CN105039207B - A kind of bacillus (Bacillus sp.) ZY and its application - Google Patents
A kind of bacillus (Bacillus sp.) ZY and its application Download PDFInfo
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- CN105039207B CN105039207B CN201510360259.1A CN201510360259A CN105039207B CN 105039207 B CN105039207 B CN 105039207B CN 201510360259 A CN201510360259 A CN 201510360259A CN 105039207 B CN105039207 B CN 105039207B
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Abstract
The invention belongs to microorganism field, and in particular to a kind of bacillus (Bacillus sp.) ZY, further relate to application of the above-mentioned bacterial strain in winter jujube ring spot is suppressed.The present invention bacillus (Bacillus sp.) ZY bacterial strains, " China Committee for Culture Collection of Microorganisms's common micro-organisms center " is preserved on April 19th, 2013, its preserving number is CGMCC No.7490；Bacillus (Bacillus sp.) ZY bacterial strains, fermented and cultured is carried out using LB fluid nutrient mediums, with oese from LB flat board picking ZY single bacterium colonies, it is inoculated into the test tube containing LB fluid nutrient mediums, 37 DEG C of constant-temperature table culture 18h obtain seed liquor, it is inoculated into according to 5% inoculum concentration in the 1L conical flasks containing 400mlLB culture mediums, 37 DEG C of 200rpm, culture 3.5d obtains zymotic fluid.The CGMCC No.7490 bacterium that the present invention is provided, are a kind of excellent Biocontrol Bacillus, are effectively extracted the active material in the bacterial strain fermentation liquor, the material has good inhibitory action to winter jujube Target spot pathogen.
Description
Technical field
The invention belongs to microorganism field, and in particular to a kind of bacillus (Bacillus sp.) ZY, further relate to above-mentioned
Bacterial strain suppress winter jujube ring spot in application.
Background technology
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.
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.
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.
Accelerate the research and development and application of biological pesticide, the need for meeting China's agricultural development, for ensureing that the healthy diet of people has weight
Meaning is wanted, development prospect is wide.
The content of the invention
It is an object of the invention to for winter jujube ring spot it is increasingly serious the problem of have there is provided one plant to winter jujube Target spot pathogen
The bacillus of inhibitory action（Bacillus sp.）ZY produce fermented and cultured and produce active material preparation method and
Its winter jujube ring spot preventing and treating in application.
The bacillus that the present invention is provided（Bacillus sp.）ZY bacterial strains, " China was preserved on 04 19th, 2013
Microbiological Culture Collection administration committee common micro-organisms center ", its preserving number is CGMCC No.7490, and preservation address is north
No. 3 Institute of Microorganism, Academia Sinica of institute of Jing Shi Chaoyang Districts North Star West Road 1.
Bacillus（Bacillus sp.）ZY strain isolations are from the beach soil sample of shellfish sand barrier island, on LB culture medium flat plates
Bacterium colony is larger, circular, protuberance, opaque；Young age bacterium surface is smooth, neat in edge, production mucus, culture more than 48h bacterium colonies surface
White fold is presented.Show that the bacterial strain belongs to bacillus through Physiology and biochemistry and 16SrRNA sequence analyses.
Using LB fluid nutrient mediums to bacillus（Bacillus sp.）ZY bacterial strains carry out fermented and cultured, with oese from
LB flat board picking ZY single bacterium colonies, are inoculated into the test tube containing LB fluid nutrient mediums, and 37 DEG C of constant-temperature table culture 18h obtain seed
Liquid, is inoculated into the 1L conical flasks containing 400mlLB culture mediums according to 5% inoculum concentration, 37 DEG C of 200rpm, and culture 3.5d is sent out
Zymotic fluid.
Bacillus（Bacillus sp.）The LB zymotic fluids of ZY bacterial strains centrifuge 20min, miillpore filter through 10 000 rpm
After filtering, fermented liquid supernatant is obtained.Through face-off it is demonstrated experimentally that active material is present with fermented liquid supernatant, using saturation sulfuric acid
The ammonium precipitation method extract the active material in fermented liquid supernatant, and the active material has good suppression effect to winter jujube Target spot pathogen
Really.
By qualitative and quantitative analysis, as a result show bacillus（Bacillus sp.）The active material that ZY bacterial strains are produced
Mainly it is made up of carbohydrate and protein, content is 67.1% and 20.8% respectively.
The bacterial strain is gram-positive bacteria, and rod-short produces gemma, middle life or end life；On LB culture medium flat plates bacterium colony compared with
Greatly, it is circular, protuberance, opaque.
The young age bacterium surface of the bacterial strain is smooth, neat in edge, production mucus, cultivates the white fold of more than 48h bacterium colonies.
4 ~ 55 DEG C of growth temperature, NaCl tolerance ranges are 0 ~ 7%.
Maltose, lactose, sorbierite and fructose can be utilized, L-Aspartic acid, creatine are not utilized.
Bacterial strain catalase experiment, V-P experiments, Xi Mengshi experiments, Starch Hydrolysis experiment, gelatin liquefaction experiment are sun
Property, methyl red experiment, the experiment of cellulose hydrolysising experiment, esterase, the experiment of glucose oxidative fermentation are feminine gender.
The beneficial effects of the present invention are the CGMCC No.7490 bacterium that the present invention is provided, are a kind of excellent biological and ecological methods to prevent plant disease, pests, and erosion gemma
Bacillus, is effectively extracted the active material in the bacterial strain fermentation liquor, and the material has good suppression to make to winter jujube Target spot pathogen
With.
Brief description of the drawings
Fig. 1 is bacillus（Bacillus sp.）Inhibitory action of the ZY bacterial strain fermentation liquors to winter jujube ring spot germ；
Fig. 2 is bacillus（Bacillus sp.）Suppression of the active material to winter jujube ring spot germ in ZY bacterial strain fermentation liquors
Make and use.
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 fermented and cultured of ZY bacterial strains
Fermentation medium is LB fluid nutrient mediums（Constitute as g/L：Yeast leaches cream 5g；Peptone 10g；NaCl 10g；
Distilled water 1000mL；）.With oese from LB flat board picking ZY single bacterium colonies, it is inoculated into the test tube containing LB fluid nutrient mediums, 37
DEG C constant-temperature table culture 18h obtains seed liquor, is inoculated into according to 5% inoculum concentration in the 1L conical flasks containing 400mLLB culture mediums,
37 DEG C of 200rpm, culture 3.5d obtains zymotic fluid.
Embodiment 2
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 purifying is drawn
Fermented liquid supernatant afterwards is gently added in Oxford cup, and whole process should prevent fluid seepage in Oxford cup.Finally, it will stand facing each other
Flat board is placed in 28 DEG C of constant incubators, cultivates 12h, as a result as shown in Figure 1.As can be seen from the figure ZY fermented liquid supernatant pair
The growth of winter jujube Target spot pathogen has obvious inhibiting effect.
Embodiment 3
The bacillus that the present invention is provided（Bacillus sp.）The extraction of active material in ZY bacterial strain fermentation liquors
Obtained fermented liquid supernatant is extracted using saturated ammonium sulphate method is further, obtained ZY zymotic fluid
Supernatant is 370mL.190.92g solid ammonium sulfate powder is slowly added into fermented liquid supernatant,（Whole process is entered in ice bath
OK）, there are a large amount of flocculent deposits, be placed on after 4 DEG C of refrigerator overnights, 10000r/min centrifugation 20min abandon supernatant, precipitated
Thing.By sediment first with a small amount of distilled water flushing, then with appropriate distilled water back dissolving, it is freeze-dried after dialysis, obtains 0.18g work
Property material dry powder, yield is 0.36g/L.
Embodiment 4
The bacillus that the present invention is provided（Bacillus sp.）Active material and winter jujube ring spot in ZY bacterial strain fermentation liquors
The result of face-off
The active material obtained in embodiment 3 is dissolved in distilled water, concentration is 10mg/ml, carries out face-off experiment（With real
Apply example 2）, will face-off flat board be placed in 28 DEG C it is incubated overnight, as a result as shown in Figure 2.Show effectively to be extracted bacterial strain ZY generations
Active material, have good inhibitory action to winter jujube ring spot.
Embodiment 5
The bacillus that the present invention is provided（Bacillus sp.）Active material is qualitative and quantitative in ZY bacterial strain fermentation liquors
Analysis
Using H2SO4-anthrone method, ninhydrin colour developing, ammonium molybdate-perchloric acid development process to the active matter that is obtained in embodiment 3
Matter has carried out qualitative analysis, as a result shows to contain carbohydrate, protein and lipid material in active component, quantitative analysis shows three kinds
The content of material is respectively 67.1%, 20.8% and 6.2%, main component be sugar and protide, therefore bacterial strain ZY produce active matter
Matter is a kind of glycoprotein material.
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：ZY is fermented（Fermentation condition be the same as Example
1）, 50 times of sprayings of zymotic fluid dilution, the 1st spray pesticide after jujube tree fallen flowers, interval 15 ~ 20d dispensers later 1 time, totally 4 times,
Entirely breeding time dispenser 5 times, its biocontrol effect is up to 75.6%.
Physicochemical property-thermal stability determination of above-mentioned bacterial strains zymotic fluid
By ZY bacterial strain fermentation liquor supernatants（With example 4）Locate respectively at 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 100 DEG C
30min is managed, using winter jujube ring spot germ as indicator bacteria, not thermally treated zymotic fluid is compared, containing toxic medium method（With example 5）
Activity is determined, ZY zymotic fluids have stronger heat endurance.50-80 DEG C of each processing 30min, bacteriostatic activity is kept at control
More than 85%, 100 DEG C of processing 60min, activity is the 79.6% of control.
The physicochemical property of bacterial strain fermentation liquor-pH Stability Determinations
By ZY bacterial strain fermentation liquor supernatants（With example 4）Handled respectively with 1mol/L HCl and 1mol/L NaOH to pH value 4,
5th, 6,7,8,9,10, static 2h, then recall to pH the natural ph of zymotic fluid, using winter jujube ring spot germ as indicator bacteria, not
The zymotic fluid handled through soda acid is compared, containing toxic medium method（With example 5）Determine activity.Have certain between pH5 ~ pH10
Bacteriostasis rate, with pH rise, bacteriostasis rate is in trend, but difference with insignificance is gradually reduced, and when pH is 6, fungistatic effect is best,
Up to 72%.
Claims (4)
1. a kind of bacillus (Bacillus sp.) ZY bacterial strains cultural method, it is characterised in that described bacillus
(Bacillus sp.) ZY bacterial strains on April 19th, 2013 are preserved in " the common micro- life of China Committee for Culture Collection of Microorganisms
Thing " center ", its preserving number is CGMCC No.7490；
Described bacillus (Bacillus sp.) ZY bacterial strains, fermented and cultured is carried out using LB fluid nutrient mediums, oese is used
From LB flat board picking ZY single bacterium colonies, it is inoculated into the test tube containing LB fluid nutrient mediums, 37 DEG C of constant-temperature table culture 18h are planted
Sub- liquid, is inoculated into the 1L conical flasks containing 400mLLB culture mediums according to 5% inoculum concentration, 37 DEG C of 200rpm, and culture 3.5d is obtained
Zymotic fluid.
2. bacillus as claimed in claim 1 (Bacillus sp.) ZY bacterial strains cultural method, it is characterised in that institute
The zymotic fluid stated is centrifuged after 20min, filtering with microporous membrane through 10000 rpm, obtains fermented liquid supernatant liquid；In described supernatant
There is active material.
3. bacillus as claimed in claim 2 (Bacillus sp.) ZY bacterial strains cultural method, it is characterised in that make
The active material in fermented liquid supernatant is extracted with saturated ammonium sulphate method.
4. using described in claim 3 bacillus (Bacillus sp.) ZY bacterial strains cultural method obtain active matter
Application of the matter in winter jujube Target spot pathogen is suppressed.
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| CN101463339A (en) * | 2009-01-08 | 2009-06-24 | 中国农业大学 | Inocula for preventing apple fungous diseases and increasing yield, and special strain therefore and use thereof |
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Inventor after: Fan Yanhui Inventor after: Wang Jun Inventor after: Yao Zhigang Inventor after: Shi Dongli Inventor after: Li Xueping Inventor before: Fan Yanhui Inventor before: Wang Jun Inventor before: Yao Zhigang Inventor before: Shi Dongli |
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