volumetric flask, fixing the volume to scale by using deionized water, so that IAA series standard solutions with standard concentrations of 0, 40, 80, 120, 160 and 200 mu g/mL are obtained, and storing in a dark place after preparation. And (3) taking 1mL of standard solution with each concentration, adding equivalent Salkowski colorimetric solution, carrying out dark reaction for 30min, detecting a light absorption value at 530nm by using an ultraviolet visible spectrophotometer, and drawing a standard curve by taking the light absorption value as a vertical coordinate and the IAA concentration as a horizontal coordinate.
4. Determination of IAA-producing ability of N24
The nitrogen-fixing strain N24 is inoculated into a conical flask containing 20mL of TY liquid culture medium and cultured for 48h at the temperature of 28 ℃ under the condition of 150r/min shaking. Centrifuging the culture solution at 10000rpm for 10min, sucking 1mL of supernatant, adding equal volume of Salkowski developing solution, reacting at room temperature in dark place for 30min, and measuring OD with ultraviolet spectrophotometer530。
The results showed that strain N24 was able to produce IAA at a concentration of 149.23mg/L in the broth.
Example 2 identification of Strain N24
1. Morphological characteristics
The N24 strain is prepared into a bacterial suspension, diluted and coated on an Ashby culture medium, and the bacterial colony and the bacterial morphology are observed after the culture is cultured for 24 hours at 28 ℃.
The result shows that the bacterium is negative in gram staining, oval in cell shape, capsular and free from spore production; after 24 hours of culture on the Ashby culture medium, the colony is circular, the edge is neat and semitransparent, and the colony appears dark brown when growing to the later stage.
2. Physiological and biochemical characteristics
The physiological and biochemical characteristics of the strain N24, such as VP reaction, carbon source utilization, indole test and the like, are determined by referring to a bacteria identification manual, and the result shows that the strain can grow by using glucose, citric acid and lactose as carbon sources, the indole test, catalase and catalase reaction are positive, and the VP reaction and methyl red test are negative.
3. 16S rDNA sequencing
Extracting genome DNA of the strain N24 by using a bacterial genome extraction kit, amplifying 16S rDNA by using bacterial universal primers 27F (5 '-AGAGTTTGATCCTGGCTCAG) and 1492R (5' -TACCTTGTTACGACTT), carrying out electrophoresis detection, then sending the amplified product to a company for sequencing, and comparing the sequence in an NCBI database. The 16SrDNA sequence of the strain is shown as SEQ ID NO. 1.
And (3) identification result: the strain N24 belongs to Azotobacter in classification, the similarity with Azotobacter chlorococcus is 99.92%, and phylogenetic tree of the strain N24 is shown in figure 2.
Azotobacteria (Azotobacter sp. CCNWSX1904) N24 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, with the preservation number of CCTCC NO: and M2019281. The preservation address is Wuhan university in China.
Example 3 wheat growth promotion test
Selecting plump wheat seeds, performing surface sterilization by using 70% ethanol, washing with sterile water for at least 6 times, coating the sterile water washed for the last time on a beef extract peptone solid culture medium, and checking whether the surface sterilization of the wheat is complete. The sterilized wheat seeds were sown in 6 pots of soil, the surface of which was covered with approximately 1cm of soil. Experiment set 2 treatments, 1 treatment of 1 plant inoculated with 1mL sterile medium, 2 treatment of 2 plants inoculated with N24 inoculum (about 10)8one/mL) 1mL, 3 replicates per treatment. The flowerpot is placed in a greenhouse, and the culture conditions are set to be 16h/8h under illumination and 25 ℃/18 ℃. After wheat seedlings emerge, water is poured once every 5 days, and the plant height, dry weight and nitrogen content of the plants are measured after 60 days.
As shown in Table 1 and FIG. 3, it can be seen from Table 1 that the growth vigor of wheat inoculated with azotobacter is significantly better than that of the non-inoculated control group, and the height, dry weight and nitrogen content of the inoculated plant are respectively increased by 33.7%, 41.8% and 57.7% compared with those of the blank control group.
TABLE 1 variation of plant height, dry weight and nitrogen content before and after inoculation of wheat with N24 bacterial liquid
| Plant height (cm) | Plant Dry weight (g) | Plant nitrogen content (g/kg) | |
| Control | 20.1 | 0.966 | 7.9 |
| Inoculation treatment | 26.87 | 1.37 | 12.46 |
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> northwest agriculture and forestry science and technology university
<120> nitrogen-fixing bacterium N24 with nitrogen-fixing effect and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1299
<212> DNA
<213> Azotobacter chromococcus
<400> 1
gtgagtaatg cctaggaatc tgcccgatag tgggggacaa cgtttcgaaa ggaacgctaa 60
taccgcatac gtcctacggg agaaagtggg ggctcttcgg acctcacgct atcggatgag 120
cctaggtcgg attagctagt tggtggggta aaggctcacc aaggcgacga tccgtaactg 180
gtctgagagg atgatcagtc acactggaac tgagacacgg tccagactcc tacgggaggc 240
agcagtggga atattggaca atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag 300
aaggtcttcg gattgtaaag cactttaagt tgggaggaag ggctgtaagc gaataccttg 360
cagttttgac gttaccgaca gaataagcac cggctaactt cgtgccagca gccgcggtaa 420
tacgaagggt gcaagcgtta atcggaatta ctgggcgtaa agcgcgcgta ggtggtttgg 480
taagttggat gtgaaagccc cgggctcaac ctgggaactg catccaaaac tgcctgacta 540
gagtacggta gagggtggtg gaatttcctg tgtagcggtg aaatgcgtag atataggaag 600
gaacaccagt ggcgaaggcg accacctgga ctgatactga cactgaggtg cgaaagcgtg 660
gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgtc gactagccgt 720
tgggctcctt gagagcttag tggcgcagct aacgcattaa gtcgaccgcc tggggagtac 780
ggccgcaagg ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg 840
gtttaattcg aagcaacgcg aagaacctta cctggccttg acatgctgag aactttccag 900
agatggattg gtgccttcgg gaactcagac acaggtgctg catggctgtc gtcagctcgt 960
gtcgtgagat gttgggttaa gtcccgtaac gagcgcaacc cttgtcctta gttaccagca 1020
cctcgggtgg gcactctaag gagactgccg gtgacaaacc ggaggaaggt ggggatgacg 1080
tcaagtcatc atggccctta cggccagggc tacacacgtg ctacaatggt cggtacagag 1140
ggttgccaag tcgcgaggcg gagctaatcc cagaaaaccg atcgtagtcc ggatcgcagt 1200
ctgcaactcg actgcgtgaa gtcggaatcg ctagtaatcg cgaatcagaa tgtcgcggtg 1260
aatacgttcc cgggccttgt acacaccgcc cgtcacacc 1299
Claims (9)
1. Azotobacter N24 with azotobacter fixing functionCharacterized in that the azotobacter N24 is classified and named as azotobacter chroococcum (A)Azotobacter chroococcum) And is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, with the preservation number of CCTCC NO: m2019281.
2. The use of the azotobacter N24 as claimed in claim 1 for increasing the content of available nitrogen in plant rhizosphere soil.
3. Use of the azotobacter N24 according to claim 1 for the preparation of a formulation for promoting plant growth.
4. Use of azotobacter N24 according to claim 1 for the preparation of a formulation for increasing plant height.
5. Use of azotobacter N24 according to claim 1 for the preparation of a formulation for increasing the dry weight of plants.
6. Use of azotobacter N24 according to claim 1 for the preparation of a formulation for increasing nitrogen content in plants.
7. Use according to any one of claims 2 to 6, wherein the plant is wheat.
8. A method for promoting plant growth, which is characterized in that 0.5-2 mL of bacterial liquid of azotobacteria N24 as defined in claim 1 is applied to plant rhizosphere soil, and the effective viable count in the bacterial liquid is 108～109one/mL.
9. The method according to claim 8, wherein the applied amount of the bacterial suspension is 1mL, and the effective viable cell count in the bacterial suspension is 108one/mL.
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| CN112029680B (en) * | 2020-08-21 | 2022-07-08 | 上海市农业科学院 | Azotobacter fuscus mutant with good nitrogen fixation capacity and application thereof |
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