CN105219681B - A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM and preparation method and application - Google Patents
A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM and preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of bacillus amyloliquefaciensBacillus amyloliquefaciensD2WM and preparation method and application, step are：A, bacillus isolates and purifies：After being inoculated with inclined-plane, preservation；B, the primary dcreening operation of bacillus：Using a connection and flat board face-off method, the bacterial strain for having antagonism to Erwinia and oil tea disease is successively selected.C, the secondary screening of bacillus：Using cylinder-plate method.The bacterial strain that primary dcreening operation has antagonism is cultivated in LB fluid nutrient mediums, is filtered out by antagonism fermented liquid and bacteria-free filtrate while has the bacterial strain of antagonism to Erwinia and oil tea Major Diseases.Application of the bacillus amyloliquefaciens in medicine in preparing treatment or prevention vegetable bacterial soft rot, oil tea disease, with wide spectrum antagonism, the speed of growth is fast, incubation time is substantially reduced for general bacterium, fungi, reduce production cost, can the efficiently multiple diseases such as antagonism Oil Tea Anthracnose, oil tea leaf spot, oil tea black spot, oil tea canker, and fungistatic effect is notable.
Description
Technical field
The present invention relates to plant protection art, and being more particularly to a kind of energy, antagonism vegetable bacterial soft rot and oil tea are mainly sick simultaneously
Harmful bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM, also relate to a kind of solution starch gemma bar
Bacterium Bacillus amyloliquefaciens D2WM preparation method, further relates to a kind of bacillus amyloliquefaciens Bacillus
Amyloliquefaciens D2WM are preparing the purposes in treating or preventing vegetable bacterial soft rot and oil tea medicine.
Background technology
Vegetable bacterial soft rot is also known as that water is rotten, rotten pimple, in addition to brassicaceous vegetable (such as radish, Chinese cabbage) is endangered, can also endanger
The vegetables such as evil potato, tomato, lettuce, cucumber, celery and green onion class, cause different degrees of loss.Vegetable bacterial soft rot morbidity is just
Phase, sick portion are that water soaking mode is translucent, after expand as ficelle web rot, sick portion's tissue is glutinous to be slided, and surface depressions after dehydration, is often overflowed
White bacterium is dense, and foul smelling, and later stage vegetables entirely soften, and all rot, see (easily figure vegetable bacterial soft rots [J] Hunan agricultures forever
Industry, 2015 (2)：28-29.) Erwinia (Erwinia) is the main pathogenic fungi for causing plant soft rot disease to occur, and it belongs to intestines
Bacteriaceae (Enterobacteriaceae), mainly include carrot soft rot Erwinia carrot subspecies (Erwinia
Carotovora ssp.carotovora, Ecc), black shin subspecies (the Erwinia carotovora of carrot soft rot Erwinia
Ssp.atroseptica, Eca) and three subspecies of Erwinia chrysanthemi (Erwinia chrysanthemi, Ech), see (P é
rombelon M.Potato diseases caused by soft rot Erwinias:an overview of
pathogenesis[J].Plant pathology.2002,51(1):1-12.) wherein, Eca selectivity is stronger, mainly invades
Contaminate temperate zone potato；Host range and geographical distribution are the most extensive under field conditions (factors) by Ecc, can infect potato, carrot, celery,
The plants such as cucumber, capsicum and witloof, spread all over the ground such as subtropical zone and temperate zone；Ech can then infect potato, dahlia and corn etc., please
See (Toth I K, Bell K S, Holeva M C, et al.Soft rot erwiniae:from genes to genomes
[J].Molecular plant pathology.2002,4(1):17-30.) in recent years, vegetable cultivation scale constantly expands, vegetable
Dish disease progressively draws attention, wherein occurred especially with vegetable bacterial soft rot it is serious, caused by loss it is huge.
Oil tea (Camellia oleifera) belongs to Theaceae (Theaceae) Camellia (Camellia), is that China is peculiar
A kind of advanced oil crops, be mainly grown on southern china subtropical zone high mountain and hilly country, be important woody oil
One of seeds are expected, see (village Rayleigh China oil tea [M] Beijing：China Forestry Publishing House, 2008.) Seed of Camellia oleifera is containing a large amount of excellent
The unrighted acid and vitamin E of matter, it is a kind of in high-quality, storage endurance table oil, is accounted for importantly in edible oil
Position；In addition, tea oil medicine, be industrially also widely used, can be as the raw material of soap, vaseline, antirust oil, lubricating oil etc.；
Tea seed cake can do agricultural chemicals, and high-quality organic fertilizer, or the raw material of the product such as extraction tannin extract, saponin, furfural, activated carbon, carry
Solid residue after taking can be as the concentrated feed of pig.It can be said that oil tea whole body is all precious, there is good application and development prospect,
There is very important effect to promoting increasing peasant income, accelerating mountain area comprehensive development, the national grain and oil safety of maintenance etc..
Oil tea disease species are various, and its harm is also more serious.Oil Tea Anthracnose, leaf blight, soft rot are by pathogen
Caused 3 kinds of destructive diseases, generally occur in each oil tea producing region in China, each position in ground of oil tea can be aggrieved, mainly draws
It is dead to play withered shedding, bud drop, branch, limb ulcer, even whole strain, see (the oil tea cultivation of Shu Qinglong, Zhang Liangfu China with
The prevention and control of plant diseases, pest control [M] .. Beijing：China Forestry Publishing House, 2009.) equally, oil tea root rot, gall of tea, black spot, canker
Deng also occurring often, the annual camellia oleiferaindustry economic loss huge because disease causes.In recent years, Hubei oil tea planting scale is not
Disconnected to expand, corresponding disease progressively draws attention.
However, the preventing and treating to vegetable bacterial soft rot and oil tea Major Diseases is generally prevented by planting resistant variety and chemistry at present
Control to control, excessive use chemical bactericide, which has, to be endangered human body health, medicament residue destruction ecological environment, triggers pathogen
Develop immunity to drugs, influence the shortcomings such as vegetables and tea oil quality and edible safety, be unfavorable for the green of vegetables and camellia oleiferaindustry
Sustainable development.Seek safe efficient and economic biogenic pesticide has turned into the focus of current preventing and treating vegetables and oil tea disease
And the task of top priority, wherein antagonistic microbe play highly important effect in biocontrol of plant disease.
Studied in the biological control of plant disease and mainly have bacterium class, Mycophyta and unwrapping wire using more microorganism
Bacterium.Bacillus (Bacilus) is a kind of preferably screening of biocontrol agents object, is studied in current biocontrol bacteria more.Its energy
Enough suppress the disease at each positions such as plant root, limb, leaf, flower and fruit, be of wide application.Because its can produce it is resistance to
The spore of hot, drought-enduring, uvioresistant and organic solvent, therefore it is very beneficial to Biocontrol Bacillus colonizing and breeding in the environment
And its production and processing of zymocyte liquid, see (the intelligent fragrant of class turns the soft rot resistance of aiiA gene konjakus and konjaku endophyte produces
Composition analysis [D] the Hua Zhong Agriculture University of Antagonistic protein, 2009.) and (Chai Xinli, Zhou Ying, woods are supported the army, and wait Su Yun gold brood cells
The anti-soft rot aiiA genes of bacillus turn elephant-foot yam research [J] Molecular Plant Breedings, 2007,5 (5)：613-618.).Current life
Anti- bacillus mainly includes bacillus subtilis (B.subtilis), bacillus megaterium (B.megaterlum), more viscous bud
Spore bacillus (B.polymyxa), bacillus pumilus (B.pumilus) and Bacillus cercus (B.cereus) etc..Sun Ming etc.
A kind of bacillus subtilis BSn5 and Antagonistic protein APn5 (Shen to carrot soft rot Erwinia with bacteriostatic activity is invented
Please number：200710053427.8), the Substance of the bacillus subtilis is albumin A Pn5, can only antagonism carrot soft rot
Erwinia subspecies, its biological and ecological methods to prevent plant disease, pests, and erosion scope are narrower；Huang waits disclosing a kind of bacillus amyloliquefaciens Lh-1 quietly and withered in preventing and treating cotton yellow
Application (the application number of disease, soft rot of Chinese cabbage, bacterial wilt, peach rot, canker of apple fruit etc.：
201410735508.6), bacillus amyloliquefaciens need to be utilized to be combined into microecological microbial agent with chitosan oligosaccharide, comparatively speaking only to cotton
The prevention effect of verticillium wilt is preferable；Liu Junang etc. discloses a kind of Brevibacillus brevis Z26 of antagonism oil tea disease, can be effective
Suppress Oil Tea Anthracnose bacterium, root rot, the growth (application number of the half of pathogen such as crazy：201110027957.1), the biocontrol microorganisms
Agent reaches 83.4% to the maximal percentage inhibition of Oil Tea Anthracnose, the space also further improved；Zhou Guoying etc. has invented one kind
The effectively complex microorganism source pesticide and application process (application number of preventing and treating oil tea disease：201310203845.6), open invention
It is a kind of by actinomyces styreptomyces globispotus strain ball spore subspecies and the microbial pesticide of bacterium Brevibacillus brevis and other additive compounds,
Oil tea one or more Major Diseases can be prevented and treated, the microbial pesticide preparation process of the compounding is relatively cumbersome, takes longer；Also have
Using bacillus subtilis to bacillus thuringiensis ML1 respectively in peanut rot disease preventive treatment and the related report in konjak soft rot preventing and treating
Road (application number：201310708367.4 application number：201510015310.5).However, can be on bacillus amyloliquefaciens simultaneously
Study on prevention and application report in vegetable bacterial soft rot and oil tea Major Diseases is seldom.In addition, document report is mostly for oil
The single preventing and treating of tea a certain kind disease, at present market there is no that two or more oil tea disease effects can be prevented and treated simultaneously substantially good
Single microbial inoculum product, and yet there are no the report to preventing and treating two kinds of diseases of oil tea black spot and oil tea canker.
The content of the invention
The purpose of the present invention is to be the provision of a kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens
D2WM, the bacillus amyloliquefaciens obtain from healthy oil tea rhizosphere soil separation screening, can efficiently prevent and treat the soft corruption of vegetables simultaneously
Disease and oil tea Major Diseases, it is one plant of Biocontrol Bacillus with wide spectrum antagonism, it is same has filled up single microorganism microbial inoculum
When prevent and treat the technological gap of three subspecies of Erwinia and a variety of oil tea disease funguses, have to soft rot and oil tea non-environmental pollution control
There is certain practical significance.
Another object of the present invention is to be the provision of a kind of bacillus amyloliquefaciens Bacillus
The preparation method of amyloliquefaciens D2WM biocontrol agents, it is easy to implement the method, it is easy to operate.Microbial inoculum obtains through following steps
Arrive：The ring of D2WM bacterium colonies picking one on LB flat boards or inclined-plane is transplanted to test tube activation, 35 DEG C of shaking table culture 16h, rotating speed
150rpm, LB fluid nutrient mediums are in test tube：Peptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L, pH 7.0；Activation
Bacterium solution afterwards is transplanted in the 250mL conical flasks equipped with 100mL LB fluid nutrient mediums according to 1%-3% inoculum concentration, rotating speed
150rpm, 35 DEG C of shaking table culture 10h-24h, when bacterial content is more than 2 × 108More than cfu/mL can obtain zymocyte liquid, storage
As biocontrol agent.It is easy that the biocontrol agent makes, and the speed of growth is fast, and Liquid Culture metabolite only needs liquid medium within
Middle culture 10-24h can obtain good antagonistic effect, and incubation time is substantially reduced relative to general bacterium, fungi, reduce
Production cost.
A further object of the invention is to be the provision of a kind of bacillus amyloliquefaciens Bacillus
Amyloliquefaciens D2WM are preparing the application in treating or preventing vegetable bacterial soft rot medicine, and the biocontrol bacterial strain can be simultaneously
Treat or prevent every vegetable bacterial soft rot that can be triggered by Erwinia, including green pepper, hot red pepper, lettuce, potato, bottle gourd, Chinese cabbage
Soft rot etc..To carrot soft rot Erwinia carrot subspecies, the black shin subspecies of carrot soft rot Erwinia and Erwinia chrysanthemi three
Individual subspecies have good antagonism.
The 4th purpose of the present invention is to be the provision of a kind of bacillus amyloliquefaciens Bacillus
Amyloliquefaciens D2WM are preparing the application in treating or preventing oil tea Major Diseases medicine, the biocontrol microorganisms of the bacterial strain
Agent can efficiently prevent and treat Oil Tea Anthracnose, leaf spot, black spot, canker etc., and the experiment of liquid fermentation filtrate bacteriostasis rate shows, the solution
Bacillus amyloliquefacienses Bacillus amyloliquefaciens D2WM the bacteriostasis rate of oil tea Major Diseases is reached 95% with
On.
In order to realize above-mentioned purpose, the present invention uses following technical measures：
Its technical concept is：Separated, purified and preserved using conventional method to soft rot and oil tea Major Diseases simultaneously
There is the biocontrol bacterial strain of antagonism.See (Yao Lijin, Wang Qi, Fu Xuechi, waiting the screening of wheat sharp eyespot Biocontrol Bacillus
And identification [J] Chinese biological preventing and treatings, 2008,24 (1)：53-57.).Using the Bacteria erwinia that laboratory is separated to as indicator bacteria, point
It is bacterium to be checked from obtained bacterial strain, bacterium will be isolated and purified and screened successively using a connection, primarily determine that has to Erwinia
The bacterial strain of antagonism, at the same using flat board face-off method further screening wherein can simultaneously antagonism oil tea pathogen antagonism it is thin
Bacterium.Point connection and flat board face-off method see (T,Banhegyi I,Herpai Z,et al.Isolation of
Bacillus strains from the rhizosphere of cereals and in vitro screening for
antagonism against phytopathogenic,food-borne pathogenic and spoilage micro-
organisms[J].Journal of Applied Microbiology,2000,89(5):840-846.) and then be respectively adopted
Cylinder-plate method and fermentation method carry out secondary screening, after the bacterial strain further screened is cultivated into 24h in LB fluid nutrient mediums respectively, culture
Liquid is filtrated to get culturing filtrate with 0.22 μm of biofilter.It is coated with 100 μ L soft rot nosophytes (about 1 × 107cfu/
ML the equidistant hole that a diameter of 7mm is beaten with card punch on LB agar plates).50 μ L antagonism fermented liquids are accessed in each hole,
Using sterilized water as control, each processing repeats three times.Antibacterial circle diameter is measured after cultivating 24h in 30 DEG C of insulating boxs, according to number
Value size determines antagonism degree.Filtering out has the bacterial strain of antagonism to soft rot nosophyte.Meanwhile respectively by primary dcreening operation Antagonistic Fungi
Filtrate and about 50 DEG C of PDA solid mediums are according to 1:Culture dish is poured into after 19 mixings, diameter is put into flat board center after cooling
For 7mm oil tea the main pathogenic fungi bacteria cake, using sterilized water as control, each processing repeats three times.Pathogen bacterium colony is measured after 5 days
Diameter.Filter out the bacterial strain for having antagonism to oil tea Major Diseases.It is final to determine that D2WM is main to soft rot opportunistic pathogen and oil tea
The antagonism of disease is most strong, and screening obtains positive effect and 1 plant of stable Antagonistic Fungi D2WM.Cylinder-plate method and fermentation method see
(Yao Lijin, Wang Qi, Fu Xuechi, the screening and the preventing and treating of identification [J] Chinese biologicals of wheat sharp eyespot Biocontrol Bacillus are waited,
2008,24(1):53-57.)。
A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM preparation method, its step are：
A, bacillus isolates and purifies：Take healthy oil tea Rhizosphere sampling 10g to be added to and fill 90mL sterilized waters and 30-50
In the triangular flask of bead, 1 is made：The suspension of 10 concentration, will after vibrating 20-30min in 28 DEG C, 180rpm shaking tables
Bacteria suspension is placed in 75-85 DEG C of water-bath and keeps 12-18min.L mL Soil Slurries are moved into the test tube for filling 9mL sterilized waters
In, it is made 10-2Bacteria suspension, the like, obtain 10-3, 10-4, 10-5And 10-6Bacteria suspension.After gradient dilution, 100 μ L are drawn
Extension rate is 10-3-10-6Bacteria suspension be uniformly coated on isolation medium -- on beef-protein medium, culture medium is matched somebody with somebody
Side is following (1L)：Beef extract 3.0g, peptone 10.0g, NaCl 5.0g, agar 15.0g, distilled water 1000mL, pH 7.2-
7.4.The flat board for being coated with bacteria suspension is placed in 30 DEG C of incubators 1 week, observes bacterium colony shape on isolation medium after 24h immediately
State, isolated and purified with three steps line hair, Unified number registration.Culture presevation：, can be in 4 DEG C of of short duration preservations after being inoculated with inclined-plane.
, can be in -80 DEG C of permanent preservations by the use of 50% (V/V) glycerine as protective agent.
B, the primary dcreening operation of bacillus：First using a connection, 100 μ L pathogens bacterium solutions (about 1 × 10 are taken7Cfu/ml) uniformly apply
On cloth LB flat boards, then with sterile toothpick on each flat board point connect four bacterial strains of participating in the experiment (successively select D1WM, D2WM,
4 bacterial strains in D3WM ... D30WM), each processing sets three repetitions.20- is cultivated in 30 DEG C and 37 DEG C of insulating boxs
28h.Simultaneously using flat board face-off method further screening wherein can simultaneously antagonism oil tea pathogen antagonistic bacterium, diameter will be accomplished fluently
PDA plate center is placed for 7mm cause of disease bacteria cake, then dips equidistant (the anomaly plate center of bacterium to be measured with sterile toothpick
1.5cm) point connects Antagonistic Fungi, is placed in 28 DEG C of constant incubator cultures, is observed after 5 days antibacterial with it's too late size, each processing weight
Again three times, the bacterial strain that bacteriostatic diameter is more than 5mm is further screened.
C, the secondary screening of bacillus：Using cylinder-plate method secondary screening.Primary dcreening operation there is into the bacterial strain of antagonism in LB fluid nutrient mediums
In with 150rpm thermophilic culture 20-28h, zymotic fluid centrifuges 10min with 8000rpm, supernatant with 0.22 μm of filtering with microporous membrane extremely
In sterilizing EP pipes.It is coated with 100 μ L pathogens (about 1 × 107Cfu/mL it is equidistant on LB agar plates) to be beaten with card punch
A diameter of 7mm hole.50 μ L zymotic fluids are accessed in each hole, using sterilized water as control, each processing is set to be repeated three times.
Antibacterial circle diameter is measured after cultivating 20-28h in 30 DEG C of insulating boxs, antagonism degree is determined according to numerical values recited.Filter out to soft corruption
Germ bacterial strain has the bacterial strain of antagonism.Meanwhile respectively by primary dcreening operation Antagonistic Fungi filtrate and about 50 DEG C of PDA solid mediums according to
1:Culture dish is poured into after 19 mixings, a diameter of 7mm oil tea the main pathogenic fungi bacteria cake is put into after cooling in flat board center, with sterile
Water is control, and each processing repeats three times.Pathogen colony diameter is measured after 5 days.Filter out has antagonism to oil tea Major Diseases
The bacterial strain of effect.Final screening obtains one plant of obvious and stable Antagonistic Fungi D2WM of antagonistic effect, a kind of bacillus amyloliquefaciens
Bacillus amyloliquefaciens D2WM。
Contain SEQ ID in a kind of described bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM
NO：Nucleotide sequence shown in 1.
A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM have following feature：The bacterial strain exists
In terms of form and cultural characteristic：Thalline is shaft-like, peritrichous, and has motility；Bacterium colony is milky, in subcircular, surface
Coarse to have fold, edge is irregular, does not produce soluble pigment.
Applicant screens to obtain one plant of bacterium from healthy oil tea rhizosphere soil, is reflected with reference to physiological and biochemical property and characterization of molecules
It is set to bacillus amyloliquefaciens, and is named as Bacillus amyloliquefaciens D2WM.Bacterial strain Bacillus
Amyloliquefaciens D2WM have been preserved in China typical culture collection center (referred to as on October 20th, 2015
CCTCC, Chinese Wuhan Wuhan Universitys), deposit number is CCTCC NO: M 2015623.
A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM are preparing treatment or prevention vegetables
Application in soft rot in medicine, it is using basic process：
A, bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM are real to vegetable bacterial soft rot inhibition zone
Test：Bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM zymotic fluids are investigated to Ou Wenshi using cylinder-plate method
The antagonism of bacterium.A ring bacterium colony is taken to be placed in the LB liquid medium of sterilizing respectively, 35 DEG C of shaking table culture 12h, rotating speed
150rpm, that is, obtain the black shin subspecies of carrot soft rot Erwinia carrot subspecies, carrot soft rot Erwinia and Erwinia chrysanthemi
3 kinds of pathogens and Antagonistic Fungi Bacillus amyloliquefaciens D2WM thalline zymotic fluids, 100 μ L pathogens are respectively taken to send out
Zymotic fluid is coated in LB solid mediums, and three 7mm hole is uniformly made a call to aseptic card punch, by Antagonistic Fungi bacillus amyloliquefaciens
Bacillus amyloliquefaciens D2WM zymotic fluids or zymotic fluid dilute 10 times with sterilized water, are added respectively into hole
50 μ L zymotic fluids or dilution after fermentation liquid, add sterilized water processing be used as blank control, 35 DEG C cultivate 24h after observe Antagonistic Fungi
To the size of 3 kinds of pathogen inhibition zones.
B, using flat board opposite culture method research bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM
To the antagonism of oil tea Major Diseases.The cause of disease bacteria cake for accomplishing fluently a diameter of 7mm is placed into plate center, then uses sterile toothpick
Dip bacterium to be measured equidistant (anomaly plate center 1.5cm) point and connect Antagonistic Fungi, be placed in 28 DEG C of constant incubator cultures, observed after 5 days
It is antibacterial to be repeated 3 times with it's too late size, each processing.
C, bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM couple are studied using zymotic fluid cultivation
The antagonism of oil tea Major Diseases.Bacillus amyloliquefaciens D2WM bacterium colonies are through LB fluid nutrient medium cultures
After 12h, 10000r/min room temperatures centrifugation 20min, thalline is removed with 0.22 μm of filtering with microporous membrane, takes Antagonistic Fungi filtrate with going out
About 50 DEG C of PDA solid mediums are according to 1 after bacterium:19 mix after pour into culture dish, be put into after cooling in flat board center a diameter of
7mm oil tea the main pathogenic fungi bacteria cake, using sterilized water as control, each processing repeats three times.Pathogen bacterium colony is measured after 5 days
Diameter.Bacteria-free filtrate bacteriostasis rate=(control colony diameter-processing colony diameter)/(control colony diameter -0.7) × 100%.
A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM are preparing treatment or prevention vegetables
Application in soft rot in medicine, it includes：
A kind of described bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM are preparing treatment or pre-
Application in the medicine of the anti-vegetable bacterial soft rot triggered by Erwinia, including green pepper, hot red pepper, lettuce, potato, bottle gourd, Chinese cabbage
Soft rot etc..
A kind of described bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM are preparing treatment or pre-
Application in anti-oil tea Major Diseases medicine, described oil tea disease is Oil Tea Anthracnose, oil tea leaf spot, oil tea soft rot, oil
Tea black spot, oil tea canker.
The present invention compared with prior art, has advantages below and effect：
1. the strains B. amyloliquefaciens Bacillus amyloliquefaciens D2WM of the present invention, are isolated from oil
Tea healthy plant rhizosphere soil, there is stable ecological environment and Biocontrol Effect.
2. it is easy that the biocontrol agent of the present invention makes, the speed of growth is fast, and Liquid Culture metabolite only needs to train in liquid
Good antagonistic effect can be obtained by supporting culture 10-24h in base, and incubation time is substantially reduced relative to general bacterium, fungi,
Reduce production cost.
3. bacterium field planting power is strong, while has very strong antagonism to make to various vegetables soft rot and a variety of oil tea Major Diseases
With having good development prospect.
4. the biocontrol bacterial strain bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM of the present invention, can lead to
Cross single bacterial strain while prevent and treat the class subspecies of Erwinia 3 and a variety of oil tea diseases, including the fresh oil tea leaf spot do not reported for work, oil tea
Black spot and oil tea canker, antimicrobial spectrum is extensive, can effectively reduce cost accounting.
5. flat board antagonistic experiment shows：Bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM couple
Oil tea Major Diseases have obvious inhibitory action.
6. the experiment of Liquid Culture metabolite bacteriostasis rate shows, bacillus amyloliquefaciens Bacillus
Amyloliquefaciens D2WM reach more than 95% to the bacteriostasis rate of oil tea Major Diseases.
Brief description of the drawings
Fig. 1 is a kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM bacterium colonies and microscopic morphology
Schematic diagram.
Fig. 2 is a kind of D2WM and other bacterial strains 16S rDNA gene order phylogenetic tree schematic diagrames.
Fig. 3 is a kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM bacterial strains to Erwinia
Inhibition zone design sketch；
Fig. 4 is that a kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM bacterial strains are main to oil tea
The flat board face-off antagonism schematic diagram of pathogen.
Fig. 5 is a kind of influence schematic diagrames of pH to Antagonistic Fungi fermentation bacteria-free filtrate antagonistic effect.
Fig. 6 is a kind of influence schematic diagram of temperature to Antagonistic Fungi fermentation bacteria-free filtrate antagonistic effect.
Brief description of the drawings：A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM bacterial strains are in form
In terms of cultural characteristic：Thalline is shaft-like, peritrichous, and has motility；Bacterium colony is milky, in subcircular, rough surface
There is fold, edge is irregular, does not produce soluble pigment.As shown in Figure 1.Molecular Identification, the 16S that will be measured are carried out to the bacterial strain
RDNA sequences (fragment length 1439bp) carry out BLAST analyses with sequence in GenBank databases, choose 9 plants of bacterial strains and use
ClustalX 2.1 carry out Multiple Sequence Alignment analysis, using MEGA6.0 softwares using adjacent method carry out phylogenetic tree structure and
Cluster analysis, MEGA6 Neighbor-Joining method constructing system chadograms, as shown in Figure 2.With reference to above-mentioned form and part
Physiological and biochemical property, can preliminary judgement antagonistic strain D2WM belong to bacillus amyloliquefaciens.Solution starch bud is investigated using cylinder-plate method
20 times of spore bacillus Bacillus amyloliquefaciens D2WM dilute antagonism of the after fermentation liquid to Erwinia, its
Fungistatic effect is shown in Fig. 3.As a result show, the biocontrol bacterial strain is to carrot soft rot Erwinia carrot subspecies, carrot soft rot Ou Wenshi
3 subspecies of the black shin subspecies of bacterium and Erwinia chrysanthemi are respectively provided with good fungistatic effect, the antagonism fermented liquid suppression after 10 times of dilution
Bacterium circle reaches more than 22mm.Bacillus amyloliquefaciens Bacillus is studied using flat board opposite culture method
As a result amyloliquefaciens D2WM are shown in Fig. 4, the Antagonistic Fungi is to oil tea anthrax to the antagonism of a variety of oil tea pathogens
Disease, leaf spot, black spot and canker have good antagonism, and especially Oil Tea Anthracnose, leaf spot, black spot are imitated
Fruit is notable.Fig. 5 and 6 is respectively the influence schematic diagram of pH and temperature to Antagonistic Fungi fermentation bacteria-free filtrate antagonistic effect.As a result show
The bacteria-free filtrate soda acid tolerance range is very wide, has certain inhibition to soft rot opportunistic pathogen in the range of 1-11 in pH, and
And in pH=9, its inhibition is optimal, it was demonstrated that the active ingredient that the environment of meta-alkalescence is more beneficial in bacteria-free filtrate plays short of money
Anti- effect.In addition, the bacteria-free filtrate retains bacteriostatic activity in the range of 20 DEG C to 60 DEG C and its antagonistic effect is optimal at 40 DEG C,
When temperature is higher than 80 DEG C, bacteriostatic activity is lost, and illustrates that the active ingredient in the bacteria-free filtrate is unable to high temperature resistant, it may be possible to egg
The material of white class.
Embodiment
Reagent or raw material used in the present invention, are purchased from commercial channel.
Bacterium source：Erwinia Erwinia carotovora ssp.carotovora, Erwinia
Chrysanthemi, Erwinia carotovora ssp.Atroseptica, with report bacterial strain (Ge Wei treasure's konjakus in document
Soft rot pathogenic process dynamic studies and study on prevention [M] .2013 based on QS systems)；Oil tea glue born of the same parents' anthracnose
Colletotrichum.gloeosporioides, (Meng Qingmin, Zhou Guoying, Liu Junang, oil teas are waited with report bacterial strain in document
The main antibacterial substances of anthracnose antagonistic bacterium Y13 isolate and purify and the mode of action [J] plant protection, 2014,40 (2)：36-
42.)；Oil Tea Anthracnose Colletotrichum nicotianae (Li Dongsheng, open one and raise tobacco charcoals with report bacterial strain in document
The morphological feature and Study on Pathogenicity [J] Agriculture in Hunan science of subcutaneous ulcer disease, 2009 (5)：97-98.)；Oil tea leaf spot
Phyllosticta capitalensis, with bacterial strain is reported in document, (horse is into the flat ginkgoes phyllosticta tikka of Peng Lijuan, Wu Shi
Sick pathogen identification [J] Guizhou Agricultural Sciences, 2014,42 (12):126-128.)；Oil tea soft rot Agaricodochium
Camellia, with report bacterial strain in document, (preventing and treating of Zhao Danyang, Qin Changsheng, Liao Fang the inflammation .5 kinds bactericide to oil tea soft rot is ground
Study carefully [J] Annuals, 2013,29 (2):28-31.)；Oil tea black spot Altermaria alternate are the same as in document
(Kobiler I, Akerman M, Huberman L, et al. are combined control persimmon storage to report bacterial strain with postharvest handling before adopting
During black spot caused by alternaric bacteria (Alternaria alternata) research [J] it is fresh-keeping with processing, 2011,5:
025.), oil tea canker Bothyosphaeria dothidea, with report bacterial strain (Slippers B, Crous P in document
W,Denman S,et al.Combined multiple gene genealogies and phenotypic characters
differentiate several species previously identified as Botryosphaeria
dothidea[J].Mycologia,2004,96(1):83-101.)
Embodiment 1：
A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM preparation method, its step are：
A, bacillus isolates and purifies：Take healthy oil tea Rhizosphere sampling 10g to be added to and fill 90mL sterilized waters and 30-50
In the triangular flask of bead, 1 is made：The suspension of 10 concentration, after vibrating 30min in 28 DEG C, 180rpm shaking tables, bacterium is hanged
Liquid is placed in 80 DEG C of water-baths and keeps 15min.L mL Soil Slurries are moved into the test tube for filling 9mL sterilized waters, be made 10-2
Bacteria suspension, the like, obtain 10-3, 10-4, 10-5And 10-6Bacteria suspension.After gradient dilution, drawing 100 μ L extension rates is
10-3-10-6Bacteria suspension be uniformly coated on isolation medium -- on beef-protein medium, culture medium prescription is as follows
(1L)：Beef extract 3.0g, peptone 10.0g, NaCl 5.0g, agar 15.0g, distilled water 1000mL, pH 7.2-7.4.It will apply
The flat board for being furnished with bacteria suspension is placed in 30 DEG C of incubators 1 week, observes bacterium colony state on isolation medium after 24h immediately, with three steps
Line hair is isolated and purified, and it is as follows to be separated to 30 plants of bacterial strain Unified number registrations：D1WM、D2WM、D3WM…D30WM.Strain
Preservation：, can be in 4 DEG C of of short duration preservations after being inoculated with inclined-plane.By the use of 50% (V/V) glycerine (factory of Shanghai development chemical industry one) as protective agent,
Can be in -80 DEG C of permanent preservations.
B, the primary dcreening operation of bacillus：100 μ L soft rot nosophytes bacterium solutions (about 1 × 10 are taken respectively7Cfu/ml) even spread
On LB flat boards, then with sterile toothpick, point meets four bacterial strains of participating in the experiment (selection D1WM, D2WM, D3WM ... successively on each flat board
4 bacterial strains in D30WM), each processing sets three repetitions.24h is cultivated in 30 DEG C and 37 DEG C of insulating boxs.Obtain wherein
There are 5 plants of bacterial strains D2WM, D9WM, D14WM, D21WM, D29WM of antagonism to Erwinia.Entered simultaneously using flat board face-off method
One step screening wherein can simultaneously antagonism oil tea pathogen antagonistic bacterium, by the cause of disease bacteria cake for accomplishing fluently a diameter of 7mm placement PDA put down
Plate center, then dip bacterium to be measured equidistant (anomaly plate center 1.5cm) point with sterile toothpick and connect Antagonistic Fungi, be placed in 28 DEG C of constant temperature
Incubator culture, observed after 3 days it is antibacterial be repeated 3 times with it's too late size, each processing, further screen bacteriostatic diameter and be more than
5mm bacterial strain.3 plants are obtained altogether by above-mentioned two steps primary dcreening operation while have the bacterial strain of antagonistic effect to soft rot and oil tea Major Diseases
D2WM、D9WM、D21WM。
C, the secondary screening of bacillus：There is the bacterial strain of antagonism in LB fluid nutrient mediums with 150rpm, 30-37 primary dcreening operation
24h is cultivated under the conditions of DEG C, zymotic fluid centrifuges 10min with 8000rpm, and supernatant is managed with 0.22 μm of filtering with microporous membrane to the EP that sterilizes
In.It is coated with 100 μ L pathogens (about 1 × 107Cfu/mL it is equidistant on LB agar plates) to beat a diameter of 7mm with card punch
Hole.50 μ L zymotic fluids are accessed in each hole, using sterilized water as control, each processing is set to be repeated three times.In 30 DEG C of insulating boxs
Antibacterial circle diameter is measured after middle culture 24h, antagonism degree is determined according to numerical values recited.Filter out has antagonism to Bacteria erwinia bacterial strain
The bacterial strain of effect.Meanwhile respectively by primary dcreening operation Antagonistic Fungi filtrate and about 50 DEG C of PDA solid mediums according to 1:Poured into after 19 mixings
Culture dish, be cooled to the oil tea the main pathogenic fungi bacteria cake for being put into a diameter of 7mm after 20-25 DEG C in flat board center, using sterilized water as
Control, each processing repeat three times.Pathogen colony diameter is measured after 5 days.Filter out has antagonism to oil tea Major Diseases
Bacterial strain.Final screening obtains 1 plant of obvious and stable Antagonistic Fungi bacillus amyloliquefaciens (Bacillus of antagonistic effect
amyloliquefaciens D2WM)。
A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens have been prepared by above-mentioned
D2WM, SEQ ID NO are contained in bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM：Core shown in 1
Nucleotide sequence.
Bacterial strain Bacillus amyloliquefaciens D2WM are preserved in Chinese Typical Representative on October 20th, 2015
Culture collection (abbreviation CCTCC, Luojiashan, Wuchang, Wuhan City, Hubei Province), deposit number M2015623.
Bacillus amyloliquefaciens a kind of while that there is antagonism to vegetable bacterial soft rot and oil tea Major Diseases
Bacillus amyloliquefaciens D2WM, CCTCC NO：M2015623 identification：
A, a kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM morphology and Physiology and biochemistry
Feature：
The bacterial strain is in terms of form and cultural characteristic：Thalline is shaft-like, peritrichous, and has motility；Bacterium colony is milky white
Color, in subcircular, rough surface has fold, and edge is irregular, does not produce soluble pigment.As shown in Figure 1.Reflected in Physiology and biochemistry
Fixed aspect：Belong to G+ bacteria, there is contact enzymatic activity, energy hydrolysis starch, D-Glucose, L- can be utilized Arabic
Sugar, D- xyloses, D-MANNOSE production acid, can utilize citrate, hydrolyzed casein and can make gelatin liquefaction, can reduce reindeer moss ox
Milk and nitrate, V-P reacting positives, without lecithin activity, it is impossible to hydrolyze hippurate and tyrosine, it is impossible to utilize Portugal
Grape sugar aerogenesis.
B, a kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM molecules identification：
To bacterial strain D2WM carry out Molecular Identification, by the 16S rDNA sequences (fragment length 1439bp) measured with
Sequence carries out BLAST analyses in GenBank databases, chooses 9 plants of bacterial strains and carries out Multiple Sequence Alignment analysis with ClustalX 2.1,
Phylogenetic tree structure and cluster analysis, MEGA6 Neighbor-Joining are carried out using adjacent method using MEGA6.0 softwares
Method constructing system chadogram, as shown in Figure 2.Accession number is HM107808.1 (Bacillus in bacterial strain D2WM and GenBank
Amyloliquefaciens strain 15) homology of bacterial strain reaches 99%.According to 16S rDNA criteria for classifications, typically recognize
It is more than 97% for 16S rDNA sequence homologies, it is believed that belong to same kind, it is special with reference to above-mentioned form and part Physiology and biochemistry
Sign, can preliminary judgement antagonistic strain D2WM belong to bacillus amyloliquefaciens.
Embodiment 2：
A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM are preparing treatment or prevention vegetables
Application in soft rot medicine, its step are：
A, a kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM are soft in treatment or prevention vegetables
In maize ear rot in medicine biocontrol agent preparation, its step is：The ring of D2WM bacterium colonies picking one on LB flat boards or inclined-plane is transplanted
Activated to test tube, 35 DEG C of shaking table culture 16h, rotating speed 150rpm, LB fluid nutrient mediums are in test tube：Peptone 10g/L, yeast carry
Take thing 5g/L, sodium chloride 5g/L, pH 7.0；Bacterium solution after activation is transplanted to equipped with 100mL LB according to 1%-3% inoculum concentration
In the 250mL conical flasks of fluid nutrient medium, rotating speed 150rpm, 35 DEG C of shaking table culture 10h-24h, when bacterial content be more than 2 ×
108More than cfu/mL can obtain zymocyte liquid, save as biocontrol agent.
B, bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM biocontrol agents press down to vegetable bacterial soft rot
Bacterium circle is tested：Bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM zymotic fluids pair are investigated using cylinder-plate method
The antagonism of Erwinia.Take a ring bacterium colony to be placed in the LB liquid medium of sterilizing respectively, 35 DEG C of shaking table culture 12h, turn
Fast 150rpm, that is, obtain the black shin subspecies of carrot soft rot Erwinia carrot subspecies, carrot soft rot Erwinia and chrysanthemum Ou Wenshi
3 kinds of cause of disease fermented liquids of bacterium, 100 μ L cause of disease fermented liquids are respectively taken to be coated in LB solid mediums, it is uniform with aseptic card punch
Three 7mm hole is made a call to, adds 50 μ L bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM into hole respectively
10 times of dilutions of biocontrol agent, the processing of sterilized water is added as blank control, Antagonistic Fungi is observed to 3 after 35 DEG C of culture 24h
The size of kind pathogen inhibition zone, its fungistatic effect are shown in Fig. 3.As a result show, the biocontrol bacterial strain is to carrot soft rot Erwinia Hu
3 subspecies of the black shin subspecies of radish subspecies, carrot soft rot Erwinia and Erwinia chrysanthemi are respectively provided with good fungistatic effect, antibacterial
Circle reaches more than 22mm.
Described vegetables are green pepper, hot red pepper, lettuce, potato, bottle gourd, Chinese cabbage etc..
Embodiment 3：
A kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM are preparing six kinds for the treatment of or prevention
Application in oil tea pathogen medicine, its step are：
A, a kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM are treating or preventing six kinds of oil
The preparation of biocontrol agent, its step are in tea pathogen medicine：The ring of D2WM bacterium colonies picking one on LB flat boards or inclined-plane is moved
Plant test tube to activate, 35 DEG C of shaking table culture 16h, rotating speed 150rpm, LB fluid nutrient mediums are in test tube：Peptone 10g/L, yeast
Extract 5g/L, sodium chloride 5g/L, pH 7.0；Bacterium solution after activation is transplanted to equipped with 100mL according to 1%-3% inoculum concentration
In the 250mL conical flasks of LB fluid nutrient mediums, rotating speed 150rpm, 35 DEG C of shaking table culture 10-24h, when bacterial content be more than 2 ×
108More than cfu/mL can obtain zymocyte liquid, save as biocontrol agent.
B, a kind of bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM and a variety of oil tea cause of disease thalline
Outer interaction research：Using flat board opposite culture method.The cause of disease bacteria cake for accomplishing fluently a diameter of 7mm is placed into plate center, then with nothing
Bacterium toothpick dips bacterium to be measured equidistant (anomaly plate center 1.5cm) point and connects Antagonistic Fungi, is placed in 28 DEG C of constant incubator cultures, 5 days
After observe and antibacterial be repeated 3 times with it's too late size, each processing.It the results are shown in Table 1 and Fig. 4.As a result Bacillus is shown
Amyloliquefaciens D2WM have good antagonism to Oil Tea Anthracnose, leaf spot, soft rot, black spot and canker
Effect, especially to Oil Tea Anthracnose, leaf spot, black spot and canker significant effect.
Suppression diameters of the Bacillus amyloliquefaciens D2WM of table 1 to oil tea Major Diseases
Data are mean+SD (similarly hereinafter) in table, suppress cause of disease after diameter=untreated pathogen diameter-processing
Bacterium diameter
Using zymotic fluid cultivation.The biocontrol agent that step A obtains is used by 10000r/min room temperatures centrifugation 20min
0.22 μm of filtering with microporous membrane removes thalline, and about 50 DEG C of PDA solid mediums are according to 1 after taking Antagonistic Fungi filtrate and sterilizing:
Culture dish is poured into after 19 mixings, a diameter of 7mm oil tea the main pathogenic fungi bacteria cake is put into after cooling in flat board center, with sterilized water
For control, each processing repeats three times.Pathogen colony diameter is measured after 5 days.Bacteria-free filtrate bacteriostasis rate=(control bacterium colony is straight
Footpath-processing colony diameter)/(control colony diameter -0.7) × 100%.
Table 2 shows that Bacillus amyloliquefaciens D2WM Antagonistic Fungis are equal to the bacteriostasis rate of oil tea Major Diseases
Reach more than 95%, 100% especially is reached to the bacteriostasis rate of phyllosticta.Series result shows Bacillus
Amyloliquefaciens D2WM has a broad antifungal spectrum, it is one plant of efficient biocontrol bacterial strain for being directed to oil tea Major Diseases.
Bacteriostasis rate of the Bacillus amyloliquefaciens D2WM bacteria-free filtrates of table 2 to oil tea Major Diseases
Described oil tea pathogen is specially Oil Tea Anthracnose bacterium, oil tea charcoal glue born of the same parents' subcutaneous ulcer germ, oil tea leaf spot fungi, oil tea
Bacteria erwinia, oil tea alternaria, oil tea ulcer bacteria.
Embodiment 4：
Bacillus amyloliquefaciens Bacillus amyloliquefaciens D2WM zymotic fluid antibacterial material characteristic research.
Ph stability determines：Antagonism fermented liquid is filtrated to get bacteria-free filtrate through sterile filters, 5mL is drawn and is transferred to nothing
In bacterium test tube, pH value is adjusted to 1,3,5,7,9,11,13, after standing 1h, to test it to soft respectively with 1M sodium hydroxides or hydrochloric acid
The inhibitory action of maize ear rot opportunistic pathogen.Using original fermentation bacteria-free filtrate as control, each processing is repeated 3 times.To soft rot opportunistic pathogen
Antagonistic effect it is as shown in Figure 5.The bacteria-free filtrate soda acid tolerance range is very wide, has to soft rot opportunistic pathogen in the range of pH1-11
Certain inhibition, and in pH=9, its inhibition is optimal, it was demonstrated that and the environment of meta-alkalescence is more beneficial for bacteria-free filtrate
In active ingredient play antagonism.
Heat stability testing：Antagonism fermented liquid is filtrated to get bacteria-free filtrate through sterile filters, takes bacteria-free filtrate 5mL to turn
Enter in sterile test tube, handle 2h at being respectively 20 DEG C, 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C in temperature, test it to soft rot opportunistic pathogen
Inhibitory action.Each processing is repeated 3 times.It is as shown in Figure 6 to the antagonistic effect of soft rot opportunistic pathogen.As a result the bacteria-free filtrate is shown
Retain bacteriostatic activity in the range of 20 DEG C to 60 DEG C and its antagonistic effect is optimal at 40 DEG C, it is antibacterial when temperature is higher than 80 DEG C
Loss of activity, illustrate that the active ingredient in the bacteria-free filtrate is unable to high temperature resistant, it may be possible to the material of protide.
SEQUENCE LISTING
<110>Hubei Engineering University
<120>A kind of bacillus amyloliquefaciensBacillus amyloliquefaciens D2WM and preparation method and
Using
<130>A kind of bacillus amyloliquefaciensBacillus amyloliquefaciens D2WM and preparation method and
Using
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 1439
<212> DNA
<213>Bacillus amyloliquefaciens
<400> 1
cgtgctatac atgcaagtcg agcggacaga tgggagcttg ctccctgatg ttagcggcgg 60
acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg ggaaaccggg 120
gctaataccg gatggttgtc tgaaccgcat ggttcagaca taaaaggtgg cttcggctac 180
cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg 240
acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac 360
gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagtg 420
ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc taactacgtg 480
ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg 540
ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt 600
ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat 660
gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct 720
gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca cgccgtaaac 780
gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac gcattaagca 840
ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg gggcccgcac 900
aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca 960
tcctctgaca atcctagaga taggacgtcc ccttcggggg cagagtgaca ggtggtgcat 1020
ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt 1080
gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga caaaccggag 1140
gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac 1200
aatggacaga acaaagggca gcgaaaccgc gaggttaagc caatcccaca aatctgttct 1260
cagttcggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag taatcgcgga 1320
tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag 1380
agtttgtaac acccgaagtc ggtgaggtaa cctttatgag ccagccgccg aaggtgatc 1439
Claims (3)
- A kind of 1. bacillus amyloliquefaciens, it is characterised in that：The bacterial strain is bacillus amyloliquefaciensBacillus amyloliquefaciensD2WM, CCTCC No：M2015623.
- A kind of 2. bacillus amyloliquefaciens described in claim 1Bacillus amyloliquefaciens It is prepared by D2WM Treat or prevent the application in vegetable bacterial soft rot medicine；Described vegetables are green pepper, hot red pepper, lettuce, potato, bottle gourd, Chinese cabbage.
- A kind of 3. bacillus amyloliquefaciens described in claim 1Bacillus amyloliquefaciensIt is prepared by D2WM Treat or prevent the application in oil tea disease medicine；Described oil tea disease is Oil Tea Anthracnose, oil tea leaf spot, oil tea soft rot, oil tea black spot, oil tea canker.
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