CN105925507B - Bacillus cereus and the application of plant growth function are passivated and promoted with heavy metal - Google Patents
Bacillus cereus and the application of plant growth function are passivated and promoted with heavy metal Download PDFInfo
- Publication number
- CN105925507B CN105925507B CN201610391094.9A CN201610391094A CN105925507B CN 105925507 B CN105925507 B CN 105925507B CN 201610391094 A CN201610391094 A CN 201610391094A CN 105925507 B CN105925507 B CN 105925507B
- Authority
- CN
- China
- Prior art keywords
- bacillus cereus
- heavy metal
- plant growth
- phosphorus
- bacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- 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
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Soil Sciences (AREA)
- General Life Sciences & Earth Sciences (AREA)
- Materials Engineering (AREA)
- Environmental Sciences (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Environmental & Geological Engineering (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of with heavy metal passivation and promotes bacillus cereus and the application of plant growth function, belongs to microorganism fungus kind technical field.Entitled bacillus cereus (Bacillus cereus) B19 of the bacillus cereus, the China Committee for Culture Collection of Microorganisms's common micro-organisms center positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica was preserved on 04 27th, 2016, deposit number is CGMCC NO.12405.The bacterial strain has the function of heavy metal passivation and promotes plant growth, suitable for insoluble phosphorus is decomposed, for producing auxin, for salt tolerant, it is used for heavy metal tolerance, for promoting plant seed germination, for increasing soil available phosphorus, Microbial nitrogen, and for reducing soil available heavy metal.
Description
Technical field
The invention belongs to microorganism fungus kind technical fields, in particular to a kind of to have heavy metal passivation and promote plant growth
The bacillus cereus of function and application.
Background technique
Phosphorus is one of mineral element necessary to plant growth, participates in the intracorporal various Physiology and biochemistries of plant in many ways
Process, as energetic supersession, photosynthesis, respiration and nitrogen are fixed.Content of tatal phosphorus is very high in general agricultural land soil, but has
It is very low to imitate phosphorus content.95% or more phosphorus is invalid phosphorus in China's soil, and plant can not be absorbed and utilized, therefore can only pass through external source
Phosphorus element is added for plant absorption utilization；And the water soluble phosphorous fertilizer by manually applying easily is tied by Ca, Fe, Al in soil
Close Ca-P, Fe-P, Al-P precipitating for forming indissoluble state.Therefore, phosphorus have been considered as a nutrition in anthropogenic soil limitation because
Son.Research existing more than 100 years of phosphate solubilizing microorganism (Phosphate-solubilizing microoganisms, PSMs) are gone through
History, with the continuous improvement of research means, people are more deep to the understanding of phosphate solubilizing microorganism, find the micro- life of phosphorus decomposing in soil
Apparent rhizosphere effect is distributed in object, has the function of activating insoluble phosphorus and promotes the effect of plant growth, can improve
Soil environment improves available phosphorus content in soil.For many years facts have proved the work of phosphate solubilizing microorganism in agricultural production
With, but part phosphate solubilizing microorganism shows extremely strong phosphate solubilization in a laboratory environment, but apply to actual production
The effect is unsatisfactory in journey, and it may be various for tracing it to its cause, but bacterial strain is lacked competitiveness under field conditions (factors), it is difficult to raw
Deposit that colonize may be one of the main reasons.Searching out can survival ability be strong in harsh environment, is easy to colonize plant root
Phosphorus decomposing bacteria microorganism it is particularly important.Bacillus has sporiferous function, with stronger resistance external environment stress
Ability can resist the injury due to caused by drying, heat and ultraviolet radiation in survived environment, to be easy to colonize soil
Earth and play field fertilizer efficiency.Therefore, the bacillus with Soluble phosphorus effect has huge application prospect in bio-fertilizer industry.
The Soluble phosphorus bacillus reported at present, predominantly bacillus megaterium (Bacillus megaterium), Xie Dian
Afnyloliquefaciens (Bacillus amyloliquefaciens), bacillus cereus (Bacillus cereus), withered grass gemma
The research of the bacterial strains such as bacillus (Bacillus subtilis), bacillus thuringiensis (Bacillus thuringiensis).Example
Such as, Zhang Weina etc. (2012) reports that bacillus megaterium (Bacillus megaterium) JD-2 has solution calcium phosphate and ovum simultaneously
The ability of phosphatide, phosphate solubilization are 22 times and 25 times of control group respectively.Wu little Qin etc. (CN200910032681.9) report
Bacillus cereus (Bacillus cereus) JYZ-SD1 titanium pigment content in lecithin fluid nutrient medium is 1.94mg/
L is 5.17 times for compareing (0.37mg/L), poplar cuttage seeding height of seedling and stem thickness ratio CK difference after inoculation JYZ-SD1 bacterial strain 150 days
38.1% and 15.9% are increased, fresh weight and dry weight ratio CK have increased separately 83.89% and 72.22%.He Yueqiu etc.
(CN201510956941.7) bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN2010-KC is reported
After CGMCC No.5722 cultivates 4d in calcium phosphate medium, titanium pigment content is 63.82mg/L in culture solution, is control
64.46 times of culture solution.These achievements show potentiality of the bacillus in terms of soil Soluble phosphorus.But phosphate solubilizing microorganism sieves
Choosing is confined to simple function bacterial strain.
Currently, the heavy metal pollution in soil is more serious, especially heavy metal cadmium.There is efficient phosphate-solubilizing and generation simultaneously
The multiple functions bacillus cereus of long element and heavy metal-passivated cadmium is applied in the soil, not only contributes to promote plant
Growth is also beneficial to soil modification.There is heavy metal to be passivated simultaneously and the bacillus cereus of plant growth function is promoted to have no
Report.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and insufficient, provide it is a kind of have heavy metal passivation and
Promote the bacillus cereus of plant growth function.
Another object of the present invention is to provide described with heavy metal passivation and promotes the waxy bud of plant growth function
The application of spore bacillus.
The purpose of the invention is achieved by the following technical solution: one kind having the function of heavy metal passivation and promotes plant growth
Bacillus cereus, entitled bacillus cereus (Bacillus cereus) B19 was preserved on 04 27th, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number are CGMCC NO.12405, are protected
Hiding address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
The 16S rDNA nucleotides sequence with heavy metal passivation and the bacillus cereus for promoting plant growth function
Column are as shown in SEQ ID NO:1.
The morphological feature with heavy metal passivation and the bacillus cereus for promoting plant growth function is as follows: for
Gram-positive bacteria, somatic cells are rod-shaped, end side, are in short or long-chain, and bacterium colony is big, rough surface, flat, irregularly；In ox
The bacterium colony formed afterwards for 24 hours is cultivated on meat extract peptone culture medium as round or approximate circle, quality is soft, non-pigment, slightly gloss
White colony.
The physiological and biochemical property with heavy metal passivation and the bacillus cereus for promoting plant growth function is such as
Under: the cellulose decomposition positive, amylase positive, catalase positive, indoles are negative, generation film is negative, methyl red
Negative, siderophore weakly positive.
The application with heavy metal passivation and the bacillus cereus for promoting plant growth function, including for decomposing
Insoluble phosphorus is used for salt tolerant, heavy metal tolerance is used for, for promoting plant seed germination, for increasing soil for producing auxin
Earth available phosphorus, Microbial nitrogen, and for reducing soil available heavy metal.
The insoluble phosphorus refers at least one of slightly solubility calcium phosphate, ferric phosphate and aluminum phosphate.
The heavy metal is cadmium.
The present invention has the following advantages and effects with respect to the prior art:
Inventor pass through lot of experiments, from the Shaoguan City, Guangdong Province Qujiang District town Ma Ba Shi Bao grow cane ground soil
One plant of bacillus cereus (Bacillus cereus) B19 is filtered out in earth.This bacterial strain has both efficient-decomposition slightly solubility phosphoric acid
Calcium, aluminum phosphate, produces auxin aspect, salt tolerant, heavy metal tolerance aspect and promotes plant seed germination and increase soil ferric phosphate
Earth available phosphorus, Microbial nitrogen and the function of reducing soil available cadmium.
After the bacterial strain, pakchoi germination, third day root long and the 7th day stem length, root long are divided compared with the control
8%, 39%, 28%, 33% is not increased.Laboratory simulation experiment is shown: it is effective to be a significant increase soil after application microbial inoculum
Phosphorus content, after different cadmium concentration soil application microbial inoculums available phosphorus than do not apply microbial inoculum increased separately 30%, 22% and
22%.It is a significant increase Microbial content after application microbial inoculum, available phosphorus ratio is not after different cadmium concentration soil application microbial inoculums
Application microbial inoculum has increased separately 24%, 21% and 22%；Microorganism nitrogen than do not apply microbial inoculum increased separately 32%, 24% with
And 29%.Effective cadmium content of soil is significantly reduced after application microbial inoculum, after different cadmium concentration soil application microbial inoculums effectively
Phosphorus reduces 2%, 27% and 19% than not applying microbial inoculum respectively.The above results show bacterial strain indissoluble phosphorus in improving soil
Validity and while phosphate fertilizer utilization efficiency, also can significantly promote pakchoi germination, improve Microbial nitrogen and reduction
Effective cadmium content of soil.
Detailed description of the invention
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 positive control, it is dense
Degree 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 that B19 bacterium is resistant to different salinity experimental comparison figures；Wherein, from left to right, then from top to bottom, sodium chloride is dense
Degree is respectively 0.5%, 1.5%, 3.5%, 5.5%, 7.5%, 9.5%.
Fig. 6 is that B19 bacterium is resistant to different cadmium concentration experimental comparison figures.
Fig. 7 is B19 bacterium heavy metal tolerance cadmium plate photo figure；Wherein, it is 25mg/L that figure A, which is cadmium concentration, and figure B is cadmium concentration
For 50mg/L.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Culture medium used in the present invention is as follows:
1. indissoluble Phos culture medium: glucose 10g, tricalcium phosphate 5g, ammonium sulfate 0.5g, sodium chloride 0.2g, magnesium sulfate
0.1g, potassium chloride 0.2g, yeast extract 0.5g, manganese sulfate 0.002g, ferrous sulfate 0.002g, 0.4% (w/v) bromophenol blue (pH
6.7) 6mL, distilled water are settled to l 000mL, pH 7.0, and solid medium separately adds agar 18.0g.
2. Phos screening and culturing medium A is made of following components: glucose 10g, calcium phosphate 5g, magnesium chloride 5g, magnesium sulfate
0.25g, potassium chloride 0.2g, ammonium sulfate 0.1g, distilled water are settled to 1 000mL, pH 7.0.
3. the difference of Phos screening and culturing medium B and Phos screening and culturing medium A, which is only that, replaces calcium phosphate with ferric phosphate
As unique phosphorus source.
4. the difference of Phos screening and culturing medium C and Phos screening and culturing medium A, which is only that, replaces calcium phosphate with aluminum phosphate
As unique phosphorus source.
5. fluid nutrient medium: yeast powder 5g, peptone 10g, sodium chloride 10g, water are settled to 1L, pH7.0.
6. LB liquid medium: yeast extract 5.0g, tryptone 10.0g, sodium chloride 10.0g, distilled water are 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: beef extract 3.0g, peptone 5.0g, sodium chloride 5.0g, agar 18.0g, distillation
Water is settled to 1000mL, pH value 7.0-7.2.
9. culture medium containing heavy metal: caddy is added in LB culture medium and makes up to designed concentration, prepares liquid
When culture medium, do not need that agar is added.The above culture medium is required to 121 DEG C of sterilizing 15min.
Separation, the purifying of 1 bacterial strain of embodiment
(1) it separates
1. primary dcreening operation
Weigh the Shaoguan City, Guangdong Province Qujiang District town Ma Ba Shi Bao grow cane ground soil 10.0g, be put into equipped with 90mL sterile water
250mL triangular flask in (add bead), 25 DEG C, 200r/min shaking table vibrate 30min, stand 20~30s, take 10mL supernatant
It is placed in 50mL sterilizing triangular flask, in 90 DEG C of water-bath 10min, supernatant after 1mL water-bath is taken to be added to indissoluble Phos culture medium,
(containing 30mL culture solution in 150mL triangular flask), two repetitions are arranged in each soil sample.It is placed in shaking table and carries out enrichment culture, revolving speed
150r/min, 30 DEG C of temperature.The enrichment culture period is 5~7d, takes 1mL culture solution to be respectively added after first time enrichment culture
Second of enrichment culture is carried out into new culture medium, same operation carries out third time enrichment culture, every time after enrichment
It takes supernatant appropriate with liquid-transfering gun in superclean bench, is placed in centrifuge tube, be centrifuged, supernatant taken, using the anti-colorimetric of molybdenum antimony
Method measures water-soluble phosphorus content in supernatant.Wherein, the water-soluble phosphorus content for being named as B19 bacterium is 255.34 μ g/mL；
2. secondary screening
The preferable bacterial strain of phosphorus decomposing effect is selected, is inoculated into Phos screening and culturing medium B and Phos screening and culturing medium C respectively
In, the concentration of available phosphorus in culture solution is measured after 30 DEG C of shaking table 150r/min, culture 3d.Wherein, in B19 bacterium ferric phosphate culture medium
Water-soluble phosphorus content is 48.22 μ g/mL, and water-soluble phosphorus content is 24.27 μ g/mL in aluminum phosphate culture medium, is protected using glycerol tube
The method of depositing saves the bacterial strain.
(2) it purifies
After purification using plate streak by the B19 bacterium filtered out, it is stored in made oblique of beef-protein medium
It is spare to be placed in 4 DEG C of refrigerators for face.
2 CHARACTERISTICS IDENTIFICATION of embodiment
Embodiment 1 is screened to obtained B19 bacterium and carries out Physiology and biochemistry identification and molecular biology identification.
(1) thalli morphology characteristic
B19 bacterium is gram-positive bacteria, and somatic cells are rod-shaped, end side, is in short or long-chain, bacterium colony is big, rough surface, flat
It is flat, irregularly.Thallus Gram's staining microphoto is as shown in Figure 1, purple is presented in thallus.The dyeing of gemma peacock green dye, shows
Micro- photo is as shown in Fig. 2, green is presented in gemma.
It is round or approximate circle, quality that B19 bacterium cultivates the bacterium colony formed afterwards for 24 hours on beef-protein medium
Soft, non-pigment, slightly glossiness white colony.
(2) growth characteristics
In liquid medium, revolving speed 180rpm, under conditions of initial ph value is 7.0, is cultivated 18 hours by 37 DEG C of temperature,
Measuring viable count is 3.00 ± 0.08 × 108cfu/mL。
(3) measuring method of physiology, biochemical characteristic is with reference to " common bacteria system identification handbook " (east show pearl), qualification result
It is shown in Table 1.
1 B19 bacterium physio-biochemical characteristics of table
Note :+it is expressed as the positive；It is expressed as feminine gender；W is expressed as weakly positive；
(4) molecular biological characteristic
B19 bacterium total DNA is extracted using RNA isolation kit (being purchased from Shanghai Sheng Gong bioengineering Co., Ltd).Using 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 bacterium, the PCR reaction system of 50 μ L are as follows: DNA profiling 2 μ L, primer 2 7F (l mM) 1 μ L,
1 μ L, rTaq enzyme of primer 1492R (l mM), 25 μ L, dd H2O 21μL。
Pcr amplification reaction program: 95 DEG C of initial denaturation 3min, into thermal cycle；95 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72
DEG C extend 30s, totally 35 circulation；72 DEG C of extension 10min.
1.0% agarose gel electrophoresis detects the length and concentration of PCR product, occurs apparent item near 1000bp
Band, after pcr amplification product is recycled, sequencing is completed by Guangzhou Ai Ji biotinylated biomolecule Technology Co., Ltd., by the DNA sequence dna of acquisition
BLAST comparison is carried out on (as shown in SEQ ID NO.1) input US National Bioinformatics Institute NCBI webpage, with Blast journey
All sequences in ordered pair database are compared analysis, as a result, it has been found that the 16S rDNA sequence of bacterial strain of the present invention and with
Bacillus cereus homology with higher in GenBank, similarity 99%.In conjunction with above-mentioned thalli morphology characteristic,
Flat-plate bacterial colony feature, physio-biochemical characteristics, 16S rDNA sequence analysis as a result, the Preliminary Identification bacterial strain be bacillus cereus
(Bacillus cereus) was named as 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, Institute of Microorganism, Academia Sinica.
3 B19 bacterium of embodiment produces the effect experiment of water-soluble phosphorus under slightly solubility Phos culture medium culture
(1) preparation of prepare liquid
Picking is stored in B19 thallus on beef extract-peptone slant medium and lines in LB solid medium tablets, and 30
It is cultivated for 24 hours at DEG C.B19 thallus on plate is accessed in the 150mL triangular flask containing 20mL LB liquid medium, 30 DEG C,
150r/min, shaking table culture is for 24 hours.Culture solution 6000r/min is centrifuged 5min, thallus is resuspended with sterile water, in vortex mixed instrument
Bacteria suspension equivalent uniformly, with liquid-transfering gun is accessed the 150mL triangular flask containing 30mL Phos screening and culturing medium by upper diffusion, and 30
DEG C, 150r/min, shaking table culture 7d.
(2) water-soluble phosphorus assay
Culture solution is taken into supernatant in 10000g, 25 DEG C of centrifugation 15min, water-soluble phosphorus is measured using molybdenum antimony resistance colorimetric method
Content.If not connecing B19 bacterium, access equivalent sterile water is control, and each processing is repeated 3 times.(unit is water-soluble phosphorus changes of contents
μ g/mL indicates the micrograms of contained water-soluble phosphorus in every milliliter of sample) test result see Fig. 3.
Fig. 3's the result shows that: the water-soluble phosphorus content of B19 fungi degradation calcium phosphate is up to 255.34 μ g/mL, ferric phosphate it is water-soluble
Property phosphorus content be 48.22 μ g/mL, the water-soluble phosphorus content of aluminum phosphate is 24.27 μ g/mL.
4 B19 bacterium of embodiment produces auxin effect test
(1) B19 bacterium auxin qualitative determination: is inoculated in the training of the 20mL LB liquid containing L-Trp (100 μ g/mL)
Support base 150mL triangular flask in, 30 DEG C, 150r/min shaking table culture for 24 hours.Take 50 μ L culture drop on whiteware plate, together
When add 50 μ L Salkowski color solution (35%HClO4+1mL 0.5mol/LFeCl3), if the sterile LB medium conduct of 50 μ L
Negative control, the color solution of 10,20,50 (μ g/mL) auxin is as positive control.By whiteware plate in room temperature, be protected from light item
It is observed after placing 30min under part, the color person of reddening indicates that auxin can be produced, and test result is shown in Fig. 4, it is seen then that the color of B19 is aobvious
It is shown as pale pink, color is between control and 10 μ g/mL.
(2) auxin quantitative determines: B19 bacterium is inoculated in the 50mL LB liquid medium containing L-Trp (μ g/mL)
In, three repetitions, 30 DEG C, 150r/min shaking table culture for 24 hours.Culture solution is centrifuged 10min at 6000 × g respectively, is taken on 4mL
4mL Salkowski color solution is added in clear liquid, mixes, and stands 30min in room temperature, dark, using ultraviolet specrophotometer,
Absorbance is surveyed under 530nm, while aseptic culture medium being used to do same treatment as control, using 0,10,20,30,50 (μ g/mL's)
Auxin makees standard curve.Test result is shown in Table 2.B19 bacterium produces auxin amount and reaches 8.72 μ g/mL.
2 B19 bacterium of table produces auxin effect test result
| Strain number | It produces IAA amount (μ g/mL) |
| CK | 0 |
| B19 | 8.72 |
5 B19 bacterium Salt tolerance of embodiment
Nutrient medium: beef extract 3.0g, peptone 10.0g, NaCl 5.0g, agar 18.0g, distilled water 1000mL, pH
7.0-7.2.According to different sodium chloride concentrations, sodium chloride is added.
By B19 bacterium be crossed to sodium chloride concentration be respectively 0.5%, 1.5%, 3.5%, 5.5%, 7.5%, 9.5%,
11.5%, on 13.5% nutrient agar panel, culture for 24 hours, observes the growing state of bacterial strain.Test result is shown in Fig. 5, with chlorine
The content for changing sodium is higher, and the increment of thallus is fewer, until 9%, the bacterium colony of thallus is very sparse, until 13%, do not observe bacterium
It falls.The result shows that: the NaCl of B19 bacterium tolerance 9%.
6 B19 bacterium heavy metal tolerance cadmium test of embodiment
It takes the B19 bacterium solution of 30 μ L to be inoculated in the LB liquid medium of 3mL, cultivates 8-10h in 37 DEG C, 180rpm, carry out
Activation.B19 bacterium solution after activation is added according to 1% inoculum concentration and contains 0,1,5,10,25,30mg/L Cd2+3mL LB liquid
In culture medium, 37 DEG C, it is bent to draw heavy metal tolerance every the light absorption value A600 of 30min measurement culture medium for 180rpm shaking table culture
Line.Test result is shown in Fig. 6 and Fig. 7.The result shows that: the Cd of B19 bacterium tolerance 30mg/L2+。
7 B19 bacterium of embodiment promotes the test of plant seed germination efficacy in laboratory
(1) prepared by bacteria suspension:
1., by shaken cultivation for 24 hours, cultivation temperature is 30 DEG C, revolving speed by point access LB liquid medium after actication of culture
For 150r/min；
2. bacterium solution is instilled blood counting chamber, count under an optical microscope, obtains cell concentration in bacterium solution；
3. bacterium solution is centrifuged in supercentrifuge, revolving speed 6000r/min, it is centrifuged 5min；
4. thallus is resuspended with sterile water；
5. the bacterial strain concentration for impregnating pakchoi seed is 2.0 × 107The bacteria suspension of cfu/ml.
(2) the surface of the seed sterilization treatment: 75% ethyl alcohol of seed impregnates 1min, then is impregnated with 2% liquor natrii hypochloritis
2min is placed in naturally dry in sterilizing filter paper.
(3) Piglet s colibacillosis is carried out using B19 bacterium and blank control, being divided into 2 processing groups, (the 1st group uses 25mL
B19 bacterium bacterium re-suspension liquid), indicated with B19, the 2nd group uses 25mL sterile water water process as blank control, is indicated with CK).Everywhere
Reason group sets four repetitions, each to repeat to contain 10 pakchoi seeds (randomly selecting uniform seed after surface sterilizing).Processing
Group B19 impregnates 30min using bacteria suspension, and CK makees same processing using sterile water.Sterilizes culture dish is taken, two layers of sterile filter is added
Paper after removing soak, is put into culture dish, 10, every ware, then cover one layer of aseptic filter paper with aseptic nipper clamping seed, with
Infiltration saves moisture, is placed in 25 DEG C of incubator cultures 7 days, pours appropriate amount of water daily, and the water of every ware wants identical, uniformly, periodically surveys
Determine germination percentage, the indexs such as stem length, root long.Test result is shown in Table 3.The bacillus cereus that the present invention separates as can be seen from Table 3
(Bacillus cereus) B19 bacterium can dramatically increase Chinese cabbage seed germination, promote the growth of pakchoi seed rhizome, third
Its root long and the 7th day stem length, root long have increased separately 39%, 28%, 33% compared with the control.
3 B19 bacterium of table is to pakchoi germination facilitation effect test result
Note: data are 3 duplicate mean+SDs, and same column * indicates significant (independent in 0.05 level difference in table
Sample t-test).
8 B19 bacterium of embodiment tests soil nutrient and impact of heavy metals
(1) prepared by bacteria suspension:
1., by shaken cultivation for 24 hours, cultivation temperature is 30 DEG C, revolving speed by point access LB liquid medium after actication of culture
For 150r/min；The Liquid Culture based component are as follows: 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, obtains cell concentration in bacterium solution；
3. bacterium solution is centrifuged in supercentrifuge, revolving speed 6000r/min, it is centrifuged 5min；
4. thallus is resuspended with sterile water；
5. the bacteria suspension that the bacterial strain concentration for being inoculated with soil is 3.0 × 107cfu/mL.
(2) experimental design:
Rice soil is acquired from big Golconda Some Mining Districts, the physical and chemical index of soil is measured, the results are shown in Table 4.Add respectively to soil
Enter the Cd of 1mg/kg and 3mg/kg2+, and aging 14d.
It with B19 bacterium and bacterium is not added carries out Piglet s colibacillosis, be divided into 6 processing groups: the 1st group of original soil adds sterile water, uses T1
It indicates；B19 bacterium 3 × 10 is added in 2nd group of original soil6Cfu/g soil, is indicated with T2；3rd group of external source adds 1mg/kg Cd2+Soil adds
Enter sterile water, is indicated with T3；4th group of external source adds 1mg/kg Cd2+B19 bacterium 3 × 10 is added in soil6Cfu/g soil, with T4 table
Show；5th group of external source adds 3mg/kg Cd2+Sterile water is added in soil, is indicated with T5；6th group of external source adds 3mg/kg Cd2+Soil
B19 bacterium 3 × 10 is added in earth6Cfu/g soil, is indicated with T6.Each processing group is repeated 3 times.Appropriate amount of water is poured daily, maintains 40% soil
Earth water content.
Application takes fresh soil to do Microbial nitrogen after microbial inoculum 5 days, and remaining soil natural is air-dried, and does soil available phosphorus, has
Imitate the experiment of state cadmium.Experimental result see the table below 5 respectively.Experimental result is shown, applies available phosphorus, Microbial, micro- life after B19 microbial inoculum
Object nitrogen significantly increases, and effective cadmium content significantly reduces.
4 laboratory simulation experiment soil physico-chemical property of table
5 B19 microbial inoculum of table is to soil nutrient and effective cadmium effect test result
Note: T1 indicates that original soil, T2 expression original soil add bacterium B19, soil after T3 expression plus 1mg/kg Cd aging, and T4 indicates to add
Soil adds bacterium B19, soil after T5 expression plus 3mg/kg Cd aging again after 1mg/kg Cd aging, and T6 is indicated after adding 3mg/kg Cd aging
Soil adds bacterium B19 again.Data are 3 duplicate mean+SDs, and same column * indicates significant (solely in 0.05 level difference in table
Vertical sample t-test).
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (6)
1. a kind of bacillus cereus being passivated with heavy metal and promote plant growth function, it is characterised in that: entitled wax
Sample bacillus (Bacillus cereus) B19, it was preserved on 04 27th, 2016 positioned at BeiChen West Road, Chaoyang District, BeiJing City
The China Committee for Culture Collection of Microorganisms's common micro-organisms center of No. 3 Institute of Microorganism, Academia Sinica, No. 1 institute is protected
Hiding number is CGMCC NO.12405.
2. the bacillus cereus with heavy metal passivation and promotion plant growth function according to claim 1, feature
Be: the 16S rDNA nucleotide sequence of the bacillus cereus is as shown in SEQ ID NO:1.
3. the bacillus cereus with heavy metal passivation and promotion plant growth function according to claim 1, feature
Be: the morphological feature of the bacillus cereus is as follows: for gram-positive bacteria, somatic cells are rod-shaped, end side, in short
Or long-chain, bacterium colony is big, rough surface, and it is flat, irregularly；The bacterium colony formed afterwards for 24 hours is cultivated on beef-protein medium
For round or approximate circle, quality is soft, non-pigment, slightly glossiness white colony.
4. the bacillus cereus with heavy metal passivation and promotion plant growth function according to claim 1, feature
Be: the physiological and biochemical property of the bacillus cereus is as follows: the cellulose decomposition positive, amylase positive, peroxidating
Hydrogen enzyme positive, indoles are negative, generation film is negative, methyl red test is negative, siderophore weakly positive.
5. there is heavy metal passivation described in any one of Claims 1 to 4 and promote the bacillus cereus of plant growth function
Using, it is characterised in that: application of the bacillus cereus in following either side: for decomposing insoluble phosphorus, use
In producing auxin, it is used for salt tolerant, is used for heavy metal tolerance, for promoting plant seed germination, for increasing soil available phosphorus, micro-
Biological carbon nitrogen, and for reducing soil available heavy metal；
The heavy metal is cadmium.
6. application according to claim 5, it is characterised in that: the insoluble phosphorus refers to slightly solubility calcium phosphate, phosphorus
At least one of sour iron and aluminum phosphate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610391094.9A CN105925507B (en) | 2016-06-02 | 2016-06-02 | Bacillus cereus and the application of plant growth function are passivated and promoted with heavy metal |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610391094.9A CN105925507B (en) | 2016-06-02 | 2016-06-02 | Bacillus cereus and the application of plant growth function are passivated and promoted with heavy metal |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105925507A CN105925507A (en) | 2016-09-07 |
| CN105925507B true CN105925507B (en) | 2019-05-07 |
Family
ID=56832383
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610391094.9A Active CN105925507B (en) | 2016-06-02 | 2016-06-02 | Bacillus cereus and the application of plant growth function are passivated and promoted with heavy metal |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105925507B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112680383A (en) * | 2021-01-28 | 2021-04-20 | 山西省农业科学院农业环境与资源研究所 | Novel phosphorus-dissolving bacteria strain and application thereof |
Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043713B (en) * | 2016-11-24 | 2022-12-16 | 华南农业大学 | Bacillus cereus Y10 and application thereof in cadmium resistance and/or reduction of effective cadmium content |
| CN106591277A (en) * | 2016-12-07 | 2017-04-26 | 华南农业大学 | Immobilized Bacillus having phosphate dissolving and heavy metal passivation functions and preparation method and application thereof |
| CN107460143B (en) * | 2017-07-05 | 2020-07-28 | 湘潭大学 | Bacillus cereus capable of biologically activating and mineralizing heavy metal cadmium and application thereof |
| CN108018239B (en) * | 2017-12-25 | 2021-03-05 | 新疆农业科学院生物质能源研究所 | Salt-resistant cellulose degradation microbial inoculum and preparation method and application thereof |
| CN108587972B (en) * | 2018-05-11 | 2021-02-23 | 北京林业大学 | Bacillus thuringiensis MRP-3 for adsorbing heavy metals and application thereof |
| CN108636356A (en) * | 2018-06-05 | 2018-10-12 | 吉林农业大学 | A kind of cornstalk biological charcoal and restorative procedure that can repair nitrogen phosphorus pollution of area source |
| CN108675894A (en) * | 2018-06-21 | 2018-10-19 | 深圳市中连胜祥农业科技有限公司 | A kind of microbial organic fertilizer containing bacillus cereus |
| CN109251880B (en) * | 2018-11-26 | 2020-05-15 | 深圳中绿环境集团有限公司 | Bacillus cereus and application thereof in improving phosphorus pollution of water body |
| CN111484948A (en) * | 2019-01-28 | 2020-08-04 | 福建省农业科学院农业生物资源研究所 | Root-promoting phosphate-solubilizing nitrogen-fixing bacillus and application thereof |
| CN110106109B (en) * | 2019-04-24 | 2020-12-01 | 华中农业大学 | Bacillus welchii and application thereof |
| CN110731223B (en) * | 2019-11-15 | 2022-02-22 | 南京林业大学 | Method for promoting growth of salix psammophila under salt stress |
| CN111518723B (en) * | 2020-04-27 | 2021-11-19 | 华南农业大学 | Bacillus gorilla YC9L and application thereof |
| CN111826311B (en) * | 2020-07-10 | 2022-04-08 | 华南农业大学 | Bacillus badensis BY08 and application thereof |
| CN112094771B (en) * | 2020-09-04 | 2023-01-13 | 暨南大学 | Bacillus cereus B-28 and application thereof |
| CN114107117B (en) * | 2021-12-01 | 2024-02-23 | 江苏省农业科学院 | Rice endophyte and application thereof |
| CN113980875B (en) * | 2021-12-15 | 2022-08-26 | 广西壮族自治区南宁良凤江国家森林公园 | Bacillus cereus P1 and application thereof |
| CN114015625B (en) * | 2021-12-15 | 2023-07-25 | 广西大学 | Bacillus cereus P30 and application thereof |
| CN114395508B (en) * | 2022-01-18 | 2023-11-24 | 广东省农业科学院蚕业与农产品加工研究所 | Bacillus cereus strain SEM-15 with heavy metal adsorption and growth promotion effects and application thereof |
| CN115044502B (en) * | 2022-06-01 | 2024-02-13 | 重庆市农业科学院 | Disease-inhibiting growth-promoting bacillus cereus YT2-1C and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181715A (en) * | 2007-10-26 | 2008-05-21 | 上海大学 | Heavy-metal polluted soil united directional restoration method by using plant-microorganism |
| CN103789240A (en) * | 2014-01-25 | 2014-05-14 | 西北农林科技大学 | Fermentation medium for improving phosphate-dissolving ability of bacillus cereus and fermentation method thereof |
| CN104988101A (en) * | 2015-08-03 | 2015-10-21 | 黑龙江省科学院微生物研究所 | Bacillus cereus and application of bacillus cereus as agricultural biological agent |
-
2016
- 2016-06-02 CN CN201610391094.9A patent/CN105925507B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181715A (en) * | 2007-10-26 | 2008-05-21 | 上海大学 | Heavy-metal polluted soil united directional restoration method by using plant-microorganism |
| CN103789240A (en) * | 2014-01-25 | 2014-05-14 | 西北农林科技大学 | Fermentation medium for improving phosphate-dissolving ability of bacillus cereus and fermentation method thereof |
| CN104988101A (en) * | 2015-08-03 | 2015-10-21 | 黑龙江省科学院微生物研究所 | Bacillus cereus and application of bacillus cereus as agricultural biological agent |
Non-Patent Citations (1)
| Title |
|---|
| Bioaccumulation characterization of cadmium by growing Bacillus cereus RC-1 and its mechanism;Fei Huang等;《Chemosphere》;20141231;第109卷;134–142 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112680383A (en) * | 2021-01-28 | 2021-04-20 | 山西省农业科学院农业环境与资源研究所 | Novel phosphorus-dissolving bacteria strain and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105925507A (en) | 2016-09-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105925507B (en) | Bacillus cereus and the application of plant growth function are passivated and promoted with heavy metal | |
| CN112458012B (en) | Bacillus belgii microbial agent and application thereof | |
| CN105861359A (en) | Heterotrophic nitrification-aerobic denitrification high temperature resisting strain for producing floc, and application thereof | |
| Fu et al. | Trichoderma affects the physiochemical characteristics and bacterial community composition of saline–alkaline maize rhizosphere soils in the cold-region of Heilongjiang Province | |
| CN106434496B (en) | One plant of brevibacterium halotolerans and its application in soil ecology reparation | |
| Gen-Fu et al. | Characterization of phosphorus-releasing bacteria in a small eutrophic shallow lake, Eastern China | |
| CN103571770B (en) | A kind of Efficient peanut rhizobiumleguminosarstrain strain and application thereof | |
| CN106167776A (en) | A kind of can bacillus cereus (Bacillus cereus) TH 35 of heavy metal cadmium and application thereof in activating soil | |
| CN105331552B (en) | One plant of efficient denitrification acinetobacter calcoaceticus novel species and its application | |
| CN105925503B (en) | One plant of salt tolerant rhizosphere growth-promoting enterobacter cloacae and its application | |
| Assigbetse et al. | Soil bacterial diversity responses to root colonization by an ectomycorrhizal fungus are not root-growth-dependent | |
| Chang et al. | Maize-soybean relay strip intercropping reshapes the rhizosphere bacterial community and recruits beneficial bacteria to suppress Fusarium root rot of soybean | |
| CN104593301B (en) | One plant of wall bacillus G1 and its preparation method and application | |
| CN103371041A (en) | Method for screening bio-control bacteria of cucumber fusarium wilt | |
| CN102167448A (en) | Application of aniline degradation strain NO.3 | |
| CN110907600A (en) | Method for measuring tobacco planting soil micro-ecological environment | |
| Wang et al. | The mechanism of microbial community succession and microbial co-occurrence network in soil with compost application | |
| CN106591173A (en) | Bacillus flexus HL-37 capable of activating soil heavy metal cadmium, and applications thereof | |
| CN104805018A (en) | Agromyces sp. MT-E used for simultaneous degradation of plurality of phthalic acid esters | |
| CN104845890A (en) | Application of Agromyces sp. MT-E in degradation of various phthalates | |
| Huang et al. | The colonization of the arbuscular mycorrhizal fungus Rhizophagus irregularis affects the diversity and network structure of root endophytic bacteria in maize | |
| Ren et al. | Screening, characteristics and mechanism of Cd-tolerance Cunninghamella bertholletiae | |
| CN103343097A (en) | Nitrogen fixing micrococcus and application thereof | |
| Rasouli-Nasab et al. | Comparison of various methods for isolation of Nocardia from soil | |
| CN102978138B (en) | Mesorhizobium KDRM185 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200121 Address after: 510000 939, No. 1, Huaxu street, Tianhe District, Guangzhou City, Guangdong Province Patentee after: Guangzhou Qin'an Agricultural Technology Co., Ltd Address before: 510642 No. five, 483 mountain road, Guangzhou, Guangdong, Tianhe District Patentee before: South China Agricultural University |