CN106399148A - Kosakonia radicincitans and application thereof - Google Patents
Kosakonia radicincitans and application thereof Download PDFInfo
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Abstract
The invention relates to kosakonia radicincitans and an application thereof. The kosakonia radicincitans GXGL-4A is preserved in China General Microbiological Culture Collection Center on June 2, 2016 with registered preservation number of CGMCC No.12588, and the sequence of a 16S rRNA gene part is shown as SEQ ID NO.1. The nitrogen-fixing bacterium GXGL-4A provided by the invention has an excellent microbial nitrogen-fixing capacity; and in a non-nitrogen Ashby medium, a nitrogen-fixing enzyme activity is 232.94+/-8.82nmol of C2H4/(mL.h). In addition, the K.radicincitans GXGL-4A provided by the invention has a good ammonium-secreting capacity; and by cultivating the GXGL-4A on the non-nitrogen Ashby solid medium, the content of the detected extracellular ammonium nitrogen is 2.51 [mu]g/mL. The strain (the kosakonia radicincitans), which has a capsule, can resist bad external environmental conditions such as ultraviolet ray, drought and the like, and the strain has a significant effect of promoting the growth of such crops as corn, rice and the like. The strain can be used for preparing bacterial manure and can reduce the application amount of a nitrogen fertilizer, so as to achieve effects of increasing production, preventing eutrophication of a water body, protecting environment and the like.
Description
Technical field
The present invention relates to microbial technology field is and in particular to one plant has secretion ammoniacal nitrogen characteristic and can promote jade
The nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A of the plant growths such as rice, Oryza sativa L. and its application.
Background technology
Nitrogen is the basic component of the various key organic molecule such as protein in organism, chlorophyll and nucleic acid,
It is also requisite nutrient in plant growth and development process.In agricultural production, nitrogen is to weigh soil battalion
Form the important mark of point situation and soil fertility situation.Due to biology take away from soil every year substantial amounts of inorganic
Nitrogen, and water also can drench and wash out a big chunk soluble nitrogen, in order to supplement the loss of Nitrogen In Soils element, most convenient
Be also most efficient method be using industrial chemistry nitrogenous fertilizer.According to Food and Agricultural Organization of the United Nations (FAO) 2002
Statistics, the 40% of annual increases in grain production be by apply chemical fertilizer obtain.China is the maximum chemical fertilizer production in the whole world
And country of consumption, the global total usage amount of chemical fertilizer is 1.42 hundred million tons within 2002, and China is 4.34 × 103Ten thousand tons, exceed complete
The 1/3 of the total fertilizer amount of ball, occupies first of the world.And in the use of nitrogenous fertilizer, the average amount of application of nitrogen fertilizer in the whole nation reaches
158kg/hm2, account for 60% (FAO, 1995) of global nitrogenous fertilizer consumption figure.However, in crop production nitrogenous fertilizer
Utilization rate is only 33%, and remaining 67% is wasted, and is equivalent to the whole world and have lost 15,900,000,000 dollars every year.
Chemical fertilizer plays huge effect to promotion plant growth, raising grain yield, but excessive administration for a long time
Chemical fertilizer, can lead to soil fertility to decline and soil degradation, cause the aggravation of desertification.Meanwhile, cause soil
The deterioration of ecological environment, the decline of ecological functions, produce such as acid rain, body eutrophication, depletion of the ozone layer, mist
The formation of haze, global warm, the series of problems such as Nitrate Accumulation in drinking water and food.These environmental problems pair
China's agricultural has huge negative effect, also constitutes certain prestige to the sustainable health development of world agriculture
The side of body.
In air, nitrogen accounts for 80%, but plant can not directly utilize, and nitrogen-fixing microorganism can be by the air
Nitrogen transformation be the available ammonia of plant, referred to as microorganism fixed nitrogen process.This process is by fixed nitrogen somatic cells
Interior multiple azotases, under conditions of room temperature and normal pressure, by the nitrogen (N of in the air2) reduction ammonification (NH3).
The nitrogen fixation of nitrogen-fixing microorganism promotes the flowing of nitrogen and circulation in whole ecosystem, is also geochemistry
A part for circulation.The statistical result of FAO nineteen ninety-five shows that the nitrogen total amount fixed by microorganism every year is about
2×106Ton, if applying carbamide, needs 4 × 108Ton, can provide nitrogen for the plant in the whole world 3/4.As can be seen here
Microorganism nitrogen fixation is maximum natural green nitrogen fertilizer plant in terrestrial ecosystems.Compared with biological nitrogen fixation, industry
Synthesis ammonia condition is harsh, will carry out, this also will produce during consuming the substantial amounts of energy under high temperature, high pressure
Raw a large amount of pollutant, cause increasing threat to the environment of human survival.Meanwhile, the high concentration N oxygen of generation
Compound more leads to increasing of the global problem that warms.Additionally, unnecessary nitrogen oxides can flow into river lake with rainwater
Pool is final to import ocean, forms body eutrophication, produces red tide, water hypoxia leads to substantial amounts of fish kills,
Aquifer cultivation industry suffers from serious losing, and huge harm that feedwater environment is arrived.Therefore, by the micro- life of fixed nitrogen
The research of the fixed nitrogen process, fixed nitrogen related gene and fixed nitrogen mutant of thing, industrial chemical fertilizer usage amount, raising to reduction
World food yield, the sustainable development of mitigation water and soil pollution, balance natural, ecological and promotion agricultural have non-
Often important meaning.
The nodulation and nitrogen fixation mode of leguminous plant and root nodule bacteria is most typically is also research biological nitrogen fixation mould the most deep
Formula.But because this pattern has the host specificity of height, and cannot widely act on other crops.Therefore,
The nitrogen-fixing microorganism of association nitrogen fixation can be carried out with non-leguminous plant, become the emphasis of Biological Nitrogen Fixation Researches.To unfavorable
The interior raw combination azotobacter of environmental condition (as arid, ultraviolet irradiate) the higher non-leguminous plant of adaptability has
Good application prospect.
Content of the invention
The purpose of the present invention is exactly to overcome the defect of above-mentioned prior art presence to provide one plant to secrete ammonium nitrogen-fixing bacteria
Kosakonia radicincitans GXGL-4A and its application.Kosakonia radicincitans GXGL-4A is solid
Nitrogen bacterium is isolatable from Rhizosphere of Crops, belongs to non-leguminous plant combination azotobacter, has good fixed nitrogen and secretes ammonium ability,
And all have good growth-promoting functions to Semen Maydiss, Oryza sativa L. etc..By pod membrane, the adaptability to poor environment outside this bacterium thalline
By force, it is applicable important potential fixed nitrogen Promoting bacteria.
The purpose of the present invention can be achieved through the following technical solutions：
The Kosakonia radicincitans GXGL-4A of the present invention is deposited in China on June 2nd, 2016
Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC.Address：Court of city of BeiJing, China
Positive area North Star West Road 1 institute 3, Microbe Inst., Chinese Academy of Sciences；Preservation registration number is CGMCC No.12588.
It belongs to γ-deformation Gammaproteobacteria, enterobacteria mesh (Enterobacteriales).Latin name：Kosakonia radicincitans.
Bacterial strain name：GXGL-4A.
Secrete the 16S rRNA Gene Partial nucleotides sequence of ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A
Row are as shown in SEQ ID No.1.
The acquisition methods of above-mentioned combination azotobacter Kosakonia radicincitans GXGL-4A, walk including following
Suddenly：
A, collection non-leguminous plant Semen Maydiss, the sample of Caulis Sacchari sinensis etc., including rhizosphere soil and root system, stem eye, therefrom
Separation and concentration nitrogen-fixing bacteria；
B, in Ashby culture medium (nitrogen-free), carry out primary dcreening operation and secondary screening；
C, the microbiology morphologic observation of nitrogen-fixing bacteria and scanning electron microscope identification；
D, the 16S rRNA PCR amplification of bacterial strain and DNA sequencing；
The PCR amplification of E, nifH gene and sequencing.
The concrete grammar of step A is to adopt from the Semen Maydiss and sugarcane field on the ground such as Shanghai, Guangdong, Guangxi, Jiangxi
The root system of collection healthy plant, rhizosphere soil and healthy stem eye, are respectively charged in vinyon bottle, in 4 DEG C of ice
Case preserves.Then, root system and stem eye sample are ground and gradient dilution, in nitrogen-free Ashby culture medium
Separation and concentration nitrogen-fixing bacteria.Specifically include following steps：
(1) separation of rhizosphere soil nitrogen-fixing bacteria
Weigh the soil that 5g is attached to Semen Maydiss or Among the Sugarcane root in the balance, put in the triangular flask of dress 45mL sterilized water,
180r/min, 28 DEG C of shaking tables stand 10min in room temperature after vibration 30min, supernatant is 10-1Mixing
Mycelium dilution liquid.With sterilized water, supernatant is carried out 10 times again to be serially diluted, respectively take the 0.1mL of each multiple dilutions liquid
Coat Ashby nitrogen-free solid plate.
(2) separation of root tissue nitrogen-fixing bacteria
Take root system, wash and drain away the water.It is soaked in 70% ethanol 30s, with aseptic water washing 3
Secondary, soak 5～10min in the 5%～20%NaClO solution adding 1 surfactant.Then again
30s, aseptic water washing 5 times is soaked in 70% ethanol.The last sterilized water rinsing coats LB solid
Flat board.As no colony growth explanation sterilization is thorough, otherwise then resample and separate.By the material of sterilizing and 10mL
Phosphate buffer is put in the mortar of sterilizing and is ground, and stands 5min, take supernatant 1mL as original after grinding
Liquid, does 10 times with sterilized water and is serially diluted, then takes 0.1mL to coat Ashby nitrogen-free flat board respectively.
The concrete grammar of step B is to prepare Ashby culture medium and (contain Mannitol 10.0g, NaCl 0.2 in 1L
g、KH2PO40.2g、MgS04·7H2O 0.2g、CaS04·2H2O 0.1g、CaC035.0g, pH 7.0, steams
Distilled water is prepared.Solid medium adds 15～20g agar) 121 DEG C, sterilizing 20min.With sterilized water to sample
Do 10 times to be serially diluted, then take 0.1mL candidate nitrogen-fixing bacteria to coat Ashby nitrogen-free flat board, 28 DEG C of perseverances respectively
After 2～3d cultivated by incubator, choose the different single bacterium colony of colonial morphology, plate streaking is repeated and separates, final
To pure bacterium colony, be stored in after liquid culture -20 DEG C standby.
The concrete grammar of step C is：
(1) routine morphological is observed：The inoculating collected is in LB solid plate, 28 DEG C of constant temperature culture
After 12～24h, the single bacterium colony of appearance, observe its size, form, surface, edge, color, projecting shape,
Color of transparency, periphery of bacterial colonies and culture medium etc..
(2) scanning electron microscope identification：Inoculating LB fluid medium incubated overnight, draws 1mL bacterium solution and enters
The centrifuge tube of sterilized 1.5mL, room temperature 4000r/min is centrifuged 2min, suctions out supernatant, plus sterilized water weight
Outstanding precipitation, draws a small amount of bacterium solution immediately after on copper coin, is placed in air drying, sample fix after is dried and uses
Scanning electron microscope (Tecnai G2spirit Biotwin, 120kV Bio-TEM) observes nitrogen-fixing bacteria form.
The concrete grammar of step D is to extract nitrogen-fixing bacteria genome using bacterial genomes extraction purification test kit
DNA, with as template DNA, according to 16S rRNA conserved sequence design primer, expand purpose fragment, electricity
Swimming isolates and purifies, reclaims target DNA fragment, sequencing.Comprise the following steps that：
(1) PCR amplification the primer is 27F and 1492R7：
27F：5'-AGAGTTTGATCCTGGCTCAG-3'；1492R：
5'-GGTTACCTTGTTACGACTT-3'；The primer is synthesized by Shanghai Sheng Gong bio-engineering corporation.
(2) PCR reaction system includes：2 × Taq Master Mix 12.5 μ L, forward primer 27F (10 μm of ol/L)
1.0 μ L, reverse primer 1492R (10 μm of ol/L) 1.0 μ L, dd H2O 9.5 μ L, template DNA 1.0 μ L,
Cumulative volume 25 μ L.Wherein, 2 × Taq Master mix is Beijing Kang Wei reagent biotech company product.Composition
For：0.1U Taq polymerase/μL、500μmol/L dNTP each、20mmol/L Tris-HCl(pH 8.3)、
100mmol/L KCl、3mmol/L MgCl2, other stabilizers and reinforcing agent.
(3) PCR reaction condition is：94 DEG C of denaturations 5min；94 DEG C of degeneration 1min, 55 DEG C of annealing 1min,
72 DEG C of extension 1.5min, 30 circulations；Finally extend 10min at 72 DEG C.Pcr amplification product is about 1.2kb,
Electrophoresis detection on 1.2% agarose gel, target stripe is recovered to be sequenced after purification.
The concrete grammar of step E is to extract nitrogen-fixing bacteria genomic DNA, as template DNA, according to nifH
The conserved sequence of gene designs primer, and nifH pcr amplification primer thing is：P1
(5'-GGCTGCGATCCVAAGGCCGAYTCVACCCG-3')、P2
(5'-CTGVGCCTTGTTYTCGCGGATSGGCATGGC-3'), expand purpose fragment, through electrophoretic separation
Purification, recovery target DNA fragment, sequencing.
The Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria of the present invention can remarkably promote Semen Maydiss, Oryza sativa L. etc.
Growth, has good nitrogenase activity and secretes ammonium performance.
The described ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes is used for preparing microbial-bacterial fertilizer, changes
Good lean soil, reduces amount of application of nitrogen fertilizer, improves crop yield, prevents body eutrophication, and environmental protection is ecological.
Compared with existing nitrogen-fixing bacteria, it is an advantage of the current invention that：
(1) nitrogen fixing capacity is higher.K.radicincitans GXGL-4A bacterial strain azotase on nitrogen-free agar
Activity reaches 232.94 ± 8.82nmol C2H4/ (mL h), and control strain gas orchid moral nitrogen-fixing bacteria Azotobacter
Vinelandii CGMCC 1.1005 nitrogenase activity is only 76.79 ± 12.52nmol C2H4/ (mL h),
GXGL-4A bacterial strain is compared gas orchid moral nitrogen-fixing bacteria and is suitable for no nitrogen environment, has more preferable nitrogen fixing capacity.
(2) secrete ammonium characteristic and be easy to crop and can directly absorb.K.radicincitans GXGL-4A can be by ammonia state
Nitrogen is secreted into and extracellular directly can be absorbed by host plant.
(3) presence of pod membrane makes K.radicincitans GXGL-4A nitrogen-fixing bacteria have more preferable anti-adversity ability,
The poor environments such as arid, ultra-vioket radiation can preferably be survived.The above characteristic is excellent for developing into this bacterium
Good microbial bacterial agent is very favorable.
Brief description
Fig. 1 is the electromicroscopic photograph of Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria；
Fig. 2 be nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A inoculate " Ning Yu 721 " maize seedling after whole
Strain comparison diagram；
Fig. 3 is the stem after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A inoculates " Ning Yu 721 " maize seedling
Leaf comparison diagram；
After Fig. 4 is nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A inoculation " Ning Yu 721 " maize seedling
Root comparison diagram；
Fig. 5 is the whole strain after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23
Comparison diagram；
Fig. 6 is the stem and leaf after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23
Comparison diagram；
Fig. 7 is the root after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23
Comparison diagram.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
The acquisition of the Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria of the present invention：Semen Maydiss from Guilin
The strain separating on plant root and obtaining through repeated screening identification in the Ashby culture medium of nitrogen-free.Gram contaminates
Color is negative, and can be able to produce when absorbing glucose by the use of sucrose, glucose, Mannitol and citrate as carbon source
Sour aerogenesis.This bacterium anaerobism or amphimicrobian, reducible nitrate.C.I. 13020. (M.R) test, product indole test
Display is negative, and catalase experiment display is positive.Colonial morphology mostly is circular or ellipse, and smooth surface moistens, and is in
Thick, light yellow or colourless, journey transparence on nitrogen-free agar.Colony diameter size is different with growth time,
From 1-2mm to 4-6mm.It is viewed as shaft-like under Electronic Speculum, has pod membrane, ammonium and extracellular polysaccharide class can be secreted
Emplastic.The raw flagellum in end, easy to fall off.Thalline size is about 0.5 μm of 1.5 μ m, single or common 2 bacterium
Somatic cell is cascaded.
This Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria is deposited in China on June 2nd, 2016
Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC.Address：Court of city of BeiJing, China
Positive area North Star West Road 1 institute 3, Microbe Inst., Chinese Academy of Sciences；Preservation registration number is CGMCC No.12588.
It belongs to γ-deformation Gammaproteobacteria, enterobacteria mesh (Enterobacteriales).Latin name：Kosakonia radicincitans.
Bacterial strain name：GXGL-4A.
Further illustrate the present invention with reference to embodiment and accompanying drawing：
Embodiment 1：The screening of bacterial strain and acquisition
Take root system, wash and drain away the water.It is soaked in 70% ethanol 30s, with aseptic water washing 3
Secondary, soak 5～10min in the 5%～20%NaClO solution adding 1 surfactant.Then again
30s, aseptic water washing 5 times is soaked in 70% ethanol.The last sterilized water rinsing coats LB solid
Flat board.As no colony growth explanation sterilization is thorough, otherwise then resample and separate.By the material of sterilizing and 10mL
Phosphate buffer is put in the mortar of sterilizing and is ground, and stands 5min, take supernatant 1mL as original after grinding
Liquid, does 10 times with sterilized water and is serially diluted, then takes 0.1mL to coat Ashby nitrogen-free flat board, 28 DEG C of perseverances respectively
After 2～3d cultivated by incubator, choose the different single bacterium colony of colonial morphology, plate streaking is repeated and separates, obtain pure
Through microscopy and Molecular Identification, bacterium colony, confirms that this bacterial strain belongs to Kosakonia radicincitans kind, Strain Designation is
Kosakonia radicincitans GXGL-4A.
Embodiment 2：The microscope inspection of bacterial strain and electron microscopic observation
Under aseptic condition, inoculating loop picking Kosakonia radicincitans GXGL-4A, is applied on microscope slide, system
Piece Gram’s staining, examine under a microscope thalli morphology.
With LB fluid medium overnight culture bacterial strain, draw the centrifuge tube that 1mL bacterium solution enters sterilized 1.5mL,
Room temperature 4000r/min is centrifuged 2min, suctions out supernatant, plus the resuspended precipitation of sterilized water, draws a small amount of immediately after
Bacterium solution, on copper coin, is placed in air drying, and sample scanning electron microscope (Tecnai G2spirit fix after is dried
Biotwin, 120kV Bio-TEM) observe nitrogen-fixing bacteria form.
The electron microscope of nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A is as shown in figure 1, wherein A shows
The thalline of series connection, B shows and shows outside thalline by pod membrane, C shows single flagellum.
Embodiment 3：Bacterial strain secretes the identification of ammonium characteristic
By GXGL-4A bacterial strain in LB culture medium in 37 DEG C, 180r/min overnight incubation, culture fluid presses 10-5
It is applied to Ashby nitrogen-free cultured on solid medium 4d after diluting again.Under the conditions of nitrogen-free, nitrogen-fixing bacteria
GXGL-4A can carry out nitrogen fixation using the nitrogen of in the air, and the part nitrogen being fixed is finally with ammoniacal nitrogen
Form is secreted into extracellular.Therefore, available liquid-transfering gun (under aseptic condition, cutting Tip head with monolithic knife) is drawn slightly
With sticky secretions, after adding the sterilized water dilution of 10 times of volumes, diluent passes through 0.45 μm of sterilised membrane filter
Degerming, collect and in aseptic Eppendorf pipe, be testing sample.In sample, the concentration of ammoniacal nitrogen adopts indophenol blue
- spectrophotography is measured.
(1) making of ammonia nitrogen content standard curve
Prepare titer：Accurately weigh 0.2358g (NH4)2SO4It is dissolved in water, is dissolved to 100mL, obtain and contain ammonia
State nitrogen quantity is 500mg/L stock solution, and 10 times of dilution obtains the titer that ammonia nitrogen content is 50mg/L.
Prepare detectable：1.25% sodium nitroprusside solution (two water sodium nitroprusside 0.362 2g, plus
Water is dissolved to 25mL), (phenol 5.00g, 1.25% sodium nitroprusside solution 2.0mL's solution A add water
It is dissolved to 500mL), (NaOH 2.50g, trisodium citrate 2.0g, NaClO 3.5mL, it is fixed to add water for solution B
Hold to 400mL).
Sequentially add various reagents according to table 1, after being sufficiently mixed, put into 37 DEG C of water-bath colour developing 20min, take out
After be water-cooled to room temperature, measure its OD value under 637nm.
The making of table 1 ammonia nitrogen content standard curve
Embodiment 4：The facilitation to corn growth for the bacterial strain
(1) sterilization of corn seed and accelerating germination
" Ning Yu 721 " corn seed is soaked 4 hours in distilled water, cleaner with sterilized water cleaning down.
Then, move in 75% ethanol process 2min, after sterile water wash 2 times, then with 5% NaC1O immersion 5
Min, sterile water wash 2 times.It is placed in culture dish after the corn seed gauze of sterilization is wrapped, in 28 DEG C
Sprout 3d in the dark.Daily observation germination, and change water in time.
(2) culture of nitrogen-fixing bacteria
By nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A in LB culture medium in 37 DEG C, 160r/min
Shaken cultivation measures OD value (OD to late log phase or early stage stationary phase600For 0.8-1.0), in Centrifuge
5804R (high speed centrifuge 8000r/min) is centrifuged 10 minutes.Then, with sterilized water washing thalline 2 times, then
It is diluted to the diluent of variable concentrations with sterilized water.Set gradually as 104(cfu/ml)、106(cfu/ml)、108
(cfu/ml) 3 Concentraton gradient, matched group and each Concentraton gradient set 3 repetitions respectively.
(3) method of maize seedling Azotobacter
By organic substrate and cultivating soil according to 1:1(V:V=1:1) ratio uniform mixing, will sprout bud corn seed
Implantation flowerpot, between the seeding stage, the same time period waters and observation counts growth of cereal crop seedlings condition every two days.After emerging, by size
Basically identical " Ning Yu 721 " maize seedling of growing way is transplanted to vinyon flowerpot (diameter 20cm, high 20cm)
In, equipped with by 1: 1 sandy soil mixing and organic substrate in flowerpot, fertility is more barren.Each kind kind 12
Basin, 3 plants of every basin.After 3 days, experimental group in insemination and emergence (put on display 1-2 piece true leaf) the 1st day afterwards, the 5th
My god, the 9th day, the 13rd day, the 17th day, the 21st day, the 25th day in Zea mays root cervical region apply nitrogen-fixing bacteria Kosakonia
Radicincitans GXGL-4A (1 milliliter every plant), every plant of Semen Maydiss of matched group add equivalent sterilized water, during culture
Pour clear water to keep ground moistening.1st group (104cfu/ml)：Semen Maydiss nitrogen-fixing bacteria are not mixed in cultivating soil
Kosakonia radicincitans GXGL-4A, will water Semen Maydiss nitrogen-fixing bacteria Kosakonia during cultivating
Radicincitans GXGL-4A bacterial concentration is 104Cfu/ml inoculation is processed.2nd group (106cfu/ml)：Training
During supporting, pouring Semen Maydiss nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A bacterial concentration is 106cells/ml
Inoculation is processed.3rd group (108cfu/ml)：Semen Maydiss nitrogen-fixing bacteria Kosakonia radicincitans is watered during culture
GXGL-4A bacterial concentration is 108(cfu/ml) inoculation is processed.Matched group：During culture, pouring sterilized water is (every
1 milliliter of strain) process, as controlled trial.Total 12 plants of every group of 3 basin.Apply Semen Maydiss nitrogen-fixing bacteria Kosakonia
After radicincitans GXGL-4A processes 6 times, plant is numbered, and carefully take out from flowerpot.Take pictures,
Survey Biomass and include plant height, root length, root fresh weight, stem and leaf fresh weight, plant fresh weight etc..
Whole strain after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A inoculates " Ning Yu 721 " maize seedling is right
Than figure as shown in Fig. 2 be from left to right followed successively by Fig. 2 matched group, 104cfu/ml、106cfu/ml、108cfu/ml
Nitrogen-fixing bacteria treatment group.Figure it is seen that the nitrogen-fixing bacteria solubility of inoculation is higher, its maize seedling whole strain growth is more prosperous
Contain.
Nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A inoculates the stem and leaf pair after " Ning Yu 721 " maize seedling
Than effect as shown in figure 3, in Fig. 3 be successively from left to right matched group, 104cfu/ml、106cfu/ml、108cfu/ml
Nitrogen-fixing bacteria are processed, from figure 3, it can be seen that the nitrogen-fixing bacteria solubility of inoculation is higher, the growth of its maize seedling stem and leaf is more vigorous.
Root after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A inoculates " Ning Yu 721 " maize seedling contrasts
Effect as shown in figure 4, in Fig. 4 be successively from left to right matched group, 104cfu/ml、106cfu/ml、108cfu/ml
Nitrogen-fixing bacteria are processed, from fig. 4, it can be seen that the nitrogen-fixing bacteria solubility of inoculation is higher, its maize seedling root growth is more vigorous.
Nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A inoculates the plant height after " Ning Yu 721 " maize seedling
(cm), root length (cm), root fresh weight (g), stem and leaf weight (g), plant fresh weight (g) concrete data as shown in table 2.
The comparison of " Ning Yu 721 " corn variety Biomass under table 2 different disposal
Note：Same row difference lowercase letter is in P<Significant level is reached, different capitalizations represent on 0.05
P<Significant level is reached on 0.01.
Embodiment 5：The facilitation to paddy growth for the bacterial strain
(1) rice paddy seed sterilization and accelerating germination
Oryza sativa L. " CBB23 " seed is soaked 4 hours in distilled water, clean with distilled water cleaning down.Then,
Move in 75% ethanol process 2min, after sterile water wash 2 times, then with 5% NaC1O immersion 5min,
Sterile water wash 2 times.The rice paddy seed of sterilization is placed in culture dish, sprouts 4d in the dark in 28 DEG C, often
It is observed germination and changes water.
(2) culture of nitrogen-fixing bacteria
By nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A in LB culture medium in 37 DEG C, 160r/min
Shaken cultivation, to late log phase or early stage stationary phase, measures OD value (OD600For 0.8-1.0).In Centrifuge
5804R high speed centrifuge 8000r/min is centrifuged 10 minutes.Then, with sterilized water washing thalline 2 times, then use
Sterilized water is diluted to the diluent of variable concentrations.Set gradually as 104(cfu/ml)、106(cfu/ml)、108(cfu/ml)
3 Concentraton gradient, matched group and each Concentraton gradient set 3 repetitions respectively.
(3) method of rice seedlings Azotobacter
By organic substrate and cultivating soil according to 1:1(V:V=1:1) ratio mix homogeneously, Germinated rice seeds are planted
Enter flowerpot, during emerging, the same time period waters (12 noon) and observation counts growth of cereal crop seedlings condition daily.Transplant：
Oryza sativa L. CBB23 seedling basically identical for size growing way is transplanted to vinyon flowerpot (diameter 20cm, height
In 20cm), equipped with by 1: 1 sandy soil mixing and organic substrate in flowerpot, fertility is more barren.Oryza sativa L. CBB23
Kind kind 12 flowerpot, 3 plants of every flowerpot, plant and allow the Oryza sativa L. CBB23 seedling of transplanting to take root completely, real after 3 days
Test group in insemination and emergence (put on display 1-2 piece true leaf) the 1st day afterwards, the 5th day, the 9th day, the 13rd day, the
Apply nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A in Oryza sativa L. basal part of stem within 17 days, the 21st day, the 25th day
(1 milliliter every plant), every plant of Oryza sativa L. of matched group adds equivalent sterilized water, and during culture, 12 noon pouring clear water is to protect
Hold ground moistening.1st group (104cfu/ml)：Cultivating soil does not mix Semen Maydiss nitrogen-fixing bacteria Kosakonia
Radicincitans GXGL-4A, will water Semen Maydiss nitrogen-fixing bacteria Kosakonia radicincitans during cultivating
GXGL-4A, bacterial concentration is 104Cfu/ml, inoculation is processed.2nd group (106cfu/ml)：Pour during culture
Fill Semen Maydiss nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, bacterial concentration is 106Cfu/ml, at inoculation
Reason.3rd group (108cfu/ml)：Semen Maydiss nitrogen-fixing bacteria Kosakonia radicincitans is watered during culture
GXGL-4A, bacterial concentration is 108Cfu/ml, inoculation is processed；Matched group：During culture, pouring sterilized water is (every
1 milliliter of strain) process, as controlled trial.Every group of 3 basins, 12 plants altogether.Nitrogen-fixing bacteria Kosakonia
After radicincitans GXGL-4A processes 6 times, rice plant is numbered, and from soil, carefully take out children
Seedling, washes rice plant root soil, takes pictures, measurement plant height, root length, root fresh weight, stem and leaf fresh weight and plant
Strain fresh weight etc..
Whole strain comparison diagram after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23 is such as
Shown in Fig. 5, be from left to right followed successively by Fig. 5 matched group, 104cfu/ml、106cfu/ml、108Cfu/ml fixed nitrogen
Bacterium treatment group.Figure it is seen that the nitrogen-fixing bacteria solubility of inoculation is higher, its Oryza sativa L. CBB23 whole strain growth is got over
Vigorous.
Stem and leaf contrast effect after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23
As shown in fig. 6, in Fig. 6 be successively from left to right matched group, 104cfu/ml、106cfu/ml、108Cfu/ml is solid
Nitrogen bacterium is processed, from fig. 6, it can be seen that the nitrogen-fixing bacteria solubility of inoculation is higher, the growth of its Oryza sativa L. CBB23 stem and leaf is got over
Vigorous.
Root contrast effect after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23 is such as
Shown in Fig. 7, in Fig. 7 be successively from left to right matched group, 104cfu/ml、106cfu/ml、108Cfu/ml fixed nitrogen
Bacterium is processed, from figure 7 it can be seen that the nitrogen-fixing bacteria solubility of inoculation is higher, its Oryza sativa L. CBB23 root growth is more prosperous
Contain.
Plant height (cm) after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23, root
Long (cm), root fresh weight (g), stem and leaf weight (g), plant fresh weight (g) concrete data as shown in table 3.
The comparison of Oryza sativa L. " CBB23 " breed organism amount under table 3 different disposal
Note：Same row difference lowercase letter is in P<Significant level is reached, different capitalizations represent on 0.05
P<Significant level is reached on 0.01.
By above example as can be seen that described secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A
There is following characteristic：
(1) nitrogen fixing capacity of advantage
K.radicincitans GXGL-4A bacterial strain can reduce acetylene, azotase on nitrogen-free agar effectively
Activity reaches 232.94 ± 8.82nmol C2H4/ (mL h), and control strain gas orchid moral nitrogen-fixing bacteria Azotobacter
Vinelandii CGMCC 1.1005 nitrogenase activity is only 76.79 ± 12.52nmol C2H4/ (mL h), result table
Bright GXGL-4A bacterial strain is compared gas orchid moral nitrogen-fixing bacteria and is suitable for no nitrogen environment, has more preferable nitrogen fixing capacity.
(2) good secrete ammonium performance
Using indophenol blue-spectrophotometry nitrogen-fixing bacteria K.radicincitans GXGL-4A in no nitrogen environment
Secrete ammonium activity.After cultivating 4 days on Ashby nitrogen-free agar, the ammoniacal nitrogen of the exocytosiss of nitrogen-fixing bacteria contains
Measure as 2.51mg/L, show to have and good secrete ammonium performance.
(3) there is significant facilitation to plant growth
Using the multigroup test of single factor test (inoculum density), it is repeated 3 times, is processed as compareing with sterilized water, investigate solid
Nitrogen bacterium K.radicincitans GXGL-4A plants to corn variety " Ning Yu 721 " and rice varieties " CBB23 "
The facilitation of strain growth.Result shows to adopt 108(cfu/ml) " Ning Yu 721 " Semen Maydiss plant height that concentration is processed
For 69.6 ± 2.486cm, comparison only 60.6 ± 2.576cm, difference reaches extremely notable；The root that nitrogen-fixing bacteria are processed is fresh
It is 4.9 ± 0.465g again, compares as 4.7 ± 0.365g, difference reaches significant level；Weight nitrogen-fixing bacteria are processed stem and leaf
For 23.3 ± 0.713g, and compare as 15.0 ± 0.786g, difference reaches significant level；At plant fresh weight nitrogen-fixing bacteria
Reason for 28.2 ± 1.136g, compare as 21.9 ± 2.494g, difference reaches pole significant level.In rice varieties
Experiment conclusion on " CBB23 " identical with corn variety " Ning Yu 721 " it was demonstrated that nitrogen-fixing bacteria K.
Radicincitans GXGL-4A has significant facilitation to the growth of Oryza sativa L. and seeding corn and other crops.
(4) there is pod membrane, the bad environmental condition such as arid, ultraviolet can be resisted
By thick-walled pod film outside electron microscopic observation result display nitrogen-fixing bacteria K.radicincitans GXGL-4A thalline, this knot
Structure feature gives the ability of the bad environmental conditions such as its opposing arid, ultraviolet.
The above-mentioned description to embodiment is to be understood that for ease of those skilled in the art and use to send out
Bright.Person skilled in the art obviously easily can make various modifications to these embodiments, and here
The General Principle illustrating is applied in other embodiment without through performing creative labour.Therefore, the present invention does not limit
In above-described embodiment, those skilled in the art according to the announcement of the present invention, without departing from changing that scope is made
Entering and change all should be within protection scope of the present invention.
Claims (7)
1. secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, on June 2nd, 2016 for one plant
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation registration number is CGMCC
No.12588.
2. according to claim 1 secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, its
It is characterised by, its 16S rRNA Gene Partial sequence is as shown in SEQ ID NO.1.
3. according to claim 1 and 2 secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A,
It is characterized in that, the described ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes has pod membrane.
4. according to claim 1 and 2 secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A,
It is characterized in that, the described ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes has nitrogen fixing capacity,
Described secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A and can have on nitrogen-free agar
Effect ground reduction acetylene, nitrogenase activity reaches 232.94 ± 8.82nmol C2H4/(mL·h).
5. according to claim 1 and 2 secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A,
It is characterized in that, described secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A and have secrete ammonium performance,
The described ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes trains on Ashby nitrogen-free agar
After supporting 4 days, the ammonia nitrogen content of the exocytosiss of nitrogen-fixing bacteria is 2.51mg/L.
6. according to claim 1 and 2 secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A,
It is characterized in that, the described ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes has to plant growth
Facilitation.
7. secrete the application of ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A as claimed in claim 1,
It is characterized in that, the described ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes is used for preparing microorganism
Bacterial manure.
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| CN106086042A (en) * | 2016-06-30 | 2016-11-09 | 上海交通大学 | Nitrogen-fixing bacteria ammonium vector gene, mutant, nitrogen-fixing bacteria ammonium carrier mutant and application |
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| CN112219485A (en) * | 2020-10-12 | 2021-01-15 | 昆明理工大学 | Soil microorganism culture method for improving soil in alpine and high-altitude areas |
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| CN113462602B (en) * | 2021-07-09 | 2022-05-24 | 浙江工业大学 | Corksaiella radicata ZJPH202011 and application thereof |
| CN114350688A (en) * | 2021-12-28 | 2022-04-15 | 上海交通大学 | Application of guaA gene, plasmid and strain in expression of azotobacterin |
| CN114350688B (en) * | 2021-12-28 | 2024-03-15 | 上海交通大学 | Application of guaA gene, plasmid and strain in expression of azotobacter ferrite |
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