CN106434493B - One plant of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete and its application - Google Patents
One plant of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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
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- 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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
Abstract
The invention discloses one plant of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete and its applications.The biological and ecological methods to prevent plant disease, pests, and erosion streptomycete is blastmycin streptomycete, and it is CGMCC No.13270 in the number of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center that bacterial strain number, which is JZB130180,.The bacterial strain has significant antagonism to serious 14 kinds of plant pathogenic fungis and 6 kinds of plant pathogenetic bacterias occur in agricultural production, and antimicrobial spectrum is wide.The bacterial strain generates chitinase, cellulase, protease and thermophilic iron element, the essential characteristic for having biocontrol microorganisms, the fermentation liquid of the bacterial strain has stronger control effect to graw mold of tomato and peach brown rot, and there are apparent growth-promoting functions to tomato plant, illustrate that the bacterial strain has broad application prospects in the field of biological control of plant disease.
Description
Technical field
The invention belongs to microbial pesticide technical fields, and in particular to one plant of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete and its application.
Background technique
As people increasingly pay attention to and understand in depth to the ecological balance, organic food and green and healthy concept, biology
Means of prevention has deep society's effect because a series of problems brought by chemical pesticide can be effectively avoided in it 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.Actinomyces 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.Actinomyces are still to develop to open in current world wide
The research hotspot of new medicine, novel pesticide and biological control live bacteria agent is sent out, many kinds of metabolic function is different, is that one kind has extensively
The microbial resources of general practical use.
Peach is that one of favorite fruit of people, peach also have become the pillar industry in many areas the most.By chain nuclear disk
Brown rot caused by bacterium (Monilinia spp.) is kernel approaches in one of most important disease and world wide in peach production
Most important postharvest disease.This disease before adopting, after adopting and storage the transport phase can cause a large amount of underproduction, be particularly acute after adopting,
America and Europe's loss is up to 59-90%.But peach brown rot is a kind of common and serious disease of generation of peach fruit growth period and storage period,
In worldwide distribution.The South and the North of China has generation, especially with the peach producing region of the coastal areas such as Zhejiang, Shandong and Yangtze-Huaihe River Valley
Generation is most heavy, and since two thousand, peach brown rot generally occurs in Pekinese Pinggu District, and aggravates year by year.Many research reports,
The rotten of fruit is mainly due to caused by the intrusion of pathogenic microorganism.In order to efficiently reduce damage caused by fruit postharvest decay
It loses, people mostly use the physical measures such as low temperature, controlled atmosphere, decompression to carry out prevention and treatment brown rot etc. with the use of chemical pesticide for a long time
The generation of many fungal diseases.But main applied chemistry chemical control disease at present, though it can effectively and quickly reach prevention and treatment mesh
, but a large amount of use will cause pesticide residue, pollution environment and generate adverse effect to human health, such as long-term single use,
Germ can generate resistance, as described above, environmentally safe, pollution-free and cost is relatively low because having for biological control, meet agriculture life
The requirement of state and sustainable development becomes the hot spot researched and developed at present.
Summary of the invention
The technical problem to be solved by the present invention is to how inhibit phytopathogen and promote plant growth.
In order to solve the above technical problems, the present invention provides a streptomycetes.
Streptomycete provided by the present invention, blastmycin streptomycete (Streptomyces blastmyceticus), bacterium
Strain number is JZB130180, in the number 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.
JZB130180 is on solid medium for blastmycin streptomycete (Streptomyces blastmyceticus), bacterium
The circle in regular neat in edge is fallen, thallus is in yellow, and shrinkage, center projections, with prolonging for incubation time occurs in bacterium colony surface
It is long, there is linen sorus.Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 is
Milk can be solidified and be peptonized by gram-positive bacteria, liquefiable gelatin, catalase and oxidase positive, can be by Starch Hydrolysis, energy
It is faint to utilize citrate, do not generate 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 in atpD, recA, rpoB gene order.
In order to solve the above technical problems, the present invention provides contain blastmycin streptomycete (Streptomyces
Blastmyceticus) JZB130180 or/and blastmycin streptomycete (Streptomyces blastmyceticus)
The microbial inoculum of the metabolin of JZB130180.
Concretely cause of disease bacteria inhibitor or the disease suppression agent of above-mentioned microbial inoculum.
The active constituent of above-mentioned cause of disease bacteria inhibitor can be blastmycin streptomycete (Streptomyces
Blastmyceticus) JZB130180 or/and blastmycin streptomycete (Streptomyces blastmyceticus)
The metabolin of JZB130180, the active constituent of above-mentioned cause of disease bacteria inhibitor can also contain other biological ingredient or abiotic component,
Other active components those skilled in the art of above-mentioned cause of disease bacteria inhibitor can determine according to the inhibitory effect to pathogen.
The active constituent of above-mentioned disease suppression agent can be blastmycin streptomycete (Streptomyces
Blastmyceticus) JZB130180 or/and blastmycin streptomycete (Streptomyces blastmyceticus)
The metabolin of JZB130180, the active constituent of above-mentioned disease suppression agent can also contain other biological ingredient or abiotic component, on
The other active components those skilled in the art for stating disease suppression agent can determine according to the inhibitory effect to disease.
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 or/and blastmycin strepto-
Following any applications of the metabolin of bacterium (Streptomyces blastmyceticus) JZB130180 also belong to the present invention
Protection scope:
1) inhibiting the application in pathogen；
2) application in preparation cause of disease bacteria inhibitor；
3) application in disease suppression agent is being prepared；
4) inhibiting the application in disease.
Above, the pathogen can be following all or part of pathogens: plant botrytis bacterium, plant brown rot germ,
Rhizoctonia cerealis, fusarium graminearum, gaeumannomyces graminis, Colletotrichum capsici, grape anthracnose, plant wilt disease
Bacterium, Rhizoctonia solani Kuhn, lily pine root fungus, Botryosphaeria berengeriana f. sp, pepper scab fungus, avenae subsp.citrull, eggplant blueness are withered
Germ, wax printing fabric, soil crown gall bacillus and Black Rot on Chinese Cabbage bacterium.
The plant wilt can be all or part in following germs: cucumber fusarium axysporum, cotton-wilt fusarium,
Withered germ of water-melon and cabbage oxysporum.
The bacillus can be wax printing fabric.
In the plant brown rot germ, the fruit of the plant is drupe, such as peach.Plant brown rot germ concretely U.S. Australia
Type drupe chain sclerotinia sclerotiorum (Monilinia fructicola).
In the plant botrytis bacterium, the plant can be plant of Solanaceae, such as tomato.
Above, the disease can be for following all or part of diseases: plant botrytis, plant brown rot, wheat line are withered
Disease, wheat scab, take-all, pepper anthracnose, bitter rot or anthracnose of grape, plant wilt disease, rice sheath blight disease, lily root-rot
Disease, ring rot of apple, bacterial spot of pepper, angular leaf spot of cucumber, eggplant bacterial wilt and Black Rot on Chinese Cabbage.
The plant wilt disease can be all or part in following four kinds of diseases: cucumber fusarium axysporum, cotton wilt, watermelon
Wilt disease and Cabbage Wilt Disease.
In the plant brown rot, the fruit of the plant can be drupe, such as peach.
In the plant botrytis, the plant can be plant of Solanaceae, such as tomato.
Above, in the microbial inoculum, in addition to the active constituent, also contain carrier.The carrier can be normal for pesticide field
It and is being biologically inert carrier.The carrier can be solid carrier or liquid-carrier；The solid carrier can be
Mineral material, vegetable material or high-molecular compound；The mineral material can for clay, talcum, kaolin, montmorillonite, white carbon,
At least one of zeolite, silica and diatomite；The vegetable material can be at least one of corn flour, bean powder and starch；
The high-molecular compound can be polyvinyl alcohol or/and polyglycols；The liquid-carrier can be organic solvent, vegetable oil, mineral
Oil or water；The organic solvent can be decane or/and dodecane.
The dosage form of the microbial inoculum can be a variety of dosage forms, such as liquor, emulsion, suspending agent, pulvis, granule, wettable powder
Or water dispersible granules.
As needed, surfactant (such as polysorbas20, Tween 80), adhesive, stabilization can be also added in the microbial inoculum
Agent (such as antioxidant), pH adjusting agent.
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 or/and blastmycin strepto-
Application of the metabolin of bacterium (Streptomyces blastmyceticus) JZB130180 in promotion plant growth also belongs to
Protection scope of the present invention.
In above-mentioned application, the plant can be following any plants:
P1) seed plant；
P2) dicotyledon；
P3) plant of Solanaceae；
P4) tomato.
In above-mentioned application, the promotion plant growth 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 metabolin of blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 can
It is obtained from the fermentation liquid of blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180.Blastmycin
The metabolin of streptomycete (Streptomyces blastmyceticus) JZB130180 can be specifically prepared as follows:
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 is cultivated in fluid nutrient medium, removes liquid
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 in culture (fermentation liquid) is obtained
The metabolin of blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180.
In the application, the botrytis cinerea can be Botrytis cinerea (Botrytis cinerea).
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 in agricultural production to occurring
Serious 14 kinds of plant pathogenic fungis and 6 kinds of plant pathogenetic bacterias have significant antagonism, and antimicrobial spectrum is wide.The bacterial strain generates several
Fourth matter enzyme, cellulase, protease and thermophilic iron element, have the essential characteristic of biocontrol microorganisms, the fermentation liquid of the bacterial strain is to tomato gray mould
Disease and peach brown rot have stronger control effect, and have apparent growth-promoting functions to tomato plant, illustrate the 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 abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on November 14th, 2016
Collection is registered on the books number: CGMCC No.13270
Detailed description of the invention
Fig. 1 is separation plate.
Wherein, A-E is bacterium colony when soil separates on plate, and F is the actinomyces inclined-plane saved after purification.
Fig. 2 is antagonistic results of the plate antagonistic activity measurement to Monilinia fructicola.
Wherein, CK is Monilinia fructicola control.
Fig. 3 is cultivation conditions (left figure) of the bacterial strain JZB130180 on three kinds of culture mediums and the mycelia under 400 times of light microscopics
Volume morphing (right figure).
Fig. 4 is the PCR amplification result in bacterial strain JZB130180 identification.
M:DL2000DNA Marker, swimming lane 1 and 2: the DNA sample of extraction；Swimming lane 3-6: being followed successively by 16S rDNA,
The gene magnification 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 bacteria inhibition assay result of the bacterial strain JZB130180 to phytopathogen.
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: watermelon is withered
Wither germ, K: cabbage oxysporum, L: Rhizoctonia solani Kuhn, M: lily pine root fungus, N: Botryosphaeria berengeriana f. sp, O: capsicum scab
Germ, P: avenae subsp.citrull, Q: eggplant ralstonia solanacearum, R: wax printing fabric, S: Agrobacterium tumefaciens C58, T: Chinese cabbage
Black rot.
Fig. 7 is the detection that bacterial strain JZB130180 produces ectoenzyme related to biological and ecological methods to prevent plant disease, pests, and erosion.
Wherein, A: chitinase detection；B: protease detection；C: cellulase detection；D: thermophilic iron element detection
Fig. 8 is fungistatic effect of the sterile ferment filtrate of bacterial strain JZB130180 to Monilinia fructicola.
Fig. 9 is Monilinia fructicola growing state in toxic medium.
Wherein, concentration of 1~6 expression sterile ferment filtrate of bacterial strain JZB130180 in toxic medium is followed successively by 0 μ g/
mL12.5μg/mL、25.0μg/mL、50.0μg/mL、100μg/mL、200μg/mL。
Figure 10 is inhibitory effect of the sterile ferment filtrate of bacterial strain JZB130180 to peach brown rot.
Wherein, 1-3 row is followed successively by processing B1, processing C1 and negative control.
Figure 11 is inhibitory effect of the sterile ferment filtrate of bacterial strain JZB130180 to graw mold of tomato.
Wherein, 1-4 row is followed successively by processing B2, processing C2, processing D and negative control.
Figure 12 is that the sterile ferment filtrate of bacterial strain JZB130180 measures the growth-promoting functions of tomato plant.
Wherein, AZ10, BZ10 and CZ10 are 10 days results from sowing of seed soaking；DZ20 be seed soaking from
20 days results are sowed；DG20 is 20 days results from sowing of root irrigation；EZ30 is seed soaking from sowing
30 days results；EG30 is 30 days results from sowing of root irrigation；FZ40 is seed soaking 40 days from sowing
As a result；FG40 is 40 days results from sowing of root irrigation.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The used pathogen public can also obtain from field acquisition from Beijing City Agriculture and Forestry Institute in following embodiments
, to repeat the application experiment:
Viscous group specialized form [the Fusarium oxysporum of cabbage oxysporum --- Fusarium oxysporum
Schl.f.sp.conglutinans (Wollenw.) Snyder&Hansen] (the brassicaceous vegetable wilt disease such as Li Mingyuan and
Its Pathogen identification plant protection .2003,29 (6): 44-45)；
(the withered germ of water-melon such as Geng Lihua is raw for withered germ of water-melon (Fusarium oxysporum f.sp.niveum)
Manage foundation and the verifying China's Vegetable .2010 of Race identification technical system, (20): 52-56)
(the wheat sharp eyespot verticillium toxins such as million woodss of recording plant wheat to rhizoctonia cerealis (Rhizoctonia cerealis)
The effect Yangzhou University journal (agricultural and life science version) of strain) .2011,3 2 (3): 55-59)；
Gaeumannomyces graminis (Gaeumannomyces graminis (Sacc.) Arx&Olivier var.tritici
J.Walker) (influence hubei agricultural science of the LIU WEIGUO chemicals treatment to take-all preventive effect and its yield factors
.2012,51(16):3483-3487)；
Botrytis cinerea --- Botrytis cinerea (Botrytis cinerea Per.ex Fr.) (Beijing the such as Li Xinghong
Liquefaction resistance plant protection .2012 of area's botrytis cinerea to pyrimethanil, 38 (4): 141-143)；
Monilinia fructicola --- chain sclerotinia sclerotiorum (Monilinia fructicola (wint.) Rehm) (peach brown rot such as Wang Fei
The generation of disease and prevention and treatment fruit trees flower plants .2012,5:58)；
Colletotrichum capsici (Colletotrichum capsici) (such as Song Genmiao benziothiazolinone and difenoconazole mixture
To the synergistic effect plant protection .2012 of 4 kinds of various pathogenic bacterias, 38 (4): 171-174)；
Grape anthracnose --- colletotrichum gloeosporioides Penz (Colletortrichum gloeosporioides Penz.e t
Sacc.) (grape anthracnose S R AP analysis of genetic diversity China's agronomy such as Li Lixia is notified to .2012,28 (1 2):
230-235)；
Avenae subsp.citrull (Pseudomonas syringae pv.lachrymans) (A.T.Alleyne.et.al.I
dentification of Pseudomonas syringae pv.lachrymans in Barbados by rep-
PCR.Journal of Agricultural Science and Technology B1.2001,593-597)；
Eggplant ralstonia solanacearum (Ralstonia the solanacearum) (mirror of the eggplant Resistance to bacterial wilt material such as Feng Linlin
The fixed and the Changjiang river character observation vegetables .2000, (10): 35-37)；
Black Rot on Chinese Cabbage bacterium (Xanthomonas campestris pv.campsetris) (Chinese cabbage such as Zhai Wenhui
The humid test of black rot identification and its correlation analysis China's Vegetable .2010 in seedling stage and Adult plant resistance, (10): 59-
63)；
Lily pine root fungus-fusarium oxysporum (Fusarium oxysporum Schlecht) (the lily root-rot such as peace wisdom
Sick Pathogen identification and control method centre circle vegetables .2010, (3): 23-24)
(model is at bright equal crown gall soil by Agrobacterium tumefaciens C58 (Agrobacterium tumefaciens C58cereon)
Secretory protein signal peptide analysis microorganism journal .2005,45 (4): 561-566 in earth bacillus C58Cereon)；
Fusarium graminearum (Fusarium graminearum Schw.) (grind by Chen Ran etc., wheat scab biological control
Study carefully progress Henan Agricultural Sciences .2014,43 (12): 1-5)
Cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerinum Owen) (cucumber such as Wei Qiaojie
The screening and identification and its biological preventive effect Agricultural University Of Nanjing journal .2013 of wilt disease Antagonistic Fungi, 36 (1): 40-46)
Cotton-wilt fusarium (Fusarium oxysporum f.sp.vasinfectum) (Zhu Weiling etc., cotton wilt
Selection of Antagonistic Bacteria .2006,25 (2): 7-9 of bacterium)
Rhizoctonia solani Kuhn (Rhizoctonia solani) (Chen Siyu etc., 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 sensibility and its cross resistance Plant Pathology .2013,43 (5): 541-548 of azoles and Flusilazole)
Pepper scab fungus (Xanthomonas vesicatoria) is (to safety etc., pepper scab fungus
The plasmid of (Xanthomonas vesicatoria) and xanthomonas oryzae pv. oryzicola (X.oryzae pv.oryzicola) and
Itself and resistance to streptomycin and resistance to copper sexual intercourse Plant Pathology .2003,33 (4): 330-333)
Wax printing fabric (Bacillus cereus) (Wei Kun etc., degradation characteristic and degradation of the wax printing fabric to pyrene
Enzyme studies ACTA Scientiae Circumstantiae .2016,36 (2): 506-512)
Partial medium involved in following embodiments is as follows:
Potato dextrose agar (PDA): potato 200g is cleaned and is cut into about 1cm3Fritter boil about
With four layers of filtered through gauze after 30min, filtrate complements to 1000ml with tap water, adds glucose 20g, agar 15g, pH are naturally, 121
DEG C high pressure sterilization 30min.
PD fluid nutrient medium: potato 200g is cleaned and is cut into about 1cm3Fritter boil after about 30min with four layers of gauze mistake
Filter, filtrate complement to 1000ml with tap water, add glucose 20g, and pH is naturally, 121 DEG C of high pressure sterilization 30min.
Gause I agar medium: soluble starch 20g, NaCl 0.5g, KNO31g, FeSO4﹒ 7H2O 0.01g,
K2HPO40.5g, MgSO4﹒ 7H2O 0.5g, agar 20g, 7.2~7.4,121 DEG C of sterilizing 30min of tap water 1000ml, pH.
Actinomyces isolation medium I: glucose 10g, beef extract 2g, asparagine 0.5g, K2HPO40.5g, agar 20g,
Tap water 1000ml, pH6.8 or naturally, 121 DEG C of sterilizing 30min.
Actinomyces isolation medium II: soluble starch 20g, NaCl 0.5g, KNO31g, Fe2(SO4)30.01g,
K2HPO40.5g, MgSO4﹒ 7H2O 0.5g, agar 20g, 7.2~7.4,121 DEG C of sterilizing 30min of tap water 1000ml, pH.
Actinomyces isolation medium III: glycerol 20g, NaCl 1g, CaCO30.1g, L-arginine 2.5g, FeSO4﹒ 7H2O
0.1g, MgSO4﹒ 7H2O 0.1g, agar 20g, 6.8~7.0,121 DEG C of sterilizing 15min of tap water 1000ml, pH.
Actinomyces isolation medium IV: sucrose 15g, NaNO32g, yeast extract 4g, K2HPO40.5g, MgSO4﹒
7H2O0.5g, KCl 0.5g, FeSO4﹒ 7H2O 0.01g, agar 20g, 7.2,121 DEG C of sterilizing 20min of tap water 1000ml, pH.
Actinomyces isolation medium V: soluble starch 10g, (NH4)2SO42g, K2HPO41g, MgSO4﹒ 7H2O 1g,
NaCl 1g, CaCO33g, agar 15g, 7.2~7.4,121 DEG C of sterilizing 20min of tap water 1000ml, pH.
Actinomyces isolation medium VI: oatmeal immersion liquid 20g, mark amount salting liquid (FeSO4﹒ 7H2O 0.1g, MnCl2﹒ 4H2O
0.1g, ZnSO4﹒ 7H2O 0.1g, tap water 100ml) 1ml, agar 15g, 7.2,121 DEG C of tap water 1000ml, pH sterilizings
90min。
Actinomyces 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, brewer's wort 10.0g, glucose 4.0g, agar 20.0g, distilled water 1000mL,
PH7.2-7.4,121 DEG C of sterilizing 20min.
The separation and identification of embodiment 1, blastmycin streptomycete JZB130180
1.1 strain isolation
The pedotheque for acquiring Guizhou In China province Thunder God Mountain High aititude region passes through dry heat treatment, at drying at room temperature 1-2 weeks,
After levigate sieving, every part of soil sample is separated again after 100 DEG C of 1 hours of processing in air dry oven.It accurately weighs
Soil sample 1g is put into the sterile triangular flask of 50ml equipped with 9ml aseptic salt solution and small bead, the abundant shaken well on shaking table
Afterwards, as 10-1Concentration dilution liquid, then 10 are drawn with 1ml pipettor-1Concentration dilution liquid moves into the sterile triangular flask of 9ml, shakes up
10-2Concentration dilution liquid, and so on, it is diluted to 10-3The dilution of concentration.After sample suspension is ready to, with sterile shifting
Liquid device requires to draw 10 in strict accordance with sterile working-2Concentration and 10-3Each 0.1ml of concentration dilution liquid be respectively put into advance it is good,
(contain 20mg/l K in culture medium on the actinomyces isolation medium plate of reference numeral2Cr2O7Inhibit soil as inhibitor
Fungi and bacterium in sample), it is then shoveled with sterile coated with glass and is gently coated with suspension uniformly on plate, it will be coated
Plate be placed in constant temperature incubation in 28 DEG C of constant incubators, until growing bacterium colony.The actinomyces cultivated in constant incubator are observed,
The bacterium colony of growth and maturity is used in time on aseptic inoculation needle picking to Gause I agar medium inclined-plane, scribing line culture, to divide
From more actinomyces, the isolation medium of 25-30ml is at least toppled in each culture dish, with anti-chap thallus inactivation, culture
Time extends to 3-4 weeks, chooses bacterium in batches.The different actinomycetes strains that same pedotheque is separated in different culture medium are unified
After number, (Fig. 1) is saved on the temporary inclined-plane of 4 DEG C of refrigerators.
The screening of biocontrol bacterial strain: it using Monilinia fructicola as target pathogens, is cultivated in 25 DEG C of constant incubators on PDA
5-7 days, there is a large amount of spore layer to generate on plate at this time.It is first connect in central point on the uniform PDA plate of thickness few
Perhaps the spore of Monilinia fructicola is then few in the actinospore for putting upper four plants of separated purifying respectively at pathogen 2cm
Perhaps, the pathogen of actinomyces is not connect using surrounding as blank control.It marks to be placed in 25 DEG C of constant incubators and cultivate, to right
When being paved with entire plate according to plate, experimental result is observed and recorded.The test is in triplicate.
Test result: more than 20 strains are obtained to the stronger Antagonistic Actinomycetes bacterial strain of Monilinia fructicola bacteriostasis in result,
In one plant number be JZB130180 bacterial strain fungistatic effect it is best, antibacterial bandwidth is at 7mm or more (Fig. 2).
The identification of 1.2 bacterial 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 incubations observe and record the bacterial strain when cultivating the 2nd day, 4 days, 6 days, 8 days and 10 days respectively and exist
Culture form on each culture medium meets bacterium culture 5-7d using inserted sheet method, observes under an optical microscope and aerial hyphae of taking pictures
Form.The result shows that bacterial strain JZB130180 can be grown on above-mentioned three kinds of culture mediums, bacterium colony is in the circle of regular neat in edge
Shape, thallus are in yellow, and shrinkage occurs in bacterium colony surface, and center projections linen spore occur with the extension of incubation time
Heap.In above-mentioned three kinds of culture mediums, the bacterium grown on ISP2 culture medium it is more luxuriant, produce spore ability it is stronger, secondly for PDA train
Base is supported, the bacterium growth on Gause I culture medium is poor.From colonial morphology and microexamination, it was initially believed that the Pseudomonas is in actinomyces
(Fig. 3).
Physiological and biochemical property: to bacterial strain JZB130180 bibliography " edaphon studies principle and method ", (woods is first
Expensive, 2010) method carries out amylorrhexis, hydrogen sulfide generations, citrate utilization, gelatin liquefaction, catalase, melanin production in
Life, nitrate reduction, milk solidification are measured with some physiological-biochemical characteristics such as peptonizing.The result shows that bacterial strain JZB130180 is leather
Milk can be solidified and be peptonized by Lan Shi positive bacteria, liquefiable gelatin, catalase and oxidase positive, can be by Starch Hydrolysis, can be micro-
It is weak to utilize citrate, do not generate melanin and hydrogen sulfide.
Determination and analysis of sequence: bacterial strain JZB130180 is activated on PDA plate, and 28 DEG C of culture 5d transfer into PD liquid
Training is shaken in culture medium for 24 hours, takes 1mL bacterium solution, and centrifugation is stayed thallus, mentioned using Biomed bacterial genomes DNA rapidly extracting kit
Take the genomic DNA of JZB130180 bacterial strain.Primer sequence design and the PCR amplification program etc. for expanding selected gene are discussed in detail
It is shown in Table 1.PCR is all made of 25 μ L amplification systems: 10pmoL/L upstream and downstream primer each 1 μ L, 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, 1 μ L of genomic DNA template (10ng),
ddH2O 18μL.Pcr amplification product uses 1% agarose gel electrophoresis to detect, and target stripe after purification, connects through gel extraction
PMD-18T carrier is connect, DH5 α E. coli competent, the screening of X-gal indigo plant hickie are converted, PCR method detects positive clone molecule, will
The bacterium solution of positive clone molecule is sent to company's sequencing.Primer synthesis and sequence in this research is by the calm and peaceful biotechnology of Sino-U.S.
(Beijing) Co., Ltd completes.The 16S rRNA gene magnification of bacterial strain JZB130180 and after being sequenced, LPSN (http: //
Www.bacterio.net/ higher kind of homology of reference culture is chosen in), with
Actinoalloteichuscyanogriseus IFO 14455TFor periphery, the sequence of acquisition is in NCBI website use BLAST
Be compared, and by similar sequences in 3 software of DNAMAN 5.2.2 and MEGA 5.2.1Version and GenBank into
Row multiple sequence matches arrangement analysis, and Neighbor-joining method phylogenetic tree construction (follows for 1000 times through bootstrap
Ring) verifying phylogenetic tree reliability.The result shows that the base of the 16S rDNA, atpD, recA, rpoB of bacterial strain JZB130180
Gene-amplification result is as shown in Figure 4.The system constructed from the 16S rDNA sequence (sequence 1 in sequence table) of bacterial strain JZB130180
As can be seen that bacterial strain JZB130180 and blastmycin streptomycete Streptomyces on development tree (Fig. 5)
Blastmyceticus NRRL B-5480AY999802.1 gathers in a branch.Expand obtained bacterial strain JZB130180
House-keeping gene atpD, recA, rpoB sequence respectively as shown in sequence 2-4 in sequence table.The sequence of these house-keeping genes
By the website NCBI Blast search comparison as a result, it has been found that, and it is almost the same by the result of 16S rDNA sequence alignment analysis, because
This, is accredited as blastmycin streptomycete (Streptomyces blastmyceticus) for JZB130180 bacterial strain.
Table 1PCR amplification gene and primer sequence
Blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 is November 14 in 2016
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number CGMCC
No.13270.Hereinafter referred blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180 or bacterial strain
JZB130180。
The antimicrobial spectrum of embodiment 2, blastmycin streptomycete (Streptomyces blastmyceticus) JZB130180
Measurement
For trying culture medium:
LB agar medium: tryptone 10.0g, yeast extract 5.0g, NaCl 10.0g, agar 15.0g, distilled water
1000mL, pH 7.0~7.2；
LB liquid medium: in addition to agar is not added, other make with LB agar 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 mediums are through 121 DEG C of high pressure sterilization 20min.
Test method:
(1) tablet face-off method is used to the bacteriostasis measurement of plant pathogenic fungi.Specific steps are as follows: first by bacterium
Strain JZB130180 and various plant pathogenic fungis (table 2) cultivate 4-5d in PDA culture medium respectively, are the sterile of 7mm with diameter
Punch makes bacteria cake, and on blank PDA plate, pathogen bacteria cake is placed in plate center, and culture medium is close in mycelia face, in distance
It is labelled on culture medium at pathogen 2.5cm in 180 ° of bacteria cakes by bacterial strain JZB1301800 are counter, in 25-28 DEG C of constant incubator
Constant temperature incubation 5-7d, only to connect the plate of pathogen as blank control, measure pathogen and bacterial strain JZB130180 colony edge it
Between antibacterial bandwidth and each pathogen blank control colony diameter, every kind of pathogen repeats three times；
(2) Double layer culture method is used to the bacteriostasis measurement of plant pathogenetic bacteria, specific steps are as follows: will activation
Bacterial strain JZB130180 bacterium solution dibbling on KB plate, 28 DEG C of culture 48h, with 3mL chloroform with its steam kill bacterial strain
JZB130180 thallus stands 10~12h, so that the volatilization of chloroform steam is complete.Plant pathogenetic bacteria (table 2) is respectively in LB
Activation culture on fluid nutrient medium, 28 DEG C of shaken cultivations of 180r/min are for 24 hours.Cultured various target pathogenetic bacterias are distinguished
10 are prepared into sterile saline8Cfu/mL bacteria suspension.It draws after 3mL thawing is added in 100 μ l bacteria suspensions and is cooled to 50 DEG C
It in 1% water agar, mixes rapidly, pours into chloroform immediately and killed on the plate of bacterial strain JZB130180, be paved into uniform thin
Layer, each processing repeat three times, 28 DEG C of culture 36h, observation fungistatic effect and crossing method measurement antibacterial circle diameter.
Test result:
Antimicrobial spectrum measurement result shows and (is shown in Table 2 and Fig. 6), which almost has all plant pathogenic fungis very bright
Aobvious bacteriostasis reaches 12mm to the antibacterial bandwidth of cabbage oxysporum and rhizoctonia cerealis, the suppression to Botryosphaeria berengeriana f. sp
Bacterium effect is smaller, and antibacterial bandwidth is 0.5mm.Stronger inhibiting effect is also shown to selected plant pathogenetic bacteria, it is antibacterial
Loop diameter is between 35-50mm.Show that the bacterium has wider antimicrobial spectrum.
The antimicrobial spectrum measurement result of table 2JZB130180 bacterial strain
Note: this content is not surveyed in "-" expression；Data are the average value of 3 repetition numerical value in table, and same letter indicates difference
Not significant (P < 0.05).
Embodiment 3, the measurement of the biological and ecological methods to prevent plant disease, pests, and erosion related activity of bacterial strain JZB130180
1, for trying culture medium: CAS culture medium (detecting thermophilic iron element) is made 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: contain 1mmol/
L FeCl3·6H2The 10mmol/L HCl solution of O, pH 2.0；HDTMA solution: 72.9mg bromination hexadecane base front three ammonia is molten
Solution is in 40mL water；The HDTMA solution & stir that 40mL is added after the CAS solution of 50mL is mixed with 10mL ferrous solution is uniform,
Obtained black-and-blue sterilization of liquids, the liquid, that 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, with 50% (W/V) KOH tune
PH to 6.8 is saved, 15g agar is added and is settled to 800mL with distilled water, 50 DEG C are cooled to after high pressure sterilization；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 are settled to 750mL, cold after high pressure sterilization
But to 50 DEG C.After the solution 3 of 750mL is added to 800mL solution 2, and with 30mL solution 4 be filtration sterilization 10% (W/V)
Casamino acid mixing, finally 1580mL mixed liquor is added in 100mL solution 1, is slowly stirred and (avoids generating 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 medium (detection protease): import skimmed milk power 40g, agar 10g, deionized water
1000mL, 115 DEG C of high pressure sterilization 30min mix with LB solid medium 1:9, it is spare to be paved into plate.
Congo red culture medium (detection cellulase): 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 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: cornstarch 30.0g, glucose 10.0g, bean cake powder 20.0g, albumen
Peptone 6.0g, ammonium sulfate 2.50g, MgSO4﹒ 7H2O 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 mediums are through 121 DEG C of high pressure sterilization 20min.
2, it tests
The detection of 2.1 biological and ecological methods to prevent plant disease, pests, and erosion correlation ectoenzymes
2.1.1 the detection of thermophilic iron element
After bacterial strain JZB130180 is first cultivated 3-5d on Gause I culture medium through 28 DEG C, it is inoculated in CAS culture medium,
After 28 DEG C are cultivated 3-5 days, whether observation bacterium colony periphery generates yellow halo.Each processing sets 3 repetitions.Since thermophilic iron element competes
The iron ion that EDTA is chelated in culture medium, turns yellow culture medium by blue, therefore periphery of bacterial colonies yellow halo shows
The generation of thermophilic iron element.
2.1.2 the detection of chitinase
After bacterial strain JZB130180 is first cultivated 3-5d on Gause I culture medium through 28 DEG C, 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 occurs.Each
Processing sets 3 repetitions.
2.1.3 the detection of protease
After bacterial strain JZB130180 is first cultivated 3-5d on Gause I culture medium through 28 DEG C, it is inoculated in skim milk fine jade
Rouge culture medium, after 28 DEG C are cultivated 3-5 days, there is the production that transparent circle shows protease in the rear generation for observing bacterium colony periphery transparent circle
It is raw.Each processing sets 3 repetitions.
2.1.4 the detection of cellulase
After bacterial strain JZB130180 is first cultivated 3-5d on Gause I culture medium through 28 DEG C, it is inoculated in Congo red culture
Base, after 28 DEG C are cultivated 3-5 days, there is red hydrolysis circle and shows cellulase in the generation of observation periphery of bacterial colonies red hydrolysis circle
It generates.Each processing sets 3 repetitions.
2.1.5 the detection of phosphate
After bacterial strain JZB130180 is first cultivated 3-5d on Gause I culture medium through 28 DEG C, it is inoculated in Pikovskaya '
S agar medium after 28 DEG C are cultivated 3-5 days, observes the generation of periphery of bacterial colonies transparent circle, transparent circle occurs and show phosphate
It generates.Each processing sets 3 repetitions.
The result shows that bacterial strain JZB130180 can secrete chitinase, cellulase, thermophilic iron element and protease, do not secrete
Phosphate (Fig. 7).The plain activity of chitinase, cellulase and thermophilic iron therein is that biocontrol microorganisms have the important of biological and ecological methods to prevent plant disease, pests, and erosion function
Reference index.
The preparation of the 2.2 sterile ferment filtrates of bacterial strain JZB130180
Bacterial strain JZB130180 is on ISP2 culture medium, after 28 DEG C of 5~7d of constant temperature incubation, 2 ring thallus is taken 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~for 24 hours on shaking table takes a small amount of training
Nutrient solution observes a large amount of mycelia and do not polluted by spherical or bacillus, as seed culture fluid in microscopically observation, with
3% inoculum concentration is inoculated in fermentation medium (100mL loading amount/500mL triangular flask), 28 DEG C of 200r/min 5~7d of shaken cultivation,
Terminate after fermenting, the fermentation culture as prepared.Fermentation culture 12000rpm centrifugation 10min is taken into supernatant, is used
0.22 μm of filtering with microporous membrane supernatant degerming, obtains the sterile ferment filtrate of bacterial strain JZB130180.
Bacteriostatic activity of the 2.3 bacterial strain JZB130180 to Monilinia fructicola
Monilinia fructicola is inoculated on PDA plate, 22 DEG C of 5~7d of culture to entire plate produce spore abundant, add suitable
Spore was scraped homemade absorbent cotton filtering layer, and removed mycelia, by spore suspension nothing by the sterile water of amount to producing on spore plate
Bacterium water is diluted to 107A spore/mL is spare.Batch cultur ware (Φ=9cm) every ware pours into 25mL PDA culture medium, to be solidified
The spore suspension for drawing 200 μ L Monilinia fructicolas afterwards is spread evenly across on PDA plate, aseptically, will be in PDA plate
Upper cultured Monilinia fructicola makes bacteria cake with the aseptic card punch of Φ=7mm, takes out bacteria cake, and the step of 100 μ L is added in every hole
The rapid 2.2 sterile ferment filtrate of bacterial strain JZB130180, places 22 DEG C of 2~3d of constant temperature incubation, observes and measure antibacterial circle diameter.
The result shows that the sterile ferment filtrate of bacterial strain JZB130180 reaches the antibacterial circle diameter of Monilinia fructicola on plate
3.0cm or more (Fig. 8) illustrates in the sterile ferment filtrate of bacterial strain JZB130180 containing the object to the strong bacteriostatic activity of Monilinia fructicola
Matter.
Toxicity test of the 2.4 bacterial strain JZB130180 to Monilinia fructicola
2.4.1 the production of Monilinia fructicola bacteria cake
Monilinia fructicola is inoculated on PDA plate, 22 DEG C of 5~7d of culture to entire plate produce spore abundant, add suitable
Spore was scraped homemade absorbent cotton filtering layer, and removed mycelia, by spore suspension nothing by the sterile water of amount to producing on spore plate
Bacterium water is diluted to 107A spore/mL is spare.Batch cultur ware (Φ=9cm) every ware pours into 25mL PDA culture medium, to be solidified
The spore suspension for drawing 200 μ L Monilinia fructicolas afterwards is spread evenly across on PDA plate, aseptically, will be in PDA plate
Upper cultured Monilinia fructicola gets a certain number of Monilinia fructicola bacteria cakes with the aseptic card punch of Φ=6mm.
2.4.2 toxic medium preparation: when PDA culture medium melts cool to 45 DEG C or so, by the bacterial strain of step 2.2
The sterile ferment filtrate of JZB130180 and PDA (20mL) are mixed according to 1.25%, 2.5%, 5%, 10%, 20% ratio, bacterial strain
The ultimate density of the sterile 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
The consistent uniform plate of thin and thick is made in g/mL, obtains toxic medium.The PDA of the sterile ferment filtrate of bacterial strain JZB130180 is not added
For blank control.3 repetitions of each processing, it is stand-by after cooling.
2.4.3 indoor toxicity test
The Monilinia fructicola bacteria cake of 2.4.1 is placed on the toxic medium of 2.4.2 with sterile transfer needle, mycelia faces
Under, 1 piece of 6mm bacteria cake is placed on each plate center, is cultivated in 22 DEG C of low temperature incubators.
As a result record: after 5~7d of culture, crossing method measures colony diameter, and data analysis, which returns, finds out fermentation liquid
EC50。
Colony diameter (mm)=actual measurement colony diameter average value -6
Relative inhibition (%)=(control colony diameter-processing colony diameter)/control colony diameter × 100%
Using the content of the sterile ferment filtrate of bacterial strain JZB130180 in a pda as concentration, the logarithm of concentration be independent variable X,
Inhibiting rate probability value are dependent variable Y, find out virulence regression equation, related coefficient (r), EC50Value the results are shown in Table 3.
As known from Table 3, EC of the sterile ferment filtrate of bacterial strain JZB130180 to Monilinia fructicola50For 40.5 μ g/mL, explanation
The sterile ferment filtrate of bacterial strain JZB130180 has stronger inhibiting effect to peach brown rot original.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.
Toxicity test result of the 3 sterile ferment filtrate of bacterial strain JZB130180 of table to peach brown rot
Measurement of the 2.5 bacterial strain JZB130180 to peach brown rot and graw mold of tomato preventive effect
2.5.1 the spore suspension preparation of pathogen: Monilinia fructicola and botrytis cinerea are respectively 23~25 on PDA
After DEG C 4~5d of constant temperature incubation, scrapes its conidium and be placed in sterile water, be put on oscillator and vibrate, be made into Monilinia fructicola
(peach brown rot bacterial content is 10 to spore suspension8Cfu/mL) and botrytis cinerea spore suspension (botrytis cinerea contains
Amount is 108cfu/mL)。
In triplicate, each duplicate experimental method is as follows for experiment: selection appearance is neat, does not have pest and disease damage and mechanical damage
Peach fruits (kind: Kubo) and tamato fruit (kind: good powder 19).Fruit is first handled into 1min with 0.5%NaClO, with nothing
Bacterium water is repeatedly rinsed well.The wound of 5mm (depth) × 3mm (width) is pierced with aseptic inoculation needle in fruit waist, each fruit pricks 4
Peach fruits after wound dries, are divided into 3 groups, every group of 5 fruits, respectively processing A1 (negative control), processing B1 by hole at random
With processing C1.Tamato fruit is divided into 4 groups at random, every group of 5 fruits, respectively processing A2 (negative control), processing B2, place
Manage C2 and processing D.
The each Peach fruits for handling A1 are inoculated with 50 μ l sterile waters, and after fully absorbing, each Peach fruits are inoculated with 30 μ l steps
2.5.1 Monilinia fructicola spore suspension；Handle B1 each Peach fruits be inoculated with 50 μ l step 2.2 bacterial strain JZB130180 without
Bacterium ferment filtrate, after fully absorbing, each Peach fruits are inoculated with the Monilinia fructicola spore suspension of 30 μ l step 2.5.1；Place
The each Peach fruits for managing C1 are inoculated with 50 sterile 5 times of dilutions of ferment filtrate of μ l step 2.2 bacterial strain JZB130180 and (use sterile water dilute
Release the liquid that the sterile ferment filtrate 5 of step 2.2 bacterial strain JZB130180 obtains again), after fully absorbing, each Peach fruits inoculation
The Monilinia fructicola spore suspension of 30 μ l step 2.5.1.The each tamato fruit for handling A2 is inoculated with 50 μ l sterile waters, to complete
After absorption, each tamato fruit is inoculated with the Monilinia fructicola spore suspension of 30 μ l step 2.5.1；Handle each tomato fruit of B2
The 50 sterile ferment filtrates of μ l step 2.2 bacterial strain JZB130180 of real inoculation, after fully absorbing, each tamato fruit is inoculated with 30 μ l
The Monilinia fructicola spore suspension of step 2.5.1；The each tamato fruit for handling C2 is inoculated with 50 μ l step 2.2 bacterial strains
5 times of dilutions of the sterile ferment filtrate of JZB130180 (use the sterile ferment filtrate 5 of 2.2 bacterial strain JZB130180 of sterile water dilution step
The liquid obtained again), after fully absorbing, each tamato fruit is inoculated with the Monilinia fructicola spore suspension of 30 μ l step 2.5.1
Liquid；Each tamato fruit of processing D is inoculated with 50 sterile 10 times of dilutions of ferment filtrate of μ l step 2.2 bacterial strain JZB130180 and (uses nothing
The liquid that the sterile ferment filtrate 10 of 2.2 bacterial strain JZB130180 of bacterium water dilution step obtains again), after fully absorbing, each tomato
Fruit is inoculated with the Monilinia fructicola spore suspension of 30 μ l step 2.5.1.It is placed into plastic package box, one layer of housing fresh-keeping
Film, 23 DEG C, moisturizing (humidity is not less than 90%) culture.Incidence and lesion diameter are managed everywhere every counting for 24 hours.According to scab
Diameter calculates the control efficiency of peach brown rot and graw mold of tomato: control efficiency (%)=(negative control disease according to following formula
The respective treated lesion diameter of spot diameter -)/negative control lesion diameter × 100%.
It can be seen that the sterile ferment filtrate of step 2.2 bacterial strain JZB130180 from the disease incidence and lesion diameter of Peach fruits
(abbreviation stoste) and 5 times of dilutions of the sterile ferment filtrate of step 2.2 bacterial strain JZB130180 (referred to as 5 times of dilutions) are to brown rot
Bacterium shows inhibitory effect, and the higher inhibitory effect of concentration is preferably (table 4 and Figure 10).The fruit of stoste processing was imitated in prevention and treatment in first 2 days
Fruit is up to 100%.After on day 3, stoste is higher than 5 times of 10% or more dilutions to the preventive effect of brown rot germ.Illustrate inoculating strain
The sterile ferment filtrate of JZB130180 can postpone the generation of Peach fruits brown rot, and also have certain inhibiting effect to Spot expansion, right
The control efficiency of peach brown rot is up to 50% or more.
Control effect testing result of the 4 sterile ferment filtrate of bacterial strain JZB130180 of table to peach brown rot
The sterile ferment filtrate of step 2.2 bacterial strain JZB130180 (abbreviation stoste) and step 2.2 bacterial strain JZB130180 are sterile
5 times of dilutions of ferment filtrate (referred to as 5 times of dilutions) and step 2.2 bacterial strain JZB130180 10 times of dilutions of sterile ferment filtrate
(referred to as 10 times of dilutions) show stronger inhibitory effect (table 5 and Figure 11) to botrytis cinerea.Stoste processing performance
Preferably, it does not fall ill always in the entire experiment process, followed by 5 times of dilution processing, is finally 10 times of dilutions.Preceding 2 days institutes
Some processing lesion diameters are unobvious, but disease incidence reduces with the increase of concentration.At the 6th day, 5 times of dilutions and 10 quilts
The disease incidence of dilution is 20% and 70% significantly lower than CK (100%), and is extended with incubation time, and control efficiency is still very bright
It is aobvious.With the increase of concentration, processing group lesion diameter compared with CK is small, and disease incidence is low.Illustrate step 2.2 bacterial strain JZB130180
Sterile ferment filtrate can prevent the generation of graw mold of tomato, can effectively prevent graw mold of tomato.
Control effect testing result of the 5 sterile ferment filtrate of bacterial strain JZB130180 of table to graw mold of tomato
Embodiment 4, bacterial strain JZB130180 measure the growth-promoting functions of plant
(1) seed soaking: good 19 tomato seeds of powder use 75% alcohol surface sterilization 5 minutes, aseptic water washing 3 times,
10% liquor natrii hypochloritis surface sterilization 10min, after rinsed with sterile water 5 times, by form tomato seeds consistent as far as possible be divided into from
Water seed soaking (blank control CK1), 50 times of seed soakings, 100 times of seed soakings and 500 times of seed soakings.
50 times of fermentations dilution seed soaking (50X): it is filtered with the sterile fermentation of the step 2.2 bacterial strain JZB130180 of embodiment 3
50 times of dilutions of liquid (are obtained again with the sterile ferment filtrate 50 of step 2.2 bacterial strain JZB130180 of tap water dilution embodiment 3
Liquid) it soaks seed for 24 hours, every 12h replaces single treatment liquid.
100 times of fermentations dilution seed soaking (100X): with the sterile fermentation of step 2.2 bacterial strain JZB130180 of embodiment 3
100 times of dilutions of filtrate (are obtained with 100 times of the sterile ferment filtrate of step 2.2 bacterial strain JZB130180 of tap water dilution embodiment 3
The liquid arrived) it soaks seed for 24 hours, every 12h replaces single treatment liquid.
500 times of fermentations dilution seed soaking (500X): with the sterile fermentation of step 2.2 bacterial strain JZB130180 of embodiment 3
500 times of dilutions of filtrate (are obtained with 500 times of the sterile ferment filtrate of step 2.2 bacterial strain JZB130180 of tap water dilution embodiment 3
The liquid arrived) it soaks seed for 24 hours, every 12h replaces single treatment liquid.
Tap water seed soaking (blank control CK1): for 24 hours with isometric tap water seed soaking, every 12h replaces single treatment
Liquid.
It after seed soaking, is seeded in nutritive cube (sterilized soil+turf=1:1), is control with tap water seed soaking, is sowing
Growth-promoting effect is investigated when 10d, 20d, 30d and 40d for cultivating afterwards.Each processing sets three repetitions, 5 nutrition of each repetition
Alms bowl, every alms bowl broadcast three tomato seeds.
(2) pouring is handled: the 19 tomato seed bud of good powder sprouted by pretreatment is seeded in nutritive cube, it is long to two to seedling
Ye Qi is divided at tap water root irrigation (blank control CK2), 50 times of root irrigations, 100 times of root irrigations and 500 times of pouring roots
Reason.
50 times of fermentations dilution root irrigation (50X): with the sterile hair of step 2.2 bacterial strain JZB130180 of 20mL embodiment 3
50 times of dilutions of ferment filtrate start to carry out the pouring of first time root, later every the 10d reality for pouring same volume with first time
The 50 times of dilution roots of the sterile ferment filtrate of step 2.2 bacterial strain JZB130180 for applying example 3 are poured 1 time, are poured altogether three times.
100 times of fermentations dilution root irrigation (100X): sterile with the step 2.2 bacterial strain JZB130180 of 20mL embodiment 3
100 times of dilutions of ferment filtrate start to carry out the pouring of first time root, pour same volume with first time every 10d later
100 times of dilution roots of the sterile ferment filtrate of step 2.2 bacterial strain JZB130180 of embodiment 3 are poured 1 time, are poured altogether three times.
500 times of fermentations dilution root irrigation (500X): sterile with the step 2.2 bacterial strain JZB130180 of 20mL embodiment 3
500 times of dilutions of ferment filtrate start to carry out the pouring of first time root, pour same volume with first time every 10d later
500 times of dilution roots of the sterile ferment filtrate of step 2.2 bacterial strain JZB130180 of embodiment 3 are poured 1 time, are poured altogether three times.
Blank control root irrigation (blank control CK2): being started to carry out first time root pouring with 20mL tap water, after
It pours 1 time, is poured altogether three times every the tap water root for pouring same volume with first time 10d.
Each processing sets three repetitions, 5 nutritive cubes of each repetition, and every alms bowl broadcasts three tomato seeds.
Testing index and method: height of seedling and root long, radical, germination percentage, seedling fresh weight etc..With ruler measure each processing seedling with
The length of root, wherein root with longest one for measurement object, using the average value of all seedling of participating in the experiment of each processing as seedling
High and root long.Radical: the item number of root of the statistics all length greater than 1cm finally equally takes all seedling of participating in the experiment of each processing
Average value.
Test result:
Continuously the test result of detection 40d shows compared with the blank control of water treatment, each concentration bacterial strain
The growing way of tomato seedling is prosperous after the sterile ferment filtrate processing of JZB130180, wherein by the tomato of seed soaking, the fibrous root number of seedling
It increased significantly, main root length accounts for clear superiority；By the tomato seedling of root irrigation, stem thickness, main root and stem length, fibrous root number, plant height, plant
There were significant differences compared with blank control for strain fresh weight etc., and above-mentioned each index is soaked seed better than blank control tap water at later stages
It handles tomato seedling (table 6 and Figure 12).Comprehensively consider statistical result, selects 100 times of fermentation dilution liquid irrigating roots, 500 times of fermentation dilutions
Liquid effect of soaking seed is best.
The processing of 6 JZB130180 bacterial strain of table measures (the 20d investigation result from sowing) to the growth-promoting functions of tomato seedling
Note: the data that a.b.c.d. is arranged in table are the set duplicate average value of test.
<110>Beijing City Agriculture and Forestry Institute
<120>one plants of biological and ecological methods to prevent plant disease, pests, and erosion streptomycetes and its applications
<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 (13)
1. blastmycin streptomycete (Streptomyces blastmyceticus), bacterial strain number is JZB130180, in
The number 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 blastmycin streptomycete described in claim 1
The microbial inoculum of object.
3. cause of disease bacteria inhibitor, it is characterised in that: the cause of disease bacteria inhibitor contains blastmycin strepto- described in claim 1
The metabolin of bacterium or/and blastmycin streptomycete described in claim 1.
4. cause of disease bacteria inhibitor according to claim 3, it is characterised in that: the cause of disease bacteria inhibitor is to following pathogens
It is inhibited: plant botrytis bacterium, plant brown rot germ, rhizoctonia cerealis, fusarium graminearum, take-all
Bacterium, Colletotrichum capsici, grape anthracnose, plant wilt, Rhizoctonia solani Kuhn, lily pine root fungus, apple wheel line
Germ, pepper scab fungus, avenae subsp.citrull, eggplant ralstonia solanacearum, Bacillus cercus, soil crown gall bacillus and/or big
Seed detection bacterium.
5. disease suppression agent, it is characterised in that: the disease suppression agent contains blastmycin streptomycete described in claim 1
Or/and the metabolin of blastmycin streptomycete described in claim 1.
6. disease suppression agent according to claim 5, it is characterised in that: the disease is plant botrytis, plant brown rot
Disease, wheat sharp eyespot, wheat scab, take-all, pepper anthracnose, bitter rot or anthracnose of grape, plant wilt disease, rice banded sclerotial blight
Disease, lily root rot, ring rot of apple, bacterial spot of pepper, angular leaf spot of cucumber, eggplant bacterial wilt and/or Black Rot on Chinese Cabbage.
7. the metabolin of blastmycin streptomycete described in claim 1 or/and blastmycin streptomycete described in claim 1
Following any applications:
1) metabolin of blastmycin streptomycete described in claim 1 or/and blastmycin streptomycete described in claim 1
Inhibiting the application in pathogen；
2) metabolin of blastmycin streptomycete described in claim 1 or/and blastmycin streptomycete described in claim 1
Application in preparation cause of disease bacteria inhibitor；
3) metabolin of blastmycin streptomycete described in claim 1 or/and blastmycin streptomycete described in claim 1
Preparing the application in disease suppression agent；
4) metabolin of blastmycin streptomycete described in claim 1 or/and blastmycin streptomycete described in claim 1
Inhibiting the application in disease.
8. application according to claim 7, it is characterised in that: the pathogen is plant botrytis bacterium, plant brown rot
Bacterium, rhizoctonia cerealis, fusarium graminearum, gaeumannomyces graminis, Colletotrichum capsici, grape anthracnose, plant are withered
Germ, Rhizoctonia solani Kuhn, lily pine root fungus, Botryosphaeria berengeriana f. sp, pepper scab fungus, avenae subsp.citrull, eggplant are green
Blight bacterium, Bacillus cercus, soil crown gall bacillus and/or Black Rot on Chinese Cabbage bacterium；
The disease is plant botrytis, plant brown rot, wheat sharp eyespot, wheat scab, take-all, capsicum anthrax
Disease, bitter rot or anthracnose of grape, plant wilt disease, rice sheath blight disease, lily root rot, ring rot of apple, bacterial spot of pepper, cucumber angle spot
Disease, eggplant bacterial wilt and/or Black Rot on Chinese Cabbage.
9. the metabolin of blastmycin streptomycete described in claim 1 or/and blastmycin streptomycete described in claim 1
Promoting the application in plant growth.
10. application according to claim 9, it is characterised in that: the plant is seed plant.
11. application according to claim 9, it is characterised in that: the plant is dicotyledon.
12. application according to claim 9, it is characterised in that: the plant is plant of Solanaceae.
13. application according to claim 9, it is characterised in that: the plant is tomato.
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