CN109943500A - A kind of screening technique of the screening drop cadmium bacterium from rice paddy seed - Google Patents
A kind of screening technique of the screening drop cadmium bacterium from rice paddy seed Download PDFInfo
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- CN109943500A CN109943500A CN201910139339.2A CN201910139339A CN109943500A CN 109943500 A CN109943500 A CN 109943500A CN 201910139339 A CN201910139339 A CN 201910139339A CN 109943500 A CN109943500 A CN 109943500A
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Abstract
This application discloses a kind of to screen the screening technique for dropping cadmium bacterium from rice paddy seed.The following steps are included: surface sterilization: screening the rice paddy seed of full grains, first rinsed with the NaCLO that available chlorine content is 1%, seed is collected after rinsing, then use sterile water wash；It isolates and purifies: cultivating endogenetic bacteria in the medium, using 30 DEG C of constant incubator cultures, irregularly observe bacterium situation；The single colonie for selecting different mode of appearance, is saved；Identification: biological property analysis is carried out to all seed endophytes separated: producing siderophore function qualitative detection, producing IAA Function detection, Soluble phosphorus Function detection, fixed nitrogen function and resistance to Cd and detects；Molecular biology identification is carried out to it simultaneously；Screening: by the biological characteristics detection to all bacterial strains as a result, the bacterial strain for wherein integrating physiological function advantage is selected to save as dominant bacteria.This method can comprehensively filter out drop cadmium bacterium from rice paddy seed.
Description
Technical field
This application involves bacteria screening technical field, the screening of specifically a kind of screening drop cadmium bacterium from rice paddy seed
Method.
Background technique
Rice paddy seed is the important carrier of endogenetic bacteria, and it is all logical that the endogenetic bacteria in rice plant different parts is most of
Seed heredity is crossed, important function is played for the vital movement of rice.Compared to other traditional microorganisms, with rice
Plant have it is geneogenous be closely connected, be capable of the symbiosis of " harmony joint ", therefore rice paddy seed is that research endogenetic bacteria inhibits
The ideal object of rice Cd uptake ability.
Secondly, can be played in analyzing rice seed with the potential endogenetic bacteria diversity for inhibiting rice Cd uptake ability
Effective directive function helps to filter out more efficient function stem.
Finally, before about the research of the rice Cd uptake ability concern direct function and effect of microorganism or micro- mostly
The application method of biology.But since microorganism is sensitive to environmental condition, colonization ability is poor, and function and effect are unstable, so
Effect cannot be played in practical application well.The body that additional microorganism and rice endophyte group interact as one
System, the variation about microbiologic population in system are but rarely reported.This research will probe into Rice Endophytic Bacteria community diversity and
The variation of composition contacts the variation of rice Cd uptake ability, discloses the mechanism of action of endogenetic bacteria, and actual agricultural is applied
There is important directive significance.
In the screening technique of bacterium, bacterium is centrifugated according to bacterium volume size, needed for can quickly filtering out
Bacterium, but much only one centrifugal networks of bacteria separating apparatus, the pore size of centrifugal network is constant, cannot be adjusted, each
Bacteria separating apparatus can only also filter out the bacterium of same granular size, and bacteria separating apparatus cannot carry out multistage point to bacterium
From, be not able to satisfy the isolated requirement to a variety of bacteriums of different sizes, can not meet the demand of experiment, be unfavorable for bacterium multiplicity
The experimental study of change.
In consideration of it, a kind of screening technique for dropping cadmium bacterium of the present invention, useful Selective agar medium screen bacterium,
Have and bacterium is screened by bacterium volume size, bacterium is screened with three kinds of culture mediums, can more comprehensively be protected
The endophyte reserved seed for planting inside son, is identified using multiple standards, ensure that the high-precision of screening.
Summary of the invention
This application provides a kind of to screen the screening technique for dropping cadmium bacterium from rice paddy seed, is dropped using three kinds of culture mediums
The screening of cadmium bacterium, while being identified using a variety of screening index, screening precision is improved, while can guarantee to screen the complete of bacterium
Face property.
The present invention provides a kind of screening technique of screening drop cadmium bacterium from rice paddy seed, comprising the following steps:
Surface sterilization: screening the rice paddy seed of full grains, is first rinsed 10 minutes with the NaCLO that available chlorine content is 1%,
Seed is collected after rinsing, then with sterile water wash 5 times, every time 1 minute；
Seed grinding: according to the capacity of grinding pot, being added suitable rice paddy seed, grinding pot be put into adapter, if
Parameter is set, beveller is started, is ground 1-2 minutes；
It isolates and purifies: in the medium by seed lapping liquid coating, endogenetic bacteria is cultivated in the medium, using 30
DEG C constant incubator culture, irregularly observes bacterium situation；The single colonie for selecting different mode of appearance, is saved；
Identification: biological property analysis is carried out to all seed endophytes separated: producing the qualitative inspection of siderophore function
Survey, producing IAA Function detection, Soluble phosphorus Function detection, fixed nitrogen function, resistance to Cd detection and drop cadmium Function detection；Simultaneously to its into
Row molecular biology identification；
Screening: by the biological characteristics detection to all bacterial strains as a result, selection wherein integrates physiological function qualification
Bacterial strain with drop cadmium function is saved as dominant bacteria.
Further, during the step isolates and purifies, using raw in the various culture medium cultures of R2A, 1/2LB and 1/2PDA tri-
Seed lapping liquid is coated on tri- kinds of plating mediums of R2A, 1/2LB and 1/2PDA by bacterium.
Further, in the step identification, the production siderophore function qualitative detection process are as follows: after isolating and purifying
Seed endogenetic bacteria is inoculated on the detection plate with CAS detection liquid, is cultivated in 28 DEG C of incubator 46 hours, observation
The formation and size of the orange-yellow haloing of periphery of bacterial colonies in plate.
Further, in the step identification, the producing IAA Function detection process are as follows: by the seed after isolating and purifying
Endogenetic bacteria is inoculated in the fluid nutrient medium of the L-Trp containing 100mg/L, is carried out under the conditions of 30 DEG C and 180r/min
After shaking table culture 1 day, take 50 μ L bacteria suspensions drop on whiteware plate, while color solution is added, by whiteware plate in room temperature
Avoid light place is observed after 30 minutes, and the color person of reddening is the positive, and expression being capable of producing IAA.
Further, in step identification, the Soluble phosphorus Function detection process are as follows: by slant strains with 0.8% life
Salt water is managed, and after 40 DEG C of activation 48h, then phosphorus bacteria fertilizer dilution spread on plate, 40 DEG C of culture 5d are often preordained
When measurement Soluble phosphorus loop diameter and colony diameter, both calculate ratio, the initial characterization bacterium phosphate solubilization size, ratio is bigger, says
Bright phosphate solubilization is stronger.
Further, in the step identification, the color solution is the HCLO that volume 50ml contains 35%4Liquid and body
Product 1ml contains the FeCL of 0.5mol/L3The mixed liquor of liquid.
Further, the drop cadmium Function detection process are as follows: by drop cadmium bacterium addition in water-culturing rice liquid, in paddy growth
During observe and detect the cadmium content of rice, testing result is determined as the drop cadmium bacterium with drop cadmium function without cadmium content.
The beneficial effects of the present invention are: (1) it using three kind culture mediums can more fully retain the endophyte of seed；
It (2) is that lower layer's plate is convenient for measuring so that transparent circle is high-visible by three kinds of culture mediums of addition in the method for the present invention；(3) exist
The identification that several functions are used in the identification method of the method for the present invention design, improves the order of accuarcy for screening eugenic bacterium；Drop
The transparent circle size good relationship that cadmium bacterium screening flat board method measures, the selection result accuracy are high.
It should be understood that the above general description and the following detailed description are merely exemplary, this can not be limited
Application.
Detailed description of the invention
Fig. 1 is a kind of screening technique flow chart of the screening drop cadmium bacterium from rice paddy seed provided by the embodiment of the present application；
The drawings herein are incorporated into the specification and forms part of this specification, and is used to explain together with specification
The principle of the application.
Specific embodiment
The application is described in further detail below by specific embodiment and in conjunction with attached drawing.
As shown in Figure 1, the present invention provides a kind of screening technique of screening drop cadmium bacterium from rice paddy seed, including following step
It is rapid:
Surface sterilization: screening the rice paddy seed of full grains, is first rinsed 10 minutes with the NaCLO that available chlorine content is 1%,
Seed is collected after rinsing, then with sterile water wash 5 times, every time 1 minute；
Seed grinding: according to the capacity of grinding pot, being added suitable rice paddy seed, grinding pot be put into adapter, if
Parameter is set, beveller is started, is ground 1-2 minutes；
It isolates and purifies: in the medium by seed lapping liquid coating, endogenetic bacteria is cultivated in the medium, using 30
DEG C constant incubator culture, irregularly observes bacterium situation；The single colonie for selecting different mode of appearance, is saved；
Identification: biological property analysis is carried out to all seed endophytes separated: producing the qualitative inspection of siderophore function
Survey, producing IAA Function detection, Soluble phosphorus Function detection, fixed nitrogen function, resistance to Cd detection and drop cadmium Function detection；Simultaneously to it
Carry out molecular biology identification；
Screening: by the biological characteristics detection to all bacterial strains as a result, selection wherein integrates physiological function advantage
Bacterial strain is saved as dominant bacteria.
The specific steps of the endophyte with drop cadmium function are filtered out from rice paddy seed are as follows: screen full grains first
Rice paddy seed carries out surface sterilization, is first rinsed 10 minutes with the NaCLO that available chlorine content is 1%, seed is collected after rinsing, then use
Sterile water wash 5 times, every time 1 minute；Rice paddy seed after disinfection is ground: according to the capacity of grinding pot, being added appropriate
Rice paddy seed, grinding pot is put into adapter, be arranged parameter, start beveller, grind 1-2 minutes；
It isolates and purifies: seed lapping liquid being coated in tri- kinds of plating mediums of R2A, 1/2LB and 1/2PDA raw in cultivating
Bacterium irregularly observes bacterium situation using 30 DEG C of constant incubator cultures；The single colonie of different mode of appearance is selected, is carried out
It saves.
Identification: biological property analysis is carried out to all rice paddy seed endophytes separated: including producing siderophore function
It can qualitative detection, producing IAA Function detection, Soluble phosphorus Function detection, fixed nitrogen function, resistance to Cd detection and drop cadmium Function detection；Together
When molecular biology identification is carried out to it；
Produce siderophore function qualitative detection process are as follows: the seed endogenetic bacteria after isolating and purifying is inoculated into be examined with CAS
It surveys on the detection plate of liquid, 46 hours is cultivated in 28 DEG C of incubator, observe the orange-yellow haloing of periphery of bacterial colonies in plate
Formation and size.Yellow halo illustrates that bacterial strain has the ability for producing siderophore, and yellow halo and the ratio of bacterium colony circle size are bigger,
It is stronger to illustrate that it produces siderophore ability.Select the bacterial strain with yellow halo that there is the rice paddy seed for producing siderophore as qualified
The bacterial strain of endophyte.
The rice paddy seed endophyte that qualified the having of screening produces siderophore is subjected to producing IAA Function detection, detection process
Are as follows: the seed endogenetic bacteria after isolating and purifying is inoculated in the fluid nutrient medium of the L-Trp containing 100mg/L, at 30 DEG C
After carrying out shaking table culture 1 day under the conditions of 180r/min, take 50 μ L bacteria suspensions drop on whiteware plate, while colorimetric is added
Liquid, color solution are the HCLO that volume 50ml contains 35%4Liquid and volume 1ml contain the FeCL of 0.5mol/L3The mixing of liquid
Then liquid is observed whiteware plate after room temperature avoid light place 30 minutes, the color person of reddening is the positive, and expression can secrete
IAA.Positive color is purple powder, is purple powder as the qualified bacterial strain with producing IAA function with positive control.
The qualified rice paddy seed endophyte with producing IAA function is further subjected to Soluble phosphorus Function detection, was detected
Journey are as follows: elute 0.8% physiological saline of slant strains, and after 40 DEG C of activation 48h, then phosphorus bacteria fertilizer dilution spread
In on plate, 40 DEG C of culture 5d, daily Timing measurement Soluble phosphorus loop diameter and colony diameter, both calculating ratio, initial characterization are somebody's turn to do
Bacterium phosphate solubilization size, ratio is bigger, illustrates that phosphate solubilization is stronger.The bacterial strain with transparent haloing is selected to have as qualified
The bacterial strain of phosphate solubilization.
The qualified rice paddy seed endophyte with phosphate solubilization is finally subjected to drop cadmium Function detection, detection process are as follows:
By drop cadmium bacterium addition in water-culturing rice liquid, the cadmium content of rice, testing result are observed and detected during paddy growth
Without cadmium content, it is determined as the qualified drop cadmium bacterium with drop cadmium function.
By the biological characteristics detection to all bacterial strains as a result, selection wherein integrates the rice seed of physiological function qualification
Sub- endophyte is saved as qualified drop cadmium bacterium.
A kind of screening technique of screening drop cadmium bacterium from rice paddy seed provided herein is used compared with prior art
Three kinds of culture mediums screen bacterium, can more comprehensively retain the endophyte with drop cadmium function inside seed, adopt
Testing and appraisal is carried out with the multiple standards such as siderophore ability, producing IAA ability, phosphate solubilization and drop cadmium ability are produced, ensure that
The high-precision of screening.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (7)
1. a kind of screening technique of the screening drop cadmium bacterium from rice paddy seed, which comprises the following steps:
Surface sterilization: screening the rice paddy seed of full grains, is first rinsed 10 minutes with the NaCLO that available chlorine content is 1%, rinsing
After collect seed, then with sterile water wash 5 times, every time 1 minute；
Seed grinding: according to the capacity of grinding pot, suitable rice paddy seed is added, grinds 1-2 minutes, obtains seed lapping liquid；
It isolates and purifies: in the medium by seed lapping liquid coating, cultivating the interior life of rice paddy seed in the medium
Bacterium irregularly observes bacterium situation using 30 DEG C of constant incubator cultures；The single colonie of different mode of appearance is selected, is carried out
It saves；
Identification: biological property analysis is carried out to all seed endophytes separated: producing siderophore function qualitative detection, divide
Secrete IAA Function detection, Soluble phosphorus Function detection, fixed nitrogen Function detection, resistance to Cd detection and drop cadmium Function detection；It is carried out simultaneously
Molecular biology identification；
Screening: by the biological characteristics detection to all bacterial strains as a result, selection wherein integrates having for physiological function qualification
The bacterial strain of drop cadmium function is saved as dominant bacteria.
2. a kind of screening technique of screening drop cadmium bacterium from rice paddy seed according to claim 1, which is characterized in that described
During step isolates and purifies, using the various culture medium culture endogenetic bacterias of R2A, 1/2LB and 1/2PDA tri-, seed lapping liquid is coated with
On tri- kinds of plating mediums of R2A, 1/2LB and 1/2PDA.
3. a kind of screening technique of screening drop cadmium bacterium from rice paddy seed according to claim 1, which is characterized in that described
In step identification, the production siderophore function qualitative detection process are as follows: the seed endogenetic bacteria after isolating and purifying is inoculated into tool
On the detection plate for having CAS detection liquid, 46 hours are cultivated in 28 DEG C of incubator, and it is orange-yellow to observe periphery of bacterial colonies in plate
The formation and size of haloing.
4. a kind of screening technique of screening drop cadmium bacterium from rice paddy seed according to claim 1, which is characterized in that described
Step identification in, the producing IAA Function detection process are as follows: the seed endogenetic bacteria after isolating and purifying be inoculated in containing
In the fluid nutrient medium of the L-Trp of 100mg/L, after carrying out shaking table culture 1 day under the conditions of 30 DEG C and 180r/min, 50 μ are taken
Color solution is added on whiteware plate in L bacteria suspension drop, and whiteware plate is seen after room temperature avoid light place 30 minutes
It examines, the color person of reddening is the positive, and expression being capable of producing IAA.
5. a kind of screening technique of screening drop cadmium bacterium from rice paddy seed according to claim 1, which is characterized in that described
In step identification, the Soluble phosphorus Function detection process are as follows: elute 0.8% physiological saline of slant strains, and in 40 DEG C
After activating 48h, then phosphorus bacteria fertilizer dilution spread on plate, 40 DEG C of culture 5d, daily Timing measurement Soluble phosphorus loop diameter and bacterium
Diameter is fallen, calculates the two ratio, the initial characterization bacterium phosphate solubilization size, ratio is bigger, illustrates that phosphate solubilization is stronger.
6. a kind of screening technique of screening drop cadmium bacterium from rice paddy seed according to claim 4, which is characterized in that described
In step identification, the color solution during the producing IAA Function detection is the HCLO that volume 50ml contains 35%4Liquid and
Volume 1ml contains the FeCL of 0.5mol/L3The mixed liquor of liquid.
7. a kind of screening technique of screening drop cadmium bacterium from rice paddy seed according to claim 1, which is characterized in that described
In step identification, the drop cadmium Function detection process are as follows: by drop cadmium bacterium addition in water-culturing rice liquid, in the process of paddy growth
Middle observation and the cadmium content for detecting rice, testing result are determined as the drop cadmium bacterium with drop cadmium function without cadmium content.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114903049A (en) * | 2022-05-31 | 2022-08-16 | 湘潭大学 | Application of hydroxamic acid type siderophore as rice cadmium reduction preparation |
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| 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 |
| WO2015200852A2 (en) * | 2014-06-26 | 2015-12-30 | Ait Austrian Institute Of Technology Gmbh | Plant-endophyte combinations and uses therefor |
| 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 |
| CN106754373A (en) * | 2017-02-08 | 2017-05-31 | 浙江农林大学 | Resistance to cadmium produces separation method and the application of siderophore bacterial strain |
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2019
- 2019-02-25 CN CN201910139339.2A patent/CN109943500A/en active Pending
Patent Citations (4)
| 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 |
| WO2015200852A2 (en) * | 2014-06-26 | 2015-12-30 | Ait Austrian Institute Of Technology Gmbh | Plant-endophyte combinations and uses therefor |
| 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 |
| CN106754373A (en) * | 2017-02-08 | 2017-05-31 | 浙江农林大学 | Resistance to cadmium produces separation method and the application of siderophore bacterial strain |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114903049A (en) * | 2022-05-31 | 2022-08-16 | 湘潭大学 | Application of hydroxamic acid type siderophore as rice cadmium reduction preparation |
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