CN101319198B - Mangrove plant rhizosphere growth promoting azotobacter (DZY-N56) and uses thereof - Google Patents
Mangrove plant rhizosphere growth promoting azotobacter (DZY-N56) and uses thereof Download PDFInfo
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Abstract
The invention provides redwood plant rhizosphere promoting azotobacteria (DZY-N56) and an application thereof. The bacteria is Sphingomonas sp. DZY-N56, is sieved and separated from a redwood plant rhizosphere sediment sample in a tropical mangrove zone, contains a nif gene nifH and 16S rDNA as shown by SEQ1, and is preserved in China Center for Type Culture Collection with a preservation No. of CCTCC NO: M207120. The strain has high biological nitrogen fixation activity, and can obviously promote plant growth by using microbial inoculum which contains the bacteria for inoculation of redwood plants such as mangrove, Rhizophora stylosa, bruguiera gymnorrhiza and so on, thereby the strain can be applied to prepare biological azotobacterin for promoting the growth of the mangrove plants.
Description
Technical field:
The invention belongs to biological technical field, particularly relate to a kind of application that mangrove plant is had good short come into force arnotto woods rhizosphere azotobacter and preparation bio-azotobacter fertilizer thereof.
Background technology:
Mangrove forest is the woody plant community, xylium of NATURAL DISTRIBUTION in the torrid zone, seashore tideland, subtropics, cover about 60-75% torrid zone and subtropics seashore, the eubiosis of safeguarding the river mouth, bay is played an important role, be important component part (the Holguin et al. of fecundity of the sea, 2001), be considered to have the ecosystem than high productivity.This ecosystem has higher organic level (global annual litter is 100TgC), for the stretch of coastal water and the relevant coenosis habitat of adjoining provides organism (Holguin et al., 2001; Lugomela and Bergman, 2002).Yet the inorganic nutrients of mangrove ecosystem are poor, especially lower (Holguin et al., 1992 of inorganic nitrogen level; Vazquezet al., 2000), the biological nitrogen fixation of diazotroph is proved to be main source (Hicks and Sylvester, 1985 of mangrove ecosystem inorganic nitrogen; Kyaruzi et al 2003).Biological nitrogen fixation produces influence greatly as mangrove ecosystem and source of students element biomass geochemistry round-robin important step to mangrove ecosystem and ecotope.
Owing to human excessive exploitation, reclaim fields from the sea and behavior such as urbanization, the mangrove forest in the whole world is reduced just with surprising rapidity, according to the conservative data estimation of Food and Argriculture OrganizationFAO, mangrove forest just disappears with the speed of annual decreased average 1-2%, its speed even surpassed the disappearance speed of karang and tropical forest.In developing country, the speed of disappearance is faster, and mangrove forest be positioned at developing country more than 90%.American Red woods action items group responsible official Aheredo (1999) thinks that tropical and subtropical zone country once was distributed with mangrove forest on 3/4 shoreline, and is only remaining now less than half, and most of mangrove forest is the ecosystem of serious degradation.Scientist's prophesy, if develop as one pleases, in nearly 100 years of future, the mankind will thoroughly lose mangrove forest (Duke, 2007).
In recent years, the researchs such as child care of the maintenance of mangrove ecosystem and mangrove forest plant become popular research field, the international conference that is intended to protect mangrove forest that also has a plurality of international organizations and group to initiate.China's Agenda 21 (1994) preference is recovered mangrove forest to have included agenda in reconstruction technique in the works.The ITTO tissue, the planned target of 2002-2006 also recovers to classify as one of primary subsidy target (Liao Baowen, 2005) to mangrove ecosystem.The resuming work of visible red woods Wetlands ecosystems needed us badly and carries out energetically, all faces big difficulty but Study on Theory still is actual carrying out of resuming work.
The recovery building-up work of mangrove forest is relatively slower always, and this mainly is because the mangrove forest surrival rate of afforestation extremely low (Liao Baowen, 1999).The scientist of country such as Mexico and India utilizes plant-growth to urge living bacterium (PGPB, plant growth-promoting bacteria) mangrove forest is urged to give birth to, obtained the certain phase progress, but the short bacterial classification of giving birth to of the suitable mangrove forest of finding still is a minority, and this type of bacterial classification still lacks and be most by the collection of maintaining secrecy.
Tropical Foresty Inst., Chinese Academy of Foresty Sciences just cooperates with biological study center, Mexico northwest at present, introduce relevant bacterial classification the main mangrove of China is inoculated test and relevant microbial inoculum research, in the hope of solving the extremely low thorny problem of China's beach location mangrove forest surrival rate of afforestation.Yet it is little to introduce the surviving rate of bacterial classification in our national mangrove forest, and also exists outer kind invasion to understand the various hidden danger that produce.
Summary of the invention:
The objective of the invention is to propose strain screening and separating from the tropical mangrove forest of China, have the short growing nitrogen-fixing bacterium novel species of the active mangrove plant rhizosphere of higher biological nitrogen fixation.
Another object of the present invention is to propose the application of the short growing nitrogen-fixing bacterium of described mangrove plant rhizosphere at the preparation bio-azotobacter fertilizer.
Mangrove plant rhizosphere proposed by the invention is urged the growing nitrogen-fixing bacterium, is to gather the vinelandii novel species of separating the mangrove plant rhizosphere sediment sample from the tropical mangrove area of Sanya, Hainan Province.Its called after of classifying: Sphingol single-cell belongs to (Sphingomonas sp.) DZY-N56.This bacterium on July 28th, 2007 be preserved in Chinese typical culture collection center (CCTCC, the address: Chinese Wuhan City Wuhan University), deposit number: CCTCC NO:M207120.
The bacteria characteristic of bacterial classification of the present invention is described below:
Gram-negative, shaft-like, do not produce spore, 0.2-0.4 * 1.6-2.5 μ m, cell is single, and less (Fig. 1-3) in pairs also arranged.The oxydase reaction feminine gender, the catalase positive, aerobic or amphimicrobian.Well-grown on Ashby, LB and BUG substratum.Salt tolerance is better, can be divided into normal growth in 5% the nutrient solution at salt.About can growing for diameter 1-2cm through cultivation about 10d on the Ashby substratum, carrotiness, circle, neat in edge surface be exsiccant projection bacterium colony comparatively.Acetylene reduction method (ARA) shows that this bacterium can utilize N
2As nitrogenous source, have higher biological nitrogen fixation activity, under the pure culture condition, its nitrogenase activity 20-74umolC
2H
2h
-1Mg
-1Fresh weight.
Extract genomic dna from the pure growth of bacterial strain (Sphingomonas sp.) DZY-N56, use the specific primer PolF/PolR of nif (with reference to Poly et al., 2001) carry out pcr amplification, comparatively ideal purpose band increases, prove that this bacterial strain has biological nitrogen fixation gene (Fig. 4), its sequence is shown in SEQ ID NO:2.Utilization bacterial 16 S rDNA complete sequence universal primer 16SF/16SR (with reference to Weisenburg et al., 1998) carries out pcr amplification, obtains 16S rDNA sequence by sequencing analysis, and the complete sequence of the 16S rDNA of this bacterial strain is shown in SEQ ID NO:1.By the BLAST software among the GenBank sequence in 16S rDNA sequence and the GenBank database is compared, in database, do not find on all four sequence, be indicated as the new gene order of finding first.Successfully submitting new gene order to gene pool obtains accession number and is: EF202991.
The 16S rDNA sequence similarity higher representative gene order of having chosen part and measure in the GenBank database with Neibor-joining method constructing system evolutionary tree (Fig. 5), is carried out Phylogenetic Analysis by ClustalW software and Mega software.16S rDNA sequence with (Sphingomonas sp.) DZY-N56 is the most close with Sphingol single-cell Sphingomonas panni (AJ575818.2) homology as can be seen from phylogenetic tree (Fig. 5), is 97% (Query coverage is 96%).Result such as Physiology and biochemistry, BIOLOG shows that further these two kinds of bacterium have obvious difference (as table 1).This shows that bacterial strain (Sphingomonas sp.) DZY-N56 is the new fixed nitrogen bacterial classification of a strain.
Two kinds of bacterium indexs of table 1 Sphingomonas panni and Sphingomonas sp.strain DZY-N56 relatively
(+: expression positive reaction;-: the expression negative reaction)
Annotate: the bacterial classification character of Sphingomonas panni (AJ575818.2) is with reference to Hans-J ü rgen Busse, 2005 article Description of two novelspecies, Sphingomonas abaci sp.nov.and Sphingomonas panni sp.nov.DZY-N56 data are BIOLOG bacterium automatic analyser data.
Handle two kinds of mangroves with Sphingomonas sp.DZY-N56, Bruguiera conjugata (Bruguiera gymnorhiza) and Rhizophora stylosa (Rhizophorastylosa), the result is as shown in table 2.
Table 2Sphingomonas sp.DZY-N56 influence after 45 days to Bruguiera conjugata and Red sea Lan Pei processing
Annotate: the table intermediate value is 10 groups of parallel sample mean values.Weight refers to weight in wet base in the biomass; Remaining weight all refers to dry weight.
From table 2 we as can be seen this bacterium for Bruguiera conjugata and Rhizophora stylosa good growth-promoting functions is arranged.Compare with control group, the leaf area of Bruguiera conjugata has tangible increase, and increase rate is 152%; Content of carotenoid also significantly increases, and is 3.5 times of control group.Photosynthetic pigments content also increases to some extent, and chlorophyll a, chlorophyll b and total chlorophyll have increased by 52%, 96% and 91% respectively.Then obvious not as Bruguiera conjugata for the Rhizophora stylosa effect, leaf area has increased by 44%, and chlorophyll has increased by 148% in the photosynthetic pigments.
This bacterial strain is carried out the biological safety experiment, soil inoculation method result shows that (about 28 ℃) at ambient temperature are under the humidity 60%-75% condition, this fermented liquid does not all have pathogenecity to Hai Lan, Rhizophora stylosa, Bruguiera conjugata etc., and is safe and harmless to the growth of mangrove.By detect indexs such as strains tested recall rate, the variation of soil original position nitrogenase activity with the plate isolation method, the result can continue survival after showing this bacterium inoculation, can significantly promote the soil nitrogenase activity to increase simultaneously.Experiment showed, with the mangroves such as microbial inoculum inoculation Hai Lan, Rhizophora stylosa and Bruguiera conjugata that contain this bacterium obviously to promote plant strain growth, therefore, this bacterium can be used as the application that preparation promotes the bio-azotobacter fertilizer of mangrove plant growth.
The present invention will produce beneficial effect in the following areas: this bacterium is come into force fruit better for the short of mangrove plant, the production engineering bacterium that can be used for nature mangrove ecosystem bacterial manure, improve the productivity of mangrove ecosystem, be expected to be used for the challenge that mangrove forest recovers, afforests.
Description of drawings:
Fig. 1, Fig. 2, Fig. 3 are Sphingomonas sp.DZY-N56 bacterium colony, scanning, transmission electron microscope photo;
Fig. 4 is the electrophoresis photo of the genomic dna of Sphingomonas sp.DZY-N56 and 16S rDNA that increases and nif nifH;
Fig. 5 is Sphingomonas sp.DZY-N56 position in 16S rDNA phylogenetic tree.
Embodiment
One, materials and methods
1, material
1.1 soil sample collection
Sample picks up from littoral mangrove area Bruguiera conjugata (Bruguiera gymnorhiza) the rhizosphere settling in red Shahe, Sanya, Hainan Province, and the laboratory cryopreservation is taken back in sealing in the polyethylene bag of the sterilization of packing into behind the sample collecting.
1.2 substratum
Ashby liquid nutrient medium: N.F,USP MANNITOL 10.0g, KH
2PO
4H
2O 0.2g, MgSO
47H
2O 0.2g, NaCl 0.2g, CaSO
40.1g, CaCO
35.0g, deionized water 1000mL, pH 7.0.
Ashby solid medium: Ashby liquid nutrient medium+2.2% agar.
Improvement
No nitrogen liquid nutrient medium: sucrose 10g, oxysuccinic acid 5.0g, K
2HPO
4H
2O 0.1g, KH
2PO
4H
2O 0.4g, MgSO
47H
2O 0.2g, NaCl 0.1g, Na
2MoO
4H
2O 0.002g, FeCl30.01g, deionized water 1000ml, pH 7.0.
The LB substratum: yeast extract 5g, Tryptones 10g, NaCl 10g, agar 1-2%, deionized water 1000ml, pH 7.0.
2, method
2.1 strain separating
With get behind the sample mixing about 1g in 200ml Ashby,
Perhaps
No nitrogen liquid nutrient medium (the Ashby substratum with
Substratum was with 1: 1 mixed) in, 30 ℃ of constant temperature 48h add rich the cultivation.Then bacterium liquid is diluted to 10-4,10-5, three extent of dilution of 10-6 respectively, each dilution bacteria suspension is inoculated on the Ashby solid medium with coating method, and 30 ℃ of 48h cultivate the back and select typical single bacterium colony to be further purified.
2.2 bacterial classification purifying
With isolated bacterium colony, with inoculating needle picking typical single bacterium colony,, under opticmicroscope, observe thalli morphology through smear staining, impure as the isolating bacterium colony of institute, separate with spread plate once more after should doing serial dilution, purebred until obtaining.Inoculation behind the purifying is preserved in no nitrogen Ashby solid medium and the full substratum glycerine of LB.
2.3 nitrogenase vitality test in the liquid culture
Adopt the acetylene reduction method to measure nitrogenase activity (with reference to Capone 1993), the pure inoculation that the inclined-plane is preserved improves in 10ml liquid
In the substratum,, get 1ml respectively in the 5ml bottle of sterilization, cover soft rubber ball in 30 ℃ of shaking table shaking culture 72h.Take 5% air from container away with syringe, add the acetylene gas of equal volume then.Each sample is established 3 repetitions, and 1 blank does not add acetylene gas in the control container.All samples is hatched 12h, the growing amount of usefulness SQ-204 gas chromatographic detection ethene in 30 ℃.Acetylene reduction method (ARA) shows that this bacterium can utilize N2 as nitrogenous source, and has higher biological nitrogen fixation activity, and average nitrogenase activity is 20-74umolC under the pure culture condition
2H
2h
-1Mg
-1Fresh weight.
2.4DNA extraction and purification
Method is with reference to the elegant pearl in east " common bacteria system identification handbook " P409.
2.5NifH the amplification of gene
The nifH gene universal primer PolF/PolR that adopts nitrogen-fixing bacteria is (with reference to Poly et al., 2001) carry out pcr amplification: PoIF:5 '-TGCGA (C/T) CC (G/C) AA (A/G) GC (C/G/T) GACTC-3 ', PolR:5 '-AT (G/C) GCCATCAT (C/T) TC (A/G) CCGGA-3 '.
Suitable reaction system and reaction conditions through repeatedly experiment back foundation are as follows:
The PCR reaction system comprises: the PCR response procedures is:
Get 2 μ l PCR reaction product and detect with 1.5% agarose gel electrophoresis, remaining PCR product directly gives Invitrogen Bioisystech Co., Ltd with the order-checking of ABI Prism 3730XL DNA analysis system.Obtain the nifH sequence of 332bp.Sequence is directly submitted the Genbank database, and sequence number is: EU707338.
2.616S the amplification of rDNA complete sequence
The square method of DNA extraction and purifying is seen eastern elegant pearl " common bacteria system identification handbook " P409.Adopt bacterial 16 S rDNA complete sequence universal primer 16SF/16SR (Weisenburg et al., 1998), 16SF:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 16SR:5 '-CGGTTACCTTGTTACGACTT-3 ' carries out pcr amplification.
Suitable reaction system and reaction conditions through repeatedly experiment back foundation are as follows:
The PCR reaction system comprises: the PCR response procedures is:
Get 2 μ l PCR reaction product and detect with 1.0% agarose gel electrophoresis, remaining PCR product directly gives Invitrogen Bioisystech Co., Ltd with the order-checking of ABI Prism 3730XL DNA analysis system.The 16S rDNA sequence that obtains.Sequence is directly submitted the Genbank database, and sequence number is: EU707337.Utilize DNAMAN software that sequencing result is carried out homology relatively, utilize BLAST software that gene order and GenBank (www.ncbi.nlm.nih.gov) database that mensuration obtains carried out the sequence alignment analysis, obtain the 16S rDNA gene order of close typical strain, utilize the ClustalW among the BIOEDIT that sequence is arranged then, utilize adjacent method (Neighbor-Joining) among the Mega to set up the phylogenetic tree of 16S rDNA gene, carry out Phylogenetic Analysis, genetic distance wherein calculates with the Tamura-Nei formula, the long difference degree of having represented of branch, the numeral on each are the support per-cent of 1000 bootstrap double sampling analyses.
2.7 bacterial strain preparation efficiency assay
This bacterium (Sphingomonas sp.DZY-N56) is inoculated in the no nitrogen Ashby liquid nutrient medium, is 10 until concentration
8Cell/ml, stand-by.
Adopt 2 kinds of mangrove-Bruguiera conjugatas and Rhizophora stylosa to carry out potted plant experiment.10 parallel samples of every kind of mangrove.Add contrast.Plastic tub is placed under the open-air condition, goes into plastic tub with plantation behind the off-the-shelf bacterium liquid immersion mangrove root 3-4h.Measure each index after 45 days.Contrast experiment's index is roots of plants, leaf area, plant weight, photosynthetic pigments etc.
Experimental result is as shown in table 2.
Sequence table
<110〉Chinese Academy of Science Nanhai Ocean Research Institute
<120〉short growing nitrogen-fixing bacterium (DZY-N56) of mangrove plant rhizosphere and application thereof
<160>2
<210>1
<211>1412
<212>DNA
<213〉Sphingol single-cell belongs to (Sphingomonas sp.) DZY-N56
<400>1
TCGCCCTCGT?CGTCATAGGT?GGGATCACTC?ATGTCAAGTC?GAACGAAGCC?TTCGGGCTTA?60
GTGGCGCACG?GGTGCGTAAC?GCGTGGGAAT?CTGCCCTTTG?GTTCGGAATA?ACAGTTGGAA?120
ACGACTGCTA?ATACCGGATG?ATGACGAAAG?TCCAAAGATT?TATCGCCAGA?GGATGAGCCC?180
GCGTAGGATT?AGCTAGTTGG?TGTGGTAAAG?GCGCACCAAG?GCGACGATCC?TTAGCTGGTC?240
TGAGAGGATG?ATCAGCCACA?CTGGGACTGA?GACACGGCCC?AGACTCCTAC?GGGAGGCAGC?300
AGTGGGGAAT?ATTGGACAAT?GGGCGAAAGC?CTGATCCAGC?AATGCCGCGT?GAGTGATGAA?360
GGCCCTAGGG?TTGTAAAGCT?CTTTTACCCG?GGATGATAAT?GACAGTACCG?GGAGAATAAG?420
CCCCGGCTAA?CTCCGTGCCA?GCAGCCGCGG?TAATACGGAG?GGGGCTAGCG?TTGTTCGGAA?480
TTACTGGGCG?TAAAGCGCAC?GTAGGCGGCT?TTGTAAGTTA?GAGGTGAAAG?CCTGGAGCTT?540
AACTCCAGAA?CTGCCTTTAA?GACTGCATCG?CTTGAATCCG?GGAGAGGTGA?GTGGAATTCC?600
GAGTGTAGAG?GTGAAATTCG?TAGATATTCG?GAAGAACACC?AGTGGCGAAG?GCGGCTCACT?660
GGACCGGTAT?TGACGCTGAG?GTGCGAAAGC?GTGGGGAGCA?AACAGGATTA?GATACCCTGG?720
TAGTCCACGC?CGTAAACGAT?GATAACTAGC?TGTCCGGGGA?CTTGGTCTTT?GGGTGGCGCA?780
GCTAACGCAT?TAAGTTATCC?GCCTGGGGAG?TACGGCCGCA?AGGTTAAAAC?TCAAATGAAT?840
TGACGGGGGC?CTGCACAAGC?GGTGGAGCAT?GTGGTTTAAT?TCGAAGCAAC?GCGCAGAACC?900
TTACCAGCGT?TTGACATGTC?CGGACGACTG?GCAGAGATGC?CTTTCTTCCC?TTCGGGGACT?960
GGAACACAGG?TGCTGCATGG?CTGTCGTCAG?CTCGTGTCGT?GAGATGTTGG?GTTAAGTCCC?1020
GCAACGAGCG?CAACCCTCGC?CTTTAGTTAC?CATCATTTAG?TTGGGTACTC?TAAAGGAACC?1080
GCCGGTGATA?AGCCGGAGGA?AGGTGGGGAT?GACGTCAAGT?CCTCATGGCC?CTTACGCGCT?1140
GGGCTACACA?CGTGCTACAA?TGGCGGTGAC?AGTGGGCAGC?AATCCCGCGA?GGGTGAGCTA?1200
ATCTCCAAAA?GCCGTCTCAG?TTCGGATTGT?TCTTTGCAAC?TCGAGAGCAT?GAAGGCGGAA?1260
TCGCTAGTAA?TCGCGGATCA?GCATGCCGCG?GTGAATACGT?TCCCAGGCCT?TGTACACACC?1320
GCCCGTCACA?CCATGGGAGT?TGGATTCACC?CGAAGGCGTT?GCGCCAACCC?GCAAGGGAGC?1380
AGGCGAACCT?TTTGTGTTTT?TACTTGATAT?CT 1412
<210>2
<211>338
<212>DNA
<213〉Sphingol single-cell belongs to (Sphingomonas sp.) DZY-N56
<220>
<221>CDS
<400>2
TTGCGATCGA?TCCCAAAGCG?GACTCGACCC?GCCTGATCTT?GCACGCGAAG?GCTCAGGACA?60
CCATCTTGTC?GCTGGCCGCT?GAAGCTGGTT?CGGTGGAGGA?CCTCGAACTG?GAAGACGTGA?120
TGAAGGTCGG?GTACCGCGAC?ATCCGTTGCG?TGGAATCCGG?TGGCCCTGAG?CCTGGGGTGG?180
GCTGCGCCGG?CCGCGGCGTG?ATCACTTCGA?TCAACTTCCT?GGAAGAAAAC?GGCGCCTACG?240
AAGGCGTGGA?CTATGTGTCC?TACGACGTGC?TGGGCGACGT?GGTGTGCGGT?GGCTTTGCCA?300
TGCCCATCCG?TAGAACAAGC?ACATGCATCT?GCCCGCCC 338
Claims (3)
1. a mangrove plant rhizosphere is urged the growing nitrogen-fixing bacterium, this bacterium is that the Sphingol single-cell that screening and separating is come out from the mangrove plant rhizosphere sediment sample of tropical mangrove area belongs to (Sphingomonas sp.) DZY-N56, contain just like 16Sr dna sequence dna and the nif nifH shown in the SEQ ID NO:1, be preserved in Chinese typical culture collection center, deposit number is CCTCC NO:M207120.
2. mangrove plant rhizosphere according to claim 1 is urged the growing nitrogen-fixing bacterium, it is characterized in that the contained nif nifH of this bacterium is shown in SEQ ID NO:2.
3. the short growing nitrogen-fixing bacterium of the described mangrove plant rhizosphere of claim 1 promotes the application of the bio-azotobacter fertilizer of mangrove plant growth in preparation.
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Cited By (2)
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CN104136599A (en) * | 2011-11-24 | 2014-11-05 | 国家科学和技术研究委员会(Conicet) | Recombinant nitrogen-fixing bacterial strain, inoculum containing the same and application methods |
CN104974968A (en) * | 2015-07-28 | 2015-10-14 | 吉首大学 | Strain YAB-3 for degrading PFOA (perfluorooctanoic acid) and application thereof |
Families Citing this family (1)
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CN105176855B (en) * | 2015-04-29 | 2018-01-19 | 齐齐哈尔大学 | One plant of Sphingomonas bacterium obtained through separation and its application in continuous cropping watermelon growing is promoted |
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2008
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN104136599A (en) * | 2011-11-24 | 2014-11-05 | 国家科学和技术研究委员会(Conicet) | Recombinant nitrogen-fixing bacterial strain, inoculum containing the same and application methods |
CN104136599B (en) * | 2011-11-24 | 2017-05-24 | 国家科学和技术研究委员会(Conicet) | Restructuring nitrogen-fixing bacteria bacterial strain, the inoculum that contains described bacterial strain and application method |
CN104974968A (en) * | 2015-07-28 | 2015-10-14 | 吉首大学 | Strain YAB-3 for degrading PFOA (perfluorooctanoic acid) and application thereof |
CN104974968B (en) * | 2015-07-28 | 2017-10-17 | 吉首大学 | One kind degraded PFOA bacterial strains YAB 3 and its application |
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