CN108048351A - One plant of acyl homoserine lactones degradation bacteria and its application in disease control - Google Patents
One plant of acyl homoserine lactones degradation bacteria and its application in disease control Download PDFInfo
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Abstract
The invention discloses one plant of acyl homoserine lactones degradation bacteria and its applications in disease control.The degradation bacteria is acinetobacter calcoaceticus（Acinetobacter schindleri）, acinetobacter calcoaceticus strain X J 10 is referred in particular to, which is preserved in China typical culture collection center on November 2nd, 2017, and preserving number is CCTCC NO：M2017650.The bacterial strain, which can degrade, colony induction signaling molecule and utilizes it as sole carbon source growth, has notable and quick degradation to AHLs colony induction signaling molecules；Therefore micropopulation sensing communication can be blocked by degrading colony induction signaling molecule, inhibits the expression of the virulence factor of pathogenic bacteria, so as to achieve the purpose that controlling disease, there are huge applications potentiality in terms of prevention relies on the pathogenic pathogen harm of AHLs mediations；And chemical pesticide abuse problem can be reduced, while provide new strategy for the harm of biological control phytopathogen.
Description
Technical field
The invention belongs to technical field of biological control.More particularly, to one plant of acyl homoserine lactones degradation bacteria and
Its application in disease control.
Background technology
Microorganism perceives the variation of population density by monitoring the concentration of extracellular signaling molecule (self-induction agent), when it is reached
During to a certain threshold value, start the expression of target gene, coordinate group behavior, i.e. quorum sensing (Quorum Sensing, QS)
(Whiteley M,Diggle S P,Greenberg E P.Progress in and promise of bacterial
quorum sensing research[J].Nature.2017,551(7680):313-320.).Quorum sensing is widely present in
In micropopulation, and it is related with a variety of biological functions of microorganism, such as：Bioluminescence, symbiosis, biofilm formation, antibiosis
(Davies DG, the Parsek MR, Pearson such as plain synthesis, One-male unit, plasmid transfer, sporogenesis, gene swapping
JP,et al.The involvement of cell-to-cell signals in the development of a
bacterial biofilm[J].Science,1998,280(5361):295-298.)。
Wherein, acyl homoserine lactones class substance (N-Acyl homoserine lactones, AHLs) is gram
The distinctive colony induction signaling of negative bacterium, such substance mostly has the identical cricoid structure of homoserine lactone, and all has
There is acyl side-chain, but its acyl side-chain length, saturation degree have differences.Such as common N- (3-oxododecanoyl)-L-
Homoserine lactone (OdDHL) and N- (3-oxohexanoyl)-L-homos erine lactone (OHHL) belong to
In AHLs, difference is acyl side-chain length difference.AHLs signals are widely present in Gram-negative bacteria, are caused including plant
Germ Erwinia (Erwinia), enlightening Cattell bacterium (Dickeya), human pathogen Pseudomonas aeruginosa (Pseudomonas
Aeruginosa) etc., and it is related to pathogenic bacteria pathogenic, such as：OHHL is the group of plant pathogen Dickeya zeae EC1
Induction signal molecule, and its mediation quorum sensing phenomenon and with its pathogenic related (Hussain M B, Zhang H B, Xu
J L,et al.The acyl-homoserine lactone-type quorum-sensing system modulates ce
ll motility and virulence of Erwinia chrysanthemi pv.zeae[J].Journal of
Biotec hnology,2008,190(3):1045-1053.), OdDHL is human pathogen Pseudomonas aeruginosa (Pseudo monas
Aeruginosa colony induction signaling molecule), and the formation of the intervention school-based and biomembrane of its mediation is in close relations
(Huang J J,Han J I,Zhang L H,et al.Utilization of acyl-hom oserine lactone
quorum signals for growth by a soil pseudomonad and Pseudo monas aeruginosa
PAO1[J].Applied and Environmental Microbiology,2003,69(10):5941-5949.)。
Quorum sensing be quenched (Quorum Quenching, QQ) i.e. by inhibit the synthesis of signaling molecule, accumulation, monitoring,
Or the mechanism of enzyme degradation or modification is carried out to signaling molecule to disturb intervention school-based (Fetzner S.Quorum
quenching enzymes[J].Journal of Biotechnology,2015,201:2-14.).The study found that pass through group
The method that body-sensing should be quenched carrys out controlling disease, and not generating selection pressure to pathogen makes pathogen be not likely to produce the resistance to the action of a drug.Group
It is effective new way for preventing phytobacterial disease that sensing, which is quenched, is had easy to operate, economical and practical, environmental-friendly, efficient
And the cycle it is short the advantages that.For the development of different groups inductive signal, the research hotspot that preparation is current international coverage is quenched in group.
The content of the invention
The technical problem to be solved by the present invention is to overcome the defects of existing phytopathogen Prevention Technique and deficiency, one is provided
Kind has efficient degradation colony induction signaling molecule AHLs signals, the bacterial strain of AHLs signal analogs and its use as biocontrol microorganisms
On the way.
The object of the present invention is to provide the acinetobacter calcoaceticus of one plant of degradable AHLs colony induction signaling molecule
(Acinetobacter schindleri) strain X J-10.
Another object of the present invention is to provide the acinetobacter calcoaceticus in prevention relies on the pathogenic plant disease of AHLs mediations
Using.
Still a further object of the present invention is to provide a kind of method for preventing and relying on the pathogenic fungus diseases that AHLs causes a disease.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
Acinetobacter calcoaceticus (Acinetobacter schindleri) bacterium of one plant of degradable AHLs colony induction signaling molecule
Strain XJ-10, the bacterial strain are preserved in China typical culture collection center (CCTCC) on November 2nd, 2017, and preserving number is
CCTCC NO：M2017650, preservation address are：Wuhan, China, Wuhan University.
Soil sample of the bacterial strain from acquisition from Xinjiang, isolates and purifies acquisition, by the shape to the bacterial strain through artificial screening
State feature, physio-biochemical characteristics and 16S rDNA Phylogenetic Analysis, it is acinetobacter calcoaceticus to identify the bacterial strain
(Acinetobacter schindleri), and it is named as XJ-10.
Different antibiotics sensitivity analyses have been carried out to strain X J-10 simultaneously.J-10 is to ampicillin for the strain X
Resistance reaches more than 400 μ g/mL, and the resistance of kanamycins reaches 200 μ g/mL, and the resistance of gentamicin reaches 100 μ g/mL, right
Streptomysin, the resistance of chloramphenicol reach 20 μ g/mL.
Acinetobacter calcoaceticus (Acinetobacter schindleri) strain X J-10 provided by the invention can be with fast degradation group
Body induction signal molecule AHLs.Acinetobacter calcoaceticus can degrade gram negative bacteria quorum sensing signal, especially Erwinia
(Erwinia), the microbe groups group such as enlightening Cattell bacterium (Dickeya), Pseudomonas aeruginosa (Pseudomonas aeruginosa)
Inductive signal.The signaling molecule of the pathogenic bacteria of these Gram-negative monoids is homoserine lactone class substance.
In the application of degradation colony induction signaling molecule, shown by experimental study, acinetobacter calcoaceticus
(Acinetobacter schindleri) strain X J-10 has colony induction signaling molecule AHLs notable and quick degradation to make
With.Can in using OdDHL as the culture medium of sole carbon source normal growth, in 48h can by initial concentration be 0.2mM group
Induction signal molecule OdDHL is decomposed completely.Meanwhile the bacterial strain also there is preferable degradation to imitate colony induction signaling molecule OHHL
Fruit.There is huge application potential in terms of the pathogenic bacteria harm of prevention AHLs mediations.
Therefore, application of the acinetobacter calcoaceticus in colony induction signaling molecule AHLs and/or AHLs signal analog of degrading or
Application in the product for preparing degradation AHLs and/or AHLs signal analogs；The pathogenic plant of AHLs mediations is relied in prevention
Application in disease or the application in terms of the prevention preparation for relying on the pathogenic bacteria that AHLs causes a disease is prepared；It should all be the present invention's
Within protection domain.
Specifically, the AHLs class signals substance is homoserine lactone class substance.Including OdDHL and OHHL etc..
Preferably, the acinetobacter calcoaceticus is above-mentioned acinetobacter calcoaceticus strain X J-10.
In addition, based on above application, acinetobacter calcoaceticus (Acinetobacter schindleri) bacterial strain provided by the invention
XJ-10 has the development potentiality of biological control.
The present invention also provides a kind of methods for preventing and relying on the pathogenic fungus diseases that AHLs causes a disease, and specifically use acinetobacter calcoaceticus
Bacteria suspension crop is handled.The mode of the processing is inoculation or spraying treatment.
Preferably, the acinetobacter calcoaceticus is above-mentioned acinetobacter calcoaceticus strain X J-10.
Wherein, the pathogenic bacteria that the dependence AHLs causes a disease include：Enlightening Cattell bacterium (Dickeya), Erwinia (Erwinia)
Or Pseudomonas aeruginosa (Pseudomonas aeruginosa) etc.；The cause that acinetobacter calcoaceticus strain X J-10 causes a disease to these dependences AHLs
The disease of germ has significant Biocontrol Effect.
Preferably, the optimum medium of strain X J-10 bacteria suspensions is prepared as eutrophy culture medium, and formula is：Tryptose
Peptone 10.0g/L, yeast extract 5.0g/L, 6.8~7.2,121 DEG C of 15~25min of sterilizing of sodium chloride 10.0g/L, pH.
In addition, a kind of degradation of the degradable colony induction signaling molecule AHLs containing strain X J-10 and/or its bacteria suspension
A kind of biological prevention and control agent for the pathogenic bacteria that microbial inoculum and dependence AHLs containing strain X J-10 and/or its bacteria suspension cause a disease, also all
It should within protection scope of the present invention.
The invention has the advantages that：
The present invention has preferable degrading activity the study found that acinetobacter calcoaceticus is directed to colony induction signaling molecule AHLs, and
Degradation effect stabilization is significantly and quick, and the prevention for the pathogenic pathogenic bacteria of AHLs mediations provides a kind of new approaches and methods.
Meanwhile there is significant fast degradation activity not to colony induction signaling molecule the present invention also provides one plant
Lever bacteria strain XJ-10 has huge application potential in terms of the pathogenic pathogenic bacteria of prevention AHLs mediations, this is with biological control
It substitutes chemical prevention and is target without the therapeutic strategy of selection pressure is caused to provide new exploitation on the way to block quorum sensing
Footpath.
Description of the drawings
Fig. 1 is colonial morphology figures of the strain X J-10 of the present invention on LB culture mediums.
Fig. 2 is colonial morphology figures of the strain X J-10 of the present invention on blood plate culture medium.
Fig. 3 is the scanning electron microscope (SEM) photograph of the strain X J-10 of the present invention.
Fig. 4 is the Phylogenetic analysis figure of the strain X J-10 of the present invention.
Fig. 5 is growing state figures of the strain X J-10 of the present invention in different antibiotic.
Fig. 6 is the degrading activity measurement chart of the strain X J-10 of the present invention.
Fig. 7 is the HPLC figures (control that figure A is non-inoculating strain XJ-10 that the strain X J-10 of the present invention degrades to OdDHL
Figure, figure B, C are respectively strain X J-10 to OdDHL 12h, degradation high-efficient liquid phase chromatogram for 24 hours).
Strain X J-10, DH5 α, B23, EC1 that Fig. 8 is the present invention are individually inoculated with and are connect respectively with EC1 co-inoculations
Incidence of the kind after potato tubers 30h.
Fig. 9 be the present invention strain X J-10,3937 individually inoculation and co-inoculation in castock for 24 hours after morbidity
Situation.
Specific embodiment
It is further illustrated the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits 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.
Used material is as follows in following embodiment：
LB culture mediums：Tryptone 10.0g/L, yeast extract 5.0g/L, sodium chloride 10.0g/L, pH 6.8~7.2,
121 DEG C of 15~25min of sterilizing.LB solid culture based formulas is to add in the agar of 1.5% (w/ ν) in liquid medium.
Basal salt media (MSM)：(NH4)2SO4, 2.0g/L；CaCl2·2H2O, 0.01g/L；Na2HPO4·12H2O,
1.5g/L；KH2PO4, 1.5g/L；MgSO4·7H2O, 0.2g/L；FeSO4·7H2O, 0.001g/L；pH 6.5.
Basal medium (MM)：K2HPO4, 10.5g/L；KH2PO4, 4.5g/L；(NH4)2SO4, 2.0g/L；Mannitol,
2.0g/L；Glycerol, 2.0g/L；MgSO4·7H2O, 0.2g/L；CaCl2, 0.01g/L；
FeSO4, 0.005g/L；MnCl2, 0.002g/L；pH 6.5.
OdDHL, OHHL are purchased from the virtuous Chemical Industry Science Co., Ltd in Shanghai, and reagent needed for X-gal, culture medium is purchased certainly
The companies such as neat Hunan, Guangzhou, Hua Qisheng.
Separation, the screening of embodiment 1OdDHL degradation bacterias (acinetobacter calcoaceticus strain X J-10)
1st, the separation and screening of acinetobacter calcoaceticus strain X J-10
(1) soil sample gathers：The soil sample from Xinjiang is gathered as microbial source.
Soil sample is gathered on October 9th, 2014 from Xinjiang province farmland, is sampled, is packed, preserving as microbial source band
Return separation and screening that laboratory carries out bacterial strain.
(2) enrichment culture of bacterial strain：Basal salt media (MSM) is prepared, the MSM of 50mL is packed into 250mL triangular flasks
Culture medium, 121 DEG C of 15~25min of sterilizing, aseptically adds in OdDHL mother liquors (solvent is methanol), makes OdDHL after cooling
Ultimate density is 5 μM, in addition adds in soil sample 5g, 30 DEG C is positioned over, after 200rpm shaking table cultures 7d, by 10% inoculum concentration
It is transferred in the MSM culture mediums that OdDHL concentration is 10 μM.After the same terms culture 7d, then it is transferred to by 10% inoculum concentration
OdDHL concentration is in 20 μM of MSM culture mediums, then with the same terms culture 7d.And so on, it is continuously increased the concentration of OdDHL.
(3) strain isolation：The separation of bacterial strain is carried out using spread plate.
1mL end MSM culture medium zymotic fluids are taken, it is respectively 10 to be diluted to concentration with sterile water-1、10-2、10-3、10-4、
10-5、10-6、10-7、10-8Zymotic fluid, then draw the 100 μ L of zymotic fluid of each concentration respectively, be coated on LB solid plates
Uniformly, dry, 30 DEG C of cultures are for 24 hours.The single bacterium colony of the different shape grown on picking tablet is rule training repeatedly in LB solid plates
Purifying is supported, until isolating single strain.Single strain is stored in -80 DEG C with glycerine conservation method, treats further experiment detection degradation
Activity.
(4) bacterial strain screening：Using reporting bacterial strain (Agrobacterium tumefaciens NT1) screening there is degradation to live
The bacterial strain of property.
By bacterial strain to be screened from -80 DEG C of activation, line on LB solid medium tablets, in 30 DEG C of incubator cultures
24h.Picking single bacterium colony is seeded to LB fluid nutrient mediums, and with 30 DEG C, the condition of 200rpm is incubated overnight, and obtains bacterium solution.Take 1
OD600The thalline of value is resuspended with MSM culture mediums of the 1mL using OdDHL as sole carbon source, obtains mixture to be cultivated, MSM cultures
The concentration of OdDHL is 20 μM in base.Mixture to be cultivated is transferred in 2mL centrifuge tubes, with 30 DEG C, the condition of 200rpm is trained
It supports for 24 hours.After for 24 hours, 5 μ L reaction mixtures point samples are taken to being coated on the MM plates of 200 μ L reporting bacterial strain bacterium solutions, wherein reporting bacterial strain
The OD of bacterium solution600Contain the X-gal that concentration is 40 μ g/mL in=0.4, MM plate.The MM plates for having put sample are positioned over 28 DEG C
Incubator, for 24 hours after observation experiment result.
Interpretation of result, OdDHL have diffusivity, and the OdDHL contained in reaction mixture is more, from centered on sample point to
The distance of surrounding diffusion is more remote, then the blue circle radius occurred on MM plates is bigger, is compared with CK experimental groups, and the two blue circle radius gets over phase
It is near then illustrate that the bacterial strain is weaker or without degradation effect to OdDHL degradations, conversely, the OdDHL contained in reaction mixture is fewer,
The smaller appearance even without blue circle of blue circle diameter then generated.It is filtered out according to experimental result best to OdDHL degradation effects
Bacterial strain, be named as degradation bacteria XJ-10.
The identification of 2 acinetobacter calcoaceticus strain X J-10 of embodiment
The present embodiment has carried out morphological feature, physio-biochemical characteristics and 16S rDNA systems for degradation bacteria XJ-10 and has sent out
Analysis is educated, it is acinetobacter calcoaceticus (Acinetobacter schindleri) to identify the bacterial strain.It is specific as follows：
(1) colony morphology characteristic：48h is cultivated on LB solid plates, bacterium colony is flat, and surface is smooth opaque, and edge is whole
Together, as shown in Figure 1；Rice white is presented in the bacterial clump on blood plate, as shown in Figure 2；In diffusion in LB fluid nutrient mediums
Property muddy, aerobic, well-grown at 30 DEG C.
(2) morphological features：As shown in figure 3, thalline is in rod-shaped, and it is sometimes subsphaeroidal, there is 1 polar flagella.
(3) physio-biochemical characteristics：The bacterial strain be Gram-negative bacteria, aerobic, catalase test reacting positive, oxidizing ferment,
Hemolytic test, gelatin liquefaction, Starch Hydrolysis experiment reaction negative；Can growth temperature range be 20~41 DEG C, optimum growth temperature
For 30 DEG C, optimal pH 7.0.Its Physiology and biochemistry qualification result is as shown in Table 1 and Table 2.
(4) 16S rDNA sequences and phylogenetic analysis：The 16S rDNA gene order length of strain X J-10 is
1420bp, with ncbi database (http：//www.ncbi.nlm.nih.gov/) be compared after find, strain X J-10 with
Acinetobacter schindleri have higher homology, and systematic evolution tree is as shown in Figure 4.
In conclusion by the morphological feature of strain X J-10, physio-biochemical characteristics and 16S rDNA gene orders
Identification and analysis, identify that the bacterial strain belongs to acinetobacter calcoaceticus (Acinetobacter schindleri), are preserved in Chinese Typical Representative training
Object collection (CCTCC) is supported, preserving number is CCTCC NO：M2017650, preservation address are Wuhan, Chinas, Wuhan University.
1 strain X J-10 Physiology and biochemistry qualification results of table
Note：-：Negative reaction；+：Positive reaction.
2 strain X J-10 utilization of carbon source qualification results of table
Note："-" is expressed as negative reaction or does not utilize；"+" represents positive reaction or utilization
The antibiotics sensitivity analysis of 3 strain X J-10 of embodiment
In order to preferably study strain X J-10, the present embodiment has carried out Multiple Classes of Antibiotics sensibility to strain X J-10
Detection.
Analysis of experimental results：As shown in figure 5, the strain X J-10 resistance of ampicillin is reached 400 μ g/mL and with
On, 200 μ g/mL are reached to the resistance of kanamycins, 100 μ g/mL are reached to the resistance of gentamicin, to streptomysin, chloramphenicol
Resistance reach 20 μ g/mL.This result is beneficial to choose suitable antibiotic as reference in follow-up study.
The Activity determination of 4 strain X J-10 of embodiment degradations OdDHL
The present embodiment detects J-10 pairs of strain X using reporting bacterial strain (Agrobacterium tumefaciens NT1)
The degradation effect of OdDHL.
1st, experimental procedure：
With LB solid medium tablet activated strains XJ-10, tablet is placed in 30 DEG C of incubators, is incubated overnight.Picking list
Colony inoculation is into LB liquid medium, and with 30 DEG C, the condition of 200rpm is incubated overnight to obtain bacterium solution.Take 1 OD600The bacterium of value
Body, the resuspension thalline of the MSM culture mediums using OdDHL as sole carbon source for drawing 1mL obtain treating reaction mixture, wherein MSM trainings
The concentration for supporting OdDHL in base is 80 μM.Reaction mixture is transferred in 2mL centrifuge tubes, with 30 DEG C, the CMC model of 200rpm
24h.After for 24 hours, 5 μ L reaction mixtures points is taken then to put reporting bacterial strain successively in lower section to agar strip top
(Agrobacterium tumefaciens NT1) bacterium solution.Then the agar of reaction mixture and reporting bacterial strain will have been put
Item is positioned over 28 DEG C of incubators, observation experiment result after culture for 24 hours.Wherein, it containing concentration is 40 μ g/mL X-gal that agar strip, which is,
MM plates slitting gained.
Escherichia coli DH5 α, to bacterial strains of the OdDHL without degradation effect, are in the present embodiment feminine gender to be known
Control group；Bacillus thuringiensis subsp israelensis (Bacillus thuringiensis subsp.Israelensis) B23
It is in the present embodiment positive control (Dong Y, Xu J, Li X, et for the known bacterial strain to OdDHL with degradation effect
al.AiiA,an enzyme that inactivates the acylhomoserine lactone quorum-sensing
signal and attenuates the virulence of Erwinia carotovora[J].Proceedings of
the National Academy of Sciences,2000,97(7):3526-3531.)；CK is the blank pair of not mycetome
According to.
2nd, interpretation of result：
As shown in fig. 6, CK and DH5 α experimental groups are from agar strip top to reporting bacterial strain, the last one becomes blue to experimental result
Point illustrates that the content of OdDHL in the two experimental group reaction mixtures is close apart from basically identical, does not have degradation with DH5 α
The fact that OdDHL abilities, is consistent.The unchanged indigo plant of B23 and XJ-10 experimental groups reporting bacterial strain, illustrates that the reaction of the two experimental groups mixes
It closes in object and does not contain OdDHL, OdDHL is degraded completely, i.e. XJ-10 can degrade OdDHL.
Embodiment 5 detects the activity of strain X J-10 degradations OdDHL using HPLC
1st, step：The strain X J-10 LB solid panels activation in -80 DEG C will be frozen, after 30 DEG C of cultures for 24 hours on picking tablet
Single bacterium colony be inoculated in LB liquid medium, with 30 DEG C, the condition of 200rpm is incubated overnight to obtain bacterium solution.Take 1 OD value
Thalline, thalline are resuspended with the MSM culture mediums of 1mL, re-suspension liquid are added in 19mL MSM basal mediums, and add OdDHL mothers
Liquid (solvent is methanol), it is 0.2mM to make its ultimate density.It at 30 DEG C, is cultivated under conditions of 200rpm, in 0h, 12h and for 24 hours three
A point in time sampling, and OdDHL remaining in sample is extracted, the residual quantity that OdDHL is measured using HPLC represents bacterial strain
XJ-10 is to the degradation situation of OdDHL.
2nd, in sample OdDHL extracting method：Appropriate amount of sample is taken into 15mL centrifuge tubes, is centrifuged with the speed of 4000rpm
After 5min, 5mL supernatants are taken into 50mL separatory funnels, and isometric ethyl acetate is added in into separatory funnel, are acutely shaken
It after swinging 3min, stands, lower floor's solution is transferred in 15mL centrifuge tubes by layering, and upper liquid is filled into 50mL round bottoms through funnel and burns
In bottle, filter paper is covered in funnel.Lower floor's solution carries out 1 extraction again as stated above.Filtrate is incorporated to round-bottomed flask, 45 DEG C of constant temperature
Rotary evaporated to dryness is dry, with 2 washing round-bottomed flasks of chromatography methanol point, is settled to 2mL, through 0.45 μM of organic membrane filtration into
Sample bottle measures its residual volume using HPLC methods.
3rd, HPLC measures OdDHL residual volume methods：
HPLC instrument models：Waters 2695.Chromatographic column：C18Reverse-phase chromatographic column (250 μ m 4.6mm × 5 μm).Flow velocity
For 0.8mL/min.Column temperature is 30 DEG C.Mobile phase：Methanol：Water=70；30(v：v).Detection wavelength is 210nm.Sample size is 20 μ
L。
4th, OdDHL degradation rates computational methods：Degradation rate (%)=(1-A1/A0) × 100, A1For OdDHL after degradation bacteria processing
Residual concentration, A0For the OdDHL residual concentrations after control treatment.
5th, analysis of experimental results：
For HPLC testing results as shown in fig. 7, the compares figure that figure A is non-inoculating strain XJ-10, figure B, C are respectively strain X J-
10 couples of OdDHL 12h, degradation figure for 24 hours, degradation rate respectively reach 85.71%, 98.8%, corresponding time strain X J-10
OD600Value is respectively 0.144,0.233.Experiment shows that under the conditions of OdDHL is with existing for sole carbon source bacterial strain being capable of fast prompt drop
It solves OdDHL and utilizes its growth.
6 strain X J-10 of embodiment studies the biocontrol effect of bacterial soft rot of potato
The present embodiment is with plant soft rot disease pathogenic bacteria Dickeya zeae EC1 (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].Journal of
Biotechnology,2008,190(3):Exemplified by 1045-1053.), the pathogenic bacteria that strain X J-10 causes a disease to relying on AHLs are studied
Biocontrol effect.
1st, step：
With LB solid medium tablet activated strains XJ-10 and EC1,30 DEG C of cultures are for 24 hours.Distinguish afterwards on picking tablet for 24 hours
Single bacterium colony, be seeded in LB liquid medium, with 30 DEG C, the condition of 200rpm is incubated overnight.Seven experimental groups are set respectively：
XJ-10+LB, DH5 α+LB, B23+LB, EC1+LB, XJ-10+EC1, DH5 α+EC1, B23+EC1.
Wherein, Escherichia coli DH5 α to be known to bacterial strains of the OdDHL without degradation effect, in the present embodiment
For negative control group.Bacillus thuringiensis subsp israelensis (Bacillus thuringiensis
Subsp.Israelensis) B23 is the known bacterial strain to OdDHL with degradation effect, is in the present embodiment positive control.
In experiment, XJ-10, DH5 α, the final working solution OD of B23, EC1600=1.0.Each experimental group is inoculated with the mixing of 5 μ L with liquid-transfering gun
Bacterium solution is central to potato slices.
2nd, interpretation of result：
As shown in figure 8, the potato of XJ-10+LB, DH5 α tri- experimental groups of+LB, B23+LB does not fall ill, illustrate XJ-10,
DH5 α, B23 are to potato no pathogenicity；EC1, DH5 α+EC1, tri- experimental group potato incidences of B23+EC1 do not have bright
Significant difference is different.The potato morbidity severity of experimental group XJ-10+EC1 is substantially compared with EC1, DH5 α+EC1, tri- experiments of B23+EC1
Group morbidity severity is light.The experimental results showed that strain X J-10 has significantly soft rot caused by Dickeya zeae EC1
Biocontrol effect.
7 strain X J-10 of embodiment studies the biocontrol effect of soft rot of cabbage
The present embodiment can trigger 3937 (the Glasner J of pathogen Dickeya dadantii of soft rot of cabbage to draw
D,Yang C,Reverchon S,et al.Genome sequence of the plant-pathogenic bacterium
Dickeya dadantii 3937[J].Journal of Biotechnology,2011,193(8):2076-2077.) be
Example, the biocontrol effect for the pathogen that research strain X J-10 causes a disease to relying on AHLs.
1st, step：
With LB solid mediums tablet activated strains XJ-10 and 3937,30 DEG C of cultures are for 24 hours.Distinguish afterwards on picking tablet for 24 hours
Single bacterium colony, be seeded in LB liquid medium, with 30 DEG C, the condition of 200rpm is incubated overnight.LB is set respectively, 3937, XJ-
10, XJ-10+3937, four experimental groups make the final working concentration OD of 3937, XJ-10600=1.0.Each experimental group liquid-transfering gun
The mixed bacteria liquid of 2 μ L is inoculated with to castock.Observe incidence afterwards for 24 hours.
2nd, interpretation of result：
As shown in figure 9, can see being inoculated with and having cultivated on castock for 24 hours, two experimental groups of LB, XJ-10 connect
It does not fall ill at kind, illustrates LB, XJ-10 is to castock no pathogenicity.At 3937 inoculation of experimental group, experimental group XJ-10+
Occur at 3937 inoculation without apparent soft rotten symptom, occurring degree is lighter.The experimental results showed that strain X J-10 is to Dickeya
Soft rot caused by dadantii 3937 has significant biocontrol effect.
Claims (10)
1. the acinetobacter calcoaceticus of one plant of degradable AHLs colony induction signaling molecule（Acinetobacter schindleri）Bacterial strain
XJ-10, which is characterized in that the bacterial strain is preserved in China typical culture collection center on November 2nd, 2017, and preserving number is
CCTCC NO：M2017650.
2. prepared by application of the acinetobacter calcoaceticus in colony induction signaling molecule AHLs and/or AHLs signal analog of degrading
Application in the product of degradation AHLs and/or AHLs signal analogs.
3. application according to claim 2, which is characterized in that the AHLs class signals substance is homoserine lactone class object
Matter.
4. application of the acinetobacter calcoaceticus in prevention relies on the pathogenic plant disease of AHLs mediations relies on what AHLs caused a disease preparing
Application in terms of the prevention preparation of pathogenic bacteria.
5. the application according to claim 2 or 4, which is characterized in that the acinetobacter calcoaceticus is not lever described in claim 1
Bacteria strain XJ-10.
6. a kind of prevent the method for relying on the pathogenic fungus diseases that AHLs causes a disease, which is characterized in that with the bacteria suspension pair of acinetobacter calcoaceticus
Crop is handled.
7. according to the method described in claim 6, it is characterized in that, the acinetobacter calcoaceticus is acinetobacter calcoaceticus described in claim 1
Strain X J-10.
8. according to the method described in claim 6, it is characterized in that, the pathogenic bacteria that the dependence AHLs causes a disease include：Ou Wenshi
Bacterium（Erwinia）, enlightening Cattell bacterium（Dickeya）, Pseudomonas aeruginosa（Pseudomonas aeruginosa）.
9. a kind of degradation bacterial agent of degradable colony induction signaling molecule AHLs, which is characterized in that containing described in claim 1 not
Lever bacteria strain XJ-10 and/or its bacteria suspension.
10. the biological prevention and control agent for the pathogenic bacteria that a kind of dependence AHLs causes a disease, which is characterized in that contain not lever described in claim 1
Bacteria strain XJ-10 and/or its bacteria suspension.
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