CN105349464B - A kind of azotobacter MBC1 and its application - Google Patents

A kind of azotobacter MBC1 and its application Download PDF

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CN105349464B
CN105349464B CN201510860778.4A CN201510860778A CN105349464B CN 105349464 B CN105349464 B CN 105349464B CN 201510860778 A CN201510860778 A CN 201510860778A CN 105349464 B CN105349464 B CN 105349464B
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张宇清
邵晨曦
秦树高
冯薇
吴斌
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Beijing Forestry University
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Abstract

The invention discloses a kind of azotobacter (Pseudomonas cannabina) MBC1, its deposit number is CGMCC No.10817, 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

A kind of azotobacter MBC1 and its application
Technical field
The invention belongs to biological nitrogen fixation fields, and in particular to a kind of azotobacter MBC1 and its application.
Background technique
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 Seriously.
Nitrogen input mode in natural environment is mainly biological nitrogen fixation.Nitrogen fixation, which increases soil, can utilize nitrogen content, Supplementing nitrogen for microorganism and plant promotes it to grow and be metabolized, 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 leguminous plant 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.And these are few It measures known growing nitrogen-fixing microbial inoculum to require the environmental conditions such as environment temperature and humidity, resistance is poor, is applied to agriculture more In field or compost, it is unsuitable for the desert ecosystem of harsh environmental conditions.
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.
Summary of the invention
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 (Pseudomonas cannabina) MBC1, Its deposit number is CGMCC No.10817, and the deposit date is on May 19th, 2015, depositary institution was Chinese microorganism strain preservation Administration committee's common micro-organisms center.
Above-mentioned bacterial strains are Pseudomonas cannabina.Strain Pseudomonas cannabina (strain name)- MBC1 (bacterial strain name) is that the topsoil sterile device acquisition cryogenic conditions of Desert Area are being taken back laboratory, through excessive From, pure culture, Gram's staining, nitrogenase activity detection and 16srDNA sequencing gained.
Morphological character：Bacterial strain observation on Gram's staining slice sees that cell is not spiral in single, straight bacillus Shape.Size is generally 0.5-1 × 1.5-4 μm.Amphitrichous does not generate sheath or protrusion, without rest stage, does not form brood cell, Without pod membrane, Gram-negative.
Cultural character：The bacterial strain on Ah 's shellfish culture medium in 28 DEG C after -30 DEG C of cultures 3-7 days, well-grown, obligate need Oxygen.Colonial morphology is different, and most diameter 2-3mm, edge is irregular, flat wet.
Physiological property：Decomposing protein ability is strong, but fermenting carbohydrate ability is lower, and decomposition glucose produces acid but do not produce gas, Mannitol, lactose and sugaring are not decomposed, and can liquefy gelatin.Decomposing urea does not form indoles, and magnesia test is positive, can benefit Use citrate salt.
Functional characteristic：By inclined-plane Ah 's shellfish culture medium culture, measured with acetylene reduction method, which has gone out fixed nitrogen Activity.
The present invention also provides the microbial inoculums for containing the azotobacter MBC1.The microbial inoculum can be liquid bacterial agent, It can be solid fungicide.
Optionally, in the microbial inoculum also contain thallus adsorbent material, the thallus adsorbent 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 MBC1 is 2.1 × 108-2.9×108cfu/g。
Preferably, in the microbial inoculum, the content of azotobacter MBC1 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 MBC1 is inoculated in primary inclined plane culture medium, 28-30 DEG C culture 3-5 days, obtain one Grade bacterial strain；By the level-one strain inoculated in secondary liquid culture medium, shake culture 2-3 days under the conditions of 28-30 DEG C is obtained Second level bacterial strain；By the second level strain inoculated 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 proportion mix 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, CaCO35.0g is sweet Reveal 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, H2O 1000mL, agar 20g, pH 6.8- 7.0。
The present invention also provides the azotobacter MBC1 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 inoculum/mu.
Invention provides a kind of growing nitrogen-fixing bacterium, and it is 50.87n 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 South of Maowusu Sandland.This area's drought；Entirely Year and day and night temperature are big (Feng Wei, 2014)；Soil alkaline disaster is serious, and strongly, 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 Intensity is penetrated in 70-130 μ W/cm2Ultraviolet light under, radiated time is differed from 15s to 30min, and sterilizing rate is up to 99% or more (Lee Fragrant cloud, et al., 2006, Zhang Fengcai and Yuan and it is gloomy, 1988, Zhang Fuyun and conditions are all, 2005).Commonly sterilising conditions is ultraviolet The uitraviolet intensity of South of Maowusu Sandland summer is significantly lower than when line intensity and irradiation.The bacterial strain is in arid, the big temperature difference, salt Nitrogenase activity can be still lived and reproduced and had under alkali disaster and strong ultraviolet radiation, and especially the bacterium is to ultraviolet light Highly resistant is to increase ultraviolet radiation increasingly strong the present in Global climate change Ozone hole not available for general bacterium For it, more there is ecological significance and wide application value.Its manufactured microbial inoculum can be applied to arid, high ultraviolet light The ecosystem of radiation, and the nitrogen accumulation of these ecosystems can be increased, it can play the role of improveing soil environment, The physiological activity of microorganism and plant is improved, net primary productivity is increased.Experiment shows the microbial inoculum being seeded in Mu Us Shadi After Nan Yuan is without the naked husky surface 1 year of nitrogenase activity, surface layer can detecte nitrogenase activity, be 461.78n mol C2H2m-2h-1, and available nitrogen is also increased by 3.16mg kg-1Increase 22.1mg kg-1.Briefly, microbial inoculum made of the bacterial strain There are simple, inexpensive, drought-resistant, the high UV light resistance of production 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.10817；The classification naming of the collection suggestion is Pseudomonas cannabina；Strain number is MBC1.
Specific embodiment
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 South of Maowusu Sandland.This area's drought；It is annual It is big (Feng Wei, 2014) with day and night temperature；Strongly, especially summer uitraviolet intensity is up to 200-533 μ W/cm for ultraviolet light2 (Pu Aiping, et al., 2005), radiation index is at 8 grades or so.And under normal circumstances in radiation intensity in 70-130 μ W/cm2's Under ultraviolet light, radiated time is differed from 15s to 30min, sterilizing rate up to 99% or more (Li Xiangyun, et al., 2006, Zhang Feng Ability and Yuan and it is gloomy, 1988, Zhang Fuyun and conditions are all, 2005).It is obvious low when the uitraviolet intensity of common sterilising conditions and irradiation Uitraviolet intensity in South of Maowusu Sandland summer field, thus the bacterial strain with to ultraviolet light have 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 sufficiently 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 water, 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 Xu shellfish culture medium used in the present invention 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。
Bacterium will be screened and carry out Gram stain, then observe ne ar at microscope (OLYMPUS BX51) And it takes pictures.
Then the 50 μ L of purifying bacterium colony in culture medium is inoculated on inclined-plane Ah 's shellfish solid medium with liquid-transfering gun, is 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 It compare experiment can with same experiments from now on, sample cultivated in illumination box to guarantee accurate environmental condition.For The result is set to be more nearly true 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, injection, which has been walked, measures ethylene emanation in the gas chromatograph (163 type gas chromatograph of Hitachi) of steady baseline.It is raw with ethylene At amount (nmol C2H4/m-2h-1) nitrogenase activity of the azotobacter strain is described, for 50.87n mol/ μ Lh.
It will show then to carry out the bacterium with nitrogenase activity isolated with the method for 16S rDNA recognition sequence Identification, process are as follows：It with phenol and chloroform recovery DNA of bacteria, is dissolved with isopropanol, is saved in 4 DEG C.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 is added (2.5mmol/L each) dNTPs and 18.7 μ l ddH2O is made into 25 μ l reaction systems.This is subjected to 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 instrument.PCR is produced Object is detected with 1.5% agarose gel electrophoresis, and sample is saved in -20 DEG C.PCR product is sent to Huada gene company and is sequenced, it will The mode bacterial sequences reported in column and ncbi database are sequenced and carry out similarity system design.
Embodiment 2
Azotobacter MBC1 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 strain inoculated is in secondary liquid culture medium, the shake culture 3 days under the conditions of 30 DEG C, obtains second level bacterial strain；By second 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 Contact obtains microbial inoculum after mixing；So that the content of azotobacter MBC1 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, CaCO35.0g is sweet Reveal 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, H2O 1000mL, agar 20g, pH 6.8- 7.0。
The Rh liquid microelement：H3BO35.0g, Na3MoO45.0g, H2O 1000mL。
Embodiment 3
By the microbial inoculum prepared in embodiment 2 and sand according to mass ratio 1:10 mix after obtain mixture, then by the mixing Object is sprinkled upon South of Maowusu Sandland upper soll layer, and it is about 500g microbial inoculum/mu that the dosage of microbial inoculum, which is dosage,.
After the microbial inoculum is seeded in South of Maowusu Sandland without the naked husky surface 1 year of nitrogenase activity, surface layer can be examined Nitrogenase activity is measured, is 461.78n mol C2H2m-2h-1, and soil available nitrogen is also by 3.16mg kg-1It increases 22.1mg kg-1。
Mu Us Shadi natural environmental condition is very severe, and major casualty weather has that arid, ultraviolet light is too strong, 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 lower, and 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, often have 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 Feng Wei, 2014)；Strongly, especially summer uitraviolet intensity is up to 200-533 μ W/cm for ultraviolet light2(Pu love Duckweed, et al., 2005), radiation index is at 8 grades or so.The discovery of the bacterial strain and the effect of inoculation of its microbial inoculum are illustrated for dry Drought, the temperature difference be big, dust storm, saline and alkaline disaster and intensive ultraviolet radiation have excellent tolerance, can effectively increase soil Nitrogen input capability increases soil available nitrogen content.In particular, being 70-130 μ W/cm in ultraviolet radiation intensity2When, radiation Time is differed from 15s to 30min, sterilizing rate usually up to 99% or more (Li Xiangyun, et al., 2006, Zhang Fengcai and Yuan and Gloomy, 1988, Zhang Fuyun and conditions are all, and 2005).Hair is significantly lower than when the uitraviolet intensity of above-mentioned common sterilising conditions and irradiation The uitraviolet intensity in black element sand ground south edge summer field and time, illustrate the general overwhelming majority of the bacterial strain and microbial inoculum ratio Bacterium has 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, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Claims (11)

1. a kind of azotobacter (Pseudomonas cannabina) MBC1, deposit number is CGMCC No.10817, preservation Date is on May 19th, 2015, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. the microbial inoculum containing azotobacter MBC1 described in claim 1.
3. microbial inoculum according to claim 2, which is characterized in that also contain thallus adsorbent material, the bacterium in the microbial inoculum Body adsorbent 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 thallus adsorbent material is turf.
5. microbial inoculum according to claim 4, which is characterized in that in the microbial inoculum, the content of azotobacter MBC1 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 MBC1 is 2.9×108cfu/g。
7. the microbial inoculum according to 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 microbial inoculum as claimed in claim 2, which is characterized in that include the following steps：
The azotobacter MBC1 is inoculated in primary inclined plane culture medium, 28-30 DEG C culture 3-5 days, obtain level-one bacterium Strain；By the level-one strain inoculated in secondary liquid culture medium, shake culture 2-3 days under the conditions of 28-30 DEG C obtains second level Bacterial strain；By the second level strain inoculated in three-level solid medium, culture obtains three after 1-2 days under the conditions of 28-30 DEG C Grade bacterial strain；It according to the content of azotobacter MBC1 in microbial inoculum is 2.1 × 10 by the three-level bacterial strain and thallus adsorbent material8- 2.9×108The proportion of cfu/g obtains microbial inoculum after mixing；
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. microbial inoculum described in any one of azotobacter MBC1 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 inoculum/ Mu.
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