CN101298601B - Mangrove plant growth promoting azotobacter (DZY-X1) and use thereof - Google Patents

Mangrove plant growth promoting azotobacter (DZY-X1) and use thereof Download PDF

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CN101298601B
CN101298601B CN2008100289730A CN200810028973A CN101298601B CN 101298601 B CN101298601 B CN 101298601B CN 2008100289730 A CN2008100289730 A CN 2008100289730A CN 200810028973 A CN200810028973 A CN 200810028973A CN 101298601 B CN101298601 B CN 101298601B
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mangrove
bacterium
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vibrio
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董俊德
张燕英
杨志浩
杨斌
张偲
王友绍
孙翠慈
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South China Sea Institute of Oceanology of CAS
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Abstract

The invention provides a mangrove plant rhizosphere growth-promoting nitrogen fixing bacterium (DZY-X1) and an application thereof. The bacterium is the vibrio sp. 2006-DZY-X1 screened from the rhizosphere settlings of the tropical mangrove plants and contains 16SrDNA serial and nifH and is stored in the CCTCC; the collection number is CCTCCNO: M207118. The strain has high biological nitrogen fixing activity; the microbial inoculum with the bacterium is used to breed mangrove plants such as rhizophora stylosa and bruguiera, which can evidently promote the growth of plants, thereby being capable of preparing the biological nitrogen fixing bacterium fertilizer promoting the growth of mangrove plants.

Description

Short growing nitrogen-fixing bacterium (DZY-X1) of mangrove plant rhizosphere and application thereof
Technical field
The application's patent belongs to biological technical field, particularly relates 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 that a strain filters out from the tropical mangrove ecosystem 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 the vinelandii novel species that screens from the tropical mangrove ecosystem of Sanya, Hainan Province.Its called after of classifying: Vibrio Vibrio sp.2006-DZY-X1.This bacterium on July 28th, 2007 be preserved in Chinese typical culture collection center (CCTCC, the address: Chinese Wuhan City Wuhan University), deposit number: CCTCCNO:M207118.
The bacteria characteristic of bacterial classification of the present invention is described below:
Bacterial classification is described: Gram-negative, straight rod-short does not produce spore, 0.5 * 1.2-1.8 μ m, single life.Oxidase positive, the catalase positive, aerobic or amphimicrobian, OF fermentation.Being breathing pattern, is again the fermentating metabolism type.Aerogenesis not.Give birth to the flagellum desire with one pole and move, flagellum has a center, and has epitheca.Under bad condition, form spheroplast.Well-grown on Ashby, TCBS and BUG substratum.About can growing for diameter 3-4cm through cultivation about 5d on the Ashby substratum, tawny, circle, the ganoid flat colony of neat in edge (as Fig. 1).Well-grown and rapid.The 2%-3% salinity is advisable.V.P. negative.Most responsive to microbiotic, resistance is less.Currently reported Vibrio nitrogen fixation is as V.diazotrophicus and V.naroiegens.Acetylene reduction method (ARA) shows that this bacterium can utilize N 2As nitrogenous source, and have higher biological nitrogen fixation activity, under the pure culture condition, its nitrogenase activity 30-78umolC 2H 2h -1Mg -1Fresh weight.Use specific primer, the DNA that its pure growth is extracted carries out pcr amplification, and by the pcr amplification of nif nifH, the comparatively ideal purpose band that increases proves that this bacterial strain has biological nitrogen fixation gene (Fig. 2).
Extract genomic dna from the pure growth of bacterial strain Vibrio sp.2006-DZY-X1, specific primer PolF/PolR and the bacterial 16 S rDNA complete sequence universal primer 16SF/16SR of utilization nif carries out pcr amplification, the ideal purpose that all increases band, obtain nif sequence and 16S rDNA sequence by gene clone and sequencing analysis, by the BLAST software among the GenBank sequence in nifH sequence and 16S rDNA sequence and the GenBank database is compared, in database, do not find on all four sequence, show the new gene order of these 2 gene orders for finding first.Successfully submitting new gene order to gene pool, to obtain accession number be EF422075 and EF199929.
In the GenBank database, chosen part and measured the higher representative gene order of sequence similarity, with Neibor-joining method constructing system evolutionary tree (seeing Fig. 3,4), carried out Phylogenetic Analysis by ClustalW software and Mega software.From phylogenetic tree (Fig. 3) as can be seen the 16S rDNA sequence of bacterial strain Vibrio sp.2006-DZY-X1 have the sequence of nearer sibship all from Vibrio (Vibrio), and have 98% similarity with the 16S rDNA sequence of known bacterial classification V.diazotrophicus and V.vulnificus, have 98% homology with Vibrio sp.QY102 and the Vibrio sp.K22-41 for fixed kind in this genus.Fixed kind V.hispanicus in belonging to this in addition, V.metschnikovii and V.fluvialis have the homology of 95-97%.
Nif (nifH) sequence of bacterial strain Vibrio sp.2006-DZY-X1 and the nitrogenase gene sibship of known bacterial classification Vibrio diazotrophicus be (Fig. 4) recently, and sequence homology is 97%.
Physiology and biochemistry is the result show, this bacterium and the two strains known bacterial strain obviously different (table 1) the most close with its sibship prove a strain novel species.
Two kinds of bacterium indexs comparisons of table 1Vibrio.diazotrophicus and DZY-X1 (+: the expression positive reaction;-: the expression negative reaction)
Figure G2008100289730D00021
Figure G2008100289730D00031
Annotate: V.diazotrophicus data refer east elegant pearl " common bacteria identification handbook ".
Handle two kinds of mangroves with the bacterium liquid that contains Vibrio sp.2006-DZY-X1, Bruguiera conjugata (Bruguiera gymnorhiza) and Rhizophora stylosa (Rhizophora stylosa), the result is as shown in table 2.
Table 2Vibrio sp.2006-DZY-X1 influence after 45 days to Bruguiera conjugata and the processing of Rhizophora stylosa embryo
Figure G2008100289730D00041
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, leaf area is apparently higher than control group.Leaf area mean value is higher than Bruguiera conjugata and Rhizophora stylosa 280.9% and 74.9% respectively.The root biomass also improves, and wherein the Rhizophora stylosa raising is apparent in view, is 56%, and the Bruguiera conjugata effect is not really remarkable, has improved 11.1%.Handling the photosynthetic plain color content in back also all has apparent in view raising, and Rhizophora stylosa chlorophyll b content has improved 148.3%, and carotenoid content has improved 235.2%.Bruguiera conjugata improves not obvious, and chlorophyll b content is handled the back and improved 20%, and carotenoid content improves 35%.These results show, can obviously promote plant strain growth with the mangroves such as microbial inoculum inoculation Hai Lan, Rhizophora stylosa and Bruguiera conjugata that contain this bacterium, and 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 is Vibrio sp.2006-DZY-X1 bacterium colony growing state photo on TCBS;
Fig. 2 is Vibrio sp.2006-DZY-X1 bacterium colony growing state photo on BUG;
Fig. 3 is the transmission electron microscope photo of Vibrio sp.2006-DZY-X1 bacterial strain;
Fig. 4 be bacterial strain Vibrio sp.2006-DZY-X1 electrophorogram (1:DNA, 2:16S rDNA, 3: nif nifH, M1: λ DNA/EcoR I+Hind III, M2:DL2000);
Fig. 5 is Vibrio sp.2006-DZY-X1 site plan in 16S rDNA unrooted phylogenetic tree;
Fig. 6 is the site plan of Vibrio sp.2006-DZY-X1 in the no root development tree of setting up with nif nifH.
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
Figure G2008100289730D00051
No nitrogen liquid nutrient medium: sucrose 10g, oxysuccinic acid 5.0g, K 2HPO 4H 2O 0.1g, KH 2PO 4H 2O0.4g, MgSO 47H 2O 0.2g, NaCl 0.1g, Na 2M OO 4H 2O 0.002g, FeCl 30.01g, 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
2.1 strain separating
With get behind the sample mixing about 1g in 200ml Ashby,
Figure G2008100289730D00052
Perhaps Ashby+
Figure G2008100289730D00053
No nitrogen liquid nutrient medium (the Ashby substratum with
Figure G2008100289730D00054
Substratum was with 1: 1 mixed) in, 30 ℃ of constant temperature 48h add rich the cultivation.Then bacterium liquid is diluted to 10 respectively -4, 10 -5, 10 -6Three extent of dilution, 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
Figure G2008100289730D00055
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 N 2As nitrogenous source, and have higher biological nitrogen fixation activity, average nitrogenase activity is 30-78umolC 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 " P 409
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: PolF: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 '.
Through repeatedly having set up suitable reaction system and reaction conditions after the experiment.
Figure G2008100289730D00061
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 nffH 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 " P 409Adopt bacterial 16 S rDNA complete sequence universal primer 16SF/16SR (Weisenburg et al., 1998), 16SF:5 '-AGAGTITGATCCTGGCTCAG-3 ', 16SR:5 '-CGGTTACCTTGTTACGACTT-3 ' carries out pcr amplification.As follows through having set up suitable reaction system and reaction conditions after repeatedly testing:
Figure G2008100289730D00062
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 is directly submitted the Genbank database with sequence, 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 the efficiency assay of bacterial strain preparation in soil
This bacterium Vibrio sp.2006-DZY-X1 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.
The result is as shown in table 2.

Claims (2)

1. a mangrove plant rhizosphere is urged the growing nitrogen-fixing bacterium, this bacterium is Vibrio (Vibriosp.) 2006-DZY-X1 that screens from tropical mangrove ecosystem, contain 16SrDNA sequence and nif nifH, be preserved in Chinese typical culture collection center, deposit number is CCTCC NO:M207118.
2. the application of the short growing nitrogen-fixing bacterium of the described mangrove plant rhizosphere of claim 1 in the bio-azotobacter fertilizer of preparation promotion mangrove plant growth.
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