CN109182159A - Bacterium and its application in disease prevention and control is quenched in a kind of N- acyl homoserine lactones - Google Patents
Bacterium and its application in disease prevention and control is quenched in a kind of N- acyl homoserine lactones Download PDFInfo
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Abstract
The invention discloses one kindNBacterium and its application in disease prevention and control is quenched in acyl homoserine lactones.The research of the invention finds that, intermediate anthropi has quenching activity to a variety of AHLs colony induction signaling molecules, and it is separated to plant height effect and bacteria strain D-2 is quenched, can be degraded a variety of AHLs colony induction signaling molecules quick and significantly, including OHHL, OOHL and OdDHL etc., it can significantly mitigate simultaneously and rely on the pathogenic pathogen Disease symptoms of AHLs, have huge application potential in terms of prevention and treatment AHLs mediates pathogenic pathogenic bacteria harm.And the strain activity is stablized, to substitute chemical prevention with biological control and being target without causing the therapeutic strategy for selecting pressure to provide new development approach to block quorum sensing, relying in terms of colony induction signaling mediates pathogenic pathogen harm in prevention and treatment has huge popularization and application potentiality, and this be quenched with group has broad application prospects for the Anti-pathogenic biocontrol microorganisms of the mode of action.
Description
Technical field
The invention belongs to technical field of biological control.More particularly, to one kindNBacterium is quenched in acyl homoserine lactones
And its application in disease prevention and control.
Background technique
Carrot soft rot Pectinatus carrot subspecies (Pectobacterium carotovorum subsp. carotovora, Pcc) be plant soft rot disease pathogen Major Members (Lim J, Jee S, Lee D H, et al..
Biocontrol of Pectobacterium carotovorum subsp. carotovorum using
bacteriophage PP1[J]. Journal of Microbiology and Biotechnology, 2013, 23(8):
1147-1153.).Pcc can not only infect a variety of temperate zones, subtropical zone crops and ornamental plant, such as: crops Chinese cabbage, trailing plants
Fore-telling, tomato, potato, carrot, capsicum, celery, cauliflower etc.；Ornamental plant kaffir lily, lily, violet, narcissus, cactus
Deng.Moreover, it is also possible to infect medicinal plant Typhonium giganteum and milk thistle etc..The bacterial soft rot occurrence scope that Pcc causes is extensive,
As if having generation in multinational such as vertical pottery, Japanese, Germany, Argentina, New Zealand and China and endangering seriously, have become generation
Criticality disease and the underproduction for leading to respective crop, cause heavy economic losses.Directed toward bacteria property soft rot, it is main in production at present
It is prevented and treated using chemical pesticides such as thiophene bacterium ketone, thiophene, ambam, streptomycin sulphate and Zhongshengmycins.However chemical pesticide
A large amount of uses have had resulted in well-known environmental pollution, ecological balance destruction, pathogen drug resistance and food safety etc. one
Serial serious problems, in addition, the abuse of antibiotic also causes the generation of microorganism drug resistance.Therefore, it finds new, effective
Control strategy is extremely urgent.
Microorganism itself can produce and secrete one or more chemical substances, with the increase of micropopulation volume density
When, the concentration of chemical substance is also increasing in environment, after these compound concentrations reach certain threshold value, certain spies of microorganism
Determine the especially many Disease-causing genes of gene to start to express.This phenomenon is referred to as quorum sensing (Quorum sensing, QS),
These chemical substances be referred to as colony induction signaling molecule or self-induction agent (Autoinducers, AIs) (Whiteley M,
Diggle S P, Greenberg E P. Progress in and promise of bacterial quorum
Sensing research [J] Nature, 2017,551 (7680): 313-320.).NAcyl homoserine lactones class
Substance (N- Acyl homoserine lactones, AHLs) it is the distinctive colony induction signaling of Gram-negative bacteria, they are big
The cricoid structure of homoserine lactone all having the same and all there is acyl side-chain, but its acyl side-chain length, saturation degree are deposited
In difference.N(3-oxohexanoyl)-L-homoserine lactone (OHHL, 3OC6HSL),N-(3-
Oxooctanoyl)-L-homoserine lactone(OOHL, 3OC8HSL),N-(3-oxododecanoyl)-L-
Homoserine lactone (OdDHL, 3OC12HSL) belongs to AHLs, the difference is that acyl side-chain length is different.
Pcc is capable of the hydrolase of degrading plant cell wall by synthesis and secretion, such as: pectase, polygalacturonase
Enzyme, protease etc., the defense system for attacking plant destroy organization structure of the plant, obtain moisture and nutritional ingredient to support itself to give birth to
Long and breeding, after infecting successfully, there are soft rot related symptoms in host plant.Pcc also has quorum sensing phenomenon, and regulates and controls
To the expression of its pathogenic relevant hydrolase.Pcc can be synthesized and be secreted a kind of AHL---OHHL to environment, with Pcc
The increase of population density, OHHL concentration also constantly increases in environment, after OHHL reaches a certain concentration threshold value, OHHL and LuxR
Family's transcription activating protein combines, and virulence factor is that the gene of coding ectoenzyme starts to express, to pathogenic relevant hydrolase
Synthesis, host plant are successfully infected, and cause host that soft rot evil (Pierson L S I, Wood D W, Pierson occurs
E A. Homoserine lactone-mediated gene regulation in plant-associated bacteria
[M] Webster R K. Annual Review of Phytopathology, 1998:207-225.).
It is a kind of new disease proposed based on quorum sensing phenomenon that (Quorum quenching, QQ), which is quenched, in quorum sensing
Evil prevention and treatment new strategy, it may be assumed that carry out enzyme degradation or modification by inhibiting synthesis, accumulation, the monitoring of signaling molecule, or to signaling molecule
Mechanism interfere intervention school-based, inhibit the expression of microorganism and pathogenic relevant gene, weaken the pathogenic of microorganism
Property, to achieve the purpose that controlling disease.Bacterium or Quorum quenching enzymes (Quorum quenching is quenched using quorum sensing
Enzymes) degrading microorganism signaling molecule is that current toxicity is minimum, approach is quenched in maximally efficient quorum sensing.Quorum sensing
Being quenched is to be carried out by regulating and controlling intervention school-based come controlling disease, so selection pressure will not be generated to microorganism, theoretically
Bacterium will not develop drug resistance.It is a kind of new way for effectively preventing phytobacterial disease that quorum sensing, which is quenched, has operation letter
Just, the advantages that economical and practical, environmental-friendly, the high-efficient and period is short.Develop group for different groups inductive signal and system is quenched
Agent is the research hotspot of current international coverage.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of new having, and the micro- of colony induction signaling molecule ability is quenched
Biological inoculum, i.e. centre anthropi (Ochrobactrum intermedium), to a variety of AHLs colony induction signalings point
Son has significant and quick degradation, has in terms of the pathogenic bacteria harm that prevention and treatment colony induction signaling molecule mediates huge
Application potential, this is chemical prevention to be substituted with biological control and to block quorum sensing to select the anti-of pressure without causing as target
It controls strategy and provides new development approach.
The object of the present invention is to provide intermediate anthropi (Ochrobactrum intermedium) microorganism is being quenched
Application in colony induction signaling molecule AHLs, or the product of degrading microorganism colony induction signaling molecule AHLs is quenched in preparation
In application；And the application in the pathogenic plant disease of prevention and treatment dependence micropopulation induction signal molecule mediation, or
Preparation relies on the application in terms of the prevention and treatment preparation of the pathogenic pathogenic bacteria of micropopulation induction signal molecule.
Another object of the present invention is to provide a kind of pale bar in centre that degrading microorganism colony induction signaling molecule can be quenched
Bacteria strain D-2 and its application.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention study for the first time the intermediate anthropi of discovery (Ochrobactrum intermedium) have the micro- life of degradation is quenched
The effect of object colony induction signaling molecule, and isolated and purified screening and identification from the soil sample of power plant of Hebei province from acquisition and obtained
To a high-efficiency degradation bacterium, intermediate anthropi strain D-2, and Guangdong Province microorganism fungus kind guarantor is preserved on July 5th, 2018
Hiding center (GDMCC), deposit number are GDMCC No:60409, and preservation address is Xianlie Middle Road, Guangzhou City 100 compound 59
5 building, building.
The colony morphology characteristic of bacterial strain D-2 are as follows: 48 h are cultivated on nutrient agar panel, bacterium colony is white, and it is round, it is convex
It rises, translucent, neat in edge.
The morphological feature of Electronic Speculum observation thallus are as follows: cell is subsphaeroidal, amphitrichous.
The physio-biochemical characteristics of bacterial strain D-2 are as follows: Gram-negative bacteria, when growth are aerobic, catalase test, oxidizing ferment examination
Test reacting positive, gelatin liquefaction test reaction negative.
The bacterial strain D-2 reaches 400 μ gmL to the resistance of ampicillin-1More than, the resistance of kanamycins is reached
350 μg·mL-1, 40 μ gmL are reached to the resistance of gentamicin, streptomysin-1, 10 μ are reached to the resistance of chloramphenicol
g·mL-1, to tetracycline non-resistant.
The experimental results showed that the AHLs colony induction signaling molecule of the intermediate anthropi strain D-2 centering long-chain has
Quenching activity, can significantly and quickly degrade in long-chain AHLs.Bacterial strain D-2 can be in being felt with group at concentrations up to 0.4 mM
Induction signal molecule OHHL decomposes for normal growth in the basal salt media of sole carbon source and completely OHHL in 32 h.Anti-
The pathogenic bacteria harm aspect for controlling the mediation of AHLs types of populations induction signal molecule has huge application potential.
Therefore, applying below should all be within protection scope of the present invention:
Intermediate anthropi is in the application being quenched in micropopulation induction signal molecule AHLs.
Application of the intermediate anthropi in the product that degrading microorganism colony induction signaling molecule AHLs is quenched in preparation.
Intermediate anthropi relies on micropopulation induction signal molecule in prevention and treatment and mediates answering in pathogenic plant disease
With.
Intermediate anthropi relies on the prevention and treatment preparation side of the pathogenic pathogenic bacteria of micropopulation induction signal molecule in preparation
The application in face.
Preferably, in any of the above-described application, the intermediate anthropi is that the isolated centre of the present invention is grey
White bacillus strain D-2.
In addition specifically, the micropopulation induction signal molecule isNAcyl homoserine lactones substance (N-
Acyl homoserine lactones, AHLs).
Preferably, the micropopulation induction signal molecule is the AHLs of middle long-chain.
It is highly preferred that the micropopulation induction signal molecule is AHLs signaling molecule OHHL, OOHL and/or OdDHL.
Preferably, described to rely on the pathogenic pathogen of micropopulation induction signal molecule are as follows: enlightening Cattell bacterium (Dickeya
Sp.), Pectinatus (Pectobacterium Sp.) or Pseudomonas aeruginosa (Pseudomonas aeruginosa).
The plant disease includes soft rot, such as bacterial soft rot of potato or carrot fleshy root soft rot.More specifically by fruit
Soft rot caused by glue bacillus (such as Z3-3).
In addition, a kind of method that prevention and treatment relies on the pathogenic pathogenic fungus diseases of AHLs colony induction signaling molecule, specifically sharp
Inoculation processing is carried out to crop with the bacterium solution of intermediate anthropi, relies on what micropopulation induction signal molecule caused a disease to prevent and treat
Pathogen infection and disease, also should be within protection scope of the present invention.
Preferably, the mode of the processing is to carry out inoculation processing to crop.
Preferably, in application, the optimal pH that AHLs is quenched in the intermediate anthropi is 6.5, optimum temperature 30
℃。
In addition, a series of function microbial inoculum can be made using intermediate anthropi, it is such as a kind of to contain intermediate anthropi
And/or the microbial inoculum that is quenched that micropopulation induction signal molecule is quenched of its bacterium solution, one kind containing intermediate anthropi and/or
The biological prevention and control agent for relying on the pathogenic pathogen of micropopulation induction signal molecule of its bacterium solution, also should all protect in volume of the invention
Within the scope of shield.Preferably, the bacterial strain D-2 screened containing intermediate anthropi for the present invention.
The present invention additionally provides the preparation method of the bacterial strain D-2 bacterium solution simultaneously: being specifically that bacterial strain D-2 is lined LB to consolidate
On body culture medium flat plate, 24 h are cultivated at 30 DEG C, picking single colonie is inoculated in LB liquid medium preculture to logarithmic phase,
Obtain bacterial strain D-2 bacterium solution.The concentration of bacterium solution does not do stringent limitation, can specifically be adjusted according to practical degree of disease and application effect
It is whole.
Preferably, the LB culture medium are as follows: 10.0 gL of tryptone-1, 5.0 gL of yeast extract-1, sodium chloride
10.0 g·L-1, 6.8 ~ 7.2,121 DEG C of 20 min of sterilizing of pH.LB solid culture based formulas is to be added in liquid medium
1.5 %(w/ ν) agar.
The invention has the following advantages:
The research of the invention finds that intermediate anthropi (Ochrobactrum intermedium) a variety of AHLs quorum sensings are believed
Number molecule has quenching activity, and can degrade a variety of AHLs colony induction signaling molecules quick and significantly, including OHHL, OOHL
With OdDHL etc., there is huge application potential in terms of prevention and treatment AHLs mediates pathogenic pathogenic bacteria harm, this is to replace with biological control
It is target without causing the therapeutic strategy for selecting pressure to provide new development approach for chemical prevention and to block quorum sensing.
Meanwhile present invention screening obtains one plant of intermediate anthropi D-2, can be only in the OHHL at concentrations up to 0.4 mM
Normal growth in the culture medium of one carbon source, in 32 h can degradable colony induction signaling molecule OHHL, which has aobvious
The biodegradable effect of work can prevent and treat and rely on the diseases such as the pathogenic bacterial soft rot of AHLs, disease such as pathogenic to dependence AHLs
Opportunistic pathogen carrot soft rot Pectinatus carrot subspecies (Pectobacterium carotovorum subsp. carotovora,
Pcc disease caused by) has significant Biocontrol Effect.
According to the present invention, since bacterial strain D-2 has quorum sensing AHLs signal point in stability and high efficiency degrading plant pathogen
The characteristic of son, therefore, bacterial strain D-2 can be applied to the prevention and treatment that AHLs in natural environment mediates pathogenic pathogenic bacteria, can both reduce
Pesticide abuses problem, and brings new thinking, new way and new method to disease control.
Detailed description of the invention
Fig. 1 is the colonial morphology figure of bacterial strain D-2 of the invention on LB culture medium.
Fig. 2 is the scanning electron microscope (SEM) photograph of bacterial strain D-2 of the invention.
Fig. 3 is the Phylogenetic analysis figure of bacterial strain D-2 of the invention.
Fig. 4 is the growing state figure of bacterial strain D-2 of the invention in different antibiotic.
Fig. 5 be bacterial strain D-2 of the invention to different AHLs quenching activity result figures (E.coliFor negative control, B23 is sun
Property control), (a) be OHHL, (b) be OOHL, (c) be OdDHL.
Fig. 6 be bacterial strain D-2 of the invention using OHHL as sole carbon source when growth curve and degradation curve figure.
Fig. 7 be bacterial strain D-2 of the invention,E.coli, B23 is respectively with Z3-3 co-inoculation after 24 h of potato tubers
Incidence.
Fig. 8 be bacterial strain D-2 of the invention,E.coli, B23 respectively with Z3-3 co-inoculation in carrot be sliced 24 h after
Incidence.
Specific embodiment
The present invention is further illustrated 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 present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The acquisition and identification of the intermediate anthropi strain D-2 of embodiment 1
1, the separation, screening of bacterial strain D-2
(1) soil sample acquires: acquiring the soil sample from power plant of Hebei province as microbial source
Soil sample is located away from August 9th, 2017 out of Hebei province power plant, is sampled, packs, saving and take back as microbial source
School carries out strain isolation.
(2) enrichment culture of bacterial strain: preparing basic salt (MSM) culture medium, and 20 mL MSM culture mediums are attached to 250 mL cone
It sterilizes in shape bottle, OHHL mother liquor (acetonitrile is solvent) is aseptically added after cooling, makes 5 μM of ultimate density of OHHL,
5 g soil samples are added simultaneously, after cultivating 7 d under the conditions of 30 DEG C, 200 rpm, are transferred to second batch by the inoculum concentration of 10 %
In MSM culture medium containing 10 μM of OHHL.After 7 d of the same terms culture, then it is transferred to containing 20 μM by the inoculum concentration of 10 %
In the MSM culture medium of OHHL, continue to cultivate 7 d.And so on, it is continuously increased the concentration of OHHL.
Wherein, the formula of described basic salt (MSM) culture medium are as follows: 2 g of ammonium sulfate；0.2 gL of bitter salt-1；Two
0.01 gL of calcium chloride hydrate-1；0.001 gL of green vitriol-1；1.5 gL of disodium hydrogen phosphate dodecahydrate-1；
1.5 gL of sodium dihydrogen phosphate-1；pH 6.5.
(3) strain isolation is with purifying: being separated using dilution, spread plate.
Taking 1 mL end MSM culture medium fermentation liquid to be diluted to concentration with sterile water is 10-1、10-2、10-3、10-4、10-5、
10-6、10-7、10-8Gradient concentration fermentation liquid, the fermentation liquid for then drawing the 100 each concentration of μ L is uniformly coated to LB solid
On plate, 30 DEG C of cultures, the different single colonie of picking colony form is crossed culture purified repeatedly in LB solid plate, until point
Separate out single strain.- 80 DEG C of single strain are saved, to further experiment measurement to the degradation effect of AHLs.
(4) bacterial strain screening: reporting bacterial strain CF11(is utilizedAgrobacterium tumefaciens NT1) to from soil sample
Isolated bacterial strain is screened.
The bacterial strain to be screened for being stored in -80 DEG C is lined on LB plate, 30 DEG C of cultures carry out the activation of bacterial strain.It chooses
It takes single colonie to be seeded to LB liquid medium, is incubated overnight under the conditions of 30 DEG C, 200 rpm, obtains bacterium solution.Take certain volume
(the OD of V=1/600) bacterium solution be centrifuged with the condition of 4000 rpm, 10 min, abandon supernatant, obtain an OD600The thallus of value.
By 1 OD600The thallus of value is seeded in MSM culture medium of the 1mL using OHHL as sole carbon source, OHHL in MSM culture medium
Final concentration of 20 μM.Reaction mixture is placed in 2 mL centrifuge tubes, cultivates 24 under conditions of 30 DEG C, 200 rpm
h.After 24 h, the MM agar strip top of 5 μ L reaction mixture point samples to 1 cm wide is taken, then can detecte in one row of lower section point
The reporting bacterial strain CF11(of AHLsAgrobacterium tumefaciens NT1).Wherein the pH value of MM agar strip is 6.5, fine jade
Contain 40 μ gmL in rouge item-1X-gal.MM agar strip is placed on 28 DEG C of incubators, is observed in fact after being protected from light 24 h of culture
Test result.
Wherein, the formula of described basic (MM) culture medium are as follows: 2 gL of ammonium sulfate-1；0.2 gL of bitter salt-1；
0.01 gL of anhydrous calcium chloride-1；0.005 gL of ferrous sulfate-1；0.002 gL of manganese chloride-1；Dipotassium hydrogen phosphate 10.5
g·L-1；4.5 gL of potassium dihydrogen phosphate-1；pH 6.5.
(5) interpretation of result: as reporting bacterial strain CF11(Agrobacterium tumefaciens NT1 agar strip) is detected
In contain AHLs when, the related gene of beta galactosidase starts to express, and CF11 secretes the sweet enzyme of beta galactose into environment.β-
The sweet enzyme of galactolipin can be by the leuco-compounds X-gal contained in MM agar strip (the chloro- 3- indoles-β-D- galactolipin of the bromo- 4- of 5-
Glycosides) enzymatic hydrolysis be galactolipin and the dark blue bromo- 4- of color substance 5- it is indigo, the indigo bacterium colony for making entire reporting bacterial strain of the bromo- 4- of 5- becomes
Blue.AHLs can be spread on agar strip, and diffusion length is directly proportional to its concentration.In conclusion according on agar strip
Reporting bacterial strain becomes the blue more short bacterial strain that then corresponds to of distance from top and drops from AHLs content in the distance analysis original sample that top becomes blue
It is better to solve effect.The bacterial strain best to AHLs degradation effect is finally filtered out according to experimental result, is named as bacterial strain D-2.
2, the identification of bacterial strain D-2 and phylogenetic analysis
(1) colony morphology characteristic (as shown in Figure 1): cultivating 48 h on nutrient agar panel, and bacterium colony is white, round, protrusion,
It is translucent, neat in edge.
(2) morphological features: Electronic Speculum observation is as shown in Fig. 2, cell is subsphaeroidal, amphitrichous.
(3) physio-biochemical characteristics: Gram-negative bacteria, when growth, are aerobic, catalase test, oxidase test reaction sun
Property, gelatin liquefaction test reaction negative.Its Physiology and biochemistry qualification result is as shown in table 1.
1 bacterial strain D-2 Physiology and biochemistry qualification result of table
| Physiological and biochemical property | As a result | Physiological and biochemical property | As a result |
| Gram's staining | ? | Catalase | + |
| Anaerobic growth | ? | Oxidizing ferment | + |
| Gelatin liquefaction | ? |
Note :-: negative reaction；+: positive reaction.
(4) 16S rDNA sequence and phylogenetic analysis: the 16S rDNA gene order length of bacterial strain D-2 is 1396 bp,
Be compared with ncbi database (http://www.ncbi.nlm.nih.gov/), find the bacterial strain D-2 withOchrobactrum intermediumWith good homology (> 99%), systematic evolution tree is as shown in Figure 3.
(5) Biolog Automatic Analyzer for Microbes is identified:
Biolog microbe auto-analysis refers to by determining that microorganism is more to sugar, amine, vinegar, acid, pure and mild macromolecule polyalcohol etc.
The utilization power of carbon source is planted to identify the category kind of microorganism.It will use a kind of 96 hole identification plates when identification, taken off containing there are many on plate
Water carbon source.Four azole redox coloring agents can be reduced to purple when carrying out respiration using carbon source by microorganism,
The microorganism characteristic reaction pattern or " fingerprint " are formed on identification plate.Instrument acquires after " fingerprint " of the microorganism and data
Storehouse matching obtains rapidly qualification result.
The Biolog Automatic Analyzer for Microbes qualification result of bacterial strain D-2 of the present invention shows, D-2 and intermediate anthropi
(Ochrobactrum intermedium) is the most similar.The results are shown in Table 2 for bacterial strain D-2 utilization of carbon source.
2 bacterial strain D-2 utilization of carbon source qualification result of table
| Utilization of carbon source | As a result | Utilization of carbon source | As a result |
| Negative control | ? | Dextrin | ? |
| D-Maltose | + | D- cellobiose | + |
| Gentiobiose | + | Sucrose | + |
| D- turanose | + | Stachyose | ? |
| D- melitriose | ? | α-D- lactose | ? |
| D- melibiose | ? | β-formyl-D- glucoside | ? |
| D- salicin | ± | N- acetyl-d-glucosamine | + |
| N- acetyl-β-D-MANNOSE amine | ? | N- acetyl-D-galactosamine | ± |
| N-acetyl-neuraminate | ? | Alpha-D-glucose | + |
| D-MANNOSE | + | D-Fructose | + |
| D- galactolipin | + | 3- formyl glucose | ? |
| D-Fructose | + | L- fructose | + |
| L- rhamnose | + | Inosine | ± |
| D-glucitol | + | PEARLITOL 25C | ? |
| D-arabitol | ± | Inositol | ± |
| Glycerol | ? | D-Glucose -6- phosphoric acid | ? |
| D-Fructose -6- phosphoric acid | ? | D-Asp | ? |
| D-Ser | ± | Gelatin | ? |
| Glycyl-L-PROLINE | ± | L-Alanine | ± |
| L-arginine | ± | L-Aspartic acid | ± |
| Pidolidone | + | L- histamine | + |
| L-Glutimic acid | ? | Serine | + |
| D- galacturonic acid | + | L- galacturonic acid lactone | + |
| Maltonic acid | ± | D-Glucose aldehydic acid | + |
| Glucuronamide | ± | Glactaric acid | ? |
| Quininic acid | ? | Saccharic acid | ? |
| P- Hydroxy-phenyl-acetic acid | ? | Methyl pyruvate | ± |
| D-ALPHA-Hydroxypropionic acid methyl esters | ? | Pfansteihl | ± |
| α -one-glutaric acid | + | D-malic acid | + |
| L MALIC ACID | + | Bromo- succinic acid | ± |
| Polysorbate40 | ? | Gamma-amino-butyric acid | ± |
| Alpha-hydroxy-butyric acid | ± | Beta-hydroxy-D, L- butyric acid | ± |
| α -one-butyric acid | ± | Propionic acid | ± |
| Acetic acid | + | Formic acid | ? |
| Positive control | + | pH 5 | ? |
| 1% NaCl | + | 4% NaCl | ? |
| 8% NaCl | ? | Sodium lactate | + |
| Fusidinic acid | ± | D-Ser | ? |
| Troleandomycin | ± | Rifamycin | + |
| Minocycline | ? | Cillimycin | + |
| Guanidine hydrochloride | + | Vancomycin | + |
| Tetrazolium violet | + | Tetrazolium blue | + |
| Nalidixic acid | ± | Lithium chloride | ± |
| Potassium tellurite | + | Aztreonam | + |
| Sodium butyrate | + | Sodium bromate | ± |
Note: "-" is expressed as negative reaction or does not utilize；"+" indicates positive reaction or utilization；" ± ", indicates the weak reaction of sun.
In conclusion passing through 16S rDNA sequence, morphological feature, physio-biochemical characteristics and Biolog to bacterial strain D-2
Automatic Analyzer for Microbes result is analyzed, by bacterial strain D-2 be accredited as intermediate anthropi (Ochrobactrum intermedium), and it is preserved in Guangdong Province's Culture Collection (GDMCC) on July 5th, 2018, deposit number is
GDMCC No:60409, preservation address are 5 building, the buildings of compound the 59th of Xianlie Middle Road, Guangzhou City 100.
The antibiotics sensitivity of 2 bacterial strain D-2 of embodiment is analyzed
In order to preferably study the acquisition of embodiment 1 bacterial strain D-2 Biocontrol Potential, our biological characteristics to the bacterial strain
It conducts in-depth research.Experimental study sensibility of the bacterial strain D-2 to different antibiotic, it is specific as shown in Figure 4.
The results showed that bacterial strain D-2 reaches 400 μ gmL to the resistance of ampicillin-1More than, to card, that is mould
The resistance of element reaches 350 μ gmL-1, 40 μ gmL are reached to the resistance of gentamicin, streptomysin-1, to the resistance of chloramphenicol
Reach 10 μ gmL-1, to tetracycline non-resistant.This result is conducive in follow-up study choose suitable antibiotic as ginseng
It examines.
3 bacterial strain D-2 substrate spectrum analysis of embodiment
1, strain culturing and sample acquire:
Under the conditions of 30 DEG C, bacterial strain D-2 is activated on LB solid plate.Picking single colonie is seeded to LB liquid medium,
With 30 DEG C, 200 rpm conditions are incubated overnight to obtain bacterium solution.Take the certain volume (OD of V=1/600) bacterium solution with 4000 rpm, 10
The condition of min is centrifuged, and is abandoned supernatant, is obtained an OD600The thallus of value.By 1 OD600The thallus of value is seeded to 1mL difference
In MSM minimal medium using different AHLs as sole carbon source.Reaction mixture is respectively placed in 2 mL centrifuge tubes
In, so that centrifuge tube is cultivated 24 h under conditions of 30 DEG C, 200 rpm.After 24 h, 5 μ L reaction mixture point samples to 1 cm are taken
Wide MM agar strip top, then in lower section point reporting bacterial strain CF11(Agrobacterium tumefaciens NT1) bacterium solution.
Then the agar strip of good reaction mixture and reporting bacterial strain is placed in 28 DEG C of incubators, is observed in fact after being protected from light 24 h of culture
Test result.Wherein, it is 40 μ gmL that agar strip, which is containing concentration,-1The MM plate of X-gal cuts gained.It is reported according on agar strip
Bacterial strain from top become blue distance may determine that acyl homoserine lactones in sample (AHLs) content number.So that it is determined that
Whether bacterial strain D-2 has quenching activity to different AHLs.
In test, be known to be quenched a variety of AHLs bacillus thuringiensis subsp israelensis (Bacillus thuringiensis subsp. israelensis) B23 be positive control (Dong Y, Xu J, Li X, et al.
AiiA, an 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.), the active Escherichia coli (Escherichia of AHLs is quenched not have
Coli) it is used as negative control.
2, test result is shown:
In OHHL(Fig. 5 a), OOHL(Fig. 5 b), OdDHL(Fig. 5 c) in test group, CK and negative controlE.coliAHLs diffusion
Distance is consistent, i.e., AHLs content is close in the two reaction mixture；Report on the agar strip of positive control B23 and experimental group D-2
The unchanged indigo plant of bacterial strain is accused, AHLs is not contained in the reaction mixture of the two, AHLs has been degraded by bacterial strain D-2 complete.Show bacterium
Strain D-2 all has quenching activity to AHLs signaling molecule OHHL, OOHL, OdDHL.
The measurement of 4 bacterial strain D-2 of embodiment growth and degradation OHHL relation curve
1, picking bacterial strain D-2 single colonie is inoculated in preculture to logarithmic phase in LB culture medium and takes the certain volume (OD of V=1/600)
Bacterium solution is centrifuged with the condition of 4000 rpm, 10 min, is abandoned supernatant, is obtained an OD600The thallus of value.Thallus is seeded to
In 20 mL MSM culture mediums, and OHHL mother liquor is added, make 0.4 mM of its ultimate density, 30 DEG C, cultivates timing under 200 rpm
Sampling.It acquires the sample of different time points and measures OD600Value indicates the growing state of bacterial strain D-2, utilizes HPLC measurement OHHL's
Residual quantity indicates bacterial strain D-2 to the degradation situation of OHHL.
2, HPLC measuring method
HPLC:Waters 2695.Chromatographic column: Kinetex EVO C18 reverse-phase chromatographic column (250 μ m, 4.6 mm × 5 μ
M).Flow velocity: 0.5 mLmin-1.Column temperature: 30 DEG C.Mobile phase: acetonitrile: water=30:70(v:v).Detection wavelength: 210 nm.Sample introduction
Amount: 20 μ L.
3, interpretation of result:
As shown in fig. 6, in 32 h, OHHL's HPLC testing result has been degraded completely.
In conclusion the degradation of 3OC6HSL is positively correlated with bacterial strain D-2 growth, in the presence of OHHL, strain growth does not have
There is demurrage, quickly enters growth logarithmic phase.The bacterial strain is most fast to the degradation of OHHL in 0 ~ 24 h, strain culturing to 32 h,
OHHL is decomposed completely.Natural degradation rate in control in 24 h of OHHL is less than 40%.The result shows that D-2 pairs of intermediate anthropi
OHHL has significant and quick degradation, has huge application potential in terms of the pathogenic bacteria harm that prevention and treatment OHHL is mediated.
5 bacterial strain D-2 of embodiment studies the biocontrol effect of bacterial soft rot of potato
The present embodiment is to cause the pathogen carrot soft rot Pectinatus carrot subspecies of bacterial soft rot of potato
(Pectobacterium carotovorum subsp. carotovora, Pcc) and Z3-3(Liu Chun is beautiful, and Guo Song, Ai Li is spat
Heat gram, is waited in acinetobacteraidEGene encoding homoserine lactonase [J] bioengineering journal, 2017,33 (9):
For 1625-1639.), research bacterial strain D-2 is to the biocontrol effect for relying on the pathogenic pathogenic bacteria of AHLs.
In inoculation test, bacterium D-2, Escherichia coli is quenchedE.coli, bacillus thuringiensis B23 be safety it is non-pathogenic
Strain.
1, bacillus thuringiensis B23, Escherichia coliE.coli, bacterial strain D-2, carrot soft rot Pectinatus Z3-3 scribing line
In on LB solid plate, 30 DEG C are cultivated.Pick them separately the single colonie on plate, be seeded in LB liquid medium, with 30 DEG C,
The condition of 200 rpm is incubated overnight, and obtains bacterium solution.
With liquid LB by B23,E.coli, bacterial strain D-2 and pathogenic bacteria Z3-3 bacterium solution adjust to 1.0 × 107 cfu·mL-1.By Z3-3 respectively with B23,E.coli, D-2 and LB liquid medium be mixed in a certain proportion, and 5 μ L mixed bacteria liquids distinguished
It is seeded on potato.LB+Z3-3, Z3-3+ is respectively set E.coli, Z3-3+B23, Z3-3+D-2, four experimental groups.It will
Inoculated potato is placed on 28 DEG C of biochemical cultivation cases, its incidence is observed after 24 h and is taken pictures.
2, result is as shown in fig. 7, Z3-3+LB and Z3-3+E.coliThe potato tubers lesion area of experimental group is compared with Z3-3
+ B23 and the potato onset area of Z3-3+ D2 experimental group are significantly greater, fall ill more serious。Bacterium D-2 and pathogenic bacteria are quenched
Co-inoculation is substantially reduced compared with soft rot Disease symptoms when being individually inoculated with pathogenic bacteria.The experimental results showed that bacterial strain D-2 is to pectin bar
Bacterial soft rot of potato caused by bacterium Z3-3 has preferable biological control effect.
6 bacterial strain D-2 of embodiment studies the biocontrol effect of carrot soft maize ear rot
The present embodiment is to cause the pathogen carrot soft rot Pectinatus carrot subspecies of carrot soft maize ear rot
(Pectobacterium carotovorum subsp. carotovora, Pcc) and Z3-3(Liu Chun is beautiful, and Guo Song, Ai Li is spat
Heat gram, is waited in acinetobacteraidEGene encoding homoserine lactonase [J] bioengineering journal, 2017,33 (9):
For 1625-1639.), research bacterial strain D-2 is to the biocontrol effect for relying on the pathogenic pathogenic bacteria of AHLs.
1, bacillus thuringiensis B23, Escherichia coliE.coli, bacterial strain D-2, carrot soft rot Pectinatus Z3-3 scribing line
In on LB solid plate, 30 DEG C are cultivated.Pick them separately the single colonie on plate, be seeded in LB liquid medium, with 30 DEG C,
The condition of 200 rpm is incubated overnight, and obtains bacterium solution.
With liquid LB by B23,E.coli, bacterial strain D-2 and pathogenic bacteria Z3-3 bacterium solution adjust to 1.0 × 107 cfu·mL-1.By Z3-3 respectively with B23,E.coli, D-2 and LB liquid medium be mixed in a certain proportion, and 5 μ L mixed bacteria liquids distinguished
It is seeded on carrot fleshy root slice.LB+Z3-3, Z3-3+ is respectively set E.coli, Z3-3+B23, Z3-3+D-2, four
Experimental group.Inoculated carrot slice is placed on 28 DEG C of biochemical cultivation cases, its incidence is observed after 24 h and is taken pictures.
2, result is as shown in figure 8, Z3-3+LB and Z3-3+E.coliThe plant tissue of the carrot fleshy root of experimental group is
Through rotting, and only there is water stain shape scab in the carrot fleshy root of Z3-3+ B23 and Z3-3+ D2 experimental group, does not occur plant group
Rotten symptom is knitted, that is, carrot soft maize ear rot when bacterium D-2 is more individually inoculated with pathogenic bacteria Z3-3 with pathogenic bacteria Z3-3 co-inoculation is quenched
Disease symptoms are substantially reduced.The experimental results showed that bacterium D-2 is quenched to carrot fleshy root soft rot caused by Pectinatus Z3-3
With preferable biological control effect.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. intermediate anthropi (Ochrobactrum intermedium) in micropopulation induction signal molecule is quenched
Using, or the application in the product that micropopulation induction signal molecule is quenched in preparation.
2. intermediate anthropi relies on the application in the pathogenic plant disease of micropopulation induction signal molecule mediation in prevention and treatment,
Or the application in terms of preparation relies on the prevention and treatment preparation of the pathogenic pathogenic bacteria of micropopulation induction signal molecule.
3. one kind can be quenched degrading microorganism colony induction signaling molecule intermediate anthropi (Ochrobactrum intermedium) bacterial strain D-2, which is characterized in that the bacterial strain is preserved in Guangdong Province's Microbiological Culture Collection on July 5th, 2018
Center (GDMCC), deposit number are GDMCC No:60409, and preservation address is No. 59 building 5 of compound of Xianlie Middle Road, Guangzhou City 100
Building.
4. application according to claim 1 or claim 2, which is characterized in that the intermediate anthropi is intermediate described in claim 3
Anthropi strain D-2.
5. application according to claim 1 or claim 2, which is characterized in that the micropopulation induction signal molecule isNAcyl group
Homoserine lactone substance.
6. applying according to claim 5, which is characterized in that the micropopulation induction signal molecule is middle long-chain
AHLs。
7. applying according to claim 5, which is characterized in that the micropopulation induction signal molecule is AHLs signal
Molecule OHHL, OOHL and/or OdDHL.
8. a kind of method that prevention and treatment relies on the pathogenic cause of disease fungus diseases of micropopulation induction signal molecule, which is characterized in that use
The bacterium solution of intermediate anthropi carries out inoculation processing to crop.
9. what one kind can be quenched micropopulation induction signal molecule is quenched microbial inoculum, which is characterized in that contain intermediate anthropi
And/or its bacterium solution.
10. a kind of biological prevention and control agent for relying on the pathogenic pathogen of micropopulation induction signal molecule, which is characterized in that in containing
Between anthropi and/or its bacterium solution.
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