CN105385625B - A kind of azotobacter MBC2 and its application - Google Patents
A kind of azotobacter MBC2 and its application Download PDFInfo
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Abstract
The invention discloses a kind of azotobacter (Massilia dura) MBC2, preserving number is CGMCC No.10818, and the deposit date is on Mays 19th, 2015, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.The bacterial strain can still live and reproduce under arid, the big temperature difference, saline and alkaline disaster and strong ultraviolet radiation and have nitrogenase activity.Its manufactured microbial inoculum can be applied to the ecosystem of arid, the big temperature difference, saline and alkaline disaster and high ultraviolet radiation, and the nitrogen accumulation of these ecosystems can be increased, it can play the role of improveing soil environment, improve the physiological activity of microorganism and plant, increase net primary productivity.
Description
Technical field
The invention belongs to biological nitrogen fixation fields, and in particular to a kind of azotobacter MBC2 and its application.
Background technology
Desert ecosystem accounts for 41% (Delgado-Baquerizo, et al., 2013) of global area, net primary
Productivity accounts for about 20% (Whittaker, 1975) of terrestrial ecosystem productivity.In desert ecosystem, Soil Nitrogen
The scarcity of attainment point seriously limit system net primary productivity (Asner, et al., 2003, Crutsinger, et al.,
2013, Reich, et al., 2006).And desertification is also further developing, and results in land deterioration, and nitrogen nutrient is lost in
Seriously.
Nitrogen input mode in natural environment is mainly biological nitrogen fixation.Nitrogen fixation, which increases soil, can utilize nitrogen content,
Nitrogen is supplemented for microorganism and plant and promotes growth and metabolism, to increase the primary productivity of the ecosystem, and for life
The Nitrogen Cycling of state system also has vital effect.Biological nitrogen fixation mainly has symbiotic nitrogen fixation and growing nitrogen-fixing.Due to famine
Unconcerned area surroundings inclement condition, vegetation is rare, therefore the legume that can carry out symbiotic nitrogen fixation is also very rare, growing nitrogen-fixing
It is the main nitrogen input mode of desert ecosystem.Therefore, growing nitrogen-fixing is for desertification control work and desert ecosystem
The recovery of system is of great importance.
In order to help desertification control to work, the research and application for desert soil azotobacter are extremely urgent.Mesh
The preceding report for azotobacteria is considerably less for the report of azotobacter still based on symbiotic nitrogen-fixing bacteria.
It is therefore desirable to provide a kind of resistance it is strong, suitable for the fixed nitrogen with authigenic nitrogen fixation capacity of desert ecosystem
Bacterial strain.
Invention content
The purpose of the present invention is to provide one kind be suitable for arid, the temperature difference is big, saline and alkaline disaster and high uitraviolet intensity and
It can be with the bacterial strain of growing nitrogen-fixing, to increase soil nitrogenase activity and effective nitrogen content.
To achieve the above objectives, the present invention provides a kind of azotobacter (Massilia dura) MBC2, preserving numbers
For CGMCC No.10818, the deposit date is on May 19th, 2015, depositary institution was Chinese microorganism strain preservation conservator
It can common micro-organisms center.
Above-mentioned bacterial strains are Massilia dura.Bacterial strain Massilia dura (strain name)-MBC2 (bacterial strain name), being will be waste
The topsoil sterile device acquisition cryogenic conditions in unconcerned area are taking back laboratory, by separation, pure culture, gram dye
Color, nitrogenase activity detection and 16srDNA sequencing gained.
Morphological character：The bacterial strain is observed in Glanz stained slice sees cell in individually, and straight-bar bacterium is not spiral
Shape.Atrichia does not generate sheath or protrusion, without rest stage, does not form brood cell, no pod membrane, Glanz feminine gender.
Cultural character：The bacterial strain on Ah 's shellfish culture medium in 28 DEG C -30 DEG C culture 48h after, well-grown, obligate need
Oxygen.Colonial morphology is different, most diameter 1cm, and round, protrusion, edge are irregular.
Physiological property：NaCl (50g/L, 30 DEG C) growth experiment sample, pyrrolidinyl side's amine enzyme tests sample, sxemiquantitative is touched
Enzyme tests sample, and oxidase negative, indoles is negative, and string test is negative, nitrate reduction negative, urease negative.
Functional characteristic：By inclined-plane Ah 's shellfish medium culture, measured with acetylene reduction method, which has gone out fixed nitrogen
Activity.
The present invention also provides the microbial inoculums containing the azotobacter MBC2.The microbial inoculum can be liquid bacterial agent,
It can be solid fungicide.
Optionally, in the microbial inoculum also contain thalline sorbing material, the thalline sorbing material be selected from turf, perlite,
At least one of vermiculite power and micro mist calcium carbonate.
Optionally, in the microbial inoculum, the content of azotobacter MBC2 is 2.1 × 108-2.9×108cfu/g。
Preferably, in the microbial inoculum, the content of azotobacter MBC2 is 2.9 × 108cfu/g。
Optionally, the pH value of the microbial inoculum is 6.8-7.2.
The present invention also provides the preparation methods of the microbial inoculum, include the following steps：
The azotobacter MBC2 is inoculated in primary inclined plane culture medium, is cultivated 3-5 days at 28-30 DEG C, obtains one
Grade bacterial strain；By the level-one inoculation in secondary liquid culture medium, shake culture 2-3 days under the conditions of 28-30 DEG C obtains
Two level bacterial strain；By the two level inoculation in three-level solid medium, cultivates under the conditions of 28-30 DEG C and obtained after 1-2 days
Obtain three-level bacterial strain；By the three-level bacterial strain and turf according to (2.1 × 108-2.9×108) cfu/g proportioning mixing it is even after obtain
Microbial inoculum, preferably 2.9 × 108cfu/g；
Wherein, the formula of the primary inclined plane culture medium is：KH2PO40.2g/L, MgSO40.2g/L, NaCl 0.2g,
CaCO35.0g, mannitol 10.0g, CaSO40.1g, pH 7.0, agar 20g, distilled water 1000mL；
The formula of secondary liquid culture medium is：KH2PO40.2g/L, MgSO40.2g/L, NaCl 0.2g, CaCO3
5.0g, mannitol 10.0g, CaSO40.1g, pH 7.0, distilled water 1000mL；
The formula of three-level solid medium is：Mannitol 10.0g, yeast powder 1.0g, MgSO4·7H2O 0.2g, K2HPO4
0.5g, NaCl 0.1g, CaCl20.05g, Rh liquid microelement 4.0mL, H2O 1000mL, agar 20g, pH 6.8-7.0.
The present invention also provides the azotobacter MBC2 or growing nitrogen-fixing microbial inoculum the answering in soil improvement
With.
Optionally, the application includes by the microbial inoculum and sand in mass ratio 1:9-11 is applied to soil after mixing
Surface.
Optionally, the dosage of the microbial inoculum is 420-560g microbial inoculums/mu.
Invention provides a kind of growing nitrogen-fixing bacterium, and it is 60.91n that its nitrogenase activity is directly measured by acetylene reduction method
mol/μL·h.The bacterial strain is extracted from the desert ecosystem environment of Ningxia, China.This area's drought；It is annual and
Day and night temperature is big (Feng Wei, 2014)；Strongly, especially summer uitraviolet intensity up to arrives the serious ultraviolet light of soil alkaline disaster
200-533 especially summers are purple2(Pu Aiping, et al., 2005), radiation index is at 8 grades or so.And it is strong in radiation under normal circumstances
Degree in the case that as 70-1302Ultraviolet light under, radiated time is differed from 15s to 30min, and sterilizing rate is up to 99% or more
(Li Xiangyun, et al., 2006, Zhang Fengcai and Yuan and it is gloomy, 1988, Zhang Fuyun and conditions are all, 2005).The purple of common sterilising conditions
The uitraviolet intensity of In Summer Over Ningxia is significantly lower than when outside line intensity and irradiation.The bacterial strain arid, the big temperature difference, saline and alkaline disaster and
It can still be lived and reproduced under strong ultraviolet radiation and there is nitrogenase activity, especially highly resistant of the bacterium to ultraviolet light
It is to increase for ultraviolet radiation increasingly strong today in Global climate change Ozone hole not available for general bacterium,
More there is ecological significance and wide application value.Its manufactured microbial inoculum can be applied to arid, high ultraviolet radiation
The ecosystem, and the nitrogen accumulation of these ecosystems can be increased, it can play the role of improveing soil environment, improve micro-
The physiological activity of biology and plant increases net primary productivity.Experiment shows the microbial inoculum being seeded in Ningxia Margin of Southwest without fixed nitrogen
After 1 year, surface layer can detect nitrogenase activity, be 541.78n mol C on active naked husky surface2H2m-2h-1, and have
Effect nitrogen is also increased by 3.16mg kg-1Increase 21.7mg kg-1.Briefly, microbial inoculum made of the bacterial strain has making letter
The advantages that single, inexpensive, drought-resistant, high UV light resistance and fixed nitrogen.
Preservation information：Depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center China
General Microbiological Culture Collection Center (abbreviation CGMCC, address：Chaoyang District, Beijing City
The institute 3 of North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101)；Preservation date is on May 19th, 2015；
Collection number of registering on the books is CGMCC No.10818；The Classification And Nomenclature of the collection suggestion is Massilia
dura；Strain number is MBC2.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
In the conventional means that are well known to those skilled in the art of technological means used, raw materials used is commercial goods.
Embodiment 1
The bacterial strain is extracted from the desert ecosystem surface layer of Ningxia, China.This area's drought；It is annual and round the clock
The temperature difference is big；Strongly, for especially summer uitraviolet intensity up to 200-533 Chang Qianglie, especially summer ultraviolet light is strong for ultraviolet light
Degree up to arrives 200-533 μ W/cm2(Pu Aiping, et al., 2005), radiation index is at 8 grades or so.And under normal circumstances in spoke
Penetrate intensity in the case that as 70-1302Ultraviolet light under, radiated time is differed from 15s to 30min, sterilizing rate up to 99% with
It is upper (Li Xiangyun, et al., 2006, Zhang Fengcai and Yuan and it is gloomy, 1988, Zhang Fuyun and conditions are all, 2005).Common sterilising conditions
The uitraviolet intensity in In Summer Over Ningxia field is significantly lower than when uitraviolet intensity and irradiation, thus the bacterial strain has to ultraviolet light tool
There is highly resistant.
It weighs topsoil sample 10g and is put into the sterile water of the 100mL with bead and stand 20min, rotating
200r/min fully vibrates 30min on formula shaking table, soil supension.Draw the above-mentioned soil of 5.0mL respectively with Sterile pipette
Suspension, which is added in 45mL sterile waters, to be diluted, and is added to bacteria suspension on solid Ah 's shellfish culture medium with Sterile pipette, in 28 DEG C
Under the conditions of cultivate.Bacterial strain is purified with plate streak.The formula of Ah 's shellfish culture medium used in the present invention is：KH2PO4 0.2g/
L, MgSO40.2g/L, NaCl 0.2g, CaCO35.0g, mannitol 10.0g, CaSO40.1g, pH 7.0, agar 20g, distillation
Water 1000mL.
Bacterium will be screened and carry out Gram stain, then observe ne ar under microscope (OLYMPUS BX51)
And it takes pictures.
Then the 50 μ L of purifying bacterium colony in culture medium are inoculated on inclined-plane Ah 's shellfish solid medium with liquid-transfering gun, are placed on
It is cultivated three days for (30 DEG C) in incubator, then measures nitrogenase activity with acetylene reduction method.Preliminary result shows addition 20%
C2H2When, the nitrogenase activity highest that sample is showed, so C in this experiment2H2Amount accounts for the 20% of culture vessel volume.In order to
Enable experiment and same experiments compare from now on, sample is cultivated in illumination box to ensure accurate environmental condition.For
The result is set to be more nearly actual value, the environmental condition of condition of culture analog study area's summer.Incubation time is for 24 hours that preceding 16h has
Illumination, while temperature is 25 DEG C, rear 8 hours no lights, while temperature is 15 DEG C.50 μ L are extracted after culture from conical flask
Sample gas, note people have walked the middle measurement ethylene emanation of gas chromatograph (163 type gas chromatograph of Hitachi) of steady baseline.It is given birth to ethylene
At amount (nmol C2H4/m-2h-1) nitrogenase activity of the azotobacter strain is described, it is 60.91n mol/ μ Lh.
It will show then to carry out the bacterium with nitrogenase activity isolated with the method for 16S rDNA recognition sequences
Identification, process are as follows：It with phenol and chloroform recovery DNA of bacteria, is dissolved with isopropanol, in 4 DEG C of preservations.Then with 1.0 μ l
For (10pmol/ μ l) 27f and 1.0 μ l (10pmol/ μ l) 1496r as primer, 1.5 μ l DNA of bacteria are template, and 0.3 μ L are added
(2.5mmol/L each) dNTPs and 18.7 μ l ddH2O, is made into 25 μ l reaction systems.By this progress PCR reaction, 30 are followed
Ring, denaturation, annealing and elongating temperature are 94 DEG C, 52 DEG C and 72 DEG C respectively, which completes on ABI-2720 instruments.PCR is produced
Object is detected with 1.5% agarose gel electrophoresis, and sample is in -20 DEG C of preservations.PCR product is sent to Huada gene company and is sequenced, it will
The pattern bacterial sequences reported in row and ncbi database are sequenced and carry out similarity system design, it is recognized herein that sequence similarity is big
It is of the same race in 98% bacterial strain and pattern bacterium.
Embodiment 2
Azotobacter MBC2 is inoculated in primary inclined plane culture medium, is cultivated 4 days at 30 DEG C, level-one bacterial strain is obtained；It will
Level-one inoculation is in secondary liquid culture medium, the shake culture 3 days under the conditions of 30 DEG C, obtains two level bacterial strain；By two level bacterium
Strain is inoculated in three-level solid medium, and acquisition three-level bacterial strain after 2 days is cultivated under the conditions of 30 DEG C；By three-level bacterial strain and turf
Microbial inoculum is obtained after contact mixing；So that the content of azotobacter MBC2 is 2.9 × 10 in microbial inoculum8The pH value of cfu/g, microbial inoculum is
7.0。
Wherein, the formula of the primary inclined plane culture medium is：KH2PO40.2g/L, MgSO40.2g/L, NaCl 0.2g,
CaCO35.0g, mannitol 10.0g, CaSO40.1g, pH 7.0, agar 20g, distilled water 1000mL；
The formula of secondary liquid culture medium is：KH2PO40.2g/L, MgSO40.2g/L, NaCl 0.2g, CaCO3
5.0g, mannitol 10.0g, CaSO40.1g, pH 7.0, distilled water 1000mL；
The formula of three-level solid medium is：Mannitol 10.0g, yeast powder 1.0g, MgSO4·7H2O 0.2g, K2HPO4
0.5g, NaCl 0.1g, CaCl20.05g, Rh liquid microelement 4.0mL, H2O 1000mL, agar 20g, pH 6.8-7.0.
The Rh liquid microelements：H3BO35.0g, Na3MoO45.0g, H2O 1000mL。
Embodiment 3
By the microbial inoculum prepared in embodiment 2 and sand according to mass ratio 1:Obtain mixture after 10 mixings, then by the mixing
Object is sprinkled upon South of Maowusu Sandland upper soll layer, and it is about 500g microbial inoculums/mu that the dosage of microbial inoculum, which is dosage,.
After the microbial inoculum is seeded in naked husky surface of the South of Maowusu Sandland without nitrogenase activity 1 year, surface layer can be examined
Nitrogenase activity is measured, is 541.78n mol C2H2m-2h-1, and soil available nitrogen is also by 3.16mg kg-1It increases
21.7mg kg-1。
Mu Us Shadi natural environmental condition is very severe, and major casualty weather has too strong arid, ultraviolet light, strong wind, sand
Cruelly, hot dry wind, frost, hail etc. are unfavorable for the existence and breeding of animals and plants and microorganism.Mu Us Shadi belongs to typical medium temperature
Band continental climate, the climate controlling factor is mainly northwest circulation, the northern continent air mass especially controlled for a long time, therefore
Day and night temperature is larger, and temperature on average is relatively low, about 6-7 DEG C；This area's drought, average precipitation are about 280mm, year
Potential evaporation amount is 2100-2500mm, and frost-free period 128d, absolute frost-free period is 100d；Sandstorm is serious, and prevailing wind direction is west
North wind, annual mean wind speed are about 2.8m s-1, maximum wind velocity is up to 15-18m s-1, have more existing strong wind April November current year to next year
It, Windy Days are 45.8d, and at most up to 52d, annual sandstorm number of days is 20.6d；Soil alkaline degree is very high, and pH is
8.7-9.2；Strongly, especially summer uitraviolet intensity is up to 200-533 μ W/cm for ultraviolet light2, radiation index is on 8 grades of left sides
It is right.The discovery of the bacterial strain and the effect of inoculation of its microbial inoculum are illustrated for arid, the temperature difference is big, dust storm, saline and alkaline disaster and strong ultraviolet
Beta radiation has excellent tolerance, can effectively increase the nitrogen input capability of soil, increase soil available nitrogen and contain
Amount.In particular, being 70-130 μ W/cm in ultraviolet radiation intensity2When, radiated time is differed from 15s to 30min, and sterilizing rate is usual
Up to 99% or more.South of Maowusu Sandland summer is significantly lower than when the uitraviolet intensity of above-mentioned common sterilising conditions and irradiation
The uitraviolet intensity in field and time, illustrate the bacterial strain and microbial inoculum than general most bacteriums have it is special high
UV light resistance.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (11)
1. a kind of azotobacter (Massilia dura) MBC2, preserving number is CGMCC No.10818, the deposit date is
On May 19th, 2015, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. the microbial inoculum containing azotobacter MBC2 described in claim 1.
3. microbial inoculum according to claim 2, which is characterized in that also contain thalline sorbing material, the bacterium in the microbial inoculum
Body sorbing material is selected from least one of turf, perlite, vermiculite power and micro mist calcium carbonate.
4. microbial inoculum according to claim 3, which is characterized in that the thalline sorbing material is turf.
5. microbial inoculum according to claim 4, which is characterized in that in the microbial inoculum, the content of azotobacter MBC2 is
2.1×108-2.9×108cfu/g。
6. microbial inoculum according to claim 5, which is characterized in that in the microbial inoculum, the content of azotobacter MBC2 is
2.9×108cfu/g。
7. according to the microbial inoculum described in any one of claim 2-6, which is characterized in that the pH value of the microbial inoculum is 6.8-7.2.
8. the preparation method of the microbial inoculum described in any one of claim 2-7, which is characterized in that include the following steps：
The azotobacter MBC2 is inoculated in primary inclined plane culture medium, is cultivated 3-5 days at 28-30 DEG C, level-one bacterium is obtained
Strain；By the level-one inoculation in secondary liquid culture medium, shake culture 2-3 days under the conditions of 28-30 DEG C obtains two level
Bacterial strain；By the two level inoculation in three-level solid medium, is cultivated under the conditions of 28-30 DEG C and obtain three after 1-2 days
Grade bacterial strain；According to the content of azotobacter MBC2 in microbial inoculum it is 2.1 × 10 by the three-level bacterial strain and thalline sorbing material8-
2.9×108Microbial inoculum is obtained after the proportioning mixing of cfu/g；
Wherein, the formula of the primary inclined plane culture medium is：KH2PO40.2g/L, MgSO40.2g/L, NaCl 0.2g, CaCO3
5.0g, mannitol 10.0g, CaSO40.1g, pH 7.0, agar 20g, distilled water 1000mL；
The formula of secondary liquid culture medium is：KH2PO40.2g/L, MgSO40.2g/L, NaCl 0.2g, CaCO35.0g, sweet dew
Alcohol 10.0g, CaSO40.1g, pH 7.0, distilled water 1000mL；
The formula of three-level solid medium is：Mannitol 10.0g, yeast powder 1.0g, MgSO4·7H2O 0.2g, K2HPO40.5g,
NaCl 0.1g, CaCl20.05g, Rh liquid microelement 4.0mL, agar 20g, H2O 1000mL, pH 6.8-7.0.
9. the microbial inoculum described in any one of azotobacter MBC2 described in claim 1 or claim 2-7 changes in soil
Application in good.
10. application according to claim 9, which is characterized in that including by the microbial inoculum and sand in mass ratio 1：9-11
It is applied to soil surface after mixing.
11. application according to claim 9 or 10, which is characterized in that the dosage of the microbial inoculum be 420-560g microbial inoculums/
Mu.
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Non-Patent Citations (3)
| Title |
|---|
| Massilia aerlata sp.nov.,isolated from an air sample;Hang Yeon Weon，et al;《International Journal of Systematic and Evolutionary microbiology》;20080601;第58卷(第6期);全文 * |
| Yu-Qin Zhang，et al.Massilia dura sp. nov., Massilia albidiflava sp. nov.,.《International Journal of Systematic and Evolutionary Microbiology》.2006,第56卷(第2期), * |
| 毛乌素沙地土壤生物结皮细菌多样性分析与拮抗菌株5A5-3的筛选鉴定;刘慧娟等;《中国优秀硕士学位论文全文数据库》;20110815(第8期);第4页、11页、19页、20-27页 * |
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