CN105670964A - Bacillus atrophaeus strain BsR05 and application thereof - Google Patents
Bacillus atrophaeus strain BsR05 and application thereof Download PDFInfo
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- CN105670964A CN105670964A CN201610056373.XA CN201610056373A CN105670964A CN 105670964 A CN105670964 A CN 105670964A CN 201610056373 A CN201610056373 A CN 201610056373A CN 105670964 A CN105670964 A CN 105670964A
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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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- 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
Abstract
The invention discloses a Bacillus atrophaeus strain BsR05 and an application thereof. The Bacillus atrophaeus strain BsR05 is collected in the CGMCC (China General Microbiological Culture Collection Center) with the collection number being CGMCC NO.11665. A Bacillus atrophaeus strain BsR05 liquid suspending agent prepared from the strain has a specific control effect on cucumber gray mold, and the control effect is high and is averagely 70% or higher.
Description
Technical field
The present invention relates to agricultural microbial control technical field, particularly relate to atrophy bacillus cereus BsR05 bacterial strain and application thereof.
Background technology
Gray mold of cucumber is to be infected the world-wide prevalence sexually transmitted disease (STD) evil caused by fungus Deuteromycotina Botrytis cinerea (BotrytiscinereaPers.exFr.), is a kind of disease of throughout the year occurring of Fructus Cucumidis sativi Protectorate cultivation, occurs in recent years in the weight that becomes year by year. Causing rotten owing to fruit is frequently subjected to invade and harass, vegetable grower is usually referred to as decayed fruit disease, disease of going rotten. The gray mold disease of current Fructus Cucumidis sativi is on the rise, and causes a large amount of rotten melon, serious warmhouse booth of falling ill, generally can the underproduction 2～3 one-tenth, even uproot plants after their edible portions have been harvested in advance, have a strong impact on yield and the quality of Fructus Cucumidis sativi. One of key factor causing the big generation of gray mold of cucumber is that drug matching is unreasonable and institute's with medicament is developed immunity to drugs by Fructus Cucumidis sativi.
Adopt that Biocontrol microorganism controlling plant diseases is nontoxic, harmless, pollution-free, do not develop immunity to drugs, efficiently, not only conform with people's demand to pollution-free food, and the sustainable development for agricultural provides guarantee. Therefore the Biocontrol microorganism research of plant disease increasingly comes into one's own. Its model of action uniqueness of Biocontrol microorganism is various, is solve this kind of difficulty of the such as gray mold of cucumber important effective measures of anti-plant disease.
Atrophy bacillus cereus (Bacillusatrophaeus) non-pathogenic bacteria, a class mutation of bacillus subtilis, the culture medium containing carbohydrate can be formed solvable black colonies, mainly separate from soil and obtain. All it is widely used in fields such as agricultural, industry, scientific research, medical treatment and health. Often this bacterial strain is detected as quality control standard bacterial strain and Evaluation of Germicidal Efficacy test index bacterium in countries and regions such as the U.S., Britain, Japan, European Union. Apply this bacterium less for controlling plant diseases aspect research, deer show cloud etc. discloses a kind of atrophy bacillus cereus for preventing and treating cotton boll blight and strain (patent of invention 201210028087.4) thereof, pungent seapeaks etc. find that a strain atrophy bacillus cereus is used for preventing and treating head blight (pungent seapeak etc., ecological magazine, 2013,32(6)); Han Song etc. report a kind of endophyte and Fusarium oxysporum are had antagonism, are identified as atrophy bacillus cereus (Han Song, northwest Botany Gazette, 2011(5)). The existing beneficial microbe preventing and treating gray mold is mostly and directly uses bacterial strain or microbial inoculum controlling disease, such as Trichoderma spp. etc. but not easily preserve due to active bacteria formulation, its shelf life is difficult to ensure that.
Summary of the invention
The purpose of the present invention is aiming at the defect of above-mentioned existence and provides atrophy bacillus cereus BsR05 bacterial strain and application thereof.Gray mold is had stronger suppression by this bacterial strain, it is possible to make in the biological and ecological methods to prevent plant disease, pests, and erosion medicine of preparation preventing and treating gray mold of cucumber. Microbial bacterial agent effective ingredient prepared by the atrophy bacillus cereus BsR05 of the present invention is based on spore, can the long period preservation and activity is not easily lost, so the effect of biological control of diseases can effectively be improved and stablize, it is preferably applied to agricultural production.
The atrophy bacillus cereus BsR05 bacterial strain of the present invention and application technology scheme thereof are, atrophy bacillus cereus BsR05 bacterial strain, atrophy bacillus cereus (Bacillusatrophaeus) BsR05 bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode: 100101, preserving number: CGMCCNO.11665, preservation date is on November 16th, 2015.
Atrophy bacillus cereus BsR05 37 DEG C of cultivation 24h colony colours in tyrosine culture medium are all thin, produce without obvious melanin, and bacterium colony moistens, and neat in edge is opaque, protruding; Cultivating 48h, start to produce melanin for 37 DEG C, cultivate 96h for 37 DEG C, bacterium colony is black, and melanin gathers.
Atrophy bacillus cereus BsR05 can utilize glycerol, catalase reacting positive, nitrate reduction can be become nitrite, can be liquefied gelatin, available mannose fermentation, does not utilize arabinose, amygdaloside and mannose ferment, can grow in containing 7%NaCl tryptone water, starch can be hydrolyzed.
The application in preventing and treating gray mold of cucumber of the described atrophy bacillus cereus BsR05 bacterial strain.
Microbial bacterial agent prepared by described atrophy bacillus cereus BsR05 bacterial strain, atrophy bacillus cereus BsR05 is fermented, atrophy bacillus cereus BsR05 fermentation liquid adds interpolation 5% ammonium sulfate by weight percentage, 0.15% sodium benzoate, 0.1% fatty alcohol-polyoxyethylene ether, 0.20% xanthan gum prepares microbial bacterial agent.
In this microbial bacterial agent preparation process, various compositions interpolation order is: first have to slowly and not stop ammonium sulfate to add in atrophy bacillus cereus BsR05 fermentation liquid with stirring, after ammonium sulfate all dissolves, slowly it is sequentially added into sodium benzoate again, fatty alcohol-polyoxyethylene ether, xanthan gum, it is thus achieved that atrophy bacillus cereus BsR05 microbial bacterial agent suspending agent.
Atrophy bacillus cereus BsR05 fermentation liquid is prepared by following steps:
(1) slant strains: adopt solid LB media, be seeded in Tube propagation base by atrophy bacillus cereus BsR05, cultivates 24 hours to obtain slant strains under 30 DEG C of constant temperatures;
(2) shaking flask strain: adopt LB liquid medium, cultivates 18 hours to obtain seed liquor by single colony inoculation of slant strains on the mid-shaking table of LB liquid medium under 30 DEG C of constant temperatures;
(3) liquid fermentation: regulate medium pH 7.0-7.5,121 DEG C of sterilizings 20 minutes, seed liquor step (2) obtained is inoculated in 10 liters of fermentation tanks by inoculum concentration 2%, 30 DEG C of cultivations, ventilation per minute and fermenter volume are than for 1.2-1.5:1, mixing speed is 250r/min, and incubation time is 28-36 hour, in bacillus exfoliation 70%(spore rate (%)=grown spore number/(grown spore number+nutrition thalline number) × 100) time terminate fermentation obtain atrophy bacillus cereus BsR05 fermentation liquid.
In step (3), culture medium is by weight percentage: 2% Semen Maydis powder, 6% bean cake powder, 0.2%(NH4)2SO4、0.2%KH2PO4·2H2O、0.4%K2HPO4·2H2O、0.05%FeSO4·5H2O、0.3%CaCO3。
The atrophy bacillus cereus BsR05 bacterial strain of the present invention and application thereof have the beneficial effect that the suppression ratio of gray mold of cucumber is reached 83.24% by the atrophy bacillus cereus BsR05 of the present invention, present invention also offers the microbial bacterial agent utilizing above-mentioned atrophy fermentation of bacillus preparation preventing and treating gray mold of cucumber, this microbial bacterial agent can effectively prevent and treat gray mold of cucumber, has the advantage promoting plant growing simultaneously.Microbial bacterial agent effective ingredient prepared by the atrophy bacillus cereus BsR05 of the present invention is based on spore, can the long period preservation and activity is not easily lost, so the effect of biological control of diseases can effectively be improved and stablize, it is preferably applied to agricultural production.
Accompanying drawing illustrates:
Fig. 1 show atrophy bacillus cereus BsR05 37 DEG C of cultivation 96h figure in tyrosine culture medium.
Detailed description of the invention:
In order to be more fully understood that the present invention, describe technical scheme in detail with instantiation below, but the invention is not limited in this.
Embodiment 1:
The qualification of atrophy bacillus cereus (Bacillusatrophaeus)
Adopt 16SrDNA sequence alignment, in conjunction with physiological and biochemical property method for measuring, BsR05 is accredited as atrophy bacillus cereus (Bacillusatrophaeus). Specifically comprise the following steps that
(1) 16SrDNA sequence analysis
Extract the genomic DNA of BsR05 thalline, with bacterial 16 S rDNA universal primer (16sF:5'AGAGTTTGATCCTGGCTCAG3'; 16sR:5'TACGGCTACCTTGTTACGACT3') carry out pcr amplification, then reclaim test kit with agarose gel and reclaim. The PCR primer of purification after reclaiming is delivered to Shanghai Sheng Gong biotech company check order to obtain SEQIDNO1:
CTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTA60
CACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTT120
GGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTA180
TCGGAAGGTGCGGCTGGATCACTATTGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGG240
CGGCGTGCCTAATACATGCAAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTA300
GCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGA360
AACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTT420
CGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCAC480
CAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACG540
GCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACG600
GAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGA660
ACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAA720
CTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCG780
TAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAG840
GGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCG900
GTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAAC960
TGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGC1020
CGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCA1080
TTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGG1140
CCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGT1200
CTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGT1260
GGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC1320
AACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAA1380
ACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACAC1440
GTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAA1500
CCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAG1544
Use NCBI website that with the 16SrDNA sequence of various bacterium in data base, the 16SrDNA sequence of BsR05 bacterium is carried out similarity system design analysis, result shows that BsR0516SrDNA sequence is very high with the 16SrDNA sequence homology of atrophy bacillus cereus (Bacillusatrophaeus), reaches 99%.
(2) physiological and biochemical property
Atrophy bacillus cereus containing producing melanin in tyrosine culture medium, can will be referred to as Bacillus subtilis endophyticus to be different from bacillus subtilis again according to this characteristic by atrophy bacillus cereus original strain. Bacterial strain BsR05 37 DEG C of cultivation 24h colony colours in tyrosine culture medium are all thin, produce without obvious melanin, and bacterium colony moistens, and neat in edge is opaque, protruding. Cultivating 48h, start to produce melanin for 37 DEG C, cultivate 96h for 37 DEG C, bacterium colony is black, and melanin gathers, as shown in Figure of description Fig. 1.
In addition, BsR05 can utilize glycerol, catalase reacting positive, can become nitrite by nitrate reduction, and can liquefy gelatin, available mannose fermentation, do not utilize arabinose, amygdaloside and mannose ferment, can grow in containing 7%NaCl tryptone water, starch can be hydrolyzed, and bacillus subtilis can utilize mannitol, therefore identify that BsR05 is atrophy bacillus cereus.
Embodiment 2: the preparation of atrophy bacillus cereus (Bacillusatrophaeus) BsR05 microbial inoculum, step is as follows:
(1) slant strains: adopt solid LB media, be seeded in Tube propagation base by atrophy bacillus cereus (Bacillusatrophaeus) BsR05 test tube strains, cultivates 24 hours to obtain slant strains under 30 DEG C of constant temperatures.
(2) shaking flask strain: adopt LB liquid medium, cultivates 18 hours to obtain seed liquor by single colony inoculation of slant strains on the mid-shaking table of LB liquid medium under 30 DEG C of constant temperatures.
(3) liquid fermentation: culture medium is by weight percentage: 2% Semen Maydis powder, 6% bean cake powder, 0.2%(NH4)2SO4、0.2%KH2PO4·2H2O、0.4%K2HPO4·2H2O、0.05%FeSO4·5H2O、0.3%CaCO3PH7.0-7.5,121 DEG C of sterilizings 20 minutes, the seed liquor of step (2) is inoculated in 10 liters of fermentation tanks (inoculum concentration is 2%), 30 DEG C of cultivations, ventilation per minute and fermenter volume are than for 1.2-1.5:1, and mixing speed is 250r/min, incubation time is 28-36 hour, in bacillus exfoliation 70%(spore rate (%)=grown spore number/(grown spore number+nutrition thalline number) × 100) time terminate fermentation.
(4) preparation of microbial bacterial agent suspending agent: add 5% ammonium sulfate in the fermentation liquid after liquid fermentation completes, 0.15% sodium benzoate, 0.1% fatty alcohol-polyoxyethylene ether, 0.20% xanthan gum. Notice that adding order at the various compositions of suspending agent configuration is: first have to slowly and not stop ammonium sulfate to add in fermentation liquid with stirring, after ammonium sulfate all dissolves, be more slowly sequentially added into sodium benzoate, fatty alcohol-polyoxyethylene ether, xanthan gum. Finally prepare atrophy bacillus cereus BsR05 microbial bacterial agent suspending agent.
Embodiment 3: atrophy bacillus cereus (Bacillusatrophaeus) BsR05 antagonism to gray mold of cucumber
For examination pathogen: this Shandong Province academy sciences Biology Research Institute preservation of Botrytis cinerea (BotrytiscinereaPers.exFr.), adopt opposite culture method, first Botrytis cinerea is activated on PDA plate, make the pathogen bacterium cake of 5mm in the punching of colony edge region card punch after cultivating 5 days, pathogen bacterium cake is forwarded to PDA plate central authorities, inoculating atrophy bacillus cereus BsR05 from bacterium cake 2.0cm place's point, with the plate of only inoculating pathogen bacterium cake for comparison, each process sets 3 repetitions. 25 DEG C of incubators are cultivated, when blank covers with whole culture dish, measure the Developing restraint radius after comparison colony radius and inoculation BsR05, represent with antagonism suppression ratio (bacteriostasis rate (%)=(comparison increment-process increment)/comparison increment × 100).
Result is in Table 1, and the suppression ratio of gray mold of cucumber is reached 83.24% by atrophy bacillus cereus BsR05, transparent antibacterial bandwidth 11.5 millimeters. Illustrate that gray mold of cucumber is had obvious inhibitory action by atrophy bacillus cereus BsR05, there is the biological and ecological methods to prevent plant disease, pests, and erosion potential preventing and treating gray mold.
The table 1 atrophy bacillus cereus BsR05 antagonism to gray mold of cucumber
。
Embodiment 4: atrophy bacillus cereus (Bacillusatrophaeus) microbial bacterial agent greenhouse test to gray mold of cucumber preventive effect
Gray mold of cucumber: publish and educate neat cucumber seedling, Pre-trial Investigation incidence. After atrophy bacillus cereus BsR05 microbial bacterial agent suspension dilution 100 times, even spraying is to leaf two sides, often process 10 strains, 4 repetitions, ash arrhizus bacteria is inoculated with method after 24h, disease survey is carried out after 7d, comparison medicament is 40% pyrimethanil (Beijing Fleet Agricultural Science and Technology Co., Ltd.'s production), and dilute with water 1000 uses. Test site: Shandong Province academy sciences Biology Research Institute phjytotron.
Disease investigation and grade scale:
0 grade: blade is without scab; 1 grade: single blade has scab 3; 3 grades: single blade has scab 4 ~ 6; 5 grades: single blade has scab 7 ~ 10; 7 grades: single blade has scab 11 ~ 20; 9 grades: single blade has that scab is intensive accounts for leaf area more than 1/4.
Disease index (%)=100 × ∑ (each sick level × strain numbers at different levels)/(the highest sick level × total investigation strain number)
Disease refers to disease index before increment rate (%)=100 × (after medicine disease index before disease index-medicine)/medicine
Prevention effect (%)=100 × (comparison disease refers to that increment rate-process disease refers to increment rate)/comparison disease refers to increment rate
Result is in Table 2, and the gray mold of cucumber disease index that atrophy bacillus cereus BsR05 microbial bacterial agent processes is substantially less than blank, and slightly below the disease index of Chemical treatment; Illustrate that gray mold of cucumber is had good prevention effect by atrophy bacillus cereus BsR05 microbial bacterial agent.
Table 2 atrophy bacillus cereus BsR05 microbial bacterial agent suspending agent preventing and treating gray mold of cucumber laboratory test results
。
Embodiment 4: the atrophy bacillus cereus BsR05 microbial bacterial agent suspending agent field test to gray mold of cucumber preventive effect
(1) reagent agent: atrophy bacillus cereus BsR05 microbial bacterial agent, compares medicament: 40% pyrimethanil (Beijing Fleet Agricultural Science and Technology Co., Ltd.'s production).
(2) EXPERIMENTAL DESIGN and method
Test site completes at Wang Jia willow village, Jia Yue town, Zhucheng Fructus Cucumidis sativi (fruit phase) booth. Test sets 2 process, the 100 times of uses of dilution of atrophy bacillus cereus BsR05 microbial bacterial agent suspension, and 40% pyrimethanil 1000 times of uses of dilution, every mu sprays 60L, blank (CK). Field plot trial adopts random alignment, repeats 4 times, plot area 30m2.
Application method: carry out whole strain sprinkling with power sprayer, before being typically in 10:00 in the morning, after afternoon 16:00 point or the cloudy day spray.
Test survey and preventive effect calculate: investigation method is that 10 strains are fixed in each process, every strain upper, middle and lower 10 blades of investigation, the sick leaf disease feelings of investigation before spray medicine, and after spray medicine, 7d investigates once every time, the sick leaf disease feelings of record. Calculate disease index and prevention effect, carry out significance analysis with duncan's new multiple range method.
Grade scale: 0 grade anosis; 1 grade of lesion area accounts for less than the 5% of whole blade area; 3 grades of lesion areas account for the 6% ~ 15% of whole blade area; 5 grades of lesion areas account for the 16% ~ 25% of whole blade area; 7 grades of lesion areas account for the 26% ~ 50% of whole blade area; 9 grades of lesion areas account for more than the 50% of whole blade area.
In formula, CK0 is disease index before the dispenser of blank district; CK1 is disease index after the dispenser of blank district; PT0 is disease index before the dispenser of chemicals treatment district; PTl is disease index after the dispenser of chemicals treatment district.
Table 3 atrophy bacillus cereus BsR05 microbial bacterial agent suspending agent preventing and treating gray mold of cucumber field test results
。
SEQUENCELISTING
<110>Shandong Province academy sciences Biology Research Institute
<120>atrophy bacillus cereus BsR05 bacterial strain and application thereof
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ctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgta60
cacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtggggtaaccttttt120
ggagccagccgcctaaggtgggacagatgattggggtgaagtcgtaacaaggtagccgta180
tcggaaggtgcggctggatcactattggagagtttgatcctggctcaggatgaacgctgg240
cggcgtgcctaatacatgcaagtcgagcgaatggattaagagcttgctcttatgaagtta300
gcggcggacgggtgagtaacacgtgggtaacctgcccataagactgggataactccggga360
aaccggggctaataccggataacattttgaactgcatggttcgaaattgaaaggcggctt420
cggctgtcacttatggatggacccgcgtcgcattagctagttggtgaggtaacggctcac480
caaggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacg540
gcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacg600
gagcaacgccgcgtgagtgatgaaggctttcgggtcgtaaaactctgttgttagggaaga660
acaagtgctagttgaataagctggcaccttgacggtacctaaccagaaagccacggctaa720
ctacgtgccagcagccgcggtaatacgtaggtggcaagcgttatccggaattattgggcg780
taaagcgcgcgcaggtggtttcttaagtctgatgtgaaagcccacggctcaaccgtggag840
ggtcattggaaactgggagacttgagtgcagaagaggaaagtggaattccatgtgtagcg900
gtgaaatgcgtagagatatggaggaacaccagtggcgaaggcgactttctggtctgtaac960
tgacactgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgc1020
cgtaaacgatgagtgctaagtgttagagggtttccgccctttagtgctgaagttaacgca1080
ttaagcactccgcctggggagtacggccgcaaggctgaaactcaaaggaattgacggggg1140
cccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggt1200
cttgacatcctctgaaaaccctagagatagggcttctccttcgggagcagagtgacaggt1260
ggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgc1320
aacccttgatcttagttgccatcattaagttgggcactctaaggtgactgccggtgacaa1380
accggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacac1440
gtgctacaatggacggtacaaagagctgcaagaccgcgaggtggagctaatctcataaaa1500
ccgttctcagttcggattgtaggctgcaactcgcctacatgaag1544
Claims (8)
1. atrophy bacillus cereus BsR05 bacterial strain, it is characterized in that, atrophy bacillus atrophaeusBsR05 bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number: CGMCCNO.11665, and preservation date is on November 16th, 2015.
2. atrophy bacillus cereus BsR05 bacterial strain according to claim 1, it is characterised in that atrophy bacillus cereus BsR05 37 DEG C of cultivation 24h colony colours in tyrosine culture medium are all thin, producing without obvious melanin, bacterium colony moistens, neat in edge, opaque, protruding; Cultivating 48h, start to produce melanin for 37 DEG C, cultivate 96h for 37 DEG C, bacterium colony is black, and melanin gathers.
3. atrophy bacillus cereus BsR05 bacterial strain according to claim 1, it is characterized in that, atrophy bacillus cereus BsR05 can utilize glycerol, catalase reacting positive, can become nitrite by nitrate reduction, and can liquefy gelatin, available mannose fermentation, do not utilize arabinose, amygdaloside and mannose ferment, can grow in containing 7%NaCl tryptone water, starch can be hydrolyzed.
4. the atrophy bacillus cereus BsR05 bacterial strain as claimed in claim 1 application in preventing and treating gray mold of cucumber.
5. microbial bacterial agent prepared by the atrophy bacillus cereus BsR05 bacterial strain that a kind adopts described in claim 1, it is characterized in that, atrophy bacillus cereus BsR05 is fermented, atrophy bacillus cereus BsR05 fermentation liquid adds interpolation 5% ammonium sulfate by weight percentage, 0.15% sodium benzoate, 0.1% fatty alcohol-polyoxyethylene ether, 0.20% xanthan gum prepares microbial bacterial agent.
6. microbial bacterial agent according to claim 5, it is characterized in that, in this microbial bacterial agent preparation process, various compositions interpolation order is: first have to slowly and not stop ammonium sulfate to add in atrophy bacillus cereus BsR05 fermentation liquid with stirring, after ammonium sulfate all dissolves, slowly it is sequentially added into sodium benzoate again, fatty alcohol-polyoxyethylene ether, xanthan gum, it is thus achieved that atrophy bacillus cereus BsR05 microbial bacterial agent suspending agent.
7. microbial bacterial agent according to claim 5, it is characterised in that atrophy bacillus cereus BsR05 fermentation liquid is prepared by following steps:
(1) slant strains: adopt solid LB media, be seeded in Tube propagation base by atrophy bacillus cereus BsR05, cultivates 24 hours to obtain slant strains under 30 DEG C of constant temperatures;
(2) shaking flask strain: adopt LB liquid medium, cultivates 18 hours to obtain seed liquor by single colony inoculation of slant strains on the mid-shaking table of LB liquid medium under 30 DEG C of constant temperatures;
(3) liquid fermentation: regulate medium pH 7.0-7.5,121 DEG C of sterilizings 20 minutes, seed liquor step (2) obtained is inoculated in 10 liters of fermentation tanks by inoculum concentration 2%, 30 DEG C of cultivations, ventilation per minute and fermenter volume are than for 1.2-1.5:1, mixing speed is 250r/min, and incubation time is 28-36 hour, terminates fermentation when bacillus exfoliation 70% and obtains atrophy bacillus cereus BsR05 fermentation liquid.
8. microbial bacterial agent according to claim 7, it is characterised in that in step (3), culture medium is by weight percentage: 2% Semen Maydis powder, 6% bean cake powder, 0.2%(NH4)2SO4、0.2%KH2PO4·2H2O、0.4%K2HPO4·2H2O、0.05%FeSO4·5H2O、0.3%CaCO3。
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| CN107663511A (en) * | 2017-11-30 | 2018-02-06 | 沈国青 | A kind of culture medium of nitrite bacteria and preparation method thereof |
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