Sang Puxun streptomycete bacterial strains, its separation method and applicationTechnical 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 methodWith 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 pestsTo 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 biologyThe 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 environmentAnd 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 warpJi 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 classStreptomyces griseoviridis and Streptomyces lydicus, streptomyces are expected to turn into after bacillusWith 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 spectrumExtensively, the Sang Puxun streptomycete bacterial strains and its separation method and cultural method colonized rapidly in soil and plant physical efficiency, and pass through thisSang Puxun Streptomyces cultures obtained by cultural method, and the Sang Puxun streptomycete bacterial strains and Sang Puxun Streptomyces cultures shouldWith 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 quotationType 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 cultureTime lengthening, mycelia darken.(2) protein peptone culture basal growth, mycelia khaki, bacterium colony and culture medium junction are being examinedThere is orange pigment.(3) maize powder medium growth mycelia it is light yellow, after gradually become buff, be in Liquid CultureLight 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 tableNucleotide sequence in SEQ ID No.1.Carried out according to sequencing result and Genbank Streptomyces 16S rRNA gene orders sameSource 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 bagInclude：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 supernatantLiquid；S2, Sang Puxun streptomycete bacterial strains are isolated and purified using plate streak：Supernatant obtained by step S1 is diluted, using differenceThe 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 colonyPurify 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 useOese is diluted to obtain the more individual cells being independently distributed on plating medium surface by sectional streak, raw after cultureLength 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 byIndividual cells breeding, can using separate repeatedly repeatedly obtain it is purebred.Its principle is to train microbiological specimens in solidFoster 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 oneThe 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 separatedArea, 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 bigThe 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 boardC ＞ 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 upperSang 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 kindIn 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 hundredPoint 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 daysZymotic fluid；Wherein, preservation isolation medium is Gause I culture medium, and the composition of the Gause I culture medium includes：It is solvableProperty starch, KNO3、NaCl、K2HPO4、FeSO4、MgSO4And water, pH value 7.4-7.6；Seed culture medium is trained to examine protein peptoneBase is supported, the composition for examining protein peptone culture medium includes：Sucrose, peptone, FeSO4、MgSO4、KCl、K2HPO4And water；HairFerment culture medium is maize powder medium, and the composition of the maize powder medium includes：Corn flour, sucrose, peptone, starch, fermentFemale 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 diseasesApplication.
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 trainingSupport thing.
The Sang Puxun streptomycetes (Streptomyces sampsonii) of gained of the invention belong to streptomyces, by interiorBiological activity determination finds that it has inhibitory action to the several plant pathogen of gibberella saubinetii, rice banded sclerotial blight etc. ten.And foundThe 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 onlyPlurality 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 withEmbodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only explainingThe 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 preservationSampsonii) SY-FX-31, is preserved in China typical culture collection center, and deposit number is：CCTCC M 2014516, shouldBacterial strain 16S rRNA gene orders belong to streptomyces as shown in sequence table SEQ ID No.1, can not only prevent and treat various plantsDisease, 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 applicationThe 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-inIn 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, takesSupernatant.S2, Sang Puxun streptomycete bacterial strains are isolated and purified using plate streak：It is specially that supernatant obtained by step S1 is diluteRelease, specific dilution process can use plus the dilution of sterilized water vortex mixed；It is coated on using the dilution of different diluted concentrationsOn 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 listBacterium 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 prolongsLong, 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 respectivelyIt 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 mulberryGeneral inferior streptomycete bacterial strain activates 12-24h in storage medium at 25-33 DEG C；S2, the bacterial strain after activation is trained in seedSupport 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 sedimentPowder, KNO3、NaCl、K2HPO4、FeSO4、MgSO4And water, pH value 7.4-7.6.It is specific preferred, the composition of Gause I culture mediumIncluding：Soluble starch 5-40g, KNO30.1-5g, NaCl 0.1-3g, K2HPO4·3H2O 0.1-3g, FeSO4·7H2O0.01-1g, 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：SugarcaneSugar, 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, K2HPO40.1-5gAnd 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 beautifulGround 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-sBacteria 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, cucumberGrey 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's30min 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 successivelyRelease 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 3Parallel, the single bacterium colony on 28 DEG C of culture 2-7d, picking culture medium carries out plate streaking purifying, obtains Sang Puxun streptomycete bacteriumStrain.
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 SEQID No.1 nucleotide sequences.Homology ratio is carried out according to sequencing result and Genbank Streptomyces 16S rRNA gene ordersCompared 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 starch20g, 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 trainedSupport 2 days, obtain seed liquor；Then gained seed liquor is inoculated in fermentation medium (corn flour with 5% (v/v) inoculative proportion20g, sucrose 10g, peptone 2g, starch 5g, yeast extract 2g, NaCl 2g, K2HPO40.5g, MgSO40.5g, CaCO31g, waterIn 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 coolingInoculation 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 plateSupport 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 withIt 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 antibacterialRate.
Sang Puxun streptomycetes (Streptomyces sampsonii) SY-FX-31 is to the inhibitory action for trying disease fungusAs 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 tableThe 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 necessarilyConcentration carries out spraying treatment, clear water control, potted plant seedling naturally dry after chemicals treatment on crops sprayer.24h is followed byKind bacterium of downy mildew of cucumber spore suspension is placed in phjytotron 24h (temperature：23 DEG C, humidity：90%) moved to after in greenhouse justOften 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 denseDegree carries out spraying treatment, clear water control, potted plant seedling naturally dry after chemicals treatment on crops sprayer.Planted after 24hThe 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 illSituation.
7d investigation prevention effects are cultivated in greenhouse, grade scale performs National Standard of the People's Republic of China《Agricultural chemicals fieldBetween 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 inventionSampsonii) 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 showThe description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example descriptionPoint is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term notIdentical embodiment or example must be directed to.Moreover, specific features, structure, material or the feature of description can be with officeCombined in an appropriate manner in one or more embodiments or example.In addition, in the case of not conflicting, the skill of this areaArt personnel can be tied the different embodiments or example and the feature of different embodiments or example described in this specificationClose and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is exampleProperty, 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-mentionedEmbodiment is changed, changed, replacing and modification.