CN102250810B - Paddy rice endogenic nitrogen-fixing bacterium resisting to gibberella zeae and sclerotinia sclerotiorum and application thereof - Google Patents
Paddy rice endogenic nitrogen-fixing bacterium resisting to gibberella zeae and sclerotinia sclerotiorum and application thereof Download PDFInfo
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Abstract
The invention discloses a paddy rice endogenic nitrogen-fixing bacterium antagonistic to gibberella zeae and sclerotinia sclerotiorum and application thereof. The paddy rice endogenic azotobacterium is bacillus sp. GDSD223 which is collected in the China General Microbiological Culture Collection Center with the collection number CGMCC No.5036. The bacillus sp. GDSD223 CGMCC No.5036 provided by the invention has higher nitrogen-fixing activity, can be effectively antagonistic to gibberella zeae and sclerotinia sclerotiorum, and has wide application prospect to the production of nitrogen-fixing microbial agents and biological organic fertilizers.
Description
Technical field
The present invention relates to interior azotobacter of paddy rice of antagonism gibberellic hypha and sclerotium germ and uses thereof.
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 that Gibberella zeae bacterium (Gibberella zeae) 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 sclerotium germ (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.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.
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 can in the paddy rice body, carry out high-efficiency nitrogen-fixing, and the bacterium of antagonism crop head blight pathogenic bacteria Gibberella zeae bacterium (Gibberella zeae) and sclerotium disease pathogenic bacteria sclerotinite (Sclerotinia sclerotiorum).
Azotobacter in the paddy rice provided by the present invention, genus bacillus (Bacillus sp.) GDSD223, 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.5036.
Another object of the present invention provides a kind of microbial inoculum, and the activeconstituents of this microbial inoculum is described genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036.
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 genus bacillus (Bacillus sp.) the GDSD223 CGMCC No.5036 that comprises as activeconstituents.
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.) GDSD223 CGMCC No.5036 is in preparation following 1)-4) 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 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 the fungi for causing sclerotium disease that cause head blight; Described fungal diseases of plants can be head blight or sclerotium disease.
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).
Application and the application in the preparation biological organic fertilizer in producing nitrogenase of described genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 or the microbial inoculum take genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 as activeconstituents also belongs to protection scope of the present invention.
A further object of the present invention provides a kind of genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 or biological organic fertilizer take genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 as the microbial inoculum of activeconstituents of containing.
Another purpose of the present invention provides the method for a kind of cultivation genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036, and the method comprises the step that genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 is cultivated in cultivating bacillus culture medium.
Another purpose of the present invention provides a kind of method for preparing described microbial inoculum, and the method comprises the steps: described genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 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 genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036, this bacterial strain has very high nitrogenase activity, can antagonism crop head blight pathogenic bacteria Gibberella zeae bacterium (Gibberella zeae) and sclerotium disease pathogenic bacteria sclerotinite (Sclerotinia sclerotiorum), 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: genus bacillus
Latin name: (Bacillus sp.)
Strain number: GDSD223
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.5036
Description of drawings
Fig. 1 is the nitrogenase activity of genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036.
Fig. 2 is genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 antagonism Gibberella zeae bacterium (Gibberella zeae).The left side bacterium colony is Gibberella zeae bacterium (Gibberella zeae), and the right side bacterium colony is genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036.
Fig. 3 is genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 antagonism sclerotium germ (Sclerotinia sclerotiorum).The left side bacterium colony is sclerotium germ (Sclerotinia sclerotiorum), and the right side bacterium colony is genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036.
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.
Many carbon sources are hanged down nitrogen substratum (CCM): solution I: KH
2PO
40.2g, NaCl 0.1g, K
2HPO
40.8g, Na
2FeEDTA 28mg, Sodium orthomolybdate 25mg, yeast extract 100mg, N.F,USP MANNITOL 5g, sucrose 5g, Sodium.alpha.-hydroxypropionate 0.5mL, distilled water 900mL.Solution II: MgSO
47H
2O 0.2g, CaCl
22H
2O 0.06g, distilled water 100mL.Solution I, II are sterilized respectively, be cooled to about 50 ℃ and mix, add vitamin H (5 μ g/L) and each 0.5mL of VITAMIN (10 μ g/L).
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.
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.
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 (Sclerotinia sclerotiorum) (Chinese agriculture microbial strains preservation administrative center, ACCC30046).
Separation and the evaluation of azotobacter GDSD223 in embodiment 1, the paddy rice
One, the separation of azotobacter GDSD223 in the paddy rice
The concrete operations of the separation of azotobacter are as follows in the paddy rice: get fresh water rice plants (picking up from Heilongjiang Province of China), at first rinse well with tap water, then use successively 70% alcohol immersion 1min, 2% clorox surface sterilization sterilization 10min, aseptic water washing 3 times.Under the aseptic technique, accurately take by weighing sample 10.0g, in aseptic mortar, wear into pasty state, shift, be settled to 100ml, continue dilution and make series of diluted samples, respectively from 10
-4, 10
-5, 10
-6Get 0.1ml in the diluent and be uniformly coated on respectively on above-mentioned CCM substratum and the nitrogen-free agar flat board, be inverted for 28 ℃ and cultivate, behind 3～4d, picking list bacterium colony line purifying obtains azotobacter in the paddy rice.Last washing water are coated on to detect on the beef-protein medium and confirm the Plant samples sterilization thoroughly during simultaneously, with surface sterilization.To the wherein interior azotobacter GDSD223 of the separating obtained vinelandii called after of strain paddy rice.
Two, the evaluation of azotobacter GDSD223 in the paddy rice
Azotobacter GDSD223 in the paddy rice that the following aspects authentication step one is separated and purifying obtains:
1, Morphological Identification
To be in logarithmic phase, and the bacterium colony size is stable, the vinelandii GDSD223 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 GDSD223 that is in logarithmic phase, behind smear staining, adopt the form of observation by light microscope thalline.
The result shows, the circular tiling of the vinelandii GDSD223 bacterium colony that above-mentioned steps one is separated and purifying obtains, and oyster white, glossy, smooth surface, 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 GDSD223.
The physiological and biochemical property measurement result of described vinelandii GDSD223 is as shown in table 1:
The physiological and biochemical property of table 1 vinelandii GDSD223
Annotate: "+" expression is positive, and "-" expression is negative.
3,16s rDNA sequence homology analysis
Ordinary method is cultivated the vinelandii GDSD223 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 ' 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 azotobacter GDSD223 bacterial strain 16s rDNA sees sequence 1 in the sequence table for details in the paddy rice.
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 azotobacter GDSD223 is 28 ℃ in the described paddy rice, 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, azotobacter GDSD223 in the paddy rice that step 1 is separated and purifying obtains is accredited as modification bacterial alpha subgroup Bacillaceae bacillus (Bacillus sp.).This genus bacillus (Bacillus sp.) GDSD223 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.5036.
Embodiment 2, genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 nitrogenase activity determination
Embodiment 1 resulting genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 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 genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036,28 ℃ 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 the test tube slant is washed in the centrifuge tube, collect thalline, the NaOH boiling water that adds 3ml 0.5M in the precipitation boils 5min, and the HCl that adds 3ml 0.5M mixes, and gets 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 at eddy mixer
595, calculate tropina content according to the bovine serum albumin typical curve.
The result shows that the nitrogenase activity of the genus bacillus that screens (Bacillus sp.) GDSD223 CGMCC No.5036 is 38.725nmol C
2H
4/ hmg albumen, statistical study are 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 that genus bacillus of the present invention (Bacillus sp.) GDSD223 CGMCC No.5036 can high-efficiency nitrogen-fixing.
Embodiment 3, genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 antagonizing pathogenic fungi bacteriostasis rate are measured
Adopting 2 face-off methods that embodiment 1 resulting genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 is carried out the antagonizing pathogenic fungi bacteriostasis rate measures, concrete operations are as described below: inoculate respectively crop pathogenic fungi Gibberella zeae bacterium (Gibberella zeae) (or sclerotium germ (Sclerotinia sclerotiorum)) and genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 on 2 of distance center 2cm on the PDA flat board, 3 repetitions of each Screening Treatment, take only connect pathogenic fungi do not budder spore bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 flat board as the contrast.28 ℃ of constant temperature culture are measured the dull and stereotyped upper pathogenic fungi of face-off along the colony radius r of tested genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 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
126.6 ± 2.8mm; The r of sclerotium germ (Sclerotinia sclerotiorum)
063.0 ± 2.1mm, r
115.00 ± 1.83mm.Mean value is calculated for the above-mentioned formula of people: described genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 is 57.98% to the bacteriostasis rate of gibberellic hypha (Gibberella zeae), as shown in Figure 2; Bacteriostasis rate to sclerotium germ (Sclerotinia sclerotiorum) is 76.19%, as shown in Figure 3.This result shows described genus bacillus (Bacillus sp.) GDSD223 CGMCC No.5036 effectively antagonism crop gibberellic hypha (Gibberella zeae), sclerotium germ (Sclerotinia sclerotiorum).
Claims (10)
1. genus bacillus (Bacillus sp.) GDSD223, its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.5036.
2. microbial inoculum, its activeconstituents is genus bacillus claimed in claim 1 (Bacillus sp.) GDSD223CGMCC No.5036.
3. genus bacillus claimed in claim 1 (Bacillus sp.) GDSD223CGMCC No.5036 is in preparation following 1)-4) in application 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 generation nitrogenase.
4. according to claim 3 described application is characterized in that: described pathogenic fungi is fungi or the fungi for causing sclerotium disease that causes head blight; Described fungal diseases of plants is head blight or sclerotium disease.
5. application according to claim 4 is characterized in that: describedly cause that the fungi of head blight is Gibberella zeae bacterium (Gibberellazeae) or Fusarium graminearum (Fusarium graminearum); Describedly cause that the fungi of sclerotium disease is sclerotinite (Sclerotinia sclerotiorum).
6. genus bacillus claimed in claim 1 (Bacillus sp.) GDSD223CGMCC No.5036 or the described microbial inoculum of claim 2 are used in producing nitrogenase.
7. genus bacillus claimed in claim 1 (Bacillus sp.) GDSD223CGMCC No.5036 or the described microbial inoculum of claim 2 are used in the preparation biological organic fertilizer.
8. the biological organic fertilizer that contains the described genus bacillus of claim 1 (Bacillus sp.) GDSD223CGMCC No.5036 or the described microbial inoculum of claim 2.
9. cultivate the method for the described genus bacillus of claim 1 (Bacillus sp.) GDSD223CGMCC No.5036, comprise the step that genus bacillus (Bacillus sp.) GDSD223CGMCC No.5036 is cultivated at the substratum that is used for the cultivation genus bacillus.
10. the preparation method of the described microbial inoculum of claim 2 comprises the steps: genus bacillus claimed in claim 1 (Bacillus sp.) GDSD223CGMCC No.5036 is obtained described microbial inoculum as activeconstituents.
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Effective date of registration: 20140627 Address after: 028000 Tongliao north the Inner Mongolia Autonomous Region economic and Technological Development Zone on the north side of Huandongmeng Carsell Patentee after: Wang Zhongchen Address before: 100081 Beijing, Zhongguancun, South Street, No. 12, No. Patentee before: Institute of agricultural resources and regional planning, Chinese Academy of Agricultural Sciences |