CN102286402B - Soybean rhizosphere azotobacter for producing activated calcium carbonate (ACC) and realizing antagonistic effect on various pathogenic fungi and purpose thereof - Google Patents

Soybean rhizosphere azotobacter for producing activated calcium carbonate (ACC) and realizing antagonistic effect on various pathogenic fungi and purpose thereof Download PDF

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CN102286402B
CN102286402B CN 201110197670 CN201110197670A CN102286402B CN 102286402 B CN102286402 B CN 102286402B CN 201110197670 CN201110197670 CN 201110197670 CN 201110197670 A CN201110197670 A CN 201110197670A CN 102286402 B CN102286402 B CN 102286402B
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microbial inoculum
fungi
burkholderia
bulkholderia cepasea
verticillium
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孙建光
徐晶
高淼
胡海燕
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Institute of Agricultural Resources and Regional Planning of CAAS
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Abstract

The invention discloses a soybean rhizosphere azotobacter for producing activated calcium carbonate (ACC) and realizing the antagonistic effect on various pathogenic fungi and a purpose thereof. The soybean rhizosphere azotobacter is Burkholderia sp. GDSD571 and has the preservation number being CGMCC No.5039 in General Microbiology Center of China Microbial Strain Preservation Management Committee. The Burkholderia sp. GDSD571 CGMCC No.5039 provided by the invention has high nitrogen fixation activity and ACC ammonialyase activity, can realize the effective antagonistic effect on gibberella zeae, sclerotinia sclerotiorum and verticillium dahliae and has wide application prospects in nitrogen fixation microbial agent and microbial organic fertilizer production.

Description

Produce soybean rhizosphere vinelandii of acc deaminase and antagonism Various Diseases fungal pathogens and uses thereof
Technical field
The present invention relates to soybean rhizosphere vinelandii that produce acc deaminase and antagonism Various Diseases fungal pathogens and uses thereof, be particularly related to and a kind ofly can produce acc deaminase, antagonism crop gibberellic hypha, sclerotium germ and verticillium wilt pathogen, and have the bacterium that carries out the high-efficiency nitrogen-fixing function at soybean rhizosphere.
Background technology
Nitrogenous fertilizer is one of most important production means in the agriculture production.Produce chemical nitrogen fertilizer and consume mass energy, account for 70%～80% of total production cost.Energy day is becoming tight in recent years, and China's chemical fertilizers production soars with the coal price lattice and causes the chemical nitrogen fertilizer price increase, and the peasant is difficult to bear, and repercussion is strong.In addition, chemical nitrogen fertilizer is being brought into play great function aspect raising crop yield, the guarantee China grain security, but the unreasonable nitrogen of executing has formed serious pollution of area source, causes a series of serious harm, makes us startling such as the Taihu Lake Blue Algae Event.The urban groundwater azotate pollution has caused threat to drinking water safety.
Contain 78% nitrogen in the atmosphere, but its existence form is molecular state nitrogen, animals and plants can not directly utilize, and nature only has some prokaryotic micro-organisms to have the ability of directly utilizing nitrogen in the atmosphere, with its reduction ammonification, biological nitrogen fixation that Here it is.Select the batch production of high-efficiency nitrogen-fixing microorganism bacterial classification to produce and make microbial inoculum, or further become biological products with other material Compound Machining that is rich in plant nutrition, be applied to agriculture production, crop nitrogen nutrition can be provided, improve the crop rhizosphere ecotope, improve the soil biological fertility proterties, Here it is usually said nitrogen-fixing microorganism microbial inoculum or nitrogen-fixing microorganism fertilizer.Biological nitrogen fertilizer has multiple advantage: 1. biological nitrogen fertilizer is comprised of reproducible biochemical preparation and living microorganism, can regenerate, and does not have the resource exhaustion problem, is the Sustainable development agricultural material product.2. biological nitrogen fertilizer is environmental friendly fertilizer, and it is produced and use procedure does not all produce the pollutants such as the three wastes, and can improve soil physico-chemical property, increase soil fertility, and be ecological agriculture agricultural material product.3. biological nitrogen fertilizer production power consumption less, cost is low, and its cost only is 20%～40% of equivalent chemical nitrogen fertilizer, can save a large amount of coal and oil equal energy source strategic materials for country.4. use biological nitrogen fertilizer can significantly reduce agriculture production cost, improve quality of agricultural product, promote soil fertility, the peasant is benefited, development promotes social harmony.
The pathogenic bacteria of crop head blight is Gibberella zeae bacterium (Gibberella zeae), and asexual generation is Fusarium graminearum (Fusarium graminearum), is the important pathogenic bacteria of food crop.China's wheat scab has 95% to be that Gibberella zeae (Sch.) Petch is microbial, is the important disease of middle and lower reach of Yangtze River Winter Wheat Area and Northeasten Spring Wheat Area of China, often causes 20%～30% production loss during morbidity.In addition, this bacterium is also infected the crops such as corn, paddy rice, barley, causes that seedling is withered, stem rot, base is rotten, fringe is rotten etc.The pathogenic bacteria of sclerotium disease is sclerotinite (Sclerotinia sclerotiorum), its morphological specificity shows as: apothecium is little, be little cup-shaped, light salmon is to brown, single or severally bears from sclerotium, diameter 0.5-1cm, the handle brown is elongated, bending, long 3-5cm, gradually thin downwards, link to each other with sclerotium.Mycelium can form sclerotium, and the brown apothecium of long handle is created on the sclerotium.The sclerotium shape is various, long 3-15 μ m.Ascus is cylindrical, 120-140 μ m * 11 μ m, and common 8 of spore, single file is arranged, ellipse, 8-14 μ m * 4-8 μ m, lateral filament is elongated, and is linear, colourless, and the top is thicker.This bacterium is the global important phytopathogen of a kind of damage to crops and vegetables, infect widely, endanger the plants such as Cruciferae, pulse family, Solanaceae, Rutaceae, as cause cash crop and the vegetables sclerotium diseases such as rape, soybean, Sunflower Receptacle, cucumber, capsicum.Verticillium dahliae (verticillium dahliae Verticillium dahliae) is take the soil propagation as main, host range is very wide, cause the verticillium except infecting cotton, also infect cash crop tobacco, oil crops soybean, peanut, Sunflower Receptacle, sesame, vegetable crop potato, eggplant, capsicum, tomato, cucumber, watermelon, and forest, flowers etc., harm is serious, causes the tremendous economic loss.At present the control of above-mentioned fungal disease mainly relied on chemical pesticide, yet not only cost is high, contaminate environment in chemical prevention, and preventive effect is also undesirable, the simultaneously security of food is also had a strong impact on.
In recent years, the adverse environmental factors such as arid, flood, Soil Secondary salinization and heavy metal contamination cause the problem such as crop production reduction day by day serious, how Effective Raise plant stress-resistance ability and increase the important content that crop yield has become current agricultural sustainable development work.Ethene is the endogenous hormones of higher plant, growth and development of plants only needs the ethene of lower level usually, but when approaching ripe or run into arid, waterflooding, high temperature, physical abuse, disease and pest invasion and attack, can produce in a large number ethene, this be plant to a kind of physiological stress of environment, but excessive ethene can cause growth and development of plants to be obstructed even dead.1-amino-cyclopropane-1-carboxylic acid (1-aminocyclopropane-1-carboxylate, ACC) be the synthetic precursor substance of ethene, discovered in recent years, many plant-growth promoting rhizobacterias (plant growth promoting bacteria, PGPB) has the acc deaminase activity, can decomposing ammonification and α-batanone acid to ACC, to reduce ethene synthetic, thereby reduce plant to the susceptibility of adverse circumstance, improve the plant stress-resistance ability, and can promote the phytoremediation of Organic pollutants and heavy-metal contaminated soil, so people adopt the method that detects acc deaminase to screen plant-growth promoting rhizobacteria.
Bacterial classification is the basis of microbial fertilizer production application.At present, the bottleneck of restriction China microbial fertilizer industry development is exactly the seed selection problem of high-efficiency strain.Agriculture production active demand nitrogen fixation efficiency height, antagonizing pathogenic fungi, strong stress resistance, the long microbial fertilizer production bacterial classification of shelf-lives.
Summary of the invention
The purpose of this invention is to provide a strain and have 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase active, can carry out high-efficiency nitrogen-fixing at crop rhizosphere, and the bacterium of antagonism crop gibberellic hypha (Gibberella zeae), sclerotium germ (Sclerotinia sclerotiorum) and verticillium dahliae (Verticillium dahliae).
Bacterium provided by the present invention is bulkholderia cepasea (Burkholderia sp.) GD571, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 6th, 2011 and (is called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.5039.
Another object of the present invention provides a kind of microbial inoculum, and the activeconstituents of this microbial inoculum is described bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039.
This microbial inoculum also can comprise auxiliary material, such as the stalk of the ight soil of the peat composed of rotten mosses, animal, all kinds of crops, loose shell, straw, peanut skin etc. except bulkholderia cepasea (Burkholderia sp.) the GD571 CGMCC No.5039 that comprises as activeconstituents.
This microbial inoculum can be used for suppressing pathogenic fungi, control fungal diseases of plants, fixed nitrogen, Promoting plant growth, inhibition plant generation ethene, reduces plant to adverse circumstance susceptibility, raising stress resistance of plant, generation 1-amino-cyclopropane-1-carboxylic acid (ACC) desaminase and nitrogenase etc.
Described bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 is in preparation following 1)-9) in application in arbitrary microbial inoculum also belong to protection scope of the present invention:
1) for the microbial inoculum that suppresses pathogenic fungi;
2) for the microbial inoculum of preventing and treating fungal diseases of plants;
3) be used for the microbial inoculum of fixed nitrogen;
4) microbial inoculum of Promoting plant growth;
5) suppress the microbial inoculum that plant produces ethene;
6) reduce plant to the microbial inoculum of adverse circumstance susceptibility;
7) microbial inoculum of raising stress resistance of plant;
8) microbial inoculum of generation 1-amino-cyclopropane-1-carboxylic acid (ACC) desaminase;
9) microbial inoculum of generation nitrogenase.
Described pathogenic fungi can be by soil, the fertilizer in being manured into soil and/or the fungi of seed dispersal, specifically can be the fungi or fungi or the fungi for causing verticillium for causing sclerotium disease that cause head blight; Described fungal diseases of plants specifically can be head blight, sclerotium disease or verticillium; Described adverse circumstance specifically can be arid, waterflooding, high temperature, physical abuse, disease and pest invasion and attack or heavy metal contamination etc.
Describedly cause that the fungi of head blight can be Gibberella zeae bacterium (Gibberella zeae) or Fusarium graminearum (Fusarium graminearum); Describedly cause that the fungi of sclerotium disease can be sclerotinite (Sclerotinia sclerotiorum); Describedly cause that the fungi of verticillium can be verticillium dahliae (Verticillium dahliae).
Application and the application in the preparation biological organic fertilizer in producing nitrogenase or 1-Aminocyclopropane-1-carboxylate deaminase of described bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 or the microbial inoculum take this bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 as activeconstituents also belongs to protection scope of the present invention.
A further object of the present invention provides a kind of bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 or biological organic fertilizer take this bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 as the microbial inoculum of activeconstituents of containing.
Another purpose of the present invention provides the method for a kind of cultivation bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039, comprises the step that bulkholderia cepasea (Burkholderia sp.) GD571 CGMCCNo.5039 is cultivated at the substratum that is used for the cultivation bulkholderia cepasea.
Another purpose of the present invention provides a kind of method for preparing described microbial inoculum, and the method comprises the steps: described bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 to obtain described microbial inoculum as activeconstituents.
Experimental results show that, the present invention is through layer by layer screening from pedotheque, finishing screen has been selected bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039, this bacterial strain can produce 1-amino-cyclopropane-1-carboxylic acid (ACC) desaminase, and nitrogenase activity is very high, can antagonism crop head blight pathogenic bacteria Gibberella zeae bacterium (Gibberella zeae), sclerotium disease pathogenic bacteria sclerotinite (Sclerotinia sclerotiorum) and verticillium dahliae (verticillium dahliae Verticillium dahliae), compete adaptable, effect of inoculation is good, has broad application prospects in nitrogen-fixing microorganism microbial inoculum and biological organic fertilizer production.
The preservation explanation
Strain name: bulkholderia cepasea
Latin name: (Burkholderia sp.)
Strain number: GD571
Preservation mechanism: China Committee for Culture Collection of Microorganisms common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on July 6th, 2011
The preservation center numbering of registering on the books: CGMCC No.5039
Description of drawings
Fig. 1 is the nitrogenase activity of bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039.
Fig. 2 is bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 antagonism gibberellic hypha Gibberella zeae bacterium (Gibberella zeae).The left side bacterium colony is bulkholderia cepasea (Burkholderia sp.) GD571CGMCC No.5039, and the right side bacterium colony is gibberellic hypha Gibberella zeae bacterium (Gibberella zeae).
Fig. 3 is bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 antagonism sclerotium germ (Sclerotinia sclerotiorum).The left side bacterium colony is bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039, and the right side bacterium colony is sclerotium germ (Sclerotinia sclerotiorum).
Fig. 4 is bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 antagonism verticillium dahliae (Verticillium dahliae).The left side bacterium colony is bulkholderia cepasea (Burkholderia sp.) GD571CGMCC No.5039, and the right side bacterium colony is verticillium dahliae (Verticillium dahliae).
Fig. 5 is the 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase active of bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Vinelandii enrichment culture liquid ACCC55 composition: sucrose 10g, K 2HPO 43H 2O 0.5g, NaCl 0.2g, CaCO 31g, MgSO 47H 2O 0.2g, distilled water 1000ml, pH 7.0～7.2.
The ACCC55 vinelandii solid medium composition of improvement: sucrose 10g, K 2HPO 43H 2O 0.5g, NaCl 0.2g, CaCO 31g, MgSO 47H 2O 0.2g, yeast extract paste 0.5g, distilled water 1000ml, agar 1.5%～2.0%, pH 7.0～7.2.
Nitrogen-free agar: sucrose 10g, NaCl 0.12g, K 2HPO 43H 2O 0.5g, CaCO 31g, MgSO 47H 2O 0.2g, distilled water 1000mL, pH7.2.
DF substratum: KH 2PO 44.0g, Na 2HPO 46.0g, MgSO 47H 2O 0.2g, glucose 2.0g, Sunmorl N 60S 2.0g, citric acid 2.0g, (NH 4) 2SO 42.0g, component one, each 0.1ml of component two solution, H 2O 1000mL, pH 7.2; Wherein component one: H 3BO 310mg, MnSO 4H 2O 11.19mg, ZnSO 47H 2O 124.6mg, CuSO 45H 2O 78.22mg, MoO 310mg is dissolved in the 100mL sterile purified water; Component two: FeSO 47H 2O 100mg is dissolved in the 10mL sterile purified water.
The ADF substratum: ACC (1-amino-cyclopropane-1-carboxylic acid) is dissolved in ultrapure water, and filtration sterilization is added to and does not contain (NH 4) 2SO 4Sterilization DF substratum in, final concentration is 3.0mM.
Azotobacter chroococcum (Azotobacter chroococcum) ACCC11103 (referring to " Sun Jianguang etc. the screening of high-efficiency nitrogen-fixing genus bacillus and biological characteristics thereof. Scientia Agricultura Sinica, 2009,42 (6): 2043-2051 ").
Gibberellic hypha-Gibberella zeae bacterium (Gibberella zeae) (Chinese agriculture microbial strains preservation administrative center, ACCC31053).
Sclerotium germ-sclerotinite (Sclerotinia sclerotiorum) (Chinese agriculture microbial strains preservation administrative center, ACCC30046).
Verticillium dahliae-Garden Dahlia wheel branch spore (Verticillium dahliae) (Chinese agriculture microbial strains preservation administrative center, ACCC30308).
Separation and the evaluation of embodiment 1, soybean rhizosphere vinelandii GD571
One, the enrichment of soybean rhizosphere vinelandii with separate
Getting 10g soil sample (picking up from China Dark Longjiang) puts into 90ml sterilized water shaking table vibration 20min and makes dirty solution, draw 5ml and put into 30ml vinelandii enrichment culture liquid ACCC55,100rpm, 28 ℃ are carried out the shaking table shaking culture, renew bright nutrient solution behind the 72h and continue to cultivate.Carrying out the fixed nitrogen sporeformer after repeating to cultivate 3 times separates.The above-mentioned vinelandii enrichment culture of absorption 1ml thing is put in the 9ml sterilized water makes 10 -1Extent of dilution continues dilution and makes 10 -2, 10 -3, 10 -4, 10 -5Dilution bacteria suspension heats 10min in 100 ℃ of boiling water, rear each extent of dilution of cooling is got 0.1ml and is coated on the vinelandii isolation medium flat board, and 29 ℃ leave standstill cultivation.2～3d carries out purifying agaric with plate streak on the ACCC55 vinelandii culture medium flat plate of improvement after bacterium colony forms.
Two, acc deaminase positive strain screening
Soybean rhizosphere vinelandii to the step 1 gained carry out the screening of acc deaminase positive strain, and concrete grammar is as described below: the interior azotobacter that is separated to, and in the access 5mL liquid nitrogen-free agar, 30 ℃, 200r/min shaking culture 24h; Draw above-mentioned nutrient solution 0.1mL and be seeded to 5mL DF substratum shaking culture 24h; Draw above-mentioned nutrient solution 0.1mL and be seeded to shaking culture 24～48h in the 5mL ADF substratum; The bacterial classification that to grow in ADF repeats switching, cultivates, and with the ADF substratum as negative control, the bacterial strain that can grow take ACC as only nitrogen source is the acc deaminase positive strain.Strain acc deaminase positive strain called after soybean rhizosphere vinelandii GD571 with the screening gained.
Three, the evaluation of soybean rhizosphere vinelandii GD571
Also screen the soybean rhizosphere vinelandii GD571 that obtains from the following aspects authentication step one and two separation and purification:
1, Morphological Identification
To be in logarithmic phase, and the bacterium colony size is stable, the vinelandii GD571 that above-mentioned steps one is separated and purifying obtains carries out single bacterium colony state description, mainly comprises size, color, transparency, wettability, bacterium colony condition of surface (whether smooth, projection, fold, depression etc.), the colony edge state (whether neat, irregular, radial etc.) of bacterium colony.
For the described vinelandii GD571 that is in logarithmic phase, behind smear staining, adopt the form of observation by light microscope thalline.
The result shows, the circular umbilical projection of the vinelandii GD571 bacterium colony that above-mentioned steps one and two separation and purification and screening obtain, and oyster white, glossy, smooth surface is moistening, neat in edge; The thalline oval, 0.5 * 1.0 μ m, Gram-negative.
2, analysis of physio biochemical characteristics
With reference to " common bacteria system identification handbook (eastern elegant pearl, Cai Miaoying. common bacteria system identification handbook. Beijing: Science Press, 2011.) and " Microbiology Experiment " (Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiment (third edition). Beijing: Higher Education Publishing House, 1999.) measure the physiological and biochemical property of above-mentioned vinelandii GD571.
The physiological and biochemical property measurement result of described vinelandii GD571 is as follows:
Catalase reaction: feminine gender
Oxydase reaction: feminine gender
Growth temperature: 4 ℃ of growths, 28 ℃ of growths, 37 ℃ of growths or not for 60 ℃
The salt tolerance test: the 2%NaCl growth, 5%NaCl does not grow, and 7%NaCl does not grow, and 10%NaCl does not grow
Phenylalanine deaminase test: feminine gender
Utilize Citrate trianion: the positive
Hydrolyzed starch: feminine gender
Yolk lecithin enzyme: the positive
Methyl red test: the positive
V.P test: the positive
PH 5.7 growths: the positive
The sugar alcohol fermentation and acid: glucose is positive, and N.F,USP MANNITOL is positive, and lactose is positive, and sucrose is positive
3,16s rDNA sequence homology analysis
Ordinary method is cultivated the vinelandii GDSD571 that above-mentioned steps one and two separation and purification and screening obtain, extract total DNA of bacterial strain as the gene amplification template, with bacterium 16s rDNA universal primer, 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492r:5 '-TACGGTTACCTTGTTACGACTT-3 ' carry out the PCR reaction.Reaction system adopts Shanghai biotechnology company limited pcr amplification test kit.Response procedures is: 95 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ of extensions 2min, totally 30 circulations.Dna sequencing is finished by Beijing three rich polygala root biotech companies, sequence assembly and similarity analysis use DNAStar software to finish, and sequence alignment is finished online by American National biotechnology information center ncbi database (http://www.ncbi.nlm.nih.gov).
The sequence of soybean rhizosphere vinelandii GD571 bacterial strain 16s rDNA sees sequence 1 in the sequence table for details.
4, growth characteristics analysis
Bacterial strain optimum temperuture and optimal pH growth experiment have been carried out.Adopt nitrogen-free agar, at the thermal adaptability of 4 ℃, 28 ℃, 37 ℃, 60 ℃ cultivations, observation, record bacterial strain, each is processed 3 times and repeats respectively.Adjust acidity and be respectively pH3, pH4, pH5, pH6, pH7, pH8, pH9, pH10, pH11, each is processed 3 times and repeats, the optimal pH of cultivation, observation, record strain growth.
The result shows that the optimum growth temperature of described soybean rhizosphere vinelandii GD571 is 28 ℃, and the most suitable growth pH is pH7～8.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis result, the soybean rhizosphere vinelandii GD571 that step 1 and two separation and purification and screening are obtained is accredited as modification bacterium β subgroup bulkholderia cepasea section bulkholderia cepasea and belongs to (Burkholderia sp.).This soybean rhizosphere vinelandii GD571 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 6th, 2011 and (is called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.5039.
Embodiment 2, bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 nitrogenase activity determination
Embodiment 1 resulting bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 is carried out nitrogenase activity determination, concrete grammar is as described below: add 5ml improvement fixed nitrogen substratum bevel in 15 * 150mm screw socket Glass tubing, inoculation bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039,28 ℃ of cultivations.With the positive contrast of microbial fertilizer production commonly used bacterial classification azotobacter chroococcum (Azotobacter chroococcum) ACCC11103, do not inoculate the negative contrast in blank inclined-plane simultaneously, establish 3 repetitions.After cultivating 72h, change rubber plug, it is 10% that the injection acetylene gas makes final concentration, seal with medical proof fabric, continue to cultivate 72h, get 100 μ l reactant gasess, with gas chromatograph for determination ethene growing amount, calculate the nitrogenase activity of bacterial strain (bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 or azotobacter chroococcum (Azotobacter chroococcum) ACCC11103) according to following formula.Nitrogenase activity (nmol/mgh)=C 2H 4Nmol/[tropina amount (mg) * reaction times (h)], C wherein 2H 4Nmol=1000 * C 2H 4Volume (μ l) * 273 * P/[22.4 * (273+t) * 760], wherein P is air pressure (mm mercury column), t be temperature of reaction (℃).
Wherein, the tropina content assaying method is as described below: with 5ml physiological saline the lawn on the test tube slant is washed in the centrifuge tube, collect thalline (bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 or azotobacter chroococcum (Azotobacter chroococcum) ACCC11103), the NaOH boiling water that adds 3ml 0.5M in the precipitation boils 5min, the HCl that adds 3ml 0.5M mixes, get supernatant 1.0ml after centrifugal, add 5ml Xylene Brilliant Cyanine G solution, mix at eddy mixer, colour developing 3min, the light absorption value A at mensuration 595nm place 595, calculate tropina content according to the bovine serum albumin typical curve.
The result shows that the nitrogenase activity of the bulkholderia cepasea that screens (Burkholderia sp.) GD571 CGMCC No.5039 is 32.268nmol C 2H 4/ hmg albumen, statistical study are significantly higher than the microbial fertilizer nitrogenase activity 25.100nmol/hmg albumen of producing bacterial classification azotobacter chroococcum ACCC11103 commonly used, as shown in Figure 1.This result shows that bulkholderia cepasea of the present invention (Burkholderia sp.) GD571 CGMCC No.5039 can high-efficiency nitrogen-fixing.
Embodiment 3, bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 antagonizing pathogenic fungi bacteriostasis rate are measured
Adopting 2 face-off methods that embodiment 1 resulting bulkholderia cepasea (Burkholderia sp.) GD571CGMCC No.5039 is carried out the antagonizing pathogenic fungi bacteriostasis rate measures, concrete operations are as described below: inoculate respectively crop gibberellic hypha (Gibberella zeae) (perhaps sclerotium germ (Sclerotinia sclerotiorum) on 2 of distance center 2cm on the PDA flat board, perhaps verticillium dahliae (Verticillium dahliae)) and bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039,3 repetitions of each Screening Treatment do not connect the flat board of bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 as contrast only to connect pathogenic fungi.28 ℃ of constant temperature culture are measured the dull and stereotyped upper pathogenic fungi of face-off along the colony radius r of tested bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 direction with the millimeter graduated scale behind the 15d 1, and the colony radius r of the dull and stereotyped upper pathogenic fungi of contrast 0Pathogenic fungi growth inhibition ratio (%)=(contrast radius r 0Pathogenic fungi colony radius r is cultivated in-face-off 1)/contrast radius r 0* 100%.
The result shows, the r of gibberellic hypha (Gibberella zeae) 063.3 ± 2.8mm, r 133.9 ± 2.3mm; The r of sclerotium germ (Sclerotinia sclerotiorum) 063.0 ± 2.1mm, r 131.3 ± 2.5mm; The r of verticillium dahliae (Verticillium dahliae) 041.0 ± 1.9mm, r 125.0 ± 1.5mm.The above-mentioned formula of mean value substitution is calculated: described bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 is 46.45% to the bacteriostasis rate of gibberellic hypha (Gibberella zeae), as shown in Figure 2; Bacteriostasis rate to sclerotium germ (Sclerotinia sclerotiorum) is 50.32%, as shown in Figure 3; Bacteriostasis rate to verticillium dahliae (Verticillium dahliae) is 39.02%, as shown in Figure 4.This result shows described bulkholderia cepasea (Burkholderia sp.) GD571CGMCC No.5039 effectively antagonism crop gibberellic hypha (Gibberella zeae), sclerotium germ (Sclerotinia sclerotiorum) and verticillium dahliae (Verticillium dahliae).
Embodiment 4, bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.50391-amino-cyclopropane-1-carboxylic acid (ACC) deaminase activity determination
Embodiment 1 resulting bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 is carried out the acc deaminase determination of activity, concrete grammar is as described below: ACCC11103 organizes in contrast with azotobacter chroococcum (Azotobacter chroococcum), and bulkholderia cepasea (Burkholderia sp.) the GD571 CGMCC No.5039 of embodiment 1 screening gained is as experimental group., draw the 0.5ml nutrient solution and be inoculated in the 60ml nutrient solution without nitrogen liquid nutrient medium activation bacterial strain with 5ml, cultivate 24～48h for 30 ℃, 4 ℃, the centrifugal 10min collection of 8000rpm thalline do not contain (NH with 15ml 4) 2SO 4DF liquid nutrient medium centrifuge washing thalline 2 times, thalline is resuspended in the 24ml ADF substratum, cultivate 24h for 30 ℃, collect also record thalline weight.With 0.1M Tris-HCl damping fluid (pH 7.6) centrifuge washing thalline 2 times, with the thalline average mark in 3 EP pipes ,-20 ℃ of storages.Get the storage thalline and be resuspended in 1ml 0.1M Tris-HCl damping fluid (pH 7.6), the centrifugal 5min of 12000rmp collects thalline, be resuspended in the 600 μ l0.1M Tris-HCl damping fluids (pH 8.5), add 30 μ l toluene, the 30s smudge cells vibrates rapidly, get 4 ℃ of storages of 100 μ l crude enzyme liquids, be used for measuring protein concentration; All the other crude enzyme liquids carry out the acc deaminase determination of activity.Get crude enzyme liquid 200 μ l and add the ACC solution mixing that 20 μ l concentration are 0.5M, place 30 ℃ of water-bath 15min, add 1ml 0.56MHCl termination reaction, the centrifugal 5min of 12000rmp, get supernatant 1ml, add 800 μ l 0.56M HCl and 300 μ l 0.2%2,4-dinitrobenzene hydrazine solution (dissolving among the 2M HCl), 30 ℃ of insulation 30min; Add 2ml 2M NaOH mixing, 540nm surveys absorbance.Contrast α-ketone butyric acid typical curve and protein determination typical curve calculate the enzymic activity of bacterial strain.The acc deaminase method for expressing is: under the reaction conditions, every milligram of tropina per hour catalysis ACC deamination forms micromole's number of α-batanone acid, and unit is (μ mol α-batanone acid/hmg albumen).Method of protein measurement is with embodiment 2.Measurement result is 3 repetition mean values.
The result shows, the acc deaminase activity of described bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 is 5.393 μ mol α-batanone acid/hmg albumen, far above the enzymic activity of control group azotobacter chroococcum (Azotobacter chroococcum) ACCC11103, as shown in Figure 5.This result shows, described bulkholderia cepasea (Burkholderia sp.) GD571 CGMCC No.5039 has the acc deaminase activity, and then can improve the potential that crop is resisted the adverse environmental factors such as arid, waterflooding, high temperature, physical abuse, disease and pest invasion and attack or heavy metal contamination.
Figure IDA0000075929810000011
Figure IDA0000075929810000021

Claims (10)

1. bulkholderia cepasea (Burkholderia sp.) GD571, its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.5039.
2. microbial inoculum, its activeconstituents is bulkholderia cepasea claimed in claim 1 (Burkholderia sp.) GD571CGMCC No.5039.
3. microbial inoculum according to claim 2, it is characterized in that: described microbial inoculum is following 1)-9) in arbitrary microbial inoculum:
1) for the microbial inoculum that suppresses pathogenic fungi;
2) for the microbial inoculum of preventing and treating fungal diseases of plants;
3) be used for the microbial inoculum of fixed nitrogen;
4) microbial inoculum of Promoting plant growth;
5) suppress the microbial inoculum that plant produces ethene;
6) reduce plant to the microbial inoculum of adverse circumstance susceptibility;
7) microbial inoculum of raising stress resistance of plant;
8) microbial inoculum of generation 1-Aminocyclopropane-1-carboxylate deaminase;
9) microbial inoculum of generation nitrogenase;
Described pathogenic fungi is fungi or fungi or the fungi for causing verticillium for causing sclerotium disease that causes head blight; Described fungal diseases of plants is head blight, sclerotium disease or verticillium; Described adverse circumstance is arid, waterflooding, high temperature, physical abuse, disease and pest invasion and attack or heavy metal contamination.
4. bulkholderia cepasea claimed in claim 1 (Burkholderia sp.) GD571CGMCC No.5039 is in preparation following 1)-9) in application in the arbitrary microbial inoculum:
1) for the microbial inoculum that suppresses pathogenic fungi;
2) for the microbial inoculum of preventing and treating fungal diseases of plants;
3) be used for the microbial inoculum of fixed nitrogen;
4) microbial inoculum of Promoting plant growth;
5) suppress the microbial inoculum that plant produces ethene;
6) reduce plant to the microbial inoculum of adverse circumstance susceptibility;
7) microbial inoculum of raising stress resistance of plant;
8) microbial inoculum of generation 1-Aminocyclopropane-1-carboxylate deaminase;
9) microbial inoculum of generation nitrogenase;
Described pathogenic fungi is fungi or fungi or the fungi for causing verticillium for causing sclerotium disease that causes head blight; Described fungal diseases of plants is head blight, sclerotium disease or verticillium; Described adverse circumstance is arid, waterflooding, high temperature, physical abuse, disease and pest invasion and attack or heavy metal contamination.
5. microbial inoculum according to claim 3 is characterized in that: describedly cause that the fungi of head blight is Gibberella zeae bacterium (Gibberella zeae) or Fusarium graminearum (Fusarium graminearum); Describedly cause that the fungi of sclerotium disease is sclerotinite (Sclerotinia sclerotiorum); Describedly cause that the fungi of verticillium is verticillium dahliae (Verticillium dahliae).
6. application according to claim 4 is characterized in that: describedly cause that the fungi of head blight is Gibberella zeae bacterium (Gibberella zeae) or Fusarium graminearum (Fusarium graminearum); Describedly cause that the fungi of sclerotium disease is sclerotinite (Sclerotinia sclerotiorum); Describedly cause that the fungi of verticillium is verticillium dahliae (Verticillium dahliae).
7. bulkholderia cepasea claimed in claim 1 (Burkholderia sp.) GD571CGMCC No.5039 or claim 2,3 or 5 described microbial inoculums are used in producing nitrogenase or 1-Aminocyclopropane-1-carboxylate deaminase; Or,
Bulkholderia cepasea claimed in claim 1 (Burkholderia sp.) GD571CGMCC No.5039 or claim 2, the application of 3 or 5 described microbial inoculums in preparation diazotroph fertilizer.
8. the diazotroph fertilizer that contains the described bulkholderia cepasea of claim 1 (Burkholderia sp.) GD571CGMCC No.5039 or claim 2,3 or 5 described microbial inoculums.
9. cultivate the method for bulkholderia cepasea (Burkholderia sp.) GD571CGMCC No.5039, comprise the step that bulkholderia cepasea (Burkholderia sp.) GD571CGMCC No.5039 is cultivated at the substratum that is used for the cultivation bulkholderia cepasea.
10. the preparation method of claim 2,3 or 5 described microbial inoculums comprises the steps: bulkholderia cepasea claimed in claim 1 (Burkholderia sp.) GD571CGMCC No.5039 is obtained described microbial inoculum as activeconstituents.
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