CN105925507A - Bacillus cereus with heavy metal passivating and plant growth promoting functions and application of bacillus cereus - Google Patents
Bacillus cereus with heavy metal passivating and plant growth promoting functions and application of bacillus cereus Download PDFInfo
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- CN105925507A CN105925507A CN201610391094.9A CN201610391094A CN105925507A CN 105925507 A CN105925507 A CN 105925507A CN 201610391094 A CN201610391094 A CN 201610391094A CN 105925507 A CN105925507 A CN 105925507A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/085—Bacillus cereus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C09K17/00—Soil-conditioning materials or soil-stabilising materials
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Abstract
The invention discloses bacillus cereus with heavy metal passivating and plant growth promoting functions and application of the bacillus cereus, and belongs to the technical field of microbial strains. The name of the bacillus cereus is bacillus cereus B19, and is preserved in the China General Microbiological Culture Collection Management Committee Centre of Institute of Microbiology, Chinese Academy of Sciences, 3#, No.1 Yard, West Beicheng Road, Chaoyang District, Beijing, on April 27th, 2016; the preservation number is CGMCC NO. 12405. The bacillus cereus has the functions of passivating heavy metals and promoting plant growth, is suitable for decomposing difficultly soluble phosphorus, and is used for producing auxin, resisting salt, resisting the heavy metals, promoting plant seed germination, increasing effective phosphorus and microbial carbon and nitrogen of soil, and reducing available heavy metals in the soil.
Description
Technical field
The invention belongs to microorganism fungus kind technical field, particularly to one, there is heavy metal passivation and promote plant
The bacillus cereus of growth function and application.
Background technology
Phosphorus is one of mineral element necessary to plant growing, in many ways the various physiology in involved in plant body
Biochemical process, as energy metabolism, photosynthesis, Repiration and nitrogen are fixed.Full phosphorus in general agricultural land soil
Content is the highest, but available phosphorus content is the lowest.In China's soil, the phosphorus of more than 95% is invalid phosphorus, and plant cannot
Absorb, therefore can only add phosphorus element by external source and utilize for plant absorption；And by manually applying
Ca-P, Fe-P, Al-P that water soluble phosphorous fertilizer is easily combined formation indissoluble state by Ca, Fe, the Al in soil sink
Form sediment.Therefore, a limiting nutrient during phosphorus has been considered as anthropogenic soil.Phosphate solubilizing microorganism
The history in research existing more than 100 years of (Phosphate-solubilizing microoganisms, PSMs), with
Improving constantly of research means, people are the most deep to the understanding of phosphate solubilizing microorganism, find phosphorus decomposing in soil
Microbial profile has obvious rhizosphere effect, has the effect of activation insoluble phosphorus and promotes the effect of plant growth
Really, it is possible to improve soil environment, available phosphorus content in soil is made to improve.For many years facts have proved that phosphorus decomposing is micro-
Biological effect in agricultural production, but part phosphate solubilizing microorganism shows extremely strong molten in a laboratory environment
Phosphorus ability, but it is unsatisfactory to apply to effect in actual production process, and tracing it to its cause, it is many to be probably,
But bacterial strain is lacked competitiveness under field conditions (factors), it is difficult to existence is surely grown and is probably one of the main reasons.Search out
Can in adverse circumstances, survival ability be strong, the phosphate solubilizing bacteria microorganism that is prone to surely grow plant root is particularly important.
Bacillus cereus has sporiferous function, has the ability that stronger opposing external environment is coerced, it is possible to opposing institute
The injury caused due to dry, heat and ultraviolet radiation in the environment of existence, thus be prone to surely grow soil and send out
Wave field fertilizer efficiency.Therefore, have before the bacillus cereus of molten phosphorus effect has huge application in bio-fertilizer industry
Scape.
The molten Bacillus phosphorus reported at present, predominantly bacillus megaterium (Bacillus megaterium),
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus cereus (Bacillus cereus),
The bacterium such as bacillus subtilis (Bacillus subtilis), bacillus thuringiensis (Bacillus thuringiensis)
Strain is studied.Such as, Zhang Weina etc. (2012) report bacillus megaterium (Bacillus megaterium) JD-2
Tool solves calcium phosphate and the ability of lecithin simultaneously, and its phosphate solubilization is 22 times and 25 times of matched group respectively.Wu
Little celery etc. (CN200910032681.9) report bacillus cereus (Bacillus cereus) JYZ-SD1 is at ovum
In phospholipid fluid medium, titanium pigment content is 1.94mg/L, is 5.17 times of comparison (0.37mg/L),
Inoculation JYZ-SD1 bacterial strain after 150 days willow cutting seedling height of seedling and stem slightly increase 38.1% He respectively than CK
15.9%, fresh weight and dry weight add 83.89% and 72.22% respectively than CK.He Yueqiu etc.
(CN201510956941.7) report bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
After YN2010-KC CGMCC No.5722 cultivates 4d in calcium phosphate medium, titanium pigment in culture fluid
Content is 63.82mg/L, is 64.46 times of comparison culture fluid.These achievements show that bacillus cereus is at soil
Potentiality in terms of molten phosphorus.But, phosphate solubilizing microorganism screening is confined to simple function bacterial strain more.
At present, the heavy metal pollution in soil is more serious, particularly heavy metal cadmium.There is efficient phosphate-solubilizing simultaneously
Apply in soil, not only with the several functions bacillus cereus producing auxin and heavy metal-passivated cadmium
May advantageously facilitate plant growing, be also beneficial to soil amendment.There is heavy metal passivation simultaneously and promote plant growing
The bacillus cereus of function has no report.
Summary of the invention
The primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, it is provided that it is blunt that one has heavy metal
Change and promote the bacillus cereus of plant growing function.
Another object of the present invention is to that there is described in offer heavy metal passivation and the wax of promotion plant growing function
The application of sample bacillus cereus.
The purpose of the present invention is achieved through the following technical solutions: one has heavy metal passivation and promotes plant growing
The bacillus cereus of function, entitled bacillus cereus (Bacillus cereus) B19, in 2016
Within 04 month 27 days, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC),
Deposit number is CGMCC NO.12405, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica.
The 16S rDNA core of the described bacillus cereus with heavy metal passivation and promotion plant growing function
Nucleotide sequence is as shown in SEQ ID NO:1.
The morphological characteristic of the described bacillus cereus with heavy metal passivation and promotion plant growing function is such as
Under: for gram positive bacteria, somatic cells is shaft-like, end side, and in short or long-chain, bacterium colony is big, and surface is thick
Rough, flat, irregularly；Beef-protein medium is cultivated the bacterium colony formed after 24h be circle or
Sub-circular, quality are soft, non-pigment, the most glossiness white colony.
The Physiology and biochemistry of the described bacillus cereus with heavy metal passivation and promotion plant growing function is special
Levy as follows: the cellulose decomposition positive, amylase positive, catalase positive, indole feminine gender,
Produce biofilm negative, methyl red test feminine gender, the weak positive of siderophore.
The application of the described bacillus cereus with heavy metal passivation and promotion plant growing function, including using
In decomposing insoluble phosphorus, it is used for producing auxin, for salt tolerant, for heavy metal tolerance, is used for promoting plant
Germination, is used for increasing soil available phosphorus, Microbial nitrogen, and is used for reducing a soil available huge sum of money
Belong to.
Described insoluble phosphorus refers at least one in slightly solubility calcium phosphate, iron phosphate and aluminum phosphate.
Described heavy metal is cadmium.
The present invention has such advantages as relative to prior art and effect:
Inventor is through lot of experiments, and from Ma Ba town, Qujiang District, Shaoguan City, Guangdong Province, Shi Bao grows cane
Ground soil filters out a strain bacillus cereus (Bacillus cereus) B19.This bacterial strain has efficient-decomposition concurrently
Slightly solubility calcium phosphate, iron phosphate, aluminum phosphate, product auxin aspect, salt tolerant, heavy metal tolerance aspect and rush
Enter plant seed germination and increase soil available phosphorus, Microbial nitrogen and reduce the function of soil available cadmium.
After applying this bacterial strain, Plantula Brassicae chinensis germination, the 3rd day root length and the 7th day stem length, root length and compare
Compare and add 8%, 39%, 28%, 33% respectively.Laboratory simulation experiment display: notable after using microbial inoculum
Add soil available phosphorus content, after different cadmium concentration soil application microbial inoculums, available phosphorus divides than not using microbial inoculum
Do not add 30%, 22% and 22%.Microbial content it is a significant increase, in difference after using microbial inoculum
After cadmium concentration soil application microbial inoculum, available phosphorus ratio is not used microbial inoculum and is added 24%, 21% and 22% respectively；Micro-
Biological nitrogen ratio is not used microbial inoculum and is added 32%, 24% and 29% respectively.Significantly reduce after using microbial inoculum
Effective cadmium content of soil, after different cadmium concentration soil application microbial inoculums, available phosphorus reduces respectively than not using microbial inoculum
2%, 27% and 19%.The above results shows this bacterial strain indissoluble P availability and phosphorus in improving soil
While fertile utilization rate, also can remarkably promote Plantula Brassicae chinensis germination, improve Microbial nitrogen and reduce soil
Available Cd content.
Accompanying drawing explanation
Fig. 1 is the Gram’s staining displaing micro photo figure of B19 bacterium.
Fig. 2 is the spore staining displaing micro photo figure of B19 bacterium.
Fig. 3 is the comparison diagram that B19 bacterium produces water-soluble phosphorus concentration under indissoluble Phos culture medium.
Fig. 4 is that B19 bacterium produces auxin qualitative experiment comparison diagram；Wherein, from left to right, the 1st and 2 it is classified as sun
Property comparison, concentration is respectively 0,10,20,50,50 μ g/mL；3rd is classified as negative control, and the 4th is classified as
B19, sets 3 parallel holes respectively.
Fig. 5 is B19 bacterium tolerance different salinity Experimental comparison figure；Wherein, from left to right, more from top to bottom,
Sodium chloride concentration is respectively 0.5%, 1.5%, 3.5%, 5.5%, 7.5%, 9.5%.
Fig. 6 is B19 bacterium tolerance different cadmium concentration Experimental comparison figure.
Fig. 7 is B19 bacterium heavy metal tolerance cadmium flat board photo figure；Wherein, figure A be cadmium concentration be 25mg/L,
Figure B be cadmium concentration be 50mg/L.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention
It is not limited to this.
Culture medium used by the present invention is as follows:
1. indissoluble Phos culture medium: glucose 10g, tricalcium phosphate 5g, ammonium sulfate 0.5g, sodium chloride 0.2
G, magnesium sulfate 0.1g, potassium chloride 0.2g, yeast extract 0.5g, manganese sulfate 0.002g, ferrous sulfate 0.002
G, 0.4% (w/v) bromophenol blue (pH 6.7) 6mL, distilled water is settled to l 000mL, pH 7.0, solid
Culture medium separately adds agar 18.0g.
2. Phos screening culture medium A is made up of following component: glucose 10g, calcium phosphate 5g, magnesium chloride
5g, magnesium sulfate 0.25g, potassium chloride 0.2g, ammonium sulfate 0.1g, distilled water is settled to 1 000mL, pH 7.0.
3. Phos screening culture medium B and differing only in of Phos screening culture medium A replace phosphorus with iron phosphate
Acid calcium is as unique phosphorus source.
4. Phos screening culture medium C and differing only in of Phos screening culture medium A replace phosphorus with aluminum phosphate
Acid calcium is as unique phosphorus source.
5. fluid medium: yeast powder 5g, peptone 10g, sodium chloride 10g, water is settled to 1L, pH7.0.
6. LB fluid medium: yeast extract 5.0g, tryptone 10.0g, sodium chloride 10.0g, distillation
Water is settled to l 000mL, pH 7.0.
7. LB solid medium: yeast extract 5.0g, tryptone 10.0g, sodium chloride 10.0g, agar
Powder 18.0g, distilled water is settled to l 000mL, pH 7.0-7.2.
8. beef-protein medium: Carnis Bovis seu Bubali cream 3.0g, peptone 5.0g, sodium chloride 5.0g, agar 18.0
G, distilled water is settled to 1000mL, pH value 7.0-7.2.
9. the culture medium Han heavy metal: add Caddy (Cleary) in LB culture medium and make up to designed concentration, join
During fluid medium processed, it is not necessary to add agar.Above culture medium is required to 121 DEG C of sterilizing 15min.
The separation of embodiment 1 bacterial strain, purification
(1) separate
1. primary dcreening operation
Weigh Ma Ba town, Qujiang District, Shaoguan City, Guangdong Province Shi Bao grow cane ground soil 10.0g, put into equipped with 90mL
In the 250mL triangular flask of sterilized water (adding bead), 25 DEG C, 200r/min shaking table vibration 30min, stand
20～30s, take 10mL supernatant and be placed in 50mL sterilizing triangular flask, in 90 DEG C of water-bath 10min, take 1
After mL water-bath, supernatant joins indissoluble Phos culture medium, (cultivates containing 30mL in 150mL triangular flask
Liquid), each soil sample arranges two repetitions.It is placed in shaking table and carries out enrichment culture, rotating speed 150r/min, temperature 30
℃.The enrichment culture cycle is 5～7d, takes 1mL culture fluid and be each added to after enrichment culture terminates for the first time
Carrying out second time enrichment culture in new culture medium, same operation carries out third time enrichment culture, is enriched with every time
In superclean bench, take supernatant with liquid-transfering gun after end appropriate, be placed in centrifuge tube, centrifugal, take supernatant,
Molybdenum antimony resistance colorimetric method is used to measure water-soluble phosphorus content in supernatant.Wherein, the water-soluble phosphorus of named B19 bacterium
Content is 255.34 μ g/mL；
Sieve the most again
Select the preferable bacterial strain of phosphorus decomposing effect, be inoculated into Phos screening culture medium B and Phos screening training respectively
Support in base C, 30 DEG C of shaking table 150r/min, measure the concentration of available phosphorus in culture fluid after cultivating 3d.Wherein,
In B19 bacterium iron phosphate culture medium, water-soluble phosphorus content is 48.22 μ g/mL, and in aluminum phosphate culture medium, water-soluble phosphorus contains
Amount is 24.27 μ g/mL, uses glycerol pipe preservation method to preserve this bacterial strain.
(2) purification
Utilize plate streak after purification the B19 bacterium filtered out, be stored in beef-protein medium made
Inclined-plane, be placed in 4 DEG C of refrigerators standby.
Embodiment 2 CHARACTERISTICS IDENTIFICATION
The B19 bacterium that embodiment 1 screening obtains is carried out Physiology and biochemistry qualification and molecular biology identification.
(1) thalli morphology characteristic
B19 bacterium is gram positive bacteria, and somatic cells is shaft-like, end side, and in short or long-chain, bacterium colony is big, table
Face is coarse, flat, irregularly.Thalline Gram’s staining microphotograph is as it is shown in figure 1, thalline presents purple.
Spore peacock green dye dyes, and microphotograph is as in figure 2 it is shown, spore presents green.
B19 bacterium on beef-protein medium, cultivate 24h after formed bacterium colony be circle or sub-circular,
Quality is soft, non-pigment, the most glossiness white colony.
(2) growth characteristics
In liquid medium within, rotating speed 180rpm, temperature 37 DEG C, under conditions of initial ph value is 7.0, training
Supporting 18 hours, recording viable count is 3.00 ± 0.08 × 108cfu/mL。
(3) assay method reference " common bacteria system identification handbook " (the elegant pearl in east) of physiology, biochemical characteristic,
Qualification result is shown in Table 1.
Table 1 B19 bacterium physio-biochemical characteristics
Note :+it is expressed as the positive；-it is expressed as feminine gender；W is expressed as the weak positive；
(4) molecular biological characteristic
RNA isolation kit (purchased from Shanghai Sheng Gong biological engineering company limited) is used to extract B19 bacterium STb gene.Adopt
With bacterial 16 S rDNA universal primer 27F (AGA GTT TGA TCC TGG CTC AG) and 1492R
(TAC GGC TAC CTT GTT ACG ACT T), the 16S rDNA of PCR amplification antibacterial, 50 μ L's
PCR reaction system is: DNA profiling 2 μ L, primer 2 7F (l mM) 1 μ L, primer 1492R (l mM)
1 μ L, rTaq enzyme 25 μ L, dd H2O 21μL。
Pcr amplification reaction program: 95 DEG C of denaturations 3min, enters thermal cycle；95 DEG C of degeneration 30s, 52 DEG C
Annealing 30s, 72 DEG C extend 30s, totally 35 circulations；72 DEG C extend 10min.
The length of 1.0% agarose gel electrophoresis detection PCR primer and concentration, occur substantially near 1000bp
Band, by pcr amplification product reclaim after, order-checking completed by Guangzhou Ai Ji biotinylated biomolecule Technology Co., Ltd.,
DNA sequence (as shown in SEQ ID NO.1) the input US National Bioinformatics Institute NCBI that will obtain
Carry out BLAST comparison on webpage, with Blast program, all sequences in data base is compared analysis, knot
Fruit finds the 16S rDNA sequence of bacterial strain of the present invention and has higher with bacillus cereus in GenBank
Homology, its similarity is 99%.In conjunction with above-mentioned thalli morphology characteristic, flat-plate bacterial colony feature, physiology life
Changing characteristic, the result of 16S rDNA sequence analysis, this bacterial strain of Preliminary Identification is bacillus cereus (Bacillus
Cereus), named bacillus cereus (Bacillus cereus) B19, in preservation on the 27th in 04 month in 2016
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is
CGMCC NO.12405, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology of institute.
Embodiment 3 B19 bacterium produces the effect experimental of water-soluble phosphorus under slightly solubility Phos culture medium culturing
(1) preparation of liquid to be measured
Picking is stored in B19 thalline on beef extract-peptone slant medium and lines LB solid medium flat board
On, cultivate 24h at 30 DEG C.B19 thalline on flat board is accessed the 150mL containing 20mL LB fluid medium
In triangular flask, 30 DEG C, 150r/min, 24h cultivated by shaking table.Culture fluid 6000r/min is centrifuged 5min, uses
The resuspended thalline of sterilized water, on vortex mixed instrument, diffusion is uniformly, accesses bacteria suspension equivalent containing 30 with liquid-transfering gun
The 150mL triangular flask of mL Phos screening culture medium, 30 DEG C, 150r/min, 7d cultivated by shaking table.
(2) water-soluble phosphorus assay
By culture fluid at 10000g, 25 DEG C of centrifugal 15min, take supernatant, use molybdenum antimony resistance colorimetric method to measure
Water-soluble phosphorus content.If not connecing B19 bacterium, accessing equivalent sterilized water is comparison, and each process is repeated 3 times.Water
Soluble phosphorus content change (unit is μ g/mL, i.e. represents the micrograms of contained water-soluble phosphorus in every milliliter of sample)
Result of the test see Fig. 3.
The result of Fig. 3 shows: the water-soluble phosphorus content of B19 fungi degradation calcium phosphate is up to 255.34 μ g/mL, phosphoric acid
The water-soluble phosphorus content of ferrum is 48.22 μ g/mL, and the water-soluble phosphorus content of aluminum phosphate is 24.27 μ g/mL.
Embodiment 4 B19 bacterium produces auxin effect test
(1) auxin qualitative determination: B19 bacterium is inoculated in the 20mL containing L-Trp (100 μ g/mL)
In the 150mL triangular flask of LB fluid medium, 30 DEG C, 150r/min shaking table cultivation 24h.Take 50 μ L trainings
Nutrient solution is dripped on whiteware plate, adds 50 μ L Salkowski color solution (35%HClO simultaneously4+1mL 0.5
mol/LFeCl3), if the sterile LB medium of 50 μ L is as negative control, 10,20,50 (μ g/mL) is raw
The color solution of long element is as positive control.After whiteware plate is placed 30min under the conditions of room temperature, lucifuge
Observing, the color person of reddening represents can produce auxin, and result of the test is shown in Fig. 4, it is seen then that the color of B19 shows
For pale pink, color is between comparison and 10 μ g/mL.
(2) auxin quantitative determination: B19 bacterium is inoculated in the 50mL LB containing L-Trp (μ g/mL)
In fluid medium, three repetitions, 30 DEG C, 150r/min shaking table cultivation 24h.By culture fluid respectively at 6000 × g
Under be centrifuged 10min, take 4mL supernatant add 4mL Salkowski color solution, mixing, room temperature, dark
Middle standing 30min, uses ultraviolet spectrophotometer, surveys absorbance, use aseptic culture simultaneously under 530nm
Base does same treatment and makees standard curve as comparison, the auxin of employing 0,10,20,30,50 (μ g/mL).
Result of the test is shown in Table 2.B19 bacterium produces auxin amount and reaches 8.72 μ g/mL.
Table 2 B19 bacterium produces auxin effect test result
| Strain number | Produce IAA amount (μ g/mL) |
| CK | 0 |
| B19 | 8.72 |
Embodiment 5 B19 bacterium Salt tolerance
Nutrient medium: Carnis Bovis seu Bubali cream 3.0g, peptone 10.0g, NaCl 5.0g, agar 18.0g, distillation
Water 1000mL, pH 7.0-7.2.According to different sodium chloride concentrations, add sodium chloride.
B19 bacterium is scoring to sodium chloride concentration be respectively 0.5%, 1.5%, 3.5%, 5.5%, 7.5%, 9.5%,
11.5%, on the nutrient agar panel of 13.5%, cultivate 24h, observe the growing state of bacterial strain.Result of the test is shown in
Fig. 5, along with the content of sodium chloride is the highest, the increment of thalline is the fewest, and to 9%, the bacterium colony of thalline is the dilutest
Dredge, to 13%, do not observe bacterium colony.Result shows: the NaCl of B19 bacterium tolerance 9%.
Embodiment 6 B19 bacterium heavy metal tolerance cadmium test
The B19 bacterium solution taking 30 μ L is inoculated in the LB fluid medium of 3mL, in 37 DEG C, 180rpm training
Support 8-10h, activate.B19 bacterium solution after activation is added containing 0 according to the inoculum concentration of 1%, 1,5,10,
25、30mg/L Cd2+3mL LB fluid medium in, 37 DEG C, 180rpm shaking table cultivate, every 30min
Measure the light absorption value A600 of culture medium, draw heavy metal tolerance curve.Result of the test is shown in Fig. 6 and Fig. 7.Result
Show: the Cd of B19 bacterium tolerance 30mg/L2+。
Embodiment 7 B19 bacterium promotes the test of plant seed germination efficacy in laboratory
(1) prepared by bacteria suspension:
1. point after actication of culture being accessed LB fluid medium, through 24h shaken cultivation, cultivation temperature is
30 DEG C, rotating speed is 150r/min；
2. bacterium solution is instilled blood counting chamber, count under an optical microscope, show that in bacterium solution, somatic cells is dense
Degree；
3. bacterium solution is centrifuged in high speed centrifuge, rotating speed 6000r/min, centrifugal 5min；
4. by resuspended for thalline sterilized water；
5. the bacterial strain concentration being used for soaking Plantula Brassicae chinensis seed is 2.0 × 107The bacteria suspension of cfu/ml.
(2) the surface of the seed sterilization treatment: seed is by 75% soak with ethanol 1min more molten with 2% sodium hypochlorite
Immersion bubble 2min, is placed on sterilizing filter paper and naturally dries.
(3) use B19 bacterium and blank to carry out Piglet s colibacillosis, be divided into 2 process groups (the 1st group
Use 25mL B19 bacterium bacterium re-suspension liquid), represent with B19, the 2nd group uses 25mL sterilized water water to process work
For blank, represent with CK).Each process group sets four repetitions, respectively repeat containing 10 Plantula Brassicae chinensis seeds (with
Machine is uniform seed after choosing surface sterilizing).Process group B19 uses bacteria suspension to soak 30min, CK and adopts
Same process is made with sterilized water.Take sterilizing culture dish, add two-layer aseptic filter paper, after removing soak, by nothing
Bacterium tweezers gripping seed is put in culture dish, 10, every ware, then adds a cover one layer of aseptic filter paper, to infiltrate preservation
Moisture, is placed in 25 DEG C of incubators and cultivates 7 days, water every day in right amount, and the water yield of every ware is identical, uniformly, fixed
Phase measures germination percentage, the index such as stem length, root length.Result of the test is shown in Table 3.The present invention divides as can be seen from Table 3
From bacillus cereus (Bacillus cereus) B19 bacterium can dramatically increase Chinese cabbage seed germinate, promote little
The growth of Chinese cabbage seed rhizome, the 3rd day root length and the 7th day stem length, root length add the most respectively
39%, 28%, 33%.
Table 3 B19 bacterium is to Plantula Brassicae chinensis germination facilitation effect result of the test
Note: data are 3 mean+SD repeated, in table, same column * represents in the notable (independent sample t of 0.05 level difference
Inspection).
Soil nutrient and impact of heavy metals are tested by embodiment 8 B19 bacterium
(1) prepared by bacteria suspension:
1. point after actication of culture being accessed LB fluid medium, through 24h shaken cultivation, cultivation temperature is
30 DEG C, rotating speed is 150r/min；Described liquid culture based component is: yeast extract 5.0g/L, tryptone
10.0g/L, sodium chloride 10.0g/L, pH 7.0；
2. bacterium solution is instilled blood counting chamber, count under an optical microscope, show that in bacterium solution, somatic cells is dense
Degree；
3. bacterium solution is centrifuged in high speed centrifuge, rotating speed 6000r/min, centrifugal 5min；
4. by resuspended for thalline sterilized water；
5. for inoculating the bacteria suspension that bacterial strain concentration is 3.0 × 107cfu/mL of soil.
(2) EXPERIMENTAL DESIGN:
Gather rice soil from big Golconda Some Mining Districts, measure the physical and chemical index of soil, the results are shown in Table 4.To soil
It is separately added into the Cd of 1mg/kg and 3mg/kg2+, and aging 14d.
With B19 bacterium be not added with bacterium and carry out Piglet s colibacillosis, it is divided into 6 process groups: the 1st group of original soil adds aseptic
Water, represents with T1；2nd group of original soil adds B19 bacterium 3 × 106Cfu/g soil, represents with T2；3rd group
External source adds 1mg/kg Cd2+Soil adds sterilized water, represents with T3；4th group of external source adds 1mg/kg Cd2+
Soil adds B19 bacterium 3 × 106Cfu/g soil, represents with T4；5th group of external source adds 3mg/kg Cd2+Soil
Earth adds sterilized water, represents with T5；6th group of external source adds 3mg/kg Cd2+Soil adds B19 bacterium 3 × 106
Cfu/g soil, represents with T6.Each process group is repeated 3 times.Water every day in right amount, maintain 40% soil to contain
The water yield.
Take fresh soil after using microbial inoculum 5 days and do Microbial nitrogen, and residue soil natural is air-dried, do soil effective
Phosphorus, available Cd are tested.Experimental result see table 5 respectively.Experimental result shows, has after using B19 microbial inoculum
Effect phosphorus, Microbial, microorganism nitrogen the most significantly increase, and effective cadmium content significantly reduces.
Table 4 laboratory simulation experiment soil physico-chemical property
Table 5 B19 microbial inoculum is to soil nutrient and effective cadmium effect test result
Note: T1 represents that original soil, T2 represent that original soil adds bacterium B19, T3 represent add 1mg/kg Cd aging after soil, T4 represents and adds 1mg/kg
Cd is aging, and rear soil adds bacterium B19 again, T5 represent add 3mg/kg Cd aging after soil, T6 represents and adds the aging rear soil of 3mg/kg Cd again
Add bacterium B19.Data are 3 mean+SD repeated, and in table, same column * represents at notable (the independent sample of 0.05 level difference
This t checks).
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-mentioned enforcement
The restriction of example, the change made, modifies, replaces under other any spirit without departing from the present invention and principle
In generation, combine, simplify, all should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (7)
1. one kind has heavy metal passivation and promotes the bacillus cereus of plant growing function, it is characterised in that:
Entitled bacillus cereus (Bacillus cereus) B19, was preserved on 04 27th, 2016 and is positioned at north
The Chinese microorganism strain preservation of North Star West Road, Jing Shi Chaoyang District No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute
Administration committee's common micro-organisms center, deposit number is CGMCC NO.12405.
The most according to claim 1, there is heavy metal passivation and promote the waxy spore bar of plant growing function
Bacterium, it is characterised in that: the 16S rDNA nucleotide sequence such as SEQ ID NO:1 of described bacillus cereus
Shown in.
The most according to claim 1, there is heavy metal passivation and promote the waxy spore bar of plant growing function
Bacterium, it is characterised in that: the morphological characteristic of described bacillus cereus is as follows: for gram positive bacteria, bacterium
Somatic cell is shaft-like, end side, and in short or long-chain, bacterium colony is big, and rough surface is flat, irregularly；Cattle
Cultivating the bacterium colony formed after 24h on meat extract protein culture medium be that circle or sub-circular, quality are soft, colourless
White colony plain, the most glossiness.
The most according to claim 1, there is heavy metal passivation and promote the waxy spore bar of plant growing function
Bacterium, it is characterised in that: the physiological and biochemical property of described bacillus cereus is as follows: the cellulose decomposition positive,
Amylase positive, catalase positive, indole are negative, produce biofilm negative, methyl red test
Negative, the weak positive of siderophore.
5. there is described in any one of Claims 1 to 4 heavy metal passivation and promote the waxy of plant growing function
The application of bacillus cereus, it is characterised in that: the application in following either side of the described bacillus cereus:
For decomposing insoluble phosphorus, it is used for producing auxin, for salt tolerant, for heavy metal tolerance, plants for promotion
Thing germination, is used for increasing soil available phosphorus, Microbial nitrogen, and is used for reducing soil available weight
Metal.
Application the most according to claim 5, it is characterised in that: described insoluble phosphorus refers to indissoluble
At least one in property calcium phosphate, iron phosphate and aluminum phosphate.
Application the most according to claim 5, it is characterised in that: described heavy metal is cadmium.
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