CN107893040A - A kind of micropopulation induction signal molecule degradation bacteria and its application in disease control - Google Patents
A kind of micropopulation induction signal molecule degradation bacteria and its application in disease control Download PDFInfo
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Abstract
The invention discloses a kind of micropopulation induction signal molecule degradation bacteria and its application in disease control.The Pseudomonas nitroreducens are Pseudomonas nitroreducens（Pseudomonas nitroreducens）Bacterial strain W 7, the bacterial strain are preserved in China typical culture collection center on November 2nd, 2017, and preserving number is CCTCC NO：M2017649.The strain activity is stable, it can be grown using the colony induction signaling molecule such as OdDHL AHLs as sole carbon source, there is notable and quick degradation to signaling molecule, simultaneously, the bacterial strain is respectively provided with preferable degradation effect to other colony induction signaling molecules such as OHHL and DSF, and the bacterial strain also has that cultural method is simple, the speed of growth is fast, the characteristics of being not easy to make a variation, be adaptable, in the potentiality that colony is quenched direction, suppresses soft rot disease, has huge popularization and application in terms of preventing and treating relies on the pathogenic pathogen harm of micropopulation induced signal mediation.The present invention can reduce agricultural chemicals abuse problem, while provide new approaches for biological controlling plant diseases.
Description
Technical field
The invention belongs to technical field of biological control.Dropped more particularly, to a kind of micropopulation induction signal molecule
Solve bacterium and its application in disease control.
Background technology
Effective preventing and treating of phytopathogen is always the major issue faced in agricultural production, such as Erwinia
(Erwinia sp.), enlightening Cattell Pseudomonas (Dickeya sp.) bacterium, is Gram-negative phytopathogen.Wherein enlightening Cattell
Pseudomonas is that the plant pathogenetic bacteria set up in recent years newly belongs to, and its pattern bacterium is original chrysanthemum erwinia (Erwinia
chrysanthemi).Enlightening Cattell Pseudomonas (Dickeya sp.) can trigger the weight of a variety of important cereal crops and industrial crops
Major disease, such as Characters of Erwinia chrysanthemi pv. zeae, banana bacterial soft rot, potato bacterial soft rot, cause related farming
The underproduction of thing, causes heavy economic losses.Soft rot is a kind of Important Agricultural disease, there is no highly effective control in production at present
Measure processed.
The prophylactico-therapeutic measures both at home and abroad used in soft rot pathogenic bacteria is mainly chemical prevention at present, such as：Thiophene bacterium ketone, thiophene are withered
The chemical pesticides such as azoles, ambam, streptomycin sulphate and Zhongshengmycin, but a large amount of uses of chemical pesticide have had resulted in many institutes
A series of serious problems such as known environmental pollution, ecological balance destruction, the pathogen resistance to the action of a drug and food security, in addition, antibiosis
The abuse of element also causes the generation of microorganism drug resistance.Therefore, find that new, effectively preventing strategy is extremely urgent.
Quorum sensing (Quorum Sensing, QS) refers to carry out by signaling molecule (self-induction agent) between microbial cell
A kind of phenomenon of information interchange, QS are widely present in micropopulation, and can be regulated and controled specific gene and especially much be caused
The expression of ospc gene, such as：The phenomenon of quorum sensing and pathogenic relevant with its be present in plant pathogen Dickeya zeae EC1,
Signaling molecule is acyl homoserine lactones class material (N-Acyl homoserine lactones, AHLs) (Hussain M
B, Zhang H B, Xu J L, et al.The acyl-homoserine lactone-type quorum-sensing
system modulates cell motility and virulence of Erwinia chrysanthemi pv.zeae
[J] .J Bacteriol, 2008,190 (3)：1045-1053.).AHLs is the distinctive colony induction signaling of Gram-negative bacteria,
Their structures mostly with identical homoserine lactone ring-type and all with acyl side-chain, but it is its acyl side-chain length, full
Had differences with degree.N-(3-oxododecanoyl)-L-homoserine lactone(OdDHL)、N-(3-
Oxohexanoyl)-L-homoserine lactone (OHHL) belong to AHLs, and its difference is acyl side-chain length difference.
AHLs signals are widely present in Gram-negative bacteria, including plant pathogen Erwinia (Erwinia), enlightening Cattell bacterium
(Dickeya), human pathogen Pseudomonas aeruginosa (Pseudomonas aeruginosa) etc..DSF(Diffusible Signal
Factor) it is black rot pathogenic bacteria --- xanthomonas campestris (Xanthomonas campestris pv.campestris,
Xcc colony induction signaling molecule), its quorum sensing mediated and pathogenic relevant (Tang J L, Liu Y N, Barber C
E, et al.Genetic and molecular analysis of a cluster of rpf genes involved in
positive regulation of synthesis of extracellular enzymes and polysaccharide
In Xanthomonas campestris pathovar campestris [J] .Mol Gen Genet, 1991,226 (3)：
409-417.).DSF signals are present in Xanthomonas campestris (Xanthomonas), Burkholderia (Burkholderia), Pseudomonas aeruginosa
(P.aeruginosa) etc..
(Quorum Quenching, QQ) is quenched i.e. by the way that the signaling molecule of pathogen is quenched to prevent stop signal in quorum sensing
Molecule effectively accumulates, when signaling molecule concentration cannot activate the expression of pathogen Disease-causing gene less than a certain threshold value, so as to broken
Exchange between bad cell, destroy its intervention school-based.Research finds, the method being quenched by quorum sensing come controlling disease,
Not producing selection pressure to pathogen makes pathogen be not likely to produce the resistance to the action of a drug.For the biological control of micropopulation induction system
Strategy (being referred to as colony to be quenched) is the new way of effective preventing and treating phytobacterial disease, has easy to operate, economical and practical, environment
Friendly, the advantages that efficiency high and cycle are short.It is current international coverage that preparation, which is quenched, for different groups induced signal development colony
Study hotspot.
The content of the invention
The technical problem to be solved in the present invention is the defects of overcoming above-mentioned prior art and technical deficiency, there is provided one kind has
The Pseudomonas nitroreducens (Pseudomonas nitroreducens) of efficient degradation colony induction signaling molecule ability, its
There is notable and quick degradation to colony induction signaling molecules such as AHLs, DSF, in preventing and treating colony induction signaling molecule mediation
Pathogenic bacteria harm in terms of have huge application potential, this be with biological control substitute chemical prevention and using block quorum sensing as
Target is without causing the control strategy of selection pressure to provide new development approach.
It is an object of the invention to provide the Pseudomonas nitroreducens of one plant of degradable micropopulation induction signal molecule
(Pseudomonas nitroreducens) bacterial strain W-7.
The present invention is another object is that application of the Pseudomonas nitroreducens in degrading microorganism colony induction signaling molecule.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
Pseudomonas nitroreducens (the Pseudomonas of one plant of degradable micropopulation induction signal molecule
Nitroreducens) bacterial strain W-7, the bacterial strain were preserved in China typical culture collection center, preservation on November 2nd, 2017
Number it is CCTCC NO：M2017649.
The bacterial strain isolates and purifies acquisition, by the bacterium from collection from Guangzhou effluent sewerage sample through artificial screening
Morphological feature, physio-biochemical characteristics, 16S rDNA Phylogenetic Analysis and the Biolog Automatic Analyzer for Microbes of strain, mirror
It is Pseudomonas nitroreducens (Pseudomonas nitroreducens) to determine bacterial strain W-7.
Bacterial strain W-7 colony morphology characteristic is：48h is cultivated on nutrient agar panel, bacterium colony is in faint yellow, and bacterium colony is put down,
Surface is smooth translucent, neat in edge：48h is cultivated in nutrient broth medium, it is muddy in diffusivity.
The morphological feature of electron microscopic observation thalline is：Thalline is in shaft-like, has 1 polar flagella.
Bacterial strain W-7 physio-biochemical characteristics are：It is aerobic for Gram-negative bacteria, catalase test, oxidase test,
Fluorchrome experiment, nitrate reduction test, nitrate aerogenesis experiment reacting positive, hemolytic test, gelatin liquefaction test, vulcanization
Hydrogen experiment, Starch Hydrolysis experiment reaction negative；Optimum growth temperature is 30 DEG C, optimal pH 7.0.
The bacterial strain W-7 reaches more than 400 μ g/mL to the resistance of ampicillin, to gentamicin, streptomysin, chlorine
The resistance of mycin reaches more than 200 μ g/mL, and 10 μ g/mL are reached to tetracyclin resistance.
Shown by experimental study, the Pseudomonas nitroreducens bacterial strain W-7 has aobvious to colony induction signaling molecule OdDHL
Write and quick degradation, can the OdDHL at concentrations up to 0.2mM be sole carbon source culture medium in normal growth, in 48h
Interior energy decomposes the colony induction signaling molecule OdDHL that initial concentration is 0.2mM completely.Meanwhile the bacterial strain is felt to other colonies
Induction signal molecule such as OHHL and DSF are respectively provided with preferable degradation effect.In preventing and treating AHLs types of populations induction signal molecule and colony's sense
There is huge application potential in terms of the pathogenic bacteria harm of induction signal molecule DSF mediations.
Therefore, application of the Pseudomonas nitroreducens in colony induction signaling molecule AHLs and/or DSF signal of degrading,
Or the application in the product for preparing degraded AHLs and/or DSF signals falls within protection scope of the present invention.
Pseudomonas nitroreducens are in the pathogenic plant disease of the colony induction signaling molecule mediation such as preventing and treating AHLs or DSF
Application, or in terms of the preventing and treating preparation for relying on the pathogenic pathogenic bacteria of the colony induction signaling molecule such as AHLs or DSF is prepared should
With falling within protection scope of the present invention.
Preferably, in any of the above-described described application, the Pseudomonas nitroreducens are Pseudomonas nitroreducens bacterium
Strain W-7.
It is a kind of to prevent and treat the method for relying on the pathogenic pathogenic fungus diseases of the colony induction signaling molecules such as AHLs or DSF, it is to use nitre
Base reduction pseudomonad strain W-7 bacteria suspension is handled crop, and infecting for the pathogenic pathogenic bacteria of AHLs is relied on prevention.
Preferably, the mode of the processing for crop is sprayed or inoculation processing.
Preferably, during application, described Pseudomonas nitroreducens degraded AHLs optimal pH is 6.5~7.2, most thermophilic
Spend for 28 DEG C~30 DEG C.
Experiment display, Pseudomonas nitroreducens bacterial strain W-7 is to Erwinia (Erwinia), enlightening Cattell bacterium (Dickeya)
Or the disease that Pseudomonas aeruginosa (Pseudomonas aeruginosa) etc. relies on the pathogenic pathogens of AHLs has significant biological and ecological methods to prevent plant disease, pests, and erosion
Effect, the disease of the pathogenic pathogens of DSF is relied on Xanthomonas campestris (Xanthomonas) or Burkholderia (Burkholderia) etc.
Evil is respectively provided with significant Biocontrol Effect.Therefore specifically, it is described to rely on the pathogenic pathogenic bacteria of micropopulation sensitive signal
Including：Erwinia (Erwinia), enlightening Cattell bacterium (Dickeya), Xanthomonas campestris (Xanthomonas), Burkholderia
Or Pseudomonas aeruginosa (Pseudomonas aeruginosa) etc. (Burkholderia).
A kind of degradable colony induction signaling containing above-mentioned Pseudomonas nitroreducens bacterial strain W-7 and/or its bacteria suspension
Molecule AHLs or DSF degradation bacterial agent, and a kind of dependence AHLs or DSF containing bacterial strain W-7 and/or its bacteria suspension cause a disease
The biological prevention and control agent of pathogen, also all should be within protection scope of the present invention.
The present invention additionally provides the preparation method of the bacterial strain W-7 bacteria suspensions described in eutrophy culture medium simultaneously：Specifically
Bacterial strain W-7 is lined on LB solid medium flat boards, 12~36h is cultivated at 28 DEG C~30 DEG C, picking single bacterium colony is inoculated in LB
Preculture to logarithmic phase, gained thalline is rinsed and is resuspended with 0.9% sterile saline, hanged as seed in fluid nutrient medium
Liquid, then seed suspension is seeded to the culture of LB fluid nutrient mediums extremely according to the inoculum concentration of volume ratio 0.5%~5% (preferably 1%)
Logarithmic phase, thalline are resuspended to obtain bacterial strain W-7 bacteria suspension with PBS, and the concentration of bacteria suspension is not done strict limitation, specifically may be used
It is adjusted according to actual degree of disease and application effect.
Preferably, the LB culture mediums are：Tryptone 10.0g/L, yeast extract 5.0g/L, sodium chloride 10.0g/L,
6.8~7.2,121 DEG C of 15~25min of sterilizing of pH.LB solid culture based formulas is 1.5% (w/v) of addition in liquid medium within
Agar.
The invention has the advantages that：
The present invention research find, Pseudomonas nitroreducens can efficient degradation colony induction signaling molecule AHLs and DSF,
Degradation effect is significantly and quick, has huge application potential in terms of the pathogenic pathogenic bacteria harm of preventing and treating AHLs and DSF mediations, this
To substitute chemical prevention with biological control and being that target selects the therapeutic strategy of pressure to provide without causing to block quorum sensing
New development approach.
Meanwhile present invention screening obtains a nitro-reductive pseudomonads (Pseudomonas nitroreducens)
W-7, can the OdDHL at concentrations up to 0.2mM be sole carbon source culture medium in normal growth, can effectively degrade in a short time
Colony induction signaling molecule OdDHL, 48h interior energy divides the colony induction signaling molecule OdDHL that initial concentration is 0.2mM completely
Solution, there is significant biodegradable effect.Meanwhile the bacterial strain is respectively provided with to other colony induction signaling molecules such as OHHL and DSF
Preferable degradation effect.
According to the present invention, because there is bacterial strain W-7 quorum sensing AHLs or DSF in stability and high efficiency degrading plant pathogen to believe
The characteristic of number molecule, therefore, bacterial strain W-7 can be applied to the anti-of the plant pathogen that AHLs or DSF mediations are pathogenic in natural environment
Control, can both reduce agricultural chemicals abuse problem, and new thinking, new way and new method are brought to control of plant disease.
Brief description of the drawings
Fig. 1 is colonial morphology figures of the bacterial strain W-7 of the present invention on LB culture mediums.
Fig. 2 is colonial morphology figures of the bacterial strain W-7 of the present invention on blood plate culture medium.
Fig. 3 is the bacterial strain W-7 of present invention scanning electron microscope (SEM) photograph.
Fig. 4 is the bacterial strain W-7 of present invention Phylogenetic analysis figure.
Fig. 5 is growing state figures of the bacterial strain W-7 of the present invention in different antibiotic.
The degrading activity measure figure that Fig. 6 is the bacterial strain W-7 of the present invention (DH5 α are negative control, and B23 is positive control).
Fig. 7 is HPLC figures (compares figure that figure A is non-inoculating strain W-7, the figure that the bacterial strain W-7 of the present invention degrades to OdDHL
B, C, D, E are respectively bacterial strain W-7 to OdDHL 12h, 24h, 36h, the high-efficient liquid phase chromatogram of 48h degradeds).
Fig. 8 be the present invention bacterial strain W-7 using OdDHL as sole carbon source when growth curve and degradation curve figure.
Fig. 9 is the bacterial strain W-7 of the present invention using DSF as the DSF that degraded on the solid MM minimal medium flat boards of sole carbon source
Produce hydrolysis figure.
The bacterial strain W-7, DH5 α, B23, EC1 that Figure 10 is the present invention are individually inoculated with and respectively with EC1 co-inoculations in horse
Incidence after bell potato wedge stem 30h.
Figure 11 is the individually morbidity feelings of inoculation and co-inoculation after castock 24h of the bacterial strain W-7 of the present invention, 3937
Condition.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment and Figure of description, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are purchased in market.
The Pseudomonas nitroreducens bacterial strain W-7 of embodiment 1 acquisition and identification
1st, bacterial strain W-7 separation, screening
(1) soil sample gathers：The effluent sewerage sample from Guangzhou is gathered as microbial source
Soil sample is located away from the October in 2014 of the sewer near the Agricultural University Of South China of Guangzhou, Guangdong on the 9th, is taken
Sample, pack, preserve and take back school's progress strain isolation as microbial source.
(2) enrichment culture of bacterial strain：MSM culture mediums are prepared, 50mL basal medium is attached in 250mL triangular flasks and gone out
Bacterium, AHLs mother liquors (methanol is solvent) are aseptically added after cooling, the ultimate density for making AHLs is 5 μM, is added simultaneously
Soil sample 5g is transferred to MSM of the second batch containing 10 μM of AHLs after 30 DEG C, 200rpm shaking table cultures 7d, by 10% inoculum concentration and trained
Support in base.After the same terms culture 7d, then it is transferred in the MSM culture mediums containing 20 μM of AHLs by 10% inoculum concentration, continues
Cultivate 7d.By that analogy, it is continuously increased AHLs concentration.
(3) strain isolation and purifying：Separated using dilution, spread plate.
Take 1mL ends MSM culture medium zymotic fluid sterilized water by its concentration successively gradient dilution be 10-1、10-2、10-3、10-4、
10-5、10-6、10-7、10-8Zymotic fluid, the zymotic fluid for then drawing each concentration gradient that 100 μ L have diluted equably is coated with
On LB solid plates, 30 DEG C of cultures, the single bacterium colony for the different colonial morphologies that picking is grown, rule repeatedly training in LB solid plates
Purifying is supported, until isolating single strain.By -80 DEG C of preservations of single strain, treat that further experiment determines AHLs degradation effect.
(4) bacterial strain screening：Using reporting bacterial strain (Agrobacterium tumefaciens NT1) to being separated from soil sample
Obtained bacterial strain is screened.
It will freeze and lined in -80 DEG C of bacterial strain to be screened on LB solid medium flat boards, 30 DEG C are incubated overnight.Picking
Single bacterium colony, with LB fluid nutrient medium overnight incubations, obtain bacterium solution.Take 1 OD600The thalline of value, be seeded to 1mL using AHLs as
In the MSM minimal mediums of sole carbon source, final concentration of 20 μM of AHLs in MSM minimal mediums.By reactant mixture
It is positioned in 2mL centrifuge tubes, centrifuge tube is cultivated 24h under conditions of 200rpm at 30 DEG C.After 24h, negate and answer 5 μ L reactions mixed
Compound point sample is to being coated with 200 μ L Agrobacterium tumefaciens NT1 bacterium solutions (OD600=0.4) on MM plates, its
Contain the X-gal that concentration is 40 μ g/mL in middle MM plates.The MM plates 28 DEG C of incubators of placement for having sample will be put, after cultivating 24h
Observation experiment result.
Interpretation of result：AHLs is diffusivity small molecule, and the AHLs contained in reactant mixture is more, the indigo plant occurred on MM plates
Loop diameter is bigger, illustrates that the bacterial strain, without degradation effect, conversely, the AHLs contained in reactant mixture is fewer, then produces to AHLs
The smaller appearance even without blue circle of blue circle diameter.Being filtered out according to experimental result to AHLs has the best bacterium of degradation effect
Strain, is named as bacterial strain W-7.
2nd, bacterial strain W-7 identification and phylogenetic analysis
(1) colony morphology characteristic：Above-mentioned bacterial strains W-7 is lined into LB solid mediums, it is permanent in 30 DEG C of biochemical cultivation cases
Temperature cultivates 24h, and 48h is cultivated on nutrient agar panel, and bacterium colony is in faint yellow, and bacterium colony is put down, and surface is smooth translucent, neat in edge,
As shown in Figure 1；Bacterial strain W-7 is muddy in diffusivity in LB fluid nutrient mediums, aerobic, well-grown at 30 DEG C.As shown in Fig. 2
The bacterial clump is presented faint yellow on blood plate.
(2) morphological features：As shown in figure 3, thalline is in shaft-like, there is 1 polar flagella.
(3) physio-biochemical characteristics：Bacterial strain W-7 is Gram-negative bacteria, aerobic, catalase test, oxidase test, fluorescence
Pigment experiment, nitrate reduction test, nitrate aerogenesis experiment reacting positive, hemolytic test, gelatin liquefaction test, hydrogen sulfide examination
Test, Starch Hydrolysis experiment reaction negative；Optimum growth temperature is 30 DEG C, optimal pH 7.0.Its Physiology and biochemistry qualification result such as table
Shown in 1.
(4) 16S rDNA sequences and phylogenetic analysis：Bacterial strain W-7 16S rDNA gene orders length is 1411bp,
With ncbi database (http：//www.ncbi.nlm.nih.gov/) be compared, find the bacterial strain W-7 with
Pseudomonas nitroreducens have a good homology (＞ 99%), and its systematic evolution tree is as shown in Figure 4.
(5) Biolog Automatic Analyzer for Microbes is identified：
Biolog microbe auto-analysis is a kind of method of microbial identification, and the method is to carry out data by computer
Analysis, qualification process is quick, as a result accurate credible.DIS (Distance) and SIM (Similarity) is 2 of qualification result
Important parameter, represent the matching degree of test result data corresponding with database.As DIS ＜ 5.0, SIM values closer to 1,
The reliability of qualification result is higher.
Bacterial strain W-7 of the present invention Biolog Automatic Analyzer for Microbes qualification results show that W-7 is false single with nitro reduction
Born of the same parents bacterium (Pseudomonas nitroreducens) is the most similar, and DIS ＜ 5.0, SIM value be 0.741, illustrate bacterial strain W-7 and
It is good to reduce pseudomonad (Pseudomonas nitroreducens) matching.Bacterial strain W-7 utilization of carbon source results are as shown in table 2.
In summary, by bacterial strain W-7 morphological feature, physio-biochemical characteristics, 16S rDNA gene sequencings
And the identification of Biolog Automatic Analyzer for Microbes, bacterial strain W-7 is accredited as Pseudomonas nitroreducens (Pseudomonas
Nitroreducens), and China typical culture collection center (CCTCC) is preserved in, preserving number is CCTCC NO：
M2017649, preservation address are Wuhan, China universities.
The bacterial strain W-7 Physiology and biochemistry qualification results of table 1
Note：-：Negative reaction；+：Positive reaction.
The bacterial strain W-7 utilization of carbon source qualification results of table 2
Note："-" is expressed as negative reaction or not utilized；"+" represents positive reaction or utilization.
The bacterial strain W-7 of embodiment 2 antibiotics sensitivity analysis
In order to preferably study the bacterial strain W-7 of the acquisition of embodiment 1 Biocontrol Potential, our lifes to bacterial strain W-7
Thing characteristic conducts in-depth research.Experimental study sensitiveness of the bacterial strain W-7 to different antibiotic, it is specific as shown in Figure 5.
Test result indicates that：Bacterial strain W-7 reaches more than 400 μ g/mL to the resistance of ampicillin, to gentamicin,
Streptomysin, the resistance of chloramphenicol reach more than 200 μ g/mL, and 10 μ g/mL are reached to tetracyclin resistance.After this result is beneficial to
Suitable antibiotic is chosen in continuous research as reference.
The bacterial strain W-7 of embodiment 3 determines to OdDHL degradation capability
1st, strain culturing and sample collection：Bacterial strain W-7 is lined on LB solid medium flat boards, 30 DEG C are incubated overnight.
Picking single bacterium colony, with LB fluid nutrient mediums overnight incubation to obtaining bacterium solution.Take 1 OD600The thalline of value, be seeded to 1mL with
OdDHL is as in the MSM minimal mediums of sole carbon source, final concentration of 80 μM of OdDHL in MSM minimal mediums.Will
Reactant mixture is positioned in 2mL centrifuge tubes, centrifuge tube is cultivated 24h under conditions of 200rpm at 30 DEG C.After 24h, negating should
5 μ L reactant mixtures point samples are to agar strip top, then in lower section point reporting bacterial strain (Agrobacterium tumefaciens
NT1) bacterium solution.Then the culture dish of good reactant mixture and reporting bacterial strain is placed into 28 DEG C of incubators, observed after cultivating 24h
Experimental result.Wherein, agar strip is to cut gained containing the MM plates that concentration is 40 μ g/mL X-gal.
In experiment contrast, DH5 α are negative control, to AHLs without degradation；B23 is positive control, has degraded to AHLs
Effect.Bacillus thuringiensis subsp israelensis (Bacillus thuringiensis subsp.Israelensis) B23 contains
There is coding AHLs digestive enzyme aiiA gene, there is degradation (Dong Y, Xu J, Li X, et al.AiiA, an to AHLs
enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and
Attenuates the virulence of Erwinia carotovora [J] .Proc Natl Acad Sci USA,
2000,97 (7)：3526-3531.).Experimental result shows (Fig. 6) that the reporting bacterial strain of CK and DH5 α experimental groups becomes blue and distance
It is basically identical, illustrate that remaining OdDHL contents are close in the two experimental group reactant mixtures.B23 and W-7 experimental groups report bacterium
The unchanged indigo plant of strain, illustrates not containing OdDHL in the reactant mixture of the two experimental groups, OdDHL has been degraded completely.
The bacterial strain W-7 of embodiment 4 grows and the measure of degraded OdDHL relation curves
1st, picking bacterial strain W-7 single bacterium colonies are inoculated in LB culture mediums preculture to logarithmic phase, take the thalline of 1 OD value, bacterium
Body is resuspended with MSM basal mediums, and re-suspension liquid is added in 19mL MSM basal mediums, and adds OdDHL mother liquors, makes it most
Final concentration of 0.2mM, 30 DEG C, timing sampling is cultivated under 200rpm.Gather the sample of different time points and determine OD600Value represents
Bacterial strain W-7 growing state, the residual quantity that OdDHL is determined using HPLC represent degraded situations of the bacterial strain W-7 to OdDHL.
2nd, HPLC assay methods
HPLC：Waters 2695.Chromatographic column：Kinetex EVO C18 reverse-phase chromatographic columns (250 μ m 4.6mm × 5 μm).
Flow velocity：0.8mL/min.Column temperature：30℃.Mobile phase：Methanol: water=70；30(v∶v).Detection wavelength：210nm.Sample size：20μ
L。
3rd, interpretation of result：HPLC testing results scheme B, C, D, E as shown in fig. 7, the compares figure that figure A is non-inoculating strain W-7
Respectively bacterial strain W-7 respectively reaches 63.6%, 85.9% to OdDHL 12h, 24h, 36h, 48h degraded figure, degradation rate,
91.9%, 100%, the OD of corresponding time bacterial strain W-7600Value is respectively 0.054,0.132,0.162,0.165.Growth curve and
Degradation curve figure is as shown in figure 8, OdDHL degraded is proportionate with strain growth, and in the presence of OdDHL, strain growth does not have
There is demurrage, quickly enter growth logarithmic phase.Degraded of the bacterial strain in 0~24h to OdDHL is most fast, strain culturing to 48h,
OdDHL is decomposed completely.Natural degradation rate in control in OdDHL 24h is less than 40%.
As a result show, Pseudomonas nitroreducens W-7 has notable and quick degradation to OdDHL, in preventing and treating OdDHL
There is huge application potential in terms of the pathogenic bacteria harm of mediation.
The bacterial strain W-7 of embodiment 5 determines to DSF degrading activity
Originally example is applied to live to determine degradeds of the bacterial strain W-7 to DSF by the use of the MM minimal mediums using DSF as sole carbon source
Property.
1st, the preparation using DSF as the MM minimal mediums of sole carbon source：Configuring DSF mother liquors, (solvent is methanol, and concentration is
100mM), the MM minimal mediums without any carbon source.Added in the MM minimal mediums without any carbon source certain
The DSF mother liquors of amount, mixing, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, dry so that the final concentration of 5mM of DSF.
2nd, the bacterial strain W-7 in -80 DEG C will be frozen, LB liquid medium is inoculated according to the inoculum concentration of volume ratio 1: 100, with
30 DEG C, 200rpm condition is incubated overnight.By the bacterium solution obtained after culture with 4000rpm, after 10min condition is centrifuged,
Supernatant discarding, thalline is resuspended with MSM, obtains the MSM re-suspension liquids of bacterial strain to be screened.The MSM weights of 0.5 μ L bacterial strains to be screened are taken respectively
Suspension point plate is on the MM minimal medium flat boards using 5mM DSF as sole carbon source.There is provided MSM, DH5 α experimental groups simultaneously
As control.
3rd, interpretation of result：As shown in figure 9, MSM, DH5 α experimental groups are enclosed without hydrolysis, illustrate MSM on this experiment without influence, DH5
α can not degrade DSF.W-7 experimental groups have obvious hydrolysis circle, illustrate that bacterial strain W-7 can degrade and be used as sole carbon by the use of DSF
Source.
The bacterial strain W-7 of embodiment 6 is studied the biocontrol effect of bacterial soft rot of potato
The present embodiment is to cause pathogen Dickeya zeae EC1 (Hussain the M B, Zhang of bacterial soft rot of potato
H B, Xu J L, et al.The acyl-homoserine lactone-type quorum-sensing system
modulates cell motility and virulence of Erwinia chrysanthemi pv.zeae[J].J
Bacteriol, 2008,190 (3)：Exemplified by 1045-1053.), the biological and ecological methods to prevent plant disease, pests, and erosion for the pathogenic bacteria that research bacterial strain W-7 causes a disease to relying on AHLs
Effect.
1st, by bacterial strain W-7 with rely on AHLs cause a disease pathogen --- Dickeya zeae EC1 line LB and consolidated respectively
On body culture medium flat plate, 30 DEG C are incubated overnight.Picking single bacterium colony respectively, with preculture in LB culture mediums to OD600=2.0.Respectively
Set W-7+LB ,+EC1, B23+EC1, seven experimental groups of DH5 α+LB, B23+LB, EC1+LB, W-7+EC1, DH5 α, make W-7,
DH5 α, the final working concentration OD of B23, EC1600=1.0.In experimental group, DH5 α are negative control without degradation to AHLs,
B23 has degradation to AHLs, is positive control.Each experimental group is inoculated with 5 μ L mixed bacteria liquid to potato with liquid-transfering gun.
2nd, as shown in Figure 10, interpretation of result：The potato of W-7+LB, DH5 α tri- experimental groups of+LB, B23+LB does not fall ill,
Illustrate W-7, DH5 α, B23 to potato had no pathogenicity；EC1, DH5 α+EC1, tri- experimental group potato incidences of B23+EC1
There is not notable difference.Experimental group W-7+EC1 potato falls ill the order of severity substantially compared with EC1, DH5 α+EC1, B23+EC1 tri-
The individual test group morbidity order of severity is light.Test result indicates that bacterial strain W-7 has significantly to soft rot caused by enlightening Cattell bacterium EC1
Biocontrol effect.
The bacterial strain W-7 of embodiment 7 is studied the biocontrol effect of soft rot of cabbage
The present embodiment with cause the pathogen Dickeya dadantii 3937 of soft rot of cabbage (Glasner J D,
Yang C, Reverchon S, et al.Genome sequence of the plant-pathogenic bacterium
[J] the .J Bacteriol of Dickeya dadantii 3937,2011,193 (8)：2076-2077.) exemplified by, study bacterial strain W-7
Biocontrol effect to relying on the pathogenic pathogens of AHLs.
1st, by bacterial strain W-7 with rely on AHLs cause a disease pathogen --- Dickeya dadantii 3937 are lined respectively
On LB solid medium flat boards, 30 DEG C are incubated overnight.Picking single bacterium colony respectively, with preculture in LB culture mediums to OD600=2.0.
LB, 3937, W-7, W-7+3937 are set respectively, four experimental groups, make the final working concentration OD of 3937, W-7600=1.0.Each
Experimental group is inoculated with 2 μ L mixed bacteria liquid to castock with liquid-transfering gun.
2nd, as shown in figure 11, interpretation of result：It can be seen that, two experimental groups of LB, W-7 connect on vaccinated castock
Do not fallen ill at kind, illustrate LB, W-7 is to castock had no pathogenicity.It is inoculated with experimental group W-7+3937 inoculation with experimental group 3937
Place compares to be occurred without obvious soft rotten symptom.Test result indicates that bacterial strain W-7 is to soft caused by Dickeya dadantii 3937
Maize ear rot has significant biocontrol effect.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. it is a kind of can efficient degradation micropopulation induction signal molecule Pseudomonas nitroreducens（Pseudomonas nitroreducens）Bacterial strain W-7, it is characterised in that the bacterial strain is preserved in Chinese Typical Representative culture guarantor on November 2nd, 2017
Tibetan center, preserving number are CCTCC NO：M2017649.
2. application of the Pseudomonas nitroreducens in degrading microorganism colony induction signaling molecule, or preparing micropopulation
Application in the product of induction signal molecule.
3. Pseudomonas nitroreducens are relied in preventing and treating in the pathogenic plant disease of micropopulation induction signal molecule mediation
Using, or the application in terms of the preventing and treating preparation for relying on the pathogenic pathogenic bacteria of micropopulation induction signal molecule is prepared.
4. the application according to Claims 2 or 3, it is characterised in that the Pseudomonas nitroreducens are claim 1 institute
State Pseudomonas nitroreducens bacterial strain W-7.
5. applied according to Claims 2 or 3, it is characterised in that the micropopulation induction signal molecule be AHLs or
DSF。
6. a kind of prevent and treat the method for relying on the pathogenic cause of disease fungus diseases of micropopulation induced signal, it is characterised in that uses nitro
The bacteria suspension of reduction pseudomonad is handled crop.
7. according to the method for claim 6, it is characterised in that the micropopulation induction signal molecule be AHLs or
DSF。
8. according to the method for claim 6, it is characterised in that described to rely on what micropopulation induced signal caused a disease
Pathogen includes：Erwinia（Erwinia）, enlightening Cattell bacterium（Dickeya）, Xanthomonas campestris（Xanthomonas）Burkholderia
（Burkholderia）Or Pseudomonas aeruginosa（Pseudomonas aeruginosa）.
9. a kind of degradation bacterial agent of degradable micropopulation induction signal molecule, it is characterised in that containing described in claim 1
Bacterial strain W-7 and/or its bacteria suspension.
10. a kind of biological prevention and control agent for relying on the pathogenic pathogen of micropopulation induction signal molecule, it is characterised in that containing having the right
Profit requires the 1 bacterial strain W-7 and/or its bacteria suspension.
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