CN106520607A - Bacillus amyloliquefaciens and application thereof in inhibition of plasmopara viticola - Google Patents
Bacillus amyloliquefaciens and application thereof in inhibition of plasmopara viticola Download PDFInfo
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- CN106520607A CN106520607A CN201610973916.4A CN201610973916A CN106520607A CN 106520607 A CN106520607 A CN 106520607A CN 201610973916 A CN201610973916 A CN 201610973916A CN 106520607 A CN106520607 A CN 106520607A
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- Prior art keywords
- bacillus amyloliquefaciens
- culture
- application
- fermented product
- plasmopara viticola
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses bacillus amyloliquefaciens and application thereof in inhibiting plasmopara viticola. The Bacillus amyloliquefaciens (KB) has the preservation registration number of CGMCC NO. 8179. Cultures of Bacillus amyloliquefaciens KB also fall within the scope of the present invention. The fermentation product of bacillus amyloliquefaciens KB also belongs to the scope of the present invention. The invention also protects the application of the bacillus amyloliquefaciens KB or the culture or the fermentation product in inhibiting the botrytis cinerea, preventing and treating the botrytis cinerea and preventing and treating plant diseases caused by the botrytis cinerea. The invention provides a bacillus amyloliquefaciens strain with an inhibiting effect on grape downy mildew, and has great application value for preventing and treating grape downy mildew.
Description
Technical field
The invention belongs to technical field of microbe application, and in particular to bacillus amyloliquefaciens and its suppress Fructus Vitis viniferae
Application in Pseudoperonospora cubensis.
Background technology
Downy mildew of garpe is a kind of fungal disease.The a large amount of underproduction for generally causing fruit of downy mildew of garpe, sternly
The edibility and market value of fruit is have impact on again, and the income that result in orchard worker is reduced, and seriously constrains China's Fructus Vitis viniferae
The development of industry.No matter in developed country, or in developing country, due to lacking necessary effective prevention and control medicament, oenin
The loss amount that mildew causes accounts for more than the 20% of total amount.The generation of downy mildew of garpe produces especially high-end Portugal to modern Fructus Vitis viniferae
Grape production is caused with processing and is had a strong impact on.
Downy mildew of garpe main harm blade, can also infect the tender tissues such as young sprout young fruit.There is downy mildew of garpe in blade
Afterwards, the clear fleck of faint yellow water stain shape blur margin of coming into being, gradually expands as brown irregular shape or polygon scab later,
Number speckle is connected and becomes the big speckle of irregular shape；During wet weather, white downy mildew shape thing (the i.e. sporangiophore of pathogenic bacteria is produced in the scab back side
And Sporangium)；Serious seasonal disease leaf of falling ill is early withered caducous.After tender tip occurs downy mildew of garpe, water soaked spots is formed, is then changed into
The slightly concave sunken scab of brown, when moist, scab also produces white downy mildew；Young sprout distortion when seriously ill, growth stop, or even withered.Volume
Palpus, cob, petiole can also occur downy mildew of garpe sometimes, and its symptom is similar to tender tip.After there is downy mildew of garpe in young fruit, sick portion
Fade, be hardened sagging, upper raw white downy mildew, very easy atrophy come off；Aggrieved when fruit grain is medium-sized, sick portion's brown is to dead color, soft rotten early
Fall；No longer infect after fruit color.
The pathogen of downy mildew of garpe is P. viticola, belongs to Mastigomycotina, Oomycete Peronosporales, Plasmopara.Portugal
Grape Pseudoperonospora cubensis is survived the winter in diseased tissuess with oospore, or is residued in soil with sick leaf and survived the winter.Next year ovum under optimum conditions
Spore germination produces sporangium, then produces zoospore by sporangium, propagates by means of wind and rain, invades from blade back pore, carries out first
Infect.Through the incubation period of 7-12 days, sporangiophore and Sporangium are produced in sick portion, spore germination produces zoospore and carries out again
Infect.It is 10 DEG C～15 DEG C that Sporangium sprouts preference temperature.The preference temperature that zoospore is sprouted is 18 DEG C -24 DEG C.Autumn is low
Temperature, rainy many dew, easily causes plant disease epidemic.Orchard hypsography low-lying, frame face improper ventilation tree vigo(u)r are weak, are conducive to disease to occur.
China's vinegrowing area alreadys exceed 800,000 hectares within 2014, more than France, is only second to Spain, is in the world
Effective the second big country of cultivated area of Fructus Vitis viniferae, and grape -growing areas are also being stepped up.At present, downy mildew of garpe is generally sent out
Life becomes a key factor of restriction China Fructus Vitis viniferae industry development.In producing at present to the preventing and treating of downy mildew of garpe still with
Based on chemoprevention, but " 3R " negative effect for thus bringing is very serious.The a large amount of of chemical pesticide use the ecological ring of heavy damage
Border, causes substantial amounts of pesticide residues in grape fruit, affects people healthy.In China viny region, in the harvesting of Fructus Vitis viniferae
Before, to the means of prevention of downy mildew of garpe also based on chemical pesticide, this not only affects, and the people's is healthy,
Limit the processing and outlet of grape product.Development biological pesticide and pollution-free food are listed in by China《21 st century in China is discussed
Journey》, construction requirements Beijing of Beijing modern agriculture Science and Technology City and surrounding area crop pest realize green prevention and control, agricultural products
Carry out green production.
The content of the invention
It is an object of the invention to provide bacillus amyloliquefaciens and its application in Plasmopara viticola is suppressed.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB that the present invention is provided, in 2013 09
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 12nd, and (abbreviation CGMCC, address is：Beijing
The institute 3 of city Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC NO.8179.Solution
Bacillus amyloliquefacienses (Bacillus amyloliquefaciens) KB CGMCC NO.8179, abbreviation bacillus amyloliquefaciens
KB。
The culture of bacillus amyloliquefaciens KB falls within protection scope of the present invention.The preparation method of the culture is such as
Under：Bacillus amyloliquefaciens KB is seeded to into LB fluid mediums, is cultivated, obtain culture.The preparation of the culture
Method is specific as follows：The initial concentration that bacillus amyloliquefaciens KB is seeded to LB fluid mediums and makes which in cultivating system
For 105Cfu/ milliliters, are cultivated, and obtain culture.Concretely 28-30 DEG C of the condition of the culture, 180-220rpm shake
Swing culture 48 hours.The culture concretely terminates the whole system after cultivating.
The fermented product of bacillus amyloliquefaciens KB falls within protection scope of the present invention.
The present invention also protects bacillus amyloliquefaciens KB or described cultures or the fermented product suppressing downy mildew of garpe
Application in bacterium.
The present invention also protects bacillus amyloliquefaciens KB or described cultures or the fermented product preventing and treating downy mildew of garpe
In application.
The present invention also protects bacillus amyloliquefaciens KB or described cultures or the fermented product preventing and treating downy mildew of garpe
Application in microbial plant disease.The plant concretely Fructus Vitis viniferae, such as grape variety " red milk ".
The present invention also protect it is a kind of suppress Plasmopara viticola preparation, its active component be bacillus amyloliquefaciens KB or
The culture or the fermented product.
The present invention also protects a kind of preparation for preventing and treating downy mildew of garpe, its active component to be bacillus amyloliquefaciens KB or institute
State culture or the fermented product.
The present invention also protects a kind of preparation for preventing and treating the plant disease that Plasmopara viticola causes, and its active component is Xie Dian
Afnyloliquefaciens KB or described cultures or the fermented product.The plant concretely Fructus Vitis viniferae, such as grape variety " red milk ".
Bacillus cereuss (Bacillus spp.) generally existing in soil, is to be present in soil and plant microecosystem
In one of superior microorganism population, due to producing endogenous spore, the ability with stronger opposing ambient pressure environment can
Because of the injury caused by drying, thermal and uv radiation in the survived environment of opposing, maintain its own existence ability unaffected.This
Individual biological property makes bacillus cereuss have very good application prospect, particularly in the biological product using active bacteria formulation
Show powerful vitality.Bacillus cereuss species is various, enormous amount, can produce diversified biological active substanceies and
Metabolite, with extensive prospects for commercial application.
The invention provides one plant of bacillus amyloliquefaciens inhibited to Plasmopara viticola, for oenin
The preventing and treating of mildew is with great using value.
Description of the drawings
Fig. 1 is the photo after bacillus amyloliquefaciens KB Gram’s staining.
Fig. 2 is inhibitions of the bacillus amyloliquefaciens KB in Fructus Vitis viniferae leaf dish to Plasmopara viticola.
Specific embodiment
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, if no special instructions, is conventional method.Test material used in following embodiments, if no special instructions, is certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetitions and tests, as a result make even
Average.
Plasmopara viticola (Plasmopara viticola)：Huang Xiaoqing, Kong Xiang wait so long the oenin in different hosts source
Mildew bacterium Pathogenic Tests and Sporangium size compare；《Plant protection》,2015,41(3):178-182.
Grape variety " red milk "：Principality is outstanding, red milk Fructus Vitis viniferae；《Beijing Agriculture》, 2009,22 phases.
Embodiment 1, the acquisition of bacillus amyloliquefaciens KB, identification and preservation
First, the acquisition of bacterial strain
Fructus Persicae and Fructus actinidiae chinensiss are gathered from the organic orchard of north of China.The isolated strains from fruit are dilute using conventional gradients
Release coating to separate, respectively 28 on LB solid mediums, NA solid mediums, KB solid mediums and CM0002 solid mediums
DEG C culture, picking colony morphological differencess it is larger and can pure culture bacterium colony, the purification storage on LB solid mediums, and with Fructus Vitis viniferae
Pseudoperonospora cubensis carries out the primary dcreening operation and multiple secondary screening of Antagonistic Fungi for target pathogens, finally gives one plant of strong bacterial strain of bacteriostatic activity,
It is named as bacterial strain KB.
2nd, the identification of bacterial strain
With reference to《Soil microorganism studies principle and method》(Lin Xiangui. soil microorganism studies principle and method [M], high
Deng education publishing house, 2010) described in method carry out the measure of Physiology and biochemistry and environmental resistance characteristic.
The morphological feature of bacterial strain KB：Somatic cells are in rod-short in 400 times of optical microscope hypothallus cells, straight or near
Directly, life, column or ellipse in spore, with mobility；The not chromogenic element on LB solid mediums, bacterium colony are impermeable for white
Bright, flat or circular, matt, as incubation time increases, the thickening exsiccation of bacterium colony, edge are irregular, have fold on bacterium colony, become
For creamy.
The physiological and biochemical property of bacterial strain KB：Gram-positive (photo is shown in Fig. 1), forms Mycoderma, C.I. 13020., acetyl first
Base methanol, nitrate reduction, indole reaction, contact enzyme positive, urine enzyme positive, hydrolyzable gelatin, cellulose, starch and seven leaves
Glycosides, oxydase reaction, to decompose L-Tyrosine negative, produces hydrogen sulfide, using citrate, does not utilize malonate, arginine, relies
Propylhomoserin, ODC Ornithine decarboxylase reacting positive, phenylalanine deaminase reaction negative, glucose, sucrose produce sour aerogenesis, using Portugal
Grape sugar, maltose, Sorbitol, rhamnose, inulin, galactose, inositol, xylose, mannose, Mannitol, ribose, sucrose etc., no
Using arabinose and sorbose.
The environmental resistance sex expression of bacterial strain KB：Can be just in the culture medium containing 1%-7% (mass content) Sodium Chloride
It is frequently grown, can not grows in the culture medium containing 10% (mass content) Sodium Chloride；In the culture medium of pH4.5-9.0
Normal growth, weak in pH4.0 growth potentials, pH 9.5 can not grow；It is that growth potential is weak at 4 DEG C in ambient temperature, at 55 DEG C -80 DEG C
Lower process 15min, still can normal growth.
Bacterial strain KB produces thermophilic ferrum element, cellulase, protease and chitinase.
The 16S rDNA sequences of bacterial strain KB are as shown in the sequence 1 of sequence table.
Summary result, is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) by identification of strains.
3rd, the preservation of bacterial strain
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB, was preserved on 09 12nd, 2013
(abbreviation CGMCC, address is for China Committee for Culture Collection of Microorganisms's common micro-organisms center：The Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC NO.8179.Bacillus amyloliquefaciens
(Bacillus amyloliquefaciens) KB CGMCC NO.8179, abbreviation bacillus amyloliquefaciens KB.
The bacteriostatic activity of embodiment 2, bacterial strain
1st, the fresh blade of grape variety " red milk " is gathered, is first rinsed with tap water, is then disappeared with 75% ethanol water
Poison 30 seconds, then with aseptic water washing, is then punched with card punch, obtains the leaf dish of diameter 1.5cm.
2nd, the leaf dish that step 1 is obtained is taken, is positioned over and is covered with the culture dish of sterile gauze, culture dish is placed in into sterile working
In platform, 4-5 minutes are blown.
3rd, packet transaction
Test group：After completing step 2, leaf dish is taken, soaked the 30-60 seconds in biocontrol microorganisms bacterium solution.
The preparation method of biocontrol microorganisms bacterium solution：Bacillus amyloliquefaciens KB is seeded to into LB fluid mediums (initial bacteria concentration
105Cfu/ milliliters), 28-30 DEG C, 180-220rpm shaken cultivation 48 hours take whole system, save backup at 4 DEG C.
Matched group：After completing step 2, leaf dish is taken, soaked the 30-60 seconds in sterilized water.
4th, after completing step 3, leaf dish is taken, is positioned over and is covered with the culture dish of sterile gauze (leaf dish is back side up), will training
Foster ware is placed in aseptic operating platform, blows 1-2 minutes.
5th, after completing step 4, leaf dish is taken, each leaf dish is overleaf inoculated with 20 μ l pathogen bacterium solutions (with aseptic aqueous suspension Portugal
The spore of grape Pseudoperonospora cubensis, obtains bacteria concentration for 104The pathogen bacterium solution of cfu/ml), 5-10 minutes are placed, 22 are subsequently placed in
DEG C, cultivate 10 days in 80-90% relative humiditys, the environment of alternation of light and darkness (illumination 12 hours/dark 12 hours).Observation leaf dish Portugal
Grape downy mildew incidence, according to the leaf dish number do not fallen ill account for total inoculation leaf dish number number, calculate disease suppression rate.
Carry out five repetitions to test, five repetitions are set in repeating every time to test and are processed, results averaged.
Photo after cultivating 10 days is shown in Fig. 2.The disease suppression rate of test group is 100%.As a result show, solve starch spore bar
Bacterium KB has preferably suppression function to Plasmopara viticola.
Sequence table
<160> 1
<210> 1
<211> 1456
<212> DNA
<213>Bacillus amyloliquefaciens（Bacillus amyloliquefaciens）
<400> 1
gtaaagggcg gggtgctata catgcaagtc gagcggacag atgggagctt gctccctgat 60
gttagcggcg gacgggtgag taacacgtgg gtaacctgcc tgtaagactg ggataactcc 120
gggaaaccgg ggctaatacc ggatggttgt ttgaaccgca tggttcagac ataaatggtg 180
gcttcggcta ccacttacag atggacccgc ggcgcattag ctagttggtg aggtaacggc 240
tcaccaaggc gacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga 300
cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct 360
gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct gttgttaggg 420
aagaacaagt gccgttcaaa tagggcggca ccttgacggt acctaaccag aaagccacgg 480
ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggaattattg 540
ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg 600
ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa ttccacgtgt 660
agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact ctctggtctg 720
taactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc 780
acgccgtaaa cgatgagtgc taagtgttag ggggtttccg ccccttagtg ctgcagctaa 840
cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg 900
ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 960
aggtcttgac atcctctgac aatcctagag ataggacgtc cccttcgggg gcagagtgac 1020
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080
gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg actgccggtg 1140
acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1200
acacgtgcta caatggacag aacaaagggc agcgaaaccg cgaggttaag ccaatcccac 1260
aaatctgttc tcagttcgga tcgcagtctg caactcgact gcgtgaagct ggaatcgcta 1320
gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380
cacaccacga gagtttgtaa cacccgaagt cggtgaggta acctttatgg agccagccgc 1440
cgaagtgaca gattgg 1456
Claims (10)
1. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB, its deposit number are CGMCC NO.8179.
2. the culture of bacillus amyloliquefaciens described in claim 1.
3. culture as claimed in claim 2, it is characterised in that：The preparation method of the culture is as follows：By claim 1
The bacillus amyloliquefaciens are seeded to LB fluid mediums, are cultivated, and obtain culture.
4. the fermented product of bacillus amyloliquefaciens described in claim 1.
5. bacillus amyloliquefaciens described in claim 1 or culture described in claim 2 or culture described in claim 3 or
Application of the fermented product described in claim 4 in Plasmopara viticola is suppressed.
6. bacillus amyloliquefaciens described in claim 1 or culture described in claim 2 or culture described in claim 3 or
Application of the fermented product described in claim 4 in downy mildew of garpe is prevented and treated.
7. bacillus amyloliquefaciens described in claim 1 or culture described in claim 2 or culture described in claim 3 or
Application of the fermented product described in claim 4 in the plant disease that preventing and treating Plasmopara viticola causes.
8. it is a kind of suppress Plasmopara viticola preparation, its active component be bacillus amyloliquefaciens or power described in claim 1
Profit requires culture described in culture described in 2 or claim 3 or fermented product described in claim 4.
9. a kind of preparation for preventing and treating downy mildew of garpe, its active component are bacillus amyloliquefaciens or right described in claim 1
Require culture or culture described in claim 3 or fermented product described in claim 4 described in 2.
10. the preparation of plant disease that a kind of preventing and treating Plasmopara viticola causes, its active component is Xie Dian described in claim 1
Culture described in culture described in afnyloliquefaciens or claim 2 or claim 3 or fermented product described in claim 4.
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| CN103087951A (en) * | 2013-01-14 | 2013-05-08 | 北京市农林科学院 | Bacillus amyloliquefaciens and application thereof |
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| CN103642727A (en) * | 2013-12-02 | 2014-03-19 | 北京市农林科学院 | Bacillus amyloliquefaciens inhibiting fungi and application thereof |
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| CN103087951A (en) * | 2013-01-14 | 2013-05-08 | 北京市农林科学院 | Bacillus amyloliquefaciens and application thereof |
| CN103589655A (en) * | 2013-05-20 | 2014-02-19 | 山西农业大学 | Preparation method for Bacillus amyloliquefaciens HRH 317 and antibacterial substances thereof |
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