CN105462894B - Application of the sophora tonkinensis Gapnep endogenetic bacteria B29 in prevention and treatment Radix Notoginseng anthracnose - Google Patents
Application of the sophora tonkinensis Gapnep endogenetic bacteria B29 in prevention and treatment Radix Notoginseng anthracnose Download PDFInfo
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- CN105462894B CN105462894B CN201510979695.7A CN201510979695A CN105462894B CN 105462894 B CN105462894 B CN 105462894B CN 201510979695 A CN201510979695 A CN 201510979695A CN 105462894 B CN105462894 B CN 105462894B
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
Abstract
The invention discloses a kind of sophora tonkinensis Gapnep endogenetic bacteria B29, the classification naming of sophora tonkinensis Gapnep endophyte B29 is bulkholderia cepasea (Burkholderia sp.) B29, the 16S rDNA gene order table of bacterial strain is as described in SEQ ID NO.1, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, preservation date: on May 13rd, 2015, deposit number: CGMCC No.10807.The present invention for the first time from the root of medicinal plant sophora tonkinensis Gapnep separation screening to one plant of endogenetic bacteria (Burkholderia sp.) B29, the bacterium has very strong inhibiting effect to Radix Notoginseng anthrax bacteria, brings wide application prospect for the field of biological control of Radix Notoginseng fungal disease.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of sophora tonkinensis Gapnep endogenetic bacteria B29 is in prevention and treatment Radix Notoginseng anthracnose
In application.
Background technique
Modern agriculture is overly dependent upon the use of chemical pesticide, the release of a large amount of chemical pesticides to the prevention and treatment of plant disease
Not only there is long-term destruction to ecological environment, also quality of agricultural product is caused to decline, excessive pesticide residues, pathogen
Drug resistance and to problems such as people and animals' nocuousness.Safer, effective pest control method is found with great meaning
Justice can effectively be solved the above problems using the method for biology come controlling plant diseases.
Radix Notoginseng (Panax notoginseng F.H.chen) also known as pseudo-ginseng, invaluable etc. have significant promoting blood circulationization
The stasis of blood, detumescence ding-tong effect are a kind of Chinese tradition rare medicinal herbs.It is Chinese patent drug made of primary raw material as " Yunnan is white using Radix Notoginseng
Medicine " and " Pien Tze Huang " etc. are wide-spread.In recent years, the demand to Radix Notoginseng raw material is growing.But it is true in cultivation
Fungus diseases have seriously affected Panax notoginseng Growth.Currently, prevention and treatment fungal disease depends on chemical pesticide unduly, largely on notoginseng planting
The use of chemical pesticide not only affects the quality of Radix Notoginseng, also results in a large amount of pesticide residues of pseudo-ginseng.
Radix Notoginseng anthracnose is one of Panax notoginseng Growth common disease, and seedling stage, Adult plant can fall ill, the performance of blade early stage
For the raw yellowish-brown spot of blade, later period disease portion easily perforates rupture.It will form yellowish-brown recess spot, fruit after petiole and stem infection
Browning color rots after infection, and the cause of disease of the disease is Colletotriehum gloeosporioides (abbreviation
C.gloeosporioides)。
This fungal disease is one of the main reason for causing Radix Notoginseng product quality for many years and yield to decline.In Guangxi
Traditional Genuine producing area of Radix Notoginseng, due to low altitude area and the meteorological condition of high temperature and humidity, this disease is often simultaneous,
The a large amount of underproduction for causing Radix Notoginseng are even had no harvest, and are had brought tremendous economic losses to farmer.In order to control this nosomycosis
Evil, a large amount of chemical pesticide are used, and a large amount of pesticide residues of Radix Notoginseng product are caused, serious to polluted environment, greatly
People's health is threatened.Therefore, carrying out biological control Radix Notoginseng fungal disease is very urgent to the sustainable development of Radix Notoginseng industry
Cut with it is necessary.The Antagonistic Fungi for filtering out energy while this disease of antagonism thus is of great significance, and screens and then develops and uses
Antagonistic Fungi is also one of effective control most potential control measure of Radix Notoginseng fungal disease.
The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without
It should be considered as recognizing or implying in any form that the information constitutes existing skill already known to those of ordinary skill in the art
Art.
Summary of the invention
The purpose of the present invention is to provide a kind of sophora tonkinensis Gapnep endogenetic bacteria B29 to prevent and treat the application in Radix Notoginseng anthracnose, from
And the Radix Notoginseng anthracnose occurred during energy effectively preventing notoginseng planting, to improve the yield and quality of Radix Notoginseng.
To achieve the above object, technical solution provided by the invention is as follows:
The classification naming of a kind of sophora tonkinensis Gapnep endogenetic bacteria B29, sophora tonkinensis Gapnep endophyte B29 are bulkholderia cepasea
(Burkholderia sp.) B29, the 16S rDNA gene order table of bacterial strain is as described in SEQ ID NO.1, depositary institution: in
State's Microbiological Culture Collection administration committee common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number Institute of Microorganism, Academia Sinica, preservation date: on May 13rd, 2015, deposit number: CGMCC No.10807.
Preferably, the sophora tonkinensis Gapnep endogenetic bacteria B29 metabolite the preparation method comprises the following steps: by sophora tonkinensis Gapnep endogenetic bacteria
B29 is inoculated in NB fluid nutrient medium, and being placed in temperature is 28 DEG C, fermented and cultured 10 days under the conditions of revolving speed is 130r/min, gained
Then fermentation material ultrasound 40min is extracted with ethyl acetate 2 times, ethyl acetate phase is taken to carry out being concentrated under reduced pressure to give strain fermentation object
Ethyl acetate extract, as sophora tonkinensis Gapnep endogenetic bacteria B29 metabolite.
Preferably, the sophora tonkinensis Gapnep endogenetic bacteria B29 is inoculated in the fluid nutrient medium of NB containing 1000ml.
Preferably, NB fluid nutrient medium 3g containing beef extract, yeast extract 1g, peptone 5g, the sucrose 10g, training
Supporting base pH value is 7.0.
Application of the metabolite of sophora tonkinensis Gapnep endogenetic bacteria B29 as obtained by above-mentioned preparation in prevention and treatment Radix Notoginseng anthracnose.
Compared with prior art, the invention has the following beneficial effects:
The present invention for the first time from the root of medicinal plant sophora tonkinensis Gapnep separation screening to one plant of endogenetic bacteria (Burkholderia
Sp.) B29, the bacterium have very strong inhibiting effect to Radix Notoginseng anthrax bacteria, are the field of biological control band of Radix Notoginseng fungal disease
Carry out wide application prospect.
Detailed description of the invention
Fig. 1 is the plate opposite culture of sophora tonkinensis Gapnep endogenetic bacteria B29 of the present invention Yu Radix Notoginseng anthracnose, and wherein T is Radix Notoginseng charcoal
Subcutaneous ulcer germ (Colletotriehum gloeosporioides), a are blank control, and b is experimental group.
Fig. 2 is sophora tonkinensis Gapnep endogenetic bacteria B29 strain morphology feature of the present invention, wherein a1 is colonial morphology, and b1 is thallus
Form.
Fig. 3 is the phylogenetic tree that sophora tonkinensis Gapnep endogenetic bacteria B29 bacterial strain is constructed based on 16S rDNA gene order.
Fig. 4 is that the metabolite of bacterial strain sophora tonkinensis Gapnep endogenetic bacteria B29 according to the present invention is raw to the mycelia of Radix Notoginseng anthrax bacteria
Long inhibitory effect；Wherein the 1st, the 5th for blank control be not drug containing PDA plate；2nd, the 3rd, the 4th is positive right
According to powder of carbendazim；6th, the 7th, the 8th is the metabolite of bacterial strain B29, and concentration is respectively 2mg/ml, 4mg/ml, 8mg/
ml；T is Radix Notoginseng anthrax bacteria Colletotriehum gloeosporioides.
Specific embodiment
It is described in detail With reference to embodiment, it is to be understood that protection scope of the present invention is not had
The limitation of body embodiment.Experimental method used in following embodiments is conventional method unless otherwise specified.It is following
Material used in embodiment, reagent etc., are commercially available unless otherwise specified.Ethyl acetate is commercially available point
Analyse pure ethyl acetate.2X TagMasterMix is purchased from precious bioengineering Co., Ltd (Takara), Primer-1, Primer-2
By Hua Da gene chemical synthesis.
Embodiment 1
Separation, the screening and identification of bacterial strain
One, the separation of bacterial strain
Material to be tested: the wild sophora tonkinensis Gapnep of Guangxi Tiandeng County In Limestone Area is picked up from.
Strains tested: it is provided by plant pathology research institute, Guangxi University.
Culture medium: 1000ml NA culture medium: beef extract 3g, yeast extract 1g, peptone 5g, sucrose 10g, agar 15g,
pH 7.0。
Surface sterilization: a length of 6-8cm, the sophora tonkinensis Gapnep root flowing water that width is 1-2cm fresh and healthy are rinsed into 30min to rush
Clean silt air-dries surface moisture, moves on to superclean bench then by clean sophora tonkinensis Gapnep root with rinsed with sterile water 2 times,
It is that 75% ethyl alcohol impregnates 1min by sophora tonkinensis Gapnep root volumetric concentration, rinsed with sterile water 2 times, sodium hypochlorite (has under aseptic condition
Imitate chlorine 1%) 2min is impregnated, sterile washing 3 times, it is spare that sterile blotting paper blots surface.
The separation of bacterial strain purifies: the good sophora tonkinensis Gapnep root of surface sterilization, under aseptic condition, scrapes off epidermis with sterile wood chip,
The both ends of sophora tonkinensis Gapnep root are cut off with sterile secateurs, the sterile pocket knife of remaining part and aseptic nipper separate xylem and bast,
The tissue block for taking 0.5cm long respectively is shredded with sterile secateurs and sterile water is added to grind, and after grinding sufficiently plus sterile water is settled to
5ml simultaneously stands 10min, takes 0.5ml supernatant gradient dilution 10~105Times, take 100 μ L dilutions to be applied to NA plate respectively
On, each diluted concentration is repeated 3 times, and takes last time rinsing liquid to be coated on NA plate, as negative control.NA plate is set
In 28 DEG C of 24~96h of constant temperature incubation of incubator, picking different shape bacterium colony is crossed purifying repeatedly, by bacterial strain, that is, Vietnam of purifying
Chinese scholartree endogenetic bacteria is saved with 20% glycerol.
The sophora tonkinensis Gapnep endogenetic bacteria being separated to is inoculated on LA plate, it is stand-by after being cultivated at 28 DEG C for 24 hours.By Radix Notoginseng charcoal
Subcutaneous ulcer germ is inoculated in PDA plate, stand-by after cultivating 3 days at 28 DEG C.
Two, it screens
Using tablet face-off method to the primary dcreening operation for carrying out thallus bacteriostatic activity for examination sophora tonkinensis Gapnep endogenetic bacteria.
Firstly, 6mm bacteria cake is made in Radix Notoginseng anthrax bacteria with sterilization punchers under aseptic condition；It, will in processing group
Radix Notoginseng anthrax bacteria bacteria cake is transferred to the center of the culture dish containing NA, then in the position apart from bacteria cake 2cm, gets over what is be separated to
Southern Chinese scholartree endogenetic bacteria bacterial strain connects a bacterium line, and every plant of endogenetic bacteria repeats 3 wares；In control group, Radix Notoginseng anthracnose is only connect
Bacterium bacteria cake does not connect sophora tonkinensis Gapnep endogenetic bacteria, repeats 3 wares.Then, it is cultivated at 28 DEG C, periodic observation.To fungi in control group
When covering with culture dish, in processing group, Radix Notoginseng anthrax bacteria bacteria cake center is measured to three between sophora tonkinensis Gapnep endogenetic bacteria line center
The growth radius of seven anthrax bacterias is processing growth radius；In control group, the growth radius of Radix Notoginseng anthrax bacteria is control
Grow radius.Finally, according to the following formula, calculating bacteriostasis rate:
It compares increment=control and grows radius-bacteria cake radius
It handles increment=processing and grows radius-bacteria cake radius
As a result 3 plants are obtained to all very strong antagonism sophora tonkinensis Gapnep endogenetic bacteria bacterial strain of Radix Notoginseng anthrax bacteria inhibiting effect,
In one plant of entitled B29, be 64% to the inhibiting rate of Radix Notoginseng anthrax bacteria.
Three, it identifies
(1) strain morphology feature
Colony morphology characteristic observation: by strain inoculated to be identified on NA culture medium, 28 DEG C of cultures are placed in for 24 hours, observation
The colony morphology characteristic of bacterial strain.
Morphological features observation: the above-mentioned bacterial strain cultivated on NA culture medium for 24 hours is taken to carry out Gram's staining and mirror
Inspection, by observed form result microphotograph, as shown in Figure 2.
(2) bacterial strain 16S rDNA sequence and its phylogenetic analysis
The preparation of DNA profiling:
Reagent: (1) lysis buffer: Tris-Ac (pH 7.8) 40mM, NaAc 20mM, EDTA 1mM, SDS1% (w/
v)；
(2) 5M NaCl solution.
DNA is extracted:
(a) 1.5ml overnight bacterial culture solution culture is placed in microcentrifugal tube (EP pipe), 12000rpm centrifugation
0.5min discards supernatant liquid, retains sediment；
(b) 400 μ l lysis buffers are added in sediment, is blown and beaten repeatedly with suction pipe and is allowed to be resuspended；
(c) 200ul 5M NaCl is added, mixes well, 12000rpm is centrifuged 10min, takes 600 μ l supernatants；
(d) isometric phenol/chloroform (1:1) is added, mixes, is centrifuged (12000rpm, 10min), supernatant is transferred to separately
In one clean EP pipe；
(e) isometric chloroform is added in the supernatant obtained into step (d), mixes, centrifugation (12000rpm,
10min), supernatant is transferred in another clean EP pipe；
(f) isometric isopropanol is added in gained supernatant into step (e), mixes, is placed in room temperature 10min, be centrifuged
(12000rpm, 15min) discards supernatant liquid, retains sediment；
(g) it is 70% ethanol washing step (f) gained sediment with volumetric concentration, dries, as DNA；
(h) DNA done in step (g) is dissolved in 30 μ l distilled water (ddH2O in), -20 DEG C of preservations.
PCR amplification 16S rDNA sequence:
(1) PCR instrument: ABI 3730-XL DNA sequencer (Applied Biosystems, USA)；
(2) amplimer: 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') is as shown in SEQ ID NO.2 and 1492R
(5 '-GGTTACCTTGTTACGACT-3 ') are as shown in SEQ ID NO.3；
(3) amplification system:
16S rDNA sequence PCR amplification system is as shown in table 1:
Table 1.
| Reactant | Sample-adding amount |
| 2X TagMasterMix | 25μL |
| Primer-1 | 1μL |
| Primer-2 | 1μL |
| Template DNA | 0.5μL |
| ddH2O | 22.5μL |
| React total volume | 50μL |
Note: the Template DNA in table 1 is that DNA profiling is made in DNA obtained by above-mentioned steps (h).
PCR reaction condition is as shown in table 2:
Table 2.
Note: step 2 carries out 30 circulations in table 2.
The electrophoresis detection of pcr amplification product:
The Ago-Gel (the 5 μ l/100ml containing Goldview) that deposition condition is 1%, 1 × TBE electrophoretic buffer, 90V
Electrophoresis 1 hour, PCR product applied sample amount was 3 μ L, point sample after mixing with 1 μ L Loading dye.In the ultraviolet lower sight of 254nm
It examines as a result, determining that amplified fragments are long using the DL1000DNA Marker of TaKaRa company as nucleic acid standard molecular weight object of reference
Degree.Amplified production band should be on the position of reference substance 400-700bp.
PCR product purifying and sequencing: it is carried out by Shenzhen Huada Genetic Technology Co., Ltd.
The building of systematic evolution tree:
The 16S rDNA sequence of the bacterium measured in the 16S rDNA sequence surveyed and GenBank gene pool is carried out
It compares, recalls the 16S rDNA sequence with the higher bacterial strain of its sequence homology.Using Clustal-W software to homologous sequence
More matching arrangements are carried out, the building systematic evolution tree of systematic evolution tree are carried out by 6.0 software of MEGA, as shown in Figure 3.
As a result:
(1) strain morphology feature
Colony morphology characteristic: bacterial strain bacterium colony is in yellow green on NA plate, and surface is smooth, protuberance, and neat in edge is round,
It is 1-2mm that 5 days diameters are cultivated on NA plate, as shown in a1 in Fig. 2.
Morphological features: Gram-negative is negative staining, no gemma, thallus direct rod shape, and size is (0.41~0.45)
μ m (0.96~1.29) μm, as shown in b1 in Fig. 2.
(2) bacterial strain 16S rDNA sequence and its phylogenetic analysis
Using primer 2 7F and 1492R, the segment of a 1300-1400bp is amplified from strain gene group DNA, through surveying
Sequence is simultaneously compared sequencing result by BLASTn in GenBank, utilizes the adjoining algorithm (Neighbor-joining of MEGA6
NJ it) carries out network analysis and constructs systematic evolution tree.The result shows that 16S rDNA sequence and Burkholderia
The corresponding sequence homology of sp.SAP27_1 reaches 99%.Comprehensive morphological and molecular biological characteristics, by bacterial strain Preliminary Identification
For Burkholderia sp..
Embodiment 2
Sophora tonkinensis Gapnep endogenetic bacteria B29 metabolite to the inhibiting effect one of Radix Notoginseng anthrax bacteria, the fermented and cultured of bacterial strain and
The extraction of metabolite
By sophora tonkinensis Gapnep endogenetic bacteria B29 be inoculated in the fluid nutrient medium of NB containing 1000ml (beef extract 3g, yeast extract 1g,
Peptone 5g, sucrose 10g, pH 7.0) 2 liters of conical flasks in, be placed in temperature is 28 DEG C, revolving speed is 130r/min shaking table hair
Ferment culture 10 days, then gained fermentation material ultrasonic echography 40min is extracted with ethyl acetate 2 times, takes gained acetic acid twice
Ethyl ester is mutually concentrated under reduced pressure to give the ethyl acetate extract of strain fermentation object, as sophora tonkinensis Gapnep endogenetic bacteria B29 metabolite.
Two, inhibiting effect of the sophora tonkinensis Gapnep endogenetic bacteria B29 metabolite to the mycelia growth of Radix Notoginseng anthrax bacteria
Ethyl acetate extract with mycelia growth method measurement bacterial strain is sophora tonkinensis Gapnep endogenetic bacteria B29 metabolite, right
The inhibitory activity of Radix Notoginseng anthrax bacteria mycelia growth,.By the metabolite of bacterial strain B29 and powder of carbendazim (positive control) point
It is not made containing 2mg/mL, 4mg/mL, the drug containing tablet of the metabolite of 8mg/mL concentration B29.Aseptically, with punching
6mm bacteria cake is made in disease fungus by device, and Radix Notoginseng anthrax bacteria bacteria cake is connected to each drug containing tablet center for processing, with not drug containing
The Radix Notoginseng anthrax bacteria bacteria cake that plate center connects is negative control, and in triplicate, all plates are placed in 28 for all processing and control
DEG C culture.Drug concentration is arranged by the Field information concentration of powder of carbendazim.When negative control covers with culture dish, survey respectively
Amount control bacterium colony and the growth diameter for handling bacterium colony, and according to the following formula, calculate inhibiting rate:
Negative control increment=negative control growth diameter-bacteria cake diameter
Handle increment=processing growth diameter-bacteria cake diameter
Three, minimal inhibitory concentration of the sophora tonkinensis Gapnep endogenetic bacteria B29 metabolite to Radix Notoginseng anthrax bacteria
With the metabolite of broth dilution method determination bacterial strain B29 to the minimal inhibitory concentration of Radix Notoginseng anthrax bacteria.By PDA
The Radix Notoginseng anthrax bacteria pure culture biscuits involvng inoculation of culture 5 days is in the PDB fluid nutrient medium containing 0.2% Tween 80 (v/v), and 28 DEG C,
150r/min shaking table culture 7 days, culture is then gone into triangular flask, and pours into the aseptic double-distilled water containing 0.2% Tween 80,
30min is stirred with magnetism stick, solution is filtered with sterile gauze, obtains spores solution, and with blood counting chamber by spore concentration
Adjust every milliliter 104A spore is spare.The metabolite of bacterial strain B29 is produced with the metabolism that 1%DMSO is dissolved into 80mg/mL
Object concentration for the treatment of takes 5 sterile test tubes to be sequentially arranged on rack for test tube, and number is 1,2,3,4,5 respectively, and every pipe is separately added into
1ml contains the aseptic double-distilled water of 0.2% Tween 80, and number then is added in the metabolite solution of the bacterial strain of 1ml 80mg/mL and is
Twice of serial dilution in 1 test tube and is successively carried out in each pipe.The above-mentioned gained spores solution (10 of 1ml4A/ml) add respectively
Enter in each pipe, to obtain the metabolite concentration for the treatment of of final B29: 20mg/ml (1 test tube of number), 10mg/ml (number 2
Test tube), 5mg/ml (3 test tube of number), 2.5mg/ml (4 test tube of number), 1.25mg/ml (5 test tube of number).The 1% of 1ml
The 8mg/mL Flusilazole of DMSO and 1ml replace the metabolite of B29 to execute above-mentioned treatment process respectively and as negative right
According to and positive control, it is all processing and control in triplicate.Finally, 28 DEG C of each pipe, 150r/min shaking table culture 5 days.Sophora tonkinensis Gapnep
The MIC value of the metabolite of endogenetic bacteria B29 is to completely inhibit the minimum crude extract concentration of Radix Notoginseng anthrax bacteria visible growth.
As a result:
In the percent inhibition such as table 3 that bacterial strain sophora tonkinensis Gapnep endophyte B29 metabolite grows Radix Notoginseng anthrax bacteria mycelia
It is shown:
Table 3.
Note: positive control powder of carbendazim contains 50% carbendazim in table；* indicates that data pass through single factor test variance point in table
The LSD of analysis relatively after, the metabolite and positive control powder of carbendazim of bacterial strain B29 is under same concentrations, in P0.05In level
Has significant difference.
The suppression result that the metabolite of bacterial strain sophora tonkinensis Gapnep endophyte B29 grows Radix Notoginseng anthrax bacteria mycelia shows bacterium
The metabolite of strain sophora tonkinensis Gapnep endophyte B29 all has extraordinary inhibitory effect to the mycelia growth of Radix Notoginseng anthrax bacteria, from
Table 3 is it can be seen that the percent inhibition that the metabolite of B29 grows Radix Notoginseng anthrax bacteria C. gloeosporioides mycelia
It is 100%.Compared with positive control powder of carbendazim, the metabolite of bacterial strain B29 is to Radix Notoginseng anthrax bacteria
C.gloeosporioides mycelia growth inhibitory effect with compare equally.
The minimal inhibitory concentration that bacterial strain B29 metabolite grows Radix Notoginseng anthrax bacteria is as shown in table 4:
Table 4.
| Processing | MIC(mg/ml) |
| C.gloeosporioides | |
| Flusilazole | 0.25 |
| B29 | 2.5 |
Note: positive control Flusilazole active constituent content is 400mg/mL in table.
The minimal inhibitory concentration test knot that the metabolite of bacterial strain sophora tonkinensis Gapnep endophyte B29 grows Radix Notoginseng anthrax bacteria
Fruit shows that the metabolite of bacterial strain sophora tonkinensis Gapnep endophyte B29 all has stronger inhibiting effect to Radix Notoginseng anthrax bacteria, from table 4
It can be seen that the metabolite of sophora tonkinensis Gapnep endophyte B29 is antibacterial dense to the minimum of Radix Notoginseng anthrax bacteria C.gloeosporioides
Degree is 2.5mg/ml, is 10 times of positive control.
As may be known from Table 3 and Table 4, very strong restraining epiphyte is contained in the metabolite of bacterial strain sophora tonkinensis Gapnep endophyte B29 or is killed true
The ingredient of bacterium, therefore, in the bionomic control of Radix Notoginseng anthrax bacteria, bacterial strain sophora tonkinensis Gapnep endophyte B29 has potentiality outstanding.
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These are retouched
It states and is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can carry out very much
Change and changes.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and in fact
Border application, so that those skilled in the art can be realized and utilize a variety of different exemplary embodiment party of the invention
Case and various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (3)
1. a kind of application of metabolite of sophora tonkinensis Gapnep endogenetic bacteria B29 in prevention and treatment Radix Notoginseng anthracnose, it is characterised in that: Vietnam
The classification naming of Chinese scholartree endophyte B29 be bulkholderia cepasea (BurkholderiaSp.) B29, deposit number: CGMCC No.
10807；
The preparation method of the metabolite of the sophora tonkinensis Gapnep endogenetic bacteria B29: sophora tonkinensis Gapnep endogenetic bacteria B29 is inoculated in NB liquid
In body culture medium, being placed in temperature is 28 DEG C, fermented and cultured 10 days under the conditions of revolving speed is 130r/min, gained fermentation material ultrasound
Then 40min is extracted with ethyl acetate 2 times, ethyl acetate phase is taken to carry out the ethyl acetate for being concentrated under reduced pressure to give strain fermentation object
Crude extract, as sophora tonkinensis Gapnep endogenetic bacteria B29 metabolite.
2. application of the metabolite of sophora tonkinensis Gapnep endogenetic bacteria B29 in prevention and treatment Radix Notoginseng anthracnose according to claim 1,
Be characterized in that: the sophora tonkinensis Gapnep endogenetic bacteria B29 is inoculated in 1000mL NB fluid nutrient medium.
3. application of the metabolite of sophora tonkinensis Gapnep endogenetic bacteria B29 in prevention and treatment Radix Notoginseng anthracnose according to claim 2,
It is characterized in that: 3g containing beef extract, yeast extract 1g, peptone 5g, sucrose 10g in the 1000mL NB fluid nutrient medium, culture
Base pH value is 7.0.
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| CN104877943A (en) * | 2015-06-11 | 2015-09-02 | 福建农林大学 | Antagonistic bacterium for controlling radix rehmannia root rot and application of antagonistic bacterium |
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| CN105462896A (en) * | 2015-12-23 | 2016-04-06 | 广西大学 | Application of sophora tonkinensis endophytic bacterium B29 in preventing and controlling panax notoginseng black spot |
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| CN104877943A (en) * | 2015-06-11 | 2015-09-02 | 福建农林大学 | Antagonistic bacterium for controlling radix rehmannia root rot and application of antagonistic bacterium |
| CN105462893A (en) * | 2015-12-23 | 2016-04-06 | 广西大学 | Application of sophora tonkinensis endophytic bacterium B29 in preventing and controlling panax notoginseng root rot |
| CN105462896A (en) * | 2015-12-23 | 2016-04-06 | 广西大学 | Application of sophora tonkinensis endophytic bacterium B29 in preventing and controlling panax notoginseng black spot |
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