CN104974957B - Bacillus（Bacillus sp.）ZY bacterial strains and its application - Google Patents
Bacillus（Bacillus sp.）ZY bacterial strains and its application Download PDFInfo
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- CN104974957B CN104974957B CN201510364150.5A CN201510364150A CN104974957B CN 104974957 B CN104974957 B CN 104974957B CN 201510364150 A CN201510364150 A CN 201510364150A CN 104974957 B CN104974957 B CN 104974957B
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- bacillus
- bacterial strains
- winter jujube
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- ring spot
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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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- 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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- 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
Abstract
The invention belongs to microorganism field, and in particular to a kind of bacillus（Bacillus sp.）ZY bacterial strains, further relate to above-mentioned bacterial strain and are suppressing the application of winter jujube ring spot.The bacillus of the present invention（Bacillus sp.）ZY bacterial strains, were preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms center " on April 19th, 2013, and deposit number is CGMCC No.7490.The beneficial effects of the present invention are what the present invention was provided（Bacillus sp.）ZY bacterial strains, are a kind of excellent Biocontrol Bacillus, and the bacterial strain and its liquid fermentation liquid can produce the active metabolite for having inhibitory action to winter jujube Target spot pathogen, to the biocontrol effect of winter jujube ring spot up to more than 75.6%, with good application prospect.
Description
Technical field
The invention belongs to microorganism field, and in particular to a kind of bacillus（Bacillus sp.）ZY bacterial strains, are further related to
Above-mentioned bacterial strain is suppressing the application of winter jujube ring spot.
Background technology
The fresh and tender succulence of winter jujube mouthfeel, local flavor sour-sweet is agreeable to the taste, is to eat the outstanding person in kind raw, to wide since the popularizing planting
Big planting household brings huge economic interests, but increasing sharply with cultivated area, and the generation of winter jujube disease is more and more
Weight, wherein winter jujube ring spot are relatively conventional and endanger serious fungal disease, and winter jujube ring spot not only directly contributes winter jujube production
Amount declines to be reduced with quality, and disease fungus can secrete the toxin and metabolin for producing and being harmful to people and animals, to the health of people
Constitute great threat.The preventing and treating of winter jujube ring spot is mainly prevented and treated by spraying chemicals, but long-term unreasonable useization
Medicine of learning to farm generates problems.Such as：The high poison of chemical pesticide, high residue cause harm to the health of people, animal, to soil,
Water body, air cause severe contamination, destroy the ecological balance；The continuous enhancing of the pathogen property of medicine, the usage amount of agricultural chemicals and makes
Continued to increase with frequency, occur in that the mutually incremental vicious circle of dosage and disease.Therefore exploitation to the mankind and it is environment-friendly,
And have good prevention effect, new control of plant disease method it is extremely urgent.
With the food-safe pay attention to day by day of people, the need for sustainable disease and pest control development, biological control by
National governments and the attention of plant protection expert.Biological control controls harmful organism based on ecology, is integrated control
An indispensable part in system.It avoids chemical pesticide and uses a series of plant protection, environment and the energy brought
In terms of source the problem of, it is to avoid harm of the residues of pesticides to people and animals.One has been increasingly becoming using biological pesticide substituting chemical pesticide
The trend of kind.
Bacillus population is huge, and fertility is strong, heat-resisting, acid and alkali-resistance, stable in physicochemical property, and antimicrobial spectrum extensively, is being planted
Occupy critical role in thing biological control of diseases, be main biological and ecological methods to prevent plant disease, pests, and erosion strain, including bacillus subtilis (Bacillus subtilis), Bacillus cercus (Bacillus cereus), bacillus licheniformis (Bacillus licheniformis)、
Bacillus megaterium(Bacillus megaterium), bacillus amyloliquefaciens (Bacillus amyloliquefaciens)、
Bacillus polymyxa (Bacillus potymyxa) and bacillus pumilus (Bacillus pumilis) etc..Both at home and abroad to bud
The research and development of spore bacillus have been put into production practice, are made commercial preparation, are achieved efficient biocontrol effect, tested crop yield.
However, the extensive development studied with biocontrol microorganisms, new bacterium finds to be increasingly difficult to.Shell bank is by living in intertidal zone
Shellfish it is dead after housing and chip, carry, accumulated near high-water mark through wave.Huanghe delta Accessories during Binzhou
The shell bank that Wudi, Zhanhua seashore have been formed since nearly 5000 or six thousand years during Cheng Lu, ecological environment is unique, with the U.S. easily this Anna
The shell bank of state and South America Surinam and the referred to as big Chenier in the world three, and be worldwide largest, only the old and new
And the shell bank deposited.The unique ecological environment of shell bank imparts the distinctive living resources in this area, thin for the new biological and ecological methods to prevent plant disease, pests, and erosion of excavation
Bacterium and exploitation biological pesticide provide good resource.Accelerate the research and development and application of biological pesticide, meet China's agricultural development
Need, for ensureing that the healthy diet of people is significant, development prospect is wide.
The content of the invention
For winter jujube ring spot it is increasingly serious the problem of, the present invention provides a kind of bacillus（Bacillus sp.）ZY,
The bacterial strain serves good effect in the application in the preventing and treating of winter jujube ring spot.
The bacillus that the present invention is provided（Bacillus sp.）ZY bacterial strains, were preserved in " Chinese micro- on April 19th, 2013
Biological inoculum preservation administration committee common micro-organisms center ", deposit number is CGMCC No.7490.
Above-mentioned bacillus（Bacillus sp.）ZY bacterial strains, its 16S rDNA nucleotide sequence such as sequence table institute
Show.
Above-mentioned bacillus（Bacillus sp.）ZY bacterial strains are suppressing the application of winter jujube ring spot, are also institute of the present invention
Scope of protection.To the biocontrol effect of winter jujube ring spot up to 75.6% after.Detailed process is：Carried out with LB culture mediums
Fermentation → carrying out zymotic fluid to experimental plot to spray → is measured to prevention effect.
Bacillus（Bacillus sp.）The screening of ZY bacterial strains
The soil sample gathered from shellfish sand barrier island beach is weighed into 3g, is aseptically ground, is put into and fills sterile physiological salt
In the conical flask of water, add after a certain amount of bead, vibration 48h is carried out in 37 DEG C of constant-temperature tables, makes soil abundant with water
Mix.Soil supension is subjected to gradient dilution, the soil liquid of the different dilution factors of 100 μ l is taken respectively in Zuo Beier culture mediums and LB
It is coated with culture medium, each strength solution, which is done, once to be repeated, takes out flat board after 37 DEG C of culture 24h, observe the bacterium colony on each flat board
Count and record.Aseptically, the variant single bacterium colony of picking is pure in new flat lining out point, until bacterium colony is pure on flat board
Change.The different each single bacterium colony of morphological feature is done into preliminary antagonistic experiment with winter jujube Target spot pathogen respectively, each antibacterial effect of bacterium colony is observed
Fruit simultaneously makes a record, and final acquisition fungistatic effect is bestBacillus sp. ZY。
Bacillus（Bacillus sp.）ZY bacterial strain colony characteristicses：Bacterium colony is larger, circular, grand on LB culture medium flat plates
Rise, it is opaque；Young age bacterium surface is smooth, neat in edge, production mucus, and white fold is presented in culture more than 48h bacterium colonies surface.
Bacillus（Bacillus sp.）The cell morphological characteristic of ZY bacterial strains：Gram-positive bacteria, rod-short has bud
Spore, middle life or end life.
Bacillus（Bacillus sp.）The physiological and biochemical property of ZY bacterial strains：4 ~ 55 DEG C of growth temperature, NaCl tolerance models
Enclose is 0 ~ 7%；Maltose, lactose, sorbierite and fructose can be utilized, L-Aspartic acid, creatine are not utilized；Catalase experiment, V-P
Experiment, Xi Mengshi experiments, Starch Hydrolysis experiment, gelatin liquefaction experiment are the positive, methyl red experiment, cellulose hydrolysising experiment,
Esterase experiment, the experiment of glucose oxidative fermentation are feminine gender.
The fermented liquid produces active material, and fermentation LB culture medium compositions are g/L：Yeast leaches cream 5g；Peptone 10g；
NaCl 10g；Distilled water 1000mL；pH 7.0-7.2.
Bacillus（Bacillus sp.）The 16S rDNA gene sequence characteristics of ZY bacterial strains：CGMCC No.7490 are inoculated with
In LB culture mediums, thalline is collected by centrifugation in 120 rpm, 37 DEG C of concussion and cultivate 16h, and lysozyme and SDS broken walls are added after suspension, is adopted
Genomic DNA is extracted with phenol-chloroform method, with positive primer 2 7F（5’-GAGAGTT TGATCCTGGCTCAG-3’）, and reversely draw
Thing 1541R (5’-AAGGAGGTGATC CAGCC-3’), performing PCR amplification is entered to its 16S rDNA gene, by the product of amplification by north
Win company and be sequenced in capital three.PCR conditions are：94 DEG C, 10min；94 DEG C, 45s；55 DEG C, 45s；72 DEG C, 90s；30 circulations；
72 DEG C of 10min, 4 DEG C of preservations.16S rDNA gene orders length is 1452bp, and sequencing result is contrasted with ncbi database, is found
Its withBacillus amyloliquefaciens,Bacillus methylotrophicus,Bacillus subtilis's
Homology is higher, wherein, its homology highest isBacillus methylotrophicus, homology is up to 99%, its 16S
RDNA sequence is as shown in sequence table 1.
Reference《Bergey’s Mannual of Systematic Bacteriology》Vol. VIII content, according to it
Morphological feature and physiological and biochemical property, and 16S rDNA gene order results, are a new bacterium through Bacillus subtilis ZY, belong to
Bacillus（Bacillus sp. ZY）.
（Bacillus sp.）ZY bacterial strains can be in nutrient medium, such as：Grown in common beef broth, LB, nutrient agar,
Can also be in Zuo Beier（Zobell）In the minimal medium of 2216E culture mediums, PDA culture medium or addition glucose or sucrose
Growth.
The beneficial effects of the present invention are what the present invention was provided（Bacillus sp.）ZY bacterial strains, are a kind of excellent lifes
Anti- bacillus, the bacterial strain and its liquid fermentation liquid can produce the active metabolism for having inhibitory action to winter jujube Target spot pathogen
Product, to the biocontrol effect of winter jujube ring spot up to more than 75.6%, with good application prospect.
Brief description of the drawings
Fig. 1 is bacillus（Bacillus sp.）ZY colonial morphology figures；
Fig. 2 is bacillus（Bacillus sp.）The influence figure that ZY grows to winter jujube Target spot pathogen；
Fig. 3 is bacillus（Bacillus sp.）The influence figure that ZY bacterial strain fermentation liquors grow to winter jujube Target spot pathogen.
Embodiment
Come to be further described the present invention with reference to the accompanying drawings and detailed description, so as to the skill of this area
Art personnel know more about the present invention, but do not limit the present invention with this.
Embodiment 1
The bacillus that the present invention is provided（Bacillus sp.）The separation screening of ZY bacterial strains.
The soil sample gathered from shellfish sand barrier island beach is weighed into 3g, is aseptically ground, is put into and fills sterile physiological salt
In the conical flask of water, add after a certain amount of bead, vibration 48h is carried out in 37 DEG C of constant-temperature tables, makes soil abundant with water
Mix.Soil supension is subjected to gradient dilution, the soil liquid of the different dilution factors of 100 μ l is taken respectively in Zuo Beier culture mediums（Egg
White peptone 5g；NaCl 19.45g；Ironic citrate 0.1g；Yeast extract 1.0g；KBr 0.08g；Sodium metasilicate 0.004g；NH4NO3
0.0016g；Na2SO43.24g；NaHCO30.16g；Na2HPO40.008g；CaCl21.8g；MgCl28.8g；KCl
0.55g；Sodium fluoride 0.0024g；Strontium chloride 0.034g；Agar 15.0g；Distilled water 1000ml；pH 7.4-7.8）With LB culture mediums
（Yeast leaches cream 5g；Peptone 10g；NaCl 10g；Distilled water 1000mL；pH 7.0-7.2）Upper coating, each strength solution
Do and once repeat, take out flat board after 37 DEG C of culture 24h, observe clump count and record on each flat board.Aseptically, choose
Take variant single bacterium colony pure in new flat lining out point, until bacterium colony purifying on flat board.By the different each single bacterium of morphological feature
Fall respectively with filamentous fungi（Winter jujube ring spot and cotton spoting verticillium wilt）Preliminary antagonistic experiment is done, each bacterium colony fungistatic effect is observed simultaneously
Make a record, it is final to obtain the best Bacillus sp. ZY of fungistatic effect, as a result as shown in Figure 1.
Embodiment 2：
The bacillus that the present invention is provided（Bacillus sp.）The morphological feature and physiological and biochemical property of ZY bacterial strains.
Reference《Bergey’s Mannual of Systematic Bacteriology》Vol. VIII test method is entered
OK, its Gram's staining, bacterium colony size and form are detected, growth temperature NaCl tolerances are Starch Hydrolysis, cellulose hydrolysis, bright
Dispergation, and utilization of carbon source situation.
As a result show, the bacterium is gram-positive bacteria, rod-short produces gemma, middle life or end life.On LB culture medium flat plates
Bacterium colony is larger, circular, protuberance, opaque；Young age bacterium surface is smooth, neat in edge, production mucus, and culture more than 48h bacterium colonies are in white
Color fold（See Fig. 2）.4 DEG C ~ 55 DEG C of growth temperature, NaCl tolerance ranges are 0% ~ 7%.Can using maltose, lactose, sorbierite and
Fructose, L-Aspartic acid, creatine are not utilized；Catalase experiment, V-P experiments, Xi Mengshi experiments, Starch Hydrolysis experiment, gelatin solution
It is the positive to change experiment, and methyl red experiment, the experiment of cellulose hydrolysising experiment, esterase, the experiment of glucose oxidative fermentation are feminine gender.
Embodiment 3：
The bacillus that the present invention is provided（Bacillus sp.）The PCR amplifications of the 16S rDNA genes of ZY bacterial strains and sequence
Determine.By the inoculation in LB culture mediums, 120rpm, 37 DEG C of concussion and cultivate 16h, be collected by centrifugation after thalline, suspension add it is molten
Bacterium enzyme and SDS broken walls, extract genomic DNA, with positive primer 2 7F (5 '-GAGAGTTTGATCCTG using phenol-chloroform method
GCTCAG-3 '), and reverse primer 1541R (5 '-AAGG AGGTGATCCAGCC-3 '), performing PCR is entered to its 16S rDNA gene
Amplification, wins company by Beijing three by the product of amplification and is sequenced.PCR conditions are：94 DEG C, 10min；94 DEG C, 45s；55 DEG C,
45s；72 DEG C, 90s；30 circulations；72 DEG C of 10min, 4 DEG C of preservations.16S rDNA gene orders length is 1452bp, sequencing knot
Fruit contrasts with ncbi database, finds itself and Bacillus amyloliquefaciens, Bacillus
Methylotrophicus, Bacillus subtilis homology are higher, wherein, its homology highest is Bacillus
Methylotrophicus, homology is up to 99%, and its 16S rDNA sequence is as shown in sequence table 1.
Embodiment 4：
The bacillus that the present invention is provided（Bacillus sp.）The fermented and cultured of ZY bacterial strains
Fermentation medium is LB fluid nutrient mediums.With oese from LB flat board picking ZY bacterium single bacterium colonies, be inoculated into containing
In the test tube of LB fluid nutrient mediums, 37 DEG C of constant-temperature table culture 18h obtain seed liquors, according to 5% inoculum concentration be inoculated into containing
In the 1L triangular flasks of 400mlLB culture mediums, 37 DEG C of 200rpm, culture 3.5d obtains zymotic fluid.
Embodiment 5：
The bacillus that the present invention is provided（Bacillus sp.）Suppression of the ZY bacterial strain fermentation liquors to winter jujube Target spot pathogen is made
With
Tunning in embodiment 4 is centrifuged into 20min through 10 000 rpm, by 0.22 μm micro- of supernatant sterilizing
Hole membrane filtration, obtains fermented liquid supernatant.First punched with card punch on the winter jujube ring spot fungi flat board activated, by bacteria cake
PDA plate center is transferred to, 48h is cultivated, then the Oxford cup after sterilizing is positioned over flat board relevant position, 200 μ l fermentations are drawn
Liquid supernatant is gently added in Oxford cup, and whole process should prevent fluid seepage in Oxford cup.Finally, face-off flat board is placed in
In 28 DEG C of constant incubators, 12h is cultivated, as a result as shown in Figure 3.As can be seen from the figure ZY fermented liquid supernatant is to winter jujube wheel
The growth of line germ has obvious inhibiting effect.
Embodiment 6
The bacillus that the present invention is provided（Bacillus sp.）Application of the ZY bacterial strains in No. 4 Tanakas in winter jujube experimental plot.
Winter jujube ring spot can be prevented and treated by zymotic fluid, detailed process is：It is right（Bacillus sp.）Fermented（Fermentation
Condition be the same as Example 4）, zymotic fluid dilute 50 times spraying, the 1st spray pesticide after jujube tree fallen flowers, later be spaced 15 ~ 20d apply
Medicine 1 time, totally 4 times, whole breeding time dispenser 5 times, its biocontrol effect is up to 75.6%.
Claims (2)
1. a kind of bacillus（Bacillus sp.）ZY bacterial strains, " Chinese microorganism strain guarantor is preserved on April 19th, 2013
Hide administration committee's common micro-organisms center ", deposit number is CGMCC No.7490.
2. bacillus as claimed in claim 1（Bacillus sp.）Application of the ZY bacterial strains in winter jujube ring spot is suppressed.
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| CN114181850B (en) * | 2021-11-18 | 2023-01-31 | 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) | UV radiation resistant bacillus and application thereof |
| CN114045247B (en) * | 2021-12-28 | 2023-04-25 | 滨州学院 | Bacillus seawater and application thereof in salinized farmland production |
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Effective date of registration: 20201026 Address after: No. 83, Peijia village, Liguo Township, Zhanhua District, Binzhou City, Shandong Province Patentee after: Shandong Xinzeng biological fertilizer Co., Ltd Address before: 256603 No. five, No. 391, the Yellow River Road, Shandong, Binzhou Patentee before: BINZHOU University |
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