CN102747018B - Bacillus megaterium and application thereof - Google Patents
Bacillus megaterium and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus megaterium and an application thereof, and belongs to the field of microorganisms. A plant growth promoting bacterium (bacillus megaterium JX15) is collected in the China General Microbiological Culture Collection Center on Dec.20, 2011, the class name is bacillus megaterium and the collection number is CGMCC No.5622. The bacillus megaterium has the beneficial effects that a strain can produce indoleacetic acid with high yield, can be used for nitrogen fixation and can grow by using sparingly-soluble phosphates as phosphorous sources, so as to improve the utilization rates of fertilizers, promote plant root development and absorption of the fertilizers, and increases the contents of soil mineral nitrogen and available phosphorus; the strain has a good effect on promoting growth of peanuts; the high-yield indoleacetic acid promotes the growth and the development of the peanuts; and the nitrogen and phosphate fertilizer utilization rates of the peanuts are relatively high due to the increasing of the contents of the soil mineral nitrogen and the available phosphorus.
Description
Technical field
The invention belongs to agriculture microorganism field, relate to a kind of bacillus megaterium and application thereof.
Background technology
Moisture soil is that fluvial deposit is influenced by ground water movement and farming activity and the soil that forms, gains the name because the Evening Tide phenomenon is arranged.In China, be distributed in the Yellow River more, the river plain in downstream and on the south in the plains region and the Yangtze valley in Jiangsu, Anhui, river, lake plain and the area, delta in downstream.Moisture soil range of distribution physical features is smooth, and soil layer is deep, and hydrothermal resources is abundanter, and it is wide to make kind of property, is the main upland soil of China, abounds with grain and cotton.But the Yellow River and Huai He River sea plain of moisture soil distribution area maximum, drought and waterlogging happens occasionally, and saline and alkaline harm is still arranged, and soil nutrient is low or lack in addition, and in most of the genus, low productive soil, crop yield is low and unstable.Must strengthen reasonable utilization and the improvement of moisture soil.
Many microorganisms can be dissolved indissoluble attitude phosphorus in soil, promote plant to the absorption of phosphorus, increase crop yield and improve crop quality, with phosphate solubilizing microorganism as bio-feritlizer, not only can improve the utilising efficiency of phosphorus in the soil, the fertile volume increase of joint, and can improve Soil structure, improve soil organic matter content.Plant growth-promoting bacteria (Plant Growth Promoting Bacteria, be called for short PGPB) is defined as the free living that is conducive to plant-growth under certain condition on the bacterium of soil, rhizosphere, root table, phyllosphere.These bacteriums can fixed nitrogen, molten phosphorus, molten iron, and produce plant hormone, as growth hormone, Plant hormones regulators,gibberellins, phytokinin and ethene.In addition, they can also improve the resistance of plant, comprise arid, high salt, heavy metallic poison and agricultural chemicals.But present most of plant growth-promoting bacteria also can effectively be applied to the bacterial classification that promotes plant-growth and have the molten phosphorus effect of better fixed nitrogen for taming into, may not adapt to the edatope of moisture soil.Therefore from moisture soil, separate obtaining plant growth-promoting bacteria and crop formation syntaxial system, utilize biological restoration to improve moisture soil, become the focus of current research.
Summary of the invention
The purpose of this invention is to provide a kind of bacillus megaterium.
Another object of the present invention provides the application of this bacillus megaterium.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of plant growth-promoting bacteria JX15, on December 20th, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, classification bacillus megaterium (Bacillus megaterium) by name, preserving number CGMCC No.5622.
JX15(CGMCC No.5622), be Gram-positive, sporiferous irregular shaft-like.Bacterium colony is less to be white, protuberance, neat in edge, and smooth surface is moistening, opaque (Fig. 1).Strict aerobic, chemoheterotrophy.Optimum growth temperature is 30 ℃.The catalase positive, M.R and VP test are negative, the starch hydrolysis positive, gelatin liquefaction positive, the nitrate reduction positive, Citrate trianion utilizes positive.Secretion IAA ability is strong, reaches 27.55 μ gmL
-1, nitrogenase activity reaches 19.8nmol C
2H
4/ hml has nitrogen fixing capacity preferably, grows for the phosphorus source with the insoluble phosphate, and is translated into soluble phosphate.
The major nitrogen source of using when plant growth-promoting bacteria of the present invention is cultivated includes but not limited to peptone, yeast powder, saltpetre, ammonium nitrate, ammonium sulfate, urea, L-Ala; The main carbon source of using includes but not limited to sucrose, wood sugar, N.F,USP MANNITOL, maltose, lactose, fructose, glucose; The inorganic component that uses includes but not limited to Repone K, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, dipotassium hydrogen phosphate, tricalcium phosphate, Calcium dichloride dihydrate, bitter salt, seven water and ferrous sulfate.Plant growth-promoting bacteria fermentation of the present invention can be carried out under the environment of pH5 ~ 9 at 28 ~ 32 ℃.
Described plant growth-promoting bacteria JX15(CGMCC No.5622) application in promoting plant-growth.
Described plant optimization peanut.
The application of described plant growth-promoting bacteria JX15 on peanut cultivation.
Described plant growth-promoting bacteria can also can utilize insoluble phosphate to grow for the phosphorus source by the high yield indolylacetic acid.
The ability of plant growth-promoting bacteria secretion indolylacetic acid of the present invention (IAA) is strong, reaches 27.55 μ gmL
-1Indolylacetic acid is a kind of of plant hormone, can promote the growth of root.Produce the bacterial classification of indolylacetic acid, often be attached to root system of plant or leaf surface, produce IAA and a small amount of GA when utilizing plant metabolism to produce secretory product
3Influence physiological process and the metamorphosis of plant Deng plant hormone.Show as the elongation of direct promotion root, thus increased with soil in the chance that contacts of nutritive substance; Can improve the content of plant materials Endogenous IAA; Inducing plant defence expression of gene, it is disease-resistant to improve plant materials, resistance such as drought resisting.As prioritization scheme of the present invention, the fermentation of short the livings bacterium JX15 of described rhizosphere time is carried out in pH7 ~ 8, produces IAA under this environment and measures the highest.
As further optimization of the present invention, the carbon source that described plant growth-promoting bacteria JX15 adopts is N.F,USP MANNITOL, and the nitrogenous source of employing is yeast powder or peptone or both combinations.The substratum that utilizes above-mentioned carbon source and nitrogenous source to make, the amount that the plant growth-promoting bacteria JX15 that cultivates produces IAA is the highest.
Plant growth-promoting bacteria JX15 of the present invention grows for the phosphorus source with the insoluble phosphate, and is translated into soluble phosphate.Shake under bottle condition in the laboratory, the inversion quantity of the tricalcium phosphate of JX15 reaches 41.86mgL
-1With respect to the result who adopts the inoculation inactivated bacteria as blank, the inversion quantity of the tricalcium phosphate of JX15 exceeds 36.45mgL than blank
-1Nitrogenase activity reaches 19.8nmol C
2H
4/ hml has nitrogen fixing capacity preferably.
Beneficial effect: high yield indolylacetic acid plant growth-promoting bacteria JX15(CGMCC No.5622 provided by the invention), effective fixed nitrogen and insoluble phosphate is converted into soluble phosphate, improve fertilizer utilization ratio, promote plant root system development and to the absorption of fertilizer, increase soil mineral nitrogen and available phosphorus content; This bacterial strain has good promotes growth effect at peanut, and the indolylacetic acid of high yield promotes growing of peanut, and the raising of soil mineral nitrogen and available phosphorus content also makes peanut higher to the utilization ratio of nitrogenous fertilizer and phosphate fertilizer.
Description of drawings
Fig. 1 is the bacterium colony figure of bacterial strain JX15 of the present invention;
Fig. 2 represents that different liquid amounts produce the influence of IAA to bacterial strain JX15;
Fig. 3 represents the influence that the JX15 bacterial strain of different initial pH produces IAA;
Fig. 4 represents that different carbon sources are to the influence of bacterial strain JX15 product IAA;
Fig. 5 represents that different nitrogen sources is to the influence of JX15 bacterial strain product IAA;
Fig. 6 represents the situation of utilizing of the insoluble phosphorus of bacterial strain JX15
Fig. 7 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of peanut fresh weight;
Fig. 8 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of peanut plant height;
Fig. 9 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of the full N content of peanut plant;
Figure 10 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of the full P content of peanut;
Figure 11 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of peanut root length overall;
Figure 12 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of peanut root surface area;
Figure 13 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of peanut average root diameter;
Figure 14 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of peanut tip of a root number;
Figure 15 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of soil IAA content;
Figure 16 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of soil mineral N content;
Figure 17 represents to plant peanut and inoculates the JX15 bacterial strain after 30 days to the influence of the effective P content of soil;
Biomaterial preservation information
Plant growth-promoting bacteria JX15, classification bacillus megaterium (Bacillus megaterium) by name, in on December 20th, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, the address is No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number CGMCC No.5622.
Embodiment
Below in conjunction with accompanying drawing the present invention is done further explanation.
At first prepare following three kinds of substratum.
LB substratum: peptone 10g, yeast extract 5g, sodium-chlor 10g, agar 20g, distilled water 1000ml, pH7.0-7.2,121 ℃ of sterilizations, 20min.
The LB liquid nutrient medium: do not add agar, other condition is the same.
Inorganic phosphorus bacteria substratum (PKO substratum): tricalcium phosphate 5g, glucose 10g, ammonium sulfate 0.5g, sodium-chlor 0.3g, bitter salt 0.3g, Repone K 0.3g, manganous sulfate 0.03g, ferrous sulfate 0.03g, pH7.0 agar 20g, distilled water 1000ml, pH7.0 ~ 7.2.121 ℃ of sterilizations, 20min.
Minimal medium: ammonium sulfate 2.0g; SODIUM PHOSPHATE, MONOBASIC 0.5g; Dipotassium hydrogen phosphate 0.5g; Magnesium sulfate heptahydrate 0.2g; Calcium dichloride dihydrate 0.1g, distilled water 1000mL, pH7.0,121 ℃ of sterilizations, 20min.
To choose from the moisture soil that the Nanjing slab bridge is taked and take by weighing the triangular flask that l0g places the 250ml that fills the 100ml aqua sterilisa after root sieves, in shaking table, 30 ℃, 150rmin
-1Vibration 20min leaves standstill 10min, obtains soil suspension.Contain some kinds of short livings bacterium in this soil suspension, be applied to the LB substratum after adopting the dilution method dilution, flat board is inverted, in 30 ℃, in the thermostat container behind the cultivation 24h, the single bacterium colony of the dissimilar typical cases of picking, behind dull and stereotyped purifying, 4 ℃ to be kept at the LB inclined-plane stand-by.
Filter out the plant growth-promoting bacterium that can secrete indolylacetic acid by qualitative test and quantitative assay more below.
Qualitative test: the microbionation after the separation and purification is contained the LB liquid nutrient medium of L-tryptophane (100mg/L) in employing, 30 ℃, 180rmin
-1Shaking table is cultivated 1d.Get 50 μ L bacteria suspensions and drip on the whiteware plate, add 50 μ L Salkowski color solution (50mL35%HClO simultaneously
4+ 1mL0.5M FeCl
3).To add the color solution of 50 μ L50mg/L indolylacetic acids as positive control.The whiteware plate is observed after the room temperature lucifuge is placed 30min, and the color person of reddening represents to secrete indolylacetic acid.
Quantitative assay: the bacterium of the secretion IAA that primary dcreening operation is obtained carries out quantitative assay, and culture condition is the same.At first use the OD of spectrophotometry bacteria suspension
600The value, then with bacteria suspension with 10000rmin
-1Centrifugal 10min gets supernatant liquor and adds equal-volume Salkowski color solution, and lucifuge leaves standstill 30min, measures its OD
530Value.Calculate bacteria concentration OD
600Value is 1 o'clock, the content of indolylacetic acid in the unit volume fermented liquid.Analytically pure indolylacetic acid gradient dilution preparation is adopted in the drafting of typical curve.
Carry out the screening assay of molten phosphorus situation measuring the product IAA bacterium that obtains by qualitative, quantitative screening, strains tested is inoculated in the 150mL triangular flask that fills 30mL inorganic phosphorus bacteria liquid nutrient medium, 30 ℃, 180rmin
-1After cultivating 4d, with the nutrient solution 4 ℃ of following 10000rmin of centrifuge tube that pack into
-1Centrifugal, 15min.Get supernatant liquor and measure available phosphorus content with molybdenum blue colorimetric method.And adopt the acetylene reduction method to measure its nitrogenase activity.
Filter out a plant height by above mensuration and produce indolylacetic acid, fixed nitrogen and the strong bacterial strain of molten phosphorus ability, called after JX15.This bacterial strain shakes under bottle condition in the laboratory, and described plant growth-promoting bacteria reaches 41.86mgL to the inversion quantity of tricalcium phosphate
-1With respect to the result who adopts the inoculation inactivated bacteria as blank, described plant growth-promoting bacteria exceeds 36.45mgL to the inversion quantity of tricalcium phosphate than blank
-1(Fig. 6).
The bacterial strain that the aforesaid method screening and separating is gone out, the handsome biotechnology company limited order-checking through Shanghai, sequencing result according to 16SrDNA, analyze in http://www.ncbi.nlm.nih.gov online query, utilize Blast software in GenBank, to carry out the homology comparison with other 16S rDNA sequence, select the 16SrDNA systematic evolution tree of the sequence usefulness MEGAversion3 software building JX15 of close sequence and JX15.According to the physiological and biochemical property of this bacterial strain, be accredited as bacillus megaterium (Bacillus megaterium), homology is 98%.With this bacterial strain on December 20th, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number CGMCC No.5622.
The aerobic test
The LB substratum of the bacterium of going out is poured in 3 sterilized test tubes, greatly about 2/3 place, on the aseptic technique platform, with the JX15(CGMCC No.5622 of inoculating needle picking slant culture), percutaneous puncture-inoculation (must be punctured to the pipe end) in above-mentioned substratum.30 ℃ of cultivations are respectively 3 days to 7 days observationss.Giving birth to the elder on the agar column surface is aerobic bacteria, is anerobe or facultative anaerobe as giving birth to the elder along the puncture line.Test-results shows, JX15(CGMCC No.5622) bacterium colony is along the agar column surface growth, the aseptic length of being born in the puncture line, for strictness aerobic.
Catalatic mensuration
Drip 1 3%H at clean slide
2O
2, the LB slant culture 1 of getting 18 ~ 24h encircles, at H
2O
2In smear, if there have bubble to produce to be then positive, otherwise negative.Test-results shows JX15(CGMCC No.5622) be the catalase positive.
Methyl red test (M.R test)
A. substratum and reagent: peptone 5g, glucose 5g, sodium-chlor 5g, distilled water 1000mL regulates pH7.0 ~ 7.2, the packing test tube, every pipe is adorned 4 ~ 5mL, 121 ℃ of sterilization 20min.Reagent: methyl red 0.1g, 95% alcohol 300mL, distilled water 200mL.
B. spawn culture and result observe inoculating strain JX15(CGMCC No.5622) in above-mentioned nutrient solution, cultivate l ~ 2 day for 30 ℃.In nutrient solution, add several methyl red reagent, present redness as nutrient solution, be the methyl red positive, yellow negative (methyl red color change interval 4.4 redness ~ 6.0 yellow).
Test-results shows JX15(CGMCC No.5622) be the M.R feminine gender.
Second phthalein carbinol methine test (VP test)
A. the same methyl red test of culture medium culturing base.
B. spawn culture and result observe inoculation and cultivate same methyl red test.Do VP when test, get JX15(CGMCCNo.5622) nutrient solution (about 2mL) mixes mutually with the 40%NaOH of equivalent, adds a small amount of creatine, behind the 2 ~ 5min that fully vibrates, as nutrient solution appearance redness, is the VP positive.
Test-results shows JX15(CGMCC No.5622) be the VP feminine gender.
The starch hydrolysis experiment
A. substratum and reagent add 0.2% Zulkovsky starch in meat soup peptone agar, the packing triangular flask, and 121 ℃ of sterilization 20min are standby.Road Ge Shi iodine liquid: crystalline flake of iodine 1g, potassiumiodide 2g, earlier with a small amount of (3 ~ 5mL) dissolved in distilled water potassiumiodides now add the crystalline flake of iodine, treat that iodine dissolves fully after, thin up is to 300mL.
B. spawn culture and result observe and to get JX15(CGMCC No.5622) the bacterial classification point is connected on the flat board, cultivated 3 days for 30 ℃, after forming bacterium colony, drip road Ge Shi iodine liquid at flat board, to be paved with periphery of bacterial colonies degree of being, it is blue that flat board is, and periphery of bacterial colonies is irised out now if any water white transparency, illustrates that starch is hydrolyzed.The size of the big or small general remark hydrolyzed starch ability of transparent circle.
Test-results shows JX15(CGMCC No.5622) be the starch hydrolysis positive.
The gelatin hydrolysis test
A. substratum and reagent peptone 5g, gelatin 120g, distilled water 1000mL.Regulate pH7.2 ~ 7.4, the packing test tube, the substratum height is about 4 ~ 5cm, 121 ℃ of sterilization 20min.
B. spawn culture and result observe and are seeded in test tube central authorities with puncture method.In 30 ℃ of incubators, cultivated one month, observe gelatin and whether liquefy.
Test-results shows JX15(CGMCCNo.5622) be gelatin liquefaction positive.
Nitrate reduction test
A. substratum and reagent peptone 10g, KNO
31g, distilled water 1000mL, pH7.0 ~ 7.4.Ge Lisishi (Gries) reagent: A liquid: Sulphanilic Acid 0.5g, dilute acetic acid (about 10%) 150mL; B liquid: Cai's amine 0.1g, distilled water 20mL, dilute acetic acid (about 10%) 150mL.Pentanoic reagent: pentanoic 0.5g is dissolved in the l00mL vitriol oil, uses the 20mL distilled water diluting.
B. spawn culture and result observe JX15(CGMCC No.5622) be inoculated in the nitrate liquid nutrient medium, cultivated 1,3,5 day for 30 ℃.In white porcelain dish aperture, pour a little nutrient solution into, drip 1 reagent A and B liquid then therein respectively, when nutrient solution become pink, rose, when orange or brown etc., expression has nitrite to exist, and is the nitrate reduction positive, otherwise negative.
Test-results shows JX15(CGMCC No.5622) be the nitrate reduction positive.
The utilization of Citrate trianion
A. substratum and reagent Trisodium Citrate 2g, NaCl5g, MgSO
47H
2O0.2g, (NH
4)
2 HPO
41g, 1% Australia thymol blue aqueous solution 10mL, agar 20g, distilled water 1000mL, pH6.8-7.0,121 ℃ of sterilization 20min.
B. spawn culture and result observe and to get JX15(CGMCC No.5622) will cultivate bacterial classification inoculation about 12h on the inclined-plane, to cultivate 3-7 days for 30 ℃, substratum is the positive reaction of alkaline (blueness) person, and constant person is then negative.
The test-results that Citrate trianion utilizes shows JX15(CGMCC No.5622) positive.
Embodiment 3
The plant growth-promoting bacteria JX15(CGMCC No.5622 that obtains for further checking embodiment 1) ability and the optimum condition of producing indolylacetic acid explored the influence to indolylacetic acid output at different pH, liquid amount, different carbon source, different nitrogen sources below.
To contain L-tryptophane (100mg/L) LB liquid nutrient medium and press 25ml, 50ml, 75ml, 100ml, 150ml are loaded in the triangular flask of 250mL, by 1%(v/v) inoculum size inoculation is in the JX15(CGMCC No.5622 of logarithmic phase) after, place 30 ℃, 180rmin
-1Shaking table is cultivated 24h, measures the amount of producing IAA by the method for quantitative assay.The result as shown in Figure 2 because bacterial strain JX15(CGMCC No.5622) be to support metabolism, air flow influence the efficient that bacterial strain produces IAA, during the 50mL liquid amount, bacterial strain produces the IAA amount at most, along with the liquid amount increase, output is more few afterwards.
The LB substratum that will contain L-tryptophane (100mg/L) is adjusted to different pH(4,5,6,7,8,9,10 respectively), get in the triangular flask that 50mL is loaded on 250mL, by 1%(v/v) inoculum size inoculation is in the JX15(CGMCCNo.5622 of logarithmic phase) after, place 30 ℃, 180rmin
-1Shaking table is cultivated 24h, method by quantitative assay is measured the amount of producing IAA, the result as shown in Figure 3, show JX15(CGMCC No.5622) be 4 and do not produce IAA at 10 o'clock at pH, in the strong acid and strong base environment, thalline can't carry out growth metabolism, produces IAA more than sour environment in little alkali environment, and the optimal pH of this bacterial classification high yield IAA is 7 ~ 8.
In containing L-tryptophane (100mg/L) minimal medium, add 1%(w/v respectively) carbon source, carbon source has glucose, wood sugar, sucrose, fructose, N.F,USP MANNITOL, lactose, maltose etc., get in the triangular flask that 50ml is loaded on 250ml, by 1%(v/v) inoculum size inoculation is in the JX15(CGMCC No.5622 of logarithmic phase) after, place 30 ℃, 180rmin
-1Shaking table is cultivated 24h, measures the amount of producing IAA by the method for quantitative assay.The result as shown in Figure 4, this bacterial strain is when supplying with N.F,USP MANNITOL, the ability of producing IAA is the strongest, secondly is lactose, the utilization ratio of wood sugar is minimum, produces IAA hardly.
In containing L-tryptophane (100mg/L) minimal medium (not comprising ammonium sulfate), add 0.1%(w/v respectively) nitrogenous source, nitrogenous source comprises ammonium nitrate, ammonium sulfate, saltpetre, peptone, yeast powder, urea, L-Ala etc., get in the triangular flask that 50ml is loaded on 250ml by 1%(v/v) the inoculum size inoculation is in the JX15(CGMCC No.5622 of logarithmic phase) after, place 30 ℃, 180rmin
-1Shaking table is cultivated 24h, measures the amount of producing IAA by the method for quantitative assay.The result as shown in Figure 5, when illustrating that getting yeast powder is nitrogenous source, the amount of producing IAA is maximum.
Prepare following substratum:
Ashby nitrogen-free agar: N.F,USP MANNITOL 10g, KH
2PO
40.29, MgSO
47H
2O0.29, NaCI0.29, CaSO
42H
2O0.29, CaCO
35g, PH7.2.
With the JX15(CGMCC No.5622 behind the purifying) inoculation do not have in the nitrogen solid medium to Ashby, cultivate 4d under 28 ° of C, can see that obvious bacterium colony is arranged, judge that tentatively JX15 has certain nitrogen fixing capacity, below its nitrogenase activity is measured, concrete steps are as follows:
With JX15(CGMCC No.5622) insert in the no nitrogen liquid nutrient medium, collect mycetocyte with centrifuging behind the cultivation 24h, adding 45mL sterilized water and 5mL do not have the nitrogen liquid nutrient medium in the mycetocyte of collecting again, form (every milliliter about 10 of 50mL vinelandii suspension
7Individual mycetocyte) as waiting to inoculate bacterium liquid.
Nitrogenase activity adopts the acetylene reduction method to measure, and concrete operations are as follows: with JX15(CGMCC No.5622), be seeded in and 2ml is housed does not have in the penicillin bottle of nitrogen liquid nutrient medium, cultivate 18～24h down for 32 ℃; Tampon is changed to anti-chewing-gum plug sealing, has the penicillin bottle of bacterium to extract the 0.5ml air with the syringe of good airproof performance is first from cultivation, 0.5mlC reinjects
2H
2, seal pinprick with adhesive plaster; After continuing to cultivate 24h, get 100 μ l gas samples and measure C at gas chromatograph
2H
4Peak value, standard gas C
2H
4Concentration is 130mg/L.
The HP6890 type gas chromatograph that adopts U.S. Hewlett-Packard Corporation to produce, its working conditions is set to: flame ionization ditector, 250 ° of C of temperature, H
2Flow 30ml/min, pressure 20kPa; Chromatographic column is kapillary, column length 15.0m, internal diameter 320 μ m, 30 ° of C of furnace temperature; Carrier gas N
2, flow 30ml/min, pressure 20kPa; Air flow quantity 250ml/min; Preceding 250 ° of C of injector temperature, pressure 20kPa.Acetyiene reduction activity (ARA), method of calculation are:
(unit: nmol C
2H
4/ hml)
Calculate after measured, the nitrogenase activity of JX15 reaches 19.8nmol C
2H
4/ hml illustrates that it has nitrogen fixing capacity preferably.
The present invention has obvious growth promoting function to peanut, describes below by pot experiment.
Gather the fresh moisture soil of 0 ~ 20cm soil layer under the natural condition, cross the 5mm sieve, every basin is adorned native 200g, the plantation peanut, regulate water content to 60% of maxmun field capacity, 30 days post-samplings, with root system scanner (LA1600+scanner, Canada) after scanning obtains the root system image, (Winrhizo2003b Canada) carries out the related root index analysis, measures soil IAA content with the HPLC method with the root system analysis software, measure soil mineral nitrogen content with the flux analysis instrument, measure soil available phosphorus content with molybdenum blue colorimetric method.
Peanut seed: peanut seed carries out 20% hydrogen peroxide surface sterilization 20min, aseptic water washing repeatedly, vernalization 2d, it is standby to choose the consistent seed that germinates.
Connecing bacterium handles: with JX15(CGMCC No.5622 of the present invention) be inoculated in the LB liquid nutrient medium, 30 ℃, 180rmin
-1Shaking table is cultivated, and cultivates bacterium and grows to logarithmic phase, then with bacteria suspension 10000rmin
-1Centrifugal 10min uses sterilized water resuspended again.Operate triplicate equally, and bacteria suspension is evenly sprayed in soil, inoculum size is 10
8CFUg
-1, i.e. every gram dry ground inoculation 10
8CFU JX15.
Control treatment: in contrast, soil does not spray JX15 bacterium liquid, adds the equivalent sterilized water.
Result such as Fig. 7 ~ shown in Figure 17.From Fig. 7 Fig. 8 as can be seen, after connecing the bacterium processing, the plant height of plant and overground part fresh weight have remarkable increase, have promoted the peanut growth; As can be seen from Figure 9, after inoculation was handled, the total nitrogen content of peanut plant increased to some extent than control group; As can be seen from Figure 10, after inoculation was handled, the content of tatal phosphorus of peanut plant obviously increased than control group, exceeds about 1 times than control group; From Figure 11 ~ Figure 14 as can be seen, inoculation JX15 does not handle with not connecing the bacterium processing and compares, and peanut system general length, average root diameter, root surface area and tip of a root number all significantly increase, and have promoted the growth of peanut root system; As can be seen from Figure 15, after connecing the bacterium processing, soil IAA content significantly increases, and exceeds about 2 times than control group; From Figure 16 and 17 as can be seen, after connecing the bacterium processing, soil mineral nitrogen content and available phosphorus content all increase, and wherein the soil mineral nitrogen has exceeded about 2 times than control group, and this has verified that also JX15 has the ability of fixed nitrogen phosphorus decomposing preferably.In conjunction with above result as can be seen, the growth of the foundation of plant growth-promoting bacteria JX15 of the present invention, growth have positive effect, and fixed nitrogen phosphorus decomposing ability is good, and IAA output height can effectively promote crop growth.
The above only is preferred implementation of the present invention; be noted that for those skilled in the art; under the prerequisite that does not break away from the principle of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. plant growth-promoting bacteria JX15, classification called after bacillus megaterium (
Bacillus megaterium), on December 20th, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number CGMCC No.5622.
2. the application of the described plant growth-promoting bacteria JX15 of claim 1 in promoting the peanut growth.
3. the application of the described plant growth-promoting bacteria JX15 of claim 1 on peanut cultivation.
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| CN103992963B (en) * | 2014-03-19 | 2016-06-22 | 南京博农生物科技有限公司 | A kind of bacillus megaterium and application thereof |
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| MX2019008037A (en) * | 2017-01-04 | 2019-10-07 | Novozymes Bioag As | Bacillus isolates and uses thereof. |
| CN110628659B (en) * | 2018-06-21 | 2021-10-01 | 上海交通大学 | Bacillus megaterium, preparation of microbial inoculum thereof and application of bacillus megaterium in soil heavy metal remediation |
| CN109097302A (en) * | 2018-08-16 | 2018-12-28 | 浙江农林大学 | A kind of bacillus is promoting the application in plant growth |
| CN110016445B (en) * | 2019-04-10 | 2022-01-18 | 南京农业大学 | Bacillus megaterium with nitrogen fixation capacity and application thereof |
| CN110447494A (en) * | 2019-09-02 | 2019-11-15 | 广东海粤盛农业有限公司 | A kind of high yield cultivation method of peanut |
| CN112143687B (en) * | 2020-11-05 | 2022-10-21 | 黑龙江八一农垦大学 | Bacillus megaterium and application of fermentation liquor thereof in promoting plant germination |
| CN114437965B (en) * | 2021-12-31 | 2022-12-20 | 河北科技师范学院 | Peanut rhizosphere microbial strain and method for improving and repairing soil by using biological bacterial fertilizer thereof |
| CN115029278B (en) * | 2022-06-23 | 2023-03-17 | 河北农业大学 | Bacillus megaterium and application thereof in promoting crop growth |
| CN117586914A (en) * | 2023-11-22 | 2024-02-23 | 上海交通大学 | Salt-tolerant phosphate-dissolving nitrogen-fixing bacterium for soil and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102391960A (en) * | 2011-10-27 | 2012-03-28 | 南京农业大学 | Arthrobacter chlorophenolicus L4 and application thereof |
-
2012
- 2012-07-16 CN CN 201210245861 patent/CN102747018B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102391960A (en) * | 2011-10-27 | 2012-03-28 | 南京农业大学 | Arthrobacter chlorophenolicus L4 and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| 崔巍巍.植物根际促生菌的研究进展.《辽宁农业科学》.2010,35-39. |
| 根际微型土壤动物—原生动物和线虫的生态功能;陈小云 等;《生态学报》;20070831;第27卷(第8期);3132-3143 * |
| 植物根际促生菌(PGPR)的研究与应用前景;胡江春 等;《应用生态学报》;20041031;第15卷(第10期);1963-1966 * |
| 植物根际促生菌的研究进展;崔巍巍;《辽宁农业科学》;20101231;35-39 * |
| 胡江春 等.植物根际促生菌(PGPR)的研究与应用前景.《应用生态学报》.2004,第15卷(第10期),1963-1966. |
| 陈小云 等.根际微型土壤动物—原生动物和线虫的生态功能.《生态学报》.2007,第27卷(第8期),3132-3143. |
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