CN106434493A - Strain of biocontrol Streptomyces and application thereof - Google Patents
Strain of biocontrol Streptomyces and application thereof Download PDFInfo
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- CN106434493A CN106434493A CN201611091581.XA CN201611091581A CN106434493A CN 106434493 A CN106434493 A CN 106434493A CN 201611091581 A CN201611091581 A CN 201611091581A CN 106434493 A CN106434493 A CN 106434493A
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
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- C12N1/205—Bacterial isolates
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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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
Abstract
The invention discloses a strain of Streptomyces and application thereof. The biocontrol Streptomyces is blastmycin Streptomyces and its strain number is JZB130180, which is registered in the general microbiological center of China Committee for Culture Collection of Microorganisms with CGMCC No. 13270. The strain had significant antagonistic effect on 14 kinds of plant pathogenic fungi and 6 kinds of plant pathogenic bacteria in agricultural production, and has wide antimicrobial spectrum. The strain produces chitinase, cellulase, protease and selenium, and has the basic characteristics of biocontrol bacteria. The fermentation broth of this strain has strong control effect on tomato gray mold and peach brown rot, and has significant promotion action on the tomato plants, which indicated that the strain had a broad application prospect in the field of biological control of plant diseases.
Description
Technical field
The invention belongs to microbial pesticide technical field is and in particular to one plant of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete and its application.
Background technology
With people, the concept of ecological balance, Organic food and green health is increasingly paid attention to and understand in depth, biological
Means of prevention can be effectively prevented from, because of it, the series of problems that chemical pesticide is brought, and have deep society's effect simultaneously again
Benefit, ecological benefits and long-term interest, have obtained extensive and unprecedented concern in agricultural production, sustainable in agricultural sciences
Under the overall background of development, Biological control is also the only way of agricultural development.Actinomycetes are the biological and ecological methods to prevent plant disease, pests, and erosions being most widely used earliest
Microorganism, has played great function in the Biological control of plant disease.Actinomycetes remain to develop in current world wide and open
Send out the study hotspot of new medicine, novel pesticide and Biological control live bacteria agent, the various metabolic function of its species is different, is that a class has extensively
The microbial resources of general practical use.
Fructus Persicae is one of fruit that people like the most, and peach tree has also become the pillar industry in much areas.By chain nuclear disk
The brown rot that bacterium (Monilinia spp.) causes is one of most important disease during Fructus Persicae produces, and is also kernel approaches in world wide
Most important postharvest disease.This disease before adopting, adopt after and storage the transport phase all can cause a large amount of underproduction, be particularly acute after adopting,
American-European loss is up to 59-90%.But peach brown rot is a kind of common and serious disease of generation of peach fruit trophophase and storage period,
In worldwide distribution.The South and the North of China all has generation, especially with the Fructus Persicae producing region of the coastal areas such as Zhejiang, Shandong and Yangtze-Huaihe River Valley
Occur the heaviest, since two thousand, peach brown rot generally occurs in Pekinese Pinggu District, and increases year by year.A lot of research reports,
Caused by the rotten intrusion being mainly due to pathogenic microorganism of fruit.In order to be able to efficiently reduce the damage that fruit postharvest decay causes
Lose, people are many for a long time carries out preventing and treating brown rot etc. with the use of chemical pesticide using physical measure such as low temperature, controlled atmosphere, decompressions
The generation of many fungal diseases.But main this disease of applied chemistry chemical control at present, though preventing and treating mesh can effectively and quickly be reached
, but a large amount of use can cause pesticide residues, pollution environment and human health is produced with harmful effect, such as single use for a long time,
Pathogenic bacteria can produce resistance, as described above, Biological control is because possessing, and environmentally safe, pollution-free and cost is relatively low, meets agriculture life
State and the requirement of sustainable development, become the focus of current research and development.
Content of the invention
The technical problem to be solved is how Suppressing phytopathogens promote plant growing.
For solving above technical problem, the invention provides a streptomycete.
Streptomycete provided by the present invention, blastmycin streptomycete (Streptomyces blastmyceticus), its bacterium
Strain number is JZB130180, and it is in the numbering of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center
For CGMCC No.13270.Hereinafter referred to as blastmycin streptomycete (Streptomyces blastmyceticus)
JZB130180 or bacterial strain JZB130180.
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 on solid medium, bacterium
The circle falling in regular neat in edge, thalline is in yellow, and shrinkage, center projections, prolonging with incubation time in bacterium colony surface
Long, linen sorus occurs.Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 is
Gram positive bacteria, liquefiable gelatin, catalase and oxidase positive, by milk solidification and can peptonize, can be by Starch Hydrolysis, energy
Faint utilization citrate, does not produce melanin and hydrogen sulfide.Blastmycin streptomycete (Streptomyces
Blastmyceticus) JZB130180 has the 16S rDNA sequence of the sequence 1 in sequence table, has sequence 2-4 in sequence table
Shown atpD, recA, rpoB gene order.
For solving above technical problem, the invention provides containing blastmycin streptomycete (Streptomyces
Blastmyceticus) JZB130180 or/and blastmycin streptomycete (Streptomyces blastmyceticus)
The microbial inoculum of the metabolite of JZB130180.
Concretely cause of disease bacteria inhibitor or the disease suppression agent of above-mentioned microbial inoculum.
The active component of above-mentioned cause of disease bacteria inhibitor can be blastmycin streptomycete (Streptomyces
Blastmyceticus) JZB130180 or/and blastmycin streptomycete (Streptomyces blastmyceticus)
The metabolite of JZB130180, the active component of above-mentioned cause of disease bacteria inhibitor also can contain other biological composition or abiotic component,
Other active components those skilled in the art of above-mentioned cause of disease bacteria inhibitor can be according to the inhibition determination to pathogen.
The active component of above-mentioned disease suppression agent can be blastmycin streptomycete (Streptomyces
Blastmyceticus) JZB130180 or/and blastmycin streptomycete (Streptomyces blastmyceticus)
The metabolite of JZB130180, the active component of above-mentioned disease suppression agent also can contain other biological composition or abiotic component, on
The other active components those skilled in the art stating disease suppression agent can be according to the inhibition determination to disease.
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 or/and blastmycin strepto-
Any one application following of the metabolite of bacterium (Streptomyces blastmyceticus) JZB130180 fall within the present invention
Protection domain:
1) application in suppression pathogen;
2) application in preparation cause of disease bacteria inhibitor;
3) application in preparing disease suppression agent;
4) application in suppression disease.
Above, described pathogen can be following all or part of pathogen:Plant botrytis bacterium, plant brown rot germ,
Rhizoctonia cerealis, fusarium graminearum, gaeumannomyces graminis, Colletotrichum capsici, grape anthracnose, plant droop
Bacterium, Rhizoctonia solani Kuhn, Bulbus Lilii pine root fungus, Botryosphaeria berengeriana f. sp, pepper scab fungus, avenae subsp.citrull, Fructus Solani melongenae green grass or young crops is withered
Pathogenic bacteria, wax printing fabric, soil crown gall bacillus and Black Rot on Chinese Cabbage bacterium.
Described plant wilt can be all or part in following pathogenic bacterias:Cucumber fusarium axysporum, cotton-wilt fusarium,
Withered germ of water-melon and cabbage oxysporum.
Described bacilluss can be wax printing fabric.
In described plant brown rot germ, the fruit of described plant is drupe, such as Fructus Persicae.Plant brown rot germ concretely Australia of U.S.
Type drupe chain sclerotinia sclerotiorum (Monilinia fructicola).
In described plant botrytis bacterium, described plant can be plant of Solanaceae, such as Fructus Lycopersici esculenti.
Above, described disease can be following all or part of diseases:Plant botrytis, plant brown rot, Semen Tritici aestivi stricture of vagina is withered
Disease, wheat scab, take-all, pepper anthracnose, bitter rot or anthracnose of grape, plant droop, rice sheath blight disease, Bulbus Lilii root-rot
Disease, ring rot of apple, bacterial spot of pepper, angular leaf spot of cucumber, Fructus Solani melongenae bacterial wilt and Black Rot on Chinese Cabbage.
Described plant droop can be all or part in following four kinds of diseases:Cucumber fusarium axysporum, cotton wilt, Citrullus vulgariss
Droop and Cabbage Wilt Disease.
In described plant brown rot, the fruit of described plant can be drupe, such as Fructus Persicae.
In described plant botrytis, described plant can be plant of Solanaceae, such as Fructus Lycopersici esculenti.
Above, in described microbial inoculum, in addition to described active component, also contain carrier.Described carrier can be normal for pesticide field
And be inert carrier biologically.Described carrier can be solid carrier or liquid-carrier;Described solid carrier can be
Mineral material, vegetable material or macromolecular compound;Described mineral material can for clay, Talcum, Kaolin, montmorillonite, white carbon,
At least one in zeolite, Silicon stone and kieselguhr;Described vegetable material can be at least one in Semen Maydis powder, Semen Glycines powder and starch;
Described macromolecular compound can be polyvinyl alcohol or/and polyglycols;Described liquid-carrier can be organic solvent, vegetable oil, mineral
Oil or water;Described organic solvent can be decane or/and dodecane.
The dosage form of described microbial inoculum can be multiple dosage forms, such as liquor, Emulsion, suspending agent, powder, granule, wettable powder
Or water dispersible granules.
As needed, also can add surfactant (as polysorbas20, Tween 80 etc.), binding agent in described microbial inoculum, stablize
Agent (as antioxidant), pH adjusting agent etc..
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 or/and blastmycin strepto-
Application in promoting plant growing for the metabolite of bacterium (Streptomyces blastmyceticus) JZB130180 falls within
Protection scope of the present invention.
In above-mentioned application, described plant can be any one plant following:
P1) seed plant;
P2) dicotyledon;
P3) plant of Solanaceae;
P4) Fructus Lycopersici esculenti.
In above-mentioned application, described promotion plant growing can for improve plant main root length and/or improve plant fibrous root number and/
Or improve stem height and/or Plant weight.
Above, the metabolite of blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 can
Obtain from the fermentation liquid of blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180.Blastmycin
The metabolite of streptomycete (Streptomyces blastmyceticus) JZB130180 specifically can be prepared as follows:?
Cultivate blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 in fluid medium, remove liquid
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 in culture (fermentation liquid) obtains
The metabolite of blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180.
In the application, described botrytis cinerea can be Botrytis cinerea (Botrytis cinerea).
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 occurs in agricultural production
14 kinds of serious plant pathogenic fungis and 6 kinds of plant pathogenetic bacterias have significant antagonism, and antimicrobial spectrum is wide.This bacterial strain produces several
Fourth matter enzyme, cellulase, protease and thermophilic ferrum element, possess the basic feature of biocontrol microorganisms, the fermentation liquid of this bacterial strain is to tomato gray mould
Disease and peach brown rot have stronger control effect, and have obvious growth-promoting functions to tomato plant, illustrate this bacterial strain in plant
The field of biological control of disease has broad application prospects.
Preservation explanation
Strain name:Blastmycin streptomycete (Streptomyces blastmyceticus)
Strain number:JZB130180
Preservation mechanism:China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is referred to as:CGMCC
Address:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date:On November 14th, 2016
Collection is registered on the books numbering:CGMCC No.13270
Brief description
Fig. 1 is to separate flat board.
Wherein, the bacterium colony on flat board when A-E separates for soil, F is the actinomycetes inclined-plane preserving after purification.
Fig. 2 measures the antagonistic results to Monilinia fructicola for flat board antagonistic activity.
Wherein, CK compares for Monilinia fructicola.
Fig. 3 is cultivation conditions (left figure) in three kinds of culture medium for the bacterial strain JZB130180 and the mycelia under 400 times of light microscopics
Volume morphing (right figure).
Fig. 4 is the PCR amplification in bacterial strain JZB130180 identification.
M:DL2000DNA Marker, swimming lane 1 and 2:The DNA sample extracted;Swimming lane 3-6:It is followed successively by 16S rDNA,
The gene amplification band of atpD, recA, rpoB.
Fig. 5 is the phylogenetic tree of the 16S rDNA sequence construct based on bacterial strain JZB130180.
Fig. 6 is the bacteria inhibition assay result to phytopathogen for the bacterial strain JZB130180.
Wherein, A:Rhizoctonia cerealis, B:Fusarium graminearum, C:Gaeumannomyces graminis, D:Colletotrichum capsici, E:Portugal
Grape anthrax bacteria, F:Monilinia fructicola, G:Botrytis cinerea, H:Cucumber fusarium axysporum, I:Cotton-wilt fusarium, J:Citrullus vulgariss are withered
Wither pathogenic bacteria, K:Cabbage oxysporum, L:Rhizoctonia solani Kuhn, M:Bulbus Lilii pine root fungus, N:Botryosphaeria berengeriana f. sp, O:Fructus Capsici scab
Pathogenic bacteria, P:Avenae subsp.citrull, Q:Fructus Solani melongenae ralstonia solanacearum, R:Wax printing fabric, S:Agrobacterium tumefaciens C58, T:Chinese cabbage
Black rot.
Fig. 7 produces the detection of exoenzyme related to biological and ecological methods to prevent plant disease, pests, and erosion for bacterial strain JZB130180.
Wherein, A:Chitinase detects;B:Protease detects;C:Cellulase detects;D:Thermophilic ferrum element detection
Fig. 8 is the fungistatic effect to Monilinia fructicola for the aseptic ferment filtrate of bacterial strain JZB130180.
Fig. 9 is Monilinia fructicola growing state in toxic medium.
Wherein, 1~6 expression concentration in toxic medium for the aseptic ferment filtrate of bacterial strain JZB130180 is followed successively by 0 μ g/
mL12.5μg/mL、25.0μg/mL、50.0μg/mL、100μg/mL、200μg/mL.
Figure 10 is the inhibition to peach brown rot for the aseptic ferment filtrate of bacterial strain JZB130180.
Wherein, 1-3 row is followed successively by process B1, processes C1 and negative control.
Figure 11 is the inhibition to graw mold of tomato for the aseptic ferment filtrate of bacterial strain JZB130180.
Wherein, 1-4 row is followed successively by process B2, processes C2, processes D and negative control.
Figure 12 is that the aseptic ferment filtrate of bacterial strain JZB130180 measures to the growth-promoting functions of tomato plant.
Wherein, AZ10, BZ10 and CZ10 are the result of 10 days from sowing of seed soaking;DZ20 be seed soaking from
Sow the result of 20 days;DG20 is the result of 20 days from sowing of root irrigation;EZ30 is seed soaking from sowing
The result of 30 days;EG30 is the result of 30 days from sowing of root irrigation;FZ40 is seed soaking 40 days from sowing
Result;FG40 is the result of 40 days from sowing of root irrigation.
Specific embodiment
With reference to specific embodiment, the present invention is further described in detail, the embodiment being given is only for explaining
The bright present invention, rather than in order to limit the scope of the present invention.Experimental technique in following embodiments, if no special instructions, is
Conventional method.Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
The used pathogen public in following embodiments also can obtain from Beijing City Agriculture and Forestry Institute from field acquisition
, to repeat the application experiment:
Cabbage oxysporum Fusarium oxysporum glues group specialized form [Fusarium oxysporum
Schl.f.sp.conglutinans (Wollenw.) Snyder&Hansen] (Li Mingyuan etc. brassicaceous vegetable droop and
Its Pathogen identification. plant protection .2003,29 (6):44-45);
Withered germ of water-melon (Fusarium oxysporum f.sp.niveum) (Geng Lihua etc. withered germ of water-melon is given birth to
The foundation of reason Race identification technical system and checking. China's Vegetable .2010, (20):52-56)
Rhizoctonia cerealis (Rhizoctonia cerealis) (million woodss etc. of recording. wheat sharp eyespot verticillium toxin is planted to Semen Tritici aestivi
The effect of strain. Yangzhou University's journal (agricultural and life sciences version)) .2011,3 2 (3):55-59);
Gaeumannomyces graminis (Gaeumannomyces graminis (Sacc.) Arx&Olivier var.tritici
J.Walker) (LIU WEIGUO. the impact to take-all preventive effect and its yield factors for the chemicals treatment. hubei agricultural science
.2012,51(16):3483-3487);
Botrytis cinerea Botrytis cinerea (Botrytis cinerea Per.ex Fr.) (Li Xinghong etc. Beijing ground
The liquefaction resistance to pyrimethanil for area's botrytis cinerea. plant protection .2012,38 (4):141-143);
Monilinia fructicola chain sclerotinia sclerotiorum (Monilinia fructicola (wint.) Rehm) (Wang Fei etc. Fructus Persicae brown rot
The generation of disease and preventing and treating. fruit trees flower plants .2012,5:58);
Colletotrichum capsici (Colletotrichum capsici) (Song Genmiao etc. benziothiazolinone and Difenoconazole mixture
Potentiation to 4 kinds of various pathogenic bacteria. plant protection .2012,38 (4):171-174);
Grape anthracnose colletotrichum gloeosporioides Penz (Colletortrichum gloeosporioides Penz.e t
Sacc.) (Li Lixia etc. grape anthracnose S R AP analysis of genetic diversity. Chinese agronomy circulates a notice of .2012,28 (1 2):
230-235);
Avenae subsp.citrull (Pseudomonas syringae pv.lachrymans)
(A.T.Alleyne.et.al.Identification of Pseudomonas syringae pv.lachrymans in
Barbados by rep-PCR.Journal of Agricultural Science and Technology B1.2001,
593-597);
Fructus Solani melongenae ralstonia solanacearum (Ralstonia solanacearum) (Feng Linlin etc. the mirror of Fructus Solani melongenae Resistance to bacterial wilt material
Determine and character observation. the Changjiang river vegetable .2000, (10):35-37);
Black Rot on Chinese Cabbage bacterium (Xanthomonas campestris pv.campsetris) (Zhai Wenhui etc. Chinese cabbage
The humid test of black rot identification and its correlation analysiss of seedling stage and Adult plant resistance. China's Vegetable .2010, (10):59-
63);
Bulbus Lilii pine root fungus-fusarium oxysporum (Fusarium oxysporum Schlecht) (peace wisdom etc. Bulbus Lilii root-rot
Sick Pathogen identification and prevention and controls. centre circle vegetable .2010, (3):23-24)
Agrobacterium tumefaciens C58 (Agrobacterium tumefaciens C58cereon) (model Cheng Ming etc. crown gall soil
Secretory protein signal peptide analysis in earth bacillus C58Cereon. microorganism journal .2005,45 (4):561-566);
(Chen Ran etc., wheat scab Biological control grinds fusarium graminearum (Fusarium graminearum Schw.)
Study carefully progress. Henan Agricultural Sciences .2014,43 (12):1-5)
Cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerinum Owen) (Wei Qiaojie etc. Fructus Cucumidis sativi
The Screening and Identification of droop Antagonistic Fungi and its biological preventive effect. Agricultural University Of Nanjing journal .2013,36 (1):40-46)
Cotton-wilt fusarium (Fusarium oxysporum f.sp.vasinfectum) (Zhu Weiling etc., cotton wilt
The Selection of Antagonistic Bacteria .2006,25 (2) of bacterium:7-9)
Rhizoctonia solani Kuhn (Rhizoctonia solani) (Chen Siyu etc., the screening of Rhizoctonia solani Kuhn antagonistic bacterium
And identification. plant protection journal .2013,40 (3):211-218)
Botryosphaeria berengeriana f. sp (Botryosphaeria dothidea) (Liu Baoyou etc., Botryosphaeria berengeriana f. sp p-phenylene's first ring
The sensitivity of azoles and bis(4-fluorophenyl)methyl(1H-1,2,4-triazol-1-ylmethyl)silane and its cross resistance. Plant Pathology .2013,43 (5):541-548)
Pepper scab fungus (Xanthomonas vesicatoria) (to safety etc., pepper scab fungus
The plasmid of (Xanthomonas vesicatoria) and xanthomonas oryzae pv. oryzicola (X.oryzae pv.oryzicola) and
It is with resistance to streptomycin and resistance to copper sexual intercourse. Plant Pathology .2003,33 (4):330-333)
(Wei Kun etc., wax printing fabric is to the degradation characteristic of pyrene and degraded for wax printing fabric (Bacillus cereus)
Enzyme is studied. ACTA Scientiae Circumstantiae .2016,36 (2):506-512)
The partial medium being related in following embodiments is as follows:
Potato dextrose agar (PDA):Rhizoma Solani tuber osi 200g, cleans and is cut into about 1cm3Fritter boil about
With four layers of filtered through gauze after 30min, filtrate complements to 1000ml with tap water, plus glucose 20g, and agar 15g, pH are natural, and 121
DEG C autoclaving 30min.
PD fluid medium:Rhizoma Solani tuber osi 200g, cleans and is cut into about 1cm3Fritter boil after about 30min with four layers of gauze mistake
Filter, filtrate complements to 1000ml with tap water, plus glucose 20g, and pH is natural, 121 DEG C of autoclaving 30min.
Gause I agar culture medium:Soluble starch 20g, NaCl 0.5g, KNO31g, FeSO47H2O 0.01g,
K2HPO40.5g, MgSO47H2O 0.5g, agar 20g, 7.2~7.4,121 DEG C of sterilizing 30min of tap water 1000ml, pH.
Actinomycetes isolation medium I:Glucose 10g, Carnis Bovis seu Bubali cream 2g, asparagine 0.5g, K2HPO40.5g, agar 20g,
Tap water 1000ml, pH6.8 or nature, 121 DEG C of sterilizing 30min.
Actinomycetes isolation medium II:Soluble starch 20g, NaCl 0.5g, KNO31g, Fe2(SO4)30.01g,
K2HPO40.5g, MgSO47H2O 0.5g, agar 20g, 7.2~7.4,121 DEG C of sterilizing 30min of tap water 1000ml, pH.
Actinomycetes isolation medium III:Glycerol 20g, NaCl 1g, CaCO30.1g, L-Arginine 2.5g, FeSO47H2O
0.1g, MgSO47H2O 0.1g, agar 20g, 6.8~7.0,121 DEG C of sterilizing 15min of tap water 1000ml, pH.
Actinomycetes isolation medium IV:Sucrose 15g, NaNO32g, yeast extract 4g, K2HPO40.5g, MgSO4?
7H2O0.5g, KCl 0.5g, FeSO47H2O 0.01g, agar 20g, 7.2,121 DEG C of sterilizing 20min of tap water 1000ml, pH.
Actinomycetes isolation medium V:Soluble starch 10g, (NH4)2SO42g, K2HPO41g, MgSO47H2O 1g,
NaCl 1g, CaCO33g, agar 15g, 7.2~7.4,121 DEG C of sterilizing 20min of tap water 1000ml, pH.
Actinomycetes isolation medium VI:Oatmeal immersion 20g, mark amount saline solution (FeSO47H2O 0.1g, MnCl24H2O
0.1g, ZnSO47H2O 0.1g, tap water 100ml) 1ml, agar 15g, 7.2,121 DEG C of sterilizings of tap water 1000ml, pH
90min.
Actinomycetes isolation medium VII:Glucose 1g, soluble starch 2g, yeast extract 0.5g, N-Z Amine 0.5g,
CaCO30.1g, agar 20g, 7.2~7.4,121 DEG C of sterilizing 20min of tap water 1000ml, pH.
ISP2 culture medium:Yeast extract 4.0g, beerwort 10.0g, glucose 4.0g, agar 20.0g, distilled water 1000mL,
PH7.2-7.4,121 DEG C of sterilizing 20min.
Embodiment 1, the separation of blastmycin streptomycete JZB130180 and identification
1.1 strains separation
The pedotheque in collection Guizhou In China province Thunder God Mountain High aititude region is through dry heat treatment, all in drying at room temperature 1-2,
After levigate sieving, in air dry oven, every part of soil sample is processed in 100 DEG C and carry out again after 1 hour separating.Accurately weigh
Soil sample 1g is put into equipped with the aseptic triangular flask of 50ml of 9ml aseptic salt solution and little bead, abundant shaken well on shaking table
Afterwards, as 10-1Concentration dilution liquid, then draw 10 with 1ml pipettor-1Concentration dilution liquid moves in the aseptic triangular flask of 9ml, shakes up
10-2Concentration dilution liquid, by that analogy, is diluted to 10-3The diluent of concentration.After sample suspension is ready to, use aseptic shifting
Liquid device requires absorption 10 in strict accordance with sterile working-2Concentration and 10-3That each 0.1ml of concentration dilution liquid is respectively put into is in advance good,
(20mg/l K in culture medium, is contained on the actinomycetes isolation medium flat board of reference numeral2Cr2O7To suppress soil as inhibitor
Funguses in sample and antibacterial), then suspension is gently coated with uniformly on flat board with aseptic coated with glass shovel, will be coated
Flat board be placed in constant temperature culture in 28 DEG C of constant incubators, to growing bacterium colony.Observe the actinomycetes of culture in constant incubator,
The bacterium colony of growth and maturity is used in time aseptic inoculation pin picking on Gause I agar culture medium inclined-plane, streak culture, for dividing
At least topple over the isolation medium of 25-30ml in more actinomycetes, each culture dish, with anti-chap thalline inactivation, culture
Time lengthening, to 3-4 week, chooses bacterium in batches.The different actinomycetes strain unifications that same pedotheque is separated on different culture media
After numbering, preserve (Fig. 1) on the temporary transient inclined-plane of 4 DEG C of refrigerators.
The screening of biocontrol bacterial strain:With Monilinia fructicola as target pathogens, PDA cultivates in 25 DEG C of constant incubators
5-7 days, substantial amounts of spore layer now on flat board, is all had to produce.The PDA plate of thickness uniformity first connect few in central point
Permitted the spore of Monilinia fructicola, then few in the actinospore putting four plants of separated purification at pathogen 2cm respectively
Permitted, actinomycetic pathogen is not connect as blank with surrounding.It is placed in culture in 25 DEG C of constant incubators, it is right to treat after carrying out labelling
When being paved with whole flat board according to flat board, observe and record experimental result.This test is in triplicate.
Result of the test:Result is obtained more than the 20 strains Antagonistic Actinomycetes bacterial strain stronger to Monilinia fructicola bacteriostasis, its
In one plant of numbering be JZB130180 bacterial strain fungistatic effect preferably, its antibacterial bandwidth is at more than 7mm (Fig. 2).
1.2 identification of strains
Form cultural characteristic:By the bacterial strain JZB130180 difference streak inoculation after activation in PDA, ISP2 and Gause I
On agar plate, 28 DEG C of constant temperature culture, cultivating the 2nd day, 4 days, 6 days, 8 days and observe respectively and record this bacterial strain when 10 days and exist
Culture form in each culture medium, connects bacterium culture 5-7d, aerial hyphae of observing under an optical microscope and take pictures using inserted sheet method
Form.Result shows that bacterial strain JZB130180 all can grow in above-mentioned three kinds of culture medium, and bacterium colony is in the circle of regular neat in edge
Shape, thalline is in yellow, and shrinkage, center projections in bacterium colony surface, with the prolongation of incubation time, linen spore
Heap.Secondly in above-mentioned three kinds of culture medium, this bacterium grows more luxuriant in ISP2 culture medium, and it is stronger to produce spore ability, is that PDA trains
Foster base, the bacteria growing in Gause I culture medium is poor.From colonial morphology and microexamination, it was initially believed that this Pseudomonas is in actinomycetes
(Fig. 3).
Physiological and biochemical property:To bacterial strain JZB130180 list of references《Soil microorganism research principle and method》(woods is first
Expensive, 2010) in, method carries out amylase hydrolysis, hydrogen sulfide generation, citrate utilization, gelatin liquefaction, catalase, melanin produce
Life, nitrate reduction, milk solidification measure with some physiological-biochemical characteristics such as peptonizing.Result shows that bacterial strain JZB130180 is leather
Lan Shi positive bacteria, liquefiable gelatin, catalase and oxidase positive, by milk solidification and can peptonize, can be by Starch Hydrolysis, can be micro-
Weak utilization citrate, does not produce melanin and hydrogen sulfide.
Determination and analysis of sequence:Bacterial strain JZB130180 is activated on PDA plate, 28 DEG C of culture 5d, transfer into PD liquid
Shake training 24h in culture medium, take 1mL bacterium solution, centrifugation is stayed thalline, carried using Biomed bacterial genomes DNA rapid extraction test kit
Take the genomic DNA of JZB130180 bacterial strain.The primer sequence design of gene selected by amplification and PCR amplification program etc. are discussed in detail
It is shown in Table 1.PCR is all using 25 μ L amplification systems:The each 1 μ L of 10pmoL/L upstream and downstream primer, ultrapure dNTP Mixture (10mmol/
L) 1 μ L, 2.5U Taq archaeal dna polymerase 0.5 μ L, 10 × PCR Buffer 2.5 μ L, genomic DNA template (10ng) 1 μ L,
ddH2O 18μL.Pcr amplification product is all detected with 1% agarose gel electrophoresiies, and target stripe, through cutting glue reclaim after purification, connects
Connect pMD-18T carrier, convert DH5 α E. coli competent, X-gal indigo plant white macula screening, PCR method detection positive colony is sub, will
The bacterium solution of positive colony is sent to company's sequencing.Primer synthesis in this research and sequence are by the calm and peaceful biotechnology of Sino-U.S.
(Beijing) company limited completes.The 16S rRNA gene amplification of bacterial strain JZB130180 after being sequenced, in LPSN (http://
Www.bacterio.net/ choose the reference culture of the higher kind of homology in), with Actinoalloteichuscyanogriseu
s IFO 14455TFor periphery, the sequence of acquisition compares in NCBI website use BLAST, and passes through DNAMAN
5.2.2 carry out multiple sequence with MEGA 5.2.1Version 3 software and similar sequences in GenBank and mate arrangement analysis,
Neighbor-joining method phylogenetic tree construction, develops the reliability of tree through bootstrap (1000 circulations) checking system
Property.Result shows the 16S rDNA of bacterial strain JZB130180, and the gene amplification result of atpD, recA, rpoB is as shown in Figure 4.From bacterium
The phylogenetic tree (Fig. 5) that the 16S rDNA sequence (sequence 1 in sequence table) of strain JZB130180 builds above can be seen that bacterium
Strain JZB130180 and blastmycin streptomycete Streptomyces blastmyceticus NRRL B-5480AY999802.1
Gather in a branch.Expand the house-keeping gene atpD of the bacterial strain JZB130180 obtaining, the sequence of recA, rpoB is respectively such as
In sequence table shown in sequence 2-4.The Blast search comparison through NCBI website for the sequence of these house-keeping genes it was found that
With by 16S rDNA sequence alignment analysis result basically identical, therefore, by JZB130180 identification of strains be blastmycin strepto-
Bacterium (Streptomyces blastmyceticus).
Table 1PCR amplification gene and primer sequence
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 is in November 14 in 2016
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and preserving number is CGMCC
No.13270.Hereinafter referred blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 or bacterial strain
JZB130180.
Embodiment 2, the antimicrobial spectrum of blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180
Measure
For trying culture medium:
LB agar culture medium:Tryptone 10.0g, yeast extract 5.0g, NaCl 10.0g, agar 15.0g, distilled water
1000mL, pH 7.0~7.2;
LB fluid medium:In addition to being not added with agar, other make with LB agar culture medium.
KB culture medium:Peptone 20.0g, glycerol 10.0mL, K2HPO41.50g, MgSO4·7H2O 1.50g, agar
15.0g, distilled water 1000mL, KOH tune pH value to 7.2;
Above-mentioned all culture medium are through 121 DEG C of autoclaving 20min.
Test method:
(1) bacteriostasis of plant pathogenic fungi are measured using flat board face-off method.Concrete operation step is as follows:First by bacterium
Strain JZB130180 and various plant pathogenic fungi (table 2) cultivate 4-5d respectively in PDA culture medium, aseptic with a diameter of 7mm
Bacteria cake made by card punch, and on blank PDA plate, pathogen bacteria cake is placed in flat board central authorities, and culture medium is close in mycelia face, in distance
It is in that 180 ° of bacteria cakes by bacterial strain JZB1301800 are counter at pathogen 2.5cm to be labelled in culture medium, in 25-28 DEG C of constant incubator
Constant temperature culture 5-7d, with only connect pathogen flat board as blank, measure pathogen and bacterial strain JZB130180 colony edge it
Between antibacterial bandwidth and each pathogen blank colony diameter, three repetitions of every kind of pathogen;
(2) bacteriostasis of plant pathogenetic bacteria are measured and adopt Double layer culture method, concrete operation step is as follows:To activate
Bacterial strain JZB130180 bacterium solution dibbling on KB flat board, 28 DEG C culture 48h, with 3mL chloroform, bacterial strain is killed with its steam
JZB130180 thalline, stands 10~12h, so that the volatilization of chloroform steam is completely.Plant pathogenetic bacteria (table 2) is respectively in LB
Activation culture on fluid medium, 28 DEG C of shaken cultivation 24h of 180r/min.By cultured various target pathogenetic bacterias respectively
It is prepared into 10 with physiological saline solution8Cfu/mL bacteria suspension.Drawing 100 μ l bacteria suspensions adds 3mL to be cooled to 50 DEG C after melting
In 1% water agar, rapid mixing, pour chloroform immediately into and killed on the flat board of bacterial strain JZB130180, be paved into uniformly thin
Layer, each processes three repetitions, 28 DEG C of culture 36h, observes fungistatic effect crossing method measurement antibacterial circle diameter.
Result of the test:
Antimicrobial spectrum measurement result shows (being shown in Table 2 and Fig. 6), and this bacterium almost has very bright to all of plant pathogenic fungi
Aobvious bacteriostasis, reach 12mm to the antibacterial bandwidth of cabbage oxysporum and rhizoctonia cerealis, the suppression to Botryosphaeria berengeriana f. sp
Bacterium acts on less, a width of 0.5mm of antibacterial band.Selected plant pathogenetic bacteria is also all shown with stronger inhibitory action, antibacterial
Loop diameter is between 35-50mm.Show that this bacterium has wider antimicrobial spectrum.
The antimicrobial spectrum measurement result of table 2JZB130180 bacterial strain
Note:"-" represents does not survey this content;In table, data is the meansigma methodss of 3 repetition numerical value, and same letter represents difference
Not notable (P < 0.05).
Embodiment 3, the biological and ecological methods to prevent plant disease, pests, and erosion related activity of bacterial strain JZB130180 measure
1st, for examination culture medium:CAS culture medium (detection thermophilic ferrum element) is made up of 4 kinds of solution (individually sterilizing before mixing).Solution 1
(CAS/HDTMA solution) includes CAS solution:60.5mg CAS (chromazurine) is dissolved in 50mL water;Ferrous solution:Containing 1mmol/
L FeCl3·6H2The 10mmol/L HCl solution of O, pH is 2.0;HDTMA solution:72.9mg bromination hexadecane base front three ammonia is molten
Solution is in 40mL water;The HDTMA solution & stir of 40mL is added with 10mL ferrous solution uniformly after the CAS solution of 50mL is mixed,
The black-and-blue sterilization of liquids obtaining, this liquid is CAS/HDTMA solution;Solution 2 (Salts/Buffer solution):By 30.24g
Pipes is dissolved in 750mL Salts solution (KH2PO40.3g, NaCl0.5g, NH4Cl 1.0g) in, adjusted with 50% (W/V) KOH
Section pH to 6.8, adds 15g agar and is settled to 800mL with distilled water, be cooled to 50 DEG C after autoclaving;Solution 3 (750mL):
Glucose 2g, Mannitol 2g, MgSO4·7H2O 493mg, CaCl211mg, H3BO31.4mg, ZnSO4·7H2O 1.2mg,
MnSO4·2H2O 1.17mg, Na2MoO4·2H2O 1mg, CuSO440 μ g, distilled water is settled to 750mL, cold after autoclaving
But to 50 DEG C.The solution 3 of 750mL is added to after 800mL solution 2, and is 10% (W/V) of filtration sterilization with 30mL solution 4
Casamino acid mixes, and finally adds 1580mL mixed liquor in 100mL solution 1, is slowly stirred (avoiding producing foam),
Pave plate.
Chitin culture medium (detection chitinase):Tobacco brown spot pathogen 2.5g, K2HPO40.7g, KH2PO40.3g,
MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, agar 15g, distilled water 1000mL, pH 7.0.
Skim milk agar culture medium (detection protease):Import defatted milk powder 40g, agar 10g, deionized water
1000mL, 115 DEG C of autoclaving 30min, with LB solid medium 1:9 mixings, are paved into flat board standby.
Congo red culture medium (detection fibers element enzyme):MgSO4·7H2O 0.25g, K2HPO40.5g, cellulose 1.88g,
Congo red 0.2g, gelatin 2g, agar 4g, distilled water 1000mL, pH 7.0.
Pikovskaya ' s agar culture medium (detection phosphate):Yeast extract 0.5g, glucose 10g, Ca3
(PO4)25g, (NH4)2SO40.5g, KCl0.2g, MgCl20.1g, MnSO4·H2O0.1mg, FeSO40.1mg, agar 15g,
Distilled water 1000mL, pH 7.0.
Liquid seed culture medium and fermentation medium:Corn starch 30.0g, glucose 10.0g, bean cake powder 20.0g, albumen
Peptone 6.0g, ammonium sulfate 2.50g, MgSO47H2O 1.50g, KH2PO40.30g, NaCl 7.50g, CaCO34.0g, high-temperature starch
Enzyme 0.05g, distilled water 1000mL, pH 7.0~7.4.
Above-mentioned all culture medium are through 121 DEG C of autoclaving 20min.
2nd, test
The detection of 2.1 biological and ecological methods to prevent plant disease, pests, and erosion correlation exoenzymes
2.1.1 the detection of thermophilic ferrum element
By bacterial strain JZB130180 first after 28 DEG C of culture 3-5d in Gause I culture medium, it is inoculated in CAS culture medium,
After 28 DEG C are cultivated 3-5 days, observe whether bacterium colony periphery produces yellow halo.Each process sets 3 repetitions.Due to the element competition of thermophilic ferrum
In culture medium, the iron ion of EDTA chelating, makes culture medium by blue yellowing, showing occurs in therefore periphery of bacterial colonies yellow halo
The generation of thermophilic ferrum element.
2.1.2 the detection of chitinase
By bacterial strain JZB130180 first after 28 DEG C of culture 3-5d in Gause I culture medium, it is inoculated in chitin culture
Base, after 28 DEG C are cultivated 3-5 days, observes the generation of bacterium colony periphery transparent circle, the generation that transparent circle shows chitinase.Each
Process sets 3 repetitions.
2.1.3 the detection of protease
By bacterial strain JZB130180 first after 28 DEG C of culture 3-5d in Gause I culture medium, it is inoculated in skim milk fine jade
Fat culture medium, after 28 DEG C are cultivated 3-5 days, observes the generation of bacterium colony periphery transparent circle afterwards, the product that transparent circle shows protease
Raw.Each process sets 3 repetitions.
2.1.4 the detection of cellulase
By bacterial strain JZB130180 first after 28 DEG C of culture 3-5d in Gause I culture medium, it is inoculated in Congo red culture
Base, after 28 DEG C are cultivated 3-5 days, observes the generation of periphery of bacterial colonies red hydrolysis circle, and appearance red hydrolysis circle shows cellulase
Produce.Each process sets 3 repetitions.
2.1.5 the detection of phosphate
By bacterial strain JZB130180 first after 28 DEG C of culture 3-5d in Gause I culture medium, it is inoculated in Pikovskaya '
S agar culture medium, after 28 DEG C are cultivated 3-5 days, observes the generation of periphery of bacterial colonies transparent circle, transparent circle and show phosphate
Produce.Each process sets 3 repetitions.
Result shows, bacterial strain JZB130180 can secrete chitinase, cellulase, thermophilic ferrum element and protease, does not secrete
Phosphate (Fig. 7).Chitinase therein, cellulase and thermophilic ferrum element activity are all that biocontrol microorganisms possess the important of biological and ecological methods to prevent plant disease, pests, and erosion function
Reference index.
The preparation of the aseptic ferment filtrate of 2.2 bacterial strain JZB130180
Bacterial strain JZB130180, in ISP2 culture medium, after 28 DEG C of constant temperature culture 5~7d, takes 2 ring thalline to be inoculated into liquid strain
In sub- culture medium (50mL loading amount/500mL triangular flask), 28 DEG C of 200r/min shaken cultivation 20~24h on shaking table, take a small amount of training
Nutrient solution in basis of microscopic observation it was observed that a large amount of mycelia and not polluted by spherical or bacillus, as seed culture fluid, with
3% inoculum concentration is inoculated in fermentation medium (100mL loading amount/500mL triangular flask), 28 DEG C of 200r/min shaken cultivation 5~7d,
After terminating fermentation, the fermentation culture of as preparation.This fermentation culture 12000rpm centrifugation 10min is taken supernatant, adopts
0.22 μm of filtering with microporous membrane supernatant is degerming, obtains the aseptic ferment filtrate of bacterial strain JZB130180.
The bacteriostatic activity to Monilinia fructicola for the 2.3 bacterial strain JZB130180
Monilinia fructicola is inoculated on PDA plate, 22 DEG C of culture 5~7d produce abundant spore to whole flat board, plus suitable
The sterilized water of amount, to producing on spore flat board, spore was scraped homemade absorbent cotton filtering layer, removed mycelia, by spore suspension no
Bacterium water is diluted to 107Individual spore/mL is standby.25mL PDA culture medium poured into by the every ware of batch cultur ware (Φ=9cm), to be solidified
The spore suspension drawing 200 μ L Monilinia fructicolas afterwards is spread evenly across on PDA plate, aseptically, will be in PDA plate
The upper cultured Monilinia fructicola aseptic card punch of Φ=7mm makes bacteria cake, takes out bacteria cake, and every hole adds the step of 100 μ L
The rapid 2.2 aseptic ferment filtrate of bacterial strain JZB130180, places 22 DEG C of constant temperature culture 2~3d, observes and measure antibacterial circle diameter.
Result shows that the aseptic ferment filtrate of bacterial strain JZB130180 reaches to the antibacterial circle diameter of Monilinia fructicola on flat board
More than 3.0cm (Fig. 8), illustrates to contain the thing to the strong bacteriostatic activity of Monilinia fructicola in the aseptic ferment filtrate of bacterial strain JZB130180
Matter.
The toxicity test to Monilinia fructicola for the 2.4 bacterial strain JZB130180
2.4.1 the making of Monilinia fructicola bacteria cake
Monilinia fructicola is inoculated on PDA plate, 22 DEG C of culture 5~7d produce abundant spore to whole flat board, plus suitable
The sterilized water of amount, to producing on spore flat board, spore was scraped homemade absorbent cotton filtering layer, removed mycelia, by spore suspension no
Bacterium water is diluted to 107Individual spore/mL is standby.25mL PDA culture medium poured into by the every ware of batch cultur ware (Φ=9cm), to be solidified
The spore suspension drawing 200 μ L Monilinia fructicolas afterwards is spread evenly across on PDA plate, aseptically, will be in PDA plate
The upper cultured Monilinia fructicola aseptic card punch of Φ=6mm gets a number of Monilinia fructicola bacteria cake.
2.4.2 toxic medium preparation:When PDA culture medium melts cool to 45 DEG C about, by the bacterial strain of step 2.2
The aseptic ferment filtrate of JZB130180 and PDA (20mL) mix according to 1.25%, 2.5%, 5%, 10%, 20% ratio, bacterial strain
The ultimate density of the aseptic ferment filtrate of JZB130180 is 12.5 μ g/mL, 25.0 μ g/mL, 50.0 μ g/mL, 100 μ g/mL, 200 μ
G/mL makes the consistent uniform flat board of thin and thick, obtains toxic medium.To be not added with the PDA of the aseptic ferment filtrate of bacterial strain JZB130180
For blank.Each processes 3 repetitions, stand-by after cooling.
2.4.3 Toxicity Determination
With aseptic Inoculating needle, the Monilinia fructicola bacteria cake of 2.4.1 is placed on the toxic medium of 2.4.2, mycelia faces
Under, 1 piece of 6mm bacteria cake is placed in each flat board central authorities, cultivates in 22 DEG C of low temperature incubators.
Result record:After culture 5~7d, crossing method measures colony diameter, and data analysiss return and obtain fermentation liquid
EC50.
Colony diameter (mm)=reality measurement colony diameter meansigma methodss -6
Relative inhibition (%)=(comparison colony diameter-process colony diameter)/comparison colony diameter × 100%
With the aseptic ferment filtrate of bacterial strain JZB130180 content in a pda as concentration, the logarithm value of concentration is independent variable X,
Suppression ratio niqueMin is dependent variable Y, obtains virulence regression equation, correlation coefficient (r), EC50Value the results are shown in Table 3.
As known from Table 3, the EC to Monilinia fructicola for the aseptic ferment filtrate of bacterial strain JZB13018050For 40.5 μ g/mL, explanation
The aseptic ferment filtrate of bacterial strain JZB130180 is former to peach brown rot to have stronger inhibitory action.From Fig. 9 this it appears that with
The increase of concentration is stronger to the growth inhibition effect of peach brown rot opportunistic pathogen mycelia, and when concentration is 20%, mycelia does not grow completely.
The toxicity test result to peach brown rot for the aseptic ferment filtrate of table 3 bacterial strain JZB130180
The mensure to peach brown rot and graw mold of tomato preventive effect for the 2.5 bacterial strain JZB130180
2.5.1 the spore suspension preparation of pathogen:Monilinia fructicola and botrytis cinerea are respectively 23~25 on PDA
After DEG C constant temperature culture 4~5d, scrape its conidium and be placed in sterilized water, be put into vibration on agitator, be made into Monilinia fructicola
(peach brown rot bacterial content is 10 to spore suspension8Cfu/mL) and botrytis cinerea spore suspension (botrytis cinerea contains
Measure as 108cfu/mL).
In triplicate, the experimental technique repeating every time is as follows for experiment:Selection appearance is neat, do not have pest and disease damage and mechanical damage
Peach fruits (kind:Kubo) and tamato fruit (kind:Good powder 19).First with 0.5%NaClO, fruit is processed 1min, with no
Bacterium water is repeatedly rinsed well.In the wound of fruit waist aseptic inoculation acupuncture 5mm (deep) × 3mm (wide), each fruit pricks 4
Hole, after wound dries, Peach fruits is divided at random 3 groups, every group of 5 fruits, respectively processes A1 (negative control), processes B1
With process C1.Tamato fruit is divided at random 4 groups, every group of 5 fruits, respectively process A2 (negative control), process B2, place
Reason C2 and process D.
Each Peach fruits processing A1 inoculate 50 μ l sterilized water, and after fully absorbing, each Peach fruits inoculates 30 μ l steps
2.5.1 Monilinia fructicola spore suspension;Each Peach fruits processing B1 inoculate 50 μ l step 2.2 bacterial strain JZB130180 no
Bacterium ferment filtrate, after fully absorbing, each Peach fruits inoculates the Monilinia fructicola spore suspension of 30 μ l steps 2.5.1;Place
Each Peach fruits inoculation aseptic 5 times of diluents of ferment filtrate of 50 μ l step 2.2 bacterial strain JZB130180 of reason C1 are (dilute with sterilized water
Release the liquid that the aseptic ferment filtrate 5 of step 2.2 bacterial strain JZB130180 obtains again), after fully absorbing, each Peach fruits is inoculated
The Monilinia fructicola spore suspension of 30 μ l steps 2.5.1.Each tamato fruit processing A2 inoculates 50 μ l sterilized water, treats completely
After absorption, each tamato fruit inoculates the Monilinia fructicola spore suspension of 30 μ l steps 2.5.1;Process each Fructus Lycopersici esculenti fruit of B2
The real inoculation aseptic ferment filtrate of 50 μ l step 2.2 bacterial strain JZB130180, after fully absorbing, each tamato fruit inoculates 30 μ l
The Monilinia fructicola spore suspension of step 2.5.1;Each tamato fruit processing C2 inoculates 50 μ l step 2.2 bacterial strains
5 times of diluents of the aseptic ferment filtrate of JZB130180 are (with the aseptic ferment filtrate 5 of sterilized water dilution step 2.2 bacterial strain JZB130180
The liquid obtaining again), after fully absorbing, each tamato fruit inoculates the Monilinia fructicola spore suspension of 30 μ l steps 2.5.1
Liquid;Each tamato fruit inoculation aseptic 10 times of diluents of ferment filtrate of 50 μ l step 2.2 bacterial strain JZB130180 processing D are (with no
The liquid that the aseptic ferment filtrate 10 of bacterium water dilution step 2.2 bacterial strain JZB130180 obtains again), after fully absorbing, each Fructus Lycopersici esculenti
Fruit inoculates the Monilinia fructicola spore suspension of 30 μ l steps 2.5.1.It is placed in plastic package box, one layer of overcoat is fresh-keeping
Film, 23 DEG C, moisturizing (humidity is not less than 90%) is cultivated.Manage incidence and lesion diameter every 24h statistics everywhere.According to scab
Diameter calculates peach brown rot and the prevention effect of graw mold of tomato according to equation below:Prevention effect (%)=(negative control disease
The lesion diameter of spot diameter-respective handling)/negative control lesion diameter × 100%.
Can be seen that the aseptic ferment filtrate of step 2.2 bacterial strain JZB130180 from the sickness rate of Peach fruits with lesion diameter
(abbreviation stock solution) and 5 times of diluents of the aseptic ferment filtrate of step 2.2 bacterial strain JZB130180 (referred to as 5 times diluents) are to brown rot
Bacterium all shows inhibition, and the higher inhibition of concentration is preferably (table 4 and Figure 10).The fruit that stock solution is processed was in first 2 days preventing and treating effects
Fruit reaches 100%.After the 3rd day, stock solution is higher than 5 times of diluents more than 10% to the preventive effect of brown rot germ.Inoculating strain is described
The aseptic ferment filtrate of JZB130180 can postpone the generation of Peach fruits brown rot, and also have certain inhibitory action to Spot expansion, right
The prevention effect of peach brown rot reaches more than 50%.
The control effect testing result to peach brown rot for the aseptic ferment filtrate of table 4 bacterial strain JZB130180
The aseptic ferment filtrate of step 2.2 bacterial strain JZB130180 (abbreviation stock solution) and step 2.2 bacterial strain JZB130180 are aseptic
5 times of diluents of ferment filtrate (referred to as 5 times diluents) and 10 times of diluents of the aseptic ferment filtrate of step 2.2 bacterial strain JZB130180
(referred to as 10 times diluents) all shows stronger inhibition (table 5 and Figure 11) to botrytis cinerea.Stock solution processes performance
Preferably, do not fall ill always in whole experiment process, next to that 5 times of diluents are processed, be finally 10 times of diluents.First 2 days institutes
Some process lesion diameter are all inconspicuous, but sickness rate reduces with the increase of concentration.When the 6th day, 5 times of diluents and 10 quilts
The sickness rate of diluent is 20% and 70% all significantly lower than CK (100%), and with incubation time prolongation, prevention effect is still very bright
Aobvious.With the increase of concentration, treatment group lesion diameter compared with CK is little, and sickness rate is low.Step 2.2 bacterial strain JZB130180 is described
Aseptic ferment filtrate can prevent the generation of graw mold of tomato, can effectively prevent graw mold of tomato.
The control effect testing result to graw mold of tomato for the aseptic ferment filtrate of table 5 bacterial strain JZB130180
Embodiment 4, bacterial strain JZB130180 to plant growth-promoting functions measure
(1) seed soaking:Good powder 19 tomato seeds use 75% ethanol surface sterilization 5 minutes, aseptic water washing 3 times,
10% liquor natrii hypochloritises surface sterilization 10min, after rinsed with sterile water 5 times, by form try one's best consistent tomato seeds be divided into from
Water seed soaking (blank CK1), 50 times of seed soakings, 100 times of seed soakings and 500 times of seed soakings.
50 times of fermentations diluent seed soaking (50X):With the step 2.2 bacterial strain JZB130180 of embodiment 3 aseptic fermentation filter
50 times of diluents of liquid (are obtained again with the step 2.2 bacterial strain JZB130180 aseptic ferment filtrate 50 that tap water dilutes embodiment 3
Liquid) seed soaking 24h, every 12h replacing single treatment liquid.
100 times of fermentations diluent seed soaking (100X):The aseptic fermentation of step 2.2 bacterial strain JZB130180 with embodiment 3
100 times of diluents of filtrate (are obtained with 100 times of the aseptic ferment filtrate of step 2.2 bacterial strain JZB130180 that tap water dilutes embodiment 3
The liquid arriving) seed soaking 24h, every 12h replacing single treatment liquid.
500 times of fermentations diluent seed soaking (500X):The aseptic fermentation of step 2.2 bacterial strain JZB130180 with embodiment 3
500 times of diluents of filtrate (are obtained with 500 times of the aseptic ferment filtrate of step 2.2 bacterial strain JZB130180 that tap water dilutes embodiment 3
The liquid arriving) seed soaking 24h, every 12h replacing single treatment liquid.
Tap water seed soaking (blank CK1):With isopyknic tap water seed soaking 24h, every 12h replacing single treatment
Liquid.
After seed soaking, it is seeded in nutritive cube (sterilized soil+turf=1:1) in, with tap water seed soaking for comparison, in sowing
Growth-promoting effect is investigated when 10d, 20d, 30d and 40d of cultivating afterwards.Each process sets three repetitions, and each repeats 5 nutrition
Alms bowl, three tomato seeds broadcast by every alms bowl.
(2) pouring is processed:The good powder 19 sprouted through pretreatment Fructus Lycopersici esculenti seed bud is seeded in nutritive cube, treats Seedling length to two
Ye Qi, is divided at tap water root irrigation (blank CK2), 50 times of root irrigations, 100 times of root irrigations and 500 times of pouring roots
Reason.
50 times of fermentations diluent root irrigation (50X):Aseptic of the step 2.2 bacterial strain JZB130180 with 20mL embodiment 3
50 times of diluents of ferment filtrate proceed by the pouring of first time root, later every 10d with watering the reality of same volume with first time
The 50 times of diluent roots of the aseptic ferment filtrate of step 2.2 bacterial strain JZB130180 applying example 3 water 1 time, water three times altogether.
100 times of fermentations diluent root irrigation (100X):Step 2.2 bacterial strain JZB130180 with 20mL embodiment 3 is aseptic
100 times of diluents of ferment filtrate proceed by the pouring of first time root, later every 10d with watering same volume with first time
100 times of diluent roots of the aseptic ferment filtrate of step 2.2 bacterial strain JZB130180 of embodiment 3 water 1 time, water three times altogether.
500 times of fermentations diluent root irrigation (500X):Step 2.2 bacterial strain JZB130180 with 20mL embodiment 3 is aseptic
500 times of diluents of ferment filtrate proceed by the pouring of first time root, later every 10d with watering same volume with first time
500 times of diluent roots of the aseptic ferment filtrate of step 2.2 bacterial strain JZB130180 of embodiment 3 water 1 time, water three times altogether.
Blank root irrigation (blank CK2):Proceed by the pouring of first time root with 20mL tap water, after
Watered 1 time with the tap water root with first time pouring same volume every 10d, water three times altogether.
Each process sets three repetitions, and each repeats 5 nutritive cubes, and three tomato seeds broadcast by every alms bowl.
Testing index and method:Height of seedling and root are long, radical, germination percentage, Seedling fresh weight etc..With ruler measurement each process seedling and
The length of root, wherein root with the longest one as measurement object, using the meansigma methodss of all seedling of participating in the experiment of each process as Seedling
High and root is long.Radical:Statistics all length is more than the bar number of the root of 1cm, finally equally takes all seedling of participating in the experiment of each process
Meansigma methodss.
Result of the test:
The result of the test of continuous detecting 40d shows, compared with the blank of water treatment, each concentration bacterial strain
After the process of JZB130180 aseptic ferment filtrate, the growing way of tomato seedling is prosperous, wherein, through the Fructus Lycopersici esculenti of seed soaking, the fibrous root number of Seedling
Showed increased, main root length accounts for clear superiority;Through the tomato seedling of root irrigation, stem is thick, main root and stem length, fibrous root number, plant height, plant
There were significant differences compared with blank for strain fresh weight etc., and above-mentioned each index is better than the seed soaking of blank tap water at later stages
Process tomato seedling (table 6 and Figure 12).Consider statistical result, select 100 times of fermentation dilution liquid irrigating roots, 500 times of fermentation dilutions
Liquid effect of soaking seed is optimal.
Table 6 JZB130180 bacterial strain processes the growth-promoting functions to tomato seedling and measures (20d survey result from sowing)
Note:In table, the data of a.b.c.d. row is the meansigma methodss that set test repeats.
<110>Beijing City Agriculture and Forestry Institute
<120>One plant of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete and its application
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1535
<212> DNA
<213>Blastmycin streptomycete(Streptomyces blastmyceticus)
<400> 1
agcttgcatg cctgcaggtc gacgattgag agtttgatcc tggctcagga cgaacgctgg 60
cggcgtgctt aacacatgca agtcgaacga tgaagccttt cggggtggat tagtggcgaa 120
cgggtgagta acacgtgggc aatctgccct gcactctggg acaagccctg gaaacggggt 180
ctaataccgg ataatacctg ccgaggcatc tcggtgggtt gaaagctccg gcggtgcagg 240
atgagcccgc ggcctatcag cttgttggtg gggtgatggc ctaccaaggc gacgacgggt 300
agccggcctg agagggcgac cggccacact gggactgaga cacggcccag actcctacgg 360
gaggcagcag tggggaatat tgcacaatgg gcgaaagcct gatgcagcga cgccgcgtga 420
gggatgacgg ccttcgggtt gtaaacctct ttcagcaggg aagaagcgag agtgacggta 480
cctgcagaag aagcgccggc taactacgtg ccagcagccg cggtaatacg tagggcgcaa 540
gcgttgtccg gaattattgg gcgtaaagag ctcgtaggcg gcttgttgcg tcggatgtga 600
aagcccgggg cttaaccccg ggtctgcatt cgatacgggc aggctagagt tcggtagggg 660
agatcggaat tcctggtgta gcggtgaaat gcgcagatat caggaggaac accggtggcg 720
aaggcggatc tctgggccga tactgacgct gaggagcgaa agcgtgggga gcgaacagga 780
ttagataccc tggtagtcca cgccgtaaac gttgggaact aggtgtgggc gacattccac 840
gtcgtccgtg ccgcagctaa cgcattaagt tccccgcctg gggagtacgg ccgcaaggct 900
aaaactcaaa ggaattgacg ggggcccgca caagcagcgg agcatgtggc ttaattcgac 960
gcaacgcgaa gaaccttacc aaggcttgac atacaccgga aacggccaga gatggtcgcc 1020
cccttgtggt cggtgtacag gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1080
ggttaagtcc cgcaacgagc gcaacccttg ttctgtgttg ccagcatgcc cttcggggtg 1140
atggggactc acaggagact gccggggtca actcggagga aggtggggac gacgtcaagt 1200
catcatgccc cttatgtctt gggctgcaca cgtgctacaa tggccggtac aatgagctgc 1260
gataccgtga ggtggagcga atttcaaaaa gccggtctca gttcggattg gggtctgcaa 1320
ctcgacccca tgaagttgga gttgctagta atcgcagatc agcattgctg cggtgaatac 1380
gttcccgggc cttgtacaca ccgcccgtca cgtcacgaaa gtcggtaaca cccgaagccg 1440
gtggcccaac ccttgtggag ggagccgtcg aaggtgggac tggcgattgg gacgaagtcg 1500
taacaaggta tccgtaatct ctagaggatc cccgg 1535
<210> 2
<211> 870
<212> DNA
<213>Blastmycin streptomycete(Streptomyces blastmyceticus)
<400> 2
tccgagatca ccgagcgctg gccgatccac cgcaaggcgc cgaacttcga ccagctcgag 60
tccaagaccg agatgttcga gaccggcgtc aaggtcatcg acctgctgac cccgtacgtc 120
aagggcggca agatcggtct gttcggtggt gccggtgtcg gcaagaccgt gctgatccag 180
gaaatgatct accgcgtggc caacaaccac gacggtgtgt cggtgttcgc cggtgtcggt 240
gagcgcaccc gtgagggcaa cgacctcatc gaggaaatga ccgactcggg cgtcatcgac 300
aagacggcgc tcgtcttcgg ccagatggac gagcccccgg gcacccgtct gcgcgtcgcc 360
ctggccggtc tgaccatggc ggagtacttc cgcgatgtgc agaagcagga cgtgctcttc 420
ttcatcgaca acatcttccg ttacacccag gccggttccg aggtgtccac cctgctcggc 480
cgcatgccgt ccgcggtggg ttaccagccg aacctggcgg acgagatggg tctgctgcag 540
gagcgcatca cctcgacccg cggtcactcg atcacctcga tgcaggcgat ctacgtcccc 600
gcggacgacc tgaccgaccc ggcgccggcc accaccttcg cccacctgga cgcgacgacc 660
gttctgtcgc gtccgatctc ggagaagggc atctacccgg cggtcgaccc gctggactcg 720
acgtcccgca tcctggaccc gcgctacatc gcgcaggagc actacgactg cgccatgcgc 780
gtcaagacga tcctgcagaa gtacaaggac ctccaggaca tcatcgcgat cctcggtatc 840
gacgagctgg gcgaggagga caagctcgtc 870
<210> 3
<211> 825
<212> DNA
<213>Blastmycin streptomycete(Streptomyces blastmyceticus)
<400> 3
cctcggcgac cggccgaacg accccatcga ggtcatcccc accgggtcga ccgcactcga 60
cgtcgctctc ggcgtcggcg ggctgccccg cggccgcgtc gtggaggtgt acggaccgga 120
gtcctccggt aagaccaccc tcaccctgca cgccgtggcc aacgcccaga aggcgggcgg 180
caccgtcgcc ttcgtcgacg cggagcacgc gctcgacccg gagtacgcga aggcgctggg 240
cgtcgacacc gacaacctca tcctctccca gccggacacc ggcgagcagg cgctggagat 300
cgtggacatg ctggtccgct ccggcgccct cgacctgatc gtcatcgact ccgtcgccgc 360
gctcgtgccg cgtgcggaga tcgagggcga gatgggcgac tcccacgtcg gcctccaggc 420
gcgactgatg agccaggcgc tccgcaagat caccagcgcg ctcaaccagt ccaagaccac 480
cgcgatcttc atcaaccagc tccgcgagaa gatcggcgtc atgttcggct cgccggagac 540
gacgaccggt ggccgggccc tgaagttcta cgcctcggtg cgcctggaca tccgccgcat 600
cgagaccctc aaggacggca cggaggccgt cggcaaccgc acccgcgtca aggtcgtcaa 660
gaacaaggtc gcgccgccct tcaagcaggc cgagttcgac atcctctacg gccagggcat 720
cagccgcgag ggcggcctga tcgacatggg cgtggagcac ggcttcgtcc gcaaggccgg 780
cgcctggtac acgtacgagg gcgaccagct cggccagggc aagga 825
<210> 4
<211> 871
<212> DNA
<213>Blastmycin streptomycete(Streptomyces blastmyceticus)
<400> 4
tcaaggagtt cttcggcacc agccagctgt cccagttcat ggaccagacg aacccgctgt 60
cgggcctgac ccacaagcgc cgtctgtccg cgctgggccc cggtggtctg tcccgtgagc 120
gggccggctt cgaggtccga gacgtccacc cgtcgcacta cggccgcatg tgcccgatcg 180
agacgcccga aggcccgaac atcggtctga tcggctcgct cgcgtcgtac ggccgcgtca 240
acgcgttcgg tttcgtcgag accccgtacc gcaaggtcgt gggcggcgtc gtcaccgacg 300
acgtggacta cctgactgcc gacgaggaag accgcttcgt catcgcgcag gcgaacgccc 360
ccctcaccga ggacatgcgt tacgccgagt cgcgcgtgct ggtccgccgc cgtggcggcg 420
agatcgacta catcgccggc gacgacgtcg actacatgga cgtctccccg cgccagatgg 480
tgtcggtcgc gaccgcgatg atccccttcc tcgagcacga cgacgccaac cgcgcgctca 540
tgggatcgaa catgatgcgc caggccgttc cgctgatcaa ggcggaggcc ccgctggtcg 600
gcaccggcat ggagtaccgc tgtgcggtcg acgccggtga cgtcatcaag gcggagaagg 660
acggtgtggt ccaggaggtc tccgcggact acgtcaccgt cgccaacgac gacggcacgt 720
acaacacgta ccgggtcgcc aagttctccc gctccaacca gggcacgtcc ttcaaccaga 780
aggtcgtcgt cgacgagggc gcccgggtcg tctccggcca ggtgctcgcc gacggtccgt 840
ccacggacga gggcgagatg gccctcggca a 871
Claims (10)
1. blastmycin streptomycete (Streptomyces blastmyceticus), its bacterial strain number be JZB130180, its in
The numbering of registering on the books of state's Microbiological Culture Collection administration committee common micro-organisms center is CGMCC No.13270.
2. the metabolism containing blastmycin streptomycete described in claim 1 or/and the blastmycin streptomycete described in claim 1
The microbial inoculum of thing.
3. cause of disease bacteria inhibitor it is characterised in that:Described cause of disease bacteria inhibitor contains the blastmycin strepto- described in claim 1
The metabolite of the blastmycin streptomycete described in bacterium or/and claim 1.
4. cause of disease bacteria inhibitor according to claim 3 it is characterised in that:Described cause of disease bacteria inhibitor to following all or
Fraction of pathogens bacterium is inhibited:Plant botrytis bacterium, plant brown rot germ, rhizoctonia cerealis, fusarium graminearum is little
Wheat Pathogen of Take-all, Colletotrichum capsici, grape anthracnose, plant wilt, Rhizoctonia solani Kuhn, Bulbus Lilii pine root fungus,
Botryosphaeria berengeriana f. sp, pepper scab fungus, avenae subsp.citrull, Fructus Solani melongenae ralstonia solanacearum, wax printing fabric, soil crown gall bacillus
With Black Rot on Chinese Cabbage bacterium.
5. disease suppression agent it is characterised in that:Described disease suppression agent contains the blastmycin streptomycete described in claim 1
Or/and the metabolite of the blastmycin streptomycete described in claim 1.
6. disease suppression agent according to claim 5 it is characterised in that:Described disease is following all or part of diseases:
Plant botrytis, plant brown rot, wheat sharp eyespot, wheat scab, take-all, pepper anthracnose, bitter rot or anthracnose of grape,
Plant droop, rice sheath blight disease, Bulbus Lilii root rot, ring rot of apple, bacterial spot of pepper, angular leaf spot of cucumber, Fructus Solani melongenae bacterial wilt
And Black Rot on Chinese Cabbage.
7. the metabolite of the blastmycin streptomycete described in claim 1 or/and the blastmycin streptomycete described in claim 1
Following any one application:
1) metabolite of the blastmycin streptomycete described in claim 1 or/and the blastmycin streptomycete described in claim 1
Application in suppression pathogen;
2) metabolite of the blastmycin streptomycete described in claim 1 or/and the blastmycin streptomycete described in claim 1
Application in preparation cause of disease bacteria inhibitor;
3) metabolite of the blastmycin streptomycete described in claim 1 or/and the blastmycin streptomycete described in claim 1
Application in preparing disease suppression agent;
4) metabolite of the blastmycin streptomycete described in claim 1 or/and the blastmycin streptomycete described in claim 1
Application in suppression disease.
8. according to claim 7 application it is characterised in that:Described pathogen is following all or part of pathogen:Plant
Thing ash arrhizus bacteria, plant brown rot germ, rhizoctonia cerealis, fusarium graminearum, gaeumannomyces graminis, Colletotrichum capsici,
Grape anthracnose, plant wilt, Rhizoctonia solani Kuhn, Bulbus Lilii pine root fungus, Botryosphaeria berengeriana f. sp, bacterial spot of pepper
Bacterium, avenae subsp.citrull, Fructus Solani melongenae ralstonia solanacearum, wax printing fabric, soil crown gall bacillus and Black Rot on Chinese Cabbage bacterium;
Described disease is following all or part of diseases:Plant botrytis, plant brown rot, wheat sharp eyespot, wheat scab,
Take-all, pepper anthracnose, bitter rot or anthracnose of grape, plant droop, rice sheath blight disease, Bulbus Lilii root rot, ring rot of apple,
Bacterial spot of pepper, angular leaf spot of cucumber, Fructus Solani melongenae bacterial wilt and Black Rot on Chinese Cabbage.
9. the metabolite of the blastmycin streptomycete described in claim 1 or/and the blastmycin streptomycete described in claim 1
Application in promoting plant growing.
10. according to claim 9 application it is characterised in that:Described plant is any one plant following:
P1) seed plant;
P2) dicotyledon;
P3) plant of Solanaceae;
P4) Fructus Lycopersici esculenti.
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