CN107083349B - The white black streptomycete of one plant of disease prevention growth-promoting and its preparation and application of metabolite - Google Patents
The white black streptomycete of one plant of disease prevention growth-promoting and its preparation and application of metabolite Download PDFInfo
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Abstract
The invention discloses the preparation and application of the white black streptomycete of one plant of disease prevention growth-promoting and its metabolite.The bacterial strain number of the white black streptomycete is T22, is CGMCC No.14190 in the number of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.White black streptomycete T22 all has inhibiting effect to fruit trees and vegetables ash arrhizus bacteria, Monilinia fructicola and Colletotrichum capsici etc..The white black sterile ferment filtrate of streptomycete T22 can effectively inhibit Botrytis cinerea germ, botrytis cinerea and the growth of cucumber fusarium axysporum mycelia.White black streptomycete can form hydrolysis circle on inulinase-producing activity detection culture medium by generating cellulase and chitinase.Plant pathogenic fungi can be inhibited by generating antibiotics active material and hydrolase substance by making black streptomycete T22 clear.The white black sterile ferment filtrate of streptomycete T22 can significantly promote tomato plumular axis and/or radicle elongation after suitably diluting, enhance tomato seeds germinating energy.
Description
Technical field
The invention belongs to microbial pesticide technical fields, and in particular to the white black streptomycete of one plant of disease prevention growth-promoting and its metabolism
The preparation and application of product.
Background technique
Gray mold caused by Deuteromycotina fungi Botrytis cinerea (Botrytis cinera) is infected is made in a variety of fruits and vegetables
Occur in object cultivation universal and serious.The disease can endanger stem, leaf, flower and the fruit of plant, in moist rainy and greenhouse
It is particularly acute in environment.The prevention and treatment of China's fruits and vegetables gray mold relies primarily on chemical agent at present, but a large amount of uses for a long time
The problems such as chemical agent is easy to produce drug resistance and environmental pollution there are pesticide residue, pathogen.It explores new and effective, wide
Spectrum, low toxicity, environmentally friendly control of plant disease technology are the difficult point and hot spot of current agricultural production research.Biological control has safety
The features such as pollution-free, pathogen is not likely to produce drug resistance is the effective way of diseases of garden stuff green prevention and control.It is existing at present both at home and abroad
About bacillus subtilis (Bacillus subtilis), bacillus polymyxa (Bacillus polymyxa), Trichoderma
Research of the microorganisms such as (Trichoderma spp.) to fruit trees and vegetables gray mold control efficiency.Due in gray mold pathogen species
Variation is big, and spread speed is fast after disease occurs, effectively prevents difficulty, and the existing microbial resources of reasonable development formulate novel biocontrol
Preparation increases microbial pesticide type, is of great significance for diseases of garden stuff green prevention and control and Agricultural product without pollution.
Actinomyces (Actinomycete) are widely present in the different natural ecological environment such as soil, ocean, have kind
The features such as class is various, metabolic function is different is a kind of microbial population with practical application value.It is found from microorganism
Have 70% in bioactive substance from actinomyces, wherein about 50% is the metabolite of streptomyces.There is antagonism to plant for screening
The active actinomycetes population of object disease fungus, exploitation can gradually substitute the novel microbial Pesticidal products of chemical pesticide for fruits and vegetables
Crop gray mold green prevention and control is of great significance.
Summary of the invention
The technical problem to be solved by the present invention is to how inhibit fruit trees and vegetables pathogen and fruit trees and vegetables is promoted to grow.
In order to solve the above technical problems, the present invention provides one plant of white black streptomycetes.
White black streptomycete provided by the present invention is the white black streptomycete (Streptomyces that bacterial strain number is T22
Alboniger), it is in the number of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center
CGMCC No.14190.White black streptomycete (Streptomyces alboniger) T22 of hereinafter referred.
White black streptomycete (Streptomyces alboniger) T22 has 16S rDNA shown in sequence 1 in sequence table
Sequence, white black streptomycete (Streptomyces alboniger) T22 aerial hyphae white on Gause I culture medium, can not
With the passing of time dissolubility pigment generates faint yellow pigment (Fig. 1).White black streptomycete (Streptomyces can be observed under microscope
Alboniger) T22 aerial hyphae is until flexible, spore chaining, oval (Fig. 2).
The culture of white black streptomycete (Streptomyces alboniger) T22 also belongs to protection scope of the present invention.
The culture of white black streptomycete (Streptomyces alboniger) T22 is by white black streptomycete
The substance that (Streptomyces alboniger) T22 is cultivated in microbial liquid fermentation medium.
In order to solve the above technical problems, the present invention provides cause of disease bacteria inhibitors.
Cause of disease bacteria inhibitor provided by the present invention contains white black streptomycete (Streptomyces alboniger) T22
And/or the metabolin of white black streptomycete (Streptomyces alboniger) T22.
The active constituent of above-mentioned cause of disease bacteria inhibitor can be white black streptomycete (Streptomyces alboniger) T22
And/or the metabolin of white black streptomycete (Streptomyces alboniger) T22, the active constituent of above-mentioned cause of disease bacteria inhibitor
Other biological ingredient or abiotic component, other active components those skilled in the art of above-mentioned cause of disease bacteria inhibitor can also be contained
It can be determined according to the inhibitory effect to pathogen.
In above-mentioned cause of disease bacteria inhibitor, the cause of disease bacteria inhibitor, which can have following at least one pathogens, to be inhibited to make
With:
A, ash arrhizus bacteria；
B, drupe brown rot germ；
C, Colletotrichum capsici；
D, wilt；
E, target bacterium；
F, rhizoctonia cerealis；
G, eggplant ralstonia solanacearum；
H, avenae subsp.citrull.
In order to solve the above technical problems, the present invention provides disease suppression agent.
Disease suppression agent provided by the present invention, containing white black streptomycete (Streptomyces alboniger) T22 and/
Or the metabolin of white black streptomycete (Streptomyces alboniger) T22.
The active constituent of above-mentioned disease suppression agent can for white black streptomycete (Streptomyces alboniger) T22 and/
Or the metabolin of white black streptomycete (Streptomyces alboniger) T22, the active constituent of above-mentioned disease suppression agent may be used also
Containing other biological ingredient or abiotic component, other active components those skilled in the art of above-mentioned disease suppression agent can basis
The inhibitory effect of disease is determined.
In above-mentioned disease suppression agent, the disease is following at least one:
A, gray mold；
B, drupe brown rot；
C, pepper anthracnose；
D, wilt disease；
E, target；
F, wheat sharp eyespot；
G, eggplant bacterial wilt；
H, angular leaf spot of cucumber.
White black streptomycete (Streptomyces alboniger) T22 and/or white black streptomycete (Streptomyces
Alboniger) following any applications of the metabolin of T22 also belong to protection scope of the present invention:
1) inhibiting the application in pathogen；
2) application in preparation cause of disease bacteria inhibitor；
3) inhibiting the application in disease；
4) application in disease suppression agent is being prepared.
In above-mentioned application, the pathogen can be following at least one:
A, ash arrhizus bacteria；
B, drupe brown rot germ；
C, Colletotrichum capsici；
D, wilt；
E, target bacterium；
F, rhizoctonia cerealis；
G, eggplant ralstonia solanacearum；
H, avenae subsp.citrull.
The disease can be following at least one:
A, gray mold；
B, drupe brown rot；
C, pepper anthracnose；
D, wilt disease；
E, target；
F, wheat sharp eyespot；
G, eggplant bacterial wilt；
H, angular leaf spot of cucumber.
White black streptomycete (Streptomyces alboniger) T22 and/or white black streptomycete (Streptomyces
Alboniger) following any applications of the metabolin of T22 also belong to protection scope of the present invention:
H, promoting the application in plant seed germination；
I, promoting the application in vegetable seeds plumular axis and/or radicle growth；
J is improving the application in plant seed germination gesture.
In above-mentioned application, the plant can be following any plants:
P1) tomato；
P2) tomato platymiscium；
P3) plant of Solanaceae.
Above, the metabolin of white black streptomycete (Streptomyces alboniger) T22 can be from white black streptomycete
It is obtained in the fermentation liquid of (Streptomyces alboniger) T22.The white black streptomycete (Streptomyces
Alboniger) metabolin of T22 can be the sterile metabolin of white black streptomycete (Streptomyces alboniger) T22.It is white
The sterile metabolin (sterile ferment filtrate) of black streptomycete (Streptomyces alboniger) T22 specifically can be according to such as lower section
Method preparation: cultivating white black streptomycete (Streptomyces alboniger) T22 in liquid medium, is filtered to remove liquid training
It supports white black streptomycete (Streptomyces alboniger) T22 in object (fermentation liquid) and obtains white black streptomycete
The sterile metabolin of (Streptomyces alboniger) T22.
In the application, the ash arrhizus bacteria can be botrytis cinerea, Botrytis cinerea and/or Botrytis cinerea germ；
The drupe brown rot germ can be Monilinia fructicola；The wilt can for cucumber fusarium axysporum, cabbage oxysporum and/or
Cotton-wilt fusarium.The gray mold can be graw mold of tomato, grape grey mould and/or grey mould fruit rot of strawberry, the drupe brown rot
Disease can be peach brown rot；The wilt disease can be cucumber fusarium axysporum, Cabbage Wilt Disease and/or cotton wilt.
Above, in the cause of disease bacteria inhibitor and disease suppression agent, in addition to the active constituent, also contain carrier.Institute
Stating carrier can be pesticide field commonly and be biologically inert carrier.The carrier can carry for solid carrier or liquid
Body；The solid carrier can be mineral material, vegetable material or high-molecular compound；The mineral material can for clay, talcum,
At least one of kaolin, montmorillonite, white carbon, zeolite, silica and diatomite；The vegetable material can be corn flour, bean powder
At least one of with starch；The high-molecular compound can be polyvinyl alcohol and/or polyglycols；The liquid-carrier can be to have
Solvent, vegetable oil, mineral oil or water；The organic solvent can be decane and/or dodecane.
In the cause of disease bacteria inhibitor and disease suppression agent, white black streptomycete (Streptomyces alboniger) T22
Can exist in the form of spore, mycelia or culture containing spore and/or mycelia.
The cause of disease bacteria inhibitor and the dosage form of disease suppression agent can be a variety of dosage forms, such as liquor, emulsion, suspending agent, powder
Agent, granule, wettable powder or water dispersible granules.
As needed, surfactant can be also added in the cause of disease bacteria inhibitor and disease suppression agent (such as polysorbas20 is spat
Warm 80 etc.), adhesive, stabilizer (such as antioxidant), pH adjusting agent.
The present invention is using diseases of garden stuff disease fungus as target, and separation screening obtains one from Qinghai-Tibet special habitats soil
Strain has both microorganism fungus kind-Bai Hei streptomycete (Streptomyces alboniger) T22 of disease prevention growth-promoting effect, to including ash
Various plants disease fungus including mildew bacterium has good wide spectrum antagonistic substance, and sterile ferment filtrate can effectively facilitate
Tomato seeds germination, and there is good in vitro preventive effect to graw mold of tomato, therefore can be applied to fruits and vegetables fungal disease especially
The green prevention and control of gray mold provides new resources to prepare microbial pesticide new product.
White black streptomycete (Streptomyces alboniger) T22 to fruit trees and vegetables ash arrhizus bacteria, Monilinia fructicola and
Colletotrichum capsici etc. all has inhibiting effect (table 3, Fig. 4).White black streptomycete (Streptomyces alboniger) T22 without
Bacterium ferment filtrate can effectively inhibit Botrytis cinerea germ, botrytis cinerea and cucumber fusarium axysporum mycelia growth (Fig. 5).Together
When white black streptomycete T22 can be detected in inulinase-producing activity by generating cellulase and chitinase and form hydrolysis circle on culture medium
(Fig. 6).Pathogenic can be inhibited by generating antibiotics active material and hydrolase substance by making black streptomycete T22 clear
Fungi.The sterile ferment filtrate of white black streptomycete (Streptomyces alboniger) T22 can significantly promote kind after suitably diluting
Eggplant plumular axis and/or radicle elongation, enhance tomato seeds germinating energy.Compared with the control, white black streptomycete (Streptomyces
Alboniger) 100 times of dilutions of the sterile ferment filtrate of T22 significantly increase tomato plumular axis, radicle length, improve tomato species
Sub- germinating energy, amplification are up to 15.1%, 29.7% and 43.9% respectively；White black streptomycete (Streptomyces alboniger)
The tomato seeds of sterile 200 times of dilutions of the ferment filtrate processing of T22, plumular axis, radicle length and potentiality of seed relatively compare increasing
8.8%, 18.1% and 32.3% (table 4) is added.The sterile metabolin of white black streptomycete (Streptomyces alboniger) T22
(sterile ferment filtrate), which sprays processing tomato excised leaf, can be substantially reduced graw mold of tomato lesion diameter, white black streptomycete
The sterile metabolin of (Streptomyces alboniger) T22 (sterile ferment filtrate) processing reaches the in vitro preventive effect of graw mold of tomato
To 55.1% (table 5).It makes the sterile ferment filtrate of black streptomycete (Streptomyces alboniger) T22 clear and sprays processing pair
Tomato excised leaf gray mold has good control efficiency (Fig. 7).White black streptomycete (Streptomyces alboniger)
T22 can be cultivated manually, and condition of culture is simple, it is good to produce spore, and biocontrol agent is made suitable for industrialized production, has good open
Send out application prospect.
Preservation explanation
Strain name: white black streptomycete (Streptomyces alboniger)
Strain number: T22
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 May 25th, 2017
Collection is registered on the books number: CGMCC No.14190
Detailed description of the invention
Fig. 1 is colonial morphology of the bacterial strain T22 on Gause I agar medium.
Fig. 2 is mycelia and spore shape of the bacterial strain T22 under 40 times of optical microscopies.
Fig. 3 is the systematic evolution tree based on bacterial strain T22 16S rDNA sequence construct, wherein bacterial strain T22 and white black streptomycete
Streptomyces alboniger bacterial strain (NR043228, AB184331) gathers in same branch, and similitude is up to 99%.
Fig. 4 is bacterial strain T22 to the plate antagonistic effect of disease fungus, and wherein a, b, c are respectively bacterial strain T22 to tomato gray mould
Germ, Monilinia fructicola, Colletotrichum capsici inhibiting effect.
Fig. 5 is the sterile ferment filtrate of bacterial strain T22 to the inhibiting effect of plant pathogenic fungi, and bacterium is not added respectively in a, c, e
Cucumber fusarium axysporum, botrytis cinerea, Botrytis cinerea germ bacterium colony in strain T22 without fermented liquid PDA culture medium, b, d, f points
Cucumber fusarium axysporum, botrytis cinerea, Botrytis cinerea germ in the sterile ferment filtrate PDA culture medium of bacterial strain T22 Wei not be added
Bacterium colony.
Fig. 6 is bacterial strain T22 cellulase-producing and chitinase situation, and a, b are respectively bacterial strain T22 in the Congo red training of cellulose
Support the hydrolysis circle generated on base and the Congo red culture medium of tobacco brown spot pathogen.
Fig. 7 be the sterile ferment filtrate of bacterial strain T22 to the in vitro preventive effect of graw mold of tomato wherein, a, b are not inoculated with Liquid Culture
Base sprays+ash arrhizus bacteria, and the sterile ferment filtrate of c, d bacterial strain T22 sprays+ash arrhizus bacteria, 50% fludioxonil of e, f, 1000 times of liquid sprays
Apply+ash arrhizus bacteria.
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:
Botrytis cinerea (Botrytis cinerea), cucumber fusarium axysporum (Fusarium oxysporum
F.sp.cucumerinum), Monilinia fructicola (Monilinia fructicola), Colletotrichum capsici
It is (Colletotrichum capsici), cotton-wilt fusarium (Fusarium oxysporum f.sp.vasinfectum), sweet
Blue wilt (Fusarium oxysporum f.sp.conglutinans), rhizoctonia cerealis (Rhizoctonia
Cereali), beading reaping hook germ (Fusarium moniliforme), eggplant ralstonia solanacearum (Ralstonia
Solanacearum), Black Rot on Chinese Cabbage bacterium (Xanthomonas campestris the pv.campsetris) (such as Wang Junli
The identification and biological and ecological methods to prevent plant disease, pests, and erosion specificity analysis Chinese biological preventing and treating journal of one bacillus QD-10,2014,30 (4): 564-572.).
Target bacterium (Alternaria alternata) (Zheng, et al.Characterization of
Alternaria species associated with potato foliar diseases in China.Plant
pathology,2015,64:425-433.)。
Botrytis cinerea germ (Botrytis cinerea), Botrytis cinerea (Botrytis cinerea), cucumber angle
Pinta bacterium (Pseudomonas syringae pv.lachrymans) be (mono- plant of broccoli wilt disease antagonistic bacterium of the such as Lu Caige
Screening, identification and its Antibacterial Activity North China Agricultural Journal, 2014,29 (1): 195-202.).
The separation and identification of embodiment 1, white black streptomycete (Streptomyces alboniger) T22
1.1 strain isolation
The bacterial strain is separated from the special habitats soil sample of Qinghai Province Qinghai-Tibet Platean and is obtained.Bacterial strain is using dilution-plate method point
From the specific operation method is as follows: weighing pedotheque 10g, pour into the triangular flask equipped with small bead and 90mL sterile water, shake
5min is stood after swinging 30min, 10 times is successively diluted, is configured to 10 respectively-2、10-3、10-4、10-5、10-6Suspension, draw not
Each 0.1mL of suspension with concentration is added to Gause I culture medium and (K of final concentration of 75mg/L is added2CrO7) on plate, uniformly
Coating is placed on 28 DEG C of culture observations, and the single colonie that picking is different after 5-7 days, which is crossed, to be purified.Bacterial strain after purification is transferred to height
It is cultivated on family name's No.1 slant medium, 4 DEG C save backup.The bacterial strain (abbreviation bacterial strain T22) that number is T22 is taken to carry out following mirror
It is fixed.
The identification of 1.2 bacterial strains
1.2.1 strain morphology is observed
Bacterial strain T22 is inoculated on the culture medium of " streptomycete identification handbook " recommendation, 28 DEG C of culture 7-10d observation bacterial strains are raw
Long situation records the presence or absence of the color of aerial hyphae, the color of substrate mycelium and soluble pigment and color.By bacterial strain T22
Streak inoculation enters sterile cover slips on Gause I culture medium, with 45 degree of angle oblique cuttings, after 28 DEG C of culture 7d, takes out coverslip and exists
The morphological feature of optical microphotograph microscopic observation mycelia and spore.
Bacterial strain T22 Gause I agar, glucose asparagine agar, potato agar, yeast extract malt extract agar,
Well-grown on oatmeal agar medium, aerial hyphae white；In sucrose nitrate agar, inorganic salts starch agar medium
Upper aerial hyphae white is to light olive moccasin yellow, pale green lark.Bacterial strain T22 is in Gause I agar, sucrose nitrate fine jade
Without soluble pigment on rouge culture medium, in glucose asparagine agar, potato agar, yeast extract malt extract agar, oat
Soluble pigment dark yellow is to black (table 1) on powder agar, inorganic salts starch agar medium.It can be observed that bacterium under microscope
Strain T22 aerial hyphae is until flexible, spore chaining, oval (Fig. 2).T22 is initially identified as chain according to the above morphological feature
Mould category (Streptomyces sp.).
Wherein Gause I agar: soluble starch 20.0g, NaCl 0.5g, KNO31.0g, K2HPO40.5g,
MgSO4·7H2O 0.5g, FeSO40.02g, agar 18.0g, distilled water 1000mL, pH 7.2-7.4,121 DEG C of sterilizing 30min.
Sucrose nitrate agar: sucrose 50.0g, KNO30.2g, agar 18.0g, distilled water 1000mL, pH 7.2,121
DEG C sterilizing 30min.
Glucose asparagine agar: glucose 10.0g, asparagine 0.5g, beef extract 2.0g, K2HPO40.5g, fine jade
7.2,121 DEG C of sterilizing 30min of rouge 18.0g, distilled water 1000mL, pH.
Potato agar: potato leaching juice 200mL, sucrose 20.0g, agar 18.0g, distilled water 1000mL, pH 7.2-
7.4,121 DEG C of sterilizing 30min.
Yeast extract malt extract agar culture medium (ISP2): yeast extract 4.0g, malt extract 10.0g, glucose 4.0g, trace salt
Solution 1.0mL, agar 18.0g, 7.2,121 DEG C of sterilizing 30min of distilled water 1000mL, pH.Oatmeal agar (ISP3): oat
Piece 20.0g (filtering and moisturizing is to 1000mL), Trace salts solution 1.0mL, agar 18.0g after adding water 1000m L to boil 20min,
7.2,121 DEG C of sterilizing 30min of distilled water 1000mL, pH.
Inorganic salts starch agar (ISP4): soluble starch 10.0g, K2HPO42.0g, MgSO4·7H2O 1.0g,
(NH4)2SO42.0g, CaCO32.0g, Trace salts solution 1.0mL, agar 18.0g, 7.2,121 DEG C of distilled water 1000mL, pH
Sterilize 30min.
Trace salts solution: FeSO4·7H2O 0.1g, MnCl2·4H2O 0.1g, ZnSO4·7H2O 0.1g, distilled water
1000mL。
Morphological feature of the 1 bacterial strain T22 of table in different culture medium
| Culture medium | Aerial hyphae | Substrate mycelium | Soluble pigment |
| Gause I agar | White | It is micro- yellow to yellowish-brown | Nothing |
| Sucrose nitrate agar | White is to pale green lark | White | Nothing |
| Glucose asparagine agar | White | Black gray expandable | Black gray expandable |
| Potato agar | White | Yellow | Dark green black |
| Yeast extract malt extract agar (ISP2) | White | It is yellowish | Dark yellow |
| Oatmeal agar (ISP3) | White | It is colourless to yellowish | Dark yellow |
| Inorganic salts starch agar (ISP4) | White is to light olive moccasin yellow | It is yellowish | Black |
1.2.2 bacterial strain physio-biochemical characteristics detect
Physio-biochemical characteristics predominantly detect bacterial strain to the utilization power of primary carbon source and produce element activity.Bacterial strain T22 can
Using D-Glucose, L-arabinose, PEARLITOL 25C, inositol, maltose, gossypose, L- rhamnose, sucrose, wood cannot be utilized
Sugar, fructose.Bacterial strain T22 milk peptonizes, Starch Hydrolysis, gelatin liquefaction are positive, does not generate cellulose, melanin, hydrogen sulfide (table
2)。
2 bacterial strain T22 physio-biochemical characteristics of table
| Utilization of carbon source | As a result | Enzyme activity | As a result |
| D-Glucose | + | Milk peptonizes | + |
| L-arabinose | + | Starch Hydrolysis | + |
| PEARLITOL 25C | + | Gelatin liquefaction | + |
| Inositol | + | Cellulose | - |
| Maltose | + | Melanin | - |
| Gossypose | + | Hydrogen sulfide | - |
| L- rhamnose | - | ||
| Sucrose | - | ||
| Xylose | - | ||
| Fructose | - |
Note: "+" is the positive, and "-" is feminine gender.
1.2.3 bacterial strain molecular biology identification
Picking bacterial strain T22 is inoculated on Gause I agar medium, and 28 DEG C of culture 7d collect thallus.Using bacterial gene
After group extracts kit extracts bacterial strain T22 genomic DNA, bacterial universal primers 27f:5 '-AGA GTT TGA TCC TGG is utilized
The 16S rDNA sequence of CTC AG-3 ' and 1492r:5 '-TAC GGC TAC CTT GTT ACG ACT T-3 ' progress bacterial strain T22
PCR amplification.Pcr amplification product is recycled, after connection, conversion, identification, positive colony send Beijing Bo Maide biotech company to survey
Sequence, bacterial strain T22 have the 16S rDNA sequence of sequence 1 in sequence table.By gained sequence using Blast software GenBank into
Row tetraploid rice, Clustal X software carry out Multiple Sequence Alignment, Neighbor-Joining method building in MEGA5.0 software
Systematic evolution tree.
BLAST analyses and compares, and discovery belongs to streptomyces with the higher bacterial strain of bacterial strain T22 homology, chooses similitude
The 16S rDNA sequence of higher bacterial strain uses MEGA5.0 software building phylogenetic tree (Fig. 3).The result shows that bacterial strain with
Streptomyces alboniger (NR043228, AB184331) belongs to same branch, and similitude reaches 99%, and combines shape
Bacterial strain T22 is accredited as white black streptomycete (Streptomyces alboniger) by state feature and cultural characteristic.
White black streptomycete (Streptomyces alboniger) T22 has been preserved in the micro- life of China on May 25th, 2017
Object culture presevation administration committee common micro-organisms center, deposit number are CGMCC No.14190.Hereinafter referred to as white black streptomycete
(Streptomyces alboniger) T22 or bacterial strain T22.
The bacteriostatic activity of embodiment 2, white black streptomycete (Streptomyces alboniger) T22
2.1 agar block methods: Sterile pipette draws 5mL sterile water in for trying target pathogens culture inclined-plane, uses bamboo stick
Mycelia is scraped into preparation cause of disease bacteria suspension, being spread evenly across PDA with 1mL Sterile pipette absorption 0.1mL bacteria suspension, (cause of disease is true
Bacterium) or LB (pathogenetic bacteria) plate on.
It is botrytis cinerea (Botrytis cinerea), Botrytis cinerea germ (Botrytis for examination disease fungus
Cinerea), Botrytis cinerea (Botrytis cinerea), Monilinia fructicola (Monilinia fructicola), capsicum
Anthrax bacteria (Colletotrichum capsici), Alternaria alternata bacterium (Alternaria alternata), cucumber fusarium axysporum
Bacterium (Fusarium oxysporum f.sp.cucumerinum), cotton-wilt fusarium (Fusarium oxysporum
F.sp.vasinfectum), cabbage oxysporum (Fusarium oxysporum f.sp.conglutinans), wheat line are withered
Germ (Rhizoctonia cereali), fusarium moniliforme (Fusarium moniliforme).
It is eggplant ralstonia solanacearum (Ralstonia solanacearum), Black Rot on Chinese Cabbage bacterium for examination pathogenetic bacteria
(Xanthomonas campestris pv.campsetris), avenae subsp.citrull (Pseudomonas syringae
pv.lachrymans)。
Punch cuts white black streptomycete (Streptomyces that 7d is cultivated on No. 1 plate of Gao Shi, that diameter is 0.7cm
Alboniger) T22 agar block is inoculated on the plate for applying pathogen.25 DEG C of culture 4d of disease fungus, 28 DEG C of pathogenetic bacteria
After cultivating 2d, antagonism loop diameter is measured.The result shows that white black streptomycete (Streptomyces alboniger) T22 to include kind
Various plants pathogen including solanum cinerea bacterium shows Antagonism (Fig. 4) in good ware, and antagonism loop diameter is 12-26mm
(table 3).
White black streptomycete (Streptomyces alboniger) T22 of table 3 is to the antagonism loop diameter for trying pathogen
2.2 growth rate method
2.2.1 the preparation of the sterile ferment filtrate of white black streptomycete (Streptomyces alboniger) T22
White black streptomycete (Streptomyces alboniger) T22 grown on oese picking plate is inoculated into dress
Have in the 500mL triangular flask of 100mL liquid fermentation medium, 28 DEG C of shaking table 180rpm shaken cultivation 7d, collect fermentation liquid,
10000rpm, 4 DEG C of centrifugation 10min remove thallus, and fermented liquid supernatant liquid removes training using 0.22 μm of filtering with microporous membrane fermentation liquid
White black streptomycete (Streptomyces alboniger) the T22 thallus in object is supported, white black streptomycete (Streptomyces is obtained
Alboniger) the sterile ferment filtrate of T22 (the sterile ferment filtrate of abbreviation bacterial strain T22).Wherein, liquid fermentation medium Gao Shi
No.1 fluid nutrient medium: soluble starch 20g, KNO31g, K2HPO40.5g, MgSO4·7H2O 0.5g, NaCl 0.5g,
FeSO4·7H2O 0.01g distilled water constant volume to 1000mL, sterilize under the conditions of 121 DEG C 20min, obtains liquid fermentation medium.
2.2.2 prepared by pathogen bacteria cake
To cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerinum), the tomato ash for having cultivated 5d
Sterile water is respectively added in mildew bacterium (Botrytis cinerea) and the inclined-plane Botrytis cinerea germ (Botrytis cinerea)
Mycelia is scraped preparation pathogen bacteria suspension with bamboo stick by 4mL.0.1mL bacteria suspension, which is drawn, with 1mL Sterile pipette is coated on PDA
On plate, after 28 DEG C of culture 5d, pathogen bacterium colony covers with PDA plate, and diameter 7mm circle pathogen bacterium is made with aseptic card punch
Cake.
2.2.3 the sterile ferment filtrate bacteriostasis rate measurement of Antagonistic Fungi
By the white black streptomycete sterile ferment filtrate of (Streptomyces alboniger) T22 and it is cooled to 50 DEG C or so
Inverted plate after PDA culture medium 1:4 mixing by volume, it is above-mentioned for trying pathogen bacteria cake in plate center bacterium with sterilizing bamboo stick picking
Up, every processing is repeated 3 times, and replacing sterile ferment filtrate with sterile water is control.25 DEG C of culture 4d are surveyed with crossing method
Colony diameter is measured, bacteriostasis rate is calculated.Bacteriostasis rate %=(control colony diameter-processing colony diameter)/control colony diameter ×
100.It hardly grows on the PDA plate containing the sterile ferment filtrate of bacterial strain T22 for examination disease fungus, and is compareed in sterile water
It is grown on PDA plate normal (Fig. 5).The sterile ferment filtrate of bacterial strain T22 is to cucumber fusarium axysporum (Fusarium oxysporum
F.sp.cucumerinum), botrytis cinerea (Botrytis cinerea) and Botrytis cinerea germ (Botrytis
Cinerea) mycelia growth significantly inhibits, and bacteriostasis rate is respectively 79.1%, 84.6% and 85.5%.
Embodiment 3, white black streptomycete (Streptomyces alboniger) T22 cellulase-producing and chitinase activity
White black streptomycete (Streptomyces alboniger) T22 grown on oese picking plate is in production fiber
Plain enzyme culture medium and chitinase culture medium is produced, after 28 DEG C of culture 7d, the Congo red dye liquor 5mL of 2g/L is added into plate, contaminates
Deionized water is rinsed well after color 30min, then is decolourized with the NaCl solution of 1M, and deionized water is rinsed well, and periphery of bacterial colonies is observed
Form hydrolysis circle situation.
Cellulase-producing culture medium: cellulose powder 5.0g, (NH4)2SO42.0g, KH2PO41.0g, NaCl 0.5g,
MgSO4·7H2O 0.5g, agar 18.0g, distilled water 1000mL.
Produce chitinase culture medium: tobacco brown spot pathogen 2.5g, K2HPO40.7g, K2HPO40.3g, MgSO4·
7H2O0.5g, FeSO4·7H2O 0.01g, agar 18.0g, distilled water 1000mL.
The result shows that there is hydrolysis circle to be formed around white black streptomycete (Streptomyces alboniger) T22, make clear
Black streptomycete (Streptomyces alboniger) T22 can generate cellulase and chitinase (Fig. 6).
Embodiment 4, the sterile ferment filtrate of white black streptomycete (Streptomyces alboniger) T22 are to germination
It influences
The preparation of the 4.1 sterile ferment filtrates of white black streptomycete (Streptomyces alboniger) T22
White black streptomycete (Streptomyces alboniger) T22 grown on oese picking plate is inoculated into dress
Have in the 500mL triangular flask of 100mL liquid fermentation medium, 28 DEG C of shaking table 180rpm shaken cultivation 7d, collect fermentation liquid, uses
0.22 μm of filtering with microporous membrane fermentation liquid removes white black streptomycete (Streptomyces alboniger) the T22 bacterium in culture
Body obtains the sterile ferment filtrate of white black streptomycete (Streptomyces alboniger) T22.Wherein, liquid fermentation medium
For Gause I fluid nutrient medium: soluble starch 20g, KNO31g, K2HPO40.5g, MgSO4·7H2O 0.5g, NaCl
0.5g, FeSO4·7H2O 0.01g distilled water constant volume to 1000mL, sterilize under the conditions of 121 DEG C 20min, obtains liquid fermentation and culture
Base.
The 4.2 white black sterile ferment filtrate dilution preparations of streptomycete (Streptomyces alboniger) T22
The sterile ferment filtrate of white black streptomycete (Streptomyces alboniger) T22 of step 4.1 is used into nothing respectively
Bacterium water dilutes 10 times, 100 times, 200 times, 500 times, and it is sterile to obtain white black streptomycete (Streptomyces alboniger) T22
10 times of dilutions of ferment filtrate, white black sterile 100 times of the ferment filtrate dilutions of streptomycete (Streptomyces alboniger) T22
Liquid, the sterile 200 times of dilutions of ferment filtrate of white black streptomycete (Streptomyces alboniger) T22 and Bai Hei streptomycete
The sterile 500 times of dilutions of ferment filtrate of (Streptomyces alboniger) T22.
4.3 seed germination experiment
Full grains, tomato of the same size (good powder 1) seed are chosen, using the white black streptomycete of step 4.2
The sterile 10 times of dilutions of ferment filtrate of (Streptomyces alboniger) T22,100 times of dilutions, 200 times of dilutions and
500 times of dilutions impregnate seed, use the liquid fermentation medium (sterile) of isometric step 4.1 as control.Each processing
If repeating three times, repeat to set 10 seeds every time.28 DEG C are cultivated for 24 hours, collection seed, after the clean seed of aseptic water washing, by it
It is placed in the culture dish for being covered with two layers of sterile water-soaked filter paper, 28 DEG C of moisturizing culture 5d, counts germination percentage, measure plumular axis, radicle
Length.
The experimental results showed that the sterile fermentation of white black streptomycete (Streptomyces alboniger) T22 compared with the control
100 times of dilutions of filtrate significantly increase tomato plumular axis, radicle length, improve tomato seeds germinating energy, amplification is up to respectively
15.1%, 29.7% and 43.9%；White sterile 200 times of ferment filtrate of black streptomycete (Streptomyces alboniger) T22
The tomato seeds of dilution processing, plumular axis, radicle length and potentiality of seed increase 8.8%, 18.1% and compared with control
32.3% (table 4).
The sterile ferment filtrate of white black streptomycete (Streptomyces alboniger) T22 of table 4 germinates to tomato seeds
It influences
Embodiment 5, white black streptomycete (Streptomyces alboniger) T22 fermentation liquid prevent graw mold of tomato in vitro
Control effect
Using Leaf method, 30 tomato plant top of the same size blades are chosen, 2% (w/v) sodium hypochlorite disappears
Malicious 3min, tap water flushing are dried.Test sets 3 groups of processing, 10 blades of every group of processing, including is not inoculated with fluid nutrient medium and sprays
The processing of+ash arrhizus bacteria, bacterial strain T22 fermentation liquid spray+ash arrhizus bacteria processing, and 50% 1000 times of fludioxonil liquid sprays+ash arrhizus bacteria
It handles (Fig. 7).
1, bacterial strain T22 fermentation liquid sprays+ash arrhizus bacteria processing: with the white black streptomycete of the step 4.1 of embodiment 4
The sterile ferment filtrate of (Streptomyces alboniger) T22 carries out tomato leaf to spray processing, until being covered with blade,
Botrytis cinerea (Botrytis cinerea) pure culture biscuits involvng inoculation of diameter 7mm is cut in processing using sterilization punchers afterwards for 24 hours
Central vane.
2, it is not inoculated with fluid nutrient medium and sprays+ash arrhizus bacteria processing: with the liquid fermentation and culture of the step 4.1 of embodiment 4
The white black sterile fermentation filter of streptomycete (Streptomyces alboniger) T22 of the step 4.1 of base (sterile) alternative embodiment 4
Liquid, other operations are the same as step 1.As blank control.
3,50% fludioxonil, 1000 times of liquid spray+ash arrhizus bacteria sprays processing: it is real with 50% fludioxonil, 1000 times of liquid replacements
The sterile ferment filtrate of white black streptomycete (Streptomyces alboniger) T22 of the step 4.1 of example 4 is applied, other operations are same
Step 1.It is compareed as chemical agent.
Measurement processing and control tomato leaf lesion diameter after 7d, according to formula: (blank control is averaged scab preventive effect %=
Diameter-processing is averaged lesion diameter)/blank control is averaged lesion diameter × 100%, bacterial strain T22 fermentation liquid is calculated to tomato ash
The in vitro preventive effect of mildew.The result shows that white black streptomycete (Streptomyces alboniger) the sterile ferment filtrate of T22 is (sterile
Metabolin) spray processing tomato excised leaf can be substantially reduced graw mold of tomato lesion diameter, white black streptomycete
The in vitro preventive effect of the sterile ferment filtrate of (Streptomyces alboniger) T22 (sterile metabolin) processing graw mold of tomato reaches
To 55.1% (table 5).
Table 5, white black streptomycete (Streptomyces alboniger) the sterile ferment filtrate of T22 are in vitro to graw mold of tomato
Preventive effect
| Processing | Lesion diameter cm | Preventive effect % |
| It is not inoculated with fluid nutrient medium and sprays+ash arrhizus bacteria | 2.16±0.23 | - |
| Bacterial strain T22 fermentation liquid sprays+ash arrhizus bacteria | 0.97±0.21 | 55.1% |
| 50% 1000 times of fludioxonil liquid sprays+ash arrhizus bacteria | 0.84±0.09 | 61.1% |
<110>Beijing City Agriculture and Forestry Institute
The white black streptomycete of<120>one plants of disease prevention growth-promotings and its preparation and application of metabolite
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1310
<212> DNA
<213>white black streptomycete (Streptomyces alboniger)
<400> 1
agtggcgaac gggtgagtaa cacgtgggca atctgccctt cactctggga caagccctgg 60
aaacggggtc taataccgga taacacctcc actctcctga gtggaggtta aaagctccgg 120
cggtgaagga tgagcccgcg gcctatcagc ttgttggtga ggtaatggct caccaaggcg 180
acgacgggta gccggcctga gagggcgacc ggccacactg ggactgagac acggcccaga 240
ctcctacggg aggcagcagt ggggaatatt gcacaatggg cgaaagcctg atgcagcgac 300
gccgcgtgag ggatgacggc cttcgggttg taaacctctt tcagcaggga agaagcgaaa 360
gtgacggtac ctgcagaaga agcgccggct aactacgtgc cagcagccgc ggtaatacgt 420
agggcgcaag cgttgtccgg aattattggg cgtaaagagc tcgtaggcgg cttgtcacgt 480
cgggtgtgaa agcccggggc ttaaccccgg gtctgcattc gatacgggct agctagagtg 540
tggtagggga gatcggaatt cctggtgtag cggtgaaatg cgcagatatc aggaggaaca 600
ccggtggcga aggcggatct ctgggccatt actgacgctg aggagcgaaa gcgtggggag 660
cgaacaggat tagataccct ggtagtccac gccgtaaacg gtgggaacta ggtgttggcg 720
acattccacg tcgtcggtgc cgcagctaac gcattaagtt ccccgcctgg ggagtacggc 780
cgcaaggcta aaactcaaag gaattgacgg gggcccgcac aagcagcgga gcatgtggct 840
taattcgacg caacgcgaag aaccttacca aggcttgaca tacaccggaa agcatcagag 900
atggtgcccc ccttgtggtc ggtgtacagg tggtgcatgg ctgtcgtcag ctcgtgtcgt 960
gagatgttgg gttaagtccc gcaacgagcg caacccttgt tctgtgttgc cagcatgccc 1020
ttcggggtga tggggactca cagaagaccg ccggggtcaa ctcggaggaa ggtggggacg 1080
acgtcaagtc atcatgcccc ttatgtcttg ggctgcacac gtgctacaat ggcaggtaca 1140
atgagctgcg ataccgtgag gtggagcgaa tctcaaaaag cctgtctcag ttcggattgg 1200
ggtctgcaac tcgaccccat gaagtcggag ttgctagtaa tcgcagatca gcattgctgc 1260
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac gtcacgaaag 1310
Claims (13)
1. cause of disease bacteria inhibitor, it is characterised in that: the cause of disease bacteria inhibitor contains white black streptomycete, and the white black streptomycete is
White black streptomycete (Streptomyces alboniger), bacterial strain number are T22, are entrusted in Chinese microorganism strain preservation management
The number of registering on the books of member's meeting common micro-organisms center is CGMCC No.14190.
2. cause of disease bacteria inhibitor according to claim 1, it is characterised in that: the cause of disease bacteria inhibitor is to following at least one
Kind pathogen is inhibited:
A, ash arrhizus bacteria；
B, drupe brown rot germ；
C, Colletotrichum capsici；
D, wilt；
E, target bacterium；
F, rhizoctonia cerealis；
G, eggplant ralstonia solanacearum；
H, avenae subsp.citrull.
3. disease suppression agent, it is characterised in that: the disease suppression agent contains white black streptomycete described in claim 1.
4. disease suppression agent according to claim 3, it is characterised in that: the disease is following at least one:
A, gray mold；
B, drupe brown rot；
C, pepper anthracnose；
D, wilt disease；
E, target；
F, wheat sharp eyespot；
G, eggplant bacterial wilt；
H, angular leaf spot of cucumber.
5. white black streptomycete described in claim 1 is promoting the application in the sprouting of plant of Solanaceae seed.
6. application according to claim 5, it is characterised in that: the plant of Solanaceae is tomato platymiscium.
7. application according to claim 6, it is characterised in that: the tomato platymiscium is tomato.
8. white black streptomycete described in claim 1 is promoting the application in plant of Solanaceae seed plumular axis and/or radicle growth.
9. application according to claim 8, it is characterised in that: the plant of Solanaceae is tomato platymiscium.
10. application according to claim 9, it is characterised in that: the tomato platymiscium is tomato.
11. white black streptomycete described in claim 1 is improving the application in plant of Solanaceae potentiality of seed.
12. application according to claim 11, it is characterised in that: the plant of Solanaceae is tomato platymiscium.
13. application according to claim 12, it is characterised in that: the tomato platymiscium is tomato.
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