CN102876596B - Sclerotinia sclerotiorum-antagonizing nitrogen-fixing spore bacterium and its application - Google Patents

Sclerotinia sclerotiorum-antagonizing nitrogen-fixing spore bacterium and its application Download PDF

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CN102876596B
CN102876596B CN 201110197582 CN201110197582A CN102876596B CN 102876596 B CN102876596 B CN 102876596B CN 201110197582 CN201110197582 CN 201110197582 CN 201110197582 A CN201110197582 A CN 201110197582A CN 102876596 B CN102876596 B CN 102876596B
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nitrogen
gdsd212
bacillus
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CN102876596A (en
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周剑浩
孙建光
江峰
徐晶
张友恒
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Shanghai Guohuan Ecological Technology Co.,Ltd.
Institute of Agricultural Resources and Regional Planning of CAAS
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Shanghai Hignken Ecology Technology Co ltd
Institute of Agricultural Resources and Regional Planning of CAAS
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Abstract

The invention discloses a Sclerotinia sclerotiorum-antagonizing nitrogen-fixing spore bacterium and its application. The nitrogen-fixing spore bacterium is Bacillus sp. GDSD212, which is preserved in General microbiological center of China Committee for Culture Collection of Microorganisms with a preservation number of CGMCC No.5035. The Bacillus sp. GDSD212 CGMCC No.5035 provided in the invention has high nitrogen-fixing activity, can effectively antagonize Sclerotinia sclerotiorum, and has a broad application prospect in production of nitrogen-fixing microorganism bacterial agents and bio-organic fertilizers.

Description

Fixed nitrogen sporeformer of antagonism sclerotium germ and uses thereof
Technical field
The present invention relates to fixed nitrogen sporeformer and the application thereof of antagonism sclerotium germ.
Background technology
Nitrogenous fertilizer is one of most important production means in 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 and causes the chemical nitrogen fertilizer price increase with the coal price lattice, 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, guarantee China grain security, but the unreasonable nitrogen of executing has formed serious pollution of area source, causes a series of serious harm, as the Taihu Lake Blue Algae Event makes us startling.The urban groundwater azotate pollution has caused threat to drinking water safety.
Contain 78% nitrogen in 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 atmosphere, by 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 with other material Compound Machining that is rich in plant nutrition, become biological products, 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 is only 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.
Sclerotinite (Sclerotinia sclerotiorum) be the pathogenic bacteria of sclerotium disease, morphological specificity shows as: apothecium is little, be little cup-shaped, light salmon is to brown, single or severally from sclerotium, bears, diameter 0.5-1cm, the handle brown is elongated, bending, long 3-5cm, gradually thin downwards, with sclerotium, be connected.Mycelium can form sclerotium, and the brown apothecium of long handle is created on 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, linear, colourless, and 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.At present the control of sclerotium disease is mainly relied on to chemical pesticide, yet not only cost is high, contaminate environment in chemical prevention, and preventive effect is also undesirable, the security of the food is simultaneously also had a strong impact on.
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.The microbial fertilizer production bacterial classification that agriculture production active demand nitrogen fixation efficiency is high, antagonizing pathogenic fungi, strong stress resistance, shelf-lives are long.
Summary of the invention
The purpose of this invention is to provide a strain can carry out high-efficiency nitrogen-fixing at the farm crop rhizosphere, and the bacterium of antagonism sclerotinite (Sclerotinia sclerotiorum).
Bacterium provided by the present invention is genus bacillus (Bacillus sp.) GDSD212, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 6th, 2011 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.5035.
Another object of the present invention is to provide a kind of microbial inoculum, and the activeconstituents of this microbial inoculum is described genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035.
This microbial inoculum, except genus bacillus (Bacillus sp.) the GDSD212 CGMCC No.5035 comprised as activeconstituents, also can comprise auxiliary material, 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.
This microbial inoculum can be used for suppressing pathogenic fungi, control fungal diseases of plants, fixed nitrogen, generation nitrogenase etc.
Described genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 is in preparation following 1)-4) in application in arbitrary microbial inoculum also belong to protection scope of the present invention:
1) for suppressing the microbial inoculum of pathogenic fungi;
2) for preventing and treating the microbial inoculum of fungal diseases of plants;
3) for the microbial inoculum of fixed nitrogen;
4) produce the microbial inoculum of nitrogenase.
Described pathogenic fungi, for the fertilizer by soil, in being manured into soil and/or the fungi of seed dispersal, specifically can be the fungi that causes sclerotium disease; Described fungal diseases of plants can be sclerotium disease.
Describedly cause that the fungi of sclerotium disease can be sclerotinite (Sclerotinia sclerotiorum).
The application of the microbial inoculum that described genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 or genus bacillus (Bacillus sp.) the GDSD212 CGMCC No.5035 of take are activeconstituents in producing nitrogenase and preparing biological organic fertilizer in application also belong to protection scope of the present invention.
A further object of the present invention is to provide a kind of biological organic fertilizer that contains genus bacillus (Bacillus sp.) GDSD212 CGMCCNo.5035 or take the microbial inoculum that genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 is activeconstituents.
Another purpose of the present invention is to provide the method for cultivation genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 a kind of, comprises the step that genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 is cultivated at the substratum for cultivating genus bacillus.
Another purpose of the present invention is to provide a kind of method for preparing described microbial inoculum, and the method comprises the steps: to obtain described microbial inoculum using described genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 as activeconstituents.
Experimental results show that, the present invention is through screening layer by layer from pedotheque, finishing screen has been selected genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035, this bacterial strain has very high nitrogenase activity, can antagonism sclerotium disease pathogenic bacteria sclerotinite (Sclerotinia sclerotiorum), compete adaptablely, effect of inoculation is good, in nitrogen-fixing microorganism microbial inoculum and biological organic fertilizer production, has broad application prospects.
The preservation explanation
Strain name: genus bacillus
Latin name: (Bacillus sp.)
Strain number: GDSD212
Preservation mechanism: China Committee for Culture Collection of Microorganisms's 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.5035
The accompanying drawing explanation
The nitrogenase activity that Fig. 1 is genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035.
Fig. 2 is genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 antagonism sclerotium germ (Sclerotinia sclerotiorum).The left side bacterium colony is pathogenic fungi sclerotinite (Sclerotinia sclerotiorum), and the right side bacterium colony is genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
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.
Improvement fixed nitrogen Media Components: 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%, pH7.0～7.2.
Nitrogen-free agar: sucrose 10g, NaCl 0.12g, K 2hPO 43H 2o 0.5g, CaCO 31g, MgSO 47H 2o0.2g, distilled water 1000mL, pH7.2.
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 ").
Sclerotium germ-sclerotinite (Sclerotinia sclerotiorum) (Chinese agriculture microbial strains preservation administrative center, ACCC30046).
The separation of embodiment 1, vinelandii GDSD212 and evaluation
One, the enrichment of vinelandii GDSD212 with separate
Getting 10g soil sample (agricultural land soil that the Heilongjiang Province of China nitrogen nutrition is relatively barren) 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 after 72h and continue to cultivate.Carry out the separation of fixed nitrogen sporeformer after repeating to cultivate 3 times.The above-mentioned vinelandii enrichment culture of absorption 1ml thing is put in the 9ml sterilized water makes 10 -1extent of dilution, continue dilution and make 10 -2, 10 -3, 10 -4, 10 -5dilution bacteria suspension heats 10min in 100 ℃ of boiling water, and cooling rear each extent of dilution is got 0.1ml and is coated on vinelandii isolation medium flat board, 29 ℃ of standing cultivations.2～3d, after bacterium colony forms, on the ACCC55 vinelandii culture medium flat plate of improvement, carries out purifying agaric with plate streak.Also one of them bacterial strain called after vinelandii GDSD212 of purifying gained will be separated.
Two, the evaluation of vinelandii GDSD212
Separate from the following aspects authentication step one the vinelandii GDSD212 that also purifying obtains:
1, Morphological Identification
Will be in logarithmic phase, and the bacterium colony size is stable, the vinelandii GDSD212 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 GDSD212 in logarithmic phase, adopt the form of observation by light microscope thalline after smear staining.
Result shows, the vinelandii GDSD212 bacterium colony circular protrusion that above-mentioned steps one is separated and purifying obtains, and shallow oyster white, glossy, smooth surface is moistening, neat in edge; Thalline is shaft-like, and 0.5 * 2.0～3.0 μ m, have gemma.
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 GDSD212.
The physiological and biochemical property measurement result of described vinelandii GDSD212 is as shown in table 1:
The physiological and biochemical property of table 1 vinelandii GDSD212
Figure BDA0000075929580000051
Annotate: "+" means positive, and "-" means negative.
3,16s rDNA sequence homology analysis
Ordinary method is cultivated the vinelandii GDSD212 that above-mentioned steps one separation and purification obtains, 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 ' carries 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 completed by Beijing three rich polygala root biotech company, sequence assembly and similarity analysis are used DNAStar software to complete, and sequence alignment completes online by American National biotechnology information center ncbi database (http://www.ncbi.nlm.nih.gov).
The sequence of fixed nitrogen sporeformer GDSD212 bacterial strain 16s rDNA refers to sequence 1 in sequence table.
4, growth characteristics analysis
Bacterial strain optimum temperuture and optimal pH growth experiment have been carried out.Adopt nitrogen-free agar respectively at 4 ℃, 28 ℃, 37 ℃, the 60 ℃ thermal adaptabilities of cultivating, observing, record bacterial strain, each is processed 3 times and repeats.Adjust acidity and be respectively pH3, pH4, pH5, pH6, pH7, pH8, pH9, pH10, pH11, each is processed 3 times and repeats, and cultivates, observes, records the optimal pH of strain growth.
Result shows, the optimum growth temperature of described vinelandii GDSD212 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 vinelandii GDSD212 that step 1 is separated and purifying obtains is accredited as to modification bacterial alpha subgroup Bacillaceae bacillus (Bacillus sp.).This vinelandii GDSD212 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 6th, 2011, and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.5035.
Embodiment 2, genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 nitrogenase activity determination
Genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 to embodiment 1 gained carries out nitrogenase activity determination, concrete operations are as described below: add 5ml improvement fixed nitrogen substratum bevel in 15 * 150mm screw socket Glass tubing, inoculation genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035,28 ℃ 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, establish 3 repetitions.After cultivating 72h, change rubber plug, it is 10% that the injection acetylene gas makes final concentration, with the medical proof fabric sealing, continues to cultivate 72h, gets 100 μ l reactant gasess, with gas chromatograph for determination ethene growing amount, according to the nitrogenase activity of following formula calculating bacterial strain.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 test tube slant is washed in centrifuge tube, collect thalline, in precipitation, add the NaOH boiling water of 3ml 0.5M to boil 5min, add the HCl of 3ml 0.5M to mix, get supernatant 1.0ml after centrifugal, add 5ml Xylene Brilliant Cyanine G solution, mix colour developing 3min, the light absorption value A at mensuration 595nm place on eddy mixer 595, according to the bovine serum albumin typical curve, calculate tropina content.
The result demonstration, the nitrogenase activity of the genus bacillus screened (Bacillus sp.) GDSD212 CGMCC No.5035 is 68.403nmol C 2h 4/ hmg albumen, statistical study is significantly higher than the microbial fertilizer nitrogenase activity 25.100nmol C that produces bacterial classification azotobacter chroococcum ACCC11103 commonly used 2h 4/ hmg albumen, as shown in Figure 1.This result shows, genus bacillus of the present invention (Bacillus sp.) GDSD212 CGMCC No.5035 energy high-efficiency nitrogen-fixing.
Embodiment 3, genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 antagonizing pathogenic fungi bacteriostasis rate are measured
Adopt 2 face-off methods to carry out antagonizing pathogenic fungi bacteriostasis rate mensuration to the resulting genus bacillus of embodiment 1 (Bacillus sp.) GDSD212 CGMCC No.5035, concrete operations are as described below: inoculate respectively crop pathogenic fungi sclerotinite (Sclerotinia sclerotiorum) and genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 on the PDA flat board on two of distance center 2cm, 3 repetitions of each Screening Treatment, take that only to connect the flat board that pathogenic fungi do not connect vinelandii be contrast.28 ℃ of constant temperature culture, measure the colony radius r of the dull and stereotyped upper pathogenic fungi of face-off along tested genus bacillus (Bacillus sp.) GDSD212 CGMCC No.5035 direction with the millimeter graduated scale after 15d 1, and the colony radius r of the dull and stereotyped upper pathogenic fungi of contrast 0.Pathogenic fungi growth inhibition ratio (%)=(contrast radius r 0pathogenic fungi colony radius r is cultivated in-face-off 1)/contrast radius r 0* 100%.
Result shows, the r of sclerotium germ (Sclerotinia sclerotiorum) 063.0 ± 2.Imm, r 114.67 ± 1.13mm.Mean value is calculated for the above-mentioned formula of people: described genus bacillus (Bacillus sp.) GDSD212 CGMCCNo.5035 is 76.71% to the bacteriostasis rate of sclerotium germ (Sclerotinia sclerotiorum), as shown in Figure 2.
Figure IDA0000075929670000011
Figure IDA0000075929670000021

Claims (1)

  1. Genus bacillus ( bacillussp.) GDSD212 CGMCC No. 5035 is in preparation following 1) or 2) in application in arbitrary microbial inoculum:
    1) for suppressing the microbial inoculum of pathogenic fungi;
    2) for preventing and treating the microbial inoculum of fungal diseases of plants;
    Described pathogenic fungi is the fungi that causes sclerotium disease; Described fungal diseases of plants is sclerotium disease;
    The described fungi that causes sclerotium disease be sclerotinite ( sclerotinia sclerotiorum).
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CN108484285A (en) * 2018-06-01 2018-09-04 葛江丽 A kind of dedicated disease-resistant multi-functional bacterial manure of fixed nitrogen of the Northeast's rice
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CN1580242A (en) * 2004-05-18 2005-02-16 北京生太平生物工程技术有限公司 Laterosporo short bacillus, and its fermentating process and use

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