CN106479934A - A kind of ash arrhizus bacteria antagonistic strain and its screening technique and application - Google Patents
A kind of ash arrhizus bacteria antagonistic strain and its screening technique and application Download PDFInfo
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Abstract
The invention belongs to microbial technique and field of biological control are and in particular to a kind of bacillus amyloliquefaciens（Bacillus amyloliquefaciens）BA KA4 and its screening technique and application.The present invention isolates antibacterial from soil, is therefrom filtered out to botrytis cinerea using flat board face-off method（Botrytis cinerea）Suppression ratio be 65% and stable heredity antibacterial BA KA4.Cultural characteristic through bacterial strain, form and physiological and biochemical property combine 16S rDNA sequence andgyr1 B gene sequence results are analyzed, and bacterial strain BA KA4 is initially identified as bacillus amyloliquefaciensBacillus amyloloquefaciens.Preserve strain, be CGMCC No.12190 in the numbering of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center, preferably and the lasting period is long for its prevention effect to gray mold of cucumber, has extensive antimicrobial spectrum.
Description
Technical field
The invention belongs to microbial technique and field of biological control are and in particular to a kind of bacillus amyloliquefaciens
（Bacillus amyloliquefaciens）BA-KA4 and its screening technique and application.
Background technology
Gray mold is a kind of global plant disease, and this disease is by Deuteromycotina funguses the pathogen of Botrytis cinerea
(Botrytis cinerea) causes, and infects including various crop such as Fructus Cucumidis sativi, Fructus Lycopersici esculenti, Fructus Vitis viniferaes, agricultural production is constituted greatly
Threaten, have a strong impact on yield and the quality of vegetable and fruit.At present mainly adopt pyrimethanil, Fluoxastrobin, carbendazim, diethofencarb,
The chemical pesticides such as Sukeling are prevented and treated, a large amount of uses of chemical pesticide, not only cause environmental pollution, and cause grey Portugal
The Drug resistance to medicaments such as pyrimethanil, carbendazim, diethofencarbs for the grape spore bacterium, the research such as Wang Meiqin shows, Shanxi Jinzhong area Fructus Cucumidis sativi
Ash arrhizus bacteria is anti-during the resistance of diethofencarb and Sukeling is reached, and the resistance to carbendazim is high anti-.The research such as Li Xinghong finds,
Beijing area graw mold of tomato is very universal to the Drug resistance of pyrimethanil, and based on high antibacterial strain, production should be replaced new
Bactericidal agent for preventing and treating graw mold of tomato.So, developing Biological control approach that is safe efficient, not causing resistance is to reduce gray mold
Harm, the important channel reduced environmental pollution, ensure high and stable crop yield, increasingly become the study hotspot of gray mold preventing and treating.
At present, the many plants of bacterial strains having antagonism to the pathogen of Botrytis cinerea have been filtered out.Elad is by Trichoderma harzianum T39 bacterial strain
Make Trichoderma harzianum wettable powder for controlling the generation of the diseases such as chamber crop and grape grey mould.Dik etc. compares Kazakhstan
Thatch Trichoderma spp. T39 and Aureobasidium pullulans, the light white latent ball yeast prevention effect to Fructus Cucumidis sativi and graw mold of tomato, find Trichoderma harzianum and
Aureobasidium pullulans are processed can make stem's scab and the mortality rate of plant reduce by 40% ~ 100%, and it is antibacterial to first that its preventive effect is better than antibacterial
Spirit and iprodione.Wei Yanmin etc. determines the bacteriostasis to 5 kinds of pathogenic fungi for 26 plants of actinomycetes bacterium, and wherein 5 plants to grey mold
Pathogenic bacteria bacteriostasis are stronger, and bacteriostatic activity is apparently higher than comparison chemical agents such as 50% carbendazim.Han Siqin etc. is from Changbai Mountain soil
It is isolated to one plant of actinomycetes D2-4 in earth, find sending out of this bacterium through flat board bacteriostatic test, excised leaf test and pot experiment
Zymotic fluid has very strong suppression and prevention effect to graw mold of tomato pathogenic bacteria.So far, for gray mold study on prevention and should
Antibacterial has kind more than 10, mainly has bacilluss, pseudomonass etc., controllable Fructus Lycopersici esculenti, Fructus Capsici, Fructus Fragariae Ananssae, Fructus Mali pumilae and Fructus Vitis viniferae etc.
The generation of plant botrytis and harm.Tong Yunhui etc. separates that the Antagonism obtaining is strong, the bacillus licheniformis W l0 of has a broad antifungal spectrum,
W 3, Y 2-11-1 bacterial strain, are 70% ~ 80% to the preventive effect of graw mold of tomato blade and fruit, better than 2000 times of 50% Sukeling
Liquid.The research such as Wang Wei finds that antagonistic bacterium x-75 has preferable inhibition to graw mold of tomato.On the whole, China utilizes
It is also relatively little that antagonistic bacterium prevents and treats the research of gray mold, is separated to 1 plant of bacillus amyloliquefaciens in Wang Yingguo et al., its for
The plant pathogenic fungis such as Fusarium oxysporum, white leaf spot of strawberry bacterium all have very strong inhibitory action.Wang Yiwen et al. is first from Fructus Melo
Fruit surface is separated to 1 plant of bacillus amyloliquefaciens, to Botrytis cinerea, rod method, Fusarium oxysporum, aspergillus niger and pink single-ended
The postharvest fruit and vegetable pathogenic fungi such as spore has notable and wide spectrum antagonism.
This research and utilization single target primary dcreening operation, the screening system of many targets secondary screening, have filtered out to gray mold from pedotheque
Antagonistic effect is good and the wider bacillus amyloliquefaciens BA-KA4 of antimicrobial spectrum, provides material to the Biological control for gray mold
Material supports.
Content of the invention
It is an object of the invention to provide a kind of numbering is the bacillus amyloliquefaciens of BA-KA4, it has and can suppress crop
The effect of the growth of gray mold pathogen, and antimicrobial spectrum is wider, especially has the life that can substantially suppress gray mold of cucumber pathogen
Long effect.On the other hand present invention also offers the screening technique of above-mentioned bacterial strains and purposes.
For achieving the above object, the technical solution used in the present invention is：
A kind of ash arrhizus bacteria antagonistic strain, this bacterial strain is bacillus amyloliquefaciens BA-KA4, micro- in China on March 8th, 2016
Biological inoculum preservation administration committee General Microbiological Culture collection has carried out preservation, and preserving number is：CGMCC No.
12190.
Further, described ash arrhizus bacteria antagonistic strain, has suppression P. capsici, fusarium graminearum, little
Wheat sheath blight fungus, tobacco brown spot pathogen, Bulbus Allii hyphal cluster germ, botrytis cinerea, tomato early blight bacterium, Pear black spot bacteria growing
Activity.
A kind of screening technique of above-mentioned bacillus amyloliquefaciens BA-KA4, comprises the following steps：
（1）Antibacterial in the soil pattern of different regions is isolated and purified and preservation；
（2）Primary dcreening operation：The screening of Antagonistic Fungi is carried out using the flat board method that stands facing each other, with the card punch of diameter 5mm on botrytis cinerea pers side
Edge punches, and truffle is inoculated in PDA plate central authorities, and 4 different single bacterium colonies of picking are uniformly connected on the position apart from indicator bacteria 3cm
Put, after 22 DEG C of dark culturing 5d, observe fungistatic effect, select the bacterial strain entrance secondary screening with inhibition zone；
（3）Secondary screening：With botrytis cinerea pers as indicator bacteria, the bacterial strain point entering secondary screening is connected on by anomaly plate using crossing method
On 4 angles at central 3cm, the length that flat board central authorities are simultaneously moved into diameter 5mm has the agar block of ash arrhizus bacteria, 22 DEG C of dark trainings
After foster 5d, observe antibacterial result, measure antibacterial circle diameter, calculate bacteriostasis rate, filter out the bacterial strain of bacteriostasis rate more than 60%；Carry out
Successive transfer culture, measures hereditary stability, the bacterial strain of bacteriostasis rate more than 60% after screening and culturing 10 generation；Select P. capsici, little
Wheat gibberellic hypha, rhizoctonia cerealis, tobacco brown spot pathogen, Bulbus Allii hyphal cluster germ, botrytis cinerea, tomato early blight bacterium,
Pear black spot bacterium, as indicator bacteria, is carried out the mensure of antimicrobial spectrum, observes fungistatic effect using flat board face-off method to the bacterial strain selected,
Measure antibacterial circle diameter, calculate bacteriostasis rate, filter out all preferable bacterial strain to this 8 kinds of funguses fungistatic effects, as solve starch bud
Born of the same parents bacillus BA-KA4.
Further, step（1）Concrete operations be：Adopt 120 soil coming from Yunnan, Heilungkiang, Xinjiang, Hebei Province
Earth, using PB culture medium, applies plate method using dilution and carries out soil and separate, after culture 3d in 28 DEG C of incubators, picking bacterium
The different antibacterial of the form that falls carry out 5 times line purification, numberings, and using 15% glycerol in -80 DEG C of Refrigerator stores.
Further, described PB culture medium is：Carnis Bovis seu Bubali cream 5.0 g, peptone 10.0 g, NaCl 5.0 g, agar 15
G, pH 7.4, distilled water is settled to 1L, 121 DEG C of sterilizing 20min.
A kind of application in preventing and treating crop grey mold disease for above-mentioned bacillus amyloliquefaciens BA-KA4.
Further, its prevention and controls be with sterilized water by bacillus amyloliquefaciens BA-KA4 be diluted to concentration be 1 ×
108The bacteria suspension of CFU/mL, after 121 DEG C of sterilizing 20min, foliage-spray is in crop.
Bacillus amyloliquefaciens Bacillus amyloliquefaciens CGMCC No. 12190 of the present invention
Morphological feature be：Not chromogenesises are grown on PB culture medium, bacterium colony is flat or circular, milky is opaque, bacterium colony side
Edge is neat, and there is fold on surface.Gram-positive, microscope is viewed as shaft-like, generation spore, spore ellipse or cylindricality.Referring to figure
3.
Reference《Bai Jie detail dientification of bacteria handbook》With《Common bacteria identification handbook》Carry out the Physiology and biochemistry identification of bacterial strain, knot
As shown in table 1, Preliminary Identification Antagonistic Fungi BA-KA4 is bacillus amyloliquefaciens to fruit.
Table 1 Antagonistic Fungi Physiology and biochemistry qualification result
Note："+", represents positive, and "-" represents negative
The bacillus amyloliquefaciens BA-KA4 of the present invention is to P. capsici, fusarium graminearum, rhizoctonia cerealis, Nicotiana tabacum L.
Multiple pathogenic fungi such as brown spot pathogen, Bulbus Allii hyphal cluster germ, botrytis cinerea, tomato early blight bacterium, Pear black spot bacterium all have
There is obvious inhibitory action；And it is particularly suited for being made as Agrotechnical formulation to cause for preventing and treating the infection of crop ash arrhizus bacteria
Disease.
Biological sample preservation information
Bacillus amyloliquefaciens BA-KA4, Classification And Nomenclature is Bacillus amyloliquefaciens, in March 8 in 2016
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, address：Beijing is exposed to the sun
Area North Star West Road 1 institute 3, Institute of Microorganism, Academia Sinica, postcode 100101, culture presevation number is CGMCC No.
12190.
Brief description
The antagonistic effect to botrytis cinerea pers for Fig. 1 Antagonistic Fungi BA-KA4
Fig. 2 Antagonistic Fungi BA-KA4 antimicrobial spectrum
Fig. 3 Antagonistic Fungi BA-KA4 colonial morphology
Fig. 4 Antagonistic Fungi BA-KA4 is according to the phylogenetic tree of 16S rDNA gene order
Fig. 5 Antagonistic Fungi BA-KA4 is according to the phylogenetic tree of gyrB gene order.
Specific embodiment
Used in the present invention is the soil sample gathering from Yunnan, Heilungkiang, Xinjiang, Hebei and other places for examination soil.
It is P. capsici, fusarium graminearum, rhizoctonia cerealis, tobacco brown spot pathogen, Bulbus Allii for examination pathogen
Hyphal cluster germ, botrytis cinerea, tomato early blight bacterium, Pear black spot bacterium, are separated by this laboratory and preserve.
It is purchased from academy of agricultural sciences of Hebei province for examination cucumber seeds.
For examination culture medium it is：PDA culture medium (1 L)：Peeled potatoes 200.0 g, glucose 20.0 g, agar 15
G, distilled water is settled to 1L, 115 DEG C of sterilizing 20min；PB culture medium (1 L)：Carnis Bovis seu Bubali cream 5.0 g, peptone 10.0 g, NaCl
5.0 g, agar 15 g, pH 7.4, distilled water is settled to 1L, 121 DEG C of sterilizing 20min.
In embodiment 1 soil, antibacterial isolates and purifies and preservation
（1）Adopt, from Yunnan, Heilungkiang, Xinjiang, Hebei and other places, 120 soil coming, using PB culture medium, apply flat board using dilution
Method carries out soil and separates, and after culture 3d in 28 DEG C of incubators, it is pure that the different antibacterial of picking colony form carries out 5 line
Change, numbering, and using 15% glycerol in -80 DEG C of Refrigerator stores.
（2）The primary dcreening operation of Antagonistic Fungi is carried out using the flat board method that stands facing each other, with the card punch of diameter 5mm at botrytis cinerea pers edge
Punching, truffle is inoculated in PDA plate central authorities, and 4 different single bacterium colonies of picking are uniformly connected on the position apart from indicator bacteria 3cm,
After 22 DEG C of dark culturing 5d, observe fungistatic effect, select the bacterial strain entrance secondary screening with inhibition zone.Divide from 129 soil samples
4200 plants from the bacterial isolateses arriving, do flat board dual test respectively with botrytis cinerea pers, primary dcreening operation obtains to botrytis cinerea pers
There is 191 plants of the antibacterial of antagonism.
（3）With botrytis cinerea pers as indicator bacteria, the bacterial strain point entering primary dcreening operation is connected on by anomaly plate using crossing method
On 4 angles at central 3cm, the length that flat board central authorities are simultaneously moved into diameter 5mm has the agar block of ash arrhizus bacteria, 22 DEG C of dark trainings
After foster 5d, measurement, the size of record antibacterial circle diameter simultaneously calculate suppression ratio, filter out 10 plants of the bacterial strain of bacteriostasis rate more than 60%.
Suppression ratio（%）=（Matched group pathogen colony diameter-treatment group pathogen colony diameter）/ matched group pathogen colony diameter ×
100%.Carry out successive transfer culture, measure hereditary stability, the bacterial strain of bacteriostasis rate more than 60% after screening and culturing 10 generation, above 10 plants of bacterium
After strain 10 generations of culture, bacteriostasis rate is all more than 60%；Select P. capsici, fusarium graminearum, rhizoctonia cerealis, Nicotiana tabacum L.
8 kinds of funguses such as brown spot pathogen, Bulbus Allii hyphal cluster germ, botrytis cinerea, tomato early blight bacterium, Pear black spot bacterium are as indicator bacteria
Carry out many targets secondary screening, observe fungistatic effect, measure antibacterial circle diameter, calculate bacteriostasis rate, filter out to this 8 kinds of pathogenic fungi tools
There are best fungistatic effect bacterial strain, as bacillus amyloliquefaciens BA-KA4.Result as depicted in figs. 1 and 2, the bacteriostasis rate of BA-KA4
Highest, is 65%, and stable heredity.Antagonistic Fungi BA-KA4 is respectively provided with obvious inhibitory action to 8 kinds of pathogenic fungi, has extensively
Antimicrobial spectrum, and different to the antagonistic ability of Different Kinds of Pathogens funguses.Wherein the suppression ratio of 5 kinds of pathogenic fungi is all reached
More than 60%, be respectively：It is 75.9% to Pear black spot bacterium suppression ratio, be 65.9% to Bulbus Allii hyphal cluster germ suppression ratio, early to Fructus Lycopersici esculenti
Epidemic disease bacterium suppression ratio is 62.9%, and botrytis cinerea suppression ratio is 61.8%, and the suppression ratio to P. capsici is 60.6%.
The identification of embodiment 2 Antagonistic Fungi BA-KA4
（1）The cultural characteristic of Antagonistic Fungi and morphological characteristic
This bacterial strain BA-KA4, as shown in figure 3, growing not chromogenesises in PB culture medium, bacterium colony is flat or circular, milky not
Transparent, colony edge is irregular, there is fold on surface.Gram-positive, microscope is viewed as shaft-like, generation spore, and spore is oval
Or cylindricality.
（2）The physiological and biochemical property of Antagonistic Fungi
Reference《Bai Jie detail dientification of bacteria handbook》With《Common bacteria system identification handbook》Carry out：V-P test is positive, C.I. 13020. examination
Test feminine gender, indole test is positive, gelatin liquefaction test is positive, positive, Starch Hydrolysis test is positive, fat using testing for citrate
Fat enzyme is negative, H2S produces the test positive, nitrate reduction test is positive, malonate utilization is negative, tyrosine hydrolysis are cloudy
Property, cellulose decomposition positive.
（3）PCR amplification and sequencing
（1）16S rDNA sequence analysis are identified
Test kit using Tiangeng company carries out the extraction of bacterial genomes DNA, with this DNA as masterplate, with 27F (5 '-
AGAGTTTGATCMTGGCTCAG-3 '), sequence SEQ ID No.1 and 1492R (5 '-GGYTACCTTGTTACGACTT-3 '), sequence
Row SEQ ID No.2 is primer, carries out the amplification of bacterial strain 16S rDNA.PCR amplification condition is: 95℃ 5min；94℃
1min, 50 DEG C of 1min, 72 DEG C of 2min, 35 circulations；72℃10min.By 16S rDNA amplified production in gold only intelligence biology section
Skill company limited is sequenced, and sequencing gained sequence carries out BLAST comparison by ncbi database, by MEGA5.2 software and
Known array carries out Phylogenetic Analysis.With Antagonistic Fungi BA-KA4 genomic DNA as template, carried out with primer 2 7F and 1492R
PCR expands, and the 16S rDNA nucleotide sequence length recording this bacterial strain is 1394bp, as shown in sequence SEQ ID No.3, with
In NCBI, known 16S rDNA sequence compares, and finds Antagonistic Fungi BA-KA4 bacterial strain and Bacillus
methylotrophicus strain F29、Bacillus amyloloquefaciens strain CSM11、Bacillus
Subtilis strain MB8, similarity is 100%, builds the phylogenetic tree of 16S rDNA sequence（Fig. 4）, can see
Go out, Antagonistic Fungi BA-KA4 is accredited as bacillus amyloliquefaciens Bacillus amyloloquefaciens.
（2）GyrB gene sequencing is identified
Test kit using Tiangeng company carries out the extraction of bacterial genomes DNA, and with this DNA as masterplate, forward primer is UP1
(5 '-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3 '), sequence SEQ ID No.4, reverse primer is
UP2r（5′-AGCAGGGTACGGAT GTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3′）, sequence SEQ ID No.5, expands
Increase gyrB gene order.PCR amplification condition is： 95℃ 4min；98 DEG C of 10s, 62 DEG C of 1min, 72 DEG C of 2min, 30 are followed
Ring；72℃ 8min.GyrB gene amplification product is sequenced in Jin Wei intelligence bio tech ltd, be sequenced gained sequence
BLAST comparison is carried out by ncbi database, Phylogenetic Analysis are carried out by MEGA5.2 software and known array.With antagonism
Bacterium BA-KA4 genomic DNA is template amplification gyrB gene, and sequence length is about 1010bp, as shown in sequence SEQ ID No.6,
Compare with the 16S rDNA sequence of type strain in NCBI, find Antagonistic Fungi BA-KA4 bacterial strain and solution starch brood cell's bar
Recently, similarity is 98% to the homology of bacterium Bacillus amyloloquefaciens, builds system according to gyrB gene order
System develops tree（Fig. 5）It can be seen that Antagonistic Fungi BA-KA4 and bacillus amyloliquefaciens Bacillus amyloloquefaciens
FZB42 sibship is closest.
The cultural characteristic of comprehensive bacterial strain, form and physiological and biochemical property and 16S rDNA sequence and gyrB gene order divide
Analysis result, can show that Antagonistic Fungi BA-KA4 bacterial strain is bacillus amyloliquefaciens Bacillus amyloloquefaciens.
The potted plant efficiency test to gray mold of cucumber for the embodiment 3 bacillus amyloliquefaciens BA-KA4
Prepare seed bottle, picking BA-KA4 single bacterium colony is inoculated in equipped with 100 mL triangular flasks of 20mL PB fluid medium, 28
DEG C, 180rpm shaking table concussion and cultivate 24h.Draw 10 mL bacterium solution to be inoculated in equipped with 200mL PB fluid medium from seed bottle
500mL triangular flask in, 28 DEG C, after 180rpm shaking table concussion and cultivate 48h, be diluted to 1 × 10 with sterilized water8CFU/ mL, as
Bacteria suspension.Take 121 DEG C of sterilizing 20min of bacteria suspension of same concentrations, as without fermented liquid.Fluoxastrobin dilutes 1000 with sterilized water
Times.With card punch in the ash arrhizus bacteria edge punching of growth 3d, truffle is put in 0.9% normal saline, 22 DEG C, 180rpm
Shaking table concussion shakes up 30min, and being diluted to concentration with sterilized water is 1 × 106The spore suspension of individual/mL.Potted plant Fructus Cucumidis sativi foliage-spray
After bacteria suspension, without fermented liquid, Fluoxastrobin 6h, inoculate ash arrhizus bacteria spore suspension, sterilized water is negative control.Dark moisturizing
48h, 22 DEG C of constant temperature culture, observe 5d, 7d, 14d incidence, calculate disease index.
As shown in table 2, the without fermented liquid of bacillus amyloliquefaciens BA-KA4 and Fluoxastrobin are to Fructus Cucumidis sativi grey mold for experimental result
The prevention effect of disease can reach 100% in 5d and 7d, but the preventive effect of Fluoxastrobin declines in 14d, and without fermented liquid is prevented
Effect still can reach 97.5%.Contrast Fluoxastrobin and the treatment group of bacteria suspension, the blade more enriching after without fermented liquid process
Green.The conventional medicament Fluoxastrobin of contrast, prevention effect is suitable in a short time, but the metabolite lasting period of this bacterial strain is longer.
Table 2 bacillus amyloliquefaciens BA-KA4 tests to the control efficacy of gray mold of cucumber
Compare in gray mold of cucumber and universal area occurs, prevented and treated using antagonistic microbe, it is possible to reduce chemical pesticide
Use, solve Pesticide Residue, delay pathogenic bacteria Drug resistance to produce.
Bacillus amyloliquefaciens is a kind of and the very high antibacterial of Bacillus subtillis affinity, in the growth course of its own
In can produce a series of metabolite, these metabolites enable bacillus amyloliquefaciens to have widely to suppress true
Bacterium and the activity of antibacterial.The potted plant efficiency test of this research shows the metabolite of bacillus amyloliquefaciens BA-KA4 to Fructus Cucumidis sativi ash
The prevention effect of mildew still can reach 97.5% after can reach 100%, 14d in 5d and 7d.The conventional medicament Fluoxastrobin of contrast, short-term
Interior prevention effect is suitable, but the metabolite lasting period of this bacterial strain is longer.
SEQUENCE LISTING
<110>Biology Inst., Hebei Academy of Sciences
<120>A kind of ash arrhizus bacteria antagonistic strain and its screening technique and application
<130> 2016
<160> 6
<170> PatentIn version 3.3
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<400> 1
agagtttgat cmtggctcag 20
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<213>Artificial sequence
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ggytaccttg ttacgactt 19
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<212> DNA
<213>Bacillus amyloliquefaciens
<400> 3
ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa 60
cctgcctgta agactgggat aactccggga aaccggggct aataccggat ggttgtttga 120
accgcatggt tcagacataa aaggtggctt cggctaccac ttacagatgg acccgcggcg 180
cattagctag ttggtgaggt aacggctcac caaggcgacg atgcgtagcc gacctgagag 240
ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg 300
gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt 360
cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg gcggcacctt 420
gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 480
gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct 540
gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca 600
gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc 660
agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg 720
aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg 780
tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc 840
aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 900
ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc ctagagatag 960
gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1020
agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt 1080
tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc 1140
atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca aagggcagcg 1200
aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc agtctgcaac 1260
tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1320
tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt 1380
gaggtaacct ttag 1394
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gaagtcatca tgaccgttct gcaygcnggn ggnaarttyg a 41
<210> 5
<211> 44
<212> DNA
<213>Artificial sequence
<400> 5
agcagggtac ggatgtgcga gccrtcnacr tcngcrtcng tcat 44
<210> 6
<211> 1010
<212> DNA
<213>Bacillus amyloliquefaciens
<400> 6
tgaaaccatc gaagcgatat aaagtatccg gcggtcttcc ggtgtagggg cgtctgtcgt 60
aacgccttgt cgaccactct tgacgttacg gttcatcgtg acggaaaaat ccactatcag 120
gcgtacgagc gcggtgtacc tgtggccgat cttgaagtga tcggtgatac tgataagacc 180
ggaacgatta cgcacttcgt tccggatccg gaaattttca aagaaacaac cgaatacgac 240
tatgacctgc tttcaaaccg tgtccgggaa ttggccttcc tgacaaaagg tgtaaacatc 300
acgattgaag acaaacgtga aggacaagaa cggaaaaacg agtaccacta cgaaggcgga 360
atcaaaagct atgttgagta cttaaaccgt tccaaagaag tcgttcatga agagccgatt 420
tatatcgaag gcgagaaaga cggcataacg gttgaagttg cattgcaata caacgacagc 480
tatacaagca atatttattc tttcacaaat aatatcaaca catacgaagg cggcacgcac 540
gaagccggat ttaaaaccgg tctgacccgt gttataaacg actatgcaag aagaaaaggg 600
attttcaaag aaaatgatcc gaatttaagc ggggatgatg tgagggaagg gctgactgcc 660
attatttcaa ttaagcaccc tgatccgcaa ttcgaagggc agacgaaaac gaagctcggc 720
aactccgaag cgagaacgat cactgatacg ctgttttctt ctgcgctgga aacattcctt 780
cttgaaaatc cggactcagc ccgcaaaatc gttgaaaaag gtttaatggc cgcaagagcg 840
cggatggcag cgaaaaaagc gcgggaattg acccgccgca aaagtgcgct tgagatttcc 900
aatctgccgg gcaaactggc ggactgttct tctaaagatc cgagcatttc cgagctgtat 960
atcgtagagg gtgactctgc gggcggatca gcgaacaggg acgggaccgc 1010
Claims (7)
1. a kind of ash arrhizus bacteria antagonistic strain is it is characterised in that this bacterial strain was bacillus amyloliquefaciens BA-KA4, in 2016 3
The moon has carried out preservation in China Committee for Culture Collection of Microorganisms's General Microbiological Culture collection on 8th, and preserving number is：
CGMCC No. 12190.
2. ash arrhizus bacteria antagonistic strain according to claim 1 is it is characterised in that have suppression P. capsici
（Phytophthora capsici）, fusarium graminearum（Fusarium graminearum）, rhizoctonia cerealis
（Rhizoctonia cerealis）, tobacco brown spot pathogen（Alternaria alternata）, Bulbus Allii hyphal cluster germ
（Sclerotinia allii Saw）, botrytis cinerea（Botrytis cinerea）, tomato early blight bacterium
（Alternaria solani）, Pear black spot bacterium（Alternaria alternate）The characteristic of growth.
3. a kind of screening technique of bacillus amyloliquefaciens BA-KA4 described in any one of claim 1-2 is it is characterised in that include
Following steps：
（1）Antibacterial in the soil pattern of different regions is isolated and purified and preservation；
（2）Primary dcreening operation：The screening of Antagonistic Fungi is carried out using the flat board method that stands facing each other, with the card punch of diameter 5mm activation ash arrhizus bacteria
Edge punches, and picking truffle is inoculated in PDA plate central authorities, and 4 different single bacterium colonies of picking are uniformly connected on apart from indicator bacteria 3cm's
Position, after 22 DEG C of dark culturing 4-6d, observes fungistatic effect, selects the bacterial strain entrance secondary screening with inhibition zone；
（3）Secondary screening：With ash arrhizus bacteria as indicator bacteria, the bacterial strain point entering secondary screening is connected on by anomaly plate central authorities using crossing method
On 4 angles at 2-3cm, flat board central authorities are simultaneously moved into length the agar block of ash arrhizus bacteria, after 22 DEG C of dark culturing 5-7d,
Observe antibacterial result, measure antibacterial circle diameter, calculate bacteriostasis rate, the bacterial strain of screening bacteriostasis rate more than 60%；Measure inheritance stability
Property, the bacterial strain of bacteriostasis rate more than 60% after screening and culturing 10 generation；Select P. capsici, fusarium graminearum, wheat sharp eyespot
Bacterium, tobacco brown spot pathogen, Bulbus Allii hyphal cluster germ, botrytis cinerea, tomato early blight bacterium, Pear black spot bacterium as indicator bacteria,
Using flat board face-off method, the bacterial strain selected is carried out with the mensure of antimicrobial spectrum, observes fungistatic effect, measure antibacterial circle diameter, calculate suppression
Bacterium rate, filters out and has best fungistatic effect bacterial strain, as bacillus amyloliquefaciens BA-KA4 to this 8 kinds of pathogenic fungi.
4. screening technique according to claim 3 is it is characterised in that step（1）Concrete operations be：From Yunnan, black dragon
120 pedotheques coming are adopted in river, Xinjiang, Hebei Province, using PB culture medium, apply plate method using dilution and carry out separating,
After culture 3d in 28 DEG C of incubators, the different antibacterial of picking colony form carries out 5 line purification, numberings, and using 15%
Glycerol is in -80 DEG C of Refrigerator stores.
5. screening technique according to claim 4 is it is characterised in that described PB culture medium is：Carnis Bovis seu Bubali cream 5.0 g, egg
White peptone 10.0 g, NaCl 5.0 g, agar 15 g, pH 7.4, distilled water is settled to 1L, 115 DEG C -121 DEG C sterilizing 20min-
30min.
6. the answering in preventing and treating crop grey mold disease of the bacillus amyloliquefaciens BA-KA4 described in a kind of any one of claim 1-5
With.
7. application according to claim 6 is it is characterised in that prevention and controls are by bacillus amyloliquefaciens with sterilized water
It is 1 × 10 that BA-KA4 is diluted to concentration8-1×109The bacteria suspension of CFU/mL, after 115 DEG C -121 DEG C sterilizing 20min, foliage-spray
In crop.
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| CN108077411A (en) * | 2017-12-20 | 2018-05-29 | 山西省农业科学院农产品贮藏保鲜研究所 | The preparation method and its application method of bacillus amyloliquefaciens Y-3 bio-preservatives |
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