CN107099468B - Rhodobacter and application thereof - Google Patents
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- CN107099468B CN107099468B CN201710165834.1A CN201710165834A CN107099468B CN 107099468 B CN107099468 B CN 107099468B CN 201710165834 A CN201710165834 A CN 201710165834A CN 107099468 B CN107099468 B CN 107099468B
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- 241000191025 Rhodobacter Species 0.000 title claims abstract description 17
- 230000001737 promoting effect Effects 0.000 claims abstract description 10
- 230000012010 growth Effects 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 9
- 230000015784 hyperosmotic salinity response Effects 0.000 claims abstract description 7
- 240000008067 Cucumis sativus Species 0.000 claims description 22
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims description 17
- 241000209140 Triticum Species 0.000 claims description 6
- 235000021307 Triticum Nutrition 0.000 claims description 6
- 238000009629 microbiological culture Methods 0.000 claims description 3
- 241000598149 Roseobacter sp. Species 0.000 claims 4
- 239000002689 soil Substances 0.000 abstract description 24
- 239000003513 alkali Substances 0.000 abstract description 16
- 230000008635 plant growth Effects 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 4
- 230000009466 transformation Effects 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 239000013535 sea water Substances 0.000 description 5
- 239000008223 sterile water Substances 0.000 description 5
- 235000009849 Cucumis sativus Nutrition 0.000 description 4
- 230000035784 germination Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000002344 surface layer Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 239000005955 Ferric phosphate Substances 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000255969 Pieris brassicae Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940032958 ferric phosphate Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 1
- 229910000399 iron(III) phosphate Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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Abstract
The invention provides a rhodobacter (with the preservation number of CGMCC No. 13352)Roseobactersp.), the strain number is 3-2 of the rhodobacter, belonging to the biotechnology field. The invention also provides application of the rhodobacter 3-2 with the preservation number of CGMCC No.13352 in promoting plant growth and improving plant salt tolerance. The rhodobacter 3-2 with the preservation number of CGMCC No.13352 provided by the invention has obvious effects of promoting plant growth and improving salt tolerance, can be widely applied to the transformation of saline-alkali soil and the promotion of the growth of crops in the saline-alkali soil, and has good ecological and social benefits.
Description
Technical Field
The invention relates to a strain of rhodobacter (A)Roseobactersp.) and the use of the strain; more particularly relates to a rhodobacter with the preservation number of CGMCC No.13352 (Roseobactersp.) and also relates to said rhodobacter (F.) (Roseobactersp.) in promoting plant growth and improving plant salt tolerance.
Background
The saline-alkali soil is a general term for a series of soils affected by saline-alkali components in soil, including various saline soils, alkaline earths and other various types of soils salinized and alkalized to different degrees. The area of the global saline-alkali soil is 9.5 hundred million hectares, and the area of various saline-alkali soil in China is 9913 ten thousand hectares at present. The reasonable development and utilization of the saline-alkali soil can not only enlarge the arable area and relieve the grain crisis, but also improve the ecological environment through planting plants and improve the life quality of people.
However, saline-alkali soil contains high salt content, which causes difficulty in plant growth and low yield, and plants in serious saline-alkali soil regions can hardly survive. The screening of the salt-tolerant growth-promoting microorganisms is carried out, and the method is applied to the improvement of the saline-alkali soil, and possibly can provide a low-cost resource utilization mode for the improvement of the saline-alkali soil.
Disclosure of Invention
The invention provides an rhodobacter strain capable of improving salt resistance of plants and having a growth promoting effect.
A strain of rhodobacter, the preservation unit: the preservation date of the China general microbiological culture Collection center is 2016, 11 and 28 days, and the preservation number is: CGMCC No. 13352.
The strain is separated from a Bohai sea water sample in China, has the effect of promoting plant growth, can effectively promote crops to germinate and grow in the environment with salt stress, improves the soil microenvironment of saline-alkali soil, can reduce the damage to the soil microenvironment relative to a chemical improvement mode, is beneficial to the sustainable utilization of the saline-alkali soil, and provides an effective, environment-friendly and safe microbial method for the utilization of the saline-alkali soil. The invention can provide strain resources and utilization basis for improving saline-alkali soil by utilizing microbial resources and promoting saline-alkali soil development.
Biological sample preservation information
Rhodobacter 3-2, taxonomic nameRoseobactersp. deposited in China general microbiological culture Collection center (CGMCC), address: western road No.1, north zhou yang ward, beijing, the institute for microbiology, china academy of sciences, zip code 100101, and strain number 13352.
Detailed Description
Example 1 isolation of Marine bacteria
53 surface layer seawater samples in the sea area of the Cangzhou Bohai Bay in China are collected by a surface layer water sampler, 2216E culture medium (the formula is that peptone 5g, yeast powder 1g, ferric phosphate 0.1g, agar powder 15g and seawater 1000ml are adopted, the pH value is adjusted to 7.6 by NaOH, and the diluted plating method is used after sterilization at 121 ℃ for 20 min), so that the separation of the seawater samples is carried out. Culturing the coated plate in an incubator at 28 ℃, after the plate grows out of the bacterial colonies, selecting single bacterial colonies with different forms, and performing separation and purification for 3 times by adopting a streaking separation method to obtain pure culture bacterial colonies of the marine bacteria, thereby obtaining 126 strains of the marine bacteria. The liquid culture of single colony and 60% glycerin are mixed in the ratio of 1 to 1 and stored in an ultra-low temperature refrigerator at-80 deg.c.
Example 2 screening of Marine growth-promoting bacteria
The stored marine-isolated strains were activated on LB plates, after which the activated marine bacteria were inoculated at an inoculum size of 1% into a triangular flask containing 20ml of seawater medium and cultured at 28 ℃ for 12 hours. And (3) centrifuging the bacterial liquid at 5000rpm for 10min, pouring out supernatant, adding 5ml of sterile water, shaking and re-suspending the thalli. Then centrifuged again at 5000rpm for 10min and the supernatant was decanted off. Resuspending the cells in sterile water and adjusting OD6000.2-0.3, soaking the seeds of cucumber and wheat in the prepared bacterial solution for 4h, putting the seeds on sterilized filter paper in order, and adding 5ml of sterile water. Culturing in plant culture room at 25 + -2 deg.C for 3 days, and measuring bud length and root length at regular time. Table 1 shows the results of promoting germination of cucumber and wheat by using part of the strains, wherein the strains 3-2 have good promoting effects on germination, root length and bud length of cucumber and wheat, and compared with a control, the strains 3-2 promote the root length of cucumber to 21.9% and promote the root length and bud length of wheat to 32.0% and 16.8% respectively.
TABLE 1 promotion of cucumber and wheat germination by partial strains
Example 33-2 growth promotion experiment on soil-cultured cucumber
Mixing vermiculite with nutrient soil1:1, and adding the mixture into a square flowerpot. Placing the square flowerpot filled with soil into two large white trays, adding 3L of sterile water into one tray, and adding 3L of sterile water with OD concentration into the other tray600A bacterial solution of Rhodobacter 3-2 with a value of 0.1. After the soil is completely wet, at least two cucumber seeds are cultivated in the middle of the square flowerpot and placed in a culture room for cultivation. When the cucumber grows out of a true leaf, a cucumber seedling with consistent growth vigor is reserved in each pot. After 6 weeks in the greenhouse, the cucumber plants were removed and washed, and the plant height, dry weight and wet weight data were measured (Table 3). According to the table 2, the plant height, dry weight and wet weight of the treatment added with the strain 3-2 are higher than those of the blank control, which shows that the strain 3-2 can obviously promote the cucumber growth when applied in soil.
TABLE 2 Effect of application of Strain 3-2 on cucumber growth
Example 43-2 Effect on promoting salt tolerance of cucumber
The strain 3-2 was cultured according to the method of example 2, cucumber seeds with consistent germination were planted in hydroponic boxes containing different hydroponic solutions, and a total of 4 sets of treatments were set for the experiment, namely, blank control (tap water) and bacterial treatment (tap water with a concentration of 1 × 10)4cfu/mL strain 3-2), salt treatment (sodium chloride concentration of 50mmol/L tap water solution) and salt-added bacteria treatment (concentration of 1 × 10 in water)4cfu/mL Strain 3-2 and 50mmol/L NaCl). The 4 groups of planted cucumbers are placed in a plant culture room with the temperature of 25 +/-2 ℃ and the light-dark period of 12h:12h for culture, the growth condition of the cucumbers is observed after 20 days of culture, and the data of root length, stem length and leaf length are measured. As shown in Table 3, the root length, stem height and leaf length of cucumber seedlings in the treatment group to which the strain 3-2 was added were all higher than those of the blank control, indicating that the strain can also promote plant growth in a hydroponic environment. The growth conditions of cucumbers added with 3-2 strains in a salt-containing environment and cucumbers not added with 3-2 strains are compared, and the fact that the harm of salt stress to plant growth can be relieved by adding the 3-2 strains shows that the 3-2 strains canCan improve the salt tolerance of plants.
TABLE 3 hydroponic cucumber and salt tolerance test results
EXAMPLE 5 identification of growth-promoting salt-tolerant Strain 3-2
16SrDNA identification, namely inoculating the strain 3-2 to a marine culture medium, culturing for 24 h at 28 ℃ in a shaking way, extracting the genomic DNA of the strain by adopting a genomic DNA extraction kit of Tiangen Biochemical technology (Beijing) Co., Ltd, and then performing PCR amplification by using 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-TACGGCTACCTTGTTACGAC TT-3') primers by using the extracted total DNA as a template, wherein the PCR reaction system is 50 mu L, and the reaction system is 1 mu L of genomic DNA, 5 mu L of 10 × PCRbuffer, 1 mu L of 27f primer, 1 mu L of 1492r primer, 4 mu L of dNTPs, 1 mu L of Taq enzyme (5U/mu L), and 1 mu L of ddH enzyme2O36. mu.L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1min, annealing at 50 deg.C for 1min, extension at 72 deg.C for 2min, and circulation for 35 times; extension at 72 ℃ for 10 min. The PCR product was detected by electrophoresis using 1% agarose gel, and after sequencing the PCR product with a PCR band size of about 1500bp by Shanghai Biotechnology Co., Ltd, the obtained DNA sequence (see Seq 1) was imported into GenBank and analyzed by Blast program for comparison with all sequences in the database. Construction of phylogenetic trees was performed using MEGA 4.1. Sequence analysis shows that the strain belongs to one of the family of the erythrobacteriaceae.
Claims (3)
1. A strain of rhodobacter (Roseobacter sp.)3-2 is characterized in that: the Rhodobacter (Roseobacter sp.)3-2 is preserved in China general microbiological culture Collection center (CGMCC) at 2016, 11 and 28, with the preservation number of CGMCC No. 13352.
2. Use of the rhodobacter (Roseobacter sp.)3-2 of claim 1 for promoting the growth of cucumber and wheat.
3. Use of the rhodobacter (Roseobacter sp.)3-2 according to claim 1 for improving salt tolerance of cucumber.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710165834.1A CN107099468B (en) | 2017-03-20 | 2017-03-20 | Rhodobacter and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710165834.1A CN107099468B (en) | 2017-03-20 | 2017-03-20 | Rhodobacter and application thereof |
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| Publication Number | Publication Date |
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| CN107099468A CN107099468A (en) | 2017-08-29 |
| CN107099468B true CN107099468B (en) | 2020-08-21 |
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