CN105039207A - Bacillus sp. ZY and application thereof - Google Patents
Bacillus sp. ZY and application thereof Download PDFInfo
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- CN105039207A CN105039207A CN201510360259.1A CN201510360259A CN105039207A CN 105039207 A CN105039207 A CN 105039207A CN 201510360259 A CN201510360259 A CN 201510360259A CN 105039207 A CN105039207 A CN 105039207A
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Abstract
The invention belongs to the field of microorganisms, particularly relates to a bacillus sp. ZY, and also relates to application of the bacterial strain in inhibiting winter jujube ring rot. The bacillus sp. ZY bacterial strain provided by the invention is preserved in China General Microbiological Culture Collection Center on April 19th, 2013, and the preservation number is CGMCC No.7490. An LB liquid medium is adopted for fermental cultivation of the bacillus sp. ZY bacterial strain, and the preparation method comprises the following steps: using an inoculating loop to select a ZY single colony from an LB plate; inoculating the ZY single colony into a test tube containing the LB liquid medium; carrying out shaking culture at the constant temperature of 37 DEG C for 18 h, so as to obtain a seed solution; inoculating into a 1 L conical flask containing 400 ml of LB medium according to the inoculation quantity of 5%; culturing for 3.5 d at the temperature of 37 DEG C and the rotation speed of 200 rpm, so as to obtain fermentation liquid. The CGMCC No.7490 bacterium provided by the invention is an excellent biocontrol bacillus, and the active substance with a good inhibiting effect on winter jujube ring rot in the bacterial strain fermentation liquid is effectively extracted.
Description
Technical field
The invention belongs to microorganism field, be specifically related to a kind of genus bacillus (
bacillussp.) ZY, also relate to above-mentioned bacterial strain and suppress the application in winter jujube ring spot.
Background technology
Along with people are to the pay attention to day by day of food safety, the needs of sustainable disease and pest control development, biological control is subject to the attention of national governments and plant protection expert.Biological control controls harmful organism based on ecology, be in Synthetical control system one must indispensable integral part.It avoid chemical pesticide use bring a series of plant protection, environment and energy aspect problem, avoid the harm of pesticide residue to people and animals.Applying biological agricultural chemicals substituting chemical pesticide becomes a kind of trend gradually.
But along with extensively carrying out of biocontrol microorganisms research, new bacterium finds more and more difficult.Shell bank be by the shellfish death of living in tideland after housing and chip, through wave carrying, pile up near high water mark.Wudi, Accessories during Binzhou, the Huanghe delta, Zhanhua seashore have become the shell bank formed in the process of land since nearly 5000 or six thousand years, ecotope is unique, with the U.S. easily this Anna state and South America Suriname shell bank and be called the large Chenier in the world three, and be worldwide largest, only the old and new and the shell bank deposited.The ecotope of shell bank uniqueness imparts the distinctive Biological resources in this area, provides good resource for excavating new biocontrol bacteria and developing biological pesticide.
Genus bacillus population is huge, and fecundity is strong, heat-resisting, acid and alkali-resistance, stable in physicochemical property, and antimicrobial spectrum is extensive, occupies critical role, be main biological and ecological methods to prevent plant disease, pests, and erosion bacterial classification, comprise subtilis in biocontrol of plant disease
(Bacillussubtilis), bacillus cereus
(Bacilluscereus), Bacillus licheniformis (
bacilluslicheniformis), bacillus megaterium (
bacillusmegaterium), bacillus amyloliquefaciens (
bacillusamyloliquefaciens), bacillus polymyxa (
bacilluspotymyxa) and bacillus pumilus (
bacilluspumilis) etc.Both at home and abroad the research and development of genus bacillus are put into production practice, made commercial preparation, achieve efficient biocontrol effect, tested crop yield.Accelerate research and development and the application of biological pesticide, meet the needs of China's agricultural development, for ensureing that the healthy diet of people is significant, development prospect is wide.
Summary of the invention
The object of the invention is to for the day by day serious problem of winter jujube ring spot, provide a strain to winter jujube Target spot pathogen have inhibiting genus bacillus (
bacillussp.) ZY produce fermentation culture and generation active substance preparation method and the winter jujube ring spot control in application.
Genus bacillus provided by the invention (
bacillussp.) ZY bacterial strain, be preserved on 04 19th, 2013 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; its preserving number is CGMCCNo.7490, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Genus bacillus (
bacillussp.) ZY strains separation in the beach soil sample of shellfish sand barrier island, comparatively large, circular, the protuberance of bacterium colony, opaque on LB culture medium flat plate; Children's bacterium smooth surface in age, neat in edge, product mucus, cultivate more than 48h bacterium colony surface presentation white fold.Show through Physiology and biochemistry and 16SrRNA sequential analysis, this bacterial strain belongs to genus bacillus.
Use LB liquid nutrient medium to genus bacillus (
bacillussp.) ZY bacterial strain carries out fermentation culture, with transfering loop from the mono-bacterium colony of the dull and stereotyped picking ZY of LB, be inoculated in the test tube containing LB liquid nutrient medium, 37 DEG C of constant-temperature tables are cultivated 18h and are obtained seed liquor, be inoculated in the 1L Erlenmeyer flask containing 400mlLB substratum according to 5% inoculum size, 37 DEG C of 200rpm, cultivate 3.5d and obtain fermented liquid.
Genus bacillus (
bacillussp.) the centrifugal 20min of LB fermentation liquor 10000rpm of ZY bacterial strain, after filtering with microporous membrane, obtain fermented liquid supernatant.Test through face-off and prove, active substance exists with fermented liquid supernatant, and use the active substance in saturated ammonium sulphate method extraction fermented liquid supernatant, this active substance has good inhibition to winter jujube Target spot pathogen.
By qualitative and quantitative analysis, result show genus bacillus (
bacillussp.) ZY bacterial strain produce active substance primarily of carbohydrate and protein composition, content is 67.1% and 20.8% respectively.
This bacterial strain is gram-positive microorganism, rod-short, produces gemma, middle life or end life; Comparatively large, circular, the protuberance of bacterium colony, opaque on LB culture medium flat plate.
The bacterium smooth surface in young age of this bacterial strain, neat in edge, product mucus, cultivating more than 48h bacterium colony is white fold.
Growth temperature 4 ~ 55 DEG C, NaCl tolerance range is 0 ~ 7%.
Maltose, lactose, sorbyl alcohol and fructose can be utilized, do not utilize L-Aspartic acid, creatine.
This bacterial strain catalase experiment, V-P experiment, western Meng Shi test, Starch Hydrolysis test, gelatine liquefication experiment be the positive, methyl red experiment, cellulose hydrolysis experiment, esterase experiment, glucose oxidase fermenting experiment be feminine gender.
Beneficial effect of the present invention is, CGMCCNo.7490 bacterium provided by the invention, is a kind of excellent Biocontrol Bacillus, is effectively extracted the active substance in this bacterial strain fermentation liquor, and this material has good restraining effect to winter jujube Target spot pathogen.
Accompanying drawing explanation
Fig. 1 be genus bacillus (
bacillussp.) ZY bacterial strain fermentation liquor is to the restraining effect of winter jujube ring spot germ;
Fig. 2 be genus bacillus (
bacillussp.) in ZY bacterial strain fermentation liquor active substance to the restraining effect of winter jujube ring spot germ.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further described, so that those skilled in the art more understands the present invention, but does not limit the present invention with this.
Embodiment 1
Genus bacillus provided by the invention (
bacillussp.) fermentation culture of ZY bacterial strain
Fermention medium is LB liquid nutrient medium and (consists of g/L: yeast leaches cream 5g; Peptone 10g; NaCl10g; Distilled water 1000mL; ).With transfering loop from the mono-bacterium colony of the dull and stereotyped picking ZY of LB, be inoculated in the test tube containing LB liquid nutrient medium, 37 DEG C of constant-temperature tables are cultivated 18h and are obtained seed liquor, are inoculated in the 1L Erlenmeyer flask containing 400mLLB substratum according to 5% inoculum size, 37 DEG C of 200rpm, cultivate 3.5d and obtain fermented liquid.
Embodiment 2
Genus bacillus provided by the invention (
bacillussp.) ZY bacterial strain fermentation liquor is to the restraining effect of winter jujube Target spot pathogen
By the tunning in embodiment 4 through the centrifugal 20min of 10000rpm, by the filtering with microporous membrane of 0.22 of supernatant liquor sterilizing μm, obtain fermented liquid supernatant.First punch on the winter jujube ring spot fungi flat board activated with punch tool, bacterium cake is transferred to the dull and stereotyped central authorities of PDA, cultivate 48h, again the Oxford cup after sterilizing is positioned over dull and stereotyped corresponding position, the fermented liquid supernatant drawn after 200 μ l purifying gently joins in the cup of Oxford, and whole process should prevent fluid seepage in the cup of Oxford.Finally, face-off flat board is placed in 28 DEG C of constant incubators, cultivate 12h, result as shown in Figure 1.As can be seen from the figure the growth of fermented liquid supernatant to winter jujube Target spot pathogen of ZY has obvious restraining effect.
Embodiment 3
Genus bacillus provided by the invention (
bacillussp.) extraction of active substance in ZY bacterial strain fermentation liquor
Use saturated ammonium sulphate method to do further extraction the fermented liquid supernatant obtained, the fermented liquid supernatant of the ZY obtained is 370mL.In fermented liquid supernatant, slowly add 190.92g solid ammonium sulfate powder, there is a large amount of flocks in (whole process is carried out in ice bath), and after being placed on 4 DEG C of refrigerator overnight, the centrifugal 20min of 10000r/min, abandons supernatant, be precipitated thing.Throw out is first used a small amount of distilled water flushing, then uses appropriate distilled water back dissolving, dialysis postlyophilization, obtain 0.18g active substance dry powder, yield is 0.36g/L.
Embodiment 4
Genus bacillus provided by the invention (
bacillussp.) active substance and winter jujube ring spot stand facing each other in ZY bacterial strain fermentation liquor result
Be dissolved in distilled water by the active substance obtained in embodiment 3, concentration is 10mg/ml, and carry out face-off experiment (with embodiment 2), face-off flat board is placed in 28 DEG C of constant temperature culture and spends the night, result as shown in Figure 2.Show the active substance being effectively extracted bacterial strain ZY generation, have good restraining effect to winter jujube ring spot.
Embodiment 5
Genus bacillus provided by the invention (
bacillussp.) qualitative and quantitative analysis of active substance in ZY bacterial strain fermentation liquor
Employing H2SO4-anthrone method, triketohydrindene hydrate colour developing, ammonium molybdate-perchloric acid development process have carried out qualitative analysis to the active substance obtained in embodiment 3, result shows in activeconstituents containing carbohydrate, protein and lipid material, quantitative analysis shows that the content of three kinds of materials is respectively 67.1%, 20.8% and 6.2%, main component is sugar and protein, and the active substance that therefore bacterial strain ZY produces is a kind of glycoprotein analog material.
Embodiment 6
Genus bacillus provided by the invention (
bacillussp.) ZY bacterial strain is in the application of No. 4 Tanakas in winter jujube experimental plot.
Winter jujube ring spot can be prevented and treated by fermented liquid, detailed process is: ferment (fermentation condition is with embodiment 1) to ZY, fermented liquid dilutes 50 times of sprayings, the 1st spray pesticide after jujube tree fallen flowers, later interval 15 ~ 20d dispenser 1 time, totally 4 times, whole dispenser breeding time 5 times, its biocontrol effect can reach 75.6%.
Physico-chemical property-the thermal stability determination of above-mentioned bacterial strains fermented liquid
ZY bacterial strain fermentation liquor supernatant (with example 4) is processed 30min respectively at 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 100 DEG C, with the winter jujube ring spot germ for indicator, compare without heat treated fermented liquid, measure active containing toxic medium method (with example 5), ZY fermented liquid has stronger thermostability.50-80 DEG C respectively processes 30min, and bacteriostatic activity all maintains more than 85% of contrast, 100 DEG C of process 60min, and active is 79.6% of contrast.
Physico-chemical property-pH the Stability Determination of bacterial strain fermentation liquor
Used by ZY bacterial strain fermentation liquor supernatant (with example 4) 1mol/LHCl and 1mol/LNaOH process to pH value 4,5,6,7,8,9,10 respectively, static 2h, then pH is recalled to the natural ph of fermented liquid, with the winter jujube ring spot germ for indicator, fermented liquid without acid-alkali treatment compares, and measures active containing toxic medium method (with example 5).Between pH5 ~ pH10, have certain bacteriostasis rate, along with the rising of pH, bacteriostasis rate is in downtrending gradually, but difference with insignificance, when pH is 6, fungistatic effect is best, reaches 72%.
Claims (9)
1. a genus bacillus (
bacillussp.) ZY bacterial strain, on April 19th, 2013 be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number is CGMCCNo.7490;
Described genus bacillus (
bacillussp.) ZY bacterial strain, LB liquid nutrient medium is used to carry out fermentation culture, with transfering loop from the mono-bacterium colony of the dull and stereotyped picking ZY of LB, be inoculated in the test tube containing LB liquid nutrient medium, 37 DEG C of constant-temperature tables are cultivated 18h and are obtained seed liquor, be inoculated in the 1L Erlenmeyer flask containing 400mlLB substratum according to 5% inoculum size, 37 DEG C of 200rpm, cultivate 3.5d and obtain fermented liquid.
2. genus bacillus as claimed in claim 1 (
bacillussp.) ZY bacterial strain, it is characterized in that described fermentation liquor 10000rpm centrifugal 20min, after filtering with microporous membrane, obtains fermented liquid supernatant liquid; Active substance is there is in described supernatant liquor.
3. genus bacillus as claimed in claim 1 (
bacillussp.) ZY bacterial strain, it is characterized in that, use the active substance in saturated ammonium sulphate method extraction fermented liquid supernatant, this active substance is suppressing the application in winter jujube Target spot pathogen.
4. genus bacillus as claimed in claim 3 (
bacillussp.) ZY bacterial strain, it is characterized in that, in described fermented liquid, active substance comprises by carbohydrate and protein, and content is 67.1% and 20.8% respectively.
5. genus bacillus as claimed in claim 4 (
bacillussp.) ZY bacterial strain, this bacterial strain is gram-positive microorganism, rod-short, produces gemma, middle life or end life; Comparatively large, circular, the protuberance of bacterium colony, opaque on LB culture medium flat plate.
6. genus bacillus as claimed in claim 5 (
bacillussp.) ZY bacterial strain, the bacterium smooth surface in young age of this bacterial strain, neat in edge, product mucus, cultivating more than 48h bacterium colony is white fold.
7. genus bacillus as claimed in claim 6 (
bacillussp.) ZY bacterial strain, the growth temperature of this bacterial strain 4 ~ 55 DEG C, NaCl tolerance range is 0 ~ 7%.
8. genus bacillus as claimed in claim 7 (
bacillussp.) ZY bacterial strain, this bacterial strain can utilize maltose, lactose, sorbyl alcohol and fructose, does not utilize L-Aspartic acid, creatine.
9. genus bacillus as claimed in claim 8 (
bacillussp.) ZY bacterial strain, this bacterial strain catalase experiment, V-P experiment, western Meng Shi test, Starch Hydrolysis test, gelatine liquefication experiment be the positive, methyl red experiment, cellulose hydrolysis experiment, esterase experiment, glucose oxidase fermenting experiment be feminine gender.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828839A (en) * | 2017-10-31 | 2018-03-23 | 李爽 | A kind of production method of bacillus production immunomodulatory peptides |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828839A (en) * | 2017-10-31 | 2018-03-23 | 李爽 | A kind of production method of bacillus production immunomodulatory peptides |
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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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