CN102827794B - Pseudomonas mediterranea strain and application thereof - Google Patents
Pseudomonas mediterranea strain and application thereof Download PDFInfo
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Abstract
The invention relates to a pseudomonas mediterranea strain and application thereof. The pseudomonas mediterranea MJM-3 is preserved in the general microorganism center of the China microorganism culture preservation management committee, the preservation address is No.3 of No.1 courtyard of Beichen West road of Chaoyang district of Beijing City, the preservation date is 26th July, 2012, and the preservation number is CGMCC NO.6292. The pseudomonas mediterranea strain is used as bacterial manure for growing crops. The pseudomonas mediterranea strain has ACC (1-aminocyclopropane-1-carboxylate) deaminase activity, and can secrete auximone IAA (indole acetic acid) and siderophores. Accordingly, the concentration of ethylene in a plant under the condition of environmental stress can be reduced, growth and development of a root system of the plant are promoted, iron absorption of the plant is also promoted, and growth condition of the plant is improved. Besides, when applied to agriculturalproduction, the pseudomonas mediterranea strain can promote growth of plants, yield of crops is increased, and stress resistance of the crops is strengthened.
Description
Technical field
The present invention relates to strain Mediterranean Sea pseudomonas and an application thereof.
Background technology
Exist the saline and alkaline soil of big area and secondary salinization soil in the world.Along with the aggravation of population, grain, soil and energy contradiction, especially along with the building of some large hydraulic engineerings, the secondary salinization problem is more and more serious, therefore to the exploitation in salinification soil with to prevent and treat the secondary salinization problem very urgent.
Salting of soil is one of ecological crisis of facing of the mankind.Salt stress not only can influence growth and development of plants and output, and high salt concentration is coerced and can be caused phytomass to reduce even dead.The salt alkaline stress almost can influence each important physiological process of plant, under the condition of salt stress, plant-growth is suppressed, metabolism is affected, photosynthetic chain is destroyed in the photosynthesis, cause accumulating in a large number electronics, cause and produce a large amount of endogenous activity oxygen on plastosome, chloroplast(id) and the peroxidase precursor in the vegetable cell, active oxygen comes to harm plastosome and chloroplast(id), destroy biomacromolecules such as albumen, nucleic acid in the body, bring out the cell membrane lipid peroxidation, destroy the cell plasma balance, cause metabolic disturbance.Wherein alkaline stress has also increased high pH on the basis of salt stress, thus more serious than salt to the influence of plant, even cause plant death.
At present the method for soil remediation mainly contains the method for physics, chemistry and biology, takes physics, that chemical method is removed method is often costly, and can be unsuitable for handling large-area pollution with the generation of secondary pollution.The microbial treatment method needs specific the cultivation and technical qualification, implements and does not often reach the expection experiment effect.The researchist is by conventional means seed selection resistance kinds such as interbreeds substantially at present, and existing plant species is carried out the resistance screening, and the approach that utilizes modern biotechnology to create new resistance kind is carried out the resistance breeding work.Take exercise by seed and seedling being carried out resistance, the rational application of fertilizer, chemical regulation and other measures improve cultivation step and improve stress resistance of plant.And in the last few years, the research by the inoculation beneficial microorganism improves stress resistance of plant more and more causes people's attention.Inoculation mycorrhizal fungi (mycorrhizal fungi).Mycorhiza (mycorrhiza) is the reciprocal symbiosis body that the root system of a class fungi in the soil and higher plant is set up, and participates in the fungi that mycorhiza forms and is called mycorrhizal fungi.And some fungi can't pure culture, and the production cycle of microbial inoculum is long, and is wasteful, the inoculation technique performance difficulty can not be sowed, and can only transplant its host plant in a large number, and factor such as effect of inoculation instability under the natural condition is arranged, restricted its large-area actual applying to a certain extent.
Summary of the invention
The objective of the invention is for strain Mediterranean Sea pseudomonas and an application thereof is provided.
Strain Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 of the present invention, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on June 26th, 2012, and preserving number is CGMCCNO.6292.
A strain Mediterranean Sea pseudomonas of the present invention is used for arable farming as bacterial manure.
Mediterranean Sea of the present invention pseudomonas (Pseudomonnas mediterranea) MJM-3 is Gram-negative bacteria, this bacterial strain the ADF substratum form circular, opaque, faint yellow, projection is smooth, neat in edge, sticking bacterium colony (as shown in Figure 1).
According to " common bacteria system identification handbook " with " the outstanding bacterium handbook of uncle is carried out detection and the evaluation of gramstaining, catalase, methyl red, acetyl methyl carbinol, starch hydrolysis, indoles, the experiment of Citrate trianion effect Physiology and biochemistry to isolated bacterial strain MJM-9.The result shows that Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 is Gram-negative bacteria, show the catalase positive, starch hydrolysis feminine gender, the indoles experiment shows as the positive, and methyl red, acetyl methyl carbinol, Citrate trianion effect experiment show as feminine gender.
The present invention comprises following beneficial effect:
Mediterranean Sea of the present invention pseudomonas (Pseudomonas mediterranea) MJM-3 has the acc deaminase activity, produces plant growth hormones IAA, synthesizes and has a liking for the iron element; Can in salt alkaline stress environment, promote plant nutrition absorption, coordinate plant growth effectively and improve plant anti-adversity ability under adverse environmental factor.Its application in arable farming can be promoted fertilizer efficiency, improves output.
Mediterranean Sea of the present invention pseudomonas (Pseudomonas mediterranea) MJM-3 can suppress or alleviate Plant diseases plant is produced detrimentally affect by modes such as indirect secretion siderophores, and reduces ethylene levels and directly stimulate and coordinate plant growth by generation plant hormone, enzymolysis.1-amino-cyclopropane-1-carboxylic acid (1-aminocyclopropane-1-carboxylate that Mediterranean Sea of the present invention pseudomonas (Pseudomonas mediterranea) MJM-3 contains, ACC) the direct precursor ACC of biosynthesizing ethene in the PGPR of the desaminase energy hydrolyzing plant body, produce hydroxybutyric acid and ammonia, stop the release of ethene.When this bacterium is attached to the seed epidermis, it can hydrolysis ACC, reduce seed a large amount of plant growth inhibitor and plant senescence agent-ethylene concentrations that produce under adverse environmental factor, promote elongation and the plant-growth of root.
Description of drawings
Fig. 1 is the colonial morphology photo that Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 grows at the ADF substratum;
Fig. 2 represents the synthetic content histogram of Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 IAA under different concns tryptophane condition;
Fig. 3 represents that Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 has a liking for the dull and stereotyped effect photo of the plain detection of iron;
Fig. 4 represent Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 under saline and alkaline stress conditions to the short fruit photo that comes into force of clover; Wherein, the CK fruit photo that comes into force for the control group clover is short, MJM-3 is the short fruit photo that comes into force of Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 clover;
Fig. 5 represent Mediterranean Sea pseudomonas (Pseudomonas meditrranea) MJM-3 under saline and alkaline stress conditions to the short fruit photo that comes into force of wheat; Wherein, the CK fruit photo that comes into force for the control group wheat is short; MJM-3 is the Mediterranean Sea pseudomonas
The short fruit photo that comes into force of (Pseudomonas mediterranea) MJM-3 wheat.
Embodiment
Embodiment one: strain Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 of present embodiment, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on June 26th, 2012, and preserving number is CGMCC NO.6292.
Present embodiment Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 is Gram-negative bacteria, this bacterial strain the ADF substratum form circular, opaque, faint yellow, projection is smooth, neat in edge, sticking bacterium colony (as shown in Figure 1).
According to " common bacteria system identification handbook " with " the outstanding bacterium handbook of uncle is carried out detection and the evaluation of gramstaining, catalase, methyl red, acetyl methyl carbinol, starch hydrolysis, indoles, the experiment of Citrate trianion effect Physiology and biochemistry to isolated bacterial strain MJM-9.The result shows that Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 is Gram-negative bacteria, show the catalase positive, starch hydrolysis feminine gender, the indoles experiment shows as the positive, and methyl red, acetyl methyl carbinol, Citrate trianion effect experiment show as feminine gender.
The Mediterranean Sea pseudomonas of present embodiment (Pseudomonas mediterranea) MJM-3 has the acc deaminase activity, produces plant growth hormones IAA, synthesizes and has a liking for the iron element; Can in salt alkaline stress environment, promote plant nutrition absorption, coordinate plant growth effectively and improve plant anti-adversity ability under adverse environmental factor.Its application in arable farming can be promoted fertilizer efficiency, improves output.
The Mediterranean Sea pseudomonas of present embodiment (Pseudomonas mediterranea) MJM-3 can suppress or alleviate Plant diseases plant is produced detrimentally affect by modes such as indirect secretion siderophores, and reduces ethylene levels and directly stimulate and coordinate plant growth by generation plant hormone, enzymolysis.1-amino-cyclopropane-1-carboxylic acid (1-aminocyclopropane-1-carboxylate that Mediterranean Sea of the present invention pseudomonas (Pseudomonas mediterranea) MJM-3 contains, ACC) the direct precursor ACC of biosynthesizing ethene in the PGPR of the desaminase energy hydrolyzing plant body, produce hydroxybutyric acid and ammonia, stop the release of ethene.When this bacterium is attached to the seed epidermis, it can hydrolysis ACC, reduce seed a large amount of plant growth inhibitor and plant senescence agent-ethylene concentrations that produce under adverse environmental factor, promote elongation and the plant-growth of root.
Embodiment two: a strain Mediterranean Sea pseudomonas of present embodiment is used for arable farming as bacterial manure.
The Mediterranean Sea pseudomonas of present embodiment (Pseudomonas mediterranea) MJM-3 has the acc deaminase activity, produces plant growth hormones IAA, synthesizes and has a liking for the iron element; Can in salt alkaline stress environment, promote plant nutrition absorption, coordinate plant growth effectively and improve plant anti-adversity ability under adverse environmental factor.Its application in arable farming can be promoted fertilizer efficiency, improves output.
(Pseudomonas mediterranea MJM-3 can suppress or alleviate Plant diseases plant is produced detrimentally affect the Mediterranean Sea pseudomonas of present embodiment by modes such as indirect secretion siderophores, and reduces ethylene levels and directly stimulate and coordinate plant growth by generation plant hormone, enzymolysis.1-amino-cyclopropane-1-carboxylic acid (1-aminocyclopropane-1-carboxylate that Mediterranean Sea of the present invention pseudomonas (Pseudomonas mediterranea) MJM-3 contains, ACC) the direct precursor ACC of biosynthesizing ethene in the PGPR of the desaminase energy hydrolyzing plant body, produce hydroxybutyric acid and ammonia, stop the release of ethene.When this bacterium is attached to the seed epidermis, it can hydrolysis ACC, reduce seed a large amount of plant growth inhibitor and plant senescence agent-ethylene concentrations that produce under adverse environmental factor, promote elongation and the plant-growth of root.
By following verification experimental verification beneficial effect of the present invention:
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Test 1, bacterial strain screening
A strain Mediterranean Sea pseudomonas of the present invention obtains by the following steps screening:
(1) gets rhizosphere farmland, region, the Heilungkiang soil of 1g alfalfa planting, put into the PAF substratum of 50mL, shaking culture 24h under 28 ℃ of temperature;
(2) get PAF nutrient solution after 1mL step (1) is cultivated, put into the new PAF substratum of 50mL, under 28 ℃ of temperature, continue shaking culture 24h;
(3) get PAF nutrient solution after 1mL step (2) is cultivated, put into 50mL DF salt nutrient solution A, shaking culture 24h under 28 ℃ of temperature;
(4) get DF salt nutrient solution after 1mL step (3) is cultivated, put into the 50mLADF nutrient solution, shaking culture 48h under 28 ℃ of temperature;
(5) get 1mL step (4) after cultivating nutrient solution, coat on the ADF nutrient agar, shaking culture 72h under 28 ℃ of temperature gets single bacterium colony of growing on the substratum and carries out purifying;
(6) bacterium behind step (5) purifying is numbered, picking list bacterium colony, be transferred on the ADF substratum preserve standby;
Wherein, the PAF substratum described in step (1) and (2) is: by the peptone of 10g, the casein hydrolysate of 10g, the MgSO of 1.5g
4, 1.5g K
2HPO
4Form with the glycerine of 10mL;
DF salt nutrient solution described in the step (3) is: the KH that takes by weighing 4.0g
2PO
4, 6.0g Na
2HPO
4, 0.2g MgSO
47H
2The FeSO of O, 0.1g
47H
2The citric acid of the glucose of O, 2.0g, the gluconic acid of 2.0g, the 2.0g, (NH of 2.0g
4)
2SO
4, 0.01mg H
3BO
3, 0.0112mg MnSO
4, 0.1246mg ZnSO
4, 0.0782mg CuSO
4MoO with 0.01mg
3After dissolving in the above-mentioned DF salt nutrient solution composition adding distilled water, adjust pH value to 7.5, be settled to 1000mL;
ADF nutrient solution described in step (4) and (6) is: the KH that takes by weighing 4.0g
2PO
4, 6.0g Na
2HPO
4, 0.2g MgSO
47H
2The FeSO of O, 0.1g
47H
2The citric acid of the glucose of O, 2.0g, the gluconic acid of 2.0g, 2.0g, the H of 0.01mg
3BO
3, 0.0112mg MnSO
4, 0.1246mg ZnSO
4, 0.0782mg CuSO
4MoO with 0.01mg
3After adding dissolved in distilled water in the said components, adjust pH value to 7.5, be settled to 1000mL, adding 1-amino-cyclopropane-1-carboxylic acid (ACC) behind the high-temperature sterilization, making its final concentration is 3.0mmol/L.
ADF nutrient agar in the step (5) is: add 15g agar to the step (4) of 1L and the ADF nutrient solution of (6).
Test 2, identification of strains:
Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 that screening is obtained carries out the Physiology and biochemistry evaluation, and Molecular Identification.
2.1 Physiology and biochemistry is identified
Mediterranean Sea of the present invention pseudomonas (Pseudomonas mediterranea) MJM-3 is Gram-negative bacteria, this bacterial strain the ADF substratum form circular, opaque, faint yellow, projection is smooth, neat in edge, sticking bacterium colony (as shown in Figure 1).
According to " common bacteria system identification handbook " with " the outstanding bacterium handbook of uncle is carried out detection and the evaluation of gramstaining, catalase, methyl red, acetyl methyl carbinol, starch hydrolysis, indoles, the experiment of Citrate trianion effect Physiology and biochemistry to isolated bacterial strain MJM-9.The result shows that Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 is Gram-negative bacteria, show the catalase positive, starch hydrolysis feminine gender, the indoles experiment shows as the positive, and methyl red, acetyl methyl carbinol, Citrate trianion effect experiment show as feminine gender.
2.2 Molecular Identification
Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 that above-mentioned experiment sieving is obtained carries out Molecular Identification and carries out according to following steps: adopt alkaline lysis to carry out the short DNA extraction of giving birth to bacterial strain of clover rhizosphere of acc deaminase to the above-mentioned Mediterranean Sea pseudomonas that obtains (Pseudomonas mediterranea) MJM-3 purifying bacterial strain, utilize primers F 8 and R1541, DNA with extraction is masterplate, carry out pcr amplification, entrust the order-checking of Beijing Invitrogen life Science and Technology Ltd. then; Wherein, pcr amplification primer sequence is as follows:
F8：5'-AGAGTTTGATCCTGGCTCAG-3'，
F1541：5'-AAGGAGGTGATCCAGCCGCA-3'，
The pcr amplification condition is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 90s, 30 circulations; 72 ℃ are extended 10min eventually.
The 16S rDNA length of Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 is that 830bp(is shown in sequence table Seq ID No:1), its sequence is committed to GenBank, to determine the race relation of bacterial strain.Homology analysis is the result show, the conserved regions similarity that the 16S rDNA of Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 and Pseudomonas fluorescens belong to the 16S rDNA gene order of (Pseudomonas mediterranea) reaches 99%.Comprehensive physiological and biochemical property and colony morphology characteristic, determine that Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 belongs to Pseudomonas fluorescens and belongs to (Pseudomonas mediterranea), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation on June 26th, 2012, preserving number is CGMCC NO.6292.
Above-mentioned PCR primer is bought spontaneous worker's biotechnology Shanghai company limited.
The acc deaminase determination of activity of test 3, Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3
3.1 substratum preparation:
The TSB substratum is: the glucose of NaCl, the 2.5g of the Tryptones of 17g, the soya peptone of 3g, 5g, the K of 2.5g
2HPO
4Be dissolved in the 1000mL distilled water, adjust pH value to 7.5;
ADF substratum: KH
2PO
44.0g, Na
2HPO
46.0g, MgSO
47H
2O 0.2g, FeSO
47H
2The citric acid of the glucose of O 0.1g, 2.0g, the gluconic acid of 2.0g, 2.0g, the H of 0.01mg
3BO
3, 0.0112mg MnSO
4, 0.1246mg ZnSO
4, 0.0782mg CuSO
4MoO with 0.01mg
3After adding dissolved in distilled water in the said components, adjust pH value to 7.5, be settled to 1000mL, adding 1-amino-cyclopropane-1-carboxylic acid (ACC) behind the high-temperature sterilization, making its final concentration is 3.0mmol/L;
The DF substratum is: the KH of 4.0g
2PO
4, 6.0g Na
2HPO
4, 0.2g MgSO
47H
2The FeSO of O, 0.1g
47H
2The citric acid of the glucose of O, 2.0g, the gluconic acid of 2.0g, 2.0g, the H of 0.01mg
3BO
3, 0.0112mg MnSO
4, 0.1246mg ZnSO
4, 0.0782mg CuSO
4MoO with 0.01mg
3After adding dissolved in distilled water in the said components, adjust pH value to 7.5, be settled to 1000mL.
3.2 enzyme activity determination:
The concrete steps of enzyme activity determination are as follows:
(1) picking in 50mL TSB substratum, is that 28 ℃, rotating speed be the condition of 180rpm under cultivate 12h in temperature through the single bacterium colony of the Mediterranean Sea pseudomonas after the pure culture (Pseudomonas meaditerranea) MJM-3;
(2) Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 after step (1) is cultivated is that 4 ℃, rotating speed are centrifugal 10min under the condition of 8000g in temperature, collects thalline, does not contain (NH with 50mL
4)
2SO
4DF substratum washing thalline 3 times, then with whole thalline by being inoculated in the ADF substratum, be that 28 ℃, rotating speed are to cultivate 48h under the condition of 180rpm to induce it to produce acc deaminase in temperature;
(3) the bacterium liquid after step (2) is cultivated is that 4 ℃, rotating speed are centrifugal 10min under the condition of 8000g in temperature, collect thalline, it is that 0.1mol/L, pH are in 7.6 the Tris-HCl solution that somatic cells is suspended in 1mL concentration, is transferred to then in the centrifuge tube of 1.5mL, stand-by;
(4) be centrifugal 5min under the condition of 1600g at rotating speed with the centrifuge tube in the step (3), remove supernatant, collect thalline, it is that 0.1mol/L, pH are in 8.5 the Tris-HCl solution that thalline is resuspended in 600 μ L concentration, add 30 μ L toluene again, whirlpool concussion 30s gets bacterium liquid; Get 200 μ L bacterium liquid then and do enzyme activity determination (residue bacterium liquid is done protein determination);
Simultaneously, blank is set: be that 0.1mol/L, pH add 30 μ L toluene among 8.5 the Tris-HCl in 600 μ L concentration;
(5) get the bacterium liquid of the 200 μ L that step (4) obtains, after adding 20 μ L concentration and be the ACC solution concussion 30s of 0.5mol/L, be to cultivate 15min under 30 ℃ the condition in temperature, the HCl solution that adds 1mL concentration then and be 0.56mol/L mixes, at the centrifugal 5min of room temperature (25 ℃), collect supernatant liquor, stand-by; Shake centrifugal 5min in room temperature, collect supernatant, stand-by;
(6) get 1mL step (5) supernatant of collecting and the HCl solution that 800 μ L concentration are 0.56mol/L and mix, add the mixing of 300 μ L 2,4 dinitrophenyl hydrazines again, get mixing solutions; After cultivating 30min under 30 ℃ of temperature, to wherein adding the NaOH solution termination reaction that 2mL concentration is 2mol/L, be 0,0.1,0.3,0.7,1 and 2 μ molL with 1mL concentration then with mixing solutions
-1α-batanone acid be reference liquid, be that the 540nm place carries out enzyme activity determination at light absorption value, record the ACC typical curve, be equation with y=3.7396x-0.0385, carry out the calculating of ACC resultant quantity;
(7) protein content adopts the Xylene Brilliant Cyanine G method to measure;
Wherein, the activity of acc deaminase shows that with the scale that every milligram of albumen in surveying enzyme system per hour forms α-batanone acid unit is μ mol α-KA (mgPrh)
-1Enzyme assay is all deducted the blank back of sample contrast and is calculated, and repeats 3 times.
The result shows that Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 has higher acc deaminase activity, up to 29.78 μ mol α-KA (mgPrh)
-1
The synthetic assay of the IAA of test 4, Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3:
(1) getting Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 in the DF substratum, is that 28 ℃, rotating speed are to cultivate 2d under the condition of 180rpm in temperature;
(2) getting in the step (1) Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 nutrient solution 100 μ L is transferred to respectively and contains different concns (0,50,100,200 and 500 μ gL-TrpmL
-1) in the DF substratum of tryptophane (L-Trp), be that 28 ℃, rotating speed are to cultivate 2d under the condition of 180rpm in temperature then;
(3) get middle Mediterranean Sea pseudomonas (Pseudomonas mediterranea) the MJM-3 nutrient solution of step (2) and carry out OD
600PH-value determination pH is centrifugal 10min under the condition of 8000rpm then at room temperature, rotating speed with all the other nutrient solutions, gets the Salkowsk reagent that 500 μ L supernatant liquors add 2mL, behind the incubated at room temperature 20min, at 535nm place survey light absorption value;
Simultaneously, use and adopt the method for step (3) to carry out same treatment to the DF substratum that does not contain Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 to organize in contrast;
(4) with different concns (0.01,0.05,0.25 and 0.5mgmL
-1) IAA solution be reference liquid, return to zero with control group, survey light absorption value at the 535nm place, record typical curve, with the y=0.1729x-0.0098 equation, carry out the calculating of ACC resultant quantity;
Wherein, the DF substratum is: the KH that takes by weighing 4.0g
2PO
4, 6.0g Na
2HPO
4, 0.2g MgSO
47H
2The FeSO of O, 0.1g
47H
2The citric acid of the glucose of O, 2.0g, the gluconic acid of 2.0g, the 2.0g, (NH of 2.0g
4)
2SO
4, 0.01mg H
3BO
3, 0.0112mg MnSO
4, 0.1246mg ZnSO
4, 0.0782mg CuSO
4MoO with 0.01mg
3After adding dissolved in distilled water in the said components, adjust pH value to 7.5, be settled to 1000mL.
The result as shown in Figure 2, as shown in Figure 2, Mediterranean Sea pseudomonas (Pseudomonds mediterranea) MJM-3 can produce indolylacetic acid, and synthesis of indole acetic acid amount increases along with the increase of L-Trp concentration, is 0,50,100,200,500 μ g L-TrpmL in concentration specifically
-1In the indolylacetic acid amount be respectively 0.55,7.49,12.07,26.65,36.24 μ g (mLOD
600)
-1
The iron element of having a liking for of test 5, Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 bacterial strain synthesizes assay
5.1 have a liking for the plain synthetic content qualitative detection of iron: with reference to the method for Schwyn and Neilands, Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 is connected on chromium pure CAS (the chrome azurol S) plate culture medium difficult to understand, under the condition of 28 ℃ of temperature, cultivate 48 ~ 72h, observe the colour-change of periphery of bacterial colonies, have orange chromosphere to produce, then proof has the plain generation of the iron of having a liking for;
Wherein, chromium pure CAS plate culture medium preparation method difficult to understand is as follows:
The blue dye liquor of CAS:
Solution A: the CAS of 0.06g is dissolved in the 50mL deionized water, and adding 10mL concentration again is 1mmolL
-1FeCl
3Solution (contains 10mmolL
-1HCl);
Solution B: (Hexadecyl trimethyl ammonium bromide HDTMA) is dissolved in the 40mL deionized water with the cetyl trimethylammonium bromide of 0.073g;
Solution A is slowly joined in the solution B along walls of beaker, rock mixing solution A and solution B gently, obtain the blue dye liquor of CAS, standby;
Solution a: with the KH of 15g
2PO
4, the NaCl of 25g and the NH of 50g
4Cl is dissolved in the 500mL deionized water;
Solution b: the quality percentage composition is 20% glucose solution: 20g glucose is dissolved in the 100mL deionized water;
Solution c: the NaOH of 25g is dissolved in the 150mL deionized water;
Acid casein hydrolyzate solution: 3g acid casein hydrolyzate is dissolved in the 27mL deionized water, adopts the membrane filtration sterilization of 0.22 μ m then;
Being formulated as follows of chromium pure CAS plate culture medium difficult to understand: measure 100mL solution a and mix with the 750mL deionized water, to the PIPES that wherein adds 32.24g (piperazine-1,4-two ethyl sulfonic acids); Add the special-purpose agar of microbial culture (buying from hundred special biotech firms) 15g, under 120 ℃ of temperature, 15min sterilizes then; Treat that solution is cooled to 50 ℃ and adds 30mL acid casein hydrolyzate solutions, the solution b of 10mL slowly adds the blue dye liquor of CAS of 100mL again, and abundant mixing falls dull and stereotypedly, namely gets the CAS plate culture medium; Wherein, when adding PIPES (piperazine-1,4-two ethyl sulfonic acids), because PIPES (piperazine-1,4-two ethyl sulfonic acids) less than insoluble below 5, answers the regulator solution potential of hydrogen slowly adding PIPES at pH, and constantly stir, finally with solution c solution is transferred to pH=6.8.
5.2 have a liking for the plain synthetic content quantitative analysis of iron: single colony inoculation that can produce orange chromosphere in above-mentioned " having a liking for the plain synthetic content qualitative detection of iron " in the MKB substratum, is put into shaking table then, is that 28 ℃, rotating speed are 180rmin in temperature
-1Condition under cultivate 48h, bacterium liquid after will cultivating then is that 28 ℃, rotating speed are centrifugal 10min under the condition of 1000rpm in temperature, get supernatant liquor, volume ratio with 1:1 adds CAS detection liquid in supernatant liquor, abundant mixing is behind the static 1h, survey light absorption value (A) at the 630nm place, after detecting the liquid equal-volume and mixes with deionized water and CAS, surveying light absorption value (Ar) at 630nm place is to contrast, and has a liking for the relative content of iron element in the A/Ar representative sample; This value is more low, shows that to have a liking for the iron cellulose content more high;
Wherein, the MKB substratum is: take by weighing the acidolysis casein of 5g, the glycerine of 15mL, the K of 2.5g
2HPO
4, the MgSO of 2.5g
47H
2O; Said components joined in the distilled water dissolve, regulate pH to 7.2, be settled to 1000mL, then under 121 ℃ of temperature, sterilization 20min;
CAS detects being prepared as follows of liquid:
1,0.182g CTAB is dissolved in the 50mL deionized water;
2, with 0.0054g FeCl6H
2O is dissolved in the HCl aqueous solution that 2mL concentration is 10mM;
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, and mixes with the solution that 6mL step 1 obtains again, obtains mixed solution;
5, with the PIPES of 4.307g with 20 ~ 30mL deionized water dissolving, add the dense HCl of 6.25mL, transferring pH is 5.6;
6, the solution that obtains of the solution that Overall Steps 4 is obtained and Overall Steps 5 mixes, and is settled to molten 100mL with deionized water, namely gets CAS and detects liquid.
The result as shown in Figure 3, periphery of bacterial colonies has orange chromosphere to produce, and proves that pseudomonas (Pseudomona sp.) MJM-9 can synthesize to have a liking for the iron element.
The relative content A/Ar that has a liking for the iron element in the sample is 1.15, shows that Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 has certain synthetic ability of having a liking for the iron element.
Test 6, short the giving birth to of the potted plant clover of Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 are tested
6.1 soil sample collection
The used soil sample of the present invention is all taken near the saline-alkali soil oil recovery factory, Daqing, Heilongjiang Province.5 sample prescriptions are set in each zone during collected specimens, and each sample prescription area is 1 * 1m
2, collect topsoil (0 ~ 20cm), after the mixing with soil, in the clean freshness protection package of the previously prepd of packing into, rapidly soil sample is taken back testing laboratory, soil is through pulverizing, mixing, the preservation of sieving after air-dry.Physico-chemical property for examination soil sees Table 1.
Table 1 is for the examination soil physico-chemical property
6.2 the short test method of giving birth to of potted plant clover
(1) Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-9 that preserves is activated at the ADF solid medium;
(2) the single bacterium colony after the activation is that 28 ℃, rotating speed are shaking culture 24h under the condition of 180rpm in temperature in the LB substratum in the picking step (1), regulates bacterial concentration to 1 * 10 with sterilized water
10Cfu/mL is suspension A, and is standby;
(3) use 10% (V/V) clorox to carry out surface sterilization 10min in advance alfalfa seed (dragon is herded 803 clovers, buy from the academy of agricultural sciences, Heilongjiang Province), again with the sterilized water washing more than 3 times, put into the suspension A that step (2) obtains then and soak 1h;
Simultaneously with alfalfa seed through sterilized water immersion treatment 1h, (CK) in contrast;
(4) step (3) seeds treated evenly is seeded in the flowerpot of same specification, every basin 20 strains repeat incubated at room temperature 5 times;
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 potted plant effect as shown in Figure 4.
The biomass of the clover growth-promoting functions of table 2 Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 is measured
As shown in Table 2, under the effect of Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3, the percentage of germination of alfalfa seed has improved nearly 30%, the plant height of wheat plant has increased nearly 25%, root length has increased nearly 22%, over-ground part plant fresh weight has increased by 31% respectively, dry weight has increased closely 52%, and that underground part root system fresh weight has increased is nearly 32%, dry weight has increased nearly 1.4 times, shortly comes into force fruit significantly.
As shown in Figure 4, connecing the potted plant growing way that the potted plant plant of bacterium do not connect bacterium will get well, and plant is more healthy and strong, shortly comes into force fruit obviously.
Test 8, short the giving birth to of the potted plant wheat of Mediterranean Sea pseudomonas (Pseudomonas meaditerranea) MJM-3 are tested
This tests used soil sample test 7.
The short test method of giving birth to of the potted plant wheat of this test is as follows:
(1) Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 that preserves is activated at the ADF solid medium;
(2) the single bacterium colony of picking Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 is 28 ℃ in temperature in the LB substratum, and rotating speed is shaking culture 24h under the 180rpm condition, and making bacterial concentration with the sterilized water adjusting is 1 * 10
10Cfu/mL is suspension A, and is standby;
(3) wheat seed (China spring, Heilongjiang Institute of Agricultural Sciences) is used in advance behind 10% (V/V) clorox surface sterilization 10min and, be soaked among the suspension A and handle 1h more than 3 times with the sterilized water washing;
Simultaneously with wheat seed through sterilized water immersion treatment 1h, (CK) in contrast;
(4) seeds treated in the step (3) evenly is seeded in the flowerpot of same specification, every basin 10 strains repeat 5 times, and room temperature (25 ℃) is cultivated; Regularly the suspension A for preparing is regulated with sterilized water and make bacterial concentration to 1 * 10
9Cfu/mL namely gets suspension B, gets 50mL suspension B and evenly waters around the stem and leaf of Wheat root;
Control group (CK) is treated to: seeds treated in the step (3) evenly is seeded in the flowerpot of same specification, and every basin 10 strains repeat 5 times, and room temperature (25 ℃) is cultivated; With with the pouring of water gaging such as suspension B;
Measure germinating energy in (5) one weeks, measure plant height, root length, ground plant dry weight, underground weight of root system after one month;
ADF substratum in this test is: the KH that takes by weighing 4.0g
2PO
4, 6.0g Na
2HPO
4, 0.2g MgSO
47H
2The FeSO of O, 0.1g
47H
2The citric acid of the glucose of O, 2.0g, the gluconic acid of 2.0g, 2.0g, the H of 0.01mg
3BO
3, 0.0112mg MnSO
4, 0.1246mg ZnSO
4, 0.0782mg CuSO
4MoO with 0.01mg
3Behind said components adding dissolved in distilled water, adjust pH value to 7.5, be settled to 1000mL, add 1-amino-cyclopropane-1-carboxylic acid (ACC) behind the high-temperature sterilization, making 1-amino-cyclopropane-1-carboxylic acid (ACC) final concentration is 3.0mmol/L;
The LB substratum is: take by weighing the yeast powder of 5g, and the peptone of 10g, the NaCl of 10g is settled to 1L, regulates pH value to 7.2.
Statistics is as shown in table 3, and potted plant effect as shown in Figure 5.
The biomass of the wheat growth-promoting functions of table 3 Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 is measured
As shown in Table 3, under the effect of Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3, the percentage of germination of wheat seed has improved nearly 23%, the plant height of wheat plant has increased nearly 11%, root length has increased nearly 28%, that over-ground part plant fresh weight has increased respectively is nearly 21%, dry weight has increased closely 29%, and underground part root system fresh weight has increased nearly 4 times, dry weight and increased closely 81%, shortly comes into force fruit significantly.
As shown in Figure 5, connecing the potted plant growing way that the potted plant plant of bacterium do not connect bacterium will get well, and plant is more healthy and strong, shortly comes into force fruit obviously.
Hence one can see that, and the wheat of Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 has significant growth-promoting functions.The wheat that mensuration is handled through Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3 compared with the control, percentage of germination, plant height, root length, fresh weight, dry weight all are significantly increased.
Claims (2)
1. a strain Mediterranean Sea pseudomonas, it is characterized in that it is Mediterranean Sea pseudomonas (Pseudomonas mediterranea) MJM-3, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on June 26th, 2012, and preserving number is CGMCC NO.6292.
2. the purposes of a strain Mediterranean Sea pseudomonas as claimed in claim 1 is characterized in that described Mediterranean Sea pseudomonas is used for arable farming as bacterial manure.
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| BR112017016161A2 (en) * | 2015-01-29 | 2019-11-12 | Miranda Valencia Jose Luis | granulated solid fertilizer formulated with mineral clays, siderophores chelating agents, secondary nutrients and micronutrients |
| CN104818231A (en) * | 2015-05-07 | 2015-08-05 | 安徽农业大学 | Antagonistic and growth-promoting pseudomonas producing ACC deaminase and application thereof |
| CN111304275A (en) * | 2020-02-14 | 2020-06-19 | 济宁利马菌业股份有限公司 | Culture medium for detecting black rot of flammulina velutipes and preparation method thereof |
| CN113073067A (en) * | 2021-04-17 | 2021-07-06 | 扬州大学 | Pseudomonas and application thereof |
| CN114774300B (en) * | 2021-12-31 | 2023-06-30 | 西北农林科技大学 | Pseudomonas koraiensis and application thereof |
| CN114933999B (en) * | 2022-06-21 | 2023-04-21 | 南京林业大学 | Pseudomonas and application thereof |
| PL442029A1 (en) * | 2022-08-17 | 2024-02-19 | Uniwersytet Warszawski | Composition containing siderophores produced by the Pseudomonas H12B strain, use of siderophores produced by this strain, method of preparing a composition containing siderophores and substrates for producing such composition |
| CN116024118B (en) * | 2022-08-22 | 2023-06-23 | 哈尔滨师范大学 | Saline-alkali tolerant microbial agent SYM-6 and application thereof |
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