CN101298600B - Mangrove rhizosphere growth promoting azotobacter (DZY-HS14) and use thereof - Google Patents

Abstract

The invention provides a mangrove plant rhizosphere growth-promoting nitrogen fixing bacterium (DZY-HS14) and an application thereof. The bacterium is the bacillus sp. DZY-HS14 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: M207043. 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-HS14) of mangrove plant rhizosphere and application thereof

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 that a strain filters out from the tropical mangrove root system 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 area mangrove plant rhizosphere sediment sample of Sanya, Hainan Province.Its called after of classifying: Bacillus Bacillus sp.DZY-HS14.This bacterium on April 13rd, 2007 be preserved in Chinese typical culture collection center (CCTCC, the address: Chinese Wuhan City Wuhan University), deposit number: CCTCC NO:M207043.

The bacteria characteristic of bacterial classification of the present invention is described below:

Gram-positive, shaft-like, produce oval gemma (Fig. 1--Fig. 4), peritrichous is arranged.0.1 * 4-9 μ m, individual difference very big (as Fig. 6).Oxidase positive, the catalase positive, aerobic or amphimicrobian.Well-grown on Ashby, LB and BUG substratum.About can growing for diameter 4-5cm through cultivation about 5d on the Ashby substratum, the flat colony (as Fig. 1) of white, circle, neat in edge.Most responsive to microbiotic, resistance is less.Genus bacillus is common fixed nitrogen bacterial classification.This genus bacterium was also found in mangrove forest, and reported nitrogen fixation.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 25-80umolC ₂H ₂h ^-1Mg ^-1Fresh weight.

Extract genomic dna from the pure growth of bacterial strain Bacillus sp.DZY-HS14, use the specific primer PolF/PolR of nif (with reference to Poly et al., 2001) and bacterial 16 S rDNA complete sequence universal primer 16SF/16SR (with reference to Weisenburg et al., 1998) carry out pcr amplification, the ideal purpose that all increases band proves that this bacterial strain has nif (Fig. 5).Obtain nif nifH sequence and 16S rDNA sequence by 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 obtains accession number and is: EU707337 and EU707338.

Part and the higher representative gene order of measuring of 16S rDNA sequence similarity in the GenBank database, have been chosen, with Neibor-joining method constructing system evolutionary tree (see figure 7), carry out Phylogenetic Analysis by ClustalW software and Mega software.Has the sequence of nearer sibship all from bacillus (Bacillus) with the 16S rDNA sequence of bacterial strain Bacillus sp.DZY-HS14 as can be seen from phylogenetic tree (Fig. 4), and have 98% similarity with the 16S rDNA sequence of known bacterial strain B.licheniformis, have 97% homology with known bacterial strain B.niabensis in this genus.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, and this shows that bacterial strain Bacillus sp.DZY-HS14 is a strain novel species.

Table 1 B.licheniformis and the comparison of Bacillus sp.DZY-HS14 index (+: the expression positive reaction;-: the expression negative reaction)

Annotate: pearl " common bacteria identification handbook " is liked in B.licheniformis data refer east.The B.niabensis data are from Bacillus niabensis sp.nov., isolated from cotton-waste composts for mushroom cultivation Int.J.Syst.Evol.Microbiol.57 (8), 1909-1913 (2007)

Handle two kinds of mangrove bruguieras (Bruguiera gymnorhiza) and Rhizophora stylosa (Rhizophora stylosa) with the bacterium liquid that contains Bacillus sp.DZY-HS14, the result is as shown in table 2.

Table 2 Bacillus sp.DZY-HS14 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, photosynthetic pigments content mean value is bright all apparently higher than control group.Total chlorophyll content mean value is higher than Bruguiera conjugata control group 139.7%, is higher than Rhizophora stylosa control group 34.01%.Carotene carotene content is also respectively than Bruguiera conjugata and Rhizophora stylosa control group high 183.4% and 20.6%.Growth-promoting effect for leaf area is also apparent in view, promotes Bruguiera conjugata and Rhizophora stylosa leaf area 180.7% and 72.6% respectively.Ground and subterranean promotion for different seeds are then inequality, promote that for Bruguiera conjugata the underground part effect is quite a lot of, promote that for Rhizophora stylosa the over-ground part effect is quite a lot of.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 bacterial classification screening and separating from the tropical mangrove root system of China is come out, be fit to local various envrionment conditionss, 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 and Fig. 4 are respectively the transmission electron microscope photos of Bacillus sp.DZY-HS1 bacterium colony different amplification;

Fig. 5 is the electrophorogram of bacterial strain Bacillus sp.DZY-HS1, among the figure, and 1: genomic dna, 2:16S rDNA, 3: the nif nifH that increases, M1: λ DNA/EcoR I+Hind III, M2:DL2000; ) genomic dna, 16S and the nif nifH that increases;

Fig. 6 shows bacterial strain Bacillus sp.DZY-HS14 transmissioning electric mirror determining size;

Fig. 7 is bacterial strain Bacillus sp.DZY-HS1 position in 16S rDNA phylogenetic tree.

Embodiment

One, materials and methods

1, material

1.1 soil sample collection

Sample picks up from red Shahe, Sanya, Hainan Province littoral mangrove area mangrove plant rhizosphere settling, 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, pH7.0.

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 ₂O0.4g, MgSO ₄7H ₂O 0.2g, NaCl 0.1g, Na ₂M _OO ₄H ₂O 0.002g, FeCl ₃0.01g, deionized water 1000ml, pH7.0.

LB substratum: yeast extract 5g, Tryptones 10g, NaCl 10g, agar 1-2%, deionized water 1000ml, pH7.0.

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 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 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 25-80 μ mol C 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.

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.The nifH sequence that obtains is directly submitted the Genbank database, and sequence number is: EU707338.

2.6 the amplification of 16S rDNA complete sequence

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.As follows through having set up suitable reaction system and reaction conditions after repeatedly testing:

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 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 the efficiency assay of bacterial strain preparation in soil

This bacterium Bacillus sp.DZY-HS1 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 Bacillus (Bacillus sp.) DZY-HS14 that screens from tropical mangrove area mangrove plant rhizosphere sediment sample, contain 16SrDNA sequence and nif nifH, be preserved in Chinese typical culture collection center, deposit number is CCTCC NO:M207043.

2. 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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