CN103333837B - One strain fixed nitrogen Arthrobacter and uses thereof - Google Patents
One strain fixed nitrogen Arthrobacter and uses thereof Download PDFInfo
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- 241000186063 Arthrobacter Species 0.000 title claims abstract description 41
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 19
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 31
- 244000005700 microbiome Species 0.000 claims abstract description 13
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- 235000013311 vegetables Nutrition 0.000 claims abstract description 5
- 108010020943 Nitrogenase Proteins 0.000 claims description 13
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
Abstract
The invention discloses strain fixed nitrogen Arthrobacter and uses thereof.Does is this fixed nitrogen Arthrobacter Arthrobacter (Arthrobacter? sp.) GD601, does is it CGMCC at the preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center? No.7779.Arthrobacter (Arthrobacter provided by the present invention? sp.) GD601CGMCC? No.7779 can grow surely at crop rhizosphere, there is very high fixed nitrogen potential, have broad application prospects in Vegetables Factory Plantation Growing Seedlings Inoculant, nitrogen-fixing microorganism microbial inoculum and biological organic fertilizer are produced.
Description
Technical field
The present invention relates to strain fixed nitrogen Arthrobacter and uses thereof.
Background technology
Nitrogenous fertilizer is one of most important production means in agriculture production.Produce chemical nitrogen fertilizer and consume mass energy, account for 70% ~ 80% of total production cost.Energy day is becoming tight in recent years, and chemical fertilizers production coal price lattice soar and cause chemical nitrogen fertilizer price increase, and peasant is difficult to bear, and repercussion is strong.Chemical nitrogen fertilizer has played great function in raising crop yield, guarantee China grain security, but unreasonable nitrogen of executing has defined serious pollution of area source, causes a series of serious harm, as Taihu Lake Blue Algae Event makes us startling.Urban groundwater azotate pollution causes threat to drinking water safety.
Containing the nitrogen of 78% in air, but its existence form is molecular state nitrogen, and animals and plants can not directly utilize, and nature only has some prokaryotic micro-organisms to have the ability directly utilizing nitrogen in air, is reduced ammonification, biological nitrogen fixation that Here it is.High-efficiency nitrogen-fixing microorganism bacterial classification factorial praluction is selected to make microbial inoculum, or the material Compound Machining being rich in plant nutrition further with other becomes biological products, be applied to agriculture production, crop nitrogen nutrition can be provided, improve crop rhizosphere ecotope, improve soil biological fertility proterties, Here it is usually said nitrogen-fixing microorganism microbial inoculum or nitrogen-fixing microorganism fertilizer.Biological nitrogen fertilizer has multiple advantage: 1. biological nitrogen fertilizer is made up of reproducible biochemical preparation and living microorganism, can regenerate, and there is not resource exhaustion problem, is Sustainable development agricultural material product.2. biological nitrogen fertilizer is environmental friendly fertilizer, and it is produced and use procedure does not all produce the pollutants such as the three wastes, and can improve soil physico-chemical property, and increasing soil fertility, is ecological agriculture agricultural material product.3. biological nitrogen fertilizer produce power consumption less, cost is low, its cost is only 20% ~ 40% of equivalent chemical nitrogen fertilizer, can be country's a large amount of coal of saving and oil equal energy source strategic materials.4. use biological nitrogen fertilizer significantly can reduce agriculture production cost, improve quality of agricultural product, promote soil fertility, peasant is benefited, and promote social harmony development.
Bacterial classification is the basis of microbial fertilizer production application.At present, the bottleneck of restriction China microbial fertilizer industry development is exactly high-efficiency strain, and agriculture production is in the urgent need to can the production bacterial strain of high-efficiency nitrogen-fixing.
Summary of the invention
The object of this invention is to provide the bacterium that a strain has nitrogen fixation.
Bacterium provided by the invention is Arthrobacter (Arthrobactersp.) GD601, and this bacterial strain is CGMCCNo.7779 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Another object of the present invention is to provide a kind of microbial inoculum, and the activeconstituents of this microbial inoculum is described Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779.
This microbial inoculum, except Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779 comprised as activeconstituents, also can comprise auxiliary material, as the stalk of the ight soil of the peat composed of rotten mosses, animal, all kinds of crop, loose shell, straw, Pericarppium arachidis hypogaeae etc.Described microbial inoculum also can comprise carrier.Described carrier can be solid carrier or liquid vehicle.Described solid carrier is mineral material, vegetable material or macromolecular compound; Described mineral material is at least one in clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica and diatomite; Described vegetable material is at least one in Semen Maydis powder, bean powder and starch; Described macromolecular compound is polyvinyl alcohol and/or polyglycol.Described liquid vehicle is organic solvent, vegetables oil, mineral oil or water; Described organic solvent is decane and/or dodecane.In described microbial inoculum, described activeconstituents can with by cultivate viable cell, the fermented liquid of viable cell, the filtrate of cell culture or cell and filtrate the form of mixture exist.The formulation of described composition can be multiple formulation, as liquor, emulsion, suspension agent, pulvis, granule, wettable powder or water dispersible granules.
This microbial inoculum can be used for fixed nitrogen, Promoting plant growth, raising stress resistance of plant, produces nitrogenase etc.
Described Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779 is in preparation following 1)-5) in application in arbitrary microbial inoculum also belong to protection scope of the present invention:
1) for the microbial inoculum of fixed nitrogen;
2) microbial inoculum of nitrogenase is produced;
3) microbial inoculum of Promoting plant growth;
4) microbial inoculum of farming area in Zuogong soil;
5) microbial inoculum of stress resistance of plant is improved.
In above-mentioned application, described resistance is the resistance to adverse circumstance, and described adverse circumstance is arid, waterflooding, high temperature, physical abuse, disease and pest invasion and attack or heavy metal contamination etc.
Described Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779 or take Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779 as the application and also belong to protection scope of the present invention preparing the application in biological organic fertilizer in production nitrogenase of the microbial inoculum of activeconstituents.
Another object of the present invention is to provide a kind of containing described Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779 or take described Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779 as the biological organic fertilizer of the microbial inoculum of activeconstituents.
Another object of the present invention is to provide a kind of method of cultivating Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779, comprises Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779 for cultivating the step of cultivating in the substratum of Arthrobacter.
Another object of the present invention is to provide a kind of method preparing described microbial inoculum, and the method to comprise the steps: described Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779, as activeconstituents, to obtain described microbial inoculum.
The present invention is separated vinelandii from the barley rhizosphere soil of farmland, the bacterial strain of the higher nitrogenase activity of further screening, finishing screen chooses high-efficiency nitrogen-fixing Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779, and this bacterial strain nitrogenase activity is 30.570nmolC
2h
4/ hmg albumen, statistical study pole is significantly higher than microbial fertilizer conventional production bacterial classification azotobacter chroococcum ACCC11103, has broad application prospects in Vegetables Factory Plantation Growing Seedlings Inoculant, nitrogen-fixing microorganism microbial inoculum and biological organic fertilizer are produced.
preservation explanation
Strain name: Arthrobacter
Latin name: (Arthrobactersp.)
Strain number: GD601
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on June 20th, 2013
Register on the books numbering: CGMCCNo.7779 at preservation center
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Substratum used in following embodiment is as follows:
Vinelandii enrichment culture liquid ACCC55: sucrose 10g, K
2hPO
43H
2o0.5g, NaCl0.2g, CaCO
31g, MgSO
47H
2o0.2g, be settled to 1000ml, pH7.0 ~ 7.2 with distilled water.
The ACCC55 vinelandii solid medium of improvement: sucrose 10g, K
2hPO
43H
2o0.5g, NaCl0.2g, CaCO
31g, MgSO
47H
2o0.2g, yeast extract paste 0.5g, agar 15-20g, be settled to 1000ml with distilled water, pH7.0 ~ 7.2.
Nitrogen-free agar: sucrose 10g, NaCl0.12g, K
2hPO
43H
2o0.5g, CaCO
31g, MgSO
47H
2o0.2g, is settled to 1000ml with distilled water, pH7.2.
Beef-protein medium: extractum carnis 5g, peptone 10g, NaCl5g, agar 15-20g, be settled to 1000ml with distilled water, pH7.2.
Improvement fixed nitrogen substratum: sucrose 10g, K
2hPO
43H
2o0.5g, NaCl0.2g, CaCO
31g, MgSO
47H
2o0.2g, yeast extract paste 0.5g, distilled water 1000ml, agar 15-20g, pH7.0 ~ 7.2.
Azotobacter chroococcum (Azotobacterchroococcum) ACCC11103(used in following embodiment see " Sun Jianguang etc. high-efficiency nitrogen-fixing genus bacillus screening and biological characteristics. Scientia Agricultura Sinica; 2009,42 (6): 2043-2051 ").The public can obtain this bacterial strain from Chinese Academy of Agricultural Sciences's agricultural resource and agricultural regionalization institute, to repeat the experiment of the application.
The Isolation and ldentification of embodiment 1, Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779
One, crop rhizosphere vinelandii GD601 enrichment be separated
Get 10g soil sample (picking up from inner mongolia autonomous region) put into 90ml sterilized water shaking table vibration 20min make dirty solution, draw 5ml and put into 30ml vinelandii enrichment culture liquid ACCC55,100rpm, 28 DEG C are carried out shaking table shaking culture, change fresh medium and continue to cultivate after 72h.The separation of fixed nitrogen sporeformer is carried out after repeating cultivation 3 times.Absorption 1ml above-mentioned vinelandii enrichment culture thing is put in 9ml sterilized water makes 10
-1extent of dilution, continues dilution and makes 10
-2, 10
-3, 10
-4, 10
-5dilution bacteria suspension, heats 10min in 100 DEG C of boiling water, and the rear each extent of dilution of cooling is got 0.1ml and is coated on nitrogen-free agar flat board, 29 DEG C of quiescent culture.2 ~ 3d, after bacterium colony is formed, the ACCC55 vinelandii solid medium flat board of improvement carries out purifying agaric with plate streak.Be crop rhizosphere vinelandii GD601 by one of them Strain Designation of separation and purification gained.
Two, the qualification of crop rhizosphere vinelandii GD601
The crop rhizosphere vinelandii GD601 obtained also is screened from the following aspects authentication step one separation and purification:
1, Morphological Identification
Logarithmic phase will be in and bacterium colony size is stablized, above-mentioned steps one is separated and the crop rhizosphere vinelandii GD601 that obtains of purifying carries out single bacterium colony state observation, mainly comprises the size of bacterium colony, color, transparency, wettability, bacterium colony condition of surface (whether smooth, projection, fold, depression etc.), colony edge state (whether neat, irregular, radial etc.).
For the described crop rhizosphere vinelandii GD601 being in logarithmic phase, after smear staining, adopt the form of observation by light microscope thalline.
Result shows, above-mentioned steps one is separated the crop rhizosphere vinelandii GD601 bacterium colony circular protrusions that also purifying obtains, oyster white, and glossy, smooth surface is moistening, neat in edge; Thalline rod-short; Gram-positive.
2, analysis of physio biochemical characteristics
With reference to " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying. common bacteria system identification handbook. Beijing: Science Press, 2011.) and " Microbiology Experiment " (Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiment (third edition). Beijing: Higher Education Publishing House, 1999.) measure the physiological and biochemical property of above-mentioned crop rhizosphere vinelandii GD601.
The physiological and biochemical property measurement result of described crop rhizosphere vinelandii GD601 is as follows:
Catalase reacts: positive;
Oxydase reaction: negative;
Growth temperature: 4 DEG C of growths, 28 DEG C of growths, 37 DEG C do not grow, and 60 DEG C do not grow;
Salt tolerance is tested: 2%NaCl grows, and 5%NaCl grows, and 7%NaCl grows, and 10%NaCl grows;
Phenylalanine deaminase is tested: negative;
Utilize Citrate trianion: positive;
Hydrolyzed starch: positive;
Yolk lecithin enzyme: positive;
Methyl red test: negative;
V.P tests: negative;
PH5.7 grows: negative;
Sugar alcohol fermentation and acid: glucose is positive, N.F,USP MANNITOL is positive, and lactose is negative, and sucrose is positive.
3,16srDNA sequence homology analysis
Ordinary method cultivates the crop rhizosphere vinelandii GD601 that above-mentioned steps one separation and purification obtains, extract the STb gene of bacterial strain as gene amplification template, with bacterium 16srDNA universal primer, 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492r:5 '-TACCTTGTTACGACTT-3 ' carries out PCR reaction.Reaction system adopts Shanghai biotechnology company limited pcr amplification test kit.Response procedures is: 95 DEG C of sex change 30s, 55 DEG C of annealing 1min, 72 DEG C of extensions 2min, totally 30 circulations.DNA sequencing is won polygala root biotech company by Beijing three and is completed, sequence assembly and similarity analysis use DNAStar software to complete, and gene comparison is completed online by American National Biotechnology Information center ncbi database (http://www.ncbi.nlm.nih.gov) and EzTaxon.
The 16SrDNA gene fragment that pcr gene amplification obtains crop rhizosphere vinelandii GD601 is about 1.4kb, online sequence analysis is carried out with published 16SrDNA sequence in NCBI and EzTaxon database, result display GD601 and paddy rice Arthrobacter ArthrobacteroryzaeKV-651 after measuring sequence
tthe homology of (GenBank accession number AB279889) is the highest, reaches 99.645%.
The 16srDNA sequence of crop rhizosphere vinelandii GD601 refers to sequence 1 in sequence table.
4, growth characteristics analysis
Optimum temperuture and the optimal pH growth experiment of crop rhizosphere vinelandii GD601 are carried out.Adopt nitrogen-free agar, respectively at the thermal adaptability of 4 DEG C, 28 DEG C, 37 DEG C, 60 DEG C cultivations, observation, record bacterial strain, each process repeats for 3 times.Adjustment pH is respectively 4,5,6,7,8,9,10, and each process repeats for 3 times, cultivates, observes, records the optimal pH of strain growth.
Result shows, the optimum growth temperature of described crop rhizosphere vinelandii GD601 is 28 DEG C, and the most suitable growth pH is pH7 ~ 8.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16srDNA sequence homology analysis result, crop rhizosphere vinelandii GD601 step one separation and purification obtained is accredited as bacterium territory actinomycete group micrococcaceae (Micrococcaceae) genus arthrobacter (Arthrobactersp.).This Arthrobacter (Arthrobactersp.) GD601 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 20th, 2013 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCCNo.7779.
Embodiment 2, Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779 nitrogenase activity determination
Nitrogenase activity determination is carried out to Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779 that embodiment 1 obtains, concrete grammar is as described below: in 15 × 150mm screw socket Glass tubing, add 5mL improve fixed nitrogen substratum bevel, inoculation Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779,28 DEG C of cultivations.With conventional bacterial classification azotobacter chroococcum (Azotobacterchroococcum) ACCC11103 that produces of microbial fertilizer for positive control, not inoculating blank inclined-plane is negative control, and 3 repetitions are established in each process.After cultivating 72h, change rubber plug, inject acetylene gas and make final concentration be 10%, with medical proof fabric sealing, continue to cultivate 72h, get 100 μ L reactant gasess, use gas chromatograph for determination ethylene emanation, according to the nitrogenase activity of following formulae discovery bacterial strain.Nitrogenase activity (nmol/mgh)=C
2h
4nmol/ [tropina amount (mg) × reaction times (h)], wherein C
2h
4nmol=1000 × C
2h
4volume (μ l) × 273 × P/ [22.4 × (273+t DEG C) × 760], wherein P is air pressure (mm mercury column), and t is temperature of reaction.
Wherein, tropina content assaying method is as described below: wash in centrifuge tube with 5mL physiological saline by the lawn on test tube slant, collect thalline, the NaOH boiling water adding 3mL0.5M in thalline boils 5min, adds the HCl mixing of 3mL0.5M, gets supernatant 1.0mL after centrifugal, add 5mL Coomassie Brillant Blue solution, eddy mixer mixes, and colour developing 3min, measures the light absorption value A at 595nm place
595, calculate tropina content according to bovine serum albumin typical curve.
Result shows, and the nitrogenase activity of Arthrobacter (Arthrobactersp.) GD601CGMCCNo.7779 screened is 30.570nmolC
2h
4/ hmg albumen, statistical study pole is significantly higher than microbial fertilizer, and to commonly use the nitrogenase activity of producing bacterial classification azotobacter chroococcum ACCC11103 be 25.100nmolC
2h
4/ hmg albumen.This result shows, Arthrobacter of the present invention (Arthrobactersp.) GD601CGMCCNo.7779 has very high fixed nitrogen potential, has broad application prospects in Vegetables Factory Plantation Growing Seedlings Inoculant, nitrogen-fixing microorganism microbial inoculum and biological organic fertilizer are produced.
Claims (8)
1. Arthrobacter (Arthrobactersp.) GD601, it is CGMCCNo.7779 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. a microbial inoculum, its activeconstituents is Arthrobacter according to claim 1 (Arthrobactersp.) GD601.
3. Arthrobacter according to claim 1 (Arthrobactersp.) GD601 is in preparation following 1)-3) in application in arbitrary microbial inoculum:
1) for the microbial inoculum of fixed nitrogen;
2) microbial inoculum of nitrogenase is produced;
3) microbial inoculum of Promoting plant growth.
4. Arthrobacter according to claim 1 (Arthrobactersp.) GD601 or microbial inoculum according to claim 2 are applied in production nitrogenase.
5. application in Vegetables Factory Plantation Growing Seedlings of Arthrobacter according to claim 1 (Arthrobactersp.) GD601 or microbial inoculum according to claim 2 or preparing the application in biological organic fertilizer.
6. contain the biological organic fertilizer of Arthrobacter (Arthrobactersp.) GD601 or microbial inoculum according to claim 2 described in claim 1.
7. cultivating the method for Arthrobacter (Arthrobactersp.) GD601 described in claim 1, comprising Arthrobacter (Arthrobactersp.) GD601 for cultivating the step of cultivating in the substratum of Arthrobacter.
8. the preparation method of microbial inoculum described in claim 2, to comprise the steps: Arthrobacter according to claim 1 (Arthrobactersp.) GD601, as activeconstituents, to obtain described microbial inoculum.
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| CN102286402A (en) * | 2011-07-15 | 2011-12-21 | 中国农业科学院农业资源与农业区划研究所 | Soybean rhizosphere azotobacter for producing activated calcium carbonate (ACC) and realizing antagonistic effect on various pathogenic fungi and purpose thereof |
| CN102286401A (en) * | 2011-07-15 | 2011-12-21 | 中国农业科学院农业资源与农业区划研究所 | Rice endotrophic azotobacter for producing activated calcium carbonate (ACC) and realizing antagonistic effect on fusarium graminearum and purpose thereof |
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| CN102286402A (en) * | 2011-07-15 | 2011-12-21 | 中国农业科学院农业资源与农业区划研究所 | Soybean rhizosphere azotobacter for producing activated calcium carbonate (ACC) and realizing antagonistic effect on various pathogenic fungi and purpose thereof |
| CN102286401A (en) * | 2011-07-15 | 2011-12-21 | 中国农业科学院农业资源与农业区划研究所 | Rice endotrophic azotobacter for producing activated calcium carbonate (ACC) and realizing antagonistic effect on fusarium graminearum and purpose thereof |
Non-Patent Citations (2)
| Title |
|---|
| 一株具有ACC脱氨酶活性固氮酶的筛选与鉴定;陈倩,等;《植物营养与肥料学报》;20111231;第17卷(第6期);1515-1521 * |
| 小麦、水稻、玉米、白菜、芹菜内生固氮菌及其系统发育;孙建光，等;《中国农业科学》;20121231;第45卷(第7期);1303-1317 * |
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