CN106520607B - 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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- CN106520607B CN106520607B CN201610973916.4A CN201610973916A CN106520607B CN 106520607 B CN106520607 B CN 106520607B CN 201610973916 A CN201610973916 A CN 201610973916A CN 106520607 B CN106520607 B CN 106520607B
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- bacillus amyloliquefaciens
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- downy mildew
- grape
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 10
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- Medicinal Chemistry (AREA)
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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 a bacillus amyloliquefaciens and its inhibit grape
Application in Pseudoperonospora cubensis.
Background technique
Downy mildew of garpe is a kind of fungal disease.Downy mildew of garpe generally causes a large amount of underproduction of fruit, sternly
The edible value and market value for affecting fruit again, the income for resulting in orchard worker reduce, and seriously constrain China's grape
The development of industry.No matter in developed country, or in developing country, due to lacking necessary effective prevention and control medicament, oenin
Loss amount caused by mildew accounts for 20% of total amount or more.The generation of downy mildew of garpe has given modern grape to produce especially high-end Portugal
Grape production is caused with processing to be seriously affected.
Downy mildew of garpe main harm blade, can also infect the tender tissues such as young sprout young fruit.Downy mildew of garpe occurs for blade
Afterwards, the clear fleck of faint yellow water stain shape blur margin of coming into being is gradually expanded later as brown irregular shape or polygonal scab,
Number spot, which is connected, becomes the big spot of irregular shape；When wet weather, white downy mildew shape object (the i.e. sporangiophore of germ is generated in the scab back side
And sporangium)；Disease leaf is early withered caducous when falling ill serious.After downy mildew of garpe occurs for tender tip, water soaked spots is formed, is then become
The slightly concave sunken scab of brown, scab also generates white downy mildew when moist；When seriously ill young sprout distort, growth stop in addition it is withered.Volume
Downy mildew of garpe can also occur sometimes for palpus, cob, petiole, and symptom is similar to tender tip.After downy mildew of garpe occurs for young fruit, sick portion
It fades, is hardened sagging, upper raw white downy mildew, very easy atrophy falls off；Aggrieved when fruit is medium-sized, sick portion's brown to dead color is soft rotten early
It falls；It is no longer infected after fruit color.
The pathogen of downy mildew of garpe is P. viticola, belongs to Mastigomycotina, Oomycete Peronosporales, Plasmopara.Portugal
Grape Pseudoperonospora cubensis is overwintering in diseased tissues with egg spore, or remains on sick leaf overwintering in soil.Next year ovum under optimum conditions
Spore germination generates sporangium, then generates zoospore by sporangium, and wind and rain is borrowed to propagate, and invades from blade back stomata, carries out first
It infects.By 7-12 days incubation periods, sporangiophore and sporangium are generated in sick portion, spore germination generates zoospore and carries out again
It infects.It is 10 DEG C~15 DEG C that sporangium, which sprouts preference temperature,.The preference temperature that zoospore is sprouted is 18 DEG C -24 DEG C.Autumn is low
Temperature, rainy more dew, Yi Yinqi plant disease epidemic.Orchard hypsography low-lying, frame face improper ventilation tree vigo(u)r are weak, are conducive to disease.
China's vinegrowing area alreadys exceed 800,000 hectares within 2014, is more than France, is only second to Spain, is in the world
Effective the second big country of cultivated area of grape, and grape -growing areas is also being stepped up.Currently, the generally hair of downy mildew of garpe
It is raw to have become a key factor for restricting the industry development of China's grape.At present in production to the prevention and treatment of downy mildew of garpe still with
Based on chemoprevention, but thus bring " 3R " negative effect is very serious.A large amount of use of chemical pesticide seriously destroys ecological ring
Border leads to a large amount of pesticide residue in grape fruit, influences people's health.In China viny region, in the picking of grape
Before, to the means of prevention of downy mildew of garpe also based on chemical pesticide, this not only influences the health of the people,
Limit the processing and outlet of grape product.China will develop biological pesticide and green food has been included in " 21 st century in China view
Journey ", construction requirements Beijing of Beijing modern agriculture Science and Technology City and surrounding area crop disease realize green prevention and control, agricultural products
Carry out green production.
Summary of the invention
The object of the present invention is to provide a bacillus amyloliquefaciens and its inhibiting the application in Plasmopara viticola.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB provided by the invention, in 2013 09
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address are as follows: Beijing on the 12nd
The institute 3 of city, North Star West Road, Chaoyang District 1, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC NO.8179.Solution
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB CGMCC NO.8179, abbreviation bacillus amyloliquefaciens
KB。
The culture of bacillus amyloliquefaciens KB also belongs to protection scope of the present invention.The preparation method of the culture is such as
Under: bacillus amyloliquefaciens KB is seeded to LB liquid medium, is cultivated, obtains culture.The preparation of the culture
Method is specific as follows: the initial concentration for being seeded to LB liquid medium and make it in cultivating system bacillus amyloliquefaciens KB
It is 105It cfu/ milliliters, is cultivated, obtains culture.Concretely 28-30 DEG C of the condition of the culture, 180-220rpm vibration
Swing culture 48 hours.The culture concretely terminates the whole system after culture.
The fermentation material of bacillus amyloliquefaciens KB also belongs to protection scope of the present invention.
The present invention also protects bacillus amyloliquefaciens KB or the culture or the fermentation material inhibiting downy mildew of garpe
Application in bacterium.
The present invention also protects bacillus amyloliquefaciens KB or the culture or the fermentation material in prevention and treatment downy mildew of garpe
In application.
The present invention also protects bacillus amyloliquefaciens KB or the culture or the fermentation material in prevention and treatment downy mildew of garpe
Application in microbial plant disease.The plant concretely grape, such as grape variety " red milk ".
The present invention also protect it is a kind of inhibit Plasmopara viticola preparation, active constituent be bacillus amyloliquefaciens KB or
The culture or the fermentation material.
The present invention also protects a kind of preparation for preventing and treating downy mildew of garpe, and active constituent is bacillus amyloliquefaciens KB or institute
State culture or the fermentation material.
The present invention also protects a kind of preparation for preventing and treating the microbial plant disease of downy mildew of garpe, active constituent Xie Dian
Afnyloliquefaciens KB or the culture or the fermentation material.The plant concretely grape, such as grape variety " red milk ".
Bacillus (Bacillus spp.) is generally existing in the soil, is to be present in soil and plant microecosystem
In one of superior microorganism population, can with the stronger ability for resisting ambient pressure environment due to generating endogenous spore
It resists in survived environment because of the injury caused by drying, thermal and uv radiation, maintains own existence ability unaffected.This
A biological property makes bacillus have very good application prospect, especially in the biological products using active bacteria formulation
Show powerful vitality.Bacillus is many kinds of, enormous amount, can generate diversified physiological activator and
Metabolite has extensive prospects for commercial application.
The present invention provides one plant of bacillus amyloliquefaciens inhibited to Plasmopara viticola, for oenin
The prevention and treatment of mildew has great application value.
Detailed description of the invention
Fig. 1 is the photo after bacillus amyloliquefaciens KB Gram's staining.
Fig. 2 is for bacillus amyloliquefaciens KB to the inhibitory effect of Plasmopara viticola in grape leaf dish.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Plasmopara viticola (Plasmopara viticola): Huang Xiaoqing, Kong Xiang wait so long the oenin in different host sources
Mildew bacterium Pathogenic Tests and sporangium size compare；" plant protection ", 2015,41 (3): 178-182.
Grape variety " red milk ": principality is outstanding, red milk grape；" Beijing Agriculture ", 2009,22 phases.
Embodiment 1, the acquisition of bacillus amyloliquefaciens KB, identification and preservation
One, the acquisition of bacterial strain
Peach and Kiwi berry are acquired from the organic orchard of north of China.The isolated strains from fruit are dilute using conventional gradients
Coating separation is released, respectively 28 on LB solid medium, NA solid medium, KB solid medium and CM0002 solid medium
DEG C culture, picking colony morphological differences it is larger and can pure culture bacterium colony, the purification storage on LB solid medium, and with grape
Primary dcreening operation and multiple secondary screening of the Pseudoperonospora cubensis for target pathogens progress Antagonistic Fungi, finally obtain one plant of strong bacterial strain of bacteriostatic activity,
It is named as bacterial strain KB.
Two, the identification of bacterial strain
With reference to " edaphon studies principle and method ", (Lin Xiangui edaphon studies principle and method [M], high
Equal education publishing house, 2010) description method carries out the measurement of Physiology and biochemistry and environmental resistance characteristic in.
The morphological feature of bacterial strain KB: somatic cells are in rod-short in 400 times of optical microscopy hypothallus cells, straight or close
Directly, raw in gemma, column or ellipse have motility；The not chromogenic element on LB solid medium, bacterium colony are that white is impermeable
Bright, flat or round, matt, as incubation time increases, bacterium colony thickens exsiccations, and edge is irregular, has fold on bacterium colony, change
For creamy.
The physiological and biochemical property of bacterial strain KB: Gram-positive (photo is shown in Fig. 1) forms mycoderm, methyl red, acetyl first
Base methanol, nitrate reduction, indole reaction, contact enzyme positive, urine enzyme positive, hydrolyzable gelatin, cellulose, starch and seven leaves
Tyrosine feminine gender is decomposed in glycosides, oxydase reaction, generates hydrogen sulfide, using citrate, does not utilize malonate, arginine relies
Propylhomoserin, ornithine decarboxylase reacting positive, phenylalanine deaminase reaction negative, glucose, sucrose produce acid and produce gas, using Portugal
Grape sugar, maltose, sorbierite, rhamnose, synanthrin, galactolipin, inositol, xylose, mannose, mannitol, ribose, sucrose etc., no
Utilize arabinose and sorbose.
The environmental resistance sex expression of bacterial strain KB: can be just in the culture medium of Yu Hanyou 1%-7% (mass content) sodium chloride
It is frequently grown, can not be grown in the culture medium of Yu Hanyou 10% (mass content) sodium chloride；In the culture medium of pH4.5-9.0
Normal growth, weak in pH4.0 growth potential, pH 9.5 can not be grown；Growth potential is weak at being 4 DEG C in environment temperature, at 55 DEG C -80 DEG C
Lower processing 15min, still can normal growth.
Bacterial strain KB produces thermophilic iron element, cellulase, protease and chitinase.
The 16S rDNA sequence of bacterial strain KB is as shown in the sequence 1 of sequence table.
In summary as a result, bacterial strain is accredited as bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
Three, the preservation of bacterial strain
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB, was preserved on 09 12nd, 2013
China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address are as follows: 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
1, the fresh blade for acquiring grape variety " red milk ", is first rinsed with tap water, is then disappeared with 75% ethanol water
Then poison 30 seconds is used aseptic water washing, is then punched with punch, the leaf dish of diameter 1.5cm is obtained.
2, the leaf dish for taking step 1 to obtain, is placed in the culture dish for being covered with sterile gauze, and culture dish is placed in sterile working
In platform, blow 4-5 minutes.
3, packet transaction
Test group: after completing step 2, leaf dish is taken, is impregnated 30-60 seconds in biocontrol microorganisms bacterium solution.
The preparation method of biocontrol microorganisms bacterium solution: bacillus amyloliquefaciens KB is seeded to LB liquid medium (initial bacteria concentration
105Cfu/ milliliters), 28-30 DEG C, 180-220rpm shaken cultivation 48 hours take whole system, save backup at 4 DEG C.
Control group: after completing step 2, leaf dish is taken, is impregnated 30-60 seconds in sterile water.
4, after completing step 3, leaf dish is taken, (leaf dish is back side up) is placed in the culture dish for being covered with sterile gauze, will be trained
Feeding ware is placed in aseptic operating platform, is blown 1-2 minutes.
5, after completing step 4, leaf dish is taken, each leaf dish is overleaf inoculated with 20 μ l pathogen bacterium solutions (with sterile aqueous suspension Portugal
The spore of grape Pseudoperonospora cubensis, obtaining bacteria concentration is 104The pathogen bacterium solution of cfu/ml), it places 5-10 minutes, is subsequently placed in 22
DEG C, cultivate 10 days in the environment of 80-90% relative humidity, alternation of light and darkness (illumination 12 hours/dark 12 hours).Observe leaf dish Portugal
Grape downy mildew incidence, according to the leaf dish number Zhan not fallen ill be always inoculated with leaf dish number number, calculate disease suppression rate.
It carries out five repetitions to test, repeats five reprocessings of setting in test every time, results are averaged.
Photo after culture 10 days is shown in Fig. 2.The disease suppression rate of test group is 100%.The result shows that solution starch gemma bar
Bacterium KB has the function of preferably to inhibit 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 (6)
1. the culture of bacillus amyloliquefaciens or the bacillus amyloliquefaciens is inhibiting the application in Plasmopara viticola；
The bacillus amyloliquefaciens are bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB, and preservation is compiled
Number be CGMCC NO.8179.
2. application as described in claim 1, it is characterised in that: the preparation method of the culture of the bacillus amyloliquefaciens is such as
Under: the bacillus amyloliquefaciens are seeded to LB liquid medium, is cultivated, obtains culture.
3. application of the culture of bacillus amyloliquefaciens or the bacillus amyloliquefaciens in prevention and treatment downy mildew of garpe；
The bacillus amyloliquefaciens are bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB, and preservation is compiled
Number be CGMCC NO.8179.
4. application as claimed in claim 3, it is characterised in that: the preparation method of the culture of the bacillus amyloliquefaciens is such as
Under: the bacillus amyloliquefaciens are seeded to LB liquid medium, is cultivated, obtains culture.
5. the culture of bacillus amyloliquefaciens or the bacillus amyloliquefaciens is in the prevention and treatment microbial plant of downy mildew of garpe
Application in disease；
The bacillus amyloliquefaciens are bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KB, and preservation is compiled
Number be CGMCC NO.8179.
6. application as claimed in claim 5, it is characterised in that: the preparation method of the culture of the bacillus amyloliquefaciens is such as
Under: the bacillus amyloliquefaciens are seeded to LB liquid medium, is cultivated, obtains culture.
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| 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 |
| 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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| 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 |
| CN103642727A (en) * | 2013-12-02 | 2014-03-19 | 北京市农林科学院 | Bacillus amyloliquefaciens inhibiting fungi and application thereof |
Non-Patent Citations (2)
| Title |
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| Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani.;G.Y Yu et al.;《Soil Biology and Biochemistry》;20020311;第34卷(第7期);第955-963页 * |
| 生防解淀粉芽孢杆菌（Bacillus amyloliquefaciens）最新研究进展;李红晓等;《微生物学杂志》;20160430;第36卷(第2期);第87-92页 * |
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