CN103333837A - Nitrogen-fixing arthrobacterium and application thereof - Google Patents
Nitrogen-fixing arthrobacterium and application thereof Download PDFInfo
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- CN103333837A CN103333837A CN2013102722065A CN201310272206A CN103333837A CN 103333837 A CN103333837 A CN 103333837A CN 2013102722065 A CN2013102722065 A CN 2013102722065A CN 201310272206 A CN201310272206 A CN 201310272206A CN 103333837 A CN103333837 A CN 103333837A
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- 241000186073 Arthrobacter sp. Species 0.000 claims abstract description 32
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- 239000002068 microbial inoculum Substances 0.000 claims description 41
- 241000186063 Arthrobacter Species 0.000 claims description 34
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 32
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
Abstract
The invention discloses a nitrogen-fixing arthrobacterium and application thereof. The nitrogen-fixing arthrobacterium is Arthrobacter sp. GD601, which is preserved in China General Microbiological Culture Collection Center by a preservation number of CGMCC No.7779. The Arthrobacter sp. GD601 CGMCC No.7779 provided by the invention can be colonized in rhizosphere of a crop, and has a quite high nitrogen-fixing potential; the nitrogen-fixing arthrobacterium has a wide application prospect in inoculation agent, microbial agent and bio-organic fertilizer production for vegetable industrialized seedling production.
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 the agriculture production.Produce a large amount of energy of chemical nitrogen fertilizer consumption, account for 70%～80% of total production cost.Energy day is becoming tight in recent years, and chemical fertilizers production soars with the coal price lattice and causes the chemical nitrogen fertilizer price increase, and the peasant is difficult to bear, and repercussion is strong.Chemical nitrogen fertilizer is being brought into play huge effect aspect raising crop yield, the guarantee China grain security, but the unreasonable nitrogen of executing has formed serious pollution of area source, causes a series of serious harm, makes us startling as Taihu Lake blue-green algae event.The urban groundwater azotate pollution has caused threat to drinking water safety.
Contain 78% nitrogen in the atmosphere, but its existence form is molecular state nitrogen, animals and plants can not directly utilize, and nature has only some prokaryotic micro-organisms to have the ability of directly utilizing nitrogen in the atmosphere, with its reduction ammonification, biological nitrogen fixation that Here it is.Select for use the batch production of high-efficiency nitrogen-fixing microorganism bacterial classification to produce and make microbial inoculum, or further become biological products with other material Compound Machining that is rich in plant nutrition, be applied to agriculture production, crop nitrogen nutrition can be provided, improve the crop rhizosphere ecotope, improve the soil organisms fertility property, 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 does not have the resource exhaustion problem, is the Sustainable development agricultural material product.2. biological nitrogen fertilizer is environmental friendly fertilizer, and it is produced and use does not all produce pollutants such as the three wastes, and can improve soil physico-chemical property, increase soil fertility, and be ecological agriculture agricultural material product.3. biological nitrogen fertilizer production power consumption less, cost is low, and its cost only is 20%～40% of equivalent chemical nitrogen fertilizer, can save a large amount of coal and oil equal energy source strategic materials for country.4. use biological nitrogen fertilizer can significantly reduce agriculture production cost, improve quality of agricultural product, promote soil fertility, the peasant is benefited, development promotes social harmony.
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, agriculture production press for can high-efficiency nitrogen-fixing the production bacterial strain.
Summary of the invention
The purpose of this invention is to provide the bacterium that a strain has nitrogen fixation.
Bacterium provided by the invention is Arthrobacter (Arthrobacter sp.) GD601, and the deposit number of this bacterial strain at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.7779.
Another object of the present invention provides a kind of microbial inoculum, and the activeconstituents of this microbial inoculum is described Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779.
This microbial inoculum also can comprise auxiliary material, as the stalk of the ight soil of the peat composed of rotten mosses, animal, all kinds of crops, loose shell, straw, Pericarppium arachidis hypogaeae etc. except Arthrobacter (Arthrobacter sp.) the GD601CGMCC No.7779 that comprises as activeconstituents.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 a in clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica and the diatomite; Described vegetable material is at least a in Semen Maydis powder, bean powder and the 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 the described microbial inoculum, described activeconstituents can be to be existed by the form of the mixture of the filtrate of the fermented liquid of the viable cell cultivated, viable cell, cell culture or cell and filtrate.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, promotes plant-growth, improves stress resistance of plant, produces nitrogenase etc.
Described Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779 is in preparation following 1)-5) in application in the arbitrary microbial inoculum also belong to protection scope of the present invention:
1) is used for the microbial inoculum of fixed nitrogen;
2) microbial inoculum of generation nitrogenase;
3) microbial inoculum of promotion plant-growth;
4) microbial inoculum of improvement fertilizing soil;
5) microbial inoculum of raising stress resistance of plant.
In the 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 (Arthrobacter sp.) GD601CGMCC No.7779 or be that the microbial inoculum of activeconstituents is used in producing nitrogenase and the application in the preparation biological organic fertilizer also belongs to protection scope of the present invention with Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779.
Another object of the present invention provides and a kind ofly contains described Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779 or be the biological organic fertilizer of the microbial inoculum of activeconstituents with described Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779.
Another purpose of the present invention provides the method for a kind of cultivation Arthrobacter (Arthrobacter sp.) GD601CGMCCNo.7779, comprises the step that Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779 is cultivated at the substratum that is used for the cultivation Arthrobacter.
Another purpose of the present invention provides a kind of method for preparing described microbial inoculum, and this method comprises the steps: described Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779 is obtained described microbial inoculum as activeconstituents.
The present invention separates vinelandii from the barley rhizosphere soil of farmland, further screen the bacterial strain of higher nitrogenase activity, finishing screen is chosen high-efficiency nitrogen-fixing Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779, and this bacterial strain nitrogenase activity is 30.570nmol C
2H
4/ hmg albumen, the statistical study utmost point are significantly higher than the microbial fertilizer bacterial classification azotobacter chroococcum ACCC11103 that produces commonly used, have broad application prospects in vegetable factoryization is grown seedlings Inoculant, nitrogen-fixing microorganism microbial inoculum and biological organic fertilizer production.
The preservation explanation
Strain name: Arthrobacter
Latin name: (Arthrobacter sp.)
Strain number: GD601
Preservation mechanism: China Committee for Culture Collection of Microorganisms 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
The preservation center numbering of registering on the books: CGMCC No.7779
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Substratum used among the 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 are 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 are 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.
Among the following embodiment used azotobacter chroococcum (Azotobacter chroococcum) ACCC11103(referring to " Sun Jianguang etc. the screening of high-efficiency nitrogen-fixing genus bacillus and biological characteristics thereof. 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 application's experiment.
Separation and the evaluation of embodiment 1, Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779
One, the enrichment of crop rhizosphere vinelandii GD601 with separate
Getting 10g soil sample (picking up from inner mongolia autonomous region) puts into 90ml sterilized water shaking table vibration 20min and makes muddy liquid, draw 5ml and put into 30ml vinelandii enrichment culture liquid ACCC55,100rpm, 28 ℃ are carried out the shaking table shaking culture, renew bright nutrient solution behind the 72h and continue to cultivate.Carrying out the fixed nitrogen sporeformer after repeating to cultivate 3 times separates.The above-mentioned vinelandii enrichment culture of absorption 1ml thing is put in the 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 ℃ of boiling water, each extent of dilution of cooling back is got 0.1ml and is coated on the nitrogen-free agar flat board, and 29 ℃ leave standstill cultivation.After 2～3d treats that bacterium colony forms, on the ACCC55 vinelandii solid medium flat board of improvement, carry out the bacterial classification purifying with plate streak.One of them bacterial strain called after crop rhizosphere vinelandii GD601 with the separation and purification gained.
Two, the evaluation of crop rhizosphere vinelandii GD601
Also screen the crop rhizosphere vinelandii GD601 that obtains from one separation and purification of the following aspects authentication step:
1, morphology is identified
It is stable to be in logarithmic phase and bacterium colony size, the crop rhizosphere vinelandii GD601 that above-mentioned steps one is separated and purifying obtains carries out single bacterium colony state observation, mainly comprises size, color, transparency, wettability, bacterium colony condition of surface (whether smooth, projection, fold, depression etc.), the colony edge state (whether neat, irregular, radial etc.) of bacterium colony.
For the described crop rhizosphere vinelandii GD601 that is in logarithmic phase, behind smear staining, adopt the form of observation by light microscope thalline.
The result shows, the crop rhizosphere vinelandii GD601 bacterium colony circular protrusions that above-mentioned steps one is separated and purifying obtains, and oyster white, glossy, smooth surface is moistening, neat in edge; The 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 reaction: the positive;
Oxydase reaction: feminine gender;
Growth temperature: 4 ℃ of growths, 28 ℃ of growths or not for 37 ℃, do not grow for 60 ℃;
Salt tolerance test: 2%NaCl growth, 5%NaCl growth, 7%NaCl growth, 10%NaCl growth;
Phenylalanine deaminase test: feminine gender;
Utilize Citrate trianion: the positive;
Hydrolyzed starch: the positive;
Yolk lecithin enzyme: the positive;
Methyl red test: feminine gender;
V.P test: feminine gender;
PH5.7 growth: feminine gender;
Sugar alcohol fermentation and acid: the glucose positive, the N.F,USP MANNITOL positive, lactose feminine gender, the sucrose positive.
3,16s rDNA sequence homology analysis
Ordinary method is cultivated the crop rhizosphere vinelandii GD601 that above-mentioned steps one separation and purification obtains, extract total DNA of bacterial strain as the gene amplification template, with bacterium 16s rDNA universal primer, 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492r:5 '-TACCTTGTTACGACTT-3 ' carry out the PCR reaction.Reaction system adopts Shanghai biotechnology company limited pcr amplification test kit.Response procedures is: 95 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ of extensions 2min, totally 30 circulations.Dna sequencing is finished by Beijing three rich polygala root biotech companies, sequence assembly and similarity analysis use DNAStar software to finish, and gene is compared by American National biotechnology information center ncbi database (http://www.ncbi.nlm.nih.gov) and EzTaxon is online finishes.
The pcr gene amplification obtains the about 1.4kb of 16S rDNA gene fragment of crop rhizosphere vinelandii GD601, measure after the sequence with NCBI and EzTaxon database in disclosed 16S rDNA sequence carry out online homology comparison, the result shows GD601 and paddy rice Arthrobacter Arthrobacter oryzae KV-651
TThe homology of (GenBank accession number AB279889) is the highest, reaches 99.645%.
The 16s rDNA sequence of crop rhizosphere vinelandii GD601 sees sequence 1 in the sequence table for details.
4, growth characteristics analysis
Optimum temperuture and the optimal pH growth experiment of crop rhizosphere vinelandii GD601 have been carried out.Adopt nitrogen-free agar, at the thermal adaptability of 4 ℃, 28 ℃, 37 ℃, 60 ℃ cultivations, observation, record bacterial strain, each is handled 3 times and repeats respectively.Adjust pH and be respectively 4,5,6,7,8,9,10, each is handled 3 times and repeats, the optimal pH of cultivation, observation, record strain growth.
The result shows that the optimum growth temperature of described crop rhizosphere vinelandii GD601 is 28 ℃, and the suitableeest growth pH is pH7～8.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis result, the crop rhizosphere vinelandii GD601 that the step 1 separation and purification is obtained is accredited as bacterium territory actinomycete group micrococcaceae (Micrococcaceae) genus arthrobacter (Arthrobacter sp.).This Arthrobacter (Arthrobacter sp.) GD601 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 20th, 2013 and (is called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.7779.
Embodiment 2, Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779 nitrogenase activity determination
GD601CGMCC No.7779 carries out nitrogenase activity determination to embodiment 1 resulting Arthrobacter (Arthrobacter sp.), concrete grammar is as described below: add 5mL improvement fixed nitrogen substratum bevel in 15 * 150mm screw socket Glass tubing, inoculation Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779,28 ℃ of cultivations.With the positive contrast of microbial fertilizer production commonly used bacterial classification azotobacter chroococcum (Azotobacter chroococcum) ACCC11103, do not inoculate the negative contrast in blank inclined-plane, 3 repetitions are established in each processing.After cultivating 72h, change rubber plug, it is 10% that the injection acetylene gas makes final concentration, with the medical proof fabric sealing, continues to cultivate 72h, gets 100 μ L reactant gasess, with gas chromatograph for determination ethene growing amount, according to the nitrogenase activity of following formula calculating bacterial strain.Nitrogenase activity (nmol/mgh)=C
2H
4Nmol/[tropina amount (mg) * reaction times (h)], C wherein
2H
4Nmol=1000 * C
2H
4Volume (μ l) * 273 * P/[22.4 * (273+t ℃) * 760], wherein P is air pressure (mm mercury column), t is temperature of reaction.
Wherein, the tropina content assaying method is as described below: with 5mL physiological saline the lawn on the test tube slant is washed in the centrifuge tube, collect thalline, the NaOH boiling water that adds 3mL0.5M in the thalline boils 5min, and the HCl that adds 3mL0.5M mixes, and gets supernatant 1.0mL after centrifugal, add 5mL Xylene Brilliant Cyanine G solution, mix colour developing 3min, the light absorption value A at mensuration 595nm place at eddy mixer
595, calculate tropina content according to the bovine serum albumin typical curve.
The result shows that the nitrogenase activity of the Arthrobacter that screens (Arthrobacter sp.) GD601CGMCC No.7779 is 30.570nmol C
2H
4It is 25.100nmol C that/hmg albumen, the statistical study utmost point are significantly higher than microbial fertilizer nitrogenase activity of producing bacterial classification azotobacter chroococcum ACCC11103 commonly used
2H
4/ hmg albumen.This result shows that Arthrobacter of the present invention (Arthrobacter sp.) GD601CGMCC No.7779 has very high fixed nitrogen potential, has broad application prospects in vegetable factoryization is grown seedlings Inoculant, nitrogen-fixing microorganism microbial inoculum and biological organic fertilizer production.
Claims (10)
1. Arthrobacter (Arthrobacter sp.) GD601, its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.7779.
2. microbial inoculum, its activeconstituents is the described Arthrobacter of claim 1 (Arthrobacter sp.) GD601CGMCC No.7779.
3. microbial inoculum according to claim 2, it is characterized in that: described microbial inoculum is following 1)-5) in arbitrary microbial inoculum:
1) is used for the microbial inoculum of fixed nitrogen;
2) microbial inoculum of generation nitrogenase;
3) microbial inoculum of promotion plant-growth;
4) microbial inoculum of improvement fertilizing soil;
5) microbial inoculum of raising stress resistance of plant.
4. the described Arthrobacter of claim 1 (Arthrobacter sp.) GD601CGMCC No.7779 is in preparation following 1)-5) in application in the arbitrary microbial inoculum:
1) is used for the microbial inoculum of fixed nitrogen;
2) microbial inoculum of generation nitrogenase;
3) microbial inoculum of promotion plant-growth;
4) microbial inoculum of improvement fertilizing soil;
5) microbial inoculum of raising stress resistance of plant.
5. the described application of microbial inoculum according to claim 3 or claim 4 is characterized in that: 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.
6. the described Arthrobacter of claim 1 (Arthrobacter sp.) GD601CGMCC No.7779 or claim 2,3 or 5 described microbial inoculums are used in producing nitrogenase.
7. the described Arthrobacter of claim 1 (Arthrobacter sp.) GD601CGMCC No.7779 or claim 2,3 or 5 described microbial inoculums application or the application in the preparation biological organic fertilizer in vegetable factoryization is grown seedlings.
8. the biological organic fertilizer that contains the described Arthrobacter of claim 1 (Arthrobacter sp.) GD601CGMCC No.7779 or claim 2,3 or 5 described microbial inoculums.
9. cultivate the method for the described Arthrobacter of claim 1 (Arthrobacter sp.) GD601CGMCC No.7779, comprise the step that Arthrobacter (Arthrobacter sp.) GD601CGMCC No.7779 is cultivated at the substratum that is used for the cultivation Arthrobacter.
10. the preparation method of claim 2,3 or 5 described microbial inoculums comprises the steps: the described Arthrobacter of claim 1 (Arthrobacter sp.) GD601CGMCC No.7779 is obtained described microbial inoculum as activeconstituents.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106358661A (en) * | 2016-08-27 | 2017-02-01 | 山东省潍坊市农业科学院 | Method for cultivating mother strain of nodule bacteria |
| WO2023279551A1 (en) * | 2021-07-06 | 2023-01-12 | 石河子大学 | Compound microbial fertilizer, preparation method therefor and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN102286400A (en) * | 2011-07-15 | 2011-12-21 | 上海环垦生态科技有限公司 | Rice endotrophic azotobacter for improving disease resistance and stress resistance of crops and purpose thereof |
-
2013
- 2013-07-01 CN CN201310272206.5A patent/CN103333837B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN102286400A (en) * | 2011-07-15 | 2011-12-21 | 上海环垦生态科技有限公司 | Rice endotrophic azotobacter for improving disease resistance and stress resistance of crops and purpose thereof |
Non-Patent Citations (2)
| Title |
|---|
| 孙建光，等: "小麦、水稻、玉米、白菜、芹菜内生固氮菌及其系统发育", 《中国农业科学》 * |
| 陈倩,等: "一株具有ACC脱氨酶活性固氮酶的筛选与鉴定", 《植物营养与肥料学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106358661A (en) * | 2016-08-27 | 2017-02-01 | 山东省潍坊市农业科学院 | Method for cultivating mother strain of nodule bacteria |
| CN106358661B (en) * | 2016-08-27 | 2020-04-17 | 山东省潍坊市农业科学院 | Method for cultivating rhizobium mother strain |
| WO2023279551A1 (en) * | 2021-07-06 | 2023-01-12 | 石河子大学 | Compound microbial fertilizer, preparation method therefor and application thereof |
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