CN104388356B - Sang Puxun streptomycete bacterial strains, its separation method and application - Google Patents
Sang Puxun streptomycete bacterial strains, its separation method and application Download PDFInfo
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- CN104388356B CN104388356B CN201410705973.5A CN201410705973A CN104388356B CN 104388356 B CN104388356 B CN 104388356B CN 201410705973 A CN201410705973 A CN 201410705973A CN 104388356 B CN104388356 B CN 104388356B
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- 241001655322 Streptomycetales Species 0.000 title claims abstract description 48
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 41
- 238000000926 separation method Methods 0.000 title abstract description 11
- 241000187747 Streptomyces Species 0.000 claims abstract description 21
- 201000010099 disease Diseases 0.000 claims abstract description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 17
- 241000142906 Streptomyces sampsonii Species 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 13
- 108020004465 16S ribosomal RNA Proteins 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 239000003905 agrochemical Substances 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 6
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 239000002609 medium Substances 0.000 claims description 24
- 108010080698 Peptones Proteins 0.000 claims description 21
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 21
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 20
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- 235000019698 starch Nutrition 0.000 claims description 14
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 11
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- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims description 8
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 8
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 8
- 229910052564 epsomite Inorganic materials 0.000 claims description 7
- 229910052603 melanterite Inorganic materials 0.000 claims description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 5
- 240000003768 Solanum lycopersicum Species 0.000 claims description 5
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- 235000005822 corn Nutrition 0.000 claims description 5
- 235000013312 flour Nutrition 0.000 claims description 5
- 238000011218 seed culture Methods 0.000 claims description 5
- 241000233679 Peronosporaceae Species 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 241000223218 Fusarium Species 0.000 claims description 3
- 229920000742 Cotton Polymers 0.000 claims description 2
- 239000006071 cream Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 239000000575 pesticide Substances 0.000 abstract description 11
- 239000002689 soil Substances 0.000 abstract description 10
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- 241000894006 Bacteria Species 0.000 description 16
- 238000010790 dilution Methods 0.000 description 8
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- 241000589516 Pseudomonas Species 0.000 description 6
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- 241000233866 Fungi Species 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
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- 241001530056 Athelia rolfsii Species 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000000443 biocontrol Effects 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000005352 clarification Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000006353 environmental stress Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 240000008574 Capsicum frutescens Species 0.000 description 1
- 244000241257 Cucumis melo Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
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- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 241001558929 Sclerotium <basidiomycota> Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 239000005838 Streptomyces K61 (formerly S. griseoviridis) Substances 0.000 description 1
- 241000191251 Streptomyces griseoviridis Species 0.000 description 1
- 241000218483 Streptomyces lydicus Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
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- 238000009395 breeding Methods 0.000 description 1
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- 239000001390 capsicum minimum Substances 0.000 description 1
- 238000000205 computational method Methods 0.000 description 1
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- 238000001816 cooling Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000001053 orange pigment Substances 0.000 description 1
- 235000010603 pastilles Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
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- 239000007787 solid Substances 0.000 description 1
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- 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
- C12R2001/465—Streptomyces
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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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- 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
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Abstract
The present invention relates to microbial pesticide technical field, a kind of Sang Puxun streptomycete bacterial strains and its separation method and cultural method are specifically provided, and the Sang Puxun Streptomyces cultures as obtained by the cultural method, and application and the agricultural chemicals containing them of the Sang Puxun streptomycete bacterial strains and Sang Puxun Streptomyces cultures.The Sang Puxun streptomycete bacterial strains, the entitled Sang Puxun streptomycetes of preservation (Streptomyces sampsonii) SY FX 31, are preserved in China typical culture collection center, deposit number is：CCTCC M2014516, bacterial strain 16S rRNA gene orders can prevent and treat plurality of plant diseases as shown in sequence table SEQ ID No.1, and the speed of growth is fast, sporulation quantity is big, antimicrobial spectrum is wide, is colonized rapidly in soil and plant physical efficiency.
Description
Technical field
The present invention relates to microbial pesticide technical field, and in particular to a kind of Sang Puxun streptomycete bacterial strains and its separation method
With application.
Background technology
Microbial pesticide is not likely to produce drug-fast characteristic because its is green, in terms of controlling crop diseases and insect pests
To more and more extensive attention and application.The promotion and application technology of microbial pesticide is just gradually tending to be ripe, and country is in biology
The policy support dynamics of prevention and control field increases year by year, and the use of microbial pesticide can overcome pollution of the chemical pesticide to ecological environment
And the residual in agricultural byproducts Pesticides is reduced, the quality and price of agricultural byproducts are lifted, not only with environmental benefit but also with warp
Ji benefit.
At present, the major microorganisms pesticide species that in the market is promoted concentrate on bacillus and pseudomonas.Strepto-
Pseudomonas is a kind of microorganism Pseudomonas that can produce antibiotic, is in the kind that US and European is registered as agricultural fungicidal class
Streptomyces griseoviridis and Streptomyces lydicus, streptomyces are expected to turn into after bacillus
With it is another kind of in the wide variety of microbial pesticide of field of biological control after pseudomonas.
The content of the invention
The invention provides it is a kind of it is new can prevent and treat plurality of plant diseases, and the speed of growth is fast, sporulation quantity is big, antimicrobial spectrum
Extensively, the Sang Puxun streptomycete bacterial strains and its separation method and cultural method colonized rapidly in soil and plant physical efficiency, and pass through this
Sang Puxun Streptomyces cultures obtained by cultural method, and the Sang Puxun streptomycete bacterial strains and Sang Puxun Streptomyces cultures should
With with the agricultural chemicals containing them.
The first aspect of the present invention provides a kind of Sang Puxun streptomycete bacterial strains (Streptomyces sampsonii) SY-
FX-31, the entitled Sang Puxun streptomycetes SY-FX-31 of preservation (Streptomyces sampsonii), depositary institution：Chinese allusion quotation
Type culture collection, depositary institution address：Wuhan University's Life Science College, preservation date are：On October 27th, 2014,
Deposit number is：CCTCC M 2014516, bacterial strain 16S rRNA gene orders are as shown in sequence table SEQ ID No.1.
The bacterial strain has following biological characteristics：(1) in Gause I culture basal growth, mycelia is light yellow, with culture
Time lengthening, mycelia darken.(2) protein peptone culture basal growth, mycelia khaki, bacterium colony and culture medium junction are being examined
There is orange pigment.(3) maize powder medium growth mycelia it is light yellow, after gradually become buff, be in Liquid Culture
Light yellow hypha body.
Its 16S rRNA gene order sequencing result shows that the 16S rRNA gene orders of SY-FX-31 bacterial strains are sequence table
Nucleotide sequence in SEQ ID No.1.Carried out according to sequencing result and Genbank Streptomyces 16S rRNA gene orders same
Source property compares, the results showed that Sang Puxun streptomycete of the SY-FX-31 Pseudomonas in streptomyces (Streptomyces)
(Streptomyces sampsonii)。
The second aspect of the present invention provides the separation method for separating above-mentioned Sang Puxun streptomycete bacterial strains, its step bag
Include：S1, soil is gathered from river bed, add sterilized water soil is mixed with sterilized water after vibrating and scatter, it is static to make its clarification, take supernatant
Liquid；S2, Sang Puxun streptomycete bacterial strains are isolated and purified using plate streak：Supernatant obtained by step S1 is diluted, using difference
The dilution of diluted concentration is coated on Gause I culture medium flat plate after 10-40 DEG C is cultivated 2-7d, the separation of picking single bacterium colony
Purify get Sang Puxun streptomycete bacterial strains；Wherein, the composition of Gause I culture medium includes：Soluble starch, KNO3、NaCl、
K2HPO4、FeSO4、MgSO4And water, pH value 7.4-7.6.
Wherein, plate streak refers to the different cells in microorganism mixed in together or same micropopulation to use
Oese is diluted to obtain the more individual cells being independently distributed on plating medium surface by sectional streak, raw after culture
Length is multiplied into single bacterium colony, generally this single bacterium colony as the purebred of microorganism to be separated.Sometimes this single bacterium colony not all by
Individual cells breeding, can using separate repeatedly repeatedly obtain it is purebred.Its principle is to train microbiological specimens in solid
Foster primary surface repeatedly makees " by putting to line " dilution and reaches separation purpose.The form of line has a variety of, is specifically as follows one
The cell that individual flat board is divided into four different areas is rule, and the firstth area (A areas) area is minimum, the bacterium source as bacterium to be separated
Area, second and the 3rd area (B, C area) be the transition region diluted step by step, the 4th area (D areas) is then key area, and Shi Gai areas occur big
The single bacterium colony purebred use for selection of amount.In order to obtain more typical single bacterium colony, the distribution of four area's areas should be D ＞ on flat board
C ＞ B ＞ A.
The third aspect of the present invention provides the cultural method of above-mentioned Sang Puxun streptomycete bacterial strains, and its step includes S1, will be upper
Sang Puxun streptomycete bacterial strains are stated in storage medium, 12-24h is activated at 25-33 DEG C；S2, by the bacterial strain after activation in kind
In sub- culture medium, in 25-33 DEG C, 100-150r/min is cultivated 1-2 days, obtains seed liquor；S3, by gained seed liquor with volume hundred
Point content 4-8% inoculative proportions are inoculated in fermentation medium, and at 25-33 DEG C, 100-150r/min shaken cultivations obtain for 2-4 days
Zymotic fluid；Wherein, preservation isolation medium is Gause I culture medium, and the composition of the Gause I culture medium includes：It is solvable
Property starch, KNO3、NaCl、K2HPO4、FeSO4、MgSO4And water, pH value 7.4-7.6；Seed culture medium is trained to examine protein peptone
Base is supported, the composition for examining protein peptone culture medium includes：Sucrose, peptone, FeSO4、MgSO4、KCl、K2HPO4And water；Hair
Ferment culture medium is maize powder medium, and the composition of the maize powder medium includes：Corn flour, sucrose, peptone, starch, ferment
Female cream, NaCl, K2HPO4、MgSO4、CaCO3And water.
The fourth aspect of the present invention provides a kind of Sang Puxun Streptomyces cultures, is made by above-mentioned cultural method.
The fifth aspect of the present invention provides above-mentioned Sang Puxun streptomycete bacterial strains and/or its culture in controlling plant diseases
Application.
The sixth aspect of the present invention provides a kind of agricultural chemicals, and the agricultural chemicals includes above-mentioned Sang Puxun streptomycete bacterial strains and/or its training
Support thing.
The Sang Puxun streptomycetes (Streptomyces sampsonii) of gained of the invention belong to streptomyces, by interior
Biological activity determination finds that it has inhibitory action to the several plant pathogen of gibberella saubinetii, rice banded sclerotial blight etc. ten.And found
The Sang Puxun streptomycete speeds of growth it is fast, sporulation quantity is big, antimicrobial spectrum is wide, colonized rapidly in soil and plant physical efficiency.Can not only
Plurality of plant diseases is prevented and treated, and plant resistance to environment stress can be improved, positive role is respectively provided with to improving crop yield and lifting quality,
It is a kind of biocontrol strains for having application prospect.
Embodiment
In order that technical problem solved by the invention, technical scheme and beneficial effect are more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only explaining
The present invention, it is not intended to limit the present invention.
The invention provides a kind of Sang Puxun streptomycete bacterial strains, the entitled Sang Puxun streptomycetes (Streptomyces of preservation
Sampsonii) SY-FX-31, is preserved in China typical culture collection center, and deposit number is：CCTCC M 2014516, should
Bacterial strain 16S rRNA gene orders belong to streptomyces as shown in sequence table SEQ ID No.1, can not only prevent and treat various plants
Disease, and plant resistance to environment stress can be improved, positive role is respectively provided with to improving crop yield and lifting quality, is that one kind has application
The biocontrol strains of prospect.
Invention also provides the separation method for separating above-mentioned Sang Puxun streptomycete bacterial strains, its step includes：S1,
Gather soil from river bed, add sterilized water to make soil mix with sterilized water to scatter after vibrating, be specifically as follows soil is added it is built-in
In the container of sterilized water and bead, soil is set to scatter in vibration a period of time on shaking table, vibration end is static to make its clarification, takes
Supernatant.S2, Sang Puxun streptomycete bacterial strains are isolated and purified using plate streak：It is specially that supernatant obtained by step S1 is dilute
Release, specific dilution process can use plus the dilution of sterilized water vortex mixed；It is coated on using the dilution of different diluted concentrations
On Gause I culture medium flat plate, be specifically as follows each concentration be coated with 3 it is parallel, after 10-40 DEG C is cultivated 2-7d, picking list
Bacterium colony isolates and purifies get Sang Puxun streptomycete bacterial strains.Wherein, the composition of Gause I culture medium includes：Soluble starch, KNO3、
NaCl、K2HPO4、FeSO4、MgSO4And water, pH value 7.4-7.6.
Wherein, Sang Puxun streptomycete bacterial strains are in Gause I culture basal growth, and mycelia is light yellow, as incubation time prolongs
Long, mycelia darkens.
It is preferred that the composition of Gause I culture medium includes：Soluble starch 5-40g, KNO30.1-5g, NaCl 0.1-
3g, K2HPO4·3H2O 0.1-3g, FeSO4·7H2O 0.01-1g, MgSO4·7H2O 0.1-3g and water 1-10L.
It is preferred that three concentration of dilution, the concentration of dilution includes supernatant diluting 10-2、10-3、10-4Times, it is uniform respectively
It is coated on Gause I culture medium flat plate.
Invention also provides the cultural method of above-mentioned Sang Puxun streptomycete bacterial strains, its step includes S1, by above-mentioned mulberry
General inferior streptomycete bacterial strain activates 12-24h in storage medium at 25-33 DEG C；S2, the bacterial strain after activation is trained in seed
Support in base, in 25-33 DEG C, 100-150r/min is cultivated 1-2 days, obtains seed liquor；S3, by gained seed liquor with 4-8% (v/v)
Inoculative proportion is inoculated in fermentation medium, and at 25-33 DEG C, 100-150r/min shaken cultivations obtain zymotic fluid in 2-4 days.
Wherein, storage medium is Gause I culture medium, and the composition of the Gause I culture medium includes：Solubility is formed sediment
Powder, KNO3、NaCl、K2HPO4、FeSO4、MgSO4And water, pH value 7.4-7.6.It is specific preferred, the composition of Gause I culture medium
Including：Soluble starch 5-40g, KNO30.1-5g, NaCl 0.1-3g, K2HPO4·3H2O 0.1-3g, FeSO4·7H2O
0.01-1g, MgSO4·7H2O 0.1-3g and water 1-10L.
Wherein, to examine protein peptone culture medium, the composition for examining protein peptone culture medium includes seed culture medium：Sugarcane
Sugar, peptone, FeSO4、MgSO4、KCl、K2HPO4And water；It is preferred that examining the composition of protein peptone culture medium includes：Sucrose 5-
50g, peptone 1-15g, FeSO4·7H2O 0.01-1g, MgSO4·7H2O 0.1-5g, KCl 0.1-5g, K2HPO40.1-5g
And water 1-10L.
Wherein, fermentation medium is maize powder medium, and the composition of the maize powder medium includes：Corn flour, sucrose,
Peptone, starch, yeast extract, NaCl, K2HPO4、MgSO4、CaCO3And water；It is preferred that the composition of maize powder medium includes：It is beautiful
Ground rice 5-50g, sucrose 1-20g, peptone 1-10g, starch 1-20g, yeast extract 1-10g, NaCl 1-20g, K2HPO40.1-5g,
MgSO40.1-5g, CaCO30.1-5g and water 1-10L.
Invention also provides a kind of Sang Puxun Streptomyces cultures, it is made by above-mentioned cultural method.
Invention also provides the application of above-mentioned Sang Puxun streptomycete bacterial strains and/or its culture, the Sang Puxun strepto-s
Bacteria strain and/or its culture are used for controlling plant diseases.
Wherein, plant disease includes the corruption of white muskmelon melon, capsicum is withered, tomato early epidemic, cucumber anthracnose, cotton yellow wither, cucumber
Grey mold, rice bakanae disease, tomato green grass or young crops are withered, rice banded sclerotial blight, the black shin of tobacco, Root Rot of Wheat, rice rice blast, gibberella saubinetii, rape sclerotium,
One or more in soybean root rot, tomato gray mould, cucumber foxiness or cucumber downy mildew, can prevent a variety of pest and disease damages, and antimicrobial spectrum is wide,
Application prospect is wide.
Invention also provides a kind of agricultural chemicals, the agricultural chemicals includes above-mentioned Sang Puxun streptomycete bacterial strains and/or its culture,
For microbial pesticide, it can obtain or carry out formulation for zymotic fluid obtained above directly dilution and obtain.
The present invention is further described below in conjunction with specific embodiment.
Embodiment 1
Sang Puxun streptomycetes (Streptomyces sampsonii) SY-FX-31 separation and identification
Sang Puxun streptomycetes 1. (Streptomyces sampsonii) SY-FX-31 separation
Soil 10g is gathered from river bed, is added in the triangular flask of built-in 100mL sterilized waters and bead, in 120r/min's
30min is vibrated on shaking table, vibration stands 1h after terminating, and takes supernatant 0.5mL to add in 4.5mL sterilized waters, vortex mixed is dilute successively
Release 10-2、10-3、10-4Times, take 150 μ L dilutions to be coated on Gause I culture medium flat plate respectively, each concentration is coated with 3
Parallel, the single bacterium colony on 28 DEG C of culture 2-7d, picking culture medium carries out plate streaking purifying, obtains Sang Puxun streptomycete bacterium
Strain.
Sang Puxun streptomycetes 2. (Streptomyces sampsonii) SY-FX-31 identification
16S rRNA gene order sequencing results show that the 16S rRNA gene orders of SY-FX-31 bacterial strains are sequence table SEQ
ID No.1 nucleotide sequences.Homology ratio is carried out according to sequencing result and Genbank Streptomyces 16S rRNA gene orders
Compared with, the results showed that Sang Puxun streptomycete (Streptomyces of the SY-FX-31 Pseudomonas in streptomyces (Streptomyces)
sampsonii)。
Embodiment 2
Sang Puxun streptomycetes (Streptomyces sampsonii) SY-FX-31 is tested disease fungus isolated activity
By Sang Puxun streptomycetes (Streptomyces sampsonii) SY-FX-31 in storage medium (soluble starch
20g, KNO31g, NaCl 0.5g, K2HPO4·3H2O 0.5g, FeSO4·7H2O 0.01g, MgSO4·7H2O 0.5g, water 1L,
PH7.4-7.6 in), at 25 DEG C activate 20h after, by the bacterial strain after activation in seed culture medium (sucrose 30g, peptone 5g,
FeSO4·7H2O 0.01g, MgSO4·7H2O 0.5g, KCl 0.5g, K2HPO41g, water 1L) in, in 25 DEG C, 150r/min is trained
Support 2 days, obtain seed liquor；Then gained seed liquor is inoculated in fermentation medium (corn flour with 5% (v/v) inoculative proportion
20g, sucrose 10g, peptone 2g, starch 5g, yeast extract 2g, NaCl 2g, K2HPO40.5g, MgSO40.5g, CaCO31g, water
In 1L), at 25 DEG C, 150r/min shaken cultivations obtain zymotic fluid in 2 days.
Zymotic fluid is configured to certain density mother liquor, using containing toxic medium method, PDA culture medium (200 grams of potato,
20 grams of glucose, 20 grams of agar, water 1L) in add the mother liquor for preparing in advance, pastille culture medium is made, in sterile bar after cooling
Inoculation is used as control for examination pathogen by the use of the PDA culture medium for adding sterilized water under part.For trying sick fungi training is activated in PDA plate
Support and form bacterium colony, bacteria cake is produced with card punch (5mm), mycelia is connect to bacterium down in PDA plate center.The flat board that will be inoculated with
It is placed in 28 DEG C of biochemical cultivation cases after cultivating 4-7 days and investigates, colony growth diameter is measured using crossing method, and calculate antibacterial
Rate.
Sang Puxun streptomycetes (Streptomyces sampsonii) SY-FX-31 is to the inhibitory action for trying disease fungus
As a result it is as shown in table 1.
Table 1
SY-FX-31 pairs of Sang Puxun streptomycetes (Streptomyces sampsonii) of the invention as can be seen from the above table
The inhibitory action of disease fungus is good.
Embodiment 3
Sang Puxun streptomycetes (Streptomyces sampsonii) SY-FX-31 living body biological determination of activity
The potted plant cucumber seedling (heart stage of 1 leaf 1) of growth selection neat and consistent, zymotic fluid obtained above is configured to necessarily
Concentration carries out spraying treatment, clear water control, potted plant seedling naturally dry after chemicals treatment on crops sprayer.24h is followed by
Kind bacterium of downy mildew of cucumber spore suspension is placed in phjytotron 24h (temperature：23 DEG C, humidity：90%) moved to after in greenhouse just
Often management.Greenhouse experiment：22-30 DEG C of temperature, humidity 40-60%.
The pot rice seedling (3 leaf phase) of growth selection neat and consistent, zymotic fluid obtained above is configured to certain dense
Degree carries out spraying treatment, clear water control, potted plant seedling naturally dry after chemicals treatment on crops sprayer.Planted after 24h
The strain withered mycelia block of bottom Inoculated Rice line, is placed in 5-7 days (temperature of phjytotron：28 DEG C, humidity：80%), the observation period falls ill
Situation.
7d investigation prevention effects are cultivated in greenhouse, grade scale performs National Standard of the People's Republic of China《Agricultural chemicals field
Between test of pesticide effectiveness criterion (one)》, prevention effect is calculated with disease index.As a result it is as shown in table 2.
Drug effect computational methods
Table 2
Preventing and treating result, which is tested, from live body can be seen that Sang Puxun streptomycetes (Streptomyces of the invention
Sampsonii) SY-FX-31 zymotic fluid has certain prevention effect to rice banded sclerotial blight, cucumber downy mildew.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.Moreover, specific features, structure, material or the feature of description can be with office
Combined in an appropriate manner in one or more embodiments or example.In addition, in the case of not conflicting, the skill of this area
Art personnel can be tied the different embodiments or example and the feature of different embodiments or example described in this specification
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changed, replacing and modification.
Claims (6)
1. a kind of Sang Puxun streptomycete bacterial strains, the entitled Sang Puxun streptomycetes of preservation (Streptomyces sampsonii)SY-
FX-31, is preserved in China typical culture collection center, and deposit number is：CCTCC M 2014516, the bacterial strain 16S rRNA
Gene order is as shown in sequence table SEQ ID No.1.
2. a kind of cultural method of Sang Puxun streptomycete bacterial strains as claimed in claim 1, it is characterised in that including step：
S1, by the Sang Puxun streptomycete bacterial strains described in claim 1 in storage medium, 12-24h is activated at 25-33 DEG C；
S2, by the bacterial strain after activation in seed culture medium, in 25-33 DEG C, 100-150r/min is cultivated 1-2 days, obtains seed
Liquid；
S3, gained seed liquor is inoculated in fermentation medium with volumn concentration 4-8% inoculative proportions, at 25-33 DEG C,
100-150r/min shaken cultivations obtain zymotic fluid in 2-4 days；
The storage medium is Gause I culture medium, and the composition of the Gause I culture medium includes：Soluble starch,
KNO3、NaCl、K2HPO4、FeSO4、MgSO4And water, pH value 7.4-7.6；
To examine protein peptone culture medium, the composition for examining protein peptone culture medium includes the seed culture medium：Sucrose, albumen
Peptone, FeSO4、MgSO4、KCl、K2HPO4And water；
The fermentation medium is maize powder medium, and the composition of the maize powder medium includes：Corn flour, sucrose, albumen
Peptone, starch, yeast extract, NaCl, K2HPO4、MgSO4、CaCO3And water.
3. cultural method according to claim 2, it is characterised in that
The composition of the Gause I culture medium includes：Soluble starch 5-40g, KNO30.1-5g, NaCl 0.1-3g,
K2HPO4·3H2O 0.1-3g, FeSO4·7H2O 0.01-1g, MgSO4·7H2O 0.1-3g and water 1-10L；
The composition for examining protein peptone culture medium includes：Sucrose 5-50g, peptone 1-15g, FeSO4·7H2O 0.01-1g,
MgSO4·7H2O 0.1-5g, KCl 0.1-5g, K2HPO40.1-5g and water 1-10L；
The composition of the maize powder medium includes：Corn flour 5-50g, sucrose 1-20g, peptone 1-10g, starch 1-20g, ferment
Female cream 1-10g, NaCl 1-20g, K2HPO40.1-5g, MgSO40.1-5g, CaCO30.1-5g and water 1-10L.
4. a kind of Sang Puxun Streptomyces cultures, are made as the cultural method described in Claims 2 or 3.
5. the Sang Puxun Streptomyces cultures described in Sang Puxun streptomycete bacterial strains and/or claim 4 described in claim 1 exist
Application in controlling plant diseases；The plant disease include tomato early epidemic, cotton yellow wither, cucumber grey mold, rice bakanae disease, rice
Line is withered, the one or more in gibberella saubinetii, tomato gray mould or cucumber downy mildew.
6. a kind of agricultural chemicals, it is characterised in that the agricultural chemicals includes the Sang Puxun streptomycete bacterial strains and/or power described in claim 1
Profit requires the Sang Puxun Streptomyces cultures described in 4.
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| US10136646B2 (en) | 2013-06-26 | 2018-11-27 | Indigo Ag, Inc. | Agricultural endophyte-plant compositions, and methods of use |
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| EP3393225A4 (en) | 2015-12-21 | 2019-11-06 | Indigo AG, Inc. | ENDOPHYTIC COMPOSITIONS AND METHODS FOR ENHANCING PLANT CHARACTERS IN PLANTS OF AGRONOMIC IMPORTANCE |
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