CN104988100A - Bacillus amyloliquefaciens CD-17 strain and application thereof - Google Patents
Bacillus amyloliquefaciens CD-17 strain and application thereof Download PDFInfo
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- CN104988100A CN104988100A CN201510456183.2A CN201510456183A CN104988100A CN 104988100 A CN104988100 A CN 104988100A CN 201510456183 A CN201510456183 A CN 201510456183A CN 104988100 A CN104988100 A CN 104988100A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
Abstract
The present invention provides a Bacillus amyloliquefaciens CD-17 strain (accession number in CGMCC of CGMCC No.7968) and a preparation method of a microbial agent from broth. The preparation can restore wound plant roots while effectively controlling strawberry wilt and verticillium wilt.
Description
Invention field
The present invention relates to a kind of microorganism and purposes, more specifically to the preparation method of a kind of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) CD-17 bacterial strain, purposes and preparation thereof, belong to biological pesticide technical field.
Background technology
The assorted disease caused is invaded, as samping off, blight, verticillium etc. cause heavy losses in agricultural and garden crop are produced by Soil-born plant pathogenic fungi.Many soil-borne diseases are broken out and crop can be made to have no harvest aborning, reduce the output of 1-5 layer under condition of often oestrusing.For controlling or reduce the generation of such disease, current the most frequently used method has chemical pesticide filling, chemical fumigant is to disinfecting soil, the vexed canopy of summer high temperature and utilize some microbiobacterial agents to fill with the methods such as root after crop is transplanted.But these methods need drop into a large amount of human and material resources and financial resources, and process bad meeting and cause and burn seedling and a large amount of chemical pesticides and remain.
Along with the application for many years of chemical pesticide, phytopathogen creates serious resistance to chemical pesticide.Current almost phytopathogen anti-(resistance to) property of medicine in various degree is all created to conventional chemical pesticide.In order to effective controlling plant diseases, the measure that peasant household often adopts be strengthen agricultural chemicals application concentration, improve the frequency of usage of agricultural chemicals to obtain temporary transient validity.But the use year in year out of these measures, make the resistance flora of plant pathogenic fungi in a short time journey break out stream trend.Due to using in a large number of chemical pesticide, make food safety risk more and more higher.
Compared with chemical pesticide, biological pesticide has corresponding advantage, and especially on high added value garden crop produces, biological pesticide can play good effect.Under the effect of system biological prophylactico-therapeutic measures, biological pesticide can provide favourable living environment for plant, as biocontrol microorganisms is formed with beneficial microorganism group at crop seed or rhizosphere, surely grows formation microbial film at plant surface, produce broad variety bacteriostatic compound simultaneously, suppress the breeding etc. of pathogenic bacteria.In addition, biocontrol microorganisms with the struggle for existence of pathogenic bacteria in effectively can utilize spontaneous high fecundity, occupy rapidly the energy of a large amount of living spaces and existence region material, create the disadvantageous living environment of phytopathogen, reached by above various ways and control noxious plant pathogenic bacteria infecting farm crop.The various result of this mode of action makes phytopathogen not easily develop immunity to drugs, and the feature of biocontrol microorganisms environmentally safe, make it obtain becoming possibility in sizable application in high-efficiency agriculture.The developed countries such as the current U.S., in agricultural chemicals classification, are classified as living microorganism control without harm level (OMRI), can be applied to the control of plant disease measure in Organic farming production estimation.
Catch proper control time to the control key of soil-borne disease.Due to inevitably damage can be caused to its root system in crop transplanting process, the microtrauma mouth that root system damage is formed is the main path that soil-borne disease infects, phytopathogen has microtrauma mouth to infect rapidly at root, once after infecting, more prevention and control measures are also difficult to remedy.Therefore, instant repair microtrauma mouth and form in wound circumference the First aggression that beneficial microbe colony suppresses pathogenic bacteria, is an effectively preventing measure.The invention provides a kind of microorganism roots soaking agent, form microbial film hindering around root while utilizing this agent immediately to repair to hinder root, effectively suppress the First aggression of pathogenic bacteria.Test-results shows, this bioremediation agents effectively can prevent and treat plurality of plant diseases.Preparation provided by the invention effectively can hinder technology and the application method thereof of the microbial preparation of root and diseases prevention by rehabilitation plant, does not mention in the prior art and discloses.
Summary of the invention
The present invention solves the deficiency that prior art exists, and provides a strain to be newly separated genus bacillus from grass carp intestinal Bacterial diversity: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CD-17 bacterial strain.The present invention also provides method and the utilisation technology thereof of hindering the microbial preparation of root reparation and diseases prevention with this bacillus amyloliquefaciens CD-17 bacterial strain processing plant.
The present invention is achieved by the following technical solutions:
Bacillus amyloliquefaciens of the present invention (Latin name is Bacillus amyloliquefaciens) CD-17 bacterial strain, on July 26th, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is: CGMCCNo.7968.Bacterial classification of the present invention has following feature:
(1) cellular form of CD-17 bacterial strain and physiological and biochemical property (see table 1):
Table 1. CD-17 strain cell form and physicochemical characteristics
| Experimental project | Result | Experimental project | Result |
| Gramstaining | Positive | Carbohydrate produces acid | |
| Cell shape | Shaft-like | Glucose | + |
| Cell dia > 1 μm | - | Wood sugar | + |
| Formation bud is embraced | + | L mono-pectinose | + |
| Bud is embraced and is expanded | - | N.F,USP MANNITOL | + |
| Bud embraces circle | - | Lactose | + |
| Companion embraces crystal | - | Glucose fermentation aerogenesis | - |
| Catalase | + | Utilize Citrate trianion | + |
| Oxydase | + | 50 DEG C of growths | - |
| Anaerobic growth | - | PH5.7 grows | + |
| VP tests | + | 7%NaCI grows | - |
| VP<pH6 | - | Starch Hydrolysis | + |
| VP>pH7 | - | Decompose dead drunk element | + |
| Methyl red test | - | Nitrate reduction | + |
| Urase | - | The two hydrolysis of arginine | - |
(2) CD-17 bacterial strain 16S rDNA gene order is (i.e. SEQ ID NO:1):
CCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATAGGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTC
(3) CD-17 bacterial strain gyrB gene order is (i.e. SEQ ID NO:2):
TCCACTATCAGGCGTACGAGCGCGGTGTACCTGTGGCCGATCTTGAAGTGATCGGTGATACTGATAAGACCGGAACGATTACGCACTTCGTTCCGGATCCGGAAATTTTCAAAGAAACAACCGAATACGACTATGACCTGCTTTCAAACCGTGTCCGGGAATTGGCTTCCTGACAAAAGGTGTAAACATCACGATTGAAGACAAACGTGAAGGACAAGAACGGAAAAACGAGTACCACTACGAAGGCGGAATCAAAAGCTATGTTGAGTACTTAAACCGTTCCAAAGAAGTCGTTCATGAAGAGCCGArTTATATCGAAGGCGAGAAAGACGGCATAACGGTTGAAGTTGCATTGCAATACAACGACAGCTATACAAGCAATATTTATTCTTTCACAAATAATATCAACACATACGAAGGCGGCACGCACGAAGCCGGATTTAAAACCGGTCTGACCCGTGTTATAAACGACTATGCAAGAAGAAAAGGGATTTTCAAAGAAAATGATCCGAATTTAAGCGGGGATGATGTGAGGGAAGGGCTGACTGCCATTATTTCAATTAAGCACCCTGATCCGCAATTCGAAGGGCAGACGAAAACGAAGCTCGGCAACTCCGAAGCGAGAACGATCACTGATACGCTGTTTTCTTCTGCGCTGGAAACATTCCTTCTTGAAAATCCGGACTCAGCCCGCAAAATCGTTGAAAAAGGTTTAATGGCCGCAAGAGCGCGGATGGCAGCGAAAAAAGCGCGGGAATTGACCCGCtGCAAAAGTGCGCTTGAGATTTCCAATCTGCCGGGC
With microbial preparation prepared by the CD-17 bacterial strain that plant of the present invention hinders root reparation and diseases prevention, its preparation method comprises the following steps:
(A) acquisition of bacterium liquid
Picking is cultured CD-17 bacterial strain list bacterium colony spot on PDA solid medium in advance, being inoculated in the volume that 5mL PD liquid medium (PDA substratum does not add agar) is housed is in the triangular flask of 20mL, in 30 DEG C, cultivate 16h under 200rpm condition, nutrient solution 5mL being all inoculated in the volume that 400mL PD nutrient solution is housed is in the triangular flask of 1000mL, cultivates 16h under 200rpm, 30 DEG C of conditions; The 400mL nutrient solution of gained is inoculated in the fermentor tank (east, Zhenjiang GUS-30) that the volume that 20L nutrient solution is housed is 30L, and in 20L fermentation culture, solid tolerant content is: analysis for soybean powder 100g, water soluble starch 200g, sucrose 25g, yeast powder 25g, CaCO
320g, MnSO
41.0g, arranging fermentation condition is: dissolved oxygen 100%, and stirring velocity is 350rpm, leavening temperature 33 DEG C, fermentation time 24h, pH 7.0-7.2;
(B) processing of micro-bacteria-promoting agent
The procedure of processing of microbiobacterial agent: (1) is by above-mentioned fermented liquid centrifugal 10min under 8000rpm condition, abandon supernatant, throw out 1000mL 20% glycerin solution (V/V) Eddy diffusion (is dissolved with 16 grams of NH in 20% glycerin solution
4cl), measure the living spores content of suspension, regulate the living spores number of suspension to whole spore content to be 1.0-2.8 × 10 according to measurement result 20% glycerin solution (V/V)
10cfu/mL; (2) in the above-mentioned suspension regulated, add pancreas wheat-flour by the consumption of 40g/L (W/V), and stir, the living spores number obtaining finished product microbial preparation is about 1.0 × 10
10cfu/mL, puts 5 DEG C of cryopreservation.
The invention has the beneficial effects as follows:
Bacillus amyloliquefaciens CD-17 bacterial strain provided by the invention is the biocontrol bacteria that a strain effectively can suppress various plants pathogenic fungi.The microbial preparation that the plant provided in the present invention hinders root reparation and diseases prevention is prepared from through processing by the tunning of this bacterial strain.The present invention provides in detail and utilizes CD-17 bacterial strain to prepare the gordian techniquies such as the auxiliary agent component content of the processing technology of microbiobacterial agent, application process and preparation.Microbial preparation provided by the invention is closed after application and has been repaired root microtrauma mouth, thus well can block the route of entry of soil surface characters from root wound, in addition, biocontrol bacteria in microbial preparation by the available nutrient that utilizes preparation to provide at plant rhizosphere Fast-propagation, establish beneficial microorganism group, suppress infecting of noxious plant pathogenic bacteria.
Biomaterial of the present invention, its Classification And Nomenclature is: bacillus amyloliquefaciens CD-17 (Bacillus amyloliquefaciensCD-17), (be called for short CGMCC on July 26th, 2013 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica) preservation, preserving number is: CGMCC No.7968.
Embodiment
Embodiment
Bacillus amyloliquefaciens of the present invention (Latin name is Bacillus amyloliquefaciens) bacterial strain CD-17, on July 26th, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is: CGMCCNo.7968.The cellular form of bacterial strain of the present invention and physiological and biochemical property are in table 1.16S rDNA gene order and the gyrB gene order of the bacillus amyloliquefaciens CD-17 bacterial strain of the present embodiment are shown in " summary of the invention ".
With microbial preparation prepared by the CD-17 bacterial strain that plant of the present invention hinders root reparation and diseases prevention, its preparation method comprises the following steps:
(A) acquisition of bacterium liquid
Picking is cultured CD-17 bacterial strain list bacterium colony spot on PDA solid medium in advance, being inoculated in the volume that 5mL PD liquid medium (PDA substratum does not add agar) is housed is in the triangular flask of 20mL, in 30 DEG C, cultivate 16h under 200rpm condition, nutrient solution 5mL being all inoculated in the volume that 400mL PD nutrient solution is housed is in the triangular flask of 1000mL, cultivates 16h under 200rpm, 30 DEG C of conditions; The 400mL nutrient solution of gained is inoculated in the fermentor tank (east, Zhenjiang GUS-30) that the volume that 20L nutrient solution is housed is 30L, and in 20L fermentation culture, solid tolerant content is: analysis for soybean powder 100g, water soluble starch 200g, sucrose 25g, yeast powder 25g, CaCO
320g, MnSO
41.0g, arranging fermentation condition is: dissolved oxygen 100%, and stirring velocity is 350rpm, leavening temperature 33 DEG C, fermentation time 24h, pH 7.0-7.2;
(B) processing of micro-bacteria-promoting agent
The procedure of processing of microbiobacterial agent: (1) is by above-mentioned fermented liquid centrifugal 10min under 8000rpm condition, abandon supernatant, throw out 1000mL 20% glycerin solution (V/V) Eddy diffusion (is dissolved with 16 grams of NH in 20% glycerin solution
4cl), measure the living spores content of suspension, regulate the living spores number of suspension to whole spore content to be 1.0-2.8 × 10 according to measurement result 20% glycerin solution (V/V)
10cfu/mL; (2) in the above-mentioned suspension regulated, add pancreas wheat-flour by the consumption of 40g/L (W/V), and stir, the living spores number obtaining finished product microbial preparation is about 1.0 × 10
10cfu/mL, puts 5 DEG C of cryopreservation.
Implementation result
(1) CD-17 microbiobacterial agent is to the field controling test of strawberry blight
Test treatment process: this test is carried out in cloud rabbitweed certain kind of berries cooperative society's greenhouse gardening garden, industry, and strawberry cultivars is " Ning Yu ".Chemicals treatment: (1) CD-17 microbiobacterial agent 100 times of liquid; (2) 1.0 × 10
10cfu/g subtilis wettable powder 100 times of liquid; (3) contrast clear water.Strawberry Seedlings is dug out clean root soil from nursery, clean Strawberry Seedlings root is immersed transfer after 3 minutes in above-mentioned chemicals treatment; Strawberry Fusarium Wilt conidium liquid (1.0 × 10 is watered by the strawberry processed and contrast after transplanting
5cfu/mL) 100mL/ strain.Often process leaching 400 Strawberry Seedlings, 4 repetitions are established in test, transplant on September 6th, 2013, and after transplanting, 60d checks and calculates preventive effect.
Result: field test results (table 2) shows, the process of CD-17 microbiobacterial agent 100 times of immersion roots is 92.35% to the prevention effect of strawberry blight, subtilis wettable powder 100 times of immersion roots are only reach statistics level difference between 68.81%, two medicaments to the prevention effect of strawberry blight by contrast.
Table 2. CD-17 microbiobacterial agent is to the field controling test result of strawberry blight
Note: different capitalization represents P<0.01.
(2) CD-17 microbiobacterial agent is to the field controling test of strawberry Verticillium wilt
Test treatment process: this test is carried out in cloud rabbitweed certain kind of berries cooperative society's greenhouse gardening garden, industry, and strawberry cultivars is " Ning Yu ".Chemicals treatment: (1) CD-17 microbiobacterial agent 100 times of liquid; (2) 1.0 × 10
10cfu/g subtilis wettable powder 100 times of liquid; (3) contrast clear water.Strawberry Seedlings is dug out clean root soil from nursery, clean Strawberry Seedlings root is immersed transfer after 3 minutes in above-mentioned process; Strawberry Verticillium wilt bacterium conidium liquid (1.0 × 10 is watered by the strawberry processed and contrast after transplanting
5cfu/mL) 100mL/ strain.Often process leaching 400 Strawberry Seedlings, 4 repetitions are established in test, transplant on September 6th, 2013, and after transplanting, 60d checks and calculates preventive effect.
Result: field test results (table 3) shows, the process of CD-17 microbiobacterial agent 100 times of immersion roots to strawberry Verticillium wilt prevention effect be 84.71%, subtilis wettable powder 100 times of immersion roots are only reach statistics level difference between 40.06%, two medicaments to the prevention effect of strawberry Verticillium wilt by contrast.
Table 3. CD-17 microbiobacterial agent is to the field controling test result of strawberry Verticillium wilt
Note: different capitalization represents P<0.01.
Sequence table
<110> Jiangsu Hilly Ground Zhenjiang Agriculture Science Research Institute
<120> bacillus amyloliquefaciens CD-17 bacterial strain and application
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1459
<212> DNA
<213> Bacillus amyloliquefaciens CD-17
<400> 1
ccctaataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga 60
cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg 120
ctaataccgg atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc 180
acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga 240
cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac 300
tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg 360
ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc 420
cgttcaaata gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc 480
cagcagccgc ggtaataggt aggtggcaag cgttgtccgg aattattggg cgtaaagggc 540
tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg 600
gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg 660
cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg 720
aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780
atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac 840
tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca 900
agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat 960
cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg 1020
gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 1080
atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg 1140
aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca 1200
atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc 1260
agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat 1320
cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga 1380
gtttgtaaca cccgaagtcg gtgaggtaac cttttaggag ccagccgccg aaggtgggac 1440
agatgattgg ggtgaagtc 1459
<210> 2
<211> 802
<212> DNA
<213> Bacillus amyloliquefaciens gyrB gene
<400> 2
tccactatca ggcgtacgag cgcggtgtac ctgtggccga tcttgaagtg atcggtgata 60
ctgataagac cggaacgatt acgcacttcg ttccggatcc ggaaattttc aaagaaacaa 120
ccgaatacga ctatgacctg ctttcaaacc gtgtccggga attggcttcc tgacaaaagg 180
tgtaaacatc acgattgaag acaaacgtga aggacaagaa cggaaaaacg agtaccacta 240
cgaaggcgga atcaaaagct atgttgagta cttaaaccgt tccaaagaag tcgttcatga 300
agagccgart tatatcgaag gcgagaaaga cggcataacg gttgaagttg cattgcaata 360
caacgacagc tatacaagca atatttattc tttcacaaat aatatcaaca catacgaagg 420
cggcacgcac gaagccggat ttaaaaccgg tctgacccgt gttataaacg actatgcaag 480
aagaaaaggg attttcaaag aaaatgatcc gaatttaagc ggggatgatg tgagggaagg 540
gctgactgcc attatttcaa ttaagcaccc tgatccgcaa ttcgaagggc agacgaaaac 600
gaagctcggc aactccgaag cgagaacgat cactgatacg ctgttttctt ctgcgctgga 660
aacattcctt cttgaaaatc cggactcagc ccgcaaaatc gttgaaaaag gtttaatggc 720
cgcaagagcg cggatggcag cgaaaaaagc gcgggaattg acccgctgca aaagtgcgct 780
tgagatttcc aatctgccgg gc 802
Claims (6)
1. a bacillus amyloliquefaciens bacterial strain, its Classification And Nomenclature is bacillus amyloliquefaciens CD-17
(Bacillus amyloliquefacienscD-17), it is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and preserving number is: CGMCC No. 7968.
2. Bacillus amyloliquefaciens strain according to claim 1, is characterized in that the 16S rDNA gene order of this bacterial strain is as shown in SEQ ID NO:1.
3. Bacillus amyloliquefaciens strain according to claim 1, is characterized in that this bacterial strain
gyr1 B gene sequence is as shown in SEQ ID NO:2.
4. the application of Bacillus amyloliquefaciens strain according to claim 1 in control strawberry blight and verticillium.
5. a microbial preparation utilizing Bacillus amyloliquefaciens strain described in claim 1 to be prepared from.
6. prepare a method for microbial preparation according to claim 5, it is characterized in that comprising the following steps:
(A) acquisition of bacterium liquid
Picking is cultured CD-17 bacterial strain list bacterium colony spot on PDA solid medium in advance, being inoculated in the volume that 5 mL PD liquid mediums (PDA substratum does not add agar) are housed is in the triangular flask of 20 mL, in 30 DEG C, cultivate 16 h under 200 rpm conditions, it is in the triangular flask of 1000 mL that nutrient solution 5 mL is all inoculated in the volume that 400 mL PD nutrient solutions are housed, and cultivates 16 h under 200 rpm, 30 DEG C of conditions; It is the fermentor tank of 30L that the 400mL nutrient solution of gained is inoculated in the volume that 20 L nutrient solutions are housed;
The processing of the micro-bacteria-promoting agent of B
(1) by step (A) gained fermented liquid centrifugal 10min under 8000 rpm conditions, abandon supernatant, throw out 1000 mL 20% glycerin solution (V/V) Eddy diffusions, measure the living spores content of suspension, regulate the living spores number of suspension to whole spore content to be 1.0-2.8 × 1010 cfu/mL according to measurement result 20% glycerin solution (V/V);
(2) by 40 g/L(W/V in suspension above-mentioned steps (1) regulated) consumption add pancreas wheat-flour, and stir, the living spores number obtaining finished product microbial preparation is about 1.0 × 1010 cfu/mL, puts 5 DEG C of cryopreservation.
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