CN105219681A - 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
Do you the invention discloses a kind of bacillus amyloliquefaciens <i>Bacillus? amyloliquefaciens</i>? D2WM and preparation method and application, step is: the separation and purification of A, genus bacillus: behind inoculation inclined-plane, preservation; The primary dcreening operation of B, genus bacillus: adopt some connection and dull and stereotyped face-off method, successively select bacterial strain Erwinia and oil tea disease being had to antagonistic action.The multiple sieve of C, genus bacillus: adopt cup-plate method.There is by primary dcreening operation the bacterial strain of antagonistic action to cultivate in LB liquid nutrient medium, filtered out the bacterial strain simultaneously Erwinia and oil tea Major Diseases being had to antagonistic action by Antagonistic Fungi fermented liquid and bacteria-free filtrate.This bacillus amyloliquefaciens is treated in preparation or is prevented the application in vegetable bacterial soft rot, oil tea disease Chinese traditional medicine, there is wide spectrum antagonistic action, fast growth, incubation time is substantially reduced for general bacterium, fungi, reduce production cost, the multiple diseases such as energy efficient antagonism Oil Tea Anthracnose, Camellia Leaves pinta, oil tea black spot, oil tea Peptic Ulcers, and fungistatic effect is remarkable.
Description
Technical field
The present invention relates to plant protection art; more specifically relate to a kind of can the bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM of simultaneously antagonism vegetable bacterial soft rot and oil tea Major Diseases; also relate to the preparation method of a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM simultaneously, also relate to the purposes of a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM in preparation treatment or prevention vegetable bacterial soft rot and oil tea medicine.
Background technology
Rotten also known as water, the rotten pimple of vegetable bacterial soft rot, except harm brassicaceous vegetable (as radish, Chinese cabbage etc.), also can endanger the vegetables such as potato, tomato, lettuce, cucumber, celery and green onion class, cause loss in various degree.Vegetable bacterial soft rot their early stage, disease portion is that water soaking mode is translucent, after expand as ficelle web rot, disease portion tissue is glutinous sliding, sink in dehydration rear surface, the white bacterium of normal spilling is dense, and foul smelling, later stage, vegetables were whole softening, all rot, ask for an interview (Yi Tuyong. vegetable bacterial soft rot [J]. Agriculture in Hunan, 2015 (2): 28-29.) Erwinia (Erwinia) is the main pathogenic fungi causing plant soft rot disease to occur, it belongs to enterobacteriaceae (Enterobacteriaceae), mainly comprise carrot soft rot Erwinia Radix Dauci Sativae subspecies (Erwiniacarotovorassp.carotovora, Ecc), the black shin subspecies of carrot soft rot Erwinia (Erwiniacarotovorassp.atroseptica, and Erwinia chrysanthemi (Erwiniachrysanthemi Eca), Ech) three subspecies, ask for an interview (P é rombelonM.PotatodiseasescausedbysoftrotErwinias:anovervi ewofpathogenesis [J] .Plantpathology.2002, 51 (1): 1-12.) wherein, the specificity of Eca is stronger, mainly infect temperate zone potato, Ecc under field conditions (factors) host range and regional distribution is the most extensive, can infect the plants such as potato, Radix Dauci Sativae, celery, cucumber, capsicum and witloof, spread all over the ground such as subtropics and temperate zone, Ech then can infect potato, Garden Dahlia and corn etc., ask for an interview (TothIK, BellKS, HolevaMC, etal.Softroterwiniae:fromgenestogenomes [J] .Molecularplantpathology.2002,4 (1): 17-30.) in recent years, growing vegetables scale constantly expands, vegetable disease progressively draws attention, and wherein especially occur serious with vegetable bacterial soft rot, the loss caused is huge.
Oil tea (Camelliaoleifera) belongs to Theaceae (Theaceae) Camellia (Camellia), the senior oil crops of the distinctive one of China, primary growth is in southern china subtropical zone high mountain and hilly country, it is one of important woody oil tree species, ask for an interview (Rayleigh, the village. Chinese oil tea [M]. Beijing: China Forestry Publishing House, 2008.) Seed of Camellia oleifera contains unsaturated fatty acids and the vitamin-E of a large amount of high-quality, be a kind of table oil at high-quality, storage endurance, in edible oil, account for critical role; In addition, tea oil is also widely used medical, industrial, can be used as the raw material of soap, Vaseline, slushing oil, lubrication wet goods; Tea is withered does agricultural chemicals, is also the organic fertilizer of high-quality, or extracts the raw material of the products such as tannin extract, saponin, furfural, gac, and the solid residue after extraction can be used as the concentrated feed of pig.Can say, oil tea whole body is all precious, has good application and development prospect, to promotion increasing peasant income, accelerates mountain area comprehensive development, safeguards that national grain and oil safely etc. have very important effect in aspect.
Oil tea disease species is various, and its harm is also more serious.Oil Tea Anthracnose, leaf blight, soft rot are the 3 kinds of destructive diseases caused by pathogen, generally occur in each oil tea producing region of China, each position, ground of oil tea all can be injured, mainly cause that shedding, bud drop, branch are withered, limb ulcer, even whole strain be dead, ask for an interview (Shu Qinglong, Zhang Liangfu. Chinese oil tea cultivation and the prevention and control of plant diseases, pest control [M] .. Beijing: China Forestry Publishing House, 2009.) same, oil tea root rot, gall of tea, black spot, Peptic Ulcers etc. also occur often, the financial loss that annual camellia oleiferaindustry is huge because disease causes.In recent years, Hubei oil tea planting scale constantly expands, and corresponding disease progressively draws attention.
But, at present the control of vegetable bacterial soft rot and oil tea Major Diseases is generally controlled by plantation resistant variety and chemical prevention, excessive use chemical bactericide has that harm humans is healthy, drug residue destroys ecotope, cause pathogen develops immunity to drugs, affects vegetables and the shortcomings such as tea oil quality and edible safety, is unfavorable for the Green Sustainable of vegetables and camellia oleiferaindustry.Seek focus and the task of top priority that safe, efficient and economic biogenic pesticide has become current control vegetables and oil tea disease, wherein antagonistic microbe plays very important effect in biocontrol of plant disease.
The microorganism that investigation and application is more in the biological control of Plant diseases mainly contains bacterium class, Mycophyta and actinomycetes.Genus bacillus (Bacilus) is the desirable screening of biocontrol agents object of a class, studies more in current biocontrol bacteria.It can suppress the disease at each positions such as plant root, limb, leaf, flower and fruit, is of wide application.Because it can produce the spore of heat-resisting, drought-enduring, uvioresistant and organic solvent, therefore the Biocontrol Bacillus process for processing determining to grow with breeding and zymocyte liquid thereof in the environment is extremely beneficial to, ask for an interview (the intelligent virtue of class. turn the soft rot resistance of aiiA gene konjaku and the compositional analysis [D] of konjaku endophyte generation Antagonistic protein. Hua Zhong Agriculture University, 2009.) and (Chai Xinli, Zhou Ying, woods is supported the army, Deng. the anti-soft rot aiiA gene of Tribactur turns elephant-foot yam research [J]. Molecular Plant Breeding, 2007,5 (5): 613-618.).Current Biocontrol Bacillus mainly comprises subtilis (B.subtilis), bacillus megaterium (B.megaterlum), bacillus polymyxa (B.polymyxa), bacillus pumilus (B.pumilus) and bacillus cereus (B.cereus) etc.Sun Ming etc. have invented a kind of subtilis BSn5 and Antagonistic protein APn5 (application number: 200710053427.8) carrot soft rot Erwinia to bacteriostatic activity, the Substance of this 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 is narrower; Huang waits quietly disclosing the application (application number: 201410735508.6) of a kind of bacillus amyloliquefaciens Lh-1 in control cotton verticillium wilt, soft rot of Chinese cabbage, bacterial wilt, peach canker, canker of apple fruit etc., bacillus amyloliquefaciens and oligochitosan need be utilized to be combined into microecological microbial agent, comparatively speaking only better to the prevention effect of cotton verticillium wilt; Liu Junang etc. disclose a kind of Brevibacillus brevis Z26 of antagonism oil tea disease, effectively can suppress the growth (application number: 201110027957.1) of Oil Tea Anthracnose bacterium, root rot, the half of pathogenic bacteria such as crazy, the maximal percentage inhibition of this biocontrol fungicide to Oil Tea Anthracnose reaches 83.4%, also has the space of improving further; Zhou Guoying etc. have invented a kind of complex microorganism source pesticide and application method (application number: 201310203845.6) of effective control oil tea disease, openly invent a kind of microbial pesticide by actinomycetes styreptomyces globispotus strain ball spore subspecies and bacterium Brevibacillus brevis and other additive compounds, one or more Major Diseases of oil tea can be prevented and treated, this composite microbial pesticide preparation process is relatively loaded down with trivial details, consuming time longer; Also have and utilize subtilis and bacillus thuringiensis ML1 relevant report (application number: 201310708367.4, application number: 201510015310.5) respectively in peanut rot disease preventive treatment and konjak soft rot control.But, about bacillus amyloliquefaciens can study on prevention simultaneously in vegetable bacterial soft rot and oil tea Major Diseases and application report little.In addition, bibliographical information mostly is the single control for a certain disease of oil tea, current market does not have to prevent and treat the good single microbial inoculum product of two or more oil tea disease effect substantially simultaneously, and yet there are no the report to control oil tea black spot and oil tea Peptic Ulcers two kinds of diseases.
Summary of the invention
The object of the invention is to there are provided a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM, this bacillus amyloliquefaciens obtains from healthy oil tea rhizosphere soil separation screening, vegetable bacterial soft rot and oil tea Major Diseases can be prevented and treated efficiently simultaneously, it is the Biocontrol Bacillus that a strain has wide spectrum antagonistic action, fill up the technological gap that single microorganism microbial inoculum prevents and treats Erwinia three subspecies and multiple oil tea pathogenic fungi simultaneously, to soft rot and oil tea non-environmental pollution control, there is certain practical significance.
Another object of the present invention is the preparation method that there are provided a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM biocontrol fungicide, easy to implement the method, easy and simple to handle.Microbial inoculum obtains through following steps: D2WM bacterium colony picking one ring on LB flat board or inclined-plane is transplanted to test tube activation, and 35 DEG C of shaking tables cultivate 16h, rotating speed 150rpm, in test tube, LB liquid nutrient medium is: peptone 10g/L, yeast extract 5g/L, sodium-chlor 5g/L, pH7.0; Bacterium liquid after activation is transplanted to according to the inoculum size of 1%-3% and is equipped with in the 250mL Erlenmeyer flask of 100mLLB liquid nutrient medium, rotating speed 150rpm, and 35 DEG C of shaking tables cultivate 10h-24h, when bacterial content is greater than 2 × 10
8more than cfu/mL can obtain zymocyte liquid, saves as biocontrol fungicide.This biocontrol fungicide makes easily, and fast growth, liquid culture meta-bolites only needs to cultivate 10-24h in liquid medium within can obtain good antagonistic effect, substantially reduces incubation time, reduce production cost relative to general bacterium, fungi.
A further object of the invention there are provided the application of a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM in preparation treatment or prevention vegetable bacterial soft rot medicine, every vegetable bacterial soft rot that can be caused by Erwinia can be treated simultaneously or prevent to this biocontrol strain, comprises green pepper, hot red pepper, lettuce, potato, bottle gourd, soft rot of cabbage etc.Good antagonistic action is all had to carrot soft rot Erwinia Radix Dauci Sativae subspecies, carrot soft rot Erwinia black shin subspecies and Erwinia chrysanthemi three subspecies.
4th object of the present invention there are provided the application of a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM in preparation treatment or prevention oil tea Major Diseases medicine, the biocontrol fungicide of this bacterial strain can high-efficiency prevention and control Oil Tea Anthracnose, leaf spot, black spot, Peptic Ulcers etc., the experiment of liquid fermenting filtrate bacteriostasis rate shows, the bacteriostasis rate of this bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM to oil tea Major Diseases reaches more than 95%.
In order to realize above-mentioned object, the present invention adopts following technical measures:
Its technical conceive is: adopt that conventional method carries out being separated, purifying and preservation have the biocontrol strain of antagonistic action to soft rot and oil tea Major Diseases simultaneously.Ask for an interview (Yao Lijin, Wang Qi, Fu Xuechi, etc. the screening of wheat hypochnus Biocontrol Bacillus and qualification [J]. Chinese biological is prevented and treated, and 2008,24 (1): 53-57.).The Bacteria erwinia be separated to laboratory is indicator, being separated the bacterial strain obtained is bacterium to be checked, point connection is utilized to screen successively to bacterium separation and purification, tentatively determine bacterial strain Erwinia being had to antagonistic action, adopt dull and stereotyped face-off method to screen further wherein can the antagonistic bacterium of simultaneously antagonism oil tea pathogenic bacteria simultaneously.Point connection and dull and stereotyped face-off method are asked for an interview (
t, BanhegyiI, HerpaiZ, etal.IsolationofBacillusstrainsfromtherhizosphereofcerea lsandinvitroscreeningforantagonismagainstphytopathogenic, food-bornepathogenicandspoilagemicro-organisms [J] .JournalofAppliedMicrobiology, 2000, 89 (5): 840-846.) cup-plate method and fermentation method is then adopted to carry out multiple sieve respectively, after the bacterial strain screened further is cultivated 24h respectively in LB liquid nutrient medium, nutrient solution 0.22 μm of bacterial filter obtains culturing filtrate.100 μ L soft rot nosophytes (about 1 × 10 will be coated with
7cfu/mL) on LB agar plate, the hole that diameter is 7mm beaten by equidistant punch tool.Accessing 50 μ L Antagonistic Fungi fermented liquids in each hole, take sterilized water as contrast, and each process repeats for three times.Antibacterial circle diameter is measured, according to numerical values recited determination antagonism degree cultivate 24h in 30 DEG C of thermostat containers after.Filter out bacterial strain soft rot nosophyte being had to antagonistic action.Meanwhile, pouring culture dish into, put into the oil tea the main pathogenic fungi bacterium cake that diameter is 7mm after cooling in dull and stereotyped central authorities after primary dcreening operation Antagonistic Fungi filtrate and the PDA solid medium of about 50 DEG C mixs according to 1:19 respectively, take sterilized water as contrast, each process three repetitions.Within 5 days, measure pathogenic bacteria colony diameter afterwards.Filter out bacterial strain oil tea Major Diseases being had to antagonistic action.Finally determine that the antagonistic action of D2WM to the former bacterium of soft rot and oil tea Major Diseases is the strongest, screening obtains successful and 1 stable strain Antagonistic Fungi D2WM.Cup-plate method and fermentation method are asked for an interview (Yao Lijin, Wang Qi, Fu Xuechi, etc. the screening of wheat hypochnus Biocontrol Bacillus and qualification [J]. Chinese biological is prevented and treated, and 2008,24 (1): 53-57.).
A preparation method of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM, the steps include:
The separation and purification of A, genus bacillus: get healthy oil tea Rhizosphere sampling 10g and join in the triangular flask filling 90mL sterilized water and 30-50 granulated glass sphere, make the suspension of 1:10 concentration, at 28 DEG C, vibrate in 180rpm shaking table after 20-30min, bacteria suspension is placed in 75-85 DEG C of water-bath and keeps 12-18min.LmL Soil Slurry is moved into and fills in the test tube of 9mL sterilized water, make 10
-2bacteria 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 following (1L): extractum carnis 3.0g, peptone 10.0g, NaCl5.0g, agar 15.0g, distilled water 1000mL, pH7.2-7.4.The flat board being coated with bacteria suspension is placed in 30 DEG C of incubators 1 week, observes bacterium colony state on isolation medium after 24h immediately, send out with three step line and carry out separation and purification, Unified number is registered.Culture presevation: behind inoculation inclined-plane, can 4 DEG C of of short duration preservations.With 50% (V/V) glycerine as protective material, can-80 DEG C of permanent preservations.
The primary dcreening operation of B, genus bacillus: first adopt some connection, get 100 μ L pathogenic bacteria bacterium liquid (about 1 × 10
7cfu/ml), on even spread LB flat board, then connect four bacterial strains of participating in the experiment with sterile toothpick point on each flat board and (select D1WM, D2WM, D3WM successively ... 4 bacterial strains in D30WM), each process arranges three repetitions.20-28h is cultivated in 30 DEG C and 37 DEG C of thermostat containers.Adopt dull and stereotyped face-off method to screen further wherein can the antagonistic bacterium of simultaneously antagonism oil tea pathogenic bacteria simultaneously, the pathogenic bacteria cake placement PDA plate center that diameter is 7mm will be accomplished fluently, then dip bacterium to be measured equidistant (anomaly plate central authorities 1.5cm) point with sterile toothpick and connect Antagonistic Fungi, be placed in 28 DEG C of constant incubators to cultivate, observe antibacterial with it's too late size after 5 days, in triplicate, screening bacteriostatic diameter is greater than the bacterial strain of 5mm further in each process.
The multiple sieve of C, genus bacillus: adopt cup-plate method to sieve again.Primary dcreening operation is had the bacterial strain of antagonistic action in LB liquid nutrient medium with 150rpm thermophilic cultivate 20-28h, fermented liquid with the centrifugal 10min of 8000rpm, supernatant with 0.22 μm of filtering with microporous membrane in sterilizing EP pipe.100 μ L pathogenic bacterias (about 1 × 10 will be coated with
7cfu/mL) on LB agar plate, the hole that diameter is 7mm beaten by equidistant punch tool.Access 50 μ L fermented liquids in each hole, take sterilized water as contrast, each process arranges three repetitions.Antibacterial circle diameter is measured, according to numerical values recited determination antagonism degree cultivate 20-28h in 30 DEG C of thermostat containers after.Filter out bacterial strain Bacteria erwinia bacterial strain being had to antagonistic action.Meanwhile, pouring culture dish into, put into the oil tea the main pathogenic fungi bacterium cake that diameter is 7mm after cooling in dull and stereotyped central authorities after primary dcreening operation Antagonistic Fungi filtrate and the PDA solid medium of about 50 DEG C mixs according to 1:19 respectively, take sterilized water as contrast, each process three repetitions.Within 5 days, measure pathogenic bacteria colony diameter afterwards.Filter out bacterial strain oil tea Major Diseases being had to antagonistic action.Final screening obtains the obvious and stable strain Antagonistic Fungi D2WM of antagonistic effect, a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM.
Containing the nucleotide sequence shown in SEQIDNO:1 in described a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM.
A kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM has following feature: this bacterial strain is in form and cultural characteristic: thalline is shaft-like, peritrichous, and has mobility; Bacterium colony is oyster white, and in subcircular, surface irregularity has fold, and edge is irregular, does not produce soluble pigment.
Applicant obtains a strain bacterium from healthy oil tea rhizosphere soil screening, is accredited as bacillus amyloliquefaciens in conjunction with physiological and biochemical property and characterization of molecules, and called after BacillusamyloliquefaciensD2WM.This bacterial strain BacillusamyloliquefaciensD2WM on October 20th, 2015 be preserved in China typical culture collection center (be called for short CCTCC, China. Wuhan. Wuhan University), deposit number is CCTCCNO:M2015623.
The application of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM in preparation treatment or prevention vegetable bacterial soft rot Chinese traditional medicine, its application primary process is:
A, bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM are to vegetable bacterial soft rot Bactericidal test: adopt cup-plate method to investigate bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM fermented liquid to the antagonistic action of Erwinia.Get the LB liquid medium that a ring bacterium colony is placed in sterilizing respectively, 12h cultivated by 35 DEG C of shaking tables, rotating speed 150rpm, namely carrot soft rot Erwinia Radix Dauci Sativae subspecies are obtained, the black shin subspecies of carrot soft rot Erwinia and Erwinia chrysanthemi 3 kinds of pathogenic bacterias and Antagonistic Fungi BacillusamyloliquefaciensD2WM thalline fermented liquid, respectively getting 100 μ L pathogenic bacteria fermented liquids coats in LB solid medium, the hole of three 7mm is evenly made a call to aseptic punch tool, Antagonistic Fungi bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM fermented liquid or fermented liquid sterilized water are diluted 10 times, respectively to adding 50 μ L fermented liquids or dilution secondary fermentation liquid in hole, add the process of sterilized water as blank, the size of Antagonistic Fungi to 3 kinds of pathogenic bacteria inhibition zones is observed after 35 DEG C of cultivation 24h.
B, adopt dull and stereotyped opposite culture method research bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM to the antagonistic action of oil tea Major Diseases.The pathogenic bacteria cake placement plate center that diameter is 7mm will be accomplished fluently, then dip bacterium to be measured equidistant (anomaly plate central authorities 1.5cm) point with sterile toothpick and connect Antagonistic Fungi, be placed in 28 DEG C of constant incubators to cultivate, observe antibacterial with it's too late size after 5 days, each process repeats 3 times.
C, employing fermented liquid culture method research bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM are to the antagonistic action of oil tea Major Diseases.BacillusamyloliquefaciensD2WM bacterium colony is cultivated after 12h through LB liquid nutrient medium, the centrifugal 20min of 10000r/min room temperature, with the filtering with microporous membrane removing thalline of 0.22 μm, after getting Antagonistic Fungi filtrate and sterilizing, the PDA solid medium of about 50 DEG C pours culture dish into after mixing according to 1:19, put in dull and stereotyped central authorities the oil tea the main pathogenic fungi bacterium cake that diameter is 7mm after cooling, take sterilized water as contrast, each process repeats for three times.Within 5 days, measure pathogenic bacteria colony diameter afterwards.Bacteria-free filtrate bacteriostasis rate=(contrast colony diameter-process colony diameter)/(contrast colony diameter-0.7) × 100%.
The application of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM in preparation treatment or prevention vegetable bacterial soft rot Chinese traditional medicine, it comprises:
Described a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM treats in preparation or prevents the application in the medicine of the vegetable bacterial soft rot caused by Erwinia, comprises green pepper, hot red pepper, lettuce, potato, bottle gourd, soft rot of cabbage etc.
The application of described a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM in preparation treatment or prevention oil tea Major Diseases medicine, described oil tea disease is Oil Tea Anthracnose, Camellia Leaves pinta, oil tea soft rot, oil tea black spot, oil tea Peptic Ulcers.
The present invention compared with prior art, has the following advantages and effect:
1. strains B. amyloliquefaciens BacillusamyloliquefaciensD2WM of the present invention, is separated from oil tea healthy plant rhizosphere soil, has stable ecotope and Biocontrol Effect.
2. biocontrol fungicide of the present invention makes easily, fast growth, liquid culture meta-bolites only needs to cultivate 10-24h in liquid medium within can obtain good antagonistic effect, substantially reduces incubation time, reduce production cost relative to general bacterium, fungi.
3. this bacterium field planting power is strong, all has very strong antagonistic action simultaneously, have good development prospect to various vegetables soft rot and multiple oil tea Major Diseases.
4. biocontrol strain bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM of the present invention, prevent and treat Erwinia 3 class subspecies and multiple oil tea disease by single bacterial strain simultaneously, comprise fresh Camellia Leaves pinta, oil tea black spot and oil tea Peptic Ulcers of not reporting for work, antimicrobial spectrum is extensive, effectively can reduce cost accounting.
5. dull and stereotyped antagonistic experiment shows: this bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM has obvious restraining effect to oil tea Major Diseases.
6. the experiment of liquid culture meta-bolites bacteriostasis rate shows, the bacteriostasis rate of this bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM to oil tea Major Diseases reaches more than 95%.
Accompanying drawing explanation
Fig. 1 is a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM bacterium colony and microscopic morphology schematic diagram.
Fig. 2 is the 16SrDNA gene order phylogenetic tree schematic diagram of a kind of D2WM and other bacterial strains.
Fig. 3 is that a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM bacterial strain is to the inhibition zone design sketch of Erwinia;
Fig. 4 is the flat board face-off antagonistic action schematic diagram of a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM bacterial strain to oil tea the main pathogenic fungi.
What Fig. 5 was a kind of pH on Antagonistic Fungi fermentation bacteria-free filtrate antagonistic effect affects schematic diagram.
What Fig. 6 was a kind of temperature on Antagonistic Fungi fermentation bacteria-free filtrate antagonistic effect affects schematic diagram.
Accompanying drawing illustrates: a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM bacterial strain is in form and cultural characteristic: thalline is shaft-like, peritrichous, and has mobility; Bacterium colony is oyster white, and in subcircular, surface irregularity has fold, and edge is irregular, does not produce soluble pigment.As shown in Figure 1.Molecular Identification is carried out to this bacterial strain, the 16SrDNA sequence recorded (fragment length is 1439bp) and sequence in GenBank database are carried out BLAST analysis, choose 9 strain bacterial strain ClustalX2.1 and carry out Multiple Sequence Alignment analysis, MEGA6.0 software is utilized to adopt adjacent method to carry out phylogenetic tree structure and cluster analysis, the Neighbor-Joining method constructing system evolutionary tree of MEGA6, as shown in Figure 2.In conjunction with above-mentioned form and part physiological and biochemical property, bacillus amyloliquefaciens can be belonged to by preliminary judgement antagonistic strain D2WM.Adopt cup-plate method to investigate bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM20 and doubly dilute the antagonistic action of secondary fermentation liquid to Erwinia, its fungistatic effect is shown in Fig. 3.Result shows, this biocontrol strain all has good fungistatic effect to carrot soft rot Erwinia Radix Dauci Sativae subspecies, carrot soft rot Erwinia black shin subspecies and Erwinia chrysanthemi 3 subspecies, and the Antagonistic Fungi fermented liquid inhibition zone after diluting 10 times reaches more than 22mm.Dull and stereotyped opposite culture method is adopted to study the antagonistic action of this bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM to multiple oil tea pathogenic bacteria, the results are shown in Figure 4, this Antagonistic Fungi all has good antagonistic action to Oil Tea Anthracnose, leaf spot, black spot and Peptic Ulcers, especially to Oil Tea Anthracnose, leaf spot, black spot Be very effective.Fig. 5 and 6 is respectively pH and temperature affects schematic diagram to Antagonistic Fungi fermentation bacteria-free filtrate antagonistic effect.Result shows that this bacteria-free filtrate soda acid tolerance range is very wide, within the scope of 1-11, certain inhibition is all had to the former bacterium of soft rot at pH, and when pH=9, its inhibition is best, prove that the effective constituent that the environment of meta-alkalescence is more conducive in bacteria-free filtrate plays antagonistic action.In addition, this bacteria-free filtrate within the scope of 20 DEG C to 60 DEG C, retain bacteriostatic activity and 40 DEG C time its antagonistic effect best, when temperature is higher than 80 DEG C, bacteriostatic activity is lost, and illustrating that the effective constituent in this bacteria-free filtrate can not be high temperature resistant, is likely the material of protein.
Embodiment
The reagent that the present invention is used or raw material, equal available from commercial channel.
Bacterium source: Erwinia Erwiniacarotovorassp.carotovora, Erwiniachrysanthemi, Erwiniacarotovorassp.Atroseptica, with report in document bacterial strain (Ge Weizhen. konjak soft rot pathogenic process dynamic studies and study on prevention [M] .2013 based on QS system), oil tea glue born of the same parents anthrax Colletotrichum.gloeosporioides, with reporting bacterial strain (Meng Qingmin in document, Zhou Guoying, Liu Junang, Deng. the main antibacterial substance separation and purification of Oil Tea Anthracnose antagonistic bacterium Y13 and the mode of action [J]. plant protection, 2014,40 (2): 36-42.), Oil Tea Anthracnose Colletotrichumnicotianae with report in document bacterial strain (Li Dongsheng, Zhang Yiyang. the morphological specificity of Colletotricum destructivum and Study on Pathogenicity [J]. Agriculture in Hunan science, 2009 (5): 97-98.), Camellia Leaves pinta Phyllostictacapitalensis, with report in document bacterial strain (horse become, Peng Lijuan, Wu Shiping. ginkgo phyllosticta Leaf blotch pathogeny qualification [J]. Guizhou Agricultural Sciences, 2014,42 (12): 126-128.), oil tea soft rot Agaricodochiumcamellia, with report in document bacterial strain (Zhao Danyang, Qin Changsheng, Liao Fang scorching .5 kind sterilant is to the study on prevention [J] of oil tea soft rot. Annual, 2013,29 (2): 28-31.), oil tea black spot Altermariaalternate is with reporting bacterial strain (KobilerI in document, AkermanM, HubermanL, etal. the research [J] controlling the black spot that alternaric bacteria (Alternariaalternata) causes in persimmon storage is combined with postharvest handling before adopting. fresh-keeping with processing, 2011, 5:025.), oil tea Peptic Ulcers Bothyosphaeriadothidea, with reporting bacterial strain (SlippersB in document, CrousPW, DenmanS, etal.Combinedmultiplegenegenealogiesandphenotypiccharact ersdifferentiateseveralspeciespreviouslyidentifiedasBotr yosphaeriadothidea [J] .Mycologia, 2004, 96 (1): 83-101.)
Embodiment 1:
A preparation method of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM, the steps include:
The separation and purification of A, genus bacillus: get healthy oil tea Rhizosphere sampling 10g and join in the triangular flask filling 90mL sterilized water and 30-50 granulated glass sphere, make the suspension of 1:10 concentration, at 28 DEG C, after the 30min that vibrates in 180rpm shaking table, bacteria suspension is placed in 80 DEG C of water-baths and keeps 15min.LmL Soil Slurry is moved into and fills in the test tube of 9mL sterilized water, make 10
-2bacteria 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 following (1L): extractum carnis 3.0g, peptone 10.0g, NaCl5.0g, agar 15.0g, distilled water 1000mL, pH7.2-7.4.The flat board being coated with bacteria suspension is placed in 30 DEG C of incubators 1 week, after 24h, observes bacterium colony state on isolation medium immediately, send out with three step line and carry out separation and purification, be separated to 30 strain bacterial strain Unified number registrations as follows: D1WM, D2WM, D3WM ... D30WM.Culture presevation: behind inoculation inclined-plane, can 4 DEG C of of short duration preservations.With 50% (V/V) glycerine (Shanghai development chemical industry one factory) as protective material, can-80 DEG C of permanent preservations.
The primary dcreening operation of B, genus bacillus: get 100 μ L soft rot nosophyte bacterium liquid (about 1 × 10 respectively
7cfu/ml), on even spread LB flat board, then connect four bacterial strains of participating in the experiment with sterile toothpick point on each flat board and (select D1WM, D2WM, D3WM successively ... 4 bacterial strains in D30WM), each process arranges three repetitions.24h is cultivated in 30 DEG C and 37 DEG C of thermostat containers.Obtain 5 strain bacterial strain D2WM, D9WM, D14WM, D21WM, the D29WM wherein Erwinia being had to antagonistic action.Adopt dull and stereotyped face-off method to screen further wherein can the antagonistic bacterium of simultaneously antagonism oil tea pathogenic bacteria simultaneously, the pathogenic bacteria cake placement PDA plate center that diameter is 7mm will be accomplished fluently, then dip bacterium to be measured equidistant (anomaly plate central authorities 1.5cm) point with sterile toothpick and connect Antagonistic Fungi, be placed in 28 DEG C of constant incubators to cultivate, observe antibacterial with it's too late size after 3 days, each process repetition 3 times, screening bacteriostatic diameter is greater than the bacterial strain of 5mm further.3 strains have antagonistic effect simultaneously bacterial strain D2WM, D9WM, D21WM to soft rot and oil tea Major Diseases is obtained altogether by above-mentioned two step primary dcreening operations.
The multiple sieve of C, genus bacillus: primary dcreening operation is had the bacterial strain of antagonistic action to cultivate 24h under 150rpm, 30-37 DEG C of condition in LB liquid nutrient medium, fermented liquid with the centrifugal 10min of 8000rpm, supernatant with 0.22 μm of filtering with microporous membrane in sterilizing EP pipe.100 μ L pathogenic bacterias (about 1 × 10 will be coated with
7cfu/mL) on LB agar plate, the hole that diameter is 7mm beaten by equidistant punch tool.Access 50 μ L fermented liquids in each hole, take sterilized water as contrast, each process arranges three repetitions.Antibacterial circle diameter is measured, according to numerical values recited determination antagonism degree cultivate 24h in 30 DEG C of thermostat containers after.Filter out bacterial strain Bacteria erwinia bacterial strain being had to antagonistic action.Simultaneously, culture dish is poured into after primary dcreening operation Antagonistic Fungi filtrate and the PDA solid medium of about 50 DEG C being mixed according to 1:19 respectively, putting in dull and stereotyped central authorities the oil tea the main pathogenic fungi bacterium cake that diameter is 7mm after being cooled to 20-25 DEG C, take sterilized water as contrast, and each process repeats for three times.Within 5 days, measure pathogenic bacteria colony diameter afterwards.Filter out bacterial strain oil tea Major Diseases being had to antagonistic action.Final screening obtains 1 obvious and stable strain Antagonistic Fungi bacillus amyloliquefaciens (BacillusamyloliquefaciensD2WM) of antagonistic effect.
A kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM has been prepared, containing the nucleotide sequence shown in SEQIDNO:1 in bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM by above-mentioned.
This bacterial strain BacillusamyloliquefaciensD2WM is preserved in China typical culture collection center (being called for short CCTCC, Luojiashan, Wuchang, Wuhan City, Hubei Province) on October 20th, 2015, and deposit number is M2015623.
Vegetable bacterial soft rot and oil tea Major Diseases are had to a bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM for antagonistic action, the qualification of CCTCCNO:M2015623 simultaneously:
The morphology of A, a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM and physiological and biochemical property:
This bacterial strain is in form and cultural characteristic: thalline is shaft-like, peritrichous, and has mobility; Bacterium colony is oyster white, and in subcircular, surface irregularity has fold, and edge is irregular, does not produce soluble pigment.As shown in Figure 1.In Physiology and biochemistry qualification: belong to G+ bacteria, there is catalase activity, energy hydrolyzed starch, can utilize D-Glucose, L-arabinose, D-wood sugar, D-MANNOSE to produce acid, can utilize Citrate trianion, can hydrolyzed casein make gelatine liquefication, can reduce litmus milk and nitrate, V-P reacting positive, does not have lecithin activity, hippurate and tyrosine can not be hydrolyzed, glucose aerogenesis can not be utilized.
The molecules qualification of B, a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM:
Molecular Identification is carried out to bacterial strain D2WM, the 16SrDNA sequence recorded (fragment length is 1439bp) and sequence in GenBank database are carried out BLAST analysis, choose 9 strain bacterial strain ClustalX2.1 and carry out Multiple Sequence Alignment analysis, MEGA6.0 software is utilized to adopt adjacent method to carry out phylogenetic tree structure and cluster analysis, the Neighbor-Joining method constructing system evolutionary tree of MEGA6, as shown in Figure 2.In bacterial strain D2WM and GenBank, accession number is that the homology of HM107808.1 (Bacillusamyloliquefaciensstrain15) bacterial strain reaches 99%.According to 16SrDNA criteria for classification, it is generally acknowledged that 16SrDNA sequence homology is greater than 97%, can think and belong to same kind, in conjunction with above-mentioned form and part physiological and biochemical property, bacillus amyloliquefaciens can be belonged to by preliminary judgement antagonistic strain D2WM.
Embodiment 2:
The application of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM in preparation treatment or prevention vegetable bacterial soft rot medicine, the steps include:
The preparation of A, a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM biocontrol fungicide in treatment or prevention vegetable bacterial soft rot Chinese traditional medicine, the steps include: that D2WM bacterium colony picking one ring on LB is dull and stereotyped or inclined-plane is transplanted to test tube activation, 16h cultivated by 35 DEG C of shaking tables, rotating speed 150rpm, in test tube, LB liquid nutrient medium is: peptone 10g/L, yeast extract 5g/L, sodium-chlor 5g/L, pH7.0; Bacterium liquid after activation is transplanted to according to the inoculum size of 1%-3% and is equipped with in the 250mL Erlenmeyer flask of 100mLLB liquid nutrient medium, rotating speed 150rpm, and 35 DEG C of shaking tables cultivate 10h-24h, when bacterial content is greater than 2 × 10
8more than cfu/mL can obtain zymocyte liquid, saves as biocontrol fungicide.
B, bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM biocontrol fungicide are to vegetable bacterial soft rot Bactericidal test: adopt cup-plate method to investigate bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM fermented liquid to the antagonistic action of Erwinia.Get the LB liquid medium that a ring bacterium colony is placed in sterilizing respectively, 12h cultivated by 35 DEG C of shaking tables, rotating speed 150rpm, namely carrot soft rot Erwinia Radix Dauci Sativae subspecies are obtained, the black shin subspecies of carrot soft rot Erwinia and Erwinia chrysanthemi 3 kinds of pathogenic bacteria fermented liquids, respectively getting 100 μ L pathogenic bacteria fermented liquids coats in LB solid medium, the hole of three 7mm is evenly made a call to aseptic punch tool, respectively to the 10 times of diluents adding 50 μ L bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM biocontrol fungicides in hole, add the process of sterilized water as blank, the size of Antagonistic Fungi to 3 kinds of pathogenic bacteria inhibition zones is observed after 35 DEG C of cultivation 24h, its fungistatic effect is shown in Fig. 3.Result shows, this biocontrol strain all has good fungistatic effect to carrot soft rot Erwinia Radix Dauci Sativae subspecies, carrot soft rot Erwinia black shin subspecies and Erwinia chrysanthemi 3 subspecies, and inhibition zone reaches more than 22mm.
Described vegetables are green pepper, hot red pepper, lettuce, potato, bottle gourd, Chinese cabbage etc.
Embodiment 3:
The application of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM in preparation treatment or prevention six kinds of oil tea pathogenic bacteria medicines, the steps include:
The preparation of A, a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM biocontrol fungicide in treatment or prevention six kinds of oil tea pathogenic bacteria medicines, the steps include: that D2WM bacterium colony picking one ring on LB is dull and stereotyped or inclined-plane is transplanted to test tube activation, 16h cultivated by 35 DEG C of shaking tables, rotating speed 150rpm, in test tube, LB liquid nutrient medium is: peptone 10g/L, yeast extract 5g/L, sodium-chlor 5g/L, pH7.0; Bacterium liquid after activation is transplanted to according to the inoculum size of 1%-3% and is equipped with in the 250mL Erlenmeyer flask of 100mLLB liquid nutrient medium, rotating speed 150rpm, and 35 DEG C of shaking tables cultivate 10-24h, when bacterial content is greater than 2 × 10
8more than cfu/mL can obtain zymocyte liquid, saves as biocontrol fungicide.
B, a kind of bacillus amyloliquefaciens BacillusamyloliquefaciensD2WM and multiple oil tea pathogenic bacteria is external studies mutually: adopt dull and stereotyped opposite culture method.The pathogenic bacteria cake placement plate center that diameter is 7mm will be accomplished fluently, then dip bacterium to be measured equidistant (anomaly plate central authorities 1.5cm) point with sterile toothpick and connect Antagonistic Fungi, be placed in 28 DEG C of constant incubators to cultivate, observe antibacterial with it's too late size after 5 days, each process repeats 3 times.The results are shown in Table 1 and Fig. 4.Result shows that BacillusamyloliquefaciensD2WM all has good antagonistic action to Oil Tea Anthracnose, leaf spot, soft rot, black spot and Peptic Ulcers, especially to Oil Tea Anthracnose, leaf spot, black spot and Peptic Ulcers Be very effective.
Table 1BacillusamyloliquefaciensD2WM is to the suppression diameter of oil tea Major Diseases
In table, data are mean+SD (lower same), suppress the rear pathogenic bacteria diameter of diameter=untreated pathogenic bacteria diameter-process
Adopt fermented liquid culture method.The biocontrol fungicide that step A obtains is through the centrifugal 20min of 10000r/min room temperature, with the filtering with microporous membrane removing thalline of 0.22 μm, after getting Antagonistic Fungi filtrate and sterilizing, the PDA solid medium of about 50 DEG C pours culture dish into after mixing according to 1:19, put in dull and stereotyped central authorities the oil tea the main pathogenic fungi bacterium cake that diameter is 7mm after cooling, take sterilized water as contrast, each process repeats for three times.Within 5 days, measure pathogenic bacteria colony diameter afterwards.Bacteria-free filtrate bacteriostasis rate=(contrast colony diameter-process colony diameter)/(contrast colony diameter-0.7) × 100%.
Table 2 shows the bacteriostasis rate of BacillusamyloliquefaciensD2WM Antagonistic Fungi to oil tea Major Diseases and all reaches more than 95%, especially reaches 100% to the bacteriostasis rate of phyllosticta.Series result shows BacillusamyloliquefaciensD2WM has a broad antifungal spectrum, is the efficient biocontrol strain of a strain for oil tea Major Diseases.
Table 2BacillusamyloliquefaciensD2WM bacteria-free filtrate is to the bacteriostasis rate of oil tea Major Diseases
Described oil tea pathogenic bacteria 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 BacillusamyloliquefaciensD2WM fermented liquid antimicrobial substance characteristic research.
Ph stability measures: Antagonistic Fungi fermentation liquor sterile filters filtered and obtain bacteria-free filtrate, drawing 5mL proceeds in sterile test tube, with 1M sodium hydroxide or hydrochloric acid adjust pH respectively to 1,3,5,7,9,11,13, leave standstill after 1h, test its restraining effect to the former bacterium of soft rot.With original fermentation bacteria-free filtrate in contrast, each process repetition 3 times.To the antagonistic effect of the former bacterium of soft rot as shown in Figure 5.This bacteria-free filtrate soda acid tolerance range is very wide, within the scope of pH1-11, all have certain inhibition to the former bacterium of soft rot, and when pH=9, its inhibition is best, proves that the effective constituent that the environment of meta-alkalescence is more conducive in bacteria-free filtrate plays antagonistic action.
Heat stability testing: Antagonistic Fungi fermentation liquor sterile filters is filtered and obtains bacteria-free filtrate, getting bacteria-free filtrate 5mL proceeds in sterile test tube, at temperature is 20 DEG C, 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C, process 2h respectively, test its restraining effect to the former bacterium of soft rot.Each process repetition 3 times.To the antagonistic effect of the former bacterium of soft rot as shown in Figure 6.Result show this bacteria-free filtrate within the scope of 20 DEG C to 60 DEG C, retain bacteriostatic activity and 40 DEG C time its antagonistic effect best, when temperature is higher than 80 DEG C, bacteriostatic activity is lost, and illustrating that the effective constituent in this bacteria-free filtrate can not be high temperature resistant, is likely the material of protein.
SEQUENCELISTING
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<120> bacillus amyloliquefaciens
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bacillusamyloliquefaciensd2WM and preparation method and application
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Claims (4)
1. a bacillus amyloliquefaciens, is characterized in that: this bacterial strain is bacillus amyloliquefaciens
bacillusamyloliquefaciensd2WM, CCTCCNo:M2015623.
2. a kind of bacillus amyloliquefaciens according to claim 1
bacillusamyloliquefaciensd2WM, is characterized in that: containing the nucleotide sequence shown in SEQIDNO:1 in described bacillus amyloliquefaciens.
3. a kind of bacillus amyloliquefaciens according to claim 1
bacillusamyloliquefaciensthe application of D2WM in preparation treatment or prevention vegetable bacterial soft rot medicine;
Described vegetables are green pepper, hot red pepper, lettuce, potato, bottle gourd, Chinese cabbage.
4. a kind of bacillus amyloliquefaciens according to claim 1
bacillusamyloliquefaciensthe application of D2WM in preparation treatment or prevention oil tea disease medicine;
Described oil tea disease is Oil Tea Anthracnose, Camellia Leaves pinta, oil tea soft rot, oil tea black spot, oil tea Peptic Ulcers.
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