CN102796684B - Alfalfa growthpromoting rhizobacteria MJM-11 and application thereof - Google Patents
Alfalfa growthpromoting rhizobacteria MJM-11 and application thereof Download PDFInfo
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- 241000589157 Rhizobiales Species 0.000 title abstract description 7
- 240000004658 Medicago sativa Species 0.000 title 1
- 241001217893 Enterobacter ludwigii Species 0.000 claims abstract description 58
- 241000196324 Embryophyta Species 0.000 claims abstract description 39
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an alfalfa growth-promoting rhizobacteria MJM-11 and application thereof. An Enterobacter ludwigii strain MJM-11 has a preservation number of CGMCC No.6295. The Enterobacter ludwigii strain MJM-11 can be applied in at least one of the following four ways: 1, preparation of an ACC (1-aminocyclo propane-1-carboxylate) ammonialyase; 2, preparation of indoleacetic acid; 3, preparation of siderophores; and 4, promotion of the growth of plants under the saline-alkali stress. Experiments prove that the alfalfa growth-promoting rhizobacteria MJM-11 which is obtained through separation can be used for synthesizing the ACC ammonialyase and the IAA (Indole Acetic Acid) siderophores and is capable of effectively promoting the alimentation of plants in a saline-alkali stress environment, regulating the growth of plants and enhancing the stress resistance capacity of plants in adverse conditions.
Description
Technical field
The present invention relates to microbial technology field, relate in particular to the short living bacterium MJM-11 of a strain rhizosphere of alfalfa and application thereof.
Background technology
Clover is perennial leguminous plants, is rich in amino acid, multivitamin and the mineral substance of various necessity, and the main grass seeds of producing as livestock industry, extensively planted all over the world, its cultivation history has had more than 2000 year, and outstanding advantages shows on feeding and is: grass yield is high, utilize a year limit for length, reproducibility is strong, anti-cradling, herbaceous stem is good, palatability is strong, nutritious, the volume increase of fertilizing the soil, the effect such as conserve water and soil, be a kind of Multifunctional pasture, have the good reputation of " King of Pasture ".
Clover is the leguminous forage that distributes in the world the comparison salt tolerant the widest, but in its salt tolerance raising of still needing in the actual cultivation in heavy salinized ground.China approximately has saltings approximately to have 0.12 hundred million hectare, mainly is distributed in northeast, North China, Northwest inland area and North of Yangtze River coastal area.The improvement in saltings is a global difficult problem, and saltings has also become the biggest obstacle factor of restriction China's improvement of the ecological environment and agricultural sustainable development simultaneously.Therefore understand the mechanism of plant salt damage and the strategy of reply salt stress thereof, significant for further quickening improvement of the ecological environment and agricultural sustainable development.
Plant rhizosphere is urged endophytic bacteria (plant growth-promoting rhizobacteria, be called for short PGPR) but is referred to free living at soil or grow nonparasitically upon another plant in a class Promoting plant growth of plant rhizosphere, prevent and treat disease, increase the useful mushroom of crop yield.The inoculation plant growth-promoting rhizobacteria, can make invalid nutrition effectuation in soil, prevention and control corps diseases, reduce the use of agricultural chemicals and chemical fertilizer, be solve soil, water source and food contamination at all by way of, be generally considered a kind of method of environmental friendliness, cost-effective raising crop yield.Wherein containing ACC(1-amino-cyclopropane-1-carboxylic acid 1-aminocyclopropane-1-carboxylate, be called for short ACC) PGPR of desaminase has been subject to various countries scholars' extensive concern.Acc deaminase is the short biomass of a kind of external source of effective reduction stress ethylene content, and this enzyme can slow down ethene accumulation in plant materials under adverse environmental factor, and the growth of Promoting plant growth, especially plant root significantly improves the resistance of farm crop and increases output.
Summary of the invention
An object of the present invention is to provide strain Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11.
Lu Shi enterobacteria provided by the invention (Enterobacter ludwigii) bacterial strain MJM-11, its deposit number is CGMCC No.6295.
Above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11 is following 1)-4) application at least a is also the scope of protection of the invention:
1) prepare acc deaminase;
2) prepare indolylacetic acid;
3) the iron element is had a liking in preparation;
4) Promoting plant growth; Described Promoting plant growth specifically carries out under the Saline Alkali Stress condition.
Another object of the present invention is to provide a kind of method for preparing product.
Method provided by the invention, comprise the steps: the Lu Shi enterobacteria of fermenting above-mentioned (Enterobacter ludwigii) bacterial strain MJM-11, namely obtains product; Described product is acc deaminase, indolylacetic acid or has a liking for the iron element.
In aforesaid method, if when described product is acc deaminase, the substratum that described fermentation is adopted is TSB substratum and ADF substratum; Wherein, TSB substratum: Tryptones 17g, soya peptone 3g, NaCl5g, glucose 2.5g, K
2HPO
42.5g be dissolved in 1000ml distilled water, adjust the pH value to pH=7.5.
ADF substratum: KH
2PO
44.0g, Na
2HPO
46.0g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.1g, glucose 2.0g, gluconic acid 2.0g, citric acid 2.0g, micro-0.1mL, add in distilled water and to dissolve after, adjust the pH value to pH=7.5, be settled to 1000ml, add 1-amino-cyclopropane-1-carboxylic acid (ACC) after high-temperature sterilization, making its final concentration is that 3.0mmol/L(can first prepare mother liquor 0.5mol/L); Wherein, trace element (100mL): H
3BO
310mg, MnSO
411.2mg, ZnSO
4124.6mg, CuSO
478.2mg, MoO
310mg.
When if described product is indolylacetic acid, the substratum that described fermentation is adopted is DF substratum or the DF substratum that contains tryptophane; DF substratum: KH wherein
2PO
44.0g, Na
2HPO
46.0g, MgSO
47H
2O 0.2g, FeSO
47H
2O0.1g, glucose 2.0g, gluconic acid 2.0g, citric acid 2.0g, (NH
4)
2SO
42.0g, micro-0.1mL, add in distilled water and to dissolve after, adjust the pH value to pH=7.5, be settled to 1000ml.The trace element preparation is the same.
If described product is for having a liking for iron when element, the substratum that described fermentation is adopted is the MKB substratum, wherein MKB substratum (1L): acidolysis casein 5g, glycerine 15mL, K
2HPO
42.5g, MgSO
47H
2O 2.5g, pH 7.2.121 ℃, 20min is sterilizing.
The above-mentioned method for preparing acc deaminase comprises the steps:
1) cultivate above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11 in described TSB substratum, collect thalline, obtain cultivating bacterium 1;
2) described cultivation bacterium 1 is inoculated into inducing culture in described ADF substratum, collects the inducing culture product, namely obtain acc deaminase.
In the above-mentioned method for preparing acc deaminase, in step 1), described culture condition is 28 ℃, and 180rpm cultivates 12h;
Step 2) in, described inducing culture is 28 ℃, and 180rpm cultivates 48h.
The above-mentioned method for preparing acc deaminase also comprises the steps: first with described thalline, 0.1mol/L Tris-HCl(pH=8.5), toluene and 0.5mol/L ACC mix, and cultivates, and obtains the toluene culture; Add 0.56mol/L HCl to mix described toluene culture again, centrifugal collection supernatant liquor, obtain acc deaminase.
Specifically comprise the steps: in the above-mentioned method for preparing acc deaminase
Above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) the bacterial strain MJM-11 of picking in 50ml TSB substratum 28 ℃, after 180rpm cultivates 12h, 4 ℃ of centrifugal collection thalline, with 50ml DF substratum washing three times, thalline is transferred in the ADF substratum 28 ℃, 180rpm inducing culture 48h, collect the inducing culture product;
With the centrifugal collection thalline of inducing culture product, then thalline is suspended in 1ml 0.1mol/L Tris-HCl(pH=7.6) in be transferred in the centrifuge tube of 1.5ml, the centrifugal supernatant that goes of 1600g, collect thalline; Resuspended thalline is in 600 μ l0.1mol/L Tris-HCl(pH=8.5 again) in, add 30 μ l toluene whirlpool concussion 30s, obtain the toluene cell, after adding the 20 of short duration concussions of μ l 0.5mol/L ACC in the toluene cell, cultivate 15min for 30 ℃, add afterwards 1ml 0.56mol/L HCl to mix the centrifugal 5min of concussion room temperature (25 ℃), collect supernatant liquor, obtain acc deaminase.
The above-mentioned method for preparing indolylacetic acid comprises the steps: to cultivate above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11 in described DF substratum or the described DF substratum that contains tryptophane, collect tunning, namely obtain indolylacetic acid.
In the above-mentioned method for preparing indolylacetic acid, in the described DF substratum that contains tryptophane, the final concentration of tryptophane is 0-500 μ g/mL, and is not 0.
Culture condition in the above-mentioned method for preparing indolylacetic acid is 28 ℃, and 180rpm cultivated 2 days;
The above-mentioned method for preparing indolylacetic acid also comprises the steps: described tunning centrifugal, collects supernatant liquor, obtains indolylacetic acid.
The above-mentioned method for preparing indolylacetic acid specifically comprises the steps:
The single bacterium colony MJM-11 of above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) in the DF liquid nutrient medium 28 ℃, 180rpm cultivates 2d, is transferred to and adds different concns (0,50,100,200,500 μ gmL
-1) in the DF substratum of tryptophane, 28 ℃, 180rpm cultivates 2d, collects tunning, and is then that tunning 8000rpm is centrifugal, collects supernatant liquor, obtains IAA.
The method that the iron element is had a liking in above-mentioned preparation comprises the steps: to cultivate above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11 in the MKB substratum, collects tunning, namely obtains having a liking for the iron element.
The culture condition that the method for iron element is had a liking in above-mentioned preparation is 28 ℃, and 180rpm cultivates 48h;
Above-mentioned preparation is had a liking for and is also specifically comprised the steps: described tunning centrifugally in the method for iron element, collects supernatant liquor, obtains having a liking for the iron element.
The method that the iron element is had a liking in above-mentioned preparation specifically comprises the steps:
Above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11 is inoculated in the MKB substratum, and 28 ℃ of shaking tables are cultivated (180rmin
-1) 48h; Collect tunning, tunning centrifuging and taking supernatant liquor, obtain having a liking for the iron element.
The 3rd purpose of the present invention is to provide a kind of method of Promoting plant growth.
Method provided by the invention, before comprising the steps: sowing, be dipped into plant seed in above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11 bacteria suspension; After planting, with the described plant of above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11 bacteria suspension pouring; Realize Promoting plant growth.
In aforesaid method, described plant-growth is carried out under the Saline Alkali Stress condition; What use in embodiments of the invention is the saline-alkali soil shown in table 1 index.
Described plant is specially monocotyledons or dicotyledons;
Described monocotyledons further is specially wheat or clover.
Above-mentioned Promoting plant growth is embodied in and improves percentage of germination, plant height, root length, ground plant fresh weight, ground plant fresh weight dry weight, underground root system fresh weight and/or underground weight of root system.
In aforesaid method, the root of bacteria suspension irrigating plant after planting.
Bacteria suspension (bacterial suspension A) concentration before above-mentioned sowing is 1 * 10
10Cfu/ml; Bacteria suspension after planting (bacteria suspension B) concentration is 1 * 10
9Cfu/ml.
Above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11 bacterial suspension A is prepared as follows: Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11 is inoculated in the LB substratum, in 28 ℃, shaking culture 24h under the 180rpm condition, collect bacterium liquid, regulating bacterial concentration with sterilized water is 1 * 10
10Cfu/ml, obtain bacterial suspension A.
Above-mentioned Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11 bacteria suspension B is prepared as follows: transferring to concentration with sterilized water adjusting bacterial suspension A is 1 * 10
9Cfu/ml, obtain bacteria suspension B.
The method of above-mentioned Promoting plant growth specifically comprises the steps:
Before sowing, plant seed is used in advance after 10% (V/V) clorox surface sterilization 10min and, is soaked in bacterial suspension A and processes 1h more than three times with the sterilized water washing; After planting, bacteria suspension B is evenly watered around the plantling root.
Above-mentioned bacterial strains MJM-11(Enterobacter ludwigii) be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 26th, 2012 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6295, and Classification And Nomenclature is Lu Shi enterobacteria (Enterobacter ludwigii).
Of the present invention experimental results show that, the present invention's separation obtains the short living bacterium MJM-11 of a strain rhizosphere of alfalfa, it can synthesize acc deaminase, IAA has a liking for the iron element, and can effectively promote plant nutrition absorption, coordinate plant growth and improve plant anti-adversity ability under adverse environmental factor in the Saline Alkali Stress environment, this bacterial strain can be applicable to arable farming, can promote fertilizer efficiency, improve output.
Description of drawings
Fig. 1 is bacterial strain MJM-11(Enterobacter ludwigii) colonial morphology of growing on the ADF substratum
Fig. 2 is bacterial strain MJM-11(Enterobacter ludwigii) the synthetic content of IAA under different tryptophane concentration
Fig. 3 is bacterial strain MJM-11(Enterobacter ludwigii) have a liking for the dull and stereotyped design sketch of iron element qualitative detection
Fig. 4 is bacterial strain MJM-11(Enterobacter ludwigii) under Saline Alkali Stress, clover is urged to give birth to design sketch
Fig. 5 is bacterial strain MJM-11(Enterobacter ludwigii) under Saline Alkali Stress, wheat is urged to give birth to design sketch
Embodiment
The experimental technique that uses in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The screening and identification of embodiment 1, the short living bacterium of rhizosphere of alfalfa
1, screening
(1) get the 1g Rhizosphere sampling in 50mL PAF substratum, 28 ℃ of shaking culture.Contain peptone in this PAF substratum, casein hydrolysate, MgSO
4, K
2HPO
4, glycerine.
(2) get PAF nutrient solution after 1mL concussion, in 50mL PAF substratum, 28 ℃ of shaking culture.
(3) get the PAF nutrient solution that 1mL obtains, in 50mL DF salt nutrient solution, 28 ℃ of shaking culture.
This DF salt nutrient solution contains KH
2PO
4, Na
2HPO
4, MgSO
4, FeSO
4, glucose, gluconic acid, citric acid, (NH
4)
2SO
4, H
3BO
3, MnSO
4, ZnSO
4, CuSO
4, MoO
3
(4) get the DF salt nutrient solution that 1mL obtains, in 50mL, do not contain (NH
4)
2SO
4, but contain in the above-mentioned DF salt nutrient solution of 3mM1-amino-cyclopropane-1-carboxylic acid (ACC) 28 ℃ of shaking culture.
(5) get the nutrient solution that 1mL obtains, coat on the DF salt nutrient agar that contains 3mM ACC 28 ℃ of cultivations.Get single bacterium colony purifying of growing on substratum.
(6) bacterium after purifying is numbered, picking list bacterium colony, be transferred on the ADF substratum and save backup, called after MJM-11.
2, colony characteristics and thalli morphology
This bacterial strain MJM-11 ADF substratum (formula sees below) upper form circular, opaque, faint yellow, projection is smooth, neat in edge, sticking bacterium colony.As shown in Figure 1.
3, physiological and biochemical property
According to " common bacteria system identification handbook is with " the outstanding bacterium handbook of uncle is carried out detection and the evaluation of Physiology and biochemistry to isolated MJM-11.MJM-11(Enterobacter ludwigii) show the catalase positive, Starch Hydrolysis is positive, and the indoles experiment is positive.
4,16S rDNA sequential analysis
The 16S rDNA gene order of MJM-11, see shown in nucleotides sequence list 1.The 16S rDNA gene order of MJM-11 is carried out the BLAST compare of analysis with number Genbank according to the sequence in storehouse, and result shows: the similarity of the 16S rDNA gene order of MJM-11 and Lu Shi enterobacteria (Enterobacter ludwigii) reaches 94%.
Above-mentioned bacterial strains MJM-11(Enterobacter ludwigii) be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 26th, 2012 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6295, and Classification And Nomenclature is Lu Shi enterobacteria (Enterobacter ludwigii).
Embodiment 2, bacterial strain MJM-11(Enterobacter ludwigii) application in preparing acc deaminase
1, prepare acc deaminase
TSB substratum: Tryptones 17g, soya peptone 3g, NaCl5g, glucose 2.5g, K
2HPO
42.5g be dissolved in 1000ml distilled water, adjust the pH value to pH=7.5.
ADF substratum: KH
2PO
44.0g, Na
2HPO
46.0g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.1g, glucose 2.0g, gluconic acid 2.0g, citric acid 2.0g, micro-0.1mL, add in distilled water and to dissolve after, adjust the pH value to pH=7.5, be settled to 1000ml, add 1-amino-cyclopropane-1-carboxylic acid (ACC) after high-temperature sterilization, making its final concentration is that 3.0mmol/L(can first prepare mother liquor 0.5mol/L); Wherein, trace element (100mL): H
3BO
310mg, MnSO
411.2mg, ZnSO
4124.6mg, CuSO
478.2mg, MoO
310mg.
Without (NH
4)
2SO
4The DF substratum: KH
2PO
44.0g, Na
2HPO
46.0g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.1g, glucose 2.0g, gluconic acid 2.0g, citric acid 2.0g, micro-0.1mL, add in distilled water and to dissolve after, adjust the pH value to pH=7.5, be settled to 1000ml.The trace element preparation is the same.
The MJM-11(Enterobacter ludwigii that picking is obtained by embodiment 1) the single bacterium colony of CGMCC NO.6295 is in 50ml TSB substratum 28 ℃, after 180rpm cultivates 12h, 4 ℃, the centrifugal collection thalline of 8000g, centrifugal 10min, with 50ml without (NH
4)
2SO
4DF substratum washing three times, thalline is transferred in the ADF substratum 28 ℃, 180rpm inducing culture 48h, collect the inducing culture product.4 ℃ of inducing culture products, 8000g, centrifugal 10min are collected thalline.Thalline is suspended in 1ml 0.1mol/L Tris-HCl(pH=7.6) in be transferred in the centrifuge tube of 1.5ml, 1600g is centrifugal, and 5min removes supernatant, collects thalline.
Resuspended thalline is in 600 μ l 0.1mol/L Tris-HCl(pH=8.5) in, add 30 μ l toluene whirlpool concussion 30s, get the toluene cell of its 200 μ l and do enzyme activity determination (all the other do protein determination); Add the 20 of short duration concussions of μ l 0.5mol/L ACC in the toluene cell after, cultivate 15min for 30 ℃, add afterwards 1ml 0.56mol/L HCl to mix the centrifugal 5min of concussion room temperature (25 ℃), collect supernatant liquor, obtain acc deaminase.Blank is 600 μ l0.1mol/L Tris-HCl(pH=8.5) in add 30 μ l toluene, after adding again the 20 of short duration concussions of μ l 0.5mol/L ACC, cultivate 15min for 30 ℃, add afterwards 1ml 0.56mol/L HCl to mix the centrifugal 5min of concussion room temperature (25 ℃), collect supernatant liquor (contrast).
2, acc deaminase determination of activity
Get supernatant liquor 1ml and mix with 800 μ l 0.56mol/L HCl, then add 300 μ l 2,4 dinitrophenyl hydrazines to mix, after 30 ℃ of mixing solutionss are cultivated 30min, add wherein 2ml 2mol/L NaOH to make its termination reaction.Record light absorption value in the 540nm place.Take 1mL concentration as 0,0.1,0.3,0.7,1 and 2 μ molL
-1α-batanone acid be reference liquid, add 800 μ l 0.56mol/L HCl to mix, add again 300 μ l 2, the 4-dinitrophenylhydrazine mixes, after 30 ℃ of mixing solutionss are cultivated 30min, add wherein 2ml 2mol/L NaOH to make its termination reaction, survey light absorption value under the 540nm wavelength, measure ACC typical curve y=3.7396x-0.0385.Blank take supernatant liquor (contrast) as contrast.
Determining the protein quantity adopts the Xylene Brilliant Cyanine G method to measure 311.34 μ g/ml supernatant liquors.
The activity of acc deaminase per hour forms α-batanone acid with every milligram of albumen in surveying enzyme system, and (scale of α-KA) shows, unit is μ mol α-KA (mgPrh)
-1Blank rear calculating of sample contrast all deducted in enzyme assay, repeats 3 times.
Result shows, MJM-11(Enterobacter ludwigii) acc deaminase that produces of bacterial strain has higher activity, up to 27.64 μ mol α-KA (mgPrh)
-1
Embodiment 3, bacterial strain MJM-11(Enterobacter ludwigii) application in preparation IAA is synthetic
1, preparation IAA
DF substratum: KH
2PO
44.0g, Na
2HPO
46.0g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.1g, glucose 2.0g, gluconic acid 2.0g, citric acid 2.0g, (NH
4)
2SO
42.0g, micro-0.1mL, add in distilled water and to dissolve after, adjust the pH value to pH=7.5, be settled to 1000ml.The trace element preparation is the same.
Strains tested MJM-11(Enterobacter ludwigii) in the DF substratum 28 ℃, 180rpm cultivated 2 days, then was transferred to and adds different concns (0,50,100,200,500 μ gL-TrpmL
-1) in the fresh DF substratum of tryptophane (L-Trp), 28 ℃, 180rpm cultivates 2d, collects tunning.Sampling records the OD of bacterium liquid
600Value, then, with all the other tunnings (bacterium liquid) room temperatures (25 ℃) lower 8000rpm, 10min, collect supernatant liquor, obtains IAA.
2, detect IAA content
Get its 500 μ l supernatant liquor and add 2ml Salkowsk reagent, room temperature (25 ℃) records light absorption value in the 535nm place after cultivating 20min.Return to zero in contrast with the blank substratum same treatment of DF.With different concns (0.01,0.05,0.25,0.5mgmL
-1) the IAA reference liquid, same treatment records typical curve y=0.1729x-0.0098, calculates.
Result as shown in Figure 2, bacterial strain MJM-11(Enterobacter ludwigii) can produce indolylacetic acid, and synthesis of indole acetic acid amount increases along with the increase of L-Trp concentration, in concentration, is specifically 0,50,100,200,500 μ gL-TrpmL
-1In the indolylacetic acid amount be respectively 3.75,5.40,6.88,10.67,17.04 μ g (mlOD
600)
-1
Embodiment 4, bacterial strain MJM-11(Enterobacter ludwigii) have a liking for application in the iron element in preparation
1, have a liking for the synthetic qualitative detection of iron element
The difficult to understand pure CAS plate culture medium preparation of chromium:
The blue dye liquor of CAS:
Solution A: 0.06g CAS is dissolved in the 50mL deionized water, then adds 10mL 1mmolL
-1FeCl
3Solution (contains 10mmolL
-1HCl);
Solution B: the cetyl trimethylammonium bromide of 0.073g (Hexadecyl trimethyl ammonium bromide, HDTMA) is dissolved in the 40mL deionized water;
Solution A walls of beaker slowly join in solution B, rock gently and mix solution A and solution B, obtain the blue dye liquor of CAS.
Solution a:15g KH
2PO
4, 25gNaCl, 50gNH
4Cl is dissolved in the 500ml deionized water;
20% glucose solution: 20g glucose is dissolved in the 100ml deionized water;
Solution c:25gNaOH is dissolved in the 150ml deionized water;
The acid casein hydrolyzate solution: 3g acid casein hydrolyzate is dissolved in the 27ml deionized water, the using filter film filtration sterilization;
Measure 100ml solution a and mix with the 750ml deionized water, add wherein 32.24gPIPES (piperazine-Isosorbide-5-Nitrae-two ethyl sulfonic acid; PIPES, less than insoluble below 5, answers the regulator solution potential of hydrogen slowly adding PIPES at pH, and constantly stirs, and finally with solution c, solution is transferred to pH=6.8), add microbial culture special use agar 15g, 120 ℃, 15min are sterilizing.Treat that solution is cooled to 50 ℃ of left and right and adds 30ml acid casein hydrolyzate solution, the glucose solution of 10ml 20%, more slowly add the blue dye liquor of 100ml, and fully mix, be down flat plate.
Method according to Schwyn and Neilands, to separate the bacterial strain MJM-11(Enterobacter ludwigii of preservation) be connected on chromium pure CAS (chrome azurol S) substratum difficult to understand, cultivate 72h for 28 ℃, observe the colour-change of periphery of bacterial colonies, result as shown in Figure 3, have orange chromosphere to produce, this bacterial strain can produce and have a liking for the iron element.
2, have a liking for the synthetic quantitative analysis of iron element
1) the iron element is had a liking in preparation
MKB substratum (1L): acidolysis casein 5g, glycerine 15mL, K
2HPO
42.5g, MgSO
47H
2O 2.5g, pH 7.2.121 ℃, 20min is sterilizing.
With bacterial strain MJM-11(Enterobacter ludwigii) be inoculated in the MKB substratum, 28 ℃ of shaking tables are cultivated (180rmin
-1) 48h, collect tunning (bacterium liquid); Tunning 1000rpm, 10min are centrifugal, get supernatant liquor, obtain having a liking for the iron element.
2) detect and have a liking for the iron element
CAS detects the preparation of liquid
1,0.182g CTAB is dissolved in the 50ml deionized water.
2, with 0.0054g FeCl6H
2O is dissolved in the 20ml 10mM HCl aqueous solution.
3, the 0.0605g chrome azurol S is dissolved in the 50ml deionized water.
4, the solution that 1.5ml step 2 is obtained mixes with the solution that 7.5ml step 3 obtains, then with the solution that 6ml step 1 obtains, mixes, and obtains mixed solution.
5, with 4.307g PIPES 20-30ml deionized water dissolving, add the dense HCl of 6.25ml, transferring pH is 5.6.
6, the solution that the solution that Overall Steps 4 is obtained and Overall Steps 5 obtain mixes, and with deionized water, is settled to molten 100ml.
Volume ratio with 1:1 adds CAS to detect liquid in supernatant liquor, fully mix.After static 1h, the 630nm place surveys light absorption value (A), and deionized water mixes with CAS detection liquid equal-volume the Ar that records and is contrast, has a liking for the relative content of iron element in the A/Ar representative sample.This value is lower, shows that to have a liking for the iron cellulose content higher.
MJM-11(Enterobacter ludwigii as a result) A/Ar is 1.11; Show MJM-11(Enterobacterludwigii) can synthesize and have a liking for the iron element.
Embodiment 5, bacterial strain MJM-11(Enterobacter ludwigii) under Saline Alkali Stress to the potted plant experiment of plant growth-promoting
One, bacterial strain MJM-11(Enterobacter ludwigii) under Saline Alkali Stress, clover is urged the potted plant experiment of giving birth to
The present invention's soil sample used is all taken near saline-alkali soil oil recovery factory, Daqing, Heilongjiang Province.5 sample prescriptions are set during collected specimens in each zone, sample area is 1 * 1m
2, collect topsoil (0 ~ 20cm), after mixing with soil, in the clean freshness protection package of the previously prepd of packing into, rapidly soil sample is taken back laboratory, soil, through pulverizing, mixes, the preservation of sieving after air-dry.Supply the physico-chemical property of examination soil in Table 1.
Table 1 is for the examination soil physico-chemical property
The MJM-11(Enterobacter ludwigii that (1) will be obtained by embodiment 1) activate on the ADF solid medium.
(2) picking list bacterium colony is in the LB substratum, and in 28 ℃, shaking culture 24h under the 180rpm condition, be 1 * 10 with sterilized water accent bacterial concentration
10Cfu/ml (suspension A), standby.
(3) alfalfa seed (dragon is herded 803 clovers) is used in advance after 10% (V/V) clorox surface sterilization 10min with the sterilized water washing more than three times.Soak 1h in suspension A, through sterilized water immersion treatment 1h is contrast (CK).
(4) seed and the seed after control treatment after suspension A processing evenly are seeded in the flowerpot of same specification, every basin 20 strains, repeat incubated at room temperature 5 times.Seed after suspension A is processed is regularly got 50ml bacteria suspension B and is evenly watered around the stem and leaf of Wheat root; Bacteria suspension B transfers bacterial concentration to 1 * 109cfu/ml (bacteria suspension B) for the bacterial suspension A that will prepare with sterilized water.Contrast use waits the water gaging pouring.
Measure germinating energy in (5) one weeks, measure plant height, root length, ground plant dry weight, underground weight of root system after 40 days.Statistics is as shown in table 2, and pot test effect as shown in Figure 4.
Table 2 bacterium MJM-11(Enterobacter ludwigii)
Biomass estimation to the clover growth-promoting functions
As seen from table, at plant growth-promoting rhizobacteria MJM-11(Enterobacter ludwigii) effect under, the percentage of germination of alfalfa seed has improved nearly 16%, the plant height of wheat plant has increased nearly 18%, root length has increased nearly 20%, that over-ground part plant fresh weight has increased respectively is nearly 29%, dry weight has increased closely 52%, and that underground part root system fresh weight has increased is nearly 34%, dry weight has increased nearly 1 times, and comprehensive various indexs are as seen short comes into force fruit significantly.
Two, bacterial strain MJM-11(Enterobacter ludwigii) under Saline Alkali Stress, wheat is urged the potted plant experiment of giving birth to
This experiment soil sample is with above-mentioned one.
The Lu Shi enterobacteria MJM-11(Enterobacter ludwigii that will be obtained by embodiment 1) activate on the ADF solid medium, picking MJM-11(Enterobacter ludwigii) single bacterium colony is in the LB substratum, in 28 ℃, shaking culture 24h under the 180rpm condition, regulating bacterial concentration with sterilized water is 1 * 10
10Cfu/ml (bacterial suspension A), standby.
Wash three times with sterilized water after wheat seed (China spring) is used 10% (V/V) clorox surface sterilization 10min in advance, be soaked in bacterial suspension A and process 1h, through sterilized water immersion treatment 1h is contrast (CK).Seed and the seed after control treatment after suspension A is processed evenly are seeded in the flowerpot of same specification.
Seed after suspension A is processed is regularly got 50ml bacteria suspension B and is evenly watered around the stem and leaf of Wheat root; Bacteria suspension B transfers bacterial concentration to 1 * 10 for the bacterial suspension A that will prepare
9Cfu/ml (bacteria suspension B).Contrast use waits the water gaging pouring.Every basin 10 strains, repeat 5 times, and room temperature (25 ℃) is cultivated.Measure germinating energy in one week, measure plant height, root length, ground plant dry weight, underground weight of root system after one month.
Mensuration is through MJM-11(Enterobacter ludwigii) wheat processed of bacterium compared with the control, percentage of germination, plant height, root length, fresh weight, dry weight all are significantly increased, statistics is as shown in table 2, pot test effect is as shown in Figure 5.
Table 2 is bacterial strain MJM-11(Enterobacter ludwigii)
Biomass estimation to the wheat growth-promoting functions
As seen from table, at plant growth-promoting rhizobacteria MJM-11(Enterobacter ludwigii) effect under, the percentage of germination of wheat seed has improved nearly 5.6%, the plant height of wheat plant has increased nearly 17%, root length has increased nearly 31%, that over-ground part plant fresh weight has increased respectively is nearly 19%, dry weight has increased closely 26%, and underground part root system fresh weight has increased nearly 1.1 times, dry weight and increased closely 45%, shortly comes into force fruit significantly.
Claims (9)
1. Lu Shi enterobacteria (Enterobacter ludwigii) bacterial strain MJM-11, its deposit number is CGMCC No.6295.
2. Lu Shi enterobacteria claimed in claim 1 (Enterobacter ludwigii) bacterial strain MJM-11 is following 1)-4) application at least a:
1) prepare acc deaminase;
2) prepare indolylacetic acid;
3) the iron element is had a liking in preparation;
4) Promoting plant growth under Saline Alkali Stress; Described plant is wheat or clover.
3. method for preparing product, Lu Shi enterobacteria claimed in claim 1 (Enterobacter ludwigii) the bacterial strain MJM-11 that comprises the steps: to ferment, namely obtain product; Described product is acc deaminase, indolylacetic acid or has a liking for the iron element;
When if described product is acc deaminase, the substratum that described fermentation is adopted is TSB substratum and ADF substratum;
When if described product is indolylacetic acid, the substratum that described fermentation is adopted is DF substratum or the DF substratum that contains tryptophane;
If described product is for having a liking for iron when element, the substratum that described fermentation is adopted is the MKB substratum.
4. method according to claim 3 is characterized in that:
The described method for preparing acc deaminase comprises the steps:
1) cultivate Lu Shi enterobacteria claimed in claim 1 (Enterobacter ludwigii) bacterial strain MJM-11 in described TSB substratum, collect thalline, obtain cultivating bacterium 1;
2) described cultivation bacterium 1 is inoculated into inducing culture in described ADF substratum, collects the inducing culture product, namely obtain acc deaminase.
5. method according to claim 3 is characterized in that:
The described method for preparing indolylacetic acid comprises the steps: to cultivate Lu Shi enterobacteria claimed in claim 1 (Enterobacter ludwigii) bacterial strain MJM-11 in described DF substratum or the described DF substratum that contains tryptophane, collect tunning, namely obtain indolylacetic acid.
6. method according to claim 5 is characterized in that:
In the described DF substratum that contains tryptophane, the final concentration of tryptophane is 0-500 μ g/mL, and is not 0.
7. method according to claim 3 is characterized in that:
The method that the iron element is had a liking in described preparation comprises the steps: to cultivate Lu Shi enterobacteria claimed in claim 1 (Enterobacter ludwigii) bacterial strain MJM-11 in the MKB substratum, collects tunning, namely obtains having a liking for the iron element.
8. the method for a Promoting plant growth, before comprising the steps: sowing, be dipped into plant seed in Lu Shi enterobacteria claimed in claim 1 (Enterobacter ludwigii) bacterial strain MJM-11 bacteria suspension; After planting, with the described plant of Lu Shi enterobacteria claimed in claim 1 (Enterobacter ludwigii) bacterial strain MJM-11 bacteria suspension pouring; Realize Promoting plant growth; Described plant is wheat or clover.
9. method according to claim 8 is characterized in that:
Described plant-growth is carried out under the Saline Alkali Stress condition.
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| CN104651286B (en) * | 2015-03-18 | 2017-11-07 | 商丘师范学院 | A kind of soybean nodulation endophyte DEnt1302 and its preparation method and application, microbial inoculum and preparation method thereof |
| CN104805045B (en) * | 2015-05-05 | 2017-12-19 | 中国科学院烟台海岸带研究所 | Plant growth-promoting rhizobacteria and its application |
| CN104818235A (en) * | 2015-05-18 | 2015-08-05 | 湖北工业大学 | Enterobacter ludwigii and application thereof |
| CN105838750A (en) * | 2016-03-29 | 2016-08-10 | 四川省兰月科技有限公司 | Application of enterobacteria LY6 in fermentation production of indoleacetic acid and application of fermenting liquid in crop rooting and growth promotion |
| CN109576177B (en) * | 2018-12-12 | 2022-07-12 | 新疆农业科学院土壤肥料与农业节水研究所(新疆维吾尔自治区新型肥料研究中心) | Chinese micro-rod strain SM8 and application thereof in salt tolerance and growth promotion |
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| CN110257277A (en) * | 2019-05-22 | 2019-09-20 | 哈尔滨师范大学 | A kind of composite bacteria agent ZLM-11 and its application |
| CN112111431B (en) * | 2020-09-27 | 2022-05-10 | 广西大学 | Plant growth promoting strain XN-K13 and application thereof |
| CN116333928B (en) * | 2023-03-06 | 2024-04-05 | 东北农业大学 | Separation and application of suaeda salsa rhizosphere growth-promoting bacteria |
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2012
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