CN106399148B - One kind secreting ammonium nitrogen-fixing bacteria and its application - Google Patents
One kind secreting ammonium nitrogen-fixing bacteria and its application Download PDFInfo
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Abstract
The present invention relates to one kind to secrete ammonium nitrogen-fixing bacteria and its application.Secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, China Committee for Culture Collection of Microorganisms's common micro-organisms center is deposited on June 2nd, 2016, preservation registration number is CGMCC No.12588, and 16S rRNA Gene Partial sequence is as shown in SEQ ID NO.1.Nitrogen-fixing bacteria GXGL-4A of the invention has good microorganism nitrogen fixing capacity, in the Ashby culture medium of nitrogen-free, 232.94 ± 8.82nmol of nitrogenase activity C2H4/(mL·h).In addition, nitrogen-fixing bacteria K.radicincitans GXGL-4A of the invention secretes ammonium ability with good, after cultivating 4 days on nitrogen-free Ashby solid medium, the extracellular ammonium nitrogen content measured is 2.51 μ g/mL.The bacterium has pod membrane, can resist bad external environmental condition such as ultraviolet light, arid etc., and have apparent facilitation to the growth of the crops such as corn, rice.It can be used for preparing microbial-bacterial fertilizer, reduce amount of application of nitrogen fertilizer, to reach the effects of increasing production and prevent water eutrophication, protection environment.
Description
Technical field
The present invention relates to microorganisms technical fields, and in particular to one plant has secretion ammoniacal nitrogen characteristic and can promote corn, water
The nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A of the plant growths such as rice and its application.
Background technique
Nitrogen is the basic component of the various crucial organic molecules such as protein in organism, chlorophyll and nucleic acid, and
The essential nutrient in plant growth and development process.In agricultural production, nitrogen is to measure ASI systematic approach situation
With the important logo of soil fertility situation.Since biology takes away a large amount of inorganic nitrogen from soil every year, and water can also be drenched and be washed out
A big chunk soluble nitrogen, in order to supplement the loss of nitrogen in soil, most convenient is also that most efficient method is using work
Industry chemical nitrogen fertilizer.According to the statistics of Food and Agricultural Organization of the United Nations (FAO) 2002, the 40% of annual increases in grain production is by applicationization
What fertilizer obtained.China is the maximum chemical fertilizer production in the whole world and country of consumption, and the global total usage amount of chemical fertilizer is 1.42 hundred million tons within 2002, I
State is 4.34 × 103It ten thousand tons, more than the 1/3 of global total fertilizer amount, occupies first of the world.And in the use of nitrogenous fertilizer, the whole nation is flat
Equal amount of application of nitrogen fertilizer reaches 158kg/hm2, 60% (FAO, 1995) of Zhan Quanqiu nitrogenous fertilizer consumption figure.However, in crop production
The utilization rate of nitrogenous fertilizer is only 33%, and remaining 67% is wasted, and is equivalent to the whole world and has lost 15,900,000,000 dollars every year.
Chemical fertilizer plays huge effect to promotion plant growth, raising grain yield, but long-term excessive application is chemical
Fertilizer will lead to soil fertility decline and soil degradation, cause the aggravation of desertification.Meanwhile causing the evil of ecological environment of soil
Change, the decline of ecological functions, generate as acid rain, water eutrophication, depletion of the ozone layer, haze formation, global warm, drink
With the series of problems such as Nitrate Accumulation in water and food.These environmental problems have huge negative effect to China's agricultural,
Certain threat is constituted to the sustainable health development of world agriculture.
Nitrogen accounts for about 80% in atmosphere, but plant cannot directly utilize, and nitrogen-fixing microorganism can be by the nitrogen in air
It is converted into the available ammonia of plant, referred to as microorganism fixed nitrogen process.This process is by a variety of solid in fixed nitrogen somatic cells
Nitrogen enzyme, under conditions of room temperature and normal pressure, by the nitrogen (N in air2) reduction ammonification (NH3).The nitrogen fixation of nitrogen-fixing microorganism
Promote the flowing of nitrogen and circulation and a part of geochemical cycle in the entire ecosystem.FAO nineteen ninety-five
Statistical result shows that the nitrogen total amount fixed every year by microorganism is about 2 × 106Ton, needs 4 × 10 if applying urea8Ton, can
Plant for the whole world 3/4 provides nitrogen.It can be seen that microorganism nitrogen fixation is maximum natural green in terrestrial ecosystems
Nitrogen fertilizer plant.Compared with biological nitrogen fixation, industry synthetic ammonia condition is harsh, to carry out under high temperature and pressure, this is consumed largely
A large amount of pollutants will be also generated during the energy, and increasing threat is caused to the environment of human survival.Meanwhile generation
High concentration nitrogen oxide more leads to the exacerbation of the global problem that warms.In addition, extra nitrogen oxides can flow into river with rainwater
Stream lake finally imports ocean, forms water eutrophication, generates red tide, and water hypoxia causes a large amount of fish dead, water body
Aquaculture is by serious loss, and to giving water environment to arrive huge harm.Therefore, pass through the fixed nitrogen mistake of nitrogen-fixing microorganism
The research of journey, fixed nitrogen related gene and fixed nitrogen mutant to the industrial chemical fertilizer usage amount of reduction, improves world food yield, mitigation
The sustainable development of water and soil pollution, balance natural ecology and promotion agricultural has very important meaning.
The nodulation and nitrogen fixation mode of leguminous plant and rhizobium is that most typically is also research biological nitrogen fixation mode the most deep.
But since the mode has the host specificity of height, and it can not widely act on other crops.It therefore, can be with non-pulse family
Plant carries out the nitrogen-fixing microorganism of association nitrogen fixation, becomes the emphasis of Biological Nitrogen Fixation Researches.To adverse environment condition (such as arid, purple
Outside line irradiation) the interior raw combination azotobacter of non-leguminous plant more adaptable has a good application prospect.
Summary of the invention
Ammonium nitrogen-fixing bacteria are secreted it is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide one plant
Kosakonia radicincitans GXGL-4A and its application.Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria
It is isolated from Rhizosphere of Crops, belongs to non-leguminous plant combination azotobacter, there is good fixed nitrogen and secretes ammonium ability, and to corn, water
Rice etc. has good growth-promoting functions.The bacterium thallus is outside applicable important latent to the adaptable of poor environment by pod membrane
In fixed nitrogen Promoting bacteria.
The purpose of the present invention can be achieved through the following technical solutions:
Kosakonia radicincitans GXGL-4A of the invention has been deposited in the micro- life of China on June 2nd, 2016
Object culture presevation administration committee common micro-organisms center, abbreviation CGMCC.Address: city, BeiJing, China, North Star West Road, Chaoyang District 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 nucleotide sequence of ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A
As shown in SEQ ID No.1.
The acquisition methods of above-mentioned combination azotobacter Kosakonia radicincitans GXGL-4A, comprising the following steps:
A, the sample of non-leguminous plant corn, sugarcane etc., including rhizosphere soil and root system, stem eye are acquired, therefrom separation is rich
Collect nitrogen-fixing bacteria；
B, primary dcreening operation and secondary screening are carried out in Ashby culture medium (nitrogen-free)；
C, the microbiology morphologic observation of nitrogen-fixing bacteria and scanning electron microscope identification；
D, the 16S rRNA PCR amplification and DNA sequencing of bacterial strain；
E, the PCR amplification and sequencing of nifH gene.
The specific method of step A is to acquire health in the corn and sugarcane field on the ground such as Shanghai, Guangdong, Guangxi, Jiangxi and plant
Root system, rhizosphere soil and the healthy stem eye of strain, are respectively charged into vinyon bottle, save in 4 DEG C of refrigerators.Then, to root system
It is ground with stem eye sample and gradient dilution, the separation and concentration nitrogen-fixing bacteria on nitrogen-free Ashby culture medium.Specifically include following step
It is rapid:
(1) separation of rhizosphere soil nitrogen-fixing bacteria
It weighs the soil that 5g is attached to corn or Among the Sugarcane root in the balance, is put into the triangular flask of dress 45mL sterile water,
10min, supernatant i.e. 10 are being stored at room temperature after vibrating 30min on 180r/min, 28 DEG C of shaking tables-1Mix thallus dilution.It uses again
Supernatant progress is serially diluted for 10 times by sterile water, respectively takes the 0.1mL of each multiple dilution to be coated on Ashby nitrogen-free solid flat
Plate.
(2) separation of root tissue nitrogen-fixing bacteria
Root system is taken, be eluted with water and is drained away the water.It is impregnated into 30s in 70% alcohol, with aseptic water washing 3 times, then
5~10min is impregnated in 5%~20%NaClO solution that surfactant is dripped in addition 1.Then it is soaked in 70% alcohol again
Steep 30s, aseptic water washing 5 times.The sterile water that last time is rinsed is coated on LB solid plate.As sterile length of being born illustrates to sterilize
Thoroughly, on the contrary then separation of resampling.The material of sterilizing and 10mL phosphate buffer are put into the mortar of sterilizing and are ground, is ground
After stand 5min, take supernatant 1mL as original liquid, do 10 times with sterile water and be serially diluted, then take 0.1mL to be coated on respectively
Ashby nitrogen-free plate.
The specific method of step B is to prepare Ashby culture medium (to contain mannitol 10.0g, NaCl 0.2g, KH in 1L2PO4
0.2g、MgS04·7H2O 0.2g、CaS04·2H2O 0.1g、CaC035.0g, pH 7.0, distilled water are prepared.Solid medium
15~20g agar is added) 121 DEG C, sterilizing 20min.10 times are done to sample with sterile water to be serially diluted, then take 0.1mL to wait respectively
It selects nitrogen-fixing bacteria to be coated on Ashby nitrogen-free plate, after 28 DEG C of 2~3d of insulating box culture, chooses the different single colonie of colonial morphology, instead
Again carry out plate streaking separation, finally obtain pure bacterium colony, be stored in after Liquid Culture -20 DEG C it is spare.
The specific method of step C is:
(1) routine morphological is observed: the inoculating being collected into is in LB solid plate, 28 DEG C of constant temperature incubations 12~for 24 hours
Afterwards, the single colonie of appearance observes its size, form, surface, edge, color, projecting shape, transparency, periphery of bacterial colonies and culture
The color etc. of base.
(2) scanning electron microscope is identified: inoculating LB liquid medium is incubated overnight, and absorption 1mL bacterium solution enters sterilized
The centrifuge tube of 1.5mL, room temperature 4000r/min are centrifuged 2min, and supernatant is sucked out, and add sterile water that precipitating is resuspended, draw immediately after
A small amount of bacterium solution dries sample scanning electron microscope (the Tecnai G2spirit after fixing on copper sheet, being placed in air drying
Biotwin, 120kV Bio-TEM) observation nitrogen-fixing bacteria form.
The specific method of step D is to extract nitrogen-fixing bacteria genomic DNA using bacterial genomes extraction purification kit, with
Be template DNA, according to 16S rRNA conserved sequence design primer, expand target fragment, electrophoretic separation purifying, recycling purpose
DNA fragmentation, sequencing.Specific step is as follows:
(1) PCR amplification the primer is 27F and 1492R7:
27F:5'-AGAGTTTGATCCTGGCTCAG-3'；1492R:5'-GGTTACCTTGTTACGACTT-3'；It is used to draw
Object 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 H29.5 μ L of O, 1.0 μ L of template DNA, 25 μ L of total volume.Wherein, 2 ×
Taq Master mix is Beijing Kang Wei reagent biotech company product.Composition are as follows: 0.1U Taq polymerase/ μ L,
500μmol/L dNTP each、20mmol/L Tris-HCl(pH 8.3)、100mmol/L KCl、3mmol/L MgCl2, its
Its stabilizer and reinforcing agent.
(3) PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 72 DEG C of extensions
1.5min, 30 circulations；Finally in 72 DEG C of extension 10min.Pcr amplification product is about 1.2kb, on 1.2% Ago-Gel
Electrophoresis detection, target stripe is recovered to be sequenced after purification.
The specific method of step E is to extract nitrogen-fixing bacteria genomic DNA, as template DNA, according to the conservative of nifH gene
Primers, nifH PCR amplification primer are as follows: P1 (5'-GGCTGCGATCCVAAGGCCGAYTCVACCCG-3'), P2
(5'-CTGVGCCTTGTTYTCGCGGATSGGCATGGC-3') expands target fragment, through electrophoretic separation purifying, recycling purpose
DNA fragmentation, sequencing.
Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria of the invention can remarkably promote corn, rice etc.
Growth has good nitrogenase activity and secretes ammonium performance.
The ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes is used to prepare microbial-bacterial fertilizer, changes
Good lean soil reduces amount of application of nitrogen fertilizer, improves crop yield, prevents water eutrophication, protects environmental ecology.
Compared with existing nitrogen-fixing bacteria, the present invention has the advantages that
(1) nitrogen fixing capacity is stronger.K.radicincitans GXGL-4A bacterial strain nitrogenase activity on nitrogen-free agar reaches
To 232.94 ± 8.82nmol C2H4/ (mLh), and control strain gas orchid moral nitrogen-fixing bacteria Azotobacter vinelandii
1.1005 nitrogenase activity of CGMCC is only 76.79 ± 12.52nmol C2H4/ (mLh), GXGL-4A bacterial strain are blue compared to gas
Moral nitrogen-fixing bacteria are suitable for no nitrogen environment, there is better nitrogen fixing capacity.
(2) secreting ammonium characteristic can directly be absorbed and utilized convenient for crop.K.radicincitans GXGL-4A can be by ammoniacal nitrogen point
Secrete extracellular can be directly absorbed and utilized by host plant.
(3) presence of pod membrane makes K.radicincitans GXGL-4A nitrogen-fixing bacteria have better anti-adversity ability, dry
It can preferably survive in the poor environments such as drought, ultraviolet irradiation.The above characteristic is for developing the bacterium at excellent microorganism
Microbial inoculum is very favorable.
Detailed description of the invention
Fig. 1 is the electromicroscopic photograph of Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria；
Fig. 2 is the whole strain after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A inoculation " Ning Yu 721 " maize seedling
Comparison diagram；
Fig. 3 is the cauline leaf after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A inoculation " Ning Yu 721 " maize seedling
Comparison diagram；
Fig. 4 is the root pair after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A inoculation " Ning Yu 721 " maize seedling
Than figure；
Fig. 5 is the whole strain comparison after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23
Figure；
Fig. 6 is the cauline leaf comparison after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23
Figure；
Fig. 7 is the root comparison after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23
Figure.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
The acquisition of Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria of the invention: the corn from Guilin
The strain for separating on plant root and being identified on the Ashby culture medium of nitrogen-free through repeated screening.Gram's staining is negative,
Acid can be produced when absorbing glucose and produces gas using sucrose, glucose, mannitol and citrate as carbon source.The bacterium anaerobism or
Amphimicrobian can restore nitrate.Methyl red (M.R) test produces indole test display feminine gender, and catalase experiment display is positive.
Colonial morphology is mostly round or ellipse, and surface is smooth wet, is in thick, light yellow or colourless, the journey on nitrogen-free agar
Transparence.Colony diameter size is with growth time difference, from 1-2mm to 4-6mm etc..It is viewed as rod-shaped under Electronic Speculum, there is pod membrane,
Ammonium and exocellular polysaccharide class emplastic can be secreted.Raw flagellum is held, it is easy to fall off.Thallus size is about 0.5 μm of 1.5 μ m, it is single or
Common 2 somatic cells are cascaded.
The Kosakonia radicincitans GXGL-4A nitrogen-fixing bacteria have been deposited in the micro- life of China on June 2nd, 2016
Object culture presevation administration committee common micro-organisms center, abbreviation CGMCC.Address: city, BeiJing, China, North Star West Road, Chaoyang District 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.
The present invention is further illustrated below with reference to embodiment and attached drawing:
Embodiment 1: the screening and acquisition of bacterial strain
Root system is taken, be eluted with water and is drained away the water.It is impregnated into 30s in 70% alcohol, with aseptic water washing 3 times, then
5~10min is impregnated in 5%~20%NaClO solution that surfactant is dripped in addition 1.Then it is soaked in 70% alcohol again
Steep 30s, aseptic water washing 5 times.The sterile water that last time is rinsed is coated on LB solid plate.As sterile length of being born illustrates to sterilize
Thoroughly, on the contrary then separation of resampling.The material of sterilizing and 10mL phosphate buffer are put into the mortar of sterilizing and are ground, is ground
After stand 5min, take supernatant 1mL as original liquid, do 10 times with sterile water and be serially diluted, then take 0.1mL to be coated on respectively
Ashby nitrogen-free plate after 28 DEG C of 2~3d of insulating box culture, chooses the different single colonie of colonial morphology, plate streaking is repeated
Separation, obtains pure bacterium colony, confirms that the bacterial strain belongs to Kosakonia radicincitans kind, bacterial strain through microscopy and Molecular Identification
It is named as Kosakonia radicincitans GXGL-4A.
Embodiment 2: microscope inspection and the Electronic Speculum observation of bacterial strain
Oese picking Kosakonia radicincitans GXGL-4A, is applied on glass slide, film-making under aseptic condition
And Gram's staining, thalli morphology is observed under the microscope.
With LB liquid medium overnight culture bacterial strain, the centrifuge tube that 1mL bacterium solution enters sterilized 1.5mL, room temperature are drawn
4000r/min is centrifuged 2min, and supernatant is sucked out, and adds sterile water that precipitating is resuspended, draws a small amount of bacterium solution immediately after on copper sheet, set
Sample scanning electron microscope (Tecnai G2spirit Biotwin, 120kV Bio- in air drying, after drying is fixed
TEM nitrogen-fixing bacteria form) is observed.
The electron microscope of nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A is as shown in Figure 1, wherein A display series connection
Thallus, B, which is shown, shows that thallus is outer by pod membrane, and C shows single flagellum.
Embodiment 3: bacterial strain secretes the identification of ammonium characteristic
By GXGL-4A bacterial strain in 37 DEG C, 180r/min overnight incubation in LB culture medium, culture solution presses 10-5After diluting again
It is applied to Ashby nitrogen-free cultured on solid medium 4d.Under the conditions of nitrogen-free, nitrogen-fixing bacteria GXGL-4A can utilize the nitrogen in air
Gas carry out nitrogen fixation, and the part nitrogen being fixed finally be secreted into the form of ammoniacal nitrogen it is extracellular.Therefore, liquid-transfering gun can be used
(under aseptic condition, cutting the head Tip with monolithic knife) draws slightly sticky secretion, and the sterile water dilution of 10 times of volumes is added
Afterwards, for dilution by 0.45 μm of sterilised membrane filter degerming, being collected into is sample to be tested in sterile Eppendorf pipe.Ammonia in sample
The concentration of state nitrogen is measured using indophenol blue-spectrophotometry.
(1) production of ammonia nitrogen content standard curve
It prepares titer: accurately weighing 0.2358g (NH4)2SO4It is dissolved in water, is dissolved to 100mL, the amount containing ammoniacal nitrogen of acquisition is
500mg/L stoste, 10 times of dilution obtain the titer that ammonia nitrogen content is 50mg/L.
Prepare detection reagent: (two water sodium nitroprussides, 0.362 2g, adds water to 1.25% sodium nitroprusside solution
It is dissolved to 25mL), solution A (phenol 5.00g, 1.25% sodium nitroprusside solution 2.0mL add water to be dissolved to 500mL) is molten
Liquid B (NaOH 2.50g, trisodium citrate 2.0g, NaClO 3.5mL, water is added to be settled to 400mL).
Various reagents are sequentially added according to table 1, after being sufficiently mixed, 37 DEG C of water-bath colour developing 20min is put into, uses water cooling after taking-up
But to room temperature, its OD value is measured at 637nm.
The production of 1 ammonia nitrogen content standard curve of table
Embodiment 4: facilitation of the bacterial strain to corn growth
(1) disinfection and vernalization of corn seed
" Ning Yu 721 " corn seed is impregnated 4 hours in distilled water, then clean with sterile water cleaning down.Then, it moves
It handles 2min into 75% ethyl alcohol, after sterile water wash 2 times, then with 5% NaC1O impregnates 5min, sterile water wash 2 times.It will
The corn seed of disinfection is wrapped with gauze to be placed in culture dish, sprouts 3d in the dark in 28 DEG C.Observation germination daily,
And water is changed in time.
(2) culture of nitrogen-fixing bacteria
Nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A is shaken in LB culture medium in 37 DEG C, 160r/min
Culture is swung to late log phase or early stage stationary phase measurement OD value (OD600For 0.8-1.0), Centrifuge 5804R (at a high speed from
Scheming 8000r/min) centrifugation 10 minutes.Then, with sterile water washing thalline 2 times, then with sterile water it is diluted to various concentration
Dilution.Set gradually is 104(cfu/ml)、106(cfu/ml)、108(cfu/ml) 3 concentration gradients, control group and each dense
Degree gradient sets 3 repetitions respectively.
(3) method of maize seedling Azotobacter
Organic substrate is mixed with cultivating soil according to the ratio uniform of 1:1 (V:V=1:1), bud corn seed will be sprouted
It is implanted into flowerpot, same period watering and observation count emergence every two days between the seeding stage.It is after emergence, size growing way is basic
It is inner that consistent " Ning Yu 721 " maize seedling is transplanted to vinyon flowerpot (diameter 20cm, high 20cm), equipped with pressing 1: 1 in flowerpot
Mixed sandy soil and organic substrate, fertility are more barren.Each 12 basin of kind kind, 3 plants of every basin.After 3 days, experimental group is in sowing out
Seedling (put on display 1-2 piece true leaf) the 1st day afterwards, the 5th day, the 9th day, the 13rd day, the 17th day, the 21st day, the 25th day in corn rootstock
Apply nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A (1 milliliter every plant), every plant of corn of control group adds equivalent sterile
Water pours clear water to keep ground moistening during culture.1st group (104Cfu/ml): not mixing corn nitrogen-fixing bacteria in cultivating soil
Kosakonia radicincitans GXGL-4A will pour corn nitrogen-fixing bacteria Kosakonia during culture
Radicincitans GXGL-4A bacterial concentration is 104Cfu/ml inoculation processing.2nd group (106Cfu/ml): being poured during culture
Corn nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A bacterial concentration is 106Cells/ml inoculation processing.3rd group
(108Cfu/ml): it is 10 that corn nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A bacterial concentration is poured during culture8
(cfu/ml) inoculation is handled.Control group: (1 milliliter every plant) of sterile water processing is poured during culture, as a control test.Every group 3
Total 12 plants of basin.After applying corn nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A processing 6 times, plant is numbered, and
Carefully taken out in flowerpot.It takes pictures, surveying biomass includes plant height, root long, root fresh weight, cauline leaf fresh weight, plant fresh weight etc..
Nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A is inoculated with the whole strain comparison after " Ning Yu 721 " maize seedling
Figure is as shown in Fig. 2, be from left to right followed successively by control group, 10 in Fig. 24cfu/ml、106cfu/ml、108The processing of cfu/ml nitrogen-fixing bacteria
Group.Figure it is seen that the nitrogen-fixing bacteria solubility of inoculation is higher, the whole strain growth of maize seedling is more vigorous.
Nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A is inoculated with the cauline leaf comparison after " Ning Yu 721 " maize seedling
Effect is as shown in figure 3, be successively from left to right control group, 10 in Fig. 34cfu/ml、106cfu/ml、108At cfu/ml nitrogen-fixing bacteria
Reason, from figure 3, it can be seen that the nitrogen-fixing bacteria solubility of inoculation is higher, the growth of maize seedling cauline leaf is more vigorous.
Nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A is inoculated with the root comparison effect after " Ning Yu 721 " maize seedling
Fruit is as shown in figure 4, be successively from left to right control group, 10 in Fig. 44cfu/ml、106cfu/ml、108The processing of cfu/ml nitrogen-fixing bacteria,
From fig. 4, it can be seen that the nitrogen-fixing bacteria solubility of inoculation is higher, maize seedling root growth is more vigorous.
Nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A be inoculated with " Ning Yu 721 " maize seedling after plant height (cm),
Root long (cm), root fresh weight (g), cauline leaf weight (g), the specific data of plant fresh weight (g) are as shown in table 2.
The comparison of " Ning Yu 721 " corn variety biomass under 2 different disposal of table
Note: same row difference lowercase indicates to reach the level of signifiance on P < 0.05, different capitalizations indicate P <
The level of signifiance is reached on 0.01.
Embodiment 5: facilitation of the bacterial strain to paddy growth
(1) rice paddy seed disinfection and vernalization
Rice " CBB23 " seed is impregnated 4 hours in distilled water, it is clean with distilled water cleaning down.Then, it moves to
It handles 2min in 75% ethyl alcohol, after sterile water wash 2 times, then with 5% NaC1O impregnates 5min, sterile water wash 2 times.It will disappear
The rice paddy seed of poison is placed in culture dish, sprouts 4d in the dark in 28 DEG C, is observed germination daily and is changed water.
(2) culture of nitrogen-fixing bacteria
Nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A is shaken in LB culture medium in 37 DEG C, 160r/min
Culture is swung to late log phase or early stage stationary phase, measures OD value (OD600For 0.8-1.0).Centrifuge 5804R high speed from
Scheming 8000r/min is centrifuged 10 minutes.Then, with sterile water washing thalline 2 times, then with sterile water it is diluted to the dilute of various concentration
Release liquid.Set gradually is 104(cfu/ml)、106(cfu/ml)、108(cfu/ml) 3 concentration gradients, control group and each concentration
Gradient sets 3 repetitions respectively.
(3) method of rice seedlings Azotobacter
Organic substrate and cultivating soil are uniformly mixed according to the ratio of 1:1 (V:V=1:1), Germinated rice seeds are planted
Enter flowerpot, same period watering (12 noon) and observation count emergence daily during emergence.Transplanting: by size growing way
It is inner that almost the same rice CBB23 seedling is transplanted to vinyon flowerpot (diameter 20cm, high 20cm), equipped with pressing 1 in flowerpot:
1 mixed sandy soil and organic substrate, fertility are more barren.12 flowerpot of rice CBB23 kind kind, 3 plants of every flowerpot plant and allow transplanting
Rice CBB23 seedling take root completely, experimental group is in insemination and emergence (put on display 1-2 piece true leaf) the 1st day afterwards, the 5th day, the 9th after 3 days
It, the 13rd day, the 17th day, the 21st day, the 25th day in rice basal part of stem apply nitrogen-fixing bacteria Kosakonia radicincitans
GXGL-4A (1 milliliter every plant), every plant of rice of control group adds equivalent sterile water, and 12 noon pours clear water to keep during culture
Ground moistening.1st group (104Cfu/ml): cultivating soil does not mix corn nitrogen-fixing bacteria Kosakonia radicincitans
GXGL-4A, will pour corn nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A during culture, bacterial concentration is
104Cfu/ml, inoculation processing.2nd group (106Cfu/ml): corn nitrogen-fixing bacteria Kosakonia is poured during culture
Radicincitans GXGL-4A, bacterial concentration 106Cfu/ml, inoculation processing.3rd group (108Cfu/ml): being poured during culture
Fill corn nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, bacterial concentration 108Cfu/ml, inoculation processing；Control
Group: (1 milliliter every plant) of sterile water processing is poured during culture, as a control test.Every group of 3 basins amount to 12 plants.Nitrogen-fixing bacteria
After Kosakonia radicincitans GXGL-4A is handled 6 times, rice plant is numbered, and is carefully taken out in soil
Seedling is eluted with water rice plant root soil, takes pictures, measurement plant height, root long, root fresh weight, cauline leaf fresh weight and plant fresh weight etc..
Whole strain comparison diagram such as Fig. 5 after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23
It is shown, control group, 10 are from left to right followed successively by Fig. 54cfu/ml、106cfu/ml、108Cfu/ml nitrogen-fixing bacteria processing group.From Fig. 2
As can be seen that the nitrogen-fixing bacteria solubility of inoculation is higher, the whole strain growth of rice CBB23 is more vigorous.
Cauline leaf contrast effect after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23 is such as
It is successively from left to right control group, 10 in Fig. 6 shown in Fig. 64cfu/ml、106cfu/ml、108Cfu/ml nitrogen-fixing bacteria processing, from figure
6 as can be seen that the nitrogen-fixing bacteria solubility of inoculation is higher, and the growth of rice CBB23 cauline leaf is more vigorous.
Root contrast effect such as Fig. 7 after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23
It is shown, it is successively from left to right control group, 10 in Fig. 74cfu/ml、106cfu/ml、108The processing of cfu/ml nitrogen-fixing bacteria, can from Fig. 7
To find out, the nitrogen-fixing bacteria solubility of inoculation is higher, and rice CBB23 root growth is more vigorous.
Plant height (cm), root long after nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A Inoculated Rice CBB23
(cm), root fresh weight (g), cauline leaf weight (g), the specific data of plant fresh weight (g) are as shown in table 3.
The comparison of rice " CBB23 " breed organism amount under 3 different disposal of table
Note: same row difference lowercase indicates to reach the level of signifiance on P < 0.05, different capitalizations indicate P <
The level of signifiance is reached on 0.01.
It can be seen from the above embodiments that, described to secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A
It has characteristics that
(1) nitrogen fixing capacity of advantage
K.radicincitans GXGL-4A bacterial strain can effectively restore acetylene, fixed nitrogen enzyme activity on nitrogen-free agar
Property reaches 232.94 ± 8.82nmol C2H4/ (mLh), and control strain gas orchid moral nitrogen-fixing bacteria Azotobacter
1.1005 nitrogenase activity of vinelandii CGMCC is only 76.79 ± 12.52nmol C2H4/ (mLh), the results showed that
GXGL-4A bacterial strain is suitable for no nitrogen environment compared to gas orchid moral nitrogen-fixing bacteria, there is better nitrogen fixing capacity.
(2) good to secrete ammonium performance
Using indophenol blue-spectrophotometry measurement nitrogen-fixing bacteria K.radicincitans GXGL-4A in no nitrogen environment
Secrete ammonium activity.After cultivating 4 days on Ashby nitrogen-free agar, the ammonia nitrogen content of the exocytosis of nitrogen-fixing bacteria is 2.51mg/L,
Show to have and good secretes ammonium performance.
(3) there is significant facilitation to plant growth
It is tested, is repeated 3 times using single factor test (inoculum density) multiple groups, be control with sterile water process, investigate nitrogen-fixing bacteria
K.radicincitans GXGL-4A makees the promotion of corn variety " Ning Yu 721 " and rice varieties " CBB23 " plant strain growth
With.The result shows that using 108(cfu/ml) " Ning Yu 721 " a height of 69.6 ± 2.486cm of corn plants of concentration processing, control is only
60.6 ± 2.576cm, difference reach extremely significant；The root fresh weight of nitrogen-fixing bacteria processing is 4.9 ± 0.465g, compare as 4.7 ±
0.365g, difference reach the level of signifiance；The processing of cauline leaf weight nitrogen-fixing bacteria is 23.3 ± 0.713g, and compare as 15.0 ±
0.786g, difference reach the level of signifiance；The processing of plant fresh weight nitrogen-fixing bacteria is 28.2 ± 1.136g, compare as 21.9 ±
2.494g, difference reach extremely significant level.In the experiment conclusion and corn variety " Ning Yu 721 " on rice varieties " CBB23 "
It is identical, it was demonstrated that nitrogen-fixing bacteria K.radicincitans GXGL-4A has significant promotion to the growth of rice and seeding corn and other crops
Effect.
(4) there is pod membrane, the undesirable environmental condition such as arid, ultraviolet light can be resisted
Nitrogen-fixing bacteria K.radicincitans GXGL-4A thallus is outer by thick-walled pod film, this structure as the result is shown for Electronic Speculum observation
Feature assigns its ability for resisting the undesirable environmental conditions such as arid, ultraviolet light.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (6)
1. one plant is secreted ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, it has been deposited in on June 2nd, 2016
State's Microbiological Culture Collection administration committee common micro-organisms center, preservation registration number are CGMCC No.12588；Its 16S
RRNA Gene Partial sequence is as shown in SEQ ID NO.1.
2. according to claim 1 secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, feature exists
In described secretes ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A with pod membrane.
3. according to claim 1 secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, feature exists
In, the ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes has nitrogen fixing capacity,
It is described to secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A and effectively be gone back on nitrogen-free agar
Former acetylene, nitrogenase activity reach 232.94 ± 8.82nmol C2H4/(mL·h)。
4. according to claim 1 secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, feature exists
In, it is described secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A and have secrete ammonium performance,
The ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes is cultivated 4 days on Ashby nitrogen-free agar
Afterwards, the ammonia nitrogen content of the exocytosis of nitrogen-fixing bacteria is 2.51mg/L.
5. according to claim 1 secrete ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, feature exists
In the ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes has facilitation to plant growth.
6. secreting the application of ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A, feature as described in claim 1
It is, the ammonium nitrogen-fixing bacteria Kosakonia radicincitans GXGL-4A that secretes is used to prepare microbial-bacterial fertilizer.
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