CN105462897B - Application of the sophora tonkinensis Gapnep endogenetic bacteria B21 in prevention and treatment Radix Notoginseng anthracnose - Google Patents
Application of the sophora tonkinensis Gapnep endogenetic bacteria B21 in prevention and treatment Radix Notoginseng anthracnose Download PDFInfo
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Abstract
The invention discloses a kind of sophora tonkinensis Gapnep endogenetic bacteria B21, the classification naming of sophora tonkinensis Gapnep endophyte B21 is bulkholderia cepasea (Burkholderia sp.) B21, 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.10806.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.) B21, 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 B21 is in prevention and treatment Radix Notoginseng anthracnose
In application.
Background technique
Modern agriculture is overly dependent upon the use of chemical pesticide to the prevention and treatment of plant disease, and the release of a large amount of chemical pesticides is not
Only there is long-term destruction to ecological environment, also quality of agricultural product is caused to decline, excessive pesticide residues, the drug resistance of pathogen
Property and to problems such as people and animals' nocuousness.It finds safer, effective pest control method to be of great significance, utilize
The method of biology, which carrys out controlling plant diseases, effectively to solve the above problems.
Radix Notoginseng (Panax notoginseng F.H.chen) also known as pseudo-ginseng, invaluable etc., have significant activating microcirculation and removing stasis medicinal,
Detumescence ding-tong effect is a kind of Chinese tradition rare medicinal herbs.Using Radix Notoginseng as Chinese patent drug made of primary raw material such as " Yunnan Baiyao "
" Pien Tze Huang " etc. is wide-spread.In recent years, the demand to Radix Notoginseng raw material is growing, but the nosomycosis in cultivation
Evil has seriously affected Panax notoginseng Growth.Currently, prevention and treatment fungal disease depends on chemical pesticide unduly on notoginseng planting, a large amount of chemistry
The use of 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, and blade early stage is shown as
Blade gives birth to yellowish-brown spot, and later period disease portion easily perforates rupture.It will form yellowish-brown recess spot, fruit infection after petiole and stem infection
Browning color rots afterwards, 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 three
Seven traditional Genuine producing area, due to low altitude area and the meteorological condition of high temperature and humidity, this disease is often simultaneous, causes
The a large amount of underproduction of Radix Notoginseng are even had no harvest, and are had brought tremendous economic losses to farmer.In order to control this fungal disease, greatly
The chemical pesticide of amount is used, and a large amount of pesticide residues of Radix Notoginseng product are caused, serious to polluted environment, is greatly threatened
People's health.Therefore, development biological control Radix Notoginseng fungal disease is very urgent to the sustainable development of Radix Notoginseng industry and must
It wants.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
It is also effectively to control one of 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 answering
When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.
Summary of the invention
The purpose of the present invention is to provide a kind of sophora tonkinensis Gapnep endogenetic bacteria B21 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 B21, sophora tonkinensis Gapnep endophyte B21 are bulkholderia cepasea
(Burkholderia sp.) B21, the 16S rDNA gene order table of bacterial strain is as described in SEQ ID NO.1, depositary institution: China
Microbiological Culture Collection administration committee 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.10806.
Preferably, the sophora tonkinensis Gapnep endogenetic bacteria B21 metabolite the preparation method comprises the following steps: by sophora tonkinensis Gapnep endogenetic bacteria
B21 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
Fermentation material is added 2 times of fermentation material of methanol and ultrasound 40min, is then centrifuged for after drying is concentrated under reduced pressure, and takes supernatant decompression dense
Contracting obtains the methanol crude extract of strain fermentation object, as sophora tonkinensis Gapnep endogenetic bacteria B21 metabolite.
Preferably, the sophora tonkinensis Gapnep endogenetic bacteria B21 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 B21 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.) B21, the bacterium have very strong inhibiting effect to Radix Notoginseng anthrax bacteria, bring for the field of biological control of Radix Notoginseng fungal disease
Wide application prospect.
Detailed description of the invention
Fig. 1 is the plate opposite culture of sophora tonkinensis Gapnep endogenetic bacteria B21 of the present invention Yu Radix Notoginseng anthracnose, and wherein F 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 B21 strain morphology feature of the present invention, wherein a1 is colonial morphology, and b1 is thallus shape
State.
Fig. 3 is the phylogenetic tree that sophora tonkinensis Gapnep endogenetic bacteria B21 bacterial strain is constructed based on 16S rDNA gene order.
Fig. 4 is that the metabolite of bacterial strain sophora tonkinensis Gapnep endogenetic bacteria B21 according to the present invention is raw to the mycelia of Radix Notoginseng anthrax bacteria
Long inhibitory effect；Wherein the 1st it is classified as the blank control i.e. not PDA plate of drug containing；1st arranges the 2nd~6 as the more bacterium of positive control
Clever pulvis；2nd arranges the B21 metabolite that the 2nd~6 is bacterial strain, and concentration is respectively 0.5mg/mL, 1mg/mL, 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 by specific
The limitation of embodiment.Material as used in the following examples, reagent etc., unless otherwise specified, commercially
It arrives.Methanol is the commercially available pure methanol of analysis.2X TagMasterMix is purchased from precious bioengineering Co., Ltd (Takara), Primer-
1, Primer-2 is 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 of 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, width is dry to rush for the sophora tonkinensis Gapnep root flowing water flushing 30min of 1-2cm fresh and healthy
Net silt air-dries surface moisture, superclean bench is moved on to, sterile then by clean sophora tonkinensis Gapnep root with rinsed with sterile water 2 times
Under the conditions of, by sophora tonkinensis Gapnep root volumetric concentration be 75% ethyl alcohol impregnate 1min, rinsed with sterile water 2 times, sodium hypochlorite (effective 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, uses
Sterile secateurs cuts off the both ends of sophora tonkinensis Gapnep root, and the sterile pocket knife of remaining part and aseptic nipper separate xylem and bast, point
The tissue block for not taking 0.5cm long is shredded with sterile secateurs and sterile water is added to grind, and after grinding sufficiently plus sterile water is settled to 5ml
And 10min is stood, take 0.5ml supernatant gradient dilution 10~105Times, take 100 μ L dilutions to be applied on NA plate respectively, often
A diluted concentration is repeated 3 times, and takes last time rinsing liquid to be coated on NA plate, as negative control.NA plate is placed in culture
28 DEG C of 24~96h of constant temperature incubation of case, picking different shape bacterium colony are crossed purifying repeatedly, by the bacterial strain of purifying, i.e., raw in sophora tonkinensis Gapnep
Bacterium 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 anthrax
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；In processes, by Radix Notoginseng
Anthrax bacteria bacteria cake is transferred to the center of the culture dish containing NA, will be in the sophora tonkinensis Gapnep that be separated to then in the position apart from bacteria cake 2cm
Endophytic bacteria bacterial strain connects a bacterium line, and every plant of endogenetic bacteria repeats 3 wares；In control group, Radix Notoginseng anthrax bacteria bacteria cake is only connect,
Sophora tonkinensis Gapnep endogenetic bacteria is not connect, repeats 3 wares.Then, it is cultivated at 28 DEG C, periodic observation.Culture is covered with to fungi in control group
When ware, in processing group, measurement Radix Notoginseng anthrax bacteria bacteria cake center to Radix Notoginseng anthracnose between sophora tonkinensis Gapnep endogenetic bacteria line center
The growth radius of bacterium is processing growth radius；In control group, the growth radius of Radix Notoginseng anthrax bacteria is control growth radius.Most
Afterwards, according to the following formula, bacteriostasis rate is calculated:
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 B21, 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: taking the above-mentioned bacterial strain cultivated on NA culture medium for 24 hours to carry out Gram's staining and microscopy,
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: 1% (w/ of Tris-Ac (pH 7.8) 40mM, NaAc 20mM, EDTA 1mM, SDS
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, be centrifuged (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.
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 observation of 254nm as a result, using the DL1000DNA Marker of TaKaRa company as nucleic acid standard molecular weight
Object of reference determines expanding fragment length.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 compared
It is right, correlated series are downloaded according to comparison result.It is by the adjoining algorithm (Neighbor-joining NJ) of MEGA 6.0
System is analyzed and constructs systematic evolution tree, as shown in Figure 3.
As a result:
(1) strain morphology feature
Colony morphology characteristic: bacterial strain bacterium colony is creamy white on NA plate, and surface is smooth, protuberance, and neat in edge is round,
It is 2-3mm 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 (0.43~0.52) μ
M × (0.96~1.32) μm, as shown in b1 in Fig. 2.
(2) bacterial strain 16S rDNA sequence and its phylogenetic analysis
Using bacterial universal primers 27F and 1492R, a 1300-1400bp size is amplified from strain gene group DNA
Segment, compared through sequencing and by sequencing result by BLASTn in GenBank.The result shows that bacterial strain B21 with
Bacterium in Burkholderia has very high base sequence similitude, therefore downloads Burkholderia in all GenBank
Reference strain sequence, be used for Phylogenetic Analysis, with the Pandoraea thiooxydans in Pandoraea
(NR116008) and Pandoraea pulmonicola (NR115186) is used as outer group.In the systematic evolution tree of building, B21
It gets together to form end branch with Burkholderia anthina (KM019865).Base similarity system design the result shows that,
B21 differs a base, sequence similarity 99.7% with Burkholderia anthina (KM019865).Comprehensive morphological
And molecular biological characteristics, bacterial strain is initially identified as Burkholderia sp..
Embodiment 2
Inhibiting effect of the Metabolite to Radix Notoginseng anthrax bacteria
One, the extraction of the fermented and cultured of bacterial strain and B21 metabolite
Sophora tonkinensis Gapnep endogenetic bacteria B21 is inoculated in the fluid nutrient medium of NB containing 1000ml (beef extract 3g, yeast extract 1g, egg
White peptone 5g, sucrose 10g, pH 7.0) 2 liters of conical flasks in, be placed in the training of temperature is 28 DEG C, revolving speed is 130r/min shaker fermentation
Support 10 days, gained fermentation material be concentrated under reduced pressure it is dry after, 2 times of fermentation material of methanol is added and uses ultrasonic echography 40min, it is ultrasonic
It is centrifuged, gained supernatant after centrifugation is taken to be concentrated under reduced pressure to give the methanol crude extract of strain fermentation object, it is raw as in sophora tonkinensis Gapnep
Bacterium B21 metabolite.
Two, inhibiting effect of the sophora tonkinensis Gapnep endogenetic bacteria B21 metabolite to the mycelia growth of Radix Notoginseng anthrax bacteria
The inhibitory activity that Radix Notoginseng anthrax bacteria mycelia is grown with the B21 metabolite of mycelia growth method measurement bacterial strain.It will
The B21 metabolite and powder of carbendazim (positive control) of bacterial strain are respectively prepared containing 0.5mg/mL, 1mg/mL, 2mg/mL, 4mg/
The drug containing tablet of mL, 8mg/mL concentration B21 metabolite.Aseptically, 6mm bacterium is made in disease fungus with punch
Cake, it is processing, the Radix Notoginseng anthracnose connect with not drug containing tablet center that Radix Notoginseng anthrax bacteria bacteria cake, which is connected to each drug containing tablet center,
Bacterium bacteria cake is negative control, and in triplicate, all plates are placed in 28 DEG C of cultures for all processing and control.Drug concentration presses carbendazim
The Field information concentration of pulvis is arranged.When negative control covers with culture dish, the life of measurement control bacterium colony and processing bacterium colony respectively
Long diameter, 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 B21 metabolite to Radix Notoginseng anthrax bacteria
With the B21 metabolite of broth dilution method determination bacterial strain to the minimal inhibitory concentration of Radix Notoginseng anthrax bacteria.PDA is trained
Feeding 5 days Radix Notoginseng anthrax bacteria pure culture biscuits involvng inoculations are 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, with magnetism
Stick stirs 30min, and solution is filtered with sterile gauze, obtains spores solution, and arrived spore concentration adjusting with blood counting chamber
Every milliliter 104A spore is spare.The metabolite of bacterial strain is dissolved into the metabolite concentration for the treatment of of 80mg/mL with 1%DMSO,
5 sterile test tubes are taken to be sequentially arranged on rack for test tube, number is 1,2,3,4,5 respectively, and every pipe is separately added into 1ml and spits containing 0.2%
The aseptic double-distilled water of temperature 80, then by the bacterial strain methanol crude extract solution of 1ml 80mg/mL be added in the test tube that number is 1 and according to
It is secondary that twice of serial dilution is carried out in each pipe.The above-mentioned gained spores solution (10 of 1ml4A/ml) it is separately added into each pipe, to obtain
Obtain B21 metabolite concentration for the treatment of finally: 20mg/ml (1 test tube of number), 10mg/ml (2 test tube of number), 5mg/ml (number
3 test tubes), 2.5mg/ml (4 test tube of number), 1.25mg/ml (5 test tube of number).The 8mg/mL fluorine silicon of the 1%DMSO and 1ml of 1ml
Azoles missible oil replace B21 metabolite to execute above-mentioned treatment process respectively and as negative control and positive control, all processing and
Control is in triplicate.Finally, 28 DEG C of each pipe, 150r/min shaking table culture 5 days.The MIC value of B21 metabolite is complete inhibition three
The minimum crude extract concentration of seven anthrax bacteria visible growths.
As a result:
In the percent inhibition such as table 3 that bacterial strain sophora tonkinensis Gapnep endophyte B21 metabolite grows Radix Notoginseng anthrax bacteria mycelia
It is shown:
Table 3.
Note: positive control powder of carbendazim contains 50% carbendazim in table 3；* indicates that data pass through single factor test variance point in table
The LSD of analysis relatively after, Metabolite and positive control powder of carbendazim are under same concentrations, in P0.05Have in level significant
Sex differernce.
The suppression result that the metabolite of bacterial strain sophora tonkinensis Gapnep endophyte B21 grows Radix Notoginseng anthrax bacteria mycelia shows bacterium
The metabolite of strain sophora tonkinensis Gapnep endophyte B21 all has extraordinary inhibitory effect to the mycelia growth of Radix Notoginseng anthrax bacteria, from
Table 3 is it can be seen that the metabolite of sophora tonkinensis Gapnep endophyte B21 is grown to Radix Notoginseng anthrax bacteria C.gloeosporioides mycelia
Percent inhibition be 88.71-100%.Compared with positive control powder of carbendazim, the metabolism of bacterial strain sophora tonkinensis Gapnep endophyte B21
Product is less than or equal to 4mg/mL in concentration to the inhibitory effect of Radix Notoginseng anthrax bacteria C.gloeosporioides mycelia growth
When, slightly below compare, it is equivalent with compareing in concentration 8mg/mL.
The minimal inhibitory concentration that bacterial strain B21 metabolite grows Radix Notoginseng anthrax bacteria is as shown in table 4:
Table 4.
| Processing | MIC(mg/ml) |
| C.gloeosporioides | |
| Flusilazole | 0.25 |
| B21 | 2.5 |
Note: positive control Flusilazole active constituent content is 400mg/mL in table.
The minimal inhibitory concentration test result that the metabolite of bacterial strain sophora tonkinensis Gapnep endophyte B21 grows Radix Notoginseng anthrax bacteria
Show that the metabolite of bacterial strain sophora tonkinensis Gapnep endophyte B21 all has stronger inhibiting effect to Radix Notoginseng anthrax bacteria, it can from table 4
To find out minimal inhibitory concentration of the metabolite to Radix Notoginseng anthrax bacteria C.gloeosporioides of sophora tonkinensis Gapnep endophyte B21
It is 10 times of positive control for 2.5mg/ml.
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 B21 or is killed true
The ingredient of bacterium, therefore, in the bionomic control of Radix Notoginseng anthrax bacteria, bacterial strain sophora tonkinensis Gapnep endophyte B21 has potentiality outstanding.
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering
With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention 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 B21 in prevention and treatment Radix Notoginseng anthracnose, it is characterised in that: Vietnam
The classification naming of Chinese scholartree endophyte B21 be bulkholderia cepasea (Burkholderia sp.) B21, deposit number: CGMCC
No.10806；
The preparation method of the metabolite of the sophora tonkinensis Gapnep endogenetic bacteria B21: sophora tonkinensis Gapnep endogenetic bacteria B21 is inoculated in liquid
In culture medium, being placed in temperature is 28 DEG C, fermented and cultured 10 days under the conditions of revolving speed is 130r/min, and gained fermentation material is through being concentrated under reduced pressure
After drying, 2 times of fermentation material of methanol and ultrasound 40min is added, is then centrifuged for, supernatant is taken to be concentrated under reduced pressure to give strain fermentation object
Methanol crude extract, as sophora tonkinensis Gapnep endogenetic bacteria B21 metabolite.
2. application of the metabolite of sophora tonkinensis Gapnep endogenetic bacteria B21 in prevention and treatment Radix Notoginseng anthracnose according to claim 1,
Be characterized in that: the sophora tonkinensis Gapnep endogenetic bacteria B21 is inoculated in fluid nutrient medium containing 1000ml.
3. application of the metabolite of sophora tonkinensis Gapnep endogenetic bacteria B21 in prevention and treatment Radix Notoginseng anthracnose according to claim 1,
Be characterized in that: the fluid nutrient medium 3g containing beef extract, yeast extract 1g, peptone 5g, sucrose 10g, Medium's PH Value are
7.0。
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| WO2010018371A2 (en) * | 2008-08-13 | 2010-02-18 | University College Cardiff Consusltants Ltd | Antimicrobial agent and method for the production thereof |
| CN105462892A (en) * | 2015-12-23 | 2016-04-06 | 广西大学 | Application of sophora tonkinensis endophytic bacterium B21 in preventing and controlling panax notoginseng black spot |
| CN105462891A (en) * | 2015-12-23 | 2016-04-06 | 广西大学 | Application of sophora tonkinensis endophytic bacterium B21 in preventing and controlling panax notoginseng root rot |
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| CN105462892A (en) * | 2015-12-23 | 2016-04-06 | 广西大学 | Application of sophora tonkinensis endophytic bacterium B21 in preventing and controlling panax notoginseng black spot |
| CN105462891A (en) * | 2015-12-23 | 2016-04-06 | 广西大学 | Application of sophora tonkinensis endophytic bacterium B21 in preventing and controlling panax notoginseng root rot |
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