CN110358695A - A kind of drought resisting growth promoting bacteria agent and its screening technique, liquid bacterial agent - Google Patents
A kind of drought resisting growth promoting bacteria agent and its screening technique, liquid bacterial agent Download PDFInfo
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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
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Abstract
The invention belongs to bacteriums；Its culture medium technical field discloses a kind of drought resisting growth promoting bacteria agent and its screening technique, liquid bacterial agent；The drought resisting Promoting bacteria is pseudomonad G3.Pseudomonas strains Pseudomonas koreenis G3 liquid bacterial agent of the invention can significantly improve jujube plant Drought resistance；Under identical drought stress degree, the jujube plant for not being inoculated with microbial inoculum can more effectively improve activities of antioxidant enzymes, increase the content of plant tumor growth hormone, enhance the photosynthesis characteristics of plant, alleviate injury of the drought stress to plant, is conducive to growth of the plant under drought environment.The present invention can provide technical support for the development and application of plant growth-promoting drought resisting microbial manure, promote the growth of commodity trees.
Description
Technical field
The invention belongs to bacteriums；Its culture medium technical field more particularly to a kind of drought resisting growth promoting bacteria agent and its screening technique,
Liquid bacterial agent.
Background technique
Currently, the prior art commonly used in the trade is such that pseudomonad is widely present in soil, main species have
Pseudomonas fluorescens, pseudomonas putida, pseudomonas aeruginosa, Pseudomonas aurantica, Pseudomonas chlororaphis etc..They are due to tool
There is Soluble phosphorus, fixed nitrogen, secretion growth hormone, generate the characteristics such as thermophilic iron element, is widely used in all kinds of agricultures such as wheat, rape, corn
In the production and biological control of crop and a small amount of forest.Orange pseudomonad is administered to tomato rhizosphere by the prior art, can be significant
Promote the growth of tomato seedling；Pseudomonas chlororaphis microbial inoculum is obvious to balsam pear and cucumber fusarium axysporum control efficiency.But alleviating
The explorative research of plant drouhgt stress is few.Currently, it is dry mainly to be covered and used antitranspirant to alleviate jujube plant using film
Drought stress.Although film covering has the function of that reducing transpiration and soil moisture is lost, and in use, is easy
It hinders natural precipitation to enter ridge body, is unfavorable for the recovery of soil damage caused by a drought to a certain extent.And the film after is not easy point
Solution, can cause damages to soil environment.Alleviate arid using antitranspirant, using effect is vulnerable to vesicular structure, cuticula
The influence of the factors such as structure, the surface tension of medicament and spray pattern.Compared with film covers and uses antitranspirant, drought resisting promotees
Bacteria-promoting agent is colonized in plant root, and by secreting plant hormone, activating soil nutrient promotes plant strain growth, thus alleviate arid,
It is a kind of more environmentally-friendly effective method.But due in jujube plant body early period and the separation screening work of rhizosphere drought resisting microorganism
Work amount is larger, and practical operation needs certain microbiology profession basis, therefore so far still without using microbial bacterial agent
Report for jujube drought resisting.
In conclusion problem of the existing technology is:
(1) it is taken time and effort using film covering, and film is not easily decomposed, and is caused damages to environment.
(2) antitranspirant alleviates arid, and using effect is influenced by many factors and environmental condition.
Supplement solves the difficulty and meaning of above-mentioned technical problem: in the research for being used for jujube drought resisting using microbial bacterial agent
In, the screening of drought resisting Promoting bacteria and its preparation of liquid bacterial agent are key techniques.The present invention mainly carries out in terms of the two
Research, it is intended to the screening technique of drought resisting Promoting bacteria and its techniqueflow of liquid bacterial agent preparation are sought, it is economic and environment-friendly to realize
Drought resisting mode provides technical support.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of drought resisting growth promoting bacteria agent and its screening techniques, liquid
Microbial inoculum.
The invention is realized in this way a kind of drought resisting Promoting bacteria, the drought resisting Promoting bacteria is pseudomonad G3, deposit number
CGMCC1.16599。
Another object of the present invention is to provide a kind of drought resisting Promoting bacterias in improving jujube plant Drought resistance
Application.
Another object of the present invention is to provide a kind of drought resisting Promoting bacterias to improve answering in plant Drought resistance
With.
Another object of the present invention is to provide a kind of screening technique of drought resisting Promoting bacteria, the drought resisting growth promoting bacteria agents
Screening technique the following steps are included:
Step 1, with LB culture medium from different pedotheques separation of bacterial；
Step 2 carries out ACC deaminase activity to separated bacterial strain and drought tolerance measures, and preliminary screening goes out drought-enduring bacterial strain；
Step 3 to separated bacterial strain progress, phosphorus decomposing, fixed nitrogen, produces IAA characteristic measurement；Filter out drought-enduring growth-promoting effect compared with
Good bacterial strain；
Step 4 carries out morphology, Physiology and biochemistry, 16S rDNA molecular biological analysis to separated obtained bacterial strain and reflects
It is fixed.
Further, in the step 1, shake obtains root after the jujube plant root of acquisition is removed surface bulk soil
Border soil；It is dissolved with sterile water, rhizosphere soil suspension is made；Using dilution-plate method, in serial soil supension 10-4、10-5With
10-6In respectively take 0.1mL be coated with LB plate, 28 DEG C of culture 36h, picking single colonie, scribing line purify；Isolated bacterial strain is saved
In the inclined-plane LB, for use.
Further, in the step 2, using the method for Penrose and Glick obtained bacterium separated to step S101
Strain carries out ACC deaminase activity measurement, filters out the higher bacterial strain of ACC deaminase activity；By the higher bacterium of ACC deaminase activity
Strain is in LB culture medium, 28 DEG C, 128r/min, shaken cultivation 36h, obtains bacteria suspension；Take 1mL bacterial suspension inoculation in 0 ,-
The TSB culture medium of 0.05, -0.30,-the 0.73MPa flow of water, filtered out out of bacterial strain that isolate with ACC deaminase activity,
The preferable bacterial strain of drought tolerance.
Further, in the step 3, the isolated bacterial strain point of step 2 is connected on PKV plate, 28 DEG C of constant temperature trainings
Support 7d；According to the bacterium colony grown on plate whether there is transparent circle tentatively to be judged the phosphorus decomposing characteristic of bacterial strain, and according to molten
The size ratio of the diameter of phosphorus circle and colony diameter judges the size of bacterial strain phosphate solubilization；Using the low nitrogen culture medium inspection of Ah Xu shellfish
Survey the Characteristics of Nitrogen Fixation of bacterial strain；It is measured using the ability that the method for Loper produces growth hormone IAA to bacterial strain；
In the step 4, the molecular biological variety identification method of 16S rDNA are as follows:
1) the bacterial genomes DNA that extraction step S103 is screened；
2) using genomic DNA as template, using universal primer:
Upstream primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 '；
Downstream primer: 5 '-AAGGAGGTGATCCAGCCGCA-3 '；
Carry out pcr amplification reaction.25 μ L PCR reaction systems include each 1 μ L of 1 μ L, 10 μm of ol/L of upper and lower primer of DNA, and 2
× Taq PCR Master Mix, uses ddH2O complements to 25 μ L；Response procedures are 94 DEG C of denaturation 5min, 94 DEG C of denaturation 30s, 47
DEG C primer renaturation 1min, 72 DEG C of extension 2min, 30 circulations；Last 72 DEG C of extensions 10min；
3) PCR product is subjected to electrophoresis detection and be sequenced, the homology for analyzing the bacterial strain is compared in NCBI, is divided
Class.
Another object of the present invention is to provide a kind of drought resisting growth promoting bacteria agents that the drought resisting Promoting bacteria obtains.
Another object of the present invention is to provide a kind of drought resisting growth promoting bacteria agents to improve jujube plant Drought resistance
In application.
Another object of the present invention is to provide a kind of drought resisting growth promoting bacteria agents in improving plant Drought resistance
Using.
In conclusion advantages of the present invention and good effect are as follows: pseudomonas strains Pseudomonas koreenis G3 liquid
Body microbial inoculum can significantly improve jujube plant Drought resistance.Under identical drought stress degree, it is not inoculated with the jujube of microbial inoculum
Plant can more effectively improve activities of antioxidant enzymes, increase the content of plant tumor growth hormone, enhance the photosynthesis characteristics of plant,
To alleviate injury of the drought stress to plant, be conducive to growth of the plant under drought environment.The present invention can be plant growth-promoting
The development and application of drought resisting microbial manure provide technical support, promote the growth of commodity trees.
Detailed description of the invention
Fig. 1 is the screening technique flow chart of drought resisting growth promoting bacteria agent provided in an embodiment of the present invention.
Fig. 2 is the phylogenetic tree where Pseudomonas koreenis G3 bacterial strain provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
It is covered for the prior art using film and alleviates jujube plant drought stress using antitranspirant and taken time and effort, and
Film is not easily decomposed, and is caused damages to environment；Alleviate arid using antitranspirant, the sustainable time is short, and what need to repeatedly be sprayed asks
Topic.The present invention can provide technical support for the development and application of plant growth-promoting drought resisting microbial manure, promote the growth of commodity trees.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
Drought resisting growth promoting bacteria agent provided in an embodiment of the present invention is pseudomonad G3, and pseudomonad G3 is preserved in China Microbiological
Culture presevation administration committee common micro-organisms center, address: the institute 3 of BeiChen West Road, Chaoyang District, BeiJing City 1, Chinese science
Institute of microbiology, institute, postcode 100101, preservation date on December 27th, 2018, deposit number CGMCC1.16599.
As shown in Figure 1, the screening technique of drought resisting growth promoting bacteria agent provided in an embodiment of the present invention the following steps are included:
S101: LB culture medium separation of bacterial from different pedotheques is used；
S102: ACC deaminase activity is carried out to separated bacterial strain and drought tolerance measures, preliminary screening goes out drought-enduring bacterial strain；
S103: to separated bacterial strain progress, phosphorus decomposing, fixed nitrogen, IAA characteristic measurement is produced；The drought-enduring growth-promoting effect of further screening
The preferable bacterial strain of fruit；
S104: morphology, Physiology and biochemistry, 16S rDNA molecular biological analysis are carried out to separated obtained bacterial strain and reflected
It is fixed.
In a preferred embodiment of the invention, in step S101, for guarantee obtained strains diversity and drought tolerance, according to
The geographical distribution in the main jujube producing region in Shaanxi Province, in conjunction with different altitude height and soil moisture content, selection is differently adopted
Sample.
In a preferred embodiment of the invention, in step S101, the jujube plant root for that will acquire removes surface bulk
Gently shake obtains rhizosphere soil after soil.It is dissolved with sterile water, rhizosphere soil suspension is made.Using dilution-plate method, it is being
Column soil supension 10-4、10-5With 10-6In respectively take 0.1mL be coated with LB plate, 28 DEG C of culture 36h, picking single colonie, scribing line it is pure
Change.Isolated bacterial strain is stored in the inclined-plane LB, for use.
In a preferred embodiment of the invention, in step S102, using the method for Penrose and Glick to step S101
Separated obtained bacterial strain carries out ACC deaminase activity measurement, filters out the higher bacterial strain of ACC deaminase activity.Concretely,
Again by the higher bacterial strain of ACC deaminase activity in LB culture medium, 28 DEG C, 128r/min, shaken cultivation 36h, it is outstanding to obtain bacterium
Liquid.Take 1mL bacterial suspension inoculation in the TSB culture medium of the different flows of water (0, -0.05, -0.30, -0.73MPa) again, from step S101
It filters out in the bacterial strain that initial gross separation goes out with ACC deaminase activity, the preferable bacterial strain of drought tolerance.
In a preferred embodiment of the invention, in step S103, the isolated bacterial strain point of step S102 is connected to PKV and is put down
On plate, 28 DEG C of constant temperature incubation 7d.According to the bacterium colony grown on plate whether there is transparent circle to carry out just to the phosphorus decomposing characteristic of bacterial strain
Step judges, and the size of bacterial strain phosphate solubilization is judged according to the size ratio of the diameter of Soluble phosphorus circle and colony diameter.Using Ah
The Characteristics of Nitrogen Fixation of the low nitrogen culture medium detection bacterial strain of palpus shellfish.It is carried out using the ability that the method for Loper produces growth hormone IAA to bacterial strain
Measurement.
In a preferred embodiment of the invention, in step S104, the molecular biological variety identification method of 16S rDNA are as follows:
1) the bacterial genomes DNA that extraction step S103 is screened；
2) using genomic DNA as template, using universal primer:
Upstream primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 '；
Downstream primer: 5 '-AAGGAGGTGATCCAGCCGCA-3 '；
Carry out pcr amplification reaction.25 μ L PCR reaction systems include DNA 1 μ L, each 1 μ L of upper and lower primer (10 μm of ol/L),
2 × TaqPCRMasterMix uses ddH2O complements to 25 μ L.Response procedures are 94 DEG C of denaturation 5min, 94 DEG C of denaturation 30s, 47 DEG C
Primer renaturation 1min, 72 DEG C of extension 2min, 30 circulations；Last 72 DEG C of extensions 10min.
3) PCR product electrophoresis detection and be sequenced, the homology for analyzing the bacterial strain is compared in NCBI, to its into
Row classification.
The bacterial strain G3 colonial morphology rule that the present invention screens, neat in edge is transparent, water profit.It is raw through morphology, physiology
Change, 16S rDNA molecular biological analysis identifies that the bacterial strain is Pseudomonas koreensis.
Application effect of the invention is explained in detail combined with specific embodiments below.
Embodiment 1:
(1) the rhizosphere soil sample of isolated strains is collected in Qingjian jujube experiment station, Xibei Univ. of Agricultural & Forest Science & Technology adult jujube
Tree.
It (2) is 1 year raw grafting of jujube (Zizyphusjujuba) kind " July is fresh " for examination plant, by northwest agricultural section
Qingjian jujube experiment station, skill university provides.
(3) Xibei Univ. of Agricultural & Forest Science & Technology, test forest farm, the Weihe River is picked up from for examination soil.Sampling depth is 0-20cm, sandy loam,
Its soil physico-chemical property are as follows: organic matter 11.25gkg-1, full nitrogen 0.84gkg-1, full phosphorus 0.32g kg-1, alkali-hydrolyzable nitrogen
47.80mg·kg-1, rapid available phosphorus 3.60mgkg-1, available potassium 42.15mgkg-1.Cross 1mm sieve after sample folk song is dry, and with mistake
The river sand of 1mm sieve is with spare after 3:1 (V:V) mixing.
(4) reagents such as peptone, yeast extract, macrogol (PEG6000), sodium chloride, calcium phosphate are purchased from Chengdu section dragon
Chemical reagent factory.
(5) TIANamp Bacteria DNA kit is purchased from TIANGEN Biotech (Beijing) Co., Ltd..
(6) screening of drought resisting pseudomonad Pseudomonas koreenis G3
1. using LB culture medium separation of bacterial from different pedotheques
2. the drought-enduring growth-promoting bacterial strain of preliminary screening
1) isolated strains drought tolerance
2) isolated strains growth-promoting characteristic
3) as a result, it has been found that, G3 bacterial strain is raw in the culture medium of the different flows of water (- 0.10MPa, -0.30MPa, -0.73MPa)
Long amount is with OD600Value expression, respectively 1.04,0.82,0.50, illustrate that the bacterial strain has certain drought tolerance.
(7) identification of drought resisting pseudomonad Pseudomonas koreenis G3
1. Morphological Identification
2. Physiology and biochemistry is identified
Referring to " common bacteria system identification handbook ", every physiological and biochemical test is carried out to bacterial strain, comprising: Starch Hydrolysis,
Lecithin degradation, catalase, tyrosine hydrolysis, citrate utilization, gelatin hydrolysis, V-P test, methyl red test, salt tolerance
Test, sole carbon source utilize test.
3 bacterial strain G3 Physiology and biochemistry qualification result of table
The Physiology and biochemistry of bacterial strain G3 the results are shown in Table 3.According to " common bacteria system identification handbook ", thus it is speculated that bacterial strain G3 is false single
Born of the same parents bacterium Pseudomonas koreenis.
The major physiological biochemical character of 3 G3 bacterial strain of table
(8) molecular biology identification of 16S rDNA
1) DNA is extracted；
2) PCR reaction amplification and sequencing；
3) building of phylogenetic tree；
(9) pseudomonad strain Pseudomonas koreenis G3 liquid fermentation liquid is to jujube plant drought stress
Relieving effect
1) preparation of strain liquid fermentation liquid；
2) method of administration of liquid fermentation liquid；
3) influence of the bacterial strain to jujube plant strain growth under drought stress；
4) influence of the bacterial strain to jujube plant chlorophyll content and photosynthesis characteristics under drought stress；
5) influence of the bacterial strain to jujube plant proline content and activities of antioxidant enzymes under drought stress；
6) influence of the bacterial strain to jujube plant hormone content under drought stress.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of drought resisting Promoting bacteria, which is characterized in that the drought resisting Promoting bacteria is pseudomonad G3, deposit number
CGMCC1.16599。
2. a kind of drought resisting Promoting bacteria as described in claim 1 is improving the application in jujube plant Drought resistance.
3. a kind of drought resisting Promoting bacteria as described in claim 1 is improving the application in plant Drought resistance.
4. a kind of screening technique of drought resisting Promoting bacteria as described in claim 1, which is characterized in that the sieve of the drought resisting growth promoting bacteria agent
Choosing method the following steps are included:
Step 1, with LB culture medium from different pedotheques separation of bacterial；
Step 2 carries out ACC deaminase activity to separated bacterial strain and drought tolerance measures, and preliminary screening goes out drought-enduring bacterial strain；
Step 3 to separated bacterial strain progress, phosphorus decomposing, fixed nitrogen, produces IAA characteristic measurement；It is preferable to filter out drought-enduring growth-promoting effect
Bacterial strain；
Step 4 carries out morphology, Physiology and biochemistry, 16S rDNA molecular biological analysis to separated obtained bacterial strain and identifies.
5. the screening technique of drought resisting Promoting bacteria as claimed in claim 4, which is characterized in that in the step 1, by acquisition
Shake obtains rhizosphere soil after jujube plant root removes surface bulk soil；It is dissolved with sterile water, rhizosphere soil suspension is made；
Using dilution-plate method, in serial soil supension 10-4、10-5With 10-6In respectively take 0.1mL be coated with LB plate, 28 DEG C of culture 36h,
Picking single colonie, scribing line purifying；Isolated bacterial strain is stored in the inclined-plane LB, for use.
6. the screening technique of drought resisting Promoting bacteria as claimed in claim 4, which is characterized in that in the step 2, use
The method of Penrose and Glick obtained bacterial strain separated to step S101 carries out ACC deaminase activity measurement, filters out ACC
The higher bacterial strain of deaminase active；By the higher bacterial strain of ACC deaminase activity in LB culture medium, 28 DEG C, 128r/min, oscillation
36h is cultivated, bacteria suspension is obtained；Take 1mL bacterial suspension inoculation in 0, -0.05, -0.30, the TSB culture medium of -0.73MPa flow of water, from
It is filtered out in the bacterial strain isolated with ACC deaminase activity, the preferable bacterial strain of drought tolerance.
7. the screening technique of drought resisting Promoting bacteria as claimed in claim 4, which is characterized in that in the step 3, by step 2
Isolated bacterial strain point is connected on PKV plate, 28 DEG C of constant temperature incubation 7d；It is transparent according to whether the bacterium colony grown on plate has
Circle tentatively judges the phosphorus decomposing characteristic of bacterial strain, and judges bacterium according to the size ratio of the diameter of Soluble phosphorus circle and colony diameter
The size of strain phosphate solubilization；Using the Characteristics of Nitrogen Fixation of the low nitrogen culture medium detection bacterial strain of Ah Xu shellfish；Using the method for Loper to bacterial strain
The ability for producing growth hormone IAA is measured；
In the step 4, the molecular biological variety identification method of 16S rDNA are as follows:
1) the bacterial genomes DNA that extraction step S103 is screened；
2) using genomic DNA as template, using universal primer:
Upstream primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 '；
Downstream primer: 5 '-AAGGAGGTGATCCAGCCGCA-3 '；
Carry out pcr amplification reaction；25 μ L PCR reaction systems include 1 μ L, upper and lower primer 10umol/L each 1 μ L, 2 × Taq of DNA
PCR Master Mix, uses ddH2O complements to 25 μ L；Response procedures are 94 DEG C of denaturation 5min, 94 DEG C of denaturation 30s, 47 DEG C of primers
Renaturation 1min, 72 DEG C of extension 2min, 30 circulations；Last 72 DEG C of extensions 10min；
3) PCR product is subjected to electrophoresis detection and be sequenced, the homology for analyzing the bacterial strain is compared in NCBI, is classified.
8. a kind of drought resisting growth promoting bacteria agent for having the right that the 1 drought resisting Promoting bacteria is required to obtain.
9. a kind of drought resisting growth promoting bacteria agent as claimed in claim 8 is improving the application in jujube plant Drought resistance.
10. a kind of drought resisting growth promoting bacteria agent as claimed in claim 9 is improving the application in plant Drought resistance.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111793680A (en) * | 2020-07-30 | 2020-10-20 | 华南农业大学 | Growth-promoting strain screening method based on high-throughput sequencing and application thereof |
| CN114774300A (en) * | 2021-12-31 | 2022-07-22 | 西北农林科技大学 | Korean pseudomonas and application thereof |
| CN114934002A (en) * | 2022-06-30 | 2022-08-23 | 塔里木大学 | Novel actinomycete species and application thereof in drought resistance and growth promotion of plants |
| CN117229963A (en) * | 2023-09-21 | 2023-12-15 | 东北林业大学 | Korean pseudomonas YBZ2 for preventing and treating walnut Jiao Shezheng and application thereof |
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